Generation of new blood vessels from pre-existing vasculature (angiogenesis) is accompanied in almost all states by increased vascular permeability. This is true in physiological as well as pathological angiogenesis, but is more marked during disease states. Physiological angiogenesis occurs during tissue growth and repair in adult tissues, as well as during development. Pathological angiogenesis is seen in a wide variety of diseases, which include all the major causes of mortality in the west: heart disease, cancer, stroke, vascular disease and diabetes. Angiogenesis is regulated by vascular growth factors, particularly the vascular endothelial growth factor family of proteins (VEGF). These act on two specific receptors in the vascular system (VEGF-R1 and 2) to stimulate new vessel growth.VEGFs also directly stimulate increased vascular permeability to water and large-molecular-weight proteins. We have shown that VEGFs increase vascular permeability in mesenteric microvessels by stimulation of tyrosine autophosphorylation of VEGF-R2 on endothelial cells, and subsequent activation of phospholipase C (PLC). This in turn causes increased production of diacylglycerol (DAG) that results in influx of calcium across the plasma membrane through store-independent cation channels. We have proposed that this influx is through DAG-mediated TRP channels. It is not known how this results in increased vascular permeability in endothelial cells in vivo. It has been shown, however, that VEGF can stimulate formation of a variety of pathways through the endothelial cell, including transcellular gaps, vesiculovacuolar organelle formation, and fenestrations. A hypothesis is outlined that suggests that these all may be part of the same process.
Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) by stimulating Ca(2+) influx into endothelial cells. To determine whether VEGF-mediated Ca(2+) influx is stimulated by release of Ca(2+) from intracellular stores, we measured the effect of Ca(2+) store depletion on VEGF-mediated increased L(p) and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) of frog mesenteric microvessels. Inhibition of Ca(2+) influx by perfusion with NiCl(2) significantly attenuated VEGF-mediated increased [Ca(2+)](i). Depletion of Ca(2+) stores by perfusion of vessels with thapsigargin did not affect the VEGF-mediated increased [Ca(2+)](i) or the increase in L(p). In contrast, ATP-mediated increases in both [Ca(2+)](i) and L(p) were inhibited by thapsigargin perfusion, demonstrating that ATP stimulated store-mediated Ca(2+) influx. VEGF also increased Mn(2+) influx after perfusion with thapsigargin, whereas ATP did not. These data showed that VEGF increased [Ca(2+)](i) and L(p) even when Ca(2+) stores were depleted and under conditions that prevented ATP-mediated increases in [Ca(2+)](i) and L(p). This suggests that VEGF acts through a Ca(2+) store-independent mechanism, whereas ATP acts through Ca(2+) store-mediated Ca(2+) influx.
Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (Lp) in vivo. To determine the signal transduction cascade through which this is mediated, we measured the effect of inhibition of various signalling pathways on VEGF‐mediated acute increases in Lp in individually perfused frog mesenteric microvessels.
VEGF receptors have previously been shown to activate phospholipase C‐γ (PLCγ), protein kinase C (PKC) and MEK, the mitogen‐activated and extracellular signal‐related kinase (ERK) kinase. To determine the role of these signalling pathways we measured the effects of inhibitors of each on the VEGF‐mediated increase in Lp.
VEGF‐mediated increases in Lp were attenuated by pre‐treatment with the PLC inhibitor U73122, but not affected by treatment with the inactive enantiomer U73343. The PLC inhibitor was also able to attenuate the increase in Lp mediated by the inflammatory mediator ATP.
Inhibition of either PKC or MEK activation using the selective inhibitors bisindolylmaleimide (BIM, 1 μm) and PD98059 (30 μm), respectively, did not change the VEGF‐mediated increase in Lp. However, PD98059, BIM and U73122 all reduced phosphorylation of ERK1/2 determined by Western blot analysis with anti‐phospho‐ERK1/2 antibodies.
Furthermore, inhibition of the conversion of diacyl glycerol (DAG) to arachidonic acid, by perfusion with the DAG lipase inhibitor RHC80267 (50 μm), did not attenuate the increase in Lp brought about by VEGF.
These data suggest that VEGF acutely increases microvascular permeability in vivo through a mechanism that is dependent on PLC stimulation, but is independent of PKC or MEK activation or production of arachidonic acid from DAG. We therefore propose that VEGF acutely acts to increase Lp through the direct actions of DAG, independently of PKC or arachidonic acid.
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