Low-Density Lipoprotein Receptor Knockout Mice Exhibit Exaggerated Microvascular Responses to Inflammatory Stimuli
Abstract The objective of this study was to determine whether genetically induced hypercholesterolemia affects leukocyte–endothelial cell interactions in postcapillary venules of the mouse cremaster muscle. Leukocyte adhesion, emigration, and other microvascular parameters were assessed in venules of normal (wild-type) and low-density lipoprotein receptor–deficient (LDLr−/−) mice maintained on either normal rodent chow or on a high cholesterol diet (HCD). Measurements were obtained under control conditions and after administration of either leukotriene B4 (LTB4), platelet-activating factor (PAF), or tumor necrosis factor-α (TNF-α). Elevated numbers of adherent and emigrated leukocytes were observed in venules of LDLr−/− (compared with wild-type) mice on HCD, both under baseline conditions and after exposure to either LTB4, PAF, or TNF-α. Plasma TNF-α levels were also elevated in LDLr−/− versus wild-type mice. Administration of blocking monoclonal antibodies demonstrated that intercellular adhesion molecule-1, but not vascular cell adhesion molecule-1, mediates the exaggerated leukocyte–endothelial cell adhesion observed in LDLr−/− mice. The results of these studies indicate that chronic hypercholesterolemia predisposes the microvasculature to intense leukocyte–endothelial cell adhesion in response to different inflammatory stimuli.
Hypercholesterolemia, a primary risk factor for the development of atherosclerosis, appears to be associated with the initiation and progression of an inflammatory response in the vascular system. The chronic inflammatory nature of atherosclerotic lesions within major arteries has been described as consisting of inflammatory cell infiltrates, cellular proliferation, enhanced cytokine production, and an increased expression of endothelial CAMs.1 2 3 4 Although these inflammatory manifestations of atherosclerosis are generally assumed to occur exclusively in major arterial vessels, the results of recent studies suggest that the inflammatory response also extends to the arterial and venous segments of the microcirculation.5 6
It has been shown that rats placed on a hypercholesterolemic diet exhibit an attenuated endothelium-dependent relaxation of arterioles to acetylcholine and an increased number of rolling leukocytes in unstimulated postcapillary venules.6 It has also been noted that inflammatory mediator-induced recruitment of adherent leukocytes in postcapillary venules of hypercholesterolemic rats is more intense than that observed in their normocholesterolemic counterparts.7 There is also evidence suggesting that the enhanced leukocyte–endothelial cell adhesion and other microvascular abnormalities associated with hypercholesterolemia may be linked to a reduction in the production of endothelial cell–derived NO.6 8 Indeed, the oxidized lipoproteins found in the plasma of hypercholesterolemic subjects have been shown to inhibit the production and/or bioavailability of NO and to enhance the production of cytokines.1 9 10 Inasmuch as NO has been shown to be a potent inhibitor of leukocyte adhesion, it has been argued that a reduction in NO generation accounts for the inflammatory responses noted in hypercholesterolemic animals.11
Leukocyte–endothelial cell adhesion involves a well-coordinated series of interactions between adhesion glycoproteins expressed on the surface of both endothelial cells and circulating leukocytes. In normocholesterolemic animals, these adhesive interactions normally occur on the venous side of the microvasculature.12 However, with hypercholesterolemia, leukocyte–endothelial cell adhesion occurs on both the arterial (lesion development)3 and venous6 sides of the circulation. Although hypercholesterolemia and the oxidation products of circulating lipoproteins are known to induce alterations in leukocyte function,13 14 it is a change in the adhesivity of endothelial cells that appears to account for the enhanced leukocyte–endothelial cell adhesion of hypercholesterolemia.15
Targeted disruption, deletion, or insertion of specific genes related to lipoprotein metabolism have led to the development of different mouse models of hypercholesterolemia and atherosclerosis.16 17 18 Many of these animals develop arterial lesions similar to those seen in humans, especially when placed on high fat diets. Consequently, the mutant mouse models are considered to be more relevant to the human form of hypercholesterolemia and atherosclerosis than rats placed on HCD, which do not develop arterial lesions. LDLr−/− mice, for example, are moderately hypercholesterolemic on a diet of normal rodent chow but develop profound hypercholesterolemia and arterial lesions (fatty streaks and plaque formation) when placed on a high fat chow.16 This animal model closely resembles familial hypercholesterolemia in humans, and it is frequently used for studies of this naturally occurring genetic mutation that leads to atherosclerosis.
The overall objective of the present study was to use LDLr−/− mice to address the following questions: (1) Does hypercholesterolemia induce a chronic inflammatory state within the microvasculature? (2) Does hypercholesterolemia result in an exaggerated inflammatory response to exogenous lipid-derived proinflammatory stimuli? (3) Which adhesion molecules on vascular endothelium mediate this response and/or exhibit an increased cell surface expression? (4) Are cytokine levels increased in chronically hypercholesterolemic mice, and do cytokines also elicit an exaggerated inflammatory response in this animal model? Intravital videomicroscopic analyses of the microvasculature in cremaster muscle were used to address these issues in LDLr−/− mice placed on either ND or HCD.
Materials and Methods
Human recombinant TNF-α was obtained from R&D Systems. LTB4 was purchased from Calbiochem. All other chemicals were acquired from Sigma Chemical Co.
Age-matched male B6129 (background strain for the LDLr−/− mice, n=96) and LDLr−/− (n=129) mice were maintained on one of three dietary regimens: (1) normal rodent chow (ND) for 8 weeks, (2) normal rodent chow for 4 weeks followed by 4 weeks of HCD (Teklad 90221 containing 1.25% cholesterol and 15.8% fat, Harlan Teklad), or (3) 8 weeks of HCD. Animals (body weight, 30 to 45 g) were anesthetized using xylazine (7.5 mg/kg IM) and ketamine hydrochloride (150 mg/kg IM). Anesthesia was maintained with supplemental doses of ketamine (15 mg/kg IM) as needed. A tracheotomy was performed to facilitate breathing throughout the procedure. The left jugular vein was cannulated for the administration of MAbs. Systemic arterial pressure was measured using a Statham P23A pressure transducer attached to a cannula inserted into the right carotid artery. Systemic arterial pressure was continuously monitored on a physiological recorder (Grass Instruments). Core body temperature was monitored with an intra-abdominally placed thermometer and maintained at 35.5±0.5°C using an infrared lamp. Animal handling procedures were approved by the LSU Medical Center institutional animal care and use committee and were in accordance with the guidelines of the American Physiological Society.
Mice were placed in dorsal recumbency on a Plexiglas microscope stage with an optically clear viewing pedestal and surgically prepared as previously described,19 with minor modifications. Briefly, the left cremaster muscle was carefully dissected free of associated fascia, incised on its ventral surface, and spread over the viewing pedestal using peripherally attached sutures. The cremaster tissue was constantly suffused with PBS (8 mL/min, 35°C, pH 7.4) bubbled with 5% CO2/95% N2.
The cremaster muscle microvasculature was visualized using an intravital microscope (Optiphot, Nikon), a ×20 objective lens (E plan 20/0.4, Nikon), and a multi-image module (Nikon) set at ×1.25 magnification. Transillumination of the tissue was provided with a 12-V, 50-W tungsten light source. A color video camera (VK-C150, Hitachi) mounted on the multi-image module projected the acquired images onto a color monitor (PVM-2030, Sony) while an interposed videocassette recorder (BR-S601MU, JVC) captured the images for off-line analysis. A video time-date generator (WJ810, Panasonic) projected the time, date, and clock function onto the on-line images.
Single branched venules of 25- to 40-μm diameter with a wall SR of >500 s−1 were chosen for study. Venular diameter was measured on-line using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station). An optical Doppler velocimeter (Microcirculation Research Institute) was likewise used on-line to determine centerline Vrbc (mm/s). Calibration of the velocimeter was performed using a rotating glass disk coated with red blood cells. Vmean was calculated as follows: Vmean=Vrbc/1.6. Using the Newtonian definition, SR was calculated as follows: SR=8(Vmean/D), where D is venular diameter.
Leukocyte flux was determined on- and off-line as the number of leukocytes per minute rolling past a specified transverse plane within the venule over a 5-minute period. Vwbc was determined as the average time required for a leukocyte to traverse a 100-μm length of venule. A leukocyte was considered adherent to the venular endothelium if it remained stationary within a specified 100-μm length of the venule for ≥30 seconds. Leukocyte adhesion was expressed as the number of adherent cells per 100-μm length of venule over that 5-minute period. Emigrated leukocytes were determined on-line as the number of interstitial leukocytes in the field of view adjacent (within 40 μm) to the venule at the end of each recording period. The Vrbc/Vwbc ratio was calculated from the previously determined values.
Experimental Protocols for Intravital Microscopy
Experimental measurements were obtained after a 20-minute stabilization period with constant PBS suffusion. Preparations were considered acceptable when an appropriate-sized vessel maintained an SR of ≥500 s−1 throughout the experiment. The anesthetics administered and surgical manipulation associated with the establishment of this preparation can potentially influence the magnitude of the inflammatory cell recruitment elicited by specific stimuli. Hence, in order to minimize such influences on the inflammatory responses to hypercholesterolemia, both wild-type and LDLr−/− mice were exposed to identical anesthetic and surgical procedures before data collection. In order to assess the basal inflammatory state associated with each treatment group (after baseline measurements in control studies), PBS suffusion was continued for the 30-minute experimental period with repeat measurements obtained at 15 and 30 minutes after baseline determinations. Studies evaluating the acute inflammatory responses in these respective treatment groups were performed using the proinflammatory agent LTB4. In this group of experiments, LTB4 (5 nmol/L) was added to the suffusate (after baseline measurements were taken), and then repeat measurements were obtained at 15 and 30 minutes of LTB4 exposure. In order to determine whether the exaggerated inflammatory responses elicited by LTB4 treatment were unique to this lipid mediator, an additional series of adhesion experiments was performed with PAF (100 nmol/L) in the superfusate. Based on results in initial studies showing nearly equivalent responses in both HCD groups, all subsequent intravital microscopic experiments were performed using the ND and 4-week HCD dietary groups with each strain of mouse.
In order to define the adhesion molecules that mediate the enhanced leukocyte adherence elicited by LTB4 suffusion in LDLr−/− mice, MAbs known to block the firm adhesion of neutrophils and/or mononuclear leukocytes to endothelial cells were administered. These MAbs included the following: YN-1 (IgG2b), a rat anti-mouse ICAM-1 MAb20 ; MK1.9.1 (IgG1), a rat anti-mouse VCAM-1 MAb21 22 ; and R35-38 (rat IgG2b), a control-matched nonbinding MAb (Pharmingen). Because of results demonstrating no differences in the flux and rolling velocity of leukocytes after LTB4 treatment, anti-selectin antibodies were not evaluated. MAbs YN-1 and MK1.9.1 were provided by Dr Mary Gerritsen (Bayer Laboratories, West Haven, Conn) and have been shown to have functional blocking activity.20 21 22 All MAbs were administered at a dose of 2 mg/kg IV.
Because of our initial findings of an exaggerated inflammatory response in hypercholesterolemic mice that was accompanied by an elevated plasma TNF-α concentration (see below), we chose to examine whether exogenous TNF-α is as effective in eliciting leukocyte–endothelial cell adhesion in microvessels of wild-type and LDLr−/− mice. In this series of intravital microscopic experiments, TNF-α was administered intraperitoneally (5 μg/kg) 5 hours before intravital microscopic examination of the cremaster muscle. After the 20-minute stabilization period, three to five randomly selected venules and arterioles (25- to 35-μm diameter) were observed and analyzed for 5 minutes each. If at least three arterioles and three venules with SR of ≥500 s−1 could not be found, the animal was excluded. All measured values obtained from the three to five arterioles and venules were averaged to yield single arteriolar and venular values for each animal.
In Vivo Determination of Endothelial CAM Expression
Constitutive and induced expression of endothelial CAMs in striated muscle was determined in vivo as described previously.23 24 Briefly, specific binding MAbs directed against ICAM-1 (YN-1), VCAM-1 (MK1.9.1), P-selectin (RB40.34), and E-selectin (9A9) were radiolabeled with 125I using the iodogen method. Similarly, P-23, a nonbinding antibody, was labeled with 131I. The left jugular vein and caudal abdominal aorta of anesthetized LDLr−/− and B6129 mice (maintained on either the 4-week ND or 4-week HCD diet) were cannulated. Accordingly, a mixture of 125I-labeled binding MAb±unlabeled binding MAb and 131I-labeled P-23 was injected intravenously. Five minutes later, a sample of blood was obtained, followed by an isovolemic exchange of bicarbonate-buffered saline between the jugular and aortic cannulas. A sample of striated muscle was harvested after copious flushing of all tissues. Accumulated activities of 125I (binding MAb) and 131I (nonbinding MAb) in the muscle tissue were determined using a 14800 Wizard 3 gamma counter (Wallac). Radioactivity levels were also obtained from the blood sample and from an aliquot of the preinjection mixture of antibodies. Adhesion molecule expression was determined by subtracting the activity of the nonbinding MAb (131I-labeled P-23) from that of the binding MAb (125I-labeled YN-1, 125I-labeled MK1.9.1, 125I-labeled RB40.34, or 125I-labeled 9A9). This difference in radioactivities (expressed as a percentage of injected dose per gram tissue) was multiplied by the total amount of injected binding MAb (in micrograms) divided by 100, and endothelial CAM expression was calculated as micrograms of MAb per gram tissue.
Each endothelial CAM was measured under constitutive (unstimulated) conditions and 5 hours after TNF-α stimulation (25 μg/kg IP injection). In order to determine whether HCD affects the expression of preformed P-selectin, measurements of this endothelial CAM were obtained 5 minutes after intravenous administration of histamine (0.5 mg).24 RB40.34 was purchased from Pharmingen, whereas 9A9 and P-23 were gifts from Drs Barry Wolitzky (Hoffman–La Roche, Nutley, NJ) and Donald C. Anderson (Pharmacia-Upjohn, Kalamazoo, Mich), respectively.
Plasma TNF-α Bioassay
Previous studies have demonstrated elevated plasma and mRNA levels of TNF-α in LPS-challenged hypercholesterolemic rabbits.1 25 Since we have previously shown that TNF-α is a potent stimulus for increased ICAM-1 expression in different vascular beds,23 we chose to determine whether this cytokine could mediate the exaggerated inflammatory response observed in LDLr−/− mice. This was addressed by comparing plasma TNF-α levels in normal and hypercholesterolemic mice. Plasma murine TNF-α concentrations were determined using a commercially available enzyme-linked immunosorbent assay kit (Endogen). Constitutive and endotoxin (LPS, Salmonella abortus equi)–stimulated (1 mg/kg IP) levels were measured from samples obtained just before and 2 hours after LPS administration, respectively. These measurements were made in both wild-type and LDLr−/− mice placed on either a normal or high cholesterol diet.
All values are reported as mean±SEM, with statistical significance set at P<.05. Statistical analysis of studies in which comparisons were made between the normocholesterolemic (B6129 ND) and other dietary groups, and also within the LDLr−/− dietary groups, was performed using one-way ANOVA with Fisher’s least significant difference post hoc test. Studies in which comparisons were made only between the B6129 ND group and specific dietary groups were evaluated using Student’s t test (PAF and TNF-α intravital microscopy studies).
Plasma cholesterol levels attained in each strain of mouse when maintained on ND or placed on HCD for either 4 or 8 weeks are seen in Fig 1⇓. Wild-type mice developed a significant rise in plasma cholesterol level after either 4 or 8 weeks of high fat chow. The LDLr−/− mice, which had higher cholesterol levels on ND relative to the B6129 mice on ND, developed profound hypercholesterolemia (>2000 mg/dL) when on HCD for either 4 or 8 weeks.
In the control experiments (no inflammatory mediator applied to the cremaster), LDLr−/− mice maintained on HCD (LDLr−/− 4- and 8-week HCD groups, data not shown) sustained elevated levels of adherent leukocytes in postcapillary venules compared with their normocholesterolemic counterparts (Fig 2⇓). This accumulation of adherent leukocytes was observed during baseline measurements and throughout the experiments. No significant differences were found between dietary treatments within each strain of mouse. The relative constancy of leukocyte adhesion depicted in Fig 2⇓ indicates that surgical preparation of the cremaster does not induce a time-dependent leukocyte–endothelial cell adhesion response. Similar to adherent leukocytes, the number of emigrated cells was significantly higher in the 4-week HCD LDLr−/− mice at all time points in the control studies (Fig 2⇓). No other measured variables were significantly different between groups in the control studies (see Table 1⇓). Thus, the control data obtained from these experiments suggest the presence of a chronic inflammatory state in the two groups of mice with the highest plasma cholesterol levels.
Superfusion of the cremaster preparation with LTB4 after baseline measurements resulted in the rapid recruitment of adherent leukocytes in the postcapillary venules (Fig 3⇓). Comparisons between groups revealed a significantly greater number of adherent leukocytes in all three LDLr−/− dietary groups (8-week HCD group is not shown) versus the normocholesterolemic group (Fig 3⇓). Likewise, at 30 minutes, the mice with the highest cholesterol level had a significantly greater number of emigrated leukocytes. No statistically significant differences were noted for all other measured variables between treatments (Table 2⇓).
To determine whether the exaggerated leukocyte adhesion and emigration responses observed in LDLr−/− are unique to LTB4, we studied the inflammatory responses to another lipid mediator, ie, PAF. In this series of experiments, the leukocyte adhesion and emigration responses were compared between normocholesterolemic B6129 ND mice and both mouse strains on HCD for 4 weeks. PAF, like LTB4, elicited an exaggerated recruitment of adherent and emigrated leukocytes (5- and 3-fold, respectively) in the LDLr−/− HCD group (Fig 4⇓).
Since our LTB4 experiments had demonstrated an exaggerated recruitment of firmly adherent leukocytes with no differences in the flux or rolling velocities of the leukocytes, we then attempted to determine which adhesion molecules were responsible for the enhanced stationary adhesion in the LDLr−/− HCD group. Consequently, CD11/CD18–ICAM-1 and VLA4–VCAM-1 interactions were targeted with anti-mouse ICAM-1 (YN-1) and VCAM-1 (MK1.9.1) MAbs. As illustrated in Fig 5⇓, MAb YN-1 (anti–ICAM-1) significantly reduced the LTB4-induced recruitment of firmly adherent leukocytes, whereas neither MAb MK1.9.1 (anti–VCAM-1) nor MAb R35-38 (isotype-matched, nonbinding) had a significant effect on this response. These responses suggest that the exaggerated leukocyte adhesion response elicited by LTB4 in LDLr−/− HCD mice is mediated in large part by ICAM-1.
Table 3⇓ summarizes the data obtained from experiments that compare the microvascular inflammatory responses of control (B6129) and LDLr−/− HCD mice to the cytokine TNF-α. These experiments revealed that (similar to the lipid mediators LTB4 and PAF) TNF-α elicited an exaggerated inflammatory response in postcapillary venules of LDLr−/− HCD mice. There were some notable differences in the responses elicited by TNF-α compared with LTB4 and PAF, including an increased flux and decreased velocity of rolling leukocytes in venules. Another more striking difference was the recruitment of adherent leukocytes in arterioles of TNF-challenged LDLr−/− mice after 4 weeks of HCD.
In order to further assess the inflammatory nature of the hypercholesterolemic state in LDLr−/− mice, constitutive and LPS-stimulated plasma TNF-α levels were measured. Fig 6⇓ shows that constitutive levels of plasma TNF-α were not detected in wild-type (B6129) mice placed on normal chow, with low levels (<0.3 ng/mL) detected under basal conditions in the LDLr−/− mice on either diet (ND or HCD) and B6129 mice on HCD for 4 weeks. After challenge with LPS, the LDLr−/− 4-week HCD group responded with augmented plasma TNF-α production (P=.05 for the B6129 4-week HCD group).
The effects of hypercholesterolemia on constitutive and induced expression of ICAM-1, VCAM-1, P-selectin, and E-selectin in skeletal muscle of wild-type and LDLr−/− mice are summarized in Table 4⇓. The results of these experiments, based on the dual-radiolabeled MAb technique, indicate that both constitutive and TNF-α–induced expression of the different endothelial CAMs was virtually identical between the wild-type and LDLr−/− mice placed on either ND or HCD. However, constitutive expression of E-selectin was significantly higher in LDLr−/− than wild-type mice placed on ND. In addition, the constitutive expression of both E-selectin and VCAM-1 was lower in LDLr−/− placed on HCD than in their counterparts on ND.
The present study demonstrates that the chronic severe hypercholesterolemia observed in LDLr−/− mice placed on HCD is accompanied by a low-grade inflammatory state that is characterized by the accumulation of adherent and emigrated leukocytes in unstimulated postcapillary venules. In addition, these mutants (LDLr−/−), when placed on either ND or HCD, exhibited an exaggerated inflammatory response when challenged with different inflammatory agents (LTB4, PAF, or TNF-α). A noteworthy feature of the latter experiments was the accumulation of adherent leukocytes on the arteriolar side of the microvasculature. These enhanced inflammatory responses were not observed in wild-type (B6129) mice maintained on HCD, suggesting that the diet alone was not sufficient to elicit an augmented inflammatory reaction in mice.
LTB4 and PAF are known for their ability to elicit microvascular responses that are characteristic of acute inflammation.26 Both inflammatory mediators have been shown to promote the recruitment of adherent and emigrated leukocytes when applied to venules in otherwise normal animals.26 The leukocyte–endothelial cell adhesion elicited by LTB4 and PAF appears to involve an interaction between the β2 integrin CD11/CD18 on leukocytes and constitutively expressed ICAM-1 on endothelial cells.26 Elevated tissue levels of both lipid mediators have been described for various neutrophil-dependent inflammatory diseases, including ischemia/reperfusion.27 28 29 30 31 Furthermore, antagonists to LTB4 and PAF appear to be very effective in attenuating the recruitment of adherent leukocytes observed in postcapillary venules exposed to acute inflammatory stimuli such as ischemia/reperfusion.29 30 31 The findings of the present study indicate that the microvasculature of LDLr−/− mice placed on HCD are particularly sensitive to the proinflammatory actions of both LTB4 and PAF. This observation supports the view that the vascular abnormalities associated with chronic hypercholesterolemia and atherosclerosis are not confined to the sites of lesion formation in large arteries.
Previous studies have shown that hypercholesterolemic rabbits exhibit more profound increases in plasma TNF-α and mRNA levels after LPS stimulation than do their normocholesterolemic counterparts.1 25 Our studies in LDLr−/− mice support this observation, inasmuch as HCD-fed LDLr−/− mice produced more TNF-α when challenged with LPS than did wild-type mice placed on the same HCD. However, our findings significantly extend this observation by demonstrating that both the arterial and venous segments of the microvasculature of hypercholesterolemic LDLr−/− mice are far more responsive to the inflammatory actions of TNF-α than are those segments in wild-type mice placed on the same dietary regimen. This observation, coupled with the exaggerated inflammatory responses elicited by LTB4 and PAF, indicates that the increased sensitivity to inflammatory stimuli in LDLr−/− mice on HCD is not likely due to an increased number and/or affinity of receptors (on leukocytes and/or endothelial cells) for a single mediator or specific class of mediators (eg, cytokines). Furthermore, the observation that exogenous TNF elicited a larger adhesion response in postcapillary venules of LDLr−/− (with its higher circulating TNF level) than in wild-type mice would argue against the elevated plasma TNF level as the principal cause of the exaggerated inflammatory response.
Although the present study does not provide definitive information concerning the specific mechanism that accounts for the exaggerated inflammatory responses in the microvasculature of LDLr−/− mice, there are some possibilities that warrant consideration. One possibility is the existence of a factor in LDLr−/− mice (when placed on HCD) that acts to prime leukocytes and/or endothelial cells to the inflammatory actions of LTB4, PAF, and TNF-α. Candidate priming agents include oxidized LDL and products of LDL oxidation, such as lysophosphatidylcholine and PAF-like lipids. Lysophosphatidylcholine has been shown to be a chemoattractant,32 to induce VCAM-1 and ICAM-1 expression,33 and to block endothelium-dependent vasorelaxation.34 Oxidized LDL, but not native LDL, has been shown to directly inactivate NO and inhibit its formation and/or release. Studies suggest that the primary oxidized lipid responsible for the decreased bioavailability of NO in hypercholesterolemia is lysophosphatidylcholine.8 34 35 Oxidized LDL has been shown to also contain oxidatively modified phospholipids with PAF-like activity that can be blocked using PAF receptor antagonists.36 Indeed, the increased leukocyte–endothelial cell adhesion seen after intravenous administration of oxidized LDL is attenuated by PAF receptor blockade, thus suggesting an important role for PAF or PAF-like lipids in the recruitment and activation of leukocytes in hypercholesterolemia.37
An alternative explanation for the enhanced recruitment of adherent leukocytes in lipid mediator–stimulated or cytokine-stimulated microvessels of LDLr−/− mice may relate to the level of expression of endothelial CAMs. Histopathological evaluation of atherosclerotic arteries have shown increased expression of VCAM-1, ICAM-1, and P- and E-selectin in the lesioned areas.38 39 40 These observations are supported by in vitro studies showing that highly oxidized LDL induces endothelial expression of VCAM-1 and ICAM-1.33 Alternatively, mildly oxidized LDL does not cause ICAM-1 or VCAM-1 expression and actually may reduce E-selectin levels.41 42 Hence, the expression of inducible endothelial CAMs appears to be dependent on the severity of LDL oxidation. The MAb-blocking data presented herein reveals a primary role for the interaction of ICAM-1 (presumably, the constitutively expressed form) with its ligand (CD11/CD18) in mediating the recruitment of adherent leukocytes in LDLr−/− after an acute inflammatory challenge with LTB4. This observation is consistent with findings of Lehr et al43 and Kurtel et al,44 which implicate CD11/CD18 in the leukocyte adhesion responses elicited by intravascular administration of oxidized LDL or oxidized chylomicrons.
We used the dual radiolabeled MAb technique to quantify the expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 in skeletal muscle vasculature of both wild-type and LDLr−/− mice that were placed on either ND or HCD. Our results indicate that there are no substantial differences in the level of endothelial CAM expression between normal and chronically hypercholesterolemic mice. Hence, these findings suggest that the increased endothelial CAM expression demonstrated by immunohistochemistry in large arteries38 40 does not appear to occur at the microvascular level.
Irrespective of the mechanisms that underlie the exaggerated inflammatory responses noted in the microvasculature of LDLr−/− mice on HCD, our findings suggest that humans with familial hypercholesterolemia may be at greater risk for developing acute inflammation and the microvascular dysfunction that generally accompanies this condition. Inasmuch as LTB4, PAF, and TNF-α have all been implicated in the microvascular and tissue injury associated with ischemia/reperfusion, our findings in LDLr−/− mice suggest that individuals with familial hypercholesterolemia may have an increased risk for not only developing ischemia (due to atherosclerotic vessel occlusion) but also for sustaining ischemia/reperfusion injury. Additional work is needed to address this possibility and to define the cellular and molecular basis for the exaggerated inflammatory responses observed in animals that are genetically deficient in the LDL receptor.
Selected Abbreviations and Acronyms
|CAM||=||cell adhesion molecule|
|HCD||=||high cholesterol diet|
|ICAM||=||intercellular adhesion molecule|
|LDLr−/−||=||LDL receptor–deficient (knockout)|
|TNF||=||tumor necrosis factor|
|VCAM||=||vascular cell adhesion molecule|
|Vmean||=||mean red blood cell velocity|
|Vrbc||=||red blood cell velocity|
|Vwbc||=||leukocyte rolling velocity|
This study was supported by a grant from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL-26441). The authors wish to thank Janice Russell for her technical help with the TNF-α bioassays and Julie Morris (statistician) for her assistance with the statistical analysis.
- Received February 20, 1997.
- Accepted May 12, 1997.
- © 1997 American Heart Association, Inc.
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