Keiji Tanigaki

Scavenger Receptor Class B Type I Is a Plasma Membrane Cholesterol Sensor

Rationale: Signal initiation by the high-density lipoprotein (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. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. Objective: The molecular basis and functional significance of SR-BI interaction with PM 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 PM cholesterol sensor. Methods and Results: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain 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-&bgr;-cyclodextrin also was 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 methyl-&bgr;-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. Conclusions: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.

C-Reactive Protein Inhibits Insulin Activation of Endothelial Nitric Oxide Synthase via the Immunoreceptor Tyrosine-Based Inhibition Motif of FcγRIIB and SHIP-1

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 FcγRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcγRIIB in NIH3T3IR 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 domain–containing inositol 5′-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 FcγRIIB 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.

C-Reactive Protein Causes Insulin Resistance in Mice Through Fcγ Receptor IIB–Mediated Inhibition of Skeletal Muscle Glucose Delivery

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.

Bazedoxifene and conjugated estrogen prevent diet-induced obesity, hepatic steatosis, and type 2 diabetes in mice without impacting the reproductive tract

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 + 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 + 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 + BZA. Whereas E₂ or CE alone invoked a uterotrophic response, BZA alone or CE + BZA had negligible impact on the uterus. Thus, CE + 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.

Coupling of Fcγ Receptor I to Fcγ Receptor IIB by Src Kinase Mediates C-Reactive Protein Impairment of Endothelial Function

Rationale: Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease risk and endothelial dysfunction. CRP antagonizes endothelial nitric oxide synthase (eNOS) through processes mediated by the IgG receptor Fc&ggr; receptor IIB (Fc&ggr;RIIB), its immunoreceptor tyrosine-based inhibitory motif, and SH2 domain-containing inositol 5′-phosphatase 1. In mice, CRP actions on eNOS blunt carotid artery re-endothelialization. Objective: How CRP activates Fc&ggr;RIIB in endothelium is not known. We determined the role of Fc&ggr; receptor I (Fc&ggr;RI) and the basis for coupling of Fc&ggr;RI to Fc&ggr;RIIB in endothelium. Methods and Results: In cultured endothelial cells, Fc&ggr;RI-blocking antibodies prevented CRP antagonism of eNOS, and CRP activated Src via Fc&ggr;RI. CRP-induced increases in Fc&ggr;RIIB immunoreceptor tyrosine-based inhibitory motif phosphorylation and SH2 domain-containing inositol 5′-phosphatase 1 activation were Src-dependent, and Src inhibition prevented eNOS antagonism by CRP. Similar processes mediated eNOS antagonism by aggregated IgG used to mimic immune complex. Carotid artery re-endothelialization was evaluated in offspring from crosses of CRP transgenic mice (TG-CRP) with either mice lacking the &ggr; subunit of Fc&ggr;RI (FcR&ggr;−/−) or Fc&ggr;RIIB−/− mice. Whereas re-endothelialization was impaired in TG-CRP vs wild-type, it was normal in both FcR&ggr;−/−; TG-CRP and Fc&ggr;RIIB−/−; TG-CRP mice. Conclusions: CRP antagonism of eNOS is mediated by the coupling of Fc&ggr;RI to Fc&ggr;RIIB by Src kinase and resulting activation of SH2 domain-containing inositol 5′-phosphatase 1, and consistent with this mechanism, both Fc&ggr;RI and Fc&ggr;RIIB are required for CRP to blunt endothelial repair in vivo. Similar mechanisms underlie eNOS antagonism by immune complex. Fc&ggr;RI and Fc&ggr;RIIB may be novel therapeutic targets for preventing endothelial dysfunction in inflammatory or immune complex-mediated conditions.

Fcγ Receptors and Ligands and Cardiovascular Disease

Fc&ggr; receptors (Fc&ggr;Rs) classically modulate intracellular signaling on binding of the Fc region of IgG in immune response cells. How Fc&ggr;R and their ligands affect cardiovascular health and disease has been interrogated recently in both preclinical and clinical studies. The stimulation of activating Fc&ggr;R 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 F&ggr;cR, Fc&ggr;RIIB, also has adverse consequences in endothelial cells, antagonizing NO production and reparative mechanisms. In preclinical disease models, activating Fc&ggr;Rs promote atherosclerosis, whereas Fc&ggr;RIIB is protective, and activating Fc&ggr;Rs also enhance thrombotic and nonthrombotic vascular occlusion. The Fc&ggr;R ligand C-reactive protein (CRP) has undergone intense study. Although in rodents CRP does not affect atherosclerosis, it causes hypertension and insulin resistance and worsens myocardial infarction. Massive data have 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 warrant further investigation. To date, studies of genetic variants of activating Fc&ggr;Rs are insufficient to implicate the receptors in coronary heart disease pathogenesis in humans. However, a link between Fc&ggr;RIIB and human hypertension may be emerging. Further knowledge of the vascular biology of Fc&ggr;R 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.

Endothelial Fcγ Receptor IIB Activation Blunts Insulin Delivery to Skeletal Muscle to Cause Insulin Resistance in Mice

Modest elevations in C-reactive protein (CRP) are associated with type 2 diabetes. We previously revealed in mice that increased CRP causes insulin resistance and mice globally deficient in the CRP receptor Fcγ receptor IIB (FcγRIIB) were protected from the disorder. FcγRIIB is expressed in numerous cell types including endothelium and B lymphocytes. Here we investigated how endothelial FcγRIIB influences glucose homeostasis, using mice with elevated CRP expressing or lacking endothelial FcγRIIB. Whereas increased CRP caused insulin resistance in mice expressing endothelial FcγRIIB, mice deficient in the endothelial receptor were protected. The insulin resistance with endothelial FcγRIIB activation was due to impaired skeletal muscle glucose uptake caused by attenuated insulin delivery, and it was associated with blunted endothelial nitric oxide synthase (eNOS) activation in skeletal muscle. In culture, CRP suppressed endothelial cell insulin transcytosis via FcγRIIB activation and eNOS antagonism. Furthermore, in knock-in mice harboring constitutively active eNOS, elevated CRP did not invoke insulin resistance. Collectively these findings reveal that by inhibiting eNOS, endothelial FcγRIIB activation by CRP blunts insulin delivery to skeletal muscle to cause insulin resistance. Thus, a series of mechanisms in endothelium that impairs insulin movement has been identified that may contribute to type 2 diabetes pathogenesis.

IgG Receptor FcγRIIB Plays a Key Role in Obesity-Induced Hypertension

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fc&ggr; receptor IIB (Fc&ggr;RIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that Fc&ggr;RIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that Fc&ggr;RIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas Fc&ggr;RIIB+/+ mice developed obesity-induced hypertension, Fc&ggr;RIIB−/− mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; Fc&ggr;RIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet–fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an Fc&ggr;RIIB-dependent process. Thus, we have identified a novel role for Fc&ggr;RIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting Fc&ggr;RIIB may potentially offer new means to treat hypertension in obese individuals.

Rasip1 is essential to blood vessel stability and angiogenic blood vessel growth

Abstract 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. 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. 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. Graphical Abstract

Hyposialylated IgG activates endothelial IgG receptor FcγRIIB to promote obesity-induced insulin resistance

Type 2 diabetes mellitus (T2DM) is a common complication of obesity. Here, we have shown that activation of the IgG receptor Fc&ggr;RIIB in endothelium by hyposialylated IgG plays an important role in obesity-induced insulin resistance. Despite becoming obese on a high-fat diet (HFD), mice lacking Fc&ggr;RIIB globally or selectively in endothelium were protected from insulin resistance as a result of the preservation of insulin delivery to skeletal muscle and resulting maintenance of muscle glucose disposal. IgG transfer in IgG-deficient mice implicated IgG as the pathogenetic ligand for endothelial Fc&ggr;RIIB in obesity-induced insulin resistance. Moreover, IgG transferred from patients with T2DM but not from metabolically healthy subjects caused insulin resistance in IgG-deficient mice via Fc&ggr;RIIB, indicating that similar processes may be operative in T2DM in humans. Mechanistically, the activation of Fc&ggr;RIIB by IgG from obese mice impaired endothelial cell insulin transcytosis in culture and in vivo. These effects were attributed to hyposialylation of the Fc glycan, and IgG from T2DM patients was also hyposialylated. In HFD-fed mice, supplementation with the sialic acid precursor N-acetyl-D-mannosamine restored IgG sialylation and preserved insulin sensitivity without affecting weight gain. Thus, IgG sialylation and endothelial Fc&ggr;RIIB may represent promising therapeutic targets to sever the link between obesity and T2DM.