Paul W.M. Fedak

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.

Endothelin Antagonism and Interleukin-6 Inhibition Attenuate the Proatherogenic Effects of C-Reactive Protein

Background—C-reactive protein (CRP) has been suggested to actively participate in the development of atherosclerosis. In the present study, we examined the role of the potent endothelium-derived vasoactive factor endothelin-1 (ET-1) and the inflammatory cytokine interleukin-6 (IL-6) as mediators of CRP-induced proatherogenic processes. Methods and Results—Saphenous vein endothelial cells (HSVECs) were incubated with human recombinant CRP (25 &mgr;g/mL, 24 hours) and the expression of vascular cell adhesion molecule (VCAM-1), intracellular adhesion molecule (ICAM-1), and monocyte chemoattractant chemokine-1 was determined. The effects of CRP on LDL uptake were assessed in macrophages using immunofluorescent labeling of CD32 and CD14. In each study, the effect of endothelin antagonism (bosentan) and IL-6 inhibition (monoclonal anti-IL-6 antibodies) was examined. The effects of CRP on the secretion of ET-1 and IL-6 from HSVECs were also evaluated. Incubation of HSVECs with recombinant human CRP resulted in a marked increase in ICAM-1 and VCAM-1 expression (P <0.001). Likewise, CRP caused a significant increase in monocyte chemoattractant chemokine-1 production, a key mediator of leukocyte transmigration (P <0.001). CRP caused a marked and sustained increase in native LDL uptake by macrophages (P <0.05). These proatherosclerotic effects of CRP were mediated, in part, via increased secretion of ET-1 and IL-6 (P <0.01) and were attenuated by both bosentan and IL-6 antagonism (P <0.01). Conclusions—CRP actively promotes a proatherosclerotic and proinflammatory phenotype. These effects are mediated, in part, via the production of ET-1 and IL-6 and are attenuated by mixed ETA/B receptor antagonism and IL-6 inhibition. Bosentan may be useful in decreasing CRP-mediated vascular disease.

Cardioprotective c-kit+ cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines

Clinical trials of bone marrow stem/progenitor cell therapy after myocardial infarction (MI) have shown promising results, but the mechanism of benefit is unclear. We examined the nature of endogenous myocardial repair that is dependent on the function of the c-kit receptor, which is expressed on bone marrow stem/progenitor cells and on recently identified cardiac stem cells. MI increased the number of c-kit+ cells in the heart. These cells were traced back to a bone marrow origin, using genetic tagging in bone marrow chimeric mice. The recruited c-kit+ cells established a proangiogenic milieu in the infarct border zone by increasing VEGF and by reversing the cardiac ratio of angiopoietin-1 to angiopoietin-2. These oscillations potentiated endothelial mitogenesis and were associated with the establishment of an extensive myofibroblast-rich repair tissue. Mutations in the c-kit receptor interfered with the mobilization of the cells to the heart, prevented angiogenesis, diminished myofibroblast-rich repair tissue formation, and led to precipitous cardiac failure and death. Replacement of the mutant bone marrow with wild-type cells rescued the cardiomyopathic phenotype. We conclude that, consistent with their documented role in tumorigenesis, bone marrow c-kit+ cells act as key regulators of the angiogenic switch in infarcted myocardium, thereby driving efficient cardiac repair.

C-Reactive Protein Upregulates Angiotensin Type 1 Receptors in Vascular Smooth Muscle

Background—Accumulating evidence suggests that C-reactive protein (CRP), in addition to predicting vascular disease, may actively facilitate lesion formation by inciting endothelial cell activation. Given the central importance of angiotensin type 1 receptor (AT1-R) in the pathogenesis of atherosclerosis, we examined the effects of CRP on AT1-R expression and kinetics in vascular smooth muscle (VSM) cells. In addition, the effects of CRP on VSM migration, proliferation, and reactive oxygen species (ROS) production were evaluated in the presence and absence of the angiotensin receptor blocker, losartan. Lastly, the effects of CRP (and losartan) on neointimal formation were examined in vivo in a rat carotid angioplasty model. Methods and Results—The effects of human recombinant CRP (0 to 100 &mgr;g/mL) on AT1-R transcript, mRNA stability, and protein expression were studied in cultured human VSM cells. AT1-R binding was assessed with 125I-labeled angiotensin II (Ang II). VSM migration was assessed with wound cell migration assays, whereas VSM proliferation was determined with [3H]-incorporation and cell number. The effects of CRP (and losartan) on Ang II–induced ROS production were evaluated by 2′,7′-dichlorofluorescein fluorescence. Lastly, the effects of CRP (and losartan) on neointimal formation, VSM cell migration, proliferation, and matrix formation were studied in vivo in a rat carotid artery balloon injury model. CRP markedly upregulated AT1-R mRNA and protein expression and increased AT1-R number on VSM cells. CRP promoted VSM migration and proliferation in vitro and increased ROS production. Furthermore, CRP potentiated the effects of Ang II on these processes. In the rat carotid artery angioplasty model, exposure to CRP resulted in an increase in cell migration and proliferation, collagen and elastin content, and AT1-R expression, as well as an increase in neointimal formation; these effects were attenuated by losartan. Conclusions—CRP, at concentrations known to predict cardiovascular events, upregulates AT1-R–mediated atherosclerotic events in vascular smooth muscle in vitro and in vivo. These data lend credence to the notion that CRP functions as a proatherosclerotic factor as well as a powerful risk marker.

Fundamentals of Reperfusion Injury for the Clinical Cardiologist

Case presentation: S.B. is a 48-year-old man who suffered an acute anterior myocardial infarction and received fibrinolytic therapy. The patient died ≈12 hours after reperfusion. K.R. is a 68-year-old diabetic woman who underwent conventional coronary artery bypass graft surgery and developed low output syndrome after reperfusion postoperatively. V.A. is a 55-year-old man who developed a stunned myocardium after percutaneous coronary reperfusion. What is reperfusion injury, and why is it important?nnReperfusion of coronary flow is necessary to resuscitate the ischemic or hypoxic myocardium. Timely reperfusion facilitates cardiomyocyte salvage and decreases cardiac morbidity and mortality. Reperfusion of an ischemic area may result, however, in paradoxical cardiomyocyte dysfunction, a phenomenon termed “reperfusion injury.” Modalities for reperfusion include not only thrombolysis, but also percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), and cardiac transplantation. Reperfusion injury has been observed in each of these situations. We discuss here the fundamental principles of reperfusion injury from a mechanistic and pharmacological standpoint.nn### What is reperfusion injury, and why is it important? nnThe myocardium can tolerate brief periods (up to 15 minutes) of severe and even total myocardial ischemia without resultant cardiomyocyte death. Although the cardiomyocytes suffer ischemic injury, the damage is reversible with prompt arterial reperfusion. Indeed, such transient periods of ischemia are encountered in the clinical situations of angina, coronary vasospasm, and balloon angioplasty, and are not associated with concomitant myocyte cell death.1,2⇓ With increasing duration and severity of ischemia, however, greater cardiomyocyte damage can develop, with a predisposition to a spectrum of reperfusion-associated pathologies, collectively called reperfusion injury.3 Reperfusion injury results in myocyte damage through myocardial stunning, microvascular and endothelial injury, and irreversible cell damage or necrosis (termed lethal reperfusion injury; Figure 1).3,4⇓ nnnnFigure 1. Mechanisms and mediators of reperfusion injury. Reperfusion strategies are required to resuscitate the ischemic myocardium. In the clinical realm, these include …

Endothelial Progenitor Cells New Hope for a Broken Heart

Mr S. is a 62-year-old restaurant owner who has had recurrent stable angina that has not improved despite maximal medical therapy and an earlier coronary bypass. On angiography, Mr S. has diffusely diseased coronary vessels and is no longer considered a candidate for further direct revascularization. An alternative treatment strategy for revascularizing ischemic tissue is therefore required. One potential strategy, therapeutic neovascularization, aims to promote the formation of natural bypasses or collaterals within the ischemic tissue by harvesting the potential of endothelial progenitor cells (EPCs). This From Bench to Bedside article will explore the current concept of an EPC, the role EPCs play in neovascularization, the strategies used to maximize EPC number and function, the experimental and clinical evidence supporting EPC utility, and the future direction of EPC research.nnSee p 2995 nnProgenitor cells are primitive bone marrow (BM) cells that have the capacity to proliferate, migrate, and differentiate into various mature cell types. EPCs, in particular, possess the ability to mature into the cells that line the lumen of blood vessels.1 The first evidence indicating the presence of EPCs in the adult circulation emerged when mononuclear blood cells from healthy human volunteers were shown to acquire an endothelial cell–like phenotype in vitro and to incorporate into capillaries in vivo.2 These putative EPCs were characterized via expression of CD34 and vascular endothelial growth factor receptor-2 (VEGFR-2), 2 antigens shared by embryonic endothelial progenitors, and hematopoietic stem cells (HSCs). Subsequent studies confirmed that CD34+ cells isolated from BM or umbilical cord blood also had the capacity to differentiate into mature endothelial cells.3–5 However, both CD34 and VEGFR-2 are expressed on mature endothelial cells.6 Thus, the search for more unique EPC markers continued.nnIn addition to CD34, early hematopoietic progenitor cells express CD133 (AC133), which is not …

Bicuspid Aortic Cusp Fusion Morphology Alters Aortic Three-Dimensional Outflow Patterns, Wall Shear Stress, and Expression of Aortopathy

Background— Aortic 3-dimensional blood flow was analyzed to investigate altered ascending aorta (AAo) hemodynamics in bicuspid aortic valve (BAV) patients and its association with differences in cusp fusion patterns (right-left, RL versus right-noncoronary, RN) and expression of aortopathy. Methods and Results— Four-dimensional flow MRI measured in vivo 3-dimensional blood flow in the aorta of 75 subjects: BAV patients with aortic dilatation stratified by leaflet fusion pattern (n=15 RL-BAV, mid AAo diameter=39.9±4.4 mm; n=15 RN-BAV, 39.6±7.2 mm); aorta size controls with tricuspid aortic valves (n=30, 41.0±4.4 mm); healthy volunteers (n=15, 24.9±3.0 mm). Aortopathy type (0–3), systolic flow angle, flow displacement, and regional wall shear stress were determined for all subjects. Eccentric outflow jet patterns in BAV patients resulted in elevated regional wall shear stress (P<0.0125) at the right-anterior walls for RL-BAV and right-posterior walls for RN-BAV in comparison with aorta size controls. Dilatation of the aortic root only (type 1) or involving the entire AAo and arch (type 3) was found in the majority of RN-BAV patients (87%) but was mostly absent for RL-BAV patients (87% type 2). Differences in aortopathy type between RL-BAV and RN-BAV patients were associated with altered flow displacement in the proximal and mid AAo for type 1 (42%–81% decrease versus type 2) and distal AAo for type 3 (33%–39% increase versus type 2). Conclusions— The presence and type of BAV fusion was associated with changes in regional wall shear stress distribution, systolic flow eccentricity, and expression of BAV aortopathy. Hemodynamic markers suggest a physiological mechanism by which the valve morphology phenotype can influence phenotypes of BAV aortopathy.

Hepatocyte Growth Factor or Vascular Endothelial Growth Factor Gene Transfer Maximizes Mesenchymal Stem Cell–Based Myocardial Salvage After Acute Myocardial Infarction

Background— Mesenchymal stem cell (MSC)-based regenerative strategies were investigated to treat acute myocardial infarction and improve left ventricular function. Methods and Results— Murine AMI was induced by coronary ligation with subsequent injection of MSCs, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), or MSCs +HGF/VEGF into the border zone. Left ventricular ejection fraction was calculated using micro–computed tomography imaging after 6 months. HGF and VEGF protein injection (with or without concomitant MSC injection) significantly and similarly improved the left ventricular ejection fraction and reduced scar size compared with the MSC group, suggesting that myocardial recovery was due to the cytokines rather than myocardial regeneration. To provide sustained paracrine effects, HGF or VEGF overexpressing MSCs were generated (MSC-HGF, MSC-VEGF). MSC-HGF and MSC-VEGF showed significantly increased in vitro proliferation and increased in vivo proliferation within the border zone. Cytokine production correlated with MSC survival. MSC-HGF– and MSC-VEGF–treated animals showed smaller scar sizes, increased peri-infarct vessel densities, and better preserved left ventricular function when compared with MSCs transfected with empty vector. Murine cardiomyocytes were exposed to hypoxic in vitro conditions. The LDH release was reduced, fewer cardiomyocytes were apoptotic, and Akt activity was increased if cardiomyocytes were maintained in conditioned medium obtained from MSC-HGF or MSC-VEGF cultures. Conclusions— This study showed that (1) elevating the tissue levels of HGF and VEGF after acute myocardial infarction seems to be a promising reparative therapeutic approach, (2) HGF and VEGF are cardioprotective by increasing the tolerance of cardiomyocytes to ischemia, reducing cardiomyocyte apoptosis and increasing prosurvival Akt activation, and (3) MSC-HGF and MSC-VEGF are a valuable source for increased cytokine production and maximize the beneficial effect of MSC-based repair strategies.

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.

C-Reactive Protein Alters Antioxidant Defenses and Promotes Apoptosis in Endothelial Progenitor Cells

Objectives—C-reactive protein (CRP) has been suggested to participate in the development of atherosclerosis, in part, by promoting endothelial dysfunction and impairing endothelial progenitor cell (EPC) survival and differentiation. In the present study, we evaluated the effects of CRP on antioxidative enzymes, reactive oxygen species production, telomerase activity, and apoptosis in human circulating EPCs. Methods and Results—EPCs, isolated from peripheral venous blood, were cultured in the absence or presence of native pentameric azide and lipopolysaccharide (LPS)-free CRP (0, 5, 15, and 20 &mgr;g/mL), N-acetylcysteine (NAC), hydrogen peroxide (H2O2) or monoclonal anti-CRP antibodies. Fluorescence-activated cell sorter (FACS) analysis was used for the measurement of intracellular H2O2 and superoxide (O2−) by loading cells with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA). Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Western blot analysis was used for the determination of manganese superoxide dismutase (MnSOD) and glutathione peroxidase expression, and polymerase chain reaction enzyme-linked immunosorbent assay was used to assess telomerase activity. Incubation of EPCs with CRP caused a concentration dependent increase in reactive oxygen species (ROS) production and apoptosis, with an effect quantitatively similar to H2O2. This effect was attenuated during coincubation with NAC or anti-CRP antibodies. Furthermore, CRP altered EPC antioxidative enzyme levels, demonstrating a reduced expression of glutathione peroxidase and a significant increase in MnSOD expression. Transfection of EPCs with MnSOD-RNAi resulted in a reduction in CRP-induced ROS production, apoptosis, and telomerase inactivation. Conclusions—CRP, at concentrations known to predict cardiovascular events, may serve to impair EPC antioxidant defenses, and promote EPC sensitivity toward oxidant-mediated apoptosis and telomerase inactivation. These data further support a direct role of CRP in the development and/or progression of atherothrombosis.