Dongqi Xing

Estrogen and Mechanisms of Vascular Protection

Estrogen has antiinflammatory and vasoprotective effects when administered to young women or experimental animals that appear to be converted to proinflammatory and vasotoxic effects in older subjects, particularly those that have been hormone free for long periods. Clinical studies have raised many important questions about the vascular effects of estrogen that cannot easily be answered in human subjects. Here we review cellular/molecular mechanisms by which estrogen modulates injury-induced inflammation, growth factor expression, and oxidative stress in arteries and isolated vascular smooth muscle cells, with emphasis on the role of estrogen receptors and the nuclear factor-kappaB (NFkappaB) signaling pathway, as well as evidence that these protective mechanisms are lost in aging subjects.

Atrial Natriuretic Peptide Inhibits Transforming Growth Factor β–Induced Smad Signaling and Myofibroblast Transformation in Mouse Cardiac Fibroblasts

This study tested the hypothesis that activation of atrial natriuretic peptide (ANP)/cGMP/protein kinase G signaling inhibits transforming growth factor (TGF)-β1–induced extracellular matrix expression in cardiac fibroblasts and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Left ventricular hypertrophy and fibrosis, collagen deposition, and myofibroblast transformation of cardiac fibroblasts in response to pressure overload by transverse aortic constriction were exaggerated in ANP-null mice compared with wild-type controls. ANP and cGMP inhibited TGF-β1–induced myofibroblast transformation, proliferation, collagen synthesis, and plasminogen activator inhibitor-1 expression in cardiac fibroblasts isolated from wild-type mice. Following pretreatment with cGMP, TGF-β1 induced phosphorylation of Smad3, but the resultant pSmad3 could not be translocated to the nucleus. pSmad3 that had been phosphorylated with recombinant protein kinase G-1α was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry. The analysis revealed phosphorylation of Ser309 and Thr388 residues, sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-β receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3. These results suggest that phosphorylation of Smad3 by protein kinase G is a potential molecular mechanism by which activation of ANP/cGMP/protein kinase G signaling disrupts TGF-β1–induced nuclear translocation of pSmad3 and downstream events, including myofibroblast transformation, proliferation, and expression of extracellular matrix molecules in cardiac fibroblasts. We postulate that this process contributes to the antifibrogenic effects of the natriuretic peptide in heart.

Estrogen Modulates Inflammatory Mediator Expression and Neutrophil Chemotaxis in Injured Arteries

Background—We have previously shown that estrogen (17&bgr;-estradiol; E2) inhibits neointima formation and migration of leukocytes, particularly neutrophils, into rat carotid arteries after acute endoluminal injury. This study tested the hypothesis that E2 inhibits expression of adhesion molecules, chemokines, and proinflammatory cytokines in rat carotid arteries in the early hours after balloon injury, thus attenuating the stimulus for leukocyte entry and negatively modulating the injury response. Methods and Results—Ovariectomized (OVX) rats were randomly assigned to treatment with E2 or vehicle (V) and subjected to balloon injury of the right carotid artery. After 2, 6, and 24 hours, rats were euthanized, and both carotid arteries were processed for real-time reverse transcription–polymerase chain reaction (2 and 24 hours), ELISA (6 hours), or neutrophil chemotaxis assay (24 hours). Expression of mRNA for adhesion molecules (P-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1), chemoattractants (cytokine-induced neutrophil chemoattractant [CINC]-2&bgr; and monocyte chemoattractant protein [MCP]-1), and proinflammatory cytokines (interleukin [IL]-1 and IL-6) was markedly increased (2 to 5000 times) in injured arteries of OVX+V rats at 2 hours and was reduced by 24 hours. E2 significantly attenuated expression of the proinflammatory mediators (by 60% to 80%) at 2 hours. ELISA confirmed injury-induced upregulation of neutrophil and monocyte/macrophage chemoattractants (CINC-2&agr;, MCP-1) in OVX+V arteries and E2-induced inhibition of CINC-2&agr; expression. E2 significantly (by 65%) inhibited neutrophil chemotactic activity of arterial homogenates. Conclusions—E2 attenuates the early vascular injury response, at least in part, by negatively modulating proinflammatory mediator expression and the resultant chemotactic activity of injured vessels for neutrophils.

Protein O-GlcNAcylation: a new signaling paradigm for the cardiovascular system

The posttranslational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide beta-N-acetylglucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification. Protein O-GlcNAcylation is rapidly emerging as a key regulator of critical biological processes including nuclear transport, translation and transcription, signal transduction, cytoskeletal reorganization, proteasomal degradation, and apoptosis. Increased levels of O-GlcNAc have been implicated as a pathogenic contributor to glucose toxicity and insulin resistance, which are both major hallmarks of diabetes mellitus and diabetes-related cardiovascular complications. Conversely, there is a growing body of data demonstrating that the acute activation of O-GlcNAc levels is an endogenous stress response designed to enhance cell survival. Reports on the effect of altered O-GlcNAc levels on the heart and cardiovascular system have been growing rapidly over the past few years and have implicated a role for O-GlcNAc in contributing to the adverse effects of diabetes on cardiovascular function as well as mediating the response to ischemic injury. Here, we summarize our present understanding of protein O-GlcNAcylation and its effect on the regulation of cardiovascular function. We examine the pathways regulating protein O-GlcNAcylation and discuss, in more detail, our understanding of the role of O-GlcNAc in both mediating the adverse effects of diabetes as well as its role in mediating cellular protective mechanisms in the cardiovascular system. In addition, we also explore the parallels between O-GlcNAc signaling and redox signaling, as an alternative paradigm for understanding the role of O-GlcNAcylation in regulating cell function.

Estradiol and Progestins Differentially Modulate Leukocyte Infiltration After Vascular Injury

Background—Inflammation plays an important role in the response to endoluminal vascular injury. Estrogen (17&bgr;-estradiol, E2) inhibits neointima formation in animal models, and the progestin medroxyprogesterone acetate (MPA) blocks this effect. This study tested the hypothesis that E2 inhibits the migration of inflammatory cells, particularly granulocytes, into the rat carotid arteries after acute endoluminal injury and that MPA blocks this effect. Methods and Results—Ovariectomized rats were randomly divided into subgroups and treated with E2, MPA, E2+MPA, or vehicle and subjected to balloon injury of the right carotid artery. After 1, 3, or 7 days, rats were euthanized, and carotid arteries (injured and control) were analyzed for inflammatory cells by flow cytometry. At 1 day, granulocytes (HIS48+ and CD45+), monocyte/macrophages (Mar1+ and CD45+), and T lymphocytes (CD3+ and CD45+) were increased 26-fold, 12-fold, and 3-fold, respectively, in injured compared with contralateral control arteries of vehicle-treated rats. Granulocytes and monocyte/macrophages decreased markedly by 3 days. E2 reduced the granulocyte and monocyte/macrophage populations of injured vessels by ≈50% and increased T lymphocytes. MPA had no independent effect on inflammatory cells but completely blocked the effect of E2. Immunohistochemical examination verified these findings and localized inflammatory cells to the adventitial and periadventitial domains of injured vessels. Conclusions—E2 may limit the neointimal response to endoluminal vascular injury, at least in part, by limiting leukocyte entry from adventitial/periadventitial tissues into injured vessels early in the injury response.

Hormone Replacement Therapy and Inflammation. Interactions in Cardiovascular Disease

Inflammation plays a central role in the pathogenesis of many forms of vascular disease, including atherosclerosis. Atherogenesis begins with endothelial damage, and the damaged endothelium expresses adhesion molecules, chemokines, and proinflammatory cytokines that direct atherosclerotic plaque formation and spill into the circulation as biomarkers of atherosclerotic disease risk. Menopausal hormone therapy, including a variety of estrogen preparations with or without a progestin, has negative modulatory effects on most of these soluble inflammatory markers, including E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha, inconsistent effects on interleukin-6, and stimulatory effects on transforming growth factor-beta, a vasoprotective cytokine. In contrast, C-reactive protein, a circulating proinflammatory cytokine produced in both liver and atherosclerotic arteries, increases in response to oral conjugated estrogens but not to transdermal estrogen. Although C-reactive protein is clearly linked to increased cardiovascular disease risk in women, the hormone-induced rise in this biomarker is not associated with increased risk and may be related to a first-pass effect of C-reactive protein production in the liver after oral estrogen absorption. Many important questions about the effects of ovarian hormones on vascular inflammation and the pathogenesis of vascular disease cannot be answered in human subjects. Insights from fundamental mechanistic studies in animal models are needed to delineate the cellular/molecular events that determine whether these hormones protect or injure blood vessels.

Increased protein O-GlcNAc modification inhibits inflammatory and neointimal responses to acute endoluminal arterial injury

Inflammation plays a major role in vascular disease. We have shown that leukocyte infiltration and inflammatory mediator expression contribute to vascular remodeling after endoluminal injury. This study tested whether increasing protein O-linked-N-acetylglucosamine (O-GlcNAc) levels with glucosamine (GlcN) and O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) inhibits acute inflammatory and neointimal responses to endoluminal arterial injury. Ovariectomized rats were treated with a single injection of GlcN (0.3 mg/g ip), PUGNAc (7 nmol/g ip) or vehicle (V) 2 h before balloon injury of the right carotid artery. O-GlcNAc-modified protein levels decreased markedly in injured arteries of V-treated rats at 30 min, 2 h, and 24 h after injury but returned to control (contralateral uninjured) levels after 14 days. Both GlcN and PUGNAc increased O-GlcNAc-modified protein levels in injured arteries compared with V controls at 30 min postinjury; the GlcN-mediated increase persisted at 24 h but was not evident at 14 days. Proinflammatory mediator expression increased markedly after injury and was reduced significantly (30-50%) by GlcN and PUGNAc. GlcN and PUGNAc also inhibited infiltration of neutrophils and monocytes in injured arteries. Chronic (14 days) treatment with GlcN reduced neointima formation in injured arteries by 50% compared with V controls. Acute GlcN and PUGNAc treatment increases O-GlcNAc-modified protein levels and inhibits acute inflammatory responses in balloon-injured rat carotid arteries; 14 day GlcN treatment inhibits neointima formation in these vessels. Augmenting O-GlcNAc modification of proteins in the vasculature may represent a novel anti-inflammatory and vasoprotective mechanism.

O -GlcNAc Modification of NFκB p65 Inhibits TNF-α-Induced Inflammatory Mediator Expression in Rat Aortic Smooth Muscle Cells

Background We have shown that glucosamine (GlcN) or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) treatment augments O-linked-N-acetylglucosamine (O-GlcNAc) protein modification and attenuates inflammatory mediator expression, leukocyte infiltration and neointima formation in balloon injured rat carotid arteries and have identified the arterial smooth muscle cell (SMC) as the target cell in the injury response. NFκB signaling has been shown to mediate the expression of inflammatory genes and neointima formation in injured arteries. Phosphorylation of the p65 subunit of NFκB is required for the transcriptional activation of NFκB. This study tested the hypothesis that GlcN or PUGNAc treatment protects vascular SMCs against tumor necrosis factor (TNF)-α induced inflammatory stress by enhancing O-GlcNAcylation and inhibiting TNF-α induced phosphorylation of NFκB p65, thus inhibiting NFκB signaling. Methodology/Principal Findings Quiescent rat aortic SMCs were pretreated with GlcN (5 mM), PUGNAc (10−4 M) or vehicle and then stimulated with TNF-α (10 ng/ml). Both treatments inhibited TNF-α-induced expression of chemokines [cytokine-induced neutrophil chemoattractant (CINC)-2β and monocyte chemotactic protein (MCP)-1] and adhesion molecules [vascular cell adhesion molecule (VCAM)-1 and P-Selectin]. Both treatments inhibited TNF-α induced NFκB p65 activation and promoter activity, increased NFκB p65 O-GlcNAcylation and inhibited NFκB p65 phosphorylation at Serine 536, thus promoting IκBα binding to NFκB p65. Conclusions There is a reciprocal relationship between O-GlcNAcylation and phosphorylation of NFκB p65, such that increased NFκB p65 O-GlcNAc modification inhibits TNF-α-Induced expression of inflammatory mediators through inhibition of NFκB p65 signaling. These findings provide a mechanistic basis for our previous observations that GlcN and PUGNAc treatments inhibit inflammation and remodeling induced by acute endoluminal arterial injury.

Aging leads to increased levels of protein O-linked N-acetylglucosamine in heart, aorta, brain and skeletal muscle in Brown-Norway rats

Changes in the levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on nucleocytoplasmic protein have been associated with a number of age-related diseases such as Alzheimer’s and diabetes; however, there is relatively little information regarding the impact of age on tissue O-GlcNAc levels. Therefore, the goal of this study was to determine whether senescence was associated with alterations in O-GlcNAc in heart, aorta, brain and skeletal muscle and if so whether there were also changes in the expression of enzymes critical in regulating O-GlcNAc levels, namely, O-GlcNAc transferase (OGT), O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase (GFAT). Tissues were harvested from 5- and 24-month old Brown-Norway rats; UDP-GlcNAc, a precursor of O-GlcNAc was assessed by HPLC, O-GlcNAc and OGT levels were assessed by immunoblot analysis and GFAT1/2, OGT, O-GlcNAcase mRNA levels were determined by RT-PCR. In the 24-month old animals serum insulin and triglyceride levels were significantly increased compared to the 5-month old group; however, glucose levels were unchanged. Protein O-GlcNAc levels were significantly increased with age (30–107%) in all tissues examined; however, paradoxically the expression of OGT, which catalyzes O-GlcNAc formation, was decreased by ∼30% in the heart, aorta and brain. In the heart increased O-GlcNAc was associated with increased UDP-GlcNAc levels and elevated GFAT mRNA while in other tissues we found no difference in UDP-GlcNAc or GFAT mRNA levels. These results demonstrate that senescence is associated with increased O-GlcNAc levels in multiple tissues and support the notion that dysregulation of pathways leading to O-GlcNAc formation may play an important role in the development of age-related diseases.

Aged rats lose vasoprotective and anti-inflammatory actions of estrogen in injured arteries.

Objective: 17&bgr;-Estradiol (E2) negatively modulates neointima formation, leukocyte infiltration, and proinflammatory mediator expression after vascular injury in young (10-wk-old) ovariectomized (OVX) rats. Trials of E2 in elderly postmenopausal women have not confirmed a vasoprotective effect. This study tested the hypothesis that responsiveness to E2 is lost in injured arteries of aged (12-mo-old) OVX rats. Design: E2- or vehicle-treated OVX rats underwent balloon injury of the carotid artery and were killed after 2 weeks for morphometric examination of arteries, after 24 hours for assessment of leukocyte infiltration, and after 2 hours for quantification of proinflammatory mediator mRNA expression. Results: Neointima formation was significantly reduced in aged compared with young vehicle-treated rats. E2 treatment had directionally opposite effects on intima/media ratios in aged (+75%) and young (−40%) rats. Injury induced increases in infiltrating total leukocytes, neutrophils, monocytes/macrophages, and expression of proinflammatory mediators in arteries of aged rats; E2 had no effect on these inflammatory responses to injury. Estrogen receptor &agr; and &bgr; protein expression were similar in carotid arteries of young and aged rats on immunofluorescence testing. Conclusions: Aged OVX rats lose the vasoprotective and anti-inflammatory responses to exogenous E2 seen in younger animals. These results may be relevant to the lack of vasoprotection observed in outcome trials of estrogen therapy in postmenopausal women.