Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and͞or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins

Transforming growth factor-β (TGF-β) is closely associated with progressive renal fibrosis. Significant progress has been accomplished in determining the cellular signaling pathways that are activated by TGF-β. This knowledge is being applied to glomerular mesangial cell models of extracellular matrix (ECM) accumulation. A central component of TGF-β-stimulated mesangial cell fibrogenesis is the TGF-β family-specific Smad signal transduction pathway. However, while Smads play an important role in collagen accumulation, recent findings indicate that cross talk among a variety of pathways is necessary for maximal stimulation of collagen expression. Further investigation of these multiple interactions will provide insight into possible ways to interrupt cellular mechanisms of glomerular fibrogenesis.

These data indicate that MAP kinase pathways can be activated by TGF-beta1 in mesangial cells and that the ERK MAP kinase plays a role in TGF-beta-stimulated collagen I expression. Because we have shown previously that SMADs mediate TGF-beta1-stimulated collagen I expression, our findings raise the possibility of interactions between the MAP kinase and the SMAD pathways.

We investigated the composition, properties, and utility of a novel copolymer of P(AAm-co-EG) designed to be an adaptable, durable, and biocompatible surface treatment of metallic, polymeric, and ceramic materials. Solution deposition and photoinitiation reactions were employed to graft a silane layer and then two sequential polymer layers (a discontinuous two stage polymerization) onto oxide surfaces. Different solvents, polymer concentrations, and cross-linker concentrations in the top polymer layer were compared. Contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to characterize layer wettability, thickness, and chemistry, respectively. A sandwich type network formed between acrylamide and poly(ethylene glycol) when acetone was used as the solvent for both layers. In contrast, an interpenetrating polymer network between acrylamide and poly(ethylene glycol) formed when acetone and methanol were used as the solvents for polymerization of the acrylamide and poly(ethylene glycol) layers, respectively. Interpenetrating polymer network configured samples were tested for protein adsorption and strength of cell attachment. Protein adsorption experiments in 15% fetal bovine serum indicated that significant amounts of protein do not adsorb to the surface of the thin polymer films (∼20 nm). Cell detachment experiments indicated that cells contacting copolymer-modified surfaces were removed by lower shear stresses than cells contacting clean and amine-terminated, (N-(2-aminoethyl)-3-aminopropyl)-trimethoxysilane modified surfaces.

Basu RK, Hubchak S, Hayashida T, Runyan CE, Schumacker PT, Schnaper HW. Interdependence of HIF-1␣ and TGF-␤/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression.

Transforming growth factor (TGF)-beta is a central stimulus of the events leading to chronic progressive kidney disease, having been implicated in the regulation of cell proliferation, hypertrophy, apoptosis and fibrogenesis. The fact that it mediates these varied events suggests that multiple mechanisms play a role in determining the outcome of TGF-beta signaling. Regulation begins with the availability and activation of TGF-beta and continues through receptor expression and localization, control of the TGF-beta family-specific Smad signaling proteins, and interaction of the Smads with multiple signaling pathways extending into the nucleus. Studies of these mechanisms in kidney cells and in whole-animal experimental models, reviewed here, are beginning to provide insight into the role of TGF-beta in the pathogenesis of renal dysfunction and its potential treatment.

Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-β-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-β in a TGF-β receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-β stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-β-stimulated increase in both hypoxia-responsive element (HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis.

Abstract. TGF-␤1 has been implicated in glomerular extracellular matrix accumulation, although the precise cellular mechanism(s) by which this occurs is not fully understood. The authors have previously shown that the Smad signaling pathway is present and functional in human glomerular mesangial cells and plays a role in activating type I collagen gene expression. It also was determined that TGF-␤1 activates ERK mitogen-activated protein kinase in mesangial cells to enhance Smad activation and collagen expression. Here, it was shown that TGF-␤1 rapidly induces cytoskeletal rearrangement in human mesangial cells, stimulating smooth muscle ␣-actin detection in stress fibers and promoting focal adhesion complex assembly and redistribution. Disrupting the actin cytoskeleton with cytochalasin D (Cyto D) selectively decreased basal and TGF-␤1-induced cell-layer collagen I and IV accumulation. The balance of matrix metalloproteinases (MMP) and inhibitors was altered by Cyto D or TGF-␤1 alone, increasing MMP activity, increasing MMP-1 expression, and decreasing tissue inhibitor of matrix metalloproteinase-2 expression. Cyto D also decreased basal and TGF-␤1-stimulated ␣1(I) collagen mRNA but did not inhibit TGF-␤-stimulated ␣1(IV) mRNA expression. A similar decrease in ␣1(I) mRNA expression caused by the actin polymerization inhibitor latrunculin B was partially blocked by the addition of jasplakinolide, which promotes actin assembly. The Rho-family GTPase inhibitor C. difficile toxin B or the Rho-associated kinase inhibitor Y-27632 also blocked TGF-␤1-stimulated ␣1(I) mRNA expression. Cytoskeletal disruption reduced Smad2 phosphorylation but had little effect on mRNA stability, TGF-␤ receptor number, or receptor affinity. Thus, TGF-␤1-mediated collagen I accumulation is associated with cytoskeletal rearrangement and Rho-GTPase signaling.