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The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores., (Copyright 2008 S. Karger AG, Basel.)

The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme β-galactosylceramidase leading to accumulation of the neurotoxic metabolite 1-β-d-galactosylsphingosine (psychosine) in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Here, we demonstrate that psychosine has anti-angiogenic properties by causing the disassembling of endothelial cell actin structures at micromolar concentrations as found in the brain of patients with globoid cell leukodystrophy. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors, defect that was corrected after transduction with a lentiviral vector harbouring the murine β-galactosylceramidase complementary DNA. Finally, RNA interference-mediated β-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case. Together these data demonstrate that β-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy. [ABSTRACT FROM PUBLISHER]

The subcutaneous Matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic molecules. However, quantification of the angiogenic response in the plug remains a problematic task. Here we report a simple, rapid, unbiased and reverse transcription-quantitative PCR (RT-qPCR) method to investigate the angiogenic process occurring in the Matrigel plug in response to fibroblast growth factor-2 (FGF2). To this purpose, a fixed amount of human cells were added to harvested plugs at the end of the in vivo experimentation as an external cell tracer. Then, mRNA levels of the pan-endothelial cell markers murine CD31 and vascular endothelial- cadherin were measured by species-specific RT-qPCR analysis of the total RNA and data were normalized for human GAPDH or β- actin mRNA levels. RT-qPCR was used also to measure the levels of expression in the plug of various angiogenesis/inflammation-related genes. The procedure allows the simultaneous, quantitative evaluation of the newly-formed endothelium and of non-endothelial/inflammatory components of the cellular infiltrate in the Matrigel implant, as well as the expression of genes involved in the modulation of the angiogenesis process. Also, the method consents the quantitative assessment of the effect of local or systemic administration of anti-angiogenic compounds on the neovascular response triggered by FGF2. [ABSTRACT FROM AUTHOR]

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase β-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31−/VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease. [ABSTRACT FROM AUTHOR]