123I-Labeled oxLDL Is Widely Distributed Throughout the Whole Body in Mice.

PURPOSE: Oxidized low-density lipoprotein (oxLDL) plays a key role in endothelial dysfunction, vascular inflammation, and atherogenesis. The aim of this study was to assess blood clearance and in vivo kinetics of radiolabeled oxLDL in mice. METHODS: We synthesized 123I-oxLDL by the iodine monochloride method, and performed an uptake study in CHO cells transfected with lectin-like oxLDL receptor-1 (LOX-1). In addition, we evaluated the consistency between the 123I-oxLDL autoradiogram and the fluorescence image of DiI-oxLDL after intravenous injection for both spleen and liver. Whole-body dynamic planar images were acquired 10 min post injection of 123I-oxLDL to generate regional time-activity curves (TACs) of the liver, heart, lungs, kidney, head, and abdomen. Regional radioactivity for those excised tissues as well as the bladder, stomach, gut, and thyroid were assessed using a gamma counter, yielding percent injected dose (%ID) and dose uptake ratio (DUR). The presence of 123I-oxLDL in serum was assessed by radio-HPLC. RESULTS: The cellular uptakes of 123I-oxLDL were identical to those of DiI-oxLDL, and autoradiograms and fluorescence images also exhibited consistent distributions. TACs after injection of 123I-oxLDL demonstrated extremely fast kinetics. The radioactivity uptake at 10 min post-injection was highest in the liver (40.8 ± 2.4% ID). Notably, radioactivity uptake was equivalent throughout the rest of the body (39.4 ± 2.7% ID). HPLC analysis revealed no remaining 123I-oxLDL or its metabolites in the blood. CONCLUSION: 123I-OxLDL was widely distributed not only in the liver, but also throughout the whole body, providing insight into the pathophysiological effects of oxLDL.

Procyanidins are potent inhibitors of LOX-1: a new player in the French Paradox.

Lectin-like oxidized LDL receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL (oxLDL) and plays multiple roles in the development of cardiovascular diseases. We screened more than 400 foodstuff extracts for identifying materials that inhibit oxLDL binding to LOX-1. Results showed that 52 extracts inhibited LOX-1 by more than 70% in cell-free assays. Subsequent cell-based assays revealed that a variety of foodstuffs known to be rich in procyanidins such as grape seed extracts and apple polyphenols, potently inhibited oxLDL uptake in Chinese hamster ovary (CHO) cells expressing LOX-1. Indeed, purified procyanidins significantly inhibited oxLDL binding to LOX-1 while other ingredients of apple polyphenols did not. Moreover, chronic administration of oligomeric procyanidins suppressed lipid accumulation in vascular wall in hypertensive rats fed with high fat diet. These results suggest that procyanidins are LOX-1 inhibitors and LOX-1 inhibition might be a possible underlying mechanism of the well-known vascular protective effects of red wine, the French Paradox.

LOX-1 mediates vascular lipid retention under hypertensive state.

OBJECTIVES: Hypertension is a powerful independent risk factor for atherosclerotic cardiovascular diseases; however, the precise molecular mechanisms whereby hypertension promotes atherosclerotic formation remain to be determined. The interaction between oxidized low-density lipoprotein (oxLDL) and its receptor lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays a critical role in atherogenesis. To clarify how hypertension promotes atherosclerosis, we investigated specific roles of LOX-1 in acceleration of lipid deposition under a hypertensive state. METHODS: We employed a model of stroke-prone spontaneously hypertensive rats (SHR-SP) that exhibits acute lipid deposition in mesenteric artery induced by high fat and salt loading. These vascular lipid deposition lesions share similar characteristics with the initial lesions of human atherosclerosis. RESULTS: The enhanced LOX-1 expression in SHR-SP was associated with oxidized LDL deposited in vascular wall. Anti-LOX-1 neutralizing antibody dramatically suppressed the lipid deposition in vivo in SHR-SP. Vitamin E decreased serum oxLDL-like LOX-1 ligands, and suppressed the vascular lipid deposition. The vascular permeability, evaluated by the leakage of Evans blue, was markedly enhanced by pretreatment of oxLDL. The enhancement of vascular permeability induced by oxLDL was suppressed by anti-LOX-1 antibody. CONCLUSION: The enhanced expression and activation of LOX-1 mediated the enhancement of vascular permeability, which contributed to the vascular lipid accumulation under hypertensive states.

Microvascular hemodynamic responses to arteriovenous shunting in rat limb.

Autologus veins have been used clinically as a bypass conduit for reconstruction of small arteries, but there are few data available for microvascular response to arteriovenous (AV) shunting. This study was aimed to evaluate microvascular hemodynamic changes induced by creating AV anastomosis in rat hindlimb. Using intravital fluorescence videomicroscopy, we measured velocities of red blood cells (RBCs) flowing in the microvascular network in the control state, in the occlusion state where the superficial femoral artery (SFA) was occluded, and in the AV shunting state where the AV anastomosis was opened after occlusion of SFA. RBC velocities were measured in 155 capillaries of 6 rats using a dual window method and a frame-by-frame technique. The mean velocity and the coefficient of variation were 0.61 mm/sec and 0.90 in the control state, 0.34 mm/sec and 1.30 in the occlusion state, 0.83 mm/sec and 1.24 in the AV shunting state, respectively. These indicated that hemodynamic heterogeneity among capillaries increased with decrease in mean velocity following the arterial occlusion, while the AV shunting augmented the heterogeneity with increase in mean velocity. Capillaries with low perfusion (<0.1 mm/sec) or high perfusion (>1.0 mm/sec) were 5.8% or 20.6%, 29.6 or 5.2%, and 22.6 or 30.3% out of all measured capillaries in the control, occlusion and AV shunting conditions, respectively. In conclusion, AV shunting increased capillary perfusion and also its spatial heterogeneity, preferentially inducing high velocity in the microvasculature.

Heterogeneity of capillary flow in the retrograde microcirculation induced in rat limb by arteriovenous shunting.

Arteriovenous (AV) fistulas have been used clinically for improving adjunctive bypass patency. Such AV shunting induces retrograde flow in the microvascular network, which may induce microvascular remodeling and angiogenesis at the chronic phase. This paper was aimed to examine heterogeneity of blood flow among capillaries in the retrograde microcirculation induced by AV shunting. An AV anastomosis was created in rat hind limb. Using a dual window method or frame-by-frame technique on the fluorescence microscopic video images, we measured velocities of red blood cells (RBCs) flowing in the capillary network in three flow conditions: control (normal flow), arterial occlusion, and AV shunting (retrograde flow). For each flow condition, RBC velocities were obtained in 155 capillaries of 6 rats. By classifying all the capillaries into four groups based on the levels of RBC velocity in the occlusion state, we evaluated the mean velocities, coefficient of variation (CV), and histograms for each group of capillaries. The mean velocity and CV in each group changed significantly from the control to AV shunting states. Especially, most significant changes appeared in capillary groups where the superficial femoral artery or its collateral arteries might have a direct influence. Though the AV shunting improved capillary perfusion in the mean level, major parts of capillaries still remained at low perfusion.

Cell morphological changes in venous remodeling induced by arteriovenous grafting in rat limb.

Vascular remodeling induced in rat limb by arteriovenous (AV) shunting was investigated by evaluating changes in vascular diameter and cell morphology. In Wistar rats, a vein graft was implanted in situ in the hind limb. Flow-rate in the grafted vein was assessed by measuring flow in the common femoral artery using an ultrasonic flowmeter. Nuclei and actin filaments of the venous wall were stained with propidium iodine and phalloidine-FITC, and the samples were observed using confocal laser microscopy. The grafted veins became circular in cross-section with increase in diameter during two weeks after AV shunting. Owing to the increase in diameter, the estimated wall shear stress was not increased so much as the flow-rate. The confocal laser microscopic observation showed that endothelial cells (ECs) and smooth muscle cells (SMCs) in the grafted veins were either aligned well (2 out of 8 samples), or ECs were denudated and SMCs were disrupted (in 6 out of 8 samples). The cell density of ECs was unchanged from the control level. In conclusion, the grafted vein was remodeled with morphological changes in ECs and SMCs during 2 weeks after AV shunting.

Capillary angiogenesis and remodeling induced in rat limb by arteriovenous shunting.

Capillary angiogenesis and remodeling induced by arteriovenous (AV) shunting in rat hind limb was investigated by evaluating changes in capillary density and diameter in the skeletal muscle subject to retrograde flow and high pressure. Wistar rats were used, and an AV anastomosis was created in the hind limb. Two weeks after AV shunting, the microvasculature in the limb was visualized by GS-lectine, and the samples were observed using confocal laser microscopy. The capillary density were increased by approximately 150% for small vessels (<13 microm in diameter) under retrograde flow condition, but no change appeared for large vessels (>13 microm in diameter). The capillary diameters were not significantly different between control and chronic condition. In conclusion, retrograde flow produced by AV shunting increased capillary density but it did not change the capillary diameter significantly.

[Immunohistochemical study for monoamine neurons in the brain of unilateral inner ear impaired rats].

Patients with inner ear impairment have complaints of vertigo and also occasionally depression. The present study was undertaken in order to evaluate changes in monoamines which have reportedly been closely related to depression, using cisplatin-induced unilateral inner-ear impaired rats. A dose of 0.5 mg/kg of cisplatin was injected into the right tympanic cavity under pentobarbital Na+ anesthesia. One or two weeks later, animals were fixed with paraformaldehyde, and thereafter immunohistochemical stainings for monoamine-containing cells in the brain were carried out. To visualize 5-hydroxytryptamine (5-HT), noradrenaline (NA) and dopamine (DA) neurons, we used mouse antibodies against 5-HT, NA, and DA syntheses, i.e., tryptophan hydroxylase (TRH), tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH). The number of TRH immunoreactive neurons significantly decreased in the lateral dorsal raphe nucleus of the ipsilateral side when compared with the contralateral side. The number of DA neurons, which were immunoreactive to TH, but not to DBH, significantly decreased in the hypothalamus of the ipsilateral side. The number of NA neurons which were immunoreactive to both TH and DBH significantly decreased in the locus coeruleus and ventral lateral pons of the ipsilateral side. An additional control study with saline-injected rats showed a lack of differences in monoamine syntheses between the injected and contralateral sides, the expressions of the synthesis on both sides being similar to that obtained in the contralateral side in cisplatin-injected rats. These results indicated the decreases in monoamine syntheses at the ipsilateral side only in the cisplatin-administered rats. We conclude that inner ear impairment may diminish the ipsilateral amount of monoamines in the brain but not the cotralateral, possibly inducing a vestibular compensation such as an upregulation of monoamine receptors.

Maturity of pericytes in cerebral neocapillaries induced by growth factors: fluorescence immuno-histochemical analysis using confocal laser microscopy.

The maturity of pericytes in cerebral neocapillaries induced by two different growth factors: basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF), was examined using an immunohistochemical staining technique. Cerebral angiogenesis was induced in mice by implanting a sandwich system of bFGF/PDGF gel and nylon-mesh over the exposed cortex. On 28th day after incubation, a small volume of cerebral tissue with the nylon-mesh was isolated and stained using tetramethyl rhodamine isothiocyanate (TRITC)-labeled secondary antibody to the primary antibody against NG_2 proteoglycan and fluorescein isothiocyanate (FITC)-labeled Griffonia simplicifolia (GS)-lectin. Using a confocal laser microscopic system, we observed the cerebral neocapillaries on the upper surface of the nylon-mesh and evaluated the maturity of pericytes stained with NG_2 based on the fluorescence immunohistological images. The pericyte appeared rich in neocapillaries induced by PDGF. It was suggested that pericytes might play a key role in the regulation of blood flow in neovessels.

Measurement of red cell velocity in microvessels using particle image velocimetry (PIV).

A new technique using particle image velocimetry (PIV) has been developed to evaluate the detailed velocity profiles of red cells flowing in microvessels. The microcirculation in rat mesentery was directly observed using high-speed videomicroscopy, and the images of red cells flowing in the mesenteric arterioles were recorded simultaneously with the arterial blood pressure. Based on the high-speed videomicroscopic images obtained, velocity vectors in single or branched arterioles were evaluated to obtain velocity profiles across the cross-section of arterioles. It was shown that in single and straight arterioles the velocity profile was blunt with a pit at the central region, and its pit was marked in bifurcation. The present technique enables us to analyze red cell velocity profiles up to 0.8 microm in the spatial resolution and 1 msec in the time interval.