Lectin-like oxidized LDL receptor 1 is involved in CRP-mediated complement activation.

BACKGROUND: C-reactive protein (CRP) is purported to be a risk factor that acts independently of LDL cholesterol in predicting all-cause mortality in patients with ischemic heart disease. Lectin-like oxidized LDL receptor 1 (LOX-1) impairs endothelial function and exacerbates myocardial injury. We recently demonstrated that CRP increased vascular permeability through direct binding to LOX-1. Here we examined, using a hypertensive rat model, whether LOX-1 is involved in CRP-induced complement activation. METHODS AND RESULTS: In the cultured LOX-1-expressing cell line hLOX-1-CHO, CRP increased complement activation, but did not do so in native CHO cells. Depleting C1q from serum abolished CRP-induced complement activation. Incubation of CRP with serum on immobilized recombinant LOX-1 similarly showed that CRP activated C1q-requiring classical complement pathway in a LOX-1-dependent manner. Interestingly, the interaction between CRP and LOX-1 was dependent on Ca²âº ion and competed with phosphocholine, suggesting that LOX-1 bound to the B-face of CRP with a phosphocholine-binding domain. This was in contrast to Fcγ receptors, to which CRP bound in A-face with complement-binding domain. In vivo, intradermal injection of CRP to hypertensive SHRSP rats induced complement activation detected by C3d deposition and leukocyte infiltration around the injected area. Anti-LOX-1 antibody reduced the extent of complement activation and leukocyte infiltration. CONCLUSIONS: LOX-1 appears to be involved in CRP-induced complement activation, and thus may serve to locate the site of CRP-induced complement activation and inflammation.

Oxidized LDL receptor LOX-1 binds to C-reactive protein and mediates its vascular effects.

BACKGROUND: C-reactive protein (CRP) exerts biological activity on vascular endothelial cells. This activity may promote atherothrombosis, but the effects of this activity are still controversial. Lectin-like oxidized LDL receptor-1 (LOX-1), the oxidized LDL receptor on endothelial cells, is involved in endothelial dysfunction induced by oxidized LDL. METHODS: We used laser confocal microscopy to examine and fluorescence cell image analysis to quantify the binding of fluorescently labeled CRP to cells expressing LOX-1. We then examined the binding of unlabeled CRP to recombinant human LOX-1 in a cell-free system. Small interfering RNAs (siRNAs) against LOX-1 were applied to cultured bovine endothelial cells to analyze the role of LOX-1 in native cells. To observe its in vivo effects, we injected CRP intradermally in stroke-prone spontaneously hypertensive (SHR-SP) rats and analyzed vascular permeability. RESULTS: CRP bound to LOX-1-expressing cells in parallel with the induction of LOX-1 expression. CRP dose-dependently bound to the cell line and recombinant LOX-1, with significant binding detected at 0.3 mg/L CRP concentration. The K(d) value of the binding was calculated to be 1.6 x 10(-7) mol/L. siRNA against LOX-1 significantly inhibited the binding of fluorescently labeled CRP to the endothelial cells, whereas control RNA did not. In vivo, intradermal injection of CRP-induced vascular exudation of Evans blue dye in SHR-SP rats, in which expression of LOX-1 is greatly enhanced. Anti-LOX-1 antibody significantly suppressed vascular permeability. CONCLUSIONS: CRP and oxidized LDL-receptor LOX-1 directly interact with each other. Two risk factors for ischemic heart diseases, CRP and oxidized LDL, share a common molecule, LOX-1, as their receptor.

Blood flow regulation in the cerebral microvasculature with an arcadal network: a numerical simulation.

Blood flow regulation in the cerebral microvasculature with an arcadal network was investigated using a numerical simulation. A mathematical model for blood flow in the arcadal network, based on in vivo data of cat cerebral microvasculature and flow velocity was developed. The network model consists of 45 vessel segments and 25 branching points. To simulate microvascular response to blood flow, non-reactive (solid), cerebral arteriole-like, or skeletal muscle arteriole-like responses to wall shear stress were taken into account. Numerical calculation was carried out in the flow condition where the inlet (arterial) pressure was changed from 60 to 120 mmHg. Flow-rate in each efferent vessel and the mean flow-rate over all efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The simulation demonstrated the wall shear stress-induced vasodilation in the arcadal network worked to maintain the blood flow at a constant level with pressure variable in a wide range. It is suggested that an individual microvessel (segment) should join in the regulatory process of flow, interacting with other microvessels (cooperative regulation).

Microembolic flow disturbances in the cerebral microvasculature with an arcadal network: a numerical simulation.

Flow disturbance due to microembolism in the cerebral microvasculature with an arcadal network was studied by a numerical simulation. A mathematical model for flow in the arcadal network was developed, based on in vivo data of cat cerebral microvasculature and flow velocity. The network model consisted of 45 vessel segments, and 25 branching points. To simulate microvascular responses to blood flow, the following three types of responses to wall shear stress were considered; non-reactive (solid-like), cerebral arteriole, and skeletal muscle arteriole-like responses. The numerical calculation was carried out in the condition where a feeding arteriole was occlused. Flow changes in efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The present simulation has demonstrated that blood flow in efferent vessels was influenced by the topology of the vascular network and the response pattern in single vessels. The arcadal structure of arterioles might be most effective in response to flow disturbances in efferent vessels.

Cerebral microvascular responses to air embolism-reperfusion in the cat using fluorescence videomicroscopy.

Cerebral microvascular changes due to air embolism-reperfusion in the cat were investigated. Air embolism-reperfusion was produced in the cerebral microvessels by an intra-carotid injection of air (0.2-0.3 ml). Air emboli in the cerebral arterioles were observed continuously from the air injection to reperfusion using fluorescence videomicroscopy. Arteriolar diameter was measured based on video images of arterioles filled with rhodamine-B isothiocyanate dextran, and red cell velocity was measured using a dual window technique with FITC-labeled red cells. Air bubbles ceased to move in the arterioles of 20-70 microm diameter and blood flow was almost stopped in distal arterioles. The air emboli were of cylindrical shape in the arterioles, with hemispherical end caps. The emboli progressed slowly at rates of 7-73 microm/sec and then flowed away. The air emboli induced ischemia-reperfusion with the ischemic duration of approximately 1 minute (10 sec-3 min) at the arteriolar level. Cerebral arterioles began to dilate after the formation of the air emboli and significantly dilated by approximately 50% after reperfusion. A transient increase in red cell velocity was observed after reperfusion. Arteriolar dilation and the increase in velocity led to a hyperemic response in arteriolar flow to ischemia-reperfusion.

The effect of long-term supplementation of vitamin C on leukocyte adhesion to the cerebral endothelium in STZ-induced diabetic rats.

The effect of long-term supplementation of vitamin C on leukocyte adhesion to the cerebral endothelium was investigated in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar Furth rats by intravenous injection of STZ. The vitamin C, ascorbic acid, was supplemented with drinking water (1 g/l). The rats were divided into control and diabetic groups without or with supplementation of vitamin C. The cerebral microcirculation was directly observed through a cranial window after different periods (12, 24 and 36 weeks) of vitamin C supplementation, using fluorescence videomicroscopy. Leukocyte adhesion to the venular endothelium was examined by labeling leukocytes with rhodamin 6G. The number density of adherent leukocytes in STZ-diabetic rats was increased significantly, compared with control rats. This increase in leukocyte adhesion was prevented by the long-term supplemented vitamin C. It was suggested that the antioxidant effect of vitamin C might be responsible for the prevention of leukocyte adhesion in diabetes mellitus.

Cerebral microcirculatory changes in rat with a cardiopulmonary bypass using fluorescence videomicroscopy.

Cerebral microcirculatory changes in rat with a cardiopulmonary bypass (CPB) at normothermia was investigated in relation to cerebrovascular disorders caused by surgical operation with CPB. The mean arterial pressure was changed from 50 to 200 mmHg by changing the pump flow-rate. A non-pulsatile flow model was developed by stopping the cardiac beat using a fibrillator. The pial microcirculation was visualized using fluorescence-labeled red cells and dextran, and was directly observed under a fluorescence videomicroscope during CPB. Based on the recorded videoimages, the arteriolar diameter and red cell velocity were measured, in which single arterioles with approximately 40 microm diameter were selected among the pial arterioles. It was shown that when the arterial pressure was changed: (1) arteriolar vasodilation or constriction appeared during pulsatile flow but it disappeared during non-pulsatile flow, and (2) the arteriolar red cell velocity increased or decreased linearly during non-pulsatile flow as well as pulsatile flow. The flow-rate was almost constant at a large range of the mean arterial pressure from 60 to 160 mmHg during pulsatile flow (autoregulation), but it increased or decreased during non-pulsatile flow with an increase or decrease in mean arterial pressure, respectively. It was suggested that pulsativity might be responsible for cerebral autoregulation.

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.

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.