The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates.

This study determined if blocking ligand occupancy of the αVβ3 integrin could inhibit the pathophysiologic changes that occur in the early stages of diabetic nephropathy (DN). Diabetic rats were treated with either vehicle or a monoclonal antibody that binds the β3 subunit of the αVβ3 integrin. After 4 weeks of diabetes the urinary albumin to creatinine ratio (UACR) increased in both diabetic animals that subsequently received vehicle and in the animals that subsequently received the anti-β3 antibody compared with control nondiabetic rats. After 8 weeks of treatment the UACR continued to rise in the vehicle-treated rats; however it returned to levels comparable to control nondiabetic rats in rats treated with the anti-β3 antibody. Treatment with the antibody prevented the increase of several profibrotic proteins that have been implicated in the development of DN. Diabetes was associated with an increase in phosphorylation of the β3 subunit in kidney homogenates from diabetic animals, but this was prevented by the antibody treatment. This study demonstrates that, when administered after establishment of early pathophysiologic changes in renal function, the anti-β3 antibody reversed the effects of diabetes normalizing albuminuria and profibrotic proteins in the kidney to the levels observed in nondiabetic control animals.

IGF-I-stimulated sarcoma viral oncogene (Src) activation during hyperglycemia is required for propagating downstream signaling. The aim of the current study was to determine the mechanism by which hyperglycemia enhances IGF-I-stimulated Src activation and the role of NADPH oxidase 4 (Nox4) and protein kinase C ζ (PKCζ) in mediating this response in vascular smooth muscle cells (VSMCs). Nox4 expression was analyzed in VSMCs exposed to hyperglycemia. The role of Nox4-derived reactive oxygen species (ROS) in IGF-I-stimulated Src activation was investigated via knockdown of Nox4. Different isoforms of PKC were screened to investigate their role in hyperglycemia-induced Nox4. The oxidation of Src was shown to be a prerequisite for its activation in response to IGF-I during hyperglycemia. Hyperglycemia induced Nox4, but not Nox1, and p22 phagocyte oxidase (p22phox) expression and IGF-I stimulated Nox4/p22phox complex formation, leading to increased ROS generation. Knockdown of Nox4 prevented ROS generation and impaired the oxidation and activation of Src in response to IGF-I, whereas knockdown of Nox1 had no effect. PKCζ was shown to mediate the hyperglycemia-induced increase in Nox4 expression. The key observations in cultured VSMCs were confirmed in the diabetic mice. Nox4-derived ROS is responsible for the enhancing effect of hyperglycemia on IGF-I-stimulated Src activation, which in turn amplifies IGF-I-linked downstream signaling and biological actions.

IGF-binding protein (IGFBP)-2 overexpression confers resistance to high-fat feeding and inhibits the differentiation of preadipocytes in vitro. However, whether administration of IGFBP-2 can regulate adipogenesis in vivo and the domains that mediate this response have not been defined. IGFBP-2 contains 2 heparin-binding domains (HBD), which are localized in the linker region (HBD1) and C-terminal region (HBD2) of IGFBP-2. To determine the relative importance of these domains, we used synthetic peptides as well as mutagenesis. Both HBD1 and HBD2 peptides inhibited preadipocyte differentiation, but the HBD2 peptide was more effective. Selective substitution of charged residues in the HBD1 or HBD2 regions attenuated the ability of the full-length protein to inhibit cell differentiation, but the HBD2 mutant had the greatest reduction. To determine their activities in vivo, pegylated forms of each peptide were administered to IGFBP-2(-/-) mice for 12 weeks. Magnetic resonance imaging scanning showed that only the HBD2 peptide significantly reduced (48 ± 9%, P < .05) gain in total fat mass. Both inguinal (32 ± 7%, P < .01) and visceral fat (44 ± 7%, P < .01) were significantly decreased by HBD2 whereas HBD1 reduced only visceral fat accumulation (24 ± 5%, P < .05). The HBD2 peptide was more effective peptide in reducing triglyceride content and serum adiponectin, but only the HBD2 peptide increased serum leptin. These findings demonstrate that the HBD2 domain of IGFBP-2 is the primary region that accounts for its ability to inhibit adipogenesis and that a peptide encompassing this region has activity that is comparable with native IGFBP-2.

Smooth muscle cells (SMC) maintained in high glucose are more responsive to IGF-I than those in normal glucose. There is significantly more thrombospondin-1 (TSP-1) in extracellular matrix surrounding SMC grown in 25 mM glucose. In this study we investigated 1) the mechanism by which glucose regulates TSP-1 levels and 2) the mechanism by which TS-1 enhances IGF-I signaling. The addition of TSP-1 to primary SMC was sufficient to enhance IGF-I responsiveness in normal glucose. Reducing TSP-1 protein levels inhibited IGF-I signaling in SMC maintained in high glucose. We determined that TSP-1 protected IAP/CD47 from cleavage and thereby facilitated its association with SHP substrate-1 (SHPS-1). We have shown previously that the hyperglycemia induced protection of IAP from cleavage is an important component of the ability of hyperglycemia to enhance IGF-I signaling. Furthermore we determined that TSP-1 also enhanced phosphorylation of the beta3 subunit of the alphaVbeta3 integrin, another molecular event that we have shown are critical for SMC response to IGF-I in high glucose. Our studies also revealed that the difference in the amount of TSP-1 in the two different glucose conditions was due, at least in part, to a difference in the cellular uptake and degradation of TSP-1.

Increased responsiveness of vascular cells to the growth factor IGF-I has been implicated in complications associated with diabetes. Here we describe the development of an assay and screening of a library of compounds for their ability to accelerate cleavage of the transmembrane protein integrin-associated protein (IAP) thereby disrupting the association between IAP and SHPS-1 which we have shown as critical for the enhanced response of vascular cells to IGF-I. The cell-based ELISA utilizes an antibody that specifically detects cleaved, but not intact, IAP. Of the 1040 compounds tested, 14 were considered active by virtue of their ability to stimulate an increase in antibody-binding indicative of IAP cleavage. In experiments with smooth muscle and retinal endothelial cell cultures in hyperglycemic conditions, each active compound was shown to accelerate the cleavage of IAP, and this was associated with a decrease in IAP association with SHPS-1 as determined by coimmunoprecipitation of the proteins from cell lysates. As a consequence of the acceleration in IAP cleavage, the compounds were shown to inhibit IGF-I-stimulated phosphorylation of key signaling molecules including Shc and ERK1/2, and this in turn was associated with a decrease in IGF-I-stimulated cell proliferation. Identification of these compounds that utilize this mechanism has the potential to yield novel therapeutic approaches for the prevention and treatment of vascular complications associated with diabetes.

Smooth muscle cell (SMC) maintained in medium containing normal levels of glucose do not proliferate in response to IGF-I, whereas cells maintained in medium containing 25 mmol/l glucose can respond. The aim of this study was to determine whether signaling events that have been shown to be required for stimulation of SMC growth were regulated by glucose concentrations in vivo.

Pain of unknown etiology is frequent in individuals with the tumor predisposition syndrome neurofibromatosis 1 (NF1), even when tumors are absent. Nerve Schwann cells (SCs) were recently shown to play roles in nociceptive processing, and we find that chemogenetic activation of SCs is sufficient to induce afferent and behavioral mechanical hypersensitivity in wild-type mice. In mouse models, animals showed afferent and behavioral hypersensitivity when SCs, but not neurons, lacked Nf1. Importantly, hypersensitivity corresponded with SC-specific upregulation of mRNA encoding glial cell line-derived neurotrophic factor (GDNF), independently of the presence of tumors. Neuropathic pain-like behaviors in the NF1 mice were inhibited by either chemogenetic silencing of SC calcium or by systemic delivery of GDNF-targeting antibodies. Together, these findings suggest that alterations in SCs directly modulate mechanical pain and suggest cell-specific treatment strategies to ameliorate pain in individuals with NF1.