Rationale Signal initiation by the HDL receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain (CTTM). The CTTM uniquely interacts with plasma membrane (PM) cholesterol. Objective The molecular basis and functional significance of SR-BI interaction with plasma membrane cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a plasma membrane cholesterol sensor. Methods and Results In studies performed in COS-M6 cells, mutation of a highly-conserved CTTM glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-β-cyclodextrin (CD) was also prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or CD in cultured enterocytes, and it is required for HDL activation of eNOS and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. Conclusion Through interaction with plasma membrane cholesterol, SR-BI serves a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.

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Fcγ receptors (FcγR) classically modulate intracellular signaling upon binding of the Fc region of IgG in immune response cells. How FcγR and their ligands impact cardiovascular health and disease has recently been interrogated in both preclinical and clinical studies. The stimulation of activating FcγR in endothelial cells, vascular smooth muscle cells and monocytes/macrophages causes a variety of cellular responses that may contribute to vascular disease pathogenesis. Stimulation of the lone inhibitory FγcR, FcγRIIB, also has adverse consequences in endothelial cells, antagonizing NO production and reparative mechanisms. In preclinical disease models, activating FcγR promote atherosclerosis whereas FcγRIIB is protective, and activating FcγR also enhance thrombotic and non-thrombotic vascular occlusion. The FcγR ligand C-reactive protein (CRP) has undergone intense study. Although in rodents CRP does not impact atherosclerosis, it causes hypertension and insulin resistance and worsens myocardial infarction. Massive data has accumulated indicating an association between increases in circulating CRP and coronary heart disease in humans. However, Mendelian randomization studies reveal that CRP is not likely a disease mediator. CRP genetics and hypertension warrants further investigation. Studies to date of genetic variants of activating FcγR are insufficient to implicate the receptors in coronary heart disease pathogenesis in humans. However, a link between FcγRIIB and human hypertension may be emerging. Further knowledge of the vascular biology of FcγR and their ligands will potentially enhance our understanding of cardiovascular disorders, particularly in patients whose greater predisposition for disease is not explained by traditional risk factors, such as individuals with autoimmune disorders.

Elevations in C-reactive protein (CRP) are associated with an increased risk of insulin resistance. Whether CRP plays a causal role is unknown. Here we show that CRP transgenic mice and wild-type mice administered recombinant CRP are insulin resistant. Mice lacking the inhibitory Fcγ receptor IIB (FcγRIIB) are protected from CRP-induced insulin resistance, and immunohistochemistry reveals that FcγRIIB is expressed in skeletal muscle microvascular endothelium and is absent in skeletal muscle myocytes, adipocytes, and hepatocytes. The primary mechanism in glucose homeostasis disrupted by CRP is skeletal muscle glucose delivery, and CRP attenuates insulin-induced skeletal muscle blood flow. CRP does not impair skeletal muscle glucose delivery in FcγRIIB−/− mice or in endothelial nitric oxide synthase knock-in mice with phosphomimetic modification of Ser1176, which is normally phosphorylated by insulin signaling to stimulate nitric oxide–mediated skeletal muscle blood flow and glucose delivery and is dephosphorylated by CRP/FcγRIIB. Thus, CRP causes insulin resistance in mice through FcγRIIB-mediated inhibition of skeletal muscle glucose delivery.

Abstract-Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor Fc␥RIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant Fc␥RIIB in NIH3T3 IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domaincontaining inositol 5Ј-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via Fc␥RIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes. Key Words: C-reactive protein Ⅲ insulin Ⅲ eNOS Ⅲ Fc␥RIIB Ⅲ SHIP1 I n addition to its critical role in metabolism, insulin promotes the cardiovascular protective functions of the endothelium. These include the activation of NO production by endothelial NO synthase (eNOS). 1 The resulting NO regulates vasodilation, angiogenesis, thrombosis, hemostasis, and vascular smooth muscle cell growth and migration, 2 and it also attenuates monocyte adhesion, which is among the initiating steps in the development of atherosclerosis. 3,4 Linking cardiovascular and metabolic homeostasis, insulin activation of eNOS also promotes blood flow that augments glucose disposal in insulin target tissues, particularly skeletal muscle. Insulin stimulates eNOS enzymatic activity by insulin receptor (IR)-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt kinase, leading to eNOS Ser1179 phosphorylation. Upstream insulin signaling entails the phosphorylation of the IR ␤ subunit (IR␤) and IR substrate-1 (IRS-1). 1 C-reactive protein (CRP) is a member of the pentraxin family and an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. 5-7 Recently, we showed that CRP inhibits eNOS activation by insulin by blunting eNO...

gated estrogen prevent diet-induced obesity, hepatic steatosis, and type 2 diabetes in mice without impacting the reproductive tract. Am J Physiol Endocrinol Metab 307: E345-E354, 2014. First published June 17, 2014 doi:10.1152/ajpendo.00653.2013.-Despite the capacity of estrogens to favorably regulate body composition and glucose homeostasis, their use to combat obesity and type 2 diabetes is not feasible, because they promote sex steroid-responsive cancers. The novel selective estrogen receptor modulator (SERM) bazedoxifene acetate (BZA) uniquely antagonizes both breast cancer development and estrogen-related changes in the female reproductive tract. How BZA administered with conjugated estrogen (CE) or alone impacts metabolism is unknown. The effects of BZA or CE ϩ BZA on body composition and glucose homeostasis were determined in ovariectomized female mice fed a Western diet for 10 -12 wk. In contrast to vehicle, estradiol (E2), CE, BZA, and CE ϩ BZA equally prevented body weight gain by 50%. In parallel, all treatments caused equal attenuation of the increase in body fat mass invoked by the diet as well as the increases in subcutaneous and visceral white adipose tissue. Diet-induced hepatic steatosis was attenuated by E2 or CE, and BZA alone or with CE provided even greater steatosis prevention; all interventions improved pyruvate tolerance tests. Glucose tolerance tests and HOMA-IR were improved by E2, CE, and CE ϩ BZA. Whereas E2 or CE alone invoked a uterotrophic response, BZA alone or CE ϩ BZA had negligible impact on the uterus. Thus, CE ϩ BZA affords protection from diet-induced adiposity, hepatic steatosis, and insulin resistance with minimal impact on the female reproductive tract in mice. These combined agents may provide a valuable new means to favorably regulate body composition and glucose homeostasis and combat fatty liver. bazedoxifene; conjugated estrogen; hepatic steatosis; obesity; type 2 diabetes IN ADDITION TO THEIR ROLES in sexual development and reproduction, estrogens contribute to the regulation of body weight and body composition and to glucose homeostasis. Insulin sensitivity is greater in women prior to menopause vs. agematched men (16,42), in women the risk for weight gain and for the development of type 2 diabetes increases with the decline in the levels of estrogens that occurs at menopause, and menopause favors an increase in total body fat and visceral fat deposition (4). In addition, hormone replacement therapy with conjugated estrogen (CE) and medroxyprogesterone acetate (MPA) in postmenopausal women causes a 21-35% decrease in diabetes occurrence (29,35). Furthermore, in female rodents and primates, ovariectomy results in impaired insulin sensitivity and glucose homeostasis, and these effects are reversed by estradiol (E 2 ) (30, 54). Studies in mice have additionally demonstrated that E 2 provides potent protection against highfat diet-induced glucose intolerance and insulin resistance (46). The metabolic actions of estrogens are mediated primarily by estrogen receptor (E...

SUMMARY Cardiovascular function depends on patent, continuous and stable blood vessel formation by endothelial cells (ECs). Blood vessel development initiates by vasculogenesis, as ECs coalesce into linear aggregates and organize to form central lumens that allow blood flow. Molecular mechanisms underlying in vivo vascular ‘tubulogenesis’ are only beginning to be unraveled. We previously showed that the GTPase-interacting protein called Rasip1 is required for the formation of continuous vascular lumens in the early embryo. Rasip1−/− ECs exhibit loss of proper cell polarity and cell shape, disrupted localization of EC-EC junctions and defects in adhesion of ECs to extracellular matrix (ECM). In vitro studies showed that Rasip1 depletion in cultured ECs blocked tubulogenesis. Whether Rasip1 is required in blood vessels after their initial formation remained unclear. Here, we show that Rasip1 is essential for vessel formation and maintenance in the embryo, but not in quiescent adult vessels. Rasip1 is also required for angiogenesis in three models of blood vessel growth: in vitro matrix invasion, retinal blood vessel growth and Directed In vivo Angiogenesis Assays (DIVAA). Rasip1 is thus necessary in growing embryonic blood vessels, postnatal angiogenic sprouting and remodeling, but is dispensable for maintenance of established blood vessels, making it a potential anti-angiogenic therapeutic target.

Background: Obesity-related hypertension is a common disorder, and attempts to combat the underlying obesity are often unsuccessful. We previously revealed that mice globally deficient in the inhibitory immunoglobulin G (IgG) receptor FcγRIIB are protected from obesity-induced hypertension. However, how FcγRIIB participates is unknown. Studies were designed to determine if alterations in IgG contribute to the pathogenesis of obesity-induced hypertension. Methods: Involvement of IgG was studied using IgG μ heavy chain–null mice deficient in mature B cells and by IgG transfer. Participation of FcγRIIB was interrogated in mice with global or endothelial cell–specific deletion of the receptor. Obesity was induced by high-fat diet (HFD), and blood pressure (BP) was measured by radiotelemetry or tail cuff. The relative sialylation of the Fc glycan on mouse IgG, which influences IgG activation of Fc receptors, was evaluated by Sambucus nigra lectin blotting. Effects of IgG on endothelial NO synthase were assessed in human aortic endothelial cells. IgG Fc glycan sialylation was interrogated in 3442 human participants by mass spectrometry, and the relationship between sialylation and BP was evaluated. Effects of normalizing IgG sialylation were determined in HFD-fed mice administered the sialic acid precursor N-acetyl-D-mannosamine (ManNAc). Results: Mice deficient in B cells were protected from obesity-induced hypertension. Compared with IgG from control chow–fed mice, IgG from HFD-fed mice was hyposialylated, and it raised BP when transferred to recipients lacking IgG; the hypertensive response was absent if recipients were FcγRIIB-deficient. Neuraminidase-treated IgG lacking the Fc glycan terminal sialic acid also raised BP. In cultured endothelial cells, via FcγRIIB, IgG from HFD-fed mice and neuraminidase-treated IgG inhibited vascular endothelial growth factor activation of endothelial NO synthase by altering endothelial NO synthase phosphorylation. In humans, obesity was associated with lower IgG sialylation, and systolic BP was inversely related to IgG sialylation. Mice deficient in FcγRIIB in endothelium were protected from obesity-induced hypertension. Furthermore, in HFD-fed mice, ManNAc normalized IgG sialylation and prevented obesity-induced hypertension. Conclusions: Hyposialylated IgG and FcγRIIB in endothelium are critically involved in obesity-induced hypertension in mice, and supportive evidence was obtained in humans. Interventions targeting these mechanisms, such as ManNAc supplementation, may provide novel means to break the link between obesity and hypertension.