Reprogramming tumor-associated macrophages to outcompete endovascular endothelial progenitor cells and suppress tumor neoangiogenesis.

Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.

PD-L1-positive circulating endothelial progenitor cells associated with immune response to PD-1 blockade in patients with head and neck squamous cell carcinoma.

A number of the inhibitors against programmed death protein 1 (PD-1) have been approved to treat recurrent or metastatic squamous cell carcinoma of head and neck (HNSCC). The interaction between PD-1 and its ligand (PD-L1) serves as an immune checkpoint that governs cytotoxic immune effectors against tumors. Numerous clinical trials of PD-1/PD-L1 inhibitors have so far been discordant about having sufficient PD-L1 expression in the tumor as a prerequisite for a successful anti-PD-1 treatment. On the other hand, vascular endothelial cells modulate immune activities through PD-L1 expression, and thus it is possible that the expressions of circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CPCs) could affect antitumor immunity as well as neoangiogenesis. Here we investigated the potential involvement of PD-L1+ CECs and PD-L1+ CPCs in PD-1 blockade treatments for HNSCC patients. We measured CD8+ T cells, CECs, and CPCs in the peripheral blood of the HNSCC patients treated by anti-PD-1 therapies. We found that their PD-L1+ CPC expression before anti-PD1 therapies was strongly correlated with treatment responses and overall survival. Moreover, if the first infusion of PD-1 inhibitors reduced ≥ 50% PD-L1+ CPCs, a significantly better outcome could be predicted. In these patients as well as in an animal model of oral cancer, Pd-l1+ CPC expression was associated with limited CD8+ T-cell infiltration into the tumors, and anti-PD-1 treatments also targeted Pd-l1+ CPCs and increased CD8+ T-cell infiltration. Our results highlight PD-L1+ CPC as a potential regulator in the anti-PD-1 treatments for HNSCC.

Ex-vivo expanded CD34+ cell transplantation alleviates fibrotic liver injury via innate immune modulation in metabolic dysfunction-associated steatohepatitis mice.

BACKGROUND: In drug-induced liver injury, vascular endothelial progenitor cells, specifically the CD34+ cell fractions, have been found to decrease liver fibrosis and promote regeneration. However, it is unclear whether CD34+ cell transplantation has anti-fibrogenic effects on MASH, which has previously been treated effectively with anti-angiogenic therapy. We investigated the efficacy of ex vivo-expanded CD34+ cells in treating MASH livers. MATERIALS AND METHODS: Diet-induced MASH mice were fed a choline-deficient, L-amino acid-defined, high-fat diet for 12 or 20 weeks, and were designated as a mild and a severe fibrosis model, respectively. Mouse bone marrow CD34+ cells were expanded for 7 days, transplanted into each mouse once or twice 2 weeks later, and sacrificed at 4 weeks after the first transplantation. RESULTS: Expanded CD34+ cell transplantation ameliorated liver fibrosis, regardless of fibrosis degree, as indicated by the decrease in α-smooth muscle actin-positive cells, hydroxyproline concentration, and fibrogenic gene expression of Col1a1 and Timp1. Furthermore, engrafted CD34+ cells reduced alanine transaminase levels, the number of TUNEL+ hepatocytes, and 8-OHdG concentration. RNA-sequencing data showed that "defense response to virus" was the most down-regulated category in the Gene Ontology analysis and subsequent analysis revealed the suppression of RIG-I-like receptors/Irf7/Stat1/Cxcl10 axis in expanded CD34+ cell-transplanted livers. Finally, the downregulation of CXCL10 expression inhibits the mobilization of inflammatory immune cells, macrophages, T cells, and natural killer cells to the MASH liver. CONCLUSIONS: These findings suggest that transplanted expanded CD34+ cells alleviate fibrotic liver injury in MASH mouse models through possible modulation of the innate immune response, which is abnormally activated by hepatocyte lipotoxicity.

Endothelial dysfunction and cardiovascular risk in post-COVID-19 patients after 6- and 12-months SARS-CoV-2 infection.

INTRODUCTION: SARS-CoV-2 infection causes severe endothelial damage, an essential step for cardiovascular complications. Endothelial-colony forming cells (ECFCs) act as a biomarker of vascular damage but their role in SARS-CoV-2 remain unclear. The aim of this study was to assess whether the number of ECFCs and angiogenic biomarkers remained altered after 6 and 12-months post-infection and whether this imbalance correlated with the presence of long-COVID syndrome and other biological parameters measured. METHODS: Seventy-two patients were recruited at different time-points after overcoming COVID-19 and thirty-one healthy controls. All subjects were matched for age, gender, BMI, and comorbidities. ECFCs were obtained from peripheral blood and cultured with specific conditions. RESULTS: The results confirm the presence of a long-term sequela in post-COVID-19 patients, with an abnormal increase in ECFC production compared to controls (82.8% vs. 48.4%, P < 0.01) that is maintained up to 6-months (87.0% vs. 48.4%, P < 0.01) and 12-months post-infection (85.0% vs. 48.4%, P < 0.01). Interestingly, post-COVID-19 patients showed a significant downregulation of angiogenesis-related proteins compared to controls indicating a clear endothelial injury. Troponin, NT-proBNP and ferritin levels, markers of cardiovascular risk and inflammation, remained elevated up to 12-months post-infection. Patients with lower numbers of ECFC exhibited higher levels of inflammatory markers, such as ferritin, suggesting that ECFCs may play a protective role. Additionally, long-COVID syndrome was associated with higher ferritin levels and with female gender. CONCLUSIONS: These findings highlight the presence of vascular sequela that last up to 6- and 12-months post-infection and point out the need for preventive measures and patient follow-up.

Endothelial Progenitor Cells and Macrophage Subsets Recruitment in Postischemic Mouse Hind Limbs.

INTRODUCTION: Peripheral arterial disease (PAD) occurs from atherosclerotic obstruction of arteries in the lower extremities. Restoration of perfusion requires angiogenesis and arteriogenesis through migration and differentiation of endothelial progenitor cells (EPCs) and macrophages at the site of injury. The time of recruitment has not been fully investigated. In this study, we investigated the infiltration of these cells in murine hind limb ischemia (HLI) model of PAD. METHODS: EPCs and M1-like and M2-like macrophages from ischemic skeletal muscles were quantified by flow cytometry at day-0, 1, 3, 7, and 14 post-HLI. RESULTS: The abundance of EPCs increased from day 1 and was highest on day 7 until day 14. M1-like population similarly increased and was highest on day 14 during the experiment. M2-like population was significantly greater than M1-like at baseline but surpassed the highest value of M1-like by day 7 during the experiment. Muscle regeneration and capillary density also increased and were highest at days 3 and 7, respectively, during the experiment. All mice achieved near full perfusion recovery by day 14. CONCLUSION: Thus, we observed a gradual increase in the percentage of EPC's and this was temporally paralleled with initial increase in M1-like followed by sustained increased in M2-like macrophages and perfusion recovered post-HLI.

Transplantation of endothelial progenitor cells improves myocardial hypertrophy in spontaneously hypertensive rats through HO-1/CREB3/AKT axis.

Hypertensive myocardial hypertrophy produces a hostile microenvironment characterized by cardiomyocyte hypertrophy, inflammation and oxidative stress, which also leads to endothelial progenitor cells (EPCs) dysfunction, preventing EPC migration, adhesion and angiogenesis. Heme oxygenase-1 (HO-1) is an intracellular protein that plays an important role in angiogenesis and cell survival. The upregulation of cAMP response element-binding protein 3 (CREB3) is closely related to the formation of endothelial cells. The purpose of this study was to evaluate the role of HO-1 and CREB3 in EPCs and their effects on hypertensive myocardial hypertrophy. EPCs were transfected with HO-1 adenoviral overexpression vector (Ad-HO-1) or together with CREB3 siRNA (si-CREB3), or transfected with CREB3 adenoviral overexpression vector (Ad-CREB3) or together with HO-1 siRNA, and then treated with 100 nM Ang Ⅱ for 12 h. Overexpressing HO-1 or CREB3 promoted adhesion to extracellular matrix, cell migration, and angiogenesis, inhibited the secretion of inflammatory factors TNF-α and IL-6, and reduced ROS level, ICAM-1 and MCP-1 mRNA expression levels in EPCs treated with Ang Ⅱ. Online prediction and Co-IP assay showed that HO-1 interacts with CREB3, and they promote expression of each other. EPC-conditioned medium supplemented with CREB3 recombinant protein decreased the levels of ANP and BNP mRNA in H9C2 cells treated with Ang Ⅱ and alleviated oxidative stress. Ad-CREB3 transfected EPCs promoted the phosphorylation of AKT in vivo and in vitro, thereby improving myocardial swelling and dysfunction in SHR rats. Taken together, transplantation of CREB3 overexpressing EPCs alleviates myocardial hypertrophy in spontaneously hypertensive rats by promoting HO-1 protein expression and AKT phosphorylation.

Endothelial progenitor cells in pregnancy-related diseases.

Placental neovascularization plays a crucial role in fetomaternal circulation throughout pregnancy and is dysregulated in several pregnancy-related diseases, including preeclampsia, gestational diabetes mellitus, and fetal growth restriction. Endothelial progenitor cells (EPCs) are a heterogeneous population of cells that differentiate into mature endothelial cells, which influence vascular homeostasis, neovascularization, and endothelial repair. Since their discovery in 1997 by Asahara et al., the role of EPCs in vascular biology has garnered a lot of interest. However, although pregnancy-related conditions are associated with changes in the number and function of EPCs, the reported findings are conflicting. This review discusses the discovery, isolation, and classification of EPCs and highlights discrepancies between current studies. Overviews of how various diseases affect the numbers and functions of EPCs, the role of EPCs as biomarkers of pregnancy disorders, and the potential therapeutic applications involving EPCs are also provided.

Cell-Extracellular Matrix Interactions Play Multiple Essential Roles in Aortic Arch Development.

RATIONALE: Defects in the morphogenesis of the fourth pharyngeal arch arteries (PAAs) give rise to lethal birth defects. Understanding genes and mechanisms regulating PAA formation will provide important insights into the etiology and treatments for congenital heart disease. OBJECTIVE: Cell-ECM (extracellular matrix) interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin α5ß1 and Fn1 (fibronectin) expressed in the Isl1 lineages regulate PAA formation. The objective of the current studies was to investigate cellular mechanisms by which integrin α5ß1 and Fn1 regulate aortic arch artery morphogenesis. METHODS AND RESULTS: Using temporal lineage tracing, whole-mount confocal imaging, and quantitative analysis of the second heart field (SHF) and endothelial cell (EC) dynamics, we show that the majority of PAA EC progenitors arise by E7.5 in the SHF and contribute to pharyngeal arch endothelium between E7.5 and E9.5. Consequently, SHF-derived ECs in the pharyngeal arches form a plexus of small blood vessels, which remodels into the PAAs by 35 somites. The remodeling of the vascular plexus is orchestrated by signals dependent on the pharyngeal ECM microenvironment, extrinsic to the endothelium. Conditional ablation of integrin α5ß1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates aortic arch artery morphogenesis at multiple steps: (1) accumulation of SHF-derived ECs in the pharyngeal arches, (2) remodeling of the EC plexus in the fourth arches into the PAAs, and (3) differentiation of neural crest-derived cells adjacent to the PAA endothelium into vascular smooth muscle cells. CONCLUSIONS: PAA formation is a multistep process entailing dynamic contribution of SHF-derived ECs to pharyngeal arches, the remodeling of endothelial plexus into the PAAs, and the remodeling of the PAAs into the aortic arch artery and its major branches. Cell-ECM interactions regulated by integrin α5ß1 and Fn1 play essential roles at each of these developmental stages.

LncRNA NEAT-2 regulate the function of endothelial progenitor cells in experimental Sepsis model.

BACKGROUND: Sepsis is a life-threatening disease with a limited effectiveness and the potential mechanism remains unclear. LncRNA NEAT-2 is reported to be involved in the regulation of cardiovascular disease. This study aimed to investigate the function of NEAT-2 in sepsis. METHODS: We built sepsis animal model with Male Balb/C mice induced by cecal ligation and puncture (CLP). A total of 54 mice were randomly assigned into eight groups: sham operation group (n = 18), CLP group (n = 18), CLP plus si-control group (n = 3), CLP plus si-NEAT2 group (n = 3), CLP plus mimic control group (n = 3), CLP plus miR-320 group (n = 3), CLP plus normal saline group (n = 3), and normal control group (n = 3). The number of peripheral endothelial progenitor cells (EPCs), the expression level of NEAT-2 and miR-320 were detected during progression of sepsis, as well as the number of peripheral EPCs and level of TNF-α, IL-6, VEGF, ALT, AST and Cr. In addition, the function of EPCs was evaluated after NEAT-2 knockdown and miR-320 overexpression in vitro. RESULTS: The number of circulating EPCs increased significantly in sepsis. NEAT-2 expression was significantly increased in the progress of sepsis, accompanied with miR-320 downregulated. NEAT-2 knockdown and miR-320 overexpression attenuated hepatorenal function and increased cytokines in sepsis. Moreover, NEAT-2 knockdown and miR-320 overexpression decreased the proliferation, migration and angiogenesis of endothelial progenitor cells in vitro. CONCLUSIONS: LncRNA-NEAT2 regulated the number and function of endothelial progenitor cells via miR-320 in sepsis, which may contribute to the development of novel potential clinical therapy for sepsis.

Sodium tanshinone IIA sulfonate ameliorates neointima by protecting endothelial progenitor cells in diabetic mice.

BACKGROUND: Endothelial progenitor cells (EPCs) transplantation is one of the effective therapies for neointima associated with endothelial injury. Diabetes impairs the function of EPCs and cumbers neointima prevention of EPC transplantation with an ambiguous mechanism. Sodium Tanshinone IIA Sulfonate (STS) is an endothelium-protective drug but whether STS protects EPCs in diabetes is still unknown. METHODS: EPCs were treated with High Glucose (HG), STS, and Nucleotide-binding Domain-(NOD) like Receptor 3 (NLRP3), caspase-1, the Receptor of Advanced Glycation End products (AGEs) (RAGE) inhibitors, Thioredoxin-Interacting Protein (TXNIP) siRNA, and EPC proliferation, differentiation functions, and senescence were detected. The treated EPCs were transplanted into db/db mice with the wire-injured Common Carotid Artery (CCA), and the CD31 expression and neointima were detected in the CCA inner wall. RESULTS: We found that STS inhibited HG-induced expression of NLRP3, the production of active caspase-1 (p20) and mature IL-1ß, the expression of catalase (CAT) cleavage, γ-H2AX, and p21 in EPCs. STS restored the expression of Ki67, CD31 and von Willebrand Factor (vWF) in EPCs; AGEs were found in the HG-treated EPCs supernatant, and RAGE blocking inhibited the expression of TXNIP and the production of p20, which was mimicked by STS. STS recovered the expression of CD31 in the wire-injured CCA inner wall and the prevention of neointima in diabetic mice with EPCs transplantation. CONCLUSION: STS inhibits the aggravated neointima hyperplasia by protecting the proliferation and differentiation functions of EPC and inhibiting EPC senescence in diabetic mice. The mechanism is related to the preservation of CAT activity by inhibiting the RAGE-TXNIP-NLRP3 inflammasome pathway.

Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells.

BACKGROUND: We evaluated the efficacy of the factor Xa inhibitor rivaroxaban on the differentiation ability of vascular endothelial progenitor cells (EPCs), which play roles in vascular injury repair and atherogenesis. Antithrombotic treatment in patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI) is challenging, and current guidelines recommend oral anticoagulant monotherapy 1 year or more after PCI. However, biological evidence of the pharmacological effects of anticoagulants is insufficient. METHODS: EPC colony-forming assays were performed using peripheral blood-derived CD34-positive cells from healthy volunteers. Adhesion and tube formation of cultured EPCs were assessed in human umbilical cord-derived CD34-positive cells. Endothelial cell surface markers were assessed using flow cytometry, and Akt and endothelial nitric oxide synthase (eNOS) phosphorylation were examined using western blot analysis of EPCs. Adhesion, tube formation and endothelial cell surface marker expression was observed in EPCs transfected with small interfering RNA (siRNA) against protease-activated receptor (PAR)-2. Finally, EPC behaviors were assessed in patients with atrial fibrillation undergoing PCI in whom warfarin was changed to rivaroxaban. RESULTS: Rivaroxaban increased the number of large EPC colonies and increased the bioactivities of EPCs, including adhesion and tube formation. Rivaroxaban also increased vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, Tie-2, and E-selectin expression as well as Akt and eNOS phosphorylation. PAR-2 knockdown increased the bioactivities of EPCs and endothelial cell surface marker expression. Patients in whom the number of large colonies increased after switching to rivaroxaban showed better vascular repair. CONCLUSIONS: Rivaroxaban increased the differentiation ability of EPCs, leading to potential advantages in the treatment of coronary artery disease.

Downregulated calmodulin expression contributes to endothelial cell impairment in diabetes.

Endothelial dysfunction, a central hallmark of cardiovascular pathogenesis in diabetes mellitus, is characterized by impaired endothelial nitric oxide synthase (eNOS) and NO bioavailability. However, the underlying mechanisms remain unclear. Here in this study, we aimed to identify the role of calmodulin (CaM) in diabetic eNOS dysfunction. Human umbilical vein endothelial cells and murine endothelial progenitor cells (EPCs) treated with high glucose (HG) exhibited downregulated CaM mRNA/protein and vascular endothelial growth factor (VEGF) expression with impeded eNOS phosphorylation and cell migration/tube formation. These perturbations were reduplicated in CALM1-knockdown cells but prevented in CALM1-overexpressing cells. EPCs from type 2 diabetes animals behaved similarly to HG-treated normal EPCs, which could be rescued by CALM1-gene transduction. Consistently, diabetic animals displayed impaired eNOS phosphorylation, endothelium-dependent dilation, and CaM expression in the aorta, as well as deficient physical interaction of CaM and eNOS in the gastrocnemius. Local CALM1 gene delivery into a diabetic mouse ischemic hindlimb improved the blunted limb blood perfusion and gastrocnemius angiogenesis, and foot injuries. Diabetic patients showed insufficient foot microvascular autoregulation, eNOS phosphorylation, and NO production with downregulated CaM expression in the arterial endothelium, and abnormal CALM1 transcription in genome-wide sequencing analysis. Therefore, our findings demonstrated that downregulated CaM expression is responsible for endothelium dysfunction and angiogenesis impairment in diabetes, and provided a novel mechanism and target to protect against diabetic endothelial injury.

GDF11 promotes wound healing in diabetic mice via stimulating HIF-1ɑ-VEGF/SDF-1ɑ-mediated endothelial progenitor cell mobilization and neovascularization.

Non-healing diabetic wounds (DW) are a serious clinical problem that remained poorly understood. We recently found that topical application of growth differentiation factor 11 (GDF11) accelerated skin wound healing in both Type 1 DM (T1DM) and genetically engineered Type 2 diabetic db/db (T2DM) mice. In the present study, we elucidated the cellular and molecular mechanisms underlying the action of GDF11 on healing of small skin wound. Single round-shape full-thickness wound of 5-mm diameter with muscle and bone exposed was made on mouse dorsum using a sterile punch biopsy 7 days following the onset of DM. Recombinant human GDF11 (rGDF11, 50 ng/mL, 10 µL) was topically applied onto the wound area twice a day until epidermal closure (maximum 14 days). Digital images of wound were obtained once a day from D0 to D14 post-wounding. We showed that topical application of GDF11 accelerated the healing of full-thickness skin wounds in both type 1 and type 2 diabetic mice, even after GDF8 (a muscle growth factor) had been silenced. At the cellular level, GDF11 significantly facilitated neovascularization to enhance regeneration of skin tissues by stimulating mobilization, migration and homing of endothelial progenitor cells (EPCs) to the wounded area. At the molecular level, GDF11 greatly increased HIF-1ɑ expression to enhance the activities of VEGF and SDF-1ɑ, thereby neovascularization. We found that endogenous GDF11 level was robustly decreased in skin tissue of diabetic wounds. The specific antibody against GDF11 or silence of GDF11 by siRNA in healthy mice mimicked the non-healing property of diabetic wound. Thus, we demonstrate that GDF11 promotes diabetic wound healing via stimulating endothelial progenitor cells mobilization and neovascularization mediated by HIF-1ɑ-VEGF/SDF-1ɑ pathway. Our results support the potential of GDF11 as a therapeutic agent for non-healing DW.

Endothelial progenitor cell derived exosomes mediated miR-182-5p delivery accelerate diabetic wound healing via down-regulating PPARG.

Diabetic wound is one of the most common and serious complications of diabetes, which is characterized by abnormal number and quality of wound repair related cells. Previous studies have shown that human endothelial progenitor cells derived exosomes (EPCs-EXO) can promote diabetic wound healing through modulating vascular endothelial cell function. The purpose of this study was to investigate the biological effects and molecular mechanisms of EPCs-EXO on diabetic wound healing. The regulation of EPCs-EXO on human immortalized epidermal cell line HaCaT in high glucose (HG) environment was evaluated. Our data showed that EPCs-EXO promoted the proliferation, migration, while inhibited apoptosis of HaCaTs challenged by HG via elevating miR-182-5p expression level in vitro. Skin wound healing was significantly enhanced by EPCs-EXO in diabetic mice. Moreover, bioinformatics analyses and luciferase reporter assay indicated that exosomal miR-182-5p was bound to PPARG 3' UTR sequence and inhibited the expression of PPARG. Collectively, our findings provided a new role of EPCs-EXO in the clinical treatment of diabetic skin wounds. Diabetic wound is one of the most common and serious complications of diabetes, which is characterized by abnormal number and quality of wound repair related cells. Previous studies have shown that human endothelial progenitor cells derived exosomes (EPCs-EXO) can promote diabetic wound healing through modulating vascular endothelial cell function. The purpose of this study was to investigate the biological effects and molecular mechanisms of EPCs-EXO on diabetic wound healing. The regulation of EPCs-EXO on human immortalized epidermal cell line HaCaT in high glucose (HG) environment was evaluated. Our data showed that EPCs-EXO promoted the proliferation, migration, while inhibited apoptosis of HaCaTs challenged by HG via elevating miR-182-5p expression level in vitro. Skin wound healing was significantly enhanced by EPCs-EXO in diabetic mice. Moreover, bioinformatics analyses and luciferase reporter assay indicated that exosomal miR-182-5p was bound to PPARG 3' UTR sequence and inhibited the expression of PPARG. Collectively, our findings provided a new role of EPCs-EXO in the clinical treatment of diabetic skin wounds.

Novel Function of Nogo-A as Negative Regulator of Endothelial Progenitor Cell Angiogenic Activity: Impact in Oxygen-Induced Retinopathy.

Endothelial Progenitor Cells (EPCs) can actively participate in revascularization in oxygen-induced retinopathy (OIR). Yet the mechanisms responsible for their dysfunction is unclear. Nogo-A, whose function is traditionally related to the inhibition of neurite function in the central nervous system, has recently been documented to display anti-angiogenic pro-repellent properties. Based on the significant impact of EPCs in retinal vascularization, we surmised that Nogo-A affects EPC function, and proceeded to investigate the role of Nogo-A on EPC function in OIR. The expression of Nogo-A and its specific receptor NgR1 was significantly increased in isolated EPCs exposed to hyperoxia, as well as in EPCs isolated from rats subjected to OIR compared with respective controls (EPCs exposed to normoxia). EPCs exposed to hyperoxia displayed reduced migratory and tubulogenic activity, associated with the suppressed expression of prominent EPC-recruitment factors SDF-1/CXCR4. The inhibition of Nogo-A (using a Nogo-66 neutralizing antagonist peptide) or siRNA-NGR1 in hyperoxia-exposed EPCs restored SDF-1/CXCR4 expression and, in turn, rescued the curtailed neovascular functions of EPCs in hyperoxia. The in vivo intraperitoneal injection of engineered EPCs (Nogo-A-inhibited or NgR1-suppressed) in OIR rats at P5 (prior to exposure to hyperoxia) prevented retinal and choroidal vaso-obliteration upon localization adjacent to vasculature; coherently, the inhibition of Nogo-A/NgR1 in EPCs enhanced the expression of key angiogenic factors VEGF, SDF-1, PDGF, and EPO in retina; CXCR4 knock-down abrogated suppressed NgR1 pro-angiogenic effects. The findings revealed that hyperoxia-induced EPC malfunction is mediated to a significant extent by Nogo-A/NgR1 signaling via CXCR4 suppression; the inhibition of Nogo-A in EPCs restores specific angiogenic growth factors in retina and the ensuing vascularization of the retina in an OIR model.

Fibroblast growth factor 21 reverses high-fat diet-induced impairment of vascular function via the anti-oxidative pathway in ApoE knockout mice.

Circulating endothelial progenitor cells (EPCs), which function in vascular repair, are the markers of endothelial dysfunction and vascular health. Fibroblast growth factor 21 (FGF21), a liver-secreted protein, plays a crucial role in glucose homeostasis and lipid metabolism. FGF21 has been reported to attenuate the progression of atherosclerosis, but its impact on EPCs under high oxidative stress conditions remains unclear. In vitro studies showed that the ß-klotho protein was expressed in cultured EPCs and that its expression was upregulated by FGF21 treatment. Hydrogen peroxide (H2 O2 )-induced oxidative stress impaired EPC function, including cell viability, migration and tube formation. Pretreatment with FGF21 restored the functions of EPCs after the exposure to H2 O2 . Administration of N(ω)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, inhibited the effects of FGF21 in alleviating oxidative injury by suppressing endothelial nitric oxide synthase (eNOS). In an in vivo study, the administration of FGF21 significantly reduced total cholesterol (TC) and blood glucose levels in apolipoprotein E (ApoE)-deficient mice that were fed a high-fat diet (HFD). Endothelial function, as reflected by acetylcholine-stimulated aortic relaxation, was improved after FGF21 treatment in ApoE-deficient mice. Analysis of mRNA levels in the aorta indicated that FGF21 increased the mRNA expression of eNOS and upregulated the expression of the antioxidant genes superoxide dismutase (SOD)1 and SOD2 in ApoE-deficient mice. These data suggest that FGF21 improves EPC functions via the Akt/eNOS/nitric oxide (NO) pathway and reverses endothelial dysfunction under oxidative stress. Therefore, administration of FGF21 may ameliorate a HFD-induced vascular injury in ApoE-deficient mice.

Endothelial colony-forming cell-derived exosomal miR-21-5p regulates autophagic flux to promote vascular endothelial repair by inhibiting SIPL1A2 in atherosclerosis.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) represents an efficient therapeutic method for atherosclerosis but conveys a risk of causing restenosis. Endothelial colony-forming cell-derived exosomes (ECFC-exosomes) are important mediators during vascular repair. This study aimed to investigate the therapeutic effects of ECFC-exosomes in a rat model of atherosclerosis and to explore the molecular mechanisms underlying the ECFC-exosome-mediated effects on ox-LDL-induced endothelial injury. METHODS: The effect of ECFC-exosome-mediated autophagy on ox-LDL-induced human microvascular endothelial cell (HMEC) injury was examined by cell counting kit-8 assay, scratch wound assay, tube formation assay, western blot and the Ad-mCherry-GFP-LC3B system. RNA-sequencing assays, bioinformatic analysis and dual-luciferase reporter assays were performed to confirm the interaction between the miR-21-5p abundance of ECFC-exosomes and SIPA1L2 in HMECs. The role and underlying mechanism of ECFC-exosomes in endothelial repair were explored using a high-fat diet combined with balloon injury to establish an atherosclerotic rat model of vascular injury. Evans blue staining, haematoxylin and eosin staining and western blotting were used to evaluate vascular injury. RESULTS: ECFC-exosomes were incorporated into HMECs and promoted HMEC proliferation, migration and tube formation by repairing autophagic flux and enhancing autophagic activity. Subsequently, we demonstrated that miR-21-5p, which is abundant in ECFC-exosomes, binds to the 3' untranslated region of SIPA1L2 to inhibit its expression, and knockout of miR-21-5p in ECFC-exosomes reversed ECFC-exosome-decreased SIPA1L2 expression in ox-LDL-induced HMEC injury. Knockdown of SIPA1L2 repaired autophagic flux and enhanced autophagic activity to promote cell proliferation in ox-LDL-treated HMECs. ECFC-exosome treatment attenuated vascular endothelial injury, regulated lipid balance and activated autophagy in an atherogenic rat model of vascular injury, whereas these effects were eliminated with ECFC-exosomes with knockdown of miR-21-5p. CONCLUSIONS: Our study demonstrated that ECFC-exosomes protect against atherosclerosis- or PTCA-induced vascular injury by rescuing autophagic flux and inhibiting SIAP1L2 expression through delivery of miR-21-5p. Video Abstract.

Endothelial progenitor cells overexpressing platelet derived growth factor-D facilitate deep vein thrombosis resolution.

To assess the therapeutic efficacy of PDGF-D-overexpressing endothelial progenitor cells (EPCs) in deep vein thrombosis. Inferior vena cava thrombosis was induced in female Sprague Dawley (SD) rats. Animals were injected via the distal vena cava with EPCs overexpressing PDGF-D after transfection with a lentiviral vector containing the PDGF-D gene. The effect on thrombosis in animals who received EPCs was evaluated using MSB staining, immunohistochemistry, immunofluorescence, and venography; the steady-state mRNA and protein levels of PDGF-D and its receptor (PDGF-Rß) were determined by RT-PCR and Western blotting, respectively; and the PDGF-D-induced mobilization of circulating EPCs was estimated by flow cytology. Compared with controls, injection of EPCs overexpressing PDGF-D was associated with increased thrombosis resolution; recanalization; PDGF-D and PDGF-Rß expression; induction of monocyte homing; and mobilization of EPCs to the venous circulation. In a rat model, transplantation of PDGF-D-overexpressing EPCs facilitated the resolution of deep vein thrombosis.

Mobilized and apheresis-collected endothelial progenitor cells with plerixafor.

BACKGROUND: Endothelial progenitor cells (EPCs) are immature cells able to proliferate and contribute to endothelial repair, vascular homeostasis, neovascularization, and angiogenesis. It therefore seems likely that circulating EPCs have therapeutic potential in ischemic and vascular diseases. In this study we evaluated the efficiency of EPC mobilization and collection by large volume leukapheresis in subjects with hematological diseases, treated with plerixafor in association with G-CSF. METHODS: Twenty-two patients with lymphoid malignancies underwent rHuG-CSF and plerixafor treatment followed by leukapheresis. Blood samples before and after treatment and apheresis liquid sample were taken and analyzed by flow cytometry in order to quantified EPC. RESULTS: The percentage of CD34+ cells and EPCs among circulating total nuclear cells (TNCs) increased significantly by approximately 2-fold and 3-fold, respectively, after plerixafor treatment. Consequently, the absolute number of CD34+ cells and EPCs were increased 4-fold after plerixafor treatment. The median PB concentration of EPCs before and after treatment were 0.77/µL (0.31-2.15) and 3.41/µL (1.78-4.54), respectively, P < .0001. The total EPCs collected per patient were 3.3×107 (0.8×107 -6.8×107 ). CONCLUSION: We have shown that plerixafor in combination with G-CSF allows the mobilization and collection of large amounts of EPCs along with CD34+ cells in lymphoid neoplasm patients. The possibility to collect and to store these cells could represent a promising therapeutic tool for the treatment of ischemic complications without the need of in vitro expansion.

Construction and a preliminary study of paracrine effect of bone marrow-derived endothelial progenitor cell sheet.

The release of paracrine factors from endothelial progenitor cell (EPC) sheet is a central mechanism of tissue repair. The purpose of this study was to constuct the rat bone marrow derived-endothelial progenitor cell (BM-EPCs) sheet and investigate invest the role of stromal cell-derived factor-1α (SDF-1α)/CXCR4 axis in the biological function of BM-EPCs sheet. BM-EPC cells were identified by the cell-surface markers-CD34/CD133/VE-cadherin/KDR using flow cytometry and dual affinity for acLDL and UEA-1. After 7 days of incubation, the BM-EPC single-cell suspensions were seeded on thermo-sensitive plate to harvest the BM-EPC cell sheets. The expression levels of SDF-1α/CXCR4 axis-associated genes and proteins were examined using RT-qPCR and western blot analysis, and enzyme-linked immunosorbent assay (ELISA) was applied to determine the concentration of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and SDF-1α in the cell culture medium. The BM-EPC cell sheets were successfully harvested. Moreover, BM-EPC cell sheets have superior migration and tube formation activity when compared with single cell suspension. When capillary-like tube were formed from EPCs sheets, the releasing of paracrine factors such as VEGF, EGF and SDF-1α were increased. To reveal the mechanism of tube formation of BM-EPCs sheets, our research showed that the activation of PI3K/AKT/eNOS pathway was involved in the process, because the phosphorylation of CXCR, PI3K, AKT and eNOS were increased. BM-EPC cell sheets have superior paracrine and tube formation activity than the BM-EPC single-cell. The strong ability to secrete paracrine factors was be potentially related to the SDF-1α/CXCR4 axis through PI3K/AKT/eNOS pathway.