Plasminogen Activator Inhibitor-1 Antisense Oligodeoxynucleotides Abrogate Mesangial Fibronectin Accumulation

Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-beta1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 microM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-beta1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-beta1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-beta1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.

Effects of Mycophenolic Acid and Rapamycin on Toll-like Receptor Expression in Hypoxic Human Proximal Tubular Epithelial Cells / 대한이식학회지

BACKGROUND: Toll like receptor (TLR), an element of innate immunity, is upregulated by Ischemia/reperfusion (IR) injury and may be involved in adaptive immune response. Immunosuppressive agents may increase or attenuate IR injury and TLR expression. To explore the involvement of TLRs in hypoxic tubular injury and modification by mycophenolic acid (MPA) rapamycin (RAP), this study examined TLR expression in hypoxia-induced human renal proximal tubular epithelial cells (HK-2). METHODS: HK-2 cells were cultured in keratinocyte-SFM media supplemented with epidermal growth factor and bovine pituitary extract. The Induction of hypoxia was achieved using GasPak pouch system. TLR 2, 3, and 4 mRNA expression was analyzed by real time RT-PCR using SYBR green and TLR 4 protein expression was evaluated by Western blot analysis. MPA at concentration of 100 nM and 1uM and RAP at concentration of 20, 50, and 100 nM were added to culture medium. RESULTS: TLR4 but noTLR2 or TLR3 mRNA expressions increased in hypoxic HK-2 cells at 24 and 48 hrs. TLR4 protein expression also increased in hypoxic HK-2 cells at 24 and 48 hrs. MPA (100 nM and 1uM) and RAP (20, 50, and 100 nM) decreased hypoxia-induced TLR4 mRNA expression in HK-2 cells compared to normoxia at 24 hrs. However, TLR4 protein expression was decreased only by RAP at 20 and 50 nM. CONCLUSIONS: The results suggest that RAP may modify hypoxic renal tubular damage by decreasing TLR4-mediated inflammatory and immune reactions.

Effects of Inosine Monophosphate Dehydrogenase Inhibition on Platelet-derived Growth Factor- Induced Fibronectin Secretion and Cellular Reactive Oxygen Species in Mouse Mesangial Cells / 대한이식학회지

PURPOSE: Mesangial cell extracellular matrix (ECM) synthesis plays an important role in various renal diseases. Mycophenolic acid (MPA), which is an inhibitor of inosine monophosphate dehydrogenase (IMPDH), inhibits mesangial cell proliferation and ECM synthesis. However, the exact mechanism of MPA has not been clearly elucidated in mesangial cells. To examine the relative importance of IMPDH on the inhibitory action of MPA, we compared the effects of MPA or IMPDH2 siRNA on platelet-derived growth factor (PDGF)-induced fibronectin secretion and cellular reactive oxygen species (ROS) in mouse mesangial cells (MMC). METHODS: MMC were stimulated with PDGF 10 ng/ml with or without MPA 0.1~10micrometer, IMPDH2 siRNA 10~50 nM, or N-acetylcystein (NAC). IMPDH2 siRNA was transiently transfected by lipofectamine for 24 hours. MPA 0.1~10micrometer, ribavirin 10~100micrometer, and NAC 5 mM were administered 1 hour before the stimulation. Cell viability was measured by methylthiazoletetrazolium (MTT) assay, fibronectin secretion by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS by flow cytometry. RESULTS: PDGF 10 ng/ml effectively increased fibronectin secretion and cellular ROS in MMC. MPA and NAC at concentration without affecting basal level of fibronectin and cellular ROS ameliorated PDGF-induced fibronectin secretion and cellular ROS. However, IMPDH2 siRNA only partially reduced PDGF- induced fibronectin secretion and cellular ROS in MMC. CONCLUSION: These results suggest that MPA may inhibit PDGF-induced fibronectin secretion partly through IMPDH2 or cellular ROS in MMC, and there may be other mechanisms on the inhibitory action of MPA in mesenchymal cells.

Effects of Mycophenolic Acid on Oleic Acid- induced Rat Vascular Smooth Muscle Cell Proliferation

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development and progression of chronic allograft vasculopathy. Mycophenolic acid (MPA) inhibits various mesenchymal cell proliferation, and reactive oxygen species (ROS) are involved in the anti-pro-liferative effect of MPA. In this study, we investigated the effects of MPA on oleic acid (OA)-induced VSMC proliferation and also the role of ROS in these processes. METHODS: Primary cultured rat VSMCs from Sprague-Dawley were stimulated with OA 100micrometer. MPA 0.1~10micrometer and N-acetylcystein (NAC) 5 mM were administered 1 hour before adding the OA. Cell proliferation was measured by Methylthiazoletetrazolium (MTT) assay, proliferating cell nuclear antigen (PCNA) expression by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS by flow cytometry. RESULTS: OA at 100micrometer significantly increased MTT level by 1.6-fold as well as PCNA expression at 48 hours in rat VSMCs. OA also induced DCF-sensitive cellular ROS by 1.6-fold at 5 minutes and the increment of cellular ROS remained for up to 1 hour. MPA at above 1micrometer inhibited OA- induced VSMC proliferation and cellular ROS in a dose-ependent manner. NAC 5 mM also inhibited OA-induced rat VSMC activation. CONCLUSION: These results suggest that MPA inhibits OA-induced VSMC proliferation partially through the inhibition of cellular ROS.

Effects of Tautomycetin on the Proliferation and Fibronectin Secretion in Vascular Smooth Muscle Cells and Glomerular Mesangial Cells / 대한이식학회지

PURPOSE: Proliferation and extracellular matrix (ECM) accumulation in the vascular smooth muscle cell (VSMC) and glomerular mesangial cell (MC) play key roles in the development and the progression of transplant glomerulosclerosis and chronic allograft nephropathy. Tautomycetin (TMC), a newly developed immunosuppressive agent, induces T-lymphocyte apoptosis through the inhibition of tyrosine kinase and protein phosphatase 1. We examined the effects of TMC on platelet-derived growth factor (PDGF)-induced proliferation and ECM synthesis in cultured VSMCs and MCs of Sprague- Dawley rats, and investigated the molecular mechanisms that are involved. METHODS: Different concentrations of TMC were administered 1 hour before the addition of PDGF 10 ng/mL into the growth-arrested and synchronized cells. Cell proliferation was assessed by methylthiazoletetrazolium (MTT) assay. Caspase-3 cleavage, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK were assessed by Western blot analysis, respectively. RESULTS: PDGF 10 ng/ mL increased cell proliferation, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK in both VSMCs and MCs. In both cultured cells, TMC at above 1 microgram/mL significantly reduced basal MTT and increased cleavage caspase-3 in a dose-dependent manner. TMC at 100 ng/mL decreased the PDGF-induced VSMC and MC proliferation without cytotoxicity. However, fibronectin secretion and the activation of Akt, ERK, and p38 MAPK were not affected at this low concentration of TMC, respectively. CONCLUSION: The present data demonstrated that low-dose TMC reduced PDGF-induced VSMC and MC proliferation without affecting the fibronectin secretion and cellular kinase activation.

The Relationship between Serum Creatinine Concentrations and Oxidative Stress or Plasma Levels of Inflammatory Cytokine after Living Donor Renal Transplantation

PURPOSE: Increased oxidative stress and chronic inflammatory process have been substantially linked with the development and progression of chronic renal failure. However, the roles of oxidative stress and inflammation in chronic renal allograft dysfunction are not yet clear. The present study examined the plasma levels of lipid peroxides (LPO), C-reactive protein (CRP), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), transforming growth factor-beta1 (TGF-beta1), and heat shock protein 70 (Hsp 70) as the representative markers of oxidative stress, the inflammatory process and fibrosis among healthy control subjects (kidney donors, n=24), end-stage renal disease (ESRD) patients (n=28), transplant recipients with a serum creatinine (Scr) less than 1.5 mg% (n=30), and transplant recipients with Scr between 1.5 and 5.0 mg% (n=32) during the time at least one year after renal transplantation. METHODS: The plasma LPO concentration was measured by a thiobarbituric acid (TBA) reaction, CRP was measured by latex-enhanced immunonephelometric assay, and TGF-beta1, IL-6, sIL-6R, and HSP 70 were measured by enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: The Plasma concentrations of LPO, TGF-beta1, Hsp 70, and IL-6 were significantly higher in both the ESRD and renal transplant recipients with Scr levels between 1.5 and 5.0 mg% than in both the healthy controls and transplant patients with Scr levels below 1.5 mg%. Plasma sIL-6R was significantly increased only in the ESRD patients. Among the other 3 groups, the concentrations of sIL-6R were not different between the groups. The plasma CRP concentrations were not different among the 4 study groups. Upon regression analysis, the plasma concentrations of LPO, TGF-beta1, Hsp 70, IL-6 and sIL-6R were positively correlated with the Scr level, respectively. CONCLUSION: Our results demonstrate that the markers of oxidative stress, inflammation and fibrosis were correlated with the Scr level which represents the graft (and native) renal function.

Effects of Mycophenolic Acid and Rapamycin on PDGF-induced Mesangial Cell Proliferation and Extracellular Matrix Production / 대한이식학회지

BACKGROUND: Excess proliferation and extracellular matrix (ECM) accumulation of mesenchymal cells such as vascular smooth muscle cells (VSMC) and glomerular mesangial cells cause chronic allograft nephropathy showing transplant vascular sclerosis and glomerulosclerosis. Mycophenolic acid (MPA) and rapamycin (RPM) are well known as strong inhibitors of VSMC proliferation, but their effects on the glomerular mesangial cells are not yet clearly understood. This study examined the effects of MPA or RPM on PDGF-induced proliferation and ECM accumulation in rat glomerular mesangial cells. METHODS: Mesangial cells isolated from the glomeruli of Sprague-Dawley rats were cultured with DMEM containing 20% fetal bovine serum. Growth arrested and synchronized cells were administered with test drugs (MPA10 nM~10micrometer, RPM 0.1 nM~1micrometer) before the addition of PDGF 10 ng/mL. Cell proliferation was assessed by [3H]thymidine incorporation, collagen by [3H]proline incorporation, and fibronectin, ERK, and p38 MAPK by Western blot analysis. RESULTS: PDGF increased mesangial cell proliferation by 4.64-fold. Compared to stimulated control, MPA above 500 nM and RPM above 10 nM showed a significant inhibitory effect in a dose- dependent manner. The IC50 of MPA and RPM against PDGF-induced mesangial cell proliferation were around 500 nM and 100 nM, respectively. The collagen synthesis was also inhibited by MPA and RPM, but the fibronectin secretion was inhibited by MPA alone. The proliferation of mesangial cell correlated with activation of ERK and p38 MAPK. MPA, but not RPM, inhibited ERK and p38 MAPK activation. CONCLUSIONS: This study demonstrated that MPA and RPM significantly inhibited PDGF-induced proliferation and ECM production in rat glomerular mesangial cells. The inhibitory effects of MPA, but not RPM, are correlated with ERK and p38 MAPK.

Rapamycin Inhibits Platelet-Derived Growth Factor- Induced Collagen, but Not Fibronectin, Synthesis in Rat Mesangial Cells

Rapamycin, a macrocyclic lactone, is effective in reducing the incidence of acute rejection after renal transplantation. The inhibitory effects of rapamycin on lymphocyte proliferation and the molecular mechanisms that were involved have been described. However, its effects on glomerular mesangial cells have not been clearly understood, and here, we examined the effect of rapamycin on platelet-derived growth factor (PDGF) - induced extracellular matrix synthesis as well as cell proliferation in mesangial cells. Rat mesangial cells were isolated from the glomeruli of Sprague-Dawley rats and cultured with Dulbecco's modified Eagles medium containing 20% fetal bovine serum. Different concentrations of rapamycin were administered 1 hour before the addition of 10 ng/ml of PDGF into growth arrested and synchronized cells. Cell proliferation was assessed by [3H]thymidine incorporation, total collagen synthesis by [3H]proline incorporation, and fibronectin secretion into the medium by Western blot analysis. In the mesangial cells, PDGF increased cell proliferation by 4.6-fold, total collagen synthesis by 1.8-fold, and fibronectin secretion by 3.2-fold. Rapamycin above 10 nM significantly inhibited PDGF-induced proliferation and collagen synthesis, but the treatment of rapamycin up to 1micrometer did not show any significant effects on PDGF-induced fibronectin secretion. These inhibitory effects of rapamycin on PDGF-induced mesangial cell proliferation and collagen synthesis reflect the potential value of rapamycin in the prevention and treatment of glomerulosclerosis in patients with chronic allograft nephropathy.

Mechanisms Involved in the Inhibitory Effects of Mycophenolic Acid on the PDGF-induced Proliferation of Vascular Smooth Muscle Cells / 대한신장학회잡지

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development and progression of chronic allograft vasculopathy as in atherosclerosis. We already reported that mycophenolic acid (MPA) inhibited VSMC proliferation, cellular reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) in human VSMCs. In this study, we examined further molecular mechanisms involved in the anti-proliferative effect of MPA in rat VSMCs. METHODS: Primary rat VSMCs were stimulated with PDGF-BB 10 ng/mL in the presence or absence of MPA and various kinds of cell signaling inhibitors. Cell proliferation was assessed by [H3]- thymidine incorporation, NAD(P)H oxidase subunits mRNA expression by RT-PCR, dichlorofluorescein- sensitive cellular ROS by FACS, and the activation of PDGF receptor-beta (Tyr 751), rac1, and MAPK by Western blot analysis. RESULTS: PDGF increased cell proliferation and cellular ROS, activation of PDGF receptor-beta (Tyr 751), rac1, expression of p22phox and MOX1 mRNA, ERK 1/2, and p38 MAPK, compared to control. MPA inhibited up-regulation of rac1 phosphorylation, p22phox and MOX1 mRNA expression, cellular ROS, and phosphorylation of ERK 1/2 and p38 MAPK. However, MPA did not affect PDGF receptor-beta (Tyr 751) activation. Wortmannin, diphenyleniodonium (DPI), trolox, and NAC, each inhibited PDGF- induced ERK 1/2 and p38 MAPK activation. PD98059 and p38 MAPK inhibitor also inhibited PDGF-induced cell proliferation. CONCLUSION: These results suggest that MPA inhibits PDGF-induced VSMC proliferation through inhibiting NAD(P)H oxidase-dependent cellular ROS leading to ERK 1/2 and p38 MAPK activation.

Oxidative Stress and Chronic Allograft Nephropathy

Oxidative stress defined as outbalanced generation of reactive oxygen species (ROS) than the existing antioxidative defense mechanisms plays an important role in tissue injury. Ischemia/reperfusion accompanied during organ transplantation is well- established oxidative stress-induced tissue injury. We hypothesized that oxidative stress may also play a role in the development and progression of chronic allograft nephropathy (CAN), since that ROS are major signaling molecules of growth factors and cytokines [platelet-derived growth factors, transforming growth factor-beta1 (TGF-beta1) ] upregulated in the kidney of CAN, that ROS in turn upregulate TGF-beta1, and that mycophenolic acid may inhibit features of CAN [proliferation and extracellular matrix (ECM) accumulation in vascular smooth muscle cells and glomerular mesangial cells] through inhibiting cellular ROS. Cellular ROS activate signal transduction cascade (protein kinase C, mitogen-activated protein kinases, and janus kinases) and transcription factors (nuclear factor-kappa B, activated protein-1, specificity protein 1, and signal transducers and activators of transcription) leading to regulation of genes and proteins involved in cellular proliferation, ECM remodeling, and apoptosis accompanied in CAN. This review is intended to provide an overview of oxidative stress in renal allograft nephropathy.

Effect of Mycophenolic Acid and Rapamycin on the Proliferation and Collagen Synthesis of the Vascular Smooth Muscle Cell of Rat / 대한이식학회지

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation and extracellular matrix protein accumulation play important roles in chronic allograft vasculopathy. Mycophenolic acid (MPA) or rapamycin (RPM) was reported to inhibit VSMC proliferation in vitro and in vivo. However, effects of MPA or RPM on collagen synthesis of VSMCs, and the combined effects of MPA and RPM treatment on VSMC proliferation are not yet reported. METHODS: VSMCs isolated from the aorta of Sprague-Dawley rats were cultured with EMEM supplemented with 10% fetal bovine serum and insulin/ transferrin. Growth arrested and synchronized cells were pretreated with test drugs (alone or combination of various concentrations of MPA and RPM) 1 hour before the addition of 10 ng/ml PDGF. Cell proliferation was assessed by [H3]- thymidine incorporation, and collagen synthesis by [H3]- proline incorporation. RESULTS: PDGF increased cell proliferation and collagen synthesis by 3.4- and 2.1-fold, respectively, compared to control. MPA at above 100 nM or RPM at above 1 nM effectively inhibited PDGF-induced cell proliferation and collagen synthesis. The IC50 of MPA or RPM against PDGF-stimulated cell proliferation was between 100 nM and 1 micrometer or between 1 nM and 10 nM, respectively. The combination of MPA and RPM showed additive effects on PDGF-induced VSMC proliferation in a multiple regression analysis (R2=0.508, P<0.05). CONCLUSION: The present study demonstrated that MPA or RPM significantly inhibited PDGF-induced VSMC proliferation. These independent phenomena were well maintained as suggested by additive effects after combination treatment. PDGF-induced collagen synthesis was also effectively suppressed by the treatment of MPA or RPM.

Effect of Mycophenolic Acid and Rapamycin on the Proliferation of Glomerular Mesangial Cell of Rat

PURPOSE: Excess proliferation of mesenchymal cells such as vascular smooth muscle cells and glomerular mesangial cells, cause transplant vascular sclerosis and glomerulosclerosis, which are typical pathological lesions of chronic allograft dysfunction. Mycophenoic acid (MPA) and rapamycin (RPM) were recently reported to have strong anti-proliferative potentials toward vascular smooth muscle cells. However, the potential effects of these drugs, either alone or in combination, on glomerular mesangial cells, remain to be reported. METHODS: Primary cultured mesangial cells, from Sprague-Dawley rats, were isolated, and stimulated with 10ng/ml of PDGF. The test drugs MPA and RPM were administered at various concentrations, either alone or in combination, 15 minutes before the addition of the PDGF. The cell proliferation was assessed by [3H]-thymidine incorporation. RESULTS: The PDGF effectively stimulated the proliferation of the mesangial cells. The MPA inhibited the proliferation in a dose-dependent manner. In comparison to the stimulated control, the MPA (above 500 nM) showed a significant inhibitory effect. The IC50 of the MPA, against PDGF-stimulated mesangial cell proliferation, was between 500 nM and 1microM. The RPM, at 10 nM, showed a significant inhibitory effect. In a linear regression analysis, the RPM was supposed to suppress the mesangial proliferation in a dose-dependent manner (P<0.05). The pattern of inhibition for the MPA and RPM combination was very similar to that of either the MPA or the RPM alone. Both the MPA and RPM were shown to independently suppress the mesangial proliferation from a multiple regression analysis (R2=0.415, P<0.001). CONCLUSION: We demonstrated that MPA and RPM significantly inhibited the proliferation of glomerular mesangial cells, and that these effects were well maintained when used in combination. Our data indicate that both MPA and RPM have unique potentials in preventing the development of transplant mesangial proliferation in renal transplant recipients.

Effects of Carvedilol on PDGF-induced Collagen Synthesis and Signal Transduction in Rat Vascular Smooth Muscle Cell / 대한이식학회지

PURPOSE: Proliferation, migration, and the accumulation of extracellular matrix (ECM) protein of vascular smooth muscle cells (VSMC) play roles for transplant arteriosclerosis. We have previously reported that carvedilol (CA) inhibits the proliferation and the migration of VSMCs. The present study examined the effects of CA on platelet-derived growth factor (PDGF)-induced collagen synthesis in VSMC and the roles of reactive oxygen species (ROS), extracellular signal- regulated protein kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). METHODS: Primary cultured rat VSMCs were obtained from aorta of Sprague-Dawley rats. Growth arrested and synchronized cells were pretreated with CA (10 nM~10micrometer) at 1 hour before the addition of PDGF 10 ng/ml. Collagen synthesis was measured by 3[H]-proline incorporation, ROS by flow cytometry using ROS-sensitivedichlorofluorescein (DCF) dye, and the activation of ERK andp38 MAPK by Western blot analysis. RESULTS: PDGF significantly increased collagen synthesis by 2.0-fold, intracellular ROS by 1.6-fold, the activation of ERK 1/2 and p38 MAPK by 4.2-fold and 3.9-fold compared to control, respectively. CA above 1micrometer inhibited PDGF-induced collagen synthesis. CA also inhibited DCF-sensitive ROS and the activation of ERK and p38 MAPK. All pharmacological inhibitors of ROS, ERK, and p38 MAPK effectively inhibited PDGF-induced collagen synthesis. CONCLUSION: These data suggest that CA inhibit PDGF-induced collagen synthesis possibly through inhibiting intracellular ROS and ERK 1/2 and p38 MAPK activation.

Increased Intracellular Reactive Oxygen Species in Peripheral Blood Mononuclear Cells from Renal Transplant Recipients with Decreased Graft Function / 대한이식학회지

PURPOSE: Oxidative stress plays an important role in the development and progression of renal injury. However, the role of reactive oxygen species (ROS) in renal allograft dysfunction is not clear. The present study examined the level of intracellular ROS in healthy control (kidney donor, n=37), end-stage renal disease (ESRD) patients (n=36), transplant recipients with serum creatinine (Scr) less than 1.5 mg% (n=33), and recipients with Scr between 1.5 and 5.0 mg% (n=36) at least one year after renal transplantation. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque gradient method. Dichlorofluorescein (DCF)-sensitive ROS was measured by flow cytometry and expressed as an arbitrary unit. RESULTS: Basal ROS production in PBMC was significantly increased in ESRD patients compared to healthy control. Basal ROS production in both transplant patient groups was not significantly different from healthy control. Phorbol-12-myristate-13- acetate (PMA) and hydrogen peroxide significantly enhanced intracellular ROS in all 4 groups. PMA- and hydrogen peroxide-induced cellular ROS was significantly higher in renal recipients with Scr between 1.5 and 5.0 mg% than in both healthy control and patients with Scr below 1.5 mg%. In regression analysis all, PMA- and hydrogen peroxide- induced as well as basal intracellular ROS in PBMC was correlated with Scr. CONCLUSION: Our results demonstrate that oxidative stress correlates with the declining of renal graft function.

Effects of Mycophenolic Acid, Rapamycin, and Carvedilol on the PDGF-induced Fibronectin Secretion by Rat Vascular Smooth Muscle Cells: Implication of MAP Kinase / 대한이식학회지

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation and extra-cellular matrix (ECM) protein accumulation play important roles in transplant vascular sclerosis and re- stenosis after balloon vascular injury. Mycophenolic acid (MPA), rapamycin (RPM), and carvedilol (CA) were proven to inhibit the proliferation of VSMC. Fibronectin is a multifunctional ECM protein and induces tissue fibrosis. Since mitogen-activated protein kinases (MAPK) are upstream signaling molecules of VSMC proliferation and fibronectin production, this study examined the effects of MPA, RPM, and CA on the fibronectin secretion and MAPK activation in rat VSMC stimulated by platelet derived growth factor (PDGF). METHODS: VSMC was isolated from the aorta of male Sprague-Dawley rat, weighing 200-250 g and cultured with EMEM containing 10% fetal bovine serum and insulin/transferrin supplement. Near confluent VSMC were incubated with serum-free media for 48 hours to arrest and synchronize the cell growth. Test drugs were administered 15 minutes before the addition of PDGF 10 ng/mL. Cell proliferation, fibronectin secretion, and MAPK activation in VSMCs were measured by Western blot analysis. RESULTS: PDGF induced cell proliferation, fibronectin secretion, and extracellular- regulatary protein kinase 1/2 (ERK 1/2) and p38 MAPK activation by 1.7-, 1.5-, 3.3-, 3.9-fold, respectively, compared to control. MPA (>1 microM), CA (>100 nM), PD98059 (>30 microM), and p38 MAPK inhibitor (>10 nM) effectively inhibited PDGF-induced proliferation and fibronectin secretion. RPM, up to 100 nM, effectively inhibited cell proliferation, but did not inhibit fibronectin secretion. MPA and CA, but not RPM, inhibited PDGF-induced ERK 1/2 and p38 MAPK activation. CONCLUSION: The present study demonstrates that MPA and CA inhibit both cell proliferation and fibronectin secretion in rat VSMC stimulated by PDGF. Inhibition of both ERK 1/2 and p38 molecules are significantly associated with these events. Even though, it has a significant anti-proliferative effect on the rat VSMC, RPM neither affected the phosphorylation of ERK 1/2 and p38 nor secretion of fibronectin. These data suggest that ERK 1/2- and p38 MAPK-independent, more proximal pathway may exist for PDGF-induced proliferation of rat VSMC.

Effect of Carvedilol on Human Vascular Smooth Muscle Cell Proliferation and Its Signal Transduction

PURPOSE: Vascular smooth muscle cells (VSMCs) migration and proliferation play important roles in transplant vascular sclerosis and restenosis after balloon vascular injury. The anti-proliferative and anti- migratory effects of carvedilol (CA), a unique alpha- and beta-blocking anti-hypertensive drug, on the VSMCs were confirmed previously. Since reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) family play important roles in proliferation of VSMCs, the present study examined the effects of CA on intracellular ROS generation, activation of ERK1/2 and p38 MAPK, and proliferation of VSMCs cultured under platelet derived growth factor (PDGF). METHODS: Human VSMCs obtained from ATCC were cultured with RPMI-1640 containing 10% fetal bovine serum. Near confluent VSMCs were incubated with serum-free media for 48 hours to arrest and synchronize the cell growth. CA was administered 1 hour before the addition of PDGF. 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (DCF)-sensitive intracellular ROS was detected by FACS. Activations of ERK1/2 and p38 MAPK were measured by Western blot analysis. Proliferation of VSMCs was assessed by [3H]-thymidine incorporation. RESULTS: PDGF at 10 ng/ml, which induced human VSMCs proliferation, rapidly increased intracellular ROS by 1.6-fold (P<0.01), ERK1/2 activation by 2.1-fold (P<0.01), and p38 MAPK activation by 1.9-fold (P<0.01), respectively, as compared to the control. CA 1 and 10muM effectively inhibited PDGF-induced human VSMCs proliferation. CA also effectively inhibited PDGF-induced intracellular ROS generation as well as ERK1/2 and P38 MAPK activation. CONCLUSION: The present study suggests that CA inhibits PDGF-induced human VSMCs proliferation, possibly by inhibiting intracellular ROS generation and activation of ERK1/2 and p38 MAPK.

Effect of Carvedilol Alone or with Cyclosporine on the Migration of Cultured Rat Vascular Smooth Muscle Cell

PURPOSE: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs), which are triggered by endothelium-derived cytokines or growth factors, play a major role in the chronic transplant vasculopathy or vascular remodeling process after vascular injury. We have reported that carvedilol, a new anti-hypertensive agent, inhibits cytokine-triggered proliferation of cultured rat VSMCs. In this study, we investigate the effect of carvedilol on the migration of rat VSMCs. METHODS: Growth-arrested cultured VSMCs (passage 8-11) from the aorta of rat (Sprague-Dawley) were used. Migration was measured using a microchemotaxis chamber with a polycarbonate membrane. Platelet derived growth factor (PDGF) or angiotensin-II (ANG-II) was used as a stimulator and was added into the lower well of the chamber. A density of 1X104 cells per well with carvedilol and/or cyclosporine A (CsA) was seeded into the upper well of chamber. Degree of migration was assessed by using the number of migrated cells per high power field of light microscopy. RESULTS: PDGF and ANG-II stimulated VSMC chemotaxis effectively. Carvedilol decreased PDGF-induced migration to 88.9 (+/-16.0)% and 37.4 (+/-10.5)% at 1 microM and 10 microM, respectively. Carvedilol inhibited both PDGF and ANG-IIinduced chemotaxis in a concentration-dependent manner. The IC50 of carvedilol in PDGF and ANG-ll-induced VSMC migration was around 10 microM. CsA (100 nM) neither significantly inhibited the migration of VSMC, regardless of the kinds of cytokines, nor affected the inhibitory activities of carvedilol. The pattern of inhibition in the group with a combined addition of carvedilol and CsA was very similar to that of carvedilol alone group, regardless of the kinds of cytokines. CONCLUSION: We demonstrated that carvedilol alone or in the presence of CsA significantly inhibited the cytokine- induced migration of VSMC. These data indicate that carvedilol has a unique potential to reduce the development of chronic transplant vasculopathy when used with CsA in hypertensive renal transplant recipients.

Effect of Carvedilol Alone or with Cyclosporine on the Proliferation of Rat Vascular Smooth Muscle Cell / 대한이식학회지

PURPOSE: Typical pathologic lesions of chronic allograft rejection or transplant vascular sclerosis are similar to arteriosclerotic vascular lesion of non-transplant patients, or vascular remodeling process after vascular injury. Abnormal and excess proliferation of vascular smooth muscle cells (VSMC) which are triggered by endothelium-derived cytokines or growth factors, play a major role during these process. Effective prophylactic or therapeutic strategies against chronic rejection or transplant vasculopathy is not yet clearly established. Recent in vitro cell culture study showed that carvedilol, a novel antihypertensive agent has the significant inhibitory activities against the proliferation of VSMC. METHODS: Using in vitro VSMC culture techniques, we measured anti-proliferative activity of carvedilol alone, or in combination of cyclosporine, a basic immunosuppressive agent for transplantation. Growth-arrested early passage (3-5) cultured VSMC from the aorta of rat (Sprague-Dawley) were exposed to platelet derived growth factor (PDGF), endothelin-l, or antiotensin-ll, respectively. Carvedilol and/or cyclosporine was added as inhibitors. Proliferation was assessed by incorporated [(3)H]-thymidine activity. RESULTS: PDGF stimulated mitogenesis most effectively. Carvedilol inhibited mitogenesis in dose-dependent manner in the presence of PDGF(10ng/ml). Compared to control, proliferation was significantly decreased to 60.3 (+/-10.4)% and 18.3 (+/- 5.9)% in the presence of 1 micro M and 10 micro M of carvidilol, respectively (p<0.05, each). Carvedilol also produced significant concentration-dependent inhibitory activities against VSMC stimulated by endothelin-1 (10 nM) and angiotensin-II (100 nM). The IC50 of carvedilol in PDGF-, endothelin-l, and angiotensin-ll-stimulated VSMC were 1-10 micro M. Cyclosporine (100 nM) did not show significant inhibition of VSMC regardless of the kinds of cytokines. However, combined addition of carvedilol and cyclosporine produced significant VSMC inhibition. The pattern of inhibition in c very similar with that of carvedilol alone group regardless of the kinds of cytokines. CONCLUSION: We demonstrated that carvedilol significantly inhibited the proliferation of VSMC regardless of the kind of cytokines, and even under the presence of cyclosporine in VSMC cultures. These indicated that carvedilol has the unique potential to reduce the development of transplant vasculopathy when used with cyclosporine in hypertensive renal transplant recipients.