Noninvasive evaluation of left ventricular force-frequency relationships by measuring carotid arterial wave intensity during exercise stress.

BACKGROUND AND PURPOSE: Estimation of the contractility of the left ventricle during exercise is important in drawing up a protocol of cardiac rehabilitation. It has been demonstrated that color Doppler- and echo tracking-derived carotid arterial wave intensity is a sensitive index of global left ventricular (LV) contractility. We assessed the feasibility of measuring carotid arterial wave intensity and determining force-frequency (contractility-heart rate) relations (FFRs) during exercise totally noninvasively. METHODS: We measured carotid arterial wave intensity with a combined color Doppler and echo tracking system in 25 healthy young male volunteers (age 20.8 ± 1.2 years) at rest and during exercise. FFRs were constructed by plotting the maximum value of wave intensity (WD1) against heart rate (HR). RESULTS: We first confirmed that HR increased linearly with an increase in work load in each subject (r (2) = 0.95 ± 0.04). WD1 increased linearly with an increase in HR. The goodness-of-fit of the regression line of WD1 on HR in each subject was very high (r (2) = 0.48-0.94, p < 0.0001, respectively). The slope of the WD1-HR relation ranged 0.30-2.20 [m/s(3) (beat/min)]. CONCLUSIONS: Global LV FFRs can be generated in healthy young volunteers with an entirely noninvasive combination of exercise and wave intensity. These data should show the potential usefulness of the FFR in the context of cardiac rehabilitation.

The short-term effects of C-peptide on the early diabetes-related ultrastructural changes to the podocyte slit diaphragm of glomeruli in rats.

OBJECTIVE: This study aimed to investigate the structural changes in the slit diaphragm, caused by early diabetes, and the nephroprotective effect of C-peptide. METHODS: Streptozotocin-induced type 1 diabetic Wistar rats were divided into control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide groups. C-peptide was infused into rats for one day. The slit diaphragm component proteins podocin, CD2AP, and nephrin, were stained immunofluorescently and their distribution quantitatively analyzed by determining the overlapping area ratio. The interfoot process gap length was measured from electron microscopic images. RESULTS: Diabetic duration correlated best with the area ratio of podocin and CD2AP (r = 0.626), followed by other protein combinations, showing progressive change in the slit diaphragm structure. C-peptide-treatment did not alter area ratios. The interfoot process gap length was wider in diabetic rats (p < 0.05) and did not narrow with C-peptide-treatment in either control or diabetic rats (both p < 0.05). CONCLUSIONS: Diabetes widened the interfoot process gap length and distorted the slit diaphragm structure progressively and heterogeneously in rats with early diabetes; this was not altered by C-peptide-treatment. The nephroprotective effect of C-peptide in decreasing the glomerular filtration rate appears to be functional rather than structural.

Role of endogenous hydrogen peroxide during angiotensin type 1 receptor blockers administration in pacing-induced metabolic coronary vasodilatation in dogs in vivo.

BACKGROUND: We have previously demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in canine coronary microcirculation in vivo. However, the role of H2O2/EDHF during angiotensin type 1 receptor blockers (ARB) administration in metabolic coronary dilatation in vivo remains to be examined. We examined whether H2O2 during ARB administration is involved in pacing-induced metabolic coronary vasodilatation in dogs in vivo and if so, whether such beneficial effects of ARB administration acutely improve coronary vasodilatation in diabetes mellitus (DM). METHODS: Canine subepicardial coronary small arteries (CSA,≥ 100 µm) and arterioles (CA, <100 µm) in left anterior descending artery area were continuously observed by an intravital microscope under cyclooxygenase blockade(ibuprofen, 12.5 mg/kg, intravenous infusion, iv). Experiments were performed during paired right ventricular pacing under the following 4 conditions (n=5 each); (i) control, (ii) DM(alloxan 40 mg/ kg, iv, 1 week prior to study), (iii) DM+ARB(olmesartan, 10 µg/kg/min, 10 min, intracoronary infusion,ic)+L-NMMA (NOS inhibitor, 2 !mol/min, ic) and (iv)DM+ARB+catalase (H2O2 discomposer, 1000 U/ml, 5 min, ic). RESULTS: Cardiac tachypacing (60 to 120 bpm) caused coronary vasodilatation in both-sized arteries under control conditions. DM significantly decreased the vasodilatation compared with control in CSA and there was a residual vasodilatation for the loss of NO in CA, whereas DM+ARB+L-NMMA improved the vasodilatation compared with DM alone in CA and was significantly decreased by DM+ARB+catalase in CA. CONCLUSIONS: These results indicate that H2O2 during ARB administration is involved in pacing-induced metabolic coronary vasodilatation in DM in vivo and that there are substantial compensatory interactions between NO and H2O2.

In vivo quantitative visualization analysis of the effect of C-peptide on glomerular hyperfiltration in diabetic rats by using multiphoton microscopy.

OBJECTIVE: The purpose of this study was to visualize glomerular hyperfiltration in rats at the early stage of diabetes under in vivo conditions and to quantitatively examine the effect of C-peptide on filtration. METHODS: Type 1 diabetes was induced by streptozotocin (50 mg/kg) in Wistar rats. The rats were divided into four groups: control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide. C-peptide was continuously infused (50 pmol/kg/min). Filtration was visualized by a bolus shot of various sizes of dextrans (3 k to 70 k Da) conjugated with Texas Red and observed with a multiphoton microscope under inhaled anesthesia. Relative sieving coefficients were used to quantify filtration. RESULTS: Almost all smaller particles (3­10 k Da) were filtered both in control and diabetic rats. Filtration of larger particles (40­70 k Da) was seen in normal rats but was more apparent in diabetic rats, where it was progressive according to the duration of diabetes. C-peptide administration restored the leakage of larger particles to the level seen for the control. CONCLUSIONS: We visualized and analyzed hyperfiltration and confirmed that C-peptide has a nephroprotective effect. Furthermore, we found that the leakage of larger particles increased as the duration of diabetes increased.

Intermittent, moderate-intensity aerobic exercise for only eight weeks reduces arterial stiffness: evaluation by measurement of stiffness parameter and pressure-strain elastic modulus by use of ultrasonic echo tracking.

BACKGROUND AND PURPOSE: Aerobic exercise has been reported to be associated with reduced arterial stiffness. However, the intensity, duration, and frequency of aerobic exercise required to improve arterial stiffness have not been established. In addition, most reports base their conclusions on changes in pulse wave velocity, which is an indirect index of arterial stiffness. We studied the effects of short-term, intermittent, moderate-intensity exercise training on arterial stiffness based on measurements of the stiffness parameter (ß) and pressure-strain elastic modulus (E p), which are direct indices of regional arterial stiffness. METHODS: A total of 25 young healthy volunteers (18 men) were recruited. By use of ultrasonic diagnostic equipment we measured ß and E p of the carotid artery before and after 8 weeks of exercise training. RESULTS: After exercise training, systolic pressure (P s), diastolic pressure (P d), pulse pressure, systolic arterial diameter (D s), and diastolic arterial diameter (D d) did not change significantly. However, the pulsatile change in diameter ((D s - D d)/D d) increased significantly, and ß and E p decreased significantly. CONCLUSIONS: For healthy young subjects, ß and E p were reduced by intermittent, moderate-intensity exercise training for only 8 weeks.

Increased passive stiffness of cardiomyocytes in the transverse direction and residual actin and myosin cross-bridge formation in hypertrophied rat hearts induced by chronic ß-adrenergic stimulation.

BACKGROUND: Left ventricular (LV) hypertrophy is often present in patients with diastolic heart failure. However, stiffness of hypertrophied cardiomyocytes in the transverse direction has not been fully elucidated. The aim of this study was to assess passive cardiomyocyte stiffness of hypertrophied hearts in the transverse direction and the influence of actin-myosin cross-bridge formation on the stiffness. METHODS AND RESULTS: Wistar rats received a vehicle (control) or isoproterenol (ISO) subcutaneously. After 7 days, compared with the controls, ISO administration had significantly increased heart weight and LV wall thickness and had decreased peak early annular relaxation velocity (e') assessed by echocardiography. Elastic modulus of living cardiomyocytes in the transverse direction assessed by an atomic force microscope was significantly higher in the ISO group than in controls. We added butanedione monoxime (BDM), an inhibitor of actin-myosin interaction, and blebbistatin, a specific myosin II inhibitor, to the medium. BDM and blebbistatin significantly reduced the elastic modulus of cardiomyocytes in the ISO group. X-ray diffraction analysis showed that the reflection intensity ratio (I((1,0))/I((1,1))) at diastole was not different before and after treatment with BDM, which induces complete relaxation, in control hearts, but that I((1,0))/I((1,1)) was significantly increased after BDM treatment in the ISO group, indicating residual cross-bridge formation in hypertrophied hearts. CONCLUSIONS: Passive cardiomyocyte stiffness in the transverse direction is increased in hearts with ISO-induced hypertrophy and this is caused by residual actin-myosin cross-bridge formation.

Live-cell visualization of the trans-cellular mode of monocyte transmigration across the vascular endothelium, and its relationship with endothelial PECAM-1.

In response to atherogenic stimuli, blood monocytes transmigrate across the vascular endothelium not only through endothelial cell-cell junctions (para-cellular) but also through endothelial cells themselves (trans-cellular). The molecular mechanism of the latter is mostly unknown, because it rarely happens, especially in vitro. Although many reports have recognized trans-cellular migration from snapshot images of leukocytes halfway across the endothelium at non-junctional locations, it often produces a false-positive result, because some leukocytes that initiate trans-cellular migration withdraw and return to the apical endothelial surface. Thus, analyzing the entire process is essential. In this study, complete monocyte trans-cellular migration was successfully captured for live cells, with simultaneous visualization of endothelial PECAM-1. We suggest the possible existence of both PECAM-1-related migration at peri-junctional sites and PECAM-1-unrelated migration at sites remote from junctions. This is the first report to describe the entire process of monocyte trans-cellular migration for live cells and its relationship with endothelial PECAM-1.

Monocyte trans-endothelial migration augments subsequent transmigratory activity with increased PECAM-1 and decreased VE-cadherin at endothelial junctions.

BACKGROUND: Although the importance of monocyte trans-endothelial migration in early atherogenesis is well recognized, it is unclear whether and how one transmigration event affects endothelium to facilitate subsequent ones. In this study, we tested the hypothesis that monocyte transmigration alters endothelial junctional organization to facilitate subsequent transmigration. METHODS AND RESULTS: When human monocytes were added twice at intervals of ≈30 min to IL-1beta-prestimulated human umbilical vein endothelial cells in vitro, significant augmentation of transmigration was observed at the second addition (≈1.5-fold, analyzed from a total of 231 monocytes in 3 experiments). Endothelial surface expressions of two major junctional molecules, PECAM-1 and VE-cadherin, increased and decreased respectively, in response to monocyte addition, which could facilitate subsequent transmigration. To further investigate spatiotemporal dynamics of the increasing molecule, PECAM-1, we constructed a PECAM-1-GFP expression system and found that monocyte transmigration induced local accumulation of endothelial PECAM-1 around the transmigration spot, which was followed by transmigration of subsequent monocyte around the same location. Detailed analysis revealed that within the defined region around one transmigration event, 50% of later transmigrating monocytes used the same or similar location as the previous one (10 out of 20 transmigrating monocytes in 11 experiments). CONCLUSIONS: These findings show that monocyte trans-endothelial migration alters endothelial junctional organization to a more monocyte-permeable state (increased PECAM-1 and decreased VE-cadherin), resulting in the augmented transmigratory activity at a later stage. This positive feedback mechanism is partially associated with monocyte transmigration-induced local accumulation of endothelial PECAM-1, which promotes transmigration of following monocytes at the same location.

Erythropoietin enhances hydrogen peroxide-mediated dilatation of canine coronary collateral arterioles during myocardial ischemia in dogs in vivo.

We have previously demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) plays an important role in the canine coronary microcirculation as an endothelium-derived hyperpolarizing factor in vivo. However, it remains to be examined whether endogenous H(2)O(2) is involved in the dilatation of coronary collaterals during myocardial ischemia in vivo and, if so, whether erythropoietin (EPO) enhances the responses. Canine subepicardial native collateral small arteries (CSA; ≥ 100 µm) and arterioles (CA; <100 µm) were observed using an intravital microscope. Experiments were performed after left anterior descending coronary artery ischemia (90 min) under the following eight conditions (n = 5 each): control, EPO, EPO+catalase, EPO+N-monomethyl-l-arginine (l-NMMA), EPO+l-NMMA+catalase, EPO+l-NMMA+iberiotoxin [Ca(2+)-activated K(+) (K(Ca)) channel blocker], EPO+l-NMMA+apamin+charybdotoxin (K(Ca) channel blocker), and EPO+wortmannin (phosphatidylinositol 3-kinase inhibitor). Myocardial ischemia caused significant vasodilatation in CA but not in CSA under control conditions, which was significantly decreased by catalase in CA. After EPO, the vasodilatation was significantly increased in both sizes of arteries and was significantly decreased by catalase. The enhancing effect of EPO was reduced by l-NMMA but not by catalase in CSA and was reduced by l-NMMA+catalase in CA, where the greater inhibitory effects were noted with l-NMMA+catalase, l-NMMA+iberiotoxin, L-NMMA+apamin+charybdotoxin, or wortmannin. EPO significantly ameliorated ischemia-induced impairment of myocardial Akt phosphorylation, which was abolished by l-NMMA+catalase or wortmannin. EPO also ameliorated oxidative stress and myocardial injury, as assessed by plasma 8-hydroxydeoxyguanosine and troponin-T, respectively. These results indicate that EPO enhances H(2)O(2)-mediated dilatation of coronary collateral arterioles during myocardial ischemia in dogs in vivo.

Recent perspective on coronary bifurcation intervention: statement of the "Bifurcation Club in KOKURA".

The treatment of coronary bifurcation lesion remains a challenging issue even in the drug-eluting stent era. Frequent restenosis and stent thrombosis have been recently shown to be related not only to geometrical gap or stent structural deformation but also to rheological disturbance. Low wall shear stress at the lateral side of the bifurcation is likely to cause atherosclerotic changes due to easy access of the macrophages that induce chemical mediators. The turbulent flow over stent metal may facilitate accumulation of platelets, which results in thrombosis. The jailed strut and excess metal overlap may increase these risks. Since dramatic changes of the coronary flow pattern at the bifurcation are closely related to the genesis of atherosclerosis, future bifurcation intervention technique should be considered to restore the original physiological state as well as the anatomical structure. This article summarizes the global consensus of the members of the Asian Bifurcation Club and European Bifurcation Club at the KOKURA meeting. It also provides a perspective of basic sciences relating to bifurcation anatomy, physiology, and pathology, in the search for a best strategy for bifurcation intervention.

Altered nano/micro-order elasticity of pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a disease characterized by progressively increased resistance of pulmonary arteries. In this study, we evaluated the mechanical property of single pulmonary artery smooth muscles cells (PASMC) from patients with IPAH and tested whether the PASMC showed abnormal response to a vasodilator by use of an atomic force microscope (AFM). METHODS: PASMC were isolated and cultured from explanted lungs of 7 patients with IPAH (IPAH-PASMC). Normal vascular specimens from 3 patients with bronchogenic carcinoma were used as normal controls (normal PASMC). The nano/micro-order elasticity of five to ten living PASMC in each sample was measured by parabolic force curves of cantilever deflection/indentation obtained by using an AFM. The elasticity measurements were performed under control conditions and under condition of nitric oxide (NO) treatment (190 and 380 nmol/L). RESULTS: There was no significant difference between nano/micro-order elasticity of normal PASMC and that of IPAH-PASMC under the control conditions. In normal PASMC, NO (190 and 380 nmol/L) significantly reduced (i.e., softened) the nano/micro-order elasticity. However, NO did not reduce elasticity in IPAH-PASMC, indicating higher vasodilator-resistive nano/micro-order rigidity in IPAH-PASMC. CONCLUSION: Nano/micro-order elasticity change in PASMC in response to vasodilation induced by NO is reduced in patients with IPAH.

The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes.

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053+/-934 Pa, that of osteoid osteocytes was 7971+/-422 Pa and that of mature osteocytes was 4471+/-198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.

Visualisation of the effects of dilazep on rat afferent and efferent arterioles in vivo.

Although the effects of dilazep hydrochloride (dilazep), a nucleoside transport inhibitor, have been examined, there have been no visualisation studies on the physiological effects of dilazep on the glomerular arterioles. The purpose of this study was to visualise and evaluate the effects of dilazep and consequently the effects of adenosine, which dilazep augments by measuring glomelurar diameters, renal blood flow and resistance in rats in vivo. We time-sequentially examined afferent and efferent arteriolar diameter changes using an intravital videomicroscope and renal blood flow. We administered dilazep at a dose of 300 microg/kg intravenously. To further investigate the effects of dilazep, rats were pre-treated with 8-p-sulfophenyl theophylline (a nonselective adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (an A1 receptor antagonist), or 3,7-dimethyl-1-propargylxanthine (an A2 receptor antagonist). Dilazep constricted the afferent and efferent arterioles at the early phase and dilated them at the later phase, with the same degree of vasoconstrictive and vasodilatory effect on both arterioles. A1 blockade abolished vasoconstriction and augmented vasodilatation at the later phase and A2 blockade abolished vasodilatation and augmented vasoconstriction at the early phase. Non-selective blockade abolished both early vasoconstriction and later vasodilatation. In conclusion, adenosine augmented by dilazep constricted the afferent and efferent arterioles of the cortical nephrons at the early phase and dilated both arterioles at the later phase via A1 and A2 adenosine receptor activation, respectively. That the ratio of afferent to efferent arteriolar diameter was fairly constant suggests that intraglomerular pressure is maintained in the acute phase by adenosine despite the biphasic flow change.

Coronary microcirculation in the beating heart.

The phase opposition of velocity waveforms between coronary arteries (predominantly diastolic) and veins (systolic) is the most prominent characteristic of coronary hemodynamics. This unique arterial and venous flow patterns indicate the importance of intramyocardial capacitance vessels and variable resistance vessels during a cardiac cycle. It was shown that during diastole the intramyocardial capacitance vessels have two functional components, unstressed volume and ordinary capacitance. Unstressed volume is defined as the volume of blood in a vessel at zero transmural pressure. In vivo observation of systolic narrowing of arterioles in mid-wall and in subendocardium indicates the increase in resistance by cardiac contraction.

Role of Cu,Zn-SOD in the synthesis of endogenous vasodilator hydrogen peroxide during reactive hyperemia in mouse mesenteric microcirculation in vivo.

We have recently demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous H2O2 in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous H2O2 during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-SOD(-/-) mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, N(G)-monomethyl-L-arginine (L-NMMA) alone, and L-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or L-NMMA alone and was almost completely inhibited by L-NMMA + catalase in wild-type mice, whereas it was inhibited by L-NMMA and L-NMMA + catalase in the Cu,Zn-SOD(-/-) mice. RH-induced increase in blood flow after L-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-SOD(-/-) mice. In mesenteric arterioles of the Cu,Zn-SOD(-/-) mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular H(2)O(2) production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-SOD(-/-) mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous H(2)O(2) that contributes to RH in mouse mesenteric smaller arterioles.

Important role of endogenous hydrogen peroxide in pacing-induced metabolic coronary vasodilation in dogs in vivo.

OBJECTIVES: We examined whether endogenous hydrogen peroxide (H2O2) is involved in pacing-induced metabolic vasodilation in vivo. BACKGROUND: We have previously demonstrated that endothelium-derived H2O2 is an endothelium-derived hyperpolarizing factor in canine coronary microcirculation in vivo. However, the role of endogenous H2O2 in metabolic coronary vasodilation in vivo remains to be examined. METHODS: Canine subepicardial small coronary arteries (> or =100 microm) and arterioles (<100 microm) were continuously observed by a microscope under cyclooxygenase blockade (ibuprofen, 12.5 mg/kg intravenous [IV]) (n = 60). Experiments were performed during paired right ventricular pacing under the following 7 conditions: control, nitric oxide (NO) synthase inhibitor (N(G)-monomethyl-L-arginine [L-NMMA], 2 micromol/min for 20 min intracoronary [IC]), catalase (a decomposer of H2O2, 40,000 U/kg IV and 240,000 U/kg/min for 10 min IC), 8-sulfophenyltheophylline (SPT) (an adenosine receptor blocker, 25 mug/kg/min for 5 min IC), L-NMMA+catalase, L-NMMA+tetraethylammonium (TEA) (K(Ca)-channel blocker, 10 microg/kg/min for 10 min IC), and L-NMMA+catalase+8-SPT. RESULTS: Cardiac tachypacing (60 to 120 beats/min) caused coronary vasodilation in both-sized arteries under control conditions in response to the increase in myocardial oxygen consumption. The metabolic coronary vasodilation was decreased after L-NMMA in subepicardial small arteries with an increased fluorescent H2O2 production compared with catalase group, whereas catalase decreased the vasodilation of arterioles with an increased fluorescent NO production compared with the L-NMMA group, and 8-SPT also decreased the vasodilation of arterioles. Furthermore, the metabolic coronary vasodilation was markedly attenuated after L-NMMA+catalase, L-NMMA+TEA, and L-NMMA+catalase+8-SPT in both-sized arteries. CONCLUSIONS: These results indicate that endogenous H2O2 plays an important role in pacing-induced metabolic coronary vasodilation in vivo.