Hypercholesterolemia Enhances Thromboembolism in Arterioles but Not Venules
Complete Reversal by l-Arginine
We investigated in vivo the effect of cholesterol diet–induced hypercholesterolemia (HC) on thromboembolism in nonatherosclerotic rabbit mesenteric arterioles and venules (diameter 21 to 45 μm). After mechanical vessel wall injury, the ensuing thromboembolic reaction was studied by intravital videomicroscopy. A dramatic prolongation of embolization duration (median >600 seconds) was observed in the arterioles of the HC group compared with the arterioles of a normal chow–fed (NC) control group (142 seconds, P<0.0001); concomitantly, relative thrombus height increased (thrombus height/vessel diameter was 68% for the HC group and 58% for the NC group; P<0.05). By contrast, in venules, cholesterol did not affect embolization duration (42 seconds for HC group, 34 seconds for NC group) and thrombus height (66% for HC group, 64% for NC group). Furthermore, the role of endothelial NO synthesis was studied. In arterioles, stimulation of endogenous NO synthesis through mesenteric superfusion of l-arginine (1 mmol/L) completely reversed cholesterol-enhanced embolization (152 seconds) but did not influence thrombus height (63%). l-Arginine had no effect in venules of the HC group (51 seconds) and nor in the arterioles and venules of the NC group (177 seconds for arterioles, 43 seconds for venules). This study indicates that hypercholesterolemia selectively enhances thrombus formation and embolization in arterioles but not in venules and that stimulation of endogenous NO production antagonizes this enhancement of arteriolar thromboembolism.
Patients with hypercholesterolemia are prone to the development of atherosclerosis, ultimately leading to myocardial infarction, cerebral infarction, and/or critical limb ischemia.1,2⇓ Most of these disorders result from thromboembolic processes at the site of the lesion.3 The vascular lesion plays an important role in these processes,3,4⇓ but the role of hypercholesterolemia, as such, in thromboembolic events is less clear. Besides, the mediators involved in hypercholesterolemia-related thromboembolic events are, as yet, not well known.
Platelets derived from hypercholesterolemic patients and rabbits show in vitro an enhanced tendency to aggregate.5,6⇓ The observation that l-arginine, the substrate of endogenous NO synthase, reverses the enhanced platelet aggregation in hypercholesterolemia in vitro suggests that reduced production or bioavailability of NO is involved in hypercholesterolemia-induced thromboembolic processes.5,6⇓ An additional argument for the involvement of NO is that in vivo endothelium-dependent vasodilation has been shown to be reduced in hypercholesterolemic states, an effect that could be reversed by l-arginine.7–10⇓⇓⇓
Because studies performed in vitro on interactions between blood platelets and the vessel wall lack the contribution of natural microenvironmental factors (eg, local fluid dynamic conditions and the balance between activating and inhibiting agents), which may be of key importance for platelet–vessel wall interactions, in vivo studies are essential in evaluating the role of hypercholesterolemia in thromboembolic processes. To shed more light on this role, in vivo studies should be performed in preatherosclerotic models, excluding factors induced by the advanced atherosclerotic lesion. Therefore, the first aim of the present study was to investigate in vivo the effect of diet-induced hypercholesterolemia on thromboembolism with the use of a previously described nonatherosclerotic rabbit model.11 The second aim of the present study was to investigate whether changes in endogenous NO are involved in the effect of hypercholesterolemia on thromboembolism. The latter was studied by stimulating endogenous NO synthesis by local application of l-arginine during this process. Because in vivo the antithrombotic properties of arteriolar and venular vessel walls differ remarkably,11–14⇓⇓⇓ the present study was performed in arterioles and in venules.
Animals and Intravital Video Microscopy
Experiments were approved by the local animal ethics committee. Thirty-one male New Zealand White rabbits (weight range 2.1 to 2.9 kg) were used. Anesthesia was induced and maintained with ketamine hydrochloride and xylazine hydrochloride (for induction, 40 and 5 mg/kg body wt IM, respectively; for maintenance, 40 and 5 mg/kg IV per hour, respectively). A catheter was inserted into the femoral artery to measure mean arterial pressure and heart rate. Body temperature was kept at 37°C to 38°C by using a thermocontrolled infrared heating lamp.
After surgery, blood was collected from a central ear artery in EDTA (10%). Electronic platelet counts, hemoglobin concentration, and hematocrit were assessed by using a Coulter Counter (model ZF). Total cholesterol, LDL cholesterol (LDL), and HDL cholesterol (HDL) levels were assessed by using enzymatic colorimetry (Roche Cholesterol plus assays).
Rabbits were ventilated (animal ventilator 4601, Technical & Scientific Equipment) to maintain systemic arterial pH, Pco2, and Po2 at normal values (mean±SD)11,15⇓: pH 7.47±0.02, Pco2 36±4 mm Hg, and Po2 98±7 mm Hg (ABL 3 Radiometer). No statistical differences existed between the experimental groups.
After laparotomy, a segment of the distal ileum was exteriorized and continuously superfused with buffered Tyrode’s solution (37°C, pH 7.35 to 7.40). The mesenteric tissue was visualized with a Leitz intravital microscope by use of transillumination with a tungsten lamp and a ×25 objective (Leitz LL ×25, numerical aperture 0.35). Images were recorded on videotape. Final magnification at the front plane of the TV camera was ×40 (Grundig FA 32, 1 inch).
Vascular diameter was measured offline with an image-shearing device. Mean red blood cell velocity was measured online by using the dual-slit photometric technique. Reduced velocity, a well-known first-order approximation of wall shear rate,16 was calculated by dividing mean red blood cell velocity by vessel diameter.
Vessel Wall Puncture and Thromboembolic Reaction
Arterioles and venules with diameters between 20 and 45 μm were selected. Vessel walls were punctured with a glass micropipette (tip diameter ≈6 μm).11 Immediately after puncture, the thromboembolic reaction started. In all vessels, a white thrombus was formed, consisting of tightly packed platelets, the height and shape of which remained constant. Circulating platelets adhered to this stationary thrombus mainly on its downstream side, forming a loosely packed platelet mass that did not affect the thrombus height. In nearly all vessels, except for 2 arterioles and 5 venules, these platelet masses embolized from time to time. The 7 microvessels without embolization were evenly distributed over the experimental groups. After a certain period of time, embolization stopped in most vessels.
To quantify this thromboembolic reaction, we determined the following offline from videotape: the duration of bleeding (bleeding time), the maximal thrombus height relative to the local vessel diameter, the duration of embolization, and the number of emboli produced. Each vessel was observed continuously for 600 seconds from the moment of puncture. Vessels in which embolization continued for >600 seconds were examined again intermittently during the remainder of the experiment (maximum duration 3 hours) .
Experimental Groups and Protocol
In experimental series 1, the effect of diet-induced hypercholesterolemia on thromboembolism was studied by using 2 groups of rabbits. Rabbits of the high cholesterol–fed group (HC group, n=8) received a diet containing 0.4% cholesterol, 3% coconut oil, and 3% peanut oil for 2 weeks. The normal chow–fed (control) rabbits (NC group, n=7) received a similar but cholesterol-free diet for 2 weeks (for both groups, 80 g/d; Hope Pharms). Rabbits were randomly assigned to 1 of these 2 groups. On day 15, the rabbits were anesthetized, and the arteriolar and venular thromboembolic reactions were studied.
Experimental series 2 was performed to determine whether the role of endogenous NO as an inhibitor of thromboembolism is changed by diet-induced hypercholesterolemia. To this purpose, endogenous NO synthesis was stimulated by continuously superfusing the mesentery with excess l-arginine (1 mmol/L, molecular weight 210.7), which is the substrate for NO synthase. Before each experiment, l-arginine was dissolved in buffered Tyrode’s solution,13 which did not influence the pH of the Tyrode’s solution. Previously, we demonstrated the specificity of l-arginine superfusion compared with d-arginine superfusion in enhancing endogenous NO production.13 The rabbits of this series were randomly assigned to either 2 weeks of a high cholesterol diet (HCarg group, n=8) or 2 weeks of normal chow (NCarg group, n=8). The composition and amount of the diets were the same as those in experimental series 1.
On the experimental day, the mesentery was allowed to stabilize for 30 minutes after exteriorization under continuous superfusion with buffered Tyrode’s solution without (series 1) or with l-arginine (1 mmol/L, series 2). These superfusions were continued during the rest of the experiments. A median number of 3 vessels was punctured per experiment. Each puncture was preceded by a 4-minute period to measure mean red blood cell velocity.
Because of their nonsymmetrical distribution, data are presented as medians with interquartile ranges, unless otherwise indicated. Embolization data are presented per blood vessel; averaging of data per animal led to similar results and conclusions. Differences between 2 groups were tested by the Mann-Whitney U test. Correlations were performed with the Spearman rank correlation test (coefficient rs). The level of significance was set at 5%.
Cholesterol Levels and Other Whole-Animal Parameters
Total plasma cholesterol level, LDL concentration, LDL/HDL ratio, and (to a lesser extent) HDL concentration were significantly elevated in cholesterol-fed rabbits (HC and HCarg groups) compared with rabbits assigned to control chow (NC and NCarg groups, Figure 1). All plasma lipid concentrations in the control groups and also the plasma HDL concentration in the cholesterol-fed groups were normal for rabbits.17
Whole-animal parameters were not influenced by the increase in cholesterol levels, except for a slight increase in mean arterial blood pressure in the HC rabbits (overall median value 76 mm Hg, range 73 to 85 mm Hg) compared with the NC rabbits (median 70 mm Hg, range 63 to 82 mm Hg; P=0.049). This slight elevation of mean arterial blood pressure was completely reversed by l-arginine (for HCarg group, median 72 mm Hg, range 67 to 83 mm Hg; P=0.046 compared with HC). Overall, hemoglobin concentration (median 8.1 mmol/L, range 7.0 to 9.3 mmol/L), hematocrit (median 39.2%, range 33.7% to 45.4%), platelet count (median 449×103/μL, range 231 to 689×103/μL), heart rate (median 112 bpm, range 86 to 147 bpm), and also mean arterial blood pressure (median 72 mm Hg, range 63 to 85 mm Hg) were within normal ranges for anesthetized rabbits.11,15,17⇓⇓ No significant correlations were found between any of these whole-animal parameters and embolization parameters in either arterioles or venules.
Thromboembolic Reaction In Vivo
In all vessels, bleeding and thrombus formation started immediately after wall puncture. A thrombus started to grow within 0.1 second after puncture and reached its maximal size within 1 to 2 seconds. This time frame was not influenced by hypercholesterolemia and/or l-arginine in arterioles or in venules.
Hypercholesterolemia nonsignificantly shortened arteriolar bleeding duration (P=0.07, Table 1) and significantly increased thrombus height (P=0.021; Table 1). The tendency of hypercholesterolemia to shorten bleeding time was reversed by l-arginine (P=0.004 for HCarg versus HC). In contrast, the effect of hypercholesterolemia on thrombus height was not affected by l-arginine (P=0.103 for HCarg versus HC).
Hypercholesterolemia caused a pronounced significant increase in embolization duration (HC median >600 seconds, NC median 142 seconds; P<0.0001; Figure 2); in these periods, 21 emboli were produced in HC arterioles, and only 5 were produced in NC vessels (P<0.0001, Table 1). In the 58% of the HC arterioles in which embolization continued >600 seconds, it continued during the rest of the experiment (30 minutes to 3 hours). In contrast, embolization stopped within 600 seconds in all NC arterioles.
l-Arginine completely reversed the prolongation of embolization duration in HC rabbits (for HCarg group, 152 seconds, 9 emboli; P<0.0001 compared with HC; Figure 2). Moreover, in none of the 25 HCarg arterioles did embolization continue for >600 seconds. In NC arterioles, l-arginine had no significant effect (for NCarg group, 177 seconds, 9 emboli).
The venular bleeding duration was significantly prolonged in HC rabbits (P<0.05, Table 1); this effect could not be reversed by l-arginine. Venular thrombus height was not influenced by hypercholesterolemia or l-arginine (Table 1).
As opposed to the marked effect observed in arterioles, hypercholesterolemia did not affect embolization duration and embolus production in venules (for HC group, 42 seconds, 2 emboli; for NC group, 34 seconds, 3 emboli; Figure 2 and Table 1). In addition, l-arginine had no effect on embolization duration and embolus production in HC rabbits (for HCarg group, 51 seconds, 3 emboli) and NC rabbits (for NCarg group, 43 seconds, 2 emboli). In all but 1 venule embolization stopped within the observation period of 600 seconds.
Arterioles Versus Venules
In all 4 groups, embolization duration and embolus production were significantly greater in arterioles than in venules (Figure 2).
Cholesterol Levels and Embolization
When data for all groups were pooled, levels of total plasma cholesterol and LDL cholesterol were positively and significantly correlated with embolus production in arterioles (rs>0.271, P<0.016). In contrast, in venules, no correlations were found (rs<0.194, P>0.133).
Fluid Dynamic Conditions
Hypercholesterolemia and/or l-arginine had no statistically significant effect on diameter, red blood cell velocity, or reduced velocity in arterioles or venules (Table 2). The effects of l-arginine in hypercholesterolemic rabbits were near the level of significance, although all values were P>0.05. However, no significant correlations between fluid dynamic and thromboembolic parameters were found in these vessels, with and without l-arginine, indicating that the minimal fluid dynamic effects of l-arginine did not affect thromboembolic parameters such as thrombus height and bleeding time.
The findings in the present study show that diet-induced hypercholesterolemia differently affects the thromboembolic reaction after wall puncture in mesenteric arterioles and venules in preatherosclerotic rabbits: vessel wall injury results in greater thrombus height and increased duration of embolization with a concomitant increase in the number of emboli produced in arterioles, whereas it has no such effects in venules. In arterioles, the effects of hypercholesterolemia on embolization can be completely reversed by l-arginine, the active precursor of endogenous NO synthesis.
The observation that the increase in total plasma cholesterol mainly results from an increase in LDL cholesterol suggests that the observed effects of hypercholesterolemia on arteriolar thromboembolism are caused by LDL cholesterol. This plasma lipid may influence platelet aggregation directly18 or indirectly through an influence on arteriolar vascular cell function.19,20⇓ Although direct effects of LDL cholesterol on platelets may have activated them, this cannot explain the differences in thromboembolic effects between arterioles and venules, because the plasma concentration of LDL cholesterol was probably the same in both vessel types. Therefore, the effects of LDL cholesterol on arteriolar thromboembolism are probably indirect and a consequence of interaction of LDL cholesterol with the arteriolar vessel wall, leading to oxidation of LDL. The involvement of oxidized LDL (oxLDL) is consistent with the observation that oxLDL depletes caveolae of cholesterol, resulting in displacement of endothelial NO synthase (eNOS) from caveolae and impaired eNOS activation without affecting prostacyclin (PGI2) production.21–23⇓⇓
The dramatic stimulating effect of hypercholesterolemia on thromboembolism in arterioles is similar to the effect of complete inhibition of endogenous production of NO and prostaglandins, as observed previously.14 From this previous study and another study, 13 we concluded that in arterioles, the antithromboembolic effect of endogenous NO alone is negligible but that its effect is pronounced in the presence of endogenous prostaglandins, provided that sufficient NO is produced to synergize with the arachidonic acid/prostaglandin pathway. Therefore, the observed effects of increased plasma cholesterol on thromboembolism in arterioles may be explained by the loss of NO/prostaglandin synergism that is due to reduced NO synthesis and/or bioavailability as a consequence of LDL/oxLDL-generated reactive oxygen species (ROS), such as O2− (ie, superoxide anions).24,25⇓ This notion is supported by our finding that excess amounts of l-arginine completely reverse the increased embolization in arterioles as induced by hypercholesterolemia. The latter observation indicates that the putatively reduced NO synthesis and/or bioavailability can be overcome by a surplus of the active precursor of NO synthesis. Additional support for this explanation is provided by studies showing reduced NO synthesis and/or bioavailability in microvessels from hypercholesterolemic animals or humans, an effect that could be overcome by the addition of arginine analogues in all studies.7–10,26,27⇓⇓⇓⇓⇓
An alternative explanation for our results could be that hypercholesterolemia changes arteriolar prostaglandin production, which may also result in the loss of NO/prostaglandin synergism. The increased NO production due to l-arginine administration may have compensated for such a change in prostaglandin production. The formation of proplatelet and antiplatelet prostaglandins was found to be increased in aortic segments from hypercholesterolemic rabbits compared with those from normocholesterolemic rabbits.28,29⇓ Other studies have shown that platelets isolated from hypercholesterolemic rabbits are hyperreactive to arachidonic acid28 and thromboxane A230 and less sensitive to the inhibitory activity of prostacyclin.28,31⇓ Such effects may have contributed to the observed increase in arteriolar thromboembolism in hypercholesterolemic rabbits. Unfortunately, to the best of our knowledge, no data are available on prostaglandin production in arterioles and venules of hypercholesterolemic animals or humans. Therefore, at this point in time, it is not known to what extent changes in prostaglandin metabolism affect thromboembolism in microvessels of hypercholesterolemic rabbits.
The reversal of enhanced arteriolar embolism in hypercholesterolemia by l-arginine may be explained as follows: First, in the absence of excess l-arginine, the arteriolar concentration of ROS, which is relatively high in the arterioles of normocholesterolemic normotensive rodents,32 is further increased in hypercholesterolemic animals as a result of the stimulating effect of LDL cholesterol on endothelial ROS production.24,25⇓ By the addition of excess amounts of l-arginine, stimulation of eNOS will lead to a high level of NO and reduce ROS production by a reaction between NO and O2−, resulting in the formation of ONOO− (ie, peroxynitrite). In vitro, ONOO− has either stimulating or inhibiting effects on the aggregation of platelets isolated from humans33 or rabbits.34 The net biological effect of ONOO− on platelet aggregation in vivo probably depends on the concentration of ONOO− itself and the presence of factors facilitating the conversion of ONOO− to platelet inhibitory NO.33–35⇓⇓ Second, the endothelial formation and release of asymmetrical dimethylarginine (ADMA), an endogenous NO synthase inhibitor that competes with l-arginine,36 may be enhanced in the presence of native LDL or oxLDL.36 In support of this notion is the observation that plasma concentrations of ADMA are elevated in hypercholesterolemic patients37 and rabbits.38 The biological effect of ADMA was confirmed by the observation that ADMA reduces endothelium/NO-dependent dilation of rabbit brain arterioles, an effect prevented by l-arginine application.39 Elevation of the l-arginine/ADMA ratio by exogenous l-arginine in hypercholesterolemia, as in the present study, may restore NO formation in rabbits.40
The stimulating effect of hypercholesterolemia on arteriolar thrombus height indicates that native LDL and/or oxLDL cholesterol changes the (anti)thrombogenic properties of the arteriolar wall in primary thrombus formation. This is an intriguing observation because blocking NO synthesis, 13 prostaglandin synthesis,12 or both14 does not affect thrombus height. In vitro, elevated concentrations of LDL/oxLDL cholesterol sensitize platelets to thrombin activation.41,42⇓ In our model, thrombin very likely plays a role in thrombus formation and not in embolization (M.G.A. oude Egbrink, unpublished data, 1995). Most likely, LDL/oxLDL cholesterol and thrombin synergistically enhance platelet thrombus formation at the site of arteriolar wall injury.
The observation that hypercholesterolemia does not influence the thromboembolic process in venules, despite the fact that endogenous NO is an important antithromboembolic mediator in these vessels,13 is surprising. It is conceivable that in venules, in which the ROS concentration appears to be lower than in arterioles, 32 the effect of LDL/oxLDL cholesterol on ROS production and, hence, on NO production/bioavailability is very limited. Therefore, in these vessels, the endogenous NO concentration may be maintained at the antithromboembolic level required. The absence of an effect of l-arginine on venular thromboembolism indicates that NO synthesis is already maximal in these vessels; as a result, the low number of emboli produced cannot be reduced further.
Hypercholesterolemia induced a slight increase in mean arterial blood pressure, which was reversed by the stimulation of endogenous NO synthesis with l-arginine. Although during hypercholesterolemia, the arteriolar diameter in the transparent part of the mesentery was not different from the control diameter, it should not be concluded that there was no increase in peripheral resistance. This particular part of the rabbit mesentery is known to be a vascular bed with very limited vasoactivity.13 During hypercholesterolemia, arteriolar diameters may have been reduced in other vascular areas.
In conclusion, this is the first study providing evidence of a pronounced stimulating effect of diet-induced hypercholesterolemia, as such, on in vivo thrombus formation and subsequent embolization after vessel wall injury in arterioles but not venules. These effects were observed without the presence of atherosclerotic vascular lesions. Endogenously produced NO as stimulated by excess l-arginine is able to antagonize this cholesterol-enhanced arteriolar thromboembolism.
This study was supported by the Netherlands Heart Foundation (grant 92.339).
Received December 27, 2001; revision accepted January 25, 2002.
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- ↵Broeders MAW, Tangelder GJ, Slaaf DW, Reneman RS, oude Egbrink MGA. Endogenous nitric oxide protects against thromboembolism in venules but not in arterioles. Arterioscler Thromb Vasc Biol. 1998; 18: 139–145.
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