Vessel wall remodeling is a complex phenomenon in which the loss of differentiation of vascular smooth muscle cells (VSMCs) occurs. We investigated the role of rat macrophage chemoattractant protein (MCP)-1 on rat VSMC proliferation and migration to identify the mechanism(s) involved in this kind of activity. Exposure to very low concentrations (1-100 pg/ml) of rat MCP-1 induced a significant proliferation of cultured rat VSMCs assessed as cell duplication by the counting of total cells after exposure to test substances. MCP-1 stimulated VSMC proliferation and migration in a two-dimensional lateral sheet migration of adherent cells in culture. Endogenous vascular endothelial growth factor-A (VEGF-A) was responsible for the mitogenic activity of MCP-1, because neutralizing anti-VEGF-A antibody inhibited cell proliferation in response to MCP-1. On the contrary, neutralizing anti-fibroblast growth factor-2 and anti-platelet-derived growth factor-bb antibodies did not affect VSMC proliferation induced by MCP-1. RT-PCR and Western blot analyses showed an increased expression of either mRNA or VEGF-A protein after MCP-1 activation (10-100 pg/ml), whereas no fms-like tyrosine kinase (Flt)-1 receptor upregulation was observed. Because we have previously demonstrated that hypoxia (3% O2) can enhance VSMC proliferation induced by VEGF-A through Flt-1 receptor upregulation, the effects of hypoxia on the response of VSMCs to MCP-1 were investigated. Severe hypoxia (3% O2) potentiated the growth-promoting effect of MCP-1, which was able to significantly induce cell proliferation even at a concentration as low as 0.1 pg/ml. These findings demonstrate that low concentrations of rat MCP-1 can directly promote rat VSMC proliferation and migration through the autocrine production of VEGF-A.

We have previously shown that hypoxia makes vascular smooth muscle cells (VSMCs) responsive to placental growth factor (PlGF) through the induction of functional fms-like tyrosine kinase (Flt-1) receptors. The aim of this study was to investigate the molecular mechanisms involved in the PlGF effects on proliferation and contraction of VSMCs previously exposed to hypoxia (3% O2). In cultured rat VSMCs exposed to hypoxia, PlGF increased the phosphorylation of protein kinase B (Akt), p38 and STAT3; activation of STAT3 was higher than that of other kinases. In agreement with this finding, the proliferation of hypoxia-treated VSMCs in response to PlGF was significantly impaired by the p38 and the phosphatidylinositol 3-kinase inhibitors SB202190 and LY294002, respectively, and was almost completely prevented by AG490, a janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor. Since hypoxia was able to reverse the vasorelaxant effect of PlGF into a vasoconstrictor response, the mechanism of this latter effect was also investigated. Significant Flt-1 activity was measured in isolated preparations from rat aorta exposed to hypoxia. Inhibitors of mitogen-activated protein kinase kinase, Akt and STAT3 induced a modest inhibition of the vasoconstrictor response to PlGF, while the p38 inhibitor SB202190 markedly impaired the PlGF-induced contractile response. These effects were selectively mediated by Flt-1 without any involvement of foetal liver kinase-1 receptors. These data are the first evidence that different intracellular pathways activated by Flt-1 receptor in VSMCs are involved in diverse biological effects of PlGF: while mitogen activated protein kinase kinase/extracellular signal regulated kinase(1/2) and JAK/STAT play a role in VSMC proliferation, p38 is involved in VSMC contraction. These findings may highlight the role of PlGF in vascular pathology.

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The exact phenotype and lineage of endothelial progenitor cells (EPCs) are still a matter of debate and different expansion protocols are used to obtain them. In this study, EPC expansion from peripheral blood mononuclear cells was analyzed within the first week of culture. Both the adherent and suspended cells, of which the latter usually discarded, were considered. We provide, for the first time, a systematic study of EPC phenotype and functional features within the first 3 days of culture. Moreover, within the 2nd day, both cellular fractions displayed a significant increase in endothelial marker expression which correlated with EPC properties.

Catecholamines have been shown to be involved in vascular remodeling through the stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% O(2)) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce a trophic effect in human endothelial cells through stimulation of the alpha(1D)-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective alpha(1D)-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that alpha(1D)-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by N(G)-nitro-L-arginine methyl ester. Conversely, the alpha(1A)-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the alpha(1B)-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of alpha(1D)-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of alpha(1D)-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury.