Homing Of Endothelial Progenitor Cells Research Articles

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.

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). 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. Invivo 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. 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.

Dimethyloxalylglycine Attenuates Steroid-Associated Endothelial Progenitor Cell Impairment and Osteonecrosis of the Femoral Head by Regulating the HIF-1α Signaling Pathway

Endothelial impairment and dysfunction are closely related to the pathogenesis of steroid-associated osteonecrosis of the femoral head (SONFH). Recent studies have showed that hypoxia inducible factor-1α (HIF-1α) plays a crucial role in endothelial homeostasis maintenance. Dimethyloxalylglycine (DMOG) could suppress HIF-1 degradation and result in nucleus stabilization by repressing prolyl hydroxylase domain (PHD) enzymatic activity. Our results showed that methylprednisolone (MPS) remarkably undermined biological function of endothelial progenitor cells (EPC) by inhibiting colony formation, migration, angiogenesis, and stimulating senescence of EPCs, while DMOG treatment alleviated these effects by promoting HIF-1α signaling pathway, as evidenced by senescence-associated β-galactosidase (SA-β-Gal) staining, colony-forming unit, matrigel tube formation, and transwell assays. The levels of proteins related to angiogenesis were determined by ELISA and Western blotting. In addition, active HIF-1α bolstered the targeting and homing of endogenous EPCs to the injured endothelium in the femoral head. Histopathologically, our in vivo study showed that DMOG not only alleviated glucocorticoid-induced osteonecrosis but also promoted angiogenesis and osteogenesis in the femoral head as detected by microcomputed tomography (Micro-CT) analysis and histological staining of OCN, TRAP, and Factor Ⅷ. However, all of these effects were impaired by an HIF-1α inhibitor. These findings demonstrate that targeting HIF-1α in EPCs may constitute a novel therapeutic approach for the treatment of SONFH.

Downregulation of miR-125a-5p Promotes Endothelial Progenitor Cell Migration and Angiogenesis and Alleviates Deep Vein Thrombosis in Mice Via Upregulation of MCL-1.

Endothelial progenitor cells (EPCs) contribute to recanalization of deep vein thrombosis (DVT). MicroRNAs (miRNAs) play regulatory roles in functions of EPCs, which is becoming a promising therapeutic choice for thrombus resolution. The main purpose of this study was to explore the effect of miR-125a-5p on EPC functions in deep vein thrombosis (DVT). EPCs were isolated from the peripheral blood of patients with DVT. In DVT mouse models, DVT was induced by stenosis of the inferior vena cava (IVC). The levels of miR-125a-5p and myeloid cell leukemia sequence 1 (MCL-1) in EPCs and thrombi of DVT mice were detected by RT-qPCR. EPC migration, angiogenesis, and apoptosis were estimated by Transwell assay, tube formation assay, and flow cytometry analysis. Luciferase reporter assay was utilized for detecting the binding of miR-125a-5p and MCL-1. The phosphorylation of PI3K and AKT was estimated by western blot. DVT formation in vivo was observed through hematoxylin-eosin (H&E) staining. The expression of thrombus resolution marker, CD34 molecule (CD34), in the thrombi was measured by immunofluorescence staining. MiR-125a-5p upregulation repressed EPC migration and angiogenesis and facilitated apoptosis. MiR-125a-5p downregulation showed the opposite effect. MCL-1 was targeted and negatively regulated by miR-125a-5p. Additionally, miR-125a-5p inhibited the PI3K/AKT pathway in EPCs. Inhibition of MCL-1 or PI3K/AKT pathway reversed the effect of miR-125a-5p knockdown on EPC functions. The in vivo experiments revealed that miR-125a-5p downregulation repressed thrombus formation and promoted the homing capability of EPCs to the thrombosis site, thereby alleviating DVT mice. Downregulation of miR-125a-5p promotes EPC migration and angiogenesis by upregulating MCL-1, thereby enhancing EPC homing to thrombi and facilitating thrombus resolution.

Cyclic stretch promotes vascular homing of endothelial progenitor cells via Acsl1 regulation of mitochondrial fatty acid oxidation

Endothelial progenitor cells (EPCs) play an important role in vascular repair and re-endothelialization after vessel injury. EPCs in blood vessels are subjected to cyclic stretch (CS) due to the pulsatile pressure, but the role of CS in metabolic reprogramming of EPC, particularly its vascular homing and repair, is largely unknown. In the current study, physiological CS applied to EPCs at a magnitude of 10% and a frequency of 1 Hz significantly promoted their vascular adhesion and endothelial differentiation. CS enhanced mitochondrial elongation and oxidative phosphorylation (OXPHOS), as well as adenosine triphosphateproduction. Metabolomic study and Ultra-high performance liquid chromatography-mass spectrometry assay revealed that CS significantly decreased the content of long-chain fatty acids (LCFAs) and markedly induced long-chain fatty acyl-CoA synthetase 1 (Acsl1), which in turn facilitated the catabolism of LCFAs in mitochondria via fatty acid β-oxidation and OXPHOS. In a rat carotid artery injury model, transplantation of EPCs overexpressing Acsl1 enhanced the adhesion and re-endothelialization of EPCs invivo. MRI and vascular morphology staining showed that Acsl1 overexpression in EPCs improved vascular repair and inhibited vascular stenosis. This study reveals a mechanotransduction mechanism by which physiological CS enhances endothelial repair via EPC patency.

Genistein, a Soybean Isoflavone, Promotes Wound Healing by Enhancing Endothelial Progenitor Cell Mobilization in Rats with Hemorrhagic Shock.

Severe trauma and hemorrhaging are often accompanied by delayed cutaneous wound healing. Soybean isoflavone is a natural phytoestrogen that has attracted great attention due to its protective effects against various injuries. Endothelial progenitor cells (EPCs) are precursor cells with directional differentiation characteristics. This study is to determine whether genistein (GEN), an isoflavone in soybean products, benefits wound healing in hemorrhagic shock (HS) rats by promoting EPC homing and to investigate the underlying mechanisms. In this study, it isfound that GEN promotes skin wound healing in HS rats, which is due at least partly to the mobilization of endogenous EPCs to the injury site via angiotensin II (Ang-II), stromal cell-derived factor-1alpha (SDF-1α), and transforming growth factor beta(TGF-β) signaling.

CD34+ progenitor cells as diagnostic and therapeutic targets in Alzheimer's disease.

Alzheimer’s disease (AD) is the main neurodegenerative disease leading to dementia and cognitive impairment in the elderly. Considering AD to be an epidemic, an increase from the current 50 million to more than 150 million patients is expected by the year 2050. AD is characterized by a slow, progressive and asymptomatic onset; making it difficult to decipher the precise etiology. It is well established that AD presents two characteristic features, extracellular β-amyloid plaques and intracellular tau tangles, that eventually lead to the impairment of cognitive functions. Unfortunately, AD symptomatology shares many similarities with other dementias once is present, which makes it difficult an accurate premortem diagnosis. Although AD is mainly considered an aging-related condition that affects cognitive function, several cardio- and cerebrovascular comorbidities such as hypertension or diabetes are also risk factors for cognitive impairment. Accordingly, brain vascular-associated alterations underlie many pathophysiological mechanisms of AD. We have recently reviewed the latest evidence supporting the detrimental role of vascular and angiogenic alterations during AD (Custodia et al., 2022). Remarkably, cerebral blood-brain barrier (BBB) leakage and microbleeds are associated with cognitive decline in patients with mild cognitive impairment (MCI) and early AD. Accordingly, the two-hit vascular hypothesis points at initial damage in cerebral vasculature (hit one) that eventually induces the accumulation of β-amyloid (Aβ) in the brain (hit two; Zlokovic, 2005). CD34+ bone marrow-derived progenitor cells (BMPCs) define a group of stem and progenitor cell populations released by the bone marrow that covers different subpopulations of cells from the hematopoietic linage, including endothelial progenitor cells (EPCs). EPCs exhibit characteristics of both endothelial and stem cells, and, accordingly, proangiogenic early EPCs expressing both CD34 and CD133 (a progenitor surface marker) can be distinguished from late EPCs additionally expressing KDR and/or CD146 (endothelial markers), which participate in the process of angiogenesis and vasculogenesis (Figure 1). Therefore, EPCs participate in angiogenesis and the maintenance of the endothelium by acting as a cellular reservoir for the replacement of dysfunctional endothelial cells, or by the secretion of angiogenic growth factors.Figure 1: Beneficial roles of CD34+ BMPCs following CNS injury.CD34+ BMPCs and the EPCs subtypes, early and late, can promote both angiogenesis and vasculogenesis following CNS injury by specializing in endothelial cells, and/or indirectly by secreting free and exosome-enveloped growth factors. G-CSF is a glycoprotein that acts in the bone marrow to mobilize both EPCs and CD34+ BMPCs after damage. BMPCs: Bone marrow progenitor cells; CNS: central nervous system; EPCs: endothelial progenitor cells; G-CSF: granulocyte colony-stimulating factor. Created with BioRender.com.Given that both dysfunctional angiogenesis and compromised BBB integrity seem critical in the onset and/or progression of AD, CD34+ progenitor cells, primarily EPCs, appear as potential targets for the early diagnosis and/or treatment of the disease. In this way, early and late EPCs would work synergistically: early EPCs reach the site of injury due to the high concentration of angiogenic factors and inflammatory cytokines, from which they paracrinally release different factors promoting angiogenesis and recruiting late EPCs, which either restore the endothelium or form new vessels guided by the early EPCs. Here, we discuss recent work and ongoing human clinical trials testing the feasibility of CD34+ BMPCs and EPCs as early biomarkers of AD and pharmacological targets for future treatments. Association of circulating levels of CD34+ BMPCs and cognitive decline in healthy and MCI subjects: Several cross-sectional studies have shown that the number of circulating CD34+ BMPCs decreases with age, and this may impact cognition. In this regard, a longitudinal study regarding cognition and CD34+ BMPCs levels reported that older healthy subjects had lower levels of CD34+ BMPCs than younger counterparts at baseline measurements (Hajjar et al., 2016). Moreover, this investigation revealed that subjects with higher baseline levels of several subgroups of CD34+ BMPCs such as early and late EPCs, and CD34+/KDR+ cells, among others, had better executive-derived and working memory scores over 4 years of follow-up (Hajjar et al., 2016). Recently, a large transverse study has shown the association between CD34+ BMPCs and different memory-related tests in cognitively normal subjects with coronary artery disease (Moazzami et al., 2020). Notably, circulating numbers of late EPCs were positively correlated with a better performance in tasks assessing visual, logical, and verbal immediate/delayed memory. Therefore, the amount of circulating CD34+ BMPCs subtypes appears to be negatively correlated to the cognitive decline of both healthy subjects and patients with vascular-associated conditions. Although more longitudinal clinical studies are needed to fully confirm the harmful effect of low levels of CD34+ BMPCs on the cognitive state, and other factors may be also taking part in this cognitive decline, it is still plausible that larger amounts of circulating endothelial progenitors exert a protective effect, probably by the maintenance of vascular endothelium integrity. MCI often precedes clinical symptoms of AD, and MCI patients show an increased risk of developing dementia in the future. Thus, it is very interesting to study CD34+ BMPCs/EPCs levels in patients with MCI in order to test whether such levels can be used as potential non-invasive diagnostic biomarkers to detect cognitive decline or its progression from MCI to dementia. Some studies have observed a decrease in CD34+ BMPCs and EPCs populations from MCI patients (Nation et al., 2018; Callahan et al., 2020). In this sense, MCI patients with lower levels of circulating CD34+ BMPCs and both subtypes of EPCs exhibited worse scores in memory tests and reduced cortical thickness compared to control subjects (Nation et al., 2018). Considering the angiogenesis ratio (pro-angiogenic/non-angiogenic BMPCs, including early and late EPCs), Callahan et al. (2020) showed a positive association between angiogenesis ratio and white matter hyperintensities, but not with global cerebral blood flow, hippocampal volume, or accumulation of tau and Aβ. By contrast, measurements in an older cohort of MCI patients did not show significant changes in CD34+, early EPCs, and late EPCs circulating levels compared to control subjects (Breining et al., 2016). This discrepancy may highlight that aging decreases CD34+ BMPCs to such a reduced level that is no longer different in controls compared to MCI. In summary, it seems that the reduction in CD34+ BMPCs is directly related to vascular dysfunction, increasing brain white matter microlesions and impairing cognition in MCI patients. Association of circulating levels of CD34+ BMPCs and AD: Several studies have been performed in order to determine the relationship between CD34+ BMPCs/EPCs circulating levels and the progression of AD (Maler et al., 2006; Lee et al., 2009; Stellos et al., 2010; Bigalke et al., 2011; Kong et al., 2011; Breining et al., 2016; Callahan et al., 2020; Haiyuan et al., 2020). In this way, AD patients in the early symptomatic phase already showed lower levels of CD34+ and CD34+/KDR+ cells compared to their control counterparts (Maler et al., 2006; Haiyuan et al., 2020). Notably, CD34+ BMPCs counts have negatively correlated with levels of Aβ1–42 in cerebrospinal fluid and the Aβ ratio 42/40, two well-known biomarkers for AD, as well as with age, only in the early AD group (Maler et al., 2006). Furthermore, the homing capacity of EPCs from early AD patients was already impaired (Haiyuan et al., 2020). Overall, it is becoming clear that dysfunctional CD34+ BMPCs are related to a reduced ability to repair brain endothelial cells, which appears to mediate neurotoxicity by affecting the BBB permeability. On the other hand, different outcomes were described in studies assessing the number of progenitor cells during AD progression. Specifically, lower counts of CD34+ BMPCs and EPCs have been observed in moderate and severe AD patients compared to both early AD stage (Haiyuan et al., 2020) and control subjects (Lee et al., 2009; Kong et al., 2011; Haiyuan et al., 2020). Such studies also revealed that homing and adhesion features of EPCs from AD patients were impaired (Haiyuan et al., 2020), as well as EPCs levels were inversely correlated with the mini-mental state exam (MMSE) score (Lee et al., 2009; Stellos et al., 2010; Kong et al., 2011). Furthermore, moderate to severe AD patients displayed a reduced flow velocity of the middle cerebral artery (Kong et al., 2011). In contrast, other studies reported higher levels of CD34+ BMPCs and EPCs compared to controls (Stellos et al., 2010; Bigalke et al., 2011), or even no changes (Breining et al., 2016). Intriguingly, the work from Stellos and colleagues reported an increase in both CD34+ BMPCs and early EPCs counting when comparing moderate to severe AD patients versus control subjects; however, within the AD group, there was an inverse correlation between CD34+ BMPCs and early EPCs counting and the MMSE score. Although these results seem contrary to each other, it is noteworthy that most AD patients from this study (Stellos et al., 2010) were treated with cholinesterase inhibitor; a drug involved in EPCs proliferation. Therefore, this fact may bias the results and it could explain why cell counting in the AD group was higher than in controls, but they were inversely correlated with MMSE scores. The other study that showed increased levels of CD34+ BMPCs/EPCs (Bigalke et al., 2011) only measured the numbers of CD34+ BMPCs in early to moderate AD compared to controls, with no information regarding cholinesterase inhibitor treatment. In summary, most of the studies in later AD stages support the studies performed on MCI and early AD stages. Therefore, AD-mediated loss of CD34+ BMPCs/EPCs, as well as loss of EPCs-intrinsic features, are likely present in AD patients and may constitute novel diagnostic and therapeutic targets. Potential therapy with granulocyte colony-stimulating factor (G-CSF) in AD: The G-CSF is a glycoprotein secreted by endothelial and immune cells that acts as a hematopoietic growth factor (Figure 1). Among other beneficial mechanisms following vascular injury, the G-CSF can promote angiogenesis by mobilizing EPCs (Figure 1). Therefore, G-CSF may be a potential target to enhance vascular repair in AD patients. Indeed, it has been recently shown that a G-CSF treatment improved memory as well as reduced blood levels of amyloid and tau in mild to moderate patients of AD (Potter et al., 2021). Based on these achievements, it is currently conducting a phase2b clinical trial in order to evaluate the long-term treatment of G-CSF in AD patients (NCT04902703; ClinicalTrials.gov). It would be interesting to look at CD34+ BMPCs and EPCs levels from those clinical trials in order to elucidate whether such potential benefits promoted by G-CSF therapy are totally or partially mediated by increasing CD34+ BMPCs/EPCs mobilization. Future challenges: The body of evidence supporting a vascular component underlying AD onset and/or progression is growing. However, further studies are mandatory to elucidate whether such vascular component triggers AD, is a consequence of AD, or both. Moreover, longitudinal studies are needed to confirm the relationship between CD34+ BMPCs/EPCs levels and AD progression. Given that vascular-related diseases may influence the amount of circulating progenitor cells, especially in AD patients, comorbidities present in those subjects deserve special attention when interpreting the results. Likewise, pharmacological treatments, such as a cholinesterase inhibitor, may bias the results from studies giving uncorrected information. Despite the promising results in animal models of AD, the number of published results and clinical trials regarding the direct application of EPCs as a potential therapy in AD patients is absent. This is remarkable when there is compelling evidence that supports the role of endothelial dysfunction in the onset and progression of AD, and the potential of EPCs as a diagnostic biomarker and/or therapeutic target (Custodia et al., 2022). However, we were unable to find published data or ongoing clinical trials in humans using the application of EPCs to treat AD; as already seen in a stroke clinical trial (NCT01468064). Moreover, several recent studies have highlighted the beneficial role of EPCs secretome/exosomes by protecting and repairing the BBB following damage without using a cell-based therapy. So, clinical trials based on EPCs-derived secretome/exosomes might be a safer and more promising approach in AD research. Finally, only the GCSF-based treatment is being tested in AD patients at later stages, with modest but promising results. Given that endothelium-related impairments are already seen in MCI patients, it would be really interesting to test this GCSF-based treatment in those subjects in order to increase the benefits and protect against the progression to AD. This work was partially supported by grants from the Xunta de Galicia (IN607A2018/3 to TS, IN607D 2020/09 to TS, IN606A-2021/015 to AC; IN606B-2021/010 to DRS), and Science Ministry of Spain (RTI2018-102165-B-I00 to TS, RTC2019-007373-1 to TS). Furthermore, this work was also supported by grants from the INTERREG Atlantic Area (EAPA_791/2018_ NEUROATLANTIC project to TS), INTER-REG V A España Portugal (POCTEP) (0624_2IQBIONEURO_6_E to TS), and the European Regional Development Fund (ERDF). Moreover, DRS (CD21/00166) and TS (CPII17/00027) are recipients of research contracts from the Sara Borrell and Miguel Servet Programs, respectively, from the Instituto de Salud Carlos III. Availability of data and materials:All data generated or analyzed during this study are included in this published article and its supplementary information files. Open peer reviewers:Yali Jia, Beijing Institute of Radiation Medicine, China; Rongcan Luo, Kunming Institute of Zoology Chinese Academy of Sciences, China. Additional file:Open peer review reports 1 and 2.P-Reviewers: Jia Y, Luo R; C-Editors: Zhao M, Liu WJ, Wang Lu; T-Editor: Jia Y

Granulocyte-colony stimulating factor mediates neovascularization in acellular dermal matrix-transplanted areas by promoting endothelial progenitor cell homing.

Acellular dermal matrix (ADM) is an ideal material for tissue engineering skin construction. Accelerating the vascularization of ADM is of great significance for improving the survival of skin transplantation. The purpose of this study is to investigate the function of granulocyte-colony stimulating factor (G-CSF) in endothelial progenitor cells (EPCs)-mediated neovascularization in ADM-transplanted skin area. Male Kunming mice were subcutaneous injected with 10 μg/kg GCSF at 5 days before skin in situ replantation or porcine ADM transplantation. The surrounding tissues of implanted skin or venous blood was collected from the mice before the operation, and after the operation for 48 h, 72 h, 1 week, and 2 weeks, respectively. Cells co-expressing EPC markers, CD133, CD34, and Flk-1 were detected by flow cytometry. Immunohistochemistry of BrdU was performed to evaluate neovascularization in ADM-transplanted skin area. The results showed that G-CSF treatment increased the number of CD133+-CD34+ cells and CD133+-Flk-1+ cells in ADMimplanted area as well as the number of CD34+-Flk-1+ cells in peripheral blood. Likewise, G-CSF also increased the number of capillaries in ADM-transplanted areas. To sum up, G-CSF mobilizes EPC migration from bone marrow to peripheral blood and homing to wound sites, thus inducing neovascularization in ADM-transplanted areas.

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.

Lpar1-mediated Effects in Endothelial Progenitor Cells Are Crucial for Lung Repair in Acute Respiratory Distress Syndrome/Acute Lung Injury.

Acute respiratory distress syndrome/acute lung injury (ARDS/ALI) involves acute respiratory failure characterized by vascular endothelial and lung alveolar epithelial injury. Endothelial progenitor cells (EPCs) can mediate vasculogenesis. However, the limitations of EPCs, such as low survival and differentiation, are believed to inhibit the effectiveness of autologous cell therapies. This study demonstrated that lysophosphatidic acid (LPA), a bioactive small molecule without immunogenicity, is involved in the survival and antiapoptotic effects in human umbilical cord mesenchymal stem cells. This study aimed to explore whether LPA improves the survival of EPCs, enhancing the cellular therapeutic efficacy in ARDS, and these results will expand the application of LPA in stem cells and regenerative medicine. LPA promoted the colony formation, proliferation, and migration of EPCs and upregulated the expression of vascular endothelial-derived growth factor (VEGF) in EPCs. LPA pretreatment of transplanted EPCs improved the therapeutic effect by increasing EPC numbers in the rat lungs. LPA enhanced EPC proliferation and migration through Lpar1 coupled to Gi/o and Gq/11, respectively. Activation of extracellular signal-related kinase 1/2, or ERK1/2, was related to LPA-induced EPC proliferation but not migration. LPA/Lpar1-mediated Gi/o protein was also shown to be involved in promoting VEGF expression and inhibiting IL-1α expression in EPCs. Low LPA concentrations are present after lung injury; thus, the restoration of LPA may promote endothelial cell homeostasis and lung repair in ARDS. Inhalation of LPA significantly promoted the homing of endogenous EPCs to the lung and reduced lung injury in both rats with LPS-induced ALI and Streptococcus pneumoniae-infected mice. Taken together, these data indicated that LPA/Lpar1-mediated effects in EPCs are involved in maintaining endothelial cell homeostasis and lung tissue repair under physiological conditions.

Homing of mRNA-Modified Endothelial Progenitor Cells to Inflamed Endothelium.

Endothelial progenitor cells (EPCs) are one of the most important stem cells for the neovascularization of tissues damaged by ischemic diseases such as myocardial infarction, ischemic stroke, or critical limb ischemia. However, their low homing efficiency in the treatment of ischemic tissues limits their potential clinical applications. The use of synthetic messenger RNA (mRNA) for cell engineering represents a novel and promising technology for the modulation of cell behavior and tissue regeneration. To improve the therapeutic potential of EPCs, in this study, murine EPCs were engineered with synthetic mRNAs encoding C-X-C chemokine receptor 4 (CXCR4) and P-selectin glycoprotein ligand 1 (PSGL-1) to increase the homing and migration efficiency of EPCs to inflamed endothelium. Flow cytometric measurements revealed that the transfection of EPCs with CXCR4 and PSGL-1 mRNA resulted in increased expressions of CXCR4 and PSGL-1 on the cell surface compared with the unmodified EPCs. The transfection of EPCs with mRNAs did not affect cell viability. CXCR4-mRNA-modified EPCs showed significantly higher migration potential than unmodified cells in a chemotactic migration assay. The binding strength of the EPCs to inflamed endothelium was determined with single-cell atomic force microscopy (AFM). This showed that the mRNA-modified EPCs required a three-fold higher detachment force to be released from the TNF-α-activated endothelium than unmodified EPCs. Furthermore, in a dynamic flow model, significantly increased binding of the mRNA-modified EPCs to inflamed endothelium was detected. This study showed that the engineering of EPCs with homing factors encoding synthetic mRNAs increases the homing and migration potentials of these stem cells to inflamed endothelium. Thus, this strategy represents a promising strategy to increase the therapeutic potential of EPCs for the treatment of ischemic tissues.

Upregulated miR-206 Aggravates Deep Vein Thrombosis by Regulating GJA1-Mediated Autophagy of Endothelial Progenitor Cells.

Background Deep vein thrombosis (DVT) is the third most prevalent vascular disease worldwide. MicroRNAs (miRNAs) play regulatory roles in functions of endothelial progenitor cells (EPCs), which is becoming a promising therapeutic choice for thrombus resolution. Nevertheless, the role of miR-206 in EPCs is unclear. Methods EPCs were isolated from the peripheral blood of patients with DVT. In DVT mouse models, DVT was induced by stenosis of the inferior vena cava (IVC). The levels of miR-206 and gap junction protein alpha 1 (GJA1) in EPCs and vascular tissues of DVT mice were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The proliferation, migration, apoptosis, and angiogenesis were tested by cell counting kit-8 (CCK-8) assay, Transwell assay, flow cytometry analysis, and in vitro tube formation assay. The levels of autophagy-related proteins as well as the level of GJA1 in EPCs and vascular tissues were evaluated by western blotting. DVT formation in vivo was observed through hematoxylin-eosin (HE) staining. The expression of thrombus resolution markers, CD34 molecule (CD34) and matrix metallopeptidase 2 (MMP2), in the thrombi was measured by immunofluorescence staining. Results miR-206 overexpression inhibited proliferation, migration, and angiogenesis and promoted apoptosis of EPCs, while miR-206 knockdown exerted an opposite effect on EPC phenotypes. Downregulation of GJA1, the target of miR-206, abolished the influence of miR-206 on EPC phenotypes. Furthermore, silencing of miR-206 suppressed the autophagy of EPCs via upregulating GJA1. miR-206 knockdown repressed thrombus formation, enhanced the homing ability of EPCs to the thrombosis site, and facilitated thrombus resolution in DVT mouse models. Additionally, miR-206 was upregulated while GJA1 was downregulated in vascular tissues of DVT mice. miR-206 knockdown elevated GJA1 expression in vascular tissues of DVT mice. The expression of miR-206 was negatively correlated with that of GJA1 in DVT mice. Conclusion miR-206 knockdown upregulates GJA1 to inhibit autophagy of EPCs and then promote EPC proliferation, migration, and angiogenesis, thereby enhancing EPC homing to thrombi and facilitating thrombus resolution.

Endothelial colony forming cells' tetrahydrobiopterin level in coronary artery disease patients and its association with circulating endothelial progenitor cells.

Endothelial colony forming cells (ECFCs) participate in neovascularization. Endothelial nitric oxide synthase (eNOS) derived NO· helps in homing of endothelial progenitor cells (EPCs) at the site of vascular injury. The enzyme cofactor tetrahydrobiopterin (BH4) stabilizes the catalytic active state of eNOS. Association of intracellular ECFCs biopterins and ratio of reduced to oxidized biopterin (BH4:BH2) with circulatory EPCs and ECFCs functionality have not been studied. We investigated ECFCs biopterin levels and its association with circulatory EPCs as well as ECFCs proliferative potential in terms of day of appearance in culture. Circulatory EPCs were enumerated by flowcytometry in 53 coronary artery disease (CAD) patients and 42 controls. ECFCs were cultured, characterized, and biopterin levels assessed by high performance liquid chromatography. Appearance of ECFCs' colony and their number were recorded. Circulatory EPCs were significantly lower in CAD and ECFCs appeared in 56% and 33% of CAD and control subjects, respectively. Intracellular BH4 and BH4:BH2 were significantly reduced in CAD. BH4:BH2 was positively correlated with circulatory EPCs (p = 0.01), and negatively with day of appearance of ECFCs (p = 0.04). Circulatory EPCs negatively correlated with ECFCs appearance (p = 0.02). These findings suggest the role of biopterins in maintaining circulatory EPCs and functional integrity of ECFCs.

Compound Tongluo Decoction promotes generation and homing of endothelial progenitor cells after cerebral infarction in rats by activating Shh signaling


 
 
 
 Purpose: To investigate the protective effects of Compound Tongluo decoction (CTD)on neurological function in rats, and the mechanism involved in its angiogenesis-promoting effect.
 Methods: Rats were arbitrarily assigned to sham group, permanent middle cerebral artery infarction (pMCAO) group, and PMCAO+CTD group (pMCAO plus 7-day oral treatment with CTD). Neurological deficit scores and volume of stroke-damaged areas were measured after 7 days of treatment. The levels of bone marrow-derived endothelial progenitor cells (BMEPCs) in serum and brain tissues were determined by flow cytometry and immunofluorescence. The expression levels of sonic hedgehog (Shh), vascular endothelial growth factor (VEGF), and angiopoietin-1(Ang-1) at infarct sites and in BMEPCs were quantitated using western blot assay.
 Results: The results showed that CTD markedly ameliorated neurological deficit, reduced volume of affected areas, and promoted the production and homing of BMEPCs. Moreover, CTD upregulated Shh, VEGF and Ang-1expressions at ischemic sites and EPCs, but promoted the proliferation and metastasis of EPCs. The CTD-induced changes were significantly suppressed by Shh inhibitor cyclopamine (CP).
 Conclusion: These results demonstrate that CTD promotes angiogenesis after cerebral infarction, probably by stimulating Shh signaling and triggering production and homing of EPCs, thereby providing neuroprotection against cerebral infarction. Thus, CTD is a potential neuroprotective agent against cerebral infarction in humans.
 
 
 

Up-regulated miR-204-5p promoted the migration, invasion, and angiogenesis of endothelial progenitor cells to enhance the thrombolysis of rats with deep venous thrombosis by targeting SPRED1

Deep venous thrombosis (DVT) endangers human health. Endothelial progenitor cells (EPCs) were proven to promote thrombolysis and miR-204-5p was discovered to be low-expressed in DVT patients. This study concentrated on exploring whether miR-204-5p had a regulatory effect on EPCs and DVT. Concretely, the expression of miR-204-5p in DVT patients’ blood was detected by qRT-PCR. The target of miR-204-5p was predicted by bioinformatics and verified by dual-luciferase reporter assay. After rat EPCs were isolated, identified, and transfected with miR-204-5p agomiR, antagomiR, or SPRED1 plasmids, the viability, migration, invasion, and tube formation of EPCs were detected by MTT, wound healing, Transwell, and tube formation assays, respectively. MiR-204-5p, SPRED1, p-PI3K, PI3K, p-AKT, AKT, VEGFA, and Ang1 expressions in EPCs were measured by qRT-PCR or Western blot. EPCs transfected with miR-204-5p overexpression lentivirus plasmid were injected into the DVT rat model. The histopathology of the thrombus and the homing of EPCs to thrombus in the DVT rats were observed by hematoxylin-eosin staining and confocal microscopy, respectively. We found that miR-204-5p was low-expressed in DVT patients and SPRED1 was a target gene of miR-204-5p. MiR-204-5p agomiR promoted the viability, migration, invasion, and tube formation of EPCs, the levels of VEGFA and Ang1 and the activation of PI3K/AKT pathway in EPCs, while miR-204-5p antagomiR and SPRED1 worked oppositely. SPRED1 reversed the effect of miR-204-5p agomiR on EPCs. Up-regulated miR-204-5p inhibited thrombosis and promoted EPCs homing to thrombus in DVT rats. Collectively, up-regulated miR-204-5p enhanced the angiogenesis of EPCs and thrombolysis in DVT rats by targeting SPRED1.

CXCL13/CXCR5 axis facilitates endothelial progenitor cell homing and angiogenesis during rheumatoid arthritis progression

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. The chemokine (C-X-C motif) ligand 13 (CXCL13) plays important roles in several cellular functions such as infiltration, migration, and motility. We report significantly higher levels of CXCL13 expression in collagen-induced arthritis (CIA) mice compared with controls and also in synovial fluid from RA patients compared with human osteoarthritis (OA) samples. RA synovial fluid increased endothelial progenitor cell (EPC) homing and angiogenesis, which was blocked by the CXCL13 antibody. By interacting with the CXCR5 receptor, CXCL13 facilitated vascular endothelial growth factor (VEGF) expression and angiogenesis in EPC through the PLC, MEK, and AP-1 signaling pathways. Importantly, infection with CXCL13 short hairpin RNA (shRNA) mitigated EPC homing and angiogenesis, articular swelling, and cartilage erosion in ankle joints of mice with CIA. CXCL13 is therefore a novel therapeutic target for RA.

Role of Stromal Cell-Derived Factor-1 in Endothelial Progenitor Cell-Mediated Vascular Repair and Regeneration.

Endothelial progenitor cells (EPCs) are immature endothelial cells that participate in vascular repair and postnatal neovascularization and provide a novel and promising therapy for the treatment of vascular disease. Studies in different animal models have shown that EPC mobilization through pharmacological agents and autologous EPC transplantation contribute to restoring blood supply and tissue regeneration after ischemic injury. However, these effects of the progenitor cells in clinical studies exhibitmixed results. The therapeutic efficacy of EPCs is closely associated with the number of the progenitor cells recruited into ischemic regions and their functional abilities and survival in injury tissues. In this review, we discussed the regulating role ofstromal cell-derived factor-1(also known CXCL12, SDF-1) in EPC mobilization, recruitment, homing, vascular repair and neovascularization, and analyzedthe underlying machemisms of these functions. Application of SDF-1 to improve the regenerative function of EPCs following vascular injurywas also discussed. SDF-1plays a crucial role in mobilizing EPC from bone marrowinto peripheral circulation, recruiting the progenitor cells to target tissue and protecting against cell death under pathological conditions; thus improve EPC regenerative capacity. SDF-1 are crucial for regulating EPC regenerative function, andprovide a potential target for improve therapeutic efficacy of the progenitor cells in treatment of vascular disease.

Stromal cell-derived factor-1 may play pivotal role in distraction-stimulated neovascularization of diabetic foot ulcer

Diabetic foot ulcer (DFU) has become a major medical, social and economic concern worldwide. It is highly desirable to develop promising new solutions to effectively and appropriately treat DFU. In recent years, investigators have used an innovative technology called proximal tibial cortex transverse distraction (PTCTD) to treat DFU and have achieved satisfactory results in terms of improved wound healing and circumvention of amputation as a consequence of enhanced neovascularization and perfusion of the ulcerated feet after the operation, but the underlying mechanism has not been explored. Previous studies have suggested that in addition to stimulating osteogenesis, bone distraction also facilitates neovascularization, which may be associated with the chemokine stromal cell-derived factor-1 (SDF-1). As an important member of the chemokine family, SDF-1 is primarily responsible for the homing and migration of endothelial progenitor cells (EPCs) or bone marrow-derived mesenchymal stem cells (BMSCs), and plays a central role in the process of neovascularization. In vivo or in vitro experiments show that bone distraction can induce the expression of SDF-1 and increase its plasma concentration. Moreover, some researchers have found that an insufficient level of SDF-1 in the circulation and wounds of patients with DFU can lead to impaired neovascularization. Therefore, we believe that SDF-1 plays an important role in promoting neovascularization of DFU as a result of bone distraction. We summarize the currently relevant literature to put forward an undisclosed but meaningful mechanism of bone distraction in the treatment of DFU.

Importance of β2AR 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. β2-Adrenergic receptor (β2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated β2AR signaling contributes to EPCs dysfunction, whereas enhanced β2AR 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. β2AR 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 β2AR 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 β2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against β2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of β2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired β2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of β2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of β2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired β2-adrenergic receptor (β2AR) 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. β2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating β2AR/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 β2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

Bimodal Imaging-Visible Nanomedicine Integrating CXCR4 and VEGFa Genes Directs Synergistic Reendothelialization of Endothelial Progenitor Cells.

A major challenge to treat vascular endothelial injury is the restoration of endothelium integrity in which endothelial progenitor cells (EPCs) plays a central role. Transplantation of EPCs as a promising therapeutic means is subject to two interrelated processes, homing and differentiation of EPCs in vivo, and thus a lack of either one may greatly affect the outcome of EPC‐based therapy. Herein, a polymeric nanocarrier is applied for the codelivery of CXCR4 and VEGFa genes to simultaneously promote the migration and differentiation of EPCs. Moreover, MRI T2 contrast agent SPION and NIR dye Cy7.5 are also loaded into the nanocarrier in order to track EPCs in vivo. Based on the synergistic effect of the two codelivered genes, an improved reendothelialization of EPCs is achieved in a rat carotid denuded model. The results show the potential of this bimodal imaging‐visible nanomedicine to improve the performance of EPCs in repairing arterial injury, which may push forward the stem cell‐based therapy of cardiovascular disease.

Neurotrophin-3 accelerates reendothelialization through inducing EPC mobilization and homing.

Rapid endothelialization is an effective way to treat intimal hyperplasia after intravascular stent implantation. Blood vessels and nerves coordinate with each other in function, while neurotrophin-3 (NT-3) is an important class of nerve growth factors. Our study found that NT-3 promoted endothelial progenitor cell (EPC) mobilization, and the proportion of EPCs in peripheral blood was increased by 1.774 times compared with the control group. Besides, NT-3 promoted the expression of stromal cell-derived factor-1α (SDF-1α), matrix metalloproteinase-9 (MMP9), and chemokine (C-X-C motif) receptor 4 (CXCR4) in EPCs, which increased by 59.89%, 74.46%, and 107.7%, respectively, compared with the control group. Transwell experiments showed that NT-3 enhanced the migration of EPCs by 1.31 times. Flow chamber experiments demonstrated that NT-3 captured more circulating EPCs. As shown by ELISA results, NT-3 can promote the paracrine of vascular endothelial growth factor, interleukin-8, MMP-9, and SDF-1 from EPCs. Such increased angiogenic growth factors further accelerated the closure of endothelial cell scratches. Additionally, EPC-conditioned medium in the NT-3 group significantly inhibited the proliferation of vascular smooth muscle cells. Then animal experiments also illustrated that NT-3 prominently accelerated the endothelialization of injured carotid artery. In short, NT-3 accelerated rapid reendothelialization of injured carotid artery through promoting EPC mobilization and homing.