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HMG CoA reductase inhibition modulates VEGF-induced endothelial cell hyperpermeability by preventing RhoA activation and myosin regulatory light chain phosphorylation

Lixia Zeng^*, Hanshi Xu^*, Teng-Leong Chew, Eudora Eng^*, Mehran M. Sadeghi, Stephen Adler, Yashpal S. Kanwar^|| and Farhad R. Danesh^*,1

^* Division of Nephrology/Hypertension,
Department of Cell and Molecular Biology, Feinberg School of Medicine, Chicago, Illinois, USA;
Division of Cardiovascular Medicine, Yale School of Medicine, New Haven, Connecticut, USA;
Department of Medicine, New York Medical College, Valhalla, New York, New York, USA; and
^|| Department of Pathology, Northwestern University, Chicago, Illinois, USA

¹ Correspondence: Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Searle Building 10-563, Chicago, IL 60611, USA. E-mail: f-rahimi{at}northwestern.edu

SPECIFIC AIMS

The beneficial effects of statins are usually assumed to stem from their ability to reduce cholesterol biosynthesis. However, accumulating evidence suggests that statins may also modulate several signaling pathways through their pleiotropic effects. The major aims of this study were to 1) investigate the modulatory effects of statins on VEGF signaling, and 2) characterize the signaling cascade that leads to VEGF-induced glomerular endothelial cell (GEnC) hyperpermeability.

PRINCIPAL FINDINGS

1. Effect of simvastatin on VEGF-induced permeability
To assess the role of simvastatin on VEGF-induced hyperpermeability, we measured transendothelial electrical resistance (TEER) across GEnCs. GEnCs were grown until post-confluent on Transwell® filters, exposed to various concentrations of VEGF (25, 50, and 100 ng/mL), and the electrical resistance of the filter was subsequently measured using a Millicell-Electrical Resistance System. After 30 min of exposure to various concentrations of VEGF, GEnCs displayed a significant decrease in TEER in a dose-dependent manner. To determine the effect of simvastatin on VEGF-induced GEnC hyperpermeability, TEER across GEnCs cells was measured in untreated, VEGF-stimulated (50 ng/mL), and in cells cotreated with simvastatin (1 µM) and mevalonate (400 µM). VEGF exposure lowered basal TEER levels by 60%. Simvastatin alone did not significantly affect the permeability of GEnCs. Similarly, TEER levels did not change significantly when VEGF-stimulated cells were cotreated with simvastatin, indicating that simvastatin attenuates VEGF-induced GEnC hyperpermeability. Addition of mevalonate to cells cotreated with VEGF and simvastatin decreased TEER to the levels obtained with VEGF, suggesting that the inhibitory effect of simvastatin on VEGF-induced GEnC hyperpermeability is via a mevalonate-dependent pathway.

2. RhoA activation and VEGF signaling
We had previously shown that statins (e.g., simvastatin) may exert their beneficial effects in the diabetic milieu by inhibiting geranylgeranylation of the Rho family of small GTPases. To evaluate the role of RhoA in VEGF-induced signaling pathway, a pull-down assay using the fusion protein GST-RBD which recognizes only RhoA-GTP (the active form of RhoA) was performed. RhoA-GTP was significantly increased when cells were exposed to various concentrations of VEGF for 5 min. To elucidate the modulatory effect of simvastatin on VEGF-induced GEnC hyperpermeability, cells were exposed to VEGF (50 ng/mL) and cotreated with simvastatin (1 µM). Treatment of GEnCs with VEGF for 5 min increased RhoA-GTP by 5-fold. Simvastatin alone did not have any significant effect on RhoA activity. However, cotreatment of VEGF-stimulated cells with simvastatin significantly decreased RhoA activity.

To further support the involvement of RhoA on VEGF signaling pathway, we also compared membrane-bound RhoA (active form) to the total cytoplasmic protein expression of RhoA using Western blot analysis. Increased protein expression of membrane-bound RhoA in GEnCs was observed after 5 min of stimulation with various concentrations of VEGF with reciprocal changes in cytoplasmic RhoA protein levels. Furthermore, cotreatment of cells with simvastatin (1 µM) reversed VEGF-induced (50 ng/mL) increase in membrane-associated RhoA, suggesting that the simvastatin prevents VEGF-induced translocation of RhoA to the membrane.

3. RhoA activation and VEGF-induced GEnC hyperpermeability
We then investigated whether RhoA activation contributes to the observed VEGF-induced GEnC hyperpermeability. To answer this question, GEnCs were transfected with wild-type RhoA (wtRhoA) and dominant negative mutant of RhoA (N19RhoA). Using the monolayer resistance assay, cells transfected with wtRhoA and exposed to VEGF (50 ng/mL) showed a significant decrease in the measured TEER. Unstimulated N19RhoA transfected cells did not affect the measured resistance. Transfected GEnCs with N19RhoA and stimulated with VEGF (50 ng/mL) failed to display a significant change in the measured TEER. These findings support the role of RhoA as a key mediator of the VEGF-induced hyperpermeability in GEnCs.

4. Effects of VEGF and simvastatin on cytoskeletal remodeling
That VEGF rapidly induced the activation of RhoA and that simvastatin ameliorated the effect of VEGF suggests that VEGF and statins may affect GEnC permeability via cytoskeleton remodeling. To establish whether VEGF-induced hyperpermeability involves cytoskeletal remodeling, we characterized F-actin cytoskeletal network in GEnCs by rhodamine-conjugated phalloidin staining. GEnCs do not generally display prominent stress fibers although they exhibit an extensive cortical actin network (Fig. 1 A). Within 20 min of VEGF treatment (25, 50,100 ng/mL), cells exhibited significant stress fiber formation and sizable gaps (arrows) between endothelial cells appeared in a dose-dependent manner (Fig. 1B-D ). However, VEGF stimulation (50 ng/mL) failed to induced stress fiber formation and paracellular gap formation in dominant negative RhoA transfected GEnCs as well as in cells cotreated with simvastatin (1 µM) (Fig. 1E, F ). These observations suggest that the VEGF signaling pathway involves cytoskeletal reorganization. Moreover, our data indicate that simvastatin ameliorates VEGF-induced cytoskeletal remodeling.

View larger version (72K): [in this window] [in a new window]   Figure 1. A) Unstimulated cells with few stress fibers. However, GEnCs exposed to VEGF 25 ng/mL (B), 50 ng/mL (C), or 100 ng/mL (D) exhibited significant stress fiber formation and sizable gaps between endothelial cells appeared (arrows). VEGF stimulation (50 ng/mL) did not induce significant stress fiber formation in GEnCs transfected with dominant negative RhoA (E) as well as in cells cotreated with SMV (F). Representative of 3 separate experiments.

5. Effects of VEGF and simvastatin on myosin light chain (MLC) diphosphorylation
One of the best-characterized triggering signals for stress fiber formation is phosphorylation of the regulatory part of the myosin molecule. MLC phosphorylation plays a pivotal role in initiating actin-myosin interaction and the development of F-actin-dependent cytoskeletal contractile tension. To assess whether VEGF-induced cytoskeletal remodeling involves MLC diphosphorylation, GEnCs were induced with VEGF (50 ng/mL) and the MLC diphosphorylated state was assessed by Western blot analysis using anti-diphosphorylated MLC antibody. VEGF exposure significantly increased protein levels of diphosphorylated-MLC. Cotreatment of cells with simvastatin (1 µM) inhibited VEGF-induced MLC diphosphorylation. Addition of mevalonate (400 µM) resulted in a significant increase in diphospho-MLC, indicating that the effect of simvastatin on MLC diphosphorylation was mevalonate dependent.

To gain further insight as to whether Rho/Rho kinase pathway mediates VEGF-induced MLC diphosphorylation, GEnCs transfected with wtRhoA, dominant negative RhoA (N19RhoA), and cells cotreated with Y27632 (10 µM) were exposed to VEGF (50 ng/mL). VEGF exposure in wt RhoA transfected cells significantly increased MLC diphosphorylation. However, N19RhoA transfected cells and GEnCs cotreated with Y27632 exhibited no significant change in diphosphorylated-MLC, indicating that VEGF-induced MLC diphosphorylation in GEnCs is RhoA dependent.

6. Effects of VEGF and simvastatin on GEnC contraction
Myosin-dependent cell contraction plays a key role in endothelial cell permeability in response to inflammatory cytokines such as histamine and thrombin. To approach the central question of whether VEGF-induced GEnC hyperpermeability is directly mediated through cellular contraction, GEnC contraction was assessed by live cell imaging. GEnCs transiently expressing GFP-tagged MLC were treated with VEGF (50 ng/mL) in a Bioptechs FCS2 perfusion chamber. Images were captured on a spinning disc confocal microscope. As shown in Fig. 2 A, B, VEGF exposure (50 ng/mL) dynamically induced GEnC contraction leading to increased gaps among endothelial cells. However, cotreatment of cells with simvastatin (1 µM) significantly attenuated VEGF-mediated cellular contraction and paracellular gap formation (Fig. 2C and video 3).

View larger version (61K): [in this window] [in a new window]   Figure 2. Cells transiently expressing GFP-tagged MLC were treated with VEGF (50 ng/mL) in a Bioptechs FCS2 perfusion chamber. Images were captured on a spinning disc confocal microscope (Zesis LSM510). A) Temporal profile of VEGF-induced increase in GEnC contraction. Arrows depict active regional cellular contraction. B) Increased paracellular gap formation in GEnCs exposed to VEGF (50 ng/mL) (arrows). C) Cotreatment of GEnCs with simvastatin (1 µM) attenuated paracellular gap formation.

CONCLUSIONS AND SIGNIFICANCE

The data presented in this study demonstrated that 1) simvastatin ameliorated VEGF-induced GEnC hyperpermeability, 2) VEGF-induced activation and recruitment of RhoA from the cytosol to the membrane led to MLC diphosphorylation and cytoskeletal remodeling, and 3) simvastatin, by inhibiting RhoA activation, prevented VEGF-induced MLC diphosphorylation and stress fiber formation. We propose that the underlying molecular mechanism of VEGF-induced GEnC hyperpermeability involves RhoA activation, MLC diphosphorylation, endothelial cytoskeletal remodeling, and increased cellular contraction. Endothelial cell contraction results in increased paracellular gap formation and GEnC hyperpermeability (Fig. 3 ).

View larger version (35K): [in this window] [in a new window]   Figure 3. Proposed VEGF-induced GEnC hyperpermeability. Simvastatin inhibits VEGF-induced MLC-dependent increase in endothelial cell permeability.

Our data point to an important role for RhoA in regulating endothelial permeability. The integration of these data with pathobiology of microvascualr complications of diabetes is of paramount importance. Indeed, we previously reported the critical role of several members of Rho family of small GTPases in two other important signaling cascades in the diabetic milieu: angiotensin II and glucose-mediated signaling pathways. We now report that Rho GTPases via cytoskeletal remodeling and MLC diphosphorylation are key regulators of VEGF-induced endothelial dysfunction. We extended our earlier observations by establishing that RhoA is essential for VEGF-induced MLC diphosphorylation since GEnCs transfected with dominant negative RhoA failed to exhibit MLC diphosphorylation and increased endothelial permeability.

A highly provocative finding of this study is the modulatory role of statins on the VEGF-induced signaling pathway. Recent experimental evidence on the pleiotropic effects of statins suggests that statins may exert various modulatory effects on vascular endothelium by preventing isoprenylation of small GTPase proteins. In support of a modulatory effect of statins in the diabetic milieu, we and others recently proposed that the possible beneficial effects of statins in the diabetic milieu may be independent of their cholesterol-lowering properties and mediated at least in part by the modulatory effects of statins on Rho-dependent pathways. The present study provides a novel observation on the pleiotropic effects of statins with broad clinical implications in managing patients with diabetic nephropathy (DN). Several large clinical studies have previously suggested the beneficial effects of statins in DN, but the molecular mechanism of the beneficial effects of statins in DN remains to be elucidated. We have identified a novel pleiotropic effect of simvastatin, a hydrophobic statin, on GEnC permeability. Our data indicate that by preventing RhoA activation, simvastatin inhibits MLC phosphorylation, stress fiber formation, and cytoskeletal remodeling.

Our data provide a framework to establish the role of Rho GTPases in microvascular complications of diabetes and offer a new rationale for the use of statins in the early stages of DN, independent of their cholesterol-lowering properties.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4240fje;

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