Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells.

J Silvio Gutkind,Guido Serini,Patricio E Ray,Jharna R Das

doi:10.1371/journal.pone.0153837

Abstract

Renal endothelial cells (REc) are the first target of HIV-1 in the kidney. The integrity of REc is maintained at least partially by heparin binding growth factors that bind to heparan sulfate proteoglycans located on their cell surface. However, previous studies showed that the accumulation of two heparin-binding growth factors, Vascular Endothelial Cell Growth Factor-A (VEGF-A) and Fibroblast Growth Factor-2 (FGF-2), in combination with the viral protein Tat, can precipitate the progression of HIV-renal diseases. Nonetheless, very little is known about how these factors affect the behavior of REc in HIV+ children. We carried out this study to determine how VEGF-A, FGF-2, and HIV-Tat, modulate the cytoskeletal structure and permeability of cultured REc, identify key signaling pathways involved in this process, and develop a functional REc assay to detect HIV+ children affected by these changes. We found that VEGF-A and FGF-2, acting in synergy with HIV-Tat and heparin, affected the cytoskeletal structure and permeability of REc through changes in Rho-A, Src, and Rac-1 activity. Furthermore, urine samples from HIV+ children with renal diseases, showed high levels of VEGF-A and FGF-2, and induced similar changes in cultured REc and podocytes. These findings suggest that FGF-2, VEGF-A, and HIV-Tat, may affect the glomerular filtration barrier in HIV+ children through the induction of synergistic changes in Rho-A and Src activity. Further studies are needed to define the clinical value of the REc assay described in this study to identify HIV+ children exposed to circulating factors that may induce glomerular injury through similar mechanisms.

Highlights

HIV-infected children are at risk of developing several renal diseases, including HIV-associated nephropathy (HIVAN), Hemolytic Uremic Syndrome, Thrombotic Thrombocytopenic Purpura, and acute kidney injury [1]
The reagents described below were obtained from the following sources: human recombinant VEGF-165 (PeproTech (Rocky Hill, NJ); HIV-1 Tat protein (NIH AIDS Reagent Program); human recombinant Fibroblast Growth Factor-2 (FGF-2) (R&D Systems); Heparin derived from porcine intestinal mucosa, USP 5,000 USP (Units /ml) APP Pharmaceuticals LLC; SU6656 (Calbiochem); C3 Transferase (List Biological Labs, Campbell, CA); Y-27632, cyclic 3’5 monophosphate analog, thrombin, DAPI and Beta-actin mouse monoclonal antibody (Sigma-Aldrich, MO); RO 20– 1724 (MyBiosource San Diego), and RhoA (67B9) rabbit monoclonal antibody, phospo-p44/42 MAP kinase (Thr202/Tyr204), p44/42 MAP kinase, Src rabbit monoclonal antibody, phosphoMyosin Light Chain 2 (Thr/Ser 19) rabbit polyclonal antibody, Myosin Light Chain 2 rabbit polyclonal antibody, and VEGF receptor 2 (VEGFR2) 55B11 rabbit monoclonal antibody, were all obtained from Cell Signaling Technology (Danvers, MA)
Since primary human glomerular endothelial cells (HGEc) have a limited life span and require serum and angiogenic growth factors to survive in culture, we generated a HGEc line to assess the behavior of renal endothelial cells (REc) in the absence of serum and angiogenic growth factors

Summary

Introduction

HIV-infected children are at risk of developing several renal diseases, including HIV-associated nephropathy (HIVAN), Hemolytic Uremic Syndrome, Thrombotic Thrombocytopenic Purpura, and acute kidney injury [1]. The HIV-1 transactivator of transcription (Tat) protein, which is released by HIVinfected cells and taken up by endothelial cells, functions as a heparin binding growth factor [15, 16] In this manner, extracellular Tat can act in synergy with VEGF-A or FGF-2 to modulate the cytoskeletal structure of endothelial cells [17] and podocytes [18, 19]. HIV-1 binds to HSPG through electrostatic interactions that involve the positively charged domains of gp120 and the negative charges of HSPG on endothelial cells [20], and these interactions increase virus infectivity and facilitate the release of HIV-Tat [21] These findings provide compelling evidence to suggest that VEGF-A, FGF-2, and HIV-Tat, acting in synergy, may play important roles modulating the cytoskeletal structure and permeability of RGEc in HIV+ children

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