Deletion of the complement phagocytic receptors CR3 and CR4 does not alter susceptibility to experimental cerebral malaria.

Complement receptors for C3-derived fragments (CR1-4) play critical roles in innate and adaptive immune responses. Of these receptors, CR3 and CR4 are important in binding and phagocytosis of complement-opsonized pathogens including parasites. The role of CR3 and CR4 in malaria or in cerebral malaria (CM) has received little attention and remains poorly understood in both human disease and rodent models of malaria. CR3 and CR4 are members of the ß(2) -integrin family of adhesion molecules and are expressed on all leucocytes that participate in the development of CM, most importantly as it relates to parasite phagocytosis (monocytes/macrophages) and antigen processing and presentation (dendritic cells). Thus, it is possible that these receptors might play an important role in disease development. To address this question, we examined the role of CR3(-/-) and CR4(-/-) in experimental cerebral malaria (ECM). We found that both CR3(-/-) and CR4(-/-) mice were fully susceptible to ECM and developed disease comparable to wild-type mice. Our results indicate that CR3 and CR4 are not critical to the pathogenesis of ECM despite their role in elimination of complement-opsonized pathogens. These findings support recent studies indicating the importance of the terminal complement pathway and the membrane attack complex in ECM pathogenesis.

Cerebral leucocyte infiltration in lupus-prone MRL/MpJ-fas lpr mice--roles of intercellular adhesion molecule-1 and P-selectin.

The autoimmune disease which affects MRL/MpJ-fas(lpr) mice results in cerebral leucocyte recruitment and cognitive dysfunction. We have previously observed increased leucocyte trafficking in the cerebral microcirculation of these mice; however, the types of leucocytes recruited have not been analysed thoroughly, and the roles of key endothelial adhesion molecules in recruitment of these leucocytes have not been investigated. Therefore the aim of this study was to classify the phenotypes of leucocytes present in inflamed brains of MRL/MpJ-fas(lpr) mice, and dissect the roles of endothelial adhesion molecules in their accumulation in the brain. Immunohistochemical analysis revealed significant leucocyte infiltration in the brains of 16- and 20-week-old MRL/MpJ-fas(lpr) mice, affecting predominantly the choroid plexus. Isolation of brain-infiltrating leucocytes revealed that lymphocytes and neutrophils were the main populations present. The CD3(+) lymphocytes in the brain consisted of similar proportions of CD4(+), CD8(+) and CD4(-)/CD8(-)[double negative (DN)] populations. Assessment of MRL/MpJ-fas(lpr) mice deficient in endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1) or P-selectin indicated that cerebral leucocyte recruitment persisted in the absence of these molecules, with only minor changes in the phenotypes of infiltrating cells. Together these data indicate that the brains of MRL/MpJ-fas(lpr) mice are affected by a mixed leucocyte infiltrate, of which the unusual DN lymphocyte phenotype contributes a substantial proportion. In addition, endothelial adhesion molecules ICAM-1 and P-selectin, which modulate survival of MRL/MpJ-fas(lpr) mice, do not markedly inhibit leucocyte entry into the central nervous system.

Regulation of endothelial glutathione by ICAM-1: implications for inflammation.

The role of glutathione (GSH) in inflammation is largely discussed from the context of providing reducing equivalents to detoxify reactive oxygen and nitrogen species. Inflammation is now recognized to be an underlying cause of many vascular diseases including atherosclerosis, a disease in which endothelial GSH concentrations are decreased. However, mechanisms that control GSH levels are poorly understood. Key players in the inflammatory process are endothelial adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). This adhesion molecule is present constitutively and can be induced by a variety of inflammatory stimuli. In this study, using mouse aortic endothelial cells (MAEC) deficient in ICAM-1, we demonstrate a novel interplay between constitutive ICAM-1 and cellular GSH. Deficiency of ICAM-1 was associated with an approximately twofold increase in total GSH content. Inhibiting glutamate-cysteine ligase (GCL), the enzyme that catalyses the rate-limiting step in GSH biosynthesis, prevented the increase in GSH. In addition, the catalytic subunit of GCL was increased (approximately 1.6-fold) in ICAM-1 deficient relative to wild-type cells, suggesting that constitutive ICAM-1 represses GCL expression. Furthermore, the ratio of reduced (GSH) to oxidized (GSSG) glutathione was also increased suggesting a role for ICAM-1 in modulating cellular redox status. Interestingly, increasing cytosolic GSH in wild-type mouse endothelial cells decreased constitutive ICAM-1, suggesting the presence of an inverse and reciprocal pathway. To test the effects of inducible ICAM-1 on GSH, cells were stimulated with the proinflammatory cytokine TNF-alpha. TNF-alpha stimulated production of ICAM-1, which was however not associated with induction of GSH. In contrast, supplementation of endothelial cells with GSH before TNF-alpha addition, inhibited induction of ICAM-1. These data suggest a novel regulatory pathway between constitutive ICAM-1 and GSH synthesis in the endothelium and are discussed in the context of modulating the inflammatory response.

Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration.

Nitrotyrosine formation is a hallmark of vascular inflammation, with polymorphonuclear neutrophil-derived (PMN-derived) and monocyte-derived myeloperoxidase (MPO) being shown to catalyze this posttranslational protein modification via oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (NO(2)(*)). Herein, we show that MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism and serves as a catalyst of ECM protein tyrosine nitration. Purified MPO and MPO released by intraluminal degranulation of activated human PMNs avidly bound to aortic endothelial cell glycosaminoglycans in both cell monolayer and isolated vessel models. Cell-bound MPO rapidly transcytosed intact endothelium and colocalized abluminally with the ECM protein fibronectin. In the presence of the substrates hydrogen peroxide (H(2)O(2)) and NO(2)(-), cell and vessel wall-associated MPO catalyzed nitration of ECM protein tyrosine residues, with fibronectin identified as a major target protein. Both heparin and the low-molecular weight heparin enoxaparin significantly inhibited MPO binding and protein nitrotyrosine (NO(2)Tyr) formation in both cultured endothelial cells and rat aortic tissues. MPO(-/-) mice treated with intraperitoneal zymosan had lower hepatic NO(2)Tyr/tyrosine ratios than did zymosan-treated wild-type mice. These data indicate that MPO significantly contributes to NO(2)Tyr formation in vivo. Moreover, transcytosis of MPO, occurring independently of leukocyte emigration, confers specificity to nitration of vascular matrix proteins.

Essential role of ICAM-1 in mediating monocyte adhesion to aortic endothelial cells.

Monocyte-endothelial cell interactions have been implicated in the pathogenesis of a number of vascular diseases that target arterial and aortic endothelium, including atherosclerosis. Many different adhesion molecules, such as intercellular adhesion molecule (ICAM)-1, are thought to mediate monocyte binding to endothelial cells during the development of these diseases. However, conflicting results have been reported regarding the specific role of ICAM-1 in these events. In this study, we used a genetic approach to determine the contribution of ICAM-1 in mediating monocyte adhesion to mouse aortic endothelial cells (MAEC) derived from both wild-type and ICAM-1(-/-) mice. Treatment of wild-type MAEC with oxidized low-density lipoprotein significantly induced both WEHI 274.1 and whole blood monocyte adhesion, whereas similarly treated ICAM-1(-/-) MAEC showed a complete inhibition of monocyte binding. Dose-response treatment with tumor necrosis factor-alpha also increased monocyte adhesion to wild-type MAEC, but significant adhesion was only observed at higher doses for ICAM-1(-/-) MAEC. These data demonstrate a crucial role for ICAM-1-mediated monocyte-endothelial cell interactions in response to specific stimuli involved in inflammatory vascular diseases.

In vitro culture and characterization of gene targeted mouse endothelium.

Endothelial cells play a crucial role in maintaining cardiovascular homeostasis. Although many cardiovascular disorders involve endothelial cell dysfunction, the specific cellular and molecular mechanisms involved are not well known. We sought to establish a reproducible method of endothelial cell isolation from gene targeted mice to specifically examine endothelial pathophysiological mechanisms. Primary aortic endothelial cell cultures were established from wild type and intercellular adhesion molecule-1 (ICAM-1) deficient mice. Isolation of mouse aortic endothelial cells (MAEC) by fluorescent activated cell sorting routinely resulted in pure, homogenous, primary cultures. Wild type and ICAM-1 deficient endothelial cell morphology was similar, with both cultures showing cobblestone morphology and DiI-Ac-LDL staining. Monocyte adhesion to ICAM-1 deficient aortic endothelial cells was decreased by 86% as compared with wild type MAEC. Monocyte adhesion was also determined using YN-1, an ICAM-1 blocking antibody. YN-1 decreased monocyte adhesion to wild type aortic endothelial cells by 25%, whereas YN-1 did not further decrease monocyte adhesion to ICAM-1 deficient MAEC. These data demonstrate that gene targeted endothelial cell cultures are an effective means of identifying specific cellular and molecular mechanisms involved in endothelial cell physiology and dysfunction.

Dermal and pulmonary inflammatory disease in E-selectin and P-selectin double-null mice is reduced in triple-selectin-null mice.

In the initial phase of an inflammatory response, leukocytes marginate and roll along the endothelial surface as a result of adhesive interactions between molecules on the endothelial cells and leukocytes. To evaluate the role of the 3 selectins (E, L, and P) in leukocyte rolling and emigration, a null mutation for L-selectin was introduced into previously described embryonic stem cells with null mutations in the genes for both E-selectin and P-selectin (E/P double mutants) to produce triple-selectin-null mice (E-selectin, L-selectin, and P-selectin [E/L/P] triple mutants). Triple-selectin homozygous mutant mice are viable and fertile and only rarely develop the severe mucocutaneous infections or pulmonary inflammation characteristic of E/P double-mutant mice. Surface expression of L-selectin was undetectable in triple-mutant mice on fluorescence-activated cell-sorter analysis of peripheral neutrophils. Pathological studies revealed moderate cervical lymphadenopathy and lymphoplasmacytic infiltrate, but these were less extensive than in E/P double-mutant mice. Neutrophil emigration during thioglycolate-induced peritonitis was significantly reduced at 4, 8, and 24 hours (35%, 65%, and 46% of wild-type values, respectively). Intravital microscopy of the cremaster muscle revealed almost no rolling at times up to 6 hours after exteriorization, with or without addition of tumor necrosis factor alpha. The small amount of residual rolling was dependent on alpha(4)-integrin. The occurrence of skin and pulmonary disease in E/P double-mutant mice but not E/L/P triple-mutant mice suggests that deficiency of L-selectin alters the inflammatory response in E/P mutants. (Blood. 2001;98:727-735)

Severe inflammatory defect and reduced viability in CD18 and E-selectin double-mutant mice.

CD18-deficient mice (CD18(-/-) mice) have a severe leukocyte recruitment defect in some organs, and no detectable defect in other models. Mice lacking E-selectin (CD62E(-/-) mice) have either no defect or a mild defect of neutrophil infiltration, depending on the model. CD18(-/-)CD62E(-/-), but not CD18(-/-)CD62P(-/-), mice generated by crossbreeding failed to thrive, reaching a maximum body weight of 10-15 grams. To explore the mechanisms underlying reduced viability, we investigated lethally irradiated CD62E(-/-) mice that were reconstituted with CD18(-/-) bone marrow. These mice, but not single-mutant controls, showed tenfold-increased rolling velocities in a TNF-alpha-induced model of inflammation. Leukocyte adhesion efficiency in CD18(-/-)CD62E(-/-) mice was reduced by 95%, and hematopoiesis was drastically altered, including severe bone marrow and blood neutrophilia and elevated G-CSF and GM-CSF levels. The greatly reduced viability of CD18(-/-)CD62E(-/-) mice appears to result from an inability to mount an adequate inflammatory response. Our data show that cooperation between E-selectin and CD18 integrins is necessary for neutrophil recruitment and that alternative adhesion pathways cannot compensate for the loss of these molecules.

Leukocyte and endothelial cell adhesion molecules in a chronic murine model of myocardial reperfusion injury.

Expression of endothelial and leukocyte cell adhesion molecules is a principal determinant of polymorphonuclear neutrophil (PMN) recruitment during inflammation. It has been demonstrated that pharmacological inhibition of these molecules can attenuate PMN influx and subsequent tissue injury. We determined the temporal expression of alpha-granule membrane protein-40 (P-selectin), endothelial leukocyte adhesion molecule 1 (E-selectin), and intercellular cell adhesion molecule 1 (ICAM-1) after coronary artery occlusion and up to 3 days of reperfusion. The expression of all of these cell adhesion molecules peaked around 24 h of reperfusion. We determined the extent to which these molecules contribute to PMN infiltration by utilizing mice deficient (-/-) in P-selectin, E-selectin, ICAM-1, and CD18. Each group underwent 30 min of in vivo, regional, left anterior descending (LAD) coronary artery ischemia and 24 h of reperfusion. PMN accumulation in the ischemic-reperfused (I/R) zone was assessed using histological techniques. Deficiencies of P-selectin, E-selectin, ICAM-1, or CD18 resulted in significant (P < 0.05) attenuation of PMN infiltration into the I/R myocardium (MI/R). In addition, P-selectin, E-selectin, ICAM-1, and CD18 -/- mice exhibited significantly (P < 0.05) smaller areas of necrosis after MI/R compared with wild-type mice. These data demonstrate that MI/R induces coronary vascular expression of P-selectin, E-selectin, and ICAM-1 in mice. Furthermore, genetic deficiency of P-selectin, E-selectin, ICAM-1, or CD18 attenuates PMN sequestration and myocardial injury after in vivo MI/R. We conclude that P-selectin, E-selectin, ICAM-1, and CD18 are involved in the pathogenesis of MI/R injury in mice.

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.

Acceleration and increased severity of collagen-induced arthritis in P-selectin mutant mice.

P-selectin plays an important role in leukocyte adherence to microvascular endothelium and is expressed in synovial tissue from patients with rheumatoid arthritis (RA). However, the contribution of P-selectin to the initiation and chronicity of joint inflammation is not well understood. In these studies, collagen-induced arthritis (CIA) was induced in P-selectin mutant (-/-) mice to explore the role of P-selectin in the development of joint inflammation. Surprisingly, CIA onset was accelerated and severity was increased in P-selectin mutant mice, compared with wild-type mice (+/+). Increased levels of anti-type II collagen IgG were detected in both nonarthritic and arthritic P-selectin mutant mice from days 14-91. In addition, splenocytes isolated from immunized and nonimmunized P-selectin mutant mice produced significantly less IL-2 and IL-4, but significantly higher levels of IL-10 and IL-5 than splenocytes from wild-type mice. These observations show that P-selectin-mediated leukocyte rolling is not required for the development of murine CIA and that P-selectin expression exerts a controlling effect on the development of Ag-driven inflammatory joint disease, possibly by mediating the recruitment and/or trafficking of specific leukocyte subtypes into lymphoid tissue or inflammatory foci.

Chronic inflammatory disease alters adhesion molecule requirements for acute neutrophil emigration in mouse skin.

Mutant mice triply deficient in ICAM-1, E-selectin, and P-selectin did not develop the neutrophilic skin lesions that spontaneously arise in mutants doubly deficient in E-selectin and P-selectin. Thus, ICAM-1 is essential to skin disease resulting from endothelial selectin deficiency. During experimental dermatitis, acute neutrophil emigration was completely prevented in young mice deficient in both selectins (E/P and E/P/I mutants). However, older E/P mutants with spontaneous skin lesions displayed an endothelial selectin-independent pathway for acute neutrophil emigration. In contrast, emigration remained compromised in E/P/I mutants and CD18 mutants regardless of age or lesions. Experimentally induced chronic lesions elicited this pathway for acute emigration in young E/P mutants. Thus, an endothelial selectin-independent pathway for acute neutrophil emigration is induced in E/P mice by chronic inflammation at distant sites, and this pathway may contribute to skin disease resulting from endothelial selectin deficiency.

Combinatorial requirements for adhesion molecules in mediating neutrophil emigration during bacterial peritonitis in mice.

To investigate the requirements for adhesion molecules in neutrophil emigration during peritonitis, mice received intraperitoneal injections of Streptococcus pneumoniae while the functions of multiple adhesion molecules were blocked. Emigration after 4 h was compromised by antibodies against ICAM-1 or genetic deficiency of ICAM-1. Anti-CD11a/CD18 antibodies decreased emigration in ICAM-1 mutant mice, suggesting that ICAM-1 independent emigration requires CD11/CD18 complexes. In contrast, mice mutant in ICAM-1 plus E-selectin showed no defect in emigration, suggesting that E-selectin commits neutrophils to an ICAM-1-dependent pathway during streptococcal peritonitis. However, in mutant mice lacking the three endothelial adhesion molecules E-selectin, P-selectin, and ICAM-1, emigration after 4 h was significantly compromised. Thus, P-selectin is essential to ICAM-1- and E-selectin-independent acute peritoneal inflammation. After 24 h of peritonitis, there were no differences between WT and E-selectin/P-selectin/ICAM-1 mutant mice, demonstrating that these endothelial adhesion molecules are not essential to neutrophil emigration during later stages of peritonitis.

Importance of E-selectin for firm leukocyte adhesion in vivo.

Leukocyte adhesion under flow is preferentially mediated by the selectins. In this study we used intravital microscopy to investigate whether E-selectin may promote firm leukocyte adhesion in vivo. E-Selectin is expressed by endothelial cells activated with tumor necrosis factor-alpha (TNF-alpha) and causes slow leukocyte rolling. Microinjection of formyl-methionyl-leucyl-phenylalanine (fMLP) or macrophage inflammatory protein-2 (MIP-2) next to a venule of the TNF-alpha-treated mouse cremaster muscle significantly increased the number of adherent leukocytes. In gene-targeted mice homozygous for a null mutation in the E-selectin gene or in wild-type mice treated with an E-selectin monoclonal antibody (mAb), this response was significantly attenuated (by >80%). No such defect was seen in intercellular adhesion molecule-1 (ICAM-1)-deficient mice. E-Selectin-null mice showed more rapid leukocyte rolling than wild-type or ICAM-1-deficient mice, resulting in significantly shortened leukocyte transit times through venules. Topical application of fMLP onto the whole cremaster muscle generated the same number of adherent leukocytes in wild-type and E-selectin-deficient mice. We conclude that slow leukocyte rolling through E-selectin results in long transit times, which are essential for efficient leukocyte adhesion in response to a local chemotactic stimulus.

Gene-targeted mice reveal importance of L-selectin-dependent rolling for neutrophil adhesion.

It has not been determined whether L-selectin-mediated rolling can promote leukocyte adhesion in vivo independent of P- and E-selectin. We used intravital microscopy of E- and P-selectin double-mutant mice (E-/P-) stimulated with tumor necrosis factor-alpha for 6-8 h to investigate the importance of L-selectin-dependent rolling in cremaster muscle venules. Rolling leukocyte flux in E-/P- mice was 9 +/- 2 cells/min compared with 77 +/- 17 cells/min in wild-type (WT) mice. Pretreatment with the L-selectin monoclonal antibody MEL-14 significantly reduced rolling in both E-/P- (by 89%) and WT mice (by 79%). L-selectin-dependent rolling in E-/P- mice resulted in leukocyte adhesion comparable to that seen in WT mice. MEL-14 pretreatment of E-/P- mice reduced leukocyte adhesion by 50%. The majority (approximately 80%) of intravascular leukocytes in both WT and E-/P- mice were neutrophils. We conclude that L-selectin can mediate rolling that results in sufficient leukocyte recruitment to account for the robust inflammatory response seen in E-/P- mice at later times.

Host defense against systemic infection with Streptococcus pneumoniae is impaired in E-, P-, and E-/P-selectin-deficient mice.

Endothelial selectins mediate rolling of leukocytes on endothelium, a crucial step for leukocyte firm adhesion and emigration into sites of tissue injury and infection. To characterize the role of the endothelial selectins during bacterial sepsis in vivo, Streptococcus pneumoniae (1-10 x 10(6) colony-forming units) was inoculated intraperitoneally into wild-type mice and mice with E-, P-, or E-/P-selectin deficiencies. Mice were followed 10 d for morbidity, survival, clearance of bacteremia, and leukocyte migration to the peritoneal cavity and organs 48 h after infection. All selectin-deficient mice showed a more pronounced morbidity, a significantly higher mortality associated with persistent bacteremia, and a higher bacterial load when compared with wild-type mice. These differences were most remarkable in the E-selectin-deficient mice, which showed the highest rate of mortality and bacteremia (P

Intercellular adhesion molecule-1 deficiency protects MRL/MpJ-Fas(lpr) mice from early lethality.

MRL/MpJ-Fas(lpr) (Fas(lpr)) mice develop a rapidly fatal form of systemic autoimmune disease characterized by glomerulonephritis and vasculitis similar to severe cases of systemic lupus erythematosus in humans. To evaluate the requirement for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of tissue injury in this model, we created ICAM-1-deficient MRL/MpJ-Fas(lpr) (ICAM-1/Fas(lpr)) mice. ICAM-1 deficiency resulted in a striking improvement in the survival of Fas(lpr) mice (median +/- SEM survival of Fas(lpr) = 26 +/- 1.7 vs ICAM-1/Fas(lpr) = 47 +/- 2.4 wk, p < 0.0001) and the increased survival was associated with delayed elevations of blood urea nitrogen levels in the ICAM-1/Fas(lpr) mice. Histologic examination of the ICAM-1/Fas(lpr) mice revealed an overall reduction in glomerular disease and a significant reduction in vasculitis in the kidney, lung, skin, and salivary glands when compared with Fas(lpr). These findings indicate that ICAM-1 plays a major role in development of glomerular and vascular injury in Fas(lpr) mice.

Deficiency of inflammatory cell adhesion molecules protects against atherosclerosis in mice.

Leukocyte and endothelial cell adhesion molecules (CAMs) are essential for emigration of leukocytes, with the selectins mediating the initial step of leukocyte "rolling" and the beta 2-(CD18) integrins and intercellular adhesion molecule-1 (ICAM-1) being important for firm adhesion and emigration. On the basis of evidence for an inflammatory component in the pathogenesis of atherosclerosis, including increased expression of CAMs, cytokines, and growth factors, we tested the hypothesis that decreased expression of inflammatory CAMs would reduce susceptibility to atherosclerosis. Using C57BL/6 mice fed a high-fat diet, we observed a 50% to 75% reduction in atherosclerotic fatty streaks in mice with homozygous mutations for ICAM-1, P-selectin, CD18, both ICAM-1 and CD18, or both ICAM-1 and P-selectin. In contrast to previous evidence of increased expression of CAMs in atherosclerotic lesions, which does not prove a cause-and-effect relationship, these data indicate directly that the level of expression of CAMs can determine the susceptibility to the formation of atherosclerotic fatty streaks. The results suggest that genetic variation at these loci could influence susceptibility to atherosclerosis and that pharmacological reduction of the expression or function of these CAMs might protect against atherosclerosis.