Yoichi Izumi

Production of Angiotensin II by Homogeneous Cultures of Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats

Production of angiotensin II (Ang II) in spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) has now been investigated. A nonpeptide antagonist (CV-11974) of Ang II type 1 receptors inhibited basal DNA synthesis in VSMC from SHR, but it had no effect on cells from Wistar-Kyoto (WKY) rats. Ang II-like immunoreactivity, determined by radioimmunoassay after HPLC, was readily detected in conditioned medium and extracts of SHR-derived VSMC, whereas it was virtually undetectable in VSMC from WKY rats. Isoproterenol increased the amount of Ang II-like immunoreactivity in conditioned medium and extracts of SHR-derived VSMC, whereas the angiotensin-converting enzyme inhibitor delapril significantly reduced the amount of Ang II-like immunoreactivity in conditioned medium and extracts of these cells. Reverse transcription-polymerase chain reaction analysis revealed that the abundance of mRNAs encoding angiotensinogen, cathepsin D, and angiotensin-converting enzyme was greater in VSMC from SHR than in cells from WKY rats. The abundance of cathepsin D protein by Western blotting was greater in VSMC from SHR than in cells from WKY rats. Ang I-generating and acid protease activities were detected in VSMC from SHR, but not in cells from WKY rats. These results suggest that SHR-derived VSMC generate Ang II with increases in angiotensinogen, cathepsin D, and angiotensin-converting enzyme, which contribute to the basal growth. Production of Ang II by homogeneous cultures of VSMC is considered as a new mechanism of hypertensive vascular disease.

Haplotype Analysis of the Human Renin Gene and Essential Hypertension

Abstract—The human renin gene is an attractive candidate for involvement in the underlying cause of essential hypertension (EH). Despite extensive examination, the relation between the renin gene and hypertension remains unclear. The aims of the present study were to discover new genetic markers of EH and to investigate the relations between polymorphisms of the renin gene and EH in the Japanese. Using the polymerase chain reaction–single strand conformation polymorphism (PCR-SSCP) method, we isolated 3 novel variants of the renin gene; a single nucleotide polymorphism (SNP) in intron 4 (T+17int4G), a variable number of tandem repeats (VNTR) polymorphism in intron 7, and a missense mutation in exon 9 (G1051A). We performed an association study with these polymorphisms in 212 patients with EH and 209 age-matched normotensive (NT) subjects. The frequency of genotypes VNTR and T+17int4G did not differ significantly between the 2 groups, whereas the overall distribution of G1051A was significantly different between EH and NT. Haplotype analysis revealed that the overall distribution of haplotypes differed significantly between the EH and NT groups. PRA levels in patients with EH with the G/G genotype were significantly higher than in subjects with EH with G/A and A/A genotypes. These data suggest that the missense mutation in exon 9 may affect the enzymatic function of renin and consequently may be involved in the etiology of hypertension.

Angiotensin II upregulates transforming growth factor-β type I receptor on rat vascular smooth muscle cells

Angiotensin II (Ang II) and transforming growth factor-beta (TGF-beta) modulate cell growth and metabolism. Our objective was to evaluate the effect of Ang II on the characteristics and expression of TGF-beta receptors on vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats. The addition of TGF-beta1 elicited a biphasic response on DNA synthesis in cultured VSMC in the absence of Ang II, but TGF-beta1 did not stimulate DNA synthesis in the presence of Ang II. TGF-beta binding data showed that Ang II increased the specific binding of 125I-TGF-beta1 by enhancing the expression of lower affinity receptors and increasing the number of binding sites. Ang II alone did not stimulate DNA synthesis in these cultures. However, Ang II significantly stimulated DNA synthesis after the inhibition of endogenous TGF-beta with a neutralizing antibody. The DNA synthesis stimulated by phorbol ester milisterol (PMA) was not affected by the TGF-beta neutralizing antibody. Affinity labeling data revealed receptor-ligand complexes of 280, 85, and 70 kDa, corresponding to TGF-beta type III, II, and I receptors, respectively. Incubation of VSMC with Ang II but not with PMA markedly increased the expression of the TGF-beta type I receptor. Reverse transcription and polymerase chain reaction data also indicated that Ang II, but not PMA, significantly increased the expression of TGF-beta type I receptor mRNA. Results suggest that Ang II increases the binding of TGF-beta with upregulation of TGF-beta type I receptor via a C-kinase-independent pathway. The enhanced expression of the TGF-beta type I receptor may counteract Ang II-promoted growth of VSMC.

L-NG-monomethyl arginine inhibits the vasodilating effects of low dose of endothelin-3 on rat mesenteric arteries

We have reported that low doses of endothelin-3 (ET-3) elicited continuous vasodilation of rat mesenteric arteries, which is possibly related to endothelium-derived relaxing factor (EDRF). In order to clarify whether or not the vasodilating effects of ET-3 are associated with EDRF, we examined the effects of L-NG-monomethyl arginine (L-NMMA), an analog of L-arginine, on low-dose ET-3 induced vasodilation of rat mesente-Hc arteries. Infusion of 50 microM L-NMMA inhibited the vasodilation induced by 10(-13) M ET-3 and rather elicited an increase in perfusion pressure, which itself was decreased by infusion of 150 microM L-arginine. In the presence of 50 microM L-NMMA, 10(-13) M ET-3 did not elicit any vasodilation of the mesenteric arteries preconstricted with NE, in which 150 microM L-arginine, but not D-arginine, caused considerable vasodilation. These data suggest that the vasodilating effects of low doses of ET-3 are associated with EDRF as an endothelium-derived nitric oxide.

Inhibitory effect of an angiotensin II type 1 receptor antagonist on growth of vascular smooth muscle cells from spontaneously hypertensive rats

To investigate the role of endogenous angiotensin II (Ang II) in the growth of vascular smooth muscle cells (VSMC), we examined the effect of the novel nonpeptide Ang II type 1 (AT1) receptor antagonist CV-11974 on basal and stimulated-growth of VSMC from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). CV-11974 inhibited basal DNA synthesis by VSMC from SHR, but not from WKY, in the absence of serum. In the presence of 10% calf serum, DNA synthesis was significantly higher in VSMC from SHR than in cells from WKY, and the inhibitory effect of CV-11974 was attenuated. Ang II dose-dependently stimulated DNA synthesis by VSMC from both rat strains, and this effect was abolished by CV-11974. CV-11974 slightly but significantly suppressed proliferation of VSMC from both rat strains. CV-11974 competitively inhibited the specific binding of 125I-labeled Ang II to VSMC from both rat strains. The angiotensin-converting enzyme inhibitor (ACEI) delapril also reduced basal DNA synthesis by VSMC from SHR, but did not affect proliferation of VSMC from either rat strain. These findings suggest that VSMC from SHR may synthesize Ang II which promotes their basal growth in an autocrine/paracrine manner through the AT1 receptor, and that this system may be associated with the exaggerated growth of VSMC from SHR.

Antisense oligodeoxynucleotide complementary to platelet-derived growth factor A-chain messenger RNA inhibits the arterial proliferation in spontaneously hypertensive rats without altering their blood pressures.

Objective To evaluate the effects of the antisense oligodeoxynucleotide (ODN) to platelet-derived growth factor (PDGF) A-chain messenger RNA (mRNA) on the growth of cardiovascular organs in hypertension. Design 15-Mer antisense ODN complementary to the initiation codon region of rat PDGF-A chain mRNA and non-sense ODN of identical proportion but with a random order of bases relative to that of antisense ODN were synthesized with a DNA synthesizer. Methods We examined the effects of the antisense ODN on the growth of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and Wistar–Kyoto rats, and on the expressions of PDGF A-chain mRNA by reverse transcription and polymerase chain reaction and PDGF A-chain protein by Western blot analysis in vitro. We evaluated the distribution of 32P-labeled antisense ODN and examined the effects of the antisense ODN on the growth of cardiovascular organs in vivo. Results The antisense ODN reduced the basal DNA synthesis of VSMC from SHR significantly, but did not do so in cells from Wistar–Kyoto rats. Mutations in the antisense ODN sequence reduced the ODN-induced inhibition of DNA synthesis. Addition of serum or transforming growth factor-β1 increased the DNA synthesis in the SHR-derived VSMC that was inhibited by the antisense ODN. The antisense ODN inhibited the production of PDGF A-chain protein, but not of the PDGF A-chain mRNA. The injection of 32P-antisense ODN in vivo led to a greater accumulation of radioactivity in the aorta than in other organs. Infusion of antisense ODN for 28 days did not alter the systolic blood pressure appreciably in rats of either strain. However, in SHR, it reduced markedly the elevated DNA content, [3H]-thymidine uptake, and incorporation of [3H]-thymidine into aortic DNA, and suppressed the production of aortic PDGF A-chain protein. These results indicated that the PDGF A-chain is involved in the exaggerated growth of VSMC from SHR by which inhibition of the translation of PDGF A-chain mRNA to the protein with antisense ODN occurs in vitro, and that antisense ODN to PDGF A-chain suppresses the exaggerated arterial proliferation in SHR without alterating the high blood pressure in vivo. Conclusion These results imply that inhibition of the final responsible growth factor PDGF A-chain by antisense ODN can suppress the arterial proliferation in hypertension without altering the blood pressure, suggesting that the arterial proliferation in hypertension is independent of the high blood pressure in part, and that antisense therapy could be feasible for treating hypertension.

A haplotype of the CYP4A11 gene associated with essential hypertension in Japanese men

Objective CYP4A11, a member of the cytochrome P450 family, acts mainly as an enzyme that converts arachidonic acid to 20-hydroxyeicosatetraenoic acid, a metabolite involved in blood pressure regulation in humans. Disruption of the murine cyp4a14 and cyp4a10 genes, homologues of human CYP4A11, was reported recently to cause hypertension. The gene-disrupted male mice had higher blood pressure than the gene-disrupted female mice. The present study aimed to assess the association between the human CYP4A11 gene and essential hypertension, using a haplotype-based case–control study including separate analysis of the gender groups. Methods The 304 essential hypertension patients and 207 age-matched control individuals were genotyped for three single-nucleotide polymorphisms of the human CYP4A11 gene (rs2269231, rs1126742, rs9333025). Data were assessed for three separate groups: total participants, men and women. Results For total participants, the genotypic distribution of rs1126742 differed significantly between the two groups (P = 0.005). For total participants, men and women, the recessive model (CC versus TC + TT) of rs1126742 differed significantly between the two groups (P = 0.007, P = 0.043, and P = 0.045, respectively). Logistic regression analysis showed the TC + TT genotype was significantly higher in essential hypertension patients than in control individuals for total participants and men (P = 0.022 and P = 0.043, respectively). The A-T-G haplotype frequency (established by rs2269231, rs1126742, rs9333025) was significantly higher in essential hypertension men than in control men (P = 0.043). Conclusions Essential hypertension is associated with the TC + TT genotype of rs1126742 in the human CYP4A11 gene. The A-T-G haplotype appears a useful genetic marker of essential hypertension in Japanese men.

Role of Long-Form PDGF A-Chain in the Growth of Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit exaggerated growth relative to cells from normotensive Wistar-Kyoto (WKY) rats. Platelet-derived growth factor (PDGF) A-chain has been implicated in the exaggerated growth of VSMC from SHR. Two isoforms of PDGF A-chain mRNA that either include (long form) or exclude (short form) exon 6 are produced as a result of alternative splicing. The expression of the long-form PDGF A-chain at the mRNA level and its role in the growth of VSMC from SHR have now been investigated with the use of an antisense oligodeoxynucleotide (ODN) complementary to exon 6 of the PDGF A-chain gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with primers encompassing exon 6 of PDGF A-chain mRNA revealed bands corresponding to both long- and short-form PDGF A-chain transcripts in quiescent VSMC from both SHR and WKY rats, with the long-form mRNA more abundant in VSMC from SHR than in cells from WKY rats. Expression of the long-form of PDGF A-chain mRNA was enhanced with angiotensin II and transforming growth factor-beta1 in VSMC from SHR, but not in cells from WKY rats. The antisense ODN significantly inhibited DNA synthesis by VSMC from SHR, but not by cells from WKY rats, in the absence or presence of serum. In addition, the antisense ODN significantly inhibited serum induced proliferation of VSMC from SHR, but not those from WKY rats. The antisense ODN abolished expression of the long-form PDGF A-chain mRNA in VSMC, suggesting that its inhibitory effects on the growth of VSMC from SHR are mediated by depletion of the long-form transcripts. These results indicate that the long-form of PDGF A-chain contributes to the exaggerated growth of VSMC from SHR.

A Haplotype of the CYP4F2 Gene is Associated With Cerebral Infarction in Japanese Men

BACKGROUND CYP4F2, a member of the cytochrome P450 family, acts mainly as an enzyme and is involved not only in the metabolism of leukotriene B4, but also in that of arachidonic acid. It converts arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a metabolite involved in the regulation of the vascular tone in the brain. The aim of this study was to assess the association between the human CYP4F2 gene and cerebral infarction (CI), using a haplotype-based case-control study with separate analyses of data from the gender groups. METHODS A total of 175 CI patients and 246 control subjects were genotyped for five single-nucleotide polymorphisms (SNPs) of the human CYP4F2 gene (rs3093105, rs3093135, rs1558139, rs2108622, rs3093200). For data analysis, three separate groups were assessed: all subjects, men, and women. RESULTS In the male subjects, the G allele frequency for rs2108622 was significantly higher in CI patients as compared to control subjects (P = 0.025). The overall distribution of the haplotypes in the men was significantly different between the CI patients and the control subjects (P = 0.027). Additionally, the frequency of the T-C-G haplotype for men was significantly higher in the CI patients than in the control subjects (P = 0.008). Multiple logistic regression analysis also revealed the significance of the T-C-G haplotype in men, even after adjustment for confounding factors. CONCLUSIONS The results of this study indicate that, in Japanese men, CI is associated with the G allele of rs2108622 and, in addition, that the T-C-G haplotype appears to be a useful genetic marker for CI.

Nonsense mutation of prostacyclin synthase gene in a family

We found a nonsense mutation in exon 2 of the human prostacyclin-synthase gene in a family with essential hypertension and cerebral infarction. Prostacyclin (PGI2) is an inhibitor of platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. Prostacyclin synthase (PGIS), which catalyses the formation of PGI2 from prostaglandin H2, is widely distributed, predominantly in vascular endothelial and smooth muscle cells. We have reported the organisation of this gene. We searched for possible point mutations in the exons using peripheral blood from 100 patients with essential hypertension by PCR and single strand conformation polymorphism (PCR-SSCP) analysis. One patient had an abnormally migrating band on exon 2. Sequencing of this exon showed a nonsense mutation in codon 26 (CGA/TGA). This nucleotide change makes Bst EII the restriction site. 300 people (150 with essential hypertension and 150 healthy controls) were screened by PCR and Bst EII digestion. The mutation was found in one patient with essential hypertension and in none of the controls. The patient was shown to be heterozygous for this mutation.This mutation of the stop codon is 76 bp downstream from ATG, the start codon in cDNA, thus a large part of mRNA,