Perfused Carotid Arteries Research Articles

Role of platelet CD40 ligand for endothelial cell-monocyte interaction in the presence of flow

CD40 ligand (CD154)-induced ultra-large von Willebrand factor (vWF) multimer-mediated endothelial cell-platelet-monocyte interaction may play an important role in adaptive and maladaptive vascular remodeling processes. Here we analyzed the impact of and conditions favouring the deposition of these multimers on the endothelial cell (EC) surface by way of CD40-CD154 co-stimulation in settings mimicking different forms of blood flow. Upon exposure to low oscillatory shear stress and sCD154, a release of vWF multimers comparable to histamine stimulation was monitored on the EC surface in a string-like fashion. Moreover, ex vivo perfused carotid arteries of wild type mice at low laminar shear stress rates showed a luminal release of vWF as ultra-large vWF multimers (ULVWF) upon stimulation with sCD154 which was absent in blood vessels of CD40 knockout mice. The observed CD40- and flow-dependent vWF release from intact endothelial cells and subsequent vWF multimer formation may facilitate adhesion and subsequent activation of circulating platelets at atherosclerotic predilection sites, which are characterized by disturbed flow patterns. This in turn may amplify endothelial cell-monocyte interaction, thus possibly initiating or promoting early atherosclerotic lesion formation.

P-Selectin Glycoprotein Ligand-1 Is Highly Expressed on Ly-6C hi Monocytes and a Major Determinant for Ly-6C hi Monocyte Recruitment to Sites of Atherosclerosis in Mice

Ly-6C(hi) monocytes are key contributors to atherosclerosis in mice. However, the manner in which Ly-6C(hi) monocytes selectively accumulate in atherosclerotic lesions is largely unknown. Monocyte homing to sites of atherosclerosis is primarily initiated by rolling on P- and E-selectin expressed on endothelium. We hypothesize that P-selectin glycoprotein ligand-1 (PSGL-1), the common ligand of P- and E-selectin on leukocytes, contributes to the preferential homing of Ly-6C(hi) monocytes to atherosclerotic lesions. To test this hypothesis, we examined the expression and function of PSGL-1 on Ly-6C(hi) and Ly-6C(lo) monocytes from wild-type mice, ApoE(-/-) mice, and mice lacking both ApoE and PSGL-1 genes (ApoE(-/-)/PSGL-1(-/-)). We found that Ly-6C(hi) monocytes expressed a higher level of PSGL-1 and had enhanced binding to fluid-phase P- and E-selectin compared with Ly-6C(lo) monocytes. Under in vitro flow conditions, more Ly-6C(hi) monocytes rolled on P-, E-, and L-selectin at slower velocities than Ly-6C(lo) cells. In an ex vivo perfused carotid artery model, Ly-6C(hi) monocytes interacted preferentially with atherosclerotic endothelium compared with Ly-6C(lo) monocytes in a PSGL-1-dependent manner. In vivo, ApoE(-/-) mice lacking PSGL-1 had impaired Ly-6C(hi) monocyte recruitment to atherosclerotic lesions. Moreover, ApoE(-/-)/PSGL-1(-/-) mice exhibited significantly reduced monocyte infiltration in wire injury-induced neointima and in atherosclerotic lesions. ApoE(-/-)/PSGL-1(-/-) mice also developed smaller neointima and atherosclerotic plaques. These data indicate that PSGL-1 is a new marker for Ly-6C(hi) monocytes and a major determinant for Ly-6C(hi) cell recruitment to sites of atherosclerosis in mice.

MICROVASCULAR ANASTOMOSIS TRAINING MODEL BASED ON A TURKEY NECK WITH PERFUSED ARTERIES

The microvascular anastomosis (MA) technique remains an important aspect of modern neurosurgical practice. Specialized training is essential for mastering the anastomosis of small vessels. To minimize animal use and to create a simple but realistic training model, we developed a pulsatile perfused arteries model based on turkey carotid arteries for microvascular anastomosis training. Approximately 10-cm-long segments of turkey necks were used as a material to set up this model. The diameter of turkey carotid arteries at the neck level was found to be between 1 and 3 mm. Both carotid arteries on the neck segment were cannulated and incorporated into a closed circulation circuit consisting of silicone tubes and an infusion pump that provided continuous arterial-like flow and pulsation during the MA training. Tubing connection with both proximal and distal ends of the carotid arteries allowed circulatory patency during vessel clamping and anastomosis creation. Different fluids ranging from simple water to various colored solutions or even whole blood could be used for vascular perfusion in this experimental setup. Fluid output was maintained between 11 and 16 ml/min. Minimum pressure was set at 80 cm/H20, and maximum pressure was set at 160 cm/H20. Mean fluid flow through the arteries was 8 to 13 ml/min. End-to-end, end-to-side, and side-to-side MA may be performed on this model. This training model provides several advantages: the materials are easily obtainable and inexpensive, pulsatile flow through the arteries is realistic, the model is easy to manage, and there is no need for special facilities for animal care or anesthesia that are required for live animals. The diameter and structure of turkey carotid arteries are essentially identical to the human cortical vessels and distal branches of the superficial temporal artery. The pulsatile perfused carotid arteries model presents a realistic MA training model. It provides an inexpensive and simple setup for educational purposes. This technique can minimize live animal and anesthesia needs for MA training. Wide use of this model may enhance and popularize MA training and education.

Targeted disruption of cd73/ecto-5'-nucleotidase alters thromboregulation and augments vascular inflammatory response.

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5'-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (-14%; P<0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl3) was significantly reduced by 20% in cd73-/- mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73-/- mice (-35%). In vivo platelet cAMP levels were 0.96+/-0.46 in WT versus 0.68+/-0.27 pmol/106 cells in cd73-/- mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 micromol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73-/- compared with WT littermates (WT 98% versus cd73-/- 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73-/- mice (P<0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.

Apolipoprotein E mediates the retention of high-density lipoproteins by mouse carotid arteries and cultured arterial smooth muscle cell extracellular matrices.

Lipoprotein retention in the vascular extracellular matrix (ECM) plays a critical role in atherogenesis. Previous studies demonstrated the presence of apo A-I and E in atherosclerotic lesions, suggesting that HDL may be trapped by the artery wall. We sought to determine mechanisms by which HDL could be bound and retained by the arterial wall, and whether apo E was a principal determinant of this binding. We evaluated in situ accumulation of fluorescently labeled DiI-human HDL+/-apo E in perfused carotid arteries from apo E-null mice. Apo E was important in mediating HDL binding to the vascular wall, with a 48+/-16% increase in accumulation of DiI-labeled apo E-containing HDL (HDL3+E) compared with DiI-apo E-free HDL (HDL3-E) (P=0.003). To investigate possible mechanisms responsible for retention, we assessed binding of unlabeled HDL3-E and HDL3+E to ECM generated by cultured arterial smooth muscle cells. Similar to the in situ carotid artery data, HDL3+E bound better to the ECM than did HDL3-E (3-fold lower K(a) and 3.5-fold higher B(max) for HDL3+E versus HDL3-E). These differences were eliminated after either neutralization of arginine residues on apo E or digestion of matrix with chondroitin ABC lyase, suggesting that chondroitin and/or dermatan sulfate proteoglycans were responsible for apo E-mediated increased binding. These findings demonstrate that HDL can bind to both intact murine carotid arteries and smooth muscle cell-derived ECM, and that apo E is a principal determinant in mediating the ability of HDL to be trapped and retained via its interaction with ECM proteoglycans.

Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice.

- Flow-dependent dilation is a fundamental mechanism by which large arteries ensure appropriate blood supply to tissues. We investigated whether or not the vascular kallikrein-kinin system, especially tissue kallikrein (TK), contributes to flow-dependent dilation by comparing wild-type and TK-knockout mice in which the presence or absence of TK expression was verified. We examined in vitro changes in the outer diameter of perfused carotid arteries from TK(+/+) and TK(-/-) mice. In both groups, exogenous bradykinin caused a similar dilation that was abolished by the B(2) receptor antagonist HOE-140, as well as by the NO synthase inhibitor N:(omega)-nitro-L-arginine methyl ester. However, purified kininogen dilated only TK(+/+) arteries, demonstrating the essential role of TK in the vascular formation of kinins. In TK(+/+) arteries, increasing intraluminal flow caused a larger endothelium-dependent dilation than that seen in TK(-/-). In both strains the flow response was mediated by NO and by endothelium-derived hyperpolarizing factor, whereas in TK(-/-) vasoconstrictor prostanoids participated as well. HOE-140 impaired flow-dependent dilation in TK(+/+) arteries while showing no effect in TK(-/-). This compound reduced the flow response in TK(+/+) arteries to a level similar to that in TK(-/-). After NO synthase inhibition, HOE-140 no longer affected the response of TK(+/+). Impaired flow-dependent dilation was also observed in arteries from knockout mice lacking bradykinin B(2) receptors as compared with wild-type animals. This study demonstrates the active contribution of the vascular kallikrein-kinin system to one-third of the flow-dependent dilation response via activation of B(2) receptors coupled to endothelial NO release.

Effect of atherosclerosis on responses of the perfused rabbit carotid artery to human platelets.

Vascular responses to intraluminal and abluminal activation of human platelets were examined in carotid arteries from normal and atherosclerotic rabbits. The carotid artery was perfused in vitro, platelets were activated with thrombin (0.1 unit/ml), and changes in diameter were measured. In vessels from normal animals, intraluminal activation of platelets produced dilatation of preconstricted arteries. The dilator response was attenuated by N omega-nitro-L-arginine (10(-5) M), an inhibitor of synthesis of endothelium-derived relaxing factor-nitric oxide (EDRF-NO), and augmented by LY53,857 (10(-5) M), a 5-HT2-serotonergic antagonist. Abluminal activation of platelets produced modest constriction in quiescent arteries, which was inhibited by LY53,857. Intraluminal but not abluminal ADP produced pronounced dilatation of preconstricted arteries. In vessels from atherosclerotic animals, endothelium-dependent dilatation to intraluminal activation of platelets and to ADP was impaired and dilator responses to sodium nitroprusside were normal. These experiments indicate that 1) intraluminal activation of human platelets produces endothelium-dependent dilatation in perfused carotid arteries, whereas abluminal activation of human platelets produces vasoconstriction, which is mediated primarily by serotonin, and 2) atherosclerosis markedly impairs vasodilator responses to activation of human platelets, probably because vasodilatation to ADP released from platelets is impaired.

Vascular contraction in perfused carotid arteries of cholesterol-fed rabbits

The purpose of the present study was to investigate the effects of hypercholesterolemia on sympathetic vascular responsiveness in the perfused rabbit carotid artery. Two groups of rabbit carotid arteries were evaluated for the simultaneous measurement of noradrenaline (NA) release and vasoconstrictor response induced by electric nerve stimulation and for exogenous NA-induced vasocontraction in vitro. One group of rabbits was fed a diet containing 0.5% cholesterol for 2 weeks and the other group was fed standard rabbit chow. By scanning electron microscopy, monocytes adhering to the endothelial cells and penetrating into the subendothelium were observed. Neither endothelial denudation nor platelet adhesion could be detected. Rabbit carotid arteries were cannulated and perfused with a physiological solution at a constant flow rate. The vessels were subjected to both transmural field stimulation (TFS; 1.5–24 Hz) and exogenous NA administration. TFS caused a frequency-dependent increase in endogenous NA release with subsequent pressor responses in both groups. Exogenous NA also induced a dose-dependent pressor response, but a significant reduction was observed in the cholesterol-fed group. Methoxamine induced a similar response in both groups. It was concluded that hypercholesterolemia decreased the sensitivity of extrajunctional α-receptors in the perfused rabbit carotid artery.

P-131 - Vascular contraction in perfused carotid arteries of cholesterol-fed rabbits.

The purpose of the present study was to investigate the effects of hypercholesterolemia on sympathetic vascular responsiveness in the perfused rabbit carotid artery. Two groups of rabbit carotid arteries were evaluated for the simultaneous measurement of noradrenaline (NA) release and vasoconstrictor response induced by electric nerve stimulation and for exogenous NA-induced vasoconstriction in vitro. One group of rabbits was fed a diet containing 0.5% cholesterol for 2 weeks and the other group was fed standard rabbit chow. By scanning electron microscopy, monocytes adhering to the endothelial cells and penetrating into the subendothelium were observed. Neither endothelial denudation nor platelet adhesion could be detected. Rabbit carotid arteries were cannulated and perfused with a physiological solution at a constant flow rate. The vessels were subjected to both transmural field stimulation (TFS; 1.5-24 Hz) and exogenous NA administration. TFS caused a frequency-dependent increase in endogenous NA release with subsequent pressor responses in both groups. Exogenous NA also induced a dose-dependent pressor response, but a significant reduction was observed in the cholesterol-fed group. Methoxamine induced a similar response in both groups. It was concluded that hypercholesterolemia decreased the sensitivity of extrajunctional alpha-receptors in the perfused rabbit carotid artery.

A new method for simultaneous measurement of endogenous noradrenaline release and vascular responsiveness in perfused rabbit carotid arteries in vitro

1. A new preparation for large arteries was tested for the simultaneous measurement of noradrenaline release and vasoconstrictor response induced by nerve stimulation in vitro. 2. Preparations of rabbit carotid artery with an outflow cannula were perfused with a modified Krebs-Ringer bicarbonate solution containing propranolol (3 x 10(-6) M) and cocaine (10(-6) M), to block beta-adrenoceptors and uptake-1, respectively. 3. The perfused carotid arteries were subjected to both transmural field stimulations (TFS) and exogenous noradrenaline administrations. To evaluate the preparation, we carried out the procedures in two control groups and a group with yohimbine (10(-7) M) treatment, respectively. 4. TFS over the range of 1.5-24 Hz caused a frequency-dependent pressor response. Yohimbine significantly increased the TFS-induced pressor responses. 5. TFS also caused frequency-dependent endogenous noradrenaline release. The endogenous noradrenaline release was significantly enhanced by yohimbine treatment. When noradrenaline release was expressed as release per stimulus, it was frequency-dependent in the group with yohimbine treatment but not in control groups. 6. Exogenous noradrenaline induced a dose-dependent pressor response. The exogenous noradrenaline-induced vasoconstrictor response was significantly diminished by yohimbine treatment. 7. It is concluded that this preparation can be effectively used to study drug effects on noradrenergic neurotransmission in relatively large arteries under various pathophysiological states by simultaneously measuring catecholamine release and vascular responsiveness.

Anti-EDRF effect of tumor necrosis factor in isolated, perfused cat carotid arteries.

Cat carotid arteries that have an intact endothelium were isolated and perfused with Krebs-Henseleit solution containing recombinant human tumor necrosis factor (rhTNF). Perfused arteries were preconstricted with KCl and then dilated with acetylcholine (ACh) or acidified NaNO2. After perfusion with TNF (4 micrograms/ml) for 120 min, the ACh-induced vasodilator response was markedly blunted, but the NaNO2 vasodilator response was not significantly affected. Arteries perfused with 2 micrograms/ml TNF for 60-120 min or with 4 micrograms/ml for 60 min did not develop a significantly impaired relaxation to ACh. Moreover, perfusion with 20-100 micrograms/ml cycloheximide, an inhibitor of protein synthesis, blocked the TNF-induced impairment of the relaxation to ACh. On the other hand, the vasodilator response to acidified NaNO2 did not change in any perfused carotid arteries. These results suggest that TNF promotes the synthesis of proteins that contribute to the damage of endothelial cells directly, probably by inhibiting endothelium-derived relaxing factor release.

Inhibition of cholesterol-induced increases in arterial wall permeability by propranolol

Increased arterial permeability to [ 125I]albumin is one of the earliest abnormalities found in the cholesterol-fed rabbit model for atherosclerosis and precedes morphologic changes in the arterial wall. Since propranolol is reported to retard atherogenesis in this model, the current study was undertaken to study the effect of propranolol on arterial permeability to albumin in cholesterol-fed rabbits. Carotid permeability to [ 125I]albumin was measured after 1 week of (a) a control diet containing only trace cholesterol ( N = 9), (b) a 1.5% cholesterol diet ( N = 11), or (c) a 1.5% cholesterol diet plus intraperitoneal propranolol 5 mg/kg/day ( N = 19). The carotid artery was cannulated proximally and distally and peristaltically perfused with oxygenated Modified Eagle's Medium to which [ 125I]albumin had been added. The permeability of the carotid artery to albumin was estimated by measuring 125I activity in peripheral venous samples obtained after 0, 15, 30, 60, 120, 180, and 240 min of carotid perfusion. 125I-radioactivity of the perfused carotid artery was also counted at the end of the experiment to measure 125I retained in the arterial wall. The transfer of [ 125I]albumin across the arterial wall to the venous circulation was greater in the untreated cholesterol-fed group than in controls ( P < 0.05), but cholesterol-fed animals treated with propranolol did not differ significantly from controls. 125I-albumin in the arterial wall after 240 min of perfusion was decreased in both untreated cholesterol-fed animals (1444 dpm/cm ± 350) and in propranolol-treated animals (1629 ± 309) when these were compared to controls (3909 ± 693, P < .05). These data suggest that cholesterol feeding increases both the penetration of albumin into the arterial wall and its rate of clearance from the wall to the venous circulation. Furthermore, treatment with propranolol appears to prevent this increase in trans-carotid albumin flux by preventing increases in both penetration and clearance. These findings further support the hypothesis that propranolol's ability to retard atherogenesis is mediated by a direct effect on the arterial wall.