Sphingosine-1-phosphate and its receptors in vascular endothelial and lymphatic barrier function.

The vascular and lymphatic systems both comprise a series of structurally distinct vessels lined with an inner layer of endothelial cells that function to provide a semipermeable barrier to blood and lymph. Regulation of the endothelial barrier is critical for maintaining vascular and lymphatic barrier homeostasis. One of the regulators of endothelial barrier function and integrity is sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite secreted into the blood by erythrocytes, platelets, and endothelial cells and into the lymph by lymph endothelial cells. Binding of S1P to its G protein-coupled receptors, known as S1PR1-5, regulates its pleiotropic functions. This review outlines the structural and functional differences between vascular and lymphatic endothelium and describes current understanding of the importance of S1P/S1PR signaling in regulation of barrier functions. Most studies thus far have been primarily focused on the role of the S1P/S1PR1 axis in vasculature and have been summarized in several excellent reviews, and thus, we will only discuss new perspectives on the molecular mechanisms of action of S1P and its receptors. Much less is known about the responses of the lymphatic endothelium to S1P and the functions of S1PRs in lymph endothelial cells, and this is the major focus of this review. We also discuss current knowledge related to signaling pathways and factors regulated by the S1P/S1PR axis that control lymphatic endothelial cell junctional integrity. Gaps and limitations in current knowledge are highlighted together with the need to further understand the role of S1P receptors in the lymphatic system.

Zebularine protects against blood-brain-barrier (BBB) disruption through increasing the expression of zona occludens-1 (ZO-1) and vascular endothelial (VE)-cadherin.

Blood-brain-barrier (BBB) disruption is an important pathological characteristic of ischemic stroke (IS) and mainly results from dysfunction of brain vascular endothelial cells and tight junctions. Zebularine is a novel inhibitor of DNA methyltransferase (DNMT). Here, we assessed its effects on BBB disruption in IS. Firstly, we reported that Zebularine maintained BBB integrity in middle cerebral artery occlusion (MCAO) mice by increasing the expressions of zona occludens-1 (ZO-1) and vascular endothelial (VE)-cadherin. Importantly, we found that Zebularine reduced the production of pro-inflammatory cytokines, attenuated brain edema, and improved neurological deficits. In in vitro experiments, the bEnd.3 brain endothelial cells were exposed to oxygen and glucose deprivation/reoxygenation (OGD/R), and the protective effects of Zebularine were assessed. Our findings demonstrated that Zebularine prevented OGD/R-induced cytotoxicity by reducing the release of lactate dehydrogenase (LDH). Additionally, Zebularine protected bEnd.3 cells against OGD/R-induced hyper-permeability and reduction of trans-endothelial electrical resistance (TEER). Notably, we found that treatment with Zebularine activated the Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway by increasing the phosphorylation of adenosine monophosphate-activated protein kinase α (AMPKα). Blockage of AMPKα using its specific inhibitor compound C abolished the beneficial effects of Zebularine in mitigating endothelial hyper-permeability by reducing the expressions of ZO-1 and VE-cadherin. These findings suggest that the protective effects of Zebularine against OGD/R-induced endothelial hyper-permeability are mediated by the activation of AMPKα. In conclusion, our study sheds light on the potential application of Zebularine in the treatment of IS.

Knockdown of receptor interacting protein 140 (RIP140) alleviated lipopolysaccharide-induced inflammation, apoptosis and permeability in pulmonary microvascular endothelial cells by regulating C-terminal binding protein 2 (CTBP2).

The main pathological feature of acute lung injury (ALI) is pulmonary edema caused by increased permeability of pulmonary microvascular endothelial cells (PMVECs). LPS was has been confirmed to lead to cell damage and barrier dysfunction in PMVECs. Furthermore, receptor interacting protein 140 (RIP140) was discovered to be increased in LPS-induced human pulmonary microvascular endothelial cells (HPMECs), but the mechanism of RIP140 on LPS-induced HPMECs has not been investigated. In this study, an acute lung injury model was constructed in LPS-induced HPMECs. After RIP140 was downregulated, inflammation, apoptosis and cell permeability levels were detected by RT-qPCR, TUNEL staining and FITC-Dextran, respectively. Western blotting was used to detect the protein levels of related factors. The binding of RIP140 and C-terminal binding protein 2 (CTBP2) was predicted by database and verified by Co-IP. Subsequently, CTBP2 overexpression was transfected into cells and the above experiments were performed again. The results showed that inflammation, apoptosis and permeability levels of LPS-induced HPMECs were remarkably increased compared to the untreated control group. However, these levels were suppressed after RIP140 was silenced compared to the LPS-induced HPMECs group. Notably, the Co-IP study demonstrated that RIP140 and CTBP2 interacted with each other. Moreover, CTBP2 overexpression reversed the inhibitory effects of RIP140 silencing on LPS-induced inflammation, apoptosis and permeability levels in HPMECs. Together, the study found that interference of RIP140 could alleviate LPS-induced inflammation, apoptosis and permeability in HPMECs by regulating CTBP2.

Metformin attenuates high glucose-induced injury in islet microvascular endothelial cells.

As one of the most frequently prescribed antidiabetic drugs, metformin can lower glucose levels, improve insulin resistance manage body weight. However, the effect of metformin on islet microcirculation remains unclear. In the present study, to explore the effect of metformin on islet endothelial cells and investigated the underlying mechanism, we assessed the effects of metformin on islet endothelial cell survival, proliferation, oxidative stress and apoptosis. Our results suggest that metformin stimulates the proliferation of pancreatic islet endothelial cells and inhibits the apoptosis and oxidative stress caused by high glucose levels. By activating farnesoid X receptor (FXR), metformin increases the expression of vascular endothelial growth factor-A (VEGF-A) and endothelial nitric oxide synthase (eNOS), improves the production of nitric oxide (NO) and decreases the production of ROS. After the inhibition of FXR or VEGF-A, all of the effects disappeared. Thus, metformin appears to regulate islet microvascular endothelial cell (IMEC) proliferation, apoptosis and oxidative stress by activating the FXR/VEGF-A/eNOS pathway. These findings provide a new mechanism underlying the islet-protective effect of metformin.

LINC00460 Stimulates the Proliferation of Vascular Endothelial Cells by Downregulating miRNA-24-3p.

OBJECTIVE: To clarify the effect of LINC00460 on mediating the proliferative ability of vascular endothelial cells (ECs) by targeting microRNA-24-3p (miRNA-24-3p), thus influencing the progression of atherosclerotic diseases. METHODS: Relative levels of LINC00460 and miRNA-24-3p in ECs induced with different doses of ox-LDL (oxidized low density lipoprotein) for different time points were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Influences of LINC00460 and miRNA-24-3p on the viability of ECs were assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay. Through dual-luciferase reporter gene assay, the binding between LINC00460 and miRNA-24-3p was evaluated. At last, rescue experiments were performed to identify the function of the LINC00460/miRNA-24-3p axis in regulating the proliferative ability of ECs. RESULTS: LINC00460 was upregulated after ox-LDL treatment in a dose- and time-dependent manner. Viability of ECs gradually increased with the prolongation of ox-LDL treatment and the treatment of increased dose. The overexpression of LINC00460 enhanced the viability and EdU-positive rate in ECs treated with ox-LDL. miRNA-24-3p was the direct target of LINC00460, which was negatively regulated by LINC00460. miRNA-24-3p was downregulated with the prolongation of ox-LDL treatment. The overexpression of miRNA-24-3p could reverse the effect of LINC00460 on regulating the proliferative ability of ECs. CONCLUSIONS: LINC00460 regulates the proliferative ability of ECs and thus the occurrence and development of coronary atherosclerotic diseases by targeting miRNA-24-3p.

Identification of the Relationship between Hub Genes and Immune Cell Infiltration in Vascular Endothelial Cells of Proliferative Diabetic Retinopathy Using Bioinformatics Methods.

BACKGROUND: Diabetic retinopathy (DR) is a serious ophthalmopathy that causes blindness, especially in the proliferative stage. However, the pathogenesis of its effect on endothelial cells, especially its relationship with immune cell infiltration, remains unclear. METHODS: The dataset GSE94019 was downloaded from the Gene Expression Omnibus (GEO) database to obtain DEGs. Through aggregate analyses such as Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis, a protein-protein interaction (PPI) network was constructed to analyze the potential function of DEGs. Weighted gene coexpression network analysis (WGCNA) and Cytoscape software including molecular complex detection (MCODE) and cytoHubba plug-ins were used to comprehensively analyze and determine the hub genes. ImmuCellAI analysis was performed to further study the relationship between samples, hub genes, and 24 types of immune cell infiltration. Finally, gene-set enrichment analysis (GSEA) was employed to identify the enrichment of immune cell infiltration and endothelial cell phenotype modifications in GO biological processes (BP) based on the expression level of hub genes. RESULTS: 2393 DEGs were identified, of which 800 genes were downregulated, and 1593 genes were upregulated. The results of functional enrichment revealed that 1398 BP terms were significantly enriched in DEGs. Three hub genes, EEF1A1, RPL11, and RPS27A, which were identified by conjoint analysis using WGCNA and Cytoscape software, were positively correlated with the number of CD4 naive T cells and negatively correlated with the numbers of B cells. The number of CD4 naive T cells, T helper 2 (Th2) cells, and effector memory T (Tem) cells were significantly higher while CD8 naive T cells and B cells significantly were lower in the diabetic group than in the nondiabetic group. CONCLUSIONS: We unearthed the DEGs and Hub genes of endothelial cells related to the pathogenesis of PDR: EEF1A1, RPL11, and RPS27A, which are highly related to each other and participate in the specific biological process of inflammation-related immune cell infiltration and endothelial cell development, chemotaxis, and proliferation, thus providing new perspectives into the diagnosis of and potential "killing two birds with one stone" targeted therapy for PDR.

Central Nervous System Stimulants Limit Caffeine Transport at the Blood-Cerebrospinal Fluid Barrier.

Caffeine, a common ingredient in energy drinks, crosses the blood-brain barrier easily, but the kinetics of caffeine across the blood-cerebrospinal fluid barrier (BCSFB) has not been investigated. Therefore, 127 autopsy cases (Group A, 30 patients, stimulant-detected group; and Group B, 97 patients, no stimulant detected group) were examined. In addition, a BCSFB model was constructed using human vascular endothelial cells and human choroid plexus epithelial cells separated by a filter, and the kinetics of caffeine in the BCSFB and the effects of 4-aminopyridine (4-AP), a neuroexcitatory agent, were studied. Caffeine concentrations in right heart blood (Rs) and cerebrospinal fluid (CSF) were compared in the autopsy cases: caffeine concentrations were higher in Rs than CSF in Group A compared to Group B. In the BCSFB model, caffeine and 4-AP were added to the upper layer, and the concentration in the lower layer of choroid plexus epithelial cells was measured. The CSF caffeine concentration was suppressed, depending on the 4-AP concentration. Histomorphological examination suggested that choroid plexus epithelial cells were involved in inhibiting the efflux of caffeine to the CSF. Thus, the simultaneous presence of stimulants and caffeine inhibits caffeine transfer across the BCSFB.

A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells.

Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.

FVIII regulates the molecular profile of endothelial cells: functional impact on the blood barrier and macrophage behavior.

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by deficient activity of blood coagulation factor VIII (FVIII). In addition, hemophilia patients show associated diseases including osteopenia, altered inflammation and vascular fragility which may represent the consequence of recurrent bleeding or may be related to the direct FVIII deficiency. Nowadays, recombinant FVIII is proposed to treat hemophilia patients with no circulating FVIII inhibitor. Initially described as a coenzyme to factor IXa for initiating thrombin generation, there is emerging evidence that FVIII is involved in multiple biological systems, including bone, vascular and immune systems. The present study investigated: (i) the functional activities of recombinant human FVIII (rFVIII) on endothelial cells, and (ii) the impact of rFVIII activities on the functional interactions of human monocytes and endothelial cells. We then investigated whether rFVIII had a direct effect on the adhesion of monocytes to the endothelium under physiological flow conditions. We observed that direct biological activities for rFVIII in endothelial cells were characterized by: (i) a decrease in endothelial cell adhesion to the underlying extracellular matrix; (ii) regulation of the transcriptomic and protein profiles of endothelial cells; (iii) an increase in the vascular tubes formed and vascular permeability in vitro; and (iv) an increase in monocyte adhesion activated endothelium and transendothelial migration. By regulating vascular permeability plus leukocyte adhesion and transendothelial migration, the present work highlights new biological functions for FVIII.

Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA.

Vascular endothelium plays a crucial role in vascular homeostasis and tissue fluid balance. To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-b-PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by CDH5 promoter and guide RNA (U6 promoter) induced highly efficient genome editing in endothelial cells (ECs) of the vasculatures, including lung, heart, aorta, and peripheral vessels in adult mice. Western blotting and immunofluorescent staining demonstrated an ∼80% decrease of protein expression selectively in ECs, resulting in a phenotype similar to that of genetic knockout mice. Nanoparticle delivery of plasmid DNA could induce genome editing of two genes or genome editing and transgene expression in ECs simultaneously. Thus, nanoparticle delivery of plasmid DNA is a powerful tool to rapidly and efficiently alter expression of gene(s) in ECs for cardiovascular research and potential gene therapy.

Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells.

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.

CircFUNDC1 knockdown alleviates oxygen-glucose deprivation-induced human brain microvascular endothelial cell injuries by inhibiting PTEN via miR-375.

BACKGROUND: The maintenance of human brain microvascular endothelial cell (HBMEC) function is crucial to improve the outcomes of ischemic stroke (IS). Emerging evidence shows that circular RNAs (circRNAs) are involved in IS progression. This study aimed to investigate the role of circRNA FUN14 domain containing 1 (circFUNDC1) in oxygen-glucose deprivation (OGD)-treated HBMECs. METHODS: The expression of circFUNDC1, miR-375 and phosphatase and tensin homolog (PTEN) mRNA was detected by quantitative real-time PCR (qPCR). Cell viability, apoptosis, migration and angiogenesis were determined by CCK-8 assay, flow cytometry assay, transwell assay and tube formation assay. The protein level of PTEN was detected by western blot. The relationship between miR-375 and circFUNDC1 or PTEN was confirmed by pull-down assay, dual-luciferase reporter assay and RIP assay. Exosomes were identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). RESULTS: CircFUNDC1 expression was increased in peripheral blood of IS patients and OGD-treated HBMECs. CircFUNDC1 knockdown alleviated OGD-induced cell apoptosis and promoted OGD-blocked cell viability, migration and angiogenesis of HBMECs. MiR-375 was a target of circFUNDC1, and miR-375 restoration played similar effects with circFUNDC1 knockdown. The inhibition of miR-375 reversed the effects of circFUNDC1 knockdown. In addition, PTEN was a downstream target of miR-375, and PTEN overexpression abolished the effects of miR-375 restoration. The expression of circFUNDC1 was elevated in serum-derived exosomes of IS patients, and circFUNDC1 harbored diagnostic values. CONCLUSION: CircFUNDC1 knockdown alleviates OGD-induced HBMECs injuries by inhibiting PTEN via enriching miR-375.

ETS-Related Gene Activation Preserves Adherens Junctions and Permeability in Microvascular Endothelial Cells.

ABSTRACT: ERG (ETS-related gene) is a member of the ETS (Erythroblast-transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. A wide variety of diseases and clinical conditions including trauma-hemorrhagic shock and burn injury are associated with microvascular dysfunctions, which causes excessive microvascular permeability, tissue edema and eventually, multiple organ dysfunction and death. The main purpose of this study was to determine the specific role of ERG in regulating microvascular permeability in human lung microvascular endothelial cells (HLMEC) and to evaluate if exogenous ERG will protect the barrier. The HLMECs were grown on Transwell inserts as monolayers and were transfected with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid, recombinant ERG protein or their respective controls. Recombinant vascular endothelial growth factor (VEGF) was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Changes in barrier integrity and permeability were studied using monolayer permeability assay and immunofluorescence of adherens junction proteins (VE-cadherin and ß-catenin) respectively. CRISPR/cas9-based ERG knockdown as well as VEGF treatment induced monolayer hyperpermeability, VE-cadherin, and ß-catenin junctional relocation and cytoskeletal F-actin stress fiber formation. CRISPR based ERG activation and recombinant ERG transfection attenuated VEGF-induced monolayer hyperpermeability. ERG activation preserved the adherens junctions and cytoskeleton. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability in human lung microvascular endothelial cells and endogenously or exogenously enhancing ERG provides protection against barrier dysfunction and hyperpermeability.

Bone marrow-derived mesenchymal stem cells transplantation attenuates renal fibrosis following acute kidney injury by repairing the peritubular capillaries.

After the severe initial insults of acute kidney injury, progressive kidney tubulointerstitial fibrosis may occur, the peritubular capillary (PTC) rarefaction plays a key role in the disease progression. However, the mechanisms of PTC damage were not fully understood and potential therapeutic interventions were not explored. Previous studies of our research team and others in this field suggested that bone marrow-derived mesenchymal stem cells (BMSCs) transplanted into the AKI rat model may preserve the kidney function and pathological changes. In the current study, with the ischemia/reperfusion AKI rat model, we revealed that BMSCs transplantation attenuated the renal function decrease in the AKI model through preserving the peritubular capillaries (PTCs) function. The density of PTCs is maintained by BMSCs transplantation in the AKI model, detachment and relocation of pericytes in the PTCs diminished. Then we established that BMSCs transplantation may attenuate the renal fibrosis and preserve the kidney function after AKI by repairing the PTCs. Improving the vitality of pericytes, suppressing the detachment and trans-differentiation of pericytes, directly differentiation of BMSCs into pericytes by BMSCs transplantation all participate in the PTC repair. Through these processes, BMSCs rescued the microvascular damage and improved the density of PTCs. As a result, a preliminary conclusion can be reached that BMSCs transplantation can be an effective therapy for delaying renal fibrosis after AKI.

Locus-Conserved Circular RNA cZNF292 Controls Endothelial Cell Flow Responses.

BACKGROUND: Circular RNAs (circRNAs) are generated by back splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here, we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. METHODS AND RESULTS: Combining published endothelial RNA-sequencing data sets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localization and focal adhesion organization. Mechanistically, we identified the protein SDOS (syndesmos) to specifically interact with cZNF292 in endothelial cells by RNA-affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganization thereby recapitulating cZfp292 knockout phenotypes. CONCLUSIONS: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

Leveraging avidin-biotin interaction to quantify permeability property of microvessels-on-a-chip networks.

Microvessels-on-a-chip have enabled in vitro studies to closely simulate in vivo microvessel environment. However, assessing microvessel permeability, a functional measure of microvascular exchange, has not been attainable in nonpermeable microfluidic platforms. This study developed a new approach that enables permeability coefficients (Ps) to be quantified in microvessels developed in nonpermeable chip platforms by integrating avidin-biotin technology. Microvessels were developed on biotinylated fibronectin-coated microfluidic channels. Solute transport was assessed by perfusing microvessels with fluorescence-labeled avidin. Avidin molecules that crossed endothelium were captured by substrate biotin and recorded with real-time confocal images. The Ps was derived from the rate of avidin-biotin accumulation at the substrate relative to solute concentration difference across microvessel wall. Avidin tracers with different physiochemical properties were used to characterize the barrier properties of the microvessel wall. The measured baseline Ps and inflammatory mediator-induced increases in Ps and endothelial cell (EC) [Ca2+]i resembled those observed in intact microvessels. Importantly, the spatial accumulation of avidin-biotin at substrate defines the transport pathways. Glycocalyx layer is well formed on endothelium and its degradation increased transcellular transport without affecting EC junctions. This study demonstrated that in vitro microvessels developed in this simply designed microfluidics structurally possess in vivo-like glycocalyx layer and EC junctions and functionally recapitulate basal barrier properties and stimuli-induced responses observed in intact microvessels. This new approach overcomes the limitations of nonpermeable microfluidics and provides an easily executed highly reproducible in vitro microvessel model with in vivo microvessel functionality, suitable for a wide range of applications in blood and vascular research and drug development.NEW & NOTEWORTHY Our study developed a novel method that allows permeability coefficient to be measured in microvessels developed in nonpermeable microfluidic platforms using avidin-biotin technology. It overcomes the major limitation of nonpermeable microfluidic system and provides a simply designed easily executed and highly reproducible in vitro microvessel model with permeability accessibility. This model with in vivo-like endothelial junctions, glycocalyx, and permeability properties advances microfluidics in microvascular research, suitable for a wide range of biomedical and clinical applications.

Vascular endothelial growth factor c regulates hematopoietic stem cell fate in the dorsal aorta.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells that self-renew or differentiate to establish the entire blood hierarchy. HSPCs arise from the hemogenic endothelium of the dorsal aorta (DA) during development in a process called endothelial-to-hematopoietic transition. The factors and signals that control HSPC fate decisions from the hemogenic endothelium are not fully understood. We found that Vegfc has a role in HSPC emergence from the zebrafish DA. Using time-lapse live imaging, we show that some HSPCs in the DA of vegfc loss-of-function embryos display altered cellular behavior. Instead of typical budding from the DA, emergent HSPCs exhibit crawling behavior similar to myeloid cells. This was confirmed by increased myeloid cell marker expression in the ventral wall of the DA and the caudal hematopoietic tissue. This increase in myeloid cells corresponded with a decrease in HSPCs that persisted into larval stages. Together, our data suggest that Vegfc regulates HSPC emergence in the hemogenic endothelium, in part by suppressing a myeloid cell fate. Our study provides a potential signal for modulation of HSPC fate in stem cell differentiation protocols.

3D printing of self-standing and vascular supportive multimaterial hydrogel structures for organ engineering.

Three dimensional printable formulation of self-standing and vascular-supportive structures using multi-materials suitable for organ engineering is of great importance and highly challengeable, but, it could advance the 3D printing scenario from printable shape to functional unit of human body. In this study, the authors report a 3D printable formulation of such self-standing and vascular-supportive structures using an in-house formulated multi-material combination of albumen/alginate/gelatin-based hydrogel. The rheological properties and relaxation behavior of hydrogels were analyzed before the printing process. The suitability of the hydrogel in 3D printing of various customizable and self-standing structures, including a human ear model, was examined by extrusion-based 3D printing. The structural, mechanical, and physicochemical properties of the printed scaffolds were studied systematically. Results supported the 3D printability of the formulated hydrogel with self-standing structures, which are customizable to a specific need. In vitro cell experiment showed that the formulated hydrogel has excellent biocompatibility and vascular supportive behavior with the extent of endothelial sprout formation when tested with human umbilical vein endothelial cells. In conclusion, the present study demonstrated the suitability of the extrusion-based 3D printing technique for manufacturing complex shapes and structures using multi-materials with high fidelity, which have great potential in organ engineering.

Knockdown of Long Non-coding RNA TUG1 Suppresses Migration and Tube Formation in High Glucose-Stimulated Human Retinal Microvascular Endothelial Cells by Sponging miRNA-145.

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus. The purpose of this study was to investigate the potential functional role of long non-coding RNA TUG1 in high glucose (HG)-stimulated human retinal microvascular endothelial cells (hRMECs). The results demonstrated that following 72 h of HG stimulation, enhanced proliferation, migration, and tube formation process were observed in hRMECs. Moreover, HG treatment markedly increased TUG1 expression in hRMECs, and knockdown of TUG1 notably restrained the aberrant phenotypes of hRMECs induced by HG. Mechanistically, TUG1 may serve as a competing endogenous RNA (ceRNA) for miR-145, thereby blocking the repression on VEGF-A in hRMECs. Rescue experiments further indicated that inhibition of miR-145 abolished the beneficial role of TUG1 knockdown in HG-treated hRMECs. Our data suggested that knockdown of TUG1 protects hRMECs against HG stimulation partly by regulating miR-145/VEGF-A axis.

Designing Cardiovascular Implants Taking in View the Endothelial Basement Membrane.

Insufficient endothelialization of cardiovascular grafts is a major hurdle in vascular surgery and regenerative medicine, bearing a risk for early graft thrombosis. Neither of the numerous strategies pursued to solve these problems were conclusive. Endothelialization is regulated by the endothelial basement membrane (EBM), a highly specialized part of the vascular extracellular matrix. Thus, a detailed understanding of the structure-function interrelations of the EBM components is fundamental for designing biomimetic materials aiming to mimic EBM functions. In this review, a detailed description of the structure and functions of the EBM are provided, including the luminal and abluminal interactions with adjacent cell types, such as vascular smooth muscle cells. Moreover, in vivo as well as in vitro strategies to build or renew EBM are summarized and critically discussed. The spectrum of methods includes vessel decellularization and implant biofunctionalization strategies as well as tissue engineering-based approaches and bioprinting. Finally, the limitations of these methods are highlighted, and future directions are suggested to help improve future design strategies for EBM-inspired materials in the cardiovascular field.