Shigeo Takata

Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis

Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two‐hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity‐binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 ± 11 nM and 125 ± 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type‐1 repeat (TSP‐1) domain of CTGF binds to the exon 7‐coded region of VEGF165 and that the COOH‐terminal domain preserves the affinity to both VEGF165 and VEGF121. The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor. By in vitro tube formation assay of endothelial cells, full‐length CTGF and the deletion mutant possessing the TSP‐1 domain inhibited VEGF165‐induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF165 binds to CTGF through a protein‐to‐protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.

Four Novel KVLQT1 and Four Novel HERG Mutations in Familial Long-QT Syndrome

BACKGROUND Familial long-QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur due to ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far. We investigated mutations of these genes in LQTS families. METHODS AND RESULTS Thirty-two Japanese families with LQTS were brought together for screening for mutations. Genomic DNA from each proband was examined by the polymerase chain reaction-single-strand conformation polymorphism technique followed by direct DNA sequencing. In four of the families, comprising 16 patients, mutations were identified in KVLQT1; five other families (9 patients) segregated mutant alleles of HERG. All 25 of these patients carried the specific mutations present in their respective families, and none of 80 normal individuals carried these alleles. Mutations were confirmed by endonuclease digestion or hybridization of mutant allele-specific oligonucleotides. No mutation in SCN5A was found in any family. CONCLUSIONS We identified nine different mutations among 32 families with LQTS. Eight of these were novel and account for 25% of all types of mutations reported to date. Such a variety of mutations makes it difficult to screen high-risk groups using simple methods such as endonuclease digestion or mutant allele-specific amplification.

Effects of Insulin on Vasoconstrictive Responses to Norepinephrine and Angiotensin II in Rabbit Femoral Artery and Vein

To determine whether insulin has a vasodilator action on the artery and vein, the effects of insulin at varying concentrations (120 μU/ml, 1.2 mU/ml, 12 mU/ml, and 120 mU/ml) on vasoconstriction by norepinephrine (NE) and angiotensin II (ANG II) were studied in the isolated rabbit femoral artery and vein. Helical strips were suspended in an organ bath filled with modified Krebs solution (pH 7.4), were gassed with 95% O2/5% CO2 at 36°C, and isotonic contractions were measured. Insulin significantly and dose dependently inhibited the vasoconstriction induced by NE (10−8 M for the artery and 10−7 M for the vein) at ≥1.2 mU/ml for both the artery and vein and the vasoconstriction induced by ANG II (3 × 10−10 M for the artery and 3 × 10−9 M for the vein) at ≥1.2 mU/ml for the artery and ≥12 mU/ml for the vein. The results indicate that insulin has an inhibitory effect on NE- and ANG II–induced contraction in both the artery and vein, and this appeared to be a contributory factor in the hypotensive effect observed in diabetic patients treated with insulin.

Blood Lipid Mediator Sphingosine 1-Phosphate Potently Stimulates Platelet-derived Growth Factor-A and -B Chain Expression through S1P1-Gi-Ras-MAPK-dependent Induction of Krüppel-like Factor 5

Platelet-derived growth factors (PDGFs), potent mitogens and chemoattractants for mesenchymal cell types, play essential roles in development of several organs including blood vessels, kidney, and lung, and are also implicated in the pathogenesis of atherosclerosis and malignancies. Blood lipid mediator sphingosine 1-phosphate (S1P) regulates migration, proliferation, and apoptosis in a variety of cell types through multiple G protein-coupled receptors of the Edg family, and is necessary for vascular formation at the developmental stage. We found in the present study that S1P induced severalfold increases in the mRNA and protein levels of PDGF-A and -B chains in vascular smooth muscle cells and neointimal cells. S1P stimulation of PDGF mRNA and protein expression was abolished by the small interfering RNA duplexes targeting S1P1/Edg1 receptor subtype. S1P stimulated the small GTPase Ras in a Gi-dependent manner, and activated ERK and p38 MAPK in Gi- and Ras-dependent manners. Pertussis toxin pretreatment, adenovirus-mediated Asn17Ras expression, the MEK inhibitor PD98059, or the p38 MAPK inhibitor SB203580 markedly suppressed PDGF mRNA and protein up-regulation, indicating the involvement of Gi-Ras-ERK/p38 MAPK in S1P stimulation of PDGF expression. S1P stimulated expression of the transcription factor KLF5 in manners dependent on Gi, Ras, and ERK/p38 MAPK. Down-regulation of KLF5 by small interfering RNA duplexes abolished S1P-induced PDGF-A and -B chain expression. On the other hand, overexpression of KLF5 stimulated basal and S1P-induced PDGF expression. Either S1P stimulation or KLF5 overexpression increased the PDGF-B promoter activity in a cis-element-dependent manner. These results reveal the S1P1-triggered, Gi-Ras-ERK/p38 MAPK-KLF5-dependent, stimulatory regulation of PDGF gene transcription in vascular smooth muscle cells.

What are Arrhythmogenic Substrates in Diabetic Rat Atria

Introduction: Diabetes mellitus is one of the significant independent risk factors for the development of atrial fibrillation (AF). However, the pathophysiological mechanisms of the relationship have not been fully elucidated.

G12/13 and Gq mediate S1P2-induced inhibition of Rac and migration in vascular smooth muscle in a manner dependent on Rho but not Rho kinase

AIMS The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated S1P inhibition of Rac and cell migration in VSMCs. METHODS AND RESULTS S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P(2) receptor antagonist JTE-013. The C-terminal peptides of Galpha subunits (Galpha-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of Galpha(12)-CT, Galpha(13)-CT, and Galpha(q)-CT, but not that of Galpha(s)-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G(12/13) and G(q) classes are necessary for the S1P inhibition. The expression of Galpha(q)-CT as well as Galpha(12)-CT and Galpha(13)-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT(1), which robustly couples to G(q), did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of G(q) alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT(1) receptor fused to Galpha(12) was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway. CONCLUSION These observations together suggest that S1P(2) mediates inhibition of Rac and migration through the coordinated action of G(12/13) and G(q) for Rho activation in VSMCs.

Spectral analysis of heart rate, arterial pressure, and muscle sympathetic nerve activity in normal humans

We investigated the frequency components of fluctuations in heart rate, arterial pressure, respiration, and muscle sympathetic nerve activity (MSNA) in 11 healthy women using an autoregressive model and examined the relation among variables using Akaikes relative power contribution analysis with multivariate autoregressive model fitting. Power spectral analysis of MSNA revealed two peaks, with low-frequency (LF) and high-frequency (HF) components. The LF component of MSNA was a major determinant of the LF component of arterial pressure and R-R interval variability (0.70 +/- 0.07 and 0.18 +/- 0.05, respectively). The effect of the LF component of MSNA on arterial pressure showed no change in response to propranolol but was diminished (0.35 +/- 0.08) by phentolamine (P < 0.02). The effect of the LF component of MSNA on R-R interval was not altered by pharmacological sympathetic nerve blockade. The HF component of MSNA did not influence other variables but was influenced by R-R interval, arterial pressure, and respiration. These findings indicate that the LF component of MSNA reflects autonomic oscillations, whereas the HF component is passive and influenced by other cardiovascular variables.

Altered firing pattern of single‐unit muscle sympathetic nerve activity during handgrip exercise in chronic heart failure

Sympathetic activation in chronic heart failure (CHF) is greatly augmented at rest but the response to exercise remains controversial. We previously demonstrated that single‐unit muscle sympathetic nerve activity (MSNA) provides a more detailed description of the sympathetic response to physiological stress than multi‐unit nerve recordings. The purpose of this study was to determine whether the reflex response and discharge properties of single‐unit MSNA are altered during handgrip exercise (HG, 30% of maximum voluntary contraction for 3 min) in CHF patients (New York Heart Association functional class II or III, n= 16) compared with age‐matched healthy control subjects (n= 13). At rest, both single‐unit and multi‐unit indices of sympathetic outflow were augmented in CHF compared with controls (P < 0.05). However, the percentage of cardiac intervals that contained one, two, three or four single‐unit spikes were not different between the groups. Compared to the control group, HG elicited a larger increase in multi‐unit total MSNA (Δ1002 ± 50 compared with Δ636 ± 76 units min−1, P < 0.05) and single‐unit MSNA spike incidence (Δ27 ± 5 compared with Δ8 ± 2 spikes (100 heart beats)–1), P < 0.01) in the CHF patients. More importantly, the percentage of cardiac intervals that contained two or three single‐unit spikes was increased (P < 0.05) during exercise in the CHF group only (Δ8 ± 2% and Δ5 ± 1% for two and three spikes, respectively). These results suggest that the larger multi‐unit total MSNA response observed during HG in CHF is brought about in part by an increase in the probability of multiple firing of single‐unit sympathetic neurones.

Peripheral Circulatory Effects of Insulin in Diabetes

Peripheral circulatory effects of insulin were studied in the diabetic patients with and without autonomic neuropathy. Forearm blood flow, calf venous vol ume and calf venous distensibility were measured by strain gauge plethysmo graphy. In the diabetic patients with autonomic neuropathy, mean blood pres sure fell from 96±5 to 88±5 mmHg after an intravenous injection of 4 U of monocomponent insulin (p < 0.001). Forearm vascular resistance decreased from 53.99±8.29 to 45.88±7.76 mmHg•ml -1•100ml-1•min-1 after insulin (p < 0.01). Insulin increased calf venous volume from 1.20±0.19 to 2.23±0.44 ml/100ml (p < 0.05) and calf venous distensibility from 0.039±0.004 to 0.082±0.016 ml/mmHg (p < 0.05). In contrast, in the diabetic patients without autonomic neuropathy, there were no significant changes in the mean blood pressure, forearm vascular resistance, calf venous volume and calf venous dis tensibility. Symptoms of hypoglycaemia did not occur in any patient. These results suggest that insulin has a vasodilator action on both resistance and capacitance vessels, which may be one of the main factors in insulin-in duced hypotension.

The role of peripheral capacitance and resistance vessels in hypotension following hemodialysis

The arterial pressure (AP) response to hemodialysis was studied with echocardiography and strain gauge plethysmography in 17 patients with end-stage renal disease; mean AP was unchanged in seven (group A) and was reduced by more than 10 mm Hg in 10 patients (group B). Following dialysis, body weight decreased and heart rate increased equally in both groups. Ejection fraction did not change in the two groups. Left ventricular end-diastolic volume fell by 13 +/- 10% in group A and by 24 +/- 16% in group B. Cardiac index (CI) fell in group B, but remained unchanged in group A. Systemic vascular resistance (SVR) did not change in both groups. The change in mean AP before and after dialysis was significantly correlated with that in Cl (r = 0.49, p less than 0.05), but not with that in SVR. Calf venous pressure-volume curves were not different between the two groups before dialysis. Hemodialysis shifted the curve toward the volume axis for group B but not for group A. These results suggest that hypotension following dialysis is mainly due to the fall in cardiac output, in which increases in venous distensibility play an important role.