Lisa Hahner

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide–dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Distinct hypothalamic neurons mediate estrogenic effects on energy homeostasis and reproduction.

Estrogens regulate body weight and reproduction primarily through actions on estrogen receptor-α (ERα). However, ERα-expressing cells mediating these effects are not identified. We demonstrate that brain-specific deletion of ERα in female mice causes abdominal obesity stemming from both hyperphagia and hypometabolism. Hypometabolism and abdominal obesity, but not hyperphagia, are recapitulated in female mice lacking ERα in hypothalamic steroidogenic factor-1 (SF1) neurons. In contrast, deletion of ERα in hypothalamic pro-opiomelanocortin (POMC) neurons leads to hyperphagia, without directly influencing energy expenditure or fat distribution. Further, simultaneous deletion of ERα from both SF1 and POMC neurons causes hypometabolism, hyperphagia, and increased visceral adiposity. Additionally, female mice lacking ERα in SF1 neurons develop anovulation and infertility, while POMC-specific deletion of ERα inhibits negative feedback regulation of estrogens and impairs fertility in females. These results indicate that estrogens act on distinct hypothalamic ERα neurons to regulate different aspects of energy homeostasis and reproduction.

The imprinted antisense RNA at the igf2r locus overlaps but does not imprint Mas1

The gene encoding the insulin-like growth-factor type-2 receptor (Igf2r) is maternally expressed and imprinted. A CpG island in Igf2r intron 2 that carries a maternal-specific methylation imprint was shown in a transgenic model to be essential for Igf2r imprinting and for the production of an antisense RNA from the paternal allele. We report here that the endogenous region2 is the promoter for this antisense RNA (named Air, for antisense Igf2r RNA) and that the 3′ end lies 107,796 bp distant in an intron of the flanking, but non-imprinted, gene Mas1.

High-Density Lipoprotein Promotes Endothelial Cell Migration and Reendothelialization via Scavenger Receptor-B Type I

Vascular disease risk is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL provides vascular protection are unclear. The disruption of endothelial monolayer integrity is an important contributing factor in multiple vascular disorders, and vascular lesion severity is tempered by enhanced endothelial repair. Here, we show that HDL stimulates endothelial cell migration in vitro in a nitric oxide-independent manner via scavenger receptor B type I (SR-BI)-mediated activation of Rac GTPase. This process does not require HDL cargo molecules, and it is dependent on the activation of Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinases. Rapid initial stimulation of lamellipodia formation by HDL via SR-BI, Src kinases, and Rac is also demonstrable. Paralleling the in vitro findings, carotid artery reendothelialization after perivascular electric injury is blunted in apolipoprotein A-I−/− mice, and reconstitution of apolipoprotein A-I expression rescues normal reendothelialization. Furthermore, reendothelialization is impaired in SR-BI−/− mice. Thus, HDL stimulates endothelial cell migration via SR-BI-initiated signaling, and these mechanisms promote endothelial monolayer integrity in vivo.

Estrogen Causes Dynamic Alterations in Endothelial Estrogen Receptor Expression

Abstract— Estrogen receptor (ER)&agr; mediates many of the effects of estrogen on the vascular endothelium. The purpose of the present study was to determine whether estrogen modifies endothelial ER&agr; expression. In experiments in cultured ovine endothelial cells, physiological concentrations of 17&bgr;-estradiol (E2, 10−10 to 10−8 mol/L) caused an increase in ER&agr; protein abundance that was evident after 6 hours of hormone exposure. Shorter (2-hour) E2 treatment caused ER&agr; downregulation. In contrast to the upregulation in ER&agr; after long-term E2, the expression of the other ER isoform, ER&bgr;, was downregulated. Both nonselective ER antagonism with ICI 182,780 and the inhibition of gene transcription with actinomycin D blocked the increase in ER&agr; with E2. In studies using the human ER&agr; gene promoter P-1 coupled to luciferase, an increase in ER&agr; gene transcription was evident in endothelial cells within 4 hours of E2 exposure. The transcriptional activation was fully blocked by ICI 182,780, whereas the specific ER&bgr; antagonist RR-tetrahydrochrysene yielded partial blockade. Overexpression of ER&agr; or ER&bgr; caused comparable 10- and 8-fold increases, respectively, in ER&agr; promoter activation by E2. Thus, long-term exposure to E2 upregulates ER&agr; expression in endothelial cells through the actions of either ER&agr; or ER&bgr; on ER&agr; gene transcription; in contrast, E2 causes ER&bgr; downregulation in the endothelium. We postulate that E2-induced changes in ER&agr; and ER&bgr; expression modify the effects of the hormone on vascular endothelium.

FcγRIIB Mediates C-Reactive Protein Inhibition of Endothelial NO Synthase

C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fc&ggr; receptors, causes parallel okadaic acid–sensitive loss of eNOS function, Fc&ggr;RIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires Fc&ggr;RIIB. In Fc&ggr;RIIB+/+ mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in Fc&ggr;RIIB−/− mice. Thus Fc&ggr;RIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.

The sexually dimorphic role of adipose and adipocyte estrogen receptors in modulating adipose tissue expansion, inflammation, and fibrosis

Our data demonstrate that estrogens, estrogen receptor-α (ERα), and estrogen receptor-β (ERβ) regulate adipose tissue distribution, inflammation, fibrosis, and glucose homeostasis, by determining that αERKO mice have increased adipose tissue inflammation and fibrosis prior to obesity onset. Selective deletion of adipose tissue ERα in adult mice using a novel viral vector technology recapitulated the findings in the total body ERα null mice. Generation of a novel mouse model, lacking ERα specifically from adipocytes (AdipoERα), demonstrated increased markers of fibrosis and inflammation, especially in the males. Additionally, we found that the beneficial effects of estrogens on adipose tissue require adipocyte ERα. Lastly, we determined the role of ERβ in regulating inflammation and fibrosis, by breeding the AdipoERα into the βERKO background and found that in the absence of adipocyte ERα, ERβ has a protective role. These data suggest that adipose tissue and adipocyte ERα protects against adiposity, inflammation, and fibrosis in both males and females.

C-Reactive Protein Causes Downregulation of Vascular Angiotensin Subtype 2 Receptors and Systolic Hypertension in Mice

Background— Chronic elevations in circulating C-reactive protein (CRP) are associated with a greater risk of hypertension. Whether elevations in CRP cause hypertension is unknown. Methods and Results— Chronic, conscious blood pressure (BP) measurements were performed by radiotelemetry in wild-type CF1 control and CF1 transgenic mice expressing rabbit CRP (CF1-CRP) under the regulation of the phosphoenolpyruvate carboxykinase promoter. Compared with controls, CF1-CRP mice had hypertension that was predominantly systolic, and the severity of hypertension varied in parallel with changes in CRP levels modulated by dietary manipulation. Mice that were hemizygous for the transgene with CRP levels of 9 &mgr;g/mL were also hypertensive, indicating that modest elevations in CRP are sufficient to alter BP. CRP transgenic mice had exaggerated BP elevation in response to angiotensin II and a reduction in vascular angiotensin receptor subtype 2 (AT2) expression. In contrast, the decline in BP with angiotensin receptor subtype 1 (AT1) antagonism and vascular AT1 abundance were unaltered, which indicates a selective effect of CRP on AT2. Ex vivo experiments further showed that the CRP-induced decrease in AT2 is a direct effect on the vascular wall, not requiring systemic responses, and that it is reversed by an NO donor, which indicates a role for NO deficiency in the process. In parallel, the chronic inhibition of NO synthase in wild-type mice attenuated vascular AT2 expression without affecting AT1. Conclusions— These findings provide direct evidence for CRP-induced hypertension, and they further identify a novel underlying mechanism involving downregulation of AT2 related to NO deficiency.

C-Reactive Protein Downregulates Endothelial NO Synthase and Attenuates Reendothelialization In Vivo in Mice

C-reactive protein (CRP) is an acute-phase reactant that is positively associated with cardiovascular disease risk and endothelial dysfunction. In cell culture, CRP decreases the expression of endothelial NO synthase (eNOS), which regulates diverse endothelial cell (EC) functions including migration. To determine whether CRP alters EC gene expression and phenotype in vivo, we studied CF1 transgenic mice expressing rabbit CRP (CF1-CRP) regulated by the phosphoenolpyruvate carboxykinase promoter such that levels could be altered by changing carbohydrate intake. Compared with CF1 controls with CRP of <1 &mgr;g/mL, carotid artery reendothelialization after perivascular electric injury was blunted in CF1-CRP mice, with CRP levels as low as 9 &mgr;g/mL. eNOS mRNA and enzyme abundance in carotid arteries was also blunted by CRP at 9 &mgr;g/mL in vivo, and ex vivo studies of isolated arteries showed that this occurs via direct action on the endothelium. The impaired reendothelialization with CRP was mimicked by NOS antagonism in CF1 mice; conversely, in cultured ECs CRP attenuation of migration was prevented by exogenous NO. Studies of EC transfected with human eNOS 5′ flanking sequence fused to luciferase indicated that CRP decreases eNOS gene transcription. Both mutagenesis and electrophoretic mobility shift assays further revealed that CRP-responsive elements reside within the first 79 bp of the eNOS promoter. Thus, CRP downregulates eNOS and attenuates reendothelialization in vivo in mice, and this action of CRP on eNOS is mediated at the level of gene transcription.

GPR30 deficiency causes increased bone mass, mineralization, and growth plate proliferative activity in male mice

Estrogen regulation of the male skeleton was first clearly demonstrated in patients with aromatase deficiency or a mutation in the ERα gene. Estrogen action on the skeleton is thought to occur mainly through the action of the nuclear receptors ERα and ERβ. Recently, in vitro studies have shown that the G protein–coupled receptor GPR30 is a functional estrogen receptor (ER). GPR30‐deficient mouse models have been generated to study the in vivo function of this protein; however, its in vivo role in the male skeleton remains underexplored. We have characterized size, body composition, and bone mass in adult male Gpr30 knockout (KO) mice and their wild‐type (WT) littermates. Gpr30 KO mice weighed more and had greater nasal‐anal length (p < .001). Both lean mass and percent body fat were increased in the KO mice. Femur length was greater in Gpr30 KO mice, as was whole‐body, spine, and femoral areal bone mineral density (p < .01). Gpr30 KO mice showed increased trabecular bone volume (p < .01) and cortical thickness (p < .001). Mineralized surface was increased in Gpr30 KO mice (p < .05). Bromodeoxyuridine (BrdU) labeling showed greater proliferation in the growth plate of Gpr30 KO mice (p < .05). Under osteogenic culture conditions, Gpr30 KO femoral bone marrow cells produced fewer alkaline phosphatase–positive colonies in early differentiating osteoblast cultures but showed increased mineralized nodule deposition in mature osteoblast cultures. Serum insulin‐like growth factor 1 (IGF‐1) levels were not different. These data suggest that in male mice, GPR30 action contributes to regulation of bone mass, size, and microarchitecture by a mechanism that does not require changes in circulating IGF‐1.