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Background: Vascular smooth muscle cell (VSMC) proliferation and extracellular matrix (ECM) accumulation play important roles in the development and progression of chronic allograft vasculopathy. Although mycophenolic acid (MPA) inhibits activation of mesenchymal cells through cellular reactive oxygen species (ROS), the exact mechanisms involved in theses processes have not been clearly understood. This study explored the molecular mechanisms whereby MPA inhibits cellular ROS-mediated VSMC proliferation and ECM synthesis., Materials and Methods: Primary rat VSMCs were stimulated with platelet-derived growth factor (PDGF)-BB in the presence or absence of MPA 0.1-10 micromol/L or guanosine 100 micromol/L. Cell proliferation was assessed by methylthiazoletetrazolium and proliferating cell nuclear antigen expression, fibronectin secretion, and rac1 membrane translocation by Western blot analysis, total collagen synthesis by [(3)H]-proline incorporation, dichlorofluorescein-sensitive cellular ROS by confocal microscopy, and hydrogen peroxide (H(2)O(2)) concentration by iodometric analysis., Results: MPA inhibited PDGF-induced VSMC proliferation, ECM synthesis, and cellular ROS, and these inhibitions were partially reversed by exogenous guanosine. MPA at dose inhibiting PDGF-induced VSMC activation inhibited rac1 membrane translocation, and this inhibition was fully recovered by exogenous guanosine. Additionally, MPA rapidly reduced H(2)O(2) concentration in vitro., Conclusions: The present study suggests that MPA inhibits PDGF-induced VSMC proliferation and ECM synthesis through inhibiting rac1-dependent cellular ROS and directly scavenging ROS. Both direct and indirect inhibition of cellular ROS would be the key mechanisms involved in the inhibitory effect of MPA in VSMCs.

Background: Vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix (ECM) synthesis are major pathologic features of chronic allograft vasculopathy. Carvedilol, an anti-hypertensive agent, might be an effective agent for preventing the development and progression of chronic allograft vasculopathy, since it can inhibit VSMC proliferation and migration. The present study was designed to examine the effect of carvedilol on platelet-derived growth factor (PDGF)-induced ECM synthesis in rat VSMCs. Furthermore, we evaluated whether carvedilol inhibits PDGF-induced cellular reactive oxygen species (ROS) and the activation of mitogen-activated protein kinase (MAPK)., Methods: Primary cultured rat VSMCs were stimulated with PDGF-BB (10 ng/ml) in the presence or absence of carvedilol, and the effects of carvedilol were compared with those of ROS or MAPK inhibitors. Fibronectin secretion, proliferating cell nuclear antigen (PCNA) expression, and each MAPK activation were determined by Western blot analysis, total collagen synthesis by [3H]-proline incorporation, and cellular ROS by flow cytometry., Results: PDGF significantly increased PCNA expression, fibronectin secretion, total collagen synthesis, cellular ROS, and MAPK activation in rat VSMCs. Carvedilol at doses that inhibited PDGF-induced cell proliferation, inhibited ECM synthesis, cellular ROS, or subsequent MAPK activation. Structurally different anti-oxidants and extracellular signal-regulated protein kinase or p38 MAPK inhibitor effectively inhibited PDGF-induced fibronectin secretion and total collagen synthesis., Conclusions: These results suggest that carvedilol inhibits PDGF-induced VSMC proliferation and matrix protein synthesis by inhibiting cellular ROS and the subsequent activation of MAPK. Thus the targeted inhibition of cellular ROS and MAPK might provide an effective therapeutic strategy to treat chronic allograft vasculopathy.

Excessive deposition of extracellular matrix (ECM) in the kidney is the hallmark of diabetic nephropathy. Although the amount of ECM deposited in the kidney depends on the balance between the synthesis and degradation of ECM, the role of ECM degradation in matrix remodeling has been less well appreciated. High glucose, advanced glycation end products, angiotensin II, and TGF-beta1 all increase intracellular reactive oxygen species (ROS) in renal cells and contribute to the development and progression of diabetic renal injury. The role of ROS in increased ECM synthesis has been well documented. ROS may also play a critical role in decreased ECM degradation by mediating high glucose- and TGF-beta1-induced inhibition of the proteolytic system, plasmin, and matrix metalloproteinases in the glomeruli. A recent observation suggests that ROS play an important role in tubulointerstitial fibrosis by mediating TGF-beta1-induced epithelial-mesenchymal transition (EMT). Accelerated ECM degradation is required to disrupt tubular basement membrane and complete EMT. ROS thus seem to be involved in both decreased and increased ECM degradation. It is not clear how cells determine when and where to increase or decrease ECM degradation in response to ROS. Precise definition of ROS-activated signaling pathways leading to ECM remodeling in the kidney will provide new strategies to prevent or treat diabetic renal injury.

Diabetic nephropathy is the leading cause of end-stage renal disease worldwide, but no effective therapeutic strategy is available. Because plasminogen activator inhibitor-1 (PAI-1) is increasingly recognized as a key factor in extracellular matrix (ECM) accumulation in diabetic nephropathy, this study examined the renoprotective effects of TM5275 and TM5441, two novel orally active PAI-1 inhibitors that do not trigger bleeding episodes, in streptozotocin (STZ)-induced diabetic mice. TM5275 (50 mg/kg) and TM5441 (10 mg/kg) were administered orally for 16 weeks to STZ-induced diabetic and age-matched control mice. Relative to the control mice, the diabetic mice showed significantly increased (p < 0.05) plasma glucose and creatinine levels, urinary albumin excretion, kidney-to-bodyweight ratios, glomerular volume, and fractional mesangial area. Markers of fibrosis and inflammation along with PAI-1 were also upregulated in the kidney of diabetic mice, and treatment with TM5275 and TM5441 effectively inhibited albuminuria, mesangial expansion, ECM accumulation, and macrophage infiltration in diabetic kidneys. Furthermore, in mouse proximal tubular epithelial (mProx24) cells, both TM5275 and TM5441 effectively inhibited PAI-1-induced mRNA expression of fibrosis and inflammation markers and also reversed PAI-1-induced inhibition of plasmin activity, which confirmed the efficacy of the TM compounds as PAI-1 inhibitors. These data suggest that TM compounds could be used to prevent diabetic kidney injury. [ABSTRACT FROM AUTHOR]

Transforming growth factor β1 (TGFβ1) is a potent stimulator of epithelial-to-mesenchymal transition (EMT) and has been associated with chronic kidney diseases by activating profibrotic gene expression. In this study, we investigated the role of the KEAP1-NRF2 pathway, which is a master regulator of the cellular antioxidant system, in TGFβ1-stimulated EMT gene changes using human renal tubular epithelial HK2. Treatment with TGFβ1 enhanced the levels of reactive oxygen species (ROS) and TGFβ1-stimulated EMT gene changes, including an increase in profibrotic fibronectin-1 and collagen 1A1, were diminished by the antioxidant N-acetylcysteine. In HK2, TGFβ1 suppressed NRF2 activity and thereby reduced the expression of GSH synthesizing enzyme through the elevation of ATF3 level. Therefore, the activation of NRF2 signaling with sulforaphane effectively attenuated the TGFβ1-stimulated increase in fibronectin-1 and collagen 1A1. Conversely, the TGFβ1-EMT gene changes were further enhanced by NRF2 knockdown compared to the control cells. The relationship of NRF2 signaling and TGFβ1-EMT changes was further confirmed in a stable KEAP1-knockdown HK2, which is a model of pure activation of NRF2. The TGFβ1-mediated increase of collagen 1A1 and fibronectin-1 in KEAP1 knockdown HK2 was suppressed. In particular, TGFβ1-SMAD signaling was modulated in KEAP1 knockdown HK2: the TGFβ1-stimulated SMAD2/3 phosphorylation and SMAD transcriptional activity were repressed. Additionally, the protein level of SMAD7, an inhibitor of SMAD signaling, was elevated and the level of SMURF1, an E3 ubiquitin ligase for SMAD7 protein, was diminished in KEAP1 knockdown HK2. Finally, the inhibition of SMAD7 expression in KEAP1 knockdown HK2 restored TGFβ1 response, indicating that SMURF1-SMAD7 may be a molecular signaling linking the NRF2-GSH pathway to TGFβ1-EMT changes. Collectively, these results indicate that the KEAP1-NRF2 antioxidant system can be an effective modulator of TGFβ1-stimulated renal epithelial transition to fibroblastic cells through the SMUR1-SMAD7 signaling, and further implies the beneficial role of NRF2 in chronic renal diseases. [ABSTRACT FROM AUTHOR]

Abstract: Mesangial cell (MC) proliferation and extracellular matrix (ECM) accumulation are major pathologic features of chronic renal disease including chronic allograft nephropathy (CAN). Mycophenolic acid (MPA), a potent immunosuppressant, has emerged as a treatment to prevent CAN because it inhibits MC proliferation and ECM synthesis, but the mechanism involved has not been clarified. The present study examined relative role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) activation in inhibitory effect of MPA on MC activation. Growth arrested and synchronized primary rat MC (passages 7–11) were stimulated by PDGF 10 ng/ml in the presence and absence of clinically attainable dose of MPA (0–10 μM). Cell proliferation was assessed by [3H]thymidine incorporation, fibronectin and the activation of ERK and p38 MAPK by Western blot analysis, and total collagen by [3H]proline incorporation. PDGF increased cell proliferation by 4.6-fold, fibronectin secretion by 3.2-fold, total collagen synthesis by 1.8-fold, and the activation of ERK and 38 MAPK by 5.6-fold and 3.1-fold, respectively, compared to control. MPA, at doses inhibiting PDGF-induced MC proliferation and ECM synthesis, effectively blocked p38 MAPK activation but reduced ERK activation by 23% at maximal concentration tested (10 μM). Exogenous guanosine partially reversed the inhibition of MPA on p38 MAPK activation. Inhibitor of ERK or p38 MAPK suppressed PDGF-induced MC proliferation and ECM synthesis. In conclusion, MPA inhibits p38 MAPK activation leading to inhibiting proliferation and ECM synthesis in MC. Guanosine reduction is partially responsible for inhibitory effect of MPA on p38 MAPK activation in MC. [Copyright &y& Elsevier]

Sequential effects of high glucose on mesangial cell transforming growth factor-β1 and fibronectin synthesis. Background. Transforming growth factor (TGF)-β is recognized as the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion. To gain better understanding of the temporal relationships between high glucose (HG) and mesangial cell (MC) TGF-β1 synthesis and between TGF-β1 and extracellular matrix (ECM) synthesis, the present study examined early and sequential effects of HG on TGF-β1 and fibronectin (FN) mRNA expression and protein synthesis. Methods. Confluent primary rat MC was stimulated with 5.6 (control) or 30 (high) mM glucose after synchronizing the growth by incubation with serum-free media for 48 hours. Results. Mesangial cell TGF-β1 mRNA expression increased significantly in six hours and continued to increase until 48 hours in response to HG. The level of TGF-β1 mRNA was 1.5-fold higher than that of control glucose at six hours and 1.8-fold at 48 hours. TGF-β activity in heat-activated conditioned media under HG increased 1.5- and 1.6-fold at 24 and 48 hours, respectively, compared to control glucose. FN mRNA increased significantly at 24 and 48 hours and 1.4-fold that of control glucose at both time points. FN protein also increased 1.5-fold that of control glucose at 48 hours. Anti-TGF-β antibody completely abolished HG-induced FN synthesis. Conclusions. The present findings demonstrate that HG stimulates TGF-β1 very early and prior to FN production and that HG-induced FN production is mediated by TGF-β. This finding is consistent with the view that TGF-β mediates increased ECM accumulation by MC under high glucose conditions. [ABSTRACT FROM AUTHOR]

Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose. Background Oxidative stress is one of the important mediators of vascular complications in diabetes including nephropathy. High glucose (HG) generates reactive oxygen species (ROS) as a result of glucose auto-oxidation, metabolism, and formation of advanced glycosylation end products. The concept of ROS-induced tissue injury has recently been revised with the appreciation of new roles for ROS in signaling pathways and gene expression. Methods and Results. High glucose rapidly generated dichlorofluorescein-sensitive cytosolic ROS in rat and mouse mesangial cells. Neither L -glucose nor 3-O-methyl- D -glucose increased cytosolic ROS and cytochalasin B, an inhibitor of glucose transporter, effectively inhibited HG-induced ROS generation, suggesting that glucose uptake and subsequent metabolism are required in HG-induced cytosolic ROS generation. H 2 O 2 up-regulated fibronectin mRNA expression and protein synthesis; this up-regulation was effectively inhibited by protein kinase C (PKC) inhibitor or by depletion of PKC. The HG-induced generation of ROS was, in turn, related to activation of PKC and transcription factors nuclear factor-κ;B (NF-κ;B) and activator protein-1 (AP-1) as well as to the up-regulation of transforming growth factor-β1 (TGF-β1), fibronectin mRNA expression and protein synthesis, because antioxidants effectively inhibited HG-induced PKC, NF-κ;B, AP-1 activation, and TGF-β1 and fibronectin expression in mesangial cells cultured under HG. Conclusions Although signal transduction pathways linking HG, ROS, PKC, transcription factors, and extracellular matrix (ECM) protein synthesis in mesangial cells have not been fully elucidated, the current data provide evidence that ROS generated by glucose metabolism may act as integral signaling molecules under HG as in other membrane receptor signaling. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β1 (TGF-β1) promotes tissue fibrosis by upregulating genes encoding extracellular matrix proteins and by increasing the synthesis of plasminogen activator inhibitor-1 (PAI-1). TGF-β1 induces cellular reactive oxygen species (ROS) and PAI-1 promoter region has binding sites for redox sensitive transcription factors. We, therefore, hypothesized that TGF-β1-induced upregulation of PAI-1 is ROS-dependent. Using cultured glomerular mesangial cells, we confirmed that TGF-β1 induces cellular ROS, upregulates PAI-1 mRNA and protein expression, and suppresses plasmin activity. We further demonstrated that H2O2 stimulates PAI-1 expression and suppresses plasmin activity and that N-acetylcysteine effectively reverses TGF-β1- and H2O2-induced changes in PAI-1 expression and plasmin activity. Basal as well as TGF-β1- and H2O2-induced PAI-1 expression was upregulated by depletion of intracellular GSH. The present data demonstrate that TGF-β1-induced PAI-1 in mesangial cells is ROS-dependent and imply that cellular ROS may be potential therapeutic targets in glomerular fibrosis. [Copyright &y& Elsevier]

Abstract: 8-Hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress, has been recently rediscovered to inhibit Rac1 in neutrophils and macrophages, thereby inhibiting Rac1-linked functions of these cells, including reactive oxygen species production through NADPH oxidase activation, phagocytosis, chemotaxis, and cytokine release. In vascular smooth muscle cells (VSMCs), reactive oxygen species also induce abnormal proliferation and migration leading to progression of atherosclerosis. Based upon the involvement of reactive oxygen species in phagocytic cells and VSMCs during the atherosclerotic process, we hypothesized that 8-OHdG could have antiatherosclerotic action and tested this hypothesis in an experimentally induced atherosclerosis in mice. Partially ligated ApoE knockout mice, a more physiologically relevant model of low and oscillatory flow, developed an advanced lesion in 2 weeks, and orally administered 8-OHdG significantly reduced plaque formation along with reduced superoxide formation, monocyte/macrophage infiltration, and extracellular matrix (ECM) accumulation. The effects of 8-OHdG observed in primary VSMCs were consistent with the in vivo effects of 8-OHdG and were inhibitory to angiotensin II or platelet-derived growth factor-induced production of reactive oxygen species, proliferation, migration, and ECM production. Also, angiotensin II-induced Rac1 activity in VSMCs was significantly inhibited by 8-OHdG, and transfection of constitutively active Rac1 reversed the inhibitory effect of 8-OHdG on VSMC activation. Molecular docking study showed that 8-OHdG stabilizes Rac1–GEF complex, indicating the physical contact of 8-OHdG with Rac1. These findings highly suggest that the antiatherosclerotic effect of 8-OHdG is mediated by inhibition of Rac1 activity. In conclusion, our results show a novel action of orally active 8-OHdG in suppressing atherosclerotic plaque formation in vivo and VSMC activation in vitro through inhibition of Rac1, which emphasizes a new therapeutic avenue to benefit atherosclerosis. [Copyright &y& Elsevier]