Effect of gliclazide on oxidative stress inthe vascular wall

IstanbulApril 28, 2007

Dr. L. Rodríguez MañasSº de Geriatría y U. de InvestigaciónHospital Universitario de Getafe

EARLY STAGES

FINAL STEPS… ¿SHARED?

INVOLVED FACTORS Adrenergic tone Collagen Vascular remodelling Perivascular innervation Reparing mechanisms Neovascularization ……….

ALLA RICERCA DEL MINOTAURO

Physiological role of the vascular endothelium

Furchgott RF, Zawadki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373-376, 1980.

Vascular relaxation induced by acetylcholine is mediated by the action of a factor released by the endothelium (EDRF) that produces a relaxation of the smooth muscle cells.

Characteristics of EDRF

-Released by several vasoactive agents(ACh, BK, 5-HT, thrombin, ADP, etc).

-Very short half-life (seconds).

-Inactivated by Reactive Oxygen Species (ROS, free radicals).

-Mechanism:Estimulation of soluble guanilate cyclaseAnd increase of intracellular cGMP.

L-ArginineL-Arginine L-CitrulineL-Citruline

NONO

NO-synthaseNO-synthase

EndothelialEndothelialCellCell

NO-GMPc

NONO GCsGCs

GTPGTP

PKPK

GMPcGMPc

[Ca[Ca2+2+]]ii

VasodilationVasodilation

SmoothSmoothMuscleMusclecellcell

R-SNOR-SNO

R-SHR-SHNitrosotiolsNitrosotiols

BHBH44

-Furchgott y Louis Ignarro (1986):EDRF may be nitric oxide. -Palmer RMJ, Ferrige AG, Moncada S.Nature 327:524-526, 1987.

Palmer RMJ, Ashton DS, Moncada S.Vascular endothelial cells synthesizenitric oxide from L-arginine.Nature 333: 664-666, 1988.

Oxidative stress and NO

Endothelium

Smooth Muscle Cells

BasalMembrane

BK

NO

eNOS

O2-

GMPcGMPc

OONOOONO••

Superoxide anions

< 60 years > 60 years

Peroxinitrite(nitrotyrosine)

< 60 years > 60 years

NOS

L-arg

NO O2-

BH4

NOS uncoupling

NOS

L-arg

BH4

OONOOONO••

NOO2-

Sources of ROS

I

H+

III

H+

IV

H+

II

H+H+

NAD+ NADH FADH2

FADH2O

O2

ATP sintasa UCP

ADP+Pi ATP

e-

e-Q-

e- e-

cit c

O2 O2-

Generation of free radicals

Brownlee M. Nature 2001; 414: 813-820Brownlee M. Nature 2001; 414: 813-820

HyperglycemiaHyperglycemia

MitochondriaMitochondria

OO22--

PKCPKC

NADPH oxidaseNADPH oxidase eNOSeNOS

OO22--

NONOPeroxinitritePeroxinitrite

NitrotyrosineNitrotyrosine

Endothelial dysfunctionEndothelial dysfunction

Damaged DNADamaged DNAPARPPARP

↑ ↑ poliolspoliols

GlutationGlutation

↑ ↑ DAGDAGAGEAGE

--

DiabetesDiabetes Endothelial

Dysfunction?

HbHb CH CHCH CH

OHOH

NN RR

Schiff Base

Hb-NH 2Hb-NH 2 ++C

C

C

C OHOHH

R

H

R

H OH O

HemoglobinaHemoglobin

GlucosaGlucose

NH RNH RHb CH2 CHb CH2 C

OO

Amadori Adducts

Intermediate Glycosylation

Products

-O2

-O2

-O2

-O2

Release of superoxide anions byRelease of superoxide anions byGlycosylated hemoglobinGlycosylated hemoglobin

EndotheliumEndothelium

ACh

HbHGNONO

HbHGHbHG

O2-eNOS

(+)

O2-

Smooth Smooth MuscleMuscleCellCell

RELAXATIONRELAXATION

L. Rodriguez-Mañas et al., Circulation 1993J. Angulo et al., Hypertension 1996S. Vallejo et al., Diabetologia 2000

DIABETES

THERE IS AN IMPAIRMENT OF THE ENDOTHELIUM-DEPENDENT RESPONSESMEDIATED BY NO

ENDOTHELIUMENDOTHELIUM

ACh

HbHGNONO

HbHGHbHG

O2-eNOS

(+)

O2-SMCSMC

RELAXATIONRELAXATION

-9 -8 -7 -6 -50

20

40

60

80

100

ACh (logM)

*

DIABETIC

CONTROLS

Sodium Nitroprusside

GTPGTP

GMPcGMPcGCGC + NONO

SMOOTH MUSCLESMOOTH MUSCLE

OO22--

OO22--

OO22--

x

Diabetes ImprovementGlycemic control

x

Mch

NOSNOSL-argL-arg

NONO+

LNMMA

-

ENDOTHELIUMENDOTHELIUM

NONO

Rodríguez-Mañas et al.J Hypertension, 2003; 21: 1137-1143

Age (yr) 59.2 ± 1.2 ----SBP (mm Hg) 132.2 ± 3.7 130.0 ± 4.71 NSDBP (mm Hg) 81.0 ± 2.4 76.0 ± 4.06 NSInsulinaemia (µU/mL) 12.5 ± 3.2 9.7 ± 1.35 NSCholesterol (mmol/L) 5.8 ± 0.3 5.6 ± 0.30 NSLDL-Chol. (mmol/L) 3.9 ± 0.3 3.6 ± 0.38 NSHDL-Chol. (mmol/L) 1.3 ± 0.1 1.4 ± 0.09 NSSerum creatinine (µmol/L) 85.1 ± 4.2 87.2 ± 2.89 NSBMI (Kg/m2) 28.3 ± 2.4 ----HbA1c (%)* 9.2 ± 0.5 6.9 ± 0.18 p<0.05AGEs (mcg/mL)* 13.6 ± 1.5 4.9 ± 1.18 p<0.05

PRE-TREAT(mean±SE)

POST-TREAT(mean±SE)

* Mean of the values from the patients that achieved a good metabolic control (HbA1c<7.5%) after treatment with gliclazide during 4 months (n=7). The remaining values are obtained from the whole of the sample (n=10)

18.179.094.540

100

200

300

400

500

600

Pretreatment

Post-treatment

ACh (µgr/min)

% c

han

ge

c/n

c

Type 2 Diabetes

*

0

100

200

300

400

500

600 Pretreatment

Post-treatment

% c

han

ge

c/n

c

Type 2 Diabetes

37.812.64.2

SNP (µmol/min)

*

TREATMENT WITH GLICLAZIDE

IS ACCOMPANIED BY AN

IMPROVEMENT OF THE

ENDOTHELIAL FUNCTION

CONCLUSION

Differential effect of gliclazide and glibenclamide on forearm vascular reactivity

0

3

6

9

0

5

10

15

20

25

30

35

HbA1cHbA1c Post-ischemic Post-ischemic reactive hyperemiareactive hyperemia

(%)

(%)

(ml m

in(m

l min

-1-1/1

00m

l)/1

00m

l)

GlibGlib GliclazideGliclazideGlibGlib GliclazideGliclazide

Pre Post Pre Post Pre Post Pre Post

Wascher TC & Boes U Clin Physiol Funct Imaging. 2005;25:40

Natali A, et al. Vascular Effects of Improving Metabolic Control With Metformin or Rosiglitazone in Type 2 Diabetes. Diabetes Care 2004; 27: 1349.

IS THIS EFFECT DUE TO AN

IMPROVEMENT OF THE METABOLIC

CONTROL (INESPECIFIC) OR TO A

PARTICULAR EFFECT OF GLICLAZIDE?

QUESTION

COURAGE…… WE ARE FINISHING !!!

MicrovesselsExtraction

-8 -7 -60

20

40

60

80

100

Control10 nM 14% GHHb10 µM Ascorbate+GHHb

-8 -7 -6

Control10 nM 14% GHHb

100 U/ml SOD+GHHb

*%

co

ntr

acti

on

BK (log M)

Human mesenteric microvessels

Vallejo S et al., Diabetologia 2000; 43: 83-90.

Human mesenteric microvessels

% c

on

tra

ctio

n

BK (log M)

-8 -7 -60

20

40

60

80

100Control10 nM 14 GHHbGHHb + 0.1 µM Gliclazide

**

Vallejo S et al., Diabetologia 2000; 43: 83-90.

BK (log M)

-8 -7 -60

20

40

60

80

100 Control10 nM 14% GHHbGHHb + 1 µM Gliclazide

** †

% c

on

tra

ctio

n

BK (log M)

Human mesenteric microvessels

-8 -7 -60

20

40

60

80

100Control10 nM 14% GHHbGHHb + 10 µM Gliclazide

*

Vallejo S et al., Diabetologia 2000; 43: 83-90.

BK (log M)

Control10 nM 14% GHHbGHHb + 10 µM Glibenclamide

-8 -7 -60

20

40

60

80

100

**

Endothelium

Smooth muscle

Vascular

Membrane

Basal

BK

NONO

eNOS

O2-

O2-

(+)

O2-

NOx

HGHb

XSODVitamin CGliclazide

CONCLUSION

Vallejo S et al., Diabetologia 2000; 43: 83-90.

THE AUTHORS

Sº de GeriatrSº de Geriatríaía

Unidad de InvestigaciónUnidad de Investigación

Hospital Universitario de GetafeHospital Universitario de Getafe

Leocadio Rodríguez MañasLeocadio Rodríguez Mañas

Pedro López-DórigaPedro López-Dóriga

Susana VallejoSusana Vallejo

Roberto PetidierRoberto Petidier

Julián NevadoJulián Nevado

Mariam Assad de la FuenteMariam Assad de la Fuente

Joaquín Solís JiménezJoaquín Solís Jiménez

Manuel Montes LluchManuel Montes Lluch

Marta Castro RodrMarta Castro Rodríguezíguez

Alberto Sánchez FerrerAlberto Sánchez Ferrer

DepartamentoDepartamento

Farmacología y TerapéuticaFarmacología y Terapéutica

Universidad Autónoma de MadridUniversidad Autónoma de Madrid

Carlos F. Sánchez FerrerCarlos F. Sánchez Ferrer

Concepción PeiróConcepción Peiró

Nuria LafuenteNuria Lafuente

Nuria MatesanzNuria Matesanz

Verónica Azcutia Verónica Azcutia

Elena CercasElena Cercas