Paul E. Szmitko

C-reactive protein comes of age

Cardiovascular disease remains a leading cause of death throughout the world despite advances in its detection and treatment. Commonly used risk algorithms, such as the Framingham Risk Score fail to identify all affected individuals. Novel cardiovascular risk factors that identify these missed individuals would greatly improve overall care of patients. C-reactive protein (CRP), an inflammatory biomarker, has emerged as a leading candidate to fulfill this role. Based on the results of several prospective epidemiologic studies, CRP has emerged as one of the most powerful predictors of cardiovascular disease. This marker provides valuable information to clinicians in various clinical settings, ranging from overt cardiovascular disease, stable angina, presenting acute coronary syndromes and peripheral vascular disease, to the metabolic syndrome. Furthermore, CRP has been demonstrated to actively contribute to all stages of atherogenesis, participating in endothelial dysfunction, atherosclerotic-plaque formation, plaque maturation, plaque destabilization and eventual rupture. Thus, it might also serve as a therapeutic target. It is our contention that the future will see much wider use of CRP and CRP-driven therapies in clinical medicine, improving our ability to identify and manage cardiovascular disease.

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.

Adiponectin primes human monocytes into alternative anti-inflammatory M2 macrophages

Altered macrophage kinetics is a pivotal mechanism of visceral obesity-induced inflammation and cardiometabolic risk. Because monocytes can differentiate into either proatherogenic M1 macrophages or anti-inflammatory M2 macrophages, approaches that limit M1 while promoting M2 differentiation represent a unique therapeutic strategy. We hypothesized that adiponectin may prime human monocytes toward the M2 phenotype. Adiponectin promoted the alternative activation of human monocytes into anti-inflammatory M2 macrophages as opposed to the classically activated M1 phenotype. Adiponectin-treated cells displayed increased M2 markers, including the mannose receptor (MR) and alternative macrophage activation-associated CC chemokine-1. Incubation of M1 macrophages with adiponectin-treated M2-derived culture supernatant resulted in a pronounced inhibition of tumor necrosis factor-alpha and monocyte chemotactic protein-1 secretion. Activation of human monocytes into M2 macrophages by adiponectin was mediated, in addition to AMP-activated protein kinase and peroxisome proliferator-activated receptor (PPAR)-gamma, via PPAR-alpha. Furthermore, macrophages isolated from adiponectin knockout mice demonstrated diminished levels of M2 markers such as MR, which were restored with adiponectin treatment. We report a novel immunoregulatory mechanism through which adiponectin primes human monocyte differentiation into anti-inflammatory M2 macrophages. Conditions associated with low adiponectin levels, such as visceral obesity and insulin resistance, may promote atherosclerosis, in part through aberrant macrophage kinetics.

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.

Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis.

Improving endothelial nitric oxide synthase (eNOS) bioactivity and endothelial function is important to limit native, vein graft, and transplant atherosclerosis. Visfatin, a NAD biosynthetic enzyme, regulates the activity of the cellular survival factor, Sirt1. We hypothesized that visfatin may improve eNOS expression, endothelial function, and postnatal angiogenesis. In human umbilical vein (HUVEC) and coronary artery endothelial cells, we evaluated the effects of recombinant human visfatin on eNOS protein and transcript expression and mRNA stability, in the presence and absence of visfatin RNA silencing. We also assessed visfatin-induced protein kinase B (Akt) activation and its association with src-tyrosine kinases, phosphorylation of Ser(1177) within eNOS in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibition with LY-294002, and evaluated the contributory role of extracellular signal-regulated kinase 1/2. Finally, we determined the impact of visfatin on HUVEC migration, proliferation, inflammation-induced permeability, and in vivo angiogenesis. Visfatin (100 ng/ml) upregulated and stabilized eNOS mRNA and increased the production of nitric oxide and cGMP. Visfatin-treated HUVEC demonstrated greater proliferation, migration, and capillary-like tube formation but less tumor necrosis factor-alpha-induced permeability; these effects were decreased in visfatin gene-silenced cells. Visfatin increased total Akt and Ser(473)-phospho-Akt expression with concomitant rises in eNOS phosphorylation at Ser(1177); these effects were blocked by LY-2940002. Studies with PP2 showed that the nonreceptor tyrosine kinase, src, is an upstream stimulator of the PI 3-kinase-Akt pathway. Visfatin also activated mitogen-activated protein (MAP) kinase through PI 3-kinase, and mitogen/extracellular signal-regulated kinase inhibition attenuated visfatin-elicited Akt and eNOS phosphorylation. Visfatin-filled Matrigel implants showed an elevated number of infiltrating vessels, and visfatin treatment produced significant recovery of limb perfusion following hindlimb ischemia. These results indicate a novel effect of visfatin to stimulate eNOS expression and function in endothelial cells, via a common upstream, src-mediated signaling cascade, which leads to activation of Akt and MAP kinases. Visfatin represents a translational target to limit endothelial dysfunction, native, vein graft and transplant atherosclerosis, and improve postnatal angiogenesis.

C-Reactive Protein Upregulates Receptor for Advanced Glycation End Products Expression in Human Endothelial Cells

The receptor for advanced glycation end products (RAGE) may play an important role in inflammatory processes and endothelial activation, likely to accelerate the processes of coronary atherosclerotic development, especially in diabetic patients. The factors that regulate arterial expression of RAGE are not completely clear. C-reactive protein (CRP) is identified as a key proinflammatory cytokine in patients with atherosclerosis. Therefore, we tested the hypothesis that RAGE expression in endothelial cells can be upregulated by CRP. Human saphenous vein endothelial cells were incubated with human recombinant CRP, free of sodium azide and endotoxin. RAGE protein expression was measured by flow-cytometric analysis and Western blotting. CRP caused a significant increase in RAGE protein expression at a dose as low as 5 &mgr;g/mL, with expression peaking at 24 to 48 hours after CRP incubation. The effects of modified monomeric CRP on RAGE protein expression were comparable with that of native pentameric CRP. At the mRNA level, CRP not only increased RAGE gene expression but also attenuated the degradation of RAGE mRNA. Furthermore, RNA interference of RAGE gene expression significantly decreased the level of macrophage chemoattractant protein 1, a key downstream mediator of CRP activity. Therefore, CRP at concentrations known to predict future vascular events upregulates RAGE expression in human endothelial cells at both the protein and mRNA level. Silencing of the RAGE gene prevents CRP–induced macrophage chemoattractant protein 1 activation. These data reinforce the mechanistic links among inflammation, endothelial dysfunction, and atherothrombosis.

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.

Red Wine and Your Heart

Drinking red wine has been portrayed by the media as a means of combating heart disease. Do these claims have any real medical basis? The main health benefit of moderate alcohol use appears to be related to its effect on the development of atherosclerosis or the accumulation of fatty plaques in the blood vessels, particularly the coronary arteries that supply the heart. These deposits decrease blood flow to the heart and may promote the formation of vessel-blocking clots, which can result in anginal chest pain or even a life-threatening heart attack. In this article, we examine the scientific literature behind these claims, both epidemiological (studies focused on disease within whole populations) and biological (studies focused on how the molecular components of red wine affect atherosclerosis). The moderate consumption of alcoholic beverages, defined as 1 to 2 drinks per day, has been suggested to increase overall survival rates in a number of different population groups. One standard drink is generally considered to be 1.5 oz of liquor, 5 oz of wine, or 12 oz of beer. The patient groups that appear to benefit most from light to moderate drinking, middle-aged men and women, are also those who are at increased risk for developing cardiovascular disease. Thus, the reduction in total mortality that is associated with moderate alcohol consumption, generally a 30% reduction in risk, is believed to be the result of a reduction in the risk of developing atherosclerotic disease. When the data from 51 epidemiological studies were combined, they showed that the risk of coronary heart disease decreased by approximately 20% when 0 to 2 alcoholic drinks were consumed per day. Apparently healthy adults, patients with a history of heart attack, and patients …

BRCA2 Protein Deficiency Exaggerates Doxorubicin-induced Cardiomyocyte Apoptosis and Cardiac Failure

Background: BRCA2 is widely implicated in breast and ovarian cancers, but the role of BRCA2 in the heart is unknown. Results: Loss of BRCA2 in the heart resulted in increased doxorubicin-induced DNA damage, apoptosis, and cardiac dysfunction. Conclusion: BRCA2 is a novel regulator of cardiomyocyte genomic integrity, survival, and function. Significance: BRCA2 mutation carriers may be at a heightened risk of anthracycline-induced cardiac failure. The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2−/−) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2−/− mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2−/− mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2−/− mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2−/− mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2−/− mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2−/− mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.

Endothelial progenitor cell-coated stents under scrutiny

The vascular endothelium protects the vessel wall from inflammatory cell infiltrates, thrombus formation and smooth muscle cell proliferation, in part by synthesizing and secreting various protective substances such as nitric oxide. In the presence of cardiovascular risk factors, the natural defenses of the endothelium are stressed, setting the stage for endothelial dysfunction and initiating atherogenesis (1). Similarly, during vascular interventions, such as percutaneous coronary intervention or bypass grafting, the integrity of the vascular endothelium is violated, setting in motion accelerated endothelial dysfunction, atherosclerosis and restenosis. The lack of an endothelial layer in prosthetic vascular grafts is a strong stimulus for intimal hyperplasia, which may lead to premature graft failure. Efforts to construct more biologically friendly grafts have been pursued, and in the current issue of the Journal ( pages 1113–1116), Rotmans et al provide an overview of the applicability and the potential risks of using endothelial progenitor cells (EPCs) to seed prosthetic vascular grafts.