Vascular Endothelial Growth Factor (VEGF)-Mediated Angiogenesis Is Associated with Enhanced Endothelial Cell Survival and Induction of Bcl-2 Expression

Abstract

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor that is potently angiogenic in vivo. We report here studies that suggest that VEGF potentiates angiogenesis in vivo and prolongs the survival of human dermal microvascular endothelial cells (HDMECs) in vitro by inducing expression of the anti-apoptotic protein Bcl-2. Growth-factor-enriched and serum-deficient cultures of HDMECs grown on collagen type I gels with VEGF exhibited a 4-fold and a 1.6-fold reduction, respectively, in the proportion of apoptotic cells. Enhanced HDMEC survival was associated with a dose-dependent increase in Bcl-2 expression and a decrease in the expression of the processed forms of the cysteine protease caspase-3. Cultures of HDMECs transduced with and overexpressing Bcl-2 and deprived of growth factors showed enhanced protection from apoptosis and exhibited a twofold increase in cell number and a fourfold increase in the number of capillary-like sprouts. HDMECs overexpressing Bcl-2 when incorporated into polylactic acid sponges and implanted into SCID mice exhibited a sustained fivefold increase in the number of microvessels and a fourfold decrease in the number of apoptotic cells when examined 7 and 14 days later. These results suggest that the angiogenic activity attributed to VEGF may be due in part to its ability to enhance endothelial cell survival by inducing expression of Bcl-2.

Ablation of microvessels in vivo upon dimerization of iCaspase-9

“…Furthermore, activation of iCaspase-9 was sufficient to induce apoptosis of endothelial cells in vivo resulting in ablation of human microvessels in immunodeficient mice. These results are consistent with previous observations that showed that factors such as VEGF promote endothelial cell survival by acting on various molecules including Bcl-2 and Akt, [2][3][4] that are known to function upstream of caspase-9 in the apoptosis pathway. 24 Most anti-angiogenic strategies currently in clinical trials are based on the delivery of antibodies or drugs that inhibit endothelial cell-specific survival signals and promote apoptosis by a default mechanism.…”

“…We therefore tested if apoptosis and caspase activation induced by iCaspase-9 could be affected by VEGF and bFGF, two potent antiapoptotic factors for endothelial cells in newly formed vessels. [3][4][5][6] In these experiments, we pre-incubated HDMEC-iCasp-9 with either VEGF or bFGF for 24 h, then added AP20187 and fresh VEGF or bFGF and measured the percentage of apoptotic cells after incubation with the dimerizer drug. Treatment of HDMEC-iCasp-9 with VEGF or bFGF did not have a significant effect on the levels of apoptosis triggered by induced dimerization of caspase-9 ( Figure 4a).…”

“…We have previously shown that HDMECs implanted subcutaneously in severe combined immunodeficient (SCID) mice organize into functional human microvessels that anastomose with the host vasculature and transport mouse blood cells. 3,9,25 After the establishment of human microvessels, we injected AP20187 intraperitoneally and evaluated the microvessel density in the implants. By 24 h, visual inspection revealed that implants containing HDMECs transduced with control virus were pink-red, whereas those containing HDMEC-iCasp-9 were pale (Figure 5a), suggesting that caspase-9 dimerization induces diminution of blood flow to the implants.…”

“…3 Briefly, a 2-kb cassette containing HAtagged human F v 2-caspase-9, 21 now called iCaspase-9, was inserted into the HpaI cloning site of the retroviral vector LXSN (gift from AD Miller, Fred Hutchinson Cancer Research Center, Seattle, WA, USA). The iCaspase-9 construct, reverse orientation control, or vector alone was transfected into PA317 amphotropic packaging cells with Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN, USA) to generate recombinant retroviruses.…”

“…VEGF binding to its endothelial cellspecific receptor Flk-1/KDR, and engagement of the PI3 kinase-Akt and Bcl-2 signaling pathways, has been shown to protect endothelial cells against various apoptotic stimuli including growth factor deprivation. [2][3][4] The pro-angiogenic molecules angiopoietin-1, bFGF, and ␣ V ␤ 3 also mediate signaling cascades that result in enhanced survival of endothelial cells. [5][6][7] VEGF and the pro-angiogenic factors described above appear to function primarily at the level of newly formed vessels where they promote survival by repressing endothelial cell apoptosis.…”