Effect of Lower Limb Venous Dilation on the Autonomic Cardiac Response among Healthy Young Men.

Venous occlusion of the lower limbs, which simulates edema, can alter heart rate variability (HRV) by increasing feedback information from group III/IV sensory fibers. Our aim was to quantify this effect among healthy young men. The study group included 13 men (mean age, 20.4 years). Venous occlusion of the lower limbs was induced using a pressure cuff around both thighs. The effect of occlusion on autonomic cardiac response was quantified under occlusion pressures of 20, 60, and 100 mmHg. Compression was applied for 5 min. HRV was evaluated from changes in the low-frequency (LF) and high-frequency (HF) power of the electrocardiogram and the resulting LF/HF balance. Near-infrared spectroscopy of the leg was used to quantify the effects of occlusion on deoxyhemoglobin, measured as the area under the curve (HHb-AUC). The occlusion pressure of 100 mmHg induced a significant increase in the LF/HF ratio, compared to the baseline (p < 0.05). HHb-AUC was highest for the 100 mmHg occlusion pressure compared with the 20 and 60 mmHg pressures (p < 0.01). These findings indicate that venous dilation may elicit a shift towards sympathetic dominance in the autonomic balance.

Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity.

"Antibody-breeding" has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the VH and VL domains of parent antibodies to generate diverse libraries of single-chain Fv fragments (scFvs), from which evolved mutants are selected. We produced an scFv against estradiol-17ß with 11 amino acid substitutions and a >100-fold improved affinity constant (Ka = 1.19 × 1010 M-1) over the parent scFv, enabling immunoassays with >30-fold higher sensitivity. We systematically analyzed contributions of these substitutions to the affinity enhancement. Comparing various partial scFv revertants based on their Kas indicated that a revertant with four substitutions (VH-L100gQ, VL-I29V, -L36M, -S77G) exhibited somewhat higher affinity (Ka = 1.46 × 1010 M-1). Finally, the VH-L100gQ substitution, occurring in VH complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and VL-I29V and/or VL-L36M cooperated significantly. These findings encouraged us to reconsider the potential of VH-CDR3-targeting mutagenesis, which has been frequently attempted. The substitution(s) wherein might enable a "high rate of return" in terms of selecting mutants with dramatically enhanced affinities. The "high risk" of generating a tremendous excess of "junk mutants" can be overcome with the efficient selection systems that we developed.

Free fatty acid causes leukocyte activation and resultant endothelial dysfunction through enhanced angiotensin II production in mononuclear and polymorphonuclear cells.

Release of free fatty acid (FFA) from adipose tissue is implicated in insulin resistance and endothelial dysfunction in patients with visceral fat obesity. We demonstrated previously that increased FFA levels cause endothelial dysfunction that is prevented by inhibition of the renin-angiotensin system (RAS) in humans. However, the mechanisms for FFA-mediated activation of RAS and the resultant endothelial dysfunction were not elucidated. We investigated effects of elevated FFA on activity of circulating and vascular RAS, angiotensin II-forming activity of leukocytes, and leukocyte activation of normotensive subjects. We showed that increased FFA levels significantly enhanced angiotensin II-forming activity in human mononuclear (mean fold increase: 3.5 at 180 minutes; P=0.0016) and polymorphonuclear (2.0; P=0.0012) cells, whereas parameters of the circulating and vascular RAS were not affected. We also showed that FFA caused angiotensin II- dependent leukocyte activation, which impaired endothelial function partly via increased myeloperoxidase release and presumably enhanced adhesion of leukocytes. We propose that the enhanced production of angiotensin II by FFA in mononuclear and polymorphonuclear cells causes activation of leukocytes that consequently impairs endothelial function. RAS in leukocytes may regulate the leukocyte-vasculature interaction as the mobile RAS in humans.

Slower onset of vasodilating action of brain natriuretic peptide (BNP) compared to atrial natriuretic peptide (ANP) in human forearm resistant vessels.

PURPOSE: To investigate possible differences in the time course of vasodilating effects of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in humans. METHODS: We measured forearm blood flow (FBF) by strain gauge plethymography and cyclic GMP spillover during and after intra-arterial infusions of BNP and ANP at 16.2 pmol/min for 30 min in healthy subjects. RESULTS: The steady-state responses of FBF and cyclic GMP to the infusion were achieved approximately 20 min after commencement of the infusion of BNP, but within 5 min for ANP, with similar magnitudes of maximum responses. These parameters more slowly returned to the baseline value after withdrawal of the BNP infusion than after the ANP infusion. CONCLUSION: The onset and disappearance of the guanylate cyclase-stimulating and vasodilating effects of BNP were significantly slower than those of ANP. This differing mode of vasodilator action may be relevant to the therapeutic use of natriuretic peptides.

Sympathetic hyperreactivity to air-jet stress in the chromosome 1 blood pressure quantitative trait locus congenic rats.

A chromosome 1 blood pressure quantitative trait locus (QTL) was introgressed from the stroke-prone spontaneously hypertensive rats (SHRSP) to Wistar-Kyoto (WKY) rats. This congenic strain (WKYpch1.0) showed an exaggerated pressor response to both restraint and cold stress. In this study, we evaluated cardiovascular and sympathetic response to an air-jet stress and also examined the role of the brain renin-angiotensin system (RAS) in the stress response of WKYpch1.0. We measured mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) responses to air-jet stress in WKYpch1.0, WKY, and SHRSP. We also examined effects of intracerebroventricular administration of candesartan, an ANG II type 1 receptor blocker, on MAP and HR responses to air-jet stress. Baseline MAP in the WKYpch1.0 and WKY rats were comparable, while it was lower than that in SHRSP rats. Baseline HR did not differ among the strains. In WKYpch1.0, air-jet stress caused greater increase in MAP and RSNA than in WKY. The increase in RSNA was as large as that in SHRSP, whereas the increase in MAP was smaller than in SHRSP. Intracerebroventricular injection of a nondepressor dose of candesartan inhibited the stress-induced pressor response to a greater extent in WKYpch1.0 than in WKY. Intravenous injection of phenylephrine caused a presser effect comparable between WKYpch1.0 and WKY. These results suggest that the chromosome 1 blood pressure QTL congenic rat has a sympathetic hyperreactivity to an air-jet stress, which causes exaggerated pressor responses. The exaggerated response is at least partly mediated by the brain RAS.

Inhibition of the renin-angiotensin system prevents free fatty acid-induced acute endothelial dysfunction in humans.

OBJECTIVE: An elevated free fatty acid (FFA) level impairs endothelium-dependent vasodilation in humans, which may be pathophysiologically relevant to the development of endothelial dysfunction in patients with insulin resistance. We investigated the effect of inhibition of the renin-angiotensin system (RAS) on FFA-induced endothelial dysfunction. METHODS AND RESULTS: Changes in forearm blood flow during intra-arterial infusion of acetylcholine were measured by plethysmography before and after systemic infusion of lipid/heparin in 10 healthy subjects given a single dose of placebo, losartan (50 mg), or perindopril (8 mg). Endothelial function after lipid/heparin infusion was also investigated with the coinfusion of vitamin C or NG-monomethyl-L-arginine (L-NMMA). Elevated FFA significantly reduced the response to acetylcholine by 37.7% (P=0.0096) without L-NMMA, but not the response with L-NMMA, whereas FFA did not affect the response to nitroprusside. The single dose of either losartan or perindopril completely prevented FFA-induced endothelial dysfunction. Vitamin C also prevented FFA-induced endothelial dysfunction. CONCLUSIONS: Elevated FFA levels by lipid/heparin infusion, which may partly mimic the abnormal lipid profile in patients with insulin resistance, caused endothelial dysfunction via RAS activation and the presumably resultant oxidative stress in humans. Our results suggest the therapeutic rationale for RAS inhibition in patients with high FFA levels.

Rho-kinase inhibitor improves increased vascular resistance and impaired vasodilation of the forearm in patients with heart failure.

BACKGROUND: Rho-kinase is suggested to have an important role in enhanced vasoconstriction in animal models of heart failure (HF). Patients with HF are characterized by increased vasoconstriction and reduced vasodilator responses to reactive hyperemia and exercise. The aim of the present study was to examine whether Rho-kinase is involved in the peripheral circulation abnormalities of HF in humans with the Rho-kinase inhibitor fasudil. METHODS AND RESULTS: Studies were performed in patients with HF (HF group, n=26) and an age-matched control group (n=26). Forearm blood flow was measured with a strain-gauge plethysmograph during intra-arterial infusion of graded doses of fasudil or sodium nitroprusside. Resting forearm vascular resistance (FVR) was significantly higher in the HF group than in the control group. The increase in forearm blood flow evoked by fasudil was significantly greater in the HF group than in the control group. The increased FVR was decreased by fasudil in the HF group toward the level of the control group. By contrast, FVR evoked by sodium nitroprusside was comparable between the 2 groups. Fasudil significantly augmented the impaired ischemic vasodilation during reactive hyperemia after arterial occlusion of the forearm in the HF group but not in the control group. Fasudil did not augment the increased FVR evoked by phenylephrine in the control group significantly. CONCLUSIONS: These results indicate that Rho-kinase is involved in increased FVR and impaired vasodilation of the forearm in patients with HF.

Long-term treatment with probucol improves endothelial function in patients with coronary artery disease.

Recent studies have shown that endothelial function is impaired in patients with coronary artery disease (CAD). Probucol has been recognized to have antioxidant properties as well as lipid-lowering effects, and may improve endothelial function. The aim of this study was to evaluate the effects of probucol on endothelial function in patients with CAD. We evaluated endothelial function, based on flow-mediated vasodilation during reactive hyperemia (FMD), and the intima-media thickness (IMT) of the common carotid artery using high resolution ultrasonography in patients either with (CAD group, n=26) or without CAD (Control group, n=12). We measured the serum cholesterol concentration, including the low-density lipoprotein cholesterol (LDL-cholesterol) concentration, and the plasma concentrations of homocyst(e)ine and asymmetric dimethylarginine (ADMA). Measurements of FMD and serum cholesterol were repeated after 3 months of probucol (500 mg/day, n=9) or placebo (n=9) treatment in patients with CAD. The IMT was significantly greater (p < 0.001) and FMD was significantly lower (p < 0.001) in the CAD group than in the Control group. While the serum cholesterol concentration and plasma ADMA were similar in the two groups, the plasma homocyst(e)ine concentrations were higher in the CAD group than in the Control group (p < 0.01). After probucol therapy, FMD was significantly improved in the CAD group (p < 0.05). The serum LDL-cholesterol concentration did not significantly decrease after probucol treatment. Placebo treatment did not alter FMD or the serum cholesterol concentration. Our findings suggest that long-term treatment with probucol improves endothelial function in patients with CAD, an outcome independent of the LDL-cholesterol-lowering effects of probucol, and that homocyst(e)ine may be a better predictor of atherosclerosis than ADMA.

Hypoadiponectinemia is closely linked to endothelial dysfunction in man.

Vascular endothelial dysfunction has been demonstrated in overweight or obese patients, but the molecular basis for this link has not been clarified. We asked what the relationship was between adiponectin, an adipose-specific molecule, and endothelial function. Forearm blood flow (FBF) was measured during reactive hyperemia by using strain-gauge plethysmography in 76 Japanese subjects without a history of cardiovascular or cerebrovascular disease, diabetes mellitus, hepatic, or renal disease. The peak FBF and total reactive hyperemic flow [flow debt repayment (FDR)] during reactive hyperemia were correlated with waist circumference (r = -0.418 and -0.414, respectively) and body mass index (r = -0.597 and -0.626, respectively). After correcting for age, gender, and body mass index, the peak FBF was correlated with systolic blood pressure (r = -0.294; P = 0.010), free fatty acid (FFA) (r = -0.331; P = 0.004), and adiponectin in log 10 (r = 0.492; P < 0.001), and FDR was correlated with adiponectin in log 10 (r = 0.462; P = 0.001). In stepwise multiple regression analyses, predictive variables for peak FBF were adiponectin in log 10 (r = 0.468) and FFA (r = -0.292; r(2) = 0.487; P < 0.0001); and predictive variables for FDR were adiponectin in log 10 (r = 0.474) and FFA (r = -0.275; r(2) = 0.346, P < 0.0001). Endothelial function was impaired in proportion to the severity of obesity, and the level of severity was closely related to plasma adiponectin levels. Adiponectin may play a protective role against the atherosclerotic vascular change, and loss of effects enhances endothelial dysfunction, as in obese people.

Long-term treatment with eicosapentaenoic acid improves exercise-induced vasodilation in patients with coronary artery disease.

We have previously shown that long-term treatment with eicosapentaenoic acid (EPA) improves endothelium-dependent vasodilation of the atherosclerotic arteries in both animals and humans. The aim of the present study was to examine whether EPA treatment also improves metabolic vasodilation evoked by exercise in patients with coronary artery disease (CAD). Forearm blood flow (FBF) was measured by strain gauge plethysmography in 10 patients with stable CAD, before and 3 months after oral treatment with EPA (1,800 mg/kg). FBF was measured at rest and during intra-arterial infusion of acetylcholine or sodium nitroprusside, before and after intra-arterial infusion of NG-monomethyl-L-arginine (L-NMMA, an inhibitor of nitric oxide (NO) synthesis). A rhythmic handgrip exercise was also performed for 3 min before and after L-NMMA, and FBF was measured for 3 min just after the handgrip exercise. These protocols were repeated after the long-term treatment with EPA for 3 months. The long-term treatment with EPA significantly improved the FBF responses to acetylcholine (p < 0.01), which was significantly reduced by acute administration of L-NMMA (p < 0.01). By contrast, the EPA treatment did not affect the endothelium-independent responses to sodium nitroprusside. Metabolic increases in FBF caused by the handgrip exercise were not significantly decreased by L-NMMA before the EPA treatment. The EPA treatment significantly augmented the exercise-induced increases in FBF (p < 0.05) and L-NMMA acutely abolished this augmentation (p < 0.01). These results indicate that long-term treatment with EPA improves both endothelium-dependent and exercise-induced forearm vasodilations in patients with CAD and that NO is substantially involved in the EPA-induced improvement of the FBF responses in patients with CAD.