Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

ITRI Biofilm Prevented Thoracic Adhesion in Pigs That Received Myocardial Ischemic Induction Treated by Myocardial Implantation of EPCs and ECSW Treatment.

This study tested the hypothesis that ITRI Biofilm prevents adhesion of the chest cavity. Combined extracorporeal shock wave (ECSW) + bone marrow-derived autologous endothelial progenitor cell (EPC) therapy was superior to monotherapy for improving heart function (left ventricular ejection fraction [LVEF]) in minipigs with ischemic cardiomyopathy (IC) induced by an ameroid constrictor applied to the mid-left anterior descending artery. The minipigs (n = 30) were equally designed into group 1 (sham-operated control), group 2 (IC), group 3 (IC + EPCs/by directly implanted into the left ventricular [LV] myocardium; 3 [+]/3[-] ITRI Biofilm), group 4 (IC + ECSW; 3 [+]/[3] - ITRI Biofilm), and group 5 (IC + EPCs-ECSW; 3 [+]/[3] - ITRI Biofilm). EPC/ECSW therapy was administered by day 90, and the animals were euthanized, followed by heart harvesting by day 180. In vitro studies demonstrated that cell viability/angiogenesis/cell migratory abilities/mitochondrial concentrations were upregulated in EPCs treated with ECSW compared with those in EPCs only (all Ps < 0.001). The LVEF was highest in group 1/lowest in group 2/significantly higher in group 5 than in groups 3/4 (all Ps < 0.0001) by day 180, but there was no difference in groups 3/4. The adhesion score was remarkably lower in patients who received ITRI Biofilm treatment than in those who did not (all Ps <0.01). The protein expressions of oxidative stress (NOX-1/NOX-2/oxidized protein)/apoptotic (mitochondrial-Bax/caspase3/PARP)/fibrotic (TGF-ß/Smad3)/DNA/mitochondria-damaged (γ-H2AX/cytosolic-cytochrome-C/p-DRP1), and heart failure/pressure-overload (BNP [brain natriuretic peptide]/ß-MHC [beta myosin heavy chain]) biomarkers displayed a contradictory manner of LVEF among the groups (all Ps < 0.0001). The protein expression of endothelial biomarkers (CD31/vWF)/small-vessel density revealed a similar LVEF within the groups (all Ps < 0.0001). ITRI Biofilm treatment prevented chest cavity adhesion and was superior in restoring IC-related LV dysfunction when combined with EPC/ECSW therapy compared with EPC/ECSW therapy alone.

Self-Organization of Long-Lasting Human Endothelial Capillary-Like Networks Guided by DLP Bioprinting.

Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100 µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.

TLR4-SIRT3 Mechanism Modulates Mitochondrial and Redox Homeostasis and Promotes EPCs Recruitment and Survival.

The low survival rate of endothelial progenitor cells (EPCs) in vivo which are susceptible to adverse microenvironments including inflammation and oxidative stress has become one primary challenge of EPCs transplantation for regenerative therapy. Recent studies reported functional expression of toll-like receptor (TLR) 4 on EPCs and dose-dependent effects of lipopolysaccharide (LPS) on cellular oxidative stress and angiogenic properties. However, the involved mechanism has not yet been elucidated well, and the influence of TLR4 signaling on EPCs survival and function in vivo is unknown. In the present study, we observed the effects of LPS and TLR4/SIRT3 on EPCs mitochondrial permeability and intracellular mitochondrial superoxide. We employed the monocrotaline-induced pulmonary arteriolar injury model to observe the effects of TLR4/SIRT3 on the recruitment and survival of transplanted EPCs. We found the destructive effects of 10 µg/mL LPS on mitochondrial homeostasis, and cellular viability was mediated by TLR4/SIRT3 signals at least partially, and the TLR4 mediates the early-stage recruitment of transplanted EPCs in pulmonary arteriolar inflammation injury; however, SIRT3 has more contribution to the survival of incorporated EPCs and ameliorated arteriolar remodeling in lung vascular tissue. The study provides insights for the critical role of TLR4/SIRT3 in LPS-induced oxidative stress and mitochondrial disorder in EPCs in vitro and in vivo. The TLR4/SIRT3 signaling is important for EPCs resistance against inflammation and oxidative stress and may represent a new manipulating target for developing efficient cell therapy strategy.

Purification and characterization of human adipose-resident microvascular endothelial progenitor cells.

Human adipose tissue is a rich source of adipose-derived stem cells (ASCs) and vascular endothelial progenitor cells (EPCs). However, no standardized method has been established for the isolation and purification of adipose-derived EPCs (AEPCs). The aim of this study was to establish a method for the isolation and purification of AEPCs. The stromal vascular fraction (SVF) was extracted from human lipoaspirates, and the CD45-CD31+ fraction of the SVF was collected by magnetic-activated cell sorting (MACS). The CD45-CD31+ fraction was cultured for 4.5 days, followed by a second MACS separation to collect the CD31+ fraction. Purified AEPCs were expanded without being overwhelmed by proliferating ASCs, indicating that a high level (> 95%) of AEPC purification is a key factor for their successful isolation and expansion. AEPCs exhibited typical endothelial markers, including CD31, von Willebrand factor, and the isolectin-B4 binding capacity. AEPCs formed colonies, comparable to cultured human umbilical vein endothelial cells (HUVECs). Both AEPCs and HUVECs formed capillary-like networks in the tube formation assay, with no significant difference in network lengths. We are the first to establish a purification and expansion method to isolate these cells. Because adipose tissue is a clinically accessible and abundant tissue, AEPCs may have potential advantages as a therapeutic tool for regenerative medicine.

Human Endothelial Progenitor Cells Protect the Kidney against Ischemia-Reperfusion Injury via the NLRP3 Inflammasome in Mice.

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and progression to chronic kidney disease (CKD). However, no effective therapeutic intervention has been established for ischemic AKI. Endothelial progenitor cells (EPCs) have major roles in the maintenance of vascular integrity and the repair of endothelial damage; they also serve as therapeutic agents in various kidney diseases. Thus, we examined whether EPCs have a renoprotective effect in an IRI mouse model. Mice were assigned to sham, EPC, IRI-only, and EPC-treated IRI groups. EPCs originating from human peripheral blood were cultured. The EPCs were administered 5 min before reperfusion, and all mice were killed 72 h after IRI. Blood urea nitrogen, serum creatinine, and tissue injury were significantly increased in IRI mice; EPCs significantly improved the manifestations of IRI. Apoptotic cell death and oxidative stress were significantly reduced in EPC-treated IRI mice. Administration of EPCs decreased the expression levels of NLRP3, cleaved caspase-1, p-NF-κB, and p-p38. Furthermore, the expression levels of F4/80, ICAM-1, RORγt, and IL-17RA were significantly reduced in EPC-treated IRI mice. Finally, the levels of EMT-associated factors (TGF-ß, α-SMA, Snail, and Twist) were significantly reduced in EPC-treated IRI mice. This study shows that inflammasome-mediated inflammation accompanied by immune modulation and fibrosis is a potential target of EPCs as a treatment for IRI-induced AKI and the prevention of progression to CKD.

Biodegradable Poly-ε-Caprolactone Scaffolds with ECFCs and iMSCs for Tissue-Engineered Heart Valves.

Clinically used heart valve prostheses, despite their progress, are still associated with limitations. Biodegradable poly-ε-caprolactone (PCL) nanofiber scaffolds, as a matrix, were seeded with human endothelial colony-forming cells (ECFCs) and human induced-pluripotent stem cells-derived MSCs (iMSCs) for the generation of tissue-engineered heart valves. Cell adhesion, proliferation, and distribution, as well as the effects of coating PCL nanofibers, were analyzed by fluorescence microscopy and SEM. Mechanical properties of seeded PCL scaffolds were investigated under uniaxial loading. iPSCs were used to differentiate into iMSCs via mesoderm. The obtained iMSCs exhibited a comparable phenotype and surface marker expression to adult human MSCs and were capable of multilineage differentiation. EFCFs and MSCs showed good adhesion and distribution on PCL fibers, forming a closed cell cover. Coating of the fibers resulted in an increased cell number only at an early time point; from day 7 of colonization, there was no difference between cell numbers on coated and uncoated PCL fibers. The mechanical properties of PCL scaffolds under uniaxial loading were compared with native porcine pulmonary valve leaflets. The Young's modulus and mean elongation at Fmax of unseeded PCL scaffolds were comparable to those of native leaflets (p = ns.). Colonization of PCL scaffolds with human ECFCs or iMSCs did not alter these properties (p = ns.). However, the native heart valves exhibited a maximum tensile stress at a force of 1.2 ± 0.5 N, whereas it was lower in the unseeded PCL scaffolds (0.6 ± 0.0 N, p < 0.05). A closed cell layer on PCL tissues did not change the values of Fmax (ECFCs: 0.6 ± 0.1 N; iMSCs: 0.7 ± 0.1 N). Here, a successful two-phase protocol, based on the timed use of differentiation factors for efficient differentiation of human iPSCs into iMSCs, was developed. Furthermore, we demonstrated the successful colonization of a biodegradable PCL nanofiber matrix with human ECFCs and iMSCs suitable for the generation of tissue-engineered heart valves. A closed cell cover was already evident after 14 days for ECFCs and 21 days for MSCs. The PCL tissue did not show major mechanical differences compared to native heart valves, which was not altered by short-term surface colonization with human cells in the absence of an extracellular matrix.

Effects of adipose-derived stromal cells and endothelial progenitor cells on adipose transplant survival and angiogenesis.

BACKGROUND: A paracrine mechanism is thought to mediate the proangiogenic capacity of adipose-derived stromal/stem cells (ASCs). However, the precise mechanism by which ASCs promote the formation of blood vessels by endothelial progenitor cells (EPCs) is unclear. METHODS: The EPCs-ASCs cocultures prepared in different ratios were subjected to tube formations assay to verify whether ASCs could directly participate in the tube genesis. The supernatant from cultured ASCs was used to stimulate EPCs to evaluate the effects on the angiogenic property of EPCs, as well as capacity for migration and invasion. A coculture model with transwell chamber were used to explore the regulation of angiogenesis markers expression in EPCs by ASCs. We then mixed ASCs with EPCs and transplanted them with adipose tissue into nude mice to evaluate the effects on angiogenesis in adipose tissue grafts. RESULTS: In the EPCs-ASCs cocultures, the tube formation was significantly decreased as the relative abundance of ASCs increased, while the ASCs was found to migrate and integrated into the agglomerates formed by EPCs. The supernatant from ASCs cultures promoted the migration and invasion of EPCs and the ability to form capillary-like structures. The expression of multiple angiogenesis markers in EPCs were significantly increased when cocultured with ASCs. In vivo, ASCs combined with EPC promoted vascularization in the fat transplant. Immunofluorescence straining of Edu and CD31 indicated that the Edu labeled EPC did not directly participate in the vascularization inside the fat tissue. CONCLUSIONS: ADSC can participate in the tube formation of EPC although it cannot form canonical capillary structures. Meanwhile, Soluble factors secreted by ASCs promotes the angiogenic potential of EPCs. ASCs paracrine signaling appears to promote angiogenesis by increasing the migration and invasion of EPCs and simultaneously upregulating the expression of angiogenesis markers in EPCs. The results of in vivo experiments showed that ASCs combined with EPCs significantly promote the formation of blood vessels in the fat implant. Remarkably, EPCs may promote angiogenesis by paracrine regulation of endogenous endothelial cells (ECs) rather than direct participation in the formation of blood vessels.

Lymphatic endothelial cell fate specification in the mammalian embryo: An historical perspective.

Development of the mammalian lymphatic vasculature is a stepwise process requiring the specification of lymphatic endothelial cell progenitors in the embryonic veins, and their subsequent budding to give rise to most of the mature lymphatic vasculature. In mice, formation of the lymphatic vascular network starts inside the cardinal vein at around E9.5 when a subpopulation of venous endothelial cells gets committed into the lymphatic lineage by their acquisition of Prox1 expression. Identification of critical genes regulating lymphatic development facilitated the detailed cellular and molecular characterization of some of the cellular and molecular mechanisms regulating the early steps leading to the formation of the mammalian lymphatic vasculature. A better understanding of basic aspects of early lymphatic development, and the availability of novel tools and animal models has been instrumental in the identification of important novel functional roles of this vasculature network.

Impact of following a healthy dietary pattern with co-consuming wolfberry on number and function of blood outgrowth endothelial cells from middle-aged and older adults.

Blood outgrowth endothelial cells (BOECs) have received growing attention in relation to cardiovascular disease (CVD). However, the effect of diet intervention, a primary strategy for CVD prevention, on BOECs is not reported. This study aims to investigate the effect of following a healthy dietary pattern (HDP) with or without wolfberry consumption, healthy food with potential cardiovascular benefits, on the number and function of BOECs in middle-aged and older adults. Twenty-four subjects consumed either an HDP only (n = 9) or an HDP supplemented with 15 g day-1 wolfberries (n = 15) for 16 weeks. At pre- and post-intervention, vascular health biomarkers and composite CVD risk indicators were assessed. BOECs were derived from peripheral blood mononuclear cells and their angiogenic and migration activities were measured. Isolated BOECs have typical endothelial cobblestone morphology, express von Willebrand factor and KDR. Consuming an HDP improved the BOEC colony's growth rate, which was demonstrated by significant time effects in the colony's culture time between passages 1 and 2 (P = 0.038). Both interventions increased BOECs' tube formation capacity. Moreover, HDP intervention contributed to a time effect on BOEC migration activity (P = 0.040 for t1/2gap). Correlation analysis revealed that BOEC colony number was positively associated with blood pressure, atherogenic index, vascular age, and Framingham risk score. In conclusion, adherence to an HDP improved BOECs' function in middle-aged and older populations, while additional wolfberry consumption did not provide an enhanced effect. Our results provide mechanistic dissection on the beneficial effects on BOECs of dietary pattern modification.

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.

Experimental Study on the Effect of Allogeneic Endothelial Progenitor Cells on Wound Healing in Diabetic Mice.

Endothelial progenitor cells (EPCs) are involved in the neovascularization in traumatic and ischemic sites, but EPCs are "detained" in bone marrow under diabetic conditions, which results in reduction of the number of EPCs and their biological activity in peripheral blood. Based on our previous study to mobilize autologous bone marrow EPCs by administering AMD3100+G-CSF to realize the optimal effect, our present study is aimed at exploring the effects of transplanting EPCs locally in a wound model of diabetic mice. First, we prepared and identified EPCs, and the biological functions and molecular characteristics were compared between EPCs from DB/+ and DB/DB mice. Then, we performed full-thickness skin resection in DB/DB mice and tested the effect of local transplantation of EPCs on skin wound healing. The wound healing process was recorded using digital photographs. The animals were sacrificed on postoperative days 7, 14, and 17 for histological and molecular analysis. Our results showed that DB/+ EPCs were biologically more active than those of DB/DB EPCs. When compared with the control group, local transplantation of EPCs accelerated wound healing in DB/DB mice by promoting wound granulation tissue formation, angiogenesis, and collagen fiber deposition, but there was no significant difference in wound healing between DB/+ EPCs and DB/DB EPCs transplanted into the wound. Furthermore, local transplantation of EPCs promoted the expression of SDF-1, CXCR4, and VEGF. We speculated that EPC transplantation may promote wound healing through the SDF-1/CXCR4 axis. This point is worth exploring further. Present data are of considerable significance because they raise the possibility of promoting wound healing by isolating autologous EPCs from the patient, which provides a new approach for the clinical treatment of diabetic wounds in the future.

The decrease of endothelial progenitor cells caused by high altitude may lead to coronary heart disease.

OBJECTIVE: The purpose of this study was to explore the relationship between the number of endothelial progenitor cells (EPCs) and coronary heart disease (CHD). PATIENTS AND METHODS: A total of 24 patients with CHD were chosen from Lanzhou City and Xianyang City, and then, 24 healthy controls who matched the CHD group in gender, age and address were chosen as control group. C-reactive protein (CRP) and c-reaction protein (hs-CRP) were detected. The levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), homocysteine (Hcy), hypoxia-inducible factor-1 (HIF-1α) and stromal cell-derived factor 1 (SDF-1α) were detected. RESULTS: The number of EPCs in control groups was both increased compared with CHD group (p<0.05). The number of EPCs in Xianyang control group was increased compared with Lanzhou control group (p<0.05). Compared with the control group, the levels of TC, LDL and CRP in the CHD group were higher (p<0.05). Compared with Lanzhou control group, Hcy level was decreased in Lanzhou CHD group (p<0.05). Compared with Xianyang control group, the levels of IL-8 and VEGF were increased, but the levels of HIF-1α and Hcy were decreased in the Xianyang CHD group (p<0.05). The expressions of IL-8, VEGF, Hcy and HIF-1α were increased in Lanzhou control group than the Xianyang control group (p<0.05). In Lanzhou CHD group, Spearman correlation analysis showed that the number of EPCs was negatively related to hs-CRP content (r=-0.631, p<0.05). CONCLUSIONS: The decrease of EPCs caused by high altitude may increase the expressions of various cytokines, leading to the occurrence of CHD.

Preparation of controlled degradation of insulin-like growth factor 1/spider silk protein nanofibrous membrane and its effect on endothelial progenitor cell viability.

The present study aimed to prepare a kind of controlled-releasing insulin-like growth factor 1 (IGF-1)/spider silk protein nanofibrous membrane using a electrostatic spinning method and evaluated its effect on the cell viability of endothelial progenitor cells (EPCs). Recombinant spidroin named as GMCDRSSP-IgF-1 was electro-spun into nanofibrous membrane which can be degraded by protease and be capable of sustained-release of IGF-1. The membrane can be degraded after being treated with thrombin. The release assay results showed that IGF-1 concentration could be maintained at 20 ng/ml for a long time with treatment of Tobacco Etch Virus (TEV) protease. The viability of EPCs on GMCDRSSP-IgF-1 nanofibrous membrane was significantly increased with the presence of TEV protease. The controlled and sustained release of IGF-1 from the nanofibrous membrane could promote the adhesion and viability of EPCs. In summary, the nanofibrous membrane that exhibits controlled degradation and sustained release of IGF-1 was prepared with electrostatic spinning from genetically modified recombinant spider silk protein. The nanofibrous membrane exhibited good blood compatibility and cytocompatibility. With the presence of TEV protease, the sustained-release of IGF-1 significantly promoted the adhesion and viability of EPCs. The new nanofibrous membrane can be potentially used as a scaffold for EPCs culture in vitro and future in vivo studies.

Slight up-regulation of Kir2.1 channel promotes endothelial progenitor cells to transdifferentiate into a pericyte phenotype by Akt/mTOR/Snail pathway.

It was shown that endothelial progenitor cells (EPCs) have bidirectional differentiation potential and thus perform different biological functions. The purpose of this study was to investigate the effects of slight up-regulation of the Kir2.1 channel on EPC transdifferentiation and the potential mechanism on cell function and transformed cell type. First, we found that the slight up-regulation of Kir2.1 expression promoted the expression of the stem cell stemness factors ZFX and NS and inhibited the expression of senescence-associated ß-galactosidase. Further studies showed the slightly increased expression of Kir2.1 could also improve the expression of pericyte molecular markers NG2, PDGFRß and Desmin. Moreover, adenovirus-mediated Kir2.1 overexpression had an enhanced contractile response to norepinephrine of EPCs. These results suggest that the up-regulated expression of the Kir2.1 channel promotes EPC transdifferentiation into a pericyte phenotype. Furthermore, the mechanism of EPC transdifferentiation to mesenchymal cells (pericytes) was found to be closely related to the channel functional activity of Kir2.1 and revealed that this channel could promote EPC EndoMT by activating the Akt/mTOR/Snail signalling pathway. Overall, this study suggested that in the early stage of inflammatory response, regulating the Kir2.1 channel expression affects the biological function of EPCs, thereby determining the maturation and stability of neovascularization.

Bradykinin Protects Human Endothelial Progenitor Cells from High-Glucose-Induced Senescence through B2 Receptor-Mediated Activation of the Akt/eNOS Signalling Pathway.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown. METHODS: Cord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated ß-galactosidase (SA-ß-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot. RESULTS: B2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK. CONCLUSION: BK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.

Low-dose metronomic doxorubicin inhibits mobilization and differentiation of endothelial progenitor cells through REDD1-mediated VEGFR-2 downregulation.

Low-dose metronomic chemotherapy has been introduced as a less toxic and effective strategy to inhibit tumor angiogenesis, but its anti-angiogenic mechanism on endothelial progenitor cells (EPCs) has not been fully elucidated. Here, we investigated the functional role of regulated in development and DNA damage response 1 (REDD1), an endogenous inhibitor of mTORC1, in low-dose doxorubicin (DOX)-mediated dysregulation of EPC functions. DOX treatment induced REDD1 expression in bone marrow mononuclear cells (BMMNCs) and subsequently reduced mTORC1-dependent translation of endothelial growth factor (VEGF) receptor (Vegfr)-2 mRNA, but not that of the mRNA transcripts for Vegfr-1, epidermal growth factor receptor, and insulin-like growth factor-1 receptor. This selective event was a risk factor for the inhibition of BMMNC differentiation into EPCs and their angiogenic responses to VEGF-A, but was not observed in Redd1-deficient BMMNCs. Low-dose metronomic DOX treatment reduced the mobilization of circulating EPCs in B16 melanoma-bearing wild-type but not Redd1-deficient mice. However, REDD1 overexpression inhibited the differentiation and mobilization of EPCs in both wild-type and Redd1-deficient mice. These data suggest that REDD1 is crucial for metronomic DOX-mediated EPC dysfunction through the translational repression of Vegfr-2 transcript, providing REDD1 as a potential therapeutic target for the inhibition of tumor angiogenesis and tumor progression. [BMB Reports 2021; 54(9): 470-475].

Deferoxamine accelerates endothelial progenitor cell senescence and compromises angiogenesis.

Senescence reduces the circulating number and angiogenic activity of endothelial progenitor cells (EPCs), and is associated with aging-related vascular diseases. However, it is very time-consuming to obtain aged cells (~1 month of repeated replication) or animals (~2 years) for senescence studies. Here, we established an accelerated senescence model by treating EPCs with deferoxamine (DFO), an FDA-approved iron chelator. Four days of low-dose (3 µM) DFO induced senescent phenotypes in EPCs, including a senescent pattern of protein expression, impaired mitochondrial bioenergetics, altered mitochondrial protein levels and compromised angiogenic activity. DFO-treated early EPCs from young and old donors (< 35 vs. > 70 years old) displayed similar senescent phenotypes, including elevated senescence-associated ß-galactosidase activity and reduced relative telomere lengths, colony-forming units and adenosine triphosphate levels. To validate this accelerated senescence model in vivo, we intraperitoneally injected Sprague-Dawley rats with DFO for 4 weeks. Early EPCs from DFO-treated rats displayed profoundly senescent phenotypes compared to those from control rats. Additionally, in hind-limb ischemic mice, DFO pretreatment compromised EPC angiogenesis by reducing both blood perfusion and capillary density. DFO thus accelerates EPC senescence and appears to hasten model development for cellular senescence studies.

Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury.

Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis.

Self-assembly of differentiated progenitor cells facilitates spheroid human skin organoid formation and planar skin regeneration.

Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate. Methods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions. Results: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting. Conclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.