Endothelial Progenitor Cells Cells Research Articles

Formulation and Characterization of Nanoparticle-Protein Sirtuin 1 (NPS1) by Nanoprecipitation Technique

Atherosclerosis is a cardiovascular disease caused by endothelial dysfunction. This situation will trigger the bone marrow to immediately replace it with new endothelial progenitor cells (EPC) cells. However, some studies suggest that EPC can experience premature senescence. Sirtuin-1 (SIRT1) is a cellular post-translational protein that has the task of repairing dysfunctional EPC cells. Studies have tried to develop SIRT1 activation, but currently, there are no studies that have attempted to increase SIRT1 levels in cells. Nanoparticles (NPS) are one of the methods in nanomedicine, which has the advantage of being a drug carrier. So, further research is needed on adding exogenous SIRT1 levels, NPS, which can improve the quality of EPC cells and prevent premature senescence. This study aims to report the formulation and characterization stages of nanoparticles carrying SIRT1 (NPS1) with different solvents, such as ethanol and aquadest. The method used in this formulation uses nanoprecipitation. The characterization of nanoparticles at this stage included organoleptic tests, pH tests, and quantifying using Nanodrops in determining the presence of adsorbed proteins. The pH and organoleptic test showed that the NPS1 formulation was acidic (K1 = 5.412 ± 0.73; K2 = 3.624 ± 0.45; F1 = 5.418 ± 0.55; F2 = 4.182 ± 0.07), yellow in color, and had a characteristic odor. Thus, the formulation and characteristics of NPS1 can be used as a method in drug development for anti-senescence therapy in EPC cells in further research, both in vitro, in vivo, and evaluation of preparations that are still very possible to be developed.

Platelet-Derived Growth Factor-BB Priming Enhances Vasculogenic Capacity of Bone Marrow-Derived Endothelial Precursor Like Cells.

Human endothelial progenitor cells (EPCs) were first identified in the peripheral blood and later in the cord blood and bone marrow (BM) with different vascularization capacity and different surface marker profiles. However, their identity and functional roles in neovascularization have not been clearly demonstrated in vivo and in vitro. Characterization of BM-EPC like cells were performed by fluorescence-activated cell sorting, immunofluorescence staining, enzyme-linked immunosorbent assay, Matrigel tube formation assay,andwestern blot analysis. BM-EPC like cells were identified by selective adhesion to fibronectin and collagen from BM mononuclear cells, which generate fast-growing colonies with spindle morphology, express surface markers of CD105, vWF, UEA-I lectin binding, secrete HGF, VEGF, TGF-beta1 but can be distinguished from circulating EPC and endothelial cells by no expression of surface markers such as CD31, CD309, CD45, and CD34. These BM-EPC like cells shared many cell surface markers of BM-mesenchymal stem cells (MSC) but also can be distinguished by their vasculogenic property and other unique surface markers. Furthermore, the vasculogenic capacity of BM-EPC like cells were enhanced by co-culture of BM-MSC or PDGF-BB priming. PDGF-BB stimulated cell migration, proliferation, and secretion of laminin β-1, which was proposed as one of the mechanisms involved in the better vascularization of BM-EPC like cells. PDGF-BB priming may be applied to improve the potency and function of BM-EPC like cells as vasculogenic cell therapy for the ischemic vascular repair.

Abstract 15273: Impact of Lipid Lowering Therapy on Progenitor Endothelial Cell Phenotype and Function in Coronary Artery Disease

Introduction: Early revascularization of infarcted zone is crucial to improve patient′s outcomes. Despite their primary actions, major lipid-lowering drugs may also reduce cardiovascular events by pleiotropic mechanisms. Hypothesis: Conventional (statin) and novel lipid-lowering therapies [(PSCK9-inhibitor (PCSK9i) and eicosatetraenoic acid (EPA)] can improve endothelial progenitor cells (EPC) phenotype and favor neovascularization. Methods: EPC were isolated from peripheral blood of patients with acute coronary syndrome (n=62) who were statin-naïve (n=34) or chronic statin treatment (n=28) at time of inclusion. A set of statin-naïve EPC and mature endothelial cells (HUVEC) were treated in vitro with statin (20nM), PCSKi (1μM) and EPA (3μM). EPC phenotype was determined as EPC colony number and time of appearance, proliferative rate, and ability to form capillary-like structures. Serum PCSK9 and markers of angiogenesis and endothelial function were determined by multiplexed protein assay. Results: Frequency of EPC appearance was higher in samples of statin group (78%) compared to naïve group (66%) (Fig A). They also appeared much earlier (Fig B), in greater quantity (4.7 vs 2.1 colony, p = 0.0009) and had higher proliferative rate than EPCs of naïve group. Besides they had increased potential of tube formation and shaped more branched vessel-like structures (Fig C). No differences in serum levels of biomarkers were found. In vitro treatment of EPC with statin, PCSK9i and EPA did not modify colonies behavior (Fig D). PCSK9 levels showed negative correlation with EPC proliferation (r -0.4150, p=0.004), and PCSK9i treatment increased angiogenic potential of HUVEC (Fig E). Conclusions: Statin and PCSK9i have pleiotropic effects on EPC, boosting their potential to form endothelial cells colonies and to induce angiogenesis. These effects could contribute to the progression and outcomes of coronary artery disease. Funding: ISCIII - FEDER-ERDF (PI19/00264)

Mechanism of isosorbide dinitrate combined with exercise training rehabilitation to mobilize endothelial progenitor cells in patients with coronary heart disease

ABSTRACT it was to explore effect of isosorbide dinitrate combined with exercise training and rehabilitation on endothelial progenitor cells (EPCs) in coronary heart disease. EPCs were isolated and cultured from peripheral blood of coronary heart disease patients, and morphology and surface markers were detected. Then, 116 patients were rolled into treatment group (isosorbide dinitrate + exercise rehabilitation training) and control group (isosorbide dinitrate). Characteristics of EPCs cells after treatment were compared. The mononuclear cells were round and small in size and were not evenly distributed in the culture plate. EPCs cells grew as colonies after 8d-culture, and the surrounding cells grew outward in a germinating manner with colonies as the center, forming multiple cell populations. Positive rates of EPCs surface markers CD133, CD34, and vascular endothelial growth factor receptor (KDR) were 11.25 ± 3.07%, 48.18 ± 9.13%, and 76.36 ± 8.27%, respectively. Proliferation activity of EPCs in the treatment group was dramatically higher versus controls at day three, five, and seven (P < 0.05). Adhesion ability of EPCs in treatment group was dramatically higher than controls at day three, five, and seven (P < 0.05). Migration ability of EPCs in treatment group was dramatically higher versus control group at day three, five, and seven (P < 0.05). In short, isosorbide dinitrate plus exercise rehabilitation greatly enhanced the proliferation activity, adhesion ability, and migration ability of EPCs cells, which also played a beneficial role in the repair of endothelial injury, with notable effects. Abbreviation listAbbreviation: Full name; EPCs: endothelial progenitor cells; RPMI: roswell Park Memorial Institute;VEGF: vascular endothelial growth factor; BFGF: basic fibroblast growth factor; DiL-acLDL: DiL-labeled acetyl low density lipoprotein; PBS: phosphate buffer; FITC-UEA-I: FITC-labeled Vitex agglutinin I; EDTA: ethylenediamine tetra-acetic acid; MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium.

Gender Differences in the Pharmacological Actions of Pegylated Glucagon-Like Peptide-1 on Endothelial Progenitor Cells and Angiogenic Precursor Cells in a Combination of Metabolic Disorders and Lung Emphysema.

In clinical practice, the metabolic syndrome (MetS) is often associated with chronic obstructive pulmonary disease (COPD). Although gender differences in MetS are well documented, little is known about sex-specific differences in the pathogenesis of COPD, especially when combined with MetS. Consequently, it is not clear whether the same treatment regime has comparable efficacy in men and women diagnosed with MetS and COPD. In the present study, using sodium glutamate, lipopolysaccharide, and cigarette smoke extract, we simulated lipid metabolism disorders, obesity, hyperglycemia, and pulmonary emphysema (comorbidity) in male and female C57BL/6 mice. We assessed the gender-specific impact of lipid metabolism disorders and pulmonary emphysema on angiogenic precursor cells (endothelial progenitor cells (EPC), pericytes, vascular smooth muscle cells, cells of the lumen of the nascent vessel), as well as the biological effects of pegylated glucagon-like peptide 1 (pegGLP-1) in this experimental paradigm. Simulation of MetS/COPD comorbidity caused an accumulation of EPC (CD45−CD31+CD34+), pericytes, and vascular smooth muscle cells in the lungs of female mice. In contrast, the number of cells involved in the angiogenesis decreased in the lungs of male animals. PegGLP-1 had a positive effect on lipids and area under the curve (AUC), obesity, and prevented the development of pulmonary emphysema. The severity of these effects was stronger in males than in females. Furthermore, PegGLP-1 stimulated regeneration of pulmonary endothelium. At the same time, PegGLP-1 administration caused a mobilization of EPC (CD45−CD31+CD34+) into the bloodstream in females and migration of precursors of angiogenesis and vascular smooth muscle cells to the lungs in male animals. Gender differences in stimulatory action of pegGLP-1 on CD31+ endothelial lung cells in vitro were not observed. Based on these findings, we postulated that the cellular mechanism of in vivo regeneration of lung epithelium was at least partly gender-specific. Thus, we concluded that a pegGLP-1-based treatment regime for metabolic disorder and COPD should be further developed primarily for male patients.

The Number and Potency of Endothelial Progenitor Cells in Type 2 Diabetes Mellitus Patients

BACKGROUND: Type 2 Diabetes Mellitus (T2DM) is a metabolic disease, due to the disorder of insulin function, insulin secretion, or both. Long-term hyperglycemia conditions promote endothelial dysfunction precedes to the development of multiple organ dysfunctions. Endothelial progenitor cells (EPCs) and hematopoietic stem cell (HSCs) are the key cellular effectors of postnatal neovascularization and play central role in endothelial dysfunction. However, in T2DM condition, the number of apoptotic HSCs increase, it may cause the reduction in potency and number of EPCs. In diabetes, the circulating EPCs number decrease and their functionality is impaired, but mechanism underlie of this impairement is unknown. The purpose of this study was to examine the relationship duration diabetes with the number and potency of EPC cells in T2DM patients controlled and poorly controlled.METHODS: Thirty-eight T2DM male patients were classified into two group based on Indonesian Society of Endocrinology/Perkumpulan Endokrinologi Indonesia (PERKENI) criteria on T2DM. The first group was a controlled glycemic condition group (hemoglobin A1c (HbA1C) &lt;7.0%) and the second group was a poorly controlled glycemic condition group (HbA1C &gt;7.0%). Cluster of differentiation (CD)34+ and CD133+ expressions were used as specific marker for EPC, while quantified bright aldehyde dehydrogenase (ALDHbr) assay was used to represented the potency of EPCs.RESULTS: This study showed that in poorly controlled T2DM group the number of EPCs was lower by 24.80% (p&lt;0.05) compared to the T2DM controlled group. Similarly, the expression of ALDHbr was lower by 43.07% (p&lt;0.05) in poorly controlled group.CONCLUSION: There was a decrease in the number and potency of EPCs in poorly controlled T2DM patients compared to the controlled T2DM patients. There was also a strong negative correlation between the duration of diabetes and number of EPCs.KEYWORDS: ALDHbr, endothelial progenitor cells, type 2 diabetes mellitus

The Role of Estrone in Endothelial Progenitor Cell Number and Function

Microvascular dysfunction is a key component of cardiovascular disease and estrogens, particularly estradiol, have been shown to be vascular protective. Endothelial progenitor cells (EPCs) are a stem cell population that supports the integrity of the microvasculature and show increased proliferation in response to estradiol exposure. The plasma concentration of EPCs negatively correlates with incidence of stroke and coronary artery disease. Another estrogen, estrone, has been understudied due to its weak binding at nuclear estrogen receptors ERa and ERb. However, recent evidence suggests that estrone is biologically active through a G protein coupled estrogen receptor, GPER. In some vascular cell populations, estrone independently increases proliferation and modulates the proliferative effects of estradiol. In pre‐menopausal women with high estradiol/estrone ratio, EPCs are present in sufficient numbers to enhance angiogenesis and repair vessels at sites of damage. After menopause when the estradiol/estrone ratio is low, EPC number is decreased, the microvasculature is compromised, and CVD risk increases. In the treatment of symptoms of menopause, we use a combination estrogen therapy which is largely estrone based. Using this therapy, estradiol levels are restored pharmacologically, but the estradiol/estrone ratio remains low. During treatment, EPC numbers remain low and cardiovascular risk remains high.Here we investigate the role of estrone in EPC proliferation via in vitro exposure studies using EPCs from human female blood samples and EPCs isolated from the bone marrow of Sprague Dawley Rats. We also assess how estrone, estradiol and combination exposures effect the ability of EPCs to enhance endothelial cell tube formation (vasculogenesis in vitro). Human and rat EPCs were defined as cells derived from the mononuclear cell fraction that after 10–14 days of expansion/selection in culture adhere to fibronectin, display typical endothelial cell (EC) morphology and are positive for the EPC markers VEGFR2, CD133, cKIT, CD34, and Dil‐ac‐LDL uptake. After sample collection (human peripheral blood or rat bone marrow), the mononuclear cell layer was isolated using density centrifugation. The mononuclear cell layer was then plated in endothelial growth media for 14 days. After 14 days the EPCs cells were used for expression, proliferation and tube formation assays. The proliferation assays were performed over a 24 hour exposure to 1 &amp; 2 nmol estrone, 1 &amp; 2 nmol estradiol and 1:1 (1nmol) estrone:estradiol. In our tube formation assay, EPCs were exposed to estrone, estradiol and 1:1 estrone:estradiol for 24 hours and then added to an endothelial cell tube formation assay. We assessed GPER expression via qPCR and immunocytochemistry on EPCs without hormone treatment.In vitro EPC proliferation was enhanced by both estrone and estradiol in a dose dependent manner. When estrone and estradiol were applied in a one to one ratio, resulting EPC proliferation was below that of control. GPER is expressed on both human and rat EPCs. Further studies will use a rat model to investigate physiological effects of estrone and GPER agonism on EPC ability to enhance angiogenesis in vivo.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial dysfunction and cellular repair in heart failure with preserved ejection fraction: response to a single maximal exercise bout

Endothelial dysfunction and cellular repair in heart failure with preserved ejection fraction: response to a single maximal exercise bout

Effects of endothelial progenitor cells on proliferation and biological function of hepatic stellate cells under shear stress

To investigate the effects of endothelial progenitor cells (EPCs) under shear stress on the biological function such as proliferation, adhesion, migration, apoptosis and expression of α-smooth muscle actin (α-SMA), collagen-I and collagen-Ⅲ of hepatic stellate cells (HSCs). HSCs and EPCs were inoculated into the upper and lower layers of the co-culture chamber respectively and co-incubated for 24 hours. Then, 12 dyne/cm2 shear stress was applied to EPCs cells for another 24 hours. After that, proliferation, adhesion, migration and apoptosis of HSCs were detected by cell counting kit-8 (CCK-8) kit, cell adherent assay, Boyden cell migration assay and flow cytometry respectively. Fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expression of alpha -SMA, collagen I and collagen-Ⅲ in HSCs. Under shear stress, EPCs ecological niche could obviously inhibit the proliferation, adhesion and migration of HSCs, promote the apoptosis of HSCs, and down-regulate the mRNA and protein expression of collagen-I, collagen-Ⅲ in HSC cells. Under shear stress, EPCs ecological niche could inhibit the fibrosis development of HSCs to a certain extent.

Increased Endothelial Progenitor Cell Number in Early Stage of Endometrial Cancer.

It is generally believed that circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) reflect the state of the endothelium, its injury and/or repair possibilities. In different types of cancers, increased numbers of CECs and EPCs were found, suggesting their participation in cancer angiogenesis. The objective of this study was to determine whether, in the blood circulation of women with early endometrial cancer, CEC and EPC levels differ from those of healthy women of similar age. For CEC number evaluation, samples of peripheral blood cells of women with endometrial carcinoma and control subjects were labeled with anti-CD31 and anti-CD45 antibodies; for EPCs, with anti-VEGFR2 (vascular-endothelium growth factor receptor 2)/KDR and anti-CD34 antibodies. The CEC and EPC cells were then quantified by flow cytometry. Endothelial progenitor cell numbers (CD34, VEGFR2/KDR) in the peripheral blood of women with endometrial carcinoma were significantly augmented as compared with those of control healthy women and CEC numbers (CD31, CD45) were similar in both groups. Cancer patients were divided according to the grading into G1 and G2 groups and according to the stage into International Federation of Gynecology and Obstetrics (FIGO) stage IA and FIGO IB groups. Statistically significant augmented EPC numbers were demonstrated only in G1 and FIGO IA patients. These results strongly suggest new vessel formation from recruited endothelial precursors as being involved mainly at the early stages of tumor progression.

Circulating levels of natural killer T cells and EPC cells in pre-eclamptic pregnant women

Natural killer T cells are bone marrow origin, they represent about 70% of decidua leukocytes and play an important role in the remodeling of spiral arteries [1]. Endothelial progenitor cells (EPC) are bone-marrow derived cells able to differentiate into endothelial cells [2]. During stress conditions (burns, wounds, coronary artery bypass) a mobilization of these cells from bone-marrow in repairing wounds and during pregnancy occurs [3]. The aim of our study was to evaluate the circulating levels of NK lymphocytes and circulating EPC cells in women with overt preeclampsia. Our study population consists of 39 women in the third trimester of gestation. The patients were divided into two groups: group A with 13 women affected by pre-eclampsia diagnosed according to the criteria established by AIPE 2013 guidelines, and a group B composed of 26 patients with normal pregnancy. Of these women, EPC cells (CD34+) and NK lymphocytes (CD+) circulating were assayed by flow cytometry [4]. A significant difference between groups was observed, either in EPC values (group A: 1.61±0:29 vs group B: 2.42±0.45 cells per μL, p p

Endothelial Progenitor Cells as a Marker of Endothelial Dysfunction and Atherosclerosis in Ankylosing Spondylitis: A Cross-Sectional Study.

Endothelial progenitor cells (EPCs) have reparative potential in overcoming the endothelial dysfunction and reducing cardiovascular risk. EPC depletion has been demonstrated in the setting of established atherosclerotic diseases. We evaluated whether reduced EPCs population are associated with endothelial dysfunction, subclinical atherosclerosis, and inflammatory markers in ankylosing spondylitis (AS) patients without any known traditional cardiovascular risk factor. We performed a cross-sectional study of 30 consecutive AS patients and 25 age- and sex-matched healthy controls. Patients with traditional cardiovascular risk factors were excluded. Circulating EPCs (CD34+/CD133+) were quantified by flow cytometry. The assessment of endothelial function by brachial artery flow-mediated dilatation (FMD) and ultrasound assessment of carotid intima-media thickness (CIMT) was measured in both the groups. EPCs cells were significantly (0.020 ± 0.001 vs. 0.040 ± 0.010%, p < 0.001) reduced in patients with AS compared with healthy controls. Endothelial function (7.35 ± 2.54 vs. 10.27 ± 1.73, p = 0.002), CIMT (0.63 ± 0.01 vs. 0.35 ± 0.02, p < 0.001), and inflammatory markers were also significantly (p < 0.01) altered as compared with controls. EPCs inversely correlated with tumor necrosis factor (TNF)-α and C-reactive protein (CRP) and positively correlated with endothelial function. Present study results demonstrate depleted EPC population in AS patients compared with controls. Increased level of CRP and TNF-α appears to play a key role in EPC depletion and the latter contributes to endothelial dysfunction and atherosclerosis in AS. EPC population would, therefore, represent an attractive measure of endothelial dysfunction and accelerated atherosclerosis disease associated with AS.

Abstract 3402: Ephrin B2 and Dll-4 mediated co-dependence of tumor and endothelial progenitor cells in human hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a highly vascularized tumor, which preferentially recruits new blood vessels from the hepatic artery to fuel its growth, through mechanisms not well understood. Here, we examined the intercellular cross-talk of HepG2 (human HCC) and endothelial progenitor cells (EPC) in transwell co-cultures to mimic initial tumor parenchyma and stroma interactions. We present evidence showing that, in culture, HepG2 cells secrete factors to induce an ‘activated’ EPC phenotype, namely, an increase in survival, growth, proliferation and migration rates. EPC cells showed a significant increase in growth (∼40%) and survival (∼80%) when co-cultured with HepG2 cells. Simultaneously, activated EPCs improved the viability of HepG2 cells. A major mediator of intercellular crosstalk are the secreted microvesicles called exosomes. Exosomes constitute a set of distinct nanovesicles (30-100 nm), endosomal in origin, constitutively secreted by many cells. They are involved in intercellular communication by transferring proteins and RNA from one cell to another. Intercellular crosstalk between these HepG2 cells and EPCs cells lead to qualitative and quantitative changes in the exosomal cargo secreted by the respective cells. The exosomal cargo constituting various proteins and genetic material have been demonstrated to be involved in processes which promote remodeling of extracellular matrix (ECM), biological adhesion and pathways modulating developmental processes and progression of inflammation, leading to cancer growth and development. In other work with glioma cell-derived exosomes has shown the presence of Ephrin A and B receptors which suggests a prominent role of secreted exosomes in stimulating and guiding tumor angiogenesis, a hallmark characteristic of glioblastoma multiforme (GBM). Additionally, our results with co-cultures of tumor and endothelial cells show that angiogenesis signaling in EPC appears to be induced by HepG2 cells via elevated endothelial Ephrin B2 and DLL-4 expression. Overall, our results demonstrate the co-dependence of HCC and EPC intercellular crosstalk, likely accomplished via secreted exosomes, in the initial stages of HCC establishment and stimulation of angiogenesis and development, thus a promising target for new clinical strategies. Citation Format: Rajshekhar A. Kore, Meenakshi Upreti, Azemat Jamshidi-Parsian, Rudd P.M. Dings, Issam Makhoul, Robert J. Griffin. Ephrin B2 and Dll-4 mediated co-dependence of tumor and endothelial progenitor cells in human hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3402.

Effect of antihypertensive treatment with lercanidipine on endothelial progenitor cells and inflammation in patients with mild to moderate essential hypertension

Background: It has been demonstrated that circulating endothelial progenitor cells (EPCs) number reflects the endogenous vascular repair ability, with the EPCs pool declining in presence of cardiovascular risk factors. Several drugs, including dihydropyridine calcium channel blockers, have been reported to elicit antioxidant and anti-inflammatory properties, as well as to improve vascular remodeling and dysfunction. However, no data are available about the effects of lercanidipine on EPCs. The aim of the present study was therefore to investigate the effects of short-term treatment with lercanidipine on circulating EPCs, as well as on indices of inflammation and oxidative stress.Patients and methods: Twenty essential hypertensive patients were included in the study and treated for 4 weeks with lercanidipine 20 mg per day orally. Investigations were performed in basal condition, after appropriate wash out of previous treatments, and after 4 weeks of lercanidipine treatment. Inflammatory and oxidative stress markers were assessed by ELISA technique. Lin-/7AAD-/CD34+/CD133+/VEGFR-2 + and Lin-/7AAD-/CD34+/VEGFR-2 + cells were identified by flow cytometry and considered as EPCs. EPCs cells were expressed as number of cells per million Lin-mononuclear cells.Results: Circulating EPCs were significantly increased after lercanidipine treatment (CD34+/CD133+/VEGFR-2 + cells: 78.3 ± 64.5 vs 46.6 ± 32.8; CD34+/VEGFR-2+: 87996 ± 165116 vs 1026 ± 1559, respectively, p < 0.05). A modest reduction in circulating indices of inflammation was also observed.Conclusions: In conclusion, lercanidipine is able to increase the number of circulating EPCs, possibly through a reduction of low-grade inflammation.

The effect of interleukin 10 modified endothelial progenitor cells in rat diabetic retinopathy

Objective To observe the effect of interleukin (IL) 10 modified endothelial progenitor cells (EPC) in diabetic retinopathy (DR). Methods EPC cells were collected and cultivated from the bone marrow of rats and identified by immuno-fluorescence staining. EPC cells were infected with lentivirus (LV) of EPC-LV-IL10-GFP (EPC-LV-IL10-GFP group) or EPC-LV-NC-GFP (GFP group). EPC cells without lentivirus infection was the EPC group. Enzyme-linked immuno sorbent assay (ELISA) was used to measure the concentrations of tumor necrosis factor (TNF)-α, IL10, IL8 and vascular endothelial growth factor (VEGF) in the supernatant of these three groups. 168 male Wistar rats were divided into normal control group (28 rats), diabetes mellitus (DM) group (28 rats), DM-blank control group (56 rats) and DM-intervention group (56 rats). DM was introduced in the latter 3 groups by streptozotocin intravenous injection. Three months later, the rats in the DM-blank control group and DM-intervention group were injected with EPC-LV-NC-GFP or EPC-LV-IL10-GFP by tail vein, respectively. Immunohistochemistry was used to observe the GFP expression in rat retinas. The blood-retinal barrier breakdown was detected by Evans blue (EB) dye. The retinal histopathologic changes were observed by transmission electron microscope. The mRNA level of VEGF, matrix metallproteinases-9 (MMP-9), angiopoietin-1 (Ang-1), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in retina were measured by reverse transcription-polymerase chain reaction (RT-PCR). Results ELISA showed that the levels of TNF-α and IL8 in the supernatant significantly decreased , while the levels of IL10 and VEGF increased (P<0.05) in EPC-LV-IL10-GFP group. GFP expressed in the retina of blank control group and intervention group, mainly in the ganglion cell layer, inner nuclear layer and outer plexiform layer. The retinal blood vessel pathological change and EB permeability significantly decreased in intervention group compared with DM group (P<0.05), and blank control group (P<0.05). RT-PCR revealed that the mRNA level of VEGF, MMP-9 and Ang-1 significantly increased, and eNOS decreased in DM group compared to the normal control group (P<0.05). The mRNA level of VEGF and iNOS decreased, eNOS increased while Ang-1 and MMP-9 had not changed in DM-blank control group and DM-intervention group compared with DM group (P<0.05). Conclusions IL10 modified EPC can improve the inflammative microenvironment and suppressed the pathogenesis of DR. Furthermore, EPC transplantation can increase the number of EPC and exerted their effect. Key words: Diabetic retinopathy/physiopathology; Interleukin-10; Stem cells; Animal experimentation

Effects of Vascular Endothelial Growth Factor in Recovery Phase of Acute Lung Injury in Mice.

To test the hypothesis that exogenous administration of vascular endothelial growth factor (VEGF) promotes lung repair in acute lung injury (ALI). ALI was induced by intranasal lipopolysaccharide (LPS) administration in mice, followed by different treatment protocols for 7 days in 3 groups (n = 6, each) including the LPS, the VEGF and the anti-VEGF group. At day 7, peripheral blood and lungs were collected. Lung wet-to-dry (W/D) ratio and lung injury score were measured. Immunohistochemistry assay was employed to detect the number of pulmonary vessels. Circulating endothelial progenitor cells (EPCs) was detected using flow cytometric analysis, and the apoptosis of lung cells was determined by TUNEL staining. VEGF treatment reduced W/D ratio and pulmonary neutrophil infiltration in the VEGF group compared with the LPS group. The treatment of VEGF increased the number of pulmonary vessels, and significantly increased the number of circulating EPC cells. Moreover, administration of VEGF decreased the percentage of apoptotic cells in the VEGF group. Our results suggest that VEGF may contribute to vascular endothelial repair and function as a protective factor against ALI.

Abstract 231: Role of SDF-1α-induced Double Reciprocal E-Selectin/Ligand Pairs in Mediating Recruitment of Endothelial Progenitor Cells to Ischemic Wounds

Introduction: We previously reported that stromal cell-derived factor-1α (SDF-1α)-induced endothelial progenitor cells (EPC) homing is enhanced by up-regulation of E-selectin (E-sel) on luminal endothelial cells (EC) lining capillaries, locally within wound tissues. We now investigate how elevated SDF-1α in wounds achieves a remote effect on bone marrow-derived (BMD) circulating EPC. Methods: SDF-1α-induced cell adhesion molecules in human EPC and human microvascular endothelial cells (HMVEC) were profiled using PCR-Array and validated by immunoblot. Induction of E-sel expression on murine BMD- and circulating EPC by SDF-1α was examined by flow cytometry. The involvement of E-sel/ligand in mediating SDF-1α-induced EPC-EC interaction and EPC transendothelial migration was studied using in vitro EPC-EC binding assay. The role of SDF-1α-induced E-sel in regulating EPC homing, wound neovascularization and healing were furthered studied by testing EPC from ROSA26-LacZ+/E-sel -/- versus ROSA26-LacZ+/E-sel +/+ mice in an ischemic wound model. Results: SDF-1α enhanced EPC-EC interaction by upregulating expression of E-sel/ligand in human EPC and HMVEC. Administration of SDF-1α in wound also increased E-sel expression on BMD- and circulating EPC in mice. The effects of SDF-1α on EPC-EC interaction and EPC transendothelial migration were specifically mediated by E-sel, as E-sel antagonists could inhibit these processes. E-sel was required for SDF-1α-induced EPC homing, wound neovascularization and healing in vivo (n=6, P&lt;0.05). Conclusions: EPC-EC interaction is an essential process for EPC homing. SDF-1α induces both local luminal EC lining capillaries in wound tissue and remote BMD- and circulating EPC to express E-sel and its ligands. Up-regulated E-sel/ligand pairs reciprocally on EC and EPC serve as “Double-Lock” to secure targeted EPC-EC interactions selectively occurring within wound endothelium. These findings uncover a novel mechanism underlying the pro-angiogenic effect of SDF-1α on EPC homing and point to E-sel/ligand pairs reciprocally induced on circulating EPC and local endothelium as effective targets for ex vivo manipulation and therapeutic neovascularization.

Content of Endothelial Progenitor Cells in Autologous Stem Cell Grafts Predict Survival after Transplantation for Multiple Myeloma

Multiple myeloma (MM) is considered an incurable B cell malignancy, although many patients can benefit from high-dose therapy with autologous stem cell transplantation (ASCT) as a first-line treatment. In non-Hodgkin lymphoma (NHL), ASCT is usually performed after relapse with curative intent. Disease progression is often associated with increased angiogenesis, in which endothelial progenitor cells (EPC) may have a central role. Here, we investigated the clinical impact of EPC levels in peripheral blood stem cell (PBSC) autografts for MM and NHL patients who received ASCT. EPC were identified by flow cytometry as aldehyde dehydrogenasehi CD34+ vascular endothelial growth factor receptor 2+ CD133+ cells in both MM and NHL autografts. In MM, there was a positive correlation between EPC percentage and serum (s)-β2-microglobulin levels (r2 = .371, P = .002). Unlike for NHL patients, MM patients with high numbers of infused EPC (EPC cells per kilogram) during ASCT had significant shorter progression-free survival (PFS) (P = .035), overall survival (P = .044) and time to next treatment (P = .009). In multivariate analysis, EPC cells per kilogram was a significant independent negative prognostic indicator of PFS (P = .03). In conclusion, the presence of high number of EPC in PBSC grafts is associated with adverse prognosis after ASCT in MM.

Abstract 117: Stromal Cell-derived Factor-1 Gene Modified Endothelial Progenitor Cell Effectively Attenuated Ischemic Brain Injury in Mouse Model

Background and purpose: Stromal cell derived factor 1 (SDF-1, also know as CXCL12) is a chemokine that can attract stem cells. We have shown that post-acute SDF1 gene therapy promoted angiogenesis and neurogenesis after ischemic stroke. Transplantation of endothelial progenitor cells (EPC) alone was also beneficial for ischemic stroke recovery. However, it remains unclear whether SDF-1 gene modification of EPC (EPC-SDF-1) can further promote stroke recovery. Here we used lenti-virus (LV) to deliver SDF-1 or GFP gene into EPC, then delivered gene modified EPC cells after permanent middle cerebral artery occlusion (MCAO) to investigate the effect of EPC-SDF-1 on ischemic stroke recovery. Methods and materials: Thirty-eight adult ICR male mice received EPC-SDF-1, EPC-GFP, LV-SDF-1, or PBS stereotactic injection into the peri-infarct area one week after MCAO. Brain atrophy volume, neurobehavioral tests and immunohistochemistry were performed to evaluate the effects of EPC-SDF-1 on angiogenesis and functional repair. Results: Brain atrophy volume was significantly reduced in both gene therapy and cell therapy groups 5 weeks after ischemia (p&lt;0.05; LV-SDF-1 vs. PBS; EPC-GFP vs. PBS; EPC-SDF-1 vs. PBS; n=6), with a smaller atrophy volume in EPC-SDF-1 groups comparing to LV-SDF-1 (p&lt;0.01) and EPC-GFP (p&lt;0.001) group. Neurobehavioral tests including neurological deficits and rotarod test paralleled the result of atrophy volume, with neuronal function improved in all therapeutic groups 5 weeks after ischemic (p&lt;0.001; LV-SDF-1 vs. PBS; EPC-GFP vs. PBS; EPC-SDF-1 vs. PBS; n=8-11), with a better outcome in EPC-SDF-1 group (EPC-SDF-1 vs. LV-SDF-1, p&lt;0.05; EPC-SDF-1 vs. EPC-GFP, p&lt;0.001). The number of CD31 positive blood vessels in peri-infarct area was significantly increased in LV-SDF-1 gene therapy (p&lt;0.05, n=4) and EPC-SDF-1 cell therapy (p&lt;0.001, n=4) group comparing to PBS control. Conclusion: Our results demonstrated that transplantation of SDF-1 gene modified EPC significantly increased angiogenesis after ischemic brain injury; reduced brain atrophy volume and improved the neurological outcomes of the animals. This in vivo data suggests that SDF-1 gene modification of EPC holds great potential in the treatment of ischemic stroke.

Expansion, Differentiation and Transplantation of Human Endothelial Progenitor Cells Derived from Umbilical Cord Blood CD34+ Cells

Transplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been developed as a cell therapy for ischemic diseases and hemophilia A, due to the capacities of these cells to repair vascular damage and produce factor VIII. The strategies and the mechanisms of this type of cell therapy have been extensively investigated in the past decades. However, given the problems associated with host immune responses to grafts in allotransplantation, the difficulties in obtaining adequate numbers of suitable cells for transplantation represent additional obstacles for a successful cell therapy. In the present study, human umbilical cord blood CD34+ mesenchymal cells (MCs), which generate low immune response, have been adopted as the source of endothelial progenitors. Our major goal is to establish a highly efficient approach to stimulate the expansion and subsequent differentiation of human endothelial progenitor cells (EPCs), in order to obtain sufficient quantities of healthy cells for therapeutic use. The CD34+ cells isolated from human umbilical cord blood were cultured for 6 days in a customized basal medium supplemented with a modified cytokine cocktail, including SCF, Flt-3L, TPO, IL-3, and GM-CSF. At the end of proliferation stage, cell number was calculated with Flow Cytometry, and cell morphology was characterized as well. The numbers of CD34+ cells and CD34+/CD133+/VEGFR-2+ early EPCs cells were found to be increased by approximately 108-fold and 41.9-fold, respectively, on day 6. In the differentiation stage, the attached cells were transferred to EGM-2 basal medium supplemented with FBS and additional nutrients, including VEGF, IGF, EGF, and FGF, for another 15 days. Following this two-step culture, the generated adherent cells were found to have proliferated more than 1200 folds over the initial EPCs, and they were identified as mature endothelial cells expressing CD31, vWF and FVIII. Furthermore, we developed a nonobese diabetic, severe combined immunodeficient (NOD/SCID) mouse model with portal sinusoidal endothelium injury. The NOD/SCID mice were treated with the produced EPCs/ECs through hepatic portal vein injection at the dose of 6x10^6 cells. Tissue examination demonstrated that GFP-labeled transplanted cells migrated and integrated into liver structure, where they expressed specific endothelial cell markers. Therefore, through our unique approach, we can provide an excellent source of healthy endothelial progenitors for cell therapy. DisclosuresQin:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.