Effects of Fluvastatin on Leukocyte–Endothelial Cell Adhesion in Hypercholesterolemic Rats
Abstract The overall objective of this study was to determine whether peroral treatment with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor fluvastatin influences the leukocyte–endothelial cell adhesion (LECA) observed in postcapillary venules of hypercholesterolemic rats. Rats were fed either normal chow or a chow supplemented with 1% cholesterol for 10 days. Leukocyte adherence and extravasation, leukocyte rolling velocity, red blood cell velocity, and vessel diameter were monitored in mesenteric venules superfused with either 100 nmol/L platelet-activating factor (PAF) or 20 nmol/L leukotriene B4 (LTB4). Hypercholesterolemic rats exhibited an exaggerated LECA response compared with their normocholesterolemic counterparts. In hypercholesterolemic rats, treatment with fluvastatin significantly attenuated the leukocyte-adherence responses to PAF and LTB4 as well as the leukocyte emigration response to LTB4. Fluvastatin treatment also inhibited the PAF- and LTB4-induced reductions in leukocyte rolling velocity. These findings indicate that fluvastatin blunts the inflammatory responses elicited in postcapillary venules by lipid mediators.
- Received April 12, 1996.
- Accepted November 5, 1996.
Atherogenesis is a complex disease process that is associated with endothelial cell dysfunction and/or injury in both large and microscopic blood vessels.1 2 There is a growing body of evidence that invokes a role for leukocyte–endothelial cell interactions in the pathobiology of atherosclerosis. Indeed, the adherence of circulating monocytes and lymphocytes to the endothelial lining of large arteries is one of the earliest detectable events in human1 and experimental atherosclerosis.1 3 4 5 It has been proposed that the subsequent transendothelial migration of these adherent leukocytes and their accumulation in the intima and transformation into lipid-engorged “foam cells” results in the formation of atherosclerotic plaques.1 A role for endothelial cells in the initiation of this process is supported by numerous reports of an increased expression of adhesion molecules on endothelial cells of atherosclerotic vessels.6
Several experimental strategies have been employed to simulate the leukocyte–endothelial cell interactions of atherosclerosis in experimental animals, including intravascular administration of oxidized LDLs7 and placement of animals on high-cholesterol diets.8 It has been shown that isolated rabbit coronary arteries9 and intact rat mesenteric venules10 sustain a higher level of neutrophil–endothelial cell adhesion in hypercholesterolemic animals compared with their normocholesterolemic counterparts. The results of these studies also suggest that the increased cell-to-cell adhesion results from an increased expression of adhesion molecules (eg, P-selectin and ICAM-1) on the surface of endothelial cells and that a decreased basal release of NO from endothelial cells accounts for the increased LECA.7 8 It was proposed that correction of the hypercholesterolemia-induced alteration in NO bioavailability with NO-donating compounds should prove effective in reducing the neutrophil adhesion and consequent dysfunction of large and small vessels that is associated with atherosclerosis.
Studies of neutrophil adherence to coronary artery endothelium in hypercholesterolemic animals have revealed that administration of the HMG-CoA reductase inhibitor lovastatin profoundly reduces the hyperadhesiveness of neutrophils that is normally observed in these vessels.7 Furthermore, the HMG-CoA reductase inhibitor attenuated the reduced basal NO production by endothelial cells in hypercholesterolemic coronary arteries, thereby supporting the view that an underlying cause of the increased leukocyte adhesion in hypercholesterolemia is an altered NO bioavailability.9 While these observations suggest that HMG-CoA reductase inhibitors are effective in blunting the inflammatory responses that occur in large blood vessels of animals placed on a high-cholesterol diet, it remains unclear whether these inhibitors exert a similar protective action on the LECA that occurs in postcapillary venules of hypercholesterolemic animals. Furthermore, it is not clear whether the beneficial action of an HMG-CoA reductase inhibitor on the exaggerated inflammatory responses observed in animals on a high-cholesterol diet is directly related to its ability to lower plasma cholesterol. Hence, the overall objective of the present study was to determine whether peroral treatment of hypercholesterolemic rats with fluvastatin (6 mg/kg for 10 days), an inhibitor of HMG-CoA reductase,11 alters the LECA elicited in postcapillary venules exposed to either PAF or LTB4.
Male Sprague-Dawley rats (125 to 149 g) were maintained on a purified laboratory diet. Rats were then separated into two treatment groups: a control group that received a standard laboratory diet and a cholesterol-fed group that received a 1% cholesterol-enriched diet (Oriental Kobo Co) supplemented with 0.2% cholic acid (which increases cholesterol absorption) and 2.5% olive oil as dietary fat for 10 days before the experiment. Some of the high-cholesterol–fed animals were perorally treated for 10 days with fluvastatin sodium (Sandoz Research Institute) or pravastatin sodium (another HMG-CoA reductase inhibitor extracted from tablets of Mevalotin by Sandoz Yakuhin) at a dose of 6 mg/kg once daily. In control animals, the same volume of distilled water was administered perorally.
Rats were initially anesthetized with an intraperitoneal injection of thiobutabarbital (Research Biochemicals International) at a dose of 120 mg/kg body weight, and a tracheotomy was performed to facilitate breathing. The right carotid artery was cannulated and systemic arterial pressure was measured with a pressure transducer (Statham) connected to the carotid artery cannula. Systemic blood pressure and heart rate were continuously recorded with a Grass physiological recorder (Grass Instruments). A middle abdominal incision was made, and a segment of the ileum was exteriorized through the incision. All exposed tissue was moistened with saline-soaked gauze to minimize evaporation and tissue dehydration.
Rats were placed in a right lateral recumbent position on an adjustable Plexiglas acrylic plastic microscope stage and the mesentery was prepared for microscopic observation as described previously.12 The mesentery was draped over an optically clear viewing glass that allowed for examination of a 2-cm2 segment of tissue. The temperature of the pedestal was maintained at 37°C with a constant temperature circulator (Fisher Scientific, model 80). Rectal temperature was monitored using an electrothermometer. The exposed bowel wall and mesentery were covered with Saran Wrap (Dow Chemical Co), and the mesentery was superfused with warmed bicarbonate-buffered saline (pH 7.4) that was bubbled with a mixture of 5% CO2, 95% N2.
Venules were observed through an intravital videomicroscope (Leitz, Ortholux II). The mesentery was transilluminated, and a video camera (Hitachi, VK-C150) mounted on the microscope projected the image onto a color monitor (Sony, PVM-2030). The images were recorded with a videocassette recorder for playback analysis.
Single unbranched venules with diameters ranging between 25 and 35 μm were selected for study. Venular diameter (D) was measured by using a video caliper (Microcirculation Research Institute), and red blood cell centerline velocity was measured by using an optical Doppler velocimeter (Microcirculation Research Institute). Venular blood flow was calculated as the product of mean red blood cell velocity (Vmean=centerline velocity/1.6)13 and venular cross-sectional area (assuming cylindrical geometry). The number of adherent leukocytes was determined off-line during playback (10 minutes) of videotaped images. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for ≥30 seconds.12 Adherent cells were determined as the number per 100-μm length of venule for 10 minutes. The number of emigrated leukocytes was determined off-line during playback of videotaped images. Leukocytes that were clearly visible outside the venule were counted as emigrated cells. Leukocyte emigration was expressed as the change in the number per microscopic field (2.2×104 μm2). Rolling leukocytes were defined as white blood cells that moved at a velocity less than that of red blood cells in the same vessel. Leukocyte rolling velocity (VRL) was determined as the time required for a leukocyte to transverse a given length of venule. The flux of rolling leukocytes (FRL) was determined as the number of cells rolling past a fixed point in the vessel per minute. The number of rolling leukocytes within the vessel at any given moment was determined by dividing FRL by VRL.14 The venular wall shear rate (γ) was calculated by using the Newtonian definition, γ=8(Vmean/D).15
Serum Total Cholesterol and Nitrate/Nitrite Levels
A 0.5-mL blood sample was collected via the carotid artery. Total serum cholesterol concentration was enzymatically measured by using a commercial kit (Sigma Chemical Company). Whole-animal NO production was assessed by measurements of serum nitrite/nitrate concentration.16 Serum levels of nitrite and nitrate were determined by a modification of the method of Green et al.17 Briefly, 400 μL of water was added to 100 μL of plasma. Protein was precipitated by the addition of 25 μL 30% ZnSO4. Supernatants (500 μL) were incubated with E coli, HEPES buffer (pH 7.4), and NH4 formate (pH 7.4) for 60 minutes at 37°C to reduce nitrate to nitrite. Nitrite was then quantified by using the Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% H3PO4) to a 500-μL supernatant of the sample. Nitrite concentrations were calculated from a standard curve, using sodium nitrite (Sigma) as the standard.
After all parameters that were measured on-line (arterial pressure, erythrocyte velocity, and venular diameter) were in a steady state, images from the mesenteric preparation were recorded on videotape for 10 minutes. If there were ≥6 adherent leukocytes and/or ≥8 emigrated leukocytes over a 10-minute observation period under basal conditions, the experiment was discarded. The mesentery was superfused with either 100 nmol/L PAF (Sigma) or 20 nmol/L LTB4 (Calbiochem), dissolved in bicarbonate-buffered saline, for 30 minutes, with video recordings and repeat measurements of all parameters made during the final 10 minutes of superfusion. In control experiments, the same protocol was employed, except that bicarbonate-buffered saline was used as the perfusate for the entire procedure.
All data were analyzed using standard statistical analyses, ie, ANOVA with Scheffé’s posthoc test. All values are expressed as mean±SE, and statistical significance was set at P<.05.
Table 1⇓ summarizes the serum cholesterol and nitrate/nitrite concentrations and systemic blood pressure measured in rats placed on normal chow or a high-cholesterol diet (10 days), with and without peroral fluvastatin. Blood pressure did not differ among the three experimental groups. Serum cholesterol level was more than three times higher in rats placed on a high-cholesterol diet. Fluvastatin treatment (6 mg · kg−1 · d−1 for 10 days) did not alter the serum cholesterol levels. While there was a tendency for serum nitrate/nitrite levels to fall in hypercholesterolemic rats, this response was not statistically significant. Fluvastatin did not alter serum nitrate/nitrite levels in hypercholesterolemic rats.
Table 2⇓ compares the basal values for different inflammatory and microhemodynamic variables in mesenteric venules of control and hypercholesterolemic rats with and without fluvastatin treatment. Although blood flow, mean erythrocyte velocity, and wall shear rate did not differ among the three groups, the number of adherent and rolling leukocytes, as well as the flux of rolling leukocytes, was elevated, while leukocyte rolling velocity was reduced in venules of hypercholesterolemic animals compared with controls. The number of emigrated leukocytes did not differ between the two groups. Fluvastatin treatment in hypercholesterolemic rats reduced the number of adherent leukocytes and restored leukocyte rolling velocity to normal. In a preliminary study, it was shown that fluvastatin in a single application of high concentration suppressed PAF- or LTB4-stimulated (but not basal values of) leukocyte–endothelial cell interactions, as seen in the present study with chronic treatment.
In rats fed either normal chow or a high-cholesterol diet, superfusion of the mesentery with either PAF or LTB4 resulted in the recruitment of adherent, emigrated, and rolling leukocytes, while not altering venular hemodynamics (blood flow, shear rate, and red blood cell velocity). Although both inflammatory mediators tended to elicit larger inflammatory responses (recruitment of adherent, emigrated, and rolling leukocytes) in venules of hypercholesterolemic (versus control) animals, these differences were not statistically significant.
Fig 1⇓ illustrates that the recruitment of firmly adherent leukocytes elicited by either PAF or LTB4 in hypercholesterolemic rats was significantly attenuated by treatment with fluvastatin. Figs 2⇓ and 3⇓ demonstrate that the recruitment of emigrated leukocytes and the reduction of leukocyte rolling velocity induced by either PAF or LTB4 were blunted in hypercholesterolemic rats treated with fluvastatin. Although a trend exists, fluvastatin did not have a significant effect on PAF-induced emigration. The protective actions of fluvastatin on leukocyte recruitment in hypercholesterolemic rats was not accompanied by changes in venular hemodynamics, ie, venular blood flow, red blood cell velocity, and shear rate were unaffected by fluvastatin.
Table 3⇓ illustrates that pravastatin, another HMG-CoA reductase inhibitor, did not affect the recruitment of adherent and emigrated leukocytes and the reduction in leukocyte rolling velocity normally elicited by PAF and LTB4.
There is a growing body of evidence that implicates leukocytes in the abnormal vascular function associated with hypercholesterolemia9 10 and atherogenesis.7 While much emphasis has been given to the role of monocytes in the initiation and development of atherosclerotic lesions,1 3 4 5 recent studies have described an accelerated recruitment of granulocytes to the endothelial cell surface of large and small blood vessels in the early stages of experimental hypercholesterolemia.10 Furthermore, it has been demonstrated that interventions (eg, monoclonal antibodies against leukocyte adhesion molecules or NO donors) that interfere with the recruitment of leukocytes help to maintain vessel function and endothelial barrier integrity in hypercholesterolemic blood vessels.10 The results of the present study provide support for a beneficial action of the HMG-CoA reductase inhibitor fluvastatin in blunting the recruitment of leukocytes into lipid mediator–stimulated postcapillary venules of hypercholesterolemic rats. Our data also indicate that the protective action of this agent is independent of the serum cholesterol–lowering effect observed after prolonged treatment in other species. Hence, our findings suggest that fluvastatin therapy may represent a novel approach for the prevention or attenuation of the leukocyte–endothelial cell interactions that appear to contribute to the development of atherosclerosis.
As a species, rats (eg, compared with hamsters) are known to be tolerant to the lipid-lowering effects of HMG-CoA reductase inhibitors. The expected clinical dose of fluvastatin is 0.5 to 1.0 mg/kg, which is 6 to 12 times lower than the dosage used to inhibit LECA in our study. However, previous reports on pravastatin actions in WHHL rabbits have employed doses as high as 50 mg/kg.18 While we cannot exclude a nonspecific action of the 6-mg/kg dose of fluvastatin, the lack of effect of this dose on basal LECA and venular hemodynamics would argue against significant nonspecific actions of this concentration in rats.
The results of the present study indicate that placement of rats on a high-cholesterol diet for a period of days results in a threefold increase in serum cholesterol levels, with an accompanying recruitment of rolling and emigrated leukocytes in unstimulated mesenteric venules (Tables 1⇑ and 2⇑). These observations are consistent with the findings of a recent report by Gauthier and associates10 wherein it was shown that rats placed on a high-cholesterol diet for 2 weeks exhibit a marked increase in the percentage of postcapillary venules that express the endothelial cell adhesion molecules P-selectin and ICAM-1. P-Selectin expression on endothelial cells is generally associated with an increased recruitment of rolling leukocytes, while ICAM-1 expression favors the emigration of leukocytes across microvascular endothelium.19
In addition to the inhibitory effects of fluvastatin on LECA in inflamed venules, chronic treatment of hypercholesterolemic rats with fluvastatin lowered the basal level of adherent leukocytes, with an improved leukocyte rolling velocity. Inasmuch as fluvastatin does not alter basal leukocyte adhesion in normocholesterolemic (on normal chow) rats (unpublished observations), it may be proposed that the actions of this agent are related to plasma cholesterol levels. However, this possibility seems unlikely, since no changes in serum cholesterol and lipids were observed in hypercholesterolemic rats treated with fluvastatin. In a recent study, it was demonstrated that serum lipid peroxides in hypercholesterolemic rabbits were markedly reduced by fluvastatin.20 These observations suggest that the beneficial action of fluvastatin on leukocyte adhesion may be related to a corresponding antioxidant effect of the drug.
The mechanism(s) that underlies the enhanced recruitment of rolling leukocytes in hypercholesterolemic microvessels has not been defined. A diminished capacity of endothelial cells to produce NO has been invoked to explain the altered endothelium-dependent vasorelaxation observed in hypercholesterolemic microvessels.20 Inasmuch as inhibition of NO synthase activity in postcapillary venules promotes the recruitment of leukocytes and causes a corresponding increase in the expression of P-selectin,21 it is conceivable that a reduced bioavailability of NO accounts for the inflammatory responses that we observed in unstimulated venules of hypercholesterolemic rats. The tendency for serum nitrate/nitrite levels to fall in our hypercholesterolemic rats supports this possibility.
An interesting and important observation in our study is that orally administered fluvastatin significantly attenuates the leukocyte adherence and consequent emigration across venules that is elicited by PAF in hypercholesterolemic rats. Fluvastatin treatment also blunted the LECA interactions that result from superfusion of mesenteric venules of hypercholesterolemic animals with LTB4. The ability of fluvastatin to interfere with both PAF- and LTB4-induced inflammatory responses suggests that the HMG-CoA reductase inhibitor does not exert its inhibitory action by interacting with a specific receptor. Other potential mechanisms of action of fluvastatin that appear unlikely, on the basis of our data, are a serum cholesterol–lowering effect and/or an improvement of venular perfusion. It has previously been shown that rat serum cholesterol levels are unresponsive to the cholesterol-lowering effect of HMG-CoA reductase inhibitors.22
There are three possible modes of action of fluvastatin that could account for its antiinflammatory effects in hypercholesterolemic venules: (1) inhibition of leukocyte and/or endothelial cell adhesion molecule expression, (2) attenuation of oxygen radical formation and the resultant lipid peroxidation, and (3) prevention of formation of a proinflammatory product of cholesterol metabolism (eg, mevalonic acid). The results of a preliminary in vitro study indicate that fluvastatin acts on a human monocyte cell line (U937) to suppress the expression of lymphocyte-function associated antigen-1, without affecting the expression of ICAM-1 and vascular adhesion molecule-1, on cultured human endothelial cells. Since antioxidants such as probucol have been shown to slow the progression of atherosclerotic lesion formation in hypercholesterolemic animals24 and because lipid peroxidation products can promote LECA,25 it is possible that fluvastatin acts to attenuate the enhanced peroxidation of lipoproteins and cell membranes that accompanies high serum cholesterol levels. In the present study, fluvastatin treatment attenuated PAF- and LTB4-induced LECA, but pravastatin treatment did not. It has been demonstrated that fluvastatin and pravastatin exert different effects on smooth muscle proliferation. Fluvastatin inhibits the proliferation of vascular smooth muscle cells in vitro26 and in vivo,27 but pravastatin does not. Furthermore, fluvastatin has been shown to inhibit intimal thickening after catheterization-induced injury, an effect that has been attributed to reduced migration and proliferation of smooth muscle cells rather than a serum lipid-lowering action. Pravastatin did not show the same inhibitory effects.28 Thus, it appears unlikely that the difference in the antiatherosclerotic effect of the two enzyme inhibitors can be explained in terms of lipid-lowering effectiveness. Pravastatin inhibits HMG-CoA reductase in the liver to a greater extent than in other organs, and it readily permeates hepatocytes.29 If fluvastatin, with a greater lipophilicity than pravastatin, is more permeable to smooth muscle membranes, then this factor may explain the differential antiatherosclerotic effects of the two HMG-CoA reductase inhibitors. Finally, mevalonate has been shown to reverse some of the biological actions of HMG-CoA reductase inhibitors, particularly processes linked to cell growth.30 Hence, the chronic reduction of cellular mevalonate that is expected with prolonged treatment with an HMG-CoA reductase inhibitor may exert an action on the leukocyte and endothelial cell surface that limits their ability to adhere.
In conclusion, our findings indicate that hypercholesterolemia enhances the adhesive interaction between leukocytes and endothelial cells in postcapillary venules. The HMG-CoA reductase inhibitor fluvastatin significantly reduces the inflammatory responses elicited in venules of hypercholesterolemic rats by the lipid mediators PAF and LTB4. These observations suggest that fluvastatin may slow or prevent the initiation and progression of atherosclerosis-associated vascular dysfunction by inhibiting inflammatory cell–endothelial cell interactions.
Selected Abbreviations and Acronyms
|HMG-CoA||=||3-hydroxy-3-methylglutaryl coenzyme A|
|ICAM-1||=||intercellular adhesion molecule-1|
|LECA||=||leukocyte–endothelial cell adhesion|
This study was supported by a grant from the Heart, Lung, and Blood Institute (HL 26441).
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