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Endothelial cells and macrophages are known to engage in tight and specific interactions that contribute to the modulation of vascular function. Here we show that adult endothelial cells provide critical signals for the selective growth and differentiation of macrophages from several hematopoietic progenitors. The process features the formation of well-organized colonies that exhibit progressive differentiation from the center to the periphery and toward an M2-like phenotype,characterized by enhanced expression of Tie2 and CD206/Mrc1. These colonies are long-lived depending on the contact with the endothelium; removal of the endothelial monolayer results in rapid colony dissolution. We further found that Csf1 produced by the endothelium is critical for the expansion of the macrophage colonies and that blockade of Csf1 receptor impairs colony growth. Functional analyses indicate that these macrophages are capable of accelerating angiogenesis,promoting tumor growth,and effectively engaging in tight associations with endothelial cells in vivo. These findings uncover a critical role of endothelial cells in the induction of macrophage differentiation and their ability to promote further polarization toward a proangiogenic phenotype. This work also highlights some of the molecules underlying the M2-like differentiation,a process that is relevant to the progression of both developmental and pathologic angiogenesis. View Publication

AbstractAcute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common life‐threatening syndrome with no effective pharmacotherapy. Sepsis‐related ARDS is the main type of ARDS and is more fatal than other types. Extracellular vesicles (EVs) are considered novel mediators in the development of inflammatory diseases. Our previous research suggested that endothelial cell‐derived EVs (EC‐EVs) play a crucial role in ALI/ARDS development,but the mechanism remains largely unknown. Here,we demonstrated that the number of circulating EC‐EVs was increased in sepsis,exacerbating lung injury by targeting monocytes and reprogramming them towards proinflammatory macrophages. Bioinformatics analysis and further mechanistic studies revealed that vascular cell adhesion molecule 1 (VCAM1),overexpressed on EC‐EVs during sepsis,activated the NF‐κB pathway by interacting with integrin subunit alpha 4 (ITGA4) on the monocyte surface,rather than the tissue resident macrophage surface,thereby regulating monocyte differentiation. This effect could be attenuated by decreasing VCAM1 levels in EC‐EVs or blocking ITGA4 on monocytes. Furthermore,the number of VCAM1+ EC‐EVs was significantly increased in patients with sepsis‐related ARDS. These findings not only shed light on a previously unidentified mechanism underling sepsis‐related ALI/ARDS,but also provide potential novel targets and strategies for its precise treatment. During extra‐pulmonary sepsis,more endothelial cell‐derived extracellular vesicles (EC‐EVs) are released,which play a critical role in the development of ALI/ARDS by specifically targeting and reprogramming monocytes. VCAM1,highly expressed on these EVs,activates the NF‐κB pathway by acting on ITGA4,thus promoting the differentiation of monocytes into M1‐type macrophages. View Publication

AbstractThe fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here,we implemented a 3D model of cardiac vasculogenesis,incorporating endothelial cells (EC),stromal cells,and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues,resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis,including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs,the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast,supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly,enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization,illustrating the use of this approach in the engineering of enhanced,perfusable,microfluidic models of the myocardium. View Publication

BACKGROUND: In animal models,circulating endothelial progenitor cells (EPC) have been shown to participate in repair of damaged or degenerating vascular surfaces. In humans,reduced EPC counts correlate with cardiovascular risk and disease outcome; yet it has been difficult to establish that EPC are in fact mobilized in response to vascular injury as a physiologic response. We therefore studied early (textless12h) mobilization of EPCs into the peripheral circulation after a defined vascular manipulation,percutaneous coronary intervention (PCI) in acute coronary syndrome (ACS) and non-ACS patients. METHODS AND RESULTS: CD34/CD31 positive EPC colony forming units (EPC-CFU) were quantified by a blinded observer in peripheral blood samples from eight control patients with angiographically normal coronary arteries,and in 30 patients with coronary artery lesions before and 12h after PCI. All patients (n=38) had one or more CV risk factors. Ten patients presented with acute coronary syndrome (PCI(ACS)),and the rest (n=20) underwent elective PCI (PCI(Elect)). Despite the presence of an acute coronary syndrome,patients in the PCI(ACS) group did not present with increased EPC-CFU compared with either the PCI(Elect) or control groups (Ptextgreater0.05). In addition,EPC-CFU (colonies/ml blood) increased significantly in the PCI(Elect) group after stent placement (11.8+1.6 before versus 16.5+1.9 after,P=0.0009),while in contrast,PCI did not stimulate EPC mobilization in patients in the PCI(ACS) group (9.6+3.2 before versus 6.5+1.8,P=0.20). We found a higher presenting vascular endothelial growth factor (VEGF) level in the PCI(Elect) group compared to PCI(ACS) (78.7+25.2 versus 15.3+7.9 pg/ml blood,P=0.02). However,VEGF levels increased after PCI only in the PCI(ACS) group (15.3+7.9 to 133.3+27.5 pg/ml,P=0.003) and not in the PCI(Elect) group (78.7+25.2 to 79.7+12.2 pg/ml,P=0.97). CONCLUSION: Our findings suggest that focal coronary endothelial injury as a result of PCI triggers early mobilization of EPC into the peripheral circulation in patients presenting for an elective PCI,without a corresponding rise in VEGF levels. In contrast,patients with an acute coronary syndrome fail to respond to PCI with early EPC mobilization despite a significant rise in VEGF. The results of the present study may suggest a novel mechanism for early EPC augmentation after PCI. View Publication

BACKGROUND Protection of cerebral endothelial cells (ECs) from hypoxia/reoxygenation (H/R)-induced injury is an important strategy for treating ischemic stroke. In this study,we investigated whether co-culture with endothelial progenitor cells (EPCs) and neural progenitor cells (NPCs) synergistically protects cerebral ECs against H/R injury and the underlying mechanism. RESULTS EPCs and NPCs were respectively generated from inducible pluripotent stem cells. Human brain ECs were used to produce an in vitro H/R-injury model. Data showed: 1) Co-culture with EPCs and NPCs synergistically inhibited H/R-induced reactive oxygen species (ROS) over-production,apoptosis,and improved the angiogenic and barrier functions (tube formation and permeability) in H/R-injured ECs. 2) Co-culture with NPCs up-regulated the expression of vascular endothelial growth factor receptor 2 (VEGFR2). 3) Co-culture with EPCs and NPCs complementarily increased vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) levels in conditioned medium,and synergistically up-regulated the expression of p-Akt/Akt and p-Flk1/VEGFR2 in H/R-injured ECs. 4) Those effects could be decreased or abolished by inhibition of both VEGFR2 and tyrosine kinase B (TrkB) or phosphatidylinositol-3-kinase (PI3K). CONCLUSIONS Our data demonstrate that EPCs and NPCs synergistically protect cerebral ECs from H/R-injury,via activating the PI3K/Akt pathway which mainly depends on VEGF and BDNF paracrine. View Publication

AIMS: Endothelial progenitor cells (EPC) have been shown to repair pulmonary endothelium,although they can also migrate into the arterial intima and differentiate into smooth muscle-like (mesenchymal) cells contributing to intimal hyperplasia. The molecular mechanisms by which this process proceeds have not been fully elucidated. Here,we study whether genes involved in the endothelial-to-mesenchymal transition (EnMT) may contribute to the mesenchymal phenotype acquisition of EPC and we evaluate whether transforming growth factor β1 (TGFβ1) is involved in this process. METHODS AND RESULTS: Our results show that co-culture of EPC with smooth muscle cells (SMC) increases the expression of the mesenchymal cell markers α-smooth muscle actin,sm22-α,and myocardin,and decreases the expression of the endothelial cell marker CD31. In the same conditions,we also observed a concomitant increase in the gene expression of the EnMT-related transcription factors: slug,snail,zeb1,and endothelin-1. This indicates that mesenchymal phenotype acquisition occurred through an EnMT-like process. Inhibition of TGFβ receptor I (TGFβRI) downregulated snail gene expression,blocked the EnMT,and facilitated the differentiation of EPC to the endothelial cell lineage. Furthermore,TGFβRI inhibition decreased migration of EPC stimulated by SMC without affecting their functionality and adhesion capacity. CONCLUSION: These results indicate that EPC may differentiate into SMC-like cells through an EnMT-like process and that TGFβI plays an important role in the fate of EPC. View Publication

Endothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term EPC." It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping potency assays and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review we seek to discourage the indiscriminate use of "EPCs and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well-defined cell types that have been considered EPCs because they both promote vascular repair,albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing EPC" nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and in some cases progress toward use in cell therapy. Stem Cells Translational Medicine 2017;6:1316-1320. View Publication

The blood?brain barrier (BBB) is a critical selective interface between the central nervous system (CNS) and the blood circulation. BBB dysfunction plays an important role in the neurological damage caused by sepsis. However,the mechanisms underlying the disruption of the BBB during sepsis remain unclear. We established a human induced pluripotent stem cell (iPSC)-derived BBB model and reported that treating with sepsis patient serum leads to structural and functional disruption of the BBB. In a cecal ligation and puncture (CLP)-induced mouse model of sepsis,we also observed disruption of the BBB,inflammation in the brain,and impairments in cognition. In both models,we found that the expression of TREM-1 was significantly increased in endothelial cells. TREM-1 knockout specifically in endothelial cells alleviated BBB dysfunction and cognitive impairments. Further study revealed that TREM-1 affects the expression of genes involved in the PI3K/Akt signaling pathway. The protective effects of TREM-1 inhibition on the BBB and cognition were abrogated by PI3K inhibitors. Our findings suggest that endothelial TREM-1 induces sepsis-induced BBB disruption and cognitive impairments via the PI3K/Akt signaling pathway. Targeting endothelial TREM-1 or the PI3K/Akt signaling pathway may be a promising strategy to maintain BBB integrity and improve cognitive function in sepsis patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03469-5. View Publication

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide. Laminar shear stress from blood flow,sensed by vascular endothelial cells,protects from ASCVD by upregulating the transcription factors KLF2 and KLF4,which induces an anti-inflammatory program that promotes vascular resilience. Here we identify clustered γ-protocadherins as therapeutically targetable,potent KLF2 and KLF4 suppressors whose upregulation contributes to ASCVD. Mechanistic studies show that γ-protocadherin cleavage results in translocation of the conserved intracellular domain to the nucleus where it physically associates with and suppresses signaling by the Notch intracellular domain. γ-Protocadherins are elevated in human ASCVD endothelium; their genetic deletion or antibody blockade protects from ASCVD in mice without detectably compromising host defense against bacterial or viral infection. These results elucidate a fundamental mechanism of vascular inflammation and reveal a method to target the endothelium rather than the immune system as a protective strategy in ASCVD. Joshi et al. show that γ-protocadherins suppress the anti-inflammatory KLF2 and KLF4 pathway and that targeting them is a viable therapeutic strategy to protect against atherosclerosis. View Publication

Aging of the brain vasculature plays a key role in the development of neurovascular and neurodegenerative diseases,thereby contributing to cognitive impairment. Among other factors,DNA damage strongly promotes cellular aging,however,the role of genomic instability in brain endothelial cells (EC) and its potential effect on brain homeostasis is still largely unclear. We here investigated how endothelial aging impacts blood-brain barrier (BBB) function by using excision repair cross complementation group 1 (ERCC1)-deficient human brain ECs and an EC-specific Ercc1 knock out (EC-KO) mouse model. In vitro,ERCC1-deficient brain ECs displayed increased senescence-associated secretory phenotype expression,reduced BBB integrity,and higher sprouting capacities due to an underlying dysregulation of the Dll4-Notch pathway. In line,EC-KO mice showed more P21+ cells,augmented expression of angiogenic markers,and a concomitant increase in the number of brain ECs and pericytes. Moreover,EC-KO mice displayed BBB leakage and enhanced cell adhesion molecule expression accompanied by peripheral immune cell infiltration into the brain. These findings were confined to the white matter,suggesting a regional susceptibility. Collectively,our results underline the role of endothelial aging as a driver of impaired BBB function,endothelial sprouting,and increased immune cell migration into the brain,thereby contributing to impaired brain homeostasis as observed during the aging process. View Publication

PURPOSE This study analyzes additional mechanisms behind the ocular hypotensive effect of prostaglandin F (PGF) receptor (FP receptor) agonists PGF2alpha and fluprostenol (fluprostenol-isopropyl ester [travoprost]),which reduce intraocular pressure (IOP) in patients with glaucoma probably by enhancing uveoscleral flow. The trabecular meshwork (TM) is actively involved in IOP regulation through contractile mechanisms. Contractility of TM is induced by endothelin (ET)-1,a possible pathogenic factor in glaucoma. The involvement of FP receptor agonists in the ET-1 effects on TM function was studied. METHODS The effects of FP receptor agonists on contractility of bovine TM (BTM) were investigated using a force-length transducer. The effects of PGF2alpha on intracellular Ca2+ ([Ca2+]i) mobilization in cultured cells were measured using fura-2AM. The expression of the FP receptor protein was examined using Western blot analysis. RESULTS The ET-1-induced (10(-8) M) contraction in isolated BTM was inhibited by PGF2alpha (10(-6) M) and fluprostenol (10(-6) M). This effect was blocked by FP receptor antagonists. Carbachol-induced contraction or baseline tension was not affected by PGF2alpha or fluprostenol. In cultured TM cells,ET-1 caused a transient increase in [Ca2+]i that was reduced by PGF2alpha. No reduction occurred in the presence of the FP receptor antagonist Al-8810. Western blot analysis revealed the expression of the FP receptor in native and cultured TM. CONCLUSIONS FP receptor agonists operate by direct interaction with ET-1-induced contractility of TM. This effect is mediated by the FP receptor. Thus,FP receptor agonists may decrease IOP by enhancing aqueous humor outflow through the TM by inhibiting ET-1-dependent mechanisms. View Publication

Coronary arteriogenesis is a central step in cardiogenesis,requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present,it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro,and contribute extensively to coronary-like vessels in vivo,forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates,and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo. View Publication