Complex heterogeneous tissue constructs containing multiple cell types prepared by inkjet printing technology.

Gαq-TRPC6-mediated Ca2+ Entry Induces RhoA Activation and Resultant Endothelial Cell Shape Change in Response to Thrombin

Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+ channels.

Background: Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca 2+ entry (SOCE), which is activated by a depletion of the intracellular Ca 2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca 2+ -sensor, Stim1, and the plasmalemmal Ca 2+ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca 2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients. Methodology/Principal Findings: The present study employed Ca 2+ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La 3+ and Gd 3+ . Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca 2+ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca 2+ buffer, BAPTA, BTP-2,

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Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.

TRP channels in endothelial function and dysfunction

Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca(2+) (Ca(2+))(i) in endothelial cells, resulting in Ca(2+)-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca(2+) influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V). Bradykinin-induced Ca(2+) influx was resolved into capacitative Ca(2+) entry (CCE) and non-CCE. The non-CCE component was inhibited by a B2 receptor antagonist (HOE140; 1 microM) and a phospholipase C (PLC) inhibitor (U73122; 10 microM). The action of bradykinin could be mimicked by 1-oleoyl-2-acetyl-glycerol, an analogue of diacylglycerol (DAG), and by RHC80267, a DAG-lipase inhibitor. Immunoblots showed that TRPC6 was one of the main TRPC channels expressed in endothelial cells. Transfection of H5V cells with two siRNA constructs against TRPC6 both markedly reduced the TRPC6 protein level and, at the same time, decreased the percentage of cells displaying bradykinin-induced non-CCE. siRNA transfection also reduced the magnitude of non-CCE among the cells responding to bradykinin. Taken together, our data suggest that bradykinin-induced non-CCE is mediated via the B2-PLC pathway, and that DAG may be involved in this process. Further, TRPC6 is one of the important channels participating in bradykinin-induced non-CCE in endothelial cells.

Pan Cheung Leung

Papers

Mechanism of non-capacitative Ca2+ influx in response to bradykinin in vascular endothelial cells.