Human Coronary Endothelial Cells Research Articles

Role of endothelial TRPV4 channels in vascular actions of the endocannabinoid, 2-arachidonoylglycerol.

Metabolites of the endocannabinoid, 2-arachidonoylglycerol (2-AG) have been postulated to act as endogenous activators of TRPV4, a Ca(2+) -permeable cation channel that plays a critical role in endothelium-dependent relaxation. However, it is unclear if TRPV4 contributes to the vascular actions of 2-AG. Isometric tension recording of rat small mesenteric arteries and aortae were used to assess the effect of 2-AG and the synthetic TRPV4 activator, GSK1016790A (GSK) on vascular reactivity. Changes in intracellular Ca(2+) concentration and single-channel currents were measured in TRPV4-expressing human coronary endothelial cells. In mesenteric arteries, endothelium-dependent relaxation to both 2-AG and GSK was attenuated by structurally distinct TRPV4 antagonists, HC067047, RN1734 and ruthenium red. The responses were inhibited by KCa inhibitors (apamin + charybdotoxin) and a gap junction inhibitor (18α-glycyrrhetinic acid). In contrast to GSK, 2-AG elicited considerable relaxation independently of the endothelium or TRPV4. Inhibition of 2-AG metabolism via monoacylglycerol lipase and COX (by MAFP and indomethacin) caused potentiation, while cytochrome P450 and lipoxygenase inhibitors had no effect on 2-AG relaxation. In coronary endothelial cells, 2-AG (with and without MAFP) induced HC067047-sensitive increases in intracellular Ca(2+) concentration. 2-AG also increased TRPV4 channel opening in inside-out patches. However, in aortae, GSK induced a relaxation sensitive to HC067047 and ruthenium red, whereas 2-AG induced contractions. These data suggest that 2-AG can directly activate endothelial TRPV4, which partly contributes to the relaxant response to 2-AG. However, the functional role of TRPV4 is highly dependent on the vascular region.

GW26-e1243 Inhibitory Effects of Hirudin on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells and Endothelial Cells in Vitro

GW26-e1243 Inhibitory Effects of Hirudin on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells and Endothelial Cells in Vitro

Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+NIK+ signalosome on Rab5+ endosomes

Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB-inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5(+)endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC(+) endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt(+)NIK(+) signalosome on Rab5(+) endosomes.

Snail Is a Direct Target of Hypoxia-inducible Factor 1α (HIF1α) in Hypoxia-induced Endothelial to Mesenchymal Transition of Human Coronary Endothelial Cells

Endothelial to mesenchymal transition (EndMT) was originally described in heart development where the endocardial endothelial cells that line the atrioventricular canal undergo an EndMT to form the endocardial mesenchymal cushion that later gives rise to the septum and mitral and tricuspid valves. In the postnatal heart specifically, endothelial cells that originate from the endocardium maintain increased susceptibility to undergo EndMT as remnants from their embryonic origin. Such EndMT involving adult coronary endothelial cells contributes to microvascular rarefaction and subsequent chronification of hypoxia in the injured heart, ultimately leading to cardiac fibrosis. Although in most endothelial beds hypoxia induces tip cell formation and sprouting angiogenesis, here we demonstrate that hypoxia is a stimulus for human coronary endothelial cells to undergo phenotypic changes reminiscent of EndMT via a mechanism involving hypoxia-inducible factor 1α-induced activation of the EndMT master regulatory transcription factor SNAIL. Our study adds further evidence for the unique susceptibility of endocardium-derived endothelial cells to undergo EndMT and provides novel insights into how hypoxia contributes to progression of cardiac fibrosis. Additional studies may be required to discriminate between distinct sprouting angiogenesis and EndMT responses of different endothelial cells populations.

Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease

The aim of our study was to develop a completely scaffold-free, viable, contractile cardiac tissue capable of being grafted into the damaged native heart. Our technology is based on the fundamental characteristics of the self-assembling nature of cells. We created contractile cardiac spheroids by plating a mixture of rat neonatal ventricular cardiomyocytes, human dermal fibroblasts, and human coronary microartery endothelial cells in ultralow attachment plates. First, the optimal cell ratios for the 3 cell sources were determined. Next, approximately 1 × 10(4) optimal spheroids were fused into a patch-like construct, and the morphologic characteristics and mechanical functions of these patches were evaluated. Finally, the cardiac patches were grafted into the hearts of F344 nude rats, and histologic studies were performed after transplantation. Synchronous beating of the cardiac patch was confirmed electrophysiologically and mechanically. A micronetwork of endothelial cells was also demonstrated in the construct, and the histologic study performed 5 days after transplantation showed the grafts to be viable, with functioning microvascular structures inside the graft tissue. We consider the application of our scaffold-free 3-dimensional tissue engineering technology to cardiac regeneration therapy is feasible and expect that this technology will become a promising tool for the treatment of end-stage heart failure.

Receptor for advanced glycation end products involved in circulating endothelial cells release from human coronary endothelial cells induced by C-reactive protein.

This study was designed to investigate the effect of receptor for advanced glycation end products (RAGE), S100A12 and C-reactive protein (CRP) on the release of circulating endothelial cells (CECs) from human coronary artery endothelial cells (HCAECs). HCAECs were cultured in increasing concentration of CRP (0, 12.5, 25, 50μg/ml) or S100A12 protein (0, 4, 10, 25μg/ml) for 24 hr. CECs were measured by flow cytometry. Small interfering RNA (siRNA) was designed to decrease RAGE level. Fluorescence microscopy and real-time quantitative polymerase chain reaction were used to assess the efficiency of siRNA silencing RAGE. The release of CECs from HCAECs was further evaluated by flow cytometry. CRP caused a significant increase in the release of CECs from HCAECs. The number of CECs increased by about 2-fold in 25 μg/ml CRP-treated group compared to the control group (12.22% compared to 6.82%, P=0.032). But S100A12 failed to increase the release of CECs from HCAECs. Blockade of RAGE by siRNA significantly decreased the release of CECs induced by CRP (13.22% of CRP group compared to 8.77% of CRP+siRNA group, P=0.017). RAGE is involved in the release of CECs induced by CRP, and the effect can be attenuated by silencing RAGE. RAGE may play an important role in endothelial dysfunction in cardiovascular disease. Inhibition of RAGE may be a therapeutic target for coronary artery lesions in Kawasaki disease.

Epigenetic balance of aberrant Rasal1 promoter methylation and hydroxymethylation regulates cardiac fibrosis

Methylation of CpG island promoters is a prototypical epigenetic mechanism to stably control gene expression. The aim of this study was to elucidate the contribution of aberrant promoter DNA methylation in pathological endothelial to mesenchymal transition (EndMT) and subsequent cardiac fibrosis. In human coronary endothelial cells, TGFβ1 causes aberrant methylation of RASAL1 promoter, increased Ras-GTP activity, and EndMT. In end-stage failing vs. non-failing human myocardium, increased fibrosis was associated with significantly increased RASAL1 promoter methylation, decreased RASAL1 expression, increased Ras-GTP activity, and increased expression of markers of EndMT. In mice with pressure overload due to ascending aortic constriction, BMP7 significantly reduced RASAL1 promoter methylation, increased RASAL1 expression, and decreased EndMT markers as well as decreased cardiac fibrosis. The ten eleven translocation (TET) family enzyme TET3, which demethylates through hydroxymethylation, was significantly decreased in fibrotic mouse hearts, restored with BMP7, and BMP7 effects were absent in coronary endothelial cells with siRNA knockdown of TET3. Our study provides proof-in-principle evidence that transcriptional suppression of RASAL1 through aberrant promoter methylation contributes to EndMT and ultimately to progression of cardiac fibrosis. Such aberrant methylation can be reversed through Tet3-mediated hydroxymethylation, which can be specifically induced by BMP7. This may reflect a new treatment strategy to stop cardiac fibrosis.

Abstract 15570: A Novel Ex Vivo Test System to Assess Cardiovascular Toxicity of HIV-Nef in PBMC From HIV Positive Donors

Background: To address how cardiovascular disease is increased in HIV patients with and without therapy, we have tested the effects of HIV-infected T cell when in contact to human coronary endothelial cells (HCAEC). We found that these T cells could induce endothelial dysfunction and cell death at a much larger extent than free virus itself. We could identify HIV-Nef protein, a major pathogenic factor in HIV disease progression and HIV-induced end organ disease, as the mechanism for this finding. Because of our finding that HIV-Nef is transferred from T cells to HCAEC to induce cell death, we asked whether PBMC form HIV-infected patients can recapitulate these findings. Methods: PBMC were isolated from either healthy donors or from untreated HIV patients or from those on ART using standard Ficoll purification, and intracellular Nef was determined by flow cytometry and ELISA. PBMC were co-cultivated with HCAEC for 24 hours, washed off and HCAEC were analyzed for Nef by immunofluorescence microscopy representation and ELISA. Further, HCAEC were analyzed for apoptosis by TUNEL. Results: HIV-Nef protein was found in PBMC of untreated HIV-positive and surprisingly also in patients receiving virally suppressive anti-retroviral therapy (ART). Importantly, we could demonstrate in untreated HIV patients Nef protein transfer to HCAEC and increased induction of endothelial apoptosis (see figure, ** p<0.01). Currently, equivalent studies with PBMC from patients on ART are in progress. Conclusion: This study demonstrates that Nef-positive PBMC at least in untreated HIV patients can be transferred to HCAEC to cause endothelial cell apoptosis. This will be the basis for future studies analyzing the role of NADPH oxidases and reactive oxygen species in this toxic activity of Nef. Based on this assay system we can address the hypothesis that HIV-Nef plays a pivotal role in the development of cardiovascular diseases in HIV patients with or without ART.

Premature aging induced by radiation exhibits pro-atherosclerotic effects mediated by epigenetic activation of CD44 expression.

Age is undoubtedly a major risk factor for heart disease. However, the reason for this is not entirely clear. In the course of our investigation into the mechanism of radiation-induced cardiovascular disease, we made several unexpected findings that inform us on this question. We observed that human coronary endothelial cells, while being able to initiate repair of radiation-induced DNA damage, often fail to complete the repair and become senescent. Such radiation-induced cellular aging occurs through a mutation-independent route. Endothelial cells that aged naturally through replication or as a result of radiation exhibited indistinguishable characteristics. The promoter regions of the CD44 gene in aging endothelial cells become demethylated, and the proteins are highly expressed on the cell surface, making the cells adhesive for monocytes. Adhesion is a cardinal feature that recruits monocytes to the endothelium, allowing them to infiltrate the vessel wall and initiate atherosclerosis. The epigenetic activation of CD44 expression is particularly significant as it causes persistent elevated CD44 protein expression, making senescent endothelial cells chronically adhesive. In addition to understanding why cardiovascular disease increases with age, these observations provide insights into the puzzling association between radiation and cardiovascular disease and highlight the need to consider premature aging as an additional risk of radiation to human health.

High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.

Azelnidipine, Not Amlodipine, Induces Secretion of Vascular Endothelial Growth Factor From Smooth Muscle Cells and Promotes Endothelial Tube Formation.

BackgroundWe previously reported that the calcium channel blocker (CCB) nifedipine-induced secretion of vascular endothelial growth factor (VEGF) from human coronary smooth muscle cells (HCSMCs) promoted human coronary endothelial cell (HCEC) tube formation. Therefore, we analyzed whether other CCBs, azelnidipine and amlodipine, also induced the secretion of VEGF and promoted HCEC tube formation, and the underlying molecular mechanisms.MethodsTo evaluate the tube formation, HCECs were grown on Matrigel for 18 hours in the supernatants from HCSMCs that had been treated with different kinds of reagents. Concentrations of VEGF in cultured HCSMCs were determined by specific enzyme immunoassays. Nuclear extracts from HCSMCs were prepared, and nuclear factor-kappa B (NF-κB) activation was measured by EZ-DetectTM Transcription Factor Kits for NF-κB p50 or p65.ResultsAlthough azelnidipine dose-dependently stimulated the significant secretion of VEGF from HCSMCs and this stimulation was abolished by a protein kinase C inhibitor, amlodipine-induced secretion of VEGF was significantly lower than that induced by azelnidipine. The medium derived from azelnidipine (at up to 2 μM)-treated HCSMCs led to HCEC tube formation, whereas that obtained with amlodipine did not. Azelnidipine-induced tube formation was blocked by an inhibitor of kinase insert domain-containing receptor/fetal liver kinase-1 tyrosine kinase. Azelnidipine at up to 2 μM induced NF-κB activation.ConclusionsAzelnidipine, but not amlodipine, stimulated the secretion of VEGF from HCSMCs and induced HCEC tube formation. This secretion is mediated at least in part via the activation of NF-κB. Azelnidipine may have a novel beneficial effect in improving coronary microvascular blood flow in addition to its main effect of lowering blood pressure.

Human myocardium releases heat shock protein 27 (HSP27) after global ischemia: the proinflammatory effect of extracellular HSP27 through toll-like receptor (TLR)-2 and TLR4.

The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27.

Nilotinib Exerts Direct Pro-Atherogenic and Anti-Angiogenic Effects On Vascular Endothelial Cells: A Potential Explanation For Drug-Induced Vasculopathy In CML

The BCR/ABL1 inhibitor nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). However, nilotinib apparently induces metabolic changes, including an increase in the fasting glucose level. In addition, vascular adverse events, including progressive atherosclerosis with peripheral arterial occlusive disease (AOD) have been reported in nilotinib-treated CML patients. We reviewed and updated AOD events in our CML patients receiving nilotinib (n=34) and initiated preclinical in vitro and in vivo studies in order to dissect potential targets and mechanisms. After a median observation time (MOT) of 24 months, the frequency of AOD (26.5%) and severe AOD requiring surgical intervention and/or prolonged hospitalization (17.6%) was higher in nilotinib-treated patients compared to risk factor-, observation time-, and age-matched controls (34 imatinib-treated patients with CML, 34 with myelodysplastic syndromes, 34 with JAK2-mutated MPN and 34 with lymphoid neoplasms; <5% AOD, p<0.05). After a MOT of 36 months, the frequency of AOD amounted to 36.1% and the frequency of severe AOD was 19.4%. We next examined the in vitro effects of nilotinib on cultured human umbilical vein endothelial cells (HUVEC), human coronary artery-derived endothelial cells (HCAEC), and the human microvascular endothelial cell line HMEC-1. As determined by 3H-thymidine incorporation, nilotinib was found to inhibit the proliferation of endothelial cells in a dose-dependent manner, with pharmacologically relevant IC50 values obtained in HUVEC (1.0 µM), HCAEC (100 nM), and HMEC-1 (1.0 µM), whereas imatinib showed little effect up to 5 µM. Moreover, nilotinib was found to inhibit the migration of HUVEC in a wound-scratch assay as well as angiogenesis in a tube-formation assay (relative capillary tubes: VEGF+control: 1.8±0.1, VEGF+nilotinib (100 nM): 1.3±0.1, VEGF+imatinib (100 nM): 1.7±0.05; n=3, p<0.01 for VEGF alone vs VEGF+nilotinib). In a mouse model of hindlimb ischemia, nilotinib (75 mg/kg/day p.o. for 28 days) was found to slow blood flow-recovery after induction of ischemia whereas imatinib (100 mg/kg/day p.o. for 28 days) showed no comparable effect (laser Doppler perfusion imaging ratio ischemic/control leg: control mice: 0.81±0.03, imatinib-treated mice: 0.79±0.04, nilotinib-treated mice: 0.68±0.04; n=13/group; p<0.05 for nilotinib vs control and for nilotinib vs imatinib). The decreased blood perfusion was accompanied by an increased rate of limb necrosis (necrosis score: control: 1.15±0.08, imatinib: 1.17±0.05, nilotinib: 1.54±0.18; p<0.05 for nilotinib vs control and nilotinib vs imatinib). Moreover, microvessel density was significantly lower in the affected hind limb in nilotinib-treated mice compared to imatinib-treated mice or control-mice (p<0.05). In addition, we found that nilotinib (between 1-10 µM), but not imatinib (1-10 µM) promotes the expression of pro-atherogenic cytoadhesion molecules (CAM) on HUVEC, including ICAM-1 (CD54), VCAM-1 (CD106) and E-Selectin (CD62E). By contrast, nilotinib (up to 10 µM) showed no effects on expression of plasminogen activators or uPA receptor (CD87) in cultured endothelial cells. As assessed by chemical proteomics profiling and phospho-array analysis, several angiogenesis-related and other endothelial antigens, including Tie-2/TEK, JAK1, BRAF and EPHB2 were identified as molecular targets of nilotinib, whereas imatinib did not bind to these vascular targets in endothelial cells. As assessed by immunohistochemistry using antibodies against KIT and mast cell tryptase, we also found that in our CML patients, nilotinib induces an almost complete depletion of KIT+ mast cells, a cell type that serves as unique source of heparin and uncomplexed tPA and has been implicated as a major repair cell in vascular disorders. However, imatinib was also found to induce mast cell depletion in our patients with CML. Neither nilotinib nor imatinib showed in vitro or in vivo effects on platelet adhesion or platelet aggregation. In conclusion, nilotinib exerts multiple effects on vascular endothelial cells and other perivascular cells such as mast cells, presumably through multiple mechanisms and targets. We hypothesize that these effects may contribute to nilotinib-induced vasculopathy in CML. Disclosures:Wolf:Bristol-Meyers Squibb: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding.

Enhancement of endothelialisation of coronary stents by laser surface engineering

Coronary stents have been widely used in the treatment of coronary heart disease. However, complications have hampered the long-term success of the device. Bare-metal stents (BMS) have a high rate of restenosis and poor endothelialisation. The drug-eluting stents (DES), although dramatically reduce restenosis, significantly prevent endothelialisation leading to late thrombosis and behave the same way as BMS after drug releasing. Rapid adhesion and growth of endothelial cells on the stent surface is a key process for early vascular healing after coronary stenting which contributes to the reduction of major complications. Surface properties manipulate cell growth and directly determine the success and life-span of the implants. However, the ideal surface properties of coronary stents are not yet fully understood. The objective of this research is to understand how surface micro/nano textures and associated material chemistry changes generated by a laser beam affect the behavior of endothelial cells on bare metal 316L stents. A high power laser beam was applied to modifying the surface properties of 316L coronary stent material and the commercial coronary stents, followed by examination of the adhesion and proliferation of human coronary endothelial cells that were growing on the surfaces. Surface properties were examined by scanning electron microscopy, contact angle measurement, and X-ray photoelectron spectroscopy. A novel surface with combined micro/nano features was created on stent material 316L and coronary stent with a specific surface chemistry. This surface gives rise to a threefold increase in the adhesion and eightfold increase in the proliferation of endothelial cells. Interestingly, such effects were only observed when the surface texture was produced in the nitrogen atmosphere suggesting the importance of the surface chemistry, including the dramatic increase of chromium nitride, for the interaction of endothelial cells with the material surface. This novel surface is also super-hydrophilic with close to zero water/cell culture fluid contact angles and low cytotoxicity. A novel surface created by laser surface-engineering with a combination of defined surface texture and surface chemistry was found beneficial for the improvement of coronary stent endothelialisation. The technology presented here could work with both DES and BMS with added benefit for the improvement of the biocompatibility of current coronary stents.

Endothelial microparticle-mediated transfer of microRNA126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles

Introduction: Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. Here, we sought to investigate whether endothelial microparticles (EMP), released from apoptotic endothelial cells (ECs), influence EC repair. Methods and results: EMP were generated from human coronary endothelial cells (HCAEC) and characterized by confocal microscopy and flow cytometry. Systemic treatment of mice with EMP after electrical denudation of the endothelium accelerated reendothelialization in vivo (29.81±2.31 vs. 50.71±9.84, p<0.01, n=12-14). Human EMP incorporation by murine ECs was confirmed in subsequent experiments using flow cutometry and immunofluorescence. In vitro experiments revealed that EMP uptake in ECs promote EC migration (82.02±15.72 vs. 37.05±19.00, p<0.01, n=6) and proliferation (160.8±24.67 vs. 100.0±26.86, n=6), both critical steps in endothelial repair. In order to dissect the underlying mechanisms, Taqman microRNA-array was performed and microRNA (miR)-126 was identified as the predominantly expressed miR in EMP (n=3). Following experiments demonstrated that miR-126 was transported into recipient HCAEC by EMP and functionally regulated the target protein sprouty-related, EVH1 domain containing 1 (Spred1). Genetic downregulation of Spred1 in HCAEC increased EC migration and proliferation capacity. Knockdown of miR-126 in EMP abrogated EMP-mediated effects on HCAEC migration and proliferation in vitro and reendothelialization in vivo, confirming the crucial role of miR-126 in EMP-mediated endothelial regeneration. Next, we tested whether EMP generated under pathological hyperglycaemic conditions (defined as injured EMP, (iEMP)) differed in miR expression and function from EMP. Interestingly, Taqman microRNA-array analysis revelaed that miR-126 was the strongest regulated miR between iEMP and EMP (n=3). Subsequently, iEMP derived from glucose-treated ECs showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Conclusion: Endothelial microparticles promote vascular endothelial repair by delivering functional microRNA-126 into recipient cells. This leads to downregulation of target protein Spred1 and improves migratory and proliferative EC capacity with subsequent improvement of EC repair. In pathological hyperglycaemic conditions, EMP-mediated miR-126 induced EC repair is altered.

Endothelial Overexpression of LOX-1 increases stroke size in vivo

Background: Stroke is one of the most common causes of death and long term disability worldwide primarily affecting the elderly population. Lectin-like oxidized LDL receptor 1 (LOX-1) is the receptor for oxidized LDL identified in endothelial cells. Binding of OxLDL to LOX-1 induces several cellular events in endothelial cells, such as activation of transcription factor NF-kB, upregulation of MCP-1, and reduction in intracellular NO. Accumulating evidence suggests that LOX-1 is involved in endothelial dysfunction, inflammation, atherogenesis, myocardial infarction, and intimal thickening after balloon catheter injury. Interestingly, a recent study demonstrated that acetylsalicylic acid (aspirin), which could prevent ischemic stroke, inhibited Ox-LDL-mediated LOX-1 expression in human coronary endothelial cells. The expression of LOX-1 was increased at a transient ischemic core site in the rat middle cerebral artery occlusion model. These data suggest that LOX-1 expression induces atherosclerosis in the brain and is the precipitating cause of ischemic stroke. Therefore, the goal of the present study was to investigate the role of endothelial LOX-1 in stroke using experimental mouse model. Methods and results: 12-week-old male LOX-1TG generated recently in our group and wild-type (WT) mice were applied for a transient middle cerebral artery occlusion (MCAO) model to induce ischemia/reperfusion (I/R) brain injury after 45 min of ischemia followed by 24 h of reperfusion. LOX-1TG mice developed 24 h post-MCAO significantly larger infarcts in the brain compared to WT (81.51±8.84 vs. 46.41±10.13, n=7, p < 0.05) as assessed morphologically using Triphenyltetrazolium chloride (TTC) staining. Moreover, LOX-1TG showed higher neurological deficit in RotaRod (35.57±8.92 vs. 66.14±10.63, n=7, p < 0.05) and Bederson tests (2.22±0.14 vs. 1.25±0.30, n=9-12, p < 0.05) – two experimental physiological tests for neurological function. Conclusions: Thus, our data suggest that LOX-1 plays a critical role in the ischemic stroke when expressed at unphysiological levels. Such LOX-1-associated phenotype could be due to the endothelial dysfunction. Therefore, LOX-1 may represent novel therapeutic targets for preventing ischemic stroke.

Intermedin protects against ischaemia and reperfusion injury in human ventricular cardiomyocytes: receptor subtype involvement

Purpose: Early reperfusion following myocardial infarction is essential but results in ischaemia/reperfusion (I/R) injury, whereby myocardial cells die largely due to oxidative stress. Intermedin (IMD; adrenomedullin-2), a novel peptide related to adrenomedullin (AM), is up-regulated in cardiovascular disease and is protective against I/R injury in rodent hearts and human coronary microvascular endothelial cells and cardiac non-myocytes. Protection is dependent on calcitonin receptor-like receptor (CRLR) signalling in association with one of three receptor activity-modifying proteins (RAMPs). IMD has yet to be investigated in human cardiomyocytes for a cardioprotective role. Aims: To identify if IMD and its receptor components are expressed in human ventricular cardiomyocytes; to investigate whether IMD protects these cells against simulated I/R injury and if so, determine receptor subtype involvement. Methods: Left ventricular adult human cardiomyocytes (HCM) were subjected to normoxia or simulated ischaemia for 4 h or I/R (3 h normoxia followed by 1 h reperfusion) all in the absence or presence of IMD. Cell viability was assessed using Trypan blue dye exclusion and oxidative damage by commercial Oxyblot assay. mRNA expression and protein distribution of IMD, AM, CRLR and RAMPs1-3 were assessed by Quantitative Real-Time PCR and indirect immunofluorescence microscopy, respectively. The silencing of the peptides or their receptor components using siRNA knock-down was employed to determine endogenous peptide and receptor subtype involvement in protection against I/R. Results: IMD, AM and their receptor component mRNAs were expressed in HCM; RAMP2 was the most abundant RAMP, while IMD was less abundantly expressed than AM. Ischaemia or I/R reduced the HCM cell count by 40% and 60% respectively (p<0.05 for both). IMD (10-9 mol.L-1) protected against the deleterious effects of ischaemia and I/R injury (∼20% salvaged for both conditions, p<0.05), with preservation of cell structure when examined by phase contrast microscopy and decreased oxidative stress noted on Oxyblot (p<0.001 vs. non-IMD counterpart for ischaemia or I/R). Protection was greatest when IMD was given during reperfusion (p<0.001) and was abolished by silencing CRLR (p<0.05) and RAMP2 (p<0.001). However, silencing endogenous IMD and/or AM expression did not augment extent of I/R injury in HCM. Conclusion: Exogenous IMD is protective against I/R in HCM, attributed to AM1 receptor subtype involvement. IMD administration during cardiac reperfusion could improve levels of morbidity and mortality in patients following myocardial infarction.

Abstract 166: Endothelial Overexpression of LOX-1 Increases Stroke Size in vivo

Background— Stroke is one of the most common causes of death and long term disability worldwide primarily affecting the elderly population. Lectin-like oxidized LDL receptor 1 (LOX-1) is the receptor for oxidized LDL identified in endothelial cells. Binding of OxLDL to LOX-1 induces several cellular events in endothelial cells, such as activation of transcription factor NF-kB, upregulation of MCP-1, and reduction in intracellular NO. Accumulating evidence suggests that LOX-1 is involved in endothelial dysfunction, inflammation, atherogenesis, myocardial infarction, and intimal thickening after balloon catheter injury. Interestingly, a recent study demonstrated that acetylsalicylic acid (aspirin), which could prevent ischemic stroke, inhibited Ox-LDL-mediated LOX-1 expression in human coronary endothelial cells. The expression of LOX-1 was increased at a transient ischemic core site in the rat middle cerebral artery occlusion model. These data suggest that LOX-1 expression induces atherosclerosis in the brain and is the precipitating cause of ischemic stroke. Therefore, the goal of the present study was to investigate the role of endothelial LOX-1 in stroke using experimental mouse model. Methods and Results— 12-week-old male LOX-1TG generated recently in our group and wild-type (WT) mice were applied for a transient middle cerebral artery occlusion (MCAO) model to induce ischemia/reperfusion (I/R) brain injury. LOX-1TG mice developed 24h post-MCAO significantly larger infarcts in the brain compared to WT (81.51±8.84 vs. 46.41±10.13, n=7, p &lt; 0.05) as assessed morphologically using Triphenyltetrazolium chloride (TTC) staining. Moreover, LOX-1TG showed higher neurological deficit in RotaRod (35.57±8.92 vs. 66.14±10.63, n=7, p &lt; 0.05) and Bederson tests (2.22±0.14 vs. 1.25±0.30, n=9-12, p &lt; 0.05) - two experimental physiological tests for neurological function. Conclusions— Thus, our data suggest that LOX-1 plays a critical role in the ischemic stroke when expressed at unphysiological levels. Such LOX-1 -associated phenotype could be due to the endothelial dysfunction. Therefore, LOX-1 may represent novel therapeutic targets for preventing ischemic stroke.

Role of Endothelial TRPC3 Channels in Endoplasmic Reticulum Stress Induced Apoptosis in Human Coronary Endothelial Cells

Atherosclerosis is a chronic inflammatory disease of the arterial wall and represents the major cause of coronary artery disease. The balance between apoptosis and survival signaling pathways is crucial to maintaining endothelial integrity during vascular injury, and alteration of these processes has been associated to atherogenesis. Endoplasmic reticulum (ER) stress has emerged as a critical player in development of atherosclerotic lesions. Despite its beneficial and essential corrective functions, chronic ER stress, through persistent activation of the unfolded protein response (UPR) can alter the balance between apoptosis and survival favoring lesion progression. In recent studies, we found that in human coronary artery endothelial cells (HCAECs) Transient Receptor Potential Canonical 3 (TRPC3), a member of the larger TRP superfamily of channel forming proteins, plays a role in cell survival and apoptosis. In HCAECs knockdown of TRPC3 results in an increase in the number of apoptotic cells in response to the ER stressor thapsigargin. This correlated positively with increased expression of typical ER stress markers such as CHOP and BiP. Altogether, this data suggests that TRPC3 expression and function may have a protective role in HCAECs and that TRPC3 deficiency and/or deregulation may sensitize HCAECs to the pro‐apoptotic actions of sustained ER stress. Research support: AHA 12POST11910042 to KS, R01HL111877–01 to GV.

Decreased thioredoxin reductase activity in endothelial cells exposed to high glucose is associated with protein tyrosine nitration and interaction with caveolin 1

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. Here we wanted to determine the effects of elevated glucose levels on TrxR1activity in human endothelial cells. Human coronary and aortic endothelial cells (HCAEC and HAEC, respectively) were exposed to high glucose conditions (HG= 25mM) for 5 days and/or iso‐osmotic control (L‐glucose). We found that HG diminished TrxR1 activity (30% reduction, p&lt;0.005), stimulated its association with caveolin‐1 and its subcellular localization to caveolae. Immunoprecipitation analysis revealed that HG‐promoted TrxR1 tyrosine nitration. Pre‐treatments of the cells with the peroxynitrite decomposition catalyst FeTPPS (25μM) partially rescued TrxR1 activity and prevented its interaction with caveolin‐1. FeTPPS also attenuated HG‐induced increases in caspase 3 activity and induction of apoptosis in endothelial cells. Our results suggest that inhibition of TrxR1 activity by HG in human endothelial cells is a result of its nitration and interaction with caveolin‐1. Restoration of TrxR1 activity may be a potentially beneficial therapeutic strategy for the prevention of diabetes‐induced vascular complications.