In the present study, the potential correlation between Ang II and PlGF was investigated in cultured EA.Hy 926 endothelial cells and in HUASMCs. The major findings of this study are: (1) Ang II, via AT1R, induces PlGF gene expression and protein secretion in both VECs and VSMCs; (2) Ang II increases VEGFR-1 and -2 gene expression in these vascular cells; (3) multiple signaling pathways, including PKC, ERK1/2 and PI3-K, are involved in this Ang II-induced PlGF upregulation; (4) blockade of PlGF results in the inhibition of Ang II-induced proliferation in these cells, whereas blockade of VEGF leads to inhibition of both basal and Ang II-elicited proliferation. Our observations established a role of PlGF in mediating Ang II-induced proliferation in vascular endothelial cells and vascular smooth muscle cells. In addition, our results suggested that inhibition of PlGF or VEGF might be useful in preventing abnormal VEC or VSMC proliferation evoked by Ang II.
Numerous studies suggest that the renin-angiotensin system (RAS) contributes to the pathogenesis of atherosclerosis [4, 7]. Angiotensin II, the principal effecter of the RAS, is not only a vasoactive hormone, but also a cytokine that regulates cell proliferation, inflammation and fibrosis. Ang II elicits the inflammatory response by stimulating the production of chemokines, cytokines, and adhesion molecules . Recent study demonstrated that Ang II induced vascular endothelial cell proliferation by increasing the expression of the angiogenic factor VEGF . Consistent with previous reports [24, 25, 31], we observed that Ang II increased VEGF and its two receptors expression in our experimental model. Meanwhile, our data provided direct evidence that, in vascular endothelial cell and smooth muscle cells, Ang II, via its AT1 receptor, could up-regulate PlGF expression. These results suggested that Ang II might participate in the regulation of pathological angiogenesis.
Ang II acts through binding to its specific AT1 and AT2 receptors, which are seven transmembrane glycoproteins with 30% sequence similarity. The AT1R is a classical G protein-coupled receptor, whereas AT2R often antagonizes the effects of signaling through the AT1R. Many AT1R-induced growth responses are mediated by transactivation of growth factor receptors. AT1 receptor regulates cell proliferation, cytokines production and some pathological processes, including Ang II-induced hypertension and cardiac hypertrophy. Although AT1 receptor mediates most of the recognized cardiovascular effects of Ang II, the AT2 receptor contributes to the regulation of blood pressure and renal function. Our data demonstrated that, in cultured EA.Hy 926 endothelial cells and HUASMCs, Ang II increased PlGF expression and synthesis via its AT1 receptor.
Several pathways, e.g. PKC , ERK1/2 [7, 26, 33], and PI3K/Akt , involved in AT1R activation. Using specific inhibitors, the present study showed that Ang II activated PKC, ERK1/2 and PI-3K pathways. The activation of all these pathways contributed to PlGF up-regulation in EA.Hy 926 endothelial cells and HUASMCs.
The induction of PlGF gene expression by Ang II may be of considerable clinical significance, especially in vascular inflammation and atherosclerosis. Pro-inflammatory cytokines play a crucial role of in the development of atherosclerosis and plaque instability. Quiescent vascular endothelial cells only release minimal amounts of PlGF. In contrast, activated endothelial cells could produce large amounts of PlGF. Previous studies demonstrated that PlGF activated monocytes and increased the expression of tumor necrosis factor-α(TNF-α), interleukin-1β (IL-1β), and monocyte chemotactic protein-1(MCP-1) in monocytes[36, 37]. Consequently, when stimulated by Ang II, vascular endothelial cells produce PlGF, which activates neutrophils and monocytes, results in their adherence to endothelial cells. This might trigger the pathophysiological changes observed in atherosclerosis. Our results indicated that, the administration of PlGF-neutralizing antibody significantly inhibited the Ang II-dependent proliferation of vascular endothelial cells, suggested that PlGF might be a down-stream angiogenic mediator of RAS. The neutralizing antibodies of PlGF or VEGF are less effective in inhibiting cell proliferation than the small molecule inhibitor of AT1R, since other effects are involved besides VEGF/PlGF production. It has been largely demonstrated that the Ang II-induced VSMC proliferation is mediated by PDGF and Egr-1 [38, 39]. And the AT1R blocker might be useful as an anti-angiogenic agent.
It has been recognized that VSMC proliferation within the vessel wall is an important pathogenic feature in the development of atherosclerosis [40, 41]. Ang II has been implicated to play an important role in this cellular mechanism. When exposed to hypoxia (3% O2), the proliferation and contraction of VSMC were enhanced by PlGF treatment . Furthermore, recent study has shown that PlGF expression in human atherosclerotic carotid plaques is related to inflammation and clinical plaque instability. Present observations showed that Ang II induced PlGF expression in VSMC, suggested a role of PlGF in mediating VSMC proliferation induced by Ang II.
It has been shown that Ang II could stimulate the expression of hypoxia inducible factor-1α (HIF-1α) , and HIF-1α seems to be involved in the enhanced PlGF expression stimulated by hypoxia . The hypoxia-inducible PlGF expression is mediated through NF-κB, metal-regulatory transcription factor-1(MTF-1) and the interaction between them . Ang II-induced PlGF expression might be mediated through the HIF-1α pathways. However, future studies in vascular cells are necessary to determine the role of Ang II receptors and PlGF in atherosclerosis.
In conclusion, PlGF might be one of the downstream effectors up-regulated by Ang II in vascular diseases. Several pathways, such as PKC and ERK 1/2 activation, seem to be involved in the Ang II-induced PlGF expression in vascular cells. PlGF also mediates Ang II-induced cell proliferation in VECs and HUASMCs. Thus, our study provides new insights into PlGF as one of the Ang II-inducible genes. The link of PlGF to Ang II might be a novel molecular mechanism to target cardiovascular diseases.