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.

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.

Human Aortic Valve Interstitial Cells Display Proangiogenic Properties During Calcific Aortic Valve Disease.

OBJECTIVE: The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VICp) mesenchymal-like phenotype was confirmed by CD90+/CD73+/CD44+ expression and multipotent-like differentiation ability. When VICp were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VICp and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VICp-conditioned media and confirmed at the mRNA level in VICp compared with control VIC. Conditioned media from VICp induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VICp involvement in angiogenesis by a VEGF-A dependent mechanism. CONCLUSIONS: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VICp in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.

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.

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.

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.

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.

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.

Importance of ß2AR elevation for re-endothelialization capacity mediated by late endothelial progenitor cells in hypertensive patients.

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. ß2-Adrenergic receptor (ß2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated ß2AR signaling contributes to EPCs dysfunction, whereas enhanced ß2AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. ß2AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of ß2AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The ß2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against ß2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of ß2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired ß2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of ß2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of ß2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired ß2-adrenergic receptor (ß2AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. ß2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating ß2AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that ß2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

Ischemia-induced Netrin-4 promotes neovascularization through endothelial progenitor cell activation via Unc-5 Netrin receptor B.

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases.

Rab27a deletion impairs the therapeutic potential of endothelial progenitor cells for myocardial infarction.

Endothelial progenitor cell (EPC) transplantation has shown advantages in the treatment of myocardial infarction (MI) in animal models and clinical trials through mechanisms of direct intercellular contacts, autocrine, and paracrine. However, the effects of EPC transplantation for MI treatment remain controversial and the underlying mechanisms have not been fully elucidated. Here, we explored the role of Rab27a in the therapeutic potential of EPC transplantation in MI. We found that Rab27a knockout impaired the viability, and reduced the proliferation and tube formation function of ECPs. The recovery of cardiac function and improvement of ventricular remodeling from EPCs transplantation were significantly damaged by Rab27a deletion in vivo. Rab27a deletion inhibited the protein expression of phosphoinositide 3-kinase (PI3K) and cyclin D1 and the phosphorylation levels of Akt and FoxO3a. Therefore, Rab27a knockout suppressed the PI3K-Akt-FoxO3a/cyclin D1 signaling pathway. Furthermore, Rab27a ablation dramatically reduced exosome release in EPCs. These results demonstrated that Rab27a plays an essential role in EPC functions. The elucidation of this mechanism provides novel insights into EPC transplantation as a promising treatment for post-MI injuries.

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.

Incomplete Differentiation of Engrafted Bone Marrow Endothelial Progenitor Cells Initiates Hepatic Fibrosis in the Rat.

Normal liver sinusoidal endothelial cells (LSECs) promote quiescence of hepatic stellate cells (HSCs). Prior to fibrosis, LSECs undergo capillarization, which is permissive for HSC activation, the proximate event in hepatic fibrosis. The aims of this study were to elucidate the nature of and mechanisms leading to capillarization and to determine how LSECs promote HSC quiescence and why "capillarized LSECs" lose control of HSC activation. The contribution of bone marrow (BM) endothelial progenitor cells to capillarization was identified using rats transplanted with transgenic enhanced green fluorescent protein-positive BM. Shotgun proteomics and informatics were used to identify the LSEC mediator that maintains HSC quiescence. The study shows that capillarization is due to repair of injured LSECs by BM endothelial progenitors that engraft but fail to fully mature. Lack of maturation of BM-derived LSECs is due to cell autonomous pathways that inhibit the nitric oxide pathway. We identify heparin binding epidermal growth factor-like growth factor (HB-EGF) as the signal that maintains HSC quiescence and show that immature LSECs are unable to shed HB-EGF from the cytosolic membrane. Conclusion: Chronic liver injury can recruit BM progenitors of LSECs that engraft and fail to fully differentiate, which creates an environment that is permissive for hepatic fibrosis; elucidation of these early events in the fibrotic process will provide targets for treatment of hepatic fibrosis.

Platelet-derived growth factor-AA is a substantial factor in the ability of adipose-derived stem cells and endothelial progenitor cells to enhance wound healing.

Nonhealing wounds with various forms of complications have been a major challenge for patients with different diseases, and few data are available regarding the clinical significance of platelet-derived growth factor-AA (PDGF-AA) in the enhanced wound healing with stem cells, and the precise molecular mechanism remains unclear. The study aims to investigate the role of PDGF-AA in adipose-derived stem cells (ASCs) and endothelial progenitor cells (EPCs) enhancing wound healing. In this study, ASCs and EPCs were applied to treat wounds in an animal wound model with a wound-healing assay. We knocked down PDGF-AA expression in ASCs using the PDGF-AA short hairpin RNA technique and investigated the related molecular mechanism. The wound model and wound-healing assay of the study showed that transplantation of ASCs could enhance wound healing. The results showed that the PDGF-AA knockdown ASC group had much less improvement of wound healing than other groups treated with wild-type ASCs in wound tissues. The regulation of PDGF-AA in ASCs may contribute to improve wound healing through the PI3K/Akt/eNOS signaling pathway. The data indicated that PDGF-AA might play a vital role in ASCs and EPCs enhancing wound healing, possibly by its effects on angiogenesis. It would be a potential approach using PDGF-AA for clinical treatment of chronic wounds.-Wu, L.-W., Chen, W.-L., Huang, S.-M., Chan, J. Y.-H. Platelet-derived growth factor AA is a substantial factor in the ability of adipose-derived stem cells and endothelial progenitor cells to enhance wound healing.

Oscillating shear stress mediates mesenchymal transdifferentiation of EPCs by the Kir2.1 channel.

Although endothelial progenitor cells (EPCs) are considered to be an essential source of vascular endothelial repair, their bidirectional differentiation determines that they play a double-edged role in the restoration of endothelial injury. In this research, we investigated the effect of Kir2.1 ion channel on the transdifferentiation of endothelial progenitor cells (EPCs) under the oscillating shear stress (OSS) and the molecular mechanisms underlying the pathological vascular remodeling. EPCs were treated with OSS (± 3.5 dynes/cm2, 1 Hz) simulated with the parallel flow chamber system. The results have shown that OSS promoted the expression of α-SMA and SM22, markers of mesenchymal cells on EPCs. Moreover, OSS also increased expression of Kir2.1 in EPCs. The down-regulation of Kir2.1 reduced OSS-induced EPC mesenchymal transdifferentiation. The overexpression of Kir2.1 suppressed the angiogenic abilities of EPCs in vitro. In parallel, the overexpression of Kir2.1 on EPCs thickened the carotid tunica intima in rat carotid artery balloon injured model in vivo. Taken together, those data indicated that the OSS could facilitate the transdifferentiation of EPCs by increasing Kir2.1 expression. This study provides a novel insight into the pathogenesis of cardiovascular diseases and gives evidence for Kir2.1 as a potential therapeutic target.

Effects of endothelial progenitor cells transplantation on hyperlipidemia associated kidney damage in ApoE knockout mouse model.

BACKGROUND: Hyperlipidaemia causes kidney damage over the long term. We investigated the effect of the administration of endothelial progenitor cells (EPCs) on the progression of kidney damage in a mouse model of hyperlipidaemia. METHODS: Apolipoprotein E-knockout (ApoE-/-) mice were treated with a high-cholesterol diet after spleen resection. Twenty-four weeks later, the mice were divided into two groups and intravenously injected with PBS or EPCs. Six weeks later, the recruitment of EPCs to the kidney was monitored by immunofluorescence. The lipid, endothelial cell, and collagen contents in the kidney were evaluated by specific immunostaining. The protein expression levels of transforming growth factor-ß (TGF-ß), Smad2/3, and phospho-Smad3 (p-smad3) were detected by western blot analysis. RESULTS: ApoE-/- mice treated with a high-fat diet demonstrated glomerular lipid deposition, enlargement of the glomerular mesangial matrix, endothelial cell enlargement accompanied by vacuolar degeneration and an area of interstitial collagen in the kidney. Six weeks after EPC treatment, only a few EPCs were detected in the kidney tissues of ApoE-/- mice, mainly in the kidney interstitial area. No significant differences in TGF-ß, p-smad3 or smad2/3 expression were found between the PBS group and the EPC treatment group (TGF-ß expression, PBS group: 1.06 ± 0.09, EPC treatment group: 1.09 ± 0.17, P = 0.787; p-smad3/smad2/3 expression: PBS group: 1.11 ± 0.41, EPC treatment group: 1.05 ± 0.33, P = 0.861). CONCLUSIONS: Our findings demonstrate that hyperlipidaemia causes basement membrane thickening, glomerulosclerosis and the vascular degeneration of endothelial cells. The long-term administration of EPCs substantially has limited effect in the progression of kidney damage in a mouse model of hyperlipidaemia.

Constitutive Atg5 overexpression in mouse bone marrow endothelial progenitor cells improves experimental acute kidney injury.

BACKGROUND: Endothelial Progenitor Cells have been shown as effective tool in experimental AKI. Several pharmacological strategies for improving EPC-mediated AKI protection were identified in recent years. Aim of the current study was to analyze consequences of constitutive Atg5 activation in murine EPCs, utilized for AKI therapy. METHODS: Ischemic AKI was induced in male C57/Bl6N mice. Cultured murine EPCs were systemically injected post-ischemia, either natively or after Atg5 transfection (Adenovirus-based approach). Mice were analyzed 48 h and 6 weeks later. RESULTS: Both, native and transfected EPCs (EPCsAtg5) improved persisting kidney dysfunction at week 6, such effects were more pronounced after injecting EPCsAtg5. While matrix deposition and mesenchymal transdifferentiation of endothelial cells remained unaffected by cell therapy, EPCs, particularly EPCsAtg5 completely prevented the post-ischemic loss of peritubular capillaries. The cells finally augmented the augophagocytic flux in endothelial cells. CONCLUSIONS: Constitutive Atg5 activation augments AKI-protective effects of murine EPCs. The exact clinical consequences need to be determined.

Circulatory Rejuvenated EPCs Derived from PAOD Patients Treated by CD34+ Cells and Hyperbaric Oxygen Therapy Salvaged the Nude Mouse Limb against Critical Ischemia.

This study tested whether circulatory endothelial progenitor cells (EPCs) derived from peripheral arterial occlusive disease (PAOD) patients after receiving combined autologous CD34+ cell and hyperbaric oxygen (HBO) therapy (defined as rejuvenated EPCs) would salvage nude mouse limbs against critical limb ischemia (CLI). Adult-male nude mice (n = 40) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood prior to CD34+ cell and HBO treatment (EPCPr-T) by intramuscular injection at 3 h after CLI induction) and group 4 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood after CD34+ cell and HBO treatment (EPCAf-T) by the identical injection method). By 2, 7 and 14 days after the CLI procedure, the ischemic to normal blood flow (INBF) ratio was highest in group 1, lowest in group 2 and significantly lower in group 4 than in group 3 (p < 0.0001). The protein levels of endothelial functional integrity (CD31/von Willebrand factor (vWF)/endothelial nitric-oxide synthase (eNOS)) expressed a similar pattern to that of INBF. In contrast, apoptotic/mitochondrial-damaged (mitochondrial-Bax/caspase-3/PARP/cytosolic-cytochrome-C) biomarkers and fibrosis (Smad3/TGF-ß) exhibited an opposite pattern, whereas the protein expressions of anti-fibrosis (Smad1/5 and BMP-2) and mitochondrial integrity (mitochondrial-cytochrome-C) showed an identical pattern of INBF (all p < 0.0001). The protein expressions of angiogenesis biomarkers (VEGF/SDF-1α/HIF-1α) were progressively increased from groups 1 to 3 (all p < 0.0010). The number of small vessels and endothelial cell surface markers (CD31+/vWF+) in the CLI area displayed an identical pattern of INBF (all p < 0.0001). CLI automatic amputation was higher in group 2 than in other groups (all p < 0.001). In conclusion, EPCs from HBO-C34+ cell therapy significantly restored the blood flow and salvaged the CLI in nude mice.

Effect of MicroRNA-126a-3p on Bone Marrow Mesenchymal Stem Cells Repairing Blood-brain Barrier and Nerve Injury after Intracerebral Hemorrhage.

OBJECTIVE: Intracerebral hemorrhage (ICH) is a disease that threatens human health due to its high morbidity and mortality. On behalf of finding the better methods in the treatment of ICH, researchers pay more attention to a new technology which is finding effective genes to modify stem cells. METHODS: In this study, we isolated, cultured and identified bone marrow mesenchymal stem cells (MSCs) in vitro. Further, the MSCs (transfected with lentivirus expressing microRNA-126a-3p (miR-126)) were injected into the type Ⅶ collagenase-induced ICH rats to investigate the recovery effects of blood-brain barrier (BBB) and nerve damage in vivo. RESULTS: The MSCs surface marker molecules (CD29: 98.5%; CD90: 96.5%) were highly expressed, and the blood cell surface molecule was negatively expressed (CD45: 2%). Meanwhile, it was verified that miR-126 facilitated the differentiation of MSCs into vascular endothelial cells, owing to the rise of markers (CD31 and VE-cadherin). The modified neurological severity score, modified limb placing test score, brain water content and evans blue content were reduced after transplanted miR-126-modified MSCs. It was found that miR-126 accelerated the differentiation of MSCs into vascular endothelial cells via immunohistochemical staining in vivo. HE staining indicated the area of edema was obviously decreased compared with that in ICH + vector-MSCs group. MiR-126-modified MSCs alleviated the cell apoptosis in brain tissues by TUNEL assay. In addition, the mRNA and protein expression of protease activated receptor-1 and matrix metalloproteinase-9 were diminished, whilst the expression of zonula occludens-1 (ZO-1) and claudin-5 were enhanced in ICH+miR-126-MSCs group. Immunofluorescence assay revealed that miR-126-modified MSCs decreased the disruption of tight junction (ZO-1 and claudin-5). CONCLUSIONS: All data illustrate that miR-126-modified MSCs repair BBB and nerve injury after ICH.

Silica-coated magnetic nanoparticles labeled endothelial progenitor cells alleviate ischemic myocardial injury and improve long-term cardiac function with magnetic field guidance in rats with myocardial infarction.

Low retention of endothelial progenitor cells (EPCs) in the infarct area has been suggested to be responsible for the poor clinical efficacy of EPC therapy for myocardial infarction (MI). This study aimed to evaluate whether magnetized EPCs guided through an external magnetic field could augment the aggregation of EPCs in an ischemia area, thereby enhancing therapeutic efficacy. EPCs from male rats were isolated and labeled with silica-coated magnetic iron oxide nanoparticles to form magnetized EPCs. Then, the proliferation, migration, vascularization, and cytophenotypic markers of magnetized EPCs were analyzed. Afterward, the magnetized EPCs (1 × 106 ) were transplanted into a female rat model of MI via the tail vein at 7 days after MI with or without the guidance of an external magnet above the infarct area. Cardiac function, myocardial fibrosis, and the apoptosis of cardiomyocytes were observed at 4 weeks after treatment. In addition, EPC retention and the angiogenesis of ischemic myocardium were evaluated. Labeling with magnetic nanoparticles exhibited minimal influence to the biological functions of EPCs. The transplantation of magnetized EPCs guided by an external magnet significantly improved the cardiac function, decreased infarction size, and reduced myocardial apoptosis in MI rats. Moreover, enhanced aggregations of magnetized EPCs in the infarcted border zone were observed in rats with external magnet-guided transplantation, accompanied by the significantly increased density of microvessels and upregulated the expression of proangiogenic factors, when compared with non-external-magnet-guided rats. The magnetic field-guided transplantation of magnetized EPCs was associated with the enhanced aggregation of EPCs in the infarcted border zone, thereby improving the therapeutic efficacy of MI.