Új célmolekulák azonosítása a magas vérnyomás keltette

vaszkuláris remodeling kivédésére

Pécsi TudományegyetemÁltalános Orvostudományi KarBiokémiai és Orvosi Kémiai Intézet és Szentágothai János Kutató Központ  

Sümegi Balázs

Hypertension is a major public health problem

Hypertension is a complex disease and an important risk factor for cardiovascular outcomes, such as sudden death, myocardial infarction, heart failure, and renal diseases and stroke

The control of arterial hypertension is far from optimal

Side effects of antihypertensive drugs

Low blood pressure may lead to cognitive impairment in the elderly population

Redox-dependent mechanisms contributing to vascular remodeling in hypertension

All cell types of the vessel wall produce reactive oxygen species (ROS). Vascularsmooth muscle cells VSMC: endothelial cells (EC); Macrophages (Mϕ); T-cells whichinduces increased vascular ROS production and leads to VSMC growth (proliferationand hypertrophy), increased vasoconstriction, endothelial dysfunction, inflammation.

British Journal of Pharmacology (2009), 157, 935–943.

Most of the direct antioxidant have low efficacy to prevent oxidative stress

ROS is a significant factor in the pathogenesis of vascular diseases

Scavangering ROS can be an attractive therapy?Antioxidant drugs (for example, vitamins C and E, and β-carotene) do not appear to have the clinical efficacy to prevent cardiovascular events!What are the reasons? Antioxidant can be effective only in mM concentration range!

Targeting ROS production by blocking the activity of NADPH oxidases can be more effective?In knockout mice has some protection, but there are very few data with synthetic inhibitor and has to use high concentrations.

Angiotensin II receptor blockers! Difficult to separate blood pressure lowering effect and protective effects.

1.

2.

3.

4. Raising the protective role of PARP inhibitor which can modulates signaling, transcription factors activity, stabilize mitochondrial membrane system and protect against cell death.

Abnormal ROS metabolism and its consequences in the vascular system

Inflammation

Cell Death

Abnormal signaling inducing remodeling

Why PARP inhibitors have the potential to prevent

hypertension induced vascular remodeling

PARP inhibitor can inhibit oxidative stress induced cell

damages, and prevents the induction of inflammation.

Our previous data show that PARP inhibitor can prevent

hypertension induced heart failure in a spontaneously

hypertensive rat model . Cardiovasc Res. 2009; 83, 501-10.

We have to keep in mind that only long chronic studies

can serve as realistic model for human cases.

Designing PARP inhibitors: Collaboration with Hideg K & Kalai T groupCarboxaminobenzimidazol- 4-hydroxyquinazolin +Antioxidant groups

46 weeks treatment, 5 mg/kg/day L-2286 PARP-inhibitor

L-2286

Chemical structure of L-2286 (2-[(2-Piperidine-1-ylethyl)thio]quinazolin-4(3H)-one) PARP inhibitor

Kaplan–Meier survival curvesof SHR-C and SHR-L groups

PARP inhibitor significantly extended the life-span of SHR rats

Age (weeks)

5 10 15 20 25 30 35 40 45

Sy

sto

lic b

loo

d p

ress

ure

(H

gm

m)

100

120

140

160

180

200

220

240

260

SHRSHR+PARPCFYCFY+PARP

*

SHRSHR+PARPWKYWKY+PARP

Systolic blood pressure values of normotensive (WKY-C, WKY-L) and hypertensive (SHR-C, SHR-L) rats. Values are means ± SEM

Effect of PARP inhibitor on the vascular functions and

morphology

Iso

metr

ic v

aso

rela

xati

on

(%

)

0

20

40

60

80

100WKY-C - Ach SHR-C - Ach SHR-L - Ach

10-9 10-8 10-7 10-6 10-5

A

Dose - response isometric vasomotor responses of (WKY, SHR-C, SHR-L) rat carotid arteries to acetyl-choline

All values are normalized to KCl (60mM) responses (100%)Values are mean ± SEM

Arterial stiffness index of aorta of normotensive (WKY-C, WKY-L) and hypertensive (SHR-C, SHR-L) rats

Values are mean ± SEM

0

1

2

3

4

5

6

7

WKY 10week old

SHR 10week old

WKY-C42 week

old

WKY-L42 week

old

SHR-C42 week

old

SHR-L 42week old

#

§

ln (

SB

P/D

BP

)/[(

SD

– D

D)/

DD

]

0

5

10

15

20

25

30

35

40

WKY-C WKY-L SHR-C SHR-L

Fibr

osis

%

* #

Aorta

Representative histologic sections stained with Masson’s trichrome (n= 4) Magnifications 40x fold. Aorta(A) Wistard,(B) Wistard + L-2286,(C) SHR rats,(D) SHR rats + L-2286

A B

C D

Effect of PARP inhibition on thedeposition of interstitial collagen in rat aorta

Effect of PARP inhibitor on oxidative stress

0

10

20

30

40

50

60

WKY-C WKY-L SHR-C SHR-L

Nito

tyro

sine

(a.u

.)

Aorta *

#

A DCB

Representative immunohistochemical stainings for nitrotyrosine formation (NT, brown staining) in the aortic wall of normotensive (WKY-C, WKY-L)and hypertensive (SHR-C, SHR-L) animals. Magnification 40 x fold.

A: aortic wall of WKY-C, B: aortic wall of WKY-L, C: aortic wall of SHR-C, D: aortic wall of SHR-L.

Effect of PARP inhibitoron signaling

ACTIN

p-P38-MAPK Thr180-Gly-Tyr182 (43 kDa)

p-ERK 1/2

Thr183-Tyr185 (42,44 kDa)

JNK

Thr183-Tyr185 (46-54 kDa)

p-Akt-1

Ser473 (60 kDa)

WKY-C WKY-L SHR-C SHR-L

Anti-PAR

(116 kDa)

Effect of L-2286 treatment on the phosphorylation state of Akt-1, JNK, ERK and p38-MAPK in aortas normotensive (WKY-C, WKY-L) and hypertensive

(SHR-C, SHR-L) rats

Representative merged confocal images of the localization of MKP-1. MKP-1 immunoreactivity (red) and Hoechst nuclear staining (blue) were

presented inmerged form

A B

C D

A: aortic wall of WKY-C

B: aortic wall of WKY-L

C: aortic wall of SHR-C

D: aortic wall of SHR-L

Effect of PARP-1 on MAP kinases in vascular remodeling

Hypertension

ROS

ASK1

JNKp38

DNAbreaks

PARP-1activation

PARP-1inhibitor

MKP1Expression

Genome wide effect of PARP inhibitors on transcription

Transcription factors activation in hypertension

NF-BAP-1TGF-1 – SmadOxidative stress driven beta-catenin nuclear translocationFOXOHIF-1

PARP inhibitor inactivates NF-B by inhibiting 2 retrograde PAR dependent activation pathways andpromoting the nuclear export of p65 component of NF-B

PARP inhibitor inactivates AP-1likely by preventing its activation by JNK which can be the consequence ofPARP-inhibition induced activation of Mkp-1 (MAP kinase phosphatase-1).Racz et al. Free Rad. Biol. Med. (2010) 49, 1978-88.

Regulation of Crm1-dependent nuclear export by PARP-1

Crm1 (Exporting1) export proteins from the nucleus to cytoplasm

Multiple modes of genome wide transcriptional regulation by PARP-1

A. PARP-1 can function as a transcriptional coregulator and corepressors. B. PARP-1 can act as an enhancer-binding factor (to protein or DNA) C. DNA-binding activators or repressors, or exchange factor. D. PARP-1 function as a component of insulators, which act to limit the effects of enhancers on promoters or by preventing the spread of heterochromatin. In this mode, the PARylation of CTCF by PARP-1 is likely to be important.

Effect of Oxidative stress and PARP inhibitor on the activation of transcription factors (TransAM kit)

0

0,5

1

1,5

2

2,5

control PJ-34 H2O2 H2O2+PJ-34

p-c

-Ju

n a

cti

va

tio

n

*** ***

**

2,55

2,6

2,65

2,7

2,75

2,8

2,85

control PJ-34 H2O2 H2O2+PJ-34

ST

AT

1-

ac

tiv

ati

on

0

0,5

1

1,5

2

2,5

control PJ-34 H2O2 H2O2+PJ-34

p-A

TF

2 ac

tiva

tio

n

0

0,5

1

1,5

2

2,5

3

control PJ-34 H2O2 H2O2+PJ-34

ME

F-2

act

ivat

ion

**

Myocyte enhancer factor-2 (MEF2) Signal transducer and activator

of transcription (Stat)

ATF2 (activating transcription factor 2) P-c-Jun

Curr Opin Cell Biol. 2008 20, 294-302.Transcriptional control by PARP-1: chromatin modulation, enhancer-binding, co-regulation, and insulation. Kraus WL.

Poly-ADP-ribosylation is a genome wide regulator of gene expression in vascular remodeling

Hypertension

ROS

ASK1

JNKp38

c-Jun, ATF2, LEK1, SMAD4p53, NFAT1,4, STAT4, TDF/MEF2

p53, NFB, Creb, ATF, Chop, MSK

DNAbreaks

PARP-1act.

PARP-1inh.

MKP1act.

Remodeling

NFB

Remodeling

Racz et al. Regulation of MKP-1 expression by PARP-1. Free Radic Biol Med. (2010) 49,1978-88.

Department of Biochemistry and Medical Chemistry Ferenc Gallyas Jr., Boglárka Rácz, Alíz Szabó

1st Department of Medicine Klára Magyar, László Deres, Krisztián Erős, Kitti Bruszt, Kálmán Tóth,

Róbert Halmosi

Central Electron Microscope Laboratory László Seress

Organic and Pharmacological Chemistry Kálmán Hideg, Tamás Kálai

Department of Pathophysiology and Gerontology Ákos Koller, Zoltán Vámos

Participants: