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Dysregulation of fatty acid utilization is increasingly recognized as a significant component of diabetic kidney disease. Rho-associated, coiled-coil-containing protein kinase (ROCK) is activated in the diabetic kidney, and studies over the past decade have illuminated ROCK signaling as an essential pathway in diabetic kidney disease. Here, we confirmed the distinct role of ROCK1, an isoform of ROCK, in fatty acid metabolism using glomerular mesangial cells and ROCK1 knockout mice. Mesangial cells with ROCK1 deletion were protected from mitochondrial dysfunction and redox imbalance driven by transforming growth factor β, a cytokine upregulated in diabetic glomeruli. We found that high-fat diet-induced obese ROCK1 knockout mice exhibited reduced albuminuria and histological abnormalities along with the recovery of impaired fatty acid utilization and mitochondrial fragmentation. Mechanistically, we found that ROCK1 regulates the induction of critical mediators in fatty acid metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1α, carnitine palmitoyltransferase 1, and widespread program-associated cellular metabolism. Thus, our findings highlight ROCK1 as an important regulator of energy homeostasis in mesangial cells in the overall pathogenesis of diabetic kidney disease.

Recently, the prevention of cardiovascular events has become one of the most important aims of diabetes care. Peroxisome proliferator-activated receptor (PPAR) agonists have been reported to have vascular protective effects. Here, we examined whether pemafibrate, a selective PPAR alpha agonist, attenuated neointima formation after vascular injury and vascular smooth muscle cell (VSMC) proliferation. We performed endothelial denudation injury in mice treated with a high-fat diet (HFD) or normal chow. Orally administered pemafibrate significantly attenuated neointima formation after vascular injury in HFD and normal chow mice. Interestingly, pemafibrate increased the serum fibroblast growth factor 21 concentration and decreased serum insulin concentrations in HFD mice. In addition, body weight was slightly but significantly decreased by pemafibrate in HFD mice. Pemafibrate, but not bezafibrate, attenuated VSMC proliferation in vitro. The knockdown of PPAR alpha abolished the anti-VSMC proliferation effect of pemafibrate. BrdU assay results revealed that pemafibrate dose-dependently inhibited DNA synthesis in VSMCs. Flow cytometry analysis demonstrated that G1-to-S phase cell cycle transition was significantly inhibited by pemafibrate. Pemafibrate attenuated serum-induced cyclin D1 expression in VSMCs. However, apoptosis was not induced by pemafibrate as assessed by the TUNEL assay. Similar to the in vitro data, VSMC proliferation was also decreased by pemafibrate in mice. These data suggest that pemafibrate attenuates neointima formation after vascular injury and VSMC proliferation by inhibiting cell cycle progression., Diabetes mellitus; Vascular smooth muscle cell; Atherosclerosis; PPAR alpha; Neointima formation; Cell biology; Health sciences; Cardiology; Internal medicine; Nuclear medicine; Endocrinology.

The Rho/Rho-kinase signaling pathway has been shown to be involved in the complications of diabetes. In this study, we found that fasudil, a specific Rho-kinase inhibitor, had a beneficial effect on the motor nerve conduction velocity (MNCV), which is delayed in rats with streptozotocin (STZ)-induced diabetes. Cadherin-dependent adherens junctions (AJs) in myelinating Schwann cells, necessary for proper myelin formation and rapid propagation of action potentials, are regulated by Rho/Rho-kinase signaling. These AJ structures are maintained by E-cadherin and catenin complexes such as β-catenin and p120 catenin. To elucidate the mechanism underlying the effect of fasudil on MNCV, we examined alterations in AJ structure in the peripheral nerves of the experimental rats. Our results showed that the activities of Rho and Rho-kinase increased simultaneously in the sciatic nerves of the diabetic rats. Fasudil restored the MNCV by suppressing the up-regulation of the Rho-kinase. In the diabetic state, enhanced Rho and Rho-kinase activity reduced p120 catenin expression and altered the distribution of p120 catenin and E-cadherin, which are normally localized in the paranodal compartment of the nodes of Ranvier and Schmidt-Lanterman incisures where autotypic AJs stabilize myelin structure. Fasudil restored normal p120 catenin expression and the distribution of p120 catenin and E-cadherin in the myelin sheath. In conclusion, reduced expression and altered distribution of the adhesion molecules in the myelin sheath might contribute to the slowing of the MNCV in the diabetic rats. Fasudil, through its effect on the distribution of the adhesion-related molecules, might prevent slowing of the MNCV.

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that hypoxia-inducible factor-1α (HIF-1α) is a key regulator of renal sclerosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1α in the development of renal dysfunction have not been defined. Here, we assessed whether Rho-kinase blockade attenuates HIF-1α induction and the subsequent fibrotic response using type 2 diabetic mice and cultured mesangial cells. Fasudil, a Rho-kinase inhibitor, reduced urinary albumin excretion, mesangial matrix expansion, and the expression of fibrotic mediators in db/db mice. Mechanistically, HIF-1α accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also prevented by fasudil in the renal cortex. In mesangial cells, Rho/Rho-kinase signaling was activated under hypoxic conditions. Further in vitro studies showed that pharmacological and genetic inhibition of Rho-kinase promoted proteasomal HIF-1α degradation, which subsequently suppressed HIF-1-dependent profibrotic gene expression by upregulation of prolyl hydroxylase 2. Thus, we found a previously unrecognized renoprotective mechanism for the effects of Rho-kinase inhibition and this could be a potential therapeutic target for the treatment of diabetic nephropathy.

The process of atherosclerosis is affected by interactions among numerous biological pathways. Accumulating evidence shows that endoplasmic reticulum (ER) stress plays a crucial role in the development of atherosclerosis. Rho-kinase is an effector of small GTP-binding protein Rho, and has been implicated as an atherogenic factor. Previous studies demonstrated that fasudil, a specific Rho-kinase inhibitor, exerts a cardioprotective effect by downregulating ER stress signaling. However, the molecular link between ER stress and Rho-kinase in endothelial cells has not been elucidated. In this study, we investigated the mechanisms by which fasudil regulates endothelial inflammation during ER stress. Tunicamycin, an established ER stress inducer, increased vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. Intriguingly, fasudil inhibited VCAM-1 induction. From a mechanistic stand point, fasudil inhibited expression of activating transcription factor (ATF)4 and subsequent C/EBP homologous protein (CHOP) induction by tunicamycin. Furthermore, fasudil attenuated tunicamycin-induced phophorylation of p38MAPK that is crucial for the atherogenic response during ER stress. These findings indicate that Rho-kinase regulates ER stress-mediated VCAM-1 induction by ATF4- and p38MAPK-dependent signaling pathways. Rho-kinase inhibition by fasudil would be an important therapeutic approach against atherosclerosis, in particular, under conditions of ER stress.

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis.

Resistin is an adipocytokine which plays a role in the development of insulin resistance. In this study, we investigated the direct effect of resistin on vascular endothelial cells. Resistin induced the expression of adhesion molecules such as VCAM-1 and ICAM-1, and long pentraxin 3, a marker of inflammation. The induction of VCAM-1 by resistin was inhibited partially by pitavastatin. Moreover, the induction of VCAM-1 and ICAM-1 by resistin was inhibited by adiponectin, an adipocytokine that improves insulin resistance. Taken together, these results suggest that the balance in the concentrations of adipocytokines such as resistin and adiponectin determines the inflammation status of vasculature, and in turn the progress of atherosclerosis.