An extracellular matrix (ECM) derived from the submucosa of the porcine small intestine (SIS) has been shown to induce angiogenesis and host tissue remodeling when used as a xenogeneic bioscaffold in animal models of wound repair. In the present study, we compared the in vitro effects of SIS ECM extracts to several purified angiogenic growth factors on human dermal microvascular endothelial cell (HMEC) growth patterns. The SIS ECM was shown to induce tube formation from HMEC in a three-dimensional fibrin-based angiogenesis assay in a manner similar to that caused by the addition of vascular endothelial growth factor (VEGF). This tube formation was blocked in the presence of anti-VEGF neutralizing antibody. Western blots and ELISA procedures showed that the SIS ECM contains as much as 0.77 ng VEGF/g SIS. The closely related endothelial cell mitogen, platelet-derived growth factor (PDGF), was not detectable in the SIS extracts. We conclude that VEGF is present in the SIS extracellular matrix. The role of VEGF in SIS-induced wound repair remains unknown, but its presence in the ECM makes it a possible contributor to the angiogenic effect of SIS when this ECM is used as a tissue repair scaffold in animal models of wound repair.

The heptapeptide 1, NAc-Gly-Val-DIle-Thr-Arg-Ile-ArgNHEt, a structurally modified fragment derived from the second type-1 repeat of thrombospondin-1 (TSP-1), is known to possess antiangiogenic activity. However, therapeutic utility could not be demonstrated because this peptide has a very short half-life in rodents. To optimize the PD/PK profile of 1, we initiated a systematic SAR study. The initial structural modifications were performed at positions 5 and 7 of peptide 1 and at the N- and C-termini. Out of several hundred peptides synthesized, the nonapeptide 5 (ABT-526) emerged as a promising lead. ABT-526 inhibited VEGF-induced HMVEC cell migration and tube formation in the nanomolar range and increased apoptosis of HUAEC cells. ABT-526 showed acceptable PK in rodents, dog, and monkey. ABT-526, when incorporated in an angiogenic pellet implanted in the rat cornea at 10 microM, reduced neovascularization by 92%. Substitution of DalloIle in place of DIle in ABT-526 provided nonapeptide 6 (ABT-510), which was 30-fold less active than ABT-526 in the EC migration but 20-fold more active in the tube formation assay. In comparison to ABT-526, ABT-510 has increased water solubility and slower clearance in dog and monkey. Radiolabeled ABT-510 demonstrated saturable binding to HMVEC cells at 0.02-20 nM concentrations and was displaceable by TSP-1. ABT-510 and ABT-526 were shown to significantly increase apoptosis of HUAEC cells. ABT-510 was effective in blocking neovascularization in the mouse Matrigel plug model and inhibited tumor growth in the mouse Lewis lung carcinoma model. Previous studies had shown that ABT-510 was effective in inhibiting the outgrowth of murine melanoma metastases in syngeneic mice and in blocking the growth of human bladder carcinoma implanted in nude mice. It had been also shown that ABT-510 could regress tumor lesions in pet dogs or cause unexpected stabilization of the disease in advanced canine cancer. ABT-526 and ABT-510 are the first compounds in the class of potent inhibitors of angiogenesis that mimic the antiangiogenic function of TSP-1. ABT-510 is currently in phase II clinical studies.

. Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E611-E617, 1998.-Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulinstimulated sodium transport in a dose-dependent manner (IC 50 Ϸ 6 µM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.amiloride-sensitive sodium channel; insulin signaling; receptor tyrosine kinase; renal epithelia INSULIN INCREASES SODIUM REABSORPTION in dogs and humans, and this effect appears to be manifested predominantly on the renal distal nephron (21, 10). Models of the distal nephron, toad urinary bladder (Bufo marinus) and the A6 cell line (derived from Xenopus laevis kidney), have been used to demonstrate a direct effect of insulin on amiloride-sensitive sodium transport (12,14). Patch-clamp electrophysiology has indicated that insulin causes an increase in the open probability (P O ) of the apical sodium channel (20). However, blocker-induced noise analysis demonstrated that insulin induces an increase in active sodium channel density in the apical membrane (2, 4, 11). In addition, insulin increases the apical membrane area (11). These results suggest that insulin may induce the insertion of sodium channels into the apical membrane, a hypothesis supported by our study demonstrating that brefeldin A, an inhibitor of secretion, partially inhibits insulin-stimulated sodium transport (9).The first step in insulin signaling is the binding of the hormone to its receptor, followed by autophosphorylation of the receptor and subsequent activation of the receptor tyrosine kinase (IRTK). IRTK has several substrates, including the insulin receptor substrate (IRS) proteins (33). The IRS proteins mediate some of the pleiotropic effects of insulin through interactions with proteins containing SH2 domains, including phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme, a heterodimer, phosphorylates the D-3 position of the myo-inositol ring of phosphatidylinositols (PtdIns; 17). PI 3-kinase is required for several insulin-mediated effects, including the translocation of the insulinresponsive glucose transporter (GLUT-4) to the plasma membrane of adipocytes (7) and skeletal muscle (34), and ...