Vascular Endothelial Progenitor Cells Research Articles

101 - Clinical-Risk Factors that Influence an Optimal Hypomethylating Agents Treatment in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

The purpose of this study was to explore the therapeutic efficacy of VEGF165-overexpressing vascular endothelial progenitor cells (EPCs) in post-thrombotic syndrome.A thrombus model was developed to mimic the in-vivo setting, and adenovirus transfection was used to overexpress VEGF165 in EPCs. These cells were transplanted into the animal model, and their ability to relieve endothelial injury was evaluated using haematoxylin and eosin staining, immunohistochemistry and scanning electron microscopy.Ferric chloride was used to build rat models of the inferior vena cava thrombosis, and HEK 293A cells were used to amplify adenovirus that overexpresses VEGF165. EPCs were infected with adenovirus, and this was confirmed by fluorescence microscopy. Transplantation of VEGH165-overexpressing EPCs into injured endothelial sites led to faster repair of the post-thrombotic tunica intima than wild-type EPCs.Transplantation of VEGF165-overexpressing EPCs was found to promote repair of the tunica intima, thus improving rehabilitation after surgery.

C-Kit-Positive Adipose Tissue-Derived Mesenchymal Stem Cells Promote the Growth and Angiogenesis of Breast Cancer

Background Adipose tissue-derived mesenchymal stem cells (ASCs) improve the regenerative ability and retention of fat grafts for breast reconstruction in cancer patients following mastectomy. However, ASCs have also been shown to promote breast cancer cell growth and metastasis. For the safety of ASC application, we aimed to identify specific markers for the subpopulation of ASCs that enhance the growth of breast cancer. Methods ASCs and bone marrow-derived vascular endothelial progenitor cells (EPCs) were isolated from Balb/c mice. c-Kit-positive (c-Kit+) or c-Kit-negative (c-Kit−) ASCs were cocultured with 4T1 breast cancer cells. Orthotropic murine models of 4T1, EPCs + 4T1, and c-Kit+/-ASCs + 4T1/EPCs were established in Balb/c mice. Results In coculture, c-Kit+ ASCs enhanced the viability and proliferation of 4T1 cells and stimulated c-Kit expression and interleukin-3 (IL-3) release. In mouse models, c-Kit+ASCs + 4T1/EPCs coinjection increased the tumor volume and vessel formation. Moreover, IL-3, stromal cell-derived factor-1, and vascular endothelial growth factor A in the c-Kit+ASCs + 4T1/EPCs coinjection group were higher than those in the 4T1, EPCs + 4T1, and c-Kit−ASCs + 4T1/EPCs groups. Conclusions c-Kit+ ASCs may promote breast cancer growth and angiogenesis by a synergistic effect of c-Kit and IL-3. Our findings suggest that c-Kit+ subpopulations of ASCs should be eliminated in fat grafts for breast reconstruction of cancer patients following mastectomy.

Prominin-1/CD133 expression as potential tissue-resident vascular endothelial progenitor cells in the pulmonary circulation.

Pulmonary vascular endothelial cells could contribute to maintain homeostasis in adult lung vasculature. "Tissue-resident" endothelial progenitor cells (EPCs) play pivotal roles in postnatal vasculogenesis, vascular repair, and tissue regeneration; however, their local pulmonary counterparts remain to be defined. To determine whether prominin-1/CD133 expression can be a marker of tissue-resident vascular EPCs in the pulmonary circulation, we examined the origin and characteristics of prominin-1/CD133-positive (Prom1(+)) PVECs considering cell cycle status, viability, histological distribution, and association with pulmonary vascular remodeling. Prom1(+) PVECs exhibited high steady-state transit through the cell cycle compared with Prom1(-) PVECs and exhibited homeostatic cell division as assessed using the label dilution method and mice expressing green fluorescent protein. In addition, Prom1(+) PVECs showed more marked expression of putative EPC markers and drug resistance genes as well as highly increased activation of aldehyde dehydrogenase compared with Prom1(-) PVECs. Bone marrow reconstitution demonstrated that tissue-resident cells were the source of >98% of Prom1(+) PVECs. Immunofluorescence analyses revealed that Prom1(+) PVECs preferentially resided in the arterial vasculature, including the resistant vessels of the lung. The number of Prom1(+) PVECs was higher in developing postnatal lungs. Sorted Prom1(+) PVECs gave rise to colonies and formed fine vascular networks compared with Prom1(-) PVECs. Moreover, Prom1(+) PVECs increased in the monocrotaline and the Su-5416 + hypoxia experimental models of pulmonary vascular remodeling. Our findings indicated that Prom1(+) PVECs exhibited the phenotype of tissue-resident EPCs. The unique biological characteristics of Prom1(+) PVECs predominantly contribute to neovasculogenesis and maintenance of homeostasis in pulmonary vascular tissues.

A flow cytometric approach to analyzing mature and progenitor endothelial cells following traumatic brain injury

BackgroundTraumatic brain injury (TBI) continues to be a major source of death and disability worldwide, and one of the earliest and most profound deficits comes from vascular damage and breakdown of the blood–brain barrier (BBB). Cerebral vascular endothelial cells (cvECs) and endothelial progenitor cells (EPCs) have been shown to play essential roles in vessel repair and BBB stability, although their individual contributions remain poorly defined. New methodWe employ TruCount beads with flow cytometry to precisely quantify cvECs, EPCs, and peripheral leukocytes in the murine cortex after controlled cortical impact (CCI) injury. ResultsWe found a significant reduction in the number of cvECs at 3 days post-injury (dpi), whereas the EPCs and invading peripheral leukocytes were significantly increased compared with sham controls. Proliferation studies demonstrate that both cvECs and EPCs are undergoing cell expansion in the first week post-injury. Furthermore, analysis of protein expression using mean fluorescence intensity found increases in PECAM-1, VEGFR-2, and VE-Cadherin expression per cell at 3 dpi, which is consistent with western blot analysis. Comparison with existing methodsClassic methods of cell analysis, such as histological cell counts, in the traumatic injured brain are labor intensive, time-consuming, and potentially biased; whereas flow cytometry provides an efficient, non-biased approach to simultaneously quantify multiple cell types. However, conventional flow cytometry that employs capped events can provide misleading results in CNS injured tissues. ConclusionsWe demonstrate that TruCount quantification using flow cytometry is a powerful tool for quantifying mature and progenitor endothelial cell changes after TBI.

Dysregulation of Vascular Endothelial Progenitor Cells Lung-Homing in Subjects with COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by fixed airflow limitation and progressive decline of lung function and punctuated by occasional exacerbations. The disease pathogenesis may involve activation of the bone marrow stimulating mobilization and lung-homing of progenitor cells. We investigated the hypothesis that lower circulating numbers of vascular endothelial progenitor cells (VEPCs) are a consequence of increased lung-sequestration in COPD. Nonatopic, current or ex-smokers with diagnosed COPD and nonatopic, nonsmoking normal controls were enrolled. Blood and induced sputum extracted primitive hemopoietic progenitors (HPCs) and VEPC were enumerated by flow cytometry. Migration and adhesive responses to fibronectin were assessed. In sputum, VEPC numbers were significantly greater in COPD compared to normal controls. In blood, VEPCs were significantly lower in COPD versus normal controls. There were no differences in HPC levels between the two groups in either compartment. Functionally, there was a greater migrational responsiveness of progenitors from COPD subjects to stromal cell-derived factor-1alpha (SDF-1α) compared to normal controls. This was associated with greater numbers of CXCR4+ progenitors in sputum from COPD. Increased migrational responsiveness of progenitor cells may promote lung-homing of VEPC in COPD which may disrupt maintenance and repair of the airways and contribute to COPD disease pathogenesis.

Transplantation of VEGFl65-overexpressing vascular endothelial progenitor cells relieves endothelial injury after deep vein thrombectomy

AimThe purpose of this study was to explore the therapeutic efficacy of VEGF165-overexpressing vascular endothelial progenitor cells (EPCs) in post-thrombotic syndrome. Materials and methodsA thrombus model was developed to mimic the in-vivo setting, and adenovirus transfection was used to overexpress VEGF165 in EPCs. These cells were transplanted into the animal model, and their ability to relieve endothelial injury was evaluated using haematoxylin and eosin staining, immunohistochemistry and scanning electron microscopy. ResultsFerric chloride was used to build rat models of the inferior vena cava thrombosis, and HEK 293A cells were used to amplify adenovirus that overexpresses VEGF165. EPCs were infected with adenovirus, and this was confirmed by fluorescence microscopy. Transplantation of VEGH165-overexpressing EPCs into injured endothelial sites led to faster repair of the post-thrombotic tunica intima than wild-type EPCs. ConclusionTransplantation of VEGF165-overexpressing EPCs was found to promote repair of the tunica intima, thus improving rehabilitation after surgery.

Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis.

Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo.

Effect of aliskiren on circulating endothelial progenitor cells and vascular function in patients with type 2 diabetes and essential hypertension.

The aim of this study was to investigate the effects of aliskiren on vascular function and endothelial progenitor cells (EPCs) in patients with type 2 diabetes and essential hypertension. The study enrolled type 2 diabetic patients aged >50 years under stable glycemic control and first diagnosed mild essential hypertension. In phase A (n = 20), patients received aliskiren 150-300 mg daily for 3 months. In phase B (n = 12), hydrochlorothiazide (HCTZ) 12.5-25mg daily substituted for aliskiren for 3 more months. At baseline and at the end of each phase, we assessed (i) brachial blood pressure (BBP); (ii) central aortic systolic pressure (CSP), aortic augmentation index (Aix), and pulse wave velocity (PWV) as markers of arterial stiffness; (iii) brachial artery flow-mediated dilatation (FMD) as a marker of endothelial function; (iv) left ventricular (LV) twisting and untwisting as markers of LV function and (v) EPC numbers in culture of peripheral blood mononuclear cells. Aliskiren similarly reduced BBP and CSP, increased FMD (P < 0.001) and EPC numbers (P < 0.001), decreased PWV and Aix (P < 0.05), and improved LV twisting and untwisting (P < 0.05). Although substitution of HCTZ sustained BBP at similar levels, CSP and echocardiographic indices nearly returned at baseline levels, and the improvement of FMD, PWV, Aix, and EPC numbers was abolished. Aliskiren had a favorable effect on endothelial function and EPCs, reduced arterial stiffness, and improved LV twisting and untwisting. These effects were independent of BBP lowering, as they were not observed after the achievement of similar values of BBP with HCTZ.

Treatment options for low high-density lipoproteins

To examine the current and future therapeutic option of HDL-based therapies. The inverse association between plasma level of high-density lipoprotein cholesterol (HDL-C) is strong and coherent across the population studied. In-vitro and in-vivo studies show the strong biological plausibility for HDL as a therapeutic target. Mendelian randomization does not support HDL-C as a causal (protective) cardiovascular risk factor, and clinical data does not support the concept that raising HDL-cholesterol mass alters the outcomes. Better biomarkers of HDL function are being examined in the clinical trials. These include cellular cholesterol efflux, antioxidant and anti-inflammatory effects, effects on vascular endothelial cells (inflammation and nitric oxide release) and endothelial progenitor cells. Novel therapeutic agents that alter HDL function are in advanced phase 3 trials and in early preclinical trials. These include inhibitors of cholesteryl ester transfer protein, reconstituted proteoliposomes, apolipoprotein A-I and HDL mimetic peptides and small molecules that increase apo A-I production rate. Targeting HDL-C has, to date, not led to changes in the cardiovascular outcomes. Novel therapeutic advances target the HDL function. In keeping with the recent 2013 American College of Cardiology/American Heart Association Guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults, the major focus of prevention lies with LDL-cholesterol reduction.

New advances in the mesenchymal stem cells therapy against skin flaps necrosis.

Mesenchymal stem cells (MSCs), multipotential cells that reside within the bone marrow, can be induced to differentiate into various cells, such as osteoblasts, adipocytes, chondrocytes, vascular endothelial progenitor cells, and other cell types. MSCs are being widely studied as potential cell therapy agents due to their angiogenic properties, which have been well established by in vitro and in vivo researches. Within this context, MSCs therapy appears to hold substantial promise, particularly in the treatment of conditions involving skin grafts, pedicle flaps, as well as free flaps described in literatures. The purpose of this review is to report the new advances and mechanisms underlying MSCs therapy against skin flaps necrosis.

Identification of endothelial progenitor cells in the corpus cavernosum in rats.

The vascular wall resident progenitor cells seem to serve as a local reservoir of cells for vascular repair. It was hypothesized that the corpus cavernosum may contain vascular wall endothelial progenitor cells (EPCs). In this study, we investigated the identification and localization of EPCs in the corpus cavernosum in a rat model. Adult male Sprague-Dawley rats were used to isolate EPCs from corpora cavernosum. To verify the existence and localization of EPCs, EPC-specific markers (CD34, Flk-1, and VE-cadherin) were evaluated by flow cytometric analysis and confocal microscopy. The EPC markers were mainly expressed in the cavernosal sinusoidal endothelial space. EPC-marker-positive cells made up about 3.31% of the corpus cavernosum of normal rat by FACS analysis. As shown by confocal microscopy, CD34+/Flk-1+ and CD34+/VE-cadherin+ positive cells existed in the corpus cavernosum. Our findings imply that regulation of corpus cavernosal EPCs may be a new therapeutic strategy in the treatment of erectile dysfunction.

Induction of pulmonary hypertensive changes by extracellular vesicles from monocrotaline-treated mice

Circulating endothelium-derived extracellular vesicles (EV) levels are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. Previously, we found that lung-derived EVs (LEVs) induce bone marrow-derived progenitor cells to express lung-specific mRNA and protein. In this study, we sought to determine whether LEV or plasma-derived EV (PEV) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodelling. LEV, PEV isolated from monocrotaline (MCT-EV)- or vehicle-treated mice (vehicle-EV) were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodelling were assessed by RV-to-body weight (RV/BW) and blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT- vs. vehicle-injected mice (1.99 ± 0.09 vs. 1.04 ± 0.09 mg/g; 0.159 ± 0.002 vs. 0.062 ± 0.009%). RV/BW, WT/D ratios were higher in mice injected with MCT-EV vs. mice injected with vehicle-EV (1.63 ± 0.09 vs. 1.08 ± 0.09 mg/g; 0.113 ± 0.02 vs. 0.056 ± 0.01%). Lineage-depleted bone marrow cells incubated with MCT-EV and marrow cells isolated from mice infused with MCT-EV had greater expression of endothelial progenitor cell mRNAs and mRNAs abnormally expressed in PAH than cells incubated with vehicle-EV or isolated from vehicle-EV infused mice. MCT-EV induced an apoptosis-resistant phenotype in murine pulmonary endothelial cells and lineage-depleted bone marrow cells incubated with MCT-EV induced pulmonary hypertension when injected into healthy mice. EV from MCT-injured mice contribute to the development of MCT-induced pulmonary hypertension. This effect may be mediated directly by EV on the pulmonary vasculature or by differentiation of bone marrow cells to endothelial progenitor cells that induce pulmonary vascular remodelling.

Role of vascular endothelial progenitor cells in construction of new vascular loop

Since bone marrow-derived endothelial progenitor cells (EPCs) have been detected in adult peripheral blood, the mode of vasculogenesis in the adult tissue has caught attention in field of vascularization research. To confirm the role of EPCs in construction of new vascular loop, we took the biomaterial scaffold implanted into adult rat as an experimental model to observe and examine the actions of the EPCs in neovascularization of the material by immunohistochemistry and transmission electron microscopy. Additionally, by establishing a chemotactic migration model for vascular endothelial cells (ECs) and EPCs, the migrations of ECs and EPCs were explored in simulations. The results of 20,000 simulations showed that the number of the vascular loops assisted by the EPCs was 2–5 times that of the vascular sprouts being naturally joined. Based on the results of experiments and simulations, we conclude that the EPCs are able to assist the angiogenic sprouts in joining under the condition of plenty of the EPCs being mobilized, which aggregate at sites close to sprout tips, forming a cell cord and differentiating to ECs in situ, and become vessel segments between neighboring sprouts. This suggests that there is a difference between the adult and embryo in the manner of vasculogenesis and that a small number of EPCs can play an important role to make the new blood vessels achieve rapid functionalization.

Abstract 230: Effect of aerobic exercise on Endothelial Progenitor Cells (EPCs) in patients with prediabetes

Introduction At least 79 million Americans have pre-diabetes. Though life-style modification prevents progression from Pre-diabetes to overt diabetes, the effect on vascular endothelial progenitor cells (EPC) stem cells is unknown. Methods This is a crossover study of 16-week duration, using exercise-naive pre-diabetes patients, n=12, aged 45-65 yrs with a BMI of 25-34.9. We studied flow mediated dilatation (FMD), EPC function and gene expression and serum endothelial inflammatory levels after 6 weeks each of aerobic exercise (150min/week) and non exercise phase, in a cross over design with 4 week wash-out period between the 2 phases. EPC migration was noted in response to chemotactic factors such as Vascular Endothelial Growth Factor (VEGF-A, 0- 50ng/ml) and Stromal Cell-Derived Factor-1(SDF1α, 0-100 ng/ml). Exercise compliance was monitored by regular phone calls and downloadable accelerometers. Results FMD studies (undertaken by 3 observers) showed mean FMD in non-exercise group of 5.7+0.6% which improved to 11.2+0.9% post-exercise. No significant weight loss noted between the 2 groups, however, significant reduction in leptin, IL-6, TNFα, hs-CRP, Triglyceride, and ApoB levels was noted. EPC gene expression analysis showed decrease of eNOS (5 fold) and increase of PECAM-1 (4-fold), Endothelin-1 (3-fold), IL-6 (2-fold), TNFα (3-fold) in non-exercise which improved following exercise. EPC migration improved post exercise, particularly in response to SDF-1α as did CD34+ cell number and EPC colony formation improved significantly also. Conclusion We demonstrate that pre-diabetic state is associated with poor vascular reactivity and impaired EPC stem cell function. Pre-diabetes may be a clinical window of therapeutic intervention opportunity when short course of aerobic exercise may allow improvement of vascular reactivity and EPC function despite no statistically significant weight loss. This study also indicates EPCs may act as an important biomarker for vascular function.

Arterial and Venous Progenitors of the Major Axial Vessels Originate at Distinct Locations

Currently, it remains controversial how vascular endothelial progenitor cells (angioblasts) establish their arterial or venous fates. We show using zebrafish that the arterial progenitors of the major axial vessels originate earlier and closer to the midline than the venous progenitors. Both medial and lateral progenitor populations migrate to distinct arterial and venous positions and not into a common precursor vessel as previously suggested. Overexpression of VEGF or Hedgehog (Hh) homologs results in the partially randomized distribution of arterial and venous progenitors within the axial vessels. We further demonstrate that the function of the Etv2 transcription factor is required at earlier stages for arterial development than for venous. Our results argue that the medial angioblasts undergo arterial differentiation because they receive higher concentration of Vegf and Hh morphogens than the lateral angioblasts. We propose a revised model of arterial-venous differentiation that explains how angioblasts choose between an arterial and venous fate.

Detection of vascular endothelial progenitor cells in patients with systemic lupus erythematosus

Objective To characterize the alteration in peripheral blood endothelial progenitor cells (EPCs) of patients with systemic lupus erythematosus (SLE).Methods Venous blood samples were obtained from 82 female patients with SLE aged (35 ± 10) years and 50 healthy female controls aged (35 ± 13) years.ACL 9000 automated coagulation analyzer was used to determine the level of Von Willebrand factor antigen (vWF Ag).Flow cytometry was performed to detect peripheral blood EPCs and circulating endothelial cells (CECs).Analysis of variance was performed to assess the differences in these parameters between patients with active and stable SLE and the controls,and Pearson correlation analysis was conducted to evaluate the relationship between these parameters.Results The number of CD34+ cells,CD133+ cells and CD34+CD133+cells per 200 000 peripheral blood mononuclear cells was 35.4 ± 16.7,86.5 ± 32.1 and 361.3 ± 176.4 in patients with active SLE,significantly higher than that in the patients with stable SLE (17.1 ± 10.9,28.7 ± 21.5,107.2 ±44.3,respectively,all P ＜ 0.01)) and the controls (13.8 ± 9.6,11.2 ± 5.5,92.3 ± 50.5,respectively,all P ＜0.01).The patients with active SLE exhibited an elevated level of vWF Ag (438.9％ ± 205.3％ vs.130.2％ ±51.5％,P ＜ 0.01),an increased number of EPCs (361.3 ± 176.4 vs.107.2 ± 44.3,P ＜ 0.01) but a similar number of CECs (127±51 vs.118 ± 39,P ＞ 0.05) per 200 000 peripheral blood mononuclear cells compared with the healthy controls.No significant differences were observed in these parameters between the patients with stable SLE and the controls (all P ＞ 0.05).The number of EPCs was positively correlated with the level of vWF Ag (r =0.67,P ＜ 0.01),but uncorrelated with the number of CECs (P ＞ 0.05) in patients with active SLE.Conclusions The quantity of EPCs in peripheral blood is closely correlated with the level of the vascular injury marker vWF Ag,hinting that the number of EPCs can serve as a useful marker of disease severity.

Nestin: A novel angiogenesis marker and possible target for tumor angiogenesis

Abnormal vasculature, termed tumor vessels, is a hallmark of solid tumors. The degree of angiogenesis is associated with tumor aggressiveness and clinical outcome. Therefore, exact quantification of tumor vessels is useful to evaluate prognosis. Furthermore, selective detection of newly formed tumor vessels within cancer tissues using specific markers raises the possibility of molecular targeted therapy via the inhibition of tumor angiogenesis. Nestin, an intermediate filament protein, is reportedly expressed in repair processes, various neoplasms, and proliferating vascular endothelial cells. Nestin expression is detected in endothelial cells of embryonic capillaries, capillaries of the corpus luteum, which replenishes itself by angiogenesis, and proliferating endothelial progenitor cells, but not in mature endothelial cells. Therefore, expression of nestin is relatively limited to proliferating vascular endothelial cells and endothelial progenitor cells. Nestin expression is also reported in blood vessels within glioblastoma, prostate cancer, colorectal cancer, and pancreatic cancer, and its expression is more specific for newly formed blood vessels than other endothelial cell markers. Nestin-positive blood vessels form smaller vessels with high proliferation activity in tumors. Knockdown of nestin in vascular endothelial cells suppresses endothelial cell growth and tumor formation ability of pancreatic cancers in vivo. Using nestin to more accurately evaluate microvessel density in cancer specimens may be a novel prognostic indicator. Furthermore, nestin-targeted therapy may suppress tumor proliferation via inhibition of angiogenesis in numerous malignancies, including pancreatic cancer. In this review article, we focus on nestin as a novel angiogenesis marker and possible therapeutic target via inhibition of tumor angiogenesis.

Human adipose stromal vascular cell delivery in a fibrin spray.

Adipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system. SVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVage system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel fibrin spray system. SVF cell growth and differentiation were documented by immunofluorescence staining and photomicrography. SVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SVF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD146+ and α-smooth muscle actin+ perivascular/stromal cells. Human adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.

Transfer of Monocrotaline-Induced Pulmonary Hypertension to Healthy Mice Via Microparticles

Abstract 5190 Rationale:Circulating endothelial cell-derived microparticles (MP) are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. We have shown that lung-derived MP induce marrow cells to express lung-specific mRNA, protein. Objectives:Determine if lung, plasma-derived MP (LMP, PMP) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodeling in PAH. Methods and Main Results:LMP, PMP isolated from monocrotaline (MCT)- or vehicle-treated mice were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodeling were assessed by RV-to-body weight (RV/BW), blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT vs. vehicle-injected mice (1. 99+0. 09vs. 1. 04+0. 09mg/g; 0. 159+0. 002vs. 0. 062+0. 009%). MP from MCT and vehicle-treated mice were quantitatively similar; however, MCT-LMP had higher endothelial cell mRNA expression and higher expression of mRNAs of proteins known to be abnormally expressed in PAH vs. vehicle-LMP. RV/BW, WT/D ratios were higher in mice injected with MCT-MP vs. mice injected with vehicle-MP (1. 63+0. 09vs. 1. 08+0. 09mg/g; 0. 113+0. 02vs. 0. 056+0. 01%). Lineage-depleted bone marrow cells co-cultured with MCT-MP and bone marrow cells isolated from MCT-MP infused mice had higher endothelial progenitor cell gene expression vs. cells co-cultured with vehicle-MP or cells isolated from vehicle MP infused mice. Conclusions:MP from MCT-injured mice have increased expression of growth factors implicated in PAH pathogenesis and induce RV hypertrophy, pulmonary vascular remodeling in healthy mice. Circulating MP may contribute to the development of MCT-induced pulmonary hypertension by inducing a pathologic phenotype in pulmonary vascular endothelial and endothelial progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin Treatment Enhanced Cell Surface CXCR4 Expression On Neonatal CD34-Positive Cells

Abstract 4724Erythropoietin (EPO), which is used to treat anemic immature neonates, is thought to be effective for treating neonatal hypoxic encephalopathy by stimulating nervous system cells or vascular stem cells. On stem cells, CXCR4 is an important chemokine receptor for cell migration and proliferation. Previously, we reported that increased expression of CXCR4 on cord blood (CB)-derived CD34-positive cells, caused by low oxygen tension, enhanced cell homing activity and engraftment (Ohno et al., ASH. 2010). In human endothelial stem cells, CXCR4 expression is controlled by hypoxia-inducible factor-1α (HIF-1α), and EPO expression is enhanced by hypoxia via HIF-1α. In this study, we examined the effect of EPO treatment on CXCR4 expression on premature neonatal endothelial stem cells and cultured CB-derived CD34-positive cells.First, we examined the cell surface markers CD34, CD133, and CXCR4 in preterm infants diagnosed with anemia and treated with EPO. The 10 enrolled neonates were first administered EPO when they were over 32 weeks old and were not given oxygen therapy. Blood samples were collected twice before and once 24 h after EPO administration. Mononuclear cells were isolated from peripheral blood and stained for the hematopoietic stem cell marker CD34, vascular endothelial progenitor cell marker CD133, and surface CXCR4. The cells were analyzed by flow cytometry. No change in the proportions of CD34- and CD133-positive cells were found after the treatment (CD34 positive: 0.59%→0.57%; CD133 positive: 0.52%→0.49%). The proportion of cell surface CXCR4 on the CD133-positive cells did not change after EPO administration, whereas the proportion of cell surface CXCR4 on the CD34-positive cells increased significantly after EPO was administered (average: 7.6%→11.0%, p < 0.01). In particular, slightly CD34-positive cells showed enhanced cell surface CXCR4.Next, we examined CXCR4 enhancement in EPO-treated CB-derived CD34-positive enriched samples. Samples were divided into three aliquots and cultured. The first aliquot was incubated for 24 h in RPMI-1640 medium alone as a control; the second, for 24 h in RPMI-1640 medium with EPO (10 U/ml); and the third, for 24 h in RPMI-1640 medium with EPO (10 U/ml) containing the specific HIF-1 antagonist rapamycin. Flow cytometry revealed significantly increased surface CXCR4 expression on CD34-positive cells after incubation with EPO compared with the control expression (average: 51.7% vs. 30.3%, p< 0.05). This enhancement was inhibited completely by the addition of rapamycin. Intracellular HIF-1 was enhanced significantly in the EPO-treated cells compared with the control expression on flow cytometry.The enhanced CXCR4 expression on CD34-positive cells reflects an amplification of the chemotactic and homing abilities of stem cells. The CXCR4 enhancement caused by EPO may increase tissue migration of CD34-positive cells, although this is probably a result of an active HIF-1 pathway. Disclosures:No relevant conflicts of interest to declare.