Angiogenesis and vascular cell proliferation are pivotal in physiological and pathological processes including atherogenesis, restenosis, wound healing, and cancer development. Here we show that mammalian target of rapamycin (mTOR) signaling plays a key role in hypoxia-triggered smooth muscle and endothelial proliferation and angiogenesis in vitro. Hypoxia significantly increased DNA synthesis and proliferative responses to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) in rat and human smooth muscle and endothelial cells. In an in vitro 3-dimensional model of angiogenesis, hypoxia increased PDGF- and FGF-stimulated sprout formation from rat and mouse aortas. Hypoxia did not modulate PDGF receptor mRNA, protein, or phosphorylation. PI3K activity was essential for cell proliferation under normoxic and hypoxic conditions. Activities of PI3K-downstream target PKB under hypoxia and normoxia were comparable. However, mTOR inhibition by rapamycin specifically abrogated hypoxia-mediated amplification of proliferation and angiogenesis, but was without effect on proliferation under normoxia. Accordingly, hypoxia-mediated amplification of proliferation was further augmented in mTOR-overexpressing endothelial cells. Thus, signaling via mTOR may represent a novel mechanism whereby hypoxia augments mitogen-stimulated vascular cell proliferation and angiogenesis.

Angiogenesis is crucial for many biological and pathological processes including the ovarian cycle and tumor growth. To identify molecules relevant for angiogenesis, we performed mRNA fingerprinting and subsequent Northern blot analysis using bovine cord-forming vs. monolayer-forming endothelial cells (EC) in vitro and staged bovine corpora lutea in vivo. We detected the receptor for activated C kinase 1 (RACK1), the specific receptor for activated protein kinase C beta (PKC beta), to be up-regulated in bovine cord-forming EC in vitro and in angiogenically active stages of bovine corpora lutea in vivo. Thereafter we established and determined the complete bovine RACK1 cDNA sequence. RACK1 was massively induced in subconfluent vs. contact-inhibited bovine EC, during angiogenesis in vitro, active phases of the murine ovarian cycle, human tumor angiogenesis, and in cancer cells in vivo as assessed by quantitative PCR and in situ hybridization. RACK1 transcripts were localized to proliferating EC in vitro and the endothelium of tumor neovascularizations in vivo by in situ hybridization. PKC beta plays an important role in angiogenesis and cancer growth. Our data suggest that downstream signaling of PKC beta in angiogenically active vs. inactive tissues and endothelium is affected by the availability of RACK1.

Transplantation of pancreatic islets reconstitutes glucose homeostasis in diabetes mellitus. Before transplantation, islets are disrupted from the surrounding blood vessels by the isolation procedure, with the grafted tissue being subject to ischemic damage. The survival of transplanted islets is assumed to depend on effective revascularization. Perfusion studies suggest that newly formed microvessels supplying the graft with nutrients are exclusively rebuilt by the host. It is generally not known whether isolated islets contain endothelial cells (EC), which potentially participate in the revascularization process. Therefore, we tried to detect immature EC in isolated islets by transformation with polyoma middle T antigen. Endothelioma cells were generated, implicating the presence of de-differentiated EC within isolated islets. When embedded in a fibrin gel, the islets developed cellular cords consisting of EC, whereas FGF-2 and VEGF stimulated the formation of cord-like structures. Furthermore, we studied the presence of donor EC in islet grafts by using transgenic mice with an EC lineage-specific promotor-LacZ reporter construct (Tie-2LacZ). Following islet transplantation, Tie-2LacZ-positive EC of both donor and recipient were identified in the vicinity of or within the graft up to 3 wk after transplantation. In conclusion, EC and/or their progenitors with angiogenic capacity reside within isolated islets of different species, and their proliferative potential can be stimulated by various inducers. These graft-related endothelia persist after islet transplantation and are integrated within newly formed microvessels.

Arterial Hypertension (AH) is characterized by reduced nitric oxide (NO) biosynthesis, activation of the Renin-Angiotensin-Aldosteron-System (RAAS), vasoconstriction, and microvascular rarefaction. The latter contributes to target organ damage, especially in left ventricular hypertrophy, and may partially be due to impaired angiogenesis. Angiogenesis, the formation of new microvessels and microvascular networks from existing ones, is a highly regulated process that arises in response to hypoxia and other stimuli and that relieves tissue ischemia. In AH, angiogenesis seems impaired. However, blood pressure alone does not affect angiogenesis, and microvascular rarefaction is present in normotensive persons with a family history for AH. Normal or increased NO in several processes and diseases enables or enhances angiogenesis (e.g. in portal hypertension) and reduced NO biosynthesis (for example, in a rat model of AH, in other disease models in vivo, and in endothelial NO Synthase knock out mice) impairs angiogenesis. Angiogenic growth factors such as Vascular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor (FGF) induce NO and require NO to elicit an effect. Effector molecules and corresponding receptors of the RAAS either induce (Bradykinin, Angiotensin II) or perhaps inhibit angiogenesis. The pattern of Bradykinin- and Angiotensin II-receptor expression and the capacity to normalize NO biosynthesis may determine whether ACE-inhibitors, Angiotensin II-receptor antagonists and other substances affect angiogenesis. Reconstitution of a normally vascularized tissue by reversal of impaired angiogenesis with drugs such as ACE inhibitors and AT1 receptor antagonists may contribute to successful treatment of hypertension-associated target organ damage, e.g. left ventricular hypertrophy.

Arterial hypertension (AH) is characterized by reduced nitric oxide (NO) biosynthesis, vasoconstriction, and reduced microvascular density. In this study we asked whether AH also reduces the number of microvessels by impairing angiogenesis. AH was induced in Dahl salt-sensitive rats (DSS) with a salt diet and in Wistar-Kyoto rats by inhibiting NO formation with Nomega-nitro-L-arginine (NNA). Three weeks after induction of AH, two wound chambers containing collagen I (Vitrogen) were sutured into the mesenteric cavity of each animal. After additional 14 days, wound chamber neovascularization and the extent of vascularized connective tissue ingrowth were quantified. In NNA-induced AH, the number of newly formed vessels and the ingrowth of vascularized connective tissue into the wound chamber decreased as compared to controls. However, the number of newly formed vessels and the ingrowth of vascularized connective tissue did not change with increasing blood pressure in salt-fed DSS rats as compared to those fed a normal diet. Inhibition of NO biosynthesis, but not necessarily elevating blood pressure, reduces angiogenesis. Microvascular rarefaction in AH may be partially due to reduced angiogenesis because of impaired NO biosynthesis.