Caplice, Noel M.

Abstract —Tissue factor (TF) is a low-molecular-weight glycoprotein that initiates the extrinsic clotting cascade and is considered a major regulator of arterial thrombogenicity. TF pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. The aim of this study was to define the complex interplay between TF and TFPI and the regulation of vascular thrombogenicity in a model of vascular remodeling. To determine the levels and pattern of vascular expression of TF and TFPI associated with vascular remodeling, a murine model of flow cessation was studied. TF activity of the arteries increased after ligation ( P <0.05). Quantitative analysis of homogenates of remodeled carotid arteries revealed increased TF expression but unchanged TFPI expression compared with normal carotid arteries, resulting in enhanced TF activity. To determine the potential therapeutic role of TFPI in this thrombogenic state, mice were treated with intravascular adenoviral delivery of either murine TFPI (Ad-mTFPImyc) or a control adenovirus (Ad-ΔE1). Overexpression of TFPI decreased vascular TF activity compared with viral control ( P <0.01). Overexpression of TFPI inhibited neointimal formation ( P =0.038), resulting in enhanced luminal area ( P =0.001) 4 weeks after flow cessation. In this murine model of vascular remodeling, an imbalance between TF and TFPI expression is generated, resulting in increased TF activity. Overexpression of TFPI in this model inhibits vascular TF activity and results in attenuation of vascular remodeling associated with flow interruption.

Background— Atrioventricular (AV) nodal ablation for management of atrial fibrillation (AF) is irreversible and requires permanent pacemaker implantation. We hypothesized that as an alternative, implantation of autologous fibroblasts in the perinodal region would focally modify AV nodal conduction and that this modulation would be enhanced by pretreatment with transforming growth factor-β1 (TGF-β1), a stimulant of fibroblasts. Methods and Results— Skin biopsies were taken from 12 mongrel dogs, and derived fibroblasts were dissociated and grown in culture for 2 weeks. Multiple injections (0.25 mL) were made through an 8F NOGA catheter along the fast/slow AV nodal pathways as guided by an electroanatomic mapping system. Seven dogs received fibroblasts alone (1×10 6 cells/mL), 7 dogs received TGF-β1 (5 μg), 4 dogs received fibroblasts and TGF-β1 (1×10 6 cells/mL+5 μg), and 4 dogs received saline only. AV node function was assessed at baseline and after 4 weeks. Saline (80 mL) with assigned therapy (0.25 mL per injection) was injected into the peri-AV nodal region in each dog. At baseline, the AH interval (66±3 ms) and the average RR interval (331±17 ms) in pacing-induced AF were similar in each cohort. The increase in AH interval in normal sinus rhythm was longer after fibroblast (23±4 versus 5±5 ms; P =0.05) and fibroblast plus TGF-β1 (50±5 versus 5±5 ms; P <0.001) injections than with saline alone, with similar findings during high right atrium and distal coronary sinus pacing. The AH interval was not significantly increased after TGF-β1 injections. The AH interval was significantly longer after fibroblast plus TGF-β1 injections than with either therapy (TGF-β1 or fibroblasts) alone. The RR interval during AF was increased in dogs that received fibroblasts alone (110±36 versus −41±34 ms) and to a greater extent with the addition of TGF-β1 (294±108 versus −41±34 ms). No AV block was seen in any cohort at 4 weeks. Labeled fibroblasts that expressed vimentin were identified in all dogs that received cell injections at 4 weeks. Conclusions— AV nodal modification can be achieved with injected fibroblasts without the creation of AV block. The effect on AV node conduction is substantially enhanced by pretreatment of fibroblasts with TGF-β1. These data have therapeutic potential for the management of rapid ventricular rate during AF without pacemaker implantation.

Recent experimental studies suggest C-reactive protein (CRP) may be a potential mediator of atherosclerosis and its complications. However, there is growing criticism of in vitro CRP studies that use commercial CRP preparations containing biologically active contaminants. The effects of commercial CRP, dialyzed commercial CRP (dCRP) to remove azide, and sodium azide (NaN 3 ) alone at equivalent concentrations to the undialyzed preparation were tested at varying concentrations on human umbilical vein endothelial cells (HUVEC), circulating endothelial outgrowth cells (EOC), and endothelial progenitor cells (EPC) in vitro. CRP and NaN 3 alone exhibited equivalent concentration-dependent, proapoptotic effects on HUVEC, EOC, and EPC ( P <0.01 versus control), whereas dCRP had no such effect. Similarly, CRP and NaN 3 alone caused equivalent concentration-dependent decreases in migration, proliferation, and matrigel tube formation ( P <0.01 versus control) in EOC and HUVEC, whereas dCRP had absolutely no effect on these biological functions at any of the concentrations used. We conclude that proapoptotic, antiproliferative, antimigratory, and antiangiogenic effects of this commercial CRP preparation on a number of endothelial cell phenotypes in culture may be explained by the presence of sodium azide in this preparation. This study has implications for interpretation of in vitro studies using CRP preparations containing azide at equivalent or higher concentrations.

Abstract The field of vascular biology has been stimulated by the concept that circulating endothelial progenitor cells (EPCs) may play a role in neoangiogenesis (postnatal vasculogenesis). One problem for the field has been the difficulty in accurately defining an EPC. Likewise, circulating endothelial cells (CECs) are not well defined. The lack of a detailed understanding of the proliferative potential of EPCs and CECs has contributed to the controversy in identifying these cells and understanding their biology in vitro or in vivo. A novel paradigm using proliferative potential as one defining aspect of EPC biology suggests that a hierarchy of EPCs exists in human blood and blood vessels. The potential implications of this view in relation to current EPC definitions are discussed.

Background and Purpose— Circulating endothelial progenitor cells (EPCs) play an important role in repair of injured vascular endothelium and neovascularization. The present study was designed to determine the effect of EPCs transplantation on the regeneration of endothelium and recovery of endothelial function in denuded carotid arteries. Methods— Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium for 7 days, yielding EPCs. A rabbit model of common carotid artery denudation by passage of a deflated balloon catheter was used to evaluate the effects of EPCs on endothelial regeneration and vasomotor function. Immediately after denudation, autologous EPCs (10 5 cells in 200 μL saline) or 200 μL saline alone (control) were administered into the lumen of injured artery. Results— Four weeks after transplantation, fluorescence-labeled colonies of EPCs were found in the vessel wall. Local transplantation of EPCs as compared with saline administration accelerated endothelialization and significantly improved endothelium-dependent relaxation when assessed 4 weeks after denudation (n=4 to 5, P <0.05). Transplantation of EPCs did not affect vasomotor function of arterial smooth muscle cells. Protein array analysis of conditioned media obtained from cultured EPCs demonstrated the ability of these cells to produce and release a number of proangiogenic cytokines. Conclusions— We conclude that local delivery of cultured circulating EPCs into the lumen of denuded carotid arteries accelerates endothelialization and improves endothelial function. Paracrine effects of EPCs may contribute to regenerative properties of EPCs.

Objective— Endothelial progenitor cells (EPCs) display a unique aptitude to promote angiogenesis and restore endothelial function of injured vessels. How progenitor cells can execute a regenerative program in the unfavorable environment of injury/inflammation-induced oxidative stress is poorly understood. We hypothesized that EPCs are resistant to oxidative stress and that this resistance is due to high expression and activity of antioxidant enzymes. Methods and Results— EPCs outgrown from human blood of healthy subjects demonstrated a marked resistance to cytotoxic effect of LY83583 (an generator), tumor necrosis factor-α, and serum depletion. LY83583 inhibited in vitro tube formation by human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (CAECs), but not by EPCs. Compared with HUVECs and CAECs, EPCs exhibited ≈3- to 4-fold higher expression and activity of manganese superoxide dismutase (MnSOD), but not copper zinc superoxide dismutase (CuZnSOD) or catalase. The antioxidant profile in EPCs was associated with preservation of the mitochondrial network when exposed to LY83583. Moreover, cytotoxic effects of LY83583 on CAECs and HUVECs were reversed by adenoviral overexpression of MnSOD. Conclusions— Human EPCs are resistant to oxidative stress. High intrinsic expression of MnSOD is a critical mechanism protecting EPCs against oxidative stress.

Background— Recently, we identified circulating smooth muscle progenitor cells (SPCs) in human peripheral blood. The integrin profile of such progenitors is currently unknown and may affect their in vivo homing characteristics. In this study, we determined the integrin profile of vascular progenitors and SPC adhesion to extracellular matrix (ECM) proteins in vitro and in vivo. Methods and Results— SPCs and endothelial progenitor cells (EPCs) were isolated from peripheral blood of healthy human subjects, and expression of surface integrins and adhesion to several vascular ECM proteins were determined. Homing of SPCs in vivo to specific ECM protein was determined by intracoronary infusion of fluorescent SPCs into porcine coronary arteries containing a fibronectin-coated mesh stent. SPCs had high expression of β 1 integrin, moderate expression of α 1 , low levels of α v β 3 , and did not express α v β 5 , β 2 , α 2 β 1 , or α 4 β 1 integrins. In contrast, EPCs had high expression of α 2 β 1 , α v β 3 , α v β 5 , β 1 , and α 1 and minimal expression of α 4 β 1 . Moreover, SPCs showed increased adherence to fibronectin and collagen type I compared with vitronectin, consistent with their integrin profile, and demonstrated a similar degree of in vivo attachment to fibronectin-coated mesh. Conclusions— These data for the first time show a spectrum of integrin expression on vascular progenitors and suggest the potential importance of integrins in mediating adherence of SPCs to specific ECM both in vitro and in vivo.

Background— Hypercholesterolemia (HC) and atherosclerosis can elicit oxidative stress, coronary endothelial dysfunction, and myocardial ischemia, which may induce growth-factor expression and lead to myocardial neovascularization. We tested the hypothesis that chronic antioxidant intervention in HC would attenuate neovascularization and preserve the expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Methods and Results— Three groups of pigs (n=6 each) were studied after 12 weeks of normal or 2% HC diet or HC+antioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily). Myocardial samples were scanned ex vivo with a novel 3D micro-CT scanner, and the spatial density and tortuosity of myocardial microvessels were determined in situ. VEGF mRNA, protein levels of VEGF and VEGF receptor-1, HIF-1α, nitrotyrosine, and superoxide dismutase (SOD) were determined in myocardial tissue. The HC and HC+antioxidant groups had similar increases in serum cholesterol levels. HC animals showed an increase in subendocardial spatial density of microvessels compared with normal (160.5±11.8 versus 95.3±8.2 vessels/cm 2 , P <0.05), which was normalized in HC+antioxidant (92.5±20.5 vessels/cm 2 , P <0.05 versus HC), as was arteriolar tortuosity. In addition, HC induced upregulation of VEGF, HIF-1α, and nitrotyrosine expression and decreased SOD expression and activity, all of which were preserved by antioxidant intervention. Conclusions— Changes in myocardial microvascular architecture invoked by HC are accompanied by increases in HIF-1α and VEGF expression and attenuated by antioxidant intervention. This underscores a role of increased oxidative stress in modulating myocardial microvascular architecture in early atherogenesis.