Hypertension, hypercholesterolemia, diabetes and obesity are among a growing list of conditions that have been designated as major risk factors for cardiovascular disease (CVD). While CVD risk factors are well known to enhance the development of atherosclerotic lesions in large arteries, there is also evidence that the structure and function of microscopic blood vessels can be profoundly altered by these conditions. The diverse responses of the microvasculature to CVD risk factors include oxidative stress, enhanced leukocyte-and platelet-endothelial cell adhesion, impaired endothelial barrier function, altered capillary proliferation, enhanced thrombosis, and vasomotor dysfunction. Emerging evidence indicates that a low-grade systemic inflammatory response that results from risk factor-induced cell activation and cell-cell interactions may underlie the phenotypic changes induced by risk factor exposure. A consequence of the altered microvascular phenotype and systemic inflammatory response is an enhanced vulnerability of tissues to the deleterious effects of secondary oxidative and inflammatory stresses, such as ischemia and reperfusion. Future efforts to develop therapies that prevent the harmful effects of risk factor-induced inflammation should focus on the microcirculation.

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Patients with inflammatory bowel disease (IBD) are susceptible to microvascular thrombosis and thromboembolism. The increased incidence of thrombosis is accompanied by enhanced coagulation and abnormalities in platelet function. Clinical studies have revealed thrombocytosis, alterations in platelet activation, enhanced platelet-leukocyte interactions, and elevated plasma levels of prothrombotic cytokines. This study was directed towards determining whether the thrombocytosis, altered platelet functions, and enhanced platelet-leukocyte interactions observed in IBD patients can be recapitulated in the dextran sodium sulfate (DSS) and T-cell transfer models of murine colonic inflammation. Flow cytometry was used to characterize platelet function in heparin-anticoagulated whole blood of control mice and in mice with colonic inflammation. Platelets were identified by characteristic light scattering and membrane expression of CD41. Thiazole orange (TO) labeling was used to differentiate between immature and mature platelets. Platelet activation was monitored using the expression of an activation epitope of GPIIb/IIIa integrin. The combination of CD41, CD45.2, Gr-1, F4/80 and isotype control antibodies was used to detect and quantify aggregates of leukocytes, neutrophils and monocytes with platelets. Our results indicated that colonic inflammation is associated with thrombocytosis, leukocytosis, and the appearance of immature platelets. An increased number of circulating activated platelets was detected in colitic mice, along with the formation of aggregates of leukocytes (PLA), neutrophils (PNA) and monocytes (PMA) with platelets. Selectin blockade with fucoidin inhibited DSS-induced PLA formation. The findings of this study indicate that many features of the altered platelet function detected in human IBD can be reproduced in animal models of colonic inflammation.

Hypertension (HTN) is an established risk factor for subsequent cardiovascular diseases, with Angiotensin II (Ang-II) playing a major role in mediating thrombotic and inflammatory abnormalities. Although T cells and interleukin-6 (IL-6) play an important role in adaptive immune responses, little is known about their role(s) in the thrombo-inflammatory responses associated with Ang-II. Here we show using intravital microscopy coupled with the light/dye injury model that Rag-1 deficient (Rag-1 −/−) and IL-6 deficient (IL-6 −/−) mice are afforded protection against Ang-II induced thrombosis. Blocking IL-6 receptors (using CD126 and gp130 antibodies) significantly diminished Ang-II-mediated thrombosis and inflammatory cell recruitment in mice. Furthermore, the adoptive transfer of IL-6 −/−-derived T cells into Rag-1 −/− mice failed to accelerate Ang-II-induced thrombosis compared to Rag-1 −/− mice reconstituted with WT-derived T cells, suggesting T cell IL-6 mediates the thrombotic abnormalities associated Ang-II HTN. Interestingly, adoptive transfer of WT T cells into Rag-1 −/− /Ang-II mice resulted in increased numbers of immature platelets, which constitutes a more active platelet population i.e. pro-thrombotic and pro-inflammatory. To translate our in vivo findings, we used clinical samples to demonstrate that IL-6 also predisposes platelets to an interaction with collagen receptors, thereby increasing the propensity for platelets to aggregate and cause thrombosis. In summary, we provide compelling evidence for the involvement of IL-6, IL-6R and T cell-dependent IL-6 signaling in Ang-II induced thrombo-inflammation, which may provide new therapeutic possibilities for drug discovery programs for the management of HTN.

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Angiotensin II (AngII) contributes to the pathogenesis of hypertension and other cardiovascular diseases. AngII induces a pro-oxidative, pro-inflammatory, and pro-thrombogenic phenotype in vascular endothelial cells. Although the peptide promotes the recruitment of leukocytes and platelets and induces oxidative stress in the microvasculature, it remains unclear whether and how the blood cell recruitment is linked to the production of reactive oxygen species (ROS). In this study, we addressed the contributions of AngII type-1 receptors (AT1r), and gp91phox to the recruitment of leukocytes and platelets and ROS production in venules during chronic (2 wks) infusion of AngII in wild type (WT) and mutant mice. Intravital video microscopy was used to measure the adhesion and emigration of leukocytes, the adhesion of fluorescently labeled platelets, and dihydrorhodamine oxidation (a measure of oxidative stress) in cremaster muscle post-capillary venules. In WT mice, AngII infusion induced a time-dependent increase in the adhesion of leukocytes and platelets and enhanced ROS production in venules. These changes in blood cell adhesion and ROS production were not observed in AT1r−/− mice, in AT1r−/− bone marrow chimeras (blood cells deficient in AT1r), gp91phox−/−, gp91phox−/− chimeras (blood cells or endothelial cells deficient in gp91phox) and in WT mice rendered granulocytopenic via i.p injection of anti-mouse Gr-1 antibody. Thrombocytopenic WT mice (platelets depleted by i.p. injection of rabbit anti-mouse thrombocyte antiserum) responded similar to WT mice. These findings implicate leukocyte-associated AT1r and gp91phox in the induction of the pro-oxidative, proinflammatory and prothrombogenic phenotype assumed by microvessels that are chronically exposed to elevated AngII.

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