senescence of cultured endothelial cells and pbmc...

Presented at the 1st Int. workshop on HIV & Aging, 4 – 5 Oct. 2010, Baltimore, USA

Senescence of cultured endothelial cells and PBMC from HIV+ patients: implication of ritonavir-boosted PIs

inducing prelamin A. Reversion by statins

Jacqueline Jacqueline CapeauCapeau, Chlo, Chloéé LefLefèèvre, Martine Auclair, Franck vre, Martine Auclair, Franck BoccaraBoccara, Jean, Jean-- Philippe Philippe BastardBastard, Corinne Vigouroux, Martine Caron, Corinne Vigouroux, Martine Caron--DebarleDebarle

INSERM U938, UniversitINSERM U938, Universitéé Pierre et Marie Curie Pierre et Marie Curie FacultFacultéé de Mde Méédecine, site Saintdecine, site Saint--Antoine, Antoine,

HHôpital ôpital Tenon, Paris, FranceTenon, Paris, France


Introduction

• HIV-infected patients present an increased risk of premature aging including cardio-vascular diseases.

• Protease inhibitors may promote premature cardiovascular disease through endothelial dysfunction either indirectly via dyslipidemia or directly via alterations of endothelial cells.

• The risk of premature cardiovascular events might also result from the accelerated aging imposed by HIV infection and/or ART.

• Vascular endothelial dysfunction is a feature of the human physiological aging process.

• In aged subjects, vascular smooth muscle cells accumulate prelamin A and express senescence markers (CD Ragnauth Circulation 2010).


C Navarro Hum. Mol. Gen, 2006

Mature lamin A is present in the lamina meshwork and the nucleoplasm and is maturated form prelamin A


• The premature aging syndrome, Hutchinson Gilford progeria, with an early death from cardiovascular diseases, is linked to mutations in lamin A leading to alterations in the prelamin A maturation process

• Some other progeroid syndromes are linked to molecular alterations in the gene encoding ZMPSTE24

• In all cases toxicity is related to accumulation of farnesylated prelamin A

• Some HIV PI are able to inhibit ZMPSTE24 (Coffinier PNAS 2007) resulting in prelamin A accumulation and cell senescence in human fibroblasts (Caron Cell Death Differ 2007)

11 years-old child


Aims of the study

1.1. To study whether a longTo study whether a long--term exposure to RTV or LPV/r term exposure to RTV or LPV/r could induce accelerated senescence in human vascular could induce accelerated senescence in human vascular coronary endothelial cells in primary culture (HCAEC)coronary endothelial cells in primary culture (HCAEC)

2. To search for markers of senescence in PBMC from HIV- infected patients under PI/r

3. To evaluate whether endothelial cells exposure to PI results in increased oxidative stress and inflammation

4. To test whether pravastatin is able to improve the PI adverse effects in endothelial cells

5. To search whether senescence markers are improved in PBMC from HIV-infected patients under statin

6. To test whether antioxidants are able to improve the PI adverse effects in endothelial cells


Aim 1: Long-term exposure of endothelial cells to PI progressively decrease cell proliferation and replication

C Lefèvre ATVB in press, 2010

Cells cultured for up to 30 days in the absence or presence of PIRTV 7.5 M or LPV/r: 10/2 M (purchased from Santa Cruz Biotechnology CA, USA)


RTV and LPV/r RTV and LPV/r increaseincrease the expression of the expression of cellcell--cycle cycle arrestarrest proteinsproteins, p21 and p53, and of , p21 and p53, and of senescencesenescence--associatedassociated ßß-- galactosidase galactosidase activityactivity

Aim 1: Long-term exposure to PI trigger endothelial cell senescence


AimAim 1: long1: long--termterm exposureexposure to PI to PI induceinduce prelaminprelamin A A accumulation and accumulation and nuclearnuclear dysmorphiesdysmorphies


Aims of the study

1. To study whether a long-term exposure to RTV or LPV/r could induce accelerated senescence in human vascular coronary endothelial cells in primary culture (HCAEC)

2.2. To search for markers of senescence in PBMC from HIVTo search for markers of senescence in PBMC from HIV-- infected patients under PI/rinfected patients under PI/r






Aim 2: PBMC from HIV-infected patients under PI/r express prelamin A and senescence markers

HIV+ patients HIV+ patients underunder NRTI NRTI withoutwithout PIPI

HIV+ patients HIV+ patients underunder PI/rPI/r


Aims of the study



3.3. To evaluate whether endothelial cells exposure to PI results To evaluate whether endothelial cells exposure to PI results in increased oxidative stress and inflammationin increased oxidative stress and inflammation





AimAim 3: Long3: Long--termterm exposureexposure of of endothelialendothelial cellscells to to PI PI inducesinduces oxidativeoxidative stress and inflammationstress and inflammation

RTV and LPV/r RTV and LPV/r increaseincrease ROS and cytokines/ROS and cytokines/chemokinechemokine production by production by endothelialendothelial cellscells

Oxidative stress Secretion of cytokines and chemokines


Aims of the study




4.4. To test whether To test whether pravastatinpravastatin is able to improve the PI adverse is able to improve the PI adverse effects in endothelial cellseffects in endothelial cells




C Navarro Hum Mol Gen 2006

The process of prelamin A maturation into lamin A: effect of statins and farnesyl-transferase inhibitors (FTI)

statinstatin

FT

I

FT

I


Aim 4: Reversion of cell senescence in endothelial cells by a treatment with statin or FTI

C Lefevre ATVB, in press 2010

Statin and FTI decrease the level of farnesylated prelamin A(Pravastatin was purchased from Sigma-Aldrich, France)


Aim 4: Reversion of oxidative stress and inflammation in endothelial cells by a treatment with statin or FTI


Aims of the study





5.5. To search whether senescence markers are improved in To search whether senescence markers are improved in PBMC from HIVPBMC from HIV--infected patients under infected patients under statinstatin



Aim 5: Decreased expression of senescence markers in PBMC from HIV-infected patients under PI/r and a statin


Aims of the study






6.6. To test whether antioxidants are able to improve the PI To test whether antioxidants are able to improve the PI adverse effects in endothelial cellsadverse effects in endothelial cells


Aim 6: Reversion of oxidative stress in endothelial cells by a treatment with antioxidants

The impact of two antioxidants, N-acetyl cysteine and Mn-TBAP was evaluatedPrelamin A accumulation was unaffected by antioxidants


Aim 6: Reversion of senescence in endothelial cells by a treatment with antioxidants


Hypothetical mechanisms of RTV-boosted PI toxicityin endothelial cells

DrugsDrugs

Inhibition of ZMP‐STE24Inhibition of ZMP‐STE24

Accumulation of farnesylated

prelamin

AAccumulation of farnesylated

prelamin

A

Increased

ROSIncreased

ROS

Activation of NFkBActivation of NFkB

Increased

IL6, IL8, MCP‐1Increased

IL6, IL8, MCP‐1 Systemic

low

grade inflammationSystemic

low

grade inflammation

Cellular senescenceCellular senescence


Hypothetical mechanisms of RTV-boosted PI toxicityin endothelial cells: beneficial effect of statins

DrugsDrugs



prelamin


prelamin

A

Increased

ROSIncreased

ROS


Increased



low


low

grade inflammation

Cellular senescenceCellular senescence

StatinsStatins


Hypothetical mechanisms of RTV-boosted PI toxicityin endothelial cells: beneficial effect of antioxidants

DrugsDrugs



prelamin


prelamin

A

Increased

ROSIncreased

ROS


Increased



low


low

grade inflammation

Cellular senescenceCellular senescenceAnti‐oxidantsAnti‐oxidants


Conclusion

• RTV and RTV-boosted LPV trigger premature senescence, oxidative stress and inflammation in human endothelial cells by a mechanism involving prelamin A accumulation • Senescence markers and prelamin A are also present in PBMC from HIV-infected patients under PI/r• This suggests that PI/r might participate to the early development of cardio-vascular disease in HIV-infected patients through prelamin A accumulation• Statins were associated with improved senescence in endothelial cells and patients’ PBMC• The ability of statins to improve PI-induced vascular senescence could be beneficial in these patients

senescence of cultured endothelial cells and pbmc...

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