Shin-ichiro Yoshida

Apelin stimulates myosin light chain phosphorylation in vascular smooth muscle cells.

Objective—Physiological roles of apelin and its specific receptor APJ signaling were investigated in vascular smooth muscle cells (VSMCs). The present study determined whether apelin activates myosin light chain (MLC), a major regulatory event in initiating smooth muscle contraction. Methods and Results—To assess MLC activation, we performed Western blot and immunohistochemical studies using an antibody against the phospho-MLC. In VSMCs, apelin induces the phosphorylation of MLC in a concentration-dependent manner with a peak at 2 minutes. Pretreatment of VSMCs with pertussis toxin abolishes the apelin-induced phosphorylation of MLC. Inhibition of protein kinase C (PKC) with GF-109203X markedly attenuated the apelin-induced MLC phosphorylation. In addition, methylisobutyl amiloride, a specific inhibitor of the Na+/H+ exchanger (NHE), and KB-R7943, a potent inhibitor for the reverse mode of the Na+/Ca2+ exchanger (NCX), significantly suppressed the action of apelin. In wild-type mice, apelin phosphorylates MLC in vascular tissue, whereas it had no response in APJ-deficient mice by Western blot and immunohistochemistry. Apelin-induced phosphorylation of MLC was accompanied with myosin phosphatase target subunit phosphorylation. Conclusions—These results provide the first evidence to our knowledge for apelin-mediated MLC phosphorylation in vitro and in vivo, which is a potential mechanism of apelin-mediated vasoconstriction.

Mice Lacking Hypertension Candidate Gene ATP2B1 in Vascular Smooth Muscle Cells Show Significant Blood Pressure Elevation

We reported previously that ATP2B1 was one of the genes for hypertension receptivity in a large-scale Japanese population, which has been replicated recently in Europeans and Koreans. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1, which plays a critical role in intracellular calcium homeostasis. In addition, it is suggested that ATP2B1 plays a major role in vascular smooth muscle contraction. Because the ATP2B1 knockout (KO) mouse is embryo-lethal, we generated mice with vascular smooth muscle cell-specific KO of ATP2B1 using the Cre-loxP system to clarify the relationship between ATP2B1 and hypertension. The KO mice expressed significantly lower levels of ATP2B1 mRNA and protein in the aorta compared with control mice. KO mice showed significantly higher systolic blood pressure as measured by tail-cuff method and radiotelemetric method. Similar to ATP2B1, the expression of the Na+-Ca2+ exchanger isoform 1 mRNA was decreased in vascular smooth muscle cells of KO mice. However, ATP2B4 expression was increased in KO mice. The cultured vascular smooth muscle cells of KO mice showed increased intracellular calcium concentration not only in basal condition but also in phenylephrine-stimulated condition. Furthermore, phenylephrine-induced vasoconstriction was significantly increased in vascular rings of the femoral artery of KO mice. These results suggest that ATP2B1 plays important roles in the regulation of blood pressure through alteration of calcium handling and vasoconstriction in vascular smooth muscle cells.

Roles for host and tumor angiotensin II type 1 receptor in tumor growth and tumor-associated angiogenesis

Angiotensin II (AII) is a multifunctional bioactive peptide, and host renin-angiotensin system (RAS) is closely associated with tumor growth. Recent reports have described that AII is a proangiogenic growth factor, and that Angiotensin II type 1 (AT1) receptor antagonists reduce tumor growth and tumor-associated angiogenesis. In this paper, we investigated the participation of AT1 receptor-signaling in cancer progression using murine Lewis lung carcinoma (LLC) cells, which express AT1 receptor, and AT1a receptor gene-deficient (AT1a−/−) mice. When LLC cells were implanted subcutaneously into wild-type (WT) mice, developed tumors showed intensive angiogenesis with an induction of vascular endothelial growth factor (VEGF) a. Compared with WT mice, tumor growth and tumor-associated angiogenesis was reduced in AT1a−/− mice with reduced expression of VEGFa. In AT1a−/− mice, administration of the AT1 receptor antagonist, TCV-116, showed further reductions of tumor growth, tumor-associated angiogenesis, and VEGFa expression. In vitro study, the expression of VEGFa mRNA and the production of VEGFa protein in LLC cells were significantly increased by AII, which were cancelled by AT1 receptor antagonist, CV-11974. Although the expression of other angiogenic factors, such as angiopoietin-1, angiopoietin-2, epidermal growth factor, and VEGF receptor 2 mRNA, was also investigated in tumor tissues, the expression of VEGFa was most correlated with tumor size among those other angiogenic factors. VEGFa induction by AT1 receptor-signaling in both host and tumor tissues is one of key regulators of tumor growth and tumor-associated angiogenesis. In conclusion, tumor tissue RAS as well as host tissue RAS were found to have an important role in tumor growth. AT1 receptor-signaling blockade may be a novel and effective target in the treatment of cancer.

Regulation of androgen receptor expression through angiotensin II type 1 receptor in prostate cancer cells.

Although the local renin‐angiotensin system (RAS) of the prostate gland is related to cell proliferation and angiogenesis, the detailed mechanism remains unclear. We examined the effects of the angiotensin II type 1 receptor (AT1R) on androgen receptor (AR) expression in prostate cancer cells.

Expression of angiotensin II type 1 receptor-interacting molecule in normal human kidney and IgA nephropathy

The intrarenal renin-angiotensin system plays a crucial role in the regulation of renal circulation and sodium reabsorption through the activation of vascular, glomerular, and tubular angiotensin II type 1 (AT(1)) receptor signaling. We previously cloned a molecule that specifically interacted with the murine AT(1) receptor to inhibit AT(1) receptor signaling, which we named ATRAP (for AT(1) receptor-associated protein). Since murine ATRAP was shown to be highly expressed in the kidney, in the present study we investigated expression and distribution of human ATRAP in normal kidney and renal biopsy specimens from patients with IgA nephropathy. In the normal human kidney, both ATRAP mRNA and protein were widely and abundantly distributed along the renal tubules from Bowmans capsule to the medullary collecting ducts. In all renal tubular epithelial cells, the ATRAP protein colocalized with the AT(1) receptor. In renal biopsy specimens with IgA nephropathy, a significant positive correlation between ATRAP and AT(1) receptor gene expression was observed. There was also a positive relationship between tubulointerstitial ATRAP expression and the estimated glomerular filtration rate in patients with IgA nephropathy. Furthermore, we examined the function of the tubular AT(1) receptor using an immortalized cell line of mouse distal convoluted tubule cells (mDCT) and found that overexpression of ATRAP by adenoviral gene transfer suppressed the angiotensin II-mediated increases in transforming growth factor-β production in mDCT cells. These findings suggest that ATRAP might play a role in balancing the renal renin-angiotensin system synergistically with the AT(1) receptor by counterregulatory effects in IgA nephropathy and propose an antagonistic effect of tubular ATRAP on AT(1) receptor signaling.

Urinary Oxidative Stress Markers Closely Reflect the Efficacy of Candesartan Treatment for Diabetic Nephropathy

Backgrounds/Aims: It has been reported that urinary oxidative stress markers are higher in diabetic patients with proteinuria. We performed the present study to elucidate the relationship between urinary excretion of oxidative stress markers, albumin excretion, and histological changes, and to confirm the potential utility of oxidative stress markers for clinical treatment. Methods: Diabetic db/db mice or nondiabetic db/m mice were administered candesartan (10 mg/kg/day) or hydralazine (50 mg/kg/day) for 18 weeks. Results: Thirty-week-old male db/db mice treated with control vehicle revealed elevated urinary excretion and immunohistological levels of 8-hydroxydeoxyguanosine in glomeruli when compared to db/m mice. Treatment with candesartan, but not hydralazine, reduced these values to levels in db/m mice. Increased mesangial expansion, urinary excretion of albumin and 8-isoprostane, and glomerular immunohistological levels of nitrotyrosine in db/db mice were also decreased markedly by candesartan but not hydralazine. Interestingly, correlations between levels of albumin and oxidative stress markers in urine were very high, even when groups undergoing long-term (44 weeks) treatment were included (correlation coefficient 0.767 with respect to 8-hydroxydeoxyguanosine, 0.888 with respect to 8-isoprostane). Conclusion: It is anticipated that urinary concentrations of oxidative stress markers will be direct barometers of glomerulus-derived oxidative stress and glomerular injury in diabetic nephropathy.

Behçet's disease complicated by IgA nephropathy with nephrotic syndrome.

A 65-year-old woman with a 48-year history of Behçet’s disease associated with nephrotic syndrome is described. Immunofluorescence study revealed IgA nephropathy. Following treatment with an angiotensin II type-I receptor-blocker, an anti-platelet drug, and an HMG-CoA reductase inhibitor, accompanied by dietary restrictions of protein and sodium, proteinuria was markedly decreased. This report describes our experience with a rare entity of Behçet’s disease complicated by nephrotic syndrome due to IgA nephropathy. Routine urine examination and renal biopsy are needed for the detection and diagnosis of renal problems with Behçet’s disease.

Arterial wall hypertrophy is ameliorated by α2-adrenergic receptor antagonist or aliskiren in kidneys of angiotensinogen-knockout mice

BackgroundArterial hypertrophy and interstitial fibrosis are important characteristics in kidneys of angiotensinogen-knockout (Atg−/−) mice. In these mice, which exhibit polyuria and hypotension, sympathetic nerve signaling is estimated to be compensatorily hyperactive. Furthermore, transforming growth factor (TGF)-β1 is overexpressed in mice kidneys. To determine whether sympathetic nerve signaling and TGF-β1 exacerbate arterial hypertrophy and interstitial fibrosis, intervention studies of such signaling are required.MethodsWe performed renal denervation and administered the α2-adrenergic receptor (AR) antagonist, atipamezole, to Atg−/− mice. A renin inhibitor, aliskiren, which was preliminarily confirmed to reduce TGF-β1 gene expression in kidneys of the mice, was additionally administered to assess the effect on the arterial hypertrophy and interstitial fibrosis.ResultsNorepinephrine content in kidneys of Atg−/− mice was three times higher than in kidneys of wild-type mice. Interventions by renal denervation and atipamezole resulted in amelioration of the histological findings. Overexpression of TGF-β1 gene in kidneys of Atg−/− mice was altered in a manner linked to the histological findings. Surprisingly, aliskiren reduced α2-AR gene expression, interstitial fibrosis, and arterial hypertrophy in kidneys of Atg−/− mice, which lack renin substrate.ConclusionsAlpha2-AR signaling is one of the causes of persistent renal arterial hypertrophy in Atg−/− mice. Aliskiren also angiotensinogen-independently reduces the extent of renal arterial hypertrophy, partly thorough downregulation of α2-ARs. Although renal arterial hypertrophy in Atg−/− mice appears to be of multifactorial origin, TGF-β1 may play a key role in the persistence of such hypertrophy.

Abstract 3187: Regulation of androgen receptor expression through angiotensin 2 receptor in prostate cancer cells

Background We have investigated the involvement of renin-angiotensin system (RAS) in the development and progression of prostate cancer. Although the local RAS of prostate gland related to cell proliferation and angiogenesis has the potential in carcinogenesis of prostate, the detailed mechanism remains unclear. In the present study, we examined the effects of angiotensin 2 (Ang2) signal, especially through AT1 receptor (AT1R) in prostate cancer cells. Method Cell proliferations of prostate cancer, androgen dependent LNCaP and androgen independent DU145 cells, were examined after knocking down AT1R by AT1 small interfering RNA (siRNA) transfection. We confirmed the expression of AT1R, androgen receptor (AR) and its related proteins by Western blot analysis. Immunocytochemical staining of AR in LNCaP cells transfected with AT1R siRNA (LNCaP siRNA cells) was performed. We examined the promoter activities of AR and PSA in luciferase assay by transfection of AT1R siRNA. Result Cell proliferation of LNCaP siRNA and DU145 cells transfected with AT1R siRNA were decreased on 7 days by 75% and 52%, respectively. Western blot analysis showed the increase of cleaved PARP and survivin related to apoptosis. Interestingly, AR was dramatically suppressed in LNCaP siRNA cells. The flow cytometry of LNCaP siRNA cells showed G2 arrest or increase of apoptosis. In addition, PSA, COX2, NFκB, and c-myc expression were decreased in LNCaP siRNA cells. Luciferase assay showed that the activity of AR promoter or PSA promoter were inhibited by AT1R knocking down. In immunocytochemical staining, the suppression of AR translocation into nucleus by DHT was confirmed in LNCaP siRNA cells. Ang2 induced the cell proliferation associated with the enhancement of AR, PSA, and NFκB, and furthermore the activity of AR or PSA promoter. Discussion In this study, we focused on the involvement of RAS in the AR expression of prostate cancer cells. Knocking down AT1 receptor protein resulted in a significant inhibition of cell growth associated with a remarked decrease of AR protein. We also found that the expression level of AT1R could modulate the transcriptional level of AR by affecting c-myc and NFκB expression. Taken together, there seems to be a strong relationship between AR and Ang2-AT1R signals. These results indicate that the inhibition of AT1 receptor has a potential to influence the AR expression in prostate cells, presumably contributing to develop novel therapeutic agents for prostate cancer, especially hormone refractory cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3187.