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Background. Social deprivation or isolation accelerates the progression of atherosclerosis in several animal models of the disease. Conversely, stable social environment has been associated with reduction in the extent and severity of atherosclerosis. While positive social interactions are thought to be related to this protective effect, little is known about the physiological mechanisms responsible. Recently, the neurohypophyseal peptide, oxytocin (OT), has been found to play a role in both positive social interactions and cardiovascular homeostasis, suggesting that this neuropeptide may be responsible for mediating the beneficial effects of positive social environment on atherosclerosis. The first aim of the current study is to examine the potential anti-inflammatory effects of OT on in vitro cellular models involved in the pathophysiology of atherosclerosis. The second aim is to examine whether long-term administration of OT slows the progression of atherosclerosis in apoE-/- mice. The third aim is to obtain evidence in vivo that OT is impacting disease through novel anti-inflammatory effects on tissues important in atherogenesis. Methods. 1) Human macrophage-like (DTHP-1) cells and human aortic endothelial cells (HAECs) were stimulated with lipopolysaccharde (LPS) alone, and in the presence of different concentrations of OT, and IL-6 secretion was measured. 2) ApoE-/- mice were socially isolated at 12 weeks of age and continuously infused with OT (n=24) or vehicle (n=21) from subcutaneously implanted osmotic minipumps for 12 weeks. Plasma levels of lipids, adiponectin, insulin, and CRP were assessed pre- and post-treatment. Extent of aortic atherosclerosis (percent lesion area) was assessed post-treatment and areas of high lesion prevalence were compared between OT and vehicle (VH) control groups. Constitutive release of IL-6 from ex vivo adipose tissue samples taken from a subset (n=12/group) was compared between treatment groups. Results. 1) OT demonstrated dose-dependent inhibition of LPS-induced IL-6 secretion from macrophages (35-55%, p < 0.01) and aortic endothelial cells (15-25%, p < 0.01). 2) ApoE-/- mice continuously infused with OT displayed decreased plasma CRP levels after 6 weeks of treatment and diminished lesion area at the thoracic aorta after 12 weeks of treatment relative to vehicle control animals (37%, p < 0.05). Additionally, adipose tissue samples taken from OT infused mice showed decreased constitutive release of IL-6 (30%, p < 0.01). These findings were unrelated to changes in plasma lipids, insulin, physical activity levels, or 24-hour corticosterone secretion. Discussion and Conclusions. These findings demonstrate that OT is capable of inhibiting stimulated pro-inflammatory cytokine production in macrophages and aortic endothelial cells in vitro, and constitutive release from adipose tissue in vivo. OT also decreased circulating CRP levels and slowed the progression of early stage atherosclerosis in an aortic region of high lesion prevalence in socially isolated apoE-/- mice. Taken together, these results suggest that increased peripheral OT could be partially responsible for the beneficial effect of positive social environment on atherosclerosis.

Gene expression was examined in vascular smooth muscle cells to study the complex interaction between oxidative injury and the pathogenesis of vascular disease. Extensive vascular remodeling coupled to increased production of 8-epi-PGF2α nuclear localization of NFκB, and alterations in glutathione homeostasis were identified as major responses of the vascular wall to oxidative stress. Transcriptional profiling studies, supported by immunohistochemistry and in situ hybridization measurements, identified genes involved in adhesion and extracellular matrix deposition (α1 integrin, collagen), cytoskeletal rearrangements (α-smooth muscle actin, α-tropomyosin), and signal transduction (NFκB, osteopontin, and LINE) as targets of oxidant injury. In the case of osteopontin (OPN), elevation of OPN levels in vSMCs was shown to be mediated by redox-regulated transcriptional mechanisms. A 200bp region located in the 5' UTR of the osteopontin promoter was found to be responsive to oxidative stress. This regulatory region contained two distinct cis acting elements involved in promoter inducibility. These elements were tentatively identified as NFKB and TIEG-1 binding sites and shown to be highly responsive to hydrogen peroxide and chemical antioxidants. Collectively these studies answer central questions regarding the mechanisms underlying the vascular response to oxidative stress and the involvement of OPN in diseases of the vascular wall.

Stroke is the third leading cause of death and the most common cause of disability in the world. To relieve the heavy burden of stroke, we need to understand the mechanisms that will form the basis of improved prevention and treatment. Epidemiological studies have found evidence for a genetic influence on the common form of stroke. However the genetic of stroke is still in its infancy. Subclinical intracranial atherosclerosis is sometime a predisposing factor for ischemic stroke (IS). This study was carried out to elucidate genetic factors influencing the complex phenotype of IS and subclinical intracranial atherosclerosis. In the Belgium Stroke Study (BSS), we collected 237 middle-aged (45-60 yrs) patients with small vessel occlusion (SVO) or large vessel atherosclerosis (LVA) IS, according to the Acute Stroke Treatment (TOAST) criteria, 326 ethnicity and gender matched subjects were used as controls. We tested variants in cholesterol-related candidate genes (sterol regulatory element binding protein, SREBP, SREBP-cleavage activating protein, SCAP, Apolipoprotein E, APOE, and Proprotein convertase subtilisin/kexin type 9, PCSKA) for association with IS. Significant gene-IS associations were further tested in a Finnish autopsy collection of 1004 cases with a quantitative assessment of atherosclerosis in the circle of Willis. While we could not detect any significant association between polymorphisms in the SREBP and SCAP genes and IS, we found evidence for association at the APOE and PCSK9 loci. The APOE ε4+ genotype was related to a more severe intracranial atherosclerosis score in men, and within the most common APOE ε3/ε3 genotype group a higher risk of IS was associated with the G-allele at the -219G/T promoter polymorphisms. At PCSK9, the minor allele (G) of the tagging E670G polymorphism appeared as a significant predictor of LVA (OR = 3.52, 95% CI 1.25-9.85; p = 0.017). Accordingly, in the Finnish autopsy series, G-allele carriers tended to have more severe allele copy number-dependent (p=0.095) atherosclerosis in the circle of Willis and in its branches. Our findings in this unique combination of clinical and autopsy data suggest a multifaceted role of apoE on the risk of cerebrovascular diseases. The APOE ε4+ genotype did not predict the risk of IS, but was associated with severity of subclinical intracranial atherosclerosis in men. In contrast, the promoter variants affecting apoE expression were significant predictors of IS, suggesting that quantitative rather than qualitative variation of apoE is related to IS independently of subclinical intracranial atherosclerosis. Furthermore, we demonstrated that PCSK9 associates with the risk of LVA stroke subtype, and suggest that the risk is related to the severity of the underlying intracranial atherosclerosis. Atherogenesis is considered as an active, inflammatory process, interleukin (IL)-18 a proinflammatory cytokine, is thought to play a central role in the development of atherosclerosis and more specifically in plaque rupture. We genotyped four haplotype tagging polymorphisms at the IL18 gene in the BSS and the Finnish autopsy series. The minor alleles of the IL18 -607 and +127 polymorphisms, as well as the haplotype carrying both minor alleles, associated with IS after adjustment for all cardiovascular risk factors. No association was seen with the development of subclinical intracranial atherosclerosis. Our findings suggest that variation in the IL18 gene influences the acute atherosclerotic IS event, but not the previous development of subclinical intracranial atherosclerosis, suggesting a causal role of IL18 in the vulnerability of cerebral arterial atherosclerotic plaques to acute rupture and subsequent thrombosis.

Statement of the problem: Many methods for surrogate outcome validation require individual patient data which is often inaccessible by clinical trialists. Methods: A review was performed to identify statistical methods for surrogate outcome validation that may be implemented using summary data from published clinical trials. The methods were used to evaluate carotid intima-media thickness (CIMT) as a surrogate outcome for cardiovascular events in a systematic review of randomized trials of interventions for atherosclerosis. Results: the review of methods identified five procedures. At two or more years of follow-up, there was a marginally significant association of CIMT with myocardial infarction and a statistically significant association with cardiovascular mortality. At ≥ four years of follow-up, a statistically significant, negative relationship was observed between CIMT and stroke. Conclusions: CIMT may be a valid surrogate outcome for myocardial infarction and cardiovascular mortality. Additional data is needed to evaluate CIMT in specific drug classes.

Cellular Inhibitor of Apoptosis 2 (cIAP2) belongs to the IAP family, a group of endogenous proteins that inhibit apoptosis. However, the physiological role of cIAP2 remains poorly defined. Knock-out (KO) and wild type (WT) mice were used to examine the effect of cIAP2 protein on the progression of atherosclerosis in apoE -/- mice. Following the high-fat diet period of 4 and 12 wks, tissues were harvested and analysis focused on the aortic root, the aortic arch, the descending aorta, and the blood. Ex vivo results show a significant decrease in aortic arch lesion area in KO vs. WT in both study groups. Results also show a decrease in aortic root lesion size in KO vs. WT in both study groups. These results support that cIAP2 is an important survival factor for lesion-associated macrophages, since loss of cIAP2 expression in this mouse model reduced atherosclerotic lesion development.

In atherosclerosis, infiltrating leukocytes and vascular smooth muscle cells (VSMCs) cause progressive vascular narrowing. Platelet-mediated thrombosis ultimately causes complete vessel occlusion, resulting in heart attack or stroke. In animal models and human patients, individually blocking these events is only partially effective. Another therapeutic strategy would be to globally target these multiple cell types. Slit proteins act as developmental neuronal repellents, and Slit2 via interaction with its receptor, Robo-1, impairs inflammatory recruitment of leukocytes and VSMCs. We detected Robo-1 expression in human and murine platelets. Using static and shear assays, we demonstrate that Slit2 impaired platelet adhesion and spreading on fibrinogen, fibronectin and collagen. Slit2 mediated these effects, in part, by suppressing activation of Akt but not Rac1, Cdc42, Erk or p38 MAPK. Slit2 also prevented ADP-mediated granular secretion. In mouse tail-bleeding experiments, Slit2 dose-dependently prolonged bleeding times in vivo. These data suggest a therapeutic role of Slit2 in atherothrombosis.

In atherosclerosis, infiltrating leukocytes and vascular smooth muscle cells (VSMCs) cause progressive vascular narrowing. Platelet-mediated thrombosis ultimately causes complete vessel occlusion, resulting in heart attack or stroke. In animal models and human patients, individually blocking these events is only partially effective. Another therapeutic strategy would be to globally target these multiple cell types. Slit proteins act as developmental neuronal repellents, and Slit2 via interaction with its receptor, Robo-1, impairs inflammatory recruitment of leukocytes and VSMCs. We detected Robo-1 expression in human and murine platelets. Using static and shear assays, we demonstrate that Slit2 impaired platelet adhesion and spreading on fibrinogen, fibronectin and collagen. Slit2 mediated these effects, in part, by suppressing activation of Akt but not Rac1, Cdc42, Erk or p38 MAPK. Slit2 also prevented ADP-mediated granular secretion. In mouse tail-bleeding experiments, Slit2 dose-dependently prolonged bleeding times in vivo. These data suggest a therapeutic role of Slit2 in atherothrombosis.

Cellular Inhibitor of Apoptosis 2 (cIAP2) belongs to the IAP family, a group of endogenous proteins that inhibit apoptosis. However, the physiological role of cIAP2 remains poorly defined. Knock-out (KO) and wild type (WT) mice were used to examine the effect of cIAP2 protein on the progression of atherosclerosis in apoE -/- mice. Following the high-fat diet period of 4 and 12 wks, tissues were harvested and analysis focused on the aortic root, the aortic arch, the descending aorta, and the blood. Ex vivo results show a significant decrease in aortic arch lesion area in KO vs. WT in both study groups. Results also show a decrease in aortic root lesion size in KO vs. WT in both study groups. These results support that cIAP2 is an important survival factor for lesion-associated macrophages, since loss of cIAP2 expression in this mouse model reduced atherosclerotic lesion development.

Gene expression was examined in vascular smooth muscle cells to study the complex interaction between oxidative injury and the pathogenesis of vascular disease. Extensive vascular remodeling coupled to increased production of 8-epi-PGF2α nuclear localization of NFκB, and alterations in glutathione homeostasis were identified as major responses of the vascular wall to oxidative stress. Transcriptional profiling studies, supported by immunohistochemistry and in situ hybridization measurements, identified genes involved in adhesion and extracellular matrix deposition (α1 integrin, collagen), cytoskeletal rearrangements (α-smooth muscle actin, α-tropomyosin), and signal transduction (NFκB, osteopontin, and LINE) as targets of oxidant injury. In the case of osteopontin (OPN), elevation of OPN levels in vSMCs was shown to be mediated by redox-regulated transcriptional mechanisms. A 200bp region located in the 5' UTR of the osteopontin promoter was found to be responsive to oxidative stress. This regulatory region contained two distinct cis acting elements involved in promoter inducibility. These elements were tentatively identified as NFKB and TIEG-1 binding sites and shown to be highly responsive to hydrogen peroxide and chemical antioxidants. Collectively these studies answer central questions regarding the mechanisms underlying the vascular response to oxidative stress and the involvement of OPN in diseases of the vascular wall.