Rat Endothelial Progenitor Cells Research Articles

Microfluidic 3D platform to evaluate endothelial progenitor cell recruitment by bioactive materials

Most of the conventional in vitro models to test biomaterial-driven vascularization are too simplistic to recapitulate the complex interactions taking place in the actual cell microenvironment, which results in a poor prediction of the in vivo performance of the material. However, during the last decade, cell culture models based on microfluidic technology have allowed attaining unprecedented levels of tissue biomimicry. In this work, we propose a microfluidic-based 3D model to evaluate the effect of bioactive biomaterials capable of releasing signaling cues (such as ions or proteins) in the recruitment of endogenous endothelial progenitor cells, a key step in the vascularization process. The usability of the platform is demonstrated using experimentally-validated finite element models and migration and proliferation studies with rat endothelial progenitor cells (rEPCs) and bone marrow-derived rat mesenchymal stromal cells (BM-rMSCs). As a proof of concept of biomaterial evaluation, the response of rEPCs to an electrospun composite made of polylactic acid with calcium phosphates nanoparticles (PLA+CaP) was compared in a co-culture microenvironment with BM-rMSC to a regular PLA control. Our results show a significantly higher rEPCs migration and the upregulation of several pro-inflammatory and proangiogenic proteins in the case of the PLA+CaP. The effects of osteopontin (OPN) on the rEPCs migratory response were also studied using this platform, suggesting its important role in mediating their recruitment to a calcium-rich microenvironment. This new tool could be applied to screen the capacity of a variety of bioactive scaffolds to induce vascularization and accelerate the preclinical testing of biomaterials. Statement of significanceFor many years researchers have used neovascularization models to evaluate bioactive biomaterials both in vitro, with low predictive results due to their poor biomimicry and minimal control over cell cues such as spatiotemporal biomolecule signaling, and in vivo models, presenting drawbacks such as being highly costly, time-consuming, poor human extrapolation, and ethically controversial. We describe a compact microphysiological platform designed for the evaluation of proangiogenesis in biomaterials through the quantification of the level of sprouting in a mimicked endothelium able to react to gradients of biomaterial-released signals in a fibrin-based extracellular matrix. This model is a useful tool to perform preclinical trustworthy studies in tissue regeneration and to better understand the different elements involved in the complex process of vascularization.

Up-regulated miR-204-5p promoted the migration, invasion, and angiogenesis of endothelial progenitor cells to enhance the thrombolysis of rats with deep venous thrombosis by targeting SPRED1

Deep venous thrombosis (DVT) endangers human health. Endothelial progenitor cells (EPCs) were proven to promote thrombolysis and miR-204-5p was discovered to be low-expressed in DVT patients. This study concentrated on exploring whether miR-204-5p had a regulatory effect on EPCs and DVT. Concretely, the expression of miR-204-5p in DVT patients’ blood was detected by qRT-PCR. The target of miR-204-5p was predicted by bioinformatics and verified by dual-luciferase reporter assay. After rat EPCs were isolated, identified, and transfected with miR-204-5p agomiR, antagomiR, or SPRED1 plasmids, the viability, migration, invasion, and tube formation of EPCs were detected by MTT, wound healing, Transwell, and tube formation assays, respectively. MiR-204-5p, SPRED1, p-PI3K, PI3K, p-AKT, AKT, VEGFA, and Ang1 expressions in EPCs were measured by qRT-PCR or Western blot. EPCs transfected with miR-204-5p overexpression lentivirus plasmid were injected into the DVT rat model. The histopathology of the thrombus and the homing of EPCs to thrombus in the DVT rats were observed by hematoxylin-eosin staining and confocal microscopy, respectively. We found that miR-204-5p was low-expressed in DVT patients and SPRED1 was a target gene of miR-204-5p. MiR-204-5p agomiR promoted the viability, migration, invasion, and tube formation of EPCs, the levels of VEGFA and Ang1 and the activation of PI3K/AKT pathway in EPCs, while miR-204-5p antagomiR and SPRED1 worked oppositely. SPRED1 reversed the effect of miR-204-5p agomiR on EPCs. Up-regulated miR-204-5p inhibited thrombosis and promoted EPCs homing to thrombus in DVT rats. Collectively, up-regulated miR-204-5p enhanced the angiogenesis of EPCs and thrombolysis in DVT rats by targeting SPRED1.

Effect of “Xiaoke Tongbi granule” on the proliferation, migration and tubule-forming ability of rat endothelial progenitor cells under high glucose conditions

Purpose: To investigate the effect of Xiaoke Tongbi granule (XTG) on the proliferation, migration and tubule-forming ability of endothelial progenitor cells (EPCs) of rats under high glucose conditions.
 Methods: Six specific pathogen-free (SPF) and twenty-four healthy rats (mean weight = 200 ± 20 g) were used in this study. Twenty-four (24) healthy rats were treated with graded concentrations of XTG (0.75 – 2.25 g/mL) for 7 days, and were thereafter euthanized to obtain serum which was later used to treat EPCs isolated from bone marrow of SPF rats. The EPCs were seeded in culture plates pre-coated with human fibronectin, and cultured at 37 °C for 72 h in a humidified atmosphere of 5 % CO2 and 95 % air. Cell viability and apoptosis were assessed using 3 (4,5 dimethyl thiazol 2 yl) 2,5 diphenyl 2H tetrazolium bromide (MTT), and flow cytometric assays, respectively. The morphology of isolated EPCs was assessed by immunofluorescence.
 Results: The isolated EPCs exhibited normal morphology, and were CD34-positive. Proliferation and migration of EPCs, and number of tubular structures formed were significantly suppressed under high glucose conditions, but were significantly and concentration-dependently promoted by XTG treatment (p < 0.05). Treatment with XTG also significantly improved the morphology of isolated EPCs (p < 0.05). Apoptosis was significantly promoted by high glucose conditions, but was significantly and concentration-dependently reduced by XTG treatment (p < 0.05). The incidence of tubule formation in high glucose group was 0.63 %, but was progressively increased from 1.37 to 1.52 % after treatment with graded concentrations of XTG.
 Conclusion: These results indicate that XTG reverses the effect of high glucose environment on EPC proliferation, migration and tubule-forming ability.

Salvianic Acid A Regulates High-Glucose-Treated Endothelial Progenitor Cell Dysfunction via the AKT/Endothelial Nitric Oxide Synthase (eNOS) Pathway.

BackgroundThe primary cause of death in patients with diabetes mellitus (DM) is diabetic macroangiopathy, a complication that related to the function and number of endothelial progenitor cells (EPCs). Salvianic acid A (SAA) is a water-soluble active ingredient of Salvia miltiorrhiza, a traditional Chinese medicine used to treat cardiovascular diseases. The purpose of this study was to explore the effects of SAA on the function of rat EPCs cultured in vitro in a high-glucose environment.Material/MethodsBone marrow-derived EPCs from 40 Sprague-Dawley rats were identified by fluorescence staining. Cell viability, apoptosis, tube formation, lactated dehydrogenase (LDH) release, and nitric oxide (NO) production were detected by 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, flow cytometry, tube formation, LDH, and 3-amino,4-aminomethyl-2′,7′-difluorescein, and diacetate assays, respectively. The expression levels of proteins were examined by western blotting.ResultsCultured EPCs showed a cobblestone morphology and positive expression of Dil-ac-LDL and FITC-UEA-1. High glucose impaired cell viability. Different concentrations of SAA had no significant effect on EPC viability. SAA reduced the apoptosis rate and LDH release, but promoted tube formation, viability, and NO production in high-glucose-treated EPCs. The ratios of p-AKT/AKT and p-eNOS/eNOS in high-glucose-treated EPCs were elevated by SAA. Phosphoinositide 3-kinase inhibitor LY294002 blocked the rescue effects of SAA on high-glucose-treated EPCs.ConclusionsSAA protected EPCs against high-glucose-induced dysfunction via the AKT/eNOS pathway.

Mitochondrial fission protein 1 up-regulation ameliorates senescence-related endothelial dysfunction of human endothelial progenitor cells.

We investigated the contribution of mitochondrial dysfunction to the senescence of human endothelial progenitor cells (EPCs) expanded in vitro and the underlying molecular mechanism. Serial passage increased cell doubling time and those cells reaching the doubling time for more than 100% were defined as senescent EPCs, of which the activity of therapeutic angiogenesis was attenuated in mouse ischemic hindlimbs. The senescent cells, in medium free of glucose and bicarbonate, showed impaired activity in migration and tube formation. Flow cytometry indicated increased content of reactive oxygen species, mitochondria, and calcium, while bioenergetic analysis showed increased oxygen consumption and reduced ATP content. Examination of mitochondrial network showed that senescence increased the length of the network and ultrastructure analysis exhibited elongated mitochondria. Immunoblotting of the senescent EPCs demonstrated decreased expression level of fission protein1 (Fis1). In rat EPCs, the Fis1 level was decreased in the animals aged 24months or older, compared to those of 3months. Silencing of Fis1 in the young EPCs using Fis1-specific siRNA leads to appearance of phenotype resembling those of senescent cells, including elevated oxidative stress, disturbed mitochondrial network, reduced mitochondria membrane potential, decreasing ATP content, lower proliferation activity, and loss of therapeutic potential in ischemic hindlimbs. Fis1 over-expression in senescent EPCs reduced the oxidative stress, increased the proliferation, and restored the cobble stone-like morphology, senescence, bioenergetics, angiogenic potential, and therapeutic activity. In human EPCs, down-regulation of Fis1 is involved in mitochondrial dysfunction and contributes to the impaired activity of EPCs during the senescence process. Enhanced expression of Fis1 in senescent EPCs restores the youthful phenotype.

Abstract 410: A Novel Combination Therapy Using First Trimester Human Umbilical Cord-Derived Mesenchymal Stromal Cells and Endothelial Progenitor Cells Significantly Improves Angiogenesis and Cardiac Recovery Following Myocardial Infarction

Introduction: Umbilical cord-derived MSC show pericyte-like characteristics and demonstrate significant angiogenic potential via paracrine and direct support to the vasculature. Endothelial progenitor cells (EPC) have the potential to initiate vascular structures while maintaining paracrine properties following ischemic injury. Hypothesis: A combination therapy of first trimester umbilical cord MSC (FTM HUCPVC) with rat EPC will promote significant angiogenesis, cardiomyocyte survival and lead to functional cardiac recovery in a rat model of MI. Methods: MI was induced by LAD ligation on 8-week-old Fox rnu rats. 1 week later, rats with notable decrease (<30%) in ejection fraction (EF) were randomly separated into 4 treatment groups (n=9) and received intramyocardial injection of: G1: Media; G2: FTM HUPVC (3х10 6 cells/rat); G3: EPC (2.2х10 6 cells/rat); G4: FTM HUCPVC - EPC combination (1:2, total cells 3х10 6 cells/rat). Endpoint analysis was at 5-days and 4-weeks after injection. Results: 5-days after injection, exclusively G4 animals showed significant cardiac recovery, improved EF and FS compared to G1 - G2 - G3 ( p <0.01). IHC analysis showed significantly less apoptosis (caspase-3), more protease activity and sarcomeric actin in combination groups G4 compared to G1-G2-G3 ( p <0.001). More FTM HUCPVC and EPC colocalized adjunct to small capillaries in G4 . Significant increase of human Angpt-2 was detected in G4 compared to G1-G2-G3 (p<0.0001). Analysis at 4-weeks post-injection showed significant improvements in end-systolic volume (128μl) and EF (43%) in G4 compared to G2 (160μl) (35%) G3 (180μl) (30%) respectively (B) . LV contractility (dpdt, tau) significantly improved in G4 compared to G1 , G2 , G3 (p<0.001). Significant reduction in scar tissue and increased LV myocardial mass was found in G4 and G2. IHC analysis showed significantly more and larger (9um) capillaries localized in LAD injured myocardium in G4 compared to G1-G2-G3 ( p <0.001). Significantly more sarcomeric actinin and connexin-43 was identified in G4 compared to G1-G2-G3 . Conclusions: Our results show the superior potential of FTM HUCPVC - EPC combination cell therapy for faster and greater cardiovascular repair and recovery compared to single-cell-type treatments for MI.

A novel combination cell therapy using first trimester human umbilical cord-derived mesenchymal stromal cells and endothelial progenitor cells significantly improves cardiac recovery after myocardial infarction

Background & Aim Umbilical cord-derived MSC showed significant angiogenic potential via paracrine and direct cell-to-cell support to the vasculature. Endothelial progenitor cells (EPC) have the potential to develop vascular structures while maintaining paracrine properties following ischemic injury. Hypothesis A combination therapy of first trimester umbilical cord perivascular cells (FTM HUCPVC) with rat EPC will promote significant angiogenesis, cardiomyocyte survival and lead to functional cardiac recovery after MI. Methods, Results & Conclusion Methods MI was induced by LAD ligation on 8-week-old Foxrnu rats. 1 week later, rats with notable decrease ( Results 5-days after injection, exclusively G4 animals showed significant cardiac recovery, improved EF and FS compared to G1-G2-G3 (p Conclusions Our results indicate the superior potential of FTM HUCPVC - EPC combination cell therapy compared to single-cell-type treatments for myocardial regeneration and cardiac function recovery after MI.

SiRNA-mediated silencing of PAI-1 gene acts as a promoter over the recanalization of endothelial progenitor cells in rats with venous thrombosis.

With the changing lifestyle, venous thrombosis (VT) is becoming increasingly prevalent and poses a burden on the health economy. Endothelial progenitor cells (EPCs) are recruited into resolving VT. We aimed to investigate the effect of plasminogen activator inhibitor 1 (PAI-1) silencing on the recanalization of VT in rat EPCs. EPCs and VT rat models were cultured and treated with negative control-siRNA vector and PAI-1-siRNA vector, respectively. 4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound-healing test, and Matrigel-induced tubular experiment were performed to detect the ability of cell proliferation, migration, and EPCs lumen formation. Immunohistochemistry was used to observe the recanalization of thrombus. The messenger RNA (mRNA) and protein expression of PAI-1 and vascular endothelial growth factor (VEGF) were determined by reverse transcription quantitative polymerase chain reaction and Western blot analysis. PAI-1-siRNA enhances the luminal formation ability of EPCs and significantly promotes EPCs homing. In response to PAI-1 gene silencing, tissues from inferior vena cava displayed reduced mRNA and protein expression of PAI-1, increased VEGF expression as well as promoted lumen-like structures. PAI-1 gene silencing can promote the recanalization of VT by enhancement of the luminal formation ability of rats' EPCs.

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 & 2 nmol estrone, 1 & 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.

Cholesterol modification of SDF-1-specific siRNA enables therapeutic targeting of angiogenesis through Akt pathway inhibition

Neovascularization during ocular tissue repair can cause severe visual loss in the optical axis and is therefore an issue of considerable concern to ophthalmologists. Here, we introduced a cholesterol-modified siRNA delivery system targeting stromal cell-derived factor 1 (SDF-1) to treat ocular angiogenesis in vivo. SDF-1 expression was analyzed in rat endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) using quantitative PCR (qPCR). Migration ability of BMSC and HUVEC were assessed through transwell assay. The proliferation effect of chol-siSDF1 on HUVEC was measured by colony formation assay. In vivo anti-angiogenic effects of chol-siSDF1 were tested in a cornea alkali burn model and the area of cornea neovascularization was measured using computer-imaging analysis system. Then phosphorylated Akt and total Akt protein levels were measured through western blot. Results turned out that rat EPCs and BMSCs showed high SDF-1 mRNA expression, which can be down-regulated by using chol-siSDF-1. Chol-siSDF-1 could significantly inhibit migration of BMSC and HUVEC. In addition, chol-siSDF1 also could inhibit HUVEC proliferation and exert a significant anti-angiogenic effect in corneal alkali burn model. As for the mechanism, chol-siSDF1 may inhibit the neovascularization, proliferation and metastasis through inhibiting the Akt signaling pathway. Thus, cholesterol modification of siRNA targeting SDF-1 displays an effective inhibition of migration and angiogenesis, with a much longer duration of inhibition effect.

The Influence of Physicochemical Properties of Biomimetic Hydroxyapatite on the In Vitro Behavior of Endothelial Progenitor Cells and Their Interaction with Mesenchymal Stem Cells.

Calcium phosphate (CaP) substrates are successfully used as bone grafts due to their osteogenic properties. However, the influence of the physicochemical features of CaPs in angiogenesis is frequently neglected despite it being a crucial process for bone regeneration. The present work focuses on analyzing the effects of textural parameters of biomimetic calcium deficient hydroxyapatite (CDHA) and sintered beta-tricalcium phosphate (β-TCP), such as specific surface area, surface roughness, and microstructure, on the behavior of rat endothelial progenitor cells (rEPCs) and their crosstalk with rat mesenchymal stem cells (rMSCs). The higher reactivity of CDHA results in low proliferation rates in monocultured and cocultured systems. This effect is especially pronounced for rMSCs alone, and for CDHA with a fine microstructure. In terms of angiogenic and osteogenic gene expressions, the upregulation of particular genes is especially enhanced for needle-like CDHA compared to plate-like CDHA and β-TCP, suggesting the importance not only of the chemistry of the substrate, but also of its textural features. Moreover, the coculture of rEPCs and rMSCs on needle-like CDHA results in early upregulation of osteogenic modulator, i.e., protein deglycase 1 might be a possible cause of overexpression of osteogenic-related genes on the same substrate.

Icariin doped bioactive glasses seeded with rat adipose-derived stem cells to promote bone repair via enhanced osteogenic and angiogenic activities

AimsCell communication between mesenchymal stem cells and blood vessel cells are crucial for bone repair. We have previously shown that the phyto-molecule icariin significantly promoted osteogenic differentiation of rat adipose-derived stem cells (ASCs). In the present study, we aimed to investigate the relationship between icariin induced osteogenic differentiation of ASCs and angiogenesis of rat endothelial progenitor cells (EPCs). Besides, we used icariin doped 45S5 Bioglass seeded with ASCs to promote bone healing in rat calvarial bone defect models. Main methodsThe conditioned medium from undifferentiated ASCs (ASCs-CM) and icariin induced ASCs (Icariin-ASCs-CM) was obtained and the vascular endothelial growth factor (VEGF) protein secretion level was measured. The angiogenic capacity and molecular mechanism of ASC-CM and Icariin-ASCs-CM on rat EPCs was analyzed. Rat calvarial bone defect models were established and treated with scaffolds implantation. Micro-CT imaging, histological and immunohistological staining were performed on the isolated specimens at 12 weeks post-surgery. Key findingsVEGF protein expression was significantly increased after icariin treatment with the highest expression in the 10−7 M icariin group. Icariin-ASCs-CM obviously increased the angiogenesis of rat EPCs and this capacity was inhibited by a VEGF/VEGF receptor-specific binding inhibitor bevacizumab. Results of the in vivo investigations showed that all scaffolds promoted bone healing compared to the Control group. Icariin significantly improved the healing capacity of 45S5 Bioglass seeded with ASCs. SignificanceImplantation of Icariin/45S5 Bioglass seeded with rat ASCs could obviously promote both osteogenesis and angiogenesis and therefore represents an ideal candidate bone substitutes for bone repair and regeneration.

Effect of β-nerve growth factor on differentiation of endothelial progenitor cells in rats

Purpose: To investigate the effect of recombinant adenovirus-mediated human β-nerve growth factor (Ad-EGFP-hβ-NGF) on the differentiation of endothelial progenitor cells (EPCs) in rats.Methods: The successfully constructed Ad-EGFP-hβ-NGF and its negative control Ad-EGFP were infected into the isolated and purified rat EPCs to observe their morphological changes. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the levels of vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and basic fibroblast growth factor (bFGF) in different rat EPC culture solutions. Western blot was performed to determine the expression of tyrosine kinase receptor A (TrKA) protein in different groups of EPCs.Results: Primary fibrous EPCs were converted into epithelium-like cells. After infection with Ad-EGFPhβ- NGF for 1 week, some EPCs became round and exhibited neural stem cell-like changes. The expression levels of VEGF, vWF and bFGF in the Ad-EGFP-hβ-NGF infection group were significantly higher than those in the control group (p < 0.01). TrKA protein in Ad-EGFP-hβ-NGF infection was also significantly up-regulated compared with that in the negative control and blank control groups (p <0.01).Conclusion: β-NGF up-regulates the expression of TrKA receptor protein and secretion of angiogenic growth factors (i.e., VEGF, vWF and bFGF), thereby promoting the differentiation of rat EPCs, which may contribute to angiopoiesis or vascular repair.Keywords: β-Nerve growth factor, Endothelial progenitor cells, Angiogenic growth factors, Tyrosine kinase receptor A, Cell differentiation

Rat endothelial progenitor cells labeled with Molday IONTM EverGreen and in vitro MRI study

To investigate the feasibility of labeling endothelial progenitor cells (EPCs) with a novel dual modal contrast agent Molday IONTM EverGreen(MIEG) and its performance in vitro MRI. EPCs were isolated from rat bone marrow and labeled with 10, 20, 50 μg/mL MIEG, respectively. The labeling rates were identified by Prussian blue staining and fluorescence microscopy. The vitality of EPCs labeled with 20 μg/mL MIEG was detected by trypan blue exclusion test at 1 d, 1 w, 2 w, and 6 w after labeling. EPCs labeled with different concentrations of MIEG were scanned by 3.0T MRI with T1 weighted and T2 weighted imaging. The labeling rates for EPCs labeled with different concentrations of MIEG were greater than 98%,and the cytoplasm of labeled EPCs was present with Prussian blue staining. Although the green lighting level went down, the labeling rate at 6 w after labeling was greater than 90%. Trypan blue exclusion test showed that there was no significant difference in the vitality between EPCs labeled with MIEG at 1 d, 1 w, 2 w and 6 w after labeling and EPCs without labeling (all P>0.05). There was no difference in signal intensity on T1 weighted image among EPCs labeled with different concentrations of MIEG. However, the signal intensity on T2 weighted image was reduced in all labeled groups, and the signal reduction became more apparent with increased concentration of MIEG. EPCs can be effectively labeled by MIEG without interference on the cell viability at the labeled concentration of 20 μg/mL. The signal intensity change of labeled cells can be detected sensitively by T2 weighted imaging at 3.0T MRI.

105 Does Neoadjuvant Androgen Deprivation Therapy Impact Erectile Function Recovery Post-Radical Prostatectomy?

INTRODUCTION AND OBJECTIVES: Erectile dysfunction (ED) is one of the most common complications of diabetes mellitus (DM). The efficacy of treatments for diabetes mellitus-induced erectile dysfunction (DMED) is quite poor, and stem cell therapy is emerging as a useful method. This study evaluated the possibility and mechanisms of DMED treatment with endothelial progenitor cells (EPCs) genetically modified to express human telomerase reverse transcriptase (hTERT). METHODS: Rat EPCs were isolated and transfected with hTERT (EPCs-hTERT). Proliferation capacity, intracellular reactive oxygen species, paracrine character and resistance to oxidative stress of EPCs-hTERT were assessed in vitro. Diabetes was induced via an intraperitoneal injection of streptozotocin. Twenty-four male DMED rats were subjected to four treatments: DMED (DMED group), EPCs (EPCs group), EPCs transfected with control lentivirus (EPC-control group) and EPCs-hTERT (EPCs-hTERT group). A group of health rats were used as a normal control group. Intracavernosal pressure (ICP) was measured using electrical stimulation for each group after 2 weeks of EPC injections. Masson’s trichrome staining was performed to determine collagen content in penile tissues. The degree of apoptosis was assessed using western blotting analysis for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling pathway was detected. RESULTS: EPCs-hTERT secreted more growth factors than EPCs and EPCs-control. EPC resistance to oxidative stress and proliferation was dramatically improved by hTERT transfection. The ICP/ mean arterial pressure (MAP) ratio induced by electrical stimulation was markedly increased in the EPCs-hTERT group compared with the EPCs and EPCs-control group. Immunofluorescence demonstrated increased cell survival in penile tissues after EPCs-hTERT implantation. The smooth muscle/collagen ratio was markedly increased in the EPCshTERT group. TGF-b1 and p-Smad expression decreased significantly in DMED rats after EPCs-hTERT treatment. The degree of apoptosis in penile tissues of the EPCs-hTERT group was considerably reduced, as demonstrated by the decreased Bax/Bcl-2 ratio. NOS expression and NO and cGMP concentrations increased significantly in the EPCshTERT group. CONCLUSIONS: The enhanced paracrine effect, resistance to oxidative stress and EPCs-hTERT proliferation contributed to the improvement of erectile function in DMED rats.

In vivo study of alginate hydrogel conglutinating cells to polycaprolactone vascular scaffolds fabricated by electrospinning.

The aim of this study was to explore an innovative cell-seeding technology applied on artificial vascular scaffolds. Scaffolds were fabricated by electrospinning polycaprolactone (PCL) and seeded with rat endothelial progenitor cells differentiated from adipose-derived stem cells. Then, we modified the PCL scaffolds through the use of alginate hydrogel conglutinating cells (AHCC), a blank alginate hydrogel coating (BAHC), and natural sedimentation seeding cells (NSSC). The blank PCL (BP) scaffolds without any modifications were considered the blank control group. After modification, the scaffolds were implanted in a rat model. The implanted scaffolds were harvested and observed using histological and immunohistochemical methods and scanning electron microscopy (SEM) at 1, 2, and 4weeks after implantation, respectively. The best regeneration and configuration of the endothelium tissue and the most similar morphology to that of natural endangium was observed qualitatively in the AHCC scaffolds. The BP scaffolds had qualitatively the worst regeneration and configuration and the most dissimilar morphology at the same time point. In the AHCC group, cells could adhere directly on the inner surface of the vascular scaffolds, eliminating the time delay via the NSSC method prior to cell adhesion. AHCC are an effective method for seeding cells on vascular scaffolds and can eliminate the time delay for cell adhesion. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2443-2454, 2017.

Treatment of diabetes mellitus-induced erectile dysfunction using endothelial progenitor cells genetically modified with human telomerase reverse transcriptase.

The efficacy of treatments for diabetes mellitus-induced erectile dysfunction (DMED) is quite poor, and stem cell therapy is emerging as a useful method. In this study, we used endothelial progenitor cells (EPCs) overexpressing human telomerase reverse transcriptase (hTERT) for the treatment of DMED. Rat EPCs were transfected with hTERT (EPCs-hTERT). EPCs-hTERT secreted more growth factors and demonstrated enhanced proliferation and resistance to oxidative stress. Twenty-four male DMED rats were subjected to four treatments: DMED (DMED group), EPCs (EPCs group), EPCs transduced with control lentivirus (EPC-control group) and EPCs-hTERT (EPCs-hTERT group). A group of healthy rats were used as the normal control group. The erectile function in the EPCs-hTERT group was markedly increased compared with the EPCs and EPCs-control groups. The EPCs-hTERT group exhibited more growth factors, smooth muscle content and retained stem cells in penile tissues. The degree of apoptosis and collagen/smooth muscle ratio in penile tissues of the EPCs-hTERT group was considerably reduced. Endothelial nitric oxide synthase (eNOS) expression increased significantly in the EPCs-hTERT group. Taken together, these data showed that the enhanced paracrine effect, resistance to oxidative stress and proliferation of EPCs-hTERT may contribute to the improvements of erectile function in DMED rats.

Evaluation of in vitro and in vivo biocompatibility of a myo-inositol hexakisphosphate gelated polyaniline hydrogel in a rat model.

Recent advances in understanding the interaction between electricity and cells/biomolecules have generated great interest in developing biocompatible electrically conductive materials. In this study, we investigated the biocompatibility of a myo-inositol hexakisphosphate gelated polyaniline hydrogel using in vitro and in vivo experiments in a rat model. The polyaniline hydrogel was used to coat a polycaprolactone scaffold and was cultured with rat endothelial progenitor cells differentiated from rat adipose-derived stem cells. Compared with the control sample on a pristine polycaprolactone scaffold, the treated polyaniline hydrogel had the same non-poisonous/cytotoxicity grade, enhanced cell adhesion, and a higher cell proliferation/growth rate. In implant studies, the polyaniline hydrogel sample induced milder inflammatory responses than did the control at the same time points. Combining the advantages of a biocompatible hydrogel and an organic conductor, the inositol phosphate-gelated polyaniline hydrogel could be used in bioelectronics applications such as biosensors, neural probes, cell stimulators, medical electrodes, tissue engineering, and electro-controlled drug delivery.

MP86-14 TREATMENT OF DIABETES MELLITUS-INDUCED ERECTILE DYSFUNCTION USING ENDOTHELIAL PROGENITOR CELLS GENETICALLY MODIFIED WITH HUMAN TELOMERASE REVERSE TRANSCRIPTASE

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology I1 Apr 2016MP86-14 TREATMENT OF DIABETES MELLITUS-INDUCED ERECTILE DYSFUNCTION USING ENDOTHELIAL PROGENITOR CELLS GENETICALLY MODIFIED WITH HUMAN TELOMERASE REVERSE TRANSCRIPTASE Yan Zhang, Tao Wang, Jun Yang, Rui Li, Zhi Chen, Shaogang Wang, Jihong Liu, and Zhangqun Ye Yan ZhangYan Zhang More articles by this author , Tao WangTao Wang More articles by this author , Jun YangJun Yang More articles by this author , Rui LiRui Li More articles by this author , Zhi ChenZhi Chen More articles by this author , Shaogang WangShaogang Wang More articles by this author , Jihong LiuJihong Liu More articles by this author , and Zhangqun YeZhangqun Ye More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2322AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Erectile dysfunction (ED) is one of the most common complications of diabetes mellitus (DM). The efficacy of treatments for diabetes mellitus-induced erectile dysfunction (DMED) is quite poor, and stem cell therapy is emerging as a useful method. This study evaluated the possibility and mechanisms of DMED treatment with endothelial progenitor cells (EPCs) genetically modified to express human telomerase reverse transcriptase (hTERT). METHODS Rat EPCs were isolated and transfected with hTERT (EPCs-hTERT). Proliferation capacity, intracellular reactive oxygen species, paracrine character and resistance to oxidative stress of EPCs-hTERT were assessed in vitro. Diabetes was induced via an intraperitoneal injection of streptozotocin. Twenty-four male DMED rats were subjected to four treatments: DMED (DMED group), EPCs (EPCs group), EPCs transfected with control lentivirus (EPC-control group) and EPCs-hTERT (EPCs-hTERT group). A group of health rats were used as a normal control group. Intracavernosal pressure (ICP) was measured using electrical stimulation for each group after 2 weeks of EPC injections. Masson's trichrome staining was performed to determine collagen content in penile tissues. The degree of apoptosis was assessed using western blotting analysis for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling pathway was detected. RESULTS EPCs-hTERT secreted more growth factors than EPCs and EPCs-control. EPC resistance to oxidative stress and proliferation was dramatically improved by hTERT transfection. The ICP/mean arterial pressure (MAP) ratio induced by electrical stimulation was markedly increased in the EPCs-hTERT group compared with the EPCs and EPCs-control group. Immunofluorescence demonstrated increased cell survival in penile tissues after EPCs-hTERT implantation. The smooth muscle/collagen ratio was markedly increased in the EPCs-hTERT group. TGF-β1 and p-Smad expression decreased significantly in DMED rats after EPCs-hTERT treatment. The degree of apoptosis in penile tissues of the EPCs-hTERT group was considerably reduced, as demonstrated by the decreased Bax/Bcl-2 ratio. NOS expression and NO and cGMP concentrations increased significantly in the EPCs-hTERT group. CONCLUSIONS The enhanced paracrine effect, resistance to oxidative stress and EPCs-hTERT proliferation contributed to the improvement of erectile function in DMED rats. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1111 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yan Zhang More articles by this author Tao Wang More articles by this author Jun Yang More articles by this author Rui Li More articles by this author Zhi Chen More articles by this author Shaogang Wang More articles by this author Jihong Liu More articles by this author Zhangqun Ye More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effect of procyanidine on VEGFR-2 expression and transduction pathway in rat endothelial progenitor cells under high glucose conditions.

The protective effect of procyanidine and its oligomers against high glucose-mediated oxidative stress injury in endothelial progenitor cells (EPCs), and effect of procyanidin on vascular endothelial growth factor receptor-2 (VEGFR-2) expression and downstream signal pathway were analyzed in vitro. Rat bone marrow mononuclear cells were isolated, cultured under normal and high glucose (HG) conditions, and the changes in cell morphology observed. The EPCs were identified, and the oxidative stress products produced by EPCs (under normal and HG conditions) were quantified. Subsequently, an appropriate number of EPCs were cultured with and without procyanidin (OPC), and the MDA concentration and relative expression of VEGFR-2, AKT, IκB-α, and nuclear factor (NF)-κB were detected 1, 3, 5, and 7 days post-culture. We observed minor (round, translucent, gradually adhering) and significant (fusiform morphology/pebble distribution) cell morphological changes 3 and 7 days post-culture, respectively. Apoptosis and oxidative stress product release in EPCs cultured with HG increased significantly compared to the control group (P < 0.05). The oxidative stress product generation and relative expression of VEGFR-2, AKT, IkB-α, and NF-κB were not significantly affected by OPC addition in normal glucose conditions (P > 0.05); alternately, products generated as a result of oxidative stress were significantly reduced, the relative expression of VEGFR-2, AKT, and NF-κB protein was upregulated, and that of IκB-α was downregulated (P < 0.05) in HG + OPC EPCs. Therefore, procyanidin may promote cell proliferation by alleviating oxidative damage to EPCs under HG conditions, and upregulating VEGFR-2 expression and its downstream signal pathway.