Macrovascular Endothelial Cells Research Articles

Endothelial Heterogeneity in the Response to Autophagy Drives Small Vessel Muscularization in Pulmonary Hypertension.

Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate and how this contributes to vascular remodeling is unclear. We hypothesized that this differential response may: (1) relate to different EC subsets, namely pulmonary artery (PAECs) versus microvascular ECs (MVECs); (2) be attributable to autophagic activation in both EC subtypes; and (3) cause replacement of MVECs by PAECs with subsequent distal vessel muscularization. EC subset responses to chronic hypoxia were assessed by single-cell RNA sequencing of murine lungs. Proliferative versus apoptotic responses, activation, and role of autophagy were assessed in human and rat PAECs and MVECs, and in precision-cut lung slices of wild-type mice or mice with endothelial deficiency in the autophagy-related gene 7 (Atg7EN-KO). Abundance of PAECs versus MVECs in precapillary microvessels was assessed in lung tissue from patients with PH and animal models on the basis of structural or surface markers. In vitro and in vivo, PAECs proliferated in response to hypoxia, whereas MVECs underwent apoptosis. Single-cell RNA sequencing analyses support these findings in that hypoxia induced an antiapoptotic, proliferative phenotype in arterial ECs, whereas capillary ECs showed a propensity for cell death. These distinct responses were prevented in hypoxic Atg7EN-KO mice or after ATG7 silencing, yet replicated by autophagy stimulation. In lung tissue from mice, rats, or patients with PH, the abundance of PAECs in precapillary arterioles was increased, and that of MVECs reduced relative to controls, indicating replacement of microvascular by macrovascular ECs. EC replacement was prevented by genetic or pharmacological inhibition of autophagy in vivo. Conditioned medium from hypoxic PAECs yet not MVECs promoted pulmonary artery smooth muscle cell proliferation and migration in a platelet-derived growth factor-dependent manner. Autophagy inhibition attenuated PH development and distal vessel muscularization in preclinical models. Autophagic activation by hypoxia induces in parallel PAEC proliferation and MVEC apoptosis. These differential responses cause a progressive replacement of MVECs by PAECs in precapillary pulmonary arterioles, thus providing a macrovascular context that in turn promotes pulmonary artery smooth muscle cell proliferation and migration, ultimately driving distal vessel muscularization and the development of PH.

A novel inducible von Willebrand Factor Cre recombinase mouse strain to study microvascular endothelial cell-specific biological processes in vivo

Mouse models are invaluable to understanding fundamental mechanisms in vascular biology during development, in health and different disease states. Several constitutive or inducible models that selectively knockout or knock in genes in vascular endothelial cells exist; however, functional and phenotypic differences exist between microvascular and macrovascular endothelial cells in different organs. In order to study microvascular endothelial cell-specific biological processes, we developed a Tamoxifen-inducible von Willebrand Factor (vWF) Cre recombinase mouse in the SJL background. The transgene consists of the human vWF promoter with the microvascular endothelial cell-selective 734 base pair sequence to drive Cre recombinase fused to a mutant estrogen ligand-binding domain [ERT2] that requires Tamoxifen for activity (CreERT2) followed by a polyadenylation (polyA) signal. We initially observed Tamoxifen-inducible restricted bone marrow megakaryocyte and sciatic nerve microvascular endothelial cell Cre recombinase expression in offspring of a mixed strain hemizygous C57BL/6-SJL founder mouse bred with mT/mG mice, with >90% bone marrow megakaryocyte expression efficiency. Founder mouse offspring were backcrossed to the SJL background by speed congenics, and intercrossed for >10 generations to develop hemizygous Tamoxifen-inducible vWF Cre recombinase (vWF-iCre/+) SJL mice with stable transgene insertion in chromosome 1. Microvascular endothelial cell-specific Cre recombinase expression occurred in the sciatic nerves, brains, spleens, kidneys and gastrocnemius muscles of adult vWF-iCre/+ SJL mice bred with Ai14 mice, with retained low level bone marrow and splenic megakaryocyte expression. This novel mouse strain would support hypothesis-driven mechanistic studies to decipher the role(s) of specific genes transcribed by microvascular endothelial cells during development, as well as in physiologic and pathophysiologic states in an organ- and time-dependent manner.

Glucose Promotes EMMPRIN/CD147 and the Secretion of Pro-Angiogenic Factors in a Co-Culture System of Endothelial Cells and Monocytes.

Vascular complications in Type 2 diabetes mellitus (T2DM) patients increase morbidity and mortality. In T2DM, angiogenesis is impaired and can be enhanced or reduced in different tissues ("angiogenic paradox"). The present study aimed to delineate differences between macrovascular and microvascular endothelial cells that might explain this paradox. In a monoculture system of human macrovascular (EaHy926) or microvascular (HMEC-1) endothelial cell lines and a monocytic cell line (U937), high glucose concentrations (25 mmole/L) increased the secretion of the pro-angiogenic factors CD147/EMMPRIN, VEGF, and MMP-9 from both endothelial cells, but not from monocytes. Co-cultures of EaHy926/HMEC-1 with U937 enhanced EMMPRIN and MMP-9 secretion, even in low glucose concentrations (5.5 mmole/L), while in high glucose HMEC-1 co-cultures enhanced all three factors. EMMPRIN mediated these effects, as the addition of anti-EMMPRIN antibody decreased VEGF and MMP-9 secretion, and inhibited the angiogenic potential assessed through the wound assay. Thus, the minor differences between the macrovascular and microvascular endothelial cells cannot explain the angiogenic paradox. Metformin, a widely used drug for the treatment of T2DM, inhibited EMMPRIN, VEGF, and MMP-9 secretion in high glucose concentration, and the AMPK inhibitor dorsomorphin enhanced it. Thus, AMPK regulates EMMPRIN, a key factor in diabetic angiogenesis, suggesting that targeting EMMPRIN may help in the treatment of diabetic vascular complications.

Shear Stress and the AMP-Activated Protein Kinase Independently Protect the Vascular Endothelium from Palmitate Lipotoxicity.

Saturated free fatty acids are thought to play a critical role in metabolic disorders associated with obesity, insulin resistance, type 2 diabetes (T2D), and their vascular complications via effects on the vascular endothelium. The most abundant saturated free fatty acid, palmitate, exerts lipotoxic effects on the vascular endothelium, eventually leading to cell death. Shear stress activates the endothelial AMP-activated protein kinase (AMPK), a cellular energy sensor, and protects endothelial cells from lipotoxicity, however their relationship is uncertain. Here, we used isoform-specific shRNA-mediated silencing of AMPK to explore its involvement in the long-term protection of macrovascular human umbilical vein endothelial cells (HUVECs) against palmitate lipotoxicity and to relate it to the effects of shear stress. We demonstrated that it is the α1 catalytic subunit of AMPK that is critical for HUVEC protection under static conditions, whereas AMPK-α2 autocompensated a substantial loss of AMPK-α1, but failed to protect the cells from palmitate. Shear stress equally protected the wild type HUVECs and those lacking either α1, or α2, or both AMPK-α isoforms; however, the protective effect of AMPK reappeared after returning to static conditions. Moreover, in human adipose microvascular endothelial cells isolated from obese diabetic individuals, shear stress was a strong protector from palmitate lipotoxicity, thus highlighting the importance of circulation that is often obstructed in obesity/T2D. Altogether, these results indicate that AMPK is important for vascular endothelial cell protection against lipotoxicity in the static environment, however it may be dispensable for persistent and more effective protection exerted by shear stress.

Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase.

Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues.

Targeted Inhibition of Phosphodiesterase (PDE) 4 As a Novel Therapy to Increase Endothelial Cell cAMP and Trigger Weibel Palade Body Exocytosis

Background Desmopressin (DDAVP) is widely used in the treatment of VWD and other bleeding disorders. DDAVP activates V2 receptors (V2R) on endothelial cells (EC) to stimulate cAMP generation and Weibel-Palade body (WPB) exocytosis. However, DDAVP has inherent limitations, including lack of oral formulation, side-effects related to renal V2R agonism and sub-optimal responses in some patients. Aims To identify previously approved drugs with capacity to trigger cAMP-dependent WPB secretion in EC to repurpose as novel hemostatic therapeutic agents. Methods Candidate agents were identified by mechanistic screening of FDA and EMA-approved drug databases and rationalized for translational relevance. For each class, prototypical drugs were tested in vitro for capacity to induce VWF release from macrovascular (HUVEC) and microvascular (HMVEC-L) EC (VWF antigen [Ag], propeptide [pp] and collagen binding [CB] in supernatant at 1 hour, expressed as fold increase relative to untreated control [NC]). VWF string formation on the surface of treated EC under flow conditions was determined using fluorescent anti-VWF antibodies and confocal microscopy. Finally, the effects of candidates on platelet aggregation were determined by light transmission aggregometry (LTA). Results and Discussion Mechanistic screening and preliminary in vitro evaluation determined a lead candidate drug class: PDE-4 inhibitors. Previous studies have shown that cAMP-hydrolyzing PDE isoforms 2, 3 and 4 are expressed in EC and that non-selective PDE inhibition (PDE-I) with IBMX alone can elevate cAMP in EC sufficiently to induce VWF release. Consistently, we observed that treatment of EC with IBMX significantly enhanced VWF secretion (median fold increase VWF:Ag vs NC:1.49, p<0.0001, Fig.1). However, PDE isoforms 2 and 3 are also present in platelets, where increased cAMP activity inhibits platelet aggregation. We confirmed by LTA that IBMX significantly attenuated TRAP-6-induced platelet aggregation, limiting the therapeutic relevance of non-selective PDE-I as VWF-raising agents. We next evaluated the capacity of isoform-selective PDE-I to induce VWF release. Selective inhibition of PDE-2 (with EHNA) and PDE-3 (with Cilostamide) had no significant effect (p>0.999, Fig.1). In contrast, selective PDE-4 inhibition with Roflumilast (ROF) resulted in a dose-dependent increase in VWF:Ag secretion in both HUVEC (1.51 fold, p<0.0001, Fig.1) and HMVEC-L (1.75 fold, p=0.007). Notably, these effects were also seen with ROF concentrations in the nanomolar range, consistent with plasma levels following single oral dose in healthy subjects. In keeping with a role for PDE-4 in regulating cAMP-mediated WPB exocytosis, ROF treatment of EC resulted in a proportionate increase in VWFpp secretion (1.74 fold, p=0.0002). Furthermore, pre-incubation of HUVEC with ROF triggered VWF string formation under flow conditions, with strings visualized in most fields of view (Fig.2, 80% ROF vs 0% NC, p=0.0003). Consistently, following treatment with ROF, increased VWF:Ag was accompanied by a parallel rise in VWF:CB in the supernatant (1.50 fold, p=0.0061), confirming the secretion of hemostatically active VWF multimers. Critically, however, ROF had no inhibitory effect on platelet aggregation, consistent with lack of PDE-4 isoform activity in platelets. Finally, ROF combined with either histamine (HIS) or thrombin (THR) had synergistic effects on VWF release, suggesting ROF could prime EC to physiological agonists at times of hemostatic challenge (3.4 fold HIS vs 5.17 fold HIS+ROF, p<0.0001; 3.7 fold THR vs 4.3 fold THR+ROF, p=0.0060). In addition, ROF exhibited synergistic activity with the cAMP-raising agent Isoprenaline compared to ROF alone (1.77 vs 1.40 fold, p=0.0040, Fig.1), suggesting combination therapy as a novel therapeutic approach. Conclusion Drug repurposing may allow for rapid and cost-effective translation into clinical practice. Importantly, PDE-4 inhibitors such as ROF are currently licensed oral agents for treating Psoriasis and COPD. Our novel findings identify PDE-4 as a critical endothelial PDE isoform that regulates WPB exocytosis and VWF secretion. Moreover, we demonstrate that ROF stimulates VWF release in vitro without deleterious effects on platelet aggregation. Our data highlight the therapeutic potential of PDE-4 inhibitors, either alone or in combination with DDAVP, in the treatment of VWD.

Endothelial heterogeneity and their relevance in cardiac development and coronary artery disease

Micro- and macrovascular endothelial cells (ECs) are characterized by structural and functional heterogeneity, which is also reflected in their secretory activity. The root of this heterogeneity and related regulatory mechanisms are still poorly understood. During embryogenesis, microvascular ECs participate in organogenesis prior to the development of the fetal circulation, suggesting that ECs are capable of releasing paracrine trophogens, termed angiocrine factors (AFs). These are angiocrine growth factors, adhesion molecules, and chemokines, which are intended to promote morphogenesis and repair of the adjacent parenchyma/stroma where the vessels are located. There is a tissue and organ-specificity of AFs that traces the heterogeneity of ECs. This AF heterogeneity also traces how ECs respond to pathological conditions or exposure to cardiovascular risk factors. The study of the mechanisms that regulate endothelial and paracrine heterogeneity and that contribute to endotheliopathy represents a broad and as yet understudied area of research. A better understanding of the cellular and molecular mechanisms that regulate this heterogeneity, leading to endotheliopathy is an exciting challenge. In this brief review we will discuss experimental advances in the heterogeneity of ECs and their AF, with a focus on their involvement in the pathogenesis of coronary artery disease.

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples.

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Correlation of alpha-1 antitrypsin levels and exosome associated neutrophil elastase endothelial injury in subjects with SARS-CoV2 infection.

BackgroundSevere acute respiratory syndrome caused by a novel coronavirus 2 (SARS-CoV-2) has infected more than 18 million people worldwide. The activation of endothelial cells is a hallmark of signs of SARS-CoV-2 infection that includes altered integrity of vessel barrier and endothelial inflammation.ObjectivesPulmonary endothelial activation is suggested to be related to the profound neutrophil elastase (NE) activity, which is necessary for sterilization of phagocytosed bacterial pathogens. However, unopposed activity of NE increases alveolocapillary permeability and extracellular matrix degradation. The uncontrolled protease activity of NE during the inflammatory phase of lung diseases might be due to the resistance of exosome associated NE to inhibition by alpha-1 antitrypsin.Method31 subjects with a diagnosis of SARS-CoV2 infection were recruited in the disease group and samples from 30 voluntaries matched for age and sex were also collected for control.ResultsWe measured the plasma levels of exosome-associated NE in SARS-CoV-2 patients which, were positively correlated with sign of endothelial damage in those patients as determined by plasma levels of LDH. Notably, we also found strong correlation with plasma levels of alpha-1 antitrypsin and exosome-associated NE in SARS-CoV-2 patients. Using macrovascular endothelial cells, we also observed that purified NE activity is inhibited by purified alpha-1 antitrypsin while, NE associated with exosomes are resistant to inhibition and show less sensitivity to alpha-1 antitrypsin inhibitory activity, in vitro.ConclusionsOur results point out the role of exosome-associated NE in exacerbation of endothelial injury in SARS-CoV-2 infection. We have demonstrated that exosome-associated NE could be served as a new potential therapeutic target of severe systemic manifestations of SARS-CoV-2 infection.

Empagliflozin prevents angiotensin II-induced hypertension related micro and macrovascular endothelial cell activation and diastolic dysfunction in rats despite persistent hypertension: Role of endothelial SGLT1 and 2

SGLT2 inhibitors (SGLT2i) showed pronounced beneficial effects in patients with heart failure but the underlying mechanisms remain unclear. We evaluated the effect of empagliflozin, selective SGLT2i, on hypertension-induced cardiac and vascular dysfunction.Male Wistar rats received diet with or without empagliflozin (30 mg/kg/day). After 1 week, a hypertensive dose of Ang II (0.4 mg/kg/day) was administered using osmotic mini-pumps for 4 weeks. Systolic blood pressure was determined by sphygmomanometry, the cardiac function by echocardiography and ex vivo (coronary microvascular endothelial cell activation, LV remodeling and fibrosis responses), and the systemic micro and macrovascular endothelial cell activation ex vivo. Empagliflozin treatment did not affect the Ang II-induced hypertensive response. Ang II treatment increased LV mass and induced LV diastolic dysfunction, fibrosis, collagen I and ANP expression, and infiltration of macrophages. In the vasculature, it caused eNOS upregulation in the aorta and down-regulation in mesenteric microvessels associated with increased oxidative stress, ACE, AT1R, VCAM-1, MCP-1, MMP-2, and MMP-9 and collagen I expression, increased endothelial SGLT1 staining in the aorta, mesenteric and coronary microvessels, increased SGLT1 and 2 protein levels in the aorta. All Ang II-induced cardiac and vascular responses were reduced by the empagliflozin treatment.Thus, the SGLT2i effectively attenuated the deleterious impact of Ang II-induced hypertension on target organs including cardiac diastolic dysfunction and remodeling, and endothelial cell activation and pro-atherosclerotic, pro-fibrotic and pro-remodeling responses in macro and microvessels despite persistent hypertension.

The immunosuppressant drug Cyclosporin A aggravates irradiation effects in endothelial cells

The immunosuppressant drug Cyclosporin A (CsA) has been widely used to prevent the development of Graft-versus-Host Disease (GvHD) that can occur after transplantation, including allogeneic graft after accidental high-dose irradiation in humans. Here, we show that CsA alone stimulates ICAM-1 overexpression in human pulmonary microvascular endothelial cells (HPMECs) through Toll-Like Receptor 4 (TLR4) and NF-κB activation. In HPMECs, CsA treatment significantly worsened the overexpression of ICAM-1 induced by high-dose irradiation (15 Gy). This additive effect of CsA was also observed when ICAM-1 overexpression was induced by another pathway (Ca2+ entry) in macrovascular endothelial cells. In addition, CsA triggered apoptosis as well as rearrangement of the actin cytoskeleton and adherens junctions (VE-Cadherin) in microvascular endothelial monolayers. High-dose irradiation triggered similar deleterious effects in endothelial monolayers and, again, CsA treatment strongly aggravated the effects of irradiation. Altogether, these results suggest that post-transplant CsA treatment may exacerbate the deleterious effects of irradiation on the endothelium.

Human endothelial cells in high glucose: New clues from culture in 3D microfluidic chips.

Several studies have demonstrated the role of high glucose in promoting endothelial dysfunction utilizing traditional two-dimensional (2D) culture systems, which, however, do not replicate the complex organization of the endothelium within a vessel constantly exposed to flow. Here we describe the response to high glucose of micro- and macro-vascular human endothelial cells (EC) cultured in biomimetic microchannels fabricated through soft lithography and perfused to generate shear stress. In 3D macrovascular EC exposed to a shear stress of 0.4 Pa respond to high glucose with cytoskeletal remodeling and alterations in cell shape. Under the same experimental conditions, these effects are more pronounced in microvascular cells that show massive cytoskeletal disassembly and apoptosis after culture in high glucose. However, when exposed to a shear stress of 4 Pa, which is physiological in the microvasculature, human dermal microvascular endothelial cells (HDMEC) show alterations of the cytoskeleton but no apoptosis. This result emphasizes the sensitivity of HDMEC to different regimens of flow. No significant variations in the thickness of glycocalyx were detected in both human endothelial cells from the umbilical vein and HDMEC exposed to high glucose in 3D, whereas clear differences emerge between cells cultured in static 2D versus microfluidic channels. We conclude that culture in microfluidic microchannels unveils unique insights into endothelial dysfunction by high glucose.

Cytoskeletal Remodeling Mimics Endothelial Response to Microgravity.

Mechanical cues contribute to the maintenance of a healthy endothelium, which is essential for vascular integrity. Indeed endothelial cells are mechanosensors that integrate the forces in the form of biochemical signals. The cytoskeleton is fundamental in sensing mechanical stimuli and activating specific signaling pathways. Because the cytoskeleton is very rapidly remodeled in endothelial cells exposed to microgravity, we investigated whether the disruption of actin polymerization by cytochalasin D in 1g condition triggers and orchestrates responses similar to those occurring in micro- and macro-vascular endothelial cells upon gravitational unloading. We focused our attention on the effect of simulated microgravity on stress proteins and transient receptor potential melastatin 7 (TRPM7), a cation channel that acts as a mechanosensor and modulates endothelial cell proliferation and stress response. Simulated microgravity downregulates TRPM7 in both cell types. However, 24 h of treatment with cytochalasin D decreases the amounts of TRPM7 only in macrovascular endothelial cells, suggesting that the regulation and the role of TRPM7 in microvascular cells are more complex than expected. The 24 h culture in the presence of cytochalasin D mimics the effect of simulated microgravity in modulating stress response in micro- and macro-vascular endothelial cells. We conclude that cytoskeletal disruption might mediate some effects of microgravity in endothelial cells.

Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

The vascular endothelial growth factor receptors (VEGFRs) can shape the neovascular phenotype of vascular endothelial cells when translocated to the nucleus, however the spatial and temporal changes in the intracellular distribution and translocation of VEGFRs to the nucleus and the organelles involved in this process is unclear. This study reports the effect of exogenous VEGF on translocation of VEGFRs and organelles in micro- and macrovascular endothelial cells. We showed that VEGF is responsible for: a rapid and substantial nuclear translocation of VEGFRs; VEGFR1 and VEGFR2 exhibit distinct spatial, temporal and structural translocation characteristics both in vitro and in vivo and this determines the nuclear VEGFR1:VEGFR2 ratio which differs between microvascular and macrovascular cells; VEGFR2 nuclear translocation is associated with the endosomal pathway transporting the receptor from Golgi in microvascular endothelial cells; and an increase in the volume of intracellular organelles. In conclusion, the nuclear translocation of VEGFRs is both receptor and vessel (macro versus micro) dependent and the endosomal pathway plays a key role in the translocation of VEGFRs to the nucleus and the subsequent export to the lysosomal system. Modulating VEGF-mediated VEGFR1 and VEGFR2 intracellular transmigration pathways may offer an alternative for the development of new anti-angiogenic therapies.

Appearance of peanut agglutinin in the blood circulation after peanut ingestion promotes endothelial secretion of metastasis-promoting cytokines.

Peanut agglutinin (PNA) is a carbohydrate-binding protein in peanuts that accounts for ~0.15% peanut weight. PNA is highly resistant to cooking and digestion and is rapidly detectable in the blood after peanut consumption. Our previous studies have shown that circulating PNA mimics the actions of endogenous galactoside-binding protein galectin-3 by interaction with tumour cell-associated MUC1 and promotes circulating tumour cell metastatic spreading. The present study shows that circulating PNA interacts with micro- as well as macro-vascular endothelial cells and induces endothelial secretion of cytokines MCP-1 (CCL2) and IL-6 in vitro and in vivo. The increased secretion of these cytokines autocrinely/paracrinely enhances the expression of endothelial cell surface adhesion molecules including integrins, VCAM and selectin, leading to increased tumour cell-endothelial adhesion and endothelial tubule formation. Binding of PNA to endothelial surface MCAM (CD146), via N-linked glycans, and subsequent activation of PI3K-AKT-PREAS40 signalling is here shown responsible for PNA-induced secretion of MCP-1 and IL-6 by vascular endothelium. Thus, in addition to its influence on promoting tumour cell spreading by interaction with tumour cell-associated MUC1, circulating PNA might also influence metastasis by enhancing the secretion of metastasis-promoting MCP-1 and IL-6 from the vascular endothelium.

GIMAP5 maintains liver endothelial cell homeostasis and prevents portal hypertension.

Portal hypertension is a major contributor to decompensation and death from liver disease, a global health problem. Here, we demonstrate homozygous damaging mutations in GIMAP5, a small organellar GTPase, in four families with unexplained portal hypertension. We show that GIMAP5 is expressed in hepatic endothelial cells and that its loss in both humans and mice results in capillarization of liver sinusoidal endothelial cells (LSECs); this effect is also seen when GIMAP5 is selectively deleted in endothelial cells. Single-cell RNA-sequencing analysis in a GIMAP5-deficient mouse model reveals replacement of LSECs with capillarized endothelial cells, a reduction of macrovascular hepatic endothelial cells, and places GIMAP5 upstream of GATA4, a transcription factor required for LSEC specification. Thus, GIMAP5 is a critical regulator of liver endothelial cell homeostasis and, when absent, produces portal hypertension. These findings provide new insight into the pathogenesis of portal hypertension, a major contributor to morbidity and mortality from liver disease.

Laminar flow substantially affects the morphology and functional phenotype of glomerular endothelial cells.

Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.

RAGE silencing deters CML-AGE induced inflammation and TLR4 expression in endothelial cells

The Nε-(carboxymethyl)lysine (CML), the predominant advanced glycation end products (AGEs) in diabetes and its RAGE induced cytokine release has been well explored. But the CML mediated multiple AGEs receptor expression is still not understood and the role played by RAGE silencing in modulating CML generated pro-inflammatory cytokines in micro and macrovascular endothelial cells is yet to be studied. HUVEC and HREC cells were exposed with CML for 24 h. RAGE, AGER1, AGER2, Gal-3, TLR4, TLR2, CD36, FEEL-1, FEEL-2, and chemokine HMGB1 were quantified by either qPCR/western blotting. The receptor's expression was also determined in control vs diabetic retina. Expression of pro-inflammatory genes, ROS, and mitochondrial membrane potential change were assessed using ELISA, DCFDA, and JC-1 method respectively. RAGE expression was silenced either by Si-RAGE or neutralising antibody with anti-RAGE and expression of other AGE receptors, adaptors, and signalling pathway were studied compared with Si-Control. CML activated RAGE, TLR4, HMGB1(p < 0.001) and Gal-3 (p < 0.05) expression in both micro and macro vascular cells. Cadaveric diabetic retinal tissues also showed increased expression of RAGE, TLR4 and HMGB1 (p < 0.05). RAGE silencing significantly reduced TLR4, HMGB1 (p < 0.05) expression and inhibited the phosphorylation of NFκB and ERK1/2 in both these cells. The TLR4 adaptors MyD88 and TIRAP (p < 0.05) showed down regulation on RAGE silencing. This study shows CML induces AGE receptors expression as observed in diabetic retina and RAGE silencing down regulated TLR4 signalling and cytokine release by partly modulating TLR4 adaptors which needs further validation. From this study we speculate targeting the TLR4 adaptors like MyD88 and TIRAP can be a potential therapeutic target for reducing diabetic induced vascular complications.

Unnecessary hesitancy on human vaccine tests-Response.

20-Hydroxyeicosatetraenoic acid (20-HETE) is formed by the ω-hydroxylation of arachidonic acid by cytochrome P450 4A and 4F enzymes, and it induces angiogenic responses in vivo. To test the hypothesis that 20-HETE increases endothelial cell (EC) proliferation via vascular endothelial growth factor (VEGF), we studied the effects of WIT003 [20-hydroxyeicosa-5(Z),14(Z)-dienoic acid], a 20-HETE analog on human macrovascular or microvascular EC. WIT003, as well as pure 20-HETE, stimulated EC proliferation by ∼40%. These proliferative effects were accompanied by increased VEGF expression and release that were observed as early as 4 h after 20-HETE agonist addition. This was accompanied by increased phosphorylation of the VEGF receptor 2. The proliferative effects of 20-HETE were markedly inhibited by a VEGF-neutralizing antibody. Polyethylene glycol-superoxide dismutase (PEG-SOD) markedly inhibited both the increases in VEGF expression and the proliferative effects of 20-HETE. In contrast, administration of the NAD(P)H oxidase inhibitor apocynin had no effect to the proliferative response to 20-HETE. The 20-HETE agonist markedly increased superoxide formation as reflected by an increase in dihydroethidium staining of EC, and this increase was inhibited by PEG-SOD but not by apocynin. 20-HETE also increased the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in EC, whereas an inhibitor of MAPK [U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] suppressed the proliferative and the VEGF changes but not the pro-oxidant effects of 20-HETE. These data suggest that 20-HETE stimulates superoxide formation by pathways other than apocynin-sensitive NAD(P)H oxidase, thereby activating MAPK and then enhancing VEGF synthesis that drives EC proliferation. Thus, 20-HETE may be involved in the regulation of EC functions, such as angiogenesis.

Isolation, purification, and conditional immortalization of murine glomerular endothelial cells of microvascular phenotype

Glomerular endothelial cells (GEnC) are a specialized microvascular subset of endothelial cells that, when injured, result in many types of diseases within the kidney. Thus, techniques to study GEnC in a cell culture system are important to investigate mechanisms of GEnC injury. Studies of endothelial cell function in culture have predominately relied on using macrovascular endothelial cells from vascular areas other than the glomerulus. Over the last 15 years, glomerular endothelial cells lines have been created but were isolated by targeting cells expressing CD31. Some studies identified endothelial cells isolated from the microvasculature do not express CD31 and some suggest that CD31+ cells are phenotypically different than endothelial cells found in capillaries. Here we detail our method of isolation, purification, and conditional immortalization of mouse glomerular endothelial cells targeting endothelial cells that do not express CD31.•This method allows for isolation, purification, and conditional immortalization of glomerular endothelial cells for continued passage of GEnCs beyond that of primary cell culture.•This method can be used in genetically modified mice to investigate how a modification of a specific gene or protein affects the glomerular endothelium at the cellular level.