Enhancing endothelial colony-forming cells for treating diabetic vascular complications: challenges and clinical prospects.

Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia, leading to various vascular complications. Accumulating evidence indicates that endothelial colony-forming cells (ECFCs) have attractive prospects for repairing and restoring blood vessels. Thus, ECFCs may be a novel therapeutic option for diabetic patients with vascular complications who require revascularization therapy. However, it has been reported that the function of ECFCs is impaired in DM, which poses challenges for the autologous transplantation of ECFCs. In this review, we summarize the molecular mechanisms that may be responsible for ECFC dysfunction and discuss potential strategies for improving the therapeutic efficacy of ECFCs derived from patients with DM. Finally, we discuss barriers to the use of ECFCs in human studies in light of the fact that there are no published reports using these cells in humans.

Fetal liver CD34+ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice.

BACKGROUND: Transplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) into immunodeficient mice is an established method to generate humanized mice harbouring a human immune system. Different sources and methods for CD34+ isolation have been employed by various research groups, resulting in customized models that are difficult to compare. A more detailed characterization of CD34+ isolates is needed for a better understanding of engraftable hematopoietic and potentially non-hematopoietic cells. Here we have performed a direct comparison of CD34+ isolated from cord blood (CB-CD34+) or fetal liver (FL-CD34+ and FL-CD34+CD14-) and their engraftment into immunocompromised NOD/Shi-scid Il2rgnull (NOG) mice. METHODS: NOG mice were transplanted with either CB-CD34+, FL-CD34+ or FL-CD34+CD14- to generate CB-NOG, FL-NOG and FL-CD14--NOG, respectively. After 15-20 weeks, the mice were sacrificed and human immune cell reconstitution was assessed in blood and several organs. Liver sections were pathologically assessed upon Haematoxylin and Eosin staining. To assess the capability of allogenic tumor rejection in CB- vs. FL-reconstituted mice, animals were subcutaneously engrafted with an HLA-mismatched melanoma cell line. Tumor growth was assessed by calliper measurements and a Luminex-based assay was used to compare the cytokine/chemokine profiles. RESULTS: We show that CB-CD34+ are a uniform population of HSPC that reconstitute NOG mice more rapidly than FL-CD34+ due to faster B cell development. However, upon long-term engraftment, FL-NOG display increased numbers of neutrophils, dendritic cells and macrophages in multiple tissues. In addition to HSPC, FL-CD34+ isolates contain non-hematopoietic CD14+ endothelial cells that enhance the engraftment of the human immune system in FL-NOG mice. We demonstrate that these CD14+CD34+ cells are capable of reconstituting Factor VIII-producing liver sinusoidal endothelial cells (LSEC) in FL-NOG. However, CD14+CD34+ also contribute to hepatic sinusoidal dilatation and immune cell infiltration, which may culminate in a graft-versus-host disease (GVHD) pathology upon long-term engraftment. Finally, using an HLA-A mismatched CDX melanoma model, we show that FL-NOG, but not CB-NOG, can mount a graft-versus-tumor (GVT) response resulting in tumor rejection. CONCLUSION: Our results highlight important phenotypical and functional differences between CB- and FL-NOG and reveal FL-NOG as a potential model to study hepatic sinusoidal dilatation and mechanisms of GVT.

Endothelial Progenitor Cells: A Review of Molecular Mechanisms in the Pathogenesis and Endovascular Treatment of Intracranial Aneurysms.

This comprehensive review explores the multifaceted role of endothelial progenitor cells (EPCs) in vascular diseases, focusing on their involvement in the pathogenesis and their contributions to enhancing the efficacy of endovascular treatments for intracranial aneurysms (IAs). Initially discovered as CD34+ bone marrow-derived cells implicated in angiogenesis, EPCs have been linked to vascular repair, vasculogenesis, and angiogenic microenvironments. The origin and differentiation of EPCs have been subject to debate, challenging the conventional notion of bone marrow origin. Quantification methods, including CD34+ , CD133+ , and various assays, reveal the influence of factors, like age, gender, and comorbidities on EPC levels. Cellular mechanisms highlight the interplay between bone marrow and angiogenic microenvironments, involving growth factors, matrix metalloproteinases, and signaling pathways, such as phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK). In the context of the pathogenesis of IAs, EPCs play a role in maintaining vascular integrity by replacing injured and dysfunctional endothelial cells. Recent research has also suggested the therapeutic potential of EPCs after coil embolization and flow diversion, and this has led the development of device surface modifications aimed to enhance endothelialization. The comprehensive insights underscore the importance of further research on EPCs as both therapeutic targets and biomarkers in IAs.

Transplantation of alveolar macrophages improves the efficacy of endothelial progenitor cell therapy in mouse model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm births, which develops due to exposure to supplemental oxygen and mechanical ventilation. Published studies demonstrated that the number of endothelial progenitor cells (EPC) is decreased in mouse and human BPD lungs and that adoptive transfer of EPC is an effective approach in reversing the hyperoxia-induced lung damage in mouse model of BPD. Recent advancements in macrophage biology identified the specific subtypes of circulating and resident macrophages mediating the developmental and regenerative functions in the lungs. Several studies reported the successful application of macrophage therapy in accelerating the regenerative capacity of damaged tissues and enhancing the therapeutic efficacy of other transplantable progenitor cells. In the present study, we explored the efficacy of combined cell therapy with EPC and resident alveolar macrophages (rAM) in hyperoxia-induced BPD mouse model. rAM and EPC were purified from neonatal mouse lungs and were used for adoptive transfer to the recipient neonatal mice exposed to hyperoxia. Adoptive transfer of rAM alone did not result in engraftment of donor rAM into the lung tissue but increased the mRNA level and protein concentration of proangiogenic CXCL12 chemokine in recipient mouse lungs. Depletion of rAM by chlodronate-liposomes decreased the retention of donor EPC after their transplantation into hyperoxia-injured lungs. Adoptive transfer of rAM in combination with EPC enhanced the therapeutic efficacy of EPC as evidenced by increased retention of EPC, increased capillary density, improved arterial oxygenation, and alveolarization in hyperoxia-injured lungs. Dual therapy with EPC and rAM has promise in human BPD.NEW & NOTEWORTHY Recent studies demonstrated that transplantation of lung-resident endothelial progenitor cells (EPC) is an effective therapy in mouse model of bronchopulmonary dysplasia (BPD). However, key factors regulating the efficacy of EPC are unknown. Herein, we demonstrate that transplantation of tissue-resident alveolar macrophages (rAM) increases CXCL12 expression in neonatal mouse lungs. rAM are required for retention of donor EPC in hyperoxia-injured lungs. Co-transplantation of rAM and EPC improves the efficacy of EPC therapy in mouse BPD model.

α7nAChR Activation Combined with Endothelial Progenitor Cell Transplantation Attenuates Lung Injury in Diabetic Rats with Sepsis through the NF-κB Pathway.

Chronic diabetes mellitus compromises the vascular system, which causes organ injury, including in the lung. Due to the strong compensatory ability of the lung, patients always exhibit subclinical symptoms. Once sepsis occurs, the degree of lung injury is more severe under hyperglycemic conditions. The α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulating inflammation and metabolism and can improve endothelial progenitor cell (EPC) functions. In the present study, lung injury caused by sepsis was compared between diabetic rats and normal rats. We also examined whether α7nAChR activation combined with EPC transplantation could ameliorate lung injury in diabetic sepsis rats. A type 2 diabetic model was induced in rats via a high-fat diet and streptozotocin. Then, a rat model of septic lung injury was established by intraperitoneal injection combined with endotracheal instillation of LPS. The oxygenation indices, wet-to-dry ratios, and histopathological scores of the lungs were tested after PNU282987 treatment and EPC transplantation. IL-6, IL-8, TNF-α, and IL-10 levels were measured. Caspase-3, Bax, Bcl-2, and phosphorylated NF-κB (p-NF-κB) levels were determined by blotting. Sepsis causes obvious lung injury, which is exacerbated by diabetic conditions. α7nAChR activation and endothelial progenitor cell transplantation reduced lung injury in diabetic sepsis rats, alleviating inflammation and decreasing apoptosis. This treatment was more effective when PNU282987 and endothelial progenitor cells were administered together. p-NF-κB levels decreased following treatment with PNU282987 and EPCs. In conclusion, α7nAChR activation combined with EPC transplantation can alleviate lung injury in diabetic sepsis rats through the NF-κB signaling pathway.

Transplantation of Endothelial Progenitor Cells: Summary and prospect.

Endothelial Progenitor Cells (EPCs) are precursor cells of endothelial cells (ECs), which can differentiate into vascular ECs, protect from endothelial dysfunction and tissue ischemia, and reduce vascular hyperplasia. Due to these functions, EPCs are used as a candidate cell source for transplantation strategies. In recent years, a great progress was achieved in EPCs biology research, and EPCs transplantation has become a research hotspot. At present, transplanted EPCs have been used to treat ischemic diseases due to their powerful vasculogenesis and beneficial paracrine effects. Although EPCs transplantation has been proved to play an important role, the clinical application of EPCs still faces many challenges. This review briefly summarized the basic characteristics of EPCs, the process of EPCs transplantation promoting the healing of ischemic tissue, and the ways to improve the efficiency of EPCs transplantation. In addition, the application of EPCs in neurological improvement, cardiovascular and respiratory diseases and the challenges and problems in clinical application of EPCs were also discussed. In the end, the application of EPCs transplantation in regenerative medicine and tissue engineering was discussed.

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.

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.

Long-term corneal recovery by simultaneous delivery of hPSC-derived corneal endothelial precursors and nicotinamide.

Human pluripotent stem cells (hPSCs) hold great promise for the treatment of various human diseases. However, their therapeutic benefits and mechanisms for treating corneal endothelial dysfunction remain undefined. Here, we developed a therapeutic regimen consisting of the combination of hPSC-derived corneal endothelial precursors (CEPs) with nicotinamide (NAM) for effective treatment of corneal endothelial dysfunction. In rabbit and nonhuman primate models, intracameral injection of CEPs and NAM achieved long-term recovery of corneal clarity and thickness, similar with the therapeutic outcome of cultured human corneal endothelial cells (CECs). The transplanted human CEPs exhibited structural and functional integration with host resident CECs. However, the long-term recovery relied on the stimulation of endogenous endothelial regeneration in rabbits, but predominantly on the replacing function of transplanted cells during the 3-year follow-up in nonhuman primates, which resemble human corneal endothelium with limited regenerative capacity. Mechanistically, NAM ensured in vivo proper maturation of transplanted CEPs into functional CECs by preventing premature senescence and endothelial-mesenchymal transition within the TGF-ß-enriched aqueous humor. Together, we provide compelling experimental evidence and mechanistic insights of simultaneous delivery of CEPs and NAM as a potential approach for treating corneal endothelial dysfunction.

Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels.

OBJECTIVE: Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. Approach and Results: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) ß3, a downstream effector of Gαq. Activated PLCß3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCß3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. CONCLUSIONS: Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCß3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.

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.

Mesenchymal stem cell and endothelial progenitor cells coinjection improves LPS-induced lung injury via Tie2 activation and downregulation of the TLR4/MyD88 pathway.

Sepsis is one of the most important complications of infection with a high mortality rate. Recently, cell therapy has been widely used to reduce the symptoms of sepsis. It has been previously reported that mesenchymal stem cell (MSC) and endothelial progenitor cells (EPC) therapy have beneficial effects in experimental models of sepsis. The effects of coculture of MSC and EPC have not yet been used to treat sepsis. Therefore, the aim of this study was to investigate the therapeutic potential of EPC + MSC coculture on the residual effects of sepsis in a lipopolysaccharide (LPS)-induced mice model. Coinjections of EPC + MSC significantly enhanced the survival rate of LPS-induced mice, decreased concentrations of pro-inflammatory cytokines, and increased the level of anti-inflammatory cytokine. The LPS-induced mice that were treated with EPC + MSC showed a notable reduction in pulmonary edema, hepatic enzymes, and C-reactive protein level compared with the control group. Our results showed that coinjection of EPC + MSC up and downregulates Tie2 and TLR4/MyD88 signaling pathways in LPS-induced mice, respectively. Also, in vitro study showed that viability, adhesion, and migration in coculture cells is significantly decreased after being induced with 10 µg/ml LPS. Our results showed that LPS impaired the functional activity of the cocultured EPC + MSC via upregulation of the TLR4/MyD88 signaling pathway, which may be associated with decreased pTie2/Tie2 expression. In conclusion, coinjection of EPC and MSC modulated the TLR4/MyD88 signaling pathway that leads to reduce the inflammatory response. This study may provide promising results for the introduction of cocultured cells to manage infectious diseases and balance the immune response through immune regulatory function.

Therapeutic Potential of Endothelial Progenitor Cells in Pulmonary Diseases.

Compromised alveolar development and pulmonary vascular remodeling are hallmarks of pediatric lung diseases such as bronchopulmonary dysplasia (BPD) and alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Although advances in surfactant therapy, corticosteroids, and antiinflammatory drugs have improved clinical management of preterm infants, those who suffer with severe vascular complications still lack viable treatment options. Paucity of the alveolar capillary network in ACDMPV causes respiratory distress and leads to mortality in a vast majority of infants with ACDMPV. The discovery of endothelial progenitor cells (EPCs) in 1997 brought forth the paradigm of postnatal vasculogenesis and hope for promoting vascularization in fragile patient populations, such as those with BPD and ACDMPV. The identification of diverse EPC populations, both hematopoietic and nonhematopoietic in origin, provided a need to identify progenitor cell-selective markers that are linked to progenitor properties needed to develop cell-based therapies. Focusing on the future potential of EPCs for regenerative medicine, this review will discuss various aspects of EPC biology, beginning with the identification of hematopoietic, nonhematopoietic, and tissue-resident EPC populations. We will review knowledge related to cell surface markers, signature gene expression, and key transcriptional regulators and will explore the translational potential of EPCs for cell-based therapy for BPD and ACDMPV. The ability to produce pulmonary EPCs from patient-derived induced pluripotent stem cells in vitro holds promise for restoring vascular growth and function in the lungs of patients with pediatric pulmonary disorders.

Dual Stem Cell Therapy Improves the Myocardial Recovery Post-Infarction through Reciprocal Modulation of Cell Functions.

Mesenchymal stromal cells (MSC) are promising candidates for regenerative therapy of the infarcted heart. However, poor cell retention within the transplantation site limits their potential. We hypothesized that MSC benefits could be enhanced through a dual-cell approach using jointly endothelial colony forming cells (ECFC) and MSC. To assess this, we comparatively evaluated the effects of the therapy with MSC and ECFC versus MSC-only in a mouse model of myocardial infarction. Heart function was assessed by echocardiography, and the molecular crosstalk between MSC and ECFC was evaluated in vitro through direct or indirect co-culture systems. We found that dual-cell therapy improved cardiac function in terms of ejection fraction and stroke volume. In vitro experiments showed that ECFC augmented MSC effector properties by increasing Connexin 43 and Integrin alpha-5 and the secretion of healing-associated molecules. Moreover, MSC prompted the organization of ECFC into vascular networks. This indicated a reciprocal modulation in the functionality of MSC and ECFC. In conclusion, the crosstalk between MSC and ECFC augments the therapeutic properties of MSC and enhances the angiogenic properties of ECFC. Our data consolidate the dual-cell therapy as a step forward for the development of effective treatments for patients affected by myocardial infarction.

MicroRNA-375-3p in endothelial progenitor cells-derived extracellular vesicles relieves myocardial injury in septic rats via BRD4-mediated PI3K/AKT signaling pathway.

OBJECTIVE: Sepsis can induce myocardial dysfunctions and endothelial progenitor cells (EPCs)-derived extracellular vesicles (EVs) can attenuate sepsis. Concerning to that, this article is intended to decode whether microRNA (miR)-375-3p in EPCs-EVs could affect myocardial injury in sepsis. METHODS: Rat bone marrow-derived EPCs and EPCs-EVs were harvested. A rat model of sepsis was established by cecal ligation and puncture. Septic rats were injected with EPCs-EVs that interfered with miR-375-3p, after which cardiac function, inflammatory response, pathological damage, oxidative stress and apoptosis were detected in myocardial tissues. miR-375-3p, bromodomain 4 (BRD4), phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) expression in myocardial tissues, and their reciprocals were identified. RESULTS: Septic rats expressed reduced miR-375-3p and elevated BRD4 in myocardial tissues. EPCs-EVs improved cardiac function, suppressed inflammation, oxidative stress and apoptosis, as well as attenuated the pathological damage of myocardial tissues in septic rats. Up-regulated/down-regulated miR-375-3p in EPCs-EVs relieved/deteriorated myocardial injury in septic rats. miR-375-3p targeted BRD4 to activate PI3K/AKT pathway, thereafter to ameliorate myocardial injury in septic rats. CONCLUSION: It is illustrated that miR-375-3p in EPCs-EVs activates BRD4-mediated PI3K/AKT signaling pathway to ameliorate myocardial injury in septic rats, which provides a therapeutic target for myocardial injury in sepsis.

Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse-Rat Chimeras.

Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1+cKIT+ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1+cKIT+ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1+cKIT+ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1+cKIT+ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1+cKIT+ endothelial progenitors may be considered for treatment of human ACDMPV.

Transplantation of Apoptosis-Resistant Endothelial Progenitor Cells Improves Renal Function in Diabetic Kidney Disease.

Background Diabetic kidney disease is associated with glomerulosclerosis and poor renal perfusion. Increased capillary formation and improved perfusion may help to halt or reverse the injury. Transplanting apoptosis-resistant p53-silenced endothelial progenitor cells (p53sh-EPCs) may help improve vascularization and renal perfusion and could be more beneficial than another stem cell such as the mouse mesenchymal stromal cell (mMSC). Methods and Results Hyperglycemia and proteinuria were confirmed at 8 to 10 weeks in streptozotocin-induced type1 diabetic C57Bl/6 mice, followed by transplantation of 0.3 million p53sh-EPCs, Null-EPCs (control), or mMSC under each kidney capsule. Urine was collected weekly for creatinine and protein levels. Blood pressure was measured by direct arterial cannulation and renal perfusion was measured by renal ultrasound. The kidneys were harvested for histology and mRNA expression. Reduction of protein/creatinine (AUC) was observed in p53sh-EPC-transplanted mice more than null-EPC (1.8-fold, P=0.03) or null-mMSC (1.6-fold, P=0.04, n=4) transplanted mice. Markers for angiogenesis, such as endothelial nitric oxide synthase (1.7-fold, P=0.06), were upregulated post p53sh-EPC transplantation compared with null EPC. However, vascular endothelial growth factor-A expression was reduced (7-fold, P=0.0004) in mMSC-transplanted mice, compared with p53sh-EPC-transplanted mice. Isolectin-B4 staining of kidney section showed improvement of glomerular sclerosis when p53sh-EPC was transplanted, compared with null-EPC or mMSC. In addition, mean and peak renal blood velocity (1.3-fold, P=0.01, 1.4-fold, P=0.001, respectively) were increased in p53sh-EPC-transplanted mice, relative to null-EPC transplanted mice. Conclusions Apoptosis-resistant p53sh EPC transplantation could be beneficial in the treatment of diabetic kidney disease by decreasing proteinuria, and improving renal perfusion and glomerular architecture.

Cotransplantation of mesenchymal stem cells and endothelial progenitor cells for treating steroid-induced osteonecrosis of the femoral head.

Steroid-induced osteonecrosis of the femoral head (ONFH) is characterized by decreased osteogenesis, angiogenesis, and increased adipogenesis. While bone tissue engineering has been widely investigated to treat ONFH, its therapeutic effects remain unsatisfactory. Therefore, further studies are required to determine optimal osteogenesis, angiogenesis and adipogenesis in the necrotic area of the femoral head. In our study, we developed a carboxymethyl chitosan/alginate/bone marrow mesenchymal stem cell/endothelial progenitor cell (CMC/ALG/BMSC/EPC) composite implant, and evaluated its ability to repair steroid-induced ONFH. Our in vitro studies showed that BMSC and EPC coculture displayed enhanced osteogenic and angiogenic differentiation. When compared with single BMSC cultures, adipogenic differentiation in coculture systems was reduced. We also fabricated a three-dimensional (3D) CMC/ALG scaffold for loading cells, using a lyophilization approach, and confirmed its good cell compatibility characteristics, that is, high porosity, low cytotoxicity and favorable cell adhesion. 3D coculture of BMSCs and EPCs also promoted secretion of osteogenic and angiogenic factors. Then, we established an rabbit model of steroid-induced ONFH. The CMC/ALG/BMSC/EPC composite implant was transplanted into the bone tunnel of the rabbit femoral head after core decompression (CD) surgery. Twelve weeks later, radiographical and histological analyses revealed CMC/ALG/BMSC/EPC composite implants had facilitated the repair of steroid-induced ONFH, by promoting osteogenesis and angiogenesis, and reducing adipogenesis when compared with CD, CMC/ALG, CMC/ALG/BMSC and CMC/ALG/EPC groups. Thus, our data show that cotransplantation of BMSCs and EPCs in 3D scaffolds is beneficial in treating steroid-induced ONFH.

MicroRNA-126 enhances the biological function of endothelial progenitor cells under oxidative stress via PI3K/Akt/GSK3ß and ERK1/2 signaling pathways.

Endothelial progenitor cell (EPC) transplantation is a safe and effective method to treat acute myocardial infarction (AMI). However, oxidative stress leads to the death of a large number of EPCs in the early stage of transplantation, severely weakening the therapeutic effect. Previous studies demonstrated that microRNAs regulate the biological function of EPCs. The aim of the current study was to investigate the effect of microRNA on the biological function of EPCs under oxidative stress. Quantitative reverse transcription PCR was performed to detect the expression of miR-126, miR-508-5p, miR-150, and miR-16 in EPCs from rats, among which miR-126 showed a relatively higher expression. Treatment with H2O2 decreased miR-126 expression in EPCs in a dose-dependent manner. EPCs were further transfected with miR-126 mimics or inhibitors, followed by H2O2 treatment. Overexpression of miR-126 enhanced the proliferation, migration, and tube formation of H2O2-treated EPCs. MiR-126 overexpression also inhibited reactive oxygen species and malondialdehyde levels and enhanced superoxide dismutase levels, as well as increased angiopoietin (Ang)1 expression and decreased Ang2 expression in H2O2-treated EPCs. Moreover, miR-126 participated in the regulation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase 3ß (GSK3ß) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in EPCs, where both pathways were activated after miR-126 overexpression in H2O2-treated EPCs. Overall, we showed that miR-126 promoted the biological function of EPCs under H2O2-induced oxidative stress by activating the PI3K/Akt/GSK3ß and ERK1/2 signaling pathway, which may serve as a new therapeutic approach to treat AMI.

Stem/Progenitor Cells and Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by endothelial dysfunction and vascular remodeling. Despite significant advancement in our understanding of the pathogenesis of PAH in recent years, treatment options for PAH are limited and their prognosis remains poor. PAH is now seen as a severe pulmonary arterial vasculopathy with structural changes driven by excessive vascular proliferation and inflammation. Perturbations of a number of cellular and molecular mechanisms have been described, including pathways involving growth factors, cytokines, metabolic signaling, elastases, and proteases, underscoring the complexity of the disease pathogenesis. Interestingly, emerging evidence suggests that stem/progenitor cells may have an impact on disease development and therapy. In preclinical studies, stem/progenitor cells displayed an ability to promote endothelial repair of dysfunctional arteries and induce neovascularization. The stem cell-based therapy for PAH are now under active investigation. This review article will briefly summarize the updates in the research field, with a special focus on the contribution of stem/progenitor cells to lesion formation via influencing vascular cell functions and highlight the potential clinical application of stem/progenitor cell therapy to PAH.