Background-Bone marrow-derived cells have been shown to contribute to endothelial replacement after vascular injury.In vitro culture of peripheral blood mononuclear cells produces cells with phenotypic characteristics of endothelium. To test the hypothesis that delivery of autologous culture-modified mononuclear cells (CMMCs) to injured arteries could attenuate the vascular response to injury, a rabbit model was studied. Methods and Results-Rabbit peripheral blood mononuclear cells were cultured in endothelial growth media for 7 to 12 days, yielding highly proliferative cells with distinct endothelial phenotype (expressing CD31 and endothelial nitric oxide synthase and capable of acetylated LDL uptake). A rabbit model of balloon carotid injury was used to evaluate the effect of day 7 CMMC delivery on vascular responses. Animals underwent balloon injury and immediate delivery of autologous CMMCs or buffered saline by 20 minutes of local dwelling. Fluorescence-labeled CMMCs were detected in all vessel layers 4 weeks after delivery. Colonies of cells that localized to the lumen and stained for endothelial markers were also identified. Local CMMC administration at the time of balloon injury accelerated reendothelialization at 4 weeks compared with saline (PϽ0.05). Moreover, CMMC delivery markedly improved endothelium-dependent vasoreactivity at 4 weeks compared with saline (PϽ0.005). Finally, CMMC treatment reduced neointimal formation by 55% at 4 weeks (PϽ0.05). Conclusions-These data demonstrate that delivery of CMMCs to balloon-injured arteries is associated with accelerated reendothelialization, enhanced endothelium-dependent vasoreactivity, and reduced neointimal formation. Thus, delivery of autologous CMMCs represents a novel vasculoprotective approach to attenuate the response to acute vascular injury.

Classically activated, inflammatory macrophages are generally considered to derive from circulating monocytes, which descend from hematopoietic stem and progenitor cells (HPCs) S).The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl

Magnetic forces can be used to rapidly place endothelial cells at the site of a magnetized intravascular stent. The delivered cells are retained in the presence of blood flow and also spread to the adjacent injured vessel wall. Potential applications include delivering a cell-based therapeutic effect to the local vessel wall as well as downstream tissue.

Background Hematopoiesis originates from the dorsal aorta during embryogenesis. While adult blood vessels harbor progenitor populations for endothelial and smooth muscle cells, it is not known if they contain hematopoietic progenitor (HPCs) or stem cells (HSCs). Here, we hypothesized that the arterial wall is a source of HPCs and HSCs in postnatal life. Methods and Results Single cell aortic disaggregates were prepared from adult chow-fed C57BL/6 and ApoE−/− mice. In short- and long-term methylcellulose-based culture, aortic cells generated a broad spectrum of multipotent and lineage-specific hematopoietic colony-forming units (CFUs), with preponderance of macrophage CFUs (CFU-M). This clonogenicity was higher in lesion-free ApoE−/− mice and primarily localized to Sca-1+ cells in the adventitia. Expression of Sca-1 in the aorta co-localized with canonical HSC markers, as well as CD45 and mature leukocyte antigens. Adoptive transfer of labeled aortic cells from GFP transgenic donors to irradiated C57BL/6 recipients confirmed content of rare HSCs (1 per 4,000,000 cells), capable of self-renewal and durable, low level reconstitution of leukocytes. Moreover, the predominance of long-term macrophage precursors was evident by late recovery of GFP+ colonies from recipient bone marrow and spleen that were exclusively CFU-M. Although trafficking from bone marrow was shown to replenish some of the aorta’s hematopoietic potential following irradiation, the majority of macrophage precursors appeared to arise locally, suggesting long-term residence in the vessel wall. Conclusions The postnatal murine aorta contains rare multipotent HPCs/HSCs and is selectively enriched with Sca-1+ monocyte/macrophage precursors. These populations may represent novel, local vascular sources of inflammatory cells.

Background-Synthetic vascular grafts cannot be used in small vessels because of graft failure caused by thrombosis and neointima formation. Rapid endothelialization may overcome this limitation. We hypothesized that a magnetic graft would be able to capture and retain endothelial cells labeled with paramagnetic particles. Methods and Results-Porcine blood derived endothelial cells were allowed to endocytose superparamagnetic iron oxide microspheres. Cell survival was assessed by trypan blue exclusion and demonstrated a dose-dependent cell survival of 75% to 95%. A flexible magnetic sheet was annealed to the external surface of a knitted Dacron graft. Labeled cells (10 6 /mL) were placed within the graft for 5 minutes. Confocal and electron microscopy confirmed uniform cell capture at the magnetized surface. The effect of shear forces on the adherent cells was evaluated in a flow chamber. The cells remained attached at rates up to 300 mL/min, with cell loss commencing at 400 mL/min. Prototype magnetic grafts were implanted in porcine carotid arteries. Labeled cells were placed within the graft for 10 minutes at the time of implantation. The grafts were evaluated after one day and uniform cell coverage was noted on the magnetized surface. In comparison, relatively few labeled cells were seen attached to a nonmagnetized surface. Key Words: coronary disease Ⅲ endothelium Ⅲ grafting Ⅲ surgery Ⅲ magnet T he major limitation of prosthetic vascular grafts is their tendency to occlude after various periods of time. This occlusion rate is higher for smaller-diameter grafts and precludes their use in a significant number of medical applications, most notably in coronary artery bypass grafting. Numerous studies have shown that failure is secondary to graft occlusion, either because of thrombogenicity of the synthetic material or because of encroachment of tissue (intimal hyperplasia) into the lumen of the graft at anastomotic sites. 1 A potential way to limit graft failure would be to provide rapid, uniform, and complete coverage with a functional endothelial layer. In a pioneering study, Stump et al have shown that a Dacron patch suspended in the flow, without contact with the vessel wall, was covered with endothelial colonies within 7 days of implantation. 2 Early efforts at graft endothelialization with the use of mature endothelial cells, 3-8 although promising, were limited by difficulties related to obtaining cells in significant numbers. The recent description of circulating endothelial progenitor cells 9 has provided a new source for cellular seeding of grafts. We have previously shown that blood-derived endothelial outgrowth cells (EOCs) are effective in preventing restenosis and can restore vascular function in animal models of arterial injury. 10 Previous work used prolonged vascular occlusion to enable cell adhesion to the vessel wall, an approach that cannot be used in clinical settings. We hypothesized that local cell capture and retention could be accomplished by using magnetic forces. EOCs were rendered magn...

Delivery of a heterogeneous population of cells with endothelial phenotype derived from peripheral blood has been shown to improve vascular responses after balloon arterial injury in an endothelium-dependent manner. Refinement of culture techniques has enabled the generation of outgrowth endothelial cells (OECs), a homogeneous population of distinctly endothelial cells expanded from circulating progenitor cells. The present study tested the hypothesis that OEC delivery would confer vascular protection after balloon arterial injury in a rabbit model. Rabbit peripheral blood mononuclear cells (PBMCs) were cultured in endothelial growth medium for 4-5 wk, yielding proliferative OECs with distinct endothelial phenotype (morphology, incorporation of acetylated LDL, and expression of endothelial nitric oxide synthase and caveolin-1 but not CD14). Animals underwent balloon carotid injury immediately followed by local delivery of autologous OECs for 20 min. Fluorescent-labeled OECs were detected in all layers at 4 wk, with immunostaining revealing maintenance of endothelial phenotype (von Willebrand factor-positive and RAM-11-negative) by luminal and nonluminal cells. To evaluate functional effects, additional animals received autologous OECs, saline, or freshly harvested PBMCs as noncultured cell controls by local dwell after balloon injury. Local OEC delivery improved endothelium-dependent vasoreactivity (P < 0.05 vs. saline and PBMC) and similarly reduced neointimal formation (P < 0.05 vs. saline and PBMC). These data suggest that OECs can be detected in injured arterial segments at 4 wk. Moreover, delivery of OECs confers greater vascular protection than PBMCs or saline controls and may thus offer a novel, autologous strategy to limit the response to mechanical injury.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates the extrinsic pathway of coagulation by inhibiting the factor VIIa/tissue factor (TF) catalytic complex. TFPI is expressed by both endothelial and smooth muscle cells in the vasculature and circulates at low levels. The role of local vascular TFPI in thrombosis and the development of vascular disease is unknown. To establish an experimental animal model to directly modulate smooth muscle cell-derived TFPI on the development of arterial thrombosis, transgenic mice in which a cDNA encoding murine TFPI is expressed from the murine SM22alpha promoter were generated. Expression of transgenic mRNA was 4-fold higher than the level of endogenous TFPI mRNA in arteries from transgenic mice. In situ hybridization confirmed that expression of the transgene was limited to medial vascular smooth muscle cells. Vascular TFPI activity was increased to 2 to 3-fold in carotid homogenates. There was no difference in plasma TFPI levels or hemostatic measures (PT, aPTT and tail vein bleeding times) between these mice and their wildtype littermates. In a ferric chloride-induced model of carotid thrombosis, homozygotic transgenic mice demonstrated resistance to thrombotic occlusion compared to wildtype littermates. In transgenic mice 22% occluded within 30 minutes of application while 84% of wild type mice occluded within the same time frame (p<0.01). Heterozygotic transgenic mice had an intermediate thrombotic phenotype. Taken together, these data indicated that local VSMC-specific TFPI overexpression attenuated ferric chloride-induced thrombosis without systemic or hemostatic effects. Furthermore, this transgenic mouse model should prove useful for studying the role of TFPI in the development and progression of vascular disease.

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. A rterial thrombosis is the proximate cause of myocardial infarction and stroke. Biochemical and clinical evidence suggests the importance of the thrombogenic nature of atherosclerotic vessels in this process. Tissue factor (TF), a low-molecular-weight glycoprotein that initiates the extrinsic clotting cascade, is considered a major regulator of coagulation, hemostasis, and thrombogenicity of atherosclerotic arteries. 1-9 TF pathway inhibitor (TFPI), which provides physiological inhibition of TF-initiated coagulation by binding to factor Xa and the TF-factor VIIa complex in a 2-step process, is found in vascular endothelium and smooth muscle cells as well as in platelets, blood monocytes, and macrophages. 10 -14 In atherosclerotic carotid arteries, TF expression is abundant, whereas TFPI expression is limited in up to 30% of plaques, resulting in predominant TF activity. 15 In plaque, where TFPI expression is the greatest, TF activity is attenuated. Thus, this imbalance between TF and TFPI expression in plaque may result in the prothrombotic phenotype associated with atherosclerosis. To investigate the development and potential regulation of this imbalance, a well-defined murine model of vascular remodeling, including neointimal formation, was studied. 16 Materials and Methods Animal ModelThe murine model of vascular remodeling associated with carotid flow cessation as described by Kumar and Lindner 16 was used. All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, Md). Briefly, via a ventral longitudinal incision, the left common carotid of adult C57BL/6 mice was identified and ligated with 4-0 silk just proximal to the bifurcation. The midline incision was closed with 6-0 vicryl, and the skin was closed with a Nexaband topical skin closure kit supplied by Veterinary Products Laboratories. Warmed lactated Ringer's solution (1 mL SC) and Enrofloxacin (Bayer) (0.01 mL IM) were injected, and the mice were allowed to recover on a warm hydrothermal pad. At 1, 2, 3, and 4 weeks, the ligated carotid arteries were harvested as fresh frozen specimens for protein analysis, or they were harvested after perfusion fixation at physiological pressure with 10% formalin before euthanasia. Tissue Processing and AnalysisThe carotid arteries were obtained from mice, thoroughly rinse...