Notch2 Signaling Sensitizes Endothelial Cells to Apoptosis by Negatively Regulating the Key Protective Molecule Survivin

BACKGROUND: Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for a novel link between Notch and TNF signaling, where Notch2 is upregulated and activated in response to TNF. Forced expression of Notch2 intracellular domain in cultured ECs promotes apoptosis and allows the significant downregulation of several cell-death-related transcripts in a dose-dependent manner. In particular, activation of Notch2 led to a rapid decrease in survivin mRNA and protein expression, while survivin upregulation was obtained by the selective knockdown of Notch2 in ECs, indicating that survivin expression is controlled at the Notch level. Moreover, Notch2 silencing and ectopic expression of survivin, but not XIAP or Bcl2, rescued ECs from TNF and Notch2-mediated apoptosis, respectively. CONCLUSIONS/SIGNIFICANCE: In conclusion, TNF signaling activates Notch2 that sensitizes ECs to apoptosis via modulation of the key apoptosis regulator survivin. Overall, our findings also indicate that specific Notch receptors control distinct functions in vascular cells and inflammatory cytokines contribute to this specificity

Thin-Capped Atheromata With Reduced Collagen Content in Pigs Develop in Coronary Arterial Regions Exposed to Persistently Low Endothelial Shear Stress

Objective—The mechanisms promoting the focal formation of rupture-prone coronary plaques in vivo remain incompletely understood. This study tested the hypothesis that coronary regions exposed to low endothelial shear stress (ESS) favor subsequent development of collagen-poor, thin-capped plaques. Approach and Results—Coronary angiography and 3-vessel intravascular ultrasound were serially performed at 5 consecutive time points in vivo in 5 diabetic, hypercholesterolemic pigs. ESS was calculated along the course of each artery with computational fluid dynamics at all 5 time points. At follow-up, 184 arterial segments with previously identified in vivo ESS underwent histopathologic analysis. Compared with other plaque types, eccentric thin-capped atheromata developed more in segments that experienced lower ESS during their evolution. Compared with lesions with higher preceding ESS, segments persistently exposed to low ESS (<1.2 Pa) exhibited reduced intimal smooth muscle cell content; marked intimal smooth muscle cell phenotypic modulation; attenuated procollagen-I gene expression; increased gene and protein expression of the interstitial collagenases matrix-metalloproteinase-1, -8, -13, and -14; increased collagenolytic activity; reduced collagen content; and marked thinning of the fibrous cap. Conclusions—Eccentric thin-capped atheromata, lesions particularly prone to rupture, form more frequently in coronary regions exposed to low ESS throughout their evolution. By promoting an imbalance of attenuated synthesis and augmented collagen breakdown, low ESS favors the focal evolution of early lesions toward plaques with reduced collagen content and thin fibrous caps—2 critical determinants of coronary plaque vulnerability.Novartis (Firm)Boston Scientific CorporationBehrakis Foundation (Research Fellowship)Hellenic Heart FoundationHellenic Atherosclerosis SocietyNational Institutes of Health (U.S.) (Grant RO1 GM49039

Pitavastatin Reduces Inflammation in Atherosclerotic Plaques in Apolipoprotein E-Deficient Mice with Late Stage Renal Disease

Objectives: Chronic renal disease (CRD) accelerates atherosclerosis and cardiovascular calcification. Statins reduce low-density lipoprotein-cholesterol levels in patients with CRD, however, the benefits of statins on cardiovascular disease in CRD remain unclear. This study has determined the effects of pitavastatin, the newest statin, on arterial inflammation and calcification in atherogenic mice with CRD. Methods and Results: CRD was induced by 5/6 nephrectomy in cholesterol-fed apolipoprotein E-deficient mice. Mice were randomized into three groups: control mice, CRD mice, and CRD mice treated with pitavastatin. Ultrasonography showed that pitavastatin treatment significantly attenuated luminal stenosis in brachiocephalic arteries of CRD mice. Near-infrared molecular imaging and correlative Mac3 immunostaining demonstrated a significant reduction in macrophage accumulation in pitavastatin-treated CRD mice. Pitavastatin treatment reduced levels of osteopontin in plasma and atherosclerotic lesions in CRD mice, but did not produce a significant reduction in calcification in atherosclerotic plaques as assesses by histology. CRD mice had significantly higher levels of phosphate in plasma than did control mice, which did not change by pitavastatin. In vitro, pitavastatin suppressed the expression of osteopontin in peritoneal macrophages stimulated with phosphate or calcium/phosphate in concentrations similar to those found in human patients with CRD. Conclusion: Our study provides in vivo evidence that pitavastatin reduces inflammation within atherosclerotic lesions in CRD mice

Inhibition of BET proteins and epigenetic signaling as a potential treatment for osteoporosis

International audienceHistone modifications are important for maintaining the transcription program. BET proteins, an important class of " histone reading proteins " , have recently been described as essential in bone biology. This study presents the therapeutic opportunity of BET protein inhibition in osteoporosis. We find that the pharmacological BET protein inhibitor JQ1 rescues pathologic bone loss in a post-ovariectomy osteoporosis model by increasing the trabecular bone volume and restoring mechanical properties. The BET protein inhibition suppresses osteoclast differentiation and activity as well as the osteoblastogenesis in vitro. Moreover, we show that treated non-resorbing osteoclasts could still activate osteoblast differentiation. In addition, specific inhibition of BRD4 using RNA interference inhibits osteoclast differentiation but strongly activates osteoblast mineralization activity. Mechanistically, JQ1 inhibits expression of the master osteoclast transcription factor NFATc1 and the transcription factor of osteoblast Runx2. These findings strongly support that targeting epigenetic chromatin regulators such as BET proteins may offer a promising alternative for the treatment of bone-related disorders such as osteoporosis

Genesis and growth of extracellular vesicle-derived microcalcification in atherosclerotic plaques

Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification zones. We also show that calcification morphology and the plaque’s collagen content – two determinants of atherosclerotic plaque stability - are interlinked

Impact of Notch Signaling on Inflammatory Responses in Cardiovascular Disorders

Notch signaling is a major pathway in cell fate decisions. Since the first reports showing the major role of Notch in embryonic development, a considerable and still growing literature further highlights its key contributions in various pathological processes during adult life. In particular, Notch is now considered as a major player in vascular homeostasis through the control of key cellular functions. In parallel, confounding evidence emerged that inflammatory responses regulate Notch signaling in vitro in endothelial cells, smooth muscle cells or vascular infiltrating cells and in vivo in vascular and inflammatory disorders and in cardiovascular diseases. This review presents how inflammation influences Notch in vascular cells and, reciprocally, emphasizes the functional role of Notch on inflammatory processes, notably by regulating key cell functions (differentiation, proliferation, apoptosis/survival, activation). Understanding how the disparity of Notch receptors and ligands impacts on vasculature biology remains critical for the design of relevant and adequate therapeutic strategies targeting Notch in this major pathological context

Impact of Notch Signaling on Inflammatory Responses in Cardiovascular Disorders

Abstract: Notch signaling is a major pathway in cell fate decisions. Since the first reports showing the major role of Notch in embryonic development, a considerable and still growing literature further highlights its key contributions in various pathological processes during adult life. In particular, Notch is now considered as a major player in vascular homeostasis through the control of key cellular functions. In parallel, confounding evidence emerged that inflammatory responses regulate Notch signaling in vitro in endothelial cells, smooth muscle cells or vascular infiltrating cells and in vivo in vascular and inflammatory disorders and in cardiovascular diseases. This review presents how inflammation influences Notch in vascular cells and, reciprocally, emphasizes the functional role of Notch on inflammatory processes, notably by regulating key cell functions (differentiation, proliferation, apoptosis/survival, activation). Understanding how the disparity of Notch receptors and ligands impacts on vasculature biology remains critical for the design of relevant and adequate therapeutic strategies targeting Notch in this major pathological context. Int. J. Mol. Sci. 2013, 14 686

Importance de la voie Notch dans la dysfonction endothéliale en transplantation (régulation et fonctions des récepteurs Notch2 et Notch4)

Notch joue un rôle important dans la survie, la prolifération et la transdifférenciation des cellules endothéliales (CE). Ces mécanismes sont tout particulièrement importants dans l'artériosclérose du greffon (AG). L'objectif de ce travail est de définir la régulation et le rôle de Notch dans la dysfonction des CE associée à l'AG. Nous avons tout d'abord étudié la régulation des molécules Notch (récepteurs, ligands et effecteurs) au cours de l'activation des CE. Ainsi, le TNFa oriente le profil Notch vers une forte diminution de Notch4 et une augmentation de Notch2, respectivement par les voies NF B et PI3K. Cet effet est toutefois associé à une baisse globale de l activité CBF1. La baisse du récepteur endothélial Notch4 est, de plus, associée à l AG dans un modèle d allogreffe cardiaque chez le rat où le TNFa, TGFb et IL10 sont fortement exprimés. Le blocage de Notch4 et de hes1 par ARN interférence favorise l expression de VCAM-1, induit l apoptose et altère la cicatrisation endothéliale. L expression constitutive de Notch2ICD grâce à un adénovirus recombinant induit l apoptose des CE par une forte répression de gènes anti- (survivine, April) et pro-apoptotiques (Bim, DAPK2, HRK, DR5, CD40). La fonction pro-apoptotique de N2ICD dépend essentiellement de l inhibition de la survivine. L'extinction de Notch2 augmente l'expression de la survivine et protège les CE de l apoptose. L'ensemble de ces travaux montre une interaction entre les voies du TNF et Notch au cours de la dysfonction endothéliale, propose un rôle pour Notch4 dans la prévention de la dysfonction endothéliale dans l'AG et démontre l'importance de Notch2 dans la survie des CE par la régulation de la survivine.Notch plays major roles in endothelial cells (EC) survival, proliferation and transdifferentiation. These events are particularly implicated in transplant arteriosclerosis (TA). This work aims to define Notch molecules regulation and its functional relevance in graft EC. We first studied Notch family members (receptors, ligands and effectors) regulation during EC activation by cytokines. We show that TNFa induced a switch in Notch expression pattern by a strong down-regulation of Notch4 and an increase in Notch2, respectively dependent on NFkB and PI3K pathways. This effect is associated with a global decrease in CBF1 activity and an opposite regulation of hes1 and hey1. Decrease in EC-restricted Notch4 expression is also associated with TA in a rat cardiac allograft model where TNFa, TGFb and IL10 are strongly expressed. Inhibition of Notch4 and hes1 by siRNA enhances VCAM1 expression, induces apoptosis and impairs endothelial injury repair, suggesting that Notch4 expression is required to maintain EC survival and quiescence. To study the role of Notch2 in EC, we constructed a recombinant adenovirus for Notch2ICD (N2ICD). Constitutive expression of N2ICD leads to EC apoptosis associated with a drastic repression of pro- (survivin, april) and anti-apoptotic genes (bim, DAPK2, HRK, DR5, CD40). Effect of N2ICD is mainly dependent on survivin inhibition. Accordingly, Notch2 knock-down increases survivin expression and protects EC from anoïkis. Altogether, our results reveal crosstalks between TNF and Notch signaling during endothelial dysfunction, propose a role for Notch4 in preventing TA-related EC dysfunction and for Notch2 role in EC survival through survivin regulation.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

TLR2 and neutrophils potentiate endothelial stress, apoptosis and detachment: implications for superficial erosion

International audienceAIMS:Superficial erosion of atheromata causes many acute coronary syndromes, but arises from unknown mechanisms. This study tested the hypothesis that Toll-like receptor-2 (TLR2) activation contributes to endothelial apoptosis and denudation and thus contributes to the pathogenesis of superficial erosion.METHODS AND RESULTS:Toll-like receptor-2 and neutrophils localized at sites of superficially eroded human plaques. In vitro, TLR2 ligands (including hyaluronan, a matrix macromolecule abundant in eroded lesions) induced endothelial stress, characterized by reactive oxygen species production, endoplasmic reticulum (ER) stress, and apoptosis. Co-incubation of neutrophils with endothelial cells (ECs) potentiated these effects and induced EC apoptosis and detachment. We then categorized human atherosclerotic plaques (n = 56) based on morphologic features associated with superficial erosion, 'stable' fibrotic, or 'vulnerable' lesions. Morphometric analyses of the human atheromata localized neutrophils and neutrophil extracellular traps (NETs) near clusters of apoptotic ECs in smooth muscle cell (SMC)-rich plaques. The number of luminal apoptotic ECs correlated with neutrophil accumulation, amount of NETs, and TLR2 staining in SMC-rich plaques, but not in 'vulnerable' atheromata.CONCLUSION:These in vitro observations and analyses of human plaques indicate that TLR2 stimulation followed by neutrophil participation may render smooth muscle cell-rich plaques susceptible to superficial erosion and thrombotic complications by inducing ER stress, apoptosis, and favouring detachment of EC

Impact of Femoral Ossification on Local and Systemic Cardiovascular Patients' Condition

International audienceBackground: Vascular calcifications are associated with a high cardiovascular morbi-mortality in the coronary territory. In parallel, femoral arteries are more calcified and develop osteoid metaplasia (OM). This study was conducted to assess the predictive value of OM and local inflammation on the occurrence of mid- and long-term adverse cardiovascular events.Method: Between 2008 and 2015, 86 atheromatous samples were harvested during femoral endarterectomy on 81 patients and processed for histomorphological analyses of calcifications and inflammation (monocytes and B cells). Histological findings were compared with the long-term follow-up of patients, including major adverse cardiac event (MACE), major adverse limb event (MALE), and mortality. Frequencies were presented as percentage, and continuous data, as mean and standard deviation. A P-value < 0.05 was considered statistically significant.Results: Median follow-up was 42.4 months (26.9-58.8). Twenty-eight percent of patients underwent a MACE; a MALE occurred in 18 (21%) limbs. Survival rate was 87.2% at 36 months. OM was found in 41 samples (51%), without any significant impact on the occurrence of MACE, MALE, or mortality. Preoperative white blood cell formulae revealed a higher rate of neutrophils associated with MACE (P = 0.04) and MALE (P = 0.0008), correlated with higher B cells counts in plaque samples.Conclusions: OM is part of femoral calcifications in almost 50% of the cases but does not seem to be an independent predictive variable for MACE or MALE. However, a higher rate of B cell infiltration of the plaque and preoperative neutrophil blood count may be predictive of adverse events during follow-up