Injection Of Endothelial Progenitor Cells Research Articles

HOXD9 regulated mitophagy to promote endothelial progenitor cells angiogenesis and deep vein thrombosis recanalization and resolution

BackgroundDeep vein thrombosis (DVT) is a common vascular surgical disease caused by the coagulation of blood in the deep veins, and predominantly occur in the lower limbs. Endothelial progenitor cells (EPCs) are multi-functional stem cells, which are precursors of vascular endothelial cells. EPCs have gradually evolved into a promising treatment strategy for promoting deep vein thrombus dissolution and recanalization through the stimulation of various physical and chemical factors.MethodsIn this study, we utilized a mouse DVT model and performed several experiments including qRT-PCR, Western blot, tube formation, wound healing, Transwell assay, immunofluorescence, flow cytometry analysis, and immunoprecipitation to investigate the role of HOXD9 in the function of EPCs cells. The therapeutic effect of EPCs overexpressing HOXD9 on the DVT model and its mechanism were also explored.ResultsOverexpression of HOXD9 significantly enhanced the angiogenesis and migration abilities of EPCs, while inhibiting cell apoptosis. Additionally, results indicated that HOXD9 specifically targeted the HRD1 promoter region and regulated the downstream PINK1-mediated mitophagy. Interestingly, intravenous injection of EPCs overexpressing HOXD9 into mice promoted thrombus dissolution and recanalization, significantly decreasing venous thrombosis.ConclusionsThe findings of this study reveal that HOXD9 plays a pivotal role in stimulating vascular formation in endothelial progenitor cells, indicating its potential as a therapeutic target for DVT management.Graphical

Endothelial progenitor cells overexpressing platelet derived growth factor-D facilitate deep vein thrombosis resolution.

To assess the therapeutic efficacy of PDGF-D-overexpressing endothelial progenitor cells (EPCs) in deep vein thrombosis. Inferior vena cava thrombosis was induced in female Sprague Dawley (SD) rats. Animals were injected via the distal vena cava with EPCs overexpressing PDGF-D after transfection with a lentiviral vector containing the PDGF-D gene. The effect on thrombosis in animals who received EPCs was evaluated using MSB staining, immunohistochemistry, immunofluorescence, and venography; the steady-state mRNA and protein levels of PDGF-D and its receptor (PDGF-Rβ) were determined by RT-PCR and Western blotting, respectively; and the PDGF-D-induced mobilization of circulating EPCs was estimated by flow cytology. Compared with controls, injection of EPCs overexpressing PDGF-D was associated with increased thrombosis resolution; recanalization; PDGF-D and PDGF-Rβ expression; induction of monocyte homing; and mobilization of EPCs to the venous circulation. In a rat model, transplantation of PDGF-D-overexpressing EPCs facilitated the resolution of deep vein thrombosis.

Endothelial progenitor cells modulate the phenotype of smooth muscle cells and increase their neointimal accumulation following vascular injury

Abstract Background Smooth muscle cells (SMCs) are the main driver of neointima formation and restenosis following vascular injury. In animal models, endothelial progenitor cells (EPCs) accelerate endothelial regeneration and reduce neointima formation after arterial injury, however EPC-capture stents do not to reduce target vessel failure compared to conventional stents. Here we examined the influence of EPCs on features of SMCs pivotal for their impact on neointima formation including proliferation, migration and phenotype switch. Methods and results EPCs, their conditioned medium and EPC-derived microparticles induced proliferation of SMCs while limiting their apoptosis. In transwell membrane experiments and scratch assays EPCs stimulated migration of SMCs and accelerated their recovery from scratch-induced injury. Treatment of SMCs with EPC-derived conditioned medium or microparticles triggered transformation of SMCs towards a synthetic phenotype. However, co-cultivation of EPCs and SMCs enabling direct cell-cell contacts preserved their original phenotype and protected from the transformative effect of SMC cholesterol-loading. Adhesion of EPCs to SMCs was stimulated by SMC injury and reduced by blocking CXCR2 and CCR5. Interaction of EPCs with SMCs modulated their secretome and synergistically increased the release of selected chemokines. Following carotid wire injury in athymic mice, injection of EPCs resulted not only in reduced neointima formation but also in altered cellular composition of the neointima with augmented accumulation of SMCs. Conclusion EPCs stimulate proliferation and migration of SMCs and increase their neointimal accumulation following vascular injury. Furthermore, EPCs modify the SMC phenotype with protection from the transformative effect of cholesterol when direct cell-cell contact is established. Funding Acknowledgement Type of funding sources: None.

Endothelial Progenitor Cells Modulate the Phenotype of Smooth Muscle Cells and Increase Their Neointimal Accumulation Following Vascular Injury

Smooth muscle cells (SMCs) are the main driver of neointima formation and restenosis following vascular injury. In animal models, endothelial progenitor cells (EPCs) accelerate endothelial regeneration and reduce neointima formation after arterial injury; however, EPC-capture stents do not reduce target vessel failure compared with conventional stents. Here we examined the influence of EPCs on features of SMCs pivotal for their impact on injury-induced neointima formation including proliferation, migration, and phenotype switch. EPCs, their conditioned medium, and EPC-derived microparticles induced proliferation of SMCs while limiting their apoptosis. In transwell membrane experiments and scratch assays, EPCs stimulated migration of SMCs and accelerated their recovery from scratch-induced injury. Treatment of SMCs with an EPC-derived conditioned medium or microparticles triggered transformation of SMCs toward a synthetic phenotype. However, co-cultivation of EPCs and SMCs enabling direct cell-cell contacts preserved their original phenotype and protected from the transformative effect of SMC cholesterol loading. Adhesion of EPCs to SMCs was stimulated by SMC injury and reduced by blocking CXCR2 and CCR5. Interaction of EPCs with SMCs modulated their secretory products and synergistically increased the release of selected chemokines. Following carotid wire injury in athymic mice, injection of EPCs resulted not only in reduced neointima formation but also in altered cellular composition of the neointima with augmented accumulation of SMCs. EPCs stimulate proliferation and migration of SMCs and increase their neointimal accumulation following vascular injury. Furthermore, EPCs context-dependently modify the SMC phenotype with protection from the transformative effect of cholesterol when a direct cell-cell contact is established.

Endothelial progenitors increase vascularization and improve fibroblasts function that prevent medication-related osteonecrosis of the jaw.

In a previous rat model, MRONJ occurrence was 50%. Our aim was to investigate the potential of endothelial progenitor cells (EPCs) to improve fibroblasts function and prevent MRONJ. Human gingival fibroblasts were cultured with EPC-conditioned media (EPC-CM); endothelial growth media (EGM-2) or DMEM followed by incubation with 10µM zoledronic (ZOL) and dexamethasone (DEX). Cell proliferation and migration were assessed by XTT and scratch wound healing assays. In vivo, ten Lewis rats were treated weekly with ZOL and DEX for 11weeks. After a week, EPCs or EGM-2 were injected to the gingiva around the molars. At 3weeks, bilateral molars were extracted. After 8weeks, wound healing was assessed, and serum VEGF and blood vessels were quantified. ZOL/DEX significantly reduced fibroblasts proliferation and wound healing. Treatment with EPC-CM before ZOL/DEX improved cell proliferation, and scratch healing (p=.007, p=.023).In vivo, local EPC injection before tooth extraction increased serum VEGF (p=.01) and soft tissue vascularization (p=.05). Normal healing was similar (80%) in EPCs and EGM-2 groups. EPC rescued fibroblasts from the cytotoxic effect of ZOL/DEX and elevated serum VEGF and vessel density that might reduce MRONJ occurrence to 20% compared to 50% in a similar model.

The effectiveness of various cytotherapeutic strategies for the treatment of ischemic stroke: a Bayesian network meta-analysis of randomized controlled trials.

Cytotherapy is a potential treatment for ischemic stroke (IS) patients but lacks uniform procedures. We aimed to assess the impact of the time of intervention, cell type, dose, and route of administration on the clinical effects by network meta-analysis. We searched public electronic databases through July 7, 2019. Bayesian network meta-analyses were performed to compare differences among different cytotherapeutic strategies. Cytotherapy can significantly improve patients' activity of daily living according to the modified Rankin Scale (standard mean difference (SMD) - 0.81; 95% confidence interval (CI) - 1.58, - 0.03; p = 0.0417) and Barthel Index (SMD 0.67; 95% CI 0.05, 1.30; p = 0.036) results as well as improve neurological recovery (SMD - 0.93; 95% CI - 1.29, - 0.57; p < 0.001). Network meta-analysis showed that the intra-arterial injection of large amounts of mononuclear cells (NCs) or aldehyde dehydrogenase (ALDH)-positive cells was beneficial for improving patients' activity of daily living, while CD34+ cells through intracerebral injection had an advantage in the recovery of injured nerve function. Intravenous injection of mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs) was beneficial in reducing mortality and serious adverse event (SAE) onset. In the subacute stage, the intra-arterial injection of NCs or ALDH cells improves patients' activity of daily living. Additionally, CD34+ cells through intracerebral injection had an advantage in the recovery of injured nerve function even in the chronic stage. Intravenous injection of MSCs or EPCs is a safety delivery route that can reduce mortality and SAE onset. However, further clinical studies are still needed to confirm these results.

A preventive injection of endothelial progenitor cells prolongs lifespan in stroke-prone spontaneously hypertensive rats

There is a pressing need for new approaches to prevent stroke. Endothelial progenitor cells (EPCs) promote vascular repair and revascularization in the ischemic brain. The present study sought to evaluate whether preventive delivery of EPCs could prevent or protect against stroke. Stroke-prone spontaneously hypertensive rats (SHR-SP) received a single injection of EPCs, and their survival time was monitored. In addition, at 28 and/or 42 days after a single injection of EPCs, SHR-SP and mice were subjected to cerebral ischemia, and cerebral ischemic injury, local angiogenesis and in vivo EPC integration were determined. Other experiments examined the effects of EPC conditioned medium, and the distribution of donor EPCs taken from GFP transgenic mice. It was found that EPC-pretreated SHR-SP showed longer lifespans than untreated controls. A single preventive injection of EPCs could produce persistent protective effects against cerebral ischemic injury (lasting at least 42 days), and promote local angiogenesis in the ischemic brain, in two types of animals (SHR-SP and normotensive mice). EPCs of donor origin could be detected in the recipient peripheral blood, and integrated into the recipient ischemic brains. Furthermore, it was suggested that mouse EPCs might exert paracrine effects on cerebral ischemic injury in addition to their direct angiogenic effects. In conclusion, a single preventive injection of EPCs prolonged the lifespan of SHR-SP, and protected against cerebral ischemic injury for at least 7 weeks. It is implied that EPC injection might be a promising candidate for a preventive role in patients at high risk for stroke.

Exogenous endothelial progenitor cells contribute to hepatocellular carcinoma vasculogenesis in nude mouse

Objective To investigate the homing specificity of bone marrow (BM)-derived endothelial progenitor cells (EPCs) of transgenic mice carrying enhanced green fluorescent protein (EGFP) using a murine intraperitoneal hepatocellular carcinoma (HCC) model and its potential clinical value. Methods BM mononuclear cells were isolated by ficoll density-gradient centrifugation and serial differential adhesion method was used to purify the EPCs carrying EGFP which had been identified by morphology, uptake of DiI-Ac-LDL, binding of UEA-1 lection, flow cytometry and in vitro tube formation. Ex vivo expanded mouse BM-derived EGFP-EPCs were randomly injected into tumor-bearing nude mouse and normal nude mouse via the tail vein. Two weeks after injection of EPCs, the livers and other organs in each group were examined for distribution of transplanted EPCs. Results Fluorescence image analysis revealed that systemically transplanted EPCs 'homed’ to HCC with significantly higher specificity, respectively, in the number of cells in the experimental group were 169.40±10.87 in the livers, 15.40±2.87 in the heart, 14.70±2.02 in the lung, 1.70±0.50 in the kidney, 28.30±4.20 in the spleen, 6.90±1.31 in the pancreas, and 4.60±1.22 in the intestine, and those in the control group were respectively 76.50±4.20, 16.70±1.32, 76.00±3.31, 24.70±1.70, 77.90±4.50, 18.60±2.00 and 12.70±1.70 correspondingly. The number of cells in the liver more than lung (P=0.001), heart (P=0.032), kidney (P=0.002), spleen(P=0.036), pancreas (P=0.001)and bowel(P=0.001), the number of lung and spleen cells was higher in heart(P=0.036), pancreas (P=0.010), kidney (P=0.030) and bowel(P=0.008). as compared to other organs within the experimental group and to anatomically matched liver sections from the control groups (P=0.001). The quantify of EGFP-EPCs in lungs and spleen were higher specificity as compared to other organs (no liver). Immunohischemical showed the transplanted cells differentiate into vascular cells and recruitmented tumor angiogenesis. Conclusion The results suggested that the autologous EPCs in vivo could be recruited specifically into HCC, and take part in the angiogenesis of HCC in the nude mice. Key words: Endothelial progenitor cell; Carcinoma, hepatocellular; Cell transplantation; Vasculogenesis; Angiogenesis

Fresh Noncultured Endothelial Progenitor Cells Improve Neonatal Lung Hyperoxia-Induced Alveolar Injury

Treatment of preterm human infants with high oxygen can result in disrupted lung alveolar and vascular development. Local or systemic administration of endothelial progenitor cells (EPCs) is reported to remedy such disruption in animal models. In this study, the effects of both fresh (enriched for KDR) and cultured bone marrow (BM)‐derived cell populations with EPC characteristics were examined following hyperoxia in neonatal mouse lungs. Intraperitoneal injection of fresh EPCs into five‐day‐old mice treated with 90% oxygen resulted in full recovery of hyperoxia‐induced alveolar disruption by 56 days of age. Partial recovery in septal number following hyperoxia was observed following injection of short‐term cultured EPCs, yet aberrant tissue growths appeared following injection of long‐term cultured cells. Fresh and long‐term cultured cells had no impact on blood vessel development. Short‐term cultured cells increased blood vessel number in normoxic and hyperoxic mice by 28 days but had no impact on day 56. Injection of fresh EPCs into normoxic mice significantly reduced alveolarization compared with phosphate buffered saline‐injected normoxic controls. These results indicate that fresh BM EPCs have a higher and safer corrective profile in a hyperoxia‐induced lung injury model compared with cultured BM EPCs but may be detrimental to the normoxic lung. The appearance of aberrant tissue growths and other side effects following injection of cultured EPCs warrants further investigation. Stem Cells Translational Medicine 2017;6:2094–2105

Thymosin β4 promotes the survival and angiogenesis of transplanted endothelial progenitor cells in the infarcted myocardium.

The survival of transplanted stem cells in ischemic tissue is poor. In the present study, the effects of thymosin β4 (Tβ4) on the survival and angiogenesis of endothelial progenitor cells (EPCs) and improvement in cardiac functions after transplantation of Tβ4-treated EPCs in the infarcted myocardium were investigated. EPCs were isolated from bone marrow of adult male rats and incubated in Endothelial Basal Medium-2. Then the cells were treated with Tβ4 at different concentrations (0.05, 0.1 and 0.2 µM), and cells incubated with DMEM were set as controls. MTT assay, Transwell assay and tube formation in Matrigel were used to detect cell viability, migration and angiogenesis, respectively. For examining the protective effect of Tβ4 on EPCs, the cells were also incubated in the condition of hypoxia and serum deprivation. p-Akt expression was also examined using western blot analysis. Rat models of myocardial infarction (MI) were established by ligation of the anterior descending branch of the left coronary artery. At four weeks after intramyocardial injection of Tβ4-treated EPCs, the changes in cardiac functions, size of the scar tissue and density of microvessels were examined by echocardiography, Masson's trichrome staining, immunohistochemistry and fluorescence in situ hybridization (FISH) for the Y-chromosome. Tβ4 enhanced EPC viability, protected the cells from apoptosis in hypoxia and serum deprivation, and promoted the proliferation and migration of the cells and formation of capillary-like structures in the cells. Moreover, Tβ4 increased p-Akt expression in the cells. The cytoprotective and proangiogenic effects of Tβ4 were in a dose-dependent manner. Tβ4-treated EPCs improved cardiac function, enhanced the repair of the infarcted myocardium, and promoted angiogenesis after transplantation in the infarcted myocardium. In conclusion, pretreatment of EPCs with Tβ4 is a novel strategy for the repair of ischemic tissue after transplantation in MI.

Abstract TP275: “Touch-and-Go”: Touching of Endothelial Cells by Endothelial Progenitor Cells Increases Activation of Pro-survival ERK1/2 Signaling

Background: Cell-based therapies can potentially promote neurological repair for CNS diseases including stroke. Pre-clinical data showed improved infarct volume and neurological scores following injection of Endothelial progenitor cells (EPCs). However relatively few EPCs were found in infarct areas, and mechanisms by which injected EPCs enhance neovascularization are largely unknown. In this study, we hypothesized that circulating EPCs would positively impact intracellular signaling cascades in rat brain endothelial cells (RBECs) even by short-duration contact due to activation of pro-survival ERK1/2 cascades. Methods: Primary RBECs and EPCs were isolated from rat brain and spleen, respectively. These cells were cultured separately, and 10 days later, cultured EPCs were transferred to plates of cultured RBECs. After 1 or 10 min incubation with cell-culture plate shaking, EPCs were washed from the plates and RBECs were subjected to western blot analysis to assess ERK1/2 phosphorylation. As a negative control for EPCs, we prepared neutrophils from different rats. Results: We confirmed that our RBECs and EPCs were viable in vitro by LDH assay and these cells were positive for their cell-type markers assessed by immunostaining. Ten min incubation of EPCs phosphorylated ERK1/2 in RBECs in an EPC-number-dependent manner, whereas identical conditions of neurtrophil incubation did not. Importantly, only 1 min incubation with EPCs significantly upregulated ERK cascades in RBECs. Remaining EPCs on RBEC surfaces may not contribute to ERK1/2 phosphorylation because very few EPCs were observed after washout. In addition, experiments by the same procedure without RBECs did not show ERK phosphorylation. Conclusion: We demonstrated increased activation of pro-survival ERK1/2 signaling in RBECs following short-duration incubation of EPCs. Results suggest that circulating EPCs may not need to be integrated into existing blood vessels to promote neovascularization. Rather, short-duration interactions between EPCs and RBECs may provide a “Touch-and-Go” stimulus that supports brain endothelial cells to make favorable environments for neovascularization.

Effect of endothelial progenitor cells derived from human umbilical cord blood on oxygen-induced retinopathy in mice by intravitreal transplantation.

To investigate the effect of endothelial progenitor cells (EPCs) labeled by carboxy fluorescein diacetate succinimidyl ester (CFSE) on murine oxygen-induced retinopathy (OIR) by intravitreal transplantation. After isolated from human umbilical cord blood mononuclear cells, EPCs were cultivated and then labeled with CFSE in vitro. C57BL/6J mice were placed to 75% hyperoxia chamber from P7 to P12 to establish OIR model. At P12, OIR mice were intravitreally injected with 1 µL suspension contained 2×105 EPCs (EPCs group) or isometric phosphate buffered saline (PBS group). The contralateral eye of each mice received no injection (OIR group). Evans blue angiography and frozen section were examined to track the labeled cells in OIR group at P15 and P19. Using retina paraffin sections and adenosinediphos phatase staining at P12 and P19, the effect of EPCs on OIR mice was evaluated quantitatively and qualitatively. The retinas from EPCs group with less non-perfusion area and fewer peripheral tufts were observed at P19, comparing with that from PBS or OIR group. The retinopathy in EPCs group receded earlier with less non-ganglion cells and neovascular nuclei, together with relatively regular distribution. The counts of the neovascular nuclei at P19 were reduced by 44% or 45%, compared with those of OIR group or PBS group respectively. Three days after EPCs injection, a large number of EPCs appeared in the vitreous cavity and adhered to the retinal surface. While at one week, the cells gathered between the internal plexiform layer and the inner limiting membrane, and some EPCs appeared in retinal vessels. EPCs transplantation can participate in the reparative procedure of the neovascularization in OIR.

Injection of hTERT-Transduced Endothelial Progenitor Cells Promotes Beneficial Aortic Changes in a High-Fat Dietary Model of Early Atherosclerosis

Objectives: Cultured endothelial progenitor cells (EPCs) display troubling issues that adversely affect their applicability to endothelial regeneration. We hypothesized that transduction of the human telomerase catalytic subunit (hTERT) gene would enhance EPC function in treating dietary-induced early atherosclerosis (AS). Methods: A dietary-induced early AS model was successfully constructed in 90 healthy male rats, while 30 healthy control (HC) rats were normally fed. Four experimental groups were constructed: an untreated HC group; an untreated AS group injected with PBS; a null EPC AS group injected with null vector-transduced EPCs, and an hTERT EPC AS group injected with hTERT-transduced EPCs. Two months postinjection, abdominal aortas were extracted to validate EPC integration and comparatively assess mRNA and protein expression of the early atherosclerotic markers VCAM-1, ICAM-1, LFA-1, Mac-1, CD44, MCP-1, endothelial nitric oxide synthase (eNOS), and apolipoprotein E. Results: In vitro, hTERT transduction of EPCs resulted in a significantly superior proliferative capacity as well as significantly higher NO, iNOS, and LDH secretory capacity. In vivo injection of hTERT-transduced EPCs produced significant reductions in CD44 and MCP-1 expression as well as a significant increase in eNOS expression relative to injection with null vector-transduced EPCs (all p < 0.05). Conclusion: hTERT-transduced human EPCs may be useful in treating dietary-induced early AS.

Optimizing material and flow properties of guest-host hydrogels for injectable cell delivery

Event Abstract Back to Event Optimizing material and flow properties of guest-host hydrogels for injectable cell delivery Minna H. Chen1, Christopher B. Highley1, Christopher B. Rodell1*, Ann Gaffey2*, Chantel Venkataraman2*, Pavan Atluri2 and Jason A. Burdick1* 1 University of Pennsylvania, Department of Bioengineering, United States 2 University of Pennsylvania, Division of Cardiovascular Surgery, Department of Surgery, United States Introduction: Cell delivery by direct injection is plagued by low (<10%) retention of cells at the injection site. The use of biomaterial carriers can improve cell retention, and shear-thinning hydrogels are particularly advantageous as cells can be encapsulated within hydrogels and then delivered via syringes and catheters in a minimally invasive approach. Toward this, we recently developed a class of shear-thinning hyaluronic acid (HA) hydrogels assembled via guest (adamantane, Ad) and host (cyclodextrin, CD) modifications[1]. However, it is important to understand how material formulations and injection parameters influence potentially damaging shear forces on cells. Materials and Methods: HA was modified with either Ad or CD and combined to form shear-thinning, injectable hydrogels upon mixing. Using rheology, mechanical properties of gel formulations at differing concentrations (2.5-7.5 wt%) and modifications (20-50%) were measured. Within these gels, 5µm polystyrene beads were embedded and injection parameters (i.e., flow rate, needle gauge) investigated; bead velocity during injection was determined (Trackpy). 3T3 fibroblast cells were similarly encapsulated and injected and live/dead staining was used to quantify cell viability and distribution. Finally, endothelial progenitor cells (EPCs) were encapsulated in one formulation and injected into the border zone region of an acute myocardial infarction (AMI) in a rat[2]. After 4 weeks, ventricular remodeling and functional outcomes were evaluated using immunostaining, histology, and echocardiography. Results and Discussion: Upon mixing, Ad-HA and CD-HA formed injectable, shear-thinning hydrogels that were used to encapsulate cells (Fig. 1A). Variations in gel formation and injection parameters, such as flow rate, resulted in parabolic flow velocity profiles at low flow rates (0.1 mL/h, Fig. 1B) and more plug-like flow profiles at high flow rates (5 mL/h, Fig. 1C). Live/dead staining of 3T3 cells after injection (Fig. 1D, 1E) showed higher viability in middle regions of the extruded gel for low flow rates. A plug-like flow profile showed higher overall viability compared to a parabolic flow profile, demonstrating an ability to enhance cell viability necessary for in vivo efficacy. Injection of EPCs encapsulated in gel in a rat model of AMI resulted in enhanced cell engraftment and vasculogenesis compared to injection of EPCs alone (15.3 ± 5.8 vessels per high power field compared to 5.0 ± 2.1 vessels per high power field, p < 0.001). Decreased myocardial scar fraction (9.9 ± 5.6 versus 27.0 ± 7.6, p < 0.0001) and increased ejection fraction (68.9 ± 12.0 versus 57.3 ± 10.7, p < 0.03) were also observed after injection of cells in gel compared to cells alone. Conclusion: We developed an injectable, shear-thinning hydrogel system for cell delivery that could be tuned to enhance cell viability after injection. In vitro assessments of cell viability show that material and flow properties affect cell viability. Delivery of cells using one formulation showed positive results for recovery after AMI in a rat model, and due to the tunable nature of the gels, this system is broadly applicable in the field of injectable cellular therapeutics. This work was funded in part by NIH T32-AR007132 (to MHC), R01 HL111090 (to JAB), TSFRE Nina Starr Braunwald Research Fellowship (to ACG), AHA predoctoral fellowship (to CBR), and AATS David C. Sabiston Research Scholarship and AHA Scientist Development Grant 13SDG17230005 (to PA).

Abstract 18871: Coagulation Factor Xa Promotes Endothelial Cell Senescence Via P53 Up-regulation

Factor Xa (FXa) occupies a central place in coagulation cascade as a convergence point between intrinsic and extrinsic pathways. Apart from this activity, the non-hemostatic function of FXa through protease activated receptors (PARs) triggers an intracellular activation that can initiate signaling cascades regulating migration and survival of cells. However, the direct effect of FXa on angiogenesis in endothelial cells (ECs) and endothelial progenitor cells (EPCs) is largely unknown. Here, we demonstrated that FXa treatment on both ECs and EPCs significantly increases the expression of p53 protein through PAR2 (p&lt;0.05). Activation of p53 proteins resulted in impairment of migration and tube formation of these cells. Moreover, chronic FXa stimulation on ECs and EPCs induced cell senescence assessed by Senescence-associated beta-galactosidase (SA-β-gal) staining (p&lt;0.05). These unfavorable effect of FXa on ECs and EPCs were diminished by co-treatment with FXa inhibitor, rivaroxaban. To test whether these in vitro observation has an in vivo counterparts, chronically treated EPCs with FXa were intravenously injected immediately after femoral artery ligation in mouse ischemic hind limb model. Laser doppler image (LDI) analysis showed that EPCs enhanced the recovery of ischemic limb perfusion and this beneficial effect of EPC was significantly blunted by FXa. Additionally, the increase in capillary density by EPCs injection was inhibited by the treatment with FXa. However, treatment with rivaroxaban significantly increased ischemic limb perfusion and capillary density compared to FXa treatment group (p&lt;0.05). In summary, these observation indicates anti-angiogenic property of FXa via p53 upregulation. FXa inhibitor, rivaroxaban, might work as anti-senescence and maintain angiogenic ability of ECs and EPCs.

Restoration of erectile function with intracavernous injections of endothelial progenitor cells after bilateral cavernous nerve injury inrats.

Endothelial progenitor cells (EPCs) are bone marrow-derived endothelial cells capable of circulating, proliferating, and differentiating into mature endothelial cells. Circulating EPCs can be directly recruited to some extent at sites of injury, and their administration could accelerate repair or endothelialization of the damaged tissue. We investigated the effects of intracavernous injections of EPCs into the corpora cavernosa of rats with erectile dysfunction (ED) caused by bilateral cavernous nerve (CN) injury. Overall, 24 male Sprague-Dawley rats were randomized into three groups: sham surgery, vehicle-only, or EPC treatment. Rats in the EPC treatment and vehicle-only groups were subjected to bilateral CN injury before injection of EPCs or vehicle, respectively, into the corpora cavernosa. Four weeks after surgery, erectile function was assessed by measuring maximum intracavernosal pressure (ICP), change in ICP, area under the ICP curve, and ratio of change in ICP and mean arterial pressure (MAP; ΔICP/MAP). Penile tissue was histomorphometrically analyzed for the expression of neural nitric oxide synthase (nNOS), neurofilament-1 (NF-1), von Willebrand factor (vWF), endothelial NOS (eNOS), and smooth muscle cell content. Maximum ICP and all other functional parameters of erectile function were significantly reduced in the vehicle-only group vs. the sham and EPC treatment groups (all p<0.001). Smooth muscle cell content was decreased in the vehicle-only vs. the sham and EPC treatment groups (both p<0.01). Expressions of vWF and eNOS in the dorsal artery were significantly higher in the EPC treatment than the vehicle-only group (p<0.05). In conclusion, EPC treatment restored erectile function in a rat model of bilateral CN injury through recruitment of EPCs toward the dorsal artery and preservation of smooth muscle cells in the corpus cavernosum. These findings elucidate the therapeutic potential of EPCs for treating ED in humans.

AB035. Restoration of erectile function with intracavernous injection of endothelial or smooth muscle progenitor cells after bilateral cavernous nerve injury in rats.

Injury to the cavernous nerve (CN) is a significant concern for patients with prostate cancer after radical prostatectomy because erectile dysfunction is secondary to denervation of the penis. Loss of innervation causes severe and irreversible morphologic changes in the penis, including induction of endothelial and smooth muscle cells apoptosis, collagen induction or fibrosis in the corpus cavernosum. Currently, cell-based therapy offers a promising strategy for neuroprotection or tissue-protection after bilateral CN injury. Bone marrow-derived progenitor cells (BMPCs) such as endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SPCs) can be derived from peripheral blood and be differentiated more easily into vascular cells during arterial remodeling than stem cells. In addition, administration of EPCs can increase vascular repair in animal models and has demonstrated some therapeutic benefits for cardiovascular conditions in clinical trials. A tight cooperation between endothelial cells and smooth muscle cells is important to regulate vessel maturation and stability. Therefore, we investigate the therapeutic effects of intracavernous injections of EPCs and SPCs into the corpora cavernosa of rats with erectile dysfunction (ED) caused by bilateral CN injury. The results show maximum ICP and all other functional parameters of erectile function are significantly reduced in the vehicle-only group versus the sham, EPC treatment and SPC treatment groups (all P<0.001). Smooth muscle cell content decreases in the vehicle-only versus the sham and EPCs treatment and SPCs treatment groups (both p<0.01). Expressions of von Willebrand factor (vWF) and endothelial nitric oxide synthase (eNOS) in the dorsal artery are significantly higher in the EPC treatment than the vehicle-only group (P<0.05). In conclusion, EPCs and SPCs treatment restores erectile function in a rat model of bilateral CN injury through recruitment of EPCs toward the dorsal artery and preservation of smooth muscle cells in the corpus cavernosum, respectively. These findings elucidate the therapeutic potential of EPCs and SPCs for treating ED in humans.

Autophagy protein 5 enhances the function of rat EPCs and promotes EPCs homing and thrombus recanalization via activating AKT

Deep venous thrombosis (DVT) is one of the most common peripheral vascular diseases. The roles of bone marrow-derived endothelial progenitor cells (EPCs) on the recanalization of venous thrombosis has been suggested recently, while the underlying mechanisms are not completely understood. Our objective was to investigate the functions of autophagy protein 5 (ATG5) in rat EPCs and its potential application in DVT. We have found that silencing of ATG5 or pharmacological suppression of ATG5 in rat EPCs reduces both the migration and psudotube formation under hypoxia in vitro. In line, overexpression of ATG5 significantly enhances the EPCs migration and psudotube formation capabilities. More importantly, injection of EPCs that stably express ATG5 increases EPC homing to the ischemic site and promotes thrombus recanalization in a rat DVT model in vivo. Mechanistically, we have shown that ATG5 overexpression enhances psudotube formation via the activation of AKT. These findings suggest that ATG5-AKT signaling plays an essential role in EPC migration and psudotube formation. Regulation of ATG5-AKT signaling may provide a potential novel therapy for DVT.

Safety assessment of bone marrow derived MSC grown in platelet-rich plasma.

The injection of endothelial progenitor cells and mononuclear cells derived from bone marrow at the ischemic region of peripheral artery disease patients is reported to be effective for therapeutic angiogenesis; however, these cell therapies require large amounts of bone marrow to obtain sufficient numbers of cells. To solve this problem, we attempted to culture bone-marrow-derived mesenchymal stem cells (BM-MSC), which are supposed to secrete several cytokines that promote angiogenesis. We also focused on using platelet-rich plasma (PRP) as a supplement for cell culture instead of fetal bovine serum. Human BM-MSC obtained from healthy volunteers expanded rapidly when cultured with 10% PRP prepared from their own blood. FACS analysis revealed that these cultured human MSC were homogeneous populations, and chromosomal analysis showed a normal karyotype. Moreover, the angiogenetic effect was apparent two weeks after human BM-MSC were injected into the ischemic muscle in SCID mice. Tumor formation was not detected three months after injection into SCID mice either subcutaneously or intramuscularly. To simulate clinical settings, canine BM-MSC were grown with canine PRP and injected into their ischemic muscles. We confirmed that donor cells existed in situ two and six weeks after operation without any side effects. These results suggest that cultured human BM-MSC can be a promising cell source for therapeutic angiogenesis.

Tripterine Treatment Improves Endothelial Progenitor Cell Function via Integrin-Linked Kinase

Background/Aims: Atherosclerosis is associated with dysfunction of endothelial progenitor cells (EPCs). Tripterine, a chemical compound derived from the Chinese medicinal plant Tripterygium wilfordii Hook, displays anti-inflammatory properties in several animal models. We hypothesized that tripterine can improve EPC function and thus the efficiency of EPC transplantation. Methods and Results: Tripterine preconditioning (2.5 μM, 4 h) improved EPC proliferation, tube formation, migration, and adhesion, and reduced apoptosis in cells cultured in ox-LDL (200 µg/ml). Tripterine restored integrin-linked kinase (ILK) levels downregulated by ox-LDL in EPCs, suggesting the involvement of the ILK/Akt pathway. Small interfering RNA-mediated depletion of ILK and dominant-negative ILK transduction inhibited the phosphorylation of the ILK downstream signaling targets protein kinase B/Akt and glycogen synthase kinase 3-beta (GSK-3β), and reduced β-catenin and cyclin D1 expression. In atherosclerotic mice injected with green fluorescent protein-labeled EPCs to evaluate EPC function, tripterine decreased aortic lesions and plaque deposition, and injection of tripterine-treated EPCs restored ILK levels. Conclusion: The present results suggest that tripterine improves vascular function in atherosclerosis by enhancing EPC function through a mechanism involving the ILK signaling pathway.