Tyra A. Witt

Autologous Culture-Modified Mononuclear Cells Confer Vascular Protection After Arterial Injury

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 (P <0.05). Moreover, CMMC delivery markedly improved endothelium-dependent vasoreactivity at 4 weeks compared with saline (P <0.005). Finally, CMMC treatment reduced neointimal formation by 55% at 4 weeks (P <0.05). 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.

Magnetic Forces Enable Rapid Endothelialization of Synthetic Vascular Grafts

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 (106/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. Conclusions— Magnetic forces can be used to rapidly cover a vascular graft with paramagnetically labeled cells. This biophysical interaction is sufficient to retain cells in the presence of blood flow. Applications of this technique may include rapid endothelialization of synthetic vascular grafts and dialysis fistulas.

Identification of a Monocyte-Predisposed Hierarchy of Hematopoietic Progenitor Cells in the Adventitia of Postnatal Murine Aorta

Background— Hematopoiesis originates from the dorsal aorta during embryogenesis. Although adult blood vessels harbor progenitor populations for endothelial and smooth muscle cells, it is not known if they contain hematopoietic progenitor or stem cells. Here, we hypothesized that the arterial wall is a source of hematopoietic progenitor and stem cells in postnatal life. Methods and Results— Single-cell aortic disaggregates were prepared from adult chow-fed C57BL/6 and apolipoprotein E–null (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, with a preponderance of macrophage colony-forming units. This clonogenicity was higher in lesion-free ApoE−/− mice and localized primarily to stem cell antigen-1–positive cells in the adventitia. Expression of stem cell antigen-1 in the aorta colocalized with canonical hematopoietic stem cell markers, as well as CD45 and mature leukocyte antigens. Adoptive transfer of labeled aortic cells from green fluorescent protein transgenic donors to irradiated C57BL/6 recipients confirmed the content of rare hematopoietic stem cells (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 green fluorescent protein–positive colonies from recipient bone marrow and spleen that were exclusively macrophage colony-forming units. Although trafficking from bone marrow was shown to replenish some of the hematopoietic potential of the aorta after 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 hematopoietic progenitor/stem cells and is selectively enriched with stem cell antigen-1–positive monocyte/macrophage precursors. These populations may represent novel, local vascular sources of inflammatory cells.

Characterization of a Resident Population of Adventitial Macrophage Progenitor Cells in Postnatal Vasculature

Rationale: Macrophages regulate blood vessel structure and function in health and disease. The origins of tissue macrophages are diverse, with evidence for local production and circulatory renewal. Objective: We identified a vascular adventitial population containing macrophage progenitor cells and investigated their origins and fate. Methods and Results: Single-cell disaggregates from adult C57BL/6 mice were prepared from different tissues and tested for their capacity to form hematopoietic colony-forming units. Aorta showed a unique predilection for generating macrophage colony-forming units. Aortic macrophage colony-forming unit progenitors coexpressed stem cell antigen-1 and CD45 and were adventitially located, where they were the predominant source of proliferating cells in the aortic wall. Aortic Sca-1+CD45+ cells were transcriptionally and phenotypically distinct from neighboring cells lacking stem cell antigen-1 or CD45 and contained a proliferative (Ki67+) Lin−c-Kit+CD135−CD115+CX3CR1+Ly6C+CD11b− subpopulation, consistent with the immunophenotypic profile of macrophage progenitors. Adoptive transfer studies revealed that Sca-1+CD45+ adventitial macrophage progenitor cells were not replenished via the circulation from bone marrow or spleen, nor was their prevalence diminished by depletion of monocytes or macrophages by liposomal clodronate treatment or genetic deficiency of macrophage colony-stimulating factor. Rather adventitial macrophage progenitor cells were upregulated in hyperlipidemic ApoE−/− and LDL-R−/− mice, with adventitial transfer experiments demonstrating their durable contribution to macrophage progeny particularly in the adventitia, and to a lesser extent the atheroma, of atherosclerotic carotid arteries. Conclusions: The discovery and characterization of resident vascular adventitial macrophage progenitor cells provides new insight into adventitial biology and its participation in atherosclerosis and provokes consideration of the broader existence of local macrophage progenitors in other tissues.

The effect of vascular smooth muscle cell-targeted expression of tissue factor pathway inhibitor in a murine model of arterial thrombosis

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.

Noninvasive monitoring of oxidative stress in transplanted mesenchymal stromal cells

OBJECTIVES The goal of this study was to validate a pathway-specific reporter gene that could be used to noninvasively image the oxidative status of progenitor cells. BACKGROUND In cell therapy studies, reporter gene imaging plays a valuable role in the assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue confer oxidative stress to transplanted cells that may influence their survival and reparative function. METHODS Rat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased oxidative stress under in vitro stress. On the basis of their up-regulation of the pro-oxidant enzyme p67(phox) subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67(phox)), an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67(phox) promoter. MSCs cotransfected with NAD(P)H p67(phox)-Fluc and a cell viability reporter gene (cytomegalovirus-Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions. RESULTS After in vitro validation of the sensor during low-serum culture, transfected MSCs were transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by using bioluminescence imaging. Compared with sham controls (no IR), cardiac Fluc intensity was significantly higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h; p < 0.01), indicating increased cellular oxidative stress. This finding was corroborated by ex vivo luminometry after correcting for Renilla luciferase activity as a measure of viable MSC number (Fluc:Renilla luciferase ratio 0.011 ± 0.003 for sham vs. 0.026 ± 0.004 for IR at 48 h; p < 0.05). Furthermore, in IR animals that received MSCs preconditioned with an antioxidant agent (tempol), Fluc signal was strongly attenuated, substantiating the specificity of the oxidative stress sensor. CONCLUSIONS Pathway-specific reporter gene imaging allows assessment of changes in the oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key biological interactions between transplanted cells and their host environment in living subjects.

Vascular-Directed Tissue Factor Pathway Inhibitor Overexpression Regulates Plasma Cholesterol and Reduces Atherosclerotic Plaque Development

Rationale: Tissue factor pathway inhibitor (TFPI) is a potent regulator of the tissue factor pathway and is found in plasma in association with lipoproteins. Objective: To determine the role of TFPI in the development of atherosclerosis, we bred mice which overexpress TFPI into the apolipoprotein E–deficient (apoE−/−) background. Methods and Results: On a high-fat diet, smooth muscle 22α (SM22α)-TFPI/apoE−/− mice were shown to have less aortic plaque burden compared to apoE−/− mice. Unexpectedly, SM22α-TFPI/apoE−/− had lower plasma cholesterol levels compared to apoE−/− mice. Furthermore, SM22α-TFPI mice fed a high-fat diet had lower cholesterol levels than did wild-type mice. Because TFPI is associated with lipoproteins and its carboxyl terminus (TFPIct) has been shown to be a ligand for the very-low-density lipoprotein (VLDL) receptor, we hypothesized that TFPI overexpression may regulate lipoprotein distribution. We quantified VLDL binding and uptake in vitro in mouse aortic smooth muscle cells from SM22α-TFPI and wild-type mice. Mouse aortic smooth muscle cells from SM22α-TFPI mice demonstrated higher VLDL binding and internalization compared to those from wild-type mice. Because SM22α-TFPI mice have increased circulating levels of TFPI antigen, we examined whether TFPIct may act to alter lipoprotein distribution. In vitro, TFPIct increased VLDL binding, uptake, and degradation in murine embryonic fibroblasts. Furthermore, this effect was blocked by heparinase treatment. In vivo, systemic administration of TFPIct reduced plasma cholesterol levels in apoE−/− mice. Conclusions: These studies suggest that overexpression of TFPI lowers plasma cholesterol through the interaction of its carboxyl terminus with lipoproteins and heparan sulfate proteoglycans.

Distribution of Circulation-Derived Endothelial Progenitors Following Systemic Delivery

Cells with an endothelial phenotype can be cultured from peripheral blood. These cells include cells of a monocytic origin with endothelial features (culture-modified mononuclear cells, CMMCs) and, at later time points, blood outgrowth endothelial cells (BOECs). Both are promising candidates for systemic cell-based cardiovascular therapies and each may have unique capabilities. Indeed, the combined use of both cell types has been shown to have synergistic therapeutic features requiring simultaneous delivery. However, the majority of preclinical studies of cell delivery have used splenectomized animals to increase systemic distribution. The goal of this study was to directly compare the distribution of these two cell types following systemic delivery in an intact animal model. A similar pattern of delivery was seen following delivery of both cell types with detection in the lung, liver, bone marrow, and spleen. Taken together, the data suggest that strategies using systemic delivery of circulation-derived cells must consider the distribution and efficiency of delivery in intact animals.

Carotid Repair Using Autologous Adipose-Derived Endothelial Cells

BACKGROUND AND PURPOSE Adipose tissue is an abundant source of endothelial cells as well as stem and progenitor cells which can develop an endothelial phenotype. It has been demonstrated that these cells have distinct angiogenic properties in vitro and in vivo. However, whether these cells have the capacity to directly improve large vessel form and function after vascular injury remains unknown. To define whether delivery of adipose-derived endothelial cells (ADECs) would improve healing of injured carotid arteries, a rabbit model of acute arterial injury was used. METHODS Autologous rabbit ADECs were generated using defined culture conditions. To test the ability of ADECs to enhance carotid artery repair, cells were delivered intraarterially after acute balloon injury. Additional delivery studies were performed after functional selection of cells before delivery. RESULTS After rabbit omental fat harvest and digestion, a proliferative, homogenous, and distinctly endothelial population of ADECs was identified. Direct delivery of autologous ADECs resulted in marked reendothelialization 48 hours after acute vascular injury as compared to saline controls (82.2+/-26.9% versus 4.2+/-3.0% P<0.001). Delivery of ADECs that were selected for their ability to take up acetylated LDL significantly improved vasoreactivity and decreased intimal formation after vascular injury. CONCLUSIONS Taken together, these data suggest that ADECs represent an autologous source of proliferative endothelial cells, which demonstrate the capacity to rapidly improve reendothelialization, improve vascular reactivity, and decrease intimal formation in a carotid artery injury model.

A direct mechanical method for accurate and efficient adenoviral vector delivery to tissues

We describe a mechanical method for delivery of adenoviral vector to the adventitial surface of arteries and to other tissues. Our goal was to characterize, principally in intact carotid artery, the morphological, biochemical, and functional effects of mechanical delivery of a recombinant β-galactosidase-expressing adenoviral vector following its direct application using a small paintbrush. Our ex vivo and in vivo data demonstrate efficient, accurate, and rapid transduction of arteries without compromise of their morphological, biochemical, and functional integrity. We also demonstrate the general applicability of this technique in vivo via transduction of skeletal muscle, fibrotendinous tissue, peritoneum, serosal surface of bowel, and wounded skin. We conclude that direct mechanical delivery of an adenoviral vector to tissues using a suitable paintbrush represents an intuitive, accurate, and effective means of augmenting gene transfer efficiency, and may be a useful adjunct to other delivery methods.