Tatsuo Shimosawa

Senescence marker protein-30 knockout mouse liver is highly susceptible to tumor necrosis factor-α- and fas-mediated apoptosis

Senescence marker protein-30 (SMP30) is a calcium-binding protein that decreases in an androgen-independent manner with aging. To elucidate the physiological role of this protein, we introduced a null mutation of the SMP30 gene into the germ line of mice. Despite the complete lack of SMP30 (SMP30-/-), these mutant mice were indistinguishable from their wild-type (SMP30+/+) littermates in terms of development and fertilization capability. We then investigated the tissue susceptibility for apoptosis induced by cytokine using primary cultured hepatocytes, because SMP30 could rescue cells from cell death caused by calcium influx, using a calcium ionophore as previously described. SMP30-/- hepatocytes were found to be more susceptible to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plus actinomycin D (ActD) than SMP30+/+ hepatocytes. In addition, the TNF-alpha/ActD-induced caspase-8 activity in SMP30-/- hepatocytes was twofold greater than that in SMP30+/+ hepatocytes. In contrast, no significant difference was observed in the TNF-alpha/ActD-induced nuclear factor-kappa B activation of SMP30+/+ versus SMP30-/- hepatocytes, indicating that SMP30 is not related to TNF-alpha/ActD-induced nuclear factor-kappa B activation itself. Moreover, deletion of the SMP30 gene enhanced liver injury after treatment in vivo with anti-Fas antibody and the SMP30+/- mice showed intermediate susceptibility to Fas-induced apoptosis. Collectively, these results demonstrate that SMP30 acts to protect cells from apoptosis.

Epigenetic modulation of the renal β-adrenergic–WNK4 pathway in salt-sensitive hypertension

How high salt intake increases blood pressure is a key question in the study of hypertension. Salt intake induces increased renal sympathetic activity resulting in sodium retention. However, the mechanisms underlying the sympathetic control of renal sodium excretion remain unclear. In this study, we found that β2-adrenergic receptor (β2AR) stimulation led to decreased transcription of the gene encoding WNK4, a regulator of sodium reabsorption. β2AR stimulation resulted in cyclic AMP-dependent inhibition of histone deacetylase-8 (HDAC8) activity and increased histone acetylation, leading to binding of the glucocorticoid receptor to a negative glucocorticoid−responsive element in the promoter region. In rat models of salt-sensitive hypertension and sympathetic overactivity, salt loading suppressed renal WNK4 expression, activated the Na+-Cl− cotransporter and induced salt-dependent hypertension. These findings implicate the epigenetic modulation of WNK4 transcription in the development of salt-sensitive hypertension. The renal β2AR-WNK4 pathway may be a therapeutic target for salt-sensitive hypertension.

Diminished nocturnal decline in blood pressure in elderly hypertensive patients with left ventricular hypertrophy

To assess the circadian blood pressure (BP) changes in elderly hypertensive patients with left ventricular hypertrophy (LVH), the ambulatory BP was measured noninvasively every 30 minutes for 24 hours in those patients with LVH (n = 15) and without LVH (n = 23), and in normotensive elderly subjects (n = 11). Although the daytime systolic BP (SBP) was comparable in the two hypertensive groups, the nighttime SBP in patients with LVH tended to be higher than in patients without LVH (149.0 +/- 15.1 versus 138.4 +/- 20.1 mm Hg, p less than 0.10). The LV mass index correlated significantly with the nighttime SBP (r = 0.43, p less than 0.01), but not with the daytime SBP (r = 0.24, ns), with clinic SBP (r = 0.14, p = ns) or the SBP after handgrip exercise (r = 0.31, p = ns). The difference in the systolic BP between daytime and nighttime (D-N SBP) in patients with LVH (2.8 +/- 9.4 mm Hg) was significantly less than that in patients without LVH (12.8 +/- 16.0 mm Hg) (p less than 0.02). In addition, the D-N SBP correlated inversely with the left ventricular mass index (r = -0.33, p less than 0.05). It was concluded that hypertension in the elderly with LVH was associated with a diminished nocturnal decline in blood pressure.

Proadrenomedullin NH(2)-terminal 20 peptide, a new product of the adrenomedullin gene, inhibits norepinephrine overflow from nerve endings.

Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin, which are derived from proadrenomedullin, exhibit remarkable hypotensive action. We investigated the effect of PAMP and adrenomedullin on peripheral sympathetic neutral transmission. Using perfused rat mesenteric arteries, PAMP (0, 1, 5, and 10 pmol/ml) decreased norepinephrine overflow by periarterial electrical nerve stimulation in a dose-dependent fashion (0.244 +/- 0.043, 0.231 +/- 0.048, 0.195 +/- 0.061 and 0.168 +/- 0.051 ng/gram tissue weigh: NS, P < 0.05, and P < 0.02, respectively). In contrast to PAMP, adrenomedullin (1, 5, and 10 pmol/ml) did not change it. In contrast, vasoconstrictive response of mesenteric arteries to exogenous norepinephrine was significantly attenuated by 10 pmol/ml of adrenomedullin but not by the same dose of PAMP. Calcitonin gene-related peptide (8-37) [CGRP(8-37)], a CGRP receptor antagonist, inhibited the vasodilatory effect of adrenomedullin but could not suppress the sympathoinhibitory effect of PAMP. Neither a nicotinic antagonist, hexamethonium, nor a presynaptic alfa2 antagonist, yohimbine, blocked the sympathoinhibitory effect of PAMP. Thus, it suggests that PAMP and adrenomedullin, which are derived from the same gene, exhibit different hypotensive mechanisms: PAMP inhibits neural transmission at peripheral sympathetic nerve ending, although adrenomedullin directly dilates vascular smooth muscle, possibly through CGRP-like receptor.

Rac1 GTPase in rodent kidneys is essential for salt-sensitive hypertension via a mineralocorticoid receptor–dependent pathway

Hypertension is a leading contributor to cardiovascular mortality worldwide. Despite this, its underlying mechanism(s) and the role of excess salt in cardiorenal dysfunction are unclear. Previously, we have identified cross-talk between mineralocorticoid receptor (MR), a nuclear transcription factor regulated by the steroid aldosterone, and the small GTPase Rac1, which is implicated in proteinuric kidney disease. We here show that high-salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via an MR-dependent pathway. We found that a high-salt diet caused renal Rac1 upregulation in salt-sensitive Dahl (Dahl-S) rats and downregulation in salt-insensitive Dahl (Dahl-R) rats. Despite a reduction of serum aldosterone levels, salt-loaded Dahl-S rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hypertension and renal damage with MR repression. We further demonstrated in aldosterone-infused rats as well as adrenalectomized Dahl-S rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR overactivity and hypertension. Finally, we confirmed the key role of Rac1 in modulating salt susceptibility in mice lacking Rho GDP-dissociation inhibitor α. Therefore, our data identify Rac1 as a determinant of salt sensitivity and provide insights into the mechanism of salt-induced hypertension and kidney injury.

Plasma adrenomedullin in diabetes

Vol 350 • November 15, 1997 1449 had secondary DVT and six cancers were diagnosed among these patients during their hospital stay (1·2%). When thrombosis was idiopathic, the rate of occult cancer was significantly higher (3·9%, p<0·02). Among the 64 patients with bilateral DVT, 41 (64%) had secondary bilateral DVT and two cancers were diagnosed (4·9%). When bilateral DVT was idiopathic, the rate of cancer was significantly higher than when it was not (35%, p<0·001). To our knowledge, this is the first study to establish the increased risk of occult cancer in bilateral DVT. The low rate of bilateral DVT probably explains the lack of data in the published research. However, this was a retrospective study with possible bias. We cannot rule out the possibility that clinical examination was more meticulous in patients with bilateral DVT. It is also possible that screening tests were more extensive in patients with bilateral venous thrombosis. However, most cancers are diagnosed by clinical examination, chest radiography, or standard biological screening tests, which were routinely done in all our patients. Further followup will be required to determine the rate of undiagnosed cancers. The rate of known cancers in our series (13%) and the rate of occult cancers found during the hospital stay (3·2%) were similar to those in other studies. Symptomatic pulmonary embolism was more common in patients with bilateral DVT than in those with unilateral DVT. The bilateral thrombosis can explain an increased risk of PE. The presence of PE and the location of DVT (proximal versus inferior vena cava) were not associated with the risk of occult cancer. Our study shows an increased rate of occult cancers in bilateral DVT (mainly in idiopathic bilateral DVT), with malignant disease in one-third of the patients. These data call for an extensive search for occult cancer in this situation, that concerns less than 10% of patients with DVT.

Blockade of the Nuclear Factor-κB Pathway in the Endothelium Prevents Insulin Resistance and Prolongs Life Spans

Background— Nuclear factor-&kgr;B (NF-&kgr;B) signaling plays critical roles in physiological and pathological processes such as responses to inflammation and oxidative stress. Methods and Results— To examine the role of endothelial NF-&kgr;B signaling in vivo, we generated transgenic mice expressing dominant-negative I&kgr;B under the Tie2 promoter/enhancer (E-DNI&kgr;B mice). These mice exhibited functional inhibition of NF-&kgr;B signaling specifically in endothelial cells. Although E-DNI&kgr;B mice displayed no overt phenotypic changes when young and lean, they were protected from the development of insulin resistance associated with obesity, whether diet- or genetics-induced. Obesity-induced macrophage infiltration into adipose tissue and plasma oxidative stress markers were decreased and blood flow and mitochondrial content in muscle and active-phase locomotor activity were increased in E-DNI&kgr;B mice. In addition to inhibition of obesity-related metabolic deteriorations, blockade of endothelial NF-&kgr;B signaling prevented age-related insulin resistance and vascular senescence and, notably, prolonged life span. These antiaging phenotypes were also associated with decreased oxidative stress markers, increased muscle blood flow, enhanced active-phase locomotor activity, and aortic upregulation of mitochondrial sirtuin-related proteins. Conclusions— The endothelium plays important roles in obesity- and age-related disorders through intracellular NF-&kgr;B signaling, thereby ultimately affecting life span. Endothelial NF-&kgr;B signaling is a potential target for treating the metabolic syndrome and for antiaging strategies.

Fibroblast growth factor 23 accelerates phosphate-induced vascular calcification in the absence of Klotho deficiency

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating hormone that acts primarily on the kidney and parathyroid. With declining kidney function there is an increase in circulating FGF23 levels, which is associated with vascular calcification and mortality in chronic kidney disease. Whether FGF23 exerts direct effects on vasculature is unclear. We evaluated the expression of Klotho and FGF receptors in rat aortic rings and rat aorta vascular smooth muscle cells maintained in culture by reverse transcription-PCR, western blotting, and immunostaining. Signaling pathways underlying FGF23 effects were assessed by western blotting, and effects of FGF23 on osteogenic markers and phosphate transporters were assessed by real-time reverse transcription-PCR. We detected Klotho and FGFR1 in total aorta but not in vascular smooth muscle cells. FGF23 augmented phosphate-induced vascular calcification in the aortic rings from uremic rats and dose dependently increased ERK1/2 phosphorylation in Klotho-overexpressing but not naive vascular smooth muscle cells. FGF23-induced ERK1/2 phosphorylation was inhibited by SU5402 (FGFR1 inhibitor) and U0126 (MEK inhibitor). FGF23 enhanced phosphate-induced calcification in Klotho-overexpressing vascular smooth muscle cells and increased osteoblastic marker expression, which was inhibited by U0126. In contrast, phosphate transporter expression was not affected by phosphate or FGF23. Thus, FGF23 enhances phosphate-induced vascular calcification by promoting osteoblastic differentiation involving the ERK1/2 pathway.

Blockade of the NF-κB Pathway in the Endothelium Prevents Insulin Resistance and Prolongs Lifespans

Background— Nuclear factor-&kgr;B (NF-&kgr;B) signaling plays critical roles in physiological and pathological processes such as responses to inflammation and oxidative stress. Methods and Results— To examine the role of endothelial NF-&kgr;B signaling in vivo, we generated transgenic mice expressing dominant-negative I&kgr;B under the Tie2 promoter/enhancer (E-DNI&kgr;B mice). These mice exhibited functional inhibition of NF-&kgr;B signaling specifically in endothelial cells. Although E-DNI&kgr;B mice displayed no overt phenotypic changes when young and lean, they were protected from the development of insulin resistance associated with obesity, whether diet- or genetics-induced. Obesity-induced macrophage infiltration into adipose tissue and plasma oxidative stress markers were decreased and blood flow and mitochondrial content in muscle and active-phase locomotor activity were increased in E-DNI&kgr;B mice. In addition to inhibition of obesity-related metabolic deteriorations, blockade of endothelial NF-&kgr;B signaling prevented age-related insulin resistance and vascular senescence and, notably, prolonged life span. These antiaging phenotypes were also associated with decreased oxidative stress markers, increased muscle blood flow, enhanced active-phase locomotor activity, and aortic upregulation of mitochondrial sirtuin-related proteins. Conclusions— The endothelium plays important roles in obesity- and age-related disorders through intracellular NF-&kgr;B signaling, thereby ultimately affecting life span. Endothelial NF-&kgr;B signaling is a potential target for treating the metabolic syndrome and for antiaging strategies.

Protective Effect of Potassium Against the Hypertensive Cardiac Dysfunction: Association With Reactive Oxygen Species Reduction

Potassium supplementation has a potent protective effect against cardiovascular disease, but the precise mechanism of it against left ventricular abnormal relaxation, relatively early functional cardiac alteration in hypertensive subjects, has not been fully elucidated. In the present study, we investigated the effect of potassium against salt-induced cardiac dysfunction and the involved mechanism. Seven- to 8-week–old Dahl salt sensitive rats were fed normal diet (0.3% NaCl) or high-salt diet (8% NaCl) with or without high potassium (8% KCl) for 8 weeks. Left ventricular relaxation was evaluated by the deceleration time of early diastolic filling obtained from Doppler transmitral inflow, the slope of the pressure curve, and the time constant at the isovolumic relaxation phase. High-salt loading induced a significant elevation of blood pressure and impaired left ventricular relaxation, accompanied by augmentation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity in the cardiac tissue, measured by the lucigenin chemiluminescence method. Blood pressure lowering by hydralazine could not ameliorate NADPH oxidase activity and resulted in no improvement of left ventricular relaxation. Interestingly, although the blood pressure remained high, potassium supplementation as well as treatment with 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl, a superoxide dismutase mimetic, not only reduced the elevated NADPH oxidase activity but also improved the left ventricular relaxation. In conclusion, a high-potassium diet has a potent protective effect on left ventricular active relaxation independent of blood pressure, partly through the inhibition of cardiac NADPH oxidase activity. Sufficient potassium supplementation might be an attractive strategy for cardiac protection, especially in the salt-sensitive hypertensive subjects.