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.

HOXD9 regulated mitophagy to promote endothelial progenitor cells angiogenesis and deep vein thrombosis recanalization and resolution.

BACKGROUND: Deep vein thrombosis (DVT) is a common vascular surgical disease caused by the coagulation of blood in the deep veins, and predominantly occur in the lower limbs. Endothelial progenitor cells (EPCs) are multi-functional stem cells, which are precursors of vascular endothelial cells. EPCs have gradually evolved into a promising treatment strategy for promoting deep vein thrombus dissolution and recanalization through the stimulation of various physical and chemical factors. METHODS: In this study, we utilized a mouse DVT model and performed several experiments including qRT-PCR, Western blot, tube formation, wound healing, Transwell assay, immunofluorescence, flow cytometry analysis, and immunoprecipitation to investigate the role of HOXD9 in the function of EPCs cells. The therapeutic effect of EPCs overexpressing HOXD9 on the DVT model and its mechanism were also explored. RESULTS: Overexpression of HOXD9 significantly enhanced the angiogenesis and migration abilities of EPCs, while inhibiting cell apoptosis. Additionally, results indicated that HOXD9 specifically targeted the HRD1 promoter region and regulated the downstream PINK1-mediated mitophagy. Interestingly, intravenous injection of EPCs overexpressing HOXD9 into mice promoted thrombus dissolution and recanalization, significantly decreasing venous thrombosis. CONCLUSIONS: The findings of this study reveal that HOXD9 plays a pivotal role in stimulating vascular formation in endothelial progenitor cells, indicating its potential as a therapeutic target for DVT management.

Retracted: Osteoprotegerin (OPG) Promotes Recruitment of Endothelial Progenitor Cells (EPCs) via CXCR4 Signaling Pathway to Improve Bone Defect Repair.

The Editors of Medical Science Monitor wish to inform you that the above manuscript has been retracted from publication due to concerns with the credibility and originality of the study, the manuscript content, and the Figure images. Reference: Rongfeng Zhang, Jianwei Liu, Shengpeng Yu, Dong Sun, Xiaohua Wang, Jingshu Fu, Jie Shen, Zhao Xie. Osteoprotegerin (OPG) Promotes Recruitment of Endothelial Progenitor Cells (EPCs) via CXCR4 Signaling Pathway to Improve Bone Defect Repair. Med Sci Monit, 2019; 25: 5572-5579. DOI: 10.12659/MSM.916838.

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

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

Activation of PPARδ in bone marrow endothelial progenitor cells improves their hematopoiesis-supporting ability after myelosuppressive injury.

Dysfunctional bone marrow (BM) endothelial progenitor cells (EPCs) with high levels of reactive oxygen species (ROS) are responsible for defective hematopoiesis in poor graft function (PGF) patients with acute leukemia or myelodysplastic neoplasms post-allotransplant. However, the underlying mechanism by which BM EPCs regulate their intracellular ROS levels and the capacity to support hematopoiesis have not been well clarified. Herein, we demonstrated decreased levels of peroxisome proliferator-activated receptor delta (PPARδ), a lipid-activated nuclear receptor, in BM EPCs of PGF patients compared with those with good graft function (GGF). In vitro assays further identified that PPARδ knockdown contributed to reduced and dysfunctional BM EPCs, characterized by the impaired ability to support hematopoiesis, which were restored by PPARδ overexpression. Moreover, GW501516, an agonist of PPARδ, repaired the damaged BM EPCs triggered by 5-fluorouracil (5FU) in vitro and in vivo. Clinically, activation of PPARδ by GW501516 benefited the damaged BM EPCs from PGF patients or acute leukemia patients in complete remission (CR) post-chemotherapy. Mechanistically, we found that increased expression of NADPH oxidases (NOXs), the main ROS-generating enzymes, may lead to elevated ROS level in BM EPCs, and insufficient PPARδ may trigger BM EPC damage via ROS/p53 pathway. Collectively, we found that defective PPARδ contributes to BM EPC dysfunction, whereas activation of PPARδ in BM EPCs improves their hematopoiesis-supporting ability after myelosuppressive therapy, which may provide a potential therapeutic target not only for patients with leukemia but also for those with other cancers.

Translational Research of the Acute Effects of Negative Emotions on Vascular Endothelial Health: Findings From a Randomized Controlled Study.

BACKGROUND: Provoked anger is associated with an increased risk of cardiovascular disease events. The underlying mechanism linking provoked anger as well as other core negative emotions including anxiety and sadness to cardiovascular disease remain unknown. The study objective was to examine the acute effects of provoked anger, and secondarily, anxiety and sadness on endothelial cell health. METHODS AND RESULTS: Apparently healthy adult participants (n=280) were randomized to an 8-minute anger recall task, a depressed mood recall task, an anxiety recall task, or an emotionally neutral condition. Pre-/post-assessments of endothelial health including endothelium-dependent vasodilation (reactive hyperemia index), circulating endothelial cell-derived microparticles (CD62E+, CD31+/CD42-, and CD31+/Annexin V+) and circulating bone marrow-derived endothelial progenitor cells (CD34+/CD133+/kinase insert domain receptor+ endothelial progenitor cells and CD34+/kinase insert domain receptor+ endothelial progenitor cells) were measured. There was a group×time interaction for the anger versus neutral condition on the change in reactive hyperemia index score from baseline to 40 minutes (P=0.007) with a mean±SD change in reactive hyperemia index score of 0.20±0.67 and 0.50±0.60 in the anger and neutral conditions, respectively. For the change in reactive hyperemia index score, the anxiety versus neutral condition group by time interaction approached but did not reach statistical significance (P=0.054), and the sadness versus neutral condition group by time interaction was not statistically significant (P=0.160). There were no consistent statistically significant group×time interactions for the anger, anxiety, and sadness versus neutral condition on endothelial cell-derived microparticles and endothelial progenitor cells from baseline to 40 minutes. CONCLUSIONS: In this randomized controlled experimental study, a brief provocation of anger adversely affected endothelial cell health by impairing endothelium-dependent vasodilation.

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

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

Dulaglutide restores endothelial progenitor cell levels in diabetic mice and mitigates high glucose-induced endothelial injury through SIRT1-mediated mitochondrial fission.

Type 2 diabetes mellitus (T2DM) significantly impairs the functionality and number of endothelial progenitor cells (EPCs) and resident endothelial cells, critical for vascular repair and regeneration, exacerbating the risk of vascular complications. GLP-1 receptor agonists, like dulaglutide, have emerged as promising therapeutic agents due to their multifaceted effects, including the enhancement of EPC activity and protection of endothelial cells. This study investigates dulaglutide's effects on peripheral blood levels of CD34+ and CD133+ cells in a mouse model of lower limb ischemia and its protective mechanisms against high-glucose-induced damage in endothelial cells. Results demonstrated that dulaglutide significantly improves blood flow, reduces tissue damage and inflammation in ischemic limbs, and enhances glycemic control. Furthermore, dulaglutide alleviated high-glucose-induced endothelial cell damage, evident from improved tube formation, reduced reactive oxygen species accumulation, and restored endothelial junction integrity. Mechanistically, dulaglutide mitigated mitochondrial fission in endothelial cells under high-glucose conditions, partly through maintaining SIRT1 expression, which is crucial for mitochondrial dynamics. This study reveals the potential of dulaglutide as a therapeutic option for vascular complications in T2DM patients, highlighting its role in improving endothelial function and mitochondrial integrity.

Human Cord Blood Endothelial Progenitor Cells and Pregnancy Complications (Preeclampsia, Gestational Diabetes Mellitus, and Fetal Growth Restriction).

Hemangioblasts give rise to endothelial progenitor cells (EPCs), which also express the cell surface markers CD133 and c-kit. They may differentiate into the outgrowth endothelial cells (OECs) that control neovascularization in the developing embryo. According to numerous studies, reduced levels of EPCs in circulation have been linked to human cardiovascular disorders. Furthermore, preeclampsia and senescence have been linked to levels of EPCs produced from cord blood. Uncertainties surround how preeclampsia affects the way EPCs function. It is reasonable to speculate that preeclampsia may have an impact on the function of fetal EPCs during the in utero period; however, the present literature suggests that maternal vasculopathies, including preeclampsia, damage fetal circulation. Additionally, the differentiation potential and general activity of EPCs may serve as an indicator of the health of the fetal vascular system as they promote neovascularization and repair during pregnancy. Thus, the purpose of this review is to compare-through the assessment of their quantity, differentiation potency, angiogenic activity, and senescence-the angiogenic function of fetal EPCs obtained from cord blood for normal and pregnancy problems (preeclampsia, gestational diabetes mellitus, and fetal growth restriction). This will shed light on the relationship between the angiogenic function of fetal EPCs and pregnancy complications, which could have an effect on the management of long-term health issues like metabolic and cardiovascular disorders in offspring with abnormal vasculature development.

Patients With Coronary Microvascular Dysfunction Have Less Circulating α-Klotho.

BACKGROUND: Coronary microvascular dysfunction (CMD) represents an early functional characteristic of coronary vascular aging. Klotho (α-klotho) is a circulating protein inversely linked to physiological aging. We examined low klotho as a potential marker for vascular aging in patients with CMD and no coronary artery disease. METHODS AND RESULTS: Patients undergoing nonurgent angiogram for chest pain who had no coronary artery disease underwent invasive coronary microvascular and endothelial function testing. CMD was defined by ≤50% increase in coronary blood flow (percentage change in coronary blood flow) in response to intracoronary acetylcholine or coronary flow reserve ≤2. Fresh arterial whole blood was used to analyze circulating endothelial progenitor cells with flow cytometry. Stored arterial plasma was used for klotho analysis by ELISA. Participants with CMD (n=62) were compared with those without CMD (n=36). Those with CMD were age 55±10 years (versus 51±11 years; P=0.07) and 73% women (versus 81%; P=0.38). Traditional risk factors for coronary artery disease were similar between groups. Patients with CMD had less klotho (0.88±1.50 versus 1.75±2.38 ng/mL; P=0.03), and the odds of low klotho in CMD were significant in a logistic regression model after adjusting for traditional cardiovascular risk factors (odds ratio [OR], 0.80 [95% CI, 0.636-0.996]; P=0.05). Higher klotho was associated with higher numbers of endothelial progenitor cells with vascular regenerative potential (CD34+ and CD34+CD133+KDR+). Among a subgroup of patients with atherosclerotic cardiovascular disease risk <5% (n=58), CMD remained associated with lower klotho (OR, 0.80 [95% CI, 0.636-0.996]; P=0.047). CONCLUSIONS: Klotho may be a biomarker for CMD and may be a therapeutic target for groups of patients without significant traditional cardiovascular risk.

The bone marrow endothelial progenitor cell response to septic infection.

Early increase in the level of endothelial progenitor cells (EPCs) in the systemic circulation occurs in patients with septic infection/sepsis. The significance and underlying mechanisms of this response remain unclear. This study investigated the bone marrow EPC response in adult mice with septic infection induced by intravenous injection (i.v.) of Escherichia coli. For in vitro experiments, sorted marrow stem/progenitor cells (SPCs) including lineage(lin)-stem cell factor receptor (c-kit)+stem cell antigen-1 (Sca-1)-, lin-c-kit+, and lin- cells were cultured with or without lipopolysaccharides (LPSs) and recombinant murine vascular endothelial growth factor (VEGF) in the absence and presence of anti-Sca-1 crosslinking antibodies. In a separate set of experiments, marrow lin-c-kit+ cells from green fluorescence protein (GFP)+ mice, i.v. challenged with heat-inactivated E. coli or saline for 24 h, were subcutaneously implanted in Matrigel plugs for 5 weeks. Marrow lin-c-kit+ cells from Sca-1 knockout (KO) mice challenged with heat-inactivated E. coli for 24 h were cultured in the Matrigel medium for 8 weeks. The marrow pool of EPCs bearing the lin-c-kit+Sca-1+VEGF receptor 2 (VEGFR2)+ (LKS VEGFR2+) and LKS CD133+VEGFR2+ surface markers expanded rapidly following septic infection, which was supported by both proliferative activation and phenotypic conversion of marrow stem/progenitor cells. Increase in marrow EPCs and their reprogramming for enhancing angiogenic activity correlated with cell-marked upregulation of Sca-1 expression. Sca-1 was coupled with Ras-related C3 botulinum toxin substrate 2 (Rac2) in signaling the marrow EPC response. Septic infection caused a substantial increase in plasma levels of IFN-γ, VEGF, G-CSF, and SDF-1. The early increase in circulating EPCs was accompanied by their active homing and incorporation into pulmonary microvasculature. These results demonstrate that the marrow EPC response is a critical component of the host defense system. Sca-1 signaling plays a pivotal role in the regulation of EPC response in mice with septic infection.

Exosomal hsa_circ_0093884 derived from endothelial progenitor cells promotes therapeutic neovascularization via miR-145/SIRT1 pathway.

Therapeutic neovascularization is a strategy to promote blood vessel growth and improve blood flow, which is critical to tissue repair and regeneration in ischemic diseases. Here, we investigated the role of endothelial progenitor cell - derived exosomes (EPC-Exos) in therapeutic neovascularization and clarified the mechanism of hsa_circ_0093884 in EPC-Exos mediated neovascularization. Injection of EPC-Exos improved mouse ischemic hindlimb perfusion, promoted angiogenesis in Matrigel plugs and mouse skin wound healing. In vitro coculture with EPC-Exos improved HUVEC proliferation, angiogenic and migration ability, while alleviated hypoxia-induced apoptosis. hsa_circ_0093884 was identified from eleven types of circRNA derived from SIRT1 and proved to be enriched in EPC-Exos. Overexpression of hsa_circ_0093884 in EPC-Exos further enhanced the angiogenic capacity, while knockdown of hsa_circ_0093884 abolished the benefits. Mechanistically, EPC-Exos mediated shuttling of hsa_circ_0093884 induced cytoplasmic sponge of miR-145, thereby releasing repression of SIRT1. In vitro co-transfection indicated silence of miR-145 further strengthened the angiogenic effect of hsa_circ_0093884, while overexpression of miR-145 inhibited hsa_circ_0093884 mediated angiogenesis and abolished the beneficial effect of EPC-Exos. Furthermore, in vivo experiments using endothelial specific SIRT1 conditional knockout mice indicated hsa_circ_0093884 overexpressing EPC-Exos failed to promote therapeutic neovascularization in SIRT1cKO mice. Collectively, our results demonstrated that EPC-Exos promoted therapeutic neovascularization through hsa_circ_0093884/miR-145/SIRT1 axis.

Apoptotic Extracellular Vesicles Induced Endothelial Cell-Mediated Autologous Stem Cell Recruitment Dominates Allogeneic Stem Cell Therapeutic Mechanism for Bone Repair.

Although stem cell therapy is proved to be a promising strategy for bone repair and regeneration, transplanted allogeneic stem cells generally suffer from unfavorable apoptosis instead of differentiation into osteocytes. How the apoptotic stem cells promote bone regeneration still needs to be uncovered. In this work, we found that apoptotic extracellular vesicles released by allogeneic stem cells are critical mediators for promoting bone regeneration. Based on the results of in vivo experiments, a mechanism of apoptotic stem cells determined autologous stem cell recruitment and enhance osteogenesis was proposed. The nanoscaled apoptotic extracellular vesicles released from transplanted stem cells were endocytosed by vascular endothelial cells and preferentially distribute at endoplasmic reticular region. The oxidized phosphatidylcholine enriched in the vesicles activated the endoplasmic reticulum stress and triggered the reflective elevation of adhesion molecules, which induced the recruitment of autologous stem cells located in the blood vessels, transported them into the defect region, and promoted osteogenesis and bone repair. These findings not only reveal the mechanism of stem cell therapy of bone defects but also provide a cue for investigation of the biological process of stem cell therapy for other diseases and develop stem cell therapeutic strategies.

Tibial transverse transport induces mobilization of endothelial progenitor cells to accelerate angiogenesis and ulcer wound healing through the VEGFA/CXCL12 pathway.

BACKGROUND: Tibial transverse transport (TTT) can promote the healing of chronic foot ulcers, but the specific cellular and molecular mechanisms by which TTT promotes wound healing remain unclear. METHODS: New Zealand White rabbits were selected to induce foot ulcer models. The treatment included unilateral TTT surgery and bilateral TTT surgery. Observation of tissue neovascularization structure by HE staining and CD31 immunofluorescence detection. Collagen fiber formation was detected through the Masson staining. The mobilization of endothelial progenitor cell (EPCs) were analyzed by VEGFR2 immunofluorescence detection and flow cytometry detection of the number of VEGFR2/Tie-2-positive cells in peripheral blood. ELISA and qPCR assay were performed to detect VEGFA and CXCL12 levels. RESULTS: The complete healing time of ulcer surfaces in sham, unilateral and bilateral TTT groups was about 22 days, 17 days and 13 days, respectively. TTT treatment significantly increased the deposition of granulation tissue and epithelialization of wounds. It also led to an increase in collagen fiber content and the level of the microvascular marker CD31. Furthermore, TTT treatment upregulated the levels of VEGFA and CXCL12 in peripheral blood and wound tissues, as well as increased the expression of VEGFR2 in wound tissues and the proportion of VEGFR2/Tie-2 in peripheral blood. Moreover, these effects of TTT treatment in the bilateral group was more significant than that in the unilateral group. CONCLUSIONS: TTT may facilitate wound fibroblasts to release VEGFA and CXCL12, causing EPC mobilization, thus promoting angiogenesis and ulcer wound healing.

Characterization of Vascular Niche in Systemic Sclerosis by Spatial Proteomics.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disease that can serve as a model to study vascular changes in response to inflammation, autoimmunity, and fibrotic remodeling. Although microvascular changes are the earliest histopathologic manifestation of SSc, the vascular pathophysiology remains poorly understood. METHODS: We applied spatial proteomic approaches to deconvolute the heterogeneity of vascular cells at the single-cell level in situ and characterize cellular alterations of the vascular niches of patients with SSc. Skin biopsies of patients with SSc and control individuals were analyzed by imaging mass cytometry, yielding a total of 90 755 cells including 2987 endothelial cells and 4096 immune cells. RESULTS: We identified 7 different subpopulations of blood vascular endothelial cells (VECs), 2 subpopulations of lymphatic endothelial cells, and 3 subpopulations of pericytes. A novel population of CD34+;αSMA+ (α-smooth muscle actin);CD31+ VECs was more common in SSc, whereas endothelial precursor cells were decreased. Co-detection by indexing and tyramide signal amplification confirmed these findings. The microenvironment of CD34+;αSMA+;CD31+ VECs was enriched for immune cells and myofibroblasts, and CD34+;αSMA+;CD31+ VECs expressed markers of endothelial-to-mesenchymal transition. The density of CD34+;αSMA+;CD31+ VECs was associated with clinical progression of fibrosis in SSc. CONCLUSIONS: Using spatial proteomics, we unraveled the heterogeneity of vascular cells in control individuals and patients with SSc. We identified CD34+;αSMA+;CD31+ VECs as a novel endothelial cell population that is increased in patients with SSc, expresses markers for endothelial-to-mesenchymal transition, and is located in close proximity to immune cells and myofibroblasts. CD34+;αSMA+;CD31+ VEC counts were associated with clinical outcomes of progressive fibrotic remodeling, thus providing a novel cellular correlate for the crosstalk of vasculopathy and fibrosis.

Novel Angiogenesis Role of GLP-1(32-36) to Rescue Diabetic Ischemic Lower Limbs via GLP-1R-Dependent Glycolysis in Mice.

BACKGROUND: Restoring the capacity of endothelial progenitor cells (EPCs) to promote angiogenesis is the major therapeutic strategy of diabetic peripheral artery disease. The aim of this study was to investigate the effects of GLP-1 (glucagon-like peptide 1; 32-36)-an end product of GLP-1-on angiogenesis of EPCs and T1DM (type 1 diabetes) mice, as well as its interaction with the classical GLP-1R (GLP-1 receptor) pathway and its effect on mitochondrial metabolism. METHODS: In in vivo experiments, we conducted streptozocin-induced type 1 diabetic mice as a murine model of unilateral hind limb ischemia to examine the therapeutic potential of GLP-1(32-36) on angiogenesis. We also generated Glp1r-/- mice to detect whether GLP-1R is required for angiogenic function of GLP-1(32-36). In in vitro experiments, EPCs isolated from the mouse bone marrow and human umbilical cord blood samples were used to detect GLP-1(32-36)-mediated angiogenic capability under high glucose treatment. RESULTS: We demonstrated that GLP-1(32-36) did not affect insulin secretion but could significantly rescue angiogenic function and blood perfusion in ischemic limb of streptozocin-induced T1DM mice, a function similar to its parental GLP-1. We also found that GLP-1(32-36) promotes angiogenesis in EPCs exposed to high glucose. Specifically, GLP-1(32-36) has a causal role in improving fragile mitochondrial function and metabolism via the GLP-1R-mediated pathway. We further demonstrated that GLP-1(32-36) rescued diabetic ischemic lower limbs by activating the GLP-1R-dependent eNOS (endothelial NO synthase)/cGMP/PKG (protein kinase G) pathway. CONCLUSIONS: Our study provides a novel mechanism with which GLP-1(32-36) acts in modulating metabolic reprogramming toward glycolytic flux in partnership with GLP-1R for improved angiogenesis in high glucose-exposed EPCs and T1DM murine models. We propose that GLP-1(32-36) could be used as a monotherapy or add-on therapy with existing treatments for peripheral artery disease. REGISTRATION: URL: www.ebi.ac.uk/metabolights/; Unique identifier: MTBLS9543.

C1q/Tumor Necrosis Factor-Related Protein-9 Is a Novel Vasculoprotective Cytokine That Restores High Glucose-Suppressed Endothelial Progenitor Cell Functions by Activating the Endothelial Nitric Oxide Synthase.

BACKGROUND: This study investigated whether gCTRP9 (globular C1q/tumor necrosis factor-related protein-9) could restore high-glucose (HG)-suppressed endothelial progenitor cell (EPC) functions by activating the endothelial nitric oxide synthase (eNOS). METHODS AND RESULTS: EPCs were treated with HG (25 mmol/L) and gCTRP9. Migration, adhesion, and tube formation assays were performed. Adiponectin receptor 1, adiponectin receptor 2, and N-cadherin expression and AMP-activated protein kinase, protein kinase B, and eNOS phosphorylation were measured by Western blotting. eNOS activity was determined using nitrite production measurement. In vivo reendothelialization and EPC homing assays were performed using Evans blue and immunofluorescence in mice. Treatment with gCTRP9 at physiological levels enhanced migration, adhesion, and tube formation of EPCs. gCTRP9 upregulated the phosphorylation of AMP-activated protein kinase, protein kinase B, and eNOS and increased nitrite production in a concentration-dependent manner. Exposure of EPCs to HG-attenuated EPC functions induced cellular senescence and decreased eNOS activity and nitric oxide synthesis; the effects of HG were reversed by gCTRP9. Protein kinase B knockdown inhibited eNOS phosphorylation but did not affect gCTRP9-induced AMP-activated protein kinase phosphorylation. HG impaired N-cadherin expression, but treatment with gCTRP9 restored N-cadherin expression after HG stimulation. gCTRP9 restored HG-impaired EPC functions through both adiponectin receptor 1 and N-cadherin-mediated AMP-activated protein kinase /protein kinase B/eNOS signaling. Nude mice that received EPCs treated with gCTRP9 under HG medium showed a significant enhancement of the reendothelialization capacity compared with those with EPCs incubated under HG conditions. CONCLUSIONS: CTRP9 promotes EPC migration, adhesion, and tube formation and restores these functions under HG conditions through eNOS-mediated signaling mechanisms. Therefore, CTRP9 modulation could eventually be used for vascular healing after injury.

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

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

Crosstalk between endothelial progenitor cells and HCC through periostin/CCL2/CD36 supports formation of the pro-metastatic microenvironment in HCC.

Metastasis causes most cancer-related deaths, and the role and mechanism of periostin (POSTN) in the metastasis of hepatocellular carcinoma (HCC) remain undiscovered. In this study, DEN and HTVi HCC models were performed in hepatic-specific Postn ablation and Postn knock-in mouse to reveal the role of POSTN in HCC metastasis. Furthermore, POSTN was positively correlated with circulating EPCs level and promoted EPC mobilization and tumour infiltration. POSTN also mediated the crosstalk between HCC and EPCs, which promoted metastasis ability and upregulated CD36 expression in HCC through indirect crosstalk. Chemokine arrays further revealed that hepatic-derived POSTN induced elevated CCL2 expression and secretion in EPCs, and CCL2 promoted prometastatic traits in HCC. Mechanistic studies showed that POSTN upregulated CCL2 expression in EPCs via the αvß3/ILK/NF-κB pathway. CCL2 further induced CD36 expression via the CCR2/STAT3 pathway by directly binding to the promoter region of CD36. Finally, CD36 was verified to have a prometastatic role in vitro and to be correlated with POSTN expression, metastasis and recurrence in HCC in clinical samples. Our findings revealed that crosstalk between HCC and EPCs is mediated by periostin/CCL2/CD36 signalling which promotes HCC metastasis and emphasizes a potential therapeutic strategy for preventing HCC metastasis.

MiR-155 promotes compensatory lung growth by inhibiting JARID2 activation of CD34+ endothelial progenitor cells.

Bone marrow-derived CD34-positive (CD34+) endothelial progenitor cells (EPCs) has unique functions in the mechanism of compensatory lung growth (CLG). The content of this study is mainly to describe the effect of microRNA (miR)-155 in the mechanisms of EPCs and CLG. Our study found that transfection of miR-155 mimic could promote EPC proliferation, migration and tube formation, while transfection of miR-155 inhibitor had the opposite effect. It was also found that transfection of pc-JARID2 inhibited EPC proliferation, migration and tube formation, while transfection of si-JARID2 had the opposite effect. miR-155 can target and negatively regulate JARID2 expression. Overexpression of JARID2 weakened the promoting effects of miR-155 mimic on EPC proliferation, migration, and tubular formation, while silencing JARID2 weakened the inhibitory effects of miR-155 inhibitors on EPC proliferation, migration, and tubular formation. Transplantation of EPCs transfected with miR-155 mimic into the left lung model effectively increased lung volume, total alveolar number, diaphragm surface area, and lung endothelial cell number, while transplantation of EPCs co-transfected with miR-155 mimic and pc-JARID2 reversed this phenomenon. Overall, we found that miR-155 activates CD34+ EPC by targeting negative regulation of JARID2 and promotes CLG.