TGF-␤1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase-derived ROS in TGF-␤1-induced activation of rat kidney fibroblasts and expression of ␣-smooth muscle actin (␣-SMA) and Fn-ED-A. In vitro, TGF-␤1 stimulated formation of abundant stress fibers and increased expression of both ␣-SMA and Fn-ED-A. In addition, TGF-␤1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-␤1-induced stimulation of NADPH oxidase activity and reduced ␣-SMA and Fn-ED-A expression. Inhibition of TGF-␤1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-␤1-enhanced NADPH oxidase activity; and decreased expression of Nox4, ␣-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-␤1-induced Smad3 but reduced both ␣-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked ␣-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-␤1 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2), and this was inhibited by blocking TGF-␤1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased ␣-SMA and Fn-ED-A. Taken together, these results suggest that TGF-␤1-induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.

The small G proteins Rac1 and RhoA regulate actin cytoskeleton, cell shape, adhesion, migration, and proliferation. Recent studies in our laboratory have shown that NADPH oxidase Nox4-derived ROS are involved in transforming growth factor (TGF)-β1-induced rat kidney myofibroblast differentiation assessed by the acquisition of an α-smooth muscle actin (α-SMA) phenotype and expression of an alternatively spliced fibronectin variant (Fn-EIIIA). Rac1 and RhoA are essential in signaling by some Nox homologs, but their role as effectors of Nox4 in kidney myofibroblast differentiation is not known. In the present study, we explored a link among Rac1 and RhoA and Nox4-dependent ROS generation in TGF-β1-induced kidney myofibroblast activation. TGF-β1 stimulated an increase in Nox4 protein expression, NADPH oxidase activity, and abundant α-SMA and Fn-EIIIA expression. RhoA but not Rac1 was involved in TGF-β1 induction of Nox4 signaling of kidney myofibroblast activation. TGF-β1 stimulated active RhoA-GTP and increased Rho kinase (ROCK). Inhibition of RhoA with small interfering RNA and ROCK using Y-27632 significantly reduced TGF-β1-induced stimulation of Nox4 protein, NADPH oxidase activity, and α-SMA and Fn-EIIIA expression. Treatment with diphenyleneiodonium, an inhibitor of NADPH oxidase, did not decrease RhoA activation but inhibited TGF-β1-induced α-SMA and Fn-EIIIA expression, indicating that RhoA is upstream of ROS generation. RhoA/ROCK also regulated polymerase (DNA-directed) δ-interacting protein 2 (Poldip2), a newly discovered Nox4 enhancer protein. Collectively, these data indicate that RhoA/ROCK is upstream of Poldip2-dependent Nox4 regulation and ROS production and induces redox signaling of kidney myofibroblast activation and may broader implications in the pathophysiology of renal fibrosis.

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SummaryPlatelet protein disulphide isomerase (PDI) has a role in platelet aggregation, probably targeting a thiol-containing platelet surface protein. The thiolcontaining P2Y 12 ADP receptor is involved in aggregation induced by most agonists and may be the target of PDI. By excluding the P2Y 12 pathway and using the anti-PDI antibody RL90 this study showed that PDI targets a non-P2Y 12 thiol-protein in aggregation. Anti-PDI inhibited signallingindependent activation of the thiol-containing fibrinogen receptor aIIbb3 by Mn 2+ , suggesting that PDI directly interacts with aIIbb3. The thiolcontaining form of PDI increased on the platelet surface with platelet activation, suggesting that active PDI readily becomes available for redox regulation of aIIbb3. Finally, using purified proteins PDI had greater ability to isomerize disulphide bonds than the aIIbb3 integrin, which also has PDIlike activity. In summary, a mechanism exists in platelets to increase the functional form of surface PDI and this PDI has a non-P2Y 12 target that may be aIIbb3.

Summary Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p‐chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol‐proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol‐containing P2Y12 ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y12 receptor antagonist 2‐MeSAMP to examine thiol‐dependent reactions in the absence of contributions from this receptor. We provide evidence for a non‐P2Y12 thiol‐dependent reaction near the final αIIbβ3‐dependent events of aggregation. We then used 3‐(N‐maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in αIIbβ3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the αIIb and β3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5′‐dithiobis‐(2‐nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in αIIbβ3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling‐independent activation of αIIbβ3 by Mn2+. Our results suggest that the thiols in αIIbβ3 that are blocked by pCMBS are important in the activation of this integrin.

Atherosclerosis is one of the major complications of diabetes and involves endothelial dysfunction, matrix alteration, and most importantly migration and proliferation of vascular smooth muscle cells (VSMCs). Although hyperglycemia and hyperinsulinemia are known to contribute to atherosclerosis, little is known about the specific cellular signaling pathways that mediate the detrimental hyperinsulinemic effects in VSMCs. Therefore, we investigated the cellular mechanisms of hyperinsulinemia-induced migration and proliferation of VSMCs. VSMCs were treated with insulin (100 nM) for 6 days and subjected to various physiological and molecular investigations. VSMCs subjected to hyperinsulinemia exhibited increased migration and proliferation, and this is paralleled by oxidative stress [increased NADPH oxidase activity, NADPH oxidase 1 mRNA expression, and reactive oxygen species (ROS) generation], alterations in mitochondrial physiology (membrane depolarization, decreased mitochondrial mass, and increased mitochondrial ROS), changes in mitochondrial biogenesis-related genes (mitofusin 1, mitofusin 2, dynamin-related protein 1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, peroxisome proliferator-activated receptor gamma coactivator 1-beta, nuclear respiratory factor 1, and uncoupling protein 2), and increased Akt phosphorylation. Diphenyleneiodonium, a known NADPH oxidase inhibitor significantly inhibited migration and proliferation of VSMCs and normalized all the above physiological and molecular perturbations. This study suggests a plausible crosstalk between mitochondrial dysfunction and oxidative stress under hyperinsulinemia and emphasizes counteracting mitochondrial dysfunction and oxidative stress as a novel therapeutic strategy for atherosclerosis.

Summary. Background: Closely spaced thiols in proteins that interconvert between the dithiol form and disulfide bonds are called vicinal thiols. These thiols provide a mechanism to regulate protein function. We previously found that thiols in both aIIb and b3 of the aIIbb3 fibrinogen receptor were required for platelet aggregation. Methods and Results: Using p-chloromercuribenzene sulfonate (pCMBS) we provide evidence that surface thiols in aIIbb3 are exposed during platelet activation. Phenylarsine oxide (PAO), a reagent that binds vicinal thiols, inhibits platelet aggregation and labeling of sulfhydryls in both aIIb and b3. For the aggregation and labeling studies, binding of PAO to vicinal thiols was confirmed by reversal of PAO binding with the dithiol reagent 2,3-Dimercapto-1-propanesulfonic acid (DMPS). In contrast, the monothiol b-mercaptoethanol did not reverse the effects of PAO. Additionally, PAO did not inhibit sulfhydryl labeling of the monothiol protein albumin, confirming the specificity of PAO for vicinal thiols in aIIbb3. As vicinal thiols represent redox sensitive sites that can be regulated by reducing equivalents from the extracellular or cytoplasmic environment, they are likely to be important in regulating activation of aIIbb3. Additionally, when the labeled integrin was passed though a lectin column containing wheat germ agglutinin and lentil lectin a substantial amount of non-labeled aIIbb3 eluted separately from the labeled receptor. This suggests that two populations of integrin exist on platelets that can be distinguished by thiol labeling. Conclusion: A vicinal thiol-containing population of aIIbb3 provides redox sensitive sites for regulation of aIIbb3.

The vicious cycle between hyperinsulinemia and insulin resistance results in the progression of atherosclerosis in the vessel wall. The complex interaction between hyperglycemia and lipoprotein abnormalities promotes the development of atherogenesis. In the early phase of atherosclerosis, macrophage‐derived foam cells play an important role in vascular remodeling. Mechanistic target of rapamycin (mTOR) signaling pathway has been identified to play an essential role in the initiation, progression, and complication of atherosclerosis. Recently sestrin2, an antioxidant, was shown to modulate TOR activity and thereby regulating glucose and lipid metabolism. But the role of sestrin2 in monocyte activation is still not clearly understood. Hence, this study is focussed on investigating the role of sestrin2 in monocyte activation under hyperglycemic and dyslipidemic conditions. High‐glucose and oxidized low‐density lipoprotein (LDL) treatments mediated proinflammatory cytokine production (M1) with a concomitant decrease in the anti‐inflammatory cytokine (M2) levels in human monocytic THP1 cells. Both glucose and oxidized LDL (OxLDL) in a dose and time‐dependent manner increased the mTOR activation with a marked reduction in the levels of pAMPK and sestrin2 expression. Both high‐glucose and OxLDL treatment increased foam cell formation and adhesion of THP1 cells to endothelial cells. Experiments employing activator or inhibitor of adenosine monophosphate kinase (AMPK) as well as overexpression or silencing of sestrin2 indicated that high‐glucose mediated monocyte polarization and adhesion of monocytes to the endothelial cells were appeared to be programmed via sestrin2‐AMPK‐mTOR nexus. Our results evidently suggest that sestrin2 plays a major role in regulating monocyte activation via the AMPK–mTOR‐pathway under diabetic and dyslipidemic conditions and also AMPK regulates sestrin2 in a feedback mechanism.