Travis W. Hein

LDLs Impair Vasomotor Function of the Coronary Microcirculation Role of Superoxide Anions

Oxidized LDLs (Ox-LDLs) inhibit endothelium-dependent dilation of isolated conduit arteries in a manner comparable to the impairment demonstrated in atherosclerotic vessels. However, it is not known whether the microvessels, which do not develop atherosclerotic lesions, are susceptible to Ox-LDL. Since endothelial release of NO plays an important role in vasodilation and since its dysfunction associated with atherosclerosis has been shown to extend into the coronary microcirculation, we hypothesized that Ox-LDLs impair endothelium-dependent vasodilation of coronary arterioles by reducing the synthesis and/or release of NO. To test this hypothesis, porcine subepicardial vessels (50 to 100 microm) were isolated, cannulated, and pressurized to 60 cm H2O without flow for in vitro study. Isolated vessels developed basal tone and dilated in a dose-dependent manner to the endothelium-dependent vasodilators serotonin, ATP, and ionomycin. These vasodilatory responses were inhibited by the NO synthase inhibitor NG-monomethyl-L-arginine and were subsequently reversed by extraluminal administration of the NO precursor L-arginine (3 mmol/L), suggesting the involvement of NO in these vasomotor responses. Intraluminal incubation of the vessels with native LDL (N-LDL) or Ox-LDL (1 mg protein/mL) significantly attenuated dilations to serotonin, ATP, and ionomycin. Ox-LDL produced more severe inhibition than did N-LDL, and the inhibitory effect was comparable to that of NG-monomethyl-L-arginine. The inhibitory effects of N-LDL and Ox-LDL were reversed by exogenous L-arginine (3 mmol/L) and were prevented by sodium dihydroxybenzene disulfonate (Tiron), a cell-permeable superoxide scavenger. In contrast, administration of the cell-impermeable superoxide scavenger superoxide dismutase prevented the inhibitory effect of N-LDL but not of Ox-LDL. In addition, the inhibitory effects of LDL were not restored by D-arginine or by removal of intraluminal LDL. Neither N-LDL nor Ox-LDL altered endothelium-independent vasodilation to sodium nitroprusside. These results indicate that coronary arterioles are susceptible to LDLs that specifically impair endothelium-dependent vasodilation by reducing NO synthesis. It is suggested that the initiation of superoxide anion production and the subsequent L-arginine deficiency may be responsible for the detrimental effect of LDL.

Selective Activation of LOX-1 Mediates C-Reactive Protein Evoked Endothelial Vasodilator Dysfunction in Coronary Arterioles

Rationale: Studies in cultured endothelium implicate that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) or Fc&ggr; receptor II (CD32) contributes to the proatherogenic effects of C-reactive protein (CRP). However, the identity of the receptors linking to deleterious actions of CRP in vasomotor regulation remains unknown. Objective: We tested the hypothesis that LOX-1 contributes to adverse effects of CRP on endothelium-dependent vasomotor function in resistance arterioles. Methods and Results: Porcine coronary arterioles were isolated for vasoreactivity study, dihydroethidium fluorescence staining of superoxide, immunohistochemical localization of receptors, immunoprecipitation of receptor/CRP interaction, and protein blot. Intraluminal treatment of pressurized arterioles with a pathophysiological level of CRP (7 µg/mL; 60 minutes) attenuated endothelium-dependent nitric oxide–mediated and prostacyclin-mediated dilations to serotonin and arachidonic acid, respectively. LOX-1 and CD32 were detected in the endothelium of arterioles. Blockade of LOX-1 with either pharmacological antagonist &kgr;-carrageenan or anti–LOX-1 antibody prevented the detrimental effect of CRP on vasodilator function, whereas anti-CD32 antibody treatment was ineffective. Denudation of endothelium and blockade of LOX-1 but not CD32 prevented CRP-induced elevation of superoxide in the vessel wall. CRP was coimmunoprecipitated with LOX-1 and CD32 from CRP-treated arterioles. Similarly, LOX-1 and CD32 blockade prevented CRP-induced arteriolar expression of plasminogen activator inhibitor-1, a thrombogenic protein. Conclusions: CRP elicits endothelium-dependent oxidative stress and compromises nitric oxide–mediated and prostacyclin-mediated vasomotor function via LOX-1 activation. In contrast, both LOX-1 and CD32 mediate plasminogen activator inhibitor-1 upregulation in arterioles by CRP. Thus, activation of LOX-1 and CD32 may contribute to vasomotor dysfunction and proatherogenic actions of CRP, respectively.

Enhanced endothelin-1/Rho-kinase signalling and coronary microvascular dysfunction in hypertensive myocardial hypertrophy

AimsnHypertensive cardiac hypertrophy is associated with reduced coronary flow reserve, but its impact on coronary flow regulation and vasomotor function remains incompletely understood and requires further investigation.nnnMethods and resultsnLeft ventricular hypertrophy was induced in mice by transverse aortic coarctation (TAC) for 4u2009weeks. The left coronary artery blood velocity (LCABV) and myocardium lactate level were measured following the metabolic activation by isoproterenol. Septal coronary arterioles were isolated and pressurized for functional studies. In TAC mice, the heart-to-body weight ratio was increased by 45%, and cardiac fractional shortening and LCABV were decreased by 51 and 14%, respectively. The resting myocardial lactate level was 43% higher in TAC mice. Isoproterenol (5 µg/g, i.p.) increased heart rate by 20% in both groups of animals, but the corresponding increase in LCABV was not observed in TAC mice. The ventricular hypertrophy was associated with elevation of myocardial endothelin-1 (ET-1), increased vascular expression of rho-kinases (ROCKs), and increased superoxide production in the myocardium and vasculature. In coronary arterioles from TAC mice, the endothelial nitric oxide (NO)-mediated dilation to acetylcholine (ACh) was reversed to vasoconstriction and the vasoconstriction to ET-1 was augmented. Inhibition of ROCK by H-1152 alleviated oxidative stress and abolished enhanced vasoconstriction to ET-1. Both H-1152 and superoxide scavenger Tempol abolished coronary arteriolar constriction to ACh in a manner sensitive to NO synthase blocker NG-nitro-L-arginine methyl ester.nnnConclusionsnMyocardial hypertrophy induced by pressure overload leads to cardiac and coronary microvascular dysfunction and ischaemia possibly due to oxidative stress, enhanced vasoconstriction to ET-1 and compromised endothelial NO function via elevated ROCK signalling.

Regulation of Coronary Vasomotor Function by Reactive Oxygen Species

Overproduction of reactive oxygen species is closely associated with cardiovascular diseases in part by impairing endothelial function and consequently compromising blood flow regulation. Superoxide and hydrogen peroxide are elevated under various disease states with reduced endothelium-derived nitric oxide bioavailability. The oxidative stress elicited by angiotensin II, C-reactive protein and tumor necrosis factor-α is mediated by the activation of different redox signaling pathways in the microvasculature. The upregulation of L-arginine consuming enzyme arginase also contributes to the reduced nitric oxide bioavailability during oxidative stress. Hydrogen peroxide exhibits vasodilator function in the coronary microcirculation and plays an important role in the physiological regulation of coronary blood flow. However, excessive production of hydrogen peroxide impairs endothelial function by reducing L-arginine availability through hydroxyl radical-mediated upregulation of arginase. This review summarizes the current knowledge on the effects superoxide and hydrogen peroxide on vasomotor function regulated by the endothelium-derived nitric oxide and prostacyclin in the coronary microcirculation.

Intravitreal Stanniocalcin-1 enhances new blood vessel growth in a rat model of laser-induced choroidal neovascularization

Purpose The purpose of this study was to investigate the impact of stanniocalcin-1 (STC-1), a photoreceptor-protective glycoprotein, on the development of choroidal neovascularization (CNV) in relation to VEGF and its main receptor (VEGFR2) expression after laser injury. Methods In rats, CNV was induced by laser photocoagulation in both eyes, followed by intravitreal injection of STC-1 in the right eye and vehicle or denatured STC-1 injection in the left eye as control. Two weeks after laser injury, fundus autofluorescence (FAF) imaging and fundus fluorescein angiography (FFA) were performed. Fluorescein leakage from CNV was graded using a defined scale system. The size of CNV was quantified with spectral domain optical coherence tomography (SD-OCT), fluorescein-labeled choroid-sclera flat mounts, and hematoxylin-eosin staining. Protein expressions were evaluated by Western blot. Results Photocoagulation produced a well-circumscribed area of CNV. With STC-1 treatment, CNV lesions assessed by FAF were increased by 50% in both intensity and area. The CNV lesions were also increased with SD-OCT, flat-mount, and histologic analyses. FFA disclosed enhanced fluorescein leakage in CNV lesions in STC-1 treated eyes. The STC-1 protein was detected in the choroidal tissue and its level was increased with CNV lesions in correlation with VEGF and VEGFR2 expressions. Intravitreal administration of STC-1 significantly increased choroidal expression of both VEGF and VEGFR2 proteins. Conclusions Chorodial tissue expresses STC-1, which seemingly acts as a stress response protein by enhancing pathological new blood vessel growth in laser-induced CNV. It is likely that STC-1 promotes CNV development via VEGF signaling.

Correlation of spectral domain optical coherence tomography with histology and electron microscopy in the porcine retina

&NA; Spectral domain optical coherence tomography (SD‐OCT) is used as a non‐invasive tool for retinal morphological assessment in vivo. Information on the correlation of SD‐OCT with retinal histology in the porcine retina, a model resembling the human retina, is limited. Herein, we correlated the hypo‐ and hyper‐reflective bands on SD‐OCT with histology of the lamellar architecture and cellular constituents of the porcine retina. SD‐OCT images were acquired with the Heidelberg Spectralis HRA + OCT. Histological analysis was performed using epoxy resin embedded tissue and transmission electron microscopy. Photomicrographs from the histologic sections were linearly scaled to correct for tissue shrinkage and correlated with SD‐OCT images. SD‐OCT images correlated well with histomorphometric data. A hyper‐reflective band in the mid‐to‐outer inner nuclear layer correlated with the presence of abundant mitochondria in horizontal cell processes and adjacent bipolar cells. A concentration of cone nuclei corresponded to a relative hypo‐reflective band in the outer portion of the outer nuclear layer. The presence of 3 hyper‐reflective bands in the outer retina corresponded to: 1) the external limiting membrane; 2) the cone and rod ellipsoid zones; and 3) the interdigitation zone of photoreceptor outer segments/retinal pigment epithelium (RPE) apical cell processes and the RPE. These correlative and normative SD‐OCT data may be employed to characterize and assess the in vivo histologic changes in retinal vascular and degenerative diseases and the responses to novel therapeutic interventions in this large animal model.