Michael Kutryk

C-Reactive Protein Attenuates Endothelial Progenitor Cell Survival, Differentiation, and Function Further Evidence of a Mechanistic Link Between C-Reactive Protein and Cardiovascular Disease

Background—Myocardial ischemia provides a potent stimulus to angiogenesis, and the mobilization and differentiation of endothelial progenitor cells (EPCs) has been shown to be important in this process. An elevated level of C-reactive protein (CRP) has emerged as one of the most powerful predictors of cardiovascular disease. However, the impact of CRP on EPC biology is unknown. Methods and Results—EPCs were isolated from the peripheral venous blood of healthy male volunteers. Cells were cultured in endothelial cell basal medium-2 in the absence and presence of CRP (5 to 20 μg/mL), rosiglitazone (1 μmol/L), and/or vascular endothelial growth factor. EPC differentiation, survival, and function were assayed. CRP at concentrations ≥15 μg/mL significantly reduced EPC cell number, inhibited the expression of the endothelial cell–specific markers Tie-2, EC-lectin, and VE-cadherin, significantly increased EPC apoptosis, and impaired EPC-induced angiogenesis. EPC-induced angiogenesis was dependent on the presence of nitric oxide, and CRP treatment caused a decrease in endothelial nitric oxide synthase mRNA expression by EPCs. However, all of these detrimental CRP-mediated effects on EPCs were attenuated by pretreatment with rosiglitazone, a peroxisome proliferator–activated receptor-γ (PPARγ ) agonist. Conclusions—Human recombinant CRP, at concentrations known to predict adverse vascular outcomes, directly inhibits EPC differentiation, survival, and function, key components of angiogenesis and the response to chronic ischemia. This occurs in part via an effect of CRP to reduce EPC eNOS expression. The PPARγ agonist rosiglitazone inhibits the negative effects of CRP on EPC biology. The ability of CRP to inhibit EPC differentiation and survival may represent an important mechanism that further links inflammation to cardiovascular disease.

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. See p 2995 Progenitor 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. In addition to CD34, early hematopoietic progenitor cells express CD133 (AC133), which is not …

Bone Morphogenetic Protein Receptor-2 Signaling Promotes Pulmonary Arterial Endothelial Cell Survival Implications for Loss-of-Function Mutations in the Pathogenesis of Pulmonary Hypertension

Mutations in the bone morphogenetic protein (BMP) receptor-2 (BMPR2) have been found in patients with idiopathic pulmonary arterial hypertension (IPAH); however, the mechanistic link between loss of BMPR2 signaling and the development of pulmonary arterial hypertension is unclear. We hypothesized that, contrary to smooth muscle cells, this pathway promotes survival in pulmonary artery endothelial cells (ECs) and loss of BMPR2 signaling will predispose to EC apoptosis. ECs were treated with BMP-2 or BMP-7 (200 ng/mL) for 24 hours in regular or serum-free (SF) medium, with and without addition of tumor necrosis factor &agr;, and apoptosis was assessed by flow cytometry (Annexin V), TUNEL, or caspase-3 activity. Treatment for 24 hours in SF medium increased apoptosis, and both BMP-2 and BMP-7 significantly reduced apoptosis in response to serum deprivation to levels not different from serum controls. Transfection with 5 &mgr;g of small interfering RNAs for BMPR2 produced specific gene silencing assessed by RT-PCR and Western blot analysis. BMPR2 gene silencing increased apoptosis almost 3-fold (P=0.0027), even in the presence of serum. Circulating endothelial progenitor cells (EPCs) isolated from normal subjects or patients with IPAH were differentiated in culture for 7 days and apoptosis was determined in the presence and absence of BMPs. BMP-2 reduced apoptosis induced by serum withdrawal in EPCs from normal subjects but not in EPCs isolated from patients with IPAH. These results support the hypothesis that loss-of-function mutations in BMPR2 could lead to increased pulmonary EC apoptosis, representing a possible initiating mechanism in the pathogenesis of pulmonary arterial hypertension.

Characterization of Operator Learning Curve for Transradial Coronary Interventions

Background—Transradial percutaneous coronary intervention (TR-PCI) improves clinical outcomes compared to the transfemoral (TF) approach. However, inadequate training and experience has limited widespread adoption by interventional cardiologists. Methods and Results—Clinical and procedural characteristics for TR-PCI were prospectively collected from 1999 to 2008. To identify minimum case volume for optimum clinical benefit, single-vessel TR-PCI cases were chronologically ranked and stratified into 1 to 50, 51 to 100, 101 to 150 and 151 to 300 case volume groups for operators starting the TR approach at the study institution. Cases by operators with a >300 TR-PCI case volume comprised the control group. TR-PCI failure rates, contrast use, guide usage, and fluoroscopy time were compared among groups. A total of 1672 patients underwent TR-PCI by 28 operators. TR-PCI failure occurred in 4% and was higher in the 1 to 50 case volume group compared to the 51 to 100 (P=0.007) and control (P=0.01) groups. Contrast use was greater in the 1 to 50 group (180±79 mL) compared to the 151 to 300 (157±75 mL, P=0.02) and control (168±79 mL, P=0.05) groups. Fluoroscopy time was higher in the 1 to 50 group (15±10 minutes) compared to the 101 to 150 (13±10 minutes, P=0.04) and control (12±9 minutes, P=0.02) groups. Reasons for TR-PCI failure included spasm (38%), subclavian tortuousity (16%), poor guide support (16%), failed access (10%), and radial loop (7%). Case volume was significantly correlated with TR-PCI failure (&bgr;=−0.0076, P=0.0028), and odds of failure was reduced by 32% for each 50 increments in case volume. Conclusions—TR-PCI success depends on operator experience, and a case volume of ≥50 cases is required to achieve outcomes comparable to experienced operators. These findings have implications both for PCI operators looking to expand their skills and for defining standards for training.

Clinician Guide to Angiogenesis

Case Presentation : J.A. is a 63-year-old man with type II diabetes and hypercholesterolemia. He has suffered 2 previous myocardial infarctions and has chronic angina. Previous angiography showed diffuse 3-vessel coronary artery disease not amenable to conventional revascularization. What are the current concepts surrounding the use of therapeutic coronary angiogenesis in this setting? Since recognition of the central role of angiogenic factors in tumor growth over 30 years ago,1 physiological and pathological angiogenesis has been implicated in diverse conditions, including vascular insufficiency, inflammation, and diabetic retinopathy. Despite considerable advances in medical therapy and improvements in revascularization procedures for coronary artery disease, a substantial proportion of patients suffer from refractory angina or recurrent myocardial ischemia requiring hospitalization. In the past decade, a number of clinical trials have examined the role of therapeutic angiogenesis for myocardial ischemia. In this article, we focus on the fundamental mechanisms of angiogenesis and discuss current and future issues in therapeutic coronary angiogenesis. Three distinct mechanisms of new blood vessel formation have been identified: Vasculogenesis, angiogenesis, and arteriogenesis. Vasculogenesis refers to the formation of blood vessels from endothelial progenitor cells, a process that was initially described as occurring during embryonic development, and more recently, in adult animals.2 Angiogenesis involves the sprouting of new capillaries from preexisting vessels, whereas arteriogenesis refers to remodeling of newly formed or preexisting vascular channels into larger and well-muscularized arterioles and collateral vessels.3 The generation of new vascular channels by angiogenesis and arteriogenesis has been shown in both animal models of myocardial ischemia and in patients with coronary disease.4 Importantly, the formation of collateral vessels in acute and chronic coronary occlusion may preserve perfusion to ischemic myocardium and thereby maintain myocardial function. Angiogenesis is a dynamic process of endothelial proliferation and differentiation. The formation of a functioning vasculature requires …

Mechanism and Predictors of Failed Transradial Approach for Percutaneous Coronary Interventions

OBJECTIVES The study aimed to determine the mechanism and predictors of procedural failure in patients undergoing percutaneous coronary intervention (PCI) from the transradial approach (TR). BACKGROUND Transradial approach PCI reduces vascular complications compared with a transfemoral approach (TF). However, the mechanism and predictors of TR-PCI failure have not been well-characterized. METHODS The study population consisted of patients undergoing TR-PCI by low-to-intermediate volume operators with traditional TF guide catheters. Baseline characteristics, procedure details, and clinical outcomes were prospectively collected. Univariate and multivariate analyses were performed to determine independent predictors of TR-PCI failure. RESULTS A total of 2,100 patients underwent TR-PCI and represented 38% of PCI volume. Mean age was 64 +/- 12 years, and 17% were female. Vascular complications occurred in 22 (1%), and TR-PCI failure was observed in 98 (4.7%) patients. The mechanism of TR-PCI failure included inability to advance guide catheter to ascending aorta in 50 (51%), inadequate guide catheter support in 35 (36%), and unsuccessful radial artery puncture in 13 (13%) patients. The PCI was successful in 94 (96%) patients with TR-PCI failure by switching to TF. On multivariate analysis, age >75 years (odds ratio [OR]: 3.86; 95% confidence interval [CI]: 2.33 to 6.40, p = 0.0006), prior coronary artery bypass graft surgery (OR: 7.47; 95% CI: 3.45 to 16.19, p = 0.0002), and height (OR: 0.97; 95% CI: 0.95 to 0.99, p = 0.02) were independent predictors of TR-PCI failure. CONCLUSIONS Transradial approach PCI can be performed by low-to-intermediate volume operators with standard equipment with a low failure rate. Age >75 years, prior coronary artery bypass graft surgery, and short stature are independent predictors of TR-PCI failure. Appropriate patient selection and careful risk assessment are needed to maximize benefits offered by TR-PCI.

Local intracoronary administration of antisense oligonucleotide against c-myc for the prevention of in-stent restenosis: Results of the randomized investigation by the thoraxcenter of antisense dna using local delivery and ivus after coronary stenting (ITALICS) trial☆

OBJECTIVE This study was designed to determine whether antisense oligodeoxynucleotides (ODN) directed against the nuclear proto-oncogene c-myc could inhibit restenosis when given by local delivery immediately after coronary stent implantation. BACKGROUND Failure of conventional pharmacologic therapies to reduce the incidence of coronary restenosis after percutaneous revascularization techniques has prompted interest in the use of agents that target intracellular central regulatory mechanisms. METHODS Eighty-five patients were randomly assigned to receive either 10 mg of phosphorothioate-modified 15-mer antisense ODN or saline vehicle by intracoronary local delivery after coronary stent implantation. The primary end point was percent neointimal volume obstruction measured by computerized analysis of electrocardiogram-gated intravascular ultrasound (IVUS) at six-month follow-up. Secondary end points included clinical outcome and quantitative coronary angiography analysis. RESULTS Analysis of follow-up IVUS data was performed on 77 patients. In-stent volume obstruction was similar between groups (44 +/- 16% and 46 +/- 14%, placebo vs. ODN; p = 0.57; 95% confidence interval: -1.13 to 0.85). Minimum luminal diameter increased from 0.84 +/- 0.36 and 0.90 +/- 0.45 (p = 0.55) to 2.70 +/- 0.37 and 2.80 +/- 0.37 (p = 0.28) after stent implantation, which decreased to 1.50 +/- 0.61 and 1.50 +/- 0.53 (p = 0.98) by six months, yielding similar loss indexes (placebo vs. ODN, respectively). There were no differences in angiographic restenosis rates (38.5 and 34.2%; p = 0.81; placebo vs. ODN) or clinical outcome. CONCLUSIONS Treatment with 10 mg of phosphorothioate-modified ODN directed against c-myc does not reduce neointimal volume obstruction or the angiographic restenosis rate in this patient population.

Dysregulation of angiogenesis-related microRNAs in endothelial progenitor cells from patients with coronary artery disease.

Endothelial progenitor cells (EPCs) play an important role in vascular repair and maintenance of vascular homeostasis through re-endothelialization and neovascularization. Cardiovascular risk factors that contribute to coronary artery disease (CAD) have been shown to negatively impact EPCs, although the mechanisms are poorly understood. MicroRNAs (miRNAs) which negatively regulate gene expression at the post-transcriptional level have been shown to impact endothelial cell (EC) angiogenic actions, but little is known about their role in modulating EPC function. In this study we first investigated if EPCs expressed EC specific, angiogenesis-related miRNAs; then determined whether the expression of these miRNAs was altered in EPCs from CAD patients as compared with healthy controls. Furthermore, we examined if atorvastatin, known to increase circulating EPC numbers, had any effect on EPC miRNA expression. We found EPCs produced miR-126, miR-130a, miR-221, miR-222 and miR-92a which have thus far been identified as the most important angiogenic miRNAs. Dysregulation of these miRNAs was detected in EPCs from CAD patients and atorvastatin treatment selectively impacted miRNA expression in EPCs. Our data provide evidence that angiogenic miRNAs might play an important role in the control of EPC function, and that their dysregulation might contribute to EPC dysfunction in patients suffering from coronary artery disease. These findings might lead to the development of novel therapeutic modalities for the prevention and treatment of CAD.

Site-Specific Intracoronary Heparin Delivery in Humans After Balloon Angioplasty A Radioisotopic Assessment of Regional Pharmacokinetics

BACKGROUND Demonstration and quantification of site-specific intracoronary administration of compounds has been confined thus far to the experimental animal laboratory. The aim of this study was to describe a scintigraphic method to demonstrate site-specific intracoronary drug delivery in humans. The methods allow on-line visualization and off-line quantification of site-specifically infused gamma-emitting compounds. METHODS AND RESULTS In 12 patients after balloon angioplasty, 99mTc-labeled heparin was administered at the site of dilatation by use of a coil balloon. Both the infusion period and the washout period after the end of infusion were monitored with a gamma-camera. A curve of counts per pixel as a function of time was derived that showed an accumulation phase during infusion followed by washout phase after the end of infusion. Both phases were fitted by regression analysis and showed a linear accumulation pattern and a biexponential washout pattern. After correction for background counts, 99mTc decay, and body attenuation, peak heparin amount and regional bioavailability were calculated. Peak amount was defined as the initial point of the slow washout component of the biexponential curve (elimination component), and regional bioavailability was defined as the area under the curve of accumulation and washout phase. Half-life and retention time, define as seven half-lives, were obtained by use of the elimination component after correction for 99mTc decay. Mean peak delivered amount was 45 +/- 44 IU (236 +/- 228 micrograms), corresponding to an efficiency of delivery ranging from 1% to 8% of the totally infused dose. Total regionally bioavailable heparin reached 244 +/- 194 IU.h (1.28 +/- 1.01 mg.h). Retention time varied from 12 to 90 hours (mean, 50:33 +/- 22:50 hours:minutes). CONCLUSIONS Site-specific intracoronary heparin delivery after angioplasty by means of the coil balloon was demonstrated in humans, and regional pharmacokinetics was quantified by use of a radioisotopic technique.

Endothelial progenitor cell therapy for the treatment of coronary disease, acute MI, and pulmonary arterial hypertension: current perspectives.

Since their identification in 1997, bone marrow derived endothelial progenitor cells (EPCs) have been studied for their role in the endogenous maintenance and repair of endothelium and their potential regenerative capacity beyond the endothelium. In particular, EPCs have been tested in cell therapy approaches with the aim of developing novel therapies for conditions currently lacking effective treatment options. In this review, we discuss the scientific background and clinical experience using EPC delivery or mobilization for the treatment of post‐angioplasty restenosis, acute myocardial infarction and pulmonary arterial hypertension. Although these approaches are safe, efficacy has yet to be proven in large randomized clinical trials. Unfortunately, the biology of EPCs is still poorly understood. The success of future clinical trials depends on a better understanding of EPC biology and intelligent design.