Transforming growth factor-beta as a treatment target in renal diseases.

A number of studies have identified transforming growth factor-beta (TGF-beta) as a critical factor in renal diseases such as glomerulosclerosis and mesangioproliferative glomerulonephritis. TGF-beta stimulates proliferation of mesangial cells, production of extracellular matrix components and induces epithelial-mesenchymal transformation in renal tissue, which plays a critical role in the pathogenesis of renal injury. Thus, TGF-beta is a treatment target in renal diseases. However, progressive renal diseases cannot be cured with present medical technologies. We have developed ribozymes and a novel gene silencer pyrrole-imidazole polyamide targeted to TGF-beta that effectively ameliorate renal injury in hypertensive rats.

Effects of an ARB on endothelial progenitor cell function and cardiovascular oxidation in hypertension.

BACKGROUND: Angiotensin II (Ang II) receptor blocker (ARB) has been reported to have protective effects on the cardiovascular system independent of blood pressure reduction. Endothelial progenitor cells (EPCs) play a significant role in neovascularization of ischemic tissue. The average lifespan of EPCs was recently reported to be shortened by oxidative stress and regulated by anti-oxidative mechanisms. It has been reported that EPCs are present in peripheral blood and have the ability to repair cardiovascular damage. We investigated the effects of an ARB, candesartan, on EPC function and cardiovascular oxidation in salt-loaded, stroke-prone, spontaneously hypertensive rats (SHR-SP) in vivo. METHODS: Salt-loaded SHR-SP were treated with candesartan (1 mg/kg/day), a diuretic (trichlormethiazide, TCM, 1.6 mg/kg/day), or an antioxidant (tempol, 5 mg/kg/day) for 2 weeks. Peripheral blood mononuclear cells (MNCs) were isolated and cultured to assay EPC colony formation and migration. Oxidative stress in EPCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay. We evaluated messenger RNA (mRNA) expression of c-kit in the heart, the renin-angiotensin system (RAS) in EPC colonies, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit in cardiovascular organs. RESULTS: Candesartan and tempol, but not TCM, markedly increased EPC colony number in SHR-SP and reduced TBARS. Candesartan also significantly decreased mRNA expression of NADPH oxidase subunits in cardiovascular organs and increased cardiac c-kit mRNA expression. EPCs expressed mRNAs of renin, cathepsin D, chymase, and Ang II type 1 and type 2 receptors. CONCLUSIONS: Candesartan, an ARB, improves EPC dysfunction and increases cardiac c-kit expression through the anti-oxidative mechanism in hypertension. The local RAS induces oxidative stress and regulates the EPC functions.

Chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta1 mRNA ameliorates renal injury in hypertensive rats.

OBJECTIVE: Transforming growth factor (TGF)-beta is a critical factor in the progression of renal injury, regardless of the primary etiology. Such injury is characterized by glomerular sclerosis and tubulointerstitial fibrosis. To develop a ribozyme-based therapy for progressive renal diseases, we examined the effects of chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta1 mRNA on glomerulosclerosis in salt-loaded, stroke-prone spontaneously hypertensive rats (SHR-SP) and salt-sensitive Dahl (Dahl-S) rats. METHODS: The chimeric DNA-RNA ribozyme to TGF-beta1 was delivered by polyethylenimine to cultured mesangial cells from SHR-SP in vitro and to glomeruli in SHR-SP in vivo. The chimeric ribozyme reduced expression of TGF-beta1 mRNA and protein, which was accompanied by inhibition of expression of extracellular matrix molecules such as fibronectin and collagen type I in mesangial cells from SHR-SP in vitro. RESULTS: One intraperitoneal injection of 200 microg of chimeric DNA-RNA ribozyme to TGF-beta1 in vivo markedly ameliorated thickening of capillary artery walls and glomerulosclerosis in salt-loaded SHR-SP and Dahl-S rats without a reduction in blood pressure. The chimeric ribozyme reduced expression of TGF-beta1 and connective tissue growth factor (CTGF) mRNAs in renal cortex in salt-loaded Dahl-S rats. Chimeric ribozyme to TGF-beta1 significantly reduced levels of protein in urine in the Dahl-S rats. CONCLUSION: These results suggest that chimeric DNA-RNA ribozyme to TGF-beta1 may be useful as a gene therapy for progressive tissue injury in a wide variety of renal diseases, including hypertensive nephrosclerosis.

Transforming growth factor-beta expression in cardiovascular organs in stroke-prone spontaneously hypertensive rats with the development of hypertension.

Transforming growth factor (TGF)-beta activity is involved in several cardiovascular diseases owing to its effects on the growth of vascular smooth muscle cells and induction of extracellular matrix formation. We evaluated expression of TGF-beta in cardiovascular organs from stroke-prone spontaneously hypertensive rats (SHR-SP) which show severe cardiovascular damages with the development of hypertension. Twelve-week-old Wistar-Kyoto (WKY)/Izm rats and SHR-SP/Izm were loaded with 1% salt for 4 weeks. Aorta, heart and kidney were removed and evaluated histologically by hematoxylin-eosin staining. Expression of TGF-beta1 mRNA was evaluated by reverse transcription and polymerase chain reaction analysis in mRNA extracted with oligo dT-cellulose. Expression of TGF-beta1 protein was evaluated by Western blot analysis and immunohistochemical study in renal cortex. Whereas expression of TGF-beta1 mRNA was detected only in the heart of SHR-SP before salt loading, it was detected in the aorta, left ventricle of heart and renal cortex from both rat strains, and it was stronger in the renal cortex of SHR-SP than in the renal cortex of WKY rats. Expression of TGF-beta1 protein was markedly higher in the renal cortex of SHR-SP than in the renal cortex of WKY rats after salt loading. TGF-beta was localized at glomeruli and capillary arteries in the renal cortex, and immunostaining was stronger in SHR-SP than in WKY rats. Expression of TGF-beta1 was increased in glomeruli and capillaries of the renal cortex with the development of hypertension in SHR-SP. These results implicate TGF-beta in the renal damage observed in hypertension.

Novel mechanisms of the antiproliferative effects of amlodipine in vascular smooth muscle cells from spontaneously hypertensive rats.

The calcium channel blocker amlodipine continues to be of interest due to its potential proven ability to hinder the progression of atherosclerosis and reduce the number of clinical ischemic events. Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) are useful in the study of atherosclerosis because they show exaggerated growth with production of angiotensin II (Ang II) by conversion to the synthetic phenotype. To clarify mechanisms of the antiproliferative effects of amlodipine, we evaluated effects of the expression of growth factors, the changes in phenotype, and the proliferation of VSMC from SHR. Amlodipine significantly inhibited basal DNA synthesis and proliferation of VSMC from SHR. Amlodipine also inhibited expression of platelet-derived growth factor (PDGF) A-chain, transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) mRNAs in VSMC from SHR. Decreases in levels of PDGF A-chain and bFGF mRNAs in VSMC from SHR were greater with amlodipine than with nifedipine. Amlodipine significantly inhibited expression of the synthetic phenotype markers osteopontin and matrix Gla mRNAs, indicating that it inhibited the exaggerated growth of VSMC from SHR and suppressed the change from the contractile phenotype to the synthetic phenotype. Thus, amlodipine may be a beneficial therapeutic agent for patients with hypertensive vascular diseases.

Effects of an angiotensin II receptor blocker on the impaired function of endothelial progenitor cells in patients with essential hypertension.

BACKGROUND: Endothelial progenitor cells (EPCs) induce neovascularization and repair vascular damage. We have demonstrated that EPC function is impaired in hypertensive rats with increases in oxidative stress and that angiotensin II receptor blockers improved the impaired function of EPCs. In this study, we investigated basal EPC functions in normotensive control subjects and patients with essential hypertension and the effect of losartan on EPC function in hypertensive patients. METHODS: Eighteen normotensive control subjects and 36 patients with essential hypertension who were undergoing treatment participated in the study. Hypertensive patients were randomly selected to receive 50mg of losartan or 4 mg of trichlormethiazide daily for 4 weeks. Peripheral blood mononuclear cells were isolated and cultured to assay EPC colony formation. Blood pressure, biological examination, and oxidative stress were evaluated in all subjects. RESULTS: The number of EPC colonies was significantly lower in patients with essential hypertension than in normotensive control subjects. EPC colony number was significantly and inversely correlated with systolic and diastolic blood pressure in all subjects. EPC colony number was significantly increased by treatment with losartan in patients with essential hypertension but not affected by treatment with trichlormethiazide. CONCLUSIONS: EPC function was inversely correlated with blood pressure and was impaired in essential hypertension. Losartan significantly improved the impaired EPC function in hypertensive patients. Impaired EPC function may determine the cardiovascular complications in essential hypertension. The improvement of EPC function with the administration of angiotensin II receptor blockers is considered to be one of the cardiovascular protective effects.

Complement 3 is involved in changing the phenotype of human glomerular mesangial cells.

Complement activation contributes to tissue injury in various forms of glomerulopathy and is characterized by deposition of complement components, which accelerates the progression of chronic renal damage. We recently reported that complement 3 (C3), a critical component of the complement system, is associated with the synthetic phenotype of vascular smooth muscle cells. It is possible that C3 stimulates mesangial cells to assume the synthetic phenotype to, in turn, induce glomerular injury and sclerosis. We investigated the role of C3 in the growth and phenotype of mesangial cells. Cultured human mesangial cells (HMCs) expressed C3 mRNA and protein, and levels were increased in response to IFN-gamma and TNF-alpha. HMCs also expressed C3a receptor mRNA and protein. Exogenous C3a stimulated DNA synthesis in HMCs in a dose-dependent manner. C3a decreased expression h-caldesmon mRNA, a marker of the contractile phenotype, and increased the expression of osteopontin, matrix Gla, and collagen type1 alpha1 (collagen IV) mRNAs, which are markers of the synthetic phenotype. C3a decreased expression of alpha-smooth muscle actin in HMCs. Small interfering RNA (siRNA) targeting C3 reduced the DNA synthesis and proliferation of HMCs, increased expression of h-caldesmon mRNA, and decreased expression of osteopontin, matrix Gla, and collagen IV mRNAs in HMCs. These results indicate that C3 causes HMCs to convert to the synthetic phenotype and stimulates growth of mesangial cells, suggesting that C3 may play an important role in phenotypic regulation of mesangial cells in renal diseases.

Two novel genotypes of the thiazide-sensitive Na-Cl cotransporter (SLC12A3) gene in patients with Gitelman's syndrome.

Gitelman's syndrome is an autosomal recessive disorder marked by salt wasting and hypokalaemia resulting from loss-of-function mutations in the SLC12A3 gene that codes for the thiazide-sensitive Na-Cl cotransporter. Gitelman's syndrome is usually distinguished from Bartter's syndrome by the presence of both hypomagnesaemia and hypocalciuria. Although recent advances in molecular genetics may make it possible to both diagnose and differentiate these diseases, the phenotypes sometimes overlap. Here we report two sporadic cases of Gitelman's syndrome and two novel genotypes of SLC12A3. Patient 1 was a compound heterozygote with a known missense mutation, L849H, and a novel mutation, R852H in exon 22. Patient 2 was homozygous for the missense mutation L849H. To our knowledge, this is the first report of a patient homozygous for 849H. Interestingly, both patients were affected with autoimmune thyroid disease. Patient 1 was affected with Hashimoto's disease, and Patient 2 was affected with Graves' disease. The symptoms of Patient 2 were more serious than those of Patient 1. Although the patients both carried the 849H allele (Patient 1 as a heterozygote and Patient 2 as a homozygous), their clinical symptoms differed. The difference in the clinical features may have been due both to phenotypic differences and the fact that Gitelman's syndrome is a complicated disorder.

Development of gene silencing pyrrole-imidazole polyamide targeting the TGF-beta1 promoter for treatment of progressive renal diseases.

Pyrrole-imidazole (Py-Im) polyamides are nuclease-resistant novel compounds that inhibit gene expression by binding to the minor groove of DNA. A Py-Im polyamide that targets rat TGF-beta1 was designed as a gene-silencing agent for progressive renal diseases, and the distribution and the effects of this polyamide on renal injury were examined in Dahl-salt sensitive (Dahl-S) rats. For identification of transcription factor binding elements for activation of the rat TGF-beta1 gene, recombinant TGF-beta1 reporter plasmids were transfected into HEK-293 cells, and promoter activity was measured. Py-Im polyamide was designed to the activator protein-1 binding site of the rat TGF-beta1 promoter. This Py-Im polyamide showed strong, fast, and specific binding to the target DNA in gel mobility shift and Biacore assays. Py-Im polyamide significantly inhibited TGF-beta1 promoter activity and expression of TGF-beta1 mRNA and protein in rat mesangial cells. Intravenously administered fluorescein-labeled polyamide distributed to the kidney of rats. Py-Im polyamide significantly inhibited expression of TGF-beta1 mRNA and protein in the renal cortex of Dahl-S rats and reduced the increase in urinary protein and albumin in Dahl-S rats independent of changes in blood pressure. These results indicate that Py-Im polyamide that targets TGF-beta1 will be a novel gene-silencing agent for the TGF-beta1-associated diseases, including progressive renal diseases.

Inhibitory effect of a novel angiotensin II type 1 receptor antagonist RNH-6270 on growth of vascular smooth muscle cells from spontaneously hypertensive rats: different anti-proliferative effect to angiotensin-converting enzyme inhibitor.

The current study was undertaken to evaluate the anti-proliferative effect of a novel angiotensin II type 1 (AT1) receptor antagonist, RNH-6270, on exaggerated growth of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR), in comparison with the effects of an angiotensin-converting enzyme (ACE) inhibitor. RNH-6270 and temocapril significantly inhibited basal DNA synthesis in VSMCs from SHRs in a dose-dependent manner, but not in cells from Wistar-Kyoto (WKY) rats. SHR-derived VSMC showed a hyperresponse of DNA synthesis to serum and angiotensin II compared with that of WKY rats-derived VSMC. RNH-6270 did not affect serum-stimulated DNA synthesis in VSMCs from both rat strains. RNH-6270 abolished angiotensin II-stimulated DNA synthesis in VSMC from both rat strains. RNH-6270 significantly inhibited proliferation of VSMC from both rat strains, but the ACE inhibitor temocapril did not exert such an effect. RNH-6270 decreased the specific binding of angiotensin II to VSMC in a competitive manner for angiotensin II receptors in both rat strains. RNH-6270 and temocapril significantly decreased the expression of growth factor mRNAs and proteins in VSMC from SHR, but not in cells from WKY rats. These results suggest that RNH-6270 is a potent AT1 receptor antagonist and has anti-proliferative effects on VSMCs from SHR, which was not seen with an ACE inhibitor. The growth inhibitory effect of RNH-6270 may be associated with the inhibition of growth factors via antagonism to AT1 receptors.

Effect of AT2 receptor on expression of AT1 and TGF-beta receptors in VSMCs from SHR.

We recently reported that overexpression of the angiotensin II type 2 (AT2) receptor downregulates the AT1a receptor through the bradykinin/NO pathway in a ligand-independent manner in vascular smooth muscle cells (VSMCs). In the present study, we investigated the effect of AT2 receptor overexpression on the expression of the AT1a receptor and transforming growth factor-beta (TGF-beta) receptor subtypes in VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Transfection of the AT2 receptor gene downregulated expression of the AT1a receptor in VSMCs from WKY, but did not affect expression of the AT1a receptor in VSMCs from SHR. Transfection of the AT2 receptor abolished DNA synthesis in response to angiotensin II in VSMCs from WKY; in VSMCs from SHR, basal DNA synthesis was suppressed, but DNA synthesis in response to Ang II was not altered. The NO substrate L-arginine augmented downregulation of the AT1a receptor in VSMCs from WKY, whereas it did not affect expression of the AT1a receptor in VSMCs from SHR. In response to AT2 receptor transfection, expression of TGF-beta type I receptor mRNA was suppressed significantly in VSMCs from WKY, whereas expression of TGF-beta type I receptor was not altered in VSMCs from SHR. These results suggest that the AT2 receptor downregulates AT1a and TGF-beta type I receptors in normal VSMCs, but not in SHR-derived VSMCs. The lack of downregulation of the AT1a receptor may contribute, in part, to the exaggerated growth of VSMCs from SHR.

Angiotensin II type 2 receptor gene transfer downregulates angiotensin II type 1a receptor in vascular smooth muscle cells.

Two distinct subtypes of angiotensin (Ang) II receptors, type 1 (AT(1)) and type 2 (AT(2)), have been identified. Vascular smooth muscle cells (VSMCs) usually express AT(1) receptor. To elucidate the direct effects of the AT(2) receptor on the AT(1) receptor in VSMCs, we transfected AT(2) receptor gene into cultured rat VSMCs. Overexpression of AT(2) receptor significantly decreased expression of AT(1a) receptor at both the mRNA and protein levels in the presence and absence of Ang II in VSMCs. Overexpression of AT(2) receptor increased expression of bradykinin and inducible NO in the presence and absence of Ang II in VSMCs. Bradykinin B(2) receptor antagonist HOE-140 and NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited the decreases in AT(1a) receptor expression by the overexpression of AT(2) receptor in VSMCs. L-Arginine augmented the decrease in AT(1a) receptor expression. Overexpression of AT(2) receptor suppressed basal DNA synthesis and proliferation of VSMCs and abolished response of DNA synthesis to Ang II in VSMCs. Our results demonstrate that overexpression of the AT(2) receptor downregulates AT(1a) receptor expression in rat VSMCs in a ligand-independent manner that is mediated by the bradykinin/NO pathway. Downregulation of AT(1a) receptor is a novel mechanism by which the AT(2) receptor regulates growth and metabolism of VSMCs.

Effects of antisense peptide nucleic acid to platelet-derived growth factor A-chain on growth of vascular smooth muscle cells.

To investigate antisense peptide nucleic acid (PNA) as a gene therapy for the arterial proliferative diseases, the authors designed and examined the effects of an antisense PNA targeting platelet-derived growth factor (PDGF) A-chain on expression of PDGF A-chain and growth of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. A 15-mer antisense PNA complementary to the initiation codon of rat and human PDGF A-chain mRNA was synthesized and purified by high-performance liquid chromatography. Gel-shift assay and biomolecular interaction analysis (BIAcore) revealed that the antisense PNA bound weakly to the target RNA, whereas it bound strongly to the target DNA. Fluorescein-isothiocyanate-labeled antisense PNA to PDGF A-chain was taken up slowly and maintained in VSMCs for a prolonged period of time. Antisense PNA inhibited expression of PDGF A-chain mRNA and protein as well as DNA synthesis in VSMCs in a dose-independent manner. Inhibition of DNA synthesis by the antisense PNA was greater than that by the antisense DNA at a low concentration (0.5 micromol/L). These results suggest that antisense PNA to PDGF A-chain will be used as a gene therapy for vascular proliferative diseases such as hypertensive vascular diseases, restenosis of coronary arteries after angioplasty, and atherosclerosis.