AIDSCKS cells and primary tumor tissues also expressed high levels of Flt-1 and KDR, the receptors for VEGF, while the normal skin of the same patients did not show any expression. production and inhibit KS cell growth in a dose-dependent manner. Furthermore, growth of KS cells in nude mice was specifically inhibited by VEGF antisense oligonucleotides. These results show that VEGF is an autocrine growth factor for AIDSCKS cells. To our knowledge, this is the first report that shows that VEGF acts as a growth stimulator in a human tumor. Inhibitors of VEGF or its cognate receptors may thus be candidates for therapeutic intervention. Kaposi sarcoma (KS) is the most common tumor associated with HIV-1 contamination (1C4). KS develops in 30% of AIDS cases. The tumor frequently involves the skin and mucous membranes and can lead to tumor-associated edema and ulceration. Visceral involvement in nearly one-third of KS patients can lead to death (5, 6). Two principal features of AIDSCKS tumors include (and Studies in Immunodeficient Mice. KSY1 (1 107) cells were inoculated subcutaneously in the lower back of 5-week-old BALB/c Nu+/NU+ athymic mice. Beginning on day 2, 25 g/g body weight of AS1, AS-3, or scrambled VEGF oligonucleotides were injected i.p. daily for 5 consecutive days. The mice were examined daily, and on day 14 they were sacrificed to quantitate the tumor size. RESULTS AIDSCKS Cells Express High Levels of VEGF. We examined the expression of VEGF-specific mRNA in several AIDSCKS cell lines (KSC10, KSC29, KSC13, KSC59, and KSY1). Fig. ?Fig.11shows that a single or two closely related VEGF mRNA transcripts were expressed at high levels in all AIDSCKS cell lines. HUVEC and AoSM cells also express VEGF mRNA but at lower levels (Fig. ?(Fig.11(36) have also recently demonstrated the expression of KDR in archived tissues of AIDSCKS biopsies. Open in a separate windows Physique 2 Expression of Flt-1 and KDR mRNA in KSY1, HUVEC (HUVE), normal skin, and KS tumor tissue from an HIV+ patient, T1 (fibroblast), 23-1 (B lymphoma), and HUT-78 (T cell lymphoma). Equal amounts of RNA were reverse transcribed to generate cDNA. (and and 0.05, test). Furthermore, mice treated with either oligonucleotide did not exhibit clinical evidence of toxicity, such as a change in food intake, activity, and body weight. Open in a separate window Physique 5 Effect on tumor growth of VEGF antisense oligonucleotides in nude mice. KSY1 cells (107) were inoculated subcutaneously in the lower back of BALB/c Nu+/NU+ athymic mice. AS-1, AS-3, and scrambled (S) VEGF oligonucleotides and diluent (PBS) were injected i.p. daily for 5 days (day 2C6). Mice were sacrificed on day 14 and tumor size was measured. Data represent the mean SD of 10 mice in each group. DISCUSSION This report shows that the angiogenic factor VEGF is an autocrine growth factor for KS. To our knowledge, this is the first demonstration that VEGF is usually a growth factor for a human tumor. Because VEGF is able to induce angiogenesis and capillary permeability (17), both of which are prominent clinical features of KS, we studied its expression in KS tissues and cell lines. High VEGF expression was indeed observed in all KS cell lines and isolates examined. KS cell lines and isolates produce abundant amounts of VEGF protein compared with HUVEC and AoSM cells. We found the expression of VEGF in KS cell lines to be 15-fold higher than in endothelial cells. The predominant form of VEGF mRNA in KS cells was found to be the 3.9-kb transcript that encodes the 165-aa form (23). This form retains the heparin-binding domain name, which results in the secreted protein being retained in the interstitial fluid. The VEGF produced in KS tumors would therefore act locally in enhancing vascular proliferation and permeability. Not Rabbit polyclonal to pdk1 only.?(Fig.11(36) have also recently demonstrated the expression of KDR in archived tissues of AIDSCKS biopsies. of the same patients did not show any expression. We further demonstrate that VEGF antisense oligonucleotides AS-1 and AS-3 specifically block VEGF mRNA and protein production and inhibit KS cell growth in a dose-dependent manner. Furthermore, growth of KS cells in nude mice was specifically inhibited by VEGF antisense oligonucleotides. These results show that VEGF is an autocrine growth factor for AIDSCKS cells. To our knowledge, this is the first report that shows that VEGF acts as a growth stimulator in a human tumor. Inhibitors of VEGF or its cognate receptors may thus be candidates for therapeutic intervention. Kaposi sarcoma (KS) is the most common tumor associated with HIV-1 contamination (1C4). KS develops in 30% of AIDS cases. The tumor frequently involves the skin and mucous membranes and can lead to tumor-associated edema and ulceration. Visceral involvement in nearly one-third of KS patients can lead to death (5, 6). Two principal features of AIDSCKS tumors include (and Studies in Immunodeficient Mice. KSY1 (1 107) cells were Fmoc-PEA inoculated subcutaneously in the lower back of 5-week-old BALB/c Nu+/NU+ athymic mice. Beginning on day 2, 25 g/g body weight of AS1, AS-3, or scrambled VEGF oligonucleotides were injected i.p. daily for 5 consecutive days. The mice were examined daily, and on day 14 they were sacrificed to quantitate the tumor size. RESULTS AIDSCKS Cells Express High Levels of VEGF. We examined the expression of VEGF-specific mRNA in several AIDSCKS cell lines (KSC10, KSC29, KSC13, KSC59, and KSY1). Fig. ?Fig.11shows that a single or two closely related VEGF mRNA transcripts were expressed at high levels in all AIDSCKS cell lines. HUVEC and AoSM cells also express VEGF mRNA but at lower levels (Fig. ?(Fig.11(36) have also recently demonstrated the expression of KDR in archived tissues of AIDSCKS biopsies. Open in a separate window Figure 2 Expression of Flt-1 and KDR mRNA in KSY1, HUVEC (HUVE), normal skin, and KS tumor tissue from an HIV+ patient, T1 (fibroblast), 23-1 (B lymphoma), and HUT-78 (T cell lymphoma). Equal amounts of RNA were reverse transcribed to generate cDNA. (and and 0.05, test). Furthermore, mice treated with either oligonucleotide did not exhibit clinical evidence of toxicity, such as a change in food intake, activity, and body weight. Fmoc-PEA Open in a separate window Figure 5 Effect on tumor growth of VEGF antisense oligonucleotides in nude mice. KSY1 cells (107) were inoculated subcutaneously in the lower back of BALB/c Nu+/NU+ athymic mice. AS-1, AS-3, Fmoc-PEA and scrambled (S) VEGF oligonucleotides and diluent (PBS) were injected i.p. daily for 5 days (day 2C6). Mice were sacrificed on day 14 and tumor size was measured. Data represent the mean SD of 10 mice in each group. DISCUSSION This report shows that the angiogenic factor VEGF is an autocrine growth factor for KS. To our knowledge, this is the first demonstration that VEGF is a growth factor for a human tumor. Because VEGF is able to induce angiogenesis and capillary permeability (17), both of which are prominent clinical features of KS, we studied its expression in KS tissues and cell lines. High VEGF expression was indeed observed in all KS cell lines and isolates examined. KS cell lines and isolates produce abundant amounts of VEGF protein compared with HUVEC and AoSM cells. We found the expression of VEGF in KS cell lines to be 15-fold higher than in endothelial cells. The predominant form of VEGF mRNA in KS cells was found to be the 3.9-kb transcript that encodes the 165-aa form (23). This form retains the heparin-binding domain, which results in the secreted protein being retained in the interstitial fluid. The VEGF produced in KS tumors would therefore act locally in enhancing vascular proliferation and permeability. Not only did we show that KS cell lines, isolates, and biopsies produced high amounts of VEGF, but we also showed that VEGF was necessary for optimal cell growth. By blocking the production of VEGF protein using specific antisense oligonuceotides to VEGF mRNA, we were able to demonstrate a marked decrease in cell growth. We examined antisense oligonucleotides to several different regions of the coding region of VEGF for their effect on KS cell growth. Two oligonucleotides (AS-1 and AS-3) were highly effective in inhibiting proliferation of AIDSCKS cells. The specificity of these oligonucleotides was further confirmed by the fact that VEGF mRNA expression was decreased only in cells treated by low concentrations of antisense but not by scrambled oligonucleotides. Similarly, VEGF protein production declined precipitously in response to AS-1 and AS-3 but not to scrambled oligonucleotides. Furthermore, the addition of exogenous rhVEGF completely abrogated the inhibitory effects of antisense oligonucleotides on cell.