A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.

Selective M2 Macrophage Depletion Leads to Prolonged Inflammation in Surgical Wounds.

BACKGROUND: A prolonged inflammatory phase is seen in aberrant wound healing and in chronic wounds. Macrophages are central to wound healing. Distinct macrophage subtypes have differing roles both in initial inflammation and in later tissue repair. Broadly, these cells can be divided into M1 and M2 macrophages. M2 macrophage proliferation and differentiation is regulated by colony-stimulating factor 1 (CSF-1) signalling and can be blocked by GW2580, a competitive cFMS kinase inhibitor, thereby allowing for analysis of the effect of M2 blockade on progression of surgical wounds. MATERIALS AND METHODS: Macrophage Fas-induced apoptosis (MaFIA) transgenic mice with a macrophage-specific promoter used to express green fluorescent protein (GFP) were used to allow for cell tracking. The animals were treated by oral gavage with GW2580. Surgical wounds were created and harvested after 2 weeks for analysis. RESULTS: GW2580-treated mice had significantly more GFP+ cells in the surgical scar than vehicle-treated animals (GW2580, 68.0 ± 3.1%; vehicle, 42.8 ± 1.7%; p < 0.001), and GW2580 treatment depleted CD206+ M2 macrophages in the scar (GW2580, 1.4%; vehicle, 19.3%; p < 0.001). Treated animals showed significantly higher numbers of neutrophils (vehicle, 18.0%; GW2580, 51.3%; p < 0.01) and M1 macrophages (vehicle, 3.8%; GW2580, 12.8%; p < 0.01) in the scar compared to vehicle-treated animals. The total collagen content in the area of the scar was decreased in animals treated with GW2580 as compared to those treated with vehicle alone (GW2580, 67.1%; vehicle, 79.9%; p < 0.005). CONCLUSIONS: Depletion of M2 macrophages in surgical wounds via CSF-1 signalling blockade leads to persistent inflammation, with an increase in neutrophils and M1 macrophages and attenuated collagen deposition.

Bone Marrow-Derived Mesenchymal Stem Cells Have Innate Procoagulant Activity and Cause Microvascular Obstruction Following Intracoronary Delivery: Amelioration by Antithrombin Therapy.

Mesenchymal stem cells (MSCs) are currently under investigation as tools to preserve cardiac structure and function following acute myocardial infarction (AMI). However, concerns have emerged regarding safety of acute intracoronary (IC) MSC delivery. This study aimed to characterize innate prothrombotic activity of MSC and identify means of its mitigation toward safe and efficacious therapeutic IC MSC delivery post-AMI. Expression of the initiator of the coagulation cascade tissue factor (TF) on MSC was detected and quantified by immunofluorescence, FACS, and immunoblotting. MSC-derived TF antigen was catalytically active and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet thrombus deposition on collagen at arterial shear, an effect abolished by heparin coadministration. In a porcine AMI model, IC infusion of 25 × 10(6) MSC during reperfusion was associated with a decrease in coronary flow reserve but not when coadministered with an antithrombin agent (heparin). Heparin reduced MSC-associated thrombosis incorporating platelets and VWF within the microvasculature. Heparin-assisted therapeutic MSC delivery also reduced apoptosis in the infarct border zone at 24 hours, significantly improved infarct size, left ventricular (LV) ejection fraction, LV volumes, wall motion, and attenuated histologic evidence of scar formation at 6 weeks post-AMI. Heparin alone or heparin-assisted fibroblast control cell delivery had no such effect. Procoagulant TF activity of therapeutic MSCs is associated with reductions in myocardial perfusion when delivered IC may be successfully managed by heparin coadministration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic IC MSC delivery for AMI.

Exopolysaccharide-producing probiotic Lactobacilli reduce serum cholesterol and modify enteric microbiota in ApoE-deficient mice.

BACKGROUND: Probiotic bacteria have been associated with a reduction in cardiovascular disease risk, a leading cause of death and disability. OBJECTIVES: The aim of this study was to assess the impact of dietary administration of exopolysaccharide-producing probiotic Lactobacillus cultures on lipid metabolism and gut microbiota in apolipoprotein E (apoE)-deficient mice. METHODS: First, we examined lipid metabolism in response to dietary supplementation with recombinant ß-glucan-producing Lactobacillus paracasei National Food Biotechnology Centre (NFBC) 338 expressing the glycosyltransferase (Gtf) gene from Pediococcus parvulus 2.6 (GTF), and naturally exopolysaccharide-producing Lactobacillus mucosae Dairy Product Culture Collection (DPC) 6426 (DPC 6426) compared with the non-ß-glucan-producing isogenic control strain Lactobacillus paracasei NFBC 338 (PNZ) and placebo (15% wt:vol trehalose). Second, we examined the effects on the gut microbiota of dietary administration of DPC 6426 compared with placebo. Probiotic Lactobacillus strains at 1 × 10(9) colony-forming units/d per animal were administered to apoE(-/-) mice fed a high-fat (60% fat)/high-cholesterol (2% wt:wt) diet for 12 wk. At the end of the study, aortic plaque development and serum, liver, and fecal variables involved in lipid metabolism were analyzed, and culture-independent microbial analyses of cecal content were performed. RESULTS: Total cholesterol was reduced in serum (P < 0.001; ∼33-50%) and liver (P < 0.05; ∼30%) and serum triglyceride concentrations were reduced (P < 0.05; ∼15-25%) in mice supplemented with GTF or DPC 6426 compared with the PNZ or placebo group, respectively. In addition, dietary intervention with GTF led to increased amounts of fecal cholesterol excretion (P < 0.05) compared with all other groups. Compositional sequencing of the gut microbiota revealed a greater prevalence of Porphyromonadaceae (P = 0.001) and Prevotellaceae (P = 0.001) in the DPC 6426 group and lower proportions of Clostridiaceae (P < 0.05), Peptococcaceae (P < 0.001), and Staphylococcaceae (P < 0.01) compared with the placebo group. CONCLUSION: Ingestion of exopolysaccharide-producing lactobacilli resulted in seemingly favorable improvements in lipid metabolism, which were associated with changes in the gut microbiota of mice.

Potent endothelial progenitor cell-conditioned media-related anti-apoptotic, cardiotrophic, and pro-angiogenic effects post-myocardial infarction are mediated by insulin-like growth factor-1.

AIMS: We have previously reported the cardioprotective effects of endothelial progenitor cell (EPC)-conditioned media (CM) therapy post-myocardial infarction (MI). In the present study, we have determined the insulin-like growth factor-1 (IGF-1) contribution to EPC CM effects on cardiomyocyte survival, contractility, and angiogenesis in vivo. METHODS AND RESULTS: Conditioned media from porcine EPC were administered intracoronary in the presence and absence of specific neutralizing antibodies to IGF-1 or control IgG in a porcine model of MI. X-vivo (non-conditioned) medium was used as a control. Functional, histological, and biochemical parameters were evaluated at 24 h and 8-week post-therapy. Conditioned media therapy significantly abrogated infarct zone (IZ) apoptosis, hypocontractility, and impaired left ventricular (LV) relaxation observed in control infarcts acutely (24 h post-MI). At 8 weeks following treatment, CM therapy augmented LV contractility and relaxation, IZ angiogenesis and inhibited infarct size expansion, wall expansion, and wall thinning. All of these acute and chronic beneficial effects of CM therapy were vitiated by neutralizing antibodies to IGF-1 but not by control IgG. Moreover, the addition of neutralizing IGF-1 antibody to control medium had no effect on these structural or functional changes in the heart post-treatment. CONCLUSION: Insulin-like growth factor-1 within the EPC CM mediates potent acute myocardial repair and chronic remodelling effects post-MI. These findings may provide a rationale for comparative trials of specific growth factors vs. current progenitor cell strategies.

Network Proteins of Human Sortilin1, Its Expression and Targetability Using Lycopene.

BACKGROUND: Sortilin1 (SORT1) is a ubiquitously expressed transporter involved in sorting or clearing proteins and is pathologically linked to tissue fibrosis and calcification. Targeting SORT1 may have potential clinical efficacy in controlling or reversing cardiovascular fibrosis and/or calcification. Hence, this study assessed the protein-protein network of human SORT1 and its targetability using known nutra-/pharmaceuticals. MATERIAL AND METHODS: Network proteins of human SORT1 were identified using the String database, and the affinity of the protein-protein interaction of this network was analysed using Chimera software (Chimera-1.17.3-mac64). The tissue-specific expression profile of SORT1 was evaluated and assessed for enrichment in different cell types, including immune cells. A library of in-house small molecules and currently used therapeutics for cardiovascular diseases were screened using AutoDock Vina to assess the targetability of human SORT1. The concentration affinity (CA) ratio of the small molecules was estimated to assess the clinical feasibility of targeting SORT1. RESULTS: IGF2R, NTRK2, GRN and GGA1 were identified as high-affinity interaction networks of SORT1. Of these high-affinity interactions, IGF2R and GRN can be considered relevant networks in regulating tissue fibrosis or the microcalcification process due to their influence on T-cell activation, inflammation, wound repair, and the tissue remodelling process. The tissue cell-type enrichment indicated major expression of SORT1 in adipocytes, specialised epithelial cells, monocytes, cardiomyocytes, and thyroid glandular cells. The binding pocket analysis of human SORT1 showed twelve potential drug interaction sites with varying binding scores (0.86 to 5.83) and probability of interaction (0.004 to 0.304). Five of the drug interaction sites were observed to be targetable at the therapeutically feasible concentration of the small molecules evaluated. Empagliflozin, sitagliptin and lycopene showed a superior affinity and CA ratio compared to established inhibitors of SORT1. CONCLUSION: IGF2R and GRN are relevant networks of SORT1, regulating tissue fibrosis or the microcalcification process. SORT1 can be targeted using currently approved small-molecule therapeutics (empagliflozin and sitagliptin) or widely used nutraceuticals (lycopene), which should be evaluated in a randomised clinical trial to assess their efficacy in reducing the cardiac/vascular microcalcification process.

Differential endothelial coverage, response to injury and neointimal integration of CX3CR1/smooth muscle-like cells after carotid or femoral arterial injury.

BACKGROUND: Previously, we established the importance of the CX3CL1/CX3CR1 axis in the promotion of myeloid cell differentiation into neointimal smooth muscle-like cells (SMLC). METHODS: In this study, acute (24 h) endothelial coverage and CX3CL1 expression as well as chronic (2 weeks) vascular remodeling was examined with respect to whether myeloid CX3CR1(+) SMLC number in the neointima differed between carotid and femoral artery wire injury. RESULTS AND CONCLUSION: Twenty-four hours after injury, CX3CL1 expression was significantly elevated in injured carotid compared to femoral arteries. In mice with CX3CR1 promoter-driven expression of green fluorescent protein, neointima formation was significantly greater (p < 0.05) 2 weeks after injury in femoral versus carotid arteries as determined by the intima/media ratio. Although the percentage of F4/80/CX3CR1(+) cell integration was similar in both models, the carotid lesion had greater proportions of cells coexpressing CX3CR1 and both α-smooth muscle actin and calponin (p < 0.05). Wire injury of carotid arteries was associated with greater CX3CL1 expression in the acute phase followed by greater CX3CR1 coexpressing SMLC content in later lesions as well as less neointima formation than in femoral arteries. This may, in part, explain the variability in lesion composition after carotid versus femoral wire injury.

Analysis of attitudes toward the source of progenitor cells in tissue-engineered products for use in burns compared with other disease states.

The first trials using progenitor cells to improve burn wound healing are beginning. However, there remains a paucity of data on patients' opinions of the source of stem cells. In this study, 279 patients attending plastic surgery/burns outpatient and medical outpatient clinics were questioned to assess willingness to accept a tissue-engineered skin product derived from a variety of sources. Levels of acceptance for the use of progenitor cells derived from these sources for treatment across a range of disease states (burns, Parkinson's disease, diabetes, and for cosmetic use) were also assessed. Overall, 80% of those questioned would accept a tissue-engineered product. Autologous cells were the preferred choice of cells (acute burns 94%, diabetes 95%, Parkinson's 93.9%). Allogeneic cells were still widely accepted (acute burns 67%, diabetes 66.7%, Parkinson's 69.2%). There was no difference observed between plastic surgical patients and medical patients in acceptance of cell therapy for burns, Parkinson's disease, or diabetes. There is good potential acceptance for the use of both autologous and allogeneic cells for the treatment of acute burns and burns' scarring as well as in diabetes and Parkinson's disease. Disease state does not appear to influence overall acceptability and choice of cells.

Bone marrow-derived CX3CR1 progenitors contribute to neointimal smooth muscle cells via fractalkine CX3CR1 interaction.

Smooth muscle cells play a major role in numerous vascular diseases that contribute to remodeling, repair after injury, and arteriogenesis, and the source of these cells is thought to lie within the vessel wall and the circulating blood. Currently, the precise origin and mechanism of differentiation of extravascular smooth muscle progenitor cells (SPCs) is unclear. We show here that the CX(3)CR1 mononuclear cell population of murine bone marrow provides a source of SPCs that contributes to smooth muscle cells within the neointimal plaque after vascular injury. Moreover, CX(3)CR1-fractalkine (FKN) interaction in vivo is essential for smooth muscle cell differentiation of bone marrow-derived progenitor cells at the vessel wall level. Functional competence of bone marrow-derived CX(3)CR1 positive cells to interact with FKN is also crucial in part for neointima formation following vascular injury. Finally, in a pure preparation of bone marrow-derived CX(3)CR1 positive cells, we show that in vitro smooth muscle cell differentiation increases markedly in the presence of FKN. Our data highlight a novel functional relationship between the myeloid and vascular systems and in the context of vascular injury and repair underscores a key chemokine-receptor pathway that may regulate cell fate when smooth muscle cell differentiation is required.

Clinical potential of adult vascular progenitor cells.

Cell therapy to treat vascular and cardiovascular diseases has evolved over the past decade with improved understanding of progenitor cell mobilization, recruitment, and differentiation. The beneficial effects seen in several preclinical studies have prompted translation of adult vascular progenitor therapy to clinical trials. To date, progenitor cells isolated from bone marrow and peripheral blood have been tested in the context of acute myocardial infarction and chronic ischemic cardiomyopathy, with moderate benefit. This therapeutic effect occurs despite a relatively small number of injected progenitor cells and short-term residence in the target zone. Thus, indirect benefits, such as paracrine factors released from these cells, have been suggested as significant contributors to therapeutic efficacy. Several additional vascular progenitors of endothelial, smooth muscle, mesenchymal, and cardiac origin have been identified that may contribute to vasculogenesis. Indeed, a unifying paradigm for the most effective cell therapy strategies to date appears to be robust support of angiogenesis. Here we discuss a number of progenitor cells that currently show potential as cardiovascular therapeutics, either singly or in combination. We look at emerging cell types and disease targets that may be exploited for therapeutic benefit and future strategies that may maximize clinical efficacy.

A specific subset of mouse bone marrow cells has smooth muscle cell differentiation capacity-brief report.

OBJECTIVE: To determine whether CX(3)CR1(+) bone marrow cells have the capacity for smooth muscle cell (SMC) differentiation. METHODS AND RESULTS: CX(3)CR1(+) and CX(3)CR1(-) cells were isolated from marrow of CX(3)CR1 transgenic mice and cultured in SMC differentiation media. Phenotypic and functional analyses showed only CX(3)CR1(+) bone marrow cells exhibit colony cell outgrowth with SMC-specific protein expression, calcium signaling, and contraction responses similar to mature SMC. CONCLUSIONS: CX(3)CR1 marks a bone marrow cells population that enriches for progenitors with capacity to differentiate in vitro into SMC-like cells.

Lentiviral tracking of vascular differentiation in bone marrow progenitor cells.

Lentiviral vectors encoding for identifiable marker genes controlled by lineage-specific promoters can be used to track differentiation of bone marrow progenitors into endothelial cells and/or smooth muscle cells. Human VE-Cadherin and Smoothelin-B promoters were cloned into a self-inactivating lentiviral vector (HR-VECad and HR-SMTHB) and used to drive expression of green fluorescent protein (eGFP). These constructs demonstrated specific promoter activity in mature endothelial and smooth muscle cells respectively in vitro. Lin(-) bone marrow progenitor cells (Lin(-) BMCs) in culture were used to test vector ability to track vascular differentiation. HR-VECad transduced Lin(-) BMCs were plated on collagen-coated slides and grown in endothelial media, while HR-SMTHB transduced Lin(-) BMCs were cultured on fibronectin-coated slides and grown in smooth muscle media. For in vivo differentiation assessment, lentiviral transduced Lin(-) BMCs resuspended in Matrigel were injected subcutaneously into C57BL/6J mice. Explants were evaluated for eGFP expression. Lin(-) BMCs grown in endothelial differentiation media produced groups of polygonal endothelial-like cells by days 16-21. When transduced with HR-VECad vector, these expressed eGFP in distinct cells within the colony by days 18-21, and coexpressed VE-Cadherin and eNOS. Lin(-) BMCs grown in smooth muscle differentiation media produced spindle-shaped cells between days 10-14 in culture. When transduced with the HR-SMTHB vector, these cells showed eGFP expression at approximately 12 days, which increased over time and coexpressed alphaSMA, calponin and myosin heavy chain. Within Matrigel plugs containing HR-VECad transduced cells, eGFP(+) constituted 0.4+/-0.2% of total cells. In contrast, within Matrigel plugs containing HR-SMTHB transduced cells, eGFP(+) cells constituted 0.2+/-0.1% of total cells. These data demonstrate the feasibility of selectively marking BMC populations for cell fate determination.

Date Palm (Phoenix dactylifera) Fruits as a Potential Cardioprotective Agent: The Role of Circulating Progenitor Cells.

Context: Date palms, along with their fruits' dietary consumption, possess enormous medicinal and pharmacological activities manifested in their usage in a variety of ailments in the various traditional systems of medicine. In recent years, the identification of progenitor cells in the adult organ systems has opened an altogether new approach to therapeutics, due to the ability of these cells to repair the damaged cells/tissues. Hence, the concept of developing therapeutics, which can mobilize endogenous progenitor cells, following tissue injury, to enhance tissue repair process is clinically relevant. Objectives: The present study investigates the potential of date of palm fruit extracts in repairing tissue injury following myocardial infarction (MI) potentially by mobilizing circulating progenitor cells. Methods: Extracts of four different varieties of date palm fruits common in Saudi Arabia eastern provision were scrutinized for their total flavonoid, total phenolic, in vitro antioxidant capacity, as well as their effects on two different rodent MI models. Results: High concentrations of phenolic and flavonoid compounds were observed in date palm fruit extracts, which contributed to the promising antioxidant activities of these extracts and the observed high protective effect against various induced in vivo MI. The extracts showed ability to build up reserves and to mobilize circulating progenitor cells from bone marrow and peripheral circulation to the site of myocardial infraction. Conclusion: Date palm fruit extracts have the potential to mobilize endogenous circulating progenitor cells, which can promote tissue repair following ischemic injury.

Intraperitoneal co-administration of low dose urethane with xylazine and ketamine for extended duration of surgical anesthesia in rats.

Procedures involving complex surgical techniques in rats, such as placement of abdominal aortic graft require extended duration of surgical anesthesia, which often can be achieved by repeated administrations of xylazine-ketamine combination. However such repeated anesthetic administration, in addition to being technically challenging, may be associated with potential adverse events due to cumulative effects of anesthesia. We report here the feasibility of using urethane at low dose (~1/10 the recommended anesthetic dose) in combination with a xylazine-ketamine mix to achieve an extended duration of surgical anesthesia in rats. The anesthesia induction phase was quick and smooth with an optimal phase of surgical anesthesia achieved for up to 90 minutes, which was significantly higher compared to that achieved with use of only xylazine-ketamine combination. The rectal temperature, heart rate and respiratory rate were within the physiological range with an uneventful recovery phase. Post surgery the rats were followed up to 3 months without any evidence of tumor or any other adverse effects related to the use of the urethane anesthetic combination. We conclude that low dose urethane can be effectively used in combination with xylazine and ketamine to achieve extended duration of surgical anesthesia up to 90 minutes in rats.

Allogeneic mesenchymal stem cells, but not culture modified monocytes, improve burn wound healing.

The use of cell therapy to improve burn wound healing is limited as a validated cell source is not rapidly available after injury. Progenitor cells have shown potential to drive the intrinsic wound regeneration. Two sources of cells, allogeneic mesenchymal stem cells (MSC) and autologous culture modified monocytes (CMM), were assessed for their ability to influence burn wound healing. Both could be widely available shortly after injury. Cells were delivered in a fibrin matrix following contact burns in a porcine burns model. Application of MSC significantly decreased the area of unhealed burn compared to CMM or delivery matrix alone (6% MSC, 27% CMM, 24% Matrix, p<0.001). MSC treated wounds showed histological evidence of improved wound healing with increased collagen content (MSC 49%, CMM 42%, p<0.01), increased epidermal area (MSC 8.8%, CMM 6.1%, p<0.01) and dermal thickness (MSC 1108 µm, CMM 1007 µm, p<0.01) compared to CMM treated wounds. Labelled MSC and CMM were identified in the wounds after 2 weeks by immunohistochemistry and FACS. A single application of allogeneic MSC improves the rate of burn wound healing and improves the histological appearance of the burn wound. These cells show potential as a cell therapy that is rapidly available following burn.

A novel CX3CR1 antagonist eluting stent reduces stenosis by targeting inflammation.

We evaluated the therapeutic efficacy of a novel drug eluting stent (DES) inhibiting inflammation and smooth muscle cell (SMC) proliferation. We identified CX3CR1 as a targetable receptor for prevention of monocyte adhesion and inflammation and in-stent neointimal hyperplasia without interfering with stent re-endothelization. Efficacy of AZ12201182 (AZ1220), a CX3CR1 antagonist was evaluated in inhibition of monocyte attachment in vitro. A prototype AZ1220 eluting PLGA-based polymer coated stent developed with an optimal elution profile and dose of 1 µM/stent was tested over 4 weeks in a porcine model of coronary artery stenting. Polymer coated stents without AZ1220 and bare metal stents were used as controls. AZ1220 inhibited monocyte attachment to CX3CL1 in a dose dependent manner. AZ1220 eluted from polymer coated stents in an ex vivo flow system retained bioactivity in inhibiting monocyte attachment to CX3CL1. At 4 weeks following deployment, AZ1220 eluting stents significantly reduced (∼60%) in-stent stenosis compared to both bare metal and polymer only coated stents and markedly reduced peri-stent inflammation and monocyte/macrophage accumulation without affecting re-endothelization. Anti-CX3CR1 drug eluting stents potently inhibited in-stent stenosis and may offer an alternative to mTOR targeting by current DES, specifically inhibiting polymer-induced inflammatory response and SMC proliferation, while retaining an equivalent re-endothelization response to bare metal stents.

Intravascular cell delivery device for therapeutic VEGF-induced angiogenesis in chronic vascular occlusion.

Site specific targeting remains elusive for gene and stem cell therapies in the cardiovascular field. One promising option involves use of devices that deliver larger and more sustained cell/gene payloads to specific disease sites using the versatility of percutaneous vascular access technology. Smooth muscle cells (SMCs) engineered to deliver high local concentrations of an angiogenic molecule (VEGF) were placed in an intravascular cell delivery device (ICDD) in a porcine model of chronic total occlusion (CTO) involving ameroid placement on the proximal left circumflex (LCx) artery. Implanted SMC were retained within the ICDD and were competent for VEGF production in vitro and in vivo. Following implantation, micro-CT analyses revealed that ICDD-VEGF significantly enhanced vasa vasora microvessel density with a concomitant increase in tissue VEGF protein levels and formation of endothelial cell colonies suggesting increased angiogenic potential. ICDD-VEGF markedly enhanced regional blood flow determined by microsphere and contrast CT analysis translating to a functional improvement in regional wall motion and global left ventricular (LV) systolic and diastolic function. Our data indicate robust, clinically relevant angiogenesis can be achieved in a human scale porcine chronic vascular occlusion model following ICDD-VEGF-based delivery of angiogenic cells. This may have implications for percutaneous delivery of numerous therapeutic factors promoting creation of microvascular bypass networks in chronic vaso-occlusive diseases.

Role of CX3CR1 receptor in monocyte/macrophage driven neovascularization.

Monocyte/macrophages are implicated in initiation of angiogenesis, tissue/organ perfusion and atherosclerosis biology. We recently showed that chemokine receptor CX(3)CR1 is an essential regulator of monocyte/macrophage derived smooth muscle cell differentiation in the vessel wall after injury. Here we hypothesised the contribution of CX(3)CR1- CX(3)CL1 interaction to in vivo neovascularization and studied the functional consequences of genetic and pharmacologic targeting of CX(3)CR1 in formation, maturation and maintenance of microvascular integrity. Cells functionally deficient in CX(3)CR1 lacked matrix tunnelling and tubulation capacity in a 3D Matrigel assay. These morphogenic and cytokinetic responses were driven by CX(3)CL1-CX(3)CR1 interaction and totally abrogated by a Rho antagonist. To evaluate the role of CX(3)CR1 system in vivo, Matrigel plugs were implanted in competent CX(3)CR1(+/gfp) and functionally deficient CX(3)CR1(gfp/gfp) mice. Leaky microvessels (MV) were formed in the Matrigel implanted in CX(3)CR1(gfp/gfp) but not in CX(3)CR1(+/gfp) mice. In experimental plaque neovascularization immature MV phenotype was observed in CX(3)CR1(gfp/gfp) mice, lacking CX(3)CR1 positive smooth muscle-like cells, extracellular collagen and basement membrane (BM) laminin compared to competent CX(3)CR1(+/gfp) mice. This was associated with increased extravasation of platelets into the intima of CX(3)CR1(gfp/gfp) but not functionally competent CX(3)CR1 mice. Pharmacologic targeting using CX(3)CR1 receptor antagonist in wild type mice resulted in formation of plaque MV with poor BM coverage and a leaky phenotype. Our data indicate a hitherto unrecognised role for functional CX(3)CR1 in Matrigel and experimental plaque neovascularization in vivo, which may buttress MV collectively in favour of a more stable non-leaky phenotype.