Nada Farhat

Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress associated with cardiovascular risk factors.

Risk factors for cardiovascular diseases (CVD) increase oxidative stress, and they are proposed to hasten endothelial cell (EC) damage and dysfunction. Our objective was to elucidate the impact of chronic exposure to risk factors for CVD on senescence of EC isolated and cultured from internal mammary arterial segments of patients with severe coronary artery disease. Senescence induced by serial passages resulted in progressive telomere shortening, and short initial telomeres predicted early appearance of senescence in culture. Neither time course of senescence nor telomere length was age-dependent, suggesting that biological age, rather than chronological age, determined the dynamics. Senescence appeared earlier in patients with longer history of risk factor for CVD, and multivariate analysis suggested that hypertension hastened the onset of senescence. Risk factors for CVD override the effects of chronological aging likely by generating stress-dependent damage: senescent EC exhibited oxidative stress (increase in lipid peroxydation and caveolin-1 gene expression) and cell damage markers (loss of eNOS expression and increase in Cox2 mRNA, lower TRF1 protein level). Thus, cell senescence was triggered both by telomere-dependent and -independent pathways. In conclusion, chronic exposure to risk factors for CVD accelerated the development of endothelial senescence that could contribute to the pathogenesis of CVD.

Na+/K+ pump and endothelial cell survival: [Na+]i/[K+]i-independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na+]i.

Recent studies have demonstrated the tissue-specific effect of Na+/K+ pump inhibition by ouabain and other cardiac glycosides on cell viability. The vascular endothelium is an initial target of cardiac glycosides employed for the management of congestive heart failure as well as circulating endogenous ouabain-like substances (EOLS), the production of which is augmented in volume-expanded hypertension. This study examined the role of the Na+/K+ pump in the survival of cultured porcine aortic endothelial cells (PAEC). Complete Na+/K+ pump inhibition with ouabain led to PAEC death, indicated by cell detachment and decreased staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Based on cell swelling and resistance to benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) a pan-caspase inhibitor, this type of cell death was classified as necrosis. In contrast to ouabain, Na+/K+ pump inhibition in K+-free medium did not affect PAEC viability and sharply attenuated apoptosis triggered by 3H decay-induced DNA damage. Necrosis evoked by ouabain was preserved after dissipation of the transmembrane gradient of K+ and Na+, whereas dissipation of the Na+ gradient abolished the antiapoptotic action of K+-free medium. Comparative analysis of these results and modulation of intracellular Na+ and K+ content by the above-listed stimuli showed that interaction of ouabain with Na+/K+-ATPase triggered necrosis independently of inhibition of Na+/K+ pump-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio, whereas protection against apoptosis under Na+/K+ pump inhibition in K+-depleted medium was mediated by [Na+]i elevation. The role of Na+/K+ pump-mediated regulation of endothelial cell survival and vascular remodelling seen in hypertension should be investigated further in context of EOLS and chronic treatment with digitalis.

Chronic treatment with N-acetyl-cystein delays cellular senescence in endothelial cells isolated from a subgroup of atherosclerotic patients

Endothelial senescence may contribute to the pathogenesis of age-related vascular disorders. Furthermore, chronic exposure to risk factors for cardiovascular disease (CVD) accelerates the effects of chronological aging by generating stress-dependent damages, including oxidative stress, therefore promoting stress-induced premature senescence. Our objective was to determine whether a chronic treatment with an antioxidant (N-acetyl-cystein, NAC) could delay senescence of endothelial cells (EC) isolated and cultured from arterial segments of patients with severe coronary artery disease. If EC were considered as one population (n=26), chronic NAC treatment slightly shortened telomere attrition rate associated with senescence but did not significantly delay the onset of endothelial senescence. However, in a subgroup of NAC-treated EC (n=15) cellular senescence was significantly delayed, NAC decreased lipid peroxidation (HNE), activated the catalytic subunit of telomerase (hTERT) and inhibited telomere attrition. In contrast, in another subgroup of EC (n=11) characterized by initial short telomeres, no effect of NAC on HNE and high levels of DNA damages, the antioxidant was not beneficial on senescence, suggesting an irreversible stress-dependent damage. In conclusion, chronic exposure to NAC can delay senescence of diseased EC via hTERT activation and transient telomere stabilization, unless oxidative stress-associated cell damage has become irreversible.

Potent in Vivo Antiangiogenic Effects of GS-101 (5′-TATCCGGAGGGCTCGCCATGCTGCT-3′), an Antisense Oligonucleotide Preventing the Expression of Insulin Receptor Substrate-1

Angiogenesis is a complex phenomenon regulated by both pro- and antiangiogenic factors such as the vascular endothelial growth factor (VEGF), and inflammation may be involved in the process. Although antagonizing VEGF has been proposed as a therapeutic approach to limit corneal angiogenesis, alternative targets are needed. In this study, we demonstrate that, under proangiogenic experimental conditions, human endothelial cells (hECs) express more insulin receptor substrate (IRS)-1 proteins relative to quiescent cells. The antisense oligonucleotide, GS-101 (5′-TATCCGGAGGGCTCGCCATGCTGCT-3′), targeting IRS-1 mRNA, dose-dependently inhibited (p < 0.01) both IRS-1 expression and in vitro angiogenesis (hEC tube-like structure formation) with IC50 of 8.51 ± 3.01 μM (mean ± S.E.M.) and 2.47 ± 0.56 μM, respectively, demonstrating that partial IRS-1 down-regulation interferes with angiogenesis. The antiangiogenic effects of GS-101 were associated with a decrease in protein kinase B (Akt) activation but not mitogen-activated protein kinase-1/2 and a dose-dependent reduction in vascular endothelial growth factor-A (IC50 = 5.59 ± 2.76 μM) and the proinflammatory cytokine interleukin-1β (IC50 = 2.19 ± 1.07 μM) mRNA expression. In accordance, once daily topical application of GS-101 dose-dependently inhibited injury-dependent corneal angiogenesis in vivo (p < 0.05). GS-101 in vivo efficacy was achieved at final tissue concentrations within in vitro EC50 for IRS-1 down-regulation. In conclusion, these results suggest that IRS-1 is important for angiogenesis and that GS-101 could become a novel therapeutic tool against corneal angiogenesis.

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2 + in adult cardiac myocytes

Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca(2+) signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with peptide: N-glycosidase F did not alter the electrophoretic mobility of ETB. The absence of N-glycosylation further indicates that these receptors did not originate at the cell surface. Intracellular photolysis of a caged ET-1 analog ([Trp-ODMNB(21)]ET-1) evoked an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n) that was attenuated by inositol 1,4,5-trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and prevented by pre-treatment with ryanodine. A caged cell-permeable analog of the ETB-selective antagonist IRL-2500 blocked the ability of intracellular cET-1 to increase [Ca(2+)]n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffics directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulates nuclear Ca(2+) signaling.

Endogenous oxidative stress prevents telomerase-dependent immortalization of human endothelial cells

INTRODUCTION With aging, oxidative stress accelerates vascular endothelial cell (EC) telomere shortening-induced senescence, and may promote atherosclerosis in humans. Our aim was to investigate whether an antioxidant treatment combined with telomerase (hTERT) over-expression would prevent senescence of EC isolated from patients with severe atherosclerosis. METHODS Cells were isolated from internal mammary arteries (n=11 donors), cultured until senescence with or without N-acetylcystein (NAC) and infected, or not, with a lentivirus over-expressing hTERT. RESULTS Compared to control EC, hTERT-NAC cells had increased telomerase activity, longer telomeres and underwent more cell divisions. According to the donor, hTERT-NAC either delayed (n=5) or prevented (n=4) EC senescence, the latter leading to cell immortalization. Lack of cell immortalization by hTERT-NAC was accompanied by an absence of beneficial effect of NAC alone in paired EC. Accordingly, lack of EC immortalization by hTERT-NAC was associated with high endogenous susceptibility to oxidation. In EC where hTERT-NAC did not immortalize EC, p53, p21 and p16 expression increased with senescence, while oxidative-dependent DNA damage associated with senescence was not prevented. CONCLUSION Our data suggest that irreversible oxidative stress-dependent damages associated with cardiovascular risk factors are responsible for senescence of EC from atherosclerotic patients.

Endothelin B receptor-mediated regulation of endothelin-1 content and release in cultured porcine aorta endothelial cell.

Several cardiovascular diseases are associated with an increase in circulating levels of endothelin-1 (ET-1). Little is known about the consequences of this increase on endothelial cell responses with respect to ET-1 production and regulation. Confluent, passage 1, cultured porcine aorta endothelial cells were exposed to exogenous ET-1 (0.1 &mgr;M) for 24 h. BQ788 (1 &mgr;M, ETB receptor antagonist) but not BQ123 (1 &mgr;M, ETA receptor antagonist) significantly (p < 0.05) reduced 125I-ET-1 uptake. The effects of BQ788 were mimicked by dansylcadaverine (0.5 m M) but not nystatin (50 &mgr;g/ml). Immunoreactive ET-1 endothelial cell content doubled (p < 0.05) after 24 h of exogenous ET-1 treatment. Bosentan (10 &mgr;M, dual ETA/B receptor antagonist) reduced (p < 0.05) immunoreactive ET-1 content in control cells. Bosentan prevented exogenous ET-1-induced endothelial cell ET-1 loading, suggesting that exogenous ET-1 is partly recycled. PreproET-1 mRNA levels were reduced (p < 0.05) by exogenous ET-1 after 24 h, an effect blocked by BQ788 and bosentan. When used alone, both receptor antagonists increased mRNA levels. The results of this study suggest that part of ET-1 is recycled through ETB receptors and subsequently released to contribute to constitutive ET-1 overflow. ET-1 exerts a negative feedback on ET-1 gene transcription, which is dependent on ETB receptor activation and internalization of the complex ET-1/ETB receptor. The maintenance of this negative regulatory loop of ET-1 production may be essential for the normal endothelial physiology.

Characterization of a novel MK3 splice variant from murine ventricular myocardium.

p38 MAP kinase (MAPK) isoforms alpha, beta, and gamma, are expressed in the heart. p38alpha appears pro-apoptotic whereas p38beta is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2(-/-) mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38alpha/beta inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38alpha and p38beta, but a poor substrate for p38delta and p38gamma. GST-MK3.2 was poorly phosphorylated by p38alpha and p38beta and not phosphorylated by p38delta and p38gamma. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.

Cloning, expression and purification of functionally active human angiopoietin-like protein 2.

Angiopoietin-like protein 2 (Angptl2) is a secreted glycoprotein that has been implicated in angiogenesis, inflammation and atherosclerosis as well as enhancing the survival of human hematopoietic stem cells. Glycosylation of Angptl2 is required for biological activity and studies of angiopoietin-like protein 2 have been hindered by the lack of a source for the mature form of this protein. We describe a system that permits purification of the glycosylated form of human Angptl2 from conditioned media of stably transfected HEK 293 cells. To facilitate purification while retaining the integrity of Angptl2’s endogenous N-terminal secretion signal peptide, GST was fused downstream of the Angptl2 coding sequence. Secreted Angptl2-GST was purified using a one-step glutathione-affinity purification scheme. The purity and identity of the resulting protein were confirmed by SDS-PAGE, immunoblotting, and mass spectrometry. N-Glycosidase treatment reduced the apparent molecular mass of Angptl2-GST on SDS-PAGE, confirming its glycosylation state. Purified human Angptl2-GST stimulated both HUVEC migration and microtubule formation in vitro. The yield of Angptl2-GST obtained was in quantities suitable for multiple applications including functional in vitro and in vivo assays.