Triglyceride-rich lipoproteins in chronic kidney disease patients undergoing maintenance haemodialysis treatment.

OBJECTIVE: Plasma triglyceride (TG) levels were reported to be high in chronic kidney disease (CKD) patients undergoing haemodialysis (HD) treatment. One of the atherogenic causes of hypertriglyceridemia is the increase in TG-rich lipoprotein remnants, which are equivalent to remnant-like particle cholesterol (RLP-C). Here, we compared the plasma levels of TG, a representative indicator of TG-rich lipoproteins and RLP-C, as well as the TG/RLP-C ratio between CKD patients undergoing HD and controls, in an effort to elucidate the atherogenicity of TG-rich lipoproteins in CKD patients on HD. MATERIALS AND METHODS: Plasma lipid and apo(lipo)protein levels and the TG/RLP-C ratio were compared between 49 CKD patients undergoing HD and 627 controls. Blood sampling for lipid and apoprotein analysis was performed in a 12-h fasting state. Controls were divided into four subgroups according to TG level (from highest to lowest). RLP-C and apo(lipo)proteins were measured using the immunoprecipitation method and turbidimetric immunoassay, respectively. In addition, a comparison between HD patients and age-, gender-, and plasma TG level-matched controls was performed. RESULTS: Plasma TG levels were 107 ± 70 (mean ± SD) mg/dl in HD patients and 115 ± 72 mg/dl in controls. Plasma RLP-C levels were 6.7 ± 4.5 mg/dl in HD patients and 4.6 ± 3.5 mg/dl in the controls (p < 0.0001). RLP-C levels decreased in descending order from the highest to the lowest TG group in controls. RLP-C levels were higher in HD patients than in controls with plasma TG levels of < 150 mg/dl (p < 0.0001). TG/RLP-C ratios were 19.0 ± 12.0 in HD patients and 25.9 ± 9.5 in controls (p < 0.0001). This ratio was significantly lower in HD patients than in all four TG subgroups. The comparison between HD patients and age-, gender-, plasma TG-matched controls revealed identical results. CONCLUSION: Plasma RLP-C levels were high, and the TG/RLP-C ratio was low in CKD patients undergoing HD treatment. These findings indicate that total plasma TG-rich lipoprotein levels were not increased, but the distribution of plasma TG-rich lipoproteins were skewed towards remnant fractions in CKD patients undergoing HD treatment; these plasma TG-rich lipoproteins appear to be more atherogenic than those in controls.

Role of mineralocorticoid receptor/Rho/Rho-kinase pathway in obesity-related renal injury.

OBJECTIVE: We examined whether aldosterone/Rho/Rho-kinase pathway contributed to obesity-associated nephropathy. SUBJECTS: C57BL/6J mice were fed a high fat or low fat diet, and mice on a high fat diet were treated with a mineralocorticoid receptor antagonist, eplerenone. RESULTS: The mice on a high fat diet not only developed obesity, but also manifested renal histological changes, including glomerular hypercellularity and increased mesangial matrix, which paralleled the increase in albuminuria. Furthermore, enhanced Rho-kinase activity was noted in kidneys from high fat diet-fed mice, as well as increased expressions of inflammatory chemokines. All of these changes were attenuated by eplerenone. In high fat diet-fed mice, mineralocorticoid receptor protein levels in the nuclear fraction and SGK1, an effector of aldosterone, were upregulated in kidneys, although serum aldosterone levels were unaltered. Furthermore, aldosterone and 3ß-hydroxysteroid dehydrogenase in renal tissues were upregulated in high fat diet-fed mice. Finally, in cultured mesangial cells, stimulation with aldosterone enhanced Rho-kinase activity, and pre-incubation with eplerenone prevented the aldosterone-induced activation of Rho kinase. CONCLUSION: Excess fat intake causes obesity and renal injury in C57BL/6J mice, and these changes are mediated by an enhanced mineralocorticoid receptor/Rho/Rho-kinase pathway and inflammatory process. Mineralocorticoid receptor activation in the kidney tissue and the subsequent Rho-kinase stimulation are likely to participate in the development of obesity-associated nephropathy without elevation in serum aldosterone levels.

T-type calcium channel blockade as a therapeutic strategy against renal injury in rats with subtotal nephrectomy.

T-type calcium channel blockers have been previously shown to protect glomeruli from hypertension by regulating renal arteriolar tone. To examine whether blockade of these channels has a role in protection against tubulointerstitial damage, we used a stereo-selective T-type calcium channel blocker R(-)-efonidipine and studied its effect on the progression of this type of renal injury in spontaneously hypertensive rats that had undergone subtotal nephrectomy. Treatment with racemic efonidipine for 7 weeks significantly reduced systolic blood pressure and proteinuria. The R(-)-enantiomer, however, had no effect on blood pressure but significantly reduced proteinuria compared to vehicle-treated rats. Both agents blunted the increase in tubulointerstitial fibrosis, renal expression of alpha-smooth muscle actin and vimentin along with transforming growth factor-beta (TGF-beta)-induced renal Rho-kinase activity seen in the control group. Subtotal nephrectomy enhanced renal T-type calcium channel alpha1G subunit expression mimicked in angiotensin II-stimulated mesangial cells or TGF-beta-stimulated proximal tubular cells. Our study shows that T-type calcium channel blockade has renal protective actions that depend not only on hemodynamic effects but also pertain to Rho-kinase activity, tubulointerstitial fibrosis, and epithelial-mesenchymal transitions.

Beneficial action of candesartan cilexetil plus amlodipine or ACE inhibitors in chronic nondiabetic renal disease.

Although multiple antihypertensive agents are required to control blood pressure (BP) in chronic renal disease, it remains undetermined whether the combination therapy with angiotensin receptor blockers (ARB) plus calcium antagonists or angiotensin-converting enzyme inhibitors (ACEI) confers more preferable action on renal disease than the ARB monotherapy. In the present study, we compared the effect of the combination therapy with ARB plus calcium antagonists/ACEI on proteinuria with that of the ARB monotherapy in chronic nondiabetic renal disease. At 1 month of the drug treatment, the candesartan monotherapy (n=19) reduced BP from 154+/-3/93+/-2 to 146+/-3/88+/-2 mmHg (P<0.05), and a similar magnitude of BP reductions was observed with the combination therapy with candesartan plus ACEI/amlodipine (from 153+/-2/95+/-2 to 144+/-2/88+/-2 mmHg, P<0.05, n=39). The depressor action of these therapies was sustained throughout the 12-month treatment. In contrast, the reduction in proteinuria was greater with the combination therapy (-52+/-3% at 12 months, n=39) than with the candesartan monotherapy (-25+/-3%, n=19), although the baseline values of proteinuria were nearly the same in the candesartan monotherapy group (1.74+/-0.22 g/day) and the combination therapy group (2.10+/-0.19 g/day, P>0.2). Of note, the proteinuria-sparing effect did not differ between the candesartan+ACEI group and the candesartan+amlodipine group. In conclusion, the present study suggests more beneficial action of the combination therapy with ARB plus ACEI/amlodipine than the ARB monotherapy in nondiabetic renal disease. Since the reduction in BP was achieved to the same level, the distinct proteinuria-sparing action of these therapies is attributed to BP-independent mechanisms, which should vary depending on the agents used.

Peroxisome proliferator-activated receptor gamma ligands inhibit mitogenic induction of p21(Cip1) by modulating the protein kinase Cdelta pathway in vascular smooth muscle cells.

The cyclin-dependent kinase inhibitor p21(Cip1) is up-regulated in response to mitogenic stimulation in various cells. PPARgamma ligands troglitazone (TRO, 10 microm) and rosiglitazone (RSG, 10 microm) attenuated the induction of p21(Cip1) protein by platelet-derived growth factor (PDGF) and insulin without affecting cognate mRNA levels in rat aortic smooth muscle cells (RASMC). The protein kinase Cdelta (PKCdelta) inhibitor rottlerin also blocked the induction of p21(Cip1) protein, whereas the conventional PKC isotype inhibitor Gö 6976 had no effect. Kinetic studies using the protein synthesis inhibitor cycloheximide showed that TRO, RSG, and rottlerin shortened the half-life of p21(Cip1) protein. TRO, RSG, and rottlerin inhibited PDGF-induced expression of p21(Cip1), but they did not affect insulin-induced expression of p21(Cip1). Both ligands inhibited PKCdelta enzymatic activity in PDGF-stimulated RASMC but not in insulin-stimulated cells. Adenovirus-mediated overexpression of PKCdelta rescued the down-regulation of p21(Cip1) expression both by TRO and RSG in PDGF-treated RASMC. These data suggested that the PKCdelta pathway plays a critical role in PDGF-induced expression of p21(Cip1) in RASMC and may be the potential target for PPARgamma ligand effects. Src kinase-dependent tyrosine phosphorylation of PKCdelta was decreased substantially by TRO and RSG. Tyrosine phosphorylation and activation of c-Src in response to PDGF were unaffected by either PPARgamma ligand. Protein-tyrosine-phosphatase inhibitors sodium orthovanadate and dephostatin prevented PPARgamma ligand effects on PKCdelta tyrosine phosphorylation and enzymatic activity. Both inhibitors also reversed PPARgamma ligand effects on p21(Cip1) expression in PDGF-treated RASMC. PPARgamma ligands enhanced protein-tyrosine-phosphatase activity in RASMC, which may be the mechanism for decreased PKCdelta tyrosine phosphorylation and activity. PPARgamma ligands regulate p21(Cip1) at a post-translational level by blocking PKCdelta signaling and accelerating p21(Cip1) turnover.

Troglitazone inhibits growth of MCF-7 breast carcinoma cells by targeting G1 cell cycle regulators.

Peroxisome proliferator activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligand activation of PPARgamma has been shown to cause growth arrest in several human tumor cell types, but the underlying molecular mechanism has not been elucidated. We report here that the PPARgamma ligand troglitazone (TRO) inhibited MCF-7 cell proliferation by blocking events critical for G1 --> S progression. Flow cytometry demonstrated that TRO at 20 microM increased the percentage of cells in G1 from 51 to 69% after 24 h. Accumulation of cells in G1 was accompanied by an attenuation of Rb protein phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TRO correlates with decreased protein levels for several G1 regulators of Rb phosphorylation (cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7 cells from TRO-mediated G1 arrest. Targeting of G1 regulatory proteins, particularly cyclin D1, and the resulting induction of G1 arrest by TRO may provide a novel antiproliferative therapy for human breast cancer.

Retinoids inhibit proliferation of human coronary smooth muscle cells by modulating cell cycle regulators.

Retinoids inhibit rat vascular smooth muscle cell (VSMC) proliferation in vitro and intimal hyperplasia in vivo. We examined the mechanism of the antiproliferative effect of retinoids on human coronary artery smooth muscle cells (human CASMCs). The RAR ligands all-trans-retinoic acid (atRA) and ethyl-p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-l-propenyl]-benzoic acid (TTNPB); a pan-RXR/RAR agonist, 9-cis-retinoic acid (9cRA); and the RXR-selective ligand AGN4204 all inhibited DNA synthesis stimulated with platelet-derived growth factor and insulin (IC(50): TTNPB 63 nmol/L, atRA 120 nmol/L, AGN4204 460 nmol/L, 9cRA 1.5 micromol/L). All retinoids blocked cell cycle progression as determined by flow cytometry and inhibited retinoblastoma protein (Rb) phosphorylation. TTNPB, atRA, and AGN4204 inhibited the mitogenic induction of cyclin D1, whereas 9cRA had no effect. None of the retinoids affected the expression of CDK 2, 4, or 6 or cyclin E. All retinoids attenuated mitogen-induced downregulation of CDKI p27(Kip1), a major negative regulator of Rb phosphorylation, partly through stabilizing p27(Kip1) turnover. These data demonstrate that retinoids have antiproliferative activity by modulating G(1) --> S cell cycle regulators in human CASMCs through inhibition of Rb phosphorylation and elevation of p27(Kip1) levels.

Doxazosin inhibits monocyte chemotactic protein 1-directed migration of human monocytes.

Monocyte chemotactic protein 1 (MCP-1)-directed transendothelial migration of monocytes plays a key role in the early development of atherosclerosis. Migration of monocytes requires degradation of extracellular matrices, a process that involves matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP). Recent studies suggest that the alpha1-adrenergic receptor antagonist doxazosin (Dox) might have antiatherosclerotic effects, although the underlying mechanisms are poorly understood. The purpose of the present study was to determine the effects of Dox on MCP-1-directed monocyte migration, MMP-9 activity, and TIMP-1 expression. MCP-1 (50 ng/ml) stimulated migration of human peripheral blood monocytes (HPBM) 2.7+/-0.42-fold and THP-1 human monocytes 5.9+/-0.83-fold compared with unstimulated control. Dox inhibited MCP-1-induced migration in a dose-dependent manner, with a maximal reduction at 10 microM of 69.5+/-5.9% in HPBM and 72.2+/-3.2% in THP-1 cells. Dox blocked migration even after pretreatment with phenoxybenzamine, an irreversible alpha1-adrenergic receptor antagonist (HPBM: phenoxybenzamine 1 microM + Dox 10 microM, 71.9+/-2.2% inhibition; THP-1 cells: phenoxybenzamine 1 microM + Dox 10 microM: 78+/-7.7% inhibition), suggesting that the antimigratory activity of Dox is mediated through a novel mechanism unrelated to its blocking of the alpha1-adrenergic receptor. Dox (10 microM) inhibited MMP-9 activity by 67.6+/-10.5%, whereas MMP-9 protein levels were not affected. Also, Dox increased PMA-induced-tissue inhibitor of MMPs-1 (TIMP-1) expression by 134.4+/-6.6%. Dox 10 microM. The present study demonstrates a potential novel antiatherosclerotic action of Dox by blocking MCP-1-directed monocyte migration, which might be partly mediated by inhibition of MMP-9 activity.

Troglitazone inhibits formation of early atherosclerotic lesions in diabetic and nondiabetic low density lipoprotein receptor-deficient mice.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a ligand-activated nuclear receptor expressed in all of the major cell types found in atherosclerotic lesions: monocytes/macrophages, endothelial cells, and smooth muscle cells. In vitro, PPARgamma ligands inhibit cell proliferation and migration, 2 processes critical for vascular lesion formation. In contrast to these putative antiatherogenic activities, PPARgamma has been shown in vitro to upregulate the CD36 scavenger receptor, which could promote foam cell formation. Thus, it is unclear what impact PPARgamma activation will have on the development and progression of atherosclerosis. This issue is important because thiazolidinediones, which are ligands for PPARgamma, have recently been approved for the treatment of type 2 diabetes, a state of accelerated atherosclerosis. We report herein that the PPARgamma ligand, troglitazone, inhibited lesion formation in male low density lipoprotein receptor-deficient mice fed either a high-fat diet, which also induces type 2 diabetes, or a high-fructose diet. Troglitazone decreased the accumulation of macrophages in intimal xanthomas, consistent with our in vitro observation that troglitazone and another thiazolidinedione, rosiglitazone, inhibited monocyte chemoattractant protein-1-directed transendothelial migration of monocytes. Although troglitazone had some beneficial effects on metabolic risk factors (in particular, a reduction of insulin levels in the diabetic model), none of the systemic cardiovascular risk factors was consistently improved in either model. These observations suggest that the inhibition of early atherosclerotic lesion formation by troglitazone may result, at least in part, from direct effects of PPARgamma activation in the artery wall.

Angiotensin II induces migration and Pyk2/paxillin phosphorylation of human monocytes.

Angiotensin (Ang) II has been shown to enhance the development of atherosclerotic lesions. Migration of monocytes is an early critical step in the atherosclerotic process. To elucidate mechanisms by which Ang II promotes atherogenesis, we investigated its effects on human monocyte migration. Ang II induced migration of human peripheral blood monocytes (HPBM) and human THP-1 monocytes at concentrations between 0.01 and 1 micromol/L, with a 3.6+/-0.6-fold induction in HPBM and a 4.8+/-0.9-fold induction in THP-1 cells at 1 micromol/L Ang II (both P<0.01 versus unstimulated cells). Addition of the Ang II receptor type 1 (AT1-R) antagonist losartan (1 to 100 micromol/L) suppressed Ang II-induced migration of HPBM and THP-1 monocytes in a dose-dependent manner, demonstrating an AT1-R-mediated mechanism. Ang II-directed migration was also blocked by the Src kinase inhibitor PP2 (10 micromol/L), by the extracellular-regulated protein kinase (ERK 1/2) inhibitor PD98059 (30 micromol/L), and by the p38-MAPK inhibitor SB203580 (10 micromol/L), indicating that Src, ERK 1/2, and p38 are all involved in Ang II-induced migration of HPBM and human THP-1 monocytes. The proline-rich tyrosine kinase 2 (Pyk2) and paxillin are 2 cytoskeleton-associated proteins involved in cell movement, phosphorylated by Ang II in other cell types, and abundantly expressed in monocytes. Ang II (1 micromol/L) induced Pyk2 and paxillin phosphorylation in human THP-1 monocytes, peaking after 10 minutes for Pyk2 with a 6.7+/-0.9-fold induction and after 2 minutes for paxillin with a 3.2+/-0.4-fold induction. Ang II-induced phosphorylation of both proteins was suppressed by losartan and the Src inhibitor PP2, whereas no effect was observed with PD98059 and SB203580. This study demonstrates a novel proatherogenic action of Ang II on human monocytes by stimulating their migration, through an AT1-R-dependent process, involving signaling through Src, ERK 1/2, and p38. Furthermore, the promigratory actions of Ang II in human monocytes are associated with the phosphorylation of 2 cytoskeleton-associated proteins, Pyk2 and paxillin.

Expression and function of peroxisome proliferator-activated receptor-gamma in mesangial cells.

P:eroxisome proliferator-activated receptor-gamma (PPARgamma) is a novel nuclear receptor, which enhances insulin-mediated glucose uptake. Ligands to PPARgamma are currently used as therapy for type II diabetes. Using Western blot analysis, RNase protection assay, and immunostaining, we identified the presence of PPARgamma message and protein in cultured primary rat mesangial cells. Electrophoretic mobility of a labeled PPARgamma response element (PPRE) was retarded in the presence of mesangial cell nuclear extract, suggesting that PPARgamma is functional in these cells. The addition of unlabeled PPRE efficiently competed away the PPARgamma-PPRE protein complex, confirming specificity of binding of the PPARgamma to the PPRE. PPARgamma ligands rosiglitazone (1 to 10 micromol/L) and troglitazone (1 to 10 micromol/L) inhibited platelet-derived growth factor-induced DNA synthesis, measured as bromodeoxyuridine incorporation (P<0.01). This inhibition was dose dependent. When administered in antidiabetic doses to streptozotocin-induced diabetic rats, troglitazone substantially normalized albumin excretion at 3 months (from 687.1 to 137.6 microgram urinary albumin/mg creatinine, P:<0.05) but did not affect hyperglycemia or blood pressure in this model. This treatment also decreased glomerular plasminogen activator inhibitor-1 (PAI-1) expression. These data suggest that PPARgamma activation may directly attenuate diabetic glomerular disease, possibly by inhibiting mesangial growth, which occurs early in the process of diabetic nephropathy, or by inhibiting PAI-1 expression. PAI-1 inhibits the activation of plasmin and matrix metalloproteinase, which degrade extracellular matrix in the glomerulus. Excess glomerular PAI-1 allows the accumulation of extracellular matrix, leading to glomerulosclerosis. These results have therapeutic implications for diabetic nephropathy as well as for proliferative mesangial diseases of the kidney.

Peroxisome proliferator-activated receptor and retinoid X receptor ligands inhibit monocyte chemotactic protein-1-directed migration of monocytes.

Monocyte chemotactic protein-1 (MCP-1)-directed transendothelial migration of monocytes plays a key role in the development of inflammatory diseases. Infiltration of tissues by monocytes requires degradation of extracellular matrices, a process that involves matrix metalloproteinases. We studied the effects of peroxisome proliferator-activated receptor (PPAR) gamma, alpha, and retinoid X receptor alpha (RXRalpha) ligands on MCP-1-directed migration and matrix metalloproteinase expression of a human acute monocytic leukemia cell line (THP-1). PPARgamma ligands attenuated MCP-1-induced migration, with 50% inhibition (IC(50)) at 2.8 microM for troglitazone and 4.8 microM for rosiglitazone. PPARalpha ligands WY-14643 (IC(50): 0.9 microM) and 5,8,11,14-eicosatetranoic acid (IC(50): 9.9 microM), and the potent RXRalpha ligand AGN 4204 (IC(50): 3.6 nM) also blocked monocyte migration. Troglitazone, rosiglitazone, or AGN 4204 inhibited phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 expression. PPARalpha activators WY-14643 and 5,8,11,14-eicosatetraynoic acid, however, had no inhibitory effect. AGN 4204 increased PMA-induced tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) expression, whereas all PPAR ligands showed no effect. All PPAR and RXRalpha ligands blocked chemotaxis of THP-1 monocytes in the absence of a matrix barrier. This study demonstrates that activated PPARs and RXRalpha, block MCP-1-directed monocyte migration, mediated, at least in part, through their effects on matrix metalloproteinase-9 or TIMP-1 production, or chemotaxis.

Doxazosin inhibits retinoblastoma protein phosphorylation and G(1)-->S transition in human coronary smooth muscle cells.

Previous studies have demonstrated that the alpha(1)-adrenergic receptor antagonist doxazosin (Dox) inhibits multiple mitogenic signaling pathways in human vascular smooth muscle cells. This broad antiproliferative activity of Dox occurs through a novel mechanism unrelated to its blocking the alpha(1)-adrenergic receptor. Flow cytometry demonstrated that Dox prevents mitogen-induced G(1)-->S progression of human coronary artery smooth muscle cells (CASMCs) in a dose-dependent manner, with a maximal reduction of S-phase transition by 88+/-10.5% in 20 ng/mL platelet-derived growth factor and 1 micromol/L insulin (P+I)-stimulated cells (P<0.01 for 10 micromol/L Dox versus P+I alone) and 52+/-18.7% for 10% FBS-induced mitogenesis (P<0.05 for 10 micromol/L Dox versus 10% FBS alone). Inhibition of G(1) exit by Dox was accompanied by a significant blockade of retinoblastoma protein (Rb) phosphorylation. Hypophosphorylated Rb sequesters the E2F transcription factor, leading to G(1) arrest. Adenoviral overexpression of E2F-1 stimulated quiescent CASMCs to progress through G(1) and enter the S phase. E2F-mediated G(1) exit was not affected by Dox, suggesting that it targets events upstream from Rb hyperphosphorylation. Downregulation of the cyclin-dependent kinase inhibitory protein p27 is important for maximal activation of G(1) cyclin/cyclin-dependent kinase holoenzymes to overcome the cell cycle inhibitory activity of Rb. In Western blot analysis, p27 levels decreased after mitogenic stimulation (after P+I, 43+/-1.8% of quiescent cells [P<0.01 versus quiescent cells]; after 10% FBS, 55+/-7.7% of quiescent cells [P<0. 05 versus quiescent cells]), whereas the addition of Dox (10 micromol/L) markedly attenuated its downregulation (after P+I, 90+/-8.3% of quiescent cells [P<0.05 versus P+I alone]; after 10% FBS, 78+/-8.3% of quiescent cells [P<0.05 versus 10% FBS alone]). Furthermore, Dox inhibited cyclin A expression, an E2F regulated gene that is essential for cell cycle progression into the S phase. The present study demonstrates that Dox inhibits CASMC proliferation by blocking cell cycle progression from the G(0)/G(1) phase to the S phase. This G(1)-->S blockade likely results from an inhibition of mitogen-induced Rb hyperphosphorylation through prevention of p27 downregulation.

Peroxisome proliferator-activated receptor gamma ligands inhibit retinoblastoma phosphorylation and G1--> S transition in vascular smooth muscle cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily that is activated by binding certain fatty acids, eicosanoids, and insulin-sensitizing thiazolidinediones (TZD). The TZD troglitazone (TRO) inhibits vascular smooth muscle cell proliferation and migration both in vitro and in vivo. The precise mechanism of its antiproliferative activity, however, has not been elucidated. We report here that PPARgamma ligands inhibit rat aortic vascular smooth muscle cell proliferation by blocking the events critical for G(1) --> S progression. Flow cytometry demonstrated that both TRO and another TZD, rosiglitazone, prevented G(1) --> S progression induced by platelet-derived growth factor and insulin. Movement of cells from G(1) --> S was also inhibited by the non-TZD, natural PPARgamma ligand 15-deoxy-(12,14)Delta prostaglandin J(2) (15d-PGJ(2)), and the mitogen-activated protein kinase pathway inhibitor PD98059. Inhibition of G(1) --> S exit by these compounds was accompanied by a substantial blockade of retinoblastoma protein phosphorylation. TRO and rosiglitazone attenuated both the mitogen-induced degradation of p27(kip1) and the mitogenic induction of p21(cip1). 15d-PGJ(2) and PD98059 inhibited both the degradation of p27(kip1) and the induction of cyclin D1 in response to mitogens. These effects resulted in the inhibition of mitogenic stimulation of cyclin-dependent kinases activated by cyclins D1 and E. These data demonstrate that PPARgamma ligands are antiproliferative drugs that act by modulating cyclin-dependent kinase inhibitors; they may provide a new therapeutic approach for proliferative vascular diseases.

Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells.

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.

Renal afferent and efferent arteriolar dilation by nilvadipine: studies in the isolated perfused hydronephrotic kidney.

Although calcium antagonists are believed to exert preferential vasodilator action on the renal preglomerular afferent arteriole, we recently demonstrated that efonidipine, a novel calcium antagonist, vasodilates both afferent and efferent arterioles. Nilvadipine also is reported to increase renal blood flow and reduce filtration fraction, suggesting indirectly afferent and efferent arteriolar vasodilation. No direct investigation, however, has been conducted examining the renal microvascular action of nilvadipine. We therefore characterized the renal microvascular reactivity to nilvadipine, by using the isolated perfused rat hydronephrotic kidney. The administration of angiotensin II (0.3 nM) caused marked vasoconstriction of afferent (from 13.5 +/- 0.6 to 9.2 +/- 0.6 microm, p < 0.01, n = 6) and efferent arterioles (from 11.5 +/- 1.0 to 7.4 +/- 0.7 microm, p < 0.01; n = 5). The subsequent addition of nilvadipine (10 nM, 100 nM, and 1 microM) caused 37 +/- 5%, 91 +/- 4%, and 95 +/- 8% reversal of afferent arteriolar constriction, respectively. Similarly, efferent arterioles manifested 59 +/- 12% reversal by 1 microM nilvadipine. Thus unlike nifedipine, which we previously reported to cause modest efferent arteriolar dilation (21 +/- 1% reversal at 1 microM), nilvadipine possesses the greater ability to dilate efferent arterioles (p < 0.01 vs. nifedipine), although both antagonists cause similar magnitudes of afferent arteriolar vasodilation. Variable effects on the efferent arteriole suggest the heterogeneity in the calcium antagonist with regard to the renal microvascular action of this agent.

Two novel 1alpha-hydroxylase mutations in French-Canadians with vitamin D dependency rickets type I1.

BACKGROUND: Vitamin D dependency rickets type I (VDDR-I) is an autosomal recessive disorder in which 25-hydroxyvitamin D 1alpha-hydroxylase (1alpha-hydroxylase) activity in renal proximal tubules is deficient. VDDR-I is recognized throughout the world, but occurs more frequently in a subset of the French-Canadian population. We and others have recently cloned the human 1alpha-hydroxylase cDNA and gene, making it possible to screen for mutations. The first VDDR-I mutations were reported in one American and four Japanese patients. In this study, we screened for 1alpha-hydroxylase mutations in French-Canadian patients with VDDR-I. METHODS: The nine exons of the 1alpha-hydroxylase gene were amplified by polymerase chain reaction (PCR) from genomic DNA of four unrelated French-Canadian patients with VDDR-I and their parents, and sequenced. RESULTS: Three of the patients were homozygous for a single base-pair deletion (G) at position 262 in the cDNA that lies in exon 2, and causes a premature termination codon upstream from the putative ferredoxin- and heme-binding domains. The fourth patient was homozygous for a 7-bp insertion (CCCCCCA) at position 1323 of the cDNA that lies in exon 8, and causes a premature termination upstream from the putative heme-binding domain. In each family, obligate carriers have one copy of the mutant allele. These mutations, which could be detected by PCR-restriction fragment length polymorphism and polyacrylamide gel electrophoresis of the PCR products, were not found in 25 normal French-Canadians. CONCLUSION: We describe two novel 1alpha-hydroxylase mutations that are consistent with loss of function in four French-Canadian patients with VDDR-I and suggest that the 1alpha-hydroxylase mutations arise from more than one founder in this population.

Parathyroid hormone activation of the 25-hydroxyvitamin D3-1alpha-hydroxylase gene promoter.

The DNA flanking the 5' sequence of the mouse 1alpha-hydroxylase gene has been cloned and sequenced. A TATA box has been located at -30 bp and aCCAAT box has been located at -79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D3-1alpha-hydroxylase by acting on the promoter of the 1alpha-hydroxylase gene.

Cloning and expression of rat 25-hydroxyvitamin D3-1alpha-hydroxylase cDNA.

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1alpha-hydroxylase (P4501alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1alpha-hydroxylase activity. The sequence analysis showed that P4501alpha was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501alpha mRNA was greatly increased in the renal proximal convoluted tubules.

Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 1 alpha-hydroxylase.

The 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase) is a cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3. This enzyme plays an important role in calcium homeostasis. Here we report the molecular cloning of cDNA and gene for human 1 alpha-hydroxylase. The cDNA clone was obtained from a human kidney cDNA library by cross-hybridization with a previously cloned rat cDNA probe. The cDNA consists of 2469 bp and encodes a protein of 508 amino acids that shows 82.5% sequence identity with the rat enzyme. A computer-aided homology search revealed that 1 alpha-hydroxylase shares a relatively high homology with vitamin D3 25-hydroxylase (about 40% amino acid identity). Northern blot analysis showed that the 2.5-kb mRNA is most abundant in kidney. The gene for human 1 alpha-hydroxylase spans approximately 6 kb, is composed of nine exons, and is present as a single copy. This molecular cloning makes it possible to investigate the genetic mechanism of diseases related to calcium metabolism, including vitamin D-dependency rickets type I.