Photorejuvenation Induced by

5-Aminolevulinic Acid Photodynamic

Therapy in the Patients with Actinic

Keratosis: A Histological Analysis

by

Min Young Park

Major in Medicine

Department of Medical Sciences

The Graduate School, Ajou University

Photorejuvenation Induced by

5-Aminolevulinic Acid Photodynamic

Therapy in the Patients with Actinic

Keratosis: A Histological Analysis

by

Min Young Park

A Dissertation Submitted to The Graduate School of Ajou University

in Partial Fulfillment of the Requirements for the Degree of

Master of Medicine

Supervised by

You Chan Kim, M.D., Ph.D.

Major in Medicine

Department of Medical Sciences

The Graduate School, Ajou University

February, 2009

This certifies that the dissertation

of Min Young Park is approved.

SUPREVISORY COMMITTEE

You Chan Kim

Eun-So Lee

Seonghyang Sohn

The Graduate School, Ajou University

December 22nd, 2008

감사의감사의감사의감사의 글글글글

본 논문을 완성할 때까지 지도와 격려를 베풀어 주셨던 지도 교수이신 김

유찬 교수님께 먼저 진심으로 감사 드립니다. 또한 논문 지도를 위하여 많은

조언를 아끼지 않으신 이은소 교수님, 손성향 교수님께 감사의 마음을 전합

니다.

연구가 진행되는 동안 많은 시간과 노력으로 도와주신 피부과학 교실원 여

러분과, 실험을 위하여 필요한 조직 염색과 분석을 도와주신 김영배 선생님

께 감사의 마음을 전합니다. 그리고 언제나 저에게 아낌없는 사랑으로 힘이

되어주신 부모님, 가족들에게 깊은 감사의 마음을 전합니다.

2009년 1월

저자씀

i

- ABSTRACT-

Photorejuvenation Induced by 5-Aminolevulinic Acid

Photodynamic Therapy in the Patients with Actinic Keratosis

: A Histological Analysis

Background: Repeated exposure to ultraviolet radiation from the sun results in premature

photoaging. Photodamaged skin is histologically characterized by reduced collagen synthesis,

increased collagen destruction, and accumulation of abnormal elastotic material in the

dermis. Photodynamic therapy (PDT) is one of many treatment options available for

photoaging; it has been shown to be effective, whereas the data on most other studies is

based on clinical observation.

Objectives: I investigated whether 5-aminolevulinic acid (ALA)-PDT induced histological

changes suggesting photorejuvenation.

Materials and Methods: Fourteen patients with one to three actinic keratosis on the face

were treated twice with ALA-PDT using a 1200W metal halogen lamp at one month

intervals. Skin biopsy before and one month after the PDT was performed. Twenty five pairs

of specimens were obtained. I examined the specimens with routine and

immunohistochemical staining and evaluated the parameters associated with photoaging

using image analysis.

ii

Results: After ALA-PDT, the mean epidermis thickness and inflammatory infiltrate in the

dermis were reduced. The total collagen volume in the dermis was significantly increased

with expression of type I and III procollagen. The level of transforming growth factor β and

transforming growth factor β type II receptors were also increased. The elastotic material with

co-localizing fibrilline-1 and tropoelastin expression in the dermis tended to decrease after

treatment. The expression of matrix metalloproteinases -1, -3, and -12 was also decreased.

Conclusion: The histological evidence showed beneficial effects of ALA-PDT for

photodamaged skin. Further controlled split-face studies are required to determine the

precise mechanism underlying the effects of ALA-PDT on photoaging.

Key words: Photodynamic therapy, Photorejuvenation, Histological changes

iii

TABLE OF CONTENTS

ABSTRACT ················································································································· ⅰ

TABLE OF CONTENTS ···························································································· iii

LIST OF FIGURES ········································································································· iv

LIST OF TABLES·········································································································· v

. Ⅰ INTRODUCTION ··································································································· 1

. Ⅱ MATERIALS AND METHODS·············································································· 3

A. SUBJECTS ············································································································ 3

B. MATERIALS AND METHODS··········································································· 3

1. Photodynamic therapy ························································································ 3

2. Hematoxylin and eosin (H&E) staining······························································ 4

3. Masson-trichrome staining and Verhoeff’s elastic staining································ 5

4. Immunohistochemical staining··········································································· 5

5. Statistical analysis······························································································· 6

. Ⅲ RESULTS ··············································································································· 7

. Ⅳ DISCUSSION ······································································································· 19

. Ⅴ CONCLUSION ····································································································· 24

REFERENCES ··········································································································· 25

국문요약 ···················································································································· 29

iv

LIST OF FIGURES

Fig. 1. Clearance of AK after ALA-PDT·········································································· 9

Fig. 2. Decreased epidermal thickness and dermal inflammatory infiltrate after ALA-PDT

········································································································································ 10

Fig. 3. Marked increased collagen deposition in the dermis after ALA-PDT ················ 11

Fig. 4. Increased expression of procollagen type I and III after ALA-PDT ················· 12

Fig. 5. Increased expression of TGF-β and TβR II in the epidermis after ALA-PDT ·· 13

Fig. 6. Accumulative effect of ALA-PDT on the expression of TGF-β, TβR II, procollagen

- I, procollagen III, and total collagen volume ················································· 14

Fig. 7. Improvement of solar elastosis by ALA-PDT····················································· 15

Fig. 8. Decreased fibrillin-1 and tropoelastin after ALA-PDT······································· 16

Fig. 9. Decreased expression of MMP-1, -3, and -12 after ALA-PDT ························· 17

v

LIST OF TABLES

Table 1. Baseline characteristics of patients and course of treatment of AKs ················ 8

Table 2. Summary of recent studies of ALA-PDT in photorejuvenation ····················· 21

1

I. INTRODUCTION

Ultraviolet (UV) irradiation from the sun damages human skin and causes premature skin aging

(photoaging) (Gilchrest, 1995). Clinically, photoaged skin differs from sun-protected, naturally

aged skin; it is thickened and rough, with course wrinkles, mottled pigmentation, and

precancerous lesions including actinic keratosis (AK) (Gilchrest, 1989).

There is growing interests in reversing the signs of photoaging. Photodynamic therapy (PDT),

with a 5-aminolevulinic acid (ALA) a photosensitizing agent, using a variety of lasers and light

sources, enhances the treatment of photodamaged skin and associated AK (Alster et al., 2005;

Gold et al., 2006; Touma et al., 2004). However, the histological changes that occur after ALA-

PDT, have not been fully assessed.

The main constituent of dermal extracellular matrix (ECM) is collagen, particularly collagens I

and III, which provide skin with its strength and resilience. However, in photoaged skin, the

production of procollagen, the precursors of collagen is reduced; an impaired transforming

growth factor -β (TGF-β)/Smad pathway, caused by UV irradiation, might play a role in the

pathology (Fisher et al., 2000; Quan et al., 2004). TGF-β is the major regulator of ECM

synthesis in human skin; it stimulates fibroblast proliferation in the dermis to enhance collagen

synthesis (Inagaki et al., 1994). Down-regulation of the TGF-β type II receptor (TβRII) and

ensuing lower response of TGF-β, by UV radiation result in reduced of collagen synthesis (Quan

et al., 2004). Moreover, dermal collagen is destroyed by the UV-induced matrix

metalloproteinases (MMPs) in human skin (Fisher et al., 1996). MMP-1(fibroblast collagenase)

is the main enzyme that degradades collagen in the skin (Kahari and Saarialho-Kere, 1997).

2

Once MMP-1 breaks down collagen, further degradation is followed by MMP-3 (stromelysin 1),

and other MMPs (Sternlicht and Werb, 2001).

Solar elastosis, the deposition of dystrophic elastotic material within the reticular dermis is also

a characteristic of photoaged skin (Montagna et al., 1989). The mechanism causing this damage

remains unclear. However, it may be related to the increased production of elastic fibers which

consist of elastin and fibrillin-rich microfibrils (Bernstein et al., 1994; Seo et al., 2001). The

degradation of elastin by MMP-12 (human macrophage metalloelastase) may contribute to the

development of solar elastosis in photoaged skin (Chung et al., 2002).

In the present study, I examined pre- and post-ALA-PDT biopsy specimens for AK, and

determined whether ALA-PDT induced histological changes reversing the destructive

connective tissue events associated with photoaging.

3

II. MATERIALS AND METHODS

A. SUBJECTS

From December 2006 to February 2008, fourteen patients with one to three AKs on the face

presenting to the outpatient clinic at the Department of Dermatology, Ajou University Hospital,

Korea were treated with ALA-PDT. All patients diagnosed with AK by skin biopsy. The study

was approved by the institutional review board (IRB number: AJIRB-CRO-08-021) and all

subjects provided written informed consent. All patients were Korean with skin types III to V.

Among these subjects were five (35.8%) males and nine (64.2%) females. The age ranged from

52 to 85, with a mean age of 62.07. There was no history of previous treatment for AKs.

B. MATERIALS AND METHODS

1. Photodynamic therapy

All patients received the same therapeutic procedure. Twenty percent ALA (MEDAC GmbH,

Hamburg, Germany) was applied to the lesions under an occlusive and light-shielding dressing.

Scales and crusts were gently removed before application of the cream. Four hours later, the

dressings were removed, and the ALA was washed off with 0.9% saline solution. The lesions

were illuminated with red light from a noncoherent light source (Waldmann PDT 1200;

Waldmann-Medizin-Technik, Villingen-Schwenningen, Germany, emission wavelength

580~740m) at a light dose of 100 J/cm2 and a fluence rate of 100 mW/cm2.

The treatments were performed twice, at one month intervals. One month after the last

procedure, the response to the PDT was evaluated. Two to three millimeter punch biopsies were

4

performed for histological examination to assess the response. Patients with residual lesions

received an additional two cycles of PDT or cryotherapy. The post-cryotherapy specimens were

excluded from the analysis. Finally, 25 pairs of skin specimens were obtained.

2. Hematoxylin and eosin (H&E) staining

Skin biopsies were fixed in 10% formalin, embedded in paraffin, and sectioned into 5-µm

sections for routine H&E staining. The epidermal thickness, degree of solar elastosis, and

inflammatory infiltrate were measured. The epidermal thickness was the mean length between

the outer sides of the epidermis, excluding the stratum corneum, and the dermo-epidermal

junction through the entire section; computer-based software (Image-Pro Plus, MEDIA

CYBERNETICSA, Silver Spring, MD, USA) was used for the measurements. For analysis of

the solar elastosis and the inflammatory infiltrate, the sections were randomized, and examined

under an Olympus microscope (Tokyo, Japan) by blinded investigators. Two individual

dermatologists scored the samples using a five-point semi-quantitative scale (0 = no staining and

4 = maximal expression within the view) of three high power fields per section; the average

score was calculated for each section.

3. Masson-trichrome staining and Verhoeff’s elastic staining

Masson-trichrome stained collagen fibers and Verhoeff’s stained elastic fibers were quantified

using computer software as mentioned above. For the scanning view of the papillary and upper

reticular dermis, low magnification (x100) was used. The epidermis and epidermal appendages

were excluded from the fields. The image program assessed the degree of expression of the

5

stained target versus the unstained background, presented as the percent expression.

4. Immunohistochemical staining

Using the immunoperoxidase technique, 5 µm sections from formalin-fixed, paraffin embedded

skin samples were stained with antibodies as follows: Rabbit monoclonal anti-procollagen type I

antibody (diluted 1:1000, Chemicon, Temecula, CA, USA), rabbit polyclonal anti-procollagen

type III antibody (diluted 1: 500, CEDARLANE, Ontario, Canada), mouse monoclonal anti-

TGF-β antibody (diluted 1: 500, Gene Tex, San Antonio, TX, USA), rabbit polyclonal anti-

TβRII antibody (diluted 1: 100, Spring Bioscience, Fremont, CA, USA), rabbit polyclonal anti-

fibrillin-1 antibody (diluted 1: 50, Abcam, Cambridge, UK), rabbit polyclonal anti-tropoelastin

antibody (diluted 1: 50, Abcam, Cambridge, UK), rabbit polyclonal anti-MMP-1 antibody

(diluted 1: 50, NeoMarker, Fremont, CA, USA), rabbit monoclonal anti- MMP-3 antibody

(diluted 1: 100, Epitomics, Burlingame, CA, USA), rabbit monoclonal anti- MMP-12 antibody

(diluted 1: 100, Epitomics, Burlingame,CA, USA), and mouse monoclonal anti-TIMP-1

antibody (diluted 1: 50, Santa Cruz, CA, USA).

For quantitative evaluation of the immunoreactivity of procollagen I, III, TGF-β, and TβRII,

the previously mentioned software was used for the immunohistochemically stained sections in

the same way it was used for collagen and elastin fiber measurements. The TGF-β and TβRII,

was evaluated in the epidermis where it is the predominant expressed. The expressing pattern of

fibrillin-1, tropoelastin, MMP-1, 3, 12, and TIMP-1 were discontinuous, not appropriate to apply

computer software. They were evaluated with a semi-quantitative scale as mentioned above.

6

5. Statistical analysis

A paired Student’s t-test was used to determine statistical significance of the differences

between the staining patterns using the SPSS 11.0 statistics program (SPSS, Inc., Chicago, IL,

USA). All P values were two-tailed, and differences were considered significant when the P-

value was less than 0.05. Summary data are expressed as the mean ± standard deviation (SD).

7

III. RESULTS

1. Clearance of AKs

The ALA-PDT was tolerated in all patients. During the treatment, variable degrees of erythema

and edema occurred, but resolved within a few days. Some patients reported mild to moderate

pain, but interruption of procedure or topical anesthesia were not required.

Initially, 23AKs were detected among the patients. One month after 2 sessions of PDT, 19

lesions from 11 patients showed a complete clinical and histological response (remission rate:

82.6%) (Fig. 1A, B). Clinically, there was no significant scarring or pigmentary changes after

treatment (Fig. 1A). Among four patients with persistent lesions, one patient with two lesions

received two additional PDT treatments, the AKs in the follow up biopsy resolved. The other

two patients underwent cryotherapy twice with liquid nitrogen, and the lesions cleared (Table 1).

8

Table 1. Baseline characteristics of patients and course of treatment of AKs

Patient

No. Sex/age Location - No. of lesions

Results of

PDT Further treatment

1 M/72 Rt. cheek - 2 CR

2 F/64 Lt. cheek - 1 CR

3 F/85 Lt. cheek- 2 CR

4 F/67 Rt. cheek - 1 CR

5 F/84 Lt. cheek - 1 CR

6 F/80 forehead -1, nose -1, Lt. cheek -1 CR

7 M/70 Lt. cheek - 1 CR

8 F/79 nose - 2 Non-CR 2 sessions of PDT

-> CR

9 M/75 nose -1 CR

10 F/52 Rt. cheek - 2 CR

11 M/64 Lt. cheek -1, Rt. cheek -1 CR

12 F/77 Rt. upper eyelid -1, Lt. cheek-1 Non-CR of

cheek lesion

2 sessions of

cryotherapy -> CR

13 F/76 Rt. cheek -1 CR

14 F/67 nose-2 Non-CR of

1 lesion

2 sessions of

cryotherapy -> CR

No, number; Rt, right; Lt, left; CR, complete response; Non-CR, non-complete response

9

pre- PDT post- PDT

Fig. 1. Clearance of AK after ALA-PDT . Patient 9 with actinic keratosis on the nose showed

complete clinical clearance of lesion (A). The atypical keratinocytes in the epidermis were all

disappeared after treatment in the same patient (H&E, x 200) (B).

A.

B.

10

2. Mean epidermal thickness and inflammatory infiltrates

The pre-treatment specimens showed thick epidermis and a large number of infiltrating cells in

the dermis. However, after treatment, the mean epidermal thickness and the inflammatory

infiltrate were significantly reduced (Fig. 2 A, B).

pre- PDT post- PDT

Epidermal thickness

0

20

40

60

80

100

120

140

160

180

pre-PDT post-PDT

Mea

n ep

ider

mal

thic

knes

s (u

m)

Inflammatory infiltrate

0

0.5

1

1.5

2

2.5

pre-PDT post-PDT

Infla

mm

ator

y in

filtr

ate

sem

i-qua

ntita

tive

anal

ysis

Fig. 2. Decreased epidermal thickness and dermal inflammatory infiltrate after ALA-PDT .

The epidermal thickness and numerous dermal inflammatory infiltrate decreased after PDT

(patient 2, H&E, x 200) (A). The mean epidermal thickness in photodamaged facial skin was

significantly reduced from 146.10 ±173.20 um to 75.26±29.70 um after treatment. And in the

semi-quantitative analysis, the dermal inflammatory infiltrate also significantly decreased (B).

(*p<0.00, **p<0.02)

A.

B. * **

11

3. Total collagen volume

After PDT, almost all biopsy specimens showed impressive collagen deposition in the upper

dermis by H&E staining that was more clearly demonstrated by Masson-trichrome staining (Fig.

3 A, B). The differences in staining density were significant (Fig. 3 C).

pre- PDT post- PDT

Expression of collagen

0.0

5.0

10.0

15.0

20.0

25.0

30.0

pre-PDT post-PDT

Mea

n co

llage

n st

aini

ng (%

)

Fig. 3. Marked increased collagen deposition in the dermis after ALA-PDT. H&E (A) and

Masson-trichrome (B) stained specimens demonstrated up-regulated collagen fibers in the upper

dermis after PDT (patient 7, x 200). Staining with Masson-trichrome demonstrated a 10.49

±9.93 % average increase of collagen in staining density in the post-treatment than in the pre-

treatment specimens (p<0.00) (C).

A.

B.

C.

12

4. Procollagen type I and type III

The expression of collagen precursors was also increased. Staining with procollagen type I and

type III antibodies demonstrated an 18.03±16.41% and 11.50±9.97% average increase in their

staining density, respectively (Fig. 4 A-C).

pre- PDT post- PDT

Expression of procollagen

0

10

20

30

40

50

60

70

pre-PDT post-PDTMea

n ta

rget

ant

igen

sta

inin

g (%

)

Procollagen I

Procollagen III

Fig. 4. Increased expression of type I and III procollagen after ALA-PDT. Type I (A) and

type III (B) procollagen expression was increased in skin sections after ALA-PDT (patient 10,

lesion 1, x 200). The average increase was significantly different (C). (*p<0.05)

A.

B.

C.

*

*

13

5. TGF-ββββ and TββββR II

In the pre-treated skin sections, the expression of TGF-β and TβR II was insignificant. However, after

treatment, the mean staining density with TGF-β and TβR II antibodies showed a significant increase

mainly in the epidermis.

pre- PDT post- PDT

TGF-b

0

10

20

30

40

50

60

70

80

pre-PDT post-PDT

Mea

n ta

rget

ant

igen

sta

inin

g (%

)

TGF-b

TbRII

C. TβR II

TGF-β

Expression of TGF-β and TβR II

*

*

B)

A)

14

Fig. 5. Increased expression of TGF-ββββ and TββββR II in the epidermis after treatment. TGF-β

(patient 3, x 200) (A) and TβR II (patient 14, x 200) (B) was more expressed in the epidermis

after ALA-PDT. The mean staining with TGF-β and TβR II antibodies demonstrated a

23.57±15.16% and 6.61±9.56% average increase in staining density, respectively (C). (*p<0.05)

In one patient with two AK lesions that underwent two additional PDT sessions, the mean

expression of TGF-β, TβR II, procollagen I, procollagen III, and total collagen increased more

proportional to the number of ALA-PDT sessions. (Fig. 6)

0

10

20

30

40

50

60

70

80

0 2 4

Sessions of PDT

Mea

n ta

rget

ant

igen

sta

inin

g (%

)

TGF-b

TbRII

Procollagen I

Procollagen III

Collagen

Fig. 6. Accumulative effect of ALA-PDT on the expression of TGF-ββββ, TββββR II, procollagen I,

procollagen III, and total collagen volume. The mean expression of TGF-β, TβR II,

procollagen I, procollagen III, and total collagen volume was more and more increased with

sessions of ALA-PDT (patient 8).

TGF-β TβR II

15

6. Elastotic material

In the H&E stained sections, a marked elastotic mass was visible in the papillary and reticular

dermis of the pre-PDT specimens that were pathognomonic for photoaging. After ALA-PDT,

there was a tendency for decrease of these elastotic masses compared to the pre-treatment

specimens. For patient 8 who received four treatment sessions, the change was more pronounced

(Fig. 7A). Verhoeff’s elastic stain demonstrated that after the PDT, the thickened and amorphous

elastotic materials disappeared and were restored to more normal horizontally arranged fibers in

the dermis (Fig. 7B).

pre- PDT post- PDT

Fig. 7. Improvement of solar elastosis by ALA-PDT. After ALA-PDT, solar elastosis was

improved (patient 8, lesion 1, H&E, x 200) (A), and elastin fiber reorganization was more clearly

presented with Verhoeff’s stain (B) (same patient, x 400).

A.

B.

16

7. Fibrillin-1 and tropoelastin

In the dermis, expression of fibrillin-1 and tropoelastin were mainly co-localized with elastotic

material, found in the epidermis to a lesser extent. After treatment, the immunoreactivity of

fibrillin-1 and tropoelastin was reduced along with resolution of the solar elastosis; the epidermal

expression also decreased (Fig. 8A-C).

pre- PDT post- PDT

Expression of fibrillin-1 and tropoelastin

0

0.5

1

1.5

2

2.5

3

3.5

pre-PDT post-PDT

Mea

n ta

rget

ant

igen

sta

inin

gse

mi-q

uant

itativ

e an

alys

is

Fibrillin-1

Tropoelastin

A.

B.

C *

*

17

Fig. 8. Decreased fibrillin-1 and tropoelastin after ALA-PDT . The fibrillin-1 (A) and

tropoelastin (B) expression decreased in the dermis after treatment (patient 8, lesion 2, x 200).

The mean difference was 1.32±0.90 and 0.56±0.58 in the semi-quantitative analysis after ALA-

PDT (C). (*p<0.05)

8. MMPs and TIMP

I also investigated changes in the MMPs, degradating enzymes of ECM, and TIMP-1, an

inhibitor of MMPs. The expression of MMP-1(Fig. 9A), MMP-3(Fig. 9B), and MMP-12 (Fig.

9C) tended to decrease after treatment. The immunoreactivity of TIMP-I was minimal when

evaluated by our method.

pre- PDT post- PDT

A.

B.

18

Expression of MMP-1,-3,-12

0

0.5

1

1.5

2

2.5

pre-PDT post-PDT

Mea

n ta

rget

ant

igen

sta

inin

gse

mi-

quan

titat

ive

anal

ysis MMP-1

MMP-3

MMP-12

Fig. 9. Decreased expression of MMP-1, -3, and -12 after ALA-PDT. The degree of MMP-1, -

3, and -12 expression reduced after ALA-PDT (A-C). The differences were 0.60±0.91,

0.64±0.91, and 0.68±1.14 in the semi-quantitative analysis, respectively (D). (*p<0.05)

C.

D. *

* *

19

IV. DISCUSSION

Topical ALA-PDT was originally used for superficial non-melanoma skin cancers and their

precursors (Fritsch et al., 1998). However other benign diseases, such as acne vulgaris,

sebaceous gland hyperplasia, and hidradenitis suppurativa have been shown to improve with this

treatment (Szeimies et al., 2002). Moreover, previous studies have demonstrated the

effectiveness of ALA-based PDT treatments using a variety of lasers and light sources for

photorejuvenation (Table 2). However, most prior data has been based on clinical observation,

without histopathological confirmation. The results of the present study provide

histopathological evidence for photorejuvenation with ALA-PDT in patients with AKs.

20

Table. 2 Summary of recent studies of ALA-PDT in photorejuvenation

Author

year

Light

source Methods Results of ALA-PDT

Touma et al.

2004 (Touma

et al., 2004)

Blue

light

(417±5

nm)

18 subjects

20% ALA-solution

1,2 or 3 hours of

application

Approximately 90% resolution of AKs and

improvement skin appearance by reducing

wrinkling, shallowness, and dyspigmentation

Dover et al.*

2005 (Dover

et al., 2005)

IPL 20 subjects

20% ALA-solution

0.5 to 1 hour of

application

Significantly greater improvement in global

photodamage, mottled pigmentation, and fine

lines in the ALA-PDT-IPL group than

treatment with IPL alone group

Marmur et

al.*2005

(Marmur et

al., 2005)

IPL 7 subjects

20% ALA-solution

1 hour of application

An increase in type I collagen fibers was seen

in the ALA-PDT-IPL group than treatment

with IPL group alone in the electron

microscopic ultrastructural analysis

Lowe et al.

2005 (Lowe

and Lowe,

2005)

LED

(633nm)

6 subjects

5% 5-ALA cream

0.5 hour of application

Significant treatment response in four out of

six subjects (67%) with a reduction in fine

lines in the periorbital area, and skin softness

was shown in all subjects.

Gold et al.*

2006 (Gold et

al., 2006)

IPL 16 subjects

20% ALA-solution

0.5 to 1 hour of

application

Improvement of crow’s feet appearance, tactile

skin roughness, mottled hyperpigmentation,

and telangectasias, and clearance rate of AK

lesions were higher in the ALA-PDT-IPL

group than the in IPL-alone group

Orringer et al.

2008 Orringer

et al., 2008)

PDL 25 subjects

20% ALA-solution

3 hour of application

Upregulation of collagen production was

shown with increase in procollagen type I and

III mRNA

IPL, intense pulsed light ; LED, light emitting diodes; PDL, pulsed dye laser; * Split-face study

21

In photoaged skin, a decrease in type I and III collagen is more prominent than in intrinsic aged

skin. Ultraviolet induces MMPs that can degrade collagen in human skin. MMP-1 initiates

cleavage of type I and III collagen, and the damaged collagen can be further degraded by MMP-

3 and MMP-9 (Sternlicht and Werb, 2001). Chronic UV exposure likely causes the accumulation

of MMP-mediated collagen damage; eventually the total collagen volume in the dermis will

decline as a result. In the present study, the expression of MMP-1 and MMP-3 was found to be

decreased one month after ALA-PDT.

In addition to destruction of mature collagen, UV irradiation impairs synthesis of new collagen,

which is reflected by the down-regulation of type I and type III procollagen gene expression

(Fisher et al., 2000). In addition, the TGF-β/Smad pathway is impaired by down-regulation of

TβRII and contributes to the pathology (Quan et al., 2004). TGF-β is a major cytokine that

stimulates fibroblast proliferation important to enhancing collagen synthesis in the dermis

(Inagaki et al., 1994). TGF-β initially binds to cell surface receptors, the TGF-β type I receptor

and TβRII. Then the activated complex interacts with intracellular signal transducer Smad

proteins that conveys TGF-β signaling (Quan et al., 2002). However, UV irradiation down-

regulates TβRII, which eventually decreases type I procollagen expression in human skin (Quan

et al., 2004). In the present study, the expression of type I and III procollagen, the precursors of

collagen, were constitutionally increased after treatment, which reflect the increased synthesis of

dermal collagen. In addition, the immunoreactivity of TGF-β as well as TβR II was significantly

increased after treatment, which indicates that they contribute to the reconstitution of the ECM.

The increase of dermal collagen, TGF-β and TβR II expression was more significant in patients

who received two more additional PDT treatments. This suggested a cumulative effect of the

22

ALA-PDT on photorejuvenation.

Another prominent feature of photoaged skin is the accumulation of dystrophic elastotic

material in the reticular dermis, known as solar elastosis (Montagna et al., 1989). Elastic fibers

consist of a central core of elastin and surrounding fibrillin-rich microfibrils (Mecham RP, 1991).

Increased tropoelastin gene expression, both in the epidermal keratinocytes and fibroblasts of

human skin, has been shown in vivo with UVB irradiation (Seo et al., 2001); this may be the

process by which elastotic materials accumulate in photodamaged skin. Moreover, in the

reticular dermis of photodamaged skin, an increased expression and deposition of fibrillin were

demonstrated (Bernstein et al., 1994). In addition to the increased production of tropoelastin and

fibrillin, degradation of elastic fibers contributes to the solor elastosis. MMP-12, the active

degradating enzyme against elastin, was reported to co-localize with the elastotic material in

photoaged skin, and the expression of MMP-12 mRNA and protein was induced by UV

irradiation in human skin in vivo (Chung et al., 2002). In addition, diverse proteinases from the

chronic UV induced inflammatory infiltrate can destroy the microfibrils (Kielty et al., 1994).

Therefore, we investigated the effect of ALA-PDT on solar elastosis, and the expression of

tropoelastin, fibrillin-1, MMP-12, and inflammatory cells. ALA-PDT reduced the accumulation

of dystrophic elastotic material in the dermis, and resulted in more normal elastin fiber. In

addition, tropoelastin and fibrillin-1, which were mainly co-localized with the elastotic materials,

showed reduced expression after the PDT, and therefore represented an improvement of the solar

elastosis. The expression of MMP-12 and inflammatory infiltrates significantly decreased after

treatment, which likely was associated with the changes in the solar elastosis as well.

Our findings showed a decrease in the intensity of MMP-1, 3, and 12 immunostaining

23

following treatment with ALA-PDT. This conflicts with the findings of some other studies that

have reported on the effects of ALA-PDT on the expression of MMPs. For example, when

normal or scleroderma fibroblast cells were treated with ALA-PDT, the levels of MMP-1 and

MMP-3 proteins increased; this was interpreted as antisclerotic effects of ALA-PDT (Karrer et

al., 2003). The expressed level of MMPs increased in a time-dependent manner with the

maximal induction at 48 hours following the PDT, thereafter with a decreasing tendency.

Recently, in an in vivo study of PDT using a pulsed dye laser in human skin, MMP-1 gene

expression was acutely elevated and then returned to baseline levels within 24 hours (Orringer et

al., 2008). However, it was also shown that MMP-2 expression was down-regulated 24 hours

after Hexvix mediated PDT in a medulloblastoma cell line (TE-671) (Chu et al., 2008).

Our results regarding decreased levels of MMPs might be explained as follows. We evaluated

the histological changes one month after the PDT, suggesting that the point in time of the

assessment might be an important consideration. In addition, various cell lines used in the in

vitro studies might differ from results from our in vivo study. TGF-β might play a role as well.

TGF-β does not only up-regulate procollagen synthesis, but it also down-regulates the

expression of MMPs (Massague, 1990). In the present study, a marked increase of TGF-β

expression was noted after the PDT. In addition, a change in the inflammatory infiltrates could

affect the decrease in the MMPs. It is known that UV-induced MMPs are secreted by diverse

cells, including infiltrated inflammatory cells (Hase et al., 2000). We found that the

inflammatory infilates were significantly decreased after the PDT. Therefore, the net effects of

the induction of TFG-β and reduction of the degradating enzymes in the inflammatory cells

likely contributed to the decreased expression of MMPs after the ALA-PDT (Rijken et al., 2005).

24

V. CONCLUSION

In conclusion, this study provided histological evidence of the beneficial effects of ALA-PDT

for photodamaged skin. In the photodamagend human skin, ALA-PDT induces deposition of

collagen, type I and type III procollagen in the dermis and TGF-β and TβR II expression in the

epidermis. In addition, MMP-1 and -3 expression was decreased after ALA-PDT. Solor elastosis

was improved accompanying with decreased expression of tropoelastin, fibrillin-1 and MMP-12

as well. These results suggest that ALA-PDT could be of therapeutic benefit in the treatment of

photoaging. Further controlled split –face studies on the mechanism of the effects of ALA-PDT

on photoaging, including signaling pathways, are necessary in order to fully elucidate the

function of ALA-PDT in human skin.

25

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light alone for photodamage. Dermatol Surg 32:795-801; discussion 801-793, 2006

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collagen degradation. Br J Dermatol 142:267-273, 2000

13. Inagaki Y, Truter S, Ramirez F: Transforming growth factor-beta stimulates alpha 2(I)

collagen gene expression through a cis-acting element that contains an Sp1-binding site. J Biol

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14. Kahari VM, Saarialho-Kere U: Matrix metalloproteinases in skin. Exp Dermatol 6:199-213,

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29

-국문 요약-

광선광선광선광선 각화증각화증각화증각화증 환자에게서환자에게서환자에게서환자에게서 5-Aminolevulinic Acid 를를를를 이용한이용한이용한이용한

광역동광역동광역동광역동 치료치료치료치료 후후후후 나타난나타난나타난나타난 광회춘광회춘광회춘광회춘 효과효과효과효과: 조직학적조직학적조직학적조직학적 분석분석분석분석

아주대학교 대학원 의학과

박민영

(지도교수: 김유찬)

연구 배경 : 만성적인 자외선 노출은 내인성 노화와는 다르고 더 심한 양상을

나타내는 광노화를 유발한다. 조직학적으로 광노화된 피부는 collagen 합성의 감소,

기존 collagen 파괴의 증가와 함께 비정상적인 elastotic material의 진피 침착을

특징으로 한다. 광노화된 피부를 회복시키기 위한 여러 가지 치료법 중에서 광역동

치료가 이에 효과가 있다는 연구들이 있다. 그러나 이들 대부분의 보고는 임상적인

관찰에 근거한 것이었다.

연구 목적: 본 연구에서는 5-aminolevulinic acid (ALA)를 이용한 광역동 치료가

광회춘 효과를 나타내는 조직학적 변화를 일으키는지 확인하고자 하였다 .

30

연구 방법: 얼굴 부위에 한 개에서 세 개의 광선 각화증을 가진 환자를 대상으로,

1200W metal halogen lamp를 광원으로 하여 한 달 간격으로 2회 ALA 광역동 치료를

시행하였다. 치료 전과 최종 치료 1 달 후 조직 검사를 시행하였고 총 25 쌍의 피부

조직을 얻었다. 각각에 대하여 기본 염색과 여러 면역 조직 화학 염색을 시행하였다.

연구 결과: ALA 를 이용한 광역동 치료 후, 평균 표피 두께가 감소하였고 진피의

염증 세포 침윤이 줄어들었다. 진피의 collagen 부피의 증가가 뚜렷하였고 이는 type

I, III procollagen발현의 증가와 동반되었다. 또한 표피에는 TGF-β 와 TGF-β type II

receptor가 치료 후 더 높게 나타났다. elastotic material에 함께 위치한 fibrilline-1 과

tropoelastin의 발현이 광역동 치료 후 감소되는 소견을 보였고, MMP-1, 3, 12 또한

감소하는 경향을 보였다.

결론: 본 연구 결과는 ALA 를 이용한 광역동 치료가 광노화 된 피부의 회복에

도움을 준다는 것에 대한 조직학적 증거를 제시하였다. 5-ALA 를 이용한 광역동

치료는 앞으로 광회춘에 대한 한가지 치료 방법으로 사용될 수 있을 것이다. 아울러

추후 이에 대한 자세한 기전을 밝히기 위한 조직화된 연구가 필요하겠다 .

핵심어: 광노화, 광역동 치료, 조직학적 변화