Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen...

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-β and PDGF-BB). Expression of smooth muscle protein 22 and -smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.

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Progesterone metabolites rapidly stimulate calcium influx in human platelets by a src-dependent pathway

The effects of several steroids and their metabolites were examined for their ability to rapidly alter intracellular free calcium ([Ca2+]i) in the anucleate human platelet. Earlier studies suggested that steroids had direct and rapid non-genomic effects to alter platelet physiology. The rationale for performing this study was to investigate the signal transduction events being activated by steroids. Super-physiologic concentrations (1.0–10.0 μM) of β-estradiol and several estradiol metabolites and analogs potentiated (approximately twofold) the action of thrombin to elevate [Ca2+]i in platelets, whereas 10.0 μM progesterone inhibited the action of human thrombin by 10–15%. Progesterone and β-estradiol by themselves did not affect [Ca2+]i. Progesterone metabolites can achieve high blood concentrations. Some progesterone metabolites, particularly those in the β-conformation, were potent stimulators of Ca2+ influx and intracellular Ca2+ mobilization in platelets. They activated phospholipase C because their ability to increase [Ca2+]i was inhibited by the phospholipase C inhibitor U-73122. The ability of pregnanediol and collagen to increase [Ca2+]i was inhibited by the src tyrosine kinase inhibitor PP1, whereas the actions of thrombin and thapsigargin to increase [Ca2+]i were not affected by PP1. The effects of progesterone metabolites to increase [Ca2+]i were observed with concentrations as low as 0.1 μM. Pregnanolone synergized with thrombin to increase [Ca2+]i. It is hypothesized that human platelets possess receptors for progesterone metabolites. These receptors when stimulated will activate platelets by causing a rapid increase in [Ca2+]i. Pregnanolone, isopregnanediol and pregnanediol were the most effective stimulators of this newly identified src-dependent signal transduction system in platelets. Progesterone metabolites may regulate platelet aggregation and hence thrombosis in vivo.


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Thrombin regulates CD40 expression in microglial cells

Microglial cells are the innate immune cells of the central nervous system and quickly respond to injury by proliferation, cytokine release, and increased cell surface antigen expression. Thrombin is a multifunctional serine proteinase, which has the capability to activate microglial cells. Here, we report that pharmaceutical-grade thrombin dose-dependently increases the expression of CD40 in N9 microglial cells. This effect is blocked by a thrombin inhibitor, mimicked by thrombin receptor-activating peptide and modified by mitogen-activated protein kinase pathway inhibitors. Thrombin-induced CD40 regulation might play a role in diseases with breakdown of the blood-brain barrier such as multiple sclerosis or stroke.


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