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Background: Thrombospondin 1 (TSP-1), fibronectin (Fn), and vitronectin (Vn) promote vascular smooth muscle cell (VSMC) chemotaxis through a variety of second messenger systems, including Ras, ERK1/2, and p38. Hypothesis: Ras, ERK1/2, and p38 differentially affect TSP-1-, Fn-, and Vn-induced VSMC chemotaxis. Methods: Bovine VSMCs were transfected with Ras N17 or treated with the following inhibitors: a farnesyl protein transferase (FPT) inhibitor, PD098059 (ERK1/2 inhibitor), or SB202190 (p38 inhibitor). Thrombospondin 1, Fn, and Vn were used as chemoattractants. Results were analyzed by analysis of variance (ANOVA) with post hoc testing (P < .05). Results: Ras N17 transfection or FPT inhibitor treatment inhibited TSP-1-, Fn-, and Vn-induced chemotaxis. PD098059 or SB202190 resulted in more inhibition of VSMC migration to TSP-1 than to Fn or Vn. Conclusions: Ras appears equally relevant in the signal transduction pathways of TSP-1-, Fn-, and Vn-induced VSMC chemotaxis. Thrombospondin 1-induced migration is more dependent upon ERK1/2 and p38 than Fn- or Vn-included migration. [ABSTRACT FROM PUBLISHER]

Angiostatin(AS), an internal fragment of plasminogen, is one of the most potent specific inhibitors of angiogenesis. Angiostatin treatment has resulted in the complete regression of human tumors implanted subcutaneously into nude mice and has great therapeutic value (O''Reilly et al., Nat. Med. 2, 689–692, 1996). Despite promising therapeutic value in the treatment of cancer, the mechanism of its action is still unknown. We found that angiostatin binds to a 35-kDa protein in bovine aortic endothelial (BAE) cells (Sharma et al., Proc. Am. Assoc. Cancer Res. 42, 568, A3050, 2002). In an attempt to begin to understand angiostatin''s mechanism of action, we have purified and characterized this 35-kDa protein from BAE cells. Internal peptide sequence analysis of purified protein demonstrated (SLYYIQQDTK, SYSPYDMLESIK, and ALLYLXGGDD) 100% sequence identity with tyrosine kinase substrate annexin II. Solid phase binding analysis suggests that angiostatin specifically bound to purified annexin II immobilized on 96-well plastic plates. Hundred-fold molar excess of unlabeled AS and anti-annexin II antibody inhibited bindings 85 and 55%, respectively, suggesting specific interaction. Annexin II is a predominant receptor for angiostatin, since neutralizing the angiostatin by soluble receptor (annexin II) effectively blocks angiostatin''s anti-EC activity. Similarly, saturating the annexin II receptor by plasminogen in endothelial cells also blocks angiostatin''s activity. Both angiostatin and plasminogen bind to purified annexin II in BAE cells saturably with apparent Kd values of 101 and 164 nM, respectively, for purified annexin II and Kd values of 83 and 125 nM, respectively, for BAE cells. Anti-annexin II monoclonal antibody inhibited angiostatin and plasminogen binding to endothelial cells by 68 and 62%, respectively, supporting our in vitro studies that annexin II is a receptor for angiostatin. Angiostatin- binding protein/annexin II specifically expressed in endothelial cells but not in fibroblasts suggests its EC-specific function. &epsiv;-Aminocaproic acid, a lys analogue, effectively blocks angiostatin and annexin II interaction, indicating that the lysine-binding domain of AS is required for binding to annexin II. These results suggest that the antiangiogenic action of angiostatin may be mediated via interaction with annexin II. Identification of annexin II as a receptor for angiostatin provides further evidence that clotting and fibrinolytic pathways are directly involved in the angiogenic process. [Copyright &y& Elsevier]

Abstract: Angiocidin, a matrix bound and tumor associated protein, has been shown to inhibit tumor progression and angiogenesis. We previously demonstrated that angiocidin binds to thrombospondin-1 and alpha2beta1 integrin. We now show that angiocidin binds and is a preferred substrate for tissue transglutaminase-2 (tTgase). Angiocidin bound tTgase saturably with a Kd of 26 nM, while an angiocidin deletion mutant missing the matrix binding domain of angiocidin failed to bind tTgase. tTgase colocalized with angiocidin on endothelial cells. tTgase bound anti-angiocidin immunoprecipitates of endothelial cell lysates. Breast cancer cells expressing high levels of tTgase attached to angiocidin immobilized on tissue culture plates. Angiocidin was a preferred substrate for tTgase forming high molecular weight cross-linked multimers when treated with tTgase. Cross-linked angiocidin contained iso-peptide bonds as demonstrated by Western blotting and immunohistochemical colocalization studies using endothelial cells treated with angiocidin. Cross-linked angiocidin inhibited cell migration in contrast to monomeric angiocidin and inhibited localization of fibronectin (FN), a pro-tumorigenic matrix protein, into the extracellular matrix (ECM) of tumor and HUVE cells. Our studies provide an additional explanation for the anti-tumor activity of angiocidin suggesting that cross-linked angiocidin disrupts the tumor ECM making it less permissive for tumor growth. [Copyright &y& Elsevier]

Abstract: Thrombospondin-1 (TSP-1), a matrix-bound adhesive glycoprotein, has been shown to modulate tumor progression. We previously demonstrated that TSP-1 up-regulates matrix metalloproteinases MMP-2 and MMP-9. Our studies suggested that the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is a key determinant in tumor cell invasion. We now report that TSP-1 up-regulates TIMP-1 expression in both human breast and prostate cancer cell lines. The effect of TSP-1 on TIMP-1 expression was examined in human breast adenocarcinoma cell lines (MDA-MB-231) and human prostate cancer cell lines (PC3-NI and PC3-ML) treated with exogenous TSP-1. TIMP-1 expression was also examined in TSP-1 stably transfected breast cancer cell line (MDA-MB-435). Northern and western blot analysis revealed TIMP-1 mRNA and TIMP-1 protein expression increased with increasing concentrations of TSP-1. This effect was inhibited by antibodies against the type I repeat domain of TSP-1 further suggesting that TSP-1 mediates TIMP-1 secretion. Inhibition of TSP-1 induced TIMP-1 levels increased tumor cell invasion. We conclude that TSP-1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and our results may provide an explanation for the divergent activities reported for TSP-1 in tumor progression. [Copyright &y& Elsevier]