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The regulation of intracelMar calcium ion concentration and mitochondrial fvnction by cyclosporh A:
a putative mechanisrn for the pathogenesis of gingival overgrowth
Livia Silvestri
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Dentistry
University of Toronto
O Copyright by Livia Silvestri 2000
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Abstract
The regulation of intracellular calcium ion concentration and mitochondrial function by cyclosporin A: a putative mechanisrn for the pathogenesis of gingival overgrowth, by Livia Silvestri. Degree: Master of Science, Department of Periodontology, Faculty of Dentistry, University of Toronto, 2000
Gingival overgrowth is associated with loss of homeostasis of collagen
metabolism çubsequent to the administration of several drugs, including the
immunosuppressant cyclosporin A. Currently, the mechanisms by w hich CsA induces
gingival overgrowth have not been resolved. My hypothesis is that the induction of
gingival overgrowth by cyclosporin A (CsA) involves the deregulation of collagen
turnover due to a disturbance of calcium utilization in the mitochondria of gingival
fibroblasts. CsA (1 0nM) inhibited collagen bead phagocytosis (Ffold; p c 0.05) in
cultured human gingival fibroblasts (HGF) and Rat2 cells. Thapsigargin (Tg; 1 FM;
pc0.01) and carbonyl cyanide mchlorophenyi hydrazone (CCCP; 10 FM; pe 0.02) also
inhibited phagocytosis of collagen beads, indicating the importance of mitochondrial
function in phagocytosis. Collagen-coated beads (CCB) induced equivalent increases
of intracellular calcium [ca2'li in HGF and Rat2 cells. Tg inhibited the collagen-bead
induced [ca2']i response 3.7-fold. Pre-treatment with CsA inhibited collagen-induced
[ca2'li and [ca2']sERCA res ponses. Collagen bead-induced p hagocytosis induced a tirne-
dependent decrease in [~a~'],i~, in both Rat2 cells and Rat2 cells pre-treated with CsA.
CsA prevented the loss of mitochondrial membrane potential of cells in suspension
indicating that CsA was indeed acting upon the mitochondrial permeability transition
pore (MPTP). Phagocytosis was decreased in mitochondria-depleted Rat2 cells
compared to Rat2 cells by Bfold ( ~ ~ 0 . 0 5 ) and by at least 2-fold when they were pre-
treated with CsA (pe 0.05). Collectively, these data indicate that CsA acts at an
intracellular locus where it: (i) inhibits calcium flux through the MPTP; (ii) inhibits calcium
exchange between the SERCA and mitochondria; and (iii) inhibits collagen
phagocytosis through a mitochondrial-dependent, calcium-regulated pathway. Future
work could focus on how the interaction of mitochondria and SERCA calcium stores
contributes to the anti-phagocytic activity of CsA.
Ackno wledgements
This is truly a project that could not have been accomplished without the
intellectual leadership, technical insight and friendship of my supen/isor, Dr. Christopher
McCulloch. The exceptional generosity that he demonstrated with his tirne and
knowledge enriched my experience as a graduate student. I am also grateful for the
technical assistance of the laboratory staff, including Pam Arora for fluorescence
microscopy, Wilson Lee for flow cytometry, Cheung Lo for the culture of mitochondria-
depleted cells and Elisabeth Lukse for immunoblotting.
I would like to thank rny family for al1 their support, encouragement, concern and
love over the years. The cornfort and security that stems from a caring farnily has
allowed me to achieve rny goals. For his unfailing motivation and understanding, I thank
rny fiancé, Frank Nicolais.
iii
Table of Contents
Abstract Acknowledgements List of Tables List of Figures List of Ab breviations
1. Literature Review
A. Periodontal Diseases
1 . l nflammatory periodontal diseases
2. Drug-induced lesions a) Drugs which cause gingival overgrowth b) Cyclosporin A
3. Common mechanisms for fibrosis a) The developrnent of fibrosis
B. Mechanisms of phagocytosis in fibroblasts
1. How is phagocytosis mediated?
2. Physiological regulation a) Growth factors and cytokines b) Integrins
C. Calcium regulation of phagocytosis
1. Calcium ion regulation
2. Calcium response to collagen phagocytosis a) calcium response within the mitochondria
3. Mitochondrial calcium metabolism
4. Calcium responses to CsA
II m..
Ill
v i vii
D. Mode1 systems and rationale
II.
III.
IV*
v.
VI.
VI I
Staternent of the Problem
Materials and Methods
Results
Discussion
Conclusions
References
List of Tables
Table 1: Drugs that induce gingival overgrowth
Table II: Propeities of the most common drugs which induce gingival overgrowth
Table III: Adverse effects of CsA therapy
List of Figures
Figure 1 :
Figure 2:
Figure 3:
Figure 4:
Figure 5:
Figure 6A:
Figure 6B:
Figure 6C:
Figure 7A:
Figure 78:
Figure 7C:
Figure 7D:
Figure 8:
Figure 9A:
Figure 9B:
Examples of possible activation mechanisms of T and B lymphocytes.
Maintenance of collagen homeostasis.
Regulation of collagen degradation.
Pathways for calcium transport in mitochondria.
Model system.
Flow cytometry - Analysis of the collagen bead binding step of phagocytosis after incubation with cyclosporin A in human gingival fibroblasts (HGFs).
CsA (10 nM) causes a marked decrease in the phagocytic capacity of HGF.
CsA causes a dose-dependent inhibition of phagocytosis.
Collagen-coated beads (CCBs) cause an equivalent rise of [ca2'li in human gingival fibroblasts (HGFs) and Rat2 cells.
Treatment with increasing concentrations of CsA induces dose-dependent increases of [ca2+li in HGF.
The addition of collagen-coated beads (CCBs) to fibroblasts in a calcium-containing medium induces a substantial calcium flux (-1 30 nM ca2') which is reduced 3.4-fold by CsA (1 0 nM).
Pre-incubation of Rat2 fibroblasts with CsA induces decreased [ca2'lsERCA response to collagen-coated beads.
Effect of agents that inhibit calcium signalling on phagocytosis in Rat2 fibroblasts.
Co-localization of MitoTracker Green and rhod 2 dyes in Rat2 fibroblasts.
The addition of collagen-coated beads to Rat2 and Rat2 fibroblasts treated with 10 nM CsA induces a time-dependent decrease in [~a~+]+l,ito.
vii
Figure 10: CsA prevents the loss of mitochondrial membrane potentiai of Rat2 fibroblasts in suspension.
Figure 1 1 A: Treatment of Rat2 fibroblasts with ethidium bromide for over 30 passages of continuous culture induces inhibition of mitochondrial DNA synthesis and reduced synthesis of the mitochondria-associated protein COX 1.
Figure 1 1 €3: Phagocytic capacity of Rat2 and Rat2EtBr fibroblasts.
Figure 12A: Incubation with CCCP inhibits [ca2+li response to collagen-coated beads (CCBs) in both Rat2 and Rat2EtBr fibroblasts.
Figure 128: Pre-incubation with 10 nM cyclosporin A (CsA) induces a large decrease in [ ~ a ~ + ] ~ ~ ~ ~ ~ in Rat2 cells but not in Rat2EtBr cells responding to CCBs.
Figure 13A: Stimulation with collagen-coated beads (CCBs) in Rat2EtSr fibroblasts stained with rhod 2/AM induces a decrease in [ ~ a ~ + ] ~ i ~ ~ of the few remaining mitochondria. 64 and Rat2EtBr.
Figure 138: CCBs induce decreased [~a*+ ]mi~~ in Rat2 and RatPEtBr. 64
Figure 13C: Addition of CCBs to Rat2 or Rat2EtBr fibroblasts pre-treated with 10 nM CsA induces decreased [ ~ a ~ ' ] m i ~ ~ . 64
Figure 14: Schematic diagram of fibroblasts with and without CsA treatment.
viii
BAPTA
CCB
CCCP
COX -1
DMSO
HGF
MPTP
PBS
SERCA
mitochondrial membrane potential
dibromo-i,2-bis-(eaminophenoxy)ethane-N,N,N', N I tetraacetic acid
intraceilular calcium concentration
rnitochondrial calcium concentration
SERCA calcium concentration
collagen-coated beads
carbonyl cyanide mchlorophenyl hydrazone
cyciosporin A
cytochrorne oxidase I
dimethylsulphoxide
human gingival fibroblasts
using 5,5',6,6'-tetrachloro-l , 1 '3,3'- tetraethylbenzirnidazolecarbocyanine iodide
membrane permeability transition pore
phosphate buffered solution
Rat2 cells treated with ethidium bromide
smooth endoplasmic reticulum ca2+ ATPase pump
I. Literature Re vie w
The specific problem addressed in this thesis is the mechanism of action of
cyclosporin A (CsA) on mitochondrial calcium signalling and collagen phagocytosis in
the context of CsA-induced gingival overgrowth. As any one of these specific topics
entalis a large literature which cannot be surveyed in detail due tu çpace restrictions, I
will focus this review on those studies which are specific to the central problem. I will
begin with an introduction on the main forms of drug-induced gingival overgrowth and a
discussion of the specific characteristics of CsA as an immunosuppressant and a
rnodulator of gingival overgrowth. A brief overview of the mechanisms for fibrosis will
lead into a discussion of collagen phagocytosis and its regulation. As the ionic
regulation of phagocytosis is a lengthy topic to review, this discussion will be restricted
to regulation of phagocytosis by calcium. This last topic will be expanded to include
calcium responses to collagen phagocytosis within different organelles, rnitochondrial
calcium metabolism, and calcium response to CsA. I will conclude the review with a
discussion of collagen phagocytic model systems and their rationale.
A. Periodontal Diseases
1. lnflammatory periodontal diseases
Periodontal diseases encompass a group of conditions which affect the
su pporting structures of the teeth including alveolar bone, root cementum, periodontal
ligament, and gingiva. The clinical signs associated with periodontal diseases may
include gingival inflammation, gingival fibrosis, epithelial ulceration and resorption of
alveolar bone. A recent classification of these diseases indicates that 'gingival
diseases' are separate from 'periodontitis'. This view is based primarily on the absence
of bone resorption with gingival diseases. As the gingiva can exhibit many altered
features with disease, gingival diseases are segmented into smaller groups based on
their etiology. Gingival diseases modified by medications include the drug-influenced
gingival enlargements (Armitage, 1 999).
Although the most common cause of gingival enlargement is an inflammatory
response to bacterial plaque (Loe, 1965), there are systemic factors which predispose
to enlargement. Specifically, the administration of certain drugs produces gingival
overgrowth (Carranza, 1 996). Kimball (1 939) first described drug-induced gingival
overgrowth due to phenytoin and until 1981 this continued to be the only drug
associated with enlargement. Subsequently, other drugs, specifically anticonvulsants,
calcium channel blockers and the immunosuppressant cyclosporin A, have been shown
to produce gingival overgrowth as a side effect. Notably, gingival overgrowth was
referred to as 'gingival hyperplasia' before histological examination revealed normal
numbers of fibroblasts surrounded by an abundance of collagen (Bonnaure-Mallet et ai.,
1995)
2. Drug-induced lesions
a) Drugs which cause gingival overgrowth
The categories of drugs which are associated with gingival overgrowth are
anticonvulsants, calcium channel blockers, and immunosuppressants (which only
includes CsA). Table I provides a list of common drugs within these categories. I will
provide a brief overview of one drug from each category to introduce a possible
common link with calcium metabolism in their mode of action.
Table 1. Drugs that induce gingival overgrowth
Drug Category 1 Agent 1 Trade name . --
Anticonvulsant Hydantoins
Succinimides
Val~roic acid
Calcium channet blockers Dihydropyridine derivatives
Benzothiazine derivatives
Phenylal kylamine de rivat ives
Ethotoin Mep henytoin Phenytoin EthosuxirnIde Methsuximide P hensuxirnide Valproic acid Cyclosporine A
Amlodipine
Felodipine Nicardi pine Nifedipine
Nimodipine Nisooldipine Nitrendipine Diltiazem
Verapamil HCI
?aganone@ ~ e s a n t o i n ~ ~ilantin" ~erontin@ celontinm Not reoorted@
~otrel@ ~orvasc@ plendi? cardene@ ~dalat@ procardia@ Nimotop@ suiar@ Not reported ~ardizern" Dilacor XR@ ~iazac@ calan@ lsoptine verelana Covera HS@
Phenytoin is an anticonvulsant which blocks or interferes with calcium influx
across cell membranes. This in turn stabilizes neuronal discharge and limits the
progression of neuronal excitation, thus depressing the motor cortex of the central
nervous system (Wilson and Kornman, 1996). Nifedipine is a calcium channel blocker
used prknarily to treat acute and chronic coronary insufficiency, including angina
pectoris and refractory hypertension (Hancock and Swan, 1992). CsA is a potent
immunosuppressive drug used primarily to prevent rejection after organ transplantation
and for treatment of autoimmune diseases (Calne et al., 1979). While the exact
immunoçuppressive mechanism of action of CsA is not completely understood, it
appears selectively and reversibly to inhibit T helper cells by interfering with the
response of these cells to an elevation of intracellular calcium ([câ2+li ) (Granelli-Piperno
et a/., 1984).
The characteristic clinicat picture of drug-induced gingival overgrowth usually
includes corn bined enlargement and inflammation. This overgrowth consists of a
primary expansion of connective tissue and epithelium which is related to the effects of
the drug and a secondary inflarnmatory component. Due to the enlargement, the
gingival sulcus is deepened which impedes effective hygienic measures. Consequently
the entire lesion takes on an inflammatory appearance because of increased
colon izat ion by su b-g ingival plaque. Consequently, the pockets (or pseudopocks?r)
become deeper, the inflammatory response is more severe, and the area becomes
more difficult to debride (Carranza, 1996). CsA-specific gingival overgrowth is also
characterized by initial enlargement of the interproximal papillae which is frequently
restricted to the anterior facial areas and may include partial coverage of the crowns.
The tissue is usually pink, dense, and resilient, with a stippled or granular surface.
There is little bleeding on probing (Raeste et al., 1978). Table II summarizes the
epidemiological and clinical properties of phenytoin, nifedipine, and CsA.
Clinical management of gingival fibrosis is difficult as the treatment often employs
surgical removal of excess tissue. This approach only addresses the signs and does
not control the source of the problem. Recurrence is common as long as drug
administration persists and treatment often consists of repeated surgical excision. If the
mechanism of fibrosis associated with gingival overgrowth were to be unraveled,
treatment outcornes could likely be improved.
b) Cyclosporin A
CsA is an immunosuppressant used after bone marrow and organ
transplantation that causes gingival overgrowth (Kahan, 1984). There is controversy
with respect to the role of plaque in CsA-induced gingival overgrowth. McGaw et al.
(1 987) reported that plaque-induced gingival inflammation exacerbated the expression
of CsA-induced gingival overgrowth. However, this notion was contested by Seymour
et al. (1987) who concluded that the magnitude of CsA-induced gingival enlargement is
related more to the plasma concentration of CsA than to the patient's periodontal status.
CsA was first studied as an immunosuppressant by Borel et al. (1976). Subsequent to
successful clinical trials in the 1970s, CsA became the drug of choice to prevent
rejection after organ transplantation. The trade name of CsA was originally
andi immune? A new oral formulation ( ~ e o r a n has been developed which is better
absorbed from the digestive systern and can be monitored by blood drug
Table Il. Properties of the most cornmon drugs which induce gingival overgrowth
CsA
Phenyfoln
Niledipine
Drug
17% il CsA blood levels c 400 ndmL, 59% il CsA b l d levels 400 nglrnL (Helti el al., 1094) 70% over 2.5 years (Daley el
al., 1986) 81% (Friskopp and
Kllnlmalm, 1986)
varies from 3% Io 84.5% (Angetopoulos and Goaz, 1072; Glkkman and Lewilus, 1941; Panuska et al, 1961)
Incidence
15% Io 83% (krak el al.. 1 087; Barclay et al., 1892; Fattore et al., 1991 ; Slavin and Taylor, 1987)
Greater risk for development in adolescents and young lemales ( Daley et al., 1986; Hetli el al., 1994; Seymour and Heasman, 1988)
Gender or Age Predilectlon
more wmmon among younger patients and Instilulionalized people (Babcock, 1965; DahHoI and Modder, 1986)
spontaneous disappearance within lew monlhs after disconlinualion 01 phenyloin
Re verslbility
mild improvemenl within 1 week ol dixonlinuation ol nitedipine, reinslilulion ol dru9 lollowing wilhdrawal resuits in recurrence within 4 weeks (Lederman el al., 1984)
rnarked reduction ol grngival enlargement requm much longer (Lainson, 1986)
no recurrence alter extraction 01 teeth (Raieilschak-Pluss el al., 1983) not presenl in edentulous
patients (Friskopp and Klinimalm, 1986)
Presen t in edentulous sites
no1 presenl in edantulous spaces and the overgrowth disappears when leeth are extracied enlargement in
edentulous mouths is rare (Oreyer el al.. 1978)
does no1 aflect edenlulous areas ( Lucas et al., 1985; Wilson and Kornman. 1996) enlargemenl seen about
dental implanls (Silverstein el al., 1995)
Dose which causes overgro wlh
CsA blood levels z 400 n! (Helti el al., 1994; Seymou~ Heasman, 1988)
CsA >5M) mgiday (Daley i sas) magnitude ot CsA-induce
gingival enlargement is reli more loo Ihe plasma concentralion lhan Io the p, periodonlal stalus (Seymoi al., 1987) some reports show a dire
relationship belween tho d ot gingival enlargement am ol Ihe phenytoin (Kapur et a
1973) others show thal the occu
and severily ol gingival enlargemenl are not neces rdated Io the dosage, concentration of phenytoin serum or saliva, of duration drug ttierapy
Rate of recurrence
blood levels >800
llmL r and
el al..
d tted
,itienI's II et
1: t egree 1 dose ,dl.,
rrenco
sarily
in i 04
combined drug therapy (concomitant adminislration il niledipirie) is a significanl risk faclor for recurrence (Rossmann et al., 1994)
recurrence aiîer surgical removal of gingival enlargement can be altenuated with dillgenl plaque control (Hall, 1969) , but no1 entirely eliminated (Slaple el al., 1978)
levels more accurately. Lindholm and Kahan (1 993) showed that a 25% increase in the
bioavailability of CsA resulted in a significant increase in the one-year graft survival rate
and the number of patients who were rejection-free. As such, NeoralQ, which is
absorbed more predictably and is not degraded by bile acids, is the preferred form of
CsA (Sells, 1994).
CsA is a highly hydrophobic cyclic endecapeptide which binds to many types of
cells with low affinity (16 = 1-5 x 10 '~ M). Since lymphocytes do not possess specific
receptors for CsA, and as its binding affinity is similar to that of phospholipid vesicles,
LeGrue et al. (1 983) suggested that the hydrophobic CsA molecule intercalates into the
phospholipid bilayer of the cell membrane and is split up into intracytoplasmic lipid
droplets to facilitate crossing of the plasma membrane.
Histopathological analysis of CsA-induced gingival overgrowth reveals excessive
collagen covered by a parakeratinized, multilayered epithelium with elongated rete pegs
(Carranza, 1996). The connective tissue may appear highly vascularized with foci of
chronic inflammatory cells (Rateitschak-PIüss, 1 983), especially plasma cells (Deliliers
et al., 1986) that are presumably a response to sub-gingival plaque.
The mode of action of CsA as an immunosuppressant has been investigated and
relies on its capacity to inhibit proliferation of activated B and T lymphocytes. However
the mechanisrn of CsA-induced gingival overgrowth has not been clarified and there is
speculation on the role of CsA as an inhibitor of the membrane permeability transition
pore in mitochondria (see below).
Both arms of the lymphocytic immune response are suppressed by CsA. T-
lymphocyte-dependent immunity (cell-mediated) and B lymphocyte-dependent
immunoglobulin synthesis (humoral immunity) are inhibited by CsA (Dongworth and
Klaus, 1982), i.e., upon stimulation, these cells respond less robustly in mounting an
immune response. Lymphocytes must be activated to respond appropriately to stimuli
and two important cytokines responsible for their activation are IL-2 and 11-4, for T and
B lymphocytes, respectively. After IL-2 or IL-4 bind to their cell surface receptors,
phosphatidylinositol bisphosphate is cleaved into diacylglycerol and inositol-1,4,5-
triphosphate (Isakov et al., 1987). Diacylglycerol can contribute to the downstream
activation of protein kinase C which then induces lymphocyte activation. lnositol-1,4,5-
triphosphate induces lymphocyte activation by elevation of cytoplasmic calcium through
release of calcium from interna1 cellular stores (King, 1988) and the maintenance of
calcium stores by leaking extracellular calcium through the cell membrane (Zilberman et
ai., 1987). In view of these findings, many investigators have tried to establish a locus
of action for CsA as an irnmunosuppressant based on its role as a modulator of [ca2'li .
CsA has been reported to not inhibit the generation of calcium signals from
intracellular (Kay et al., 1983) or extracellular sources (Gelfand et al., 1987). Based on
in vitro studies, its irnmunosuppressive behavior may be dependent on the availability of
calcium in the culture medium andlor on its ability to enter the cell (Kay, 1987).
Further, lymphocyte proliferation does not generally occur without generation of a
calcium signal. Therefore it was suggested that CsA inhibits proliferation of activated T
and B lymphocytes by interfering with calcium permeability of the cell membrane (Figure
1). Since IL-2 is an important activator of T lymphocytes, and as this process is
dependent on a rise of [ca2+li, the finding of highly selective inhibition of IL-2 synthesis
by CsA in activated T lymphocytes was significant (Granelli-Piperno et ab, 1 984).
Figure 1. Examples of possible activation rnechanisms of T and B lymphocytes
Phosphatidyi in itol bisphosphate
f i Diacylglycerol lnositol-l,4,5-trisphosphate
Lymphocyte activation
Emrnel et al. (1989) characterized the CsA-induced 11-2 suppression further and
demonstrated that transcription of 11-2 was arrested due to the inhibition of
dephosphorylation of cytoplasmic nuclear factors, (e.g. NF-AT). These factors are
necessary for transcription of 11-2. CsA binds cyclophilin, a low molecular weight
protein. Cyclophilin and CsA inhibit activation of calcineurin, a calcium- and calmodulin-
dependent phosphatase which inhibits dephosphorylation of nuclear factors
(Hanschumaker et al., 1984). If this process is the route by which CsA causes
immunosuppression, then addition of exogenous IL-2 should reverse the
immunosuppression as shown by Bunjes et al. (1981). However others have shown
only a partial recovery of T lymphocyte activation after addition of 11-2 (Kay and Benzie,
1986). Therefore other signalling intermediates may be sensitive to CsA.
Aside from its main use as an immunosuppressant, CsA unfortunately exhibits
many adverse effects in vivo (Table III). While one of the more innocuous side effects is
gingival overgrowth, these lesions provide a biological system which can be analyzed in
vitro and which suggests alternative modes of action. Apart from its action on
lymphocytes, other possible cellular functions of CsA in cells have been investigated
and an important locus of cell function seems to be centered on mitochondria.
Accordingly, in cells with increased calcium and phosphate ion concentrations,
mitochondria may undergo a permeability transition in the conductance of mitochondrial
membrane channels which results in the loss of small molecules such as glutathione
and calcium. This loss can lead to mitochondrial dysfunction. CsA inhibits these
processes (Savage et al., 1991). Crompton et al. (1 988) showed that CsA binds to
cyclophilin D, a mitochondrial protein that mediates opening of the permeability
Table 111. Adverse effects of CsA therapy
Side Effect 1 Comments
Nep hrotoxicity
Hypertension Hepatotoxicity
Infection Malignant neoplasms
Dermato logical
Neurological toxicity
Gingival overgrowth
-dose-dependent -exact mechanism is unclear -reversible with dose reduction -may be secondary to nephrotoxicity -usually dose-dependent -usually reversible -related to toxicity of graft rejection -due to immunosuppression -CsA is not carcinogenic -due to imriiunosuppressive EBV proliferation - CsA enhances production of TGF-P which promotes cells to become cancerous (Nabel, 1999) -doseodependent hypeitrichosis on the face and upper trunk in 80°h of patients -tremor which resolves with dose reduction -convulsions associated with concornmitant use of steroids in children -dose-dependent
Adapted from Thiru, S., 1989, pgs 324-359.
transition pore in the mitochondrial membrane (MPTP). This interaction inhibits opening
of the MPTP thus preventing transit of small molecules and in consequence preventing
the loss of the mitochondrial membrane potential as well. If CsA regulates the traffic of
small molecules within the mitochondria, and if mitochondrial ion homeostasis is
important in the regulation of collagen metabolism, CsA may play a role in the
dereçlilation of collagen secretion or degradation seen with gingival overgrowth.
An irnbalance between collagen synthesis and collagen degradation may lead to
drug-induced gingival overgrowth (Jones, 1986). Many investigators have studied the
effects of overgrowth-inducing drugs on collagen synthesis and the results have been
inconclusive (Newell and Irwin, 1997; Bartold, 1989; McGaw and Porter, 1988; Tipton et
al., 1991 ; McGaw and Ten-Cate, 1983). Drug-induced effects on collagen degradation
have also been investigated. Tipton et al. (1 991) demonstrated a dose-dependent
decrease in collagenase activity after CsA administration, but conflicting studies from
lngman et al. (1 994) reported that matrix metalloproteinase-1 (MMP-1) and MMP-3
cannot be detected in inflamed marginal gingival tissue of patients with periodontal
disease. Thomason et al. (1998) demonstrated that MMPs are not detected within
fibroblasts from overgrown gingiva. As collagen degradation is mediated by both a
collagenase-dependent extracellular pathway and a collagenase-independent
intracellular pathway (i.e. phagocytosis), CsA-induced deregulation of collagen
phagocytosis may be important in the etiology of gingival overgrowth. Accordingly , the
mechanisms of fibrosis and phagocytosis will be reviewed below and the possible
deregulation of these processes by CsA will be introduced.
3. Cornmon mechanisms for fibrosis
a) The developrnent of fibrosis
Fibrosis is defined as the excessive deposition of matrix components that results
in distortion or disruption of normal tissue architecture and compromised tissue function.
The development of fibrosis follows a similar pathway to that of normal wound healing
but resolution (or scarring) is impaired (Mutsaers et a/., 1997). Fibrosis can be induced
by several growth factors, including insulin-like growth factor-l (Goldstein et al., 1989)
and transforming growth factor Pl (Border et al., 1992). After the administration of CsA,
the expression of both insulin-like growth factor-l and transforming growth factor Pl is
increased, thus prornoting fibrosis (Johnson et al., 1999). As mentioned above, gingival
fibrosis may be the result of an increased rate of collagen synthesis and/or decreased
rate of collagen degradation. One aspect of degradation which rnay be significant in the
context of CsA-induced gingival overgrowth is intracellular degradation by phagocytosis.
P hagocytosis is particularly important in tissue remodelling under p hysiological
conditions such as the final stages of wound healing or during scar resolution. In both
examples, the degradation of wound collagen and other matrix proteins by phagocytic
cells is mediated by phagocytosis (Miganatti et al., 1996). In pathological conditions,
such as fibrosis, the rate of collagen phagocytosis may be decreased, thereby resulting
in net collagen overgrowth.
When phenytoin- or CsA- induced fibrosis were exarnined at the ultrastructural
level with stereologic methods, both Hall and Squier (1982) and McGaw and Porter
(1 988) observed a decreased amount of phagocytosed collagen within the gingival
cells. McCulloch and Knowles (1993) provided further N, vitro support for these
observations when phagocytosis of collagen-coated beads was inhibited in fibroblasts
treated with phenytoin or nifedipine.
B. Mechanisms of phagocytosis in fibroblasts
1. How is phagocytosis mediated?
Gingival overgrowth is a term which describes the excessive accumulation of
collagen in affected tissues (Figure 2). As such it is useful to examine the mechanisms
of collagen turnover in physiological conditions.
The fibroblast is the cell responsible for the synthesis and secretion of collagen in
gingival overgrowth, however, the fibroblast can also act as a resorptive cell when it
degrades collagen by phagocytosis. lndeed phagocytosis by gingival fibroblasts is the
main mechanism of physiological collagen degradation in vivo (Melcher and Chan,
1981) and due to the ubiquitous occurrence of collagen phagocytosis by fibroblasts,
they may be considered as professional phagocytes (Rabinovitch, 1995).
Collagen degradation may be mediated by matrix metalloproteinases (MMPs;
e.g. collagenase, gelatinase, and stromelysin), cysteine proteinases (e.g. cathepsin B
and L), or serine proteinases (e.g. plasmin and plasminogen activator). As collagen
degradation is a common process in many physiological and pathological events, it is
not surprising that the remodeling of collagen exhibits wide variations of rate. For
example, MMPs have been implicated in rapid remodeling or digestion of large amounts
of collagen during the loss of tissue architecture in acute infiammatory processes
(Birkedal-Hansen et al., 1993), tumor cell invasion and metastasis (Aznavoorian et al.,
1993), and uterine involution (Brandes and Anton, 1969). This
Figure 2. Maintenance of collagen homeostasis
MMPs p hag ocytosis
Collagen Metabolisrn in the Balance
pathway is also called the collagenase-dependent extracellular pathway (Everts and
Beertsen, 1988; Sodek and Overall, 1988).
MMPs are not usually associated with equilibrium conditions in which collagen
synthesis is balanced by collagen degradation. More cornmonly, physiological turnover
of soft connective tissues involves phagocytosis of collagen fibrils and their final
intracellular lysosome-associated degradation by cysteine proteinases. This pathway
can be regulated by growth factors and cytokines and is called the collagenase-
independent intracellular pathway (Everts and Beertsen, 1988; Sodek and Overall,
1988).
Although gingival collagen does undergo rapid turnover, this does not lead to the
net loss of tissue because the collagen turnover is in a steady-state (Figure 2). Several
studies have demonstrated that collagen degradation in physiological conditions
employs the intracellular route. Svoboda et al. (1981) showed that cross-banded
collagen fibrils were present in fibroblast lysosomes, especially in gingiva and
periodontal ligament. They also demonstrated a positive correlation between the
arnount of intracellular collagen and the rate of collagen turnover. Further, calculation of
the digestion time of internalized collagen (30 minutes); (Everts et al, 1989) is consistent
with an expected half-life of collagen corresponding to the turnover time as assessed
biochemically (Everts and Beertsen, 1 988; Everts et ai., 1 989). Finally, Liu et al. (1 995)
used mutant mice with collagenase-resistant type I collagen to show that the
collagenase-dependent extracellular pathway played an insignificant role except during
periods of extensive connective tissue turnover. As mice aged, signs such as fibrosis of
the derm is and im paired postpartum involution of the uterus were evident. Therefore,
when steady state collagen turnover predominates, as is the case during growth and
development, the int racellular collagenase-inde pendent pathway prevailed, but when
rapid collagen degradation is necessary, the extracellular collagenase-dependent
pathway prevai!ed (Figure 3).
The collagenase-independent intracellular pathway of collagen phagocytosis
involves an initial recognition of the fibril by membrane-bound receptorslintegrins
(McKeown et al., 1990). This binding step initiates actin filament remodeling (Isberg
and Tran Van Nhieu, 1995) and an increase in the expression and release of gelatinase
A (Larjava et al., 1993). This step is followed in turn by segregation and partial
digestion of the fibril andlor its surrounding non-collagenous proteins by matrix
netalloproteinases and finally lysosomal fusion with the phagosome containing partially
digested collagen fibrils and eventual degradation by cysteine proteinases within the
lysosomes.
2. Physiological Regulation
a) Growth Factors and Cytokines
Van der Zee et ai. (1995) examined the effect of several growth factors and
cytokines on phagocytosis in vitro. They concluded that only two substances had an
effect on phagocytosis: (i) interleukin-lu inhibited phagocytosis and (ii) transfoning
growth factor+ enhanced phagocytosis. When combined, they were antagonistic.
Chou et al. (1 996) investigated the role of tumor necrosis factor-a and showed in hibition
of collagen adherence and phagocytosis.
Figure 3. Reg ula tion of collagen degradation
MMPs p haggcytosis
einterleukin -1 +concanavalin A +turnor necrosis factor- a +transforming growth factor+ +concanavalin A * tumor necrosis factor- a +b transforming growth factor-8 a
Hormones may also influence phagocytosis: (i) cortisol decreases collagen
phagocytosis in pig synovial tissue (Fell et al., 1986) and (ii) increased collagen
phagocytosis was associated with post-partum uterine involution (Brandes and Anton,
1969), a process which is associated with increased progesterone.
b) lntegrins
Integrins are ceIl surface heterodimeric adhesion receptors which bind to
extracellular matrix proteins. The integrins on fibroblasts which are involved in
recognition, adhesion, and signalling in response to collagen are the alPl, ct2P1, and
-Bi (Kirchhofer et al., 1990). However, in gingival fibroblasts the a2B1 is considered to
be particularly important for the process of collagen phagocytosis (Lee et al., 1996).
Using flow cytometry, they initially showed that a2 expression was ubiquitous among
phagocytic cells and collagen phagocytosis was inhibited by blocking access to the
a& The blockade of other integrins did not alter phagocytosis. To investigate the rote
of a2B1, after binding of collagen, fibroblasts were incubated with a large number of
collagen-coated beads to occupy as many a2P1 binding sites as possible. The levels of
a2 and phagocytic efficiency were decreased. Evidently, collagen must bind to the a2
subunit before being internalized. Evidence was also presented to suggest that the a:!
subunit was internalized in the process of collagen phagocytosis as bead internalization
led to a decrease in surface levels of an, which were restored within 3 hours. Further,
Chou et al. (1 996) showed that turnor necrosis factor-a inactivated a&, thereby
inhibiting collagen binding and phagocytosis. Despite these data, the presence of a2p1
alone is not sufficient to mediate phagocytosis. This is not surprising as other collagen
receptors rnay regulate different activities. Lee et al. (1996) suggested that the a1
subunit rnay regulate the initial contact of a2 with collagen since the level of collagen-
coated beads was shown to be twice as high in cells with high al staining than with high
a2 staining. The efficiency of phagocytosis was also dependent on the number of
available collagen-coated beads and a2 . With low bead:cell ratios, the presence of
apP1 did not necessarily promote phagocytosis, suggesting an effect due to collagen-
bead availability. Further, phagocytosis seemed to be inhibited unless the a2P1 integrin
was present in at least 60% of cells.
C. Calcium Regulation of Phagocytosis
1. Calcium ion regulation
Calcium levels are very tightly controlled within the cell since intracellular calcium
concentrations are approximately 100 nM and yet extracellular calcium concentration is
- 1-2 mM. To maintain calcium homeostasis the cell possesses many pathways of
regulated entry and exit for calcium but there are fundamentally two sources of calcium.
Calcium can be released from interna1 stores within the cell or can be allowed to enter
the cell from the extracellular medium when channels in the plasma membrane are
more permeable. Although the studies on calcium signalling reviewed below refer
mainly to "profcssional" phagocytes, namely macrophages and neutrophils, Rabinovitch
(1995) notes that due to the ubiquitous occurrence of collagen phagocytosis by
fibroblasts, they rnay also be considered as professional phagocytes. In the absence of
a substantial data set on calcium signalling in fibroblasts, I review data on macrophages
and neutrophils to provide some background on calcium signalling in phagocytosis.
Murine macrophages were used by Young et al. (1 984) to illustrate the need for
tight control of [ca2']i in the execution of phagocytosis. The clamping of both
intracellular and extracellular calcium to low levels led to the inhibition of phagocytosis.
When only extracellular calcium was chelated, there was only a partial decrease of
phagocytosis. This underlines the need for calcium fluxes from intracellular stores as
well. An excessive increase in [ca2']i by ionomycin treatrnent also led to inhibition of
phagocytosis. After binding of ligand to receptors, [ca2+]i was increased and the
amplitude of this increase was directly proportional to the extent of receptor
aggregation. Kobayashi et al. (1 995) obtained similar results in human neutrophils. To
induce maximum phagocytosis, both calcium entry from the extracellular medium and
release from intracellular stores were necessary.
From both of these reports it is evident that the same controls that are used to
maintain calcium homeostasis in the absence of a phagocytic target must be maintained
to execute phagocytosis. Although individual steps of phagocytosis may seem to
promote separate rises or falls of calcium in different cell compartments, many types of
cells contain signalling networks that involve a great deal of intracellular communication
(Berridge et al., 1999)
Kwiatkowska and Sobota (1 999) have described the signalling events for
phagocytosis specific to Fcy receptors. Once the receptor binds ligand, a phagocytic
signal is generated and the cytoskeleton is rernodelled to form a phagosome. Receptor
binding induces the activation of tyrosine kinase and protein kinase C. Phosphorylation
of cytoplasmic residues on Fcy serves to promote further binding of more ligand and the
phagocytic signal is amplified. In macrophages, protein kinase C accumulates around
phagosomes (Allen et al., 1996) and isoenzymes of the kinase are activated by calcium
(Mochly-Rosen and Gordon, 1998). Consequently, calcium can amplify the phagocytic
signal further, but these observations are in the context of Fcy signalling and not
integrins.
After formation of the phagosome (which contains the internalized particle, the
integrin and parts of the plasma membrane), the phagosome fuses with a lysosome
w hich will provide the acidic environment necessary for degradation by proteases.
Fusion is regulated by calcium. For example in human neutrophils, Jaconi et al. (1990)
showed a requirement for an increase in [ca2'li to promote fusion of phagosomes and
lysosomes. Initially, Bengtsson et al. (1 993) suggested that [ca2']i does not control the
initial actin polymerization seen during receptor-ligand adhesion and subsequent
phagocytosis, but [ca2'li does control the depolymerization of the actin network so as to
promote fusion of the phagosome and lysosome. Definitive evidence on this issue has
not been obtained because experimental-specific and cell-type variations of fusion
control preclude direct comparisons.
2. Calcium response to collagen phagocytosis
In the previous section, 1 have examined the regulatory effect of calcium on
phagocytosis. In this section the effect of calcium on phagocytosis in different cellular
compartments will be reviewed. Within the cytosol, the formation of receptor-ligand
complexes and pseudopods generates rises in [ca2+li within seconds. In ongoing
phagocytic processes, [ca2'li remains elevated especially around phagosomes. As
expected there is no rise in [ca2+> when both intracellular and extracellular calcium was
chelated (Bengtsson et a l , 1993). Thus [ca2+li rises do not originate solely from
intracellular stores. It is important to consider also the uptake of extracellular calcium
within endosomes. In fact, Gerasimenko et al. (1998) showed that endosomes
delivered a substantial amount of calcium into the cell within a few minutes.
Direct evidence to illustrate the behaviour of calcium within the endoplasmic
reticulum during phagocytosis is not available but several groups have shown a physical
movement of the receptors involved in calcium regulation during phagocytosis (Favre et
al., 1996). Calcium release from intracellular stores is mediated through inositol-1,4,5-
triphosphate (InsP3) and ryanodine receptors. Calcium accumulation in stores is
mediated by sarcoplasmic/endoplasmic reticulum ca2+-~~pases , some of which are
inhibited by thapsigargin. During phagocytosis in HL-60 cells, the SERCA2 receptor,
calreticulin (a calcium-buffering protein)(Stendahl et ai., 1994) and the InsPs receptor
were translocated to the periphagosomal space (Favre et al., 1996). This suggests that
calcium within the ER may play a regulatory role in phagocytosis since the receptors
which regulate calcium levels have translocated in order to remain proximal to the
phagosome and can thereby act upon it.
Putney (1986) first developed the notion that the process of emptying and
replenishing calcium stores must be linked. He proposed that empty calcium stores
must somehow trigger the opening of store-activated channels (SOCs) in the plasma
membrane and allow calcium into the cell. He thus coined the term 'capacitative
calcium entry'. The question of how the signal of empty calcium stores is transmitted to
the SOCs is still unanswered although lrvine et al. (1990) and Berridge et al. (1995)
suggested a mechanism called 'conformational coupling'. When lnsP3 binds to the
lnsPs receptor on the endoplasrnic reticulum, calcium is released from the internal
stores. The lnsPs receptor then sustains a conformational change. It is this change
that is detected by the SOCs which are located physically close to InsP3 receptors.
lndeed van Rossurn et al. (2000) have shown that the interaction of lnsPs receptors and
SOCs control calcium entry through the plasma membrane is possibly due to their close
approximation.
a) Calcium response within the mitochondria
There is a paucity of literature on the mitochondrial calcium response due to phagocytic
stimuli. However, there is indirect evidence describing the interaction and
interdependence between calcium in the mitochondria and the endoplasmic reticulum.
Subsequent to depletion of calcium in internal stores, ATP is needed to drive the refilling
of stores by the SERCA (Gamberucci et al., 1994). This energy is derived from the
energy-producing mitochondria (Gamberucci et ai., 1 994). An additional mitochondria-
endoplasmic reticulum interaction exists in the context of the lnsPs and ryanodine
receptors. These receptors indirectly influence [ ~ a ~ + ] ~ due to their capacity to release
calcium from internal stores when bound. The extent to which they cause calcium
release depends on the calcium buffering capacity of mitochondria (Bezprozvanny et
al., 1991). A lower [ca2+li will lead to binding of the lnsPs and ryanodine receptors and
thus cause release of calcium into the cytosol whereas a higher [ca2+]i leaves the
receptors unbound (Landolfi et al., 1998). These results demonstrate that mitochondrial
function is important for appropriate calcium responses within the cell and is needed to
amplify this response.
3. Mitochondriai calcium metabolism
Mitochondria have piqued the interest of researchers as these are the only
organelles (apart from the nucleus) that possess their own genetic material and are also
the essential "powerhouses" of the cell. They are excitable organelles capable of
generating calcium signals and actively participate in cellular calcium signalling (Ichas et
a l , 1997; Babcock et ai., 1997). There are several pathways for calcium influx and
efflux as shown in figure 4. The predominant pathways for calcium uptake consist of
the calcium uniporter and the rapid mode of uptake. Calcium transport by the calcium
uniporter is dependent on the rnitochondrial membrane potential that can be generated
by redox-linked H' pumps. Consequently the more that H' is pumped out of the
mitochondria, the greater is the negative charge inside the mitochondria. To preserve
the potential across the mitochondrial membrane, more ca2' can be pumped into the
mitochondria and removed from the cytosol. The calcium uniporter is thus driven by an
electroc hemical gradient comprised of a negative mitochondrial inner membrane
potential and a low concentration of calcium in the matrix. The rapid mode uptake also
transports calcium into the mitochondria but seems to show more specificity when
dealing with brief physiologic stimuli (Bernardi, 1999).
There are three pathways for calcium efflux, the Na+-independent pathway for
ca2' efflux (NICE), the Na+-dependent pathway for ca2' efflux (NCE), and the
membrane permeability transition pore (MPTP). The NICE is possibly a H'/ ca2+
antiporter which requires a transmembrane potential as part of its driving force. It
conducts very slow movernent of ions and mediates steady-state calcium cycling in
Adapted from Bernardi, 1999. Figure 4.. Pathways for calcium transport in mitochondria. H+ pump, redox-linked H+ pumps; JN, Ca2+ uni porter; Rab rapid uptake mode; NICE, Na* -independent pathway for Ca2+ efflux; NCE, Na+ "dependent pathway for Ca2+ effiux; NHE, H+/Na* exchanger; MPTP, membrane permeability transition pore, CsA, cyclosporin A; cyp, cyclophilin; [Ca2+], ; mitochondrial Ca2+ concentration; [Ca2+], ; cytosolic Ca2+ concentration t4
O\
mitochondria. The NCE
cycling through interplay
is a ~ a ' / ~ a ~ + exc hanger that mediates physiolog ical calcium
with the NHE (H'I Na' exchanger). The MPTP is a large
channel with a pore diameter of -3 nM (Haworth and Hunter, 1979). When the pore is
open, this is described as a permeability transition and solutes with molecular mass I
1500 Da are allowed to pass into and out of the mitochondria. The pore opening
depends on transmembrane potential difference, matrix pH, and mitochondrial calcium
concentration ([ca2'lm). Prolonged opening of the pore leads to dissipation of the
mitochondrial membrane potential, leakage of cytochrome c and ultimately cell death.
This can be simulated in vitro with the addition of carbonyl cyanide mchlorophenyl
hydrazone (CCCP), a protonophore which causes a short circuit of the electrochernical
potential for protons. As a result, the ion concentration gradient is dissipated and the
electrical potential can no longer be maintained. The pore favours opening with
increases of [ca2'lm and closing with CsA. As mentioned above, CsA induces
cyclophilin D to detach from the MPTP (Nicolli et al., 1996) and thus facilitates closure
of the MPTP. In view of their involvement in the maintenance of calcium levels and their
interaction with the endoplasmic reticulurn, mitochondria evidently play an important role
in calcium homeostasis and intracellular communication. lndeed using aequorin to
target and report mitochondrial calcium ([ca2'lmit0) specifically, mitochondria can sense
microdomains of high [ca2+lc which result when IP3-sensitive channels release calcium
from the ER (Riuutto et al., 1993).
As described above, CsA inhibits the MPTP and by doing so inhibits the passage
of cations via the MPTP. As a result, calcium either cannot exit the mitochondria using
this pathway or the rate of efflux is slowed down. Since dissipation of the mitochondrial
membrane potential can lead to cell death, CsA can prevent this event and this has
been shown in vitro in gingival fibroblasts (Kulkarni et al., 1998). Sullivan et al. (1999)
showed that the addition of CsA to cells subjected to traumatic brain injury is
neuroprotective by preventing mitochondrial dysf unction. When they looked at individual
synaptosomes, there was a lower in CsA-treated cells than controls. They
proposed that because CsA inhibits the MPTP, less calcium is allowed to leave the
mitochondria and this prevents the cycling of calcium across the mitochondrial
membrane using other channels. As the effect of CsA on [ ~ a ~ ' ] ~ ~ ~ ~ does not involve
inhibition of calcineurin (which is the case with immunosuppression; Nicolli et al., 1996),
the immunosuppressive effects of CsA and its ability to inhibit the MPTP are likely
separate. To date, no other mitochondrial functions other than inhibition of the MPTP
appear to be affected by CsA (Bernardi et al., 1994; Zoratti and Szabo, 1995).
4. Calcium responses to CsA
The response of [ca2']i to CsA has received little attention although rat cardiac
cells show an upregulation of angiotensin II receptors and an increased [ca2+], that is
independent of the irnmunosuppressive action of CsA, (Le., not dependent on
calcineurin; Avdonin et a/., 1999). They proposed that these data could explain CsA-
induced hypertension and nephrotoxicity due to the vasoconstriction induced by an
augmentation of angiotensin II receptors. In addition, the sarne group examined the
effect of CsA treatment on human vascular smooth muscle cells but did not detect any
change in [ca2']i .
Whereas the effect of CsA on the rnitochondria was determined to be separate
from its action as an immunosuppressant in that it binds calcineurin, this is not the case
in the sarcoplasmic reticulum. In rat hearts, CsA mediates the upregulation of
calcineurin. By binding more calcineurin, there should be reduced phosphorylation.
Park et al. (1999) used this finding to explain a decrease in the maximal ryanodine
binding and an increaçe in the dissociation constant (&) of ryanodine binding. Since
calcium-release channels in the sarcoplasrnic reticulum need to be phosphorylated in
order to function, decreased phosphorylation due to higher levels of calcineurin leads to
less ryanodine binding and consequently less calcium release from the interna1 stores.
Model systems and rationale
An in vivo model to quantify collagen phagocytosis using electron microscopic
stereology was used by Svoboda et al., (1 981). After obtaining a magnified version of a
stained micrograph of the area of interest, two grids were overlayed on this micrograph.
With the coarse grid, the point intersections was used to estimate the number of areas
falling on cytoplasm and extracellular collagen and the fine grid was used to count the
num ber of areas falling on individual collagen profiles. This analytical system provides
a good approach to assess collagen phagocytosis in vivo, but it is both labour-intensive
and carries many assumptions on uniformity of intracellular transport, intracellular
degradation rates and cellular homogeneity. In contrast, the collagen-coated bead
model is an in vitro system used in conjunction with flow cytometry. A cell which has
engulfed a collagen-coated fluorescent bead will report a larger fluorescence intensity
per cell. The advantages of using this model are; (i) the ability to analyze a large
number of samples in a very short time; (ii) the lack of bias as to whether phagocytosis
has occurred and; (iii) the ability to report how many beads are internalized per cell.
(McKeown et al., 1 990).
As mentioned above, in vivo models are very labour-intensive and cannot
provide kinetic analysis of the data. As well, investigators do not agree on whether
incomplete phagocytosis (i.e. just cytosegregation) should be considered a positive
finding. Svoboda et al., (1 981) argued that cytosegregation should be included as this
was a necessary step for phagocytosis to occur. An important limitation of the bead
model is that it is an in vitro reductionist system and, consequently some component(s)
of the more complex in vivo setting may be omitted.
McCulloch and Knowles (1993) used the collagen-coated bead model to show
that cells from fibrotic lesions have decreased phagocytic efficiency. They also
demonstrated a reduction in phagocytosis when control cells were treated with fibrosis-
inducing drugs, including phenytoin. These findings provide the basis for my studies.
11. Statement of the problem
Literature on the cellular effects of CsA suggests a defined mechanism for its main
action as an immunosuppressant. However the modes of action which mediate rnany of
the adverse effects resulting frorn CsA administration have not been described.
Gingival overgrowth is an adverse effect that can be studied in vitro using cultured
gingival fibroblasts. Notably, fibroblasts are central to the processes of collagen
synthesis and degradation and study of collagen metabolism and its deregulation by
CsA is a logical approach to understanding the pathobiology of this disorder.
CsA acts on the mitochondrial permeability transition pore and consequently
affects calcium transport. This is relevant as two other groups of drugs which cause
gingival overgrowth also affect calcium metabolism (i.e. ant iconvulsants and calcium
channel blockers). As a result, I have considered a role for calcium in the mechanisrn of
CsA-induced gingival overgrowth. My hypothesis is that the induction of gingival
overgrowth by cyclosporin A involves deregulation of collagen turnover due to a
disturbance of calcium utilization in the mitochondria of gingival fibroblasts. The
principal rationale for this study is that the mechanism by which CsA induces gingival
overgrowth is not known and consequently, rational approaches to treatment are not
possible. If CsA regulates [ca2+] and/or mitochondrial functions, this may suggest a
possible locus of action at which gingival overgrowth is initiated.
To test my hypothesis, I investigated the effect of CsA on the phagocytic capacity
of human gingival and Rat2 fibroblasts. Next, 1 studied the effect of CsA on calcium
regulation in different cellular compartments including the cytosol, mitochondria, and
SERCA stores. I used mitochondria- depleted cells to explore the functional
relationship between mitochondrial calcium regulation in other cell compartments and
phagocytosis. My specific objectives were to:
1. Determine if CsA can affect the phagocytic capacity of human gingival fibroblasts
and Rat2 fibroblasts and if mitochondria are important for phagocytosis.
2. Establish an in vitro system to measure CsA regulation of [ca2'li in gingival
fibroblasts and Rat2 fibroblasts responding to collagen-coated beads.
3. Determine if perturbation of calcium signalling in the cytosol and in mitochondria
regulates collagen bead internalization.
4. Evaluate [~â~+],il, and [ca2'lsERCA and their responses to collagen bead stimulation
and CsA deregulation and determine if integrin-induced calcium fluxes require
mitochondrial function and [ ~ â ~ ' ] , i ~ ~ fluxes.
111. Materials and Methods
Materials
Carbonyl cyanide mchlorophenyl hydrazone and cyclosporin A were obtained from
Sigma (St. Louis, MO). Anti-human cytochrome oxidase subunit I mouse monoclonal
antibodies (clone # 6403), BAPTNAM, fura UAM, JC-1, mag-fura 2/AM, MitoTracker
Green, ~ l u r o n i c ~ F-127 and rhod 2/AM were obtained from Molecular Probes (Eugene,
OR). lonomycin and thapsigargin were obtained from Calbiochem (LaJolla, CA).
Fluorescent and non-fluorescent 2 pm diameter polystyrene beads were obtained from
Polysciences (Warrington, PA) or from Molecular Probes (Eugene, OR). Bovine
collagen (>95% type 1) was obtained from Celtrix (Palo Alto, CA).
Methods
Cell Cultures Human gingival fibroblaçts (HG F; 8- 1 5'"assage) were derived from primary explant
cultures as described (Pender and McCulloch, 1991). Cells from passages 8-15 were
grown as monolayer cultures in T-75 flasks (Costar, Mississauga, ON) containing alpha
minimal essential medium, 1 5% heat-inactivated fetal bovine serum (v/v; Flow
Laboratories, Maclean, VA), and a M O dilution of an antibiotic solution (0.17% wtlvol;
penicillin V, 0.1 Oh (wüvol) gentamicin sulfate, 0.01 pg/mL amphotericin). The cells were
maintained at 37°C in a humidified incubator containing 5% CO2 and were passaged
with 0.01 % trypsin (Gibco BRL, Burlington, Ontario, Canada). Twenty-four hours before
each experiment, cells were harvested with 0.01% trypsin and -50,000 cells were
plated onto 0.1 -mm-thick, 31 -mm-diameter, round glass coverslips (VWR, Toronto,ON)
in 35-mm-diameter Petri dishes (Falcon, Becton Dickinson, Mississauga, Ontario,
Canada) for calcium measurements. Rat2 cells (ATCC CR1 1764; Amencan Type
Culture Collection, Rockville, MD) were incubated in Dulbecco's modified Eagle's
medium (DMEM; high glucose) containing 10% fetal bovine serum (FBS) and a 1 :10
dilution of the same antibiotic solution described above. The cells were propagated,
maintained and prepared for calcium measurements as described above. According to
the ATCC, Rat2 cells are phenotypically normal by al1 criteria tested and are diploid.
They have been used in these experiments because of their ease of passaging and
their phenotypic similarity to human periodontal cells based on collagen synthesis,
collagen phagocytosis, osteopontin synthesis and morphology (Hui et ai., 1997).
Mitochondrial Depletion
Rat2 cells were grown in Dulbecco's modified Eagle's medium (DMEM; high glucose)
containing 10% fetal bovine serum (FBS) and a 1 :10 dilution of the same an antibiotic
solution described above. Cells were grown in the presence of 100 ng/mL of ethidium
bromide for 20 passages as described (Biswas et al., 1999). The dose was increased
gradually to 1 O00 ng/mL between passages 21 -30. The cells were maintained at 1000
ng/mL of ethidium bromide for at least 4 weeks before using for experiments.
[~8][ measuremen t
For measurernent of whole cell [ca2+li, cells on glass cover slips were incubated at 37°C
with 3 pM fura 2/AM in bicarbonate-free medium for 25 minutes. The attached cells
were washed twice with bicarbonate-free calcium buffer which contained 145 mM NaCI,
5 mM KCI, 5 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, and 1 mM CaC12, with pH
adjusted to 7.4 and an osmolarity of 291 mosM. CaCI2 was omitted from the buffer
solution where indicated. Inspection of cells by fluorescence microscopy demonstrated
no cornpartrnentalization of dye nor were estimates of [ca2'li changed by loading of
cells with fura 2AM in Pluronic detergent. Consequently, estimates of [ca2+li using
these loading methods very likely represent cytoplasmic [ca2'li and not simply calcium
ion concentration in membrane-bound compartments such as endosomes.
Measurements of [ca2'li were made on single cells using a Nikon Diaphot II inverted
microscope optically interfaced to an epifluorescence spectrofluorimeter and analysis
system (Photon Technology International, London, ON) operating on a 386SX personal
computer. Fura Bloaded cells were excited at alternating (approximately 100 Hz)
wavelengths of 346 and 380 nm from dual monochromators with slit widths set at 2 nm.
Emitted fluorescence was collected by a 40X quartz 1.30-NA oil-immersion Nikon Fluor
objective and passed throug h a 520/30-nm barrier filter (Omega Optical, Brattleboro,
VT). Signais from the photomultiplier tube (mode1 D104; Photon Technology) were
recorded at 2 5 points/s. A variable-aperture intrabeam mask was used to restrict
measurements to single cells.
Estimates of [ca2'li independent of the precise intracellular concentration of fur&
were calculated from dual-excitation ernitted fluorescence according to the equation of
Grynkiewicz et al. (1 985) (Le., [ca2+li = & x Sf2/Sb2 x (R-R,e)/(Rm, - R) (nM), where R
is fluorescence ratio, R,, is maximum R, Rmin is minimal R, and SfZSb2 is the ratio of
the fluorescence intensity of the free and bound dye at 380-nrn excitation). The
dissociation constant (Kd ; 305 nM) and SfZSb2 ratio (4.09) were calculated from 11
excitation wavelength scans of 1 pM fura 2 free acid (Molecular Probes) in buffers with
[ca2'] ranging from 0.0 to 39.8 mM (ca2' calibration kit; Molecular Probes). R,, of
3461380 was measured after saturation of intracellular fura 2 with ca2+ by adding 3 pM
ionomycin to allow equilibration with extracellular ca2+. R,in was measured in
ionomycin-treated cells after complete dissociation of fura 2 from [ca2'li by adding 2
mM EGTA to the calcium buffer bathing the measured cell and was estimated at 0.568.
Dye loading with mag-fura U-4M
Dye loading and data collection is similar to that described for fura 2/AM except that
intensity values are not converted to [ c ~ ~ + ] ~ ~ ~ ~ A values. Instead, the ratio of
fluorescence intensity collected at 3461380 nm was used to calculate changes in
[ca2*lsERCA as described previously (Hofer et a l , 1 998).
Dye loading with rhod-UA M
RhodQ is a single wavelength excitation and single wavelength emission dye. As ratio
imaging cannot be used to overcome problems of dye leakage and photobleaching over
time with this dye, the fluorescence intensity of rhod-2 was measured in small ( ~ 4 ~ r n ~ )
sampling grids in the lamellopodia of well-spread cells. Nucleoli (which stain brightly
with rhod 2) were not included in the measurement. Background fluorescence and
adjustments for time-dependent photo-bleaching were made for each measurement
calculating the bleach-induced rate constant (Le. time-dependent loss of fluorescence)
in untreated cell samples that were not treated with collagençoated beads.
Dye loading and coimalization with rhod-2, MitoTracker Green and JC-1
Rat2 fibroblasts were CO-loaded with 100 nM MitoTracker Green and 4.5 pM Rhod-2/AM
in 0.005% pluronic gel for 30 minutes in a 37°C incubator containing 5% CO2. The
cells were washed twice and left in calcium buffer before irnaging. The magnitude of
fluorescence of stained samples was observed on an inverted microscope (Nikon)
equipped with a CCD camera (Pentamax) and analyzed using Winview (Princeton)
software program or photographs were taken.
Collagen coating of beads
Experiments with collagen-coated beads were conducted with cells grown in T-25 tissue
culture flasks. All bead incubations with cells were conducted at 37OC with 5% CO2 ,
95% humidity. Aliquots (80 pl) of either yellow-green fluorescent (excitation max = 490
nm, emission rnax = 515 nm) or non-fluorescent 2 prn diameter polystyrene beads
were incubated with 1 mL of a 2.9 mg/mL acidic bovine collagen solution to produce
collagen-coated beads. To neutralize the collagen solution to pH=7.4 and thereby
induce fibril assembly on beads, an aliquot of 1 N NaOH was added to 1 rnL of collagen
with beads and mixed by vortexing. The beads were incubated at 37OC for 10 minutes,
followed by centrifugation at 8000 x g for 2 minutes. The supernatant was removed and
the pellet of coated beads was resuspended in 1 mL phosphate-buffered saline (PBS).
After sonication of the coated beads to produce single bead suspensions, the number of
beads per mL was estimated by counting beads in an aliquot of the sample with a
hernocytometer. Cells were incubated with collagen-coated fluorescencebeads at a
ratio of 2 beads per cell for phagocytosis experiments and assessed by flow cytometry.
For calcium experirnents, non-fluorescent collagen-coated beads were added to cells at
a ratio of -4 beads per cell.
Flow cytometric analysis of phagocytosis
Binding of collagen-coated beads to cells was assessed by flow cytometry. Cell
samples were analyzed with a FACStar Plus flow cytorneter (Becton Dickinson FACS
Systems, Mountain View, CA) at a sheath pressure of 11 psi with an Innova 70 argon
laser (Coherent Laser Products, Palo Alto, CA) at a light regulation mode setting of 250
mW and a wavelength of 488 nm. Emitted fluorescence was directed through a short
pass 560 beam splitter (al1 filters and beam splitters from Omega Optical Inc.,
Brattleboro, VT) and a 530DF30 filter for green fluorescence in channel 1.
Photomultiplier tube voltage settings were determined for each experiment on the basis
of thresholds established from negative and positive control samples for each sample
that was analyzed. To reduce the inclusion of cells with loosely bound beads during the
process of phagocytosis measurernents, cells were washed with calcium and
magnesium - free PBS and trypsinized.
Flow cytometric analysis of mitochondrial membrane potential fi).
Changes in the Ym were analyzed using 5,5',6,6'-tetrachloro-1 ,113,3'-
tetraethylbenzimidazolecarbocyanine iodide (JC-1). This cyanine dye accumulates in
the mitochondrial matrix under the influence of the Ym and forms J aggregates which
have characteristic absorption and emission spectra (Smiley et al., 1991). Untreated
cells and cells treated with CsA were incubated in 3 mL of PBS supplemented with 10%
serum containing 0.5 pM JC-1 for 1 hour. As a positive control for reduction of Y,, a
group of cells were treated with the uncoupling agent carbonyl cyanide m-
chlorophenylhydrazone (CCCP) after labeling with JC-1 (Zamzami et al , 1995). Cell
suspensions were prepared for flow cytometry (Kulkarni and McCulloch, 1995) and the
488-nm line of an argon ion laser was used for excitation. Orange and green emitted
fluorescence were collected through 585142 (FL2)- and 530130-nm (FLI) bandpass
filters. The FLI-FL2 compensation was 4% and the FL2-FL1 compensation was 44%.
Flow cytometry analysis was performed on a FACSTAR Plus flow cytometer and
analyzed using LYSYS II software (Becton-Dickinson lmmunocytochemistry Systems,
Moutainview, CA). After gating out srnall-sized (i.e. noncellular debris), 10,000 events
were collected for each analysis. Bivariate plots of FL2 versus FLI were used to
analyze Y, . Fonvard scatter was used to estimate relative cell volume. Based on
foward and side scatter, and JC-1 fluorescence levels of untreated Rat2 fibroblasts,
cells were gated into high and low FL1 and FL2 populations. The percentages and
mean fluorescence intensities of these populations were cornpared to establish the
effect of increasing concentrations of CsA.
lmmunoblotting
For assessments of the mitochondrial-specific protein cytochrome oxidase 1, the
immunoblotting methods of Marusich et ai. (1997) were used. Whole ce11 extracts were
prepared by rinsing trypsinized Rat2 cells with calcium and magnesium free (CMF) PBS
containing protease inhibitors (0.5 pg/mL leupeptin, 0.5 pglmL pepstatin, and 1 mM
PMSF). Cell pellets were solubilized for 30 min at 5 x 106 cells/mL in CMF-PBS
containing the above protease inhibitors plus 1.5% lauryl maltoside at 4OC, centrifuged
for 20 minutes at 16,600 x g, and the supernatants were saved. Equivalent amounts of
protein, determined by Bradford assay, were separated on 10% polyacrylamide gels
and transferred electrophoretically ont0 0.2 pn nitrocellulose membranes in CAPS
transfer buffer (1 0% methanol in 1 0 mM 3-[cyclo hexylaminoj-1 -propanesulfonic acid, pH
11) on ice. The blots were blocked with 5% non-fat dried milk powder in CMF-PBS,
incubated overnight at 4OC with anti-human cytochrome oxidase-1 monoclonal antibody
diluted in 5% milk CMF-PBS to 1:1000, washed with 0.05% Tween in CMF-PBS,
incubated for 1 hour in horse radish peroxidase conjugated goat anti-mouse IgG at 0.2
pg/mL in 5% milk CMF-PBS and washed with 0.05% Tween in CMF-PBS. Blots were
processed for cherniluminescence, exposed to X-ray film, developed and analyzed.
Data Analysis
Means and standard errors of the means were computed for phagocytic, calcium, and
mitochondrial membrane potential experiments. Comparisons between two groups
were perfoned with the unpaired Student's t-test. Corn parisons between more than
two groups were done using ANOVA.
Mode1 System - Fibroblast
Figure 5. Model system. Upon interaction with mitochondria, CsA will alter the calcium permeability of this organelle which in turn is likely to impact on calcium fluxes in the SERCA. The central question is: what is the effect of this perturbation on collagen phagocytosis? CC8 - collagen-coated beads; CsA - cyclosporin A; SERCA - smooth endoplasrnic reticulum calcium ATPase pump.
1 V. Results
Effect of CsA on collagen bead phagocytosis
In HGF incubated with collagen-coated beads, there was abundant binding of
beads that was inhibited -2-fold with as low as 10 nM CsA (1 hour pre-incubation
with CsA; 3 hour bead incubation; Fig. 6A). The phagocytic capacity of HGFs
treated with CsA was compared to that of Rat2 fibroblasts treated with CsA to
rationalize the use of Rat2 fibroblasts for ail subsequent experiments. Rat2 cells
were incubated with increasing doses of CsA prior to three-hour incubations with
collagen-coated beads, a protocol that enables quantitative evaluation of the
bead-binding step of phagocytosis. After the addition of 10 nM CsA, there was a
significant and comparable reduction in mean O h phagocytosis in both HGFs
(pc0.05) and Rat2 fibroblasts (pc0.05; Fig. 6B). CsA doses which are
substantially lower than those used therapeutically in humans (-200yM)
produced a significant decrease in the phagocytic capacity of Rat2 fibroblasts (pc
0.05 for 10-10,000 nM CsA; Fig. 6C). Dissipation of the mitochondrial membrane
potential using CCCP ( ~ ~ 0 . 0 2 ) or inhibition of ATP formation using sodium azide
also led to significant decreases in phagocytosis similar to those seen with CsA
(pc 0.01), suggesting an involvement of mitochondrial function in the bead-
binding step of phagocytosis.
[ca2+li and [ca2TsERCn responses to collagen beads
As integrin ligation with extracellular matrix ligands stimulates calcium rises
(Schwartz, 1993) that may be important in the bead binding step of phagocytosis,
the response of [ca2'li to collagen bead-induced phagocytosis in HGFs was
10" t o ~ loS II 'O ' RlHdpht
HGF + CCB HGF + 10 nM CsA+ CCB
untreated 10 nM CsA
untreated 10 "M 100 nM 1 pM 10 pM CCCP Sodium CsA CsA CsA CsA Azide
Figure 6. A. Flow cytometry - analysis of the collagen bead binding step of phagocytosis after incubation with cyclosporin A in human gingival f i broblasts (HGFs). B. CsA (1 0 nM) causes a marked decrease in the phagocytic capacity of HGFs. Data are means I SEM (n=3). C. CsA causes a dose-dependent inhibition of phagocytosis. To study the possible involvement of mitochondria, CCCP (10 FM) and sodium azide (1%) caused a marked decrease in the phagocytic capacity of Rat2 fibroblasts. Data are means I SEM (n=3). For figs. 6 B and C, *, p< 0.05; 7, pc0.02; $, p<0.01 when compared to vehicle-treated cells. ANOVA testing for untreated tells and increasing doses of CsA revealed that the groups are significantly different at p=0.001.
examined. The ratiometric calcium-sensitive dye fura 2 was used to measure [ca2'li as
it is relatively insensitive to photobleaching and dye leakage is minimal over time. In
Fig. 7A the insert depicts a representative tracing obtained when collagen-coated beads
were added to HGF cells and [ca2']i was measured over time. As cells were incubated
with beads at a ratio of -4: 1 (beads:cell), multiple asynchronous bead-cell interactions
occur over 1500 seconds and consequently the decrease in calcium to basal levels
occurs later (not shown). The mean [ca2+li rise above baseline was similar in HGFs
(124 i 31 nM) and Rat2 fibroblasts (1 30 2 9 nM; p>0.02). The rise to maximum levels
of [ca2']i also proceeded over a similar time course. The similarity of Rat2 fibroblasts
and HGF responses to collagen beads rationalized the use of Rat2 fibroblasts for al1
subsequent experiments which measured levels of calcium within the cell.
When CsA was added to HGFs or Rat2 cells, [ca2+]i rose steadily over time and
exhibited a dose-dependent increase of [ca2']i. The addition of CsA, even in small
doses, induced significant increases of [ca2'li compared to the DMSO vehicle (p<0.05
for 10 nM CsA; Fig. 78). When Rat2 fibroblasts were pre-treated with 10 nM CsA for 1
hour, there was a significantly reduced rise of [ca2'li in response to collagen bead-
induced phagocytosis (3.4-fold; pç0.01; Fig. 7C).
The levels of [ca2+li were investigated when intracellular stores of calcium were
depleted usinç 1 yM thapsigargin or when intracellular calcium was chelated using
BAPTAIAM. After addition of thapsigargin to either Rat2 fibroblasts or Rat2 fibroblasts
pre-treated with CsA, there was a robust rise of [ca2+li in both groups. The post-
Mean change in [Car*], tïme to maxlmum [Ca hl,
r h
Time (seconds) 1
HGF + CCB Rat2 + CCB
r l Y 1 i . I . !.., w'- n o . c --, 5 - L - 8-9. - - ..\CC*"-
0 0 . N + /*-+ ,">;F- B O . E 6 0 - %l. . . .- -.
I l .;un . - 111.1
* 4 0 .
2 0 . Thne (seconds) & a
0 . - d i I DMSO, 1nM 10nM 100nM 1pM 1OpM
untreated [CSAI O
0 ; RaPflbroblasts C
O Rat2 flbroblests + 0 . Y
10 nM CsA - 8 ,
0 . + _
O .
O * Time (seconds)
* - - + CCB, P O S ~ - ~ g
untreated + CCB
Mean change In 346i380 ratio
1 Time to maximum 3461380 ratio
-r i eoo I
- 1000 '-
; 800
400
Y I n
Rat2 + CCB Rat2 + CsA +%CS -
Figure 7. A. Collagen-coated beads (CCBs) cause an equivalent rise of [Ca2+Ii in human gingival fibroblasts (HGFs) and Rat2 cells. Note that the tirne required to reach maximum values are also similar. In contrast, cells stimulated with poly-L-lysine coated beads did not show any significant rise above baseline. Data are means I SEM (n-5). Insert shows an increase of [Ca2+], induced by collagen-coated beads in HGFs. B. Treatment with increasing concentrations of CsA induces dose-dependent increases of [Ca2+], in HG F. Data are means I SEM (n=5). *, pe 0.05; $, pe0.01 when compared to DMSO. Inset demonstrates representative tracing when CsA is added to human gingival fibroblasts loaded with fura 2/AM (2 FM) and [Ca2+ji is recorded. C. The addition of CCBs to fibroblasts in a calcium-containing medium induces a substantial calcium flux (-130 nM Ca2+) which is reduced 3.4-fold by CsA (10 nM). As expected, thapsigargin (Tg; 1 PM) induces a large calcium increase which is not affected by pre-treatment with CsA (10 nM). After depletion of intracellular calcium stores by thapsigargin, the collagen-induced calcium response is reduced -3.7-fold (post-Tg + CCB). In contrast, there is no significant reduction of collagen-induced calcium increase in CsA-t reated cells that are then treated wit h thapsigargin. Evidently CsA is not acting primarily at a thapsigargin-sensitive locus in the response to collagen beads. Data are means I SEM (n=5). *, pe 0.05; *, pc0.01 when compared to Rat2 + CCB; **, pc0.05 when compared to Rat2 fibroblasts treated with IO8 M CsA. Inset shows pronounced increase in [Ca2+], produced by thapsigargin and a very small rise due to the addition of collagen-coated beads after thapsigargin. D. Pre-incubation of Rat2 fibroblasts with CsA induces decreased [Ca2+],,,, response to collagen-coated beads. Rat2 cells were loaded with [Ca2+Is,,, indicator rnag-fura 2 and measured by ratio fluorimetry. Data are means r SEM (n=5). t, pe0.02 when compared to Rat2 + CCB.
thapsigargin response to collagen bead-induced phagocytosis was small in both groups
but showed a larger reduction of [ca2'li in Rat2 fibroblasts (3.7-fold; ~ ~ 0 . 0 5 ) than in
Rat2 fibroblasts pre-treated with CsA (2-fold). Calcium chelation with BAPTAIAM (5
PM) almost obliterated the [ca2+li rise induced by beads (Fig. 7C).
When 2 pM mag-fura 2/AM was used to Joad cells the response of [ca2'lsERcn to
collagen bead-induced phagocytosis was measured with ratio fluorimetry, pre-treatment
with CsA produced a > 5-fold reduction in the rise of [ ca2 ' ] s~~c~ (p < 0.02) over a similar
time span as vehicle-treated controls (-1 100 seconds; Fig. 7D).
Dependence of phagocytic response on calcium signalling
The addition of thapsigargin to release calcium from interna1 stores and to inhibit store
uptake dramatically reduced the phagocytic capacity of Rat2 fibroblasts (1 0.2-fold;
pc0.01) a reduction that was comparable to the 8-fold decrease seen when Rat2
fibroblasts were pre-treated with CsA (pc 0.02; Fig. 8). Pre-treatment with CsA (1 0 nM)
produced no additional inhibition of phagocytosis. A similar sized reduction of
phagocytosis was obtained after pre-treatment with BAPTAIAM. These data indicated
that intracellular calcium is important in regulating the bead-binding step of
phagocytosis.
Mitochondrial calcium ([ca27,)
Rat2 fibroblasts were CO-loaded with MitoTracker Green [which specifically stains
mitochondria, Babcock et al. (1997)l and rhod 2/AM [which reportedly stains
untreated lm Tg 10 nM CsA 5 PM + 1 yM Tg BAPTAIAM
Figure 8. Effect of agents that inhibit calcium signalling on phagocytosis in Rat2 fibroblasts. Cells treated with thapsigargin (Tg; 1pM) or BAPTNAM (5 PM) show 10- fold reduction of phagocytosis. Pre-incubation with CsA produces no additional inhibition of phagocytosis compared to thapsigargin alone. Data are means I SEM (n=3). ;t, p<0.02; $, p4.01 when compared to untreated cells.
mitochondrial calcium, Babcock et al. (1997)l to determine if these dyes CO-
localize and thereby assess if rhod 2 can be used to monitor changes in
[ca2'],ito. There was marked CO-localization of rhod 2 and MitoTracker Green
(Fig. 9Ai). After staining with rhod-2/AM for 30 minutes, Rat2 fibroblasts or Rat2
fibroblasts pre-treated with CsA for 1 hour exhibited an equivalent time-
dependent decrease of [ca2'],ito in response to collagen beads at > 10 minutes
(Fig. SAii, iii). Linear regression showed that mitochondrial calcium was lost at a
similar rate in both controls and CsA-treated cells. However, in control cells (Le.
not pre-treated with CsA). there was marked and non-linear reduction of
mitochondrial calcium loss in the 5-10 minute range (Fig. 9B).
CsA prevents loss of mitochondrial membrane potential (Y,)
As CsA is thought to affect the permeability of the MPTP (Zamzami et al., 1996),
I evaluated the efficacy of CsA to block the loss of Y, that occurs following an
apoptotic sirnulus (Kulkarni et al., 1998). Suspension-induced apoptosis
(anoikis) was used as the stimulus (Kulkarni and McCulloch, 1994) and JC-1
aggregate formation was used to estimate the mitochondrial membrane potential.
The addition of increasing concentrations of CsA to Rat2 fibroblasts in
suspension was accompanied by an elevated mitochondrial membrane potential
(pc0.01 for 1 and 10 yM CsA; pc 0.05 for 100 nM CsA; Fig. 10). Thus CsA
appears to preserve Y, as described (Zamzami et al., 1996) which indicates that
an important locus of action for CsA is the MPTP of fibroblasts.
Mean Fluorescence Intensity (x 105)
Figure 9. A. (i) Co-localization of MitoTracker Green and rhod 2 dyes in Rat2 fibroblasts. Cells were CO-loaded with both dyes and then irnaged by fluorescence microscopy using filters appropriate for either dye. (ii) Stimulation using collagen- coated beads (CCB) in Rat2 fibroblasts stained with rhod 2 induces a decrease in [Ca2+],,, over tirne. Arrows indicate decreased rhod 2 staining in discrete regions of the cell. Note that in the left panel, discrete mitochondria stained with rhod 2. In the right panel, after addition of collagen beads (2 fluorescent beads can be seen on left margin of cell) there is a generalized reduction of rhod 2 fluorescence. (iii) Stimulation using CCBs induces a decrease of [Ca2+],,, in Rat2 fibroblasts treated with CsA. B. The addition of collagen-coated beads to Rat2 and Rat2 fibroblasts treated with 10 nM CsA induces a time-dependent decrease in [Ca2+],,,. Data are means I SEM (n=5). Linear regression equations and Pearson correlation values for Rat2 and Rat2 fibroblasts treated with CsA, respectively, are y=-0.060~ + 2.22, r=-0.97 and y=-0.18~ + 4.84, r=- 0.98.
i contr I 10 nM CsA
10pM CCCP CsA
Figure 10. CsA prevents the loss of mitochondrial membrane potential of Rat2 fibroblasts in suspension. Controls are untreated cells. Analysis was conducted by flow cytometry of JC-1-loaded Rat2 cells in suspension to promote dissipation of Y, and thereby induce anoikis (suspension-induced apoptosis). Data are means I SEM (n=3). $, p<0.01 when compared to untreated cells.
Mitochondrial-depleted cells
The data in Figs. 6, 9 and 10 indicated that mitochondria may be an important organelle
for regulation of SERCA ca2+ and also of collagen bead phagocytosis. Consequently,
mitochondria-depleted Rat2 cells were produced using well-established methods
(Biswas et al., 1999) to determine if reduction of the numbers of mitochondria would
affect collagen bead-induced calcium signalling and phagocytosis. I first verified if the
standard protocol for mitochondrial depletion (Biswas et al., 1999) would in fact reduce
the numbers of mitochondria.
Rat2 fibroblasts or Rat2 fibroblasts treated with 1000 ng/mL ethidium bromide
were stained with JC-1 (0.5 PM) for visualizing mitochondria with a fluorescence
microscope. The EtBr-treated cells showed much smaller numbers of mitochondria
(Fig. 11Ai). Staining with rhod 2 AM (4.5 PM) to assess mitochondrial calcium stores
also showed a large decrease of fluorescence in the EtBr-treated cells (Fig. 11Aii).
Notably, rhod 2 staining of nucleoli was unchanged, indicating that the EtBr treatment
specifically depleted mitochondrial calcium stores, presumably due to the greatly
reduced numbers of mitochondria. Western blotting demonstrated that treated cells
also contained less of the mitochondria-specific protein, cytochrome oxidase I (Fig.
1 1 Aiii).
As shown in figure 11 B, mitochondria-depleted cells exhibited 50% less collagen
bead binding than normal cells in the absence of CsA (pc0.05). The addition of CsA
caused a dose-dependent decrease in the mean % phagocytosis of collagen-coated
beads in Rat2 fibroblasts treated with ethidium bromide (pe 0.05 for 100 and 1000 nM
CsA) similar to that seen in untreated cells. The mitochondria-depleted cells
Figure 11. A. Treatment of Rat2 fibroblasts with ethidiurn bromide for over 30 passages of continuous culture induces inhibition of mitochondrial DNA synthesis and reduced synthesis of the mitochondria-associated protein COX 1. (i) Staining of Rat2 fibroblasts and Rat2 fibroblasts treated with 1000 ng/mL ethidium bromide (RaQEtBr) with the mitochondrial specific dye, JC-1. (ii) Staining of Rat2 fibroblasts and Rat2EtBr fibroblasts with rhod 2/AM shows no change in rhod 2 staining of nucleoli but greatly reduced fluorescence in the cytoplasrn. (iii) Western blot for COX I protein in Rat2 and Rat2EtBr fibroblasts shows -3-fold reduction of this mitochondrial-specific protein. B. Phagocytic capacity of Rat2 and RatPEtBr fibroblasts. All cells were incubated with coiiagen-coated beads at a ratio of 2:1 (beads:cell) for 1 hour after indicated treatment. Treatments: 1- untreated; 2- 10 nM CsA; 3- 100 nM CsA; 4- 1 FM CsA; 5- 10 pM CsA;6- 10 nM CsA + 1 pM Tg (15 mins); 7- 1 pM Tg (15 rnins.); 8- 5 pM BAPTNAM (30 mins); 9- 500 FM CCCP (30 mins); 10- 1% sodium azide (30 mins); al1 CsA treatments were conducted for 1 hour. These data shows that compared to untreated controls, CsA induces a more marked inhibition of phagocytosis in normal Rat2 fibroblasts than in ethidium bromide-treated fibroblasts. The thapsigargin. BAPTA and CCCP effects are also proportionately less in the ethidiurn bromide-treated cells. Data are means I SEM (n=3). * ~~0.05 when compared to Rat2EtBr fibroblasts, ", pc0.05 when compared to Rat2 fibroblasts.
displayed reductions of phagocytosis after treatment with thapsigargin,
BAPTAIAM, CCCP or sodium azide (Fig. 11 B) but the magnitude of the reduction
was - one-half that of normal Rat2 cells.
Calcium signalling in mitochondria-depleted cells
Rat2 or mitochondria-depleted Rat2 fibroblasts were loaded with fura 2JAM (2
FM) and treated with CCCP (10 PM) for 10 minutes to depolarize mitochondria
and block oxidative phosphorylstion. As estimated by ratio fluorimetry, CCCP
reduced [ca2'li responses to collagen beads in Rat2 fibroblasts by -90%
(pc0.01). In contrast, collagen bead-induced [ca2']i responses in mitochondria-
depleted cells were < 10 nM and were reduced only an additional 5 nM by CCCP
(Fig . 1 2A).
As shown in Fig. 7D and Fig. 128, treatment of Rat2 fibroblasts with CsA
markedly reduces the maximal [ c a 2 + ] s ~ ~ ~ ~ response to collagen beads. In
contrast, when mitochondria-depleted Rat2 fibroblasts were treated with CsA,
there was no significant reduction of maximal [C~*']SER~A in response to beads
(Fig. 12B; pç0.2). Further, there was no significant difference between Rat2
fibroblasts and mitochondria-depleted fibroblasts with or without CsA treatment
(~>0.2).
The data shown above indicate that mitochondria regulate calcium
signalling in response to collagen beads but when the numbers of mitochondria
are reduced, SERCA stores continue to exhibit a response to beads that is
unaffected by CsA. I assessed if calcium stores in the remaining mitochondria of
Fïbroblasts + CCCP + CCB
Rat2 fibroblasts Rat2EtBr fibroblasts
Mean change in 3461380 iatlo O .I
ci 0.07 * Tirne to maximum 316/380
L
i ooo 7
Rat2EtBr Rat2EtBr + CCB + CsA + CCB
Rat2 + CCB
Rat2 + CsA + CCB
Figure 12. A. Incubation with CCCP inhibits [Ca2+Ii response to collagen-coated beads (CCBs) in both Rat2 and Rat2EtBr fibroblasts. CCBs induce only a minimal [Ca2+], response in Rat2 fibroblasts cultured with 1000 ng/mL ethidium bromide (RaPEtBr). Data are means I SEM (n=5). $, pc0.01 when compared to Rat2 fibroblasts + CCB. 8. Pre-incubation with 10 nM cyclosporin A (CsA) induces a large decrease in [Ca2+],,,, in Rat2 cells but not in Rat2EtBr cells responding to CCBs. [Ca2+],,,, was measured using ratio fluorimetiy of mag-fura 2-loaded cells. Data are means I SEM (n=5). t, pc0.02 when compared to Rat2 fibroblasts + CCB.
mitochondria-depleted cells would still discharge ca2' if stimulated with collagen
beads. Mitochondria-depleted Rat2 fibroblasts were loaded with rhod 2/AM (4.5
PM) and fluorescence intensity was recorded over 30 minutes after initiation of
collagen-bead induced phagocytosis as described (Fig. 96). In cells treated with
vehicle or CsA, the fluorescence intensity of rhod 2 decreased following
stimulation with collagen-coated beads (Fig. 13A). As expected, the baseline
fluorescence intensity due to rhod 2 reporting of [ ~ a " ' ] ~ i ~ ~ was significantiy lower
in mitochondria-depleted cells compared to control Rat2 cells. Linear regression
of fluorescence intensity data showed that [ca2'lmito in Rat2 fibroblasts dissipated
at the same rate as [~a*' ]mi~~ in RatPEtBr fibroblasts between 10-30 minutes. In
contrast to mitochondria-depleted cells which showed a linear reduction of
fluorescence between 0-30 minutes, control cells exhibited a pronounced 2-fold
reduction 5-10 minutes after bead incubation which thereafter showed a similar
linear reduction of fluorescence as the ethidium bromide-treated cells (Fig. 138).
After treatment with 10 nM CsA for 1 hour, the control Rat2 fibroblasts lost
mitochondrial calcium at a linear but faster rate than Rat2EtBr fibroblasts (slope =
-0.18 vs. slope = -0.055). The marked loss of mitochondrial calcium in the 5-10
minute range after stimulation with collagen beads was notably absent after CsA
pre-treatment.
Sasdine, non-stirnulated + CC8, t=lO minutes
10 15 20 Tirne (minutes)
Time (minutes)
Figure 13. A. (i) Stimulation with collagen-coated beads (CCB) in RatZEtBr fibroblasts stained with rhod 2/AM induces a decrease in [Ca2+],,, of the few remaining mitochondria. In this micrograp h I have included fluorescence beads (bright circles at cell periphery) to illustrate bead size and binding. Arrows point to rhod 2 stained mitochondria and indicate decreased rhod 2 staining. (ii) Stimulation with CCBs induces a decrease of [Ca2+],, in Rat2EtBr treated with CsA. B. CCBs induce decreased [Ca2+],, in Rat2 and RatPEtBr. In normal Rat2 cells, CCBs induce a s 2- fold reduction of rhod 2 fluorescence between 5-10 minutes which decreases lineariy thereafter (10-30 minutes). Mitochondria-depleted cells exhibit a linear reduction of rhod 2 fluorescence which has the sarne slope as the normal cells from 10-30 minutes. Linear regression equations and Pearson correlation values for Rat2 and Rat2EtBr fibroblasts, respectively, are y=-0.060~ + 2.22, r=-0.97 and y=-0.090~ + 2.88, r=-0.90. C. Addition of CCBs to Rat2 or Rat2EtBr fibroblasts pre-treated with 10 nM CsA induces decreased [Ca2+],,io. Note the continuous linear reduction of rhod 2 fluorescence in the normal Rat2 cells and the absence of the sharp drop from 5-10 minutes. Linear regression equations and Pearson correlation values for Rat2 and Rat2 fibroblasts both treated with CsA, respectively, are y=-0.1 8x + 4.84, r=-0.98 and y=-0.055~ + 2.1 6, r=-0.78.
Control fibroblast
'l' ytos i s
Fibroblast treated with CsA bead
Figure 14. Schematic diagram of fibroblasts with and without CsA treatment. Summary changes of [Ca2ri, [Ca2+],,,,,, [Ca2+],,, and rate of phagocytosis are represented by arrows. Subsequent to collagen bead-induced phagocytosis, different calcium responses are observed after treatment with CsA. In control cells, [Ca2+],,, decreases initially at a faster rate than CsA-treated cells. CsA sharply reduces whole cell calcium and SERCA calcium responses to collagen bead stimulation. 1 propose that binding of CsA to mitochondria inhibits mitochondrial release of calcium and therefore uptake by the SERCA store and a consequent reduced calcium response in the cytosol to collagen beads. The downstream effect is decreased phagocytic efficiency in CsA- treated fibroblasts which contributes to loss of collagen homeostasis and results in gingival overgrowth.
V. Discussion
Current data indicate that administration of CsA results in gingival overgrowth in as low
as 17% and as high as 81% of cases available for study (Hefti et al., 1994; Friskopp and
Klintmalm, 1986). The mechanisrn by which CsA induces gingival overgrowth is
unknown but some of the modes of action of CsA as an immunosuppressant are
understood (Kay, 1989). CsA inhibits the MPTP (Crompton et al., 1988) in the
mitochondrial membrane, and more recently, has been shown to act upon the internal
calcium stores and inhibit calcium release (Park et al., 1999). Further, several
functional interactions between mitochondria and internal calcium stores have been
characterized (Landolfi et al., 1998). With this background in mind, I propose that upon
entry into the cell, CsA interacts with mitochondria to affect the normal functional
relationship of mitochondrial calcium stores and other internal stores of calcium (Fig. 4).
As calcium signalling is important for integrin functions related to binding of extracellular
matrix molecules (Schwartz, 1993), this deregulation is likely to affect collagen
phagocytosis. Below I will discuss in more detail the functional relationships between
calcium deregulation and phagocytic processes.
Effect of CsA on phagocytosis
HGFs have been used previously to study collagen phagocytosis in vitro (McCulloch
and Knowles, 1993). These cells are the principal synthetic and degradative cell in
normal gingiva (Narayan and Page, 1993; Everts, 1996) and consequently are closely
lin ked to the de reg ulation of collagen rnetabolism seen in drug-induced gingival
overgrowth. I used Rat2 fibroblasts primarily for my studies since I showed that these
cells exhibit very similar calcium and phagocytic responses as HGFs. As Rat2 cells is a
stable and easily propagated cell line, problems of cell death and the phenotypic
variability associated with primary isolates were rninimized. Data from previous
collagen phagocytosis studies also dernonstrate the utility of Rat2 cells for phagocytic
studies and the similarity of the two cell types (Hui et al., 1997). For example I showed
that addition of 10 nM CsA caused a significant and similar decrease in phagocytosis
(~50%) for both cell types. Further, in Rat2 fibroblasts and HGFs, I showed that
addition of CsA, in concentrations less than those administered in vivo (Kay, l989),
caused a significant decrease of phagocytosis compared to untreated cells. These data
describe the final end-point effect of CsA upon fibroblasts studied here, (i.e. due to
some mechanism, CsA inhibits the collagen phagocytic capacity of gingival fibroblasts).
CCCP, a proton ionophore which dissipates the mitochondrial membrane potential
(Zamzami et al., 1995) and eventually promotes ce11 death (Kulkarni et al., 1998), also
reduced phagocytosis as was seen with CsA treatment. The mitochondrial dysfunction
caused by CCCP suggests the importance of mitochondria in phagocytic function.
lndeed the involvement of ATP production by mitochondria was shown by treating cells
with sodium azide which also caused a similar decrease of collagen bead phagocytosis.
Phagocytosis and [ c ~ J signals
When [ca2'li was monitored during collagen bead-induced phagocytosis, HGFs and
Rat2 fibroblasts exhibited a gradua1 similar rise in [ca2'li over 30 minutes but with no
return to baseline over this monitoring period, presumably because of the prolonged
period of bead-cell interactions and the progressive cellular contacts with more than one
bead. After intracellular calcium stores were depleted by thapsigarg in, collagen bead-
induced phagocytosis was still able to invoke a small but measurable calcium rise, an
effect that was seen in both Rat2 fibroblasts and Rat2 fibroblasts pre-treated with CsA.
If calcium was released into the cytosol through a thapsigargin-insensitive route,
collagen phagocytic stimuli may effect calcium release from other calcium stores or this
increase may be due to calcium influx from the extracellular medium (Schwartz, 1993).
Notably, the post-thapsigargin response in CsA-treated cells was not as large as
untreated control cells, suggesting that CsA interacts with thapsigargin-insensitive sites
which may cause an increase of [ca2']i due to phagocytosis. I propose that since
treated cells have more of these binding sites engaged by CsA, they cannot respond
fully to phagocytic stimuli. The chelation of [ca2']i with BAPTA also caused a significant
decrease in the [ca2*]i response compared to untreated cells and the magnitude of this
response was similar to that following thapsigargin. These data are consistent with
previous findings showing that [ca2'li responses to phagocytic stimuli are not due solely
to release from SERCA stores (Bengtsson et al., 1993). lndeed as SERCA calcium
stores were actually increased after collagen bead stimulation, it is possible that a large
part of the rise of [ca2'li due to phagocytosis originates from non-SERCA stores. Small
contributions may also come from both the extracellular medium and from other, as yet
unidentified, stores within the cell.
CsA induced a relatively rapid, dose-dependent rise of [ca2']i, likely reflecting the
transport of CsA into cells and its interaction with binding sites or receptors that regulate
intracellular calcium stores (LeGrue et al., 1983). Addition of thapsigargin to cells pre-
treated with CsA induced the same robust increase in [ca2+]i as seen in untreated cells.
However, collagen bead-induced phagocytosis aRer interna! store depletion by
thapsigargin produced a reduced response of [ca2*]i in Rat2 fibroblasts (p<0.05) and a
similar rise in Rat2 fibroblasts pre-treated with CsA. This finding indicates that pre-
treatment with CsA caused calcium release from thapsigargin-insensitive stores. As
collagen beads stimulated uptake of calcium into the SERCA, and as this uptake was
inhibited by CsA, it seems likely that mitochondrial calcium is released by collagen bead
stimuli and that the released calcium is taken up by the SERCA.
[ C ~ ~ ' ] S E R C A response to collagen phagocytic stimuli
As discussed above, pre-incu bation of Rat2 fibroblasts with CsA caused a significant
decrease in the [ C ~ ~ ' ] S E R C A response to collagen beads. Previous data by Schwartz
(1993) indicate that a large part of the increase in [ca2'li may be due to calcium release
from the SERCA stores. However, when mag-fura was used to observe the behaviour
of calcium within SERCA stores, I found an increase after collagen bead-induced
phagocytosis over 30 minutes. Therefore, both [ca2+li and [ca2'lsERCA were increased
after collagen bead-induced phagocytosis. My expectation was that release of SERCA
stores of calcium would provide the increased [ca2+li seen experimentally. However,
when cellç were pre- treated with CsA, [ca2+lsERCA and [ca2+li were reduced compared
to untreated cells. Thus CsA may act initially to release calcium from intracellular stores
thereby reducing the calcium ion pool for uptake by the SERCA. In this context,
mitochondria in untreated cells rnay sense high local concentrations of calcium and act
to buffer [ca2+], as has been shown earlier (Riuuto et al., 1993). Accordingly, in
mitochondria-depleted cells, CsA treatment did not alter [ c ~ ~ + ] ~ ~ R ~ A presumably
because productive interactions between mitochondrial and SERCA-related stores were
restricted (Biswas et al., 1 999).
[~fl],,,,~~ responses to collagen phagocytic stimuli
Collagen bead-induced phagocytosis provoked a time-dependent decrease of [ca2+lmi,
in untreated Rat2 cells or Rat2 cells pre-treated with CsA but there was a much more
rapid loss at early times (5-10 minutes) in untreated cells. Pre-incubation with CsA or
mitochondrial depletion attenuated the dissipation of mitochondrial calcium in the 5-1 0
minute range. This result may be a reflection of the initial obstruction to calcium efflux
due to inhibition of the MPTP by CsA, an effect that is probably overcome as calcium
ions exit via other channels or pumps (Bernardi, 1999). My data on CsA inhibition of
suspension-induced reduction of Y, indicate that CsA was indeed acting upon the
MPTP and consequently the supposition that CsA is altering the conductance of the
MPTP seems reasonable.
Phagocytosis in rnitochondria-depleted fibroblasts
Compared to untreated Rat2 cells, Rat2 fibroblasts treated with ethidium bromide
exhibited similar reductions of phagocytic efficiency as seen in cells treated with CsA.
This suggests that mitochondria play a role in regulating collagen phagocytosis. When
the rate of dissipation of [ca2+]mito due to collagen bead-induced phagocytosis was
compared between these two ceII types, there was no significant difference. This result
appears reasonable since the ethidium bromide-treated cells have a lower nurnber of
mitochondria but the function of the remaining mitochondria is not altered, otherwise the
cells would not proliferate in culture. A similar rate of rnitochondrial calcium dissipation
was seen in Rat2 fibroblasts treated with CsA, emphasizing that although reduced in
number, these mitochondria function normally.
The major conclusion of this thesis is that mitochondrial calcium signalling is an
important determinant of collagen bead phagocytosis and that consequently, CsA-
mediated gingival overgrowth may be mediated by the same or a similar pathway. Fig.
14 illustrates a model integrating data obtained for changes of [ca2'li, [ ~ a ~ ' ] ~ ~ ~ ~ ~ ,
[ca2'],im and phagocytic efficiency. Consistent with my overall conclusion, subsequent
to collagen bead-induced phagocytosis, very different calcium responses were
obsewed in Rat2 fibroblasts or Rat2 fibroblasts treated with CsA. [~a*'],~~, decreased
at a faster rate initially in the untreated cells. Both [C~~+]SERCA and [ca2']i increased to a
greater extent in controls than CsA-treated Rat2 fibroblasts. I propose that the effect of
CsA on mitochondria is transmitted to the SERCA stores, which then "relay" the
information and dampens the phagocytosis-induced calcium signal in the cytosol. The
effect of this perturbation on the whole cell is a decrease of phagocytic efficiency which,
in the CsA-treated gingival fibroblast, results in gingival overgrowth. While the actual
sequence of events is not shown by these data, the importance of signaling interactions
between discrete calcium stores is evident. When the sequence and regulation of these
phagocytic signaling events is deciphered, and the exact effect of CsA on the
mitochondria as a fibrosis-inducing agent is outlined, modifications to CsA therapy could
be approached more rationally. For example, if CsA could be modified so as to reduce
fibrosis while retaining its immunosuppressant properties, this would be a significant
clinical advance.
VI. Conclusions
1. CsA inhibits collagen bead phagocytosis dose-dependently in human gingival
and Rat2 fibroblasts. Phagocytosis is decreased in mitochondria-depleted
RatZEtBr fibroblasts, suggesting that mitochondria play a role in the regulation of
collagen phagocytosis.
2. Collagen-coated beads induce equivalent calcium rises in both human gingival
and Rat2 fibroblasts. Part of the calcium increase is due to thapsigargin-
sensitive and thapsigargin-insensitive stores. Mitochondria depleted Rat2EtBr
fibroblasts show reduced rise of [Cali in response to collagen-coated beads. CsA
prevents the loss of mitochondrial membrane potential of Rat2 and Rat2EtBr
fibroblasts in suspension indicating that CsA blocks the mitochondrial membrane
perrneability transition pore.
3. Depletion of internal calcium stores or dissipation of mitochondrial membrane
potential greatly reduce phagocytosis in both Rat2 and mitochondria-depleted
Rat2-EtBr fibroblasts.
4. Pre-incubation of Rat2 fibroblasts with CsA causes a decrease in [C~~* ]SERCA
which is probably related to CsA-induced release of calcium from internal stores.
In Rat2-EtBr cells, CsA treatment does not alter [C~~ ' ]SERCA because productive
interactions between mitochondrial and SERCA-related stores are restricted.
Collagen bead-induced phagocytosis induces a time-dependent decrease in
[Calmito in both Rat2 and Rat2 fibroblasts treated with CsA. Calcium release from
mitochondria is provoked by collagen phagocytosis in both cell types.
Vil. References
Allen, L.-A.H. and Aderem, A. (1 996) Molecular definition of distinct cytoskeletal structures involved in complemnt- and Fc Receptor-mediated phagocytosis in macrophages. J Exp Med 184:627-637.
Angelopoulos, A.P. and Goaz, P.W. (1972) Incidence of diphenyylhydantoin gingival hyperplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 34: 898-906.
Armitage, G .C. (1 999) Developrnent of a classification system for periodontal diseases and conditions. Ann Periodontol4: 1 -6.
Avdonin, P.V., Cottet-Maire, F., Afanasjeva, GY., Loktionova, S.A. Lhote, P. and Ruegg, U.T. (1999) Cyclosporin A up-regulates angiotensin II receptors and calcium responses in human vascular smooth muscle cells. Kidney Int 55(6):2407-2414.
Aznavoorian, S., Murphy, A.N., Stetler-Stevenson, W.G. and Liotta, L.A. (1993) Molecular aspects of tumor cell invasion and metastasis. Cancer i l : 1368-1 381 .
Babcock, J. R. (1 965) l ncidence of gingival hyperplasia associated with dilantin therapy in a hospital population. J Am Dent Assoc 71 : 1447-1 450.
Babcock, D.F., Herrington, J., Goodwin, P.C., Park Y.B. and Hille B. (1997) Mitochondrial participation in the intracellular ca2+ network. Journal of Ce11 Biology 136:833-844.
Barak, S., Engelberg, I.S. and Hiss ,J. (1987) Gingival hyperplasia caused by nifedipine - histological findings. J Periodontol: 58: 639-642.
Bartold ,P. (1 989) Regulation of human gingival fibroblast growth and synthetic activity by cyclosporine-A in vitro. J Periodontol Res 24:314-321.
Bengtsson, T., Jaconi, M.E.E., Gustafson, M., Magnusson, K.-E., Theler, J.-M., Lew, D.P. and Stendhal, 0. (1 993) Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium. Eur J Cell Biol62:49-58.
Bernardi, P. (1 999) Mitochond rial transport of cations: channels, exchangers, and perrneability transition. Physiol Rev 79: 1 127-1 155.
Bernardi, P., Broekemeier, K.M. and Pfeiffer, DR. (1 994) Recent progress on regulation of the mitochondrial peneability transition pore: a cyclosporin- sensitive pore in the inner mitochondrial membrane. J Bioenerg Biomembr 26:509-517.
Berridge, M.J. (1 995) Capacitative calcium entry. Biochem J 31 2:1-11.
Berridge, M., Lipp, P. and Bootman, M. (1999) Calcium signalling. Curr Biol 9(5): R I 57-1 59.
Bezprozvanny, I., Watras, J. and Ehrlich, B. E. (1 991 ) Bell-shaped calcium response curves of Ins(1 ,4,5)P3-and calcium-gated channels frorn endoplasrnic reticulum of cerebellum. Nature 351 :75l -754.
Birkedal-Hansen, H. (1 993) Role of cytokines and inflammatory mediators in tissue destruction. J Periodontol Res 28:500-510.
Biswas, G., Adebanjo, O.A., Freedman, B.D., Anandatheerthavarada, H.K., Vijayasarathy, C., Zaidi, M., Kotlikoff, M. and Avzdhani, G. (1999) Retrograde ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle cross-talk. EM80 J 18(3):522-533.
Bonnaure-Mallet, M., Tricot-Doleux, S. and Godeau, G. (1 995) Changes in extracellular matrix macromolecules in human gingiva after treatment with drugs inducing gingival overgrowth. Arch Oral Bi01 40:393-400.
Border, W.A., Noble, N.A., Yamamoto, T., Harper, H.R., Yamaguchi, Y., Pierschbacher, M.D. and Ruoslahti, E. (1 992) Natural inhibitor of transforming growth factor-P protects against scarring in experimental kidney disease. Nature 3603361 -364.
Borel, J.F., Feurer, C., Gubler, H.U. and Stahelin, H. (1976) Biological efects of cyclosporin A: a new antilyrnphocytic agent. Agents Actions 6:468-475.
Brandes, D. and Anton, E. (1969) Lysosomes in uterine involution: intracytoplasmic degradation of myofilaments and collagen. J Gerontol24:55-69.
Bunjes, D., Hardt, C., Rollinghoff, M. and Wagner, H. (1981) Cyclosporin A mediates immunosuppression of primary cytotoxic T cell responses by impairing the release of interleukin-1 and interleu kin-2. Eur J lmmunol 1 1 :657-661.
Calne, R.Y., Rolles, K., White, D.J., Thiru, S., Evans, D.B., McMaster, P., Dunn, D.C., Craddock, G.N., Henderson, R.G., Aziz, S. and Lewis P. (1979). Cyclosporin A initially as the only immunosuppressant in 34 recipients of cadaveric organs: 32 kidneys, 2 pancreas and 2 livers. Lancet 2: 1033-1 036.
Carranza, F.A. Jr. Gingival enlargement. ln: Clinical Periodontoloav (eds. F.A. Carranza Jr and M.G. Newman, 8'h ed) Philadelphia, PA: W.B. Saunders Co., 1 996: 235-238.
Chou, D.H., Lee, W., and McCulloch, C.A.G. (1 996) TNF-alpha inhibits collagen synthesis and at high concentrations tirnulates collagenase synthesis in fibroblasts. J lmrnunol 1 56(11):4354-4362.
Crompton. M.. Ellinger. H. and Costi, A. (1988) Inhibition by cyclosporin A of a cas- dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J 255:357-360.
Daley, T.D., Wysocki, G.P. and May C. (1 986) Clinical and pharmacological correlations in cyclosporine-induced gingival hyperplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 62: 41 7-421.
Dahllof, G. and Modéer, T. (1986) The effect of a plaque control program on the development of phenytoin-induced gingival overgrowth. A 2-year longitudinal study. J Clin Periodontol13: 845-849.
Deliliers, G.L., Santoro, F., Polli, N., Bruno, E., Fumagalli, L. and Risciotti, E. (1 986) Light and electron microscopie study of cyclosporin-A induced gingival hyperplasia. J Periodontok 57: 771 -775.
Dongworth, D.W. and Klaus, G.G.B. (1982) Effects of cyclosporin on the immune system of the mouse. 1. Evidence for a direct selective effect of cyclosporin A on B cells responding to anti-immunoglobulin antibodies. Eur. J. Immunol. 12:1018- 1022.
Dreyer, W.P., Dent, H.D. and Thomas, C.J. (1 978) Diphenylhydantoin hyperplasia of the masticatory mucosa in an edentulous epileptic patient. Oral Surg Oral Med Oral Pathol Oral Radiol Endod: 45: 701 -706.
Ernrnel, E. A., C. L. Verweij, D. B. Durand, K. M. Higgins, E. Lacy, and G. R. Crabtree. (1 989) Cyclosporin A specifically inhibits function of nuclear proteins involved in T cefl activation. Science 246: 1 61 7-1 620.
Everts, V. and Beertsen, W. (1988) The cellular basis of tooth eruption: the role of collagen phagocytosis. In Bioloaical Mechanisms of Tooth Eruotion and Root Resorption (ed. Z. Davidovitch) Birmingham, AL:EBSCO Media1237-242
Everts, V., Hembry, R.M., Reynolds, J.J. and Beertsen, W. (1 989) Metalloproteinases are not involved in the phagocytosis of collagen fibrils by fibroblasts. Matrix 9:266-276.
Everts, V. van der Zee, E., Creemers, L. and Beertsen, W. (1 996) Phagocytosis and intracellular digestion of collagen, its turnover and remodelling. Histochem J 28(4):229-245.
Fattore, L., Stableirn, M., Brenfeldt, G., Semla, T., Moran, B. and Doherty- Greenberg, J .M. (1 991 ) Gingival hyperplasia: a side effect of nifedipine and diltiazem. Spec Care Dent 11 : 107-1 09.
Favre, C.J., Jerstrom, P., Foti, M., Stendhal, O., Huggler, E., Lew, D.P. and Krause. K.-H. (1 996) Organization of ca2+ stores in rnyeloid cells: association of SERCA2b and the type-1 inositol-l,,4,5-trisphosphate receptor. Biochem J 31 6: 137-1 42.
Fell, H.B., Reynolds, J.J., Lawrence, C.E., Bagga, M.R. and Glauert, A.M. (1 986) The promotion and inhibition of collagen breakdown in organ cultures of pig synovium: The requirement for serum components and the involvement of cyclic adenosine-3',5'-monophosphate (CAMP). Collagen Re1 Res 6:51-57.
Friskopp, J. and Klintmalm, G. (1 986) Gingival enlargement: a cornparison between cyclosporine and azathioprine treated renal allograft recipients. Swed Dent J: 10: 85-92.
Gamerberucci, A., Innocenti, R., Fulcri, G., Banhegyi, R., Giunti, T., Pouan, T. and Benedetti A. (1 994) Modulation of ca2' influx dependent on store depletion by intracellular adenine-guanine nucleotide levels. J Biol Chem 269:23597- 23602.
Gelfand, E.W., Cheung, R.K., and Mills, G.B. (1987) The cyclosporins inhibit lymphocyte activation at more than one site. J lmmunol138:1115-1120.
Gerasimenko, J.V., Tepikin, A.V., Petersen, O.H. and Gerasimenko, O.V. (1998) Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr Bi0l8(24): 1335-1 338.
Glickman, 1. and Lewitus, M. (1941) Hyperplasia of the gingiva associated with Dilantin (sodium diphenyl hydantoinate) therapy. J Am Dent Assoc 28: 199-203.
Goldstein, R.H., Poliks, C.F., Pilch, P.F., Smith, B.D. and Fine, A. (1 989) Stimulation of collagen formation by insulin and insulin-like growth factor 1 in cultures of human lung fibroblasts. Endocrinology l24:964:97O.
Granelli-Piperno, A., Inaba, K. and Steinman, R. (1 984) Stimulation of lymphokine release from T lymphoblasts. Requirement for mRNA synthesis and inhibition by cyclosporin A. J Ekp Med l60:1792-1802.
Grynckiewicz, G., Poenie M. and Tsien, R.Y. (1985) A new generation of ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450.
Hajnoczky, G., Robb-Gaspers, L.D., Seitz, M.B. and Thomas, A.P. (1 995) Decoding of cytosolic calcium oscillations in the mitochondria. Ce// 82: 41 5-424.
Hall ,WB. (1 969) ~ i l a n t i n ~ hyperplasia: a preventable lesion. J Periodontol Res: 4: 36-37.
Hall, B.K. and Squier, C.A. (1 982) Ultrastructural quantitation of connective tissue changes in phenytoin-induced gingival overgrowth in the ferret. J. Dent Res 61 :942-952.
Hancock, R.H. and Swan, R.H. (1992) Nifedipine-induced gingival overgrowth. Report of a case treated by controlling plaque. J Clin Periodontol19(1): 12-1 4.
Hanschumaker, R.E., Harding, M.W., Rice, J., Drugge, R.J., and Speicher, D.W. (1 984) Cyclophilin: a specific cytosolic binding protein for CsA. Science 22:544- 547.
Haworth, R.A. and Hunter, D.R. (1 979) The ca2+-induced membrane transition inmitochondria. II. Nature of the ca2' trigger site. Arch Biochem Biophys 195:460-467.
Hefti, A.F., Eshenaur, A.E., Hassell, T.M. and Stone, C. (1 994) Gingival overgrowth in cyclosporine A treated multiple sclerosis patients. J Periodontoi 65: 744-749.
Hofer, A.M., Landolfi, B., Debellis, L., Pouan, T. and Curci, S. (1 998) Free [ca2'] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process. EMBO J 1 7(7):1986-1995.
Hui, M.-& Tenen baum, H.C. and McCulloch, C.A.G. (1 997) Collagen phagocytosis and apoptosis are induced by high level alkaline phosphatase expression in rat fibro blasts. J Cell Physioi 172:323-333.
Ichas, F., Jouaville, L.S. and Mazat, J.P. (1 997) Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Celi 89: 1 1 45-53.
Ingman, T., Sorsa, T., Michaelis, J. and Konttinen, Y.T. (1 994) lmmunohistochemical study of neutrophil- and fibroblast tpe collagenases and stromelysin-1 in adult periodontitis. Scand J Dent Res 102(6):342-349.
Irvine, R.F. (1990) 'Quantal' calcium release and the control of calcium entry by inositol phosphate - a possible mechanism. FEBS Lett 263:5-9.
Isakov, N., Mally, M.I., Scholz, W. and Altman, A. (1 987) T lymphocyte activation: the role of protein kinase C and the bifurcating inositol phospholipid signal transduction pathway. lmmunol Rev. 95:89-111.
Isberg, R.R. and Tran Van Nhieu, G. (1995) The mechanism of phagocytic uptake promoted by invasin-integrin interaction. Trends Ce11 Bioi 5: 1 20-1 24.
Jaconi, M.E.E., Lew, D.P., Carpentier, J.-L., Mangnusçion, K.E., Sjogren, M. and Stendhal? 0. (1990) Cytosolic free calcium elevation mediates the phagosome- lysosome fusion during phagocytosis in human neutrophils. J Cell Bi01 110:1555- 1564.
Johnson, D.W., Saunders, H.J., Johnson, F.J., Huq, S.O., Field, M.J. and Pollock, C.A. (1 999) Fibrogenic effects of cyclosporin A on the tubulointerstitium: role of cytokines and growth factors. Exp Nephrol7:470-478.
Jones, C. (1986) Gingival hyperplasia associated with nifedipine. Br Dent J 16O:416-417.
Kapur, R.N., Grigis, S., Little, T.M. and Masotti, R.E. (1973) Diphenylhydantoin induced gingival hyperplasia: Its relation to dose and serum level. Dev Med Child Neurol15: 483-487.
Kay, J.E. (1 989) lnhibitory effects of CsA on lymphocyte activation. In: Cvclos~orin. Mode of action and clinical a~plication (ed._ A.W. Thomson) Lancaster, UK: Kluwer Academic Publisher, p 4.
Kay, J.E. and Benzie, C.R. (1986) Lymphocyte activation by OKT3: cyclosporine sensitivity and synergism with phorbol ester. lmmunology 57:195-199.
Kay, J.E. (1 987) The cyclosporin A-sensitive event in lymphocyte activation. Ann lnst Pasteur lmmunol138(4):622-625.
Kay, J.E., Meehan, R.T. and Benzie, CR. (1983) Activation of T lymphocytes by 12-O-tetradecanoylphorbol-1 Sacetate is resistant to inhibition by cyclosporin A. lmmunol Lett 7: 151 -1 56.
Kimball, 0. (1 939) The treatment of epilepsy with sodium diphenyl-hydantoinate. JAMA 112: 1244.
King, S. (1 988) An assessrnent of p hosphoinositide hydrolysis in antigenic signal transduction in lymphocytes. Immunology 65: 1 -7.
Kirchhofer, D., Languino, L.R., Ruoslahti, E., and Pierschbacher, MD. (1990) a2Bi integrins from different ceIl types show different binding specifities. J Bioi Chem 265:615-618.
Kobayashi, K., Takahashi, K. and Nagasawa, S. (1995) The role of tyrosine phosphorylation and ca2+ accumulation in Fcy receptor-mediated phagocytosis in neutrophils J Biochem 1 17: 1 156-1 161.
Kulkarni, G.V. and McCulloch, C.A.G. (1 995) Concanavalin A induced apoptosis in fibroblasts: The role of cell surface carbohydrates in lectin rnediated cytotoxicity. J Cell Physioll65: 1 1 9-1 33.
Kulkarni, G.V., Lee, W., Seth, A. and MCCUIIOC~,C.A.G. (1998) Role of mitochondrial membrane potential in concanavalin A-induced apoptosis in human fibroblasts. Exp CeIl Res 244: 170-1 78.
Kwiatkowska K, Sobota A. Signaling pathways in phagocytosis. (1 999) BioEssays 21 : 422-431 .
Lainson, P.A. (1 986) Gingival overgrowth in a patient treated with nifedipine (Procardia). Periodontal Case Rep 8: 1 4- 1 6.
Landolfi, B., Curci, S., Debellis, L., Pouan, T. and Hofer, A.M. ca2' homeostasis in the agonist-sensitive interna1 store: functional interactions between mitochondria and the ER measured in situ in intact cells. J Ceil Biol 142:1235- 1243.
Lederman, D., Lumerman, H., Reuben, S. and Freedman, P.D. (1 984) Gingival hyperplasia associated nifedipine treatment. Report of a case. Oral Surg Oral Med Oral Path 57(6):620-622.
Lee, W., Sodek, J. and McCulloch, C.A.G. (1996) Role of integrins in regulation of collagen phagocytosis by human fibroblasts. J Ce11 Physiol168:695-704.
LeGrue, S. J., Friedman, A.W., and Kahan, B.D. (1 983). Binding of cyclosporine by human lymphocytes and phospholipid vesicles. J lmmunol131:712-718.
Lindholm, A. and Kahan, B.D. (1 993) Influence of cyclosporine pharmacokinetics, trough concentrations, and AUC monitoring on outcorne after kidney transplantation. Clin Pharmacol Ther M(2): 205-21 8.
Liu, X., Wu, H., Byrne, M., Jeffrey, J., Krane, S. and Jaenisch, R. (1995) A targeted mutation at the known collagenase cleavage site in mouse type 1 collagen impairs tissue remodelling. J Ce11 Biol l30:227-237.
Lee, H., Theilade, E. and Jensen, S. (1965) Experimental gingivitis in man. J Periodontol36: 177-187.
Lucas, R.M., Howell, L.P. and Wall, B.A. (1 985) Nifedipine-induced gingival hyperplasia. A histochemical and ultrastructural study. J Periodontol56(4):211- 21 5.
Marusich, M.F., Robinson, B.H., Taanman, J.-W., Kim, S.J., Schillace, R., Smith, J.L. and Capaldi, RA.. (1 997) Expression of mtDNA and nDNA encoded respiratory chain proteins in chemically and genetically-derived Rh00 human fibroblasts: a cornparison of subunit proteins in normal fibroblasts treated with ethidium bromide and fibroblasts from a patient with mtDNA depletion syndrome.. Biochim Biophys Acta 1382: 145-1 59.
McCulloch, C.A.G. and Knowles, G.C. (1 993) Deficiencies in collagen phagocytosis by human fibroblasts in vitro: A mechanism for fibrosis? J Ce11 Physiol 1 55:461-471.
McGaw, W.T., Lam, S. and Coates, J. (1 987) Cyclosporine-induced gingival overgrowth: correlation with dental plaque scores, gingivitis scores and cyclosporine levels in serum and saliva. Oral Surg Oral Med Oral Pathol Oral Radio1 Endodo 64: 293-297.
McGaw, W.T. and Porter H. (1 988) Cyclosporine-induced gingival overgrowth: An ultrastructural stereologic study. Oral Surg Oral Med Oral Pathol65:186-90.
McGaw, W.T. and Ten-Cate, A.R. (1983) A role for collagen phagocytosis by fibroblasts in scar remodelling: An ultrastructural sterologic study. J lnvest Dermat0181 :375-78.
McKeown, M., Knowles, G. and McCulloch, C.A.G. (1 990) Role of the cellular attachment domain of fibronectin in the phagocytosis of beads by human gingival fibroblasts in vitro. Ce11 Tissue Res 262:523-530.
Melcher, A.H. and Chan, J. (1 981 ) P hagocytosis and digestion of collagen by gingival fibroblasts in vivo: A study of serial sections. J Ultrastruct Res 77:l-36.
Mignatti, P., Rifkin, D.B., Welgus, HG. and Parks, W.C. (1996) Proteinases and tissue rernodeling. In The Molecular and Cellular Bioloav of Wound Re~air. Second Edition. (ed. R.A.F.Clark)Plenurn Press, New York:pp 427-474.
Mochly-Rosen, D. and Gordon, A.S. (1 998) Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J 12:35-45.
Mutsaers, S.E., Bishop, J.E., McGrouther, G. and Laurent, G.J. (1997) Mechanism of tissue repair: from wound healing to fibrosis. lnt J Biochem Cell Bi01 29:5-17.
Nabel, G.J. (1 999) A transformed view of cyclosporin. Nature 397(6719):47l- 472.
Narayanan, A.S. and Page, R.C. (1 983) Connective tissues of the periodontium: A summary of current work. Collagen Re/ Res 3:33-64.
Newell, J. and Irwin, C R . (1 997) Comparative effects of cyclosporin on glycosaminoglycan synthesis by gingival fibroblasts. J Periodontol68(5):443-47.
Nicolli, A., Basso, E., Petronilli, V., Wenter, R.M. and Bernardi, P. (1996) Interactions of cyclophlin with the rnitochondrial inner membrane and regulation of the permeability transition pore, a cyclosporin A-sensitive channel. J Bid Chem 271 :2185-2192,
Panuska, H.J., Gorlin, R.J., Bearman, J.E. and Mitchell, D.F. (1961) The effect of anticonvulsant drugs upon the gingiva. A series of 1048 patients. II. J Periodontol 32: 15-28.
Park, K.S., Kim, T.K. and Kim, D.H. (1 999) Cyclosporin A treatment alter characte ristics of ca2'-release channel in sarcoplasmic reticulum. Am J Physiol 276 (Heart Circ Physiol45):H865-H872.
Pender, N. and McCulloch, C.A.G. (1 991) Quantitation of actin polymerization in two human fibroblast subtypes responding to mechanical stretching. J Ce// Sci 100: 1 87-1 93.
Putney, J.W. Jr. (1 986) A model for receptor-regulated calcium entry. Ce// Calcium 7: 1 -1 2.
Rabinovitch, M. (1 995) Professional and non-professional phagocytes: an introduction. Trends Celi Bio/5:85-87,
Raeste, A.M ., Collan, Y. and Kilpinen, E. (1 978) Hereditary fibrous hyperplasia of the gingiva with varying penetrance and expressivity. Scand J Dent Res 86: 357- 365.
Rateitschak-PIiiss, E.M., Hefti, A., Lortscher, R. and Thiel, G. (1983) Initial observation that cyclosporine A induces gingival enlargement in man. J Clin Periodontol 10: 237-246.
Riuuto R., Brini M., Murgia, M. and Pouan T. (1993) Microdomains with high ca2+ close to IP3-sensitive channels that are sensed by neighbouring mitochondria. Science 262:744-747.
Rossrnann, J.A., Ingles, E. and Brown, R.S. (1 994) Multirnodal treatment of drug- induced gingival hyperplasia in a kidney transplant patient. Compendium Contin Educ Dent 15: 1266-1 274.
Savage, M.K., Jones, D.P. and Reed D.J. (1 991 )Calcium- and phosphate- dependent release and loading of glutathione by liver mitochondria. Arch Biochem Biophys 290(1):51-6.
Schwartz, M.A. (1 993)Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J Ce11 Bi01 120(4):1003- 1010
Sells, R. Long-term cyclosporin: Delivering effective therapeutic levels. London, England: Royal Society of Medicine Press Ltd., 1 994: 1 -3, 56.
Seymour, R.A., Smith, D.G. and Rogers, S.R. (1 987) The comparative effects of azathioprine and cyclosporine on some gingival health parameters of renal treansplant patients. A longitudinal study. J Clin Periodontol14: 61 0-61 3.
Seymour, R. and Heasman, P. (1 988) Drugs and the periodontium. J Clin Periodontol 1 5: 1 - 1 6,
Silverstein, L.H., Koch, J.P., Lefkove, M.D., Garnick, J.J., Singh, B. and Steflik, D.E. (1 995) Nifedipine-induced gingival enlargement around dental implants: a clinical report. J Oral implant01 21 : 1 1 6-1 20.
Slavin, J. and Taylor, J. (1 987) Cyclosporine, nifedipine, and gingival hyperplasia. Lancet 2(8561): 739.
Srniley, S.T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T.W., Steele, G.D. Jr. and Chen, L.B. (1 991 ) lntracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Nat1 Acad Sci USA 88:3671-3675.
Sodek, J. and Overall, C. (1988) Matrix degradation in hard and soft connective tissues. ln The Bioloaical Mechanisms of Tooth Eru~tion and Root Resorption (ed. 2. Davidovitch) Birmingham, AL:EBSCO Media:303-311.
Staple, P.H., Reed, M.J., Mashimo, P.A., Sedransk, N. and Umemoto, T. (1 978) Diphenylhydantoin gingival hyperplasia in Macaca arctoides. Prevention by inhibition of dental plaque deposition. J Periodontol49: 31 0-325.
Stendahl, O., Krause, K.-H., Krischer, J., Jerstrdm, P., Theler, J.M., Clark, RA., Carpentier, J.L. and Lew, D.P. (1994) Redistribution of intracellular calcium stores during phagocytosis in human neutrophils. Science 265: 1439-1 441 .
Sullivan, P.G., Thompson, M.B. and Scheff, W. (1 999) Cyclosporin A attenuates acute rnitochondiral dysfunciton following traumatic brain injury. Experimental Neurology 1 60: 226-234.
Svoboda, E.L.A., Shiga, A. and Deporter, D.A. (1 981) A stereologic analysis of collagen phagocytosis by fibroblasts in three soft connective tissues with differing rates of collagen turnover. Anat Rec 199:473-480.
Thiru, S. Pathological effects of cyclosporin A in clinical practice. ln Cvclos~orin Mode of Action and Clinical A~plication (ed. AW Thomson) Lancaster. UK: Kluwer Academic Publisher, 1989: 324-359.
Thomason, J.M., Sloan, P. and Seymour, R.A. (1 998) lmmunolocalization of collagenase (MMP-1) and stromelysin (MMP-3) in the gingival tissues of organ transplant patients medicated with cyclosporin. J Clin Periodontol25:554-560.
Tipton, D.A., Stricklin, G.P. and Dabbous, M.K. (1 991) Fibroblast heterogeneity in collagenolytic response to cyclosporine. J Ce11 Biochern 46(2): 152-1 65.
Van Der Zee, E. Everts, V., Hoeben, K. and Beertsen, W. (1995) Cytokines rnodulate phagocytosis and intracellular digestion of collagen fibrils in rabbit periosteal explants. Inverse effects on procollagenase production and collagen phagocytosis. J Ce11 Sci 108: 3307-331 5.
Van Rossum, DB., Patterson, R.L., Ma, H.T. and Gill, D.L. (2000) Calcium entry mediated by store-depletion, S-nitrosylation and TRP3 channels: cornparison of coupling and function. J Bi01 Chem epub ahead of print.
Wilson TG, Kornman KS. Fundarnentals of periodontics. Carol Stream, IL: Quintessence Publishing Co., 1 996: 263-265.
Young, J.D., Ko, S.S. and Cohn, Z.A. (1 984) The increase in intracellular free calcium associated with IgG gamma Pblgamma 1 Fc receptor-ligand interactions: role in phagocytosis. Proc Nati Acad Sei USA 81 (1 7): 5430-5434.
Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Mach, A., Hirsch, T., Susin, S.A., Petit, X.A., Mignotte, B. and Droemer, G. (1995) Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed ceIl death. J Exp Med 182:367-377.
Zilberman, Y., Howe, L.R., Moore, J.P., Hesketh, T.R. and Metcalfe, J.C. (1987) Calcium regulates inositol 1,3,4,5-tetrabisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A. EMBO J6:957- 962.
Zoratti, M. and Szabo, 1. (1 995) The mitochondrial permeability transition. Biochim Biophys Acta 1241 : 1 39-1 76.