Chao-Hung Wang

A Self-Fulfilling Prophecy C-Reactive Protein Attenuates Nitric Oxide Production and Inhibits Angiogenesis

Background—Given the central importance of nitric oxide (NO) in the development and clinical course of cardiovascular diseases, we sought to determine whether the powerful predictive value of C-reactive protein (CRP) might be explained through an effect on NO production. Methods and Results—Endothelial cells (ECs) were incubated with recombinant CRP (0 to 100 &mgr;g/mL, 24 hours), and NO and cyclic guanosine monophosphate (cGMP) production was assessed. The effects of CRP on endothelial NO synthase (eNOS) protein, mRNA expression, and mRNA stability were also examined. In a separate study, the effects of CRP (25 &mgr;g/mL) on EC cell survival, apoptosis, and in vitro angiogenesis were evaluated. Incubation of ECs with CRP resulted in a significant inhibition of basal and stimulated NO release, with concomitant reductions in cGMP production. CRP caused a marked downregulation of eNOS mRNA and protein expression. Actinomycin D studies suggested that eNOS downregulation was related to decreased mRNA stability. In conjunction with a decrease in NO production, CRP inhibited both basal and vascular endothelial growth factor–stimulated angiogenesis as assessed by EC migration and capillary-like tube formation. CRP did not induce EC survival but did, however, promote apoptosis in a NO-dependent fashion. Conclusions—CRP, at concentrations known to predict adverse vascular events, directly quenches the production of the NO, in part, through posttranscriptional effect on eNOS mRNA stability. Diminished NO bioactivity, in turn, inhibits angiogenesis, an important compensatory mechanism in chronic ischemia. Through decreasing NO synthesis, CRP may facilitate the development of diverse cardiovascular diseases. Risk reduction strategies designed to lower plasma CRP may be effective by improving NO bioavailability.

Resistin Promotes Endothelial Cell Activation Further Evidence of Adipokine-Endothelial Interaction

Background—Adipocyte-derived hormones may represent a mechanism linking insulin resistance to cardiovascular disease. In the present study, we evaluated the direct effects of resistin, a novel adipocyte-derived hormone, on endothelial activation. Methods and Results—Endothelial cells (ECs) were incubated with human recombinant resistin (10 to 100 ng/ML, 24 hours), and endothelin-1 (ET-1) release, ET-1 mRNA expression, and nitric oxide (NO) production were assessed. Transient transfection assays were used to evaluate the effects of resistin on transcription of human ET-1 gene promoter. Furthermore, the effects of resistin on AP-1–mutated ET-1 promoter were evaluated. The effects of resistin on expression of vascular cell adhesion molecule (VCAM-1) and monocyte chemoattractant chemokine (MCP-1) were studied in addition to CD40 receptor, CD40 ligand–induced MCP-1 expression, and tumor necrosis factor receptor–associated factor-3 (TRAF3), an inhibitor of CD40 signaling. Incubation of ECs with resistin resulted in an increase in ET-1 release and ET-1 mRNA expression, with no change in NO production. Whereas treatment with resistin resulted in an increase in ET-1 promoter activity, the AP-1–mutated promoter was inactive after resistin stimulation. Additionally, resistin-treated cells showed increased expression of VCAM-1 and MCP-1, with concomitant reductions in TRAF-3 expression. Resistin did not alter CD40 receptor expression; however, increased CD40 ligand induced MCP-1 production. Conclusions—The novel adipokine resistin exerts direct effects to promote EC activation by promoting ET-1 release, in part by inducing ET-1 promoter activity via the AP-1 site. Furthermore, resistin upregulates adhesion molecules and chemokines and downregulates TRAF-3, an inhibitor of CD40 ligand signaling. In this fashion, resistin may be mechanistically linked to cardiovascular disease in the metabolic syndrome.

Rosiglitazone Facilitates Angiogenic Progenitor Cell Differentiation Toward Endothelial Lineage A New Paradigm in Glitazone Pleiotropy

Background—Peroxisome proliferator–activated receptor-&ggr; (PPAR-&ggr;) agonists inhibit vascular smooth muscle proliferation and migration and improve endothelial function. It is unknown whether PPAR-&ggr; agonists favorably modulate bone marrow (BM)–derived angiogenic progenitor cells (APCs) to promote endothelial lineage differentiation and early reendothelialization after vascular intervention. Methods and Results—C57/BL6 mice, treated with or without rosiglitazone (8 mg/kg per day), a PPAR-&ggr; agonist, underwent femoral angioplasty. Rosiglitazone treatment attenuated neointimal formation (intima/media ratio: 0.98±0.12 [rosiglitazone] versus 3.1±0.5 [control]; P <0.001; n=10 per group). Using a BM transplantation model, we identified that 58±12% of the cells within the neointima at 4 weeks were derived from the BM. Pure endothelial marker–positive, pure &agr;-smooth muscle actin (&agr;SMA)–positive, or double-positive APCs could be found both in mouse BM and in human peripheral blood after culture in conditional medium enriched with vascular endothelial growth factor. Rosiglitazone caused a 6-fold (P <0.001) increase in colony formation by human endothelial progenitor cells, promoted the differentiation of APCs toward the endothelial lineage in mouse BM in vivo (0.66±0.06% [control] to 0.95±0.08% [rosiglitazone]; P <0.05) and in human peripheral blood in vitro (13.2±1.5% [control] to 28.4±3.3% [rosiglitazone]; P <0.05), and inhibited the differentiation toward the smooth muscle cell lineage. Within the neointima, rosiglitazone also stimulated APCs to differentiate into mature endothelial cells and caused earlier reendothelialization compared with controls (31±5 versus 8±2 CD31-positive cells per millimeter of neointimal surface on day 14; P <0.01). Conclusions—Similar to embryonic stem cell–derived progenitors, the adult BM and peripheral blood harbor APCs that are at least bipotential and able to differentiate into endothelial and smooth muscle lineages. The PPAR-&ggr; agonist rosiglitazone promotes the differentiation of these APCs toward the endothelial lineage and attenuates restenosis after angioplasty.

Aerobic interval training improves oxygen uptake efficiency by enhancing cerebral and muscular hemodynamics in patients with heart failure

BACKGROUND Abnormal ventilatory/hemodynamic responses to exercise contribute to functional impairment in patients with heart failure (HF). This study investigates how interval and continuous exercise regimens influence functional capacity by modulating ventilatory efficiency and hemodynamic function in HF patients. METHODS Forty-five HF patients were randomized to perform either aerobic interval training (AIT; 3-minute intervals at 40% and 80% VO(2peak)) or moderate continuous training (MCT; sustained 60% VO()for 30 min/day, 3 days/week for 12 weeks, or to a control group that received general healthcare (GHC). A noninvasive bio-reactance device was adopted to measure cardiac hemodynamics, whereas a near-infrared spectroscopy was employed to assess perfusion/O2 extraction in frontal cerebral lobe (∆[THb]FC/∆[HHb]FC) and vastus lateralis (∆[THb]VL/∆[HHb]VL), respectively. RESULTS Following the 12-week intervention, the AIT group exhibited higher oxygen uptake efficiency slope (OUES) and lower VE-VCO2 slope than the MCT and GHC groups. Furthermore, AIT, but not MCT, boosted cardiac output (CO) and increased ∆[THb]FC, ∆[THb]VL, and ∆[HHb]VL during exercise. In multivariate analyses, CO was the dominant predictor of VO(2peak). ∆[THb]FC and ∆[THb]VL, which modulated the correlation between CO and OUES, were significantly correlated with OUES. Simultaneously, ∆[THb]VL was the only factor significantly associated with VE-VCO2 slope. Additionally, AIT reduced plasma brain natriuretic peptide, myeloperoxidase, and interleukin-6 levels and increased the Short Form-36 physical/mental component scores and decreased the Minnesota Living with Heart Failure questionnaire score. CONCLUSIONS AIT effectively improves oxygen uptake efficiency by enhancing cerebral/muscular hemodynamics and suppresses oxidative stress/inflammation associated with cardiac dysfunction, and also promotes generic/disease-specific qualities of life in patients with HF.

Late-Outgrowth Endothelial Cells Attenuate Intimal Hyperplasia Contributed by Mesenchymal Stem Cells After Vascular Injury

Objectives—Mesenchymal stem cells (MSCs) are one of a number of cell types undergoing extensive investigation for cardiac regeneration therapy. It has not yet been determined whether this cell therapy also substantially contributes to vascular remodeling of diseased vessels. Methods and Results—Human MSCs and a variety of progenitor and vascular cells were used for in vitro and in vivo experiments. Wire-induced vascular injury mobilized MSCs into the circulation. Compared with human aortic smooth muscle cells, MSCs exhibited a 2.8-fold increase in the adhesion capacity in vitro (P<0.001) and a 6.3-fold increase in vivo (P<0.001). In all animal models, a significant amount of MSCs contributed to intimal hyperplasia after vascular injury. MSCs were able to differentiate into cells of endothelial or smooth muscle lineage. Coculture experiments demonstrated that late-outgrowth endothelial cells (OECs) guided MSCs to differentiate toward an endothelial lineage through a paracrine effects. In vivo, cell therapy with OECs significantly attenuated the thickness of the neointima contributed by MSCs (intima/media ratio, from 3.2±0.4 to 0.4±0.1, P<0.001). Conclusions—Tissue regeneration therapy with MSCs or cell populations containing MSCs requires a strategy to attenuate the high potential of MSCs to develop intimal hyperplasia on diseased vessels.

Glitazones and Heart Failure Critical Appraisal for the Clinician

Mr S. is a 46-year-old East-Indian male with typical manifestations of the cardiovascular dysmetabolic syndrome of insulin resistance (type II diabetes, obesity, dyslipidemia, hypertension, and elevated levels of high-sensitivity C-reactive protein). His medications include ramipril, simvastatin, enteric-coated aspirin, metformin, and glyburide. He has no symptoms of cardiac ischemia or congestive heart failure and has preserved left ventricular function. Over the past few months his glycemic control has been inadequate, and a decision to initiate an insulin sensitizer (glitazone) is made. Treatment is initiated with rosiglitazone 4 mg twice daily, with marked improvement in glycemic control and other components of the cardiovascular dysmetabolic syndrome. Approximately 6 months after initiation of therapy, your junior resident receives a phone call from Mr S, who is extremely anxious and distraught about the possibility of developing heart failure on glitazone therapy. The resident and the patient request your expert opinion about the effects of glitazones on cardiac function and associated hemodynamics. Insulin resistance has been increasingly recognized as a central metabolic disturbance predisposing a patient to hypertension, hyperlipidemia, premature atherosclerosis, left ventricular hypertrophy, and endothelial dysfunction.1 In addition to being a powerful risk marker for the development of cardiovascular disease, insulin resistance is also closely related to cardiac dysfunction and heart failure.2 Thiazolidinediones (TZD; glitazones) are peroxisome proliferator-activated receptor (PPAR) agonists that specifically augment insulin sensitivity and counter insulin resistance in patients with the cardiovascular dysmetabolic syndrome. Currently, there are 2 commercially available glitazones, rosiglitazone and pioglitazone. Troglitazone was withdrawn from the market because of hepatic side effects. The PPAR family is composed of 3 subtypes. TZDs bind with high affinity to the PPARγ isoform. The PPARγ isoform is found in the heart,3–6 endothelium, vascular smooth muscle (including atherosclerotic lesions and neointima formed after angioplasty), liver, skeletal muscle, monocytes/macrophages, and many other …

Stem Cell Factor Deficiency Is Vasculoprotective: Unraveling a New Therapeutic Potential of Imatinib Mesylate

Evidence suggests that bone marrow (BM) cells may give rise to a significant proportion of smooth muscle cells (SMCs) that contribute to intimal hyperplasia after vascular injury; however, the molecular pathways involved and the timeline of these events remain poorly characterized. We hypothesized that the stem cell factor (SCF)/c-Kit tyrosine kinase signaling pathway is critical to neointimal formation by BM-derived progenitors. Wire-induced femoral artery injury in mice reconstituted with wild-type BM cells expressing yellow fluorescent protein was performed, which revealed that 66±12% of the SMCs (α-smooth muscle actin-positive [αSMA+] cells) in the neointima were from BM. To characterize the role of the SCF/c-Kit pathway, we used c-Kit deficient W/Wv and SCF-deficient Steel-Dickie mice. Strikingly, vascular injury in these mice resulted in almost a complete inhibition of neointimal formation, whereas wild-type BM reconstitution of c-Kit mutant mice led to neointimal formation in a similar fashion as wild-type animals, as did chronic administration of SCF in matrix metalloproteinase-9–deficient mice, a model of soluble SCF deficiency. Pharmacological antagonism of the SCF/c-Kit pathway with imatinib mesylate (Gleevec) or ACK2 (c-Kit antibody) also resulted in a marked reduction in intimal hyperplasia. Vascular injury resulted in the local upregulation of SCF expression. c-Kit+ progenitor cells (PCs) homed to the injured vascular wall and differentiated into αSMA+ cells. Vascular injury also caused an increase in circulating SCF levels which promoted CD34+ PC mobilization, a response that was blunted in mutant and imatinib mesylate-treated mice. In vitro, SCF promoted adhesion of BM PCs to fibronectin. Additionally, anti-SCF antibodies inhibited adhesion of BM PCs to activated SMCs and diminished SMC differentiation. These data indicate that SCF/c-Kit signaling plays a pivotal role in the development of neointima by BM-derived PCs and that the inhibition of this pathway may serve as a novel therapeutic target to limit aberrant vascular remodeling.

Adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality.

Sepsis is a multifactorial, and often fatal, disorder typically characterized by widespread inflammation and immune activation with resultant endothelial activation. In the present study, we postulated that the adipokine adiponectin serves as a critical modulator of survival and endothelial activation in sepsis. To this aim, we evaluated both loss-of-function (adiponectin gene-deficient mice) and subsequent gain-of-function (recombinant adiponectin reconstitution) strategies in two well-established inflammatory models, cecal ligation perforation (CLP) and thioglyocollate-induced peritonitis. Adipoq(-/-) mice, subjected to CLP, exhibited a profound ( approximately 8-fold) reduction in survival compared with their wild-type Adipoq(+/+) littermates after 48 h. Furthermore, compared with wild-type controls, thioglycollate challenge resulted in a markedly greater influx of peritoneal neutrophils in Adipoq(-/-) mice accompanied by an excess production of key chemoattractant cytokines (IL-12p70, TNFalpha, MCP-1, and IL-6) and upregulation of aortic endothelial adhesion molecule VCAM-1 and ICAM-1 expressions. Importantly, all of these effects were blunted by recombinant total adiponectin administration given 3 days prior to thioglycollate challenge. The protective effects of adiponectin were ascribed largely to higher-order adiponectin oligomers, since administration of recombinant C39A trimeric adiponectin did not attenuate endothelial adhesion molecule expression in thioglycollate-challenged Adipoq(-/-) mice. These data suggest a critical role of adiponectin as a modulator of survival and endothelial inflammation in experimental sepsis and a potential mechanistic link between adiposity and increased sepsis.

Effects of hormone replacement therapy on cardiovascular risk factors in postmenopausal women.

OBJECTIVE To investigate changes in plasma lipoprotein profile, hemostatic factors, platelet aggregation, endothelin-1, and cardiac function during postmenopausal sequential 6-month hormone replacement therapy (HRT). DESIGN Open longitudinal prospective study. SETTING Gynecologic department of a medical center. PATIENT(S) Twenty-one healthy hysterectomized postmenopausal women. INTERVENTION(S) Oral E2 valerate (2 mg/d) combined with medroxyprogesterone acetate (MPA) (10 mg/d) during the last 10 days of each 21-day cycle. The treatment period was 6 months. MAIN OUTCOME MEASURE(S) Plasma lipoprotein profile, hemostatic parameters, platelet aggregation, endothelin-1, and left ventricular function. RESULT(S) After 6 months of treatment, total cholesterol, triglyceride, and low density lipoprotein (LDL) cholesterol were significantly progressively reduced. Atherogenic indices of total cholesterol-to-high-density lipoprotein (HDL) cholesterol and LDL-to-HDL cholesterols also showed a significant progressive decline. The concentrations of antithrombin III were significantly increased. The maximum aggregation and slope of platelet aggregation were significantly reduced, but all parameters were more pronounced at 1 month of HRT than at 3 or 6 months. The concentrations of endothelin-1 were significantly reduced (by 16.1%). In the evaluation of left ventricular function, only peak atrial diastolic velocity was significantly reduced. CONCLUSION(S) Combined HRT had favorable effects on lipids and lipoproteins, hemostatic factors, platelet aggregation, endothelin-1, and left ventricular function. However, further study is needed to evaluate the long-term effects of combined HRT, especially on platelet aggregation and cardiac function.

Stem Cell Factor Attenuates Vascular Smooth Muscle Apoptosis and Increases Intimal Hyperplasia After Vascular Injury

Objective—Stem cell factor (SCF) through its cognate receptor, the tyrosine kinase c-kit, promotes survival and biological functions of hematopoietic stem cells and progenitors. However, whether SCF/c-kit interactions exacerbate intimal hyperplasia through attenuating VSMC apoptosis induced by vascular injury has not been thoroughly investigated. Methods and Results—VSMCs were stimulated with serum deprivation and H2O2 to induce apoptosis. The transcription of c-kit mRNA and the expression of the c-kit protein by VSMCs were estimated by Q-polymerase chain reaction and Western blotting, respectively. The interactions of SCF and c-kit were investigated by in vitro and in vivo experiments. In vitro, H2O2 stimulation significantly induced apoptosis of VSMCs as evidenced by the 3- and 3.2-fold increases of cleaved caspase-3 compared with those in the control group by Western blot and flow cytometric analyses, respectively (P<0.01). Stimulation of apoptosis also caused 3.5- and 9-fold increases in c-kit mRNA transcription and protein expression, respectively, by VSMCs compared with those in the control group. Administration of SCF (10 to 1000 ng/mL) significantly lowered the amount of cleaved caspase-3 in H2O2-treated VSMCs (P<0.01). Specifically, SCF exerted this effect through activating Akt, followed by increasing Bcl-2 and then inhibiting the release of cytochrome-c from the mitochondria to the cytosol. In vivo, the mouse femoral artery was injured with a wire in SCF mutant (Sl/Sld), c-kit mutant (W/Wv), and colony control mice. In colony control mice, confocal microscopy demonstrated that the wire-injury generated a remarkable activation of caspase-3 on medial VSMCs, coinciding with upregulation of c-kit expression. The wire-injury also caused an increase in the expression of SCF on surviving medial VSMCs and cells in the adventitia. The upregulated c-kit expression in the vessel wall also facilitated homing by circulating SCF+ cells. Compared with colony control mice, vascular injury in SCF mutant and c-kit mutant mice caused a higher number of apoptotic VSMCs on day 14 and a lower number of proliferating cells, and resulted in significantly less neointimal formation (P<0.01) on day 28. Conclusions—The interactions between SCF and the c-kit receptor play an important role in protecting VSMCs against apoptosis and in maintaining intimal hyperplasia after vascular injury.