TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

溶血卵磷脂（lysophosphatidylcholine，lysoPC）是氧化低密度脂蛋白（oxidizedlow-densitylipoprotein，ox-LDL）的主要成分。早期的研究表明，lysoPC可诱导人冠状动脉平滑肌细胞（humancoronaryarterysmoothmusclecells，HCASMCs)凋亡，但具体机制尚不清楚。近期，我们通过细胞活力测定，流式细胞术，共聚焦显微镜及分子生物学手段对lysoPC作用的分子机制进行研究发现，lysoPC可引发细胞外钙离子内流，并显著抑制HCASMCs的生长。并且经典的钙离子阻滞剂La3+、TRPC通道阻滞剂SKF-96365...Lysophosphatidylcholine (lysoPC) is a major component of oxidized low-density lipoprotein (ox-LDL). Although an earlier study showed that lysoPC induced apoptosis in human coronary artery smooth muscle cells (SMCs), the related molecular mechanisms are not fully understood. The present study investigated how lysoPC mediates apoptosis in cultured human coronary artery smooth muscle cells (SMCs) usi...学位：博士后院系专业：生命科学学院_生物学学号：201317007

Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention

Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter

A new role of transcription factor SOX17 as potential interaction partner of KLF4 and EGR-1 in human coronary artery smooth muscle cells and in differentiating mouse ES-cells

The development of the vascular network comprises tightly regulated processes, involving vasculogenesis and angiogenesis. The cells, which are mainly participating in these processes, are endothelial cells and vascular smooth muscle cells, the latter ones especially being important for the stability of blood vessels. Uncontrolled proliferation of VSMCs contributes crucially to the development of vascular disease, e.g. in the case of atherosclerosis. Two main initiator factors of these processes are Low Densitiy Lipoprotein (LDL) and Platelet Derived Growth Factor-BB (PDGF-BB). For this reason, the VSMCs and the transcriptional regulation of their proliferation, in response to LDL and PDGF-BB, build an important target for therapeutical interventions. Sox17, a member of subgroup F of the Sox family proteins, was for the first time detected in vascular smooth muscle cells in different mouse tissues, like liver, brain, heart, lung, spleen and kidney in vivo and in human coronary artery smooth muscle cells in vitro. Moreover, a new possible protein complex, consisting of SOX17, KLF4 and EGR-1, was found in human coronary artery smooth muscle cells, as well as in 4 day old embryoid bodies. All members of this complex are induced by PDGF-BB, a growth factor which becomes activated in angiogenesis and pathological vascular conditions, stimulating the migration and proliferation of vascular SMCs. By this the complex might be involved in migration and proliferation of vascular SMCs, and moreover in pathological vascular conditions, like the progression of atherosclerosis. EGR-1 is known to be the key player in mediating the transcriptional responses to PDGF-BB and LDL and has already been implicated in progression of atherosclerosis. Because of the fact, that a complex, consisting of Sox17, Klf4 and Egr-1, was also observed to be formed in differentiating ES-cells (day 4), supports a broader role of this protein complex in the differentiation of cell-specific lineages during development, in particular vascular smooth muscle cells and endoderm lineages. The complex might have an inhibitory, as well as an activating role, as Sox17, Klf4, and Egr-1 are known to behave bifunctional. Besides, Sox17 seems to bind to -catenin during EB formation. At least this could be an indication for an involvement of the complex in modulating the wnt-signaling pathway during embryonic development

Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor y

Objectives: The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and on human coronary artery endothelial cells (HCAECs) migration. Methods: Cell proliferation was evaluated by direct cell counting using trypan blue exclusion. Cell migration was assessed by wound healing “scratch” assay and by time lapse video-microscopy. Protein expression was assessed by immunoblotting, and morphological changes were studied by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist troglitazone, and the use of highly selective antagonists of PPARγ such as PGF2α and GW9662. Results: We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type specific as it does not affect migration and proliferation of endothelial cells. Conclusions: Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug eluting stents, which could lead reduced rates of restenosis and potentially other complications of DES stent implantation

Perspectives on the Trypanosoma cruzi-host cell receptor interaction

Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets

Oxygen Delivery Strategies in Tissue-Engineering Constructs

The supply of nutrients and the removal of waste products play a major role in tissue engineering. From all the nutrients necessary for cells seeded on scaffolds for tissue regeneration, oxygen is the limiting component due to its low solubility in culture media while cells consume five to six moles of oxygen for every mole of monosaccharide. The aim of the present work was to develop different strategies to improve the supply of oxygen to human coronary artery smooth muscle cells (HCASMC) seeded on three dimensional (3D) porous biostable polyurethane scaffolds. As a springboard for the study, the measured value of oxygen diffusivity through porous polyurethane scaffolds, fabricated by using pressure differential/particulate leaching technique, was used to screen the best polymer concentration. Scaffolds fabricated form 15 wt% polymer concentration not only had higher oxygen diffusivity but also have better pore interconnectivity as shown by SEM image analysis. Moreover a convective mass transfer approach showed an improvement in the infiltration of HCASMCs into the 3D scaffolds. An oxygen carrier molecule, perfluorodecalin (PFD), was found to improve dissolved oxygen concentration in culture media. PFD was shown to be not only non-toxic to HCASMC but also have no significant effect on the morphology of the HCASMCs. Therefore, higher cell density and infiltration depth into the polyurethane scaffolds were observed when HCASMCs were cultured in a media containing PFD. The final stage of this work was to introduce an oxygen vector into the skeleton of polyurethane scaffolds. For this reason, inert Zeolite Y particles were fluorinated and shown to enhance the amount of dissolved oxygen when suspended in culture media. Fluorinated Zeolite (FZ) particles were then embedded into polyurethane scaffolds without modifying the porosity and morphology of the 3D structures. Subsequently, higher cell density and infiltration depths were observed when HCASMCs were cultured on FZ particles embedded polyurethane scaffolds in contrast to bare polyurethane scaffolds. Taken together, these data show three different but equally advantageous strategies of improving the supply of oxygen to HCASMC seeded into the interstices of 3D polyurethane scaffolds

Cyclic Nucleotide-Dependent Phosphorylation Regulates BK-Ca Channel Activity in Human Coronary Artery Smooth Muscle Cells

Cardiovascular diseases (CVD) can induce dysfunction in organ systems by attenuating normal blood flow. Gonadal steroids are vasoactive hormones, but their role in contributing to cardiovascular function remains controversial. We have demonstrated that gonadal steroids can relax coronary arteries by opening the large-conductance, calcium- and voltage-activated potassium (BKca) channel in smooth muscle cells by increasing cyclic nucleotide levels; however, the signaling pathways involved remain to be elucidated. The purpose of this study was to identify how phosphorylation (via cAMP- and cGMP-dependent protein kinases) I dephosphorylation (via phosphoprotein phosphatase 2A, PP2A) regulates BKca channel activity in human coronary artery smooth muscle cells (HCASMC). BKca channel activity was recorded from single HCASMC (Lonza/Clonetics) via single-channel and whole-cell patch-clamp. Channel activity was stimulated by increasing intracellular calcium levels or by increasing either cAMP or cGMP, but the stimulatory effect was mediated predominately via the cGMP-dependent protein kinase. In addition, inhibition of PP2A decreased channel activity. These findings demonstrate that cyclic nucleotide-dependent vasodilators can regulate arterial function via phosphorylation of BKca channels, and that dephosphorylation of the channel (via PP2A) may play a role in channel activation. We propose that modulation of BKca channels via hormone- or drug-induced phosphorylation could be a novel therapeutic means of helping to lower the risk of CVD in both males and females