Platelet-associated NAD(P)H oxidase contributes to the thrombogenic phenotype induced by hypercholesterolemia

Elevated cholesterol levels promote proinflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN), and NAD(P)H oxidase-knockout (gp91(phox-/-)) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. HC elicited increased platelet and leukocyte adhesion in WT mice versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia; however, platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN-HC and gp91(phox-/-)-HC mice. Recruitment of platelets derived from WT-HC mice in venules of SOD-TgN-HC or gp91(phox-/-)-HC recipients was comparable to ND levels. Adhesion of SOD-TgN-HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN-HC recipients, but not in WT-HC recipients. However, gp91(phox-/-)-HC platelets exhibited low levels of adhesion comparable to those of WT-ND in both hypercholesterolemic gp91(phox-/-) and WT recipients. Arteriolar dysfunction was evident in WT-HC mice, compared to WT-ND. Overexpression of SOD or, to a lesser extent, gp91(phox) deficiency restored arteriolar vasorelaxation responses toward WT-ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC-induced arteriolar dysfunction.     

Role of interleukin 12 in hypercholesterolemia-induced inflammation

We have previously shown that T lymphocytes and interferon-gamma are involved in hypercholesterolemia-induced leukocyte adhesion to vascular endothelium. This study assessed the contribution of interleukin 12 (IL-12) to these hypercholesterolemia-induced inflammatory responses. Intravital videomicroscopy was used to quantify leukocyte adhesion and emigration and oxidant stress (dihydrorhodamine oxidation) in unstimulated cremasteric venules (wall shear rate > or =500 s-1) of wild-type (WT) C57Bl/6, lymphocyte-deficient [recombinase-activating gene knockout (RAG1-/-)], and IL-12-deficient (p35-/- and p40-/-; p35 and p40 are the two subunits of active IL-12) mice on either a normal (ND) or high-cholesterol (HC) diet for 2 wk. RAG1-/--HC mice received splenocytes from WT-HC (WT --> RAG1-/-), p35-/--HC (p35-/- --> RAG1-/-), or p40-/--HC (p40-/- --> RAG1-/-) mice. Compared with WT-ND mice, WT-HC mice exhibited exaggerated leukocyte adherence and emigration as well as increased dihydrorhodamine oxidation. The enhanced leukocyte recruitment was absent in the RAG1-/--ND, p35-/--ND, and p40-/--ND groups. Hypercholesterolemia-induced leukocyte adherence and emigration were attenuated in RAG1-/--HC vs. WT-HC mice but were similar to ND mice. Furthermore, compared with WT-HC animals, p35-/--HC and p40-/--HC mice showed significantly lower leukocyte adhesion and tissue oxidant stress responses, but these values were comparable to ND mice. Leukocyte adherence and emigration in WT --> RAG1-/- mice were similar to responses of WT-HC mice. However, p35-/- --> RAG1-/- mice had lower levels of adherence and emigration vs. the WT --> RAG1-/- and WT-HC groups. Elevated levels of leukocyte adherence and emigration were restored by approximately 50% toward WT-HC levels in p40-/- --> RAG1-/- mice. These findings implicate IL-12 in the inflammatory responses observed in the venules of hypercholesterolemic mice.     

Chronic intermittent hypoxia induces atherosclerosis by NF-κB-dependent mechanisms

Chronic intermittent hypoxia (CIH) causes atherosclerosis in mice fed a high cholesterol diet (HCD). The mechanisms by which CIH promotes atherosclerosis are incompletely understood. This study defined the mechanistic role of NF-κB pathway in CIH+HCD induced atherosclerosis. Wild type (WT) and mice deficient in the p50 subunit of NF-κB (p50-KO) were fed normal chow diet (ND) or HCD, and exposed to sham or CIH. Atherosclerotic lesions on the en face aortic preparation and cross-sections of aortic root were examined. In WT mice, neither CIH nor HCD exposure alone caused, but CIH+HCD caused evident atherosclerotic lesions on both preparations after 20weeks of exposure. WT mice on ND and exposed to CIH for 35.6weeks did not develop atherosclerotic lesions. P50 gene deletion diminished CIH+HCD induced NF-κB activation and abolished CIH+HCD induced atherosclerosis. P50 gene deletion inhibited vascular wall inflammation, reduced hepatic TNF-α level, attenuated the elevation in serum cholesterol level and diminished macrophage foam cell formation induced by CIH+HCD exposure. These results demonstrate that inhibition of NF-κB activation abrogates the activation of three major atherogenic mechanisms associated with an abolition of CIH+HCD induced atherosclerosis. NF-κB may be a central common pathway through which CIH+HCD exposure activates multiple atherogenic mechanisms, leading to atherosclerosis.     

Superoxide mediates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules

Platelets have been implicated in the pathogenesis of different diseases of the vascular system, including atherosclerosis, sepsis, and ischemia-reperfusion injury; however, relatively little is known about the factors that regulate the interactions between circulating platelets and the vessel wall. The objective of this study was to define the contribution of superoxide to LPS-induced platelet-endothelial cell (P/E) adhesion in murine intestinal venules. The adhesion of rhodamine-6G-labeled murine platelets was monitored by intravital fluorescence microscopy. Four hours after LPS administration in control [wild-type (WT)] mice, an approximately 10-fold increase in P/E adhesion was detected. This response did not result from LPS-induced platelet activation. The LPS-induced P/E adhesion was greatly attenuated in NAD(P)H oxidase-deficient mice and in WT mice rendered neutropenic with anti-neutrophil serum, whereas the response was unchanged in WT mice receiving a CD18 blocking MAb or in CD18-deficient mice. A chimeric form of MnSOD that exhibits the binding properties of extracellular SOD also attenuated the LPS-induced response in WT mice. These findings indicate that neutrophil-derived superoxide plays a major role in the modulation of endotoxin-induced P/E adhesion.     

Nitric oxide modulates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules

Although platelets have been implicated in the pathogenesis of vascular diseases, little is known about factors that regulate interactions between platelets and the vessel wall under physiological conditions. The objectives of this study were to 1) define the contribution of nitric oxide (NO) to endotoxin (lipopolysaccharide, LPS)-induced platelet-endothelial cell (P/E) adhesion in murine intestinal venules and 2) determine whether the antiadhesive action of NO is mediated by soluble guanylate cyclase (sGC). Adhesive interactions between platelets and endothelial cells were monitored by intravital microscopy. LPS administration into control wild-type mice (WT) resulted in a >15-fold increase in P/E adhesion. Similar responses were observed using endothelial NO synthase (eNOS)-deficient platelets. However, treatment with the NO donor diethylenetriamine-nitric oxide (DETA-NO) attenuated the P/E adhesion response to LPS, whereas the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester or eNOS deficiency resulted in an exacerbation. P/E adhesion response did not differ between LPS-treated WT and inducible NOS-deficient mice. Inhibition of sGC abolished the attenuating effects of DETA-NO, whereas the sGC activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) reduced LPS-induced P/E adhesion. These findings indicate that 1) eNOS-derived NO attenuates endotoxin-induced P/E adhesion and 2) sGC is responsible for the antiadhesive action of NO.     

Liver X receptors regulate de novo lipogenesis in a tissue-specific manner in C57BL/6 female mice

The liver X receptors (LXRs) play a key role in cholesterol and bile acid metabolism but are also important regulators of glucose metabolism. Recently, LXRs have been proposed as a glucose sensor affecting LXR-dependent gene expression. We challenged wild-type (WT) and LXRαβ(-/-) mice with a normal diet (ND) or a high-carbohydrate diet (HCD). Magnetic resonance imaging showed different fat distribution between WT and LXRαβ(-/-) mice. Surprisingly, gonadal (GL) adipocyte volume decreased on HCD compared with ND in WT mice, whereas it slightly increased in LXRαβ(-/-) mice. Interestingly, insulin-stimulated lipogenesis of isolated GL fat cells was reduced on HCD compared with ND in LXRαβ(-/-) mice, whereas no changes were observed in WT mice. Net de novo lipogenesis (DNL) calculated from Vo(2) and Vco(2) was significantly higher in LXRαβ(-/-) than in WT mice on HCD. Histology of HCD-fed livers showed hepatic steatosis in WT mice but not in LXRαβ(-/-) mice. Glucose tolerance was not different between groups, but insulin sensitivity was decreased by the HCD in WT but not in LXRαβ(-/-) mice. Finally, gene expression analysis of adipose tissue showed induced expression of genes involved in DNL in LXRαβ(-/-) mice compared with WT animals as opposed to the liver, where expression of DNL genes was repressed in LXRαβ(-/-) mice. We thus conclude that absence of LXRs stimulates DNL in adipose tissue, but suppresses DNL in the liver, demonstrating opposite roles of LXR in DNL regulation in these two tissues. These results show tissue-specific regulation of LXR activity, a crucial finding for drug development.     

Study of molecular mechanism of Prostaglandin E1 in inhibiting coronary heart disease

Prostaglandin E1 has been used clinically for improving heart diseases. In this study, we examined the effect of Prostaglandin E1 on blood lipid levels, heart protein and genes expression in coronary heart disease (CHD) rats. Female rats were fed either a control diet or hypercholesterolemic diet for 14 weeks. The feeding of a hypercholesterolemic diet (HCD) increased the serum TC, TG, and LDL-c levels, decreased the serum HDL-c, E2, P, FSH, LH and PRL levels in CHD rats. In addition, The feeding of a HCD diet markedly increased the content of serum TXA2, TXB2, and decreased the content of serum PGI2, and PGI2/TXA2, 6-Keto PGF1a. Furthermore, the feeding of a hypercholesterolemic diet markedly increased expression levels of myocardium Fas and Caspase-3 protein and mRNA levels, vascular endothelial growth factor and basic fibroblast growth factor mRNA, and decreased RyR2 mRNA in CHD rats. The feeding of Prostaglandin E1 for 14 weeks significantly reversed these abnormal biochemical indexes in rats. These findings suggest that Prostaglandin E1 play a obvious heart protective effect. The mechanisms may be related to restraining the excessive activation of Fas and Caspase-3 protein and modulating some gene expressions associated with CHD.     

Deletion of L-selectin increases atherosclerosis development in ApoE-/- mice

Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel(-/-)) animals were crossed with mice lacking Apolipoprotein E (ApoE(-/-)). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE(-/-)L-sel(-/-)) mice and the corresponding ApoE(-/-) controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE(-/-)L-sel(-/-) mice as compared to ApoE(-/-) controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE(-/-)L-sel(-/-) animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE(-/-)L-sel(-/-) and ApoE(-/-) mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE(-/-)L-sel(-/-) as compared to ApoE(-/-) mice. Leukocyte rolling on lesions in the aorta was similar in ApoE(-/-)L-sel(-/-) and ApoE(-/-) animals. ApoE(-/-)L-sel(-/-) mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE(-/-) controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis.     

Low venular shear rates promote leukocyte-dependent recruitment of adherent platelets

The influence of reductions in venular shear rate on platelet-endothelial (P/E) cell adhesion has not been previously addressed. The objectives of this study were to define the effects of reductions in venular shear rate on P/E cell adhesion and to determine the interdependence of P/E cell adhesion and leukocyte-endothelial (L/E) cell adhesion at low shear rates. Intravital videomicroscopy was used to quantify P/E and L/E cell adhesion in rat mesenteric venules exposed to shear rates ranging between 118 +/- 9 and 835 +/- 44 s(-1). Shear rate was altered in postcapillary venules by rapid, graded blood withdrawal, without retransfusion of shed blood. Reducing shear rate from >600 s(-1) to <200 s(-1) resulted in an eightfold increase in L/E cell adhesion, whereas P/E cell adhesion increased 18-fold. A blocking antibody directed against P-selectin blunted both the P/E and L/E cell adhesion elicited by low shear rates. Immunoneutralization of CD11/CD18 on leukocytes or rendering animals neutropenic also blocked the shear rate-dependent recruitment of both platelets and leukocytes. These findings indicate that 1) low shear rates promote P/E and L/E cell adhesion in mesenteric venules, and 2) adherent neutrophils (mediated by CD11/CD18) create a platform onto which platelets can bind to the venular wall at low shear rates.     

Effect of chronic L-Arginine supplementation on aortic fatty streak formation and serum nitric oxide concentration in normal and high-cholesterol fed rabbits

Several reports indicated the beneficial effects of short-term L-Arginine (L-Arg) administration on atherosclerosis processes. The aim of this study was to evaluate the effect of chronic L-Arg supplementation on serum lipid profile, aortic Fatty Streak (FS) formation, and serum Nitric oxide (NO) concentration in Normal Diet (ND) and High-Cholesterol Diet (HCD) fed rabbits. 24 male rabbits were randomly divided into four groups (n=6 in each group) (i): ND for seven months; (ii): ND for 1 month plus ND + L-Arg for six months; (iii): HCD (1%) for 1 month plus HCD (0.5%) for six months; (iv): HCD (1%) for 1 month plus HCD (0.5%) + L-Arg for six months. At the end of the study, histological evaluation of aortic FS formation was performed. Blood samples were taken for serum lipid profile and NO concentrations. L-Arg did not change serum total cholesterol, triglyceride, LDL and LDL/HDL ratio in normal and hypercholesterolemic rabbits (p>0.05). Histological examination of thoracic aortae revealed that the HCD group had higher FS formation compared to the ND group (2.1 ± 0.16 vs. 0 ± 0; respectively; p<0.05) and L-Arg supplementation did not attenuate FS formation in the HCD group (1.93 ± 0.17 compare to 2.1 ± 0.16; p>0.05). Serum NO level in the HCD group was higher than ND groups (p<0.05). Chronic L-Arg supplementation did not alter serum NO concentration either in the hypercholesterolemic or in the ND group (p>0.05). It seems that chronic L-Arg supplementation does not have beneficial effects on aortic fatty streak formation, serum lipids and NO concentrations in this model of experimental hypercholesterolemia.     

Control of cholesterol biosynthesis,uptake and storage in hepatocytes by Cideb

Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases.     

Estriol retards and stabilizes atherosclerosis through an NO-mediated system

Estriol (E3) has little effect on the female genitals. E3 is used in hormone replacement therapy, particularly in Europe and Japan, since it obviates the need for progestin administration. However, the effect of E3 on atherosclerosis has not been elucidated. In this study, we evaluated the effect of E3 on the progression of atherosclerosis in a rabbit model. Thirty-six rabbits total were used. Twenty-eight were bilaterally oophorectomized, and 8 were not. The rabbits were divided into 5 groups and treated for 12 weeks as follows. Gp I (n = 8) was fed a high cholesterol diet (HCD; standard diet plus 0.5% cholesterol); Gp II (n = 8) was fed a HCD with E3 (0.3 mg/kg/day); Gp III (n = 8) was fed a HCD with 17beta estradiol (E2) (0.1 mg/kg/day); Gp IV (n = 8), the non oophorectomized group, was fed a HCD; and Gp NC was oophorectomized (n = 4), and fed a regular diet. E3 treatment increased the plasma E2 and E3 levels in Gp II. The plasma lipid levels were not altered by the E2 or E3 treatment. A HCD diminished the acetylcholine-induced NO mediated relaxation in the thoracic aorta. The E2 treatment (Gp III) and E3 treatment (Gp II) restored the aortic basal NO release and the aortic cyclic GMP levels, particularly effectively in the E3 group. E3 treatment also decreased the atherosclerotic area, and its effect was comparable with E2 (surface involvement: 41.2 +/- 5.1% in Gp I; 10.1 +/- 2.7% in Gp II; and 6.5 +/- 1.3% in Gp III). All four hyperlipidemic groups showed an increase of eNOS mRNA in the aortae, and this was especially pronounced in Gps II and III. The level of peroxynitrite, as determined by immunohistochemical nitrotyrosine staining, was lower in Gps II and III than in Gp I. E3 strongly activates NO-mediated systems, and could play a role in retarding the progression of atherosclerosis and in stabilizing atheroma.     

A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor

Abnormal expression of thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) was found in patients with acute coronary syndrome. Ticagrelor, an oral platelet ADP P2Y12 receptor antagonist, is widely used in these patients. The aim of this study was to verify whether different doses of ticagrelor regulated plaque progression and platelet activity by modulating TSLP/TSLPR. Seventy-five ApoE-/- mice were randomly divided into five groups: (1) high-cholesterol diet (HCD, n = 15); (2) HCD plus ticagrelor 25 mg/kg/d (T1, n = 15); (3) HCD plus ticagrelor 50 mg/kg/d (T2, n = 15); (4) HCD plus ticagrelor 100 mg/kg/d (T3, n = 15); and (5) a normal diet group (ND, n = 15). At day 0 and at week 16, blood lipids and serum TSLP levels, expression of TSLPR, CD62, and CD63, platelet aggregation, platelet ATP release, PI3K/Akt signaling pathway, and plaque morphology were assessed. HCD increased TSLPR expression and atherosclerosis progression but high-dose ticagrelor (100 mg/kg) moderated this trend. TSLPR was positively correlated with Akt1, platelet aggregation, corrected plaque area, and vulnerability index in the T3 group (P<0.01). In conclusion, low-dose ticagrelor only inhibited platelet activity. Besides this inhibition, high-dose ticagrelor modulated platelet activity and atherosclerosis mediated by TSLPR, potentially through the PI3K/Akt signal pathway.     

Roles of platelet and endothelial cell COX-1 in hypercholesterolemia-induced microvascular dysfunction

Aspirin is a common preventative therapy in patients at risk for cardiovascular diseases, yet little is known about how aspirin protects the vasculature in hypercholesterolemia. The present study determines whether aspirin, nitric oxide-releasing aspirin (NCX-4016), a selective cyclooxygenase (COX)-1 inhibitor (SC560), or genetic deficiency of COX-1 prevents the inflammatory and prothrombogenic phenotype assumed by hypercholesterolemic (HC) venules. Aspirin or NCX-4016 (60 mg/kg) was administered orally for the last week of a 2-wk HC diet. COX-1-deficient (COX-1(-/-)) and wild-type (WT) mice were transplanted with WT (WT/COX-1(-/-)) or COX-1(-/-) (COX-1(-/-)/WT) bone marrow, respectively. HC-induced adhesion of platelets and leukocytes in murine intestinal venules, observed with intravital fluorescence microscopy, was greatly attenuated in aspirin-treated mice. Adhesion of aspirin-treated platelets in HC venules was comparable to untreated platelets, whereas adhesion of SC560-treated platelets was significantly attenuated. HC-induced leukocyte and platelet adhesion in COX-1(-/-)/WT chimeras was comparable to that in SC560-treated mice, whereas the largest reductions in blood cell adhesion were in WT/COX-1(-/-) chimeras. NCX-4016 treatment of platelet recipients or donors attenuated leukocyte and platelet adhesion independent of platelet COX-1 inhibition. Platelet- and endothelial cell-associated COX-1 promote microvascular inflammation and thrombogenesis during hypercholesterolemia, yet nitric oxide-releasing aspirin directly inhibits platelets independent of COX-1.     

Overexpression of CXCL16 promotes a vulnerable plaque phenotype in Apolipoprotein E-Knockout Mice

BackgroundCXCL16 has been shown to be involved in atherosclerotic lesion development, but its role in preexisting lesions is still unclear. This study aims to assess the effect of CXCL16 on the stability of preexisting lesions.MethodsWe firstly measured plasma CXCL16 level in Apolipoprotein E–Knockout (ApoE KO) mice with either high-cholesterol diet (HCD) or normal diet (ND) by enzyme-linked immunosorbent assay (ELISA). Then, silastic collars were placed around the carotid arteries in HCD-ApoE KO mice to accelerate atherosclerotic lesions. Five weeks later, CXCL16 was overexpressed by intravenous injection of lentivirus carrying CXCL16 transgene. Two weeks after infection, lesions were stained with hematoxylin and eosin (HE) and with oil red O. Biomarkers in the lesions, such as MMPs, CCL2, VCAM-1 and TNF-α were measured by real-time polymerase chain reaction (RT-PCR), which indicate the instability of plaques.ResultsThe level of CXCL16 in plasma was higher in HCD-ApoE KO mice as compared to ND-ApoE KO mice. Circulating CXCL16 overexpression does not affect the size of preexisting plaques, but it leads to vulnerable plaque morphology and increases the expression of markers of plaque destabilization.ConclusionSystemic CXCL16 becomes much higher in atherosclerosis, and it could be a potential atherogenic biomarker. Overexpression of CXCL16 promotes the evolution of preexisting lesions to vulnerable plaques in ApoE KO mice.     

P-selectin-mediated adhesion impairs endothelium-dependent arteriolar dilation in hypercholesterolemic mice

Hypercholesterolemia is associated with an attenuation of endothelium-dependent dilation in arterioles and an increase in leukocyte and platelet adhesion in venules. The proximity of closely paired arterioles and venules is thought to facilitate heat and mass transport between the two and could be involved in transport of inflammatory and/or vasoactive mediators from venule to arteriole. In the current study, we tested the hypothesis that the impaired arteriolar dilation associated with hypercholesterolemia might be dependent on P-selectin-dependent blood cell adhesion in the closely paired venules. Leukocyte and platelet recruitment in venules and the endothelium-dependent response to bradykinin in second-order arterioles were observed in the mouse intestinal submucosa using intravital microscopy. Four weeks of a high-cholesterol diet decreased bradykinin-induced arteriolar dilation more dramatically in closely paired arterioles than in distantly paired arterioles. The dysfunctional arteriolar dilation of closely paired arterioles in hypercholesterolemic mice was significantly improved when the experiments were repeated in P-selectin-deficient mice (given the high-cholesterol diet) or in hypercholesterolemic mice injected with a P-selectin monoclonal antibody. A similar improvement in dilation of closely paired arterioles was attained in hypercholesterolemic mice given the superoxide dismutase mimetic Tempol. These findings indicate that hypercholesterolemia-induced increases in venular leukocyte and platelet adhesion might contribute to the impaired endothelium-dependent dilation of closely paired arterioles via a mechanism that is distance limited and dependent on P-selectin and superoxide.