Rho-A activation increases the permeability of HGEc-1.

<p><b>(A)</b> Cultured HGEc-1 cells were transfected with different plasmids, pCEFL-mock, constitutively active RhoAQL (pCEFL-AU5-RhoAQL), or dominant negative mutant RhoAN19 (pCEFL-AU5-RhoAN19). Twenty-four hours later, the cells were treated with VEGF-A (50 ng/ml) + Tat (100 ng/ml) + Heparin (50 units/ml), all together, and exposed to FITC-dextran as described in methods. <b>(B)</b> In other experiments HGEc-1 cells were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, MLC, Src as described in Methods. Panel B shows representative western blots corresponding to the phosphorylation changes. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, MLC and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. In each group, mock, RhoAQL and RhoAN19 transfected cells were treated with either serum free media (Controls) (-), or VEGF-A + Tat + Heparin (+). Groups that were significantly different from controls (-) were labeled with <i>asterisk</i>, *p<0.05 and **p<0.01. Cells transfected with constitutively active RhoAQL that were significantly different from mock or RhoAN19 cells, were labeled with crosses: + p <0.05 and ++ p<0.01.</p

VEGF-A in combination with HIV-Tat and heparin, increases the permeability of cultured HGEc-1 through Rho-A and Src dependent pathways.

<p><b>(A)</b> Monolayers of 5 hours-starved HGEc-1 were stimulated by VEGF-A (50 ng/ml), Tat (100 ng/ml), and Heparin (50 units/ml) all combined. The Rho-A inhibitor: C3 transferase (20 ng/ml), was added 4 hours before stimulation and the inhibitor of Src family kinase SU6656 (1 μM) and ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation. The data represent FITC-dextran permeability expresses as fold increase. <b>(B)</b> Overnight-starved HGEc-1 monolayers were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, Rac1, MLC and Src, as described in Methods. Panel B shows representative Western blots corresponding to the phosphorylation changes. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units expressed as a ratio of the total activity. Values significantly different from control cells treated with serum free media were marked with <i>asterisks</i> **p<0.01, and those significantly different from the cells treated with VEGF-A + Tat + Heparin were marked with crosses, + p<0.05 and ++ p<0.01.</p

FGF-2 and VEGF-A, in combination with HIV-Tat and heparin, induce the formation of stress fibers through Rho-A dependent pathways.

<p><b>(A)</b> Panel A shows representative changes in the formation of stress fibers detected in cultured HGEc-1. Overnight-starved HGEc-1 monolayer were stimulated by thrombin (100 units/ml) as a positive control, Tat (100 ng/ml), FGF-2 (50 ng/ml), VEGF-A (50 ng/ml), and Heparin (50 units/ml) alone or in combination. The RhoA inhibitor: C3 transferase (20 ng/ml), was added 4 hrs. before stimulation and the inhibitor of Src family kinase SU6656 (1 μM) and ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation and 20 min after treatment, F-actin fibers were visualized in cells by staining with 2 μg/ml of Alexa Fluor 488-labeled phalloidin. Cell nuclei were stained with Hoechst 33342. The scale bar is 10 μm. <b>(B)</b> The graphs show mean ± SEM values corresponding to the formation of stress fibers in three different experiments. Results were expressed as % changes in stress fibers formation relative to control cells. Values significantly different from controls were marked with an <i>asterisk</i>, *p<0.05 and **p<0.01, and those different from cells treated with VEGF-A + Tat + Heparin were marked with a cross, +p <0.05 and ++p<0.01.</p

Urine samples harvested from HIV-infected children with renal diseases increase the permeability of cultured HGEc-1 through RhoA and Src mediated mechanisms.

<p><b>(A)</b> Urine samples harvested from HIV infected children with and without renal diseases (RD) were used (1:10 dilution) to stimulate monolayers of 5 hours-starved HGEc-1. The RhoA inhibitor: C3 transferase (20 ng/ml), was added 4 hrs before stimulation. The inhibitor of Src family kinase SU6656 (1 μM) and the ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation. The data represent FITC-dextran permeability expresses as fold increase. <b>(B)</b> Overnight-starved HGEc-1 monolayer were treated with urine (1:20 dilution) for 5 min as described above and then harvested to assess the phosphorylation of RhoA, Rac1, MLC, and Src, as described in Methods. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src, in cultured HGEc-1. Results were expressed in optical density units expressed as a ratio of the total activity. Values significantly different from the control group (HIV-N; n = 5) were marked with <i>asterisks</i> **p<0.01, and those significantly different from the HIV-RD group (n = 5) were marked with stars ★ p<0.05 and ★★ p<0.01 (for permeability), or crosses, + p <0.05 and ++ p<0.01 (for signaling).</p

Urine samples harvested from HIV-infected children with renal diseases increase the permeability of cultured podocytes cell line (P-2) through Rho-A and Src mediated mechanisms.

<p><b>(A)</b> Urine samples harvested from HIV infected children with and without renal diseases (RD) were used (1:10 dilution) to stimulate monolayers of 5 hours-starved P-2 cells. The data represent FITC-dextran permeability changes expressed as fold increase. <b>(B)</b> Overnight-starved P-2 monolayers were treated with urine (1:20 dilution) for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, MLC, and Src, as described in Methods. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, MLC, and Src in cultured podocytes. Results were expressed in optical density units expressed as a ratio of the total activity. Values significantly different from the serum free treated control cells (Control) were marked with <i>asterisk</i>, *p<0.05 and **p<0.01, and differences between HIV-RD (n = 5) and HIV-N (n = 5) urine samples, were marked with stars ★ p< 0.05 (for permeability) or crosses + p<0.05 (for signaling).</p

FGF-2 and VEGF-A in combination with HIV-Tat and heparin increase the permeability of cultured HGEc-1 through Rho-A and Src dependent pathways.

<p><b>(A)</b> Monolayers of 5 hours-starved HGEc-1 were stimulated by thrombin (100 units/ml) as a positive control, Tat (100 ng/ml), FGF-2 (50 ng/ml), VEGF-A (50 ng/ml), and Heparin (50 units/ml) alone or in combination. The data represent FITC-dextran permeability changes expressed as fold increase. <b>(B)</b> Overnight-starved HGEc-1 monolayer were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, Rac1, MLC, and Src, as described in Methods. Panel B shows representative Western blots corresponding to the phosphorylation changes. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. Values significantly different from control cells treated with serum free media were marked with <i>asterisk</i>, *p<0.05 and **p<0.01.</p

Rho-A activation increases the formation of stress fibers in HGEc-1.

<p><b>(A)</b> HGEc-1 were transfected with the corresponding Rho-A constructs described above. Subsequently, 24 hours later, they were seeded on coverslips, treated with (VEGF-A + Tat + Heparin), and stained with F-actin to visualize the formation of stress fibers as described above. Scale bar = 20 μm. <b>(B)</b> The graphs show mean ± SEM values corresponding to three different experiments that assessed the formation of stress fibers in cultured HGEc-1. Results were expressed as % changes in stress fibers relative to controls. Control cells (-) were treated with serum free medium. Statistically significant differences between control cells (-) vs. VEGF-A + Tat + Heparin-treated cells (+), were highlighted with an <i>asterisk</i>, *p<0.05. Difference between cells treated with VEGF-A + Tat + Heparin vs. other groups were highlighted with a cross, ++ p < 0.01.</p