We recently demonstrated that nitric oxide (NO) significantly contributes to the mitogenic effect of vascular endothelial growth factor (VEGF), suggesting a role for the NO pathway in the signaling cascade following kinase-derivative receptor activation in vascular endothelium. The aim of this study was to investigate the intracellular pathways linked to VEGF/NO-induced endothelial cell proliferation. We assessed the activity of the mitogen-activated protein kinase (MAPK) that is specifically activated by growth factors, extracellular-regulated kinase (ERK1/2), on cultured microvascular endothelium isolated from coronary postcapillary venules. ERK1/2 was immunoprecipitated, and its activity was assessed with an immunocomplex kinase assay. In endothelial cells exposed for 5 min to the NO donor drug sodium nitroprusside at a concentration of 100 microM, ERK1/2 activity significantly increased. VEGF produced a time- and concentration-dependent activation of ERK1/2. Maximal activity was obtained after 5 min of stimulation at a concentration of 10 ng/ml. The specific MAPK kinase inhibitor PD 98059 abolished ERK1/2 activation and endothelial cell proliferation in a concentration-dependent manner in response to VEGF and sodium nitroprusside. The NO synthase inhibitor Nomega-monomethyl-L-arginine, as well as the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked the activation of ERK1/2 induced by VEGF, suggesting that NO and cGMP contributed to the VEGF-dependent ERK1/2 activation. These results demonstrate for the first time that kinase-derivative receptor activation triggers the NO synthase/guanylate cyclase pathway to activate the MAPK cascade and substantiates the hypothesis that the activation of ERK1/2 is necessary for VEGF-induced endothelial cell proliferation.

We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase–polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitorN^ω-monomethyl-l-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.

The hypothesis that thrombospondin‐1 (TSP‐1) can exert opposite effects on angiogenesis depending on the functional status of its domains/fragments was investigated. In the rabbit cornea, TSP‐1 inhibited angiogenesis induced by fibroblast growth factor‐2 (FGF‐2). However, when tested per se, TSP‐1 was able to elicit an angiogenic response comparable to that induced by FGF‐2. Induction of angiogenesis was dose‐dependent (20 ng ‐ 2 μg/pellet), was prevented by anti‐TSP antibodies or by heat‐inactivation of TSP‐1, and was not due to inflammatory mediators, to FGF‐2 or to TGF‐β. Equimolar concentrations of the 25 kDa heparin binding fragment of TSP‐1 were even more efficient than the whole molecule, and promoted the angiogenic activity of FGF‐2. On the contrary, the 140 kDa fragment of TSP‐1 did not induce angiogenesis and turned off the angiogenic response to FGF‐2. The 25 kDa fragment and TSP‐1, but not the 140 kDa fragment, increased endothelial cell invasiveness and stimulated the production and activation of matrix metalloproteinase‐2 (MMP‐2). Moreover, the 25 kDa fragment reduced the synthesis of the MMP‐2 inhibitor TIMP‐2, while the 140 kDa fragment caused a twofold increase in TIMP‐2 production and inhibited MMPs stimulation by TSP‐1 and FGF‐2. We conclude that TSP‐1 is a source of smaller mediators of angiogenesis, which affect in an opposite way endothelial cell functions and proteolytic activity, thus resulting in an opposite final effect on angiogenesis.