Endothelial Cell Phenotype Research Articles

The Pathogenesis of Port Wine Stain and Sturge Weber Syndrome: Complex Interactions between Genetic Alterations and Aberrant MAPK and PI3K Activation.

Port wine stain (PWS) is a congenital vascular malformation involving human skin. Approximately 15–20% of children a facial PWS involving the ophthalmic (V1) trigeminal dermatome are at risk for Sturge Weber syndrome (SWS), a neurocutaneous disorder with vascular malformations in the cerebral cortex on the same side of the facial PWS lesions. Recently, evidence has surfaced that advanced our understanding of the pathogenesis of PWS/SWS, including discoveries of somatic genetic mutations (GNAQ, PI3K), MAPK and PI3K aberrant activations, and molecular phenotypes of PWS endothelial cells. In this review, we summarize current knowledge on the etiology and pathology of PWS/SWS based on evidence that the activation of MAPK and/or PI3K contributes to the malformations, as well as potential futuristic treatment approaches targeting these aberrantly dysregulated signaling pathways. Current data support that: (1) PWS is a multifactorial malformation involving the entire physiological structure of human skin; (2) PWS should be pathoanatomically re-defined as “a malformation resulting from differentiation-impaired endothelial cells with a progressive dilatation of immature venule-like vasculatures”; (3) dysregulation of vascular MAPK and/or PI3K signaling during human embryonic development plays a part in the pathogenesis and progression of PWS/SWS; and (4) sporadic low frequency somatic mutations, such as GNAQ, PI3K, work as team players but not as a lone wolf, contributing to the development of vascular phenotypes. We also address many crucial questions yet to be answered in the future research investigations.

Human aortic endothelial cells have osteogenic Notch-dependent properties in co-culture with aortic smooth muscle cells

Cardiovascular calcification is one of the leading reasons of morbidity and mortality in Western countries and has many similarities to osteogenesis. The role of smooth muscle calcific transformation is well established for atherogenic lesions, but mechanisms driving initial stages of proosteogenic cell fate commitment in big vessels remain poorly understood. The role of endothelial and underlying interstitial cell interaction in driving cellular decisions is emerging from recent studies. The aim of this study was to analyze co-culture of endothelial and smooth muscle cells in vitro in acquiring proosteogenic phenotype. We co-cultured human aortic endothelial cells (EC) and human aortic smooth muscle cells (SMC) and analyzed osteogenic phenotype by ALP staining and proosteogenic gene expression by qPCR in co-cultures and in separate cellular types after magnetic CD31-sorting. In EC and SMC co-cultures osteogenic phenotype was induced as well as activated expression of RUNX2, POSTIN, BMP2/4, SOX5, COL1A SMC; co-culture of EC with SMC induced NOTCH1, NOTCH3, NOTCH4 and HEY1 expression; Notch activation by lentiviral activated Notch intracellular domain induced expression of RUNX2, OPN, POSTIN in SMC; NOTCH1 and NOTCH3 and HEY1 were selectively induced in EC during co-culture. We conclude that endothelial cells are capable of driving smooth muscle calcification via cell-cell contact and activation of Notch signaling.

A Microfluidic System for Studying the Effects of Disturbed Flow on Endothelial Cells.

Arterial endothelium experience physical stress associated with blood flow and play a central role in maintaining vascular integrity and homeostasis in response to hemodynamic forces. Blood flow within vessels is generally laminar and streamlined. However, abrupt changes in the vessel geometry due to branching, sharp turns or stenosis can disturb the laminar blood flow, causing secondary flows in the form of vortices. Such disturbed flow patterns activate pro-inflammatory phenotypes in endothelial cells, damaging the endothelial layer and can lead to atherosclerosis and thrombosis. Here, we report a microfluidic system with integrated ridge-shaped obstacles for generating controllable disturbed flow patterns. This system is used to study the effect of disturbed flow on the cytoskeleton remodeling and nuclear shape and size of cultured human aortic endothelial cells. Our results demonstrate that the generated disturbed flow changes the orientation angle of actin stress fibers and reduces the nuclear size while increases the nuclear circularity.

Proteomic analysis of human mesenchymal stromal cell secretomes: a systematic comparison of the angiogenic potential

Human mesenchymal stromal cell (hMSC) secretomes have shown to influence the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair. The angiogenic potential is of particular interest for the treatment of ischemic diseases. Interestingly, hMSC secretomes isolated from different tissue sources have shown dissimilarities with respect to their angiogenic profile. This study compares angiogenesis of hMSC secretomes from adipose tissue (hADSCs), bone marrow (hBMSCs), and umbilical cord Wharton’s jelly (hWJSCs). hMSC secretomes were obtained under xenofree conditions and analyzed by liquid chromatography tandem mass spectrometry (LC/MS-MS). Biological processes related to angiogenesis were found to be enriched in the proteomic profile of hMSC secretomes. hWJSC secretomes revealed a more complete angiogenic network with higher concentrations of angiogenesis related proteins, followed by hBMSC secretomes. hADSC secretomes lacked central angiogenic proteins and expressed most detected proteins to a significantly lower level. In vivo all secretomes induced vascularization of subcutaneously implanted Matrigel plugs in mice. Differences in secretome composition were functionally analyzed with monocyte and endothelial cell (EC) in vitro co-culture experiments using vi-SNE based multidimensional flow cytometry data analysis. Functional responses between hBMSC and hWJSC secretomes were comparable, with significantly higher migration of CD14++ CD16− monocytes and enhanced macrophage differentiation compared with hADSC secretomes. Both secretomes also induced a more profound pro-angiogenic phenotype of ECs. These results suggest hWJSCs secretome as the most potent hMSC source for inflammation-mediated angiogenesis induction, while the potency of hADSC secretomes was lowest. This systematic analysis may have implication on the selection of hMSCs for future clinical studies.

An in vivo wound healing model for the characterization of the angiogenic process and its modulation by pharmacological interventions

Angiogenesis during wound healing is essential for tissue repair and also affected during cancer treatment by anti-angiogenic drugs. Here, we introduce a minimally invasive wound healing model in the mouse ear to assess angiogenesis with high spatiotemporal resolution in a longitudinal manner in vivo using two-photon microscopy in mice expressing GCaMP2 in arterial endothelial cells. The development of vascular sprouts occurred in a highly orchestrated manner within a time window of 8 days following wounding. Novel sprouts developed exclusively from the distal stump of the transsected arteries, growing towards the proximal arterial stump. This was in line with the incidence of Ca2+ transients in the arterial endothelial cells, most probably a result of VEGF stimulation, which were more numerous on the distal part. Functional analysis revealed perfusion across the wound site via arterial sprouts developed between days 6 and 8 following the incision. At day 8, proximal and distal arteries were structurally and functionally connected, though only 2/3 of all sprouts detected were actually perfused. Treatment with the FDA approved drug, sunitinib, the preclinical drug AZD4547, as well as with the combination of the two agents had significant effects on both structural and functional readouts of neo-angiogenesis. The simplicity and high reproducibility of the model makes it an attractive tool for elucidating migratory activity, phenotype and functionality of endothelial cells during angiogenesis and for evaluating specific anti-angiogenic drug interventions.

Bone Marrow Endothelial Cells Influence Function and Phenotype of Hematopoietic Stem and Progenitor Cells after Mixed Neutron/Gamma Radiation.

The bone marrow (BM) microenvironment plays a crucial role in the maintenance and regeneration of hematopoietic stem (HSC) and progenitor cells (HSPC). In particular, the vascular niche is responsible for regulating HSC maintenance, differentiation, and migration of cells in and out of the BM. Damage to this niche upon exposure to ionizing radiation, whether accidental or as a result of therapy, can contribute to delays in HSC recovery and/or function. The ability of BM derived-endothelial cells (BMEC) to alter and/or protect HSPC after exposure to ionizing radiation was investigated. Our data show that exposure of BMEC to ionizing radiation resulted in alterations in Akt signaling, increased expression of PARP-1, IL6, and MCP-1, and decreased expression of MMP1 and MMP9. In addition, global analysis of gene expression of HSC and BMEC in response to mixed neutron/gamma field (MF) radiation identified 60 genes whose expression was altered after radiation in both cell types, suggesting that a subset of genes is commonly affected by this type of radiation. Focused gene analysis by RT-PCR revealed two categories of BMEC alterations: (a) a subset of genes whose expression was altered in response to radiation, with no additional effect observed during coculture with HSPC, and (b) a subset of genes upregulated in response to radiation, and altered when cocultured with HSPC. Coculture of BMEC with CD34+ HSPC induced HSPC proliferation, and improved BM function after MF radiation. Nonirradiated HSPC exhibited reduced CD34 expression over time, but when irradiated, they maintained higher CD34 expression. Nonirradiated HSPC cocultured with nonirradiated BMEC expressed lower levels of CD34 expression compared to nonirradiated alone. These data characterize the role of each cell type in response to MF radiation and demonstrate the interdependence of each cell’s response to ionizing radiation. The identified genes modulated by radiation and coculture provide guidance for future experiments to test hypotheses concerning specific factors mediating the beneficial effects of BMEC on HSPC. This information will prove useful in the search for medical countermeasures to radiation-induced hematopoietic injury.

Oxidative stress upregulates Wnt signaling in human retinal microvascular endothelial cells through activation of disheveled.

Abnormal retinal neovascularization associated with various retinopathies can result in irreversible vision loss. Although the mechanisms involved in this occurrence is unclear, increasing evidence suggests that aberrant Wnt signaling participates in the pathogenesis of abnormal neovascularization. Because Wntsignaling upregulation can be induced by oxidative stress through the activation of disheveled (DVL), a key molecule in the Wntsignaling pathway, we investigated whether oxidative stress can activate Wnt signaling and induce angiogenic phenotypes in human retinal microvascular endothelial cells (HRMECs). We found that increased Wntsignaling activity, as well as enhanced angiogenic phenotypes, such as tube formation and cell migration, were detected in the hydrogen peroxide-treated HRMECs. Moreover, these effects were effectively suppressed by a small-molecule Wnt inhibitor targeting the PDZ domain of DVL. Therefore, we propose that targeting abnormal Wnt signaling at the DVL level with a small-molecule inhibitor may represent a novel approach in retinal neovascularization treatment and prevention.

Sphingosine 1-phosphate receptor 1 regulates cell-surface localization of membrane proteins in endothelial cells

The endothelial cell (EC) barrier disruption has been implicated in vascular leakage and pulmonary edema. Many reports have shown that the EC barrier dysfunction is regulated by the sphingosine-1-phophate (S1P)/S1P receptor-1 (S1PR1) axis. Identifying downstream effectors for the S1P/S1PR1 axis in pulmonary vasculature has been limited by mixed populations in vitro cultures that do not retain physiological EC phenotype and complex of tedious proteomics. In this study, we used a combination of in vivo biotinylation and liquid chromatograph tandem mass spectrometry on three mouse models of S1pr1 expression, namely normal, knockout (KO) and high, to identify EC membrane proteins whose cell-surface expression is S1pr1-dependent. EC-specific KO of S1pr1 caused severe pulmonary vascular disruption and reduction of many membrane proteins on ECs. Using the MaxQuant software we were able to identify novel membrane targets of S1pr1, for instance, Cd105 and Plvap, by comparison with their membrane expressions among the three EC model systems. Moreover, regulation of Cd105 and Plvap by S1pr1 were validated with Western blot and immunostaining in vivo and in vitro. Our data suggest that S1pr1 dictates cell-surface localization of several apical membrane proteins in ECs. Our results are insightful for development of novel therapeutics to specifically target EC barrier function.

Anoctamin‐1 Expression at the Mitochondrial Membrane of Pulmonary Artery Endothelial Cells

Anoctamin‐1 (Ano1) is a calcium‐activated chloride channel that can regulate cell proliferation and cell cycle in endothelial cells (ECs). According to a recent proteomics study, Ano1 interacts with mitochondrial proteins in addition to plasma‐membrane proteins. However, the expression levels of Ano1 and its functions in other cellular compartments remain unclear. We previously reported that Ano1 is overexpressed in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH) and that Ano1 activation promotes apoptosis of ECs from patients with idiopathic PAH, likely via depolarization of mitochondrial membrane potential and increased mitochondrial reactive oxygen species generation. Therefore, we hypothesized that Ano1 forms channels at the inner mitochondrial membrane (IMM) in addition to the plasma membrane, which may contribute to a hyperproliferative phenotype of ECs under PAH. Here, we determined the mitochondrial expression of Ano1 and further investigated its role in the mitochondria.Rat lung microvascular ECs (RLMVECs) were used for biochemical and cell biological assays. HEK293T cells that stably overexpress Ano1 were generated and used for binding assays with mitochondrial inner membrane proteins. RLMVEC lysates were separated via centrifugation to obtain a mitochondria‐enriched fraction (Mito) and a cytosolic fraction (Cyto) containing other cellular organelles. Cyto was further separated by high‐speed centrifugation into a soluble fraction (Sol) and the insoluble particulate fraction (Insol) that is comprised of plasma and other organelle (e.g., Golgi and endoplasmic reticulum) membranes. The purity of each fraction and the structural integrity of isolated mitochondria in the Mito were assessed by immunoblotting of mitochondrial and non‐mitochondrial protein markers.The subcellular localization of Ano1 was determined by co‐immunostaining Ano1 and a mitochondria‐localized protein, translocase of the outer mitochondrial membrane 20 (TOM20), in RLMVECs. The mitochondrial localization of Ano1 was also confirmed by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1. Biochemical assays using protein fractionation from RLMVECs showed Ano1 protein expression in the Mito as well as the Insol obtained from the Cyto. The data from these systems indicate that Ano1 is localized in both the plasma membrane and mitochondria. Moreover, using HEK293T cells stably overexpressing Ano1, we found by immunoprecipitation that Ano1 interacts with a mitochondrial fusion protein OPA1 which possesses a trans‐membrane domain at the IMM.These results indicate the existence of Ano1 protein in the mitochondria, especially at the IMM, as well as the plasma membrane. Since OPA1 is the critical regulator of mitochondrial bioenergetics and cristae formation, increased Ano1 and OPA1 interactions may be partly involved in the molecular mechanism underlying the hyperproliferation of pulmonary artery ECs during PAH.Support or Funding InformationJ.O.‐U. was supported by NIH/NHLBI R01HL136757B.S.J was supported by AHA 8CDA34110091R.C. is supported by NIH/NHLBI R01HL135236G.C is supported by R01HL128661This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tumor microenvironment regulates endothelial cell transformation via modulation of TRPV4 channels

Abnormal angiogenesis and hyper‐permeable vessels characterize the tumor vasculature. Together, these attributes contribute to atypical growth rate and metastasis of tumor cells. We have recently shown that the mechanosensitive ion channel, transient receptor vanilloid 4 (TRPV4) expression and activity is significantly reduced in tumor endothelial cells (TEC), and that activation of TRPV4 normalized tumor vasculature and improved cancer therapy. However, whether and how the tumor microenvironment downregulates TRPV4 and transforms normal endothelial cell phenotype remains unknown. To explore this, we repeatedly exposed normal human endothelial cells (NhEC) to human lung adenocarcinoma (tumor) cell conditioned media and measured phenotypic changes, angiogenesis, and functional expression of TRPV4. We found that treatment with cancer cell conditioned media changed NhEC to a tumor endothelial‐like phenotype (ThEC) as evidenced by; increased expression of tumor endothelial cell marker 8 (TEM8) and exhibition of abnormal angiogenesis on 2D‐Matrigels compared to normal NhEC. Mechanistically, we observed that expression and activity of TRPV4 was reduced in ThEC. Finally, we found that treatment with a Rho kinase inhibitor, Y‐27632 normalized abnormal tube formation exhibited by ThEC. Taken together, our results suggest that the tumor microenvironment transforms normal endothelial cells into tumor endothelial cell‐like phenotype through the downregulation of TRPV4.Support or Funding InformationNIH (1RO1HL119705), NIH (1R15CA202847‐01), and start‐up funds from NEOMED (CKT).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Shear-Induced CCN1 Promotes Atheroprone Endothelial Phenotypes and Atherosclerosis.

Atherosclerosis occurs preferentially at the blood vessels encountering blood flow turbulence. The matricellular protein CCN1 is induced in endothelial cells by disturbed flow, and is expressed in advanced atherosclerotic lesions in patients and in the Apoe-/- mouse model. The role of CCN1 in atherosclerosis remains undefined. To assess the function of CCN1 in vivo, knock-in mice carrying the integrin α6β1-binding-defective mutant allele Ccn1-dm on the Apoe-/- background were tested in an atherosclerosis model generated by carotid artery ligation. Additionally, CCN1-regulated functional phenotypes of human umbilical vein endothelial cells, or primary mouse aortic endothelial cells isolated from wild-type and Ccn1 dm/dm mice, were investigated in the in vitro shear stress experiments under unidirectional laminar shear stress (12 dyn/cm2) versus oscillatory shear stress (±5 dyn/cm2) conditions. We found that Ccn1 expression was upregulated in the arterial endothelium 3 days after ligation before any detectable structural changes, and intensified with the progression of atherosclerotic lesions. Compared with Apoe-/- controls, Ccn1 dm/dm/ Apoe-/- mice were remarkably resistant to ligation-induced plaque formation (n=6). These mice exhibited lower oxidative stress, expression of endothelin-1 and monocyte chemoattractant protein-1, and monocyte homing. CCN1/α6β1 critically mediated flow-induced activation of the pleiotropic transcription factor nuclear factor-κB and therefore the induction of atheroprone gene expression in the mouse arterial endothelium after ligation (n=6), or in cultured human umbilical vein endothelial cells or primary mouse aortic endothelial cells exposed to oscillatory shear stress (n=3 in triplicate). Interestingly, the activation of nuclear factor-κB by CCN1/α6β1 signaling prompted more production of CCN1 and α6β1. Blocking CCN1-α6β1 binding by the Ccn1-dm mutation or by T1 peptide (derived from an α6β1-binding sequence of CCN1) disrupted the positive-feedback regulation between CCN1/α6β1 and nuclear factor-κB, and prevented flow-induced atheroprone phenotypic alterations in endothelial cells or atherosclerosis in mice. These data demonstrate a causative role of CCN1 in atherosclerosis via modulating endothelial phenotypes. CCN1 binds to its receptor integrin α6β1 to activate nuclear factor-κB, thereby instigating a vicious circle to persistently promote atherogenesis. T1, a peptide antagonist selectively targeting CCN1-α6β1, can be further optimized for developing T1-mimetics to treat atherosclerosis.

Inactivation of the tyrosine phosphatase SHP-2 drives vascular dysfunction in Sepsis.

BackgroundSepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence.MethodsPolymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay.FindingsWe identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1β as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1β-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88.InterpretationOur data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation.FundGerman Research Foundation, LMU Mentoring excellence and FöFoLe Programme, Verein zur Förderung von Wissenschaft und Forschung, German Ministry of Education and Research.

Axon Guidance-Related Factor FLRT3 Regulates VEGF-Signaling and Endothelial Cell Function.

Vascular endothelial growth factors (VEGFs) are key mediators of endothelial cell (EC) function in angiogenesis. Emerging knowledge also supports the involvement of axon guidance-related factors in the regulation of angiogenesis and vascular patterning. In the current study, we demonstrate that fibronectin and leucine-rich transmembrane protein-3 (FLRT3), an axon guidance-related factor connected to the regulation of neuronal cell outgrowth and morphogenesis but not to VEGF-signaling, was upregulated in ECs after VEGF binding to VEGFR2. We found that FLRT3 exhibited a transcriptionally paused phenotype in non-stimulated human umbilical vein ECs. After VEGF-stimulation its nascent RNA and mRNA-levels were rapidly upregulated suggesting that the regulation of FLRT3 expression is mainly occurring at the level of transcriptional elongation. Blockage of FLRT3 by siRNA decreased survival of ECs and their arrangement into capillary-like structures but enhanced cell migration and wound closure in wound healing assay. Bifunctional role of FLRT3 in repulsive vs. adhesive cell signaling has been already detected during embryogenesis and neuronal growth, and depends on its interactions either with UNC5B or another FLRT3 expressed by adjacent cells. In conclusion, our findings demonstrate that besides regulating neuronal cell outgrowth and morphogenesis, FLRT3 has a novel role in ECs via regulating VEGF-stimulated EC-survival, migration, and tube formation. Thus, FLRT3 becomes a new member of the axon guidance-related factors which participate in the VEGF-signaling and regulation of the EC functions.

1,8-Cineole Ameliorates LPS-Induced Vascular Endothelium Dysfunction in Mice via PPAR-γ Dependent Regulation of NF-κB.

1,8-Cineole (eucalyptol), a monoterpene, has been widely reported for the anti-inflammatory effects. Our previous data confirmed that 1,8-cineole ameliorated the inflammatory phenotype of human umbilical vein endothelial cells (HUVECs) by mediating NF-κB expression in vitro. At present, we investigated the protection effects of 1,8-cineole on vascular endothelium in lipopolysaccharide (LPS)-induced acute inflammatory injury mice and the potential mechanisms involved in the protection in HUVECs. Results from enzyme linked immunosorbent assays revealed that 1,8-cineole suppressed the secretion of interleukin (IL)-6 and IL-8 and increased the expression of IL-10 in the serum of LPS-induced mice. 1,8-Cineole reduced the inflammatory infiltration and the expression of vascular cell adhesion molecular 1 (VCAM-1) in the sections of thoracic aorta in LPS-induced acute inflammatory mice. Western blotting indicated that 1,8-cineole significantly decreased the phosphorylation of NF-κB p65 and increased the expression of PPAR-γ in the thoracic aorta tissue. 1,8-Cineole increased the expression of PPAR-γ in LPS-induced HUVECs. 1,8-Cineole and rosiglitazone reduced the protein and mRNA levels of VCAM-1, E-selectin, IL-6, and IL-8 in LPS-induced HUVECs, which could be reversed by the action of GW9662 (inhibitor of PPAR-γ). 1,8-Cineole and rosiglitazone blocked the LPS-induced IκBα degradation and NF-κB p65 nucleus translocation, which could be reversed by the pretreatment of GW9662 or silence of PPAR-γ gene. In conclusion, 1,8-cineole attenuated LPS-induced vascular endothelial cells injury via PPAR-γ dependent modulation of NF-κB.

LSEC Fenestrae Are Preserved Despite Pro-inflammatory Phenotype of Liver Sinusoidal Endothelial Cells in Mice on High Fat Diet.

Healthy liver sinusoidal endothelial cells (LSECs) maintain liver homeostasis, while LSEC dysfunction was suggested to coincide with defenestration. Here, we have revisited the relationship between LSEC pro-inflammatory response, defenestration, and impairment of LSEC bioenergetics in non-alcoholic fatty liver disease (NAFLD) in mice. We characterized inflammatory response, morphology as well as bioenergetics of LSECs in early and late phases of high fat diet (HFD)-induced NAFLD. LSEC phenotype was evaluated at early (2–8 week) and late (15–20 week) stages of NAFLD progression induced by HFD in male C57Bl/6 mice. NAFLD progression was monitored by insulin resistance, liver steatosis and obesity. LSEC phenotype was determined in isolated, primary LSECs by immunocytochemistry, mRNA gene expression (qRT-PCR), secreted prostanoids (LC/MS/MS) and bioenergetics (Seahorse FX Analyzer). LSEC morphology was examined using SEM and AFM techniques. Early phase of NAFLD, characterized by significant liver steatosis and prominent insulin resistance, was related with LSEC pro-inflammatory phenotype as evidenced by elevated ICAM-1, E-selectin and PECAM-1 expression. Transiently impaired mitochondrial phosphorylation in LSECs was compensated by increased glycolysis. Late stage of NAFLD was featured by prominent activation of pro-inflammatory LSEC phenotype (ICAM-1, E-selectin, PECAM-1 expression, increased COX-2, IL-6, and NOX-2 mRNA expression), activation of pro-inflammatory prostaglandins release (PGE2 and PGF2α) and preserved LSEC bioenergetics. Neither in the early nor in the late phase of NAFLD, were LSEC fenestrae compromised. In the early and late phases of NAFLD, despite metabolic and pro-inflammatory burden linked to HFD, LSEC fenestrae and bioenergetics are functionally preserved. These results suggest prominent adaptive capacity of LSECs that might mitigate NAFLD progression.

MicroRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis.

Vertebrate tissues exhibit mechanical homeostasis, showing stable stiffness and tension over time and recovery after changes in mechanical stress. However, the regulatory pathways that mediate these effects are unknown. A comprehensive identification of Argonaute-2(AGO2)-associated microRNAs and mRNAs in endothelial cells identified a network of 122 microRNA families that target 73 mRNAs encoding cytoskeletal, contractile, adhesive and extracellular matrix (CAM) proteins. These microRNAs increased in cells plated on stiff vs. soft substrates, consistent with homeostasis, and suppressed targets via microRNA recognition elements (MREs) within the 3’UTRs of CAM mRNAs. Inhibition of DROSHA or AGO2, or disruption of MREs within individual target mRNAs such as Connective Tissue Growth Factor (CTGF), induced hyper-adhesive, hyper-contractile phenotypes in endothelial and fibroblast cells in vitro, and increased tissue stiffness, contractility and extracellular matrix (ECM) deposition in the zebrafish fin-fold in vivo. Thus, a network of microRNAs buffers CAM expression to mediate tissue mechanical homeostasis.

C-Abl regulates YAPY357 phosphorylation to activate endothelial atherogenic responses to disturbed flow

Local flow patterns determine the uneven distribution of atherosclerotic lesions. This research aims to elucidate the mechanism of regulation of nuclear translocation of Yes-associated protein (YAP) under oscillatory shear stress (OSS) in the atheroprone phenotype of endothelial cells (ECs). We report here that OSS led to tyrosine phosphorylation and strong, continuous nuclear translocation of YAP in ECs that is dependent on integrin α5β1 activation. YAP overexpression in ECs blunted the anti-atheroprone effect of an integrin α5β1-blocking peptide (ATN161) in Apoe-/- mice. Activation of integrin α5β1 induced tyrosine, but not serine, phosphorylation of YAP in ECs. Blockage of integrin α5β1 with ATN161 abolished the phosphorylation of YAP at Y357 induced by OSS. Mechanistic studies showed that c-Abl inhibitor attenuated the integrin α5β1-induced YAP tyrosine phosphorylation. Furthermore, the phosphorylation of c-Abl and YAPY357 was significantly increased in ECs in atherosclerotic vessels of mice and in human plaques versus normal vessels. Finally, bosutinib, a tyrosine kinase inhibitor, markedly reduced the level of YAPY357 and the development of atherosclerosis in Apoe-/- mice. The c-Abl/YAPY357 pathway serves as a mechanism for the activation of integrin α5β1 and the atherogenic phenotype of ECs in response to OSS, and provides a potential therapeutic strategy for atherogenesis.

LncRNA H19 prevents endothelial-mesenchymal transition in diabetic retinopathy.

The pathophysiology of diabetic retinopathy is linked to hyperglycaemia and its effect on retinal microvascular tissues. The resulting endothelial injury changes the endothelial cell phenotype to acquire mesenchymal properties (i.e. endothelial-mesenchymal transition [EndMT]). Such changes can be regulated by epigenetic mechanisms, including long non-coding RNAs (lncRNAs). lncRNA H19 may influence EndMT through TGF-β. We investigated the role of H19 in regulating EndMT during diabetic retinopathy. H19 was overexpressed or silenced in human retinal endothelial cells exposed to various glucose levels. The cells were examined for H19, endothelial and mesenchymal markers. We then expanded the study to retinal tissues in a mouse model of diabetic retinopathy and also examined vitreous humour samples from individuals with proliferative diabetic retinopathy. Expression of H19 was downregulated in high glucose conditions (25mmol/l). H19 overexpression prevented glucose-induced EndMT. Such changes appear to involve TGF-β through a Smad-independent mechanism. Diabetes caused downregulation of retinal H19. Using H19 knockout mice, we demonstrated similar EndMT in the retina. Examination of vitreous humour from individuals with proliferative diabetic retinopathy also reinforced the downregulation of H19 in diabetes. We therefore concluded that H19 regulates EndMT in diabetic retinopathy through specific mechanisms. The results from our previous microarray can be found online using the GEO accession number GSE122189.

Emulating endothelial dysfunction by implementing an early atherosclerotic microenvironment within a microfluidic chip.

Recent studies on endothelial dysfunction in relation to vascular diseases including atherosclerosis have highlighted the key contribution of the microenvironment of endothelial cells (ECs). By mimicking the microenvironment of early atherosclerotic lesions, here, we replicate the pathophysiological phenotype and function of ECs within microchannels. Considering the elevated deposition of fibronectin (FN) in early atherosclerotic plaques and the close correlation between the vascular stiffness and the progression of atherosclerosis, we utilized FN coated hydrogels with increased stiffness for endothelial substrates within the microchannels. As a result, we demonstrated that endothelial integrity on FN coated microchannels is likely to be undermined exhibiting a random orientation in response to the applied fluid flow, notable disruption of vascular endothelial cadherins (VE-cadherins), and higher endothelial permeability as opposed to that on microchannels coated with collagen (CL), the atheroresistant vascular model.

LncRNA CASC9 regulates cell migration and invasion in hemangioma endothelial cells by targeting miR-125a-3p/Nrg1.

BackgroundDespite being one of the most common benign tumors, the prevalence and pathogenesis of hemangiomas (HAs) are poorly understood. We aimed to identify the biological role of the long non-coding RNA (lncRNA) CASC9 in the HA-derived endothelial cell (HDECs) phenotype as well as elucidate the mechanism involved.MethodsThe expression of CASC9 was identified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). the effect of CASC9 on cell proliferation, migration and invasion of HDECs were examined by CCK8, wound healing, and transwell assay, respectively. Bioinformatics analysis and a luciferase reporter assay were utilized to investigated the mechanisms involved. The in vivo tumorigenesis capability of CASC9 on HA was also evaluated.ResultsThe expression of CASC9 was significantly elevated in HA tissue compared to normal tissue. Down-regulation of CASC9 inhibited proliferation, migration, and invasion of HDECs. The translation of cyclinD1, N-cadherin, Twist, and MMP2 was also decreased by CASC9 knockdown treatment. Furthermore, CASC9 over-expression exerted the opposite effect of proliferation, migration, and invasion of HDECs. We also found that CASC9 interacts with miR-125a-3p/Nrg1 to regulate cellular functions. Interestingly, miR-125a-3p can reverse the effect of CASC9 on proliferation, migration, and invasion of HDECs. Together, the clinical data showed that CASC9 expression is negatively correlated with miR-125a-3p expression and positively correlated with Nrg1 expression. CASC9 also exerted anti-tumorigenesis capability in vivo.ConclusionOur study indicates that CASC9 accelerates cell growth and invasion of HDECs and provides new insights for the diagnosis and molecular therapy of HA.