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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1652-1659.)
© 1995 American Heart Association, Inc.

Articles

Nitric Oxide Protects Against Leukocyte-Endothelium Interactions in the Early Stages of Hypercholesterolemia

Theresa W. Gauthier; Rosario Scalia; Toyoaki Murohara; Jin-ping Guo; Allan M. Lefer

From the Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa.

	Abstract

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Abstract We studied the effects of CAS1609, a nitric oxide donor, on leukocyte-endothelial interactions during the early stages of hypercholesterolemia in rat mesenteric microcirculation. Rats were randomly divided into four groups: (a) rats fed control diet, (b) rats fed control diet while receiving CAS1609, (c) rats fed a high-cholesterol (HC) diet and given C93-4845 (an inactive control compound), and (d) rats fed an HC diet and given CAS1609. Both HC groups developed significantly elevated plasma cholesterol levels compared with rats fed the control diet. Intravital microscopy of mesenteric venules revealed a significant increase in leukocyte rolling and adherence in the untreated HC rats compared with control rats (P<.01). This was significantly attenuated in the HC rats given CAS1609. The HC rats given C93-4845 also developed aortic endothelial dysfunction (ie, impaired relaxation to acetylcholine or ADP) that was significantly prevented by CAS1609 infusion (P<.02). Immunohistochemical staining of ileum demonstrated significantly enhanced localization of P-selectin and intercellular adhesion molecule–1 (ICAM-1) on venular endothelium in the untreated HC rats compared with control rats (P<.01). However, P-selectin and ICAM-1 expression were significantly attenuated in HC rats given CAS1609 (P<.05 and P<.01, respectively). Thus, hypercholesterolemia induces microvascular dysfunction characterized by loss of endothelium-derived nitric oxide, increased rolling and adherence of leukocytes, and increased expression of P-selectin and ICAM-1. Infusion of CAS1609 significantly attenuated these changes due to hypercholesterolemia. Our data suggest that nitric oxide plays a significant role in the prevention of the early endothelial dysfunction observed in hypercholesterolemia.

Key Words: nitric oxide donor • P-selectin • leukocyte rolling • leukocyte adherence

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Hypercholesterolemia is associated with significant endothelial dysfunction characterized by a functional loss of endothelium-derived NO.¹ Enhanced monocyte adhesion and platelet aggregation to the endothelium of hypercholesterolemic vessels are early consequences of the functional loss of NO.^{2 3} Indeed, recent studies demonstrate reversal of the atherosclerotic process after the addition of L-arginine, the active substrate for NO synthase, whereas the NO inhibitor L-NAME can further aggravate neointimal formation and monocyte adhesion in the hypercholesterolemic state.⁴

The mechanism by which hypercholesterolemia promotes initial endothelial dysfunction remains unclear. Increased superoxide anion production from hypercholesterolemic vessels implicates depletion of bioactive NO as an early key pathophysiological consequence of hypercholesterolemia.^{5 6} We have previously established a functional relationship between the loss of endothelium-derived NO and the expression of the adhesion glycoprotein P-selectin.^{7 8 9} P-selectin is involved in the early stages of the leukocyte–endothelial cell adhesion cascade, promoting leukocyte rolling, which enables subsequent leukocyte activation and adherence to the endothelium.^{10 11}

Progressive vascular derangements characterized by alterations in venular and arteriolar reactivity prior to any histological changes of atherosclerosis have been described in similar rat models of diet-induced hypercholesterolemia.^{12 13} In this study, we examined early effects of hypercholesterolemia in the rat mesenteric microcirculation by using intravital microscopy and immunohistochemistry. The primary objective of this study was to ascertain whether continuous intravenous infusion of physiological quantities of a novel NO donor (CAS1609) could attenuate the microcirculatory changes occurring in early hypercholesterolemia. We also examined the expression of two endothelial cell adhesion molecules, P-selectin and ICAM-1, involved in leukocyte rolling and adherence in order to explore the cellular mechanisms of early hypercholesterolemic leukocyte-endothelial interaction and the role of NO in these processes.

	Methods

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Osmotic Pump Implantation
Male Sprague-Dawley rats weighing 150 to 175 g were randomly divided into four groups: (a) rats fed a control diet, (b) rats fed a control diet and administered CAS1609 (a furoxan NO donor that does not induce tolerance during chronic administration¹⁴ ), (c) rats fed an HC diet and administered C93-4845 (an inactive control compound that contains the organic backbone of CAS1609 but lacks the NO moiety), or (d) rats fed an HC diet and administered CAS1609. Rats were anesthetized intraperitoneally with sodium pentobarbital (35 mg/kg). Miniosmotic infusion pumps (model 2ML1, Alza Corp) containing either CAS1609 or C93-4845 were implanted subcutaneously in the neck and connected to the right external jugular vein via a subcutaneously tunneled PE60 catheter. These pumps deliver fluid at a rate of 10 µL/h at a dose of 30 µg/d for 14 days. All incisions were aseptically reapproximated and closed. After recovery from the anesthesia, the rats were returned to the animal facility. Control rats were fed standard diet (Purina 5001) ad libitum for 2 weeks. HC rats were fed a special diet (Purina 5001 plus 1.0% cholesterol plus 0.5% cholic acid) ad libitum for 2 weeks. All rats appeared to gain a comparable degree of weight over the 2-week period, growing to approximately 250 to 275 g body weight.

Intravital Microscopy
Rats in all groups were anesthetized with sodium pentobarbital (35 mg/kg) injected intraperitoneally. A tracheostomy was performed to maintain a patent airway throughout the experiment. A polyethylene catheter was inserted in the left carotid artery to monitor MABP. MABP was recorded on a Grass model 7 oscillographic recorder with a Statham P23AC pressure transducer (Gould Inc). The abdominal cavity was opened by means of a midline laparotomy and a second incision was made through the skin and abdominal musculature on the right flank.

A loop of ileal mesentery was exteriorized through the midline incision and placed in a temperature-controlled fluid-filled Plexiglas chamber for observation of the mesenteric microcirculation by intravital microscopy. The right flank incision was used for administration of sodium pentobarbital as needed to maintain a surgical plane of anesthesia throughout the experiment. The mesentery was placed over a Plexiglas pedestal in the superfusion chamber, and the ileum was secured for stabilization of the viewing field. The ileum and mesentery were superfused throughout the experiment with a modified Krebs-Henseleit solution (containing [mmol] 118 NaCl, 4.74 KCl, 2.45 CaCl₂, 1.19 KH₂PO₄, 1.19 MgSO₄, and 12.5 NaHCO₃) warmed to 37°C and bubbled with 95% N₂ and 5% CO₂. A Microphot microscope (Nikon Corp) with a x40 objective lens and a x10 ocular was used to visualize the mesenteric microcirculation. The image was projected by a video camera (Hamamatsu) onto a black-and-white Sony high-resolution video monitor, and the image was recorded with a videocassette recorder. Red blood cell velocity was determined on-line with an optical Doppler velocimeter¹⁵ obtained from the Microcirculation Research Institute, College Station, Tex.

The rats were allowed to stabilize for 20 to 30 minutes after surgery. After stabilization, a 30- to 50-µm–diameter postcapillary venule was chosen for observation. A baseline recording was made to establish basal values for leukocyte rolling and adherence. The numbers of rolling and adhered leukocytes as well as the leukocyte rolling velocity were determined off-line by playback of the videotape. Leukocytes were considered to be rolling if they were moving at a velocity significantly slower than that of red cells. Leukocyte rolling is expressed as the number of cells moving past a designated point per minute (ie, leukocyte flux). A leukocyte was judged to be adherent if it remained stationary for more than 30 seconds.¹⁶ Adherence is expressed as the number of adherent leukocytes per 100 micrometers of vessel length. V and D were used to calculate g by use of the formula g=8 (V_mean/D), where (V_mean=V_rbc/1.6).¹⁶

After baseline recordings were completed, blood was drawn through the arterial catheter for measurement of plasma cholesterol concentration (Sigma Diagnostics). At the completion of the experiment, all miniosmotic pumps were removed and their catheters were checked for patency, and the residual volumes of either CAS1609 or C93-4845 were measured to verify delivery of drug to the animal.

Immunohistochemistry
Immunohistochemical localization of P-selectin and ICAM-1 was also determined. Rats were anesthetized with pentobarbital and surgery was performed as described above. Both the superior mesenteric artery and superior mesenteric vein were then rapidly cannulated for perfusion fixation of the small bowel.

The ileal tissue was first washed free of blood by perfusion with Krebs-Henseleit buffer warmed to 37°C and bubbled with 95% O₂ and 5% CO₂. Once the venous perfusate was free of red blood cells, perfusion was initiated with iced 4% paraformaldehyde mixed in phosphate-buffered 0.9% NaCl for 5 minutes. A segment of ileum 3 to 4 cm long was isolated from the perfused intestine and fixed in 4% paraformaldehyde for 90 minutes at 4°C. The ileum was then cut into rings and the tissue was dehydrated with graded acetone washes at 4°C. Tissue sections were imbedded in plastic (Immunobed, Polysciences Inc) and sections 4 µm thick were cut and transferred to Vectabond-coated slides (Vector Laboratories).

Immunohistochemical localization of the adhesion molecules was accomplished by use of the avidin-biotin immunoperoxidase technique (Vectastain ABC reagent: Vector Laboratories) as previously described by Beckstead et al¹⁷ and modified by Weyrich et al.¹⁸ Tissue sections were treated with 0.25% trypsin (Sigma Chemical Co) to improve reagent penetration and then incubated with 0.3% hydrogen peroxide for 30 minutes to remove endogenous peroxide. Blocking serum (horse) was applied to the tissue for 30 minutes to reduce nonspecific binding, and then the tissue sections were incubated with the primary antibody directed against either P-selectin or ICAM-1 (ie, either PB1.3 or 1A29¹ [monoclonal mouse anti–rat ICAM-1]) at a dilution of 1:100 for 24 hours. PB1.3 was a generous gift from Dr M. Forrest, Cytel Corp, and 1A29 was purchased from Genzyme. The tissue was then incubated with the biotinylated secondary antibody and the peroxidase staining was carried out by use of 3,3'-diaminobenzidine. Control preparations were made by omitting the primary antibody or the secondary antibody. Expression of adhesion molecules was determined by microscopic detection of the brown peroxidase reaction product on the venular endothelium of the tissue sections. Positive staining was defined as a venule's displaying brown reaction product on more than 50% of the circumference of its endothelium. Fifty venules per tissue section were examined and the percentage of positive staining venules was tallied.

Isolated Arterial Ring Studies
At the end of the experiment, the thoracic aortas were excised, cleaned, cut into segments 3 to 4 mm long, and placed into warmed Krebs-Henseleit solution. Carefully prepared vascular rings were then mounted on stainless steel hooks, suspended in 10-mL organ chambers, and connected to FT-03 force displacement transducers (Grass Instrument Co) for recording on a Grass model 7 oscillographic recorder. The baths were filled with 10 mL of Krebs-Henseleit solution of the following composition (mmol/L): 118 NaCl, 4.75 KCl, 2.54 CaCl₂ · 2H₂O, 1.19 KH₂PO₄, 1.19 MgSO₄ · 7H₂O, 12.5 NaHCO₃, and 10 glucose. This solution was maintained at 37°C and aerated with a gas mixture of 95% O₂ and 5% CO₂. Aortic rings were initially stretched to give a resting force of 0.5 g and equilibrated for 60 to 90 minutes. During this period, the Krebs-Henseleit solution in the tissue baths was replaced every 20 minutes. After equilibration, the rings were exposed to 10 nmol/L U46619 (Biomol Research Laboratories Inc), a thromboxane A₂ mimetic, to generate about 0.5 g of preload. After a stable plateau contraction was achieved, cumulative concentrations of an endothelium-dependent vasodilator, acetylcholine, at 0.1, 1, 10, and 100 nmol/L (Sigma Chemical Co) were added to the bath. After the response stabilized, the rings were washed and allowed to equilibrate to baseline again. The procedure was repeated with an endothelium-independent dilator, acidified NaNO₂, at concentrations of 0.1, 1, 10, and 100 µmol/L. NaNO₂ was freshly dissolved in 0.1N HCl and titrated to pH 2.0. After an additional wash and equilibration, the procedure was again repeated with cumulative concentrations of another endothelium-dependent vasodilator, ADP, in concentrations of 1, 10, 100, and 1000 µmol/L.

Data Analysis
All data are presented as mean±SEM. Data were compared by ANOVA by use of post hoc analysis with Fisher's corrected t test. P values of .05 or less were considered statistically significant.

	Results

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Rats fed a control diet while receiving CAS1609 by continuous infusion exhibited a mean plasma cholesterol concentration of 76±3 mg/dL. This was not significantly different from plasma cholesterol concentrations obtained in rats fed a control diet but not given CAS1609. In contrast, rats fed the HC diet developed a significant elevation in plasma cholesterol to a comparable degree (ie, 300 to 320 mg/dL) whether given C93-4845 or CAS1609. Thus, infusion of the active NO donor did not affect plasma cholesterol levels compared with the inactive donor. MABPs were measured throughout the intravital observation period in all groups of rats. There was no significant difference in the initial MABP among the four groups of rats, and no significant changes in MABP occurred over the 20-minute intravital microscopy observation period.

Residual volumes of either C93-4845 or CAS1609 were withdrawn and measured from the miniosmotic pumps. Pump catheters were accessed for patency and there was no difference in volume of delivery of the assigned drugs over the 2-week period.

Intravital Microscopy
Baseline leukocyte rolling was obtained after a 20-minute stabilization period following surgery. Fig 1 illustrates the baseline leukocyte rolling observed in the four experimental groups. Rats fed the control diet with or without CAS1609 demonstrated minimal baseline leukocyte rolling (ie, about 10 to 15 cells/min). In contrast, baseline leukocyte rolling was increased threefold in cholesterol-fed rats given C93-4845 (P<.01 versus either control group). However, administration of CAS1609 to rats fed the HC diet significantly attenuated baseline leukocyte rolling compared with cholesterol-fed rats given the control compound, which does not release NO (P<.05).

View larger version (25K): [in this window] [in a new window]   Figure 1. Bar graph shows number of rolling leukocytes per minute. All values are mean±SEM for the number of rolling cells in the four groups. Bar heights represent mean values, brackets indicate SEM, and numbers at bases of bars indicate numbers of rats studied.

In addition, leukocyte adherence was also measured in each group after the 20-minute stabilization period. Minimal leukocyte adherence was observed in either control group (approximately 0.5 leukocytes/100 µm). In contrast, leukocyte adherence was significantly increased (10-fold) after 2 weeks of the HC diet (P<.01). However, this adherence was significantly reduced (P<.01) by the infusion of CAS1609 to 25% of that observed in hypercholesterolemic rats given the control compound (Fig 2).

View larger version (18K): [in this window] [in a new window]   Figure 2. Bar graph shows number of adherent leukocytes on 100 µm of postcapillary venular endothelium. All values are mean±SEM for number of adherent cells for each group. Bar heights represent mean values, brackets indicate SEM, and numbers at bases of bars indicate numbers of rats studied.

The venular shear rate was also calculated in the four experimental groups (data not shown). There was no significant difference in shear rates among the four groups, indicating that the adhesive interactions observed between leukocytes and endothelial cells are not due to changes in physical hydrodynamic forces brought about by the HC diet.

Immunohistochemistry
Expression of two of the major endothelial adhesion molecules, P-selectin and ICAM-1, was investigated on the venular mesenteric endothelium in the four experimental groups of rats. Cross-reactivity of PB1.3 with the rat endothelium was confirmed by use of the avidin-biotin immunoperoxidase technique. Cross-reactivity of PB1.3 in the rat has been previously demonstrated with flow cytometry.¹⁹ P-selectin localization was exhibited almost entirely on the venular endothelium, with little or no specific staining on the arterioles. Although P-selectin is constitutively stored in the Weibel-Palade bodies of the endothelial cell, other in vivo models indicate that the interaction of PB1.3 with P-selectin appears to require endothelial activation and translocation of active P-selectin to the cell surface.^{18 20} Comparative expression of P-selectin is summarized in Fig 3. Expression of P-selectin was significantly increased in the HC group given C93-4845 by approximately threefold (P<.01). Nevertheless, administration of CAS1609 to HC rats resulted in suppression of P-selectin expression compared with hypercholesterolemic rats given the inactive control compound (P<.05).

View larger version (26K): [in this window] [in a new window]   Figure 3. Bar graph shows percent of positive-staining venules for P-selectin in the four experimental groups of rats. Bar heights represent mean values, brackets indicate SEM, and numbers at bases of bars indicate numbers of rats studied.

The degree of ICAM-1 expression was also investigated (Fig 4). There was minimal basal expression of ICAM-1 in the control groups (ie, 2%). However, after 2 weeks of the HC diet the number of positive-staining venules increased significantly to 28±2% in the untreated group (P<.01 versus control). In contrast, cholesterol-fed rats given CAS1609 exhibited a significantly lower expression of ICAM-1 (P<.01). Thus, exogenous NO significantly attenuates the increased venular surface expression of P-selectin and ICAM-1 in this model of early hypercholesterolemia (Fig 5).

View larger version (20K): [in this window] [in a new window]   Figure 4. Bar graph shows percent of positive-staining venules for ICAM-1. Bar heights represent mean values, brackets indicate SEM, and numbers at bases of bars indicate numbers of rats studied.

View larger version (2K): [in this window] [in a new window]   Figure 5. Photomicrographs show ileal tissue sections incubated with either anti–P-selectin (PB1.3) (A, B, and C) or anti–ICAM-1 (1A29) (D, E, and F) and labeled with peroxidase substrate. Brown reaction product indicates positive antigen localization on vascular endothelium. P-selectin localization: A, venule (v) from rat fed HC diet plus C93-4845; B, venule from rat fed HC diet plus CAS1609; and C, negative control venule from rat fed HC diet plus C93-4845 with only primary antibody. ICAM-1 localization; D, venule from rat fed HC diet plus C93-4845; E, venule from rat fed HC diet plus CAS1609; and F, negative control venule from rat fed HC diet plus C93-4845 with only primary antibody. All six panels are lightly counterstained with Gill's hematoxylin 2 and have the same magnification. Calibration line represents 25 µm.

Endothelial Dysfunction in Isolated Aortic Rings
To determine the degree of large arterial endothelial dysfunction in hypercholesterolemic rats, vasorelaxation of isolated aortic rings was studied in the four experimental groups. We used both endothelium-dependent and endothelium-independent vasodilators in the rat aortic rings. A summary of the vascular responses of the aortic rings to the three vasodilators is summarized in Fig 6. Aortic rings isolated from rats fed a control diet exhibited normal vasorelaxation to acetylcholine (95% to 100% relaxation). However, after 2 weeks of an HC diet a significant endothelial dysfunction was observed, as evidenced by the markedly impaired relaxation to acetylcholine (P<.05 versus control).This was also confirmed by the attenuated vasorelaxant responses to ADP, indicating that there is a generalized endothelial dysfunction. Infusion of the active NO donor during this 2-week period significantly preserved the vasorelaxant responses to acetylcholine and ADP (P<.02 versus C93-4845), with values approaching those observed in control rings. Smooth muscle function was normal in aortic rings isolated from all four groups, as demonstrated by normal relaxation to NaNO₂.

View larger version (58K): [in this window] [in a new window]   Figure 6. Bar graph shows percent relaxation of rat aortic rings isolated from the four experimental groups of rats. Bar heights represent mean values, brackets indicate SEM, and numbers at bases of bars indicate numbers of rings studied.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The pathogenesis of hypercholesterolemia begins with alterations in endothelial function and integrity.^{21 22} Previous studies have described endothelial dysfunction of arterioles and venules after a brief exposure to diet-induced hypercholesterolemia in the rat.^{12 13} The aim of this study was to characterize the early effects of hypercholesterolemia on leukocyte–endothelial cell interaction in the mesenteric microcirculation, to relate these changes to the expression of specific cell adhesion molecules, and to determine the role of NO replacement.

The loss of endothelium-derived NO has been thought to play an important role in the early development of atherosclerosis.^{4 6 23 24 25} Reduction in NO is associated with enhanced platelet aggregation, increased neutrophil and monocyte adherence, and increased chemotaxis of monocytes during hypercholesterolemia.^{2 3 26} Loss of endothelium-dependent vasorelaxation in large arteries during hypercholesterolemia is a well-known phenomenon in humans as well as in laboratory animals.^{6 23 27}

In this study, a marked hypercholesterolemia with plasma levels paralleling those occurring in human disease occurred in rats after 2 weeks of consuming an HC diet supplemented with cholic acid. Significant increases in both baseline leukocyte rolling and leukocyte adherence to the endothelium were observed in the postcapillary venules of the mesenteric microcirculation 2 weeks after the beginning of the HC diet. Increased leukocyte-endothelium interactions, characterized by both increased leukocyte rolling and adherence, have been described in other experimental conditions, including ischemia/reperfusion^{7 8} or inhibition of NO synthesis by L-NAME superfusion.⁹ Inhibition of NO synthase with L-NAME also accelerates the neointimal formation seen during hypercholesterolemia in rabbits.⁴

The increased interactions between leukocytes and the vascular endothelium observed in the present study correlate well with the immunohistochemical data obtained from the mesenteric microvasculature. Significant increases in the expression of the endothelial cell adhesion molecules P-selectin and ICAM-1 were seen after 2 weeks of the HC diet. The expression of P-selectin has previously been associated with the loss of endothelium-derived NO in ischemia/reperfusion.^{7 16 18} P-selectin is one of the adhesion glycoproteins thought to play an important role in the increased interactions between leukocytes and endothelial cells stimulated by oxidized LDLs.²⁸ Such interactions are thought to be important in the early development of atherosclerosis. Our data suggest that a relatively short exposure to hypercholesterolemia (ie, 2 weeks) is sufficient to upregulate the expression of P-selectin, triggering further adhesive interaction between leukocytes and the vascular endothelium.

In addition to increased expression of P-selectin, the expression of ICAM-1 was also increased after rats had consumed an HC diet for 2 weeks. In this regard, expression of ICAM-1 has been described in human atherosclerotic lesions^{7 29 30} and is also upregulated by lysophosphatidylcholine in oxidized LDLs.³¹ Our data confirm increased endothelial expression of ICAM-1 in early hypercholesterolemia, and this may be the consequence of increased lysophosphatidylcholine levels, although we did not measure LDL levels in our rats. Previous studies in hypercholesterolemic rabbits showing enhanced coronary vascular adhesiveness are consistent with these findings.¹

Two weeks of an HC diet resulted in impaired vascular responses of the thoracic aorta to the endothelium-dependent vasodilators acetylcholine and ADP but not to the endothelium-independent vasodilator NaNO₂. This pattern typifies a picture of impaired vasorelaxation due to endothelial dysfunction.^{6 24} This endothelial dysfunction has been described as a loss of functional NO rather than an excess of endothelium-derived contracting factors such as PGH₂.³² Loss of functional NO may be due to an absolute decrease in NO produced or to an elevated NO level accompanied by a marked elevation in superoxide radicals that results in a functional reduction of NO activity.³³ Our model of early hypercholesterolemia, prior to histological damage, correlates well with previous in vitro evidence that demonstrates impaired endothelium-dependent relaxation after exposure to low levels of oxidized LDLs.³⁴

Treatment with the active NO donor CAS1609 during feeding of the HC diet significantly attenuated baseline leukocyte rolling and adherence in the mesenteric microcirculation compared with that in rats receiving the inactive NO donor. CAS1609 had no direct effect on MABP or baseline leukocyte-endothelial interactions. NO donors can reduce leukocyte-endothelial interactions in vivo during ischemia/reperfusion⁸ or shock states.³⁵ Recent evidence also suggests that CAS1609 can reduce intimal hyperplasia and endothelial dysfunction after arterial intimal injury to the rat carotid artery.³⁶ The reduction in leukocyte rolling and adherence observed in the present study implicates NO as a key regulator of early microvascular leukocyte-endothelial interactions in the postcapillary venule during hypercholesterolemia. Another approach to enhancing NO formation in hypercholesterolemia is by addition of L-arginine, the substrate for NO synthase. L-Arginine supplementation in experimental models of hypercholesterolemia reduces platelet aggregation,² monocyte adhesiveness,³ and subsequent atheroma formation.²⁵ L-Arginine also restores vascular responsiveness to endothelium-dependent vasodilators in the hypercholesterolemic vessels of animals and humans.^{25 37}

The reduction in leukocyte-endothelial interactions observed in the present study after administration of the active NO donor CAS1609 was accompanied by significant attenuation of P-selectin and ICAM-1 expression. Previous studies have linked the expression of P-selectin with decreased NO levels in mesenteric or myocardial ischemia/reperfusion^{7 8 18} or reduced NO synthesis in response to L-NAME.⁹ Moreover, P-selectin upregulation can be reduced by infusion of an NO donor⁸ or L-arginine replacement.⁹ The present study demonstrates for the first time that NO administration during hypercholesterolemia attenuates the expression of both P-selectin and ICAM-1.

One possible mechanism for the beneficial effects of NO during hypercholesterolemia is the scavenging of free radicals. Hypercholesterolemic vessels produce increased superoxide radicals formed via the xanthine oxidase pathway that can be neutralized with NO replacement.³³ Also, activated neutrophils produce superoxide radicals via the NADPH pathway,³⁸ and these can be neutralized by the addition of NO in vitro.³⁹ Finally, NO may protect against the oxidative modification of LDLs^{40 41} and the subsequent increases in leukocyte-endothelial interactions and mast cell degranulation caused by oxidized LDLs.⁴² Thus, an NO donor like CAS1609 may function as a free radical scavenger at any of these targets (ie, endothelium, leukocyte, macrophage) in the early setting of hypercholesterolemia.

In addition to its free radical scavenger role, or as a consequence of it, NO suppresses the expression of the adhesion glycoprotein P-selectin.^{8 9} This may be an important effect because P-selectin governs the initial interaction of neutrophils with endothelial cells, allowing for rolling and tethering of neutrophils on the endothelial surface. Leukocyte rolling is followed by further neutrophil activation, firm adherence by means of ICAM-1, and production of inflammatory mediators such as platelet activating factor.¹⁰ Thus, the leukocyte-endothelial cascade stimulated by the oxidized LDL seen in hypercholesterolemia can be activated by P-selectin^{43 44} and ICAM-1,³¹ and this may be prevented by NO.

Although the microvasular alterations that occur in mesenteric venules are accompanied by endothelial dysfunction of the thoracic aorta, one cannot assume that similar mechanisms are responsible for such alterations during hypercholesterolemia. Despite venular changes in leukocyte-endothelial interactions and expression of the adhesion molecules during early hypercholesterolemia, few effects on the mesenteric arteriolar microcirculation were seen in the present study. This is in contrast with prior work by Schuschke and coworkers^{12 13} that demonstrated both impaired venular and arteriolar responses after hypercholesterolemia in the rat, also implicating a role of NO in these processes.⁴⁵ The adhesion molecule pathways regulating such interactions probably exhibit variations not only between venules and arterioles but also among different vasculatures. In this regard, adhesive properties of polymorphonuclear leukocytes modulated by means of P-selectin after ischemia-reperfusion are exhibited in mesenteric arteries to values that are 30% of those seen in mesenteric veins.⁴⁶ We assume that similar relationships occur in other vascular beds during the early course of hypercholesterolemia. The significance of these differences and their underlying mechanisms on the development of atherosclerosis must await further study.

In summary, 2 weeks of hypercholesterolemia resulted in a marked increase in leukocyte-endothelium interactions and expression of the endothelial cell adhesion molecules P-selectin and ICAM-1, accompanied by impaired endothelium-dependent relaxation. Concurrent treatment with the novel NO donor CAS1609 during the HC diet significantly reduced leukocyte rolling and adherence. This was correlated with diminished expression of P-selectin and ICAM-1 immunohistochemically, as well as restoration of endothelium-dependent relaxation of isolated aortic rings. These data provide important new insights into the early effects of hypercholesterolemia and the role of endothelium-derived NO on these alterations.

	Selected Abbreviations and Acronyms

 

D = venular diameter g = venular wall shear rate HC = high-cholesterol ICAM-1 = intercellular adhesion molecule–1 L-NAME = NG-nitro-L-arginine methyl ester MABP = mean arterial blood pressure Vrbc = red blood cell velocity

	Acknowledgments

 
This study was supported by research grant no. GM-45434 from the National Institutes of Health. Dr Gauthier is a fellow of the Department of Pediatrics, Division of Neonatology, at Jefferson Medical College. Dr Murohara is a fellow of the Japan Heart Foundation, Tokyo. We thank Dr Piero Martorana of Cassella AG, Frankfurt, Germany, for the generous supply of both CAS1609 and C93-4845 used in this study. The authors gratefully acknowledge Robert Craig, BS, for his excellent technical assistance.

	Footnotes

 
Reprint requests to Dr Allan M. Lefer, Department of Physiology, Jefferson Medical College, 1020 Locust St, Rm 425, Philadelphia, PA 19107.

Received April 26, 1995; accepted July 18, 1995.

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