Renal damage and salt-dependent hypertension in aged transgenic mice overexpressing endothelin-1.

The recent development of endothelin-1 (ET-1) antagonists and their potential use in the treatment of human disease raises questions as to the role of ET-1 in the pathophysiology of such cardiovascular ailments as hypertension, heart failure, renal failure and atherosclerosis. It is still unclear, for example, whether activation of an endogenous ET-1 system is itself the primary cause of any of these ailments. In that context, the phenotypic manifestations of chronic ET-1 overproduction may provide clues about the tissues and systems affected by ET-1. We therefore established two lines of transgenic mice overexpressing the ET-1 gene under the direction of its own promoter. These mice exhibited low body weight, diminished fur density and two- to fourfold increases in the ET-1 levels measured in plasma, heart, kidney and aorta. There were no apparent histological abnormalities in the visceral organs of young (8 weeks old) transgenic mice, nor was their blood pressure elevated. In aged (12 months old) transgenic mice, however, renal manifestations, including prominent interstitial fibrosis, renal cysts, glomerulosclerosis and narrowing of arterioles, were detected. These pathological changes were accompanied by decreased creatinine clearance, elevated urinary protein excretion and salt-dependent hypertension. It thus appears that mild, chronic overproduction of ET-1 does not primarily cause hypertension but triggers damaging changes in the kidney which lead to the susceptibility to salt-induced hypertension.

Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene.

BACKGROUND: Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. Levels of AM are markedly increased in the fetoplacental circulation during pregnancy, although its function there remains unknown. To clarify the physiological functions of AM, we chose a gene-targeting strategy in mice. METHODS AND RESULTS: Targeted null mutation of the AM gene is lethal in utero: the mortality rate among AM(-/-) embryos was >80% at E13.5. The most apparent abnormality in surviving AM(-/-) embryos at E13.5 to E14.0 was severe hemorrhage, readily observable under the skin and in visceral organs. Hemorrhage was not detectable at E12.5 to E13.0, although the yolk sac lacked well-developed vessels. Electron microscopic examination showed endothelial cells to be partially detached from the basement structure at E12.5 in vitelline vessels and hepatic capillaries, which allowed efflux of protoerythrocytes through the disrupted barrier. The basement membrane was not clearly recognizable in the aorta and cervical artery, and the endothelial cells stood out from the wall of the lumen, only partially adhering to the basement structure. AM(+/-) mice survived to adulthood but exhibited elevated blood pressures with diminished nitric oxide production. CONCLUSIONS: AM is indispensable for the vascular morphogenesis during embryonic development and for postnatal regulation of blood pressure by stimulating nitric oxide production.

Growth hormone signalling and apoptosis in neonatal rat cardiomyocytes.

Growth hormone (GH) has been reported to be useful to treat heart failure. To elucidate whether GH has direct beneficial effects on the heart, we examined effects of GH on oxidative stress-induced apoptosis in cardiac myocytes. TUNEL staining and DNA ladder analysis revealed that hydrogen peroxide (H2O2)-induced apoptosis of cardiomyocytes was significantly suppressed by the pretreatment with GH. GH strongly activated extracellular signal-regulated kinases (ERKs) in cardiac myocytes and the cardioprotective effect of GH was abolished by inhibition of ERKs. Overexpression of dominant negative mutant Ras suppressed GH-stimulated ERK activation. Overexpression of Csk that inactivates Src family tyrosine kinases also inhibited ERK activation evoked by GH. A broad-spectrum inhibitor of protein tyrosine kinases (PTKs), genistein, strongly suppressed GH-induced ERK activation and the cardioprotective effect of GH against apoptotic cell death. GH induced tyrosine phosphorylation of EGF receptor and JAK2 in cardiac myocytes, and an EGF receptor inhibitor tyrphostin AG1478 and a JAK2 inhibitor tyrphostin B42 completely inhibited GH-induced ERK activation. Tyrphostin B42 also suppressed the phosphorylation of EGF receptor stimulated by GH. These findings suggest that GH has a direct protective effect on cardiac myocytes against apoptosis and that the effect of GH is attributed at least in part to the activation of ERKs through Ras and PTKs including JAK2, Src, and EGF receptor tyrosine kinase.

The role of the natriuretic peptides in the cardiovascular system.

The discovery of the natriuretic peptide family was a breakthrough in modern cardiovascular physiology as it provided a direct link between the heart and the kidneys in the regulation of natriuresis. Along with vasopressin and the renin-angiotensin-aldosterone system, the natriuretic peptides comprise the key peptides on which our present understanding of neuroendocrine regulation of the cardiovascular system is based. Three natriuretic peptides have been identified; the A-type, B-type and C-type natriuretic peptides. The former two, the A- and B-type natriuretic peptides, function mainly in the cardiovascular system and comprise the cardiac natriuretic peptides. Together with our increased understanding of the neurohormonal regulation of the cardiovascular system in recent years, the discovery of the natriuretic peptide family was important in the establishment of the new field of cardiovascular endocrinology.

Isoproterenol activates extracellular signal-regulated protein kinases in cardiomyocytes through calcineurin.

BACKGROUND: Extracellular signal-regulated kinases (ERKs) and calcineurin have been reported to play important roles in the development of cardiac hypertrophy. We examined here the relation between calcineurin and ERKs in cardiomyocytes. METHODS AND RESULTS: Isoproterenol activated ERKs in cultured cardiomyocytes of neonatal rats, and the activation was abolished by chelation of extracellular Ca(2+) with EGTA, blockade of L-type Ca(2+) channels with nifedipine, or depletion of intracellular Ca(2+) stores with thapsigargin. Isoproterenol-induced activation of ERKs was also significantly suppressed by calcineurin inhibitors in cultured cardiomyocytes as well as in the hearts of mice. Isoproterenol failed to activate ERKs in either the cultured cardiomyocytes or the hearts of mice that overexpress the dominant negative mutant of calcineurin. Isoproterenol elevated intracellular Ca(2+) levels at both systolic and diastolic phases and dose-dependently activated calcineurin. Inhibition of calcineurin also attenuated isoproterenol-stimulated phosphorylation of Src, Shc, and Raf-1 kinase. The immunocytochemistry revealed that calcineurin was localized in the Z band, and isoproterenol induced translocation of calcineurin and ERKs into the nucleus. CONCLUSIONS: Calcineurin, which is activated by marked elevation of intracellular Ca(2+) levels by the Ca(2+)-induced Ca(2+) release mechanism, regulates isoproterenol-induced activation of ERKs in cardiomyocytes.

CREB antisense oligonucleotides induce non-apoptotic cell death in proliferating leukemia cells, but not normal hematopoietic cells, by a bizarre non-antisense mechanism.

We report that antisense phosphorothioate oligodeoxyribonucleotides (PS-ODNs) against cyclic AMP response element-binding protein (CREB) induce the death of human leukemia cell lines including HL-60, Kasumi-1 and K562, OCI-AML1a and also primary leukemia cells isolated from patients with acute myelocytic leukemia and chronic myelocytic leukemia in blastic crisis. In contrast, normal human bone marrow CD34+ cells and normal peripheral blood lymphocytes were resistant to the antisense-mediated cell death. We found that antisense-treated HL-60 cells had prominent nuclear fragmentations but lacked apoptotic features including internucleosomal DNA cleavage and TUNEL positivity. Cell cycle analysis demonstrated a remarkable reduction in G1 phase population along with a mild accumulation of S phase and good preservation of G2/M phase, indicating cells died at G2/M without cycling into G1 phase. None of the sense-sequenced PS-ODNs induced cell death. Further, neither the expression nor the message of CREB protein was reduced by antisense treatment, indicating that cell death was mediated by a non-antisense mechanism. On the other hand, no consensus oligonucleotide sequence for cell death induction was detected. Rather, we found a good correlation between the melting temperatures and the anti-proliferative activities of the oligonucleotides. Thus, CREB antisense PS-ODNs selectively induce a non-apoptotic cell death in leukemic cells by an unknown hybridization-dependent mechanism.

Biventricular hypertrophic cardiomyopathy with right ventricular outflow tract obstruction associated with Noonan syndrome in an adult.

This report describes an adult patient with Noonan syndrome accompanied by biventricular hypertrophic cardiomyopathy causing isolated right ventricular outflow tract obstruction. Biventricular hypertrophic cardiomyopathy causing right- and/or left-side outflow tract obstruction, as well as valvular pulmonary stenosis, is relatively common in infants with Noonan syndrome. However, this condition without a dysplastic pulmonary valve, or indeed any polyvalvular dysplasia, is rare in adults with Noonan syndrome. Treatment with a beta-adrenergic receptor blocking agent improved the patient's symptoms. Because neither the etiologic and prognostic relationship nor the genetic linkage between hypertrophic cardiomyopathy associated with Noonan syndrome and non-syndromic hypertrophic cardiomyopathy is clearly defined, clinicopathological findings and further follow-up may provide important evidence for the pathogenesis of hypertrophic cardiomyopathy.

Diet-induced hyperhomocysteinemia exacerbates neointima formation in rat carotid arteries after balloon injury.

BACKGROUND: Increasing evidence indicates that elevated plasma homocysteine levels are associated with an increased risk of atherosclerosis and endothelial dysfunction, although little specific information on the mechanisms responsible for the atherogenic effects of homocysteine or on the in vivo contribution made by hyperhomocysteinemia to atherosclerosis is currently available. Because homocysteine is known to exert a direct inhibitory effect on endothelial cell growth in vitro, we hypothesized that this effect contributes to the progression of atherosclerotic lesions initiated by endothelial damage caused by mechanical injury. METHODS AND RESULTS: We prepared diet-induced hyperhomocysteinemic rats in which neointima formation after balloon injury to the common carotid artery was assessed. Moderate hyperhomocysteinemia (plasma homocysteine levels 3- to 4-fold higher than control) significantly exacerbated neointima formation. Oral administration of folate, which had a homocysteine-lowering effect, diminished neointima formation induced by moderate hyperhomocysteinemia. Furthermore, the attenuation of reendothelialization was shown in diet-induced hyperhomocysteinemic rats with Evans blue staining. CONCLUSIONS: Diet-induced hyperhomocysteinemia, even mild to moderate, exacerbates neointima formation after denuding injury, making hyperhomocysteinemia a likely risk factor for postangioplasty restenosis. It may be mediated through an inhibitory effect of homocysteine on reendothelialization. Homocysteine lowering with folate supplementation can effectively ameliorate the detrimental effects of moderate hyperhomocysteinemia. Clinical trials would seem to be warranted.

Rho plays an important role in angiotensin II-induced hypertrophic responses in cardiac myocytes.

Angiotensin II (Ang II) evokes a variety of hypertrophic responses such as activation of protein kinases, reprogramming of gene expressions and an increase in protein synthesis in cardiac myocytes. In this study, we examined the role of Rho family small GTP binding proteins (G proteins) in Ang II-induced cardiac hypertrophy. Ang II strongly activated extracellular signal-regulated protein kinases (ERKs) in cardiac myocytes of neonatal rats. Although Ang II-induced activation of ERKs was completely suppressed by an Ang II type 1 receptor antagonist, CV-11974, this activation was not inhibited by the pretreatment with C3 exoenzyme, which abrogates Rho functions. Overexpression of Rho GDP dissociation inhibitor (Rho-GDI), dominant negative mutants of Rac1 (D.N.Rac1), or D.N.Cdc42 had no effects on Ang II-induced activation of transfected ERK2. The promoter activity of skeletal alpha-actin and c-fos genes was increased by Ang II, and the increase was partly inhibited by overexpression of Rho-GDI and the pretreatment with C3 exoenzyme. Ang II increased phenylalanine incorporation into cardiac myocytes by approximately 1.4 fold as compared with control, and this increase was also significantly suppressed by the pretreatment with C3 exoenzyme. These results suggest that the Rho family small G proteins play important roles in Ang II-induced hypertrophic responses in cardiac myocytes.

Homocysteine as a risk factor for restenosis after coronary angioplasty.

We examined the relationship between plasma homocysteine levels and restenosis after PTCA (Percutaneous transluminal coronary angioplasty) to investigate whether plasma homocysteine levels can be a predictor of restenosis after PTCA. One hundred and twelve male patients who have undergone a successful elective PTCA were consecutively enrolled and plasma homocysteine levels were measured at the time of follow-up angiography. Plasma homocysteine levels in patients with restenosis were significantly higher than those in patients without restenosis (15.0 +/- 3.9 vs. 13 +/- 2.9 micromol/L; P = 0.011). The difference was augmented when diabetic patients were selectively studied. The comparison between restenosis group and non-restenosis group indicated the threshold effect of hyperhomocysteinemia. These results suggest that plasma homocysteine is a potential risk factor of restenosis after PTCA, and therapeutic strategy targeted against hyperhomocysteinemia may be beneficial for preventing restenosis.

Functional analyses of three Csx/Nkx-2.5 mutations that cause human congenital heart disease.

A homeodomain-containing transcription factor Csx/Nkx-2.5 is an important regulator of cardiogenesis in mammals. Three different mutants, Gln170ter (designated A) and Thr178Met (designated B) in the helix 2 of the homeodomain and Gln198ter mutation (designated C) just after homeodomain, have been reported to cause atrial septal defect with atrial ventricular block. We here examined the functions of these three mutants of Csx/Nkx-2.5. The atrial natriuretic peptide (ANP) promoter was activated by wild type Csx/Nkx-2.5 (WT, approximately 8-fold), B ( approximately 2-fold), and C ( approximately 6-fold) but not by A. When A, B, or C was cotransfected into COS-7 cells with the same amount of WT, WT-induced activation of the ANP promoter was attenuated by A and B (A > B), whereas C further enhanced the activation. Immunocytochemical analysis using anti-Myc tag antibody indicated that transfected Myc-tagged WT, B, and C were localized in the nucleus of both COS-7 cells and cardiomyocytes of neonatal rats, whereas A was distributed diffusely in the cytoplasm and nucleus in COS-7 cells. Electrophoretic mobility shift assay showed that Csx/Nkx-2.5-binding sequences were bound strongly by WT and C, weakly by B, but not by A. Immunoprecipitation and GST pull-down assay revealed that WT and all mutants interacted with GATA-4. The synergistic activation of the ANP promoter by WT and GATA-4 was further enhanced by C but was inhibited by A and B. In the cultured cardiomyocytes, overexpression of C but not WT, A, or B, induced apoptosis. These results suggest that although the three mutants induce the same cardiac phenotype, transactivation ability and DNA binding ability are different among the three mutants and that apoptosis may be a cause for C-induced cardiac defect.

Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer.

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.

Protein-losing enteropathy associated with hypocomplementemia and anti-nuclear antibodies.

We report a case of protein-losing enteropathy associated with an autoimmune disorder, presumably systemic lupus erythematosus. Although typical manifestations of systemic lupus erythematosus were lacking, a high serum cholesterol level, a low serum complement level, positivity for anti-nuclear antibody, and positivity for anti-single-strand DNA antibody suggested an autoimmune mechanism as the cause of the condition. Although immunohistological examination of duodenal and ileal biopsy specimens failed to reveal deposits of immune complex or complement in the vessels, capillary hyperpermeability was suspected as the mechanism of the condition.

Targeted disruption of the homeobox transcription factor Bapx1 results in lethal skeletal dysplasia with asplenia and gastroduodenal malformation.

BACKGROUND: NK homeobox genes have been shown to play important roles in cell-type specification and organogenesis. Murine Bapx1, a member of NK homeobox gene family, is expressed in all the cartilageous tissues that undergo endochondral bone formation, and in gut mesentery during embryogenesis, suggesting that Bapx1 may be a key transcription factor ragulating the development of these organs. RESULTS: We generated Bapx1-deficient mice by gene targeting. Bapx1-/- mice exhibited lethal skeletal dysplasia, with abnormal development of the vertebral column and some craniofacial bones, accompanied with asplenia and gastroduodenal malformation. We showed that the proliferative activity of the sclerotome cells, forming the vertebral column, was significantly reduced in Bapx1-/- embryos. The sclerotome cells of the mutants appeared to migrate and condense normally, but subsequent differentiation into the mature vertebral bodies and intervertebral discs were affected. The sclerotome cells in the vertebral bodies failed to differentiate into hypertrophic chondrocytes, as revealed by the undetected expression of Col10a1 and Osteopontin, and the sclerotome cells in the intervertebral discs failed to express the typical extracellular matrix proteins Col2a1, Col9a2 and aggrecan. Furthermore, we investigated the effect of loss of Bapx1 on the expression of some transcription factors, identified to be expressed in the developing sclerotome and be required for normal development of the vertebral column. Among them, we found that the expression of MFH-1 (mesenchyme forkhead-1), which was reported to regulate the proliferation and differentiation of sclerotome cells, was significantly reduced in ventromedial sclerotome cells in Bapx1-/- mice. CONCLUSION: Our analysis provided evidence that Bapx1 was indispensable for normal development of ventromedial structure of vertebral column and some of craniofacial bones, splenogenesis and morphogenesis of gastroduodenal tract.

Screening for cardiac dysfunction in asymptomatic patients by measuring B-type natriuretic peptide levels.

Early diagnosis and treatment of heart failure lead to improved survival; pre-clinical detection would thus be beneficial. A non-invasive biochemical testing method would indeed be ideal to screen for the condition. In the present study, we sought to determine whether circulating levels of B-type natriuretic peptide (BNP) correlate with cardiac function in asymptomatic subjects. 294 consenting asymptomatic subjects were examined. BNP levels in elevated patients (> 18.4 pg / ml) showed significant correlation with echocardiographic parameters of the systolic and diastolic functions (EF r = -0.51, FS r = -0.50, E/A r = 0.42, p < 0.01). Moderate correlation with the CTR on chest X-ray was also seen (r = 0.23, p < 0.01). Multiple regression analysis showed numerous echocardiographic and hemodynamic parameters including those of systolic and diastolic function in addition to left ventricular wall thickness, blood pressure and serum creatinine levels to be significantly associated with raised BNP levels. Elevated BNP levels reflect cardiac function (both systolic and diastolic) in the asymptomatic population. Detection of cardiac dysfunction by the non-invasive biochemical test may prove useful in early pre-clinical diagnosis of heart failure.

The evi-1 oncoprotein inhibits c-Jun N-terminal kinase and prevents stress-induced cell death.

Evi-1 encodes a nuclear protein involved in leukemic transformation of hematopoietic cells. Evi-1 possesses two sets of zinc finger motifs separated into two domains, and its characteristics as a transcriptional regulator have been described. Here we show that Evi-1 acts as an inhibitor of c-Jun N-terminal kinase (JNK), a class of mitogen-activated protein kinases implicated in stress responses of cells. Evi-1 physically interacts with JNK, although it does not affect its phosphorylation. This interaction is required for inhibition of JNK. Evi-1 protects cells from stress-induced cell death with dependence on the ability to inhibit JNK. These results reveal a novel function of Evi-1, which provides evidence for inhibition of JNK by a nuclear oncogene product. Evi-1 blocks cell death by selectively inhibiting JNK, thereby contributing to oncogenic transformation of cells.