永井 良三

We investigated the therapeutic effect of branched chain amino acids (BCAA) on mice with glucose intolerance induced by encephalomyocarditis virus (EMCV). Male DBA/2 mice were divided into three groups: treated with BCAA, (such as valine, leucine, and isoleucine), untreated, and control. BCAA-treated and -untreated groups were inoculated intraperitoneally with the NDK25 variant of EMCV at 200 plaque-forming units per mouse. The BCAA-treated group was administered orally 0.9 g/kg/day of each BCAA from the day after viral inoculation. The control group neither received virus inoculation nor was treated with BCAA. One week after inoculation, oral glucose tolerance tests (OGTT) were performed. After the glucose loading at 1.5 g/kg of body weight, blood glucose levels in the untreated group were 92.0+/-10.0 mg/dl at baseline, 224.6+/-10.9 mg/dl at 30 min, and 169.4+/-21.4 mg/dl at 60 min, which were significantly (P<0.05) higher than those in the control group (62. 7+/-3.6 mg/dl, 167.2+/-16.4, and 83.8+/-6.0 mg/dl, respectively). Blood glucose levels in the BCAA-treated group were 54.5+/-3.7 mg/dl at baseline, 145.2+/-8.7 mg/dl at 30 min, and 128.7+/-18.3 mg/dl at 60 min after the glucose loading, which were not significantly higher than those in the control group. Immunoreactive insulin levels at 30 min after the glucose loading were lower in the untreated group than in the control group at 1 week after virus inoculation. Histological investigations showed that the grade of insulitis in the pancreas of mice of the BCAA-treated group was lower than that of the mice of the untreated group. These results suggest that oral administration of BCAA is able to improve glucose intolerance induced by EMCV.

Cardiac homeobox gene Csx/Nkx-2.5 is essential for normal heart development and morphogenesis and is the earliest marker for cardiogenesis. To elucidate the regulatory mechanisms of Csx/Nkx-2.5 expression, we have isolated and characterized the upstream regulatory region of human Csx/Nkx-2.5 (CSX1). Transfection of the reporter gene containing a 965-bp CSX1 5' flanking region indicated that this region confers cardiomyocyte-predominant expression of CSX1. Deletion and mutational analyses revealed two positive cis-regulatory elements in this region that are essential for CSX1 expression in cardiomyocytes. Electrophoretic mobility shift assay revealed that nuclear proteins prepared from cardiac myocytes bound to these elements in a sequence-specific manner. The identification of cis-regulatory sequences of the Csx/Nkx-2.5 gene will facilitate further analysis for the upstream regulatory factors that control the expression of Csx/Nkx-2.5 and the process of vertebrate heart development.

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.

Vascular endothelial growth factor (VEGF) is known to play an essential role in embryonic vascular development. The heart is one of the main organs that produce VEGF, but it is still unknown how expression of VEGF gene is regulated in embryonic cardiac myocytes. Thus, we cloned cDNAs encoding VEGF and its receptor (a KDR/flk-1 or Quek-1 homologue) from cultured 10-day-old chick embryonic ventricular myocytes (CEVM). Reverse transcription-polymerase chain reaction revealed that the chide VEGF mRNAs consisted of at least four different species corresponding to the isoforms of 190, 166, 146 and 122 amino acids. In the embryonic heart and CEVM, the isoforms of 166 and 122 amino acids were dominant. Northern blot analysis detected an abundance of VEGF mRNA in both the embryonic heart and CEVM, even at the basal state. The levels of VEGF mRNA in CEVM were significantly augmented by forskolin (100 mu M), or phorbol 12-myristate, 13-acetate (200 nM) in a time-dependent manner in CEVM. In contrast, the basal levels of VEGF mRNA were attenuated by genistein (100 mu M), but not by H89 (100 mu M) or bisindolylmaleimide (75 mu M). Northern blot analysis also detected the chick flk-1 mRNA in abundance in the embryonic heart, and to a much lesser extent in CEVM. The expression levels of VEGF and flk-1 mRNA species were continuously high in the 6, 8 and 10-day-old chick embryonic hearts. In the 10-day-old embryonic hearts, in situ hybridization confirmed that mRNA encoding VEGF was mainly expressed in ventricular myocytes. In contrast, the flk-1 mRNA was detected in the microvascular endothelial cells, and to a lesser extent in the ventricular myocytes. These data suggest that VEGF is produced in embryonic ventricular myocytes, even at the basal state, and that the levels of VEGF mRNA may be differently regulated by various protein kinases. VEGF produced by the chick embryonic heart may play important roles in embryonic cardiovascular development by acting on surrounding endothelial cells and, possibly, on ventricular myocytes themselves. (C) 2000 Academic Press.

Coronary microangiopathy is a major complication in diabetics. However, the presence of independent factors in association with coronary microangiopathy in patients with non-insulin-dependent diabetes mellitus (NIDDM) or the difference in coronary microangiopathy between diabetics with coronary artery disease (GAD) and those with microvascular angina is unclear. Methods: Nineteen patients with NIDDM and microvascular angina, 18 patients with NIDDM and CAD, and 17 age-matched control subjects were studied. Myocardial segments that were perfused by angiographically normal coronary arteries were studied. The baseline myocardial blood flow (MBF) and the MBF during dipyridamole administration were measured using PET and N-13-ammonia, after which the myocardial flow reserve (MFR) was calculated to assess coronary microangiopathy. Results: The baseline MBF was comparable among NIDDM patients with microvascular angina, NIDDM patients with GAD, and control subjects. However, the MBF during dipyridamole administration was significantly lower in NIDDM patients with microvascular angina (126 +/- 42.7 mL/min/100 g) than that in either NIDDM patients with CAD (210 +/- 70.1 mL/min/100 g; P &lt; 0.01) or control subjects (293 +/- 159 mL/min/100 g; P &lt; 0.01), as was the MFR (NIDDM with microvascular angina, 1.90 +/- 0.73; NIDDM with CAD, 2.59 +/- 0.81 [P &lt; 0.01]; control subjects, 3.69 +/- 1.09 [P &lt; 0.01]). Multivariate stepwise regression analysis showed that, among the factors considered, glycemic control was independently related to the MFR (r = 0.838; P&lt; 0.05). Conclusion: Glycemic control appears to be essential for coronary microangiopathy in NIDDM.

Objectives: To develop a simple and practical enzyme immunoassay (EIA) for oxidatively modified low density lipoprotein (Ox-LDL) in human blood, a biological marker of atherogenesis. Design and methods: A sandwich EIA suitable for the measurement of human Ox-LDL was developed using the mouse monoclonal antibody FOH1a/DLH3. This antibody, specific for oxidized phosphatidylcholine, was used as the capture antibody, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein-B (Apo-B) IgG was used for detection. Copper-oxidized human LDL, prepared under controlled conditions, was used as a standard and the results of the EIA were expressed in arbitrary units (U/mL). Results: This EIA meets all the requirements for use in routine clinical assays in terms of sensitivity (detection limit: 1 U/mL), reproducibility (total CV: 2.3-7.7%), accuracy (recovery: 90.6-103.8%), simplicity and rapidity (&lt;4 h). Clinical performance of the assay was assessed by measurement of the Ox-LDL in the plasma of normal subjects (10.8 +/- 2.8 U/mL, mean +/- SD) and patients with coronary heart disease (CHD) (19.7 +/- 10.2). The present EIA had a sensitivity of 79% and a specificity of 75% for CHD. Conclusions: We have developed a new assay, suitable for the measurement of Ox-LDL in human blood, which meets the requirements for routine clinical assay, Copyright (C) 2000 The Canadian Society of Clinical Chemists.

Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca(2+) is a major second messenger of ET-1, the role of Ca(2+) in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the beta-type myosin heavy chain gene (beta-MHC) (-354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca(2+) and the blocking of protein kinase C activity. Similarly, the beta-MHC promoter was activated by Ca(2+) ionophores and a protein kinase C activator. beta-MHC promoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. beta-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [(3)H]phenylalanine uptake and in cell size. These results suggest that Ca(2+) plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.

BACKGROUND: Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. To determine the extent to which chronic AM overproduction affects circulatory physiology under normal and pathological conditions, we used a preproendothelin-1 promoter to establish transgenic mouse lines overexpressing AM in their vasculature. METHODS AND RESULTS: Transgenic mice overexpressing AM mainly in vascular endothelial and smooth muscle cells exhibited significantly lower blood pressure (BP) and higher plasma cGMP levels than their wild-type littermates. Blockade of NO synthase with N(G)-monomethyl-L-arginine elevated BP to a greater degree in AM transgenic mice, offsetting the BP difference between the 2 groups. Despite their lower basal BP, administration of bacterial lipopolysaccharide elicited smaller declines in BP and less severe organ damage in AM transgenic mice than in wild-type mice. Furthermore, the 24-hour survival rate after induction of lipopolysaccharide shock was significantly higher in the transgenic mice. CONCLUSIONS: A chronic increase in vascular AM production reduces BP at least in part via an NO-dependent pathway. In addition, smaller responses to LPS in transgenic mice suggest that AM is protective against the circulatory collapse, organ damage, and mortality characteristic of endotoxic shock.

The mechanisms responsible for regression of left ventricular (LV) mass with antihypertensive therapy in patients with severe hypertension remain unclear. This study was designed to examine whether systolic and diastolic blood pressures are associated with changes in LV mass. Eighteen patients with essential hypertension whose average seated diastolic blood pressure was >or = 110 mm Hg were enrolled in the study. All patients were administered antihypertensive therapy and underwent M-mode echocardiography before and after 6 months of treatment. In all patients, antihypertensive treatment significantly reduced systolic blood pressure from 175 +/- 21 mm Hg at baseline to 143 +/- 22 mm Hg at 6 months (p < 0.001), and diastolic blood pressure from 116 +/- 7 mm Hg at baseline to 92 +/- 20 mm Hg at 6 months (p < 0.001). LV mass index at 6 months was significantly reduced compared to its baseline value (p < 0.05). Change (value at 6 months-value at baseline) in systolic and diastolic blood pressures correlated positively with the change in LV mass index (r = 0.61, p < 0.01 and r = 0.71, p < 0.001, respectively). The patients were divided into responders. whose LV mass regressed by > or = 10% (n = 9), and nonresponders, whose LV mass regressed by < 10% (n = 9). Systolic (p < 0.001) and diastolic (p < 0.001) blood pressures. interventricular septal thickness (p< 0.05), posterior wall thickness (p < 0.001), and LV mass index (p < 0.001) were significantly decreased in the responders, but not in the nonresponders, at 6 months compared with those at baseline. Systolic (p < 0.05) and diastolic (p < 0.05) blood pressures in nonresponders were significantly higher than those in the responders at 6 months. The changes in systolic and diastolic blood pressures did not correlate with the change in LV mass index in the responders or the nonresponders. The regression of LV mass is strongly affected by reducing blood pressure. This is the first study using antihypertensive therapy to demonstrate that a change in blood pressure correlates positively with changes in LV mass index in severely hypertensive patients.

To evaluate the effects of left ventricular (LV) dysfunction upon the sympathetic nervous and renin-aldosterone-angiotensin systems, neurohormonal factors were measured in patients with ischemic heart disease. Eleven patients were divided into two groups by their LV ejection fraction based on previous catheterization; preserved (EF > or = 60%) and impaired (EF < 60%) LV systolic function groups. They performed supine ergometer exercise and blood samples were drawn at rest and at peak exercise. After dynamic exercise, plasma norepinephrine was significantly (p < 0.05) increased in patients with preserved LV function, whereas it was not altered in patients with impaired LV function (norepinephrine 20.8 +/- 20.5 vs 45.8 +/- 41.9, respectively). We observed no differences in basal or peak levels of neurohormonal factors, including plasma renin activity, aldosterone, and brain natriuretic peptide (BNP), between the groups. Although the plasma levels of angiotensin I and II were not different in the two groups at rest or at peak exercise, their increasing ratios from rest to peak exercise were significantly higher in patients with impaired LV function compared to those with preserved LV function (angiotensin I; -18.6 +/- 31.0% vs 64.8 +/- 66.5%, p < 0.05, angiotensin II; -5.9 +/- 41.2% vs 60.7 +/- 40.4% , p < 0.05). These results suggest that the increasing ratios of angiotensin I and II are superior to BNP as predictors of LV dysfunction, and that the sympathetic nervous system has already been activated even at rest and did not respond to dynamic exercise in patients with LV dysfunction in ischemic heart disease.

The human aging process is associated with vascular endothelial dysfunction. However, humoral factors which might protect against endothelial dysfunction during aging have not yet been identified. We recently identified the klotho gene as a possible regulator of human aging. In the present study using the klotho-deficient heterozygous mouse, we examined whether the Klotho protein is a humoral factor protecting against endothelial dysfunction. We further clotted rat klotho cDNA and investigated whether klotho mRNA expression in rat kidney is altered under pathological conditions such as hypertension, hyperlipidemia, renal failure, and inflammatory stress. The Klotho protein itself, or its metabolites, promotes endothelial NO production in aorta as well as arterioles, and klotho mRNA in kidney is downregulated under sustained circulatory stress.

Background. Altered heart and skeletal glucose usage has been reported in patients with non-insulin-dependent diabetes mellitus (NIDDM), Although elevations in plasma free fatty acid (FFA) concentrations have been implicated in reduced myocardial (18)fluorine-fluoro-2-deoxy-D-glucose uptake (MFU), the specific role of whole-body insulin resistance in MFU in patients with NIDDM compared with skeletal muscle metabolism remains controversial. Purpose. MFU and skeletal muscle (18)fluorine-fluoro-2-deoxy-D-glucose uptake (SMFU) were compared with positron emission tomography and the whole-body glucose disposal rate (GDR) during hyperinsulinemic euglycemic clamping in 26 normotensive asymptomatic patients with NIDDM who were not taking medication. These factors were also compared in 12 age-matched control subjects to increase the knowledge of the influence of whole-body insulin resistance on MFU, In addition, independent factors for both SMFU and MFU were investigated. Results. GDR in control subjects (10.0 +/- 2.97 mg/min per kilogram) was significantly higher than in patients with NIDDM (4.05 +/- 2.37 mg/min per kilogram, P &lt; .01). SMFU in patients,vith NIDDM (0.826 +/- 0.604 mg/min per 100 g) was significantly lower than that in control subjects (1.86 +/- 1.06 mg/min per 100 g, P &lt; .01), MFU in patients with NIDDM (5.35 +/- 2.10 mg/min per 100 g) was also significantly lower than that of control subjects (7.05 +/- 1.66 mg/min per 100 g, P = .0182), SMFU significantly correlated with GDR (r = .727, P &lt; .01) and FFA (r = -.52, P &lt; .01) in patients with NIDDM, MFU also correlated with GDR (r = .778, P &lt; .01) and FFA (r = -.72, P &lt; .01) in patients with NIDDM, Multivariate stepwise regression analysis showed that GDR (F = 36.8) was independently related to MFU (r = .85, P &lt; .01) whereas FFA was not (F = 1.763), where F is the value for statistical analysis of multivariate stepwise regression analysis. Conclusion. Insulin resistance is the most essential factor for both heart and skeletal muscle FDG uptake in patients with NIDDM.

Insulin resistance is commonly observed both in overt diabetes and in individuals prone to, but not yet manifesting, diabetes. Hence the maintenance or restoration of insulin sensitivity may prevent the onset of this disease. We previously showed that homozygous disruption of insulin receptor substrate-1 (IRS-1) in mice resulted in insulin resistance but not diabetes. Here, we have explored the mechanism of systemic insulin resistance in these mice and used adenovirus-mediated gene therapy to restore their insulin sensitivity. Mice expressing the IRS-1transgene showed almost normal insulin sensitivity. Expression of an IRS-1 mutant (IRS-1Deltap85) lacking the binding site for the p85 subunit of phosphatidylinositol 3-kinase (PI3K) also restored insulin sensitivity, although PI3K is known to play a crucial role in insulin's metabolic responses. Protein kinase B (PKB) activity in liver was decreased in null mice compared with the wild-type and the null mice expressing IRS-1 or IRS-1Deltap85. In primary hepatocytes isolated from null mice, expression of IRS-1 enhanced both PI3K and PKB activities, but expression of IRS-1Deltap85 enhanced only PKB. These data suggest that PKB in liver plays a pivotal role in systemic glucose homeostasis and that PKB activation might be sufficient for reducing insulin resistance even without full activation of PI3K.

A disintegrin and metalloproteinase (ADAM) represents a protein family possessing both metalloproteinase and disintegrin domains. ADAMTS-1, an ADAM family member cloned from cachexigenic colon adenocarcinoma, is unusual in that it contains thrombospondin type I motifs and anchors to the extracellular matrix. To elucidate the biological role of ADAMTS-1, we developed ADAMTS-1-null mice by gene targeting. Targeted disruption of the mouse ADAMTS-1 gene resulted in growth retardation with adipose tissue malformation. Impaired female fertilization accompanied by histological changes in the uterus and ovaries also resulted. Furthermore, ADAMTS-1(-/-) mice demonstrated enlarged renal calices with fibrotic changes from the ureteropelvic junction through the ureter, and abnormal adrenal medullary architecture without capillary formation. ADAMTS-1 thus appears necessary for normal growth, fertility, and organ morphology and function. Moreover, the resemblance of the renal phenotype to human ureteropelvic junction obstruction may provide a clue to the pathogenesis of this common congenital disease.

OBJECTIVE: The proliferation of vascular smooth muscle cells surrounding a suture line is an important factor in the development of anastomotic stricture that is frequently seen after coronary artery bypass grafting. The aim of this study was to investigate the time course of intimal thickening and to examine the expression of the molecular marker of smooth muscle cell activation surrounding the suture line. METHODS: Longitudinal aortotomy was performed in the abdominal aorta of rats. The rats were put to death 1, 2, 4, and 8 weeks after aortotomy, and the percentage of the lumen occluded by intimal thickening was calculated. All tissues were stained with antibodies against basic transcription element- binding protein 2, human cyclin-dependent kinase (cdk4), and Sp1 for immunohistochemistry. Basic transcription element-binding protein 2 is a transcription factor that is involved in phenotypic modulation of vascular smooth muscle cells. Cdk4 represents a marker for G(1) phase of the cell cycle. Sp1 is a transcription factor known to be expressed in a variety of tissues. Basic transcription element-binding protein 2 messenger RNA expression was confirmed by means of reverse transcriptase-polymerase chain reaction. RESULTS: We noted significant thickening of the intimal layer 1 week after aortotomy. Immunohistochemistry demonstrated that smooth muscle cells in the neointima were strongly positive for basic transcription element-binding protein 2 and human cyclin-dependent kinase 4, which peaked 2 weeks after aortotomy. Basic transcription element-binding protein 2 expression was closely associated with human cyclin-dependent kinase 4 expression in the neointima, although Sp1 was not. Basic transcription element-binding protein 2 messenger RNA levels were significantly up-regulated early after aortotomy. CONCLUSION: The experimental rat aortotomy model is useful to investigate the proliferation of vascular smooth muscle cells around the suture line. Moreover, our results suggest the possible role of basic transcription element-binding protein 2 in the development of vascular anastomotic strictures.

A cardiac homeobox-containing gene Csx/Nkx2-5, which is essential for cardiac development, is abundantly expressed in the adult heart as well as in the heart primordia. Targeted disruption of this gene results in embryonic lethality due to abnormal heart morphogenesis. To elucidate the role of Csx/Nkx2-5 in the adult heart, we generated transgenic mice which overexpress human Csx/Nkx2-5. The transgene was expressed abundantly in the heart and the skeletal muscle. mRNA levels of several cardiac genes including natriuretic peptides, CARP, MLC2v, and endogenous Csx/Nkx2-5 were increased in the ventricle of the transgenic mice. Electron microscopic analysis revealed that the ventricular myocardium of the transgenic mice had many secretory granules, which disappeared after administration of vasopressin. These results suggest that Csx/Nkx2-5 regulates many cardiac genes and induces formation of secretory granules in the adult ventricle.

Paraoxonase 1 (PON1) is proposed to have an anti-atherogenic action. Two polymorphisms at the PON1 (M/L55 and Q/R192) have been shown to be associated with coronary artery disease (CAD). This conclusion is not drawn universally, however, and specific ethnic characteristics may be important determinants in this association. Recently two homologues of PON1 - PON2 and PON3 - were identified and Sanghera et al. demonstrated C/S311 polymorphism at PON2 was associated with the risk of CAD. Within that context, we investigated the association between the aforementioned three polymorphisms and CAD and ischemic stroke in a Japanese population. The study population included 431 control subjects, 210 CAD patients, and 235 ischemic stroke patients. Genotype distributions and allele frequencies of M/L55 and C/S311 were similar among the control and patient groups, whereas the R192 allele frequency was significantly higher (P &lt; 0.001) in CAD (75%) and ischemic stroke (76%) patients than in control subjects (65%). When confounding influences of other risk factors were controlled for by multivariate analysis, R192 remained an independent risk determinant (additive model: OR (95% CI), P value CAD: 2.01 (1.45-2.79), 0.0001; ischemic stroke: 1.84 (1.34-2.52), 0.0002 (three genotypes into calculation)). Taken together, our data indicate that the Q/R192 is principally associated with both CAD and ischemic stroke in Japanese. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

Although smooth muscle cells (SMCs) are critical components of the circulatory system, the regulatory mechanisms of SMC differentiation remain largely unknown. In the present study, we examined the mechanism of SMC differentiation by using Xenopus laevis SM22alpha (XSM22alpha) as an SMC-specific marker. XSM22alpha cDNA contained a 600-bp open reading frame, and the predicted amino acid sequences were highly conserved in evolution. XSM22alpha transcripts were first detected in heart anlage, head mesenchyme, and the dorsal side of the lateral plate mesoderm at the tail-bud stage, possibly representing the precursors of muscle lineage. At the tadpole stage, XSM22alpha transcripts were restricted to the vascular and visceral SMCs. XSM22alpha was strongly induced by basic fibroblast growth factor (FGF) in animal caps. Although expressions of Xenopus cardiac actin were not affected by the expression of a dominant-negative FGF receptor, its injection dramatically suppressed the XSM22alpha expression. These results suggest that XSM22alpha is a useful molecular marker for the SMC lineage in Xenopus and that FGF signaling plays an important role in the induction of XSM22alpha and in the differentiation of SMCs.

A decline in oxygen concentration perturbs endothelial function, which promotes local thrombosis. In this study, we determined whether hypoxia in the range of that observed in pathophysiological hypoxic states stimulates plasminogen activator inhibitor-1 (PAI-1) production in bovine aortic endothelial cells. PAI-1 production, measured by ELISA, was increased by 4.7-fold (P&lt;0.05 versus normoxic control, n=4) at 12 hours after hypoxic stimulation. Northern blot analysis showed the progressive time-dependent increase in the steady-state level of PAI-1 mRNA expression by hypoxia, which reached a 7.5-fold increase: (P&lt;0.05 versus control, n=4) at 12 hours. Deferoxamine, which has been known to bind heme protein and to reproduce the hypoxic response, induced PAI-1 production at both the mRNA and protein levels. The half-life of PAI-1 mRNA, as determined by a standard decay assay, was not affected by hypoxia, suggesting that induction of PAI-1 mRNA was regulated mainly at the transcriptional level. Transient transfection assays of the human PAI-1 promoter-luciferase construct indicates that a hypoxia-responsive region lies between -414 and -107 relative to the transcription start site, where no putative hypoxia response element is found. The hypoxia-mediated increase in PAI-1 mRNA levels was attenuated by the tyrosine kinase inhibitors genistein (50 mu mol/L) and herbimycin A (1 mu mol/L), whereas PD98059 (50 mu mol/L, MEK1 inhibitor), SB203580 (10 mu mol/L, p38 mitogen-activated protein kinase inhibitor), and calphostin C (1 mu mol/L, protein kinase C inhibitor) had no effect on the induction of PAI-1 expression by hypoxia and deferoxamine. Genistein but not daidzein blocked the production of hypoxia- and deferoxamine-induced PAI-1 protein. Thus, we conclude that hypoxia stimulates PAI-1 gene transcription and protein production through a signaling pathway involving genistein-sensitive tyrosine kinases in vascular endothelial cells.