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Sphingosine-1 phosphate (S1P) and thrombin are agents with profound but divergent effects on vascular endothelial cell (EC) barrier properties. We have previously reported that S1P-induced focal adhesion (FA) remodeling involves interactions between focal adhesion kinase (FAK), paxillin, and G-protein-coupled receptor kinase-interacting proteins GIT1 and GIT2 and suggested a critical involvement of focal adhesions in the EC barrier regulation. In this study, we examined redistribution of FA proteins (FAK, paxillin, GIT1, and GIT2) and site-specific FAK tyrosine phosphorylation in human pulmonary artery endothelial cells stimulated with thrombin. In contrast to S1P, which we have shown to induce peripheral translocation of FA proteins associated with cortical actin ring formation, thrombin caused the redistribution of FA proteins to the ends of the newly formed massive stress fibers. S1P and thrombin induced distinct patterns of FAK site-specific phosphorylation with the FAK Y576 phosphorylation site targeted by SIP challenge and phosphorylation of three FAK sites (Y397, Y576, and Y925) in response to thrombin stimulation. Pharmacological inhibition of Src with Src-specific inhibitor PP2 abolished S1P-induced translocation of FA proteins, cortical actin ring formation, and FAK [Y576] phosphorylation. However, PP2 failed to alter thrombin-induced morphological changes and exhibited only partial inhibition of FAK site-specific tyrosine phosphorylation. These observations highlight the differential mechanisms of focal adhesion protein complex remodeling and FAK activation by S1P and thrombin and link differential FA remodeling to EC barrier regulation.

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Sphingosine 1-phosphate (S1P) enhances human pulmonary endothelial monolayer integrity via Rac GTPase-dependent formation of a cortical actin ring (Garcia et al. J Clin Invest 108: 689-701, 2001). The mechanisms underlying this response are not well understood but may involve rapid redistribution of focal adhesions (FA) as attachment sites for actin filaments. We evaluate the effects of S1P on the redistribution of paxillin, FA kinase (FAK), and the G protein-coupled receptor kinase-interacting proteins (GITs). S1P induced Rac GTPase activation and cortical actin ring formation at physiological concentrations (0.5 microM), whereas 5 microM S1P caused prominent stress fiber formation and activation of Rho and Rac GTPases. S1P (0.5 microM) stimulated the tyrosine phosphorylation of FAK Y(576), and paxillin was linked to FA disruption and redistribution to the cell periphery. Furthermore, S1P induced a transient association of GIT1 with paxillin and redistribution of the GIT2-paxillin complex to the cell cortical area without affecting GIT2-paxillin association. These results suggest a role of FA rearrangement in S1P-mediated barrier enhancement via Rac- and GIT-mediated processes.

Thiazolidinedione (TZD), a ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma), exerts anti-inflammatory effects independently of the insulin-sensitizing effect. In the present study, we tested the hypothesis that TZD prevents the progression of diabetic nephropathy by modulating the inflammatory process. Five-week-old Sprague-Dawley rats were divided into three groups: 1) nondiabetic control rats (non-DM), 2) diabetic rats (DM), and 3) diabetic rats treated with pioglitazone (DM+pio). Diabetes was induced by injection with streptozotocin (STZ). The DM+pio group received 0.0002% pioglitazone mixed in chow for 8 wk after induction of diabetes. Blood glucose and HbA1c were elevated in diabetic rats but did not change by treatment with pioglitazone. Pioglitazone reduced urinary albumin excretion and glomerular hypertrophy, suppressed the expression of transforming growth factor (TGF)-beta, type IV collagen, and ICAM-1, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, renal NF-kappaB activity was increased in diabetic rats and reduced by pioglitazone. PPAR-gamma was expressed in glomerular endothelial cells in the diabetic kidney and in cultured glomerular endothelial cells. High-glucose conditions increased the expression of ICAM-1 and the activation of NF-kappaB in cultured glomerular endothelial cells. These changes were reduced by pioglitazone, ciglitazone, and pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB. However, pioglitazone did not show the changes in the presence of PPAR-gamma antagonist GW9662. Our results suggest that the preventive effects of pioglitazone may be mediated by its anti-inflammatory actions, including inhibition of NF-kappaB activation, ICAM-1 expression, and macrophage infiltration in the diabetic kidney.

Mononuclear cells, including monocytes/macrophages and T-cells, are considered to be involved in the progression of diabetic nephropathy, although the mechanism of their recruitment into diabetic glomeruli is unclear. The intercellular adhesion molecule-1 (ICAM-1) promotes the infiltration of leukocytes into atherosclerotic lesions as well as inflammatory tissues. In the present study, we investigated the expression of ICAM-1 in the glomeruli of streptozotocin-induced diabetic rats. The expression of ICAM-1 was increased significantly during the early stage of diabetes. The number of mononuclear cells, primarily monocytes/macrophages and lymphocytes, was significantly increased in diabetic glomeruli. Mononuclear cell infiltration into diabetic glomeruli was prevented by anti-ICAM-1 monoclonal antibody. Insulin treatment decreased ICAM-1 expression and mononuclear cell infiltration. The ICAM-1 expression on cultured human umbilical vein endothelial cells was not induced under high glucose culture conditions. Glomerular hyperfiltration is a characteristic change in the early stage of diabetic nephropathy. Treatment with aldose reductase inhibitor, which prevented glomerular hyperfiltration without changes in blood glucose levels, decreased ICAM-1 expression and mononuclear cell infiltration. Moreover, we examined the ICAM-1 expression in the glomeruli of the 5/6 nephrectomized rat, which is a model for glomerular hyperfiltration without hyperglycemia. The ICAM-1 expression and infiltration of mononuclear cells was significantly increased in the glomeruli of 5/6 nephrectomized rats. We conclude that ICAM-1 is upregulated and promotes the recruitment of mononuclear cells in diabetic glomeruli. Moreover, glomerular hyperfiltration that occurs in the early stage of diabetic glomeruli may be one of the potential mechanisms of ICAM-1 upregulation in diabetic nephropathy.

Microinflammation is a common major mechanism in the pathogenesis of diabetic vascular complications, including diabetic nephropathy. Macrophage scavenger receptor-A (SR-A) is a multifunctional receptor expressed on macrophages. This study aimed to determine the role of SR-A in diabetic nephropathy using SR-A–deficient (SR-A−/−) mice. Diabetes was induced in SR-A−/− and wild-type (SR-A+/+) mice by streptozotocin injection. Diabetic SR-A+/+ mice presented characteristic features of diabetic nephropathy: albuminuria, glomerular hypertrophy, mesangial matrix expansion, and overexpression of transforming growth factor-β at 6 months after induction of diabetes. These changes were markedly diminished in diabetic SR-A−/− mice, without differences in blood glucose and blood pressure levels. Interestingly, macrophage infiltration in the kidneys was dramatically decreased in diabetic SR-A−/− mice compared with diabetic SR-A+/+ mice. DNA microarray revealed that proinflammatory genes were overexpressed in renal cortex of diabetic SR-A+/+ mice and suppressed in diabetic SR-A−/− mice. Moreover, anti–SR-A antibody blocked the attachment of monocytes to type IV collagen substratum but not to endothelial cells. Our results suggest that SR-A promotes macrophage migration into diabetic kidneys by accelerating the attachment to renal extracellular matrices. SR-A may be a key molecule for the inflammatory process in pathogenesis of diabetic nephropathy and a novel therapeutic target for diabetic vascular complications.

Sphingosine 1-phosphate (S1P) is an important vascular barrier regulatory agonist which enhances the junctional integrity of human lung endothelial cell monolayers. We have now demonstrated that S1P induced cortical actin ring formation and redistribution of focal adhesion kinase (FAK) and paxillin to the cell periphery suggesting the critical role of cell–cell adhesion in endothelial barrier enhancement. Co-immunoprecipitation studies revealed increased association of VE-cadherin with FAK and paxillin in S1P-challenged human pulmonary artery endothelial cell (HPAEC) monolayers. Furthermore, S1P-induced enhancement of VE-cadherin interaction with α-catenin and β-catenin was associated with the increased formation of FAK–β-catenin protein complexes. Depletion of β-catenin (siRNA) resulted in loss of S1P-mediated VE-cadherin association with FAK and paxillin rearrangement. Furthermore, transendothelial electrical resistance (an index of barrier function) demonstrated that β-catenin siRNA significantly attenuated S1P-induced barrier enhancement. These results demonstrate a mechanism of S1P-induced endothelial barrier enhancement via β-catenin-linked adherens junction and focal adhesion interaction.