Sulfated Polymannuroguluronate, A Novel Anti-AIDS Drug Candidate, Inhibits HIV-1 Tat-induced Angiogenesis In Kaposi's Sarcoma Cells

Kaposi's sarcoma (KS), an angioproliferation disease, is a form of malignancy which occurs in elderly or immunosuppressed patients, and is often associated with AIDS. Characteristic histological features of KS include the proliferation of spindle-shaped cells (KS cells, KCS) of vascular origin considered to be the tumor cells of KS, and of normal endothelial cells (EC) forming blood vessels (angiogenesis), inflammatory cell infiltration, and edema. KS is much more frequent and aggressive in the setting of human immunodeficiency virus type 1 (HIV-1) infection, suggesting that HIV itself or molecules produced during HIV infection could play a role in KS development and progression.Tat (the HIV-1 transactivator of transcription) is a small regulatory polypeptide of 86–102 amino acids released by HIV-1-infected cells, which recently has been pointed out as a key progression factor of KS due to its involvement in all the biological steps of angiogenesis and progression. In addition to the transactivation of HIV-1 gene expression, Tat can modulate the expression of many cellular genes including those for cytokines, adhesion molecules, major histocompatibility complex class I proteins and oncogenes, as well as cellular functions such as cell survival, growth and angiogenesis. In its extracellular form, Tat stimulates proliferation, adhesion, migration and invasion of KSC and normal EC activated by inflammatory cytokines, and promotes the tube formation in vitro and angiogenesis in vivo as well. Therefore, the inhibition of Tat production or prevention of its activity could be a useful way to inhibit the development and progression of KS and other tumors which are a frequent cause of death in AIDS patients.Glycosaminoglycans (GAGs), such as heparin, have been critically identified as pathological chaperons in Tat-mediated biological response in targeted cells. And heparan sulfate proteoglycans (HSPGs) have been shown to act as Tat receptors. Conversely, free heparin inhibits the uptake of intracellular Tat and affects the cell surface interaction mediated by Tat. Growing evidence shows that heparin and lots of heparin analog represent potent extracellular Tat antagonists of possible therapeutic value.Sulfated polymannuroguluronate (SPMG), a new heparin-like sulfated polysaccharide extracted from brown alga, is rich in 1, 4-linkedβ-D-mannuronate, with an average of 1.5 sulfates and 1.0 carboxyl groups per sugar residue and an average molecular weight of 8000Da. SPMG has entered Phase II clinical trial in China, making it the first marine sulfated polysaccharide with the potential of becoming an anti-AIDS drug. Our previous in vitro and in vivo studies demonstrated that SPMG inhibits HIV replication and may exert this effect by interfering with HIV entry into host T lymphocytes. We further showed that SPMG has a high affinity for interaction with Tat (KD=8.69×10-10 M), suggesting that SPMG might exhibit its anti-angiogenic effects via targeting Tat.Here, in the present study, we examined the effects of SPMG on Tat-induced angiogenesis and its possible mechanism.First of all, recombinant GST and GST-Tat were expressed in E. coli as GST fusion proteins. GST-chimeric proteins were then purified from the cell extracts by glutathione-agarose affinity chromatography and checked by SDS-PAGE and Coomassie blue staining. And, the bioactivity of recombinant proteins was evaluated.Our results showed that the GST moiety does not affect the bioactivity of Tat and GST-Tat can substitute for Tat in our subsequent experiments.We preferentially selected SLK cells as a model due to the fact that they are derived from a Kaposi's sarcoma tumor and in particular, they are of EC origin and capable of keeping the major characteristics of EC.1. SPMG inhibits Tat-stimulated SLK proliferation, adhesion, migration, and invasionTat exerts an essential role in KS cell proliferation. In our study, Tat significantly stimulated SLK proliferation, while SPMG dose-dependently blocked SLK cell proliferation stimulated by Tat. 25μg/ml of SPMG markedly inhibited the proliferation of SLK with the percentage inhibition 56.0%, while the percentages were as high as 78.6% and 91.6% at doses 50 and 100μg/ml, respectively Given that Tat is also capable of inducing the adhesion of KS cells, we thus evaluated the effect of SPMG on cell adhesion. We found that Tat induced the adhesion of SLK cells in a dose-dependent manner, while SPMG at the concentrations as low as 12.5μg/ml significantly and dose-dependently blocked Tat-induced SLK adhesion .As HIV-1 Tat has been consistently correlated with the metastatic potential of AIDS-KS, the migration and invasive activities of SLK cells were assessed in Transwell chambers in the presence and absence of Matrigel coating, respectively. Our dada showed that the SLK cells abundantly migrated or invaded to the lower chamber upon the stimulation with Tat. However, SPMG markedly inhibited both the migration of SLK and the invasion induced by Tat. The percentage inhibitions were strong (>50%) and significant (P< 0.001)2. SPMG inhibits Tat-stimulated tube formation in vitro and angiogenesis in vivoSLK cells form capillary-like structures when cultured on polymerized Matrigel at 37℃. Tat increases the formation of this anastomosed cellular network as compared with untreated cells. Treatment with SPMG suppressed the effect of Tat significantly, and the inhibitory effects were obvious and potent, with the inhibition rate almost approaching to 50%.To determine whether these in vitro activities were applicable in vivo, we employed the Matrigel plug model. Matrigel suspensions containing GST-Tat (1μg/ml) in the presence or absence of SPMG (100μg/ml) were injected s.c. into mice. After 7-10 days, the plugs were removed and assessed by HE staining and CD31 immunostaining. The presence of Tat resulted in a strong angiogenic response with formation of new vessels that grew into the Matrigel within 7 days. SPMG showed noticeable inhibitory effect, whereas no reaction is induced by Matrigel with buffer alone. Quantification of the extent of angiogenesis by immunofluorescence staining of endothelial marker CD31 showed that SPMG treatment significantly reduced the neovascularization induced by Tat. All these findings indicated that SPMG is capable of inhibiting angiogenesis induced by Tat in Matrigel plug in vivo.3. Mechanism elucidation about the the inhibitory effect of SPMG on Tat-induced angiogenesisWe found that SPMG inhibited Tat-induced angiogenesis of SLK cells including suppression of the proliferation, migration and tube formation of SLK cells in a dose-dependent manner, functioning as a potent Tat antagonist.It is noteworthy that Tat binds and phosphorylates VEGF receptor 2 (KDR/ Flk-1) which mediates most of the VEGF angiogenic effects in vascular endothelial cells and KS cells. We therefore investigated whether SPMG blocks Tat-mediated KDR activation and thus its downstream molecules including extracellular-signal regulated kinase(ERK1/2) and c-jun amino-terminal kinase (JNK) in SLK cells.Total KDR in cell lysate was immunoprecipitated (IP) with anti-KDR antibody and phospho-KDR was immunoblotted with anti-phosphorylated tyrosine antibody.The activation of ERK1/2 and JNK was analyzed by western blot analysis using anti-phosphoERK and anti-phosphoJNK antibodies. Our data showed that Tat resulted in an increase in tyrosine phosphorylation of the KDR, ERK1/2 and JNK. SPMG at concentrations of 50 and 100μg/ml apparently blocked the phosphorylation of KDR, and subsequent the phosphorylation of ERK1/2 and JNK respectively.It is known that Tat can enhance angiogenesis by mimicking the effects of ECM proteins (FN and VN) on cell migration, invasion and adhesion through binding toα5β1 andαvβ3 integrins via its RGD domain. For this reason, we investigated whether SPMG is capable of interfering the interaction of Tat with integrinβ1. SLK cells were treated with GST-Tat in the absence or presence of SPMG at different concentrations. The interaction between Tat with integrinβ1 was observed by IP. Our studies revealed that SPMG interfered the interaction of Tat with integrinβ1.Extracellular Tat have been shown to induce the phosphorylation of tyrosine kinases localized at cellular focal adhesion plaques, which is activated by integrin triggering and has a major role in cellular adhesion and migration. We therefore investigated whether SPMG exert its inhibition effects on the FAK and Paxillin activation induced by Tat. It was demonstrated that treatment of SLK with GST-Tat resulted in an extensive phosphorylation of FAK and Paxillin, when compared with the control. SPMG apparently decreased the degree of phosphorylation of FAK and Paxillin, respectively.Besides the Tat-mediated events mentioned above, Tat can also compete with bFGF or VEGF and release bound bFGF and VEGF to soluble forms. Being potent angiogenic factors, bFGF and VEGF act as the final mediators of Tat-induced KS and EC growth. In our study, SPMG significantly combated the Tat-induced release of bound bFGF and VEGF as well into their soluble forms. All these appear to be attributed to the binding of SPMG to Tat via its basic domain. These data demonstrate that SPMG might serve as a valuable therapeutic intervention for Tat-induced profound angiogenic effects, and subsequent pathologic events of AIDS-KS.