Beneficial effect of hydroxyfasudil, a specific Rho-kinase inhibitor, on ischemia/reperfusion injury in canine coronary microcirculation in vivo.

OBJECTIVES: We examined whether hydroxyfasudil, a specific Rho-kinase inhibitor, exerts cardioprotective effect on coronary ischemia/reperfusion (I/R) injury and, if so, whether nitric oxide (NO) is involved. BACKGROUND: Recent studies have demonstrated that Rho-kinase is substantially involved in the pathogenesis of cardiovascular diseases; however, it remains to be examined whether it is also involved in ischemia/reperfusion (I/R) injury. METHODS: Canine subepicardial small arteries (SA, >or=100 microm) and arterioles (A, <100 microm) were observed by a charge-coupled device intravital microscope during I/R. Coronary vascular responses to endothelium-dependent (acetylcholine, intracoronary [IC]) and -independent (papaverine, IC) vasodilators were examined after I/R under the following four conditions: control (n = 7), NO synthase inhibitor alone (N(G)-monomethl-L-arginine [L-NMMA], IC, n = 4), hydroxyfasudil alone (IC, n = 7), and hydroxyfasudil plus L-NMMA (n = 7). RESULTS: Hydroxyfasudil significantly attenuated serotonin (IC)-induced vasoconstriction of SA (-7 +/- 1% vs. 2 +/- 1%, p < 0.01). Coronary I/R significantly impaired coronary vasodilation to acetylcholine after I/R (SA, p < 0.05; and A, p < 0.01 vs. before I/R) and L-NMMA further reduced the vasodilation, whereas hydroxyfasudil completely preserved the responses. The vasoconstriction by L-NMMA after I/R was significantly improved by hydroxyfasudil in both-sized arteries (both p < 0.01). Expression of endothelial nitric oxide synthase (eNOS) protein in the ischemic endocardium of left anterior descending coronary artery area (as determined by Western blotting) significantly decreased (79 +/- 4%) compared with the nonischemic endocardium of LCX area (100 +/- 7%), which was improved by hydroxyfasudil (105 +/- 6%, p < 0.01). Hydroxyfasudil significantly reduced myocardial infarct size, and hydroxyfasudil with L-NMMA also reduced the infarct size compared with L-NMMA alone. CONCLUSIONS: Hydroxyfasudil exerts cardioprotective effects on coronary I/R injury in vivo, in which NO-mediated mechanism may be involved through preservation of eNOS expression.

Direct observation of epicardial coronary capillary hemodynamics during reactive hyperemia and during adenosine administration by intravital video microscopy.

Using high-resolution intravital charge-coupled device video microscopy, we visualized the epicardial capillary network of the beating canine heart in vivo to elucidate its functional role under control conditions, during reactive hyperemia (RH), and during intracoronary adenosine administration. The pencil-lens video-microscope probe was placed over capillaries fed by the left anterior descending artery in atrioventricular-blocked hearts of open-chest, anesthetized dogs paced at 60-90 beats/min (n = 17). In individual capillaries under control conditions, red blood cell flow was predominant during systole or diastole, indicating that the watershed between diastolic arterial and systolic venous flows is located within the capillaries. Capillary flow increased during RH and reached a peak flow velocity (2.1 +/- 0.6 mm/s), twice as high as control (1.2 +/- 0.5 mm/s), with enhancement of intercapillary cross-connection flow and enlargement of diameter (by 17%). With adenosine, capillary flow velocity significantly increased (1.8 +/- 0.7 mm/s). However, the increase in volumetric capillary flow with adenosine estimated from red blood cell velocity and diameter was less than the increase in arterial flow, whereas that during RH was nearly equivalent to the increase in arterial flow. There was a time lag of approximately 1.5 s for refilling of capillaries during RH, indicating their function as capacitance vessels. In conclusion, the coronary capillary network functions as 1) the major watershed between diastolic-dominant arterial and systolic-dominant venous flows, 2) a capacitor, and 3) a significant local flow amplifier and homogenizer of blood supply during RH, but with adenosine the increase in capillary flow velocity was less than the increase in arterial flow.

Exogenous NO suppresses flow-induced endothelium-derived NO production because of depletion of tetrahydrobiopterin.

Exogenous nitric oxide (NO) suppresses endothelium-derived NO production. We were interested in determining whether this is also the case in flow-induced endothelium-derived NO production. If so, then is the mechanism because of intracellular depletion of tetrahydrobiopterin [BH4; a cofactor of NO synthase (NOS)], which results in superoxide production by uncoupled NOS? Isolated canine femoral arteries were perfused with 100 microM S-nitroso-N-acetylpenicillamine (SNAP; an NO donor) and/or 64 microM BH4. Perfusion of SNAP suppressed flow-induced NO production, which was evaluated as a change in the slope of the linear relationship between perfusion rate and NO production rate (P < 0.02 vs. control; n = 7). Subsequent BH4 perfusion returned the slope to the control level. Concomitant perfusion of SNAP and BH4 retained the control-level NO production (n = 7). Concomitant perfusion of SNAP and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; 1 mM; a membrane-permeable superoxide scavenger) also retained the control-level NO production (n = 7), whereas perfusion of Tiron after SNAP could not return the NO production to the control level (P < 0.02 vs. control; n = 7). We also found a significant decrease in BH4 concentration in the endothelial cells after SNAP perfusion. In conclusion, these results indicate that exogenous NO suppresses the flow-induced, endothelium-derived NO production by superoxide released from uncoupled NOS because of intracellular BH4 depletion.

Effect of nicorandil, a K+ATP -channel opener, on coronary capillary architecture and volume after early myocardial ischemia-reperfusion--a 3-dimensional confocal laser microscopic study--.

BACKGROUND: Previous experimental and clinical studies have reported that nicorandil, an adenosine triphosphate (ATP)-sensitive K+ channel opener, exerts a beneficial effect on microvascular function and clinical outcome after myocardial ischemia. The present study assessed whether intravenous administration of nicorandil affects the 3-dimensional (3-D) architectural characteristics of capillaries and their volume after early myocardial reperfusion. METHODS AND RESULTS: Using the hearts of open-chest anesthetized rats, the left anterior descending artery was occluded for 7 min followed by reperfusion. Nicorandil or saline was infused intravenously during occlusion and reperfusion. The entire coronary microvasculature was filled with contrast medium after the hearts were arrested. Capillaries were observed 3-dimensionally by confocal laser scanning microscopy in both the control area and reperfused area of all samples. The capillary volume fraction was computed from the 3-D images. The reperfused area in both the nicorandil and saline groups showed characteristic architectural changes of the capillaries. Capillary volume fraction in the reperfused area was significantly reduced in saline group, compared with nicorandil group [12.7+/-7.2% vs 18.1 +/-5.3% (p<0.01)]. CONCLUSIONS: Intravenous nicorandil administration has a beneficial effect on capillary damage after reperfusion.

Three-dimensional microstructural abnormality of the coronary capillary network after myocardial reperfusion--comparison between 'reflow' and 'no-reflow'.

BACKGROUND: The 3-dimensional (D) structural abnormalities of the coronary capillary network and capillary volume changes after myocardial reperfusion were investigated using confocal laser scanning microscopy. METHODS AND RESULTS: Using open-chest anesthetized rats' hearts, the left anterior descending artery (LAD) was occluded for 7 min followed by reperfusion. The hearts were divided into 2 groups: (1) reperfused area stained well by intravenous indocyanine green after reperfusion (Reflow), and (2) lack of staining in the reperfused area (No-reflow). The entire coronary microvasculature was filled with contrast medium using a Langendorff's perfusion system. Capillaries were observed 3-dimensionally in the control and reperfused areas in both the Reflow and No-reflow group and the capillary volume fraction was computed from the 3-D images. The reperfused area in both groups showed decreased capillary diameter with waving and shrinkage configuration. In the No-reflow group, marked interruption of capillary network was observed. In the Reflow group the capillary volume fraction was significantly reduced in the reperfused area compared with the control area (14.8+/-4.1% vs 22.2+/-5.6%, p<0.05). Capillary volume fraction was further decreased in the No-reflow group compared with Reflow (5.3+/-1.4% vs 14.8+/-4.1%, p<0.01). CONCLUSION: The morphological changes in coronary capillaries after reperfusion were characterized by waving and shrinkage, which resulted in a reduction in capillary volume.

Global heterogeneity of glomerular volume distribution in early diabetic nephropathy.

BACKGROUND: Morphologic characteristics in early stage of nephropathy of noninsulin-dependent diabetes mellitus (NIDDM) have not been determined despite the fact thatdiagnosis in this stage of the disease is important for the prognosis. We hypothesized that heterogeneity in glomerular volume-distribution may be a sensitive index of early stage of diabetic nephropathy in NIDDM. METHODS: In spontaneous diabetic rats [Otsuka Long-Evans Tokushima Fatty (OLETF) rat (N= 5)] of 27 to 28 weeks, an experimental model of early diabetic nephropathy in human NIDDDM and age-matched control rats [Long Evans Tokushima Lean (LETO) rat (N= 5)], we completely filled the kidney with contrast medium. Glomeruli were visualized as three-dimensional images using x-ray micro-computed tomography (micro-CT). Glomerular volumes (N= 400 in each kidney) were directly measured and evaluated as absolute volume and normalized values to kidney weight and body weight. Scattering of glomerular volume-distribution was evaluated as coefficient variation (CV) (SD/mean). RESULTS: The CV was significantly larger in OLETF rat (0.195) comparing to LETO rat (0.146, P < 0.01). This difference was even consistent under the normalization to kidney weight and body weight. Absolute glomerular volume was larger in OLETF rat compared to LETO rat (P < 0.005); however, when glomerular volume was normalized, this variable was comparable between two groups. CONCLUSION: We visualized three-dimensional glomerular images in the early stages of diabetic nephropathy using micro-CT and quantified the heterogeneity in glomerular volume distribution throughout the cortex by direct measurement of the individual. We propose that heterogeneity in glomerular volume distribution is a sensitive parameter to ascertain early diabetic nephropathy in NIDDM.

Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release.

Endothelium-derived nitric oxide (NO) is synthesized in response to chemical and physical stimuli. Here, we investigated a possible role of the endothelial cell glycocalyx as a biomechanical sensor that triggers endothelial NO production by transmitting flow-related shear forces to the endothelial membrane. Isolated canine femoral arteries were perfused with a Krebs-Henseleit solution at a wide range of perfusion rates with and without pretreatment with hyaluronidase to degrade hyaluronic acid glycosaminoglycans within the glycocalyx layer. NO production rate was evaluated as the product of nitrite concentration in the perfusate and steady-state perfusion rate. The slope that correlates the linear relation between perfusion rate and NO production rate was taken as a measure for flow-induced NO production. Hyaluronidase treatment significantly decreased flow-induced NO production to 19 +/- 9% of control (mean +/- SD; P < 0.0001 vs. control; n = 11), whereas it did not affect acetylcholine-induced NO production (88 +/- 17% of pretreatment level, P = not significant; n = 10). We conclude that hyaluronic acid glycosaminoglycans within the glycocalyx play a pivotal role in detecting and amplifying the shear force of flowing blood that triggers endothelium-derived NO production in isolated canine femoral arteries.

Hydrogen peroxide, an endogenous endothelium-derived hyperpolarizing factor, plays an important role in coronary autoregulation in vivo.

BACKGROUND: Recent studies in vitro have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in animals and humans. The aim of this study was to evaluate our hypothesis that endothelium-derived H2O2 is an EDHF in vivo and plays an important role in coronary autoregulation. METHODS AND RESULTS: To test this hypothesis, we evaluated vasodilator responses of canine (n=41) subepicardial small coronary arteries (> or =100 microm) and arterioles (<100 microm) with an intravital microscope in response to acetylcholine and to a stepwise reduction in coronary perfusion pressure (from 100 to 30 mm Hg) before and after inhibition of NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA). After L-NMMA, the coronary vasodilator responses were attenuated primarily in small arteries, whereas combined infusion of L-NMMA plus catalase (an enzyme that selectively dismutates H2O2 into water and oxygen) or tetraethylammonium (TEA, an inhibitor of large-conductance K(Ca) channels) attenuated the vasodilator responses of coronary arteries of both sizes. Residual arteriolar dilation after L-NMMA plus catalase or TEA was largely attenuated by 8-sulfophenyltheophylline, an adenosine receptor inhibitor. CONCLUSIONS: These results suggest that H2O2 is an endogenous EDHF in vivo and plays an important role in coronary autoregulation in cooperation with NO and adenosine.

Dynamic changes in three-dimensional architecture and vascular volume of transmural coronary microvasculature between diastolic- and systolic-arrested rat hearts.

BACKGROUND: The phase difference of coronary arterial and venous flows indicates the importance of intramyocardial capacitance vessels in storing diastolic flow and in discharging volume in systole. However, the anatomic and functional characteristics of the capacitance vessels are unclear. We aimed to clarify those characteristics with their transmural difference by 3D visualization of transmural microvessels under diastole and systole. METHODS AND RESULTS: We performed complete intracoronary filling of a contrast medium into Langendorff's Wistar rat hearts under (1) St Thomas-perfused diastolic arrest (D-mode) and (2) BaCl(2)-induced systolic arrest (S-mode). Precise transmural 3D architectures of capillaries and of pre- and post-capillary microvessels (ie, microvessels larger than capillaries) were visualized clearly with a confocal laser scanning microscope and x-ray microcomputed tomography (microCT), respectively. Vascular volume fraction (VF) and systolic-induced VF reduction rate from D- to S-mode were analyzed. The net capillary VF in D-mode (20.4 +/- 0.9%) was 10 times that of larger microvessels and was reduced in S-mode by 32% without capillary collapse. Systolic-induced VF reduction rate was smaller in capillaries than in larger microvessels (48%; P<0.05). The larger microvessel VF in D-mode (2.2 +/-0.2%) was reduced in S-mode, accompanied by complicated 3D deformation. CONCLUSIONS: Capillaries were relatively resistant to the systolic extravascular compression compared with pre- and post-capillary microvessels, conveniently beneficial for the myocardial oxygen delivery throughout a cardiac cycle. Nevertheless, a larger change in the absolute volume of capillaries may function as effective capacitance. On one hand, the pre- and post-capillary microvessels showed a larger phasic change in resistance, which may function to maintain the capillary patency during systole.