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Vascular Endothelial Growth Factor Induces Expression of the Antiapoptotic Proteins Bcl-2 and A1 in Vascular Endothelial Cells

1998; Elsevier BV; Volume: 273; Issue: 21 Linguagem: Inglês

10.1074/jbc.273.21.13313

1083-351X

Hans‐Peter Gerber, Vishva M. Dixit, Napoleone Ferrara,

Phagocytosis and Immune Regulation

We examined the role of vascular endothelial growth factor (VEGF) in preventing apoptosis in primary human umbilical vein endothelial (HUVE) cells. VEGF was capable of preventing serum starvation-induced apoptosis at concentrations between 10 and 100 ng/ml. The addition of VEGF to serum-starved HUVE cells led to a 5.2-fold induction of Bcl-2 after 36 h and to a transient, 2.4-fold induction of A1 after a 7-h incubation, as quantitated by real time reverse transcriptase-polymerase chain reaction analysis. Western blot analysis demonstrated a 2–3-fold induction of Bcl-2 protein after 18–36 h of exposure to VEGF and a transient induction of A1 after 7 h of VEGF stimulation. Moreover, overexpression of Bcl-2 by means of transient biolistic transfection experiments of HUVE cells was sufficient to prevent endothelial cells from apoptotic cell death in the absence of VEGF. These findings indicate that Bcl-2 plays an important role in mediating the survival activity of VEGF on endothelial cells. We examined the role of vascular endothelial growth factor (VEGF) in preventing apoptosis in primary human umbilical vein endothelial (HUVE) cells. VEGF was capable of preventing serum starvation-induced apoptosis at concentrations between 10 and 100 ng/ml. The addition of VEGF to serum-starved HUVE cells led to a 5.2-fold induction of Bcl-2 after 36 h and to a transient, 2.4-fold induction of A1 after a 7-h incubation, as quantitated by real time reverse transcriptase-polymerase chain reaction analysis. Western blot analysis demonstrated a 2–3-fold induction of Bcl-2 protein after 18–36 h of exposure to VEGF and a transient induction of A1 after 7 h of VEGF stimulation. Moreover, overexpression of Bcl-2 by means of transient biolistic transfection experiments of HUVE cells was sufficient to prevent endothelial cells from apoptotic cell death in the absence of VEGF. These findings indicate that Bcl-2 plays an important role in mediating the survival activity of VEGF on endothelial cells. Bcl-2 belongs to a growing family of apoptosis regulatory gene products, which may either be death antagonists (Bcl-2, Bcl-XL, Bcl-w, Bfl-1, Brag-1, Mcl-1, and A1) or death agonists (Bax, Bak, Bcl-Xs, Bad, Bid, Bik, and Hrk; for review, see Ref. 1Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1701) Google Scholar). Bcl-2 is an intracellular protein that localizes to mitochondria, endoplasmic reticulum, and the nuclear envelope (2Monaghan P. Robertson D. Amos T.A.S. Dyer M.J.S. Mason D.Y. Greaves M.F. J. Histochem. Cytochem. 1992; 40: 1819-1825Crossref PubMed Scopus (335) Google Scholar, 3Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar) and has been shown to block apoptosis without inducing cellular proliferation (4Hockenbery D. Nunez G. Milliman C. Schreiber R.D. Korsmeyer S.J. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3514) Google Scholar, 5Vaux D.L. Cory S. Adams J.M. Nature. 1988; 335: 440-442Crossref PubMed Scopus (2701) Google Scholar). In vitro, many growth factors and cytokines have been shown to promote survival in different cell lines tested by activation of the Ras–Raf–mitogen-activated protein kinase module (for review, see Refs. 6Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1653) Google Scholar and 7Hemmings B.A. Science. 1997; 275: 628-630Crossref PubMed Scopus (434) Google Scholar). However, the mechanisms by which growth factors control proliferation and regression of endothelial cells remain poorly understood. In human umbilical vein endothelial (HUVE) cells, 1The abbreviations used are: HUVE cells, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; RT-PCR, reverse transcriptase-polymerase chain reaction; GFP, green fluorescent protein.1The abbreviations used are: HUVE cells, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; RT-PCR, reverse transcriptase-polymerase chain reaction; GFP, green fluorescent protein. increased levels of apoptosis and necrosis were observed when cells were incubated with lipopolysaccharide. Vitamins C and E reduced the levels of apoptosis, which was paralleled by an increase in Bcl-2 expression and a decrease in Bax protein levels (8Haendeler J. Zeiher A.M. Dimmeler S. Eur. J. Pharmacol. 1996; 317: 407-411Crossref PubMed Scopus (129) Google Scholar). Upon incubation of HUVE cells with transforming growth factor-β, decreased levels of Bcl-2 correlated with increased levels of apoptotic cell death (9Tsukada T. Eguchi K. Migita K. Kawabe Y. Kawakami A. Matsuoka N. Takashima H. Mizokami A. Nagataki S. Biochem. Biophys. Res. Commun. 1995; 210: 1076-1082Crossref PubMed Scopus (104) Google Scholar). 2-Methoxyestradiol was found to induce apoptosis of cultured bovine pulmonary endothelial cells (10Yue T.L. Wang X. Louden C.S. Gupta S. Pillarisetti K. Gu J.L. Hart T.K. Lysko P.G. Feuerstein G.Z. Mol. Pharmacol. 1997; 51: 951-962Crossref PubMed Scopus (196) Google Scholar). A1, a homolog of the Bcl-2 family of antiapoptotic proteins originally cloned from phorbol ester-stimulated endothelial cells, was shown to be induced by the inflammatory cytokines tumor necrosis factor and interleukin-1 (11Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Infection of microvascular endothelial cells with retroviral constructs encoding A1 led to inhibition of tumor necrosis factor-α-induced cell death in the presence of actinomycin D (11Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Similar findings were reported when Bcl-2 was overexpressed by means of retroviral infection of murine aortic endothelial cells. Enforced expression of Bcl-2 prevented apoptosis of these cells when cultured in fibroblast growth factor-depleted medium (12Kondo S. Yin D. Aoki T. Takahashi J.A. Morimura T. Takeuchi J. Exp. Cell Res. 1994; 213: 428-432Crossref PubMed Scopus (61) Google Scholar). The endothelial cell-specific mitogen vascular endothelial growth factor (VEGF) has been shown to be a key positive regulator of normal and pathological angiogenesis (13Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). A growing body of evidence indicates that VEGF may also act as a survival factor for newly formed blood vessels. In the developing retina, vascular regression in response to hyperoxia has been correlated with inhibition of VEGF release by glial cells (14Alon T. Hemo I. Itin A. Pe'er J. Stone J. Keshet E. Nat. Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1400) Google Scholar). Furthermore, administration of anti-VEGF monoclonal antibodies results in regression of established tumor-associated vasculature in xenograft models (15Yuan F. Chen Y. Dellian M. Safabakhsh N. Ferrara N. Jain R.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14765-14770Crossref PubMed Scopus (597) Google Scholar). More recently, using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, it has been shown that decreased levels of VEGF production lead to detachment of endothelial cells from the walls of preformed vessels in the tumor (16Benjamin L.E. Keshet E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8761-8766Crossref PubMed Scopus (439) Google Scholar). We have found recently that VEGF can counteract endothelial cell apoptosis induced upon serum starvation of HUVE cells in culture (see Fig. 3). 2H. P. Gerber, A. McMurtrey, H. Nguyen, Y. Minhong, V. Dixit, and N. Ferrara, submitted for publication.2H. P. Gerber, A. McMurtrey, H. Nguyen, Y. Minhong, V. Dixit, and N. Ferrara, submitted for publication. This survival activity was critically dependent on the phosphatidylinositol 3-kinase/Akt pathway. In this report, we found increased expression of the antiapoptotic proteins A1 and Bcl-2 but not Bax or Bcl-XL upon challenging primary HUVE cells with VEGF. Thus Bcl-2 and A1 are two novel VEGF target genes. HUVE cells, human microvascular endothelial cells, and CS-C medium were purchased from Cell System (Kirkland, WA). HUVE cells are pooled primary isolates from 300 individual donor umbilical veins. Cells were maintained in CS-C complete medium containing 10% fetal bovine serum and mitogens, according to the recommendations of the supplier. 24 h before initiation of serum starvation, cells were treated with trypsin and plated to a density of 20–25 × 103 cells/cm2 on six-well dishes (reverse transcriptase-polymerase chain reaction (RT-PCR) analysis) or 6-cm dishes (biolistic transfection experiments). Immediately before the experiment, cells were washed twice with phosphate-buffered saline. For serum starvation, CS-C medium without serum and mitogen, complemented with 0.1% bovine serum albumin, was added for the indicated amount of time. After biolistic transfection, cells were allowed to recover for 24 h in medium containing 10% serum. After washing the cells twice with Tris-buffered saline, minimal medium (without mitogen and serum) complemented with 0.1% bovine serum albumin was added, and the levels of apoptosis were analyzed 24 h after induction of serum starvation. HUVE cells were initially expanded for 8–10 days in the presence of completed medium. Routinely, cells between passages 4 and 10 were used in the different experimental procedures. 24 h before serum starvation, cells were split and seeded in complete medium at a density of 200,000 cells/well in six-well plates, following the procedures recommended by the manufacturer. Cells were washed for 5 min before adding Cell System basic medium complemented with 0.1% bovine serum albumin. After incubations of various duration, cells were harvested by the STAT 60 method (TEL-TEST “B”, Inc., Friendswood, TX), and total RNA was prepared according to the manufacturer's recommendations. The RNA was dissolved in 50 μl of H2O, and the concentration was determined by spectrophotometer (A 260/280 nm). To monitor gene expression we used real time RT-PCR analysis. This novel approach has been described previously (18Gerber H.-P. Condorelli F. Park J. Ferrara N. J. Biol. Chem. 1997; 272: 23659-23667Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). Briefly, 100 ng of total RNA was added to a 50-μl RT-PCR (PCR-Access, Promega). The reaction master mix was prepared according to the manufacturer's protocol to give final concentrations of 1 × avian myeloblastosis virus/Tfl reaction buffer, 0.2 mm dNTPs, 1.5 mm MgSO4, 0.1 unit/ml avian myeloblastosis virus reverse transcriptase, 0.1 unit/μl Tfl DNA polymerase, a 250 nm concentration of the primers, and a 200 nm concentration of the corresponding probe. Primers and probes for real time PCR analysis Bcl-2, A1,Bax, and Bad genes were designed by the Primer Express Program according to Heid et al. (19Heid C.A. Stevens J. Livak K.J. Williams P.M. Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (4940) Google Scholar). For sequence information of all oligonucleotides, see TableI. The primers for the humanBcl-2 gene were HUMBCL-2 555.F and HUMBCL-2 639.R, and the probe was HUMBCL-2 598.FP. For A1 analysis, the following primers were used: HUMA1 14.F and HUMA1 131.R; the probe was HUMA1 59.FP. For Bcl-XL analysis we used HSBCLXL 408.F and HSBCLXL 851.R primers, the probe was HSBCLXL 585.FP. For Bax-Aanalysis, we used HUMBAXA 155.F and HUMBAXA 301.R as primers and HUMBAXA 235.FP as probe. Primers and probes were synthesized at Genentech using conventional nucleic acid synthesis chemistry. The β-actin primer and probe (TaqMan β-actin detection reagents) were purchased from Perkin-Elmer.Table ISequences of oligonucleotide primers and real time RT-PCR probesHUMBCL-2 598.FP5′(FAM)-TGT GCG CGC GTA TAA ATT GCC GA-(TAMRA)p3′HUMBCL-2 555.FAAG CGG TCC CGT GGA TAG AHUMBCL-2 639.RTCC GGT ATT CGC AGA AGT CCHSBCLXL 585.FP5′(FAM)-TGC GTG GAA AGC GTA GAC AAG GAG ATG C-(TAMRA)p3′HSBCLXL 408.FGAG GCA GGC GAC GAG TTT GAAHSBCLXL 851.RGGG GTG GGA GGG TAG AGT GGAHUMA1 59.FP5′(FAM)-TGC TCT CCA CCA GGC AGA AGA TGA CA-(TAMRA)p3′HUMA1 14.FCAG CAC ATT GCC TCA ACA GCHUMA1 131.RTGC AGA TAG TCC TGA GCC AGCHUMBAXA 235.FP5′(FAM)-ATG ATT GCC GCC GTG GAC ACA GAC TCC-(TAMRA)p3′HSBAXA 155.FAGG ATG CGT CCA CCA AGA AGHSBAXA 301.RCCA GTT GAA GTT GCC GTC AGA Open table in a new tab RT-PCRs and the resulting relative increase in reporter fluorescent dye emission were monitored in real time by the 7700 sequence detector (Perkin-Elmer). Signals were analyzed by the sequence detector 1.6 program (Perkin-Elmer). Conditions were as follows: one cycle at 48 °C for 45 min; one cycle at 94 °C for 2 min; 40 cycles at 94 °C for 30 s, 60 °C for 1 min, and 68 °C for 2 min. Data were generated as indicated in the legend to Fig.1. Cells cultured in 10-cm dishes were washed twice in Tris-buffered saline, and 0.6 ml of RIPA buffer (1 × phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl fluoride, 30 μl/ml aprotinin, and 1 μm sodium orthovanadate) containing freshly added protease inhibitors was added. Mouse monoclonal antibody directed against human Bcl-2 (Bcl-2(100)) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Affinity-purified goat polyclonal antibody against human A1 (A1(N-20)) was from the same manufacturer. The secondary antibody to Bcl-2(100) was goat anti-mouse Ig (biotin-labeled) from Southern Biotechnologies (Birmingham, AL). The secondary antibody to A1(N-20) was biotinylated anti-goat IgG from Vector Laboratories (Burlingame, CA). Total endothelial cell extract, polyacrylamide gel electrophoresis, blotting, and immunodetection were performed according to the Santa Cruz Biotechnology protocol. 60 μg of whole cell extract was loaded on each lane of a 4–16% polyacrylamide gel. The ECL reaction kit was purchased from Amersham Pharmacia Biotech, and horseradish peroxidase streptavidin from Vector Laboratories was used. Experiments were done using the Bio-Rad 500 optimization kit in the Biolistic PDS-1000/He* Particle Delivery System (Bio-Rad). In a series of pilot experiments using luciferase reporter gene DNA, conditions were established which yield the highest levels of luciferase activity in HUVE cells (data not shown). The settings used are as follows: particle size, 1.0 μm; rupture disc, 1,100 p.s.i.; bombardment chamber vacuum, 25 mmHg; and target shelf position 3 (from the top). Gold particles were prepared according to the manufacturer's recommendations and stored as 50-μl aliquots for several months at −20 °C. Aliquots were coated with DNA immediately before gene transfer following the manufacturer's recommendations. Briefly, for five samples, a 50-μl aliquot of gold particles was Vortex mixed for 3 min, and plasmid DNA (1 mg/ml) was added to a total of 10 μg. Vortexing was continued for 2 min. Thereafter, 100 μl of a 2.5m CaCl2 solution was added to the mix. After 2 min of additional vortexing, 20 μl of a freshly prepared 0.1m spermidine solution was added followed by 2 min of constant vortexing. The mixture was allowed to settle for 1 min before tubes were centrifuged for 5 s in a tabletop microcentrifuge. The supernatant was removed, and the gold pellet was washed first with 140 μl of 70% EtOH followed by 140 μl of 100% EtOH. After removing the supernatant, the gold particles were taken up in 55 μl of 100% EtOH, and 10 μl was used per biolistic transfection. Immediately after bombardment, 5 ml of complemented medium was added. To find putative VEGF target genes mediating the VEGF survival activity, we analyzed total RNA of HUVE cells treated with VEGF by quantitative RT-PCR (TaqMan). We designed primer probe sets for quantitative RT-PCR analysis of genes known to be involved in mediating antiapoptotic activities such as Bcl-2,A1, Bcl-XL, or apoptosis-inducing genes such asBax (for sequence information, see Table I). In the primary human endothelial cell types tested, umbilical vein and lung microvascular endothelial cells, we have observed induction ofBcl-2 as early as 7 h after the addition of VEGF (Fig.1 and data not shown). The levels of Bcl-2 in HUVE cells increased over time and reached 5.2-fold after 33 h of incubation with VEGF. No significant induction of A1 orBcl-2 message in response to VEGF stimulation was observed when endothelial cells were grown in the presence of 10% fetal calf serum (data not shown). Interestingly, the A1 message was transiently increased 2.4-fold after 7 h of VEGF addition and decreased after prolonged incubation time. In a previous report, Northern blots of HUVE cells did not reveal an increase in the A1 message after incubation with a mixture of VEGF and basic fibroblast growth factor for 3 h (20Karsan A. Yee E. Kaushansky K. Harlan J.M. Blood. 1996; 87: 3089-3096Crossref PubMed Google Scholar). The failure to observe increased levels for A1 message in response to VEGF may be caused by the decreased sensitivity of the Northern blot analysis, or because the cells were exposed to a mixture of basic fibroblast growth factor (4 ng/ml) and VEGF (10 ng/ml), or because of the different incubation length with VEGF (3 hversus 7 h). When we analyzed total RNA from HUVE cells for Bcl-XL andBax expression, we could not detect any significant change in their expression levels in response to VEGF under serum starvation conditions (data not shown). To verify if increased expression of A1 and Bcl-2in response to VEGF stimulation is also reflected in increased protein levels, we performed Western blot experiments with whole cell extracts of HUVE cells cultured under the same conditions as those employed for the RT-PCR analysis. We found a direct correlation between the mRNA and the protein levels (Fig. 2) of Bcl-2 and A1 in human endothelial cells in response to VEGF stimulation. These findings suggest that VEGF exerts its up-regulatory activity on the Bcl-2 and A1 genes primarily at the transcriptional level. We found decreased levels of Bcl-2 and A1 proteins in endothelial cells cultured in the absence of VEGF, suggesting that these genes may be involved in mediating survival activity of VEGF on endothelial cells (Fig.3).2 It has been shown previously that A1 levels are induced upon incubation of human endothelial cells to inflammatory mediators such as tumor necrosis factor-α and interleukin-1β, and a possible role for theA1 gene in inflammation was suggested. By taking advantage of the biolistic transfection technique to transiently transfect primary human endothelial cells, we wanted to test whether Bcl-2 was sufficient to mediate survival in our assay conditions. Transfected endothelial cells were identified by cotransfection of an expression vector for green fluorescent protein (pEGFP, CLONTECH, Palo Alto, CA) 24 h after the beginning of serum starvation and identified by fluorescence microscopy. To assess the effects of Bcl-2, we scored transfected cells in a blinded manner as healthy or apoptotic by morphology (21Li Y. Horwitz M.S. BioTechniques. 1997; 23: 1026-1030Crossref PubMed Scopus (17) Google Scholar). Healthy endothelial cells are flat and well attached to the plate. Apoptotic endothelial cells are rounded, and some are fragmenting with the small cytoplasmic blebs that are characteristic of apoptosis. When we stained the cells with the DNA dye bisbenzimide (Hoechst 33258), apoptotic endothelial cells showed pronounced nuclear condensations. As shown in Fig. 3, Bcl-2 was sufficient to inhibit endothelial cell apoptosis in the absence of VEGF. We have observed lower levels of apoptosis when cells were transfected with Bcl-2 compared with untransfected cells grown in the presence of VEGF (100 ng/ml) or 10% fetal calf serum. These findings suggest that the levels of Bcl-2 are critical for endothelial cell survival when cultured under serum starvation conditions. VEGF exerts its biological effects by binding to its respective transmembrane receptors Flt-1 and Flk-1/KDR, which are expressed mainly on endothelial cells. Stimulation of endothelial cells with VEGF and subsequent immunoblot analysis demonstrated that VEGF induces phosphorylation of phosphatidylinositol 3-kinase, Ras GTPase-activating protein, p190-rhoGAP, p62, PLC-y, the oncogenic adaptor protein NcK, p125 focal adhesion kinase (p125 FAK), paxilin, and several others (22Terman B.I. Carrion M.E. Kovacs E. Rasmussen B.A. Eddy R.L. Shows T.B. Oncogene. 1991; 6: 1677-1683PubMed Google Scholar, 23Guo D. Jia Q. Song H.-Y. Warren R.S. Donner D.B. J. Biol. Chem. 1995; 270: 6729-6733Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 24Settleman J. Narasimhan V. Foster L.C. Weinberg R.A. Cell. 1992; 69: 539-549Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 25Ellis C. Morgan M. McCormick F. Pawson T. 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The signal transduction pathways involved in mediating the various biologic functions of VEGF on endothelial cells such as migration, proliferation, or survival remain to be characterized. Both VEGF receptors, Flk-1/KDR and Flt-1, were found previously to be expressed on HUVE cells by cell binding and cross-linking studies with VEGF or the Flt-1-specific ligand PlGF (32Park J.E. Chen H.H. Winer J. Houck K.A. Ferrara N. J. Biol. Chem. 1994; 269: 25646-25654Abstract Full Text PDF PubMed Google Scholar) or by RT-PCR analysis (18Gerber H.-P. Condorelli F. Park J. Ferrara N. J. Biol. Chem. 1997; 272: 23659-23667Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). The HUVE cells used in our experiments were pooled primary isolates from 300 individual donor umbilical veins and thus are likely to represent an average population of endothelial cells. Upon incubation of primary human endothelial cells with VEGF, we found increased expression of two antiapoptotic proteins, Bcl-2 and A1. The increase in Bcl-2 message in response to VEGF can reflect the sum of an increase in the transcriptional level as well as an increase in RNA stability. The overall increase of the Bcl-2 protein observed in these cells may represent higher levels of Bcl-2 mRNA as well as the result of an increase in the protein stability. Recently, several reports identified altered Bcl-2 levels in endothelial cells engaged in physiologic as well as pathologic angiogenesis. In human corpora lutea, Bcl-2 was found to be expressed in the vascular endothelium of some luteal arterioles and venules (33Rodger F.E. Fraser H.M. Duncan W.C. Illingworth P.J. Hum. Reprod. 1995; 10: 1566-1570Crossref PubMed Scopus (50) Google Scholar). During normal luteal phase or after treatment with chorionic gonadotrophin, the corpus luteum undergoes a rapid and massive increase in size and vasculature. This process was completely inhibited by administration of a truncated soluble Flt-1 receptor in a rat model of hormonally induced ovulation (34Ferrara N. Chen H. Davis-Smyth T. Gerber H.P. Nguyen T.H. Peers D. Chisholm V. Hillan K.J. Schwall R. Nat. Med. 1998; 4: 336-340Crossref PubMed Scopus (580) Google Scholar). These findings indirectly suggested a correlation between the levels of VEGF and Bcl-2 in endothelial cells. However, the numbers of blood vessels exhibiting Bcl-2 staining showed little variation throughout the luteal phase implying that other mechanisms are involved in luteal maintenance (33Rodger F.E. Fraser H.M. Duncan W.C. Illingworth P.J. Hum. Reprod. 1995; 10: 1566-1570Crossref PubMed Scopus (50) Google Scholar). An increase in the levels of Bcl-2 expression within the vascular endothelial spindle-shaped cells in Kaposi sarcoma lesions in humans was observed, indicating that up-regulation of Bcl-2 may be important in the pathogenesis of both classical and AIDS-associated Kaposi sarcoma (35Morris C.B. Gendelman R. Marrogi A.J. Lu M. Lockyer J.M. Alperin-Lea W. Ensoli B. Am. J. Pathol. 1996; 148: 1055-1063PubMed Google Scholar). In vitro, overexpression of Bcl-2 in a bovine endothelial cell line blocked the effects of oxidative stress such as lipid peroxidation. In these cells, Bcl-2 also blocked hyperglycemia-induced formation of advanced glycation end products, which are thought to cause tissue damage observed in diabetes-associated hyperglycemia (36Giardino I. Edelstein D. Brownlee M. J. Clin. Invest. 1996; 97: 1422-1428Crossref PubMed Scopus (229) Google Scholar). The identification of two novel VEGF target genes, Bcl-2 andA1, appears timely, given the emerging concepts that VEGF is a survival factor for endothelial cells2 and that VEGF withdrawal may result in regression of the vasculature in several cases (14Alon T. Hemo I. Itin A. Pe'er J. Stone J. Keshet E. Nat. Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1400) Google Scholar, 15Yuan F. Chen Y. Dellian M. Safabakhsh N. Ferrara N. Jain R.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14765-14770Crossref PubMed Scopus (597) Google Scholar, 16Benjamin L.E. Keshet E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8761-8766Crossref PubMed Scopus (439) Google Scholar). The role of Bcl-2 in endothelial cells during pathologic and physiologic neoangiogenesis or during vessel regression remains to be analyzed. We thank the FACS laboratory at Genentech for excellent support; and M. Vasser, P. Ng, and P. Jhurani for oligonucleotide synthesis. We thank David Wood and Charles Hoffman for excellent graphic artwork.