Objective— Notch signaling pathway controls key functions in vascular and endothelial cells (EC). However, little is known about the role of Notch in allografted vessels during the development of transplant arteriosclerosis (TA). This study investigated regulation of the Notch pathway on cardiac allograft arteriosclerosis and further examined its implication in EC dysfunction. Methods and Results— Here we show that, among Notch receptors, Notch2, -3, and -4 transcript levels were markedly downregulated in TA compared to tolerant and syngeneic allografts. TA correlates with high levels of tumor necrosis factor (TNF), transforming growth factor (TGF)β, and IL10, which consistently decrease Notch4 expression in transplants and cultured ECs. We found that inhibition of Notch activity, reflected by both a reduced CBF1 activity and Hes1 expression, parallels the downregulation of Notch4 expression mediated by TNF in ECs. Notch4 and Hes1 knockdown enhances vascular cell adhesion molecule-1 expression and promotes EC apoptosis. Silencing Notch4 or Hes1 also drastically inhibits repair of endothelial injury. Overall, our results suggest that Notch4 and basal Notch activity are required to maintain EC quiescence and for optimal survival and repair in response to injury. Conclusion— Together, our findings indicate that impaired Notch4 activity in graft ECs is a key event associated with TA by triggering EC activation and apoptosis.

BackgroundNotch 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 FindingsHere, 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/SignificanceIn 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.

Background and PurposeVascular calcification, recapitulating bone formation, has a profound impact on plaque stability. The aim of the present study was to determine the influence of bone-like vascular calcification (named osteoid metaplasia = OM) and of osteoprotegerin on plaque stability.MethodsTissue from carotid endarterectomies were analysed for the presence of calcification and signs of vulnerability according to AHA grading system. Osteoprotegerin (OPG), pericytes and endothelial cells were sought using immuno-histochemistry. Symptoms and preoperative imaging findings (CT-scan, MRI and Doppler-scan) were analyzed. Human pericytes were cultured to evaluate their ability to secrete OPG and to influence mineralization in the plaque.ResultsSeventy-three carotid plaques (49 asymptomatic and 24 symptomatic) were harvested. A significantly higher presence of OM (18.4% vs 0%, p<0.01), OPG (10.2% of ROI vs 3.4% of ROI, p<0.05) and pericytes (19% of ROI vs 3.8% of ROI, p<0.05) were noted in asymptomatic compared to symptomatic plaques. Consistently, circulating OPG levels were higher in the plasma of asymptomatic patients (3.2 ng/mL vs 2.5 ng/mL, p = 0.05). In vitro, human vascular pericytes secreted considerable amounts of OPG and underwent osteoblastic differentiation. Pericytes also inhibited the osteoclastic differentiation of CD14+ cells through their secretion of OPG.ConclusionsOPG (intraplaque an plasmatic) and OM are associated with carotid plaque stability. Pericytes may be involved in the secretion of intraplaque OPG and in the formation of OM.

These results suggest that bone like arterial calcification (OM) is highly prevalent at femoral level. Pericyte cells and the OPG/RANK/RANKL triad seem to be critical to the formation of this ectopic osteoid tissue and represent interesting potential therapeutic targets to reduce the clinical impact of arterial calcification.

MHC class I-related chain A (MICA) antigens are surface glycoproteins strongly implicated in innate immunity, and the MICA gene is highly polymorphic. Clinical observations suggest a role for donor MICA antigens expressed on transplant endothelial cells in the alloimmune response, but the effect of MICA genotype is not well understood. Here, we investigated the immunologic effect of the A5.1 mutation, related to the common MICA*008 allele. Compared with wild-type endothelial cells (ECs), homozygosity for MICA A5.1 associated with an endothelial phenotype characterized by 7-to 10-fold higher levels of MICA mRNA and MICA proteins at the cell surface, as well as exclusive release in exosomes instead of enzymatic cleavage. Mechanistically, we did not detect quantitative changes in regulatory microRNAs. Functionally, A5.1 ECs enhanced NKG2D interaction and natural killer cell activation, promoting NKG2D-dependent lysis of ECs. In kidney transplant recipients, polyreactive anti-MICA sera bound preferentially to ECs from MICA A5.1 donors, suggesting that MICA*008(A5.1) molecules are the preferential antigenic determinants on ECs of grafts. Furthermore, the incidence of MICA A5.1 mismatch revealed a statistically significant association between donor MICA A5.1 and both anti-MICA sensitization and increased proteinuria in kidney recipients. Taken together, these results identify the A5.1 mutation as an immunodominant factor and a potential risk factor for transplant survival.

Elevated PCSK9 levels are equally detrimental for patients with HeFH or non-FH: a 100-ng/ml increase in PCSK9 will lead to an increase in LDL-C of 0.20 to 0.25 mmol/l in controls and HeFH alike, irrespective of their LDLR mutation. This explains why patients with non-FH or HeFH respond equally well to monoclonal antibodies targeting PCSK9.

Background. Macrophages and synovial fibroblasts (SF) are two major cells implicated in the pathogenesis of rheumatoid arthritis (RA). SF could be a source of cytokines and growth factors driving macrophages survival and activation. Here, we studied the effect of SF on monocyte viability and phenotype. Methods. SF were isolated from synovial tissue of RA patients and CD14+ cells were isolated from peripheral blood of healthy donors. SF conditioned media were collected after 24 hours of culture with or without stimulation with TNFα or IL-1β. Macrophages polarisation was studied by flow cytometry. Results. Conditioned medium from SF significantly increased monocytes viability by 60% compared to CD14+ cells cultured in medium alone (P < 0.001). This effect was enhanced using conditioned media from IL-1β and TNFα stimulated SF. GM-CSF but not M-CSF nor IL34 blocking antibodies was able to significantly decrease monocyte viability by 30% when added to the conditioned media from IL-1β and TNFα stimulated SF (P < 0.001). Finally, monocyte cultured in presence of SF conditioned media did not exhibit a specific M1 or M2 phenotype. Conclusion. Overall, rheumatoid arthritis synovial fibroblasts stimulated with proinflammatory cytokines (IL-1β and TNFα) promote monocyte viability via GM-CSF but do not induce a specific macrophage polarization.

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