Pulmonary Biodistribution of Platelet-Derived Regenerative Exosomes in a Porcine Model.

The purpose of this study was to evaluate the biodistribution of a platelet-derived exosome product (PEP), previously shown to promote regeneration in the setting of wound healing, in a porcine model delivered through various approaches. Exosomes were labeled with DiR far-red lipophilic dye to track and quantify exosomes in tissue, following delivery via intravenous, pulmonary artery balloon catheter, or nebulization in sus scrofa domestic pigs. Following euthanasia, far-red dye was detected by Xenogen IVUS imaging, while exosomal protein CD63 was detected by Western blot and immunohistochemistry. Nebulization and intravenous delivery both resulted in global uptake of exosomes within the lung parenchyma. However, nebulization resulted in the greatest degree of exosome uptake. Pulmonary artery balloon catheter-guided delivery provided the further ability to localize pulmonary delivery. No off-target absorption was noted in the heart, spleen, or kidney. However, the liver demonstrated uptake primarily in nebulization-treated animals. Nebulization also resulted in uptake in the trachea, without significant absorption in the esophagus. Overall, this study demonstrated the feasibility of pulmonary delivery of exosomes using nebulization or intravenous infusion to accomplish global delivery or pulmonary artery balloon catheter-guided delivery for localized delivery.

Exosome biopotentiated hydrogel restores damaged skeletal muscle in a porcine model of stress urinary incontinence.

Urinary incontinence afflicts up to 40% of adult women in the United States. Stress urinary incontinence (SUI) accounts for approximately one-third of these cases, precipitating ~200,000 surgical procedures annually. Continence is maintained through the interplay of sub-urethral support and urethral sphincter coaptation, particularly during activities that increase intra-abdominal pressure. Currently, surgical correction of SUI focuses on the re-establishment of sub-urethral support. However, mesh-based repairs are associated with foreign body reactions and poor localized tissue healing, which leads to mesh exposure, prompting the pursuit of technologies that restore external urethral sphincter function and limit surgical risk. The present work utilizes a human platelet-derived CD41a and CD9 expressing extracellular vesicle product (PEP) enriched for NF-κB and PD-L1 and derived to ensure the preservation of lipid bilayer for enhanced stability and compatibility with hydrogel-based sustained delivery approaches. In vitro, the application of PEP to skeletal muscle satellite cells in vitro drove proliferation and differentiation in an NF-κB-dependent fashion, with full inhibition of impact on exposure to resveratrol. PEP biopotentiation of collagen-1 and fibrin glue hydrogel achieved sustained exosome release at 37 °C, creating an ultrastructural "bead on a string" pattern on scanning electron microscopy. Initial testing in a rodent model of latissimus dorsi injury documented activation of skeletal muscle proliferation of healing. In a porcine model of stress urinary incontinence, delivery of PEP-biopotentiated collagen-1 induced functional restoration of the external urethral sphincter. The histological evaluation found that sustained PEP release was associated with new skeletal muscle formation and polarization of local macrophages towards the regenerative M2 phenotype. The results provided herein serve as the first description of PEP-based biopotentiation of hydrogels implemented to restore skeletal muscle function and may serve as a promising approach for the nonsurgical management of SUI.

[89Zr]Zr-DBN labeled cardiopoietic stem cells proficient for heart failure.

INTRODUCTION: Radiolabeling of stem cells with a positron emitting radioisotope represents a major advancement in regenerative biotherapy enabling non-invasive imaging. To assess the value of such an approach in a clinically relevant scenario, the tolerability and therapeutic aptitude of [89Zr]zirconium-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN) labeled human cardiopoietic stem cells (CPs) were evaluated in a model of ischemic heart failure. METHODS AND RESULTS: [89Zr]Zr-DBN based radiolabeling of human CPs yielded [89Zr]Zr-DBN-CPs with radioactivity yield of 0.70 ± 0.20 MBq/106 cells and excellent label stability. Compared to unlabeled cell counterparts, [89Zr]Zr-DBN-CPs maintained morphology, viability, and proliferation capacity with characteristic expression of mesodermal and pro-cardiogenic transcription factors defining the cardiopoietic phenotype. Administered in chronically infarcted murine hearts, [89Zr]Zr-DBN-CPs salvaged cardiac pump failure, documented by improved left ventricular ejection fraction not inferior to unlabeled CPs and notably superior to infarcted hearts without cell treatment. CONCLUSION: The present study establishes that [89Zr]Zr-DBN labeling does not compromise stem cell identity or efficacy in the setting of heart failure, offering a non-invasive molecular imaging platform to monitor regenerative biotherapeutics post-transplantation.

Vasculogenic properties of adventitial Sca-1+CD45+ progenitor cells in mice: a potential source of vasa vasorum in atherosclerosis.

The cellular origins of vasa vasorum are ill-defined and may involve circulating or local progenitor cells. We previously discovered that murine aortic adventitia contains Sca-1+CD45+ progenitors that produce macrophages. Here we investigated whether they are also vasculogenic. In aortas of C57BL/6 mice, Sca-1+CD45+ cells were localised to adventitia and lacked surface expression of endothelial markers (<1% for CD31, CD144, TIE-2). In contrast, they did show expression of CD31, CD144, TIE-2 and VEGFR2 in atherosclerotic ApoE-/- aortas. Although Sca-1+CD45+ cells from C57BL/6 aorta did not express CD31, they formed CD31+ colonies in endothelial differentiation media and produced interconnecting vascular-like cords in Matrigel that contained both endothelial cells and a small population of macrophages, which were located at branch points. Transfer of aortic Sca-1+CD45+ cells generated endothelial cells and neovessels de novo in a hindlimb model of ischaemia and resulted in a 50% increase in perfusion compared to cell-free control. Similarly, their injection into the carotid adventitia of ApoE-/- mice produced donor-derived adventitial and peri-adventitial microvessels after atherogenic diet, suggestive of newly formed vasa vasorum. These findings show that beyond its content of macrophage progenitors, adventitial Sca-1+CD45+ cells are also vasculogenic and may be a source of vasa vasorum during atherogenesis.

M3RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction.

IMPACT STATEMENT: The M3RNA (microencapsulated modified messenger RNA) platform is an approach to deliver messenger RNA (mRNA) in vivo, achieving a nonintegrating and viral-free approach to gene therapy. This technology was, in this study, tested for its utility in the myocardium, providing a unique avenue for targeted gene delivery into the freshly infarcted myocardial tissue. This study provides the evidentiary basis for the use of M3RNA in the heart through depiction of its performance in cultured cells, healthy rodent myocardium, and acutely injured porcine hearts. By testing the technology in large animal models of infarction, compatibility of M3RNA with current coronary intervention procedures was verified.

Nanoparticle-Mediated Cell Capture Enables Rapid Endothelialization of a Novel Bare Metal Stent.

Incomplete endothelialization of intracoronary stents has been associated with stent thrombosis and recurrent symptoms, whereas prolonged use of dual antiplatelet therapy increases bleeding-related adverse events. Facilitated endothelialization has the potential to improve clinical outcomes in patients who are unable to tolerate dual antiplatelet therapy. The objective of this study was to demonstrate the feasibility of magnetic cell capture to rapidly endothelialize intracoronary stents in a large animal model. A novel stent was developed from a magnetizable duplex stainless steel (2205 SS). Polylactic-co-glycolic acid and magnetite (Fe3O4) were used to synthesize biodegradable superparamagnetic iron oxide nanoparticles, and these were used to label autologous blood outgrowth endothelial cells. Magnetic 2205 SS and nonmagnetic 316L SS control stents were implanted in the coronary arteries of pigs (n = 11), followed by intracoronary delivery of magnetically labeled cells to 2205 SS stents. In this study, we show extensive endothelialization of magnetic 2205 SS stents (median 98.4% cell coverage) within 3 days, whereas the control 316L SS stents exhibited significantly less coverage (median 48.9% cell coverage, p < 0.0001). This demonstrates the ability of intracoronary delivery of magnetic nanoparticle labeled autologous endothelial cells to improve endothelialization of magnetized coronary stents within 3 days of implantation.

Intravascular Delivery of Biologics to the Rat Kidney.

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

Low-Dose Gamma Irradiation of Decellularized Heart Valves Results in Tissue Injury In Vitro and In Vivo.

BACKGROUND: Decellularized heart valves are emerging as a potential alternative to current bioprostheses for valve replacement. Whereas techniques of decellularization have been thoroughly examined, terminal sterilization techniques have not received the same scrutiny. METHODS: This study evaluated low-dose gamma irradiation as a sterilization method for decellularized heart valves. Incubation of valves and transmission electron microscopy evaluation after different doses of gamma irradiation were used to determine the optimal dose of gamma irradiation. Quantitative evaluation of mechanical properties was done by tensile mechanical testing of isolated cusps. Sterilized decellularized heart valves were tested in a sheep model (n = 3 [1 at 1,500 Gy and 2 at 3,000 Gy]) of pulmonary valve replacement. RESULTS: Valves sterilized with gamma radiation between 1,000 Gy and 3,000 Gy were found to be optimal with in vitro testing. However, in vivo testing showed deteriorating valve function within 2 months. On explant, the valve with 1,500 Gy gamma irradiation showed signs of endocarditis with neutrophils on hematoxylin and eosin staining, and positive gram stain resembling streptococcus infection. The 3,000 Gy valves had no evidence of infection, but the hematoxylin and eosin staining showed evidence of wound remodeling with macrophages and fibroblasts. Tensile strength testing showed decreased strength (0 Gy: 2.53 ± 0.98 MPa, 1,500 Gy: 2.03 ± 1.23 MPa, and 3,000 Gy: 1.26 ± 0.90 MPa) with increasing levels of irradiation. CONCLUSIONS: Low-dose gamma irradiation does not maintain the mechanical integrity of valves, and the balance between sterilization and damage may not be able to be achieved with gamma irradiation. Other methods of terminal sterilization must be pursued and evaluated.

Cell surface protein disulfide isomerase regulates natriuretic peptide generation of cyclic guanosine monophosphate.

RATIONALE: The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated. OBJECTIVE: We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate. METHODS AND RESULTS: Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry. CONCLUSION: These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.

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.

Tissue factor pathway inhibitor blocks angiogenesis via its carboxyl terminus.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is the primary regulator of the tissue factor (TF) coagulation pathway. As such, TFPI may regulate the proangiogenic effects of TF. TFPI may also affect angiogenesis independently of TF, through sequences within its polybasic carboxyl terminus (TFPI C terminus [TFPIct]). We aimed to determine the effects of TFPI on angiogenesis and the role of TFPIct. METHODS AND RESULTS: Transgenic overexpression of TFPI attenuated angiogenesis in the murine hindlimb ischemia model and an aortic sprout assay. In vitro, TFPI inhibited endothelial cell migration. Peptides within the human TFPIct inhibited endothelial cell cord formation and migration in response to vascular endothelial growth factor (VEGF) 165 but not VEGF121. Furthermore, exposure to human TFPIct inhibited the phosphorylation of VEGF receptor 2 at residue Lys951, a residue known to be critical for endothelial cell migration. Finally, systemic delivery of a murine TFPIct peptide inhibited angiogenesis in the hindlimb model. CONCLUSION: These data demonstrate an inhibitory role for TFPI in angiogenesis that is, in part, mediated through peptides within its carboxyl terminus. In addition to its known role as a TF antagonist, TFPI, via its carboxyl terminus, may regulate angiogenesis by directly blocking VEGF receptor 2 activation and attenuating the migratory capacity of endothelial cells.

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.

Murine strain differences in hemostasis and thrombosis and tissue factor pathway inhibitor.

INTRODUCTION: Differences among murine strains often lead to differential responses in models of human disease. The aim of the current study was to investigate whether differences exist among strains in models of hemostasis and thrombosis and whether these differences are reflected in differences in the tissue factor (TF) pathway. METHODS: We examined baseline hemostatic parameters and the response to FeCl3-induced arterial thrombosis and a tail vein bleeding model in C57BL/6J (C57), 129S1/SvImJ (129S), and Balb/cJ (BalbC) mice. Finally, we examined TF and tissue factor pathway inhibitor (TFPI) activities in blood and expression in vascular tissue to determine whether these factors covary with a thrombotic phenotype. RESULTS: No differences were observed in PT or aPTT among strains. 129S mice had lower platelet counts (p<0.001). BalbC had an increased rate of occlusion (mean occlusion time of 330+/-45 sec) in a FeCl(3)-induced model of thrombosis when compared to C57 (1182+/-349 sec) or 129 S (1442+/-281 sec) (p<0.05). Similarly, BalbC demonstrated reduced blood loss in tail bleeding experiments when compared to C57 and 129S. Vascular expression of TF and TFPI content did not correlate with the thrombotic phenotype of BalbC. However, circulating TFPI activities were lower in BalbC compared to both C57 and 129S mice. When normalized to circulating TF activities, BalbC had lower circulating TFPI activity than C57 and 129S, and there was a significant correlation between tail bleeding and normalized TFPI activity (r=0.67). CONCLUSIONS: These data suggest that there are significant differences among strains in thrombosis and hemostasis and that circulating TFPI activity correlates with these differences.

Tissue factor pathway inhibitor overexpression inhibits hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is a commonly recognized complication of chronic respiratory disease. Enhanced vasoconstriction, pulmonary vascular remodeling, and in situ thrombosis contribute to the increased pulmonary vascular resistance observed in PH associated with hypoxic lung disease. The tissue factor pathway regulates fibrin deposition in response to acute and chronic vascular injury. We hypothesized that inhibition of the tissue factor pathway would result in attenuation of pathophysiologic parameters typically associated with hypoxia-induced PH. We tested this hypothesis using a chronic hypoxia-induced murine model of PH using mice that overexpress tissue factor pathway inhibitor (TFPI) via the smooth muscle-specific promoter SM22 (TFPI(SM22)). TFPI(SM22) mice have increased pulmonary TFPI expression compared with wild-type (WT) mice. In WT mice, exposure to chronic hypoxia (28 d at 10% O(2)) resulted in increased systolic right ventricular and mean pulmonary arterial pressures, changes that were significantly reduced in TFPI(SM22) mice. Chronic hypoxia also resulted in significant pulmonary vascular muscularization in WT mice, which was significantly reduced in TFPI(SM22) mice. Given the pleiotropic effects of TFPI, autocrine and paracrine mechanisms for these hemodynamic effects were considered. TFPI(SM22) mice had less pulmonary fibrin deposition than WT mice at 3 days after exposure to hypoxia, which is consistent with the antithrombotic effects of TFPI. Additionally, TFPI(SM22) mice had a significant reduction in the number of proliferating (proliferating cell nuclear antigen positive) pulmonary vascular smooth muscle cells compared with WT mice, which is consistent with in vitro findings. These findings demonstrate that overexpression of TFPI results in improved hemodynamic performance and reduced pulmonary vascular remodeling in a murine model of hypoxia-induced PH. This improvement is in part due to the autocrine and paracrine effects of TFPI overexpression.

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 22alpha (SM22alpha)-TFPI/apoE(-/-) mice were shown to have less aortic plaque burden compared to apoE(-/-) mice. Unexpectedly, SM22alpha-TFPI/apoE(-/-) had lower plasma cholesterol levels compared to apoE(-/-) mice. Furthermore, SM22alpha-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 SM22alpha-TFPI and wild-type mice. Mouse aortic smooth muscle cells from SM22alpha-TFPI mice demonstrated higher VLDL binding and internalization compared to those from wild-type mice. Because SM22alpha-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.

Vasoprotective effects of human CD34+ cells: towards clinical applications.

BACKGROUND: The development of cell-based therapeutics for humans requires preclinical testing in animal models. The use of autologous animal products fails to address the efficacy of similar products derived from humans. We used a novel immunodeficient rat carotid injury model in order to determine whether human cells could improve vascular remodelling following acute injury. METHODS: Human CD34+ cells were separated from peripheral buffy coats using automatic magnetic cell separation. Carotid arterial injury was performed in male Sprague-Dawley nude rats using a 2F Fogarty balloon catheter. Freshly harvested CD34+ cells or saline alone was administered locally for 20 minutes by endoluminal instillation. Structural and functional analysis of the arteries was performed 28 days later. RESULTS: Morphometric analysis demonstrated that human CD34+ cell delivery was associated with a significant reduction in intimal formation 4 weeks following balloon injury as compared with saline (I/M ratio 0.79 +/- 0.18, and 1.71 +/- 0.18 for CD34, and saline-treated vessels, respectively P < 0.05). Vasoreactivity studies showed that maximal relaxation of vessel rings from human CD34+ treated animals was significantly enhanced compared with saline-treated counterparts (74.1 +/- 10.2, and 36.8 +/- 12.1% relaxation for CD34+ cells and saline, respectively, P < 0.05) CONCLUSION: Delivery of human CD34+ cells limits neointima formation and improves arterial reactivity after vascular injury. These studies advance the concept of cell delivery to effect vascular remodeling toward a potential human cellular product.

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.

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.

Modulation of the vascular response to injury by autologous blood-derived outgrowth endothelial cells.

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.