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Background— High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results— A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC –/– mice. In RBCs from HFD-fed wild-type and DARC –/– mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions— RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.

Background— Obesity causes hypertension and sympathoactivation, a process proposed to be mediated by leptin. Protein tyrosine phosphatase 1B (PTP1B), a major new pharmaceutical target in the treatment of obesity and type II diabetes mellitus, constrains the metabolic actions of leptin, but the extent to which PTP1B regulates its cardiovascular effects is unclear. This study examined the hypothesis that PTP1B is a negative regulator of the cardiovascular effects of leptin. Methods and Results— PTP1B knockout mice had lower body fat but higher mean arterial pressure (116±5 versus 105±5 mm Hg, P P P P 1 -adrenergic receptor subtypes, consistent with blunted constriction to phenylephrine. Conclusions— These data indicate that PTP1B is a key regulator of the cardiovascular effects of leptin and that reduced vascular adrenergic reactivity provides a compensatory limit to the effects of leptin on mean arterial pressure.

Background— Despite intensive investigation, how DNA methylation influences endothelial function remains poorly understood. We used methyl-CpG–binding domain protein 2 (MBD2), an interpreter for DNA methylome–encoded information, to dissect the impact of DNA methylation on endothelial function in both physiological and pathophysiological states. Methods and Results— Human umbilical vein endothelial cells under normal conditions express moderate levels of MBD2, but knockdown of MBD2 by siRNA significantly enhanced angiogenesis and provided protection against H 2 O 2 -induced apoptosis. Remarkably, Mbd2 −/− mice were protected against hind-limb ischemia evidenced by the significant improvement in perfusion recovery, along with increased capillary and arteriole formation. Loss of MBD2 activated endothelial survival and proangiogenic signals downstream of vascular endothelial growth factor signaling characterized by an increase in endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor 2 expression, along with enhanced extracellular signal-regulated kinase 1/2 activation and BCL-2 expression. Mechanistic studies confirmed the methylation of CpG elements in the eNOS and vascular endothelial growth factor receptor 2 promoter. MBD2 binds to these methylated CpG elements and suppresses eNOS promoter activity. On ischemic insult, key endothelial genes such as eNOS and vascular endothelial growth factor receptor 2 undergo a DNA methylation turnover, and MBD2 interprets the changes of DNA methylation to suppress their expressions. Moreover, MBD2 modulation of eNOS expression is likely confined to endothelial cells because nonendothelial cells such as splenocytes fail to express eNOS after loss of MBD2. Conclusions— We provided direct evidence supporting that DNA methylation regulates endothelial function, which forms the molecular basis for understanding how environmental insults (epigenetic factor) affect the genome to modify disease susceptibility. Because MBD2 itself does not affect the methylation of DNA and is dispensable for normal physiology in mice, it could be a viable epigenetic target for modulating endothelial function in disease states.

Background— Cardiovascular disease is the leading cause of death for both men and women in the United States and the world. A profound pattern exists in the time of day at which the death occurs; it is in the morning, when the endothelium is most vulnerable and blood pressure surges, that stroke and heart attack most frequently happen. Although the molecular components of circadian rhythms rhythmically oscillate in blood vessels, evidence of a direct function for the “circadian clock” in the progression to vascular disease is lacking. Methods and Results— In the present study, we found increased pathological remodeling and vascular injury in mice with aberrant circadian rhythms, Bmal1-knockout and Clock mutant. In addition, naive aortas from Bmal1-knockout and Clock mutant mice exhibit endothelial dysfunction. Akt and subsequent nitric oxide signaling, a pathway critical to vascular function, was significantly attenuated in arteries from Bmal1-knockout mice. Conclusions— Our data reveal a new role for the circadian clock during chronic vascular responses that may be of significance in the progression of vascular disease.

Statins (HMG CoA reductase inhibitors) have beneficial vascular effects independent of their ability to reduce cholesterol synthesis. We and others have previously shown that statins acutely activate endothelial cell NOS (eNOS). Our present study characterized signaling pathways by which statins activate NOS using cultured bovine aortic endothelial cells (BAEC) and COS 7 cells. It also examined involvement of scavenger receptor - B1 (SR-B1), which is expressed in endothelial cells and maintains caveolae cholesterol concentration and eNOS localization. BAEC were exposed to lovastatin (LOV, 10 −6 M) for 2–5 min after pretreatment with the end product of HMG CoA reductase (mevalonic acid, 5×10 −4 M), a Gi protein inhibitor (pertussis toxin, 10 −4 M), a PLC inhibitor (U-73122, 10 −5 M), a receptor tyrosine kinase (RTK) inhibitor (tyrphostine 51A, 2×10 −6 M) or intracellular and extracellular calcium chelators -BAPTA-AM (10 −5 M) and EGTA (10 −4 M), respectively, and levels of NO production were determined. LOV alone acutely increased NO production by 67%. NO production in response to LOV was not blocked by mevalonic acid indicating that it occurs independent of HMG CoA reductase inhibition. Inhibition of Gi protein, PLC, and RTK almost completely blocked the LOV-induced NO generation. Removal of extracellular Ca ++ prevented statin-induced NO production by 90%, while intracellular Ca ++ chelation with BAPTA-AM reduced NO production by 45%. COS 7 cells transfected with eNOS did not show increased NO production to LOV or HDL, indicating NOS activation occurs through an endothelial cell receptor. In COS 7 cells, co-transfected with eNOS and the endothelial SR-B1, LOV or HDL (10 −5 M), a known agonist of SR-B1, increased NO production by over 200%. In COS 7 cells transfected with eNOS alone or co-transfected with the bradykinin B2 receptor, neither LOV nor HDL increased NO production, indicating effects specific for the SR-B1 receptor. In conclusion, our study shows LOV acutely activates eNOS independently of HMG CoA reductase inhibition. eNOS activation appears to involve an endothelial Gi protein receptor and subsequent activation of PLC and RTK pathways, leading to entry of extracellular calcium. Furthermore, LOV can activate eNOS via the SR-B1 receptor.

Cardiovascular disease is the leading cause of death for both men and women in the United States and the world. There is a profound pattern in the time of day at which the death occurs; it is in the morning, when the endothelium is most vulnerable and blood pressure surges, that stroke and heart attack most frequently happen. Though the biological clock rhythmically oscillates in blood vessels, evidence of a direct function in the vasculature for Bmal1, an essential clock component, is lacking. We have found that chronic flow interruption in arteries of mice with targeted disruption of Bmal1 gene (Bmal1-KO) causes pathological vascular remodeling which worsens to thrombosis with ageing. In addition, naïve aortic arteries of Bmal1-KO mice exhibit endothelial dysfunction, which is improved by relief of oxidant stress. Akt signalling, a pathway critical to vascular function, was significantly attenuated in arteries of Bmal1-KO mice. Co-transfection of cultured cells with the transcription factors Bmal-Npas2 or Bmal1-Clock induced a robust activation of the Akt promoter, providing evidence of a direct link between the circadian clock and Akt pathways. Our data reveals a new role for the biological clock in the control of endothelial signalling and vascular remodeling which may be of significant impact in the progression of vascular disease.