Protective effect of trans-nerolidol on vascular endothelial cell injury induced by lipopolysaccharides / Efectos protectores del trans-nerolidol en lesiones inducidas por lipopolisacáridos en células endoteliales vasculares

This article aimed to discuss the protection of trans - nerolidol on vascular endothelial cells (ECs) injured by lipopolysac charides. ECs were divided into four groups: normal, model, low and high dose trans - nerolidol treatment groups. The cell survival rate and the contents of NO in the cell culture supernatant were determined. The protein expression and transcript level of pe roxisome proliferator - activated receptor - γ (PPARγ), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were determined by western blotting and RT - PCR respectively. Compared with the normal group, cell livability, protein e xpression and mRNA transcript level of PPARγ and eNOS decreased, NO contents, protein expression and mRNA transcript tlevel of iNOS increased in model group significantly. Compared with model group, all the changes recovered in different degree in treatmen t groups. Hence, it was concluded that trans - nerolidol can alleviate the ECs injuryby the regulation of iNOS/eNOS through activating PPARγ in a dose - dependent manner

Este artículo tiene como objetivo discutir la protección del trans - nerolidol en las células endoteliales vasculares (CE) dañadas por lipopolisacáridos. Las CE se di vidieron en cuatro grupos: normal, modelo, grupos de tratamiento con trans - nerolidol de baja y alta dosis. Se determinó la tasa de supervivencia de las células y los contenidos de óxido nítrico (NO) en el sobrenadante del cultivo celular. La expresión de p roteínas y el nivel de transcripción del receptor activado por proliferadores de peroxisomas - γ (PPARγ), el óxido nítrico sint et asa endotelial (eNOS) y el óxido nítrico sint et asa inducible (iNOS) se determinaron mediante western blot y RT - PCR, respectivamen te. En comparación con el grupo normal, la viabilidad celular, la expresión de proteínas y el nivel de transcripción de PPARγ y eNOS disminuyeron, los contenidos de NO, la expresión de proteínas y el nivel de transcripción de iNOS aumentaron significativam ente en el grupo modelo. En comparación con el grupo modelo, todos los cambios se recuperaron en diferentes grados en los grupos de tratamiento. Por lo tanto, se concluyó que el trans - nerolidol puede aliviar el daño en las CE regulando iNOS/eNOS a través d e la activación de PPARγ de manera dependiente de la dosis.

The ß2-adrenergic receptor agonist formoterol attenuates necrosis and apoptosis in the rat myocardium under experimental stress-induced cardiac injury.

BACKGROUND: Currently, there is no effective therapy for takotsubo syndrome (stress-induced cardiac injury in humans) in the clinics. It has previously been shown that ß2-adrenergic receptor (ß2-AR) agonist formoterol reduces cardiomyocyte injury in experimental takotsubo syndrome. OBJECTIVES: The aim of this study was to investigate whether formoterol prevents apoptosis and necrosis of cardiomyocytes and endothelial cells in stress-induced cardiomyopathy. METHODS: Stress-induced cardiac injury was induced by immobilization of rats for 2, 6, and 24 hours. RESULTS: The myocardium of stressed rats showed a reduction in contractility and histological manifestations of cardiomyocyte damage: karyopyknosis, perinuclear edema of cardiomyocytes and endothelial cells, and microcirculation disturbances augmented with extended exposure to stress. In addition, apoptosis of endothelial cells was detected 6 hours after the onset of stress and peaked at 24 hours. Apoptosis of cardiomyocytes significantly gained only after 24 hours of stress exposure. These morphological alterations were associated with increased levels of serum creatine kinase-MB, syndecan-1, and thrombomodulin after 24 hours of stress. Administration of ß2-AR agonist formoterol (50 µg/kg) four times during 24-hour stress exposure led to the improvement in myocardial inotropy, decrease in the severity of histological signatures, reduction in the number of TUNEL-positive cardiomyocytes, serum creatine kinase-MB, syndecan-1, and thrombomodulin levels. CONCLUSION: Present data suggest that apoptosis and necrosis of cardiomyocytes and necrosis of endothelial cells in stress-induced cardiac injury can be mitigated by activation of the ß2-AR. However, formoterol did not eliminate completely cardiomyocyte apoptosis, histological alterations, or endothelium injury markers under stress.

Cavß3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalitis.

BACKGROUND: Tight control of cytoplasmic Ca2+ in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavß3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. METHODS: We investigated the function of Cavß3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavß3-/- (Cav ß3-deficient) mice as controls. RESULTS: We identified Cavß3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavß3. After induction of experimental autoimmune encephalomyelitis, Cavß3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavß3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavß3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavß3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavß3 with inositol 1,4,5-trisphosphate receptor proteins. CONCLUSIONS: Independent of its function as a subunit of Cav channels, Cavß3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavß3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.

Recruitment of neutrophils in glomeruli in early mouse sepsis is associated with E-selectin expression and activation of endothelial NF-κB and MAPK pathways.

Sepsis is a dysregulated systemic inflammatory response to an infection, which can lead to multiple organ dysfunction syndrome that includes the kidney. Leukocyte recruitment is an important process of the host immune defense in response to sepsis. Endothelial cells (EC) actively regulate leukocyte recruitment by expressing adhesion molecules following the activation of dedicated intracellular signal transduction pathways. Previous studies reported that the expression of adhesion molecules was associated with the activation of endothelial NF-κB p65 and MAPK c-Jun pathways in vitro in response to conditions that mimic processes that occur in inflammation. This study aimed to investigate the spatiotemporal patterns of leukocyte recruitment, expression of adhesion molecules, and endothelial nuclear p65 and c-Jun localization in renal microvascular beds of septic mice. Here, we used a cecal ligation and puncture (CLP) sepsis mouse model and RT-qPCR and immunohistochemical staining. We showed that neutrophils, macrophages, and T lymphocytes were all present in the kidney, yet only neutrophils accumulated in a spatiotemporally discernible pattern, mainly in glomeruli at 4 hours after CLP-sepsis initiation. E-selectin, not VCAM-1, was expressed in glomeruli at the same time point. In a subset of mice at 72 hours after CLP-sepsis started, VCAM-1 expression was prominent in glomerular EC, which was not related to changes in mmu-microRNA(miR)-126a-3p levels, a short noncoding microRNA previously shown to inhibit the translation of VCAM-1 mRNA into protein. Nuclear localization of p65 and c-Jun occurred in EC of all microvascular segments at 4 and 7 hours after CLP-sepsis initiation. In summary, sepsis-induced recruitment of neutrophils, E-selectin expression, and NF-κB p65 and MAPK c-Jun pathway activation coincided in glomeruli at the early stage of the disease. In the other microvascular beds, sepsis led to NF-κB p65 and MAPK c-Jun pathway activation with limited expression of E-selectin and no association with VCAM-1 expression or leukocyte recruitment.

Similarities and differences between brain and skin GNAQ p.R183Q driven capillary malformations.

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~90% of syndromic and non-syndromic CM specimens and is present in CD31pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimen from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1, and were surrounded by MRC1pos/LYVE1pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM.

Single-cell transcriptomic analysis to identify endomembrane regulation of metalloproteins and motor proteins in autoimmunity.

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue.

Enhancement of endothelial function and attenuation of portal vein injury using mesenchymal stem cells carrying miRNA-25-3p.

The aims of this study were to determine whether human umbilical cord mesenchymal stem cells (hucMSCs) modified by miRNA-25-3p (miR-25-3p) overexpression could promote venous endothelial cell proliferation and attenuate portal endothelial cell injury. HucMSCs and human umbilical vein endothelial cells (HUVEC) were isolated and cultured from human umbilical cord and characterized. Lentiviral vectors expressing miRNA-25-3p were transfected into hucMSCs and confirmed by PCR. We verified the effect of miR-25-3p-modified hucMSCs on HUVEC by cell co-culture and cell supernatant experiments. Subsequently, exosomes of miR-25-3p-modified hucMSCs were isolated from cell culture supernatants and characterized by WB, NTA and TEM. We verified the effects of miR-25-3p-modified exosomes derived from hucMSCs on HUVEC proliferation, migration, and angiogenesis by in vitro cellular function experiments. Meanwhile, we further examined the downstream target genes and signaling pathways potentially affected by miR-25-3p-modified hucMSC-derived exosomes in HUVEC. Finally, we established a rat portal vein venous thrombosis model by injecting CM-DiR-labeled hucMSCs intravenously into rats and examining the homing of cells in the portal vein by fluorescence microscopy. Histological and immunohistochemical experiments were used to examine the effects of miRNA-25-3p-modified hucMSCs on the proliferation and damage of portal vein endothelial cells. Primary hucMSCs and HUVECs were successfully isolated, cultured and characterized. Primary hucMSCs were modified with a lentiviral vector carrying miR-25-3p at MOI 80. Co-culture and cell supernatant intervention experiments showed that overexpression of miRNA-25-3p in hucMSCs enhanced HUVEC proliferation, migration and tube formation in vitro. We successfully isolated and characterized exosomes of miR-25-3p-modified hucMSCs, and exosome intervention experiments demonstrated that miR-25-3p-modified exosomes derived from hucMSCs similarly enhanced the proliferation, migration, and angiogenesis of HUVECs. Subsequent PCR and WB analyses indicated PTEN/KLF4/AKT/ERK1/2 as potential pathways of action. Analysis in a rat portal vein thrombosis model showed that miR-25-3p-modified hucMSCs could homing to damaged portal veins. Subsequent histological and immunohistochemical examinations demonstrated that intervention with miR-25-3p overexpression-modified hucMSCs significantly reduced damage and attenuated thrombosis in rat portal veins. The above findings indicate suggest that hucMSCs based on miR-25-3p modification may be a promising therapeutic approach for use in venous thrombotic diseases.

Selective mural cell recruitment of pericytes to networks of assembling endothelial cell-lined tubes.

Mural cells are critically important for the development, maturation, and maintenance of the blood vasculature. Pericytes are predominantly observed in capillaries and venules, while vascular smooth muscle cells (VSMCs) are found in arterioles, arteries, and veins. In this study, we have investigated functional differences between human pericytes and human coronary artery smooth muscle cells (CASMCs) as a model VSMC type. We compared the ability of these two mural cells to invade three-dimensional (3D) collagen matrices, recruit to developing human endothelial cell (EC)-lined tubes in 3D matrices and induce vascular basement membrane matrix assembly around these tubes. Here, we show that pericytes selectively invade, recruit, and induce basement membrane deposition on EC tubes under defined conditions, while CASMCs fail to respond equivalently. Pericytes dramatically invade 3D collagen matrices in response to the EC-derived factors, platelet-derived growth factor (PDGF)-BB, PDGF-DD, and endothelin-1, while minimal invasion occurs with CASMCs. Furthermore, pericytes recruit to EC tube networks, and induce basement membrane deposition around assembling EC tubes (narrow and elongated tubes) when these cells are co-cultured. In contrast, CASMCs are markedly less able to perform these functions showing minimal recruitment, little to no basement membrane deposition, with wider and shorter tubes. Our new findings suggest that pericytes demonstrate much greater functional ability to invade 3D matrix environments, recruit to EC-lined tubes and induce vascular basement membrane matrix deposition in response to and in conjunction with ECs.

Endothelial-derived microvesicles promote pro-migratory cross-talk with smooth muscle cells by a mechanism requiring tissue factor and PAR2 activation.

Introduction: Microvesicles (MV) released by endothelial cells (EC) following injury or inflammation contain tissue factor (TF) and mediate communication with the underlying smooth muscle cells (SMC). Ser253-phosphorylated TF co-localizes with filamin A at the leading edge of migrating SMC. In this study, the influence of endothelial-derived TF-MV, on human coronary artery SMC (HCASMC) migration was examined. Methods and Results: MV derived from human coronary artery EC (HCAEC) expressing TFWt accelerated HCASMC migration, but was lower with cytoplasmic domain-deleted TF. Furthermore, incubation with TFAsp253-MV, or expression of TFAsp253 in HCASMC, reduced cell migration. Blocking TF-factor VIIa (TF-fVIIa) procoagulant/protease activity, or inhibiting PAR2 signaling on HCASMC, abolished the accelerated migration. Incubation with fVIIa alone increased HCASMC migration, but was significantly enhanced on supplementation with TF. Neither recombinant TF alone, factor Xa, nor PAR2-activating peptide (SLIGKV) influenced cell migration. In other experiments, HCASMC were transfected with peptides corresponding to the cytoplasmic domain of TF prior to stimulation with TF-fVIIa. Cell migration was suppressed only when the peptides were phosphorylated at position of Ser253. Expression of mutant forms of filamin A in HCASMC indicated that the enhancement of migration by TF but not by PDGF-BB, was dependent on the presence of repeat-24 within filamin A. Incubation of HCASMC with TFWt-MV significantly reduced the levels of Smoothelin-B protein, and upregulated FAK expression. Discussion: In conclusion, Ser253-phosphorylated TF and fVIIa released as MV-cargo by EC, act in conjunction with PAR2 on SMC to promote migration and may be crucial for normal arterial homeostasis as well as, during development of vascular disease.

Weibel-Palade bodies: function and role in thrombotic thrombocytopenic purpura and in diarrhea phase of STEC-hemolytic uremic syndrome.

Vascular endothelial cells are equipped with numerous specialized granules called Weibel-Palade bodies (WPBs). They contain a cocktail of proteins that can be rapidly secreted (3-5 min) into the vascular lumen after an appropriate stimulus such as thrombin. These proteins are ready without synthesis. Von Willebrand factor (VWF) and P-selectin are the main constituents of WPBs. Upon stimulation, release of ultralarge VWF multimers occurs and assembles into VWF strings on the apical side of endothelium. The VWF A1 domain becomes exposed in a shear-dependent manner recruiting and activating platelets. VWF is able to recruit leukocytes via direct leukocyte binding or via the activated platelets promoting NETosis. Ultralarge VWF strings are ultimately cleaved into smaller pieces by the protease ADAMTS-13 preventing excessive platelet adhesion. Under carefully performed flowing conditions and adequate dose of Shiga toxins, the toxin induces the release of ultralarge VWF multimers from cultured endothelial cells. This basic information allows insight into the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and of STEC-HUS in the diarrhea phase. In TTP, ADAMTS-13 activity is deficient and systemic aggregation of platelets will occur after a second trigger. In STEC-HUS, stimulated release of WPB components in the diarrhea phase of the disease can be presumed to be the first hit in the damage of Gb3 positive endothelial cells.

Mechanisms of triple-negative breast cancer extravasation: Impact of the physical environment and endothelial glycocalyx.

Cancer metastasis is the leading cause of death for those afflicted with cancer. In cancer metastasis, the cancer cells break off from the primary tumor, penetrate nearby blood vessels, and attach and extravasate out of the vessels to form secondary tumors at distant organs. This makes extravasation a critical step of the metastatic cascade. Herein, with a focus on triple-negative breast cancer, the role that the prospective secondary tumor microenvironment's mechanical properties play in circulating tumor cells' extravasation is reviewed. Specifically, the effects of the physically regulated vascular endothelial glycocalyx barrier element, vascular flow factors, and subendothelial extracellular matrix mechanical properties on cancer cell extravasation are examined. The ultimate goal of this review is to clarify the physical mechanisms that drive triple-negative breast cancer extravasation, as these mechanisms may be potential new targets for anti-metastasis therapy.

A Novel Animal Model for Pulmonary Hypertension: Lung Endothelial-Specific Deletion of Egln1 in Mice.

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by high blood pressure in the pulmonary arteries, which can potentially lead to heart failure over time. Previously, our lab found that endothelia-specific knockout of Egln1, encoding prolyl 4-hydroxylase-2 (PHD2), induced spontaneous pulmonary hypertension (PH). Recently, we elucidated that Tmem100 is a lung-specific endothelial gene using Tmem100-CreERT2 mice. We hypothesize that lung endothelial-specific deletion of Egln1 could lead to the development of PH without affecting Egln1 gene expression in other organs. Tmem100-CreERT2 mice were crossed with Egln1 flox/flox mice to generate Egln1 f/f ;Tmem100-CreERT2 (LiCKO) mice. Western blot and immunofluorescent staining were performed to verify the knockout efficacy of Egln1 in multiple organs of LiCKO mice. PH phenotypes, including hemodynamics, right heart size and function, pulmonary vascular remodeling, were evaluated by right heart catheterization and echocardiography measurements. Tamoxifen treatment induced Egln1 deletion in the lung endothelial cells (ECs) but not in other organs of adult LiCKO mice. LiCKO mice exhibited an increase in right ventricular systolic pressure (RVSP, ~35 mmHg) and right heart hypertrophy. Echocardiography measurements showed right heart hypertrophy, as well as cardiac and pulmonary arterial dysfunction. Pulmonary vascular remodeling, including increased pulmonary wall thickness and muscularization of distal pulmonary arterials, was enhanced in LiCKO mice compared to wild-type mice. Tmem100 promoter-mediated lung endothelial knockout of Egln1 in mice leads to development of spontaneous PH. LiCKO mice could serve as a novel mouse model for PH to study lung and other organ crosstalk.

Effects of α­solanine on human head and neck squamous cell carcinoma cells and human umbilical vein endothelial cells in vitro.

α-solanine is a glycoalkaloid that is commonly found in nightshades (Solanum) and has a toxic effect on the human organism. Among other things, it is already known to inhibit tumor cell proliferation and induce apoptosis in tumor cell lines. Due to its potential as a tumor therapeutic, the current study investigated the effect of α-solanine on head and neck squamous cell carcinoma (HNSCC). In addition, genotoxic and antiangiogenic effects on human umbilical vein endothelial cells (HUVECs) were evaluated at subtoxic α-solanine concentrations. Cytotoxicity and apoptosis rates were measured in two human HNSCC cell lines (FaDu pharynx carcinoma cells and CAL-33 tongue carcinoma cells), as well as in HUVECs. MTT and Annexin V analyses were performed 24 h after α-solanine treatment at increasing doses up to 30 µM to determine cytotoxic concentrations. Furthermore, genotoxicity at subtoxic concentrations of 1, 2, 4 and 6 µM in HUVECs was analyzed using single-cell gel electrophoresis (comet assay). The antiangiogenic effect on HUVECs was evaluated in the capillary tube formation assay. The MTT assay indicated an induction of concentration-dependent viability loss in FaDu and CAL-33 cancer cell lines, whereas the Annexin V test revealed α-solanine-induced cell death predominantly independent from apoptosis. In HUVECs, the cytotoxic effect occurred at lower concentrations. No genotoxicity or inhibition of angiogenesis were detected at subtoxic doses in HUVECs. In summary, α-solanine had a cytotoxic effect on both malignant and non-malignant cells, but this was only observed at higher concentrations in malignant cells. In contrast to existing data in the literature, tumor cell apoptosis was less evident than necrosis. The lack of genotoxicity and antiangiogenic effects in the subtoxic range in benign cells are promising, as this is favorable for potential therapeutic applications. In conclusion, however, the cytotoxicity in non-malignant cells remains a severe hindrance for the application of α-solanine as a therapeutic tumor agent in humans.

Krüppel-like Factor 2 is an endo-protective transcription factor in diabetic kidney disease.

Diabetic kidney disease (DKD) is a microvascular complication of diabetes, and glomerular endothelial cell (GEC) injury is a key driver of DKD pathogenesis. Krüppel-like factor 2 (KLF2), a shear stress-induced transcription factor, was identified among the genes that are highly upregulated in early DKD. In the kidney, KLF2 expression is mostly restricted to endothelial cells, but its expression is also found in immune cell subsets. KLF2 expression is upregulated in response to increased shear stress by the activation of mechanosensory receptors, but suppressed by inflammatory cytokines, both of which characterize the early diabetic kidney milieu. KLF2 expression is reduced in progressive DKD and hypertensive nephropathy in humans and mice, likely due to high glucose and inflammatory cytokines such as TNF-α. However, KLF2 expression is increased in settings of glomerular hyperfiltration-induced shear stress without metabolic dysregulation, such as in settings of unilateral nephrectomy. Lower KLF2 expression is associated with CKD progression in patients with unilateral nephrectomy, consistent with its endoprotective role. KLF2 confers endoprotection by inhibition of inflammation, thrombotic activation, and angiogenesis, and thus KLF2 is considered a protective factor for cardiovascular disease (CVD). Based on similar mechanisms, KLF2 also exhibits renoprotection, and its reduced expression in endothelial cells worsens glomerular injury and albuminuria in settings of diabetes or unilateral nephrectomy. Thus, KLF2 confers endo-protective effects in both CVD and DKD, and its agonists could be potentially developed as a novel class of drugs for cardio-renal protection in diabetic patients.

Lysosomal TRPML1 triggers global Ca2+ signals and nitric oxide release in human cerebrovascular endothelial cells.

Lysosomal Ca2+ signaling is emerging as a crucial regulator of endothelial Ca2+ dynamics. Ca2+ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP3) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca2+ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca2+ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) induced a significant increase in [Ca2+]i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca2+ release was sustained both by lysosomal Ca2+ release and ER Ca2+- release through inositol-1,4,5-trisphophate receptors and store-operated Ca2+ entry. Notably, interfering with TRPML1-mediated lysosomal Ca2+ mobilization led to a decrease in the free ER Ca2+ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca2+-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca2+ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca2+ store can shape endothelial Ca2+ signaling and Ca2+-dependent functions in vascular endothelial cells.

Isolation methods and characterization of primary rat neurovascular cells.

BACKGROUND: There is significant interest in isolating cells of the blood-brain barrier (BBB) for use in in vitro screening of therapeutics and analyzing cell specific roles in neurovascular pathology. Primary brain cells play an advantageous role in BBB models; however, isolation procedures often do not produce cells at high enough yields for experiments. In addition, although numerous reports provide primary cell isolation methods, the field is lacking in documentation and detail of expected morphological changes that occur throughout culturing and there are minimal troubleshooting resources. Here, we present simplified, robust, and reproducible methodology for isolating astrocytes, pericytes, and endothelial cells, and demonstrate several morphological benchmarks for each cell type throughout the process and culture timeframe. We also analyze common considerations for developing neurovascular cell isolation procedures and recommend solutions for troubleshooting. RESULTS: The presented methodology isolated astrocytes, pericytes, and endothelial cells and enabled cell attachment, maturation, and cell viability. We characterized milestones in cell maturation over 12 days in culture, a common timeline for applications of these cell types in BBB models. Phase contrast microscopy was used to show initial cell plating, attachment, and daily growth of isolated cells. Confocal microscopy images were analyzed to determine the identity of cell types and changes to cell morphology. Nuclear staining was also used to show the viability and proliferation of glial cells at four time points. Astrocyte branches became numerous and complex with increased culture time. Microglia, oligodendrocytes, and neurons were present in mixed glial cultures for 12 days, though the percentage of microglia and neurons expectedly decreased after passaging, with microglia demonstrating a less branched morphology. CONCLUSIONS: Neurovascular cells can be isolated through our optimized protocols that minimize cell loss and encourage the adhesion and proliferation of isolated cells. By identifying timepoints of viable glia and neurons within an astrocyte-dominant mixed culture, these cells can be used to evaluate drug targeting, uptake studies, and response to pathological stimulus in the neurovascular unit.

The crosstalk between neuropilin-1 and tumor necrosis factor-α in endothelial cells.

Tumor necrosis factor-α (TNFα) is a master cytokine which induces expression of chemokines and adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in endothelial cells to initiate the vascular inflammatory response. In this study, we identified neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, as a modulator of TNFα-induced inflammatory response of endothelial cells. NRP1 shRNA expression suppressed TNFα-stimulated leukocyte adhesion and expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs). Likewise, it reduced TNFα-induced phosphorylation of MAPK p38 but did not significantly affect other TNF-induced signaling pathways, such as the classical NFκB and the AKT pathway. Immunofluorescent staining demonstrated co-localization of NRP1 with the two receptors of TNF, TNFR1 and TNFR2. Co-immunoprecipitation further confirmed that NRP1 was in the same protein complex or membrane compartment as TNFR1 and TNFR2, respectively. Modulation of NRP1 expression, however, neither affected TNFR levels in the cell membrane nor the receptor binding affinities of TNFα. Although a direct interface between NRP1 and TNFα/TNFR1 appeared possible from a protein docking model, a direct interaction was not supported by binding assays in cell-free microplates and cultured cells. Furthermore, TNFα was shown to downregulate NRP1 in a time-dependent manner through TNFR1-NFκB pathway in HUVECs. Taken together, our study reveals a novel reciprocal crosstalk between NRP1 and TNFα in vascular endothelial cells.

Up-regulation of miR-126 via DNA methylation in hypoxia-preconditioned endothelial cells may contribute to hypoxic tolerance of neuronal cells.

BACKGROUND: Endothelial cells (ECs) can confer neuroprotection by secreting molecules. This study aimed to investigate whether DNA methylation contributes to the neuroprotective gene expression induced by hypoxia preconditioning (HPC) in ECs and to clarify that the secretion of molecules from HPC ECs may be one of the molecular mechanisms of neuroprotection. METHODS: Human microvascular endothelial cell-1 (HMEC-1) was cultured under normal conditions (C), hypoxia(H), and hypoxia preconditioning (HPC), followed by the isolation of culture medium (CM). SY5Y cell incubated with the isolated CM from HMEC-1 was exposed to oxygen-glucose deprivation (OGD). The DNA methyltransferases (DNMTs), global methylation level, miR-126 and its promotor DNA methylation level in HMEC-1 were measured. The cell viability and cell injury in SY5Y were detected. RESULTS: HPC decreased DNMTs level and global methylation level as well as increased miR-126 expression in HMEC-1. CM from HPC treated HMEC-1 also relieved SY5Y cell damage, while CM from HMEC-1 which over-expression of miR-126 can reduce injury in SY5Y under OGD condition. CONCLUSIONS: These findings indicate EC may secrete molecules, such as miR-126, to execute neuroprotection induced by HPC through regulating the expression of DNMTs.

Mechanisms of mitotic inhibition in human aorta endothelial cells: Molecular and morphological in vitro spectroscopic studies.

Mitotic inhibitors are drugs commonly used in chemotherapy, but their nonspecific and indiscriminate distribution throughout the body after intravenous administration can lead to serious side effects, particularly on the cardiovascular system. In this context, our investigation into the mechanism of the cytotoxic effects on endothelial cells of mitotic inhibitors widely used in cancer treatment, such as paclitaxel (also known as Taxol) and Vinca alkaloids, holds significant practical implications. Understanding these mechanisms can lead to more targeted and less harmful cancer treatments. Human aorta endothelial cells (HAECs) were incubated with selected mitotic inhibitors in a wide range of concentrations close to those in human plasma during anticancer therapy. The analysis of single cells imaged by Raman spectroscopy allowed for visualization of the nuclear, cytoplasmic, and perinuclear areas to assess biochemical changes induced by the drug's action. The results showed significant changes in the morphology and molecular composition of the nucleus. Moreover, an effect of a given drug on the cytoplasm was observed, which can be related to its mechanism of action (MoA). Raman data supported by fluorescence microscopy measurements identified unique changes in DNA form and proteins and revealed drug-induced inflammation of endothelial cells. The primary goal of mitotic inhibitors is based on the impairment of tubulin formation and the inhibition of the mitosis process. While all three drugs affect microtubules and disrupt cell division, they do so through different MoA, i.e., Vinca alkaloids inhibit microtubule formation, whereas paclitaxel stabilizes microtubules. To sum up, the work shows how a specific drug can interact with endothelial cells.