Gene Expression In Endothelial Cells Research Articles

Beyond cholesterol-pleiotropic effects of lipoprotein apheresis.

Cardiovascular disease is a leading cause of mortality worldwide, which is caused mainly by atherosclerosis, a chronic inflammatory disease of blood vessels. Therefore, atherosclerosis represents a complex disorder, which induces damage or imbalance on different levels: for example, genes, cytokines, lipoproteins, cells, vessels, and organs. Lipoprotein apheresis (LA) is a well-established extracorporeal treatment of severe hyperlipoproteinemia. In addition, LA may have simultaneously crucial effects on many other atherogenic factors during the treatments, for example, as vascular inflammation, rheology, mobilization of adult stem cells and gene expressions in blood or endothelial cells, which will be discussed in this short review. In addition, stable microRNAs besides tissues also appear in extracellular compartments, for example, vessels, involved in atherosclerotic processes, were found to be reduced by LA treatments. In summary, LA represents a complex therapeutic procedure, that provides an ideal tool for the treatment of complex disorders such as atherosclerosis.

Non-Invasive Pulsatile Shear Stress Modifies Endothelial Activation; A Narrative Review.

The monolayer of cells that line both the heart and the entire vasculature is the endothelial cell (EC). These cells respond to external and internal signals, producing a wide array of primary or secondary messengers involved in coagulation, vascular tone, inflammation, and cell-to-cell signaling. Endothelial cell activation is the process by which EC changes from a quiescent cell phenotype, which maintains cellular integrity, antithrombotic, and anti-inflammatory properties, to a phenotype that is prothrombotic, pro-inflammatory, and permeable, in addition to repair and leukocyte trafficking at the site of injury or infection. Pathological activation of EC leads to increased vascular permeability, thrombosis, and an uncontrolled inflammatory response that leads to endothelial dysfunction. This pathological activation can be observed during ischemia reperfusion injury (IRI) and sepsis. Shear stress (SS) and pulsatile shear stress (PSS) are produced by mechanical frictional forces of blood flow and contraction of the heart, respectively, and are well-known mechanical signals that affect EC function, morphology, and gene expression. PSS promotes EC homeostasis and cardiovascular health. The archetype of inducing PSS is exercise (i.e., jogging, which introduces pulsations to the body as a function of the foot striking the pavement), or mechanical devices which induce external pulsations to the body (Enhanced External Pulsation (EECP), Whole-body vibration (WBV), and Whole-body periodic acceleration (WBPA aka pGz)). The purpose of this narrative review is to focus on the aforementioned noninvasive methods to increase PSS, review how each of these modify specific diseases that have been shown to induce endothelial activation and microcirculatory dysfunction (Ischemia reperfusion injury-myocardial infarction and cardiac arrest and resuscitation), sepsis, and lipopolysaccharide-induced sepsis syndrome (LPS)), and review current evidence and insight into how each may modify endothelial activation and how these may be beneficial in the acute and chronic setting of endothelial activation and microvascular dysfunction.

Transcriptomic analysis of a 3D blood–brain barrier model exposed to disturbed fluid flow

Cerebral aneurysms are more likely to form at bifurcations in the vasculature, where disturbed fluid is prevalent due to flow separation at sufficiently high Reynolds numbers. While previous studies have demonstrated that altered shear stress exerted by disturbed flow disrupts endothelial tight junctions, less is known about how these flow regimes alter gene expression in endothelial cells lining the blood–brain barrier. Specifically, the effect of disturbed flow on expression of genes associated with cell–cell and cell–matrix interaction, which likely mediate aneurysm formation, remains unclear. RNA sequencing of immortalized cerebral endothelial cells isolated from the lumen of a 3D blood–brain barrier model reveals distinct transcriptional changes in vessels exposed to fully developed and disturbed flow profiles applied by both steady and physiological waveforms. Differential gene expression, validated by qRT-PCR and western blotting, reveals that lumican, a small leucine-rich proteoglycan, is the most significantly downregulated gene in endothelial cells exposed to steady, disturbed flow. Knocking down lumican expression reduces barrier function in the presence of steady, fully developed flow. Moreover, adding purified lumican into the hydrogel of the 3D blood–brain barrier model recovers barrier function in the region exposed to fully developed flow. Overall, these findings emphasize the importance of flow regimes exhibiting spatial and temporal heterogeneous shear stress profiles on cell–matrix interaction in endothelial cells lining the blood–brain barrier, while also identifying lumican as a contributor to the formation and maintenance of an intact barrier.

Abstract 14950: Single-Cell Transcriptomic Analysis Reveals Emergence of Inflammatory and Activated Arterial Endothelial Cells Promoted by Cross-Talk With Mural Cells During Development of Severe Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a lethal disease characterized by occlusive arterial remodelling thought to be triggered by endothelial cell (EC) injury; however, the pathobiological mechanisms are poorly understood. We employed single cell RNA sequencing (scRNAseq) to define transcriptomic changes in the SU5416/chronic hypoxia (SU/CH) rat model. Methods: Sprague-Dawley rats were injected with 20mg/kg SU subcutaneously and exposed to 3 weeks of CH (10% O 2 ). Right ventricular systolic pressure (RVSP) was measured at baseline, 1, 3, 5, and 8-weeks, then lungs were explanted, digested, and dissociated into single cells which were sequenced using the 10x genomics platform. Results: Elevation of RVSP plateaued at >100 mmHg by 5 weeks. Dimensionality reduction of the scRNAseq data was performed using Uniform Manifold Approximation and Projection (UMAP) analysis resulting in 24 lung clusters. Cell prioritization analysis performed using Augur identified vascular cells as the most affected by SU/CH, including arterial ECs, pericytes, and gCap ECs. After sub-clustering the EC populations, eight distinct clusters were identified representing all expected subtypes (capillary, arterial, venous, and lymphatic). As well, two novel EC clusters were detected. The first showed reduced expression of classical EC genes ( Cdh5 , Cldn5 ) and increased proinflammatory markers ( RT1-Da , Cd74 ), termed ‘inflammatory’ ECs. The second was an arterial cluster that exhibited reduced expression of Dll4 and increased Cxcl12 and Fn1 , termed ‘activated’ arterial ECs. Both populations emerging at 1-week post SU, persisting throughout PAH progression. While many EC populations showed marked differential gene expression (DGE) at 1 week, the activated arterial cluster was the only cluster showing progression of DGE throughout progression of PAH. Receptor-ligand analysis with NicheNet identified activated arterial ECs as a top receiver cell responding to multiple ligands, including Bmp4/5 , Col4a1 , and Vegfa/c, which were largely derived from mural fibroblasts. Conclusion: Emergence of inflammatory and activated arterial ECs likely contribute to vascular remodelling associated with PAH promoted by cross-talk with lung stromal cells.

Altered gene expression in human brain microvascular endothelial cells in response to the infection of influenza H1N1 virus

Influenza viruses not only cause respiratory illness, but also have been reported to elicit neurological manifestations following acute viral infection. The central nervous system (CNS) has a specific defense mechanism against pathogens structured by cerebral microvasculature lined with brain endothelial cells to form the blood–brain barrier (BBB). To investigate the response of human brain microvascular endothelial cells (hBMECs) to the Influenza A virus (IAV), we inoculated the cells with the A/WSN/33 (H1N1) virus. We then conducted an RNAseq experiment to determine the changes in gene expression levels and the activated disease pathways following infection. The analysis revealed an effective activation of the innate immune defense by inducing the pattern recognition receptors (PRRs). Along with the production of proinflammatory cytokines, we detected an upregulation of interferons and interferon-stimulated genes, such as IFN-β/λ, ISG15, CXCL11, CXCL3 and IL-6, etc. Moreover, infected hBMECs exhibited a disruption in the cytoskeletal structure both on the transcriptomic and cytological levels. The RNAseq analysis showed different pathways and candidate genes associated with the neuroactive ligand-receptor interaction, neuroinflammation, and neurodegenerative diseases, together with a predicted activation of the neuroglia. Likewise, some genes linked with the mitochondrial structure and function displayed a significantly altered expression. En masse, this data supports that hBMECs could be infected by the IAV, which induces the innate and inflammatory immune response. The results suggest that the influenza virus infection could potentially induce a subsequent aggravation of neurological disorders.

Hybrid nanostructured gadolinium oxide-collagen-dextran polymeric hydrogel for corneal repair and regeneration

A bio composite hydrogel containing collagen, dextran and rare earth metal nanoparticle, gadolinium oxide nanoparticle was prepared for corneal tissue regenerative application. Gadolinium oxide nanoparticles which are widely used in MRI scanning application are not much explored for tissue regenerative applications. The synthesized nanoparticles provided stability and therapeutic potential to collagen-dextran composite for achieving tissue regenerative potential. A good compatibility towards rabbit corneal fibroblast cells (SIRC) was observed with the hydrogel along with anti-proliferation property in aortic explants. The hydrogel down-regulated the VEGF-A gene expression in endothelial cells showing its ability to safe-guard the vision from corneal neovascularization. The expression of genes, ALDH1A1 and Vimentin needed for the well-being and enhanced proliferation of corneal cells was up-regulated upon treatment with the bio composite, while the expression of pro-inflammatory cytokines IL-6 and COX-2 was down regulated. Faster regeneration and migration of corneal cells into abrasion area was observed with the bio composite in vitro. The explant studies confirmed the compatibility of bio composites towards corneal tissue as well as lens crystallin proteins. The results suggest the potential use of rare-earth metal nanoparticles-based bio composite biomaterials in repairing and regenerating corneal cells when used in combination with protein- polysaccharide biomacromolecules.

Muscle transcriptomic circuits linked to periarticular physiology in end-stage osteoarthritis.

The ability of individuals with end-stage osteoarthritis (OA) to functionally recover from total joint arthroplasty is highly inconsistent. The molecular mechanisms driving this heterogeneity have yet to be elucidated. Furthermore, OA disproportionately impacts females, suggesting a need for identifying female-specific therapeutic targets. We profiled the skeletal muscle transcriptome in females with end-stage OA (n = 20) undergoing total knee or hip arthroplasty using RNA-Seq. Single-gene differential expression (DE) analyses tested for DE genes between skeletal muscle overlaying the surgical (SX) joint and muscle from the contralateral (CTRL) leg. Network analyses were performed using Pathway-Level Information ExtractoR (PLIER) to summarize genes into latent variables (LVs), i.e., gene circuits, and link them to biological pathways. LV differences in SX versus CTRL muscle and across sources of muscle tissue (vastus medialis, vastus lateralis, or tensor fascia latae) were determined with ANOVA. Linear models tested for associations between LVs and muscle phenotype on the SX side (inflammation, function, and integrity). DE analysis revealed 360 DE genes (|Log2 fold-difference| ≥ 1, FDR ≤ 0.05) between the SX and CTRL limbs, many associated with inflammation and lipid metabolism. PLIER analyses revealed circuits associated with protein degradation and fibro-adipogenic cell gene expression. Muscle inflammation and function were linked to an LV associated with endothelial cell gene expression highlighting a potential regulatory role of endothelial cells within skeletal muscle. These findings may provide insight into potential therapeutic targets to improve OA rehabilitation before and/or following total joint replacement.

Intermittent hypoxia inhibits epinephrine-induced transcriptional changes in human aortic endothelial cells

Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular disease. While intermittent hypoxia (IH) and catecholamine release play an important role in this increased risk, the mechanisms are incompletely understood. We have recently reported that IH causes endothelial cell (EC) activation, an early phenomenon in the development of cardiovascular disease, via IH-induced catecholamine release. Here, we investigated the effects of IH and epinephrine on gene expression in human aortic ECs using RNA-sequencing. We found a significant overlap between IH and epinephrine-induced differentially expressed genes (DEGs) including enrichment in leukocyte migration, cytokine-cytokine receptor interaction, cell adhesion and angiogenesis. Epinephrine caused higher number of DEGs compared to IH. Interestingly, IH when combined with epinephrine had an inhibitory effect on epinephrine-induced gene expression. Combination of IH and epinephrine induced MT1G (Metallothionein 1G), which has been shown to be highly expressed in ECs from parts of aorta (i.e., aortic arch) where atherosclerosis is more likely to occur. In conclusion, epinephrine has a greater effect than IH on EC gene expression in terms of number of genes and their expression level. IH inhibited the epinephrine-induced transcriptional response. Further investigation of the interaction between IH and epinephrine is needed to better understand how OSA causes cardiovascular disease.

INFLUENCE OF OBESITY ON VASCULAR DYSFUNCTION AFTER TRAUMATIC HEMORRHAGE.

Background: Obesity increases the risk for morbidity and mortality after trauma. These complications are associated with profound vascular damage. Traumatic hemorrhage acutely attenuates vascular responsiveness, but the impact of obesity on this dysfunction is not known. The local inflammatory response in vascular cells is also unknown. We hypothesized that obesity potentiates trauma-induced vascular inflammation and dysfunction. Methods: Male Sprague-Dawley rats (~250 g) were fed normal chow (NC; 13.5% kcal fat, n = 20) or high-fat (HF; 60% kcal fat, n = 20) diets for 6 to 8 weeks. Under anesthesia, hemorrhage was induced by a mesenteric artery laceration, a Grade V splenic injury, and hypotension (MAP = 30-40 mm Hg) for 30 minutes. Vascular responsiveness was assessed ex vivo in isolated mesenteric arteries prehemorrhage and posthemorrhage. Gene expression for IL-1β, and IL-6, prooxidant nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), and α-adrenergic receptor were assessed in carotid artery endothelial cells (ECs) and non-ECs (media + adventitia). Results: In NC rats, hemorrhage attenuated norepinephrine-induced vasoconstriction and endothelium-dependent vasodilation to acetylcholine. In HF rats, baseline norepinephrine-induced vasoconstriction was attenuated compared with NC, but vasoconstriction and endothelium-dependent vasodilation did not change prehemorrhage to posthemorrhage. Hemorrhage led to elevated IL-1β gene expression in ECs and elevated IL1β, IL-6, NOX2, and α-adrenergic receptor gene expression in the media + adventitia compared with sham. HF rats had greater EC IL-1 β and NOX2 gene expression compared with NC rats. The hemorrhage-induced elevation of IL-1β in the media + adventitia was greatest in HF rats. Conclusion: Traumatic hemorrhage attenuates vascular responsiveness and induces vascular inflammation. The attenuated vascular responsiveness after hemorrhage is absent in obese rats, while the elevated vascular inflammation persists. A HF diet amplifies the arterial inflammation after hemorrhage. Altered vascular responsiveness and vascular inflammation may contribute to worse outcomes in obese trauma patients.

Dichotomous role of integrin-β5 in lung endothelial cells.

Pulmonary arterial hypertension (PAH) is a progressive, devastating disease, and its main histological manifestation is an occlusive pulmonary arteriopathy. One important functional component of PAH is aberrant endothelial cell (EC) function including apoptosis-resistance, unchecked proliferation, and impaired migration. The mechanisms leading to and maintaining physiologic and aberrant EC function are not fully understood. Here, we tested the hypothesis that in PAH, ECs have increased expression of the transmembrane protein integrin-β5, which contributes to migration and survival under physiologic and pathological conditions, but also to endothelial-to-mesenchymal transition (EnMT).We found that elevated integrin-β5 expression in pulmonary artery lesions and lung tissue from PAH patients and rats with PH induced by chronic hypoxia and injection of CD117+ rat lung EC clones. These EC clones exhibited elevated expression of integrin-β5 and its heterodimerization partner integrin-αν and showed accelerated barrier formation. Inhibition of integrin-ανβ5 in vitro partially blocked transforming growth factor (TGF)-β1-induced EnMT gene expression in rat lung control ECs and less in rat lung EC clones and human lung microvascular ECs. Inhibition of integrin-ανβ5 promoted endothelial dysfunction as shown by reduced migration in a scratch assay and increased apoptosis in synergism with TGF-β1. In vivo, blocking of integrin-ανβ5 exaggerated PH induced by chronic hypoxia and CD117+ EC clones in rats. In summary, we found a role for integrin-ανβ5 in lung endothelial survival and migration, but also a partial contribution to TGF-β1-induced EnMT gene expression. Our results suggest that integrin-ανβ5 is required for physiologic function of ECs and lung vascular homeostasis.

TWEAK and TNFα, Both TNF Ligand Family Members and Multiple Sclerosis-Related Cytokines, Induce Distinct Gene Response in Human Brain Microvascular Endothelial Cells.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family involved in various diseases including brain inflammatory pathologies such as multiple sclerosis. It has been demonstrated that TWEAK can induce cerebrovascular permeability in an in vitro model of the blood–brain barrier. The molecular mechanisms playing a role in TWEAK versus TNFα signaling on cerebral microvascular endothelial cells are not well defined. Therefore, we aimed to identify gene expression changes in cultures of human brain microvascular endothelial cells (hCMEC/D3) to address changes initiated by TWEAK exposure. Taken together, our studies highlighted that gene involved in leukocyte extravasation, notably claudin-5, were differentially modulated by TWEAK and TNFα. We identified differential gene expression of hCMEC/D3 cells at three timepoints following TWEAK versus TNFα stimulation and also found distinct modulations of several canonical pathways including the actin cytoskeleton, vascular endothelial growth factor (VEGF), Rho family GTPases, and phosphatase and tensin homolog (PTEN) pathways. To our knowledge, this is the first study to interrogate and compare the effects of TWEAK versus TNFα on gene expression in brain microvascular endothelial cells.

SZN-413, a FZD4 Agonist, as a Potential Novel Therapeutic for the Treatment of Diabetic Retinopathy.

PurposeThere remains a high unmet need for therapies with new mechanisms of action to achieve reperfusion of ischemic retina in diabetic retinopathy. We examined whether a novel frizzled class receptor 4 (FZD4) agonist could promote regeneration of functional blood vessels in animal models of retinopathy.MethodsWe developed a novel Norrin mimetic (SZN-413-p) targeting FZD4 and low-density lipoprotein receptor-related protein 5 (LRP5) and examined its effect on retinal and brain endothelial cells in vitro. SZN-413-p was subsequently humanized, resulting in the therapeutic candidate SZN-413, and was examined in animal models of retinopathy. In an oxygen-induced retinopathy mouse model, avascular and neovascularization areas were measured. Furthermore, in a vascular endothelial growth factor (VEGF)-induced retinal vascular leakage rabbit model, the impact on vascular leakage by SZN-413 was examined by measuring fluorescein leakage.ResultsSZN-413-p induced Wnt/β-catenin signaling and upregulated blood–brain barrier/blood–retina barrier gene expressions in endothelial cells. In the oxygen-induced retinopathy mouse model, SZN-413-p and SZN-413 significantly reduced the neovascularization area size (P < 0.001) to a level comparable to, or better than the positive control aflibercept. Both agonists also showed a reduction in avascular area size compared to vehicle (P < 0.001) and aflibercept groups (P < 0.05 and P < 0.01 for SZN-413-p and SZN-413, respectively). In the VEGF-induced retinal vascular leakage rabbit model, SZN-413 reduced retinal vascular leakage by ∼80%, compared to the vehicle-treated group (P < 0.01).ConclusionsReduction of neovascular tufts and avascular areas and of VEGF-driven retinal vascular leakage suggests that SZN-413 can simultaneously address retinal non-perfusion and vascular leakage.Translational RelevanceFZD4 signaling modulation by SZN-413 is a novel mechanism of action that can offer a new therapeutic strategy for diabetic retinopathy.

Gene expression in meningeal lymphatic endothelial cells following traumatic brain injury in mice.

Meningeal lymphatic vessels transport both the cerebrospinal fluid and interstitial fluid to the deep cervical lymph nodes. Traumatic brain injury (TBI) is accompanied by meningeal injury. We hypothesized that the TBI-induced meningeal injury would damage lymphatic vessels and affect brain function. We observed altered gene expression in meningeal lymphatic endothelial cells (LECs) in a mouse model of TBI. Through flow cytometry–based cell sorting, meningeal LECs were obtained from a mouse model of controlled cortical impact 3 days after TBI. Microarray analysis, real-time polymerase chain reaction assays, and enzyme-linked immunosorbent assays were performed to determine mRNA and protein expression levels in meningeal LECs. The number of meningeal LECs was significantly lower in the injury group than in the sham group 3 days after TBI. Additionally, the mRNA expression of lymphatic vessel endothelial hyaluronan receptor 1 (a specific marker of lymphatic vessels) in meningeal LECs was significantly lower in the injury group than in the sham group. The mRNA and protein expression of FMS-like tyrosine kinase 4 and neuropilin 2 (markers of lymphangiogenesis) in meningeal LECs was significantly higher in the injury group than in the sham group. Our findings indicate that TBI is associated with the impairment of meningeal LECs and meningeal lymphangiogenesis, which implicates lymphatic vessel injury in the pathogenesis of this condition.

The role of LRP1 in Aβ efflux transport across the blood-brain barrier and cognitive dysfunction in diabetes mellitus

BackgroundThe incidence of cognitive dysfunction in diabetes is increasing yearly, which severely affects the quality of life of patients and places a heavy burden on families and society. It has been demonstrated that impaired clearance of cerebral amyloid β-protein (Aβ) is a central event in the initiation and progression of Aβ deposition and cognitive impairment in diabetic patients. However, until now, the molecular mechanism by which diabetes mellitus induces impaired clearance of Aβ has remained unclear. ObjectiveTo investigate the role and mechanism of lipoprotein receptor-related protein 1 (LRP1) in Aβ clearance impairment and cognitive function damage caused by diabetes. MethodsSPF male C57BL/6 mice were bred, and streptozotocin (STZ) (60 mg/kg/d) was intraperitoneally injected for 5 days to establish a diabetes model. The novel object recognition test and fear conditioning test were used to assess the cognitive function of mice in each group. Western blotting, qRT-PCR, ELISAs, and immunofluorescence staining were used to detect the expression levels of Aβ and Aβ clearance-related proteins in mouse brains. HBMECs were cultured in vitro to establish the blood-brain barrier model. The clearance rate of Aβ and the expression levels of LRP1 were measured under different glucose concentration culture conditions. HBMECs were transfected with lentivirus to overexpress or knock down the LRP1, and then, the changes in Aβ clearance were detected again. We injected adeno-associated virus AAV9-SP-A-LRP1 shRNA into the tail vein of DM mice to selectively knock down LRP1 gene expression in cerebral vascular endothelial cells. Then, the cognitive function and the expression levels of Aβ and Aβ clearance-related proteins in the brains of normal, DM and LRP1 knockdown mice were detected. ResultsCompared with the controls, diabetic mice showed impaired cognitive performance, increased deposition of Aβ in the brain and decreased expression of LRP1 in the brain microvasculature. In vitro experiments showed that high glucose can downregulate the expression of LRP1 in HBMECs and damage the Aβ clearance across the blood-brain barrier (BBB). The reduction in the clearance rate of Aβ induced by high glucose was reversed by LRP1 overexpression but further substantially decreased when LRP1 was knocked down. ConclusionHyperglycemia can impair Aβ efflux in the brain by downregulating the expression of LRP1 in the brain microvasculature, eventually resulting in cognitive impairment.

Effect of Antioxidants on the Production of MCP-1 Chemokine by EA.hy926 Cells in Response to IL-6.

An elevated level of circulatory interleukin 6 (IL-6) is a biomarker for cytokine storm of various etiologies, including COVID-19, and contributes to poor prognosis. Vascular endothelial cells are one of the main targets of pathological action of IL-6. IL-6 activates the trans-signaling pathway via the formation of the IL-6/sIL-6Ra/gp130 receptor complex and subsequent activation of the JAK/STAT3 signaling pathway and PI3K/AKT and MEK/ERK kinases in some cases. Previously, it was shown by the authors' group and other researchers that reactive oxygen species (ROS), including mitochondrial ROS (mito-ROS), contribute to the induction of IL-6 expression in the endothelium, mainly due to increased activation of the transcription factor NF-kB. We have also shown that the mitochondria-targeted antioxidant SkQ1 (Plastoquinolyl-10(6'-decyltriphenyl)phosphonium) prevented tumor necrosis factor (TNF)-induced cytokine storm and death in mice. In the aortas of these animals, SkQ1 also prevented the increase in the expression of NF-kB-dependent genes, including the cytokine IL-6 and the chemokine MCP-1. In the current work, the hypothesis of mito-ROS involvement in the IL-6-signaling-mediated proinflammatory gene expression in endothelial cells is tested. SkQ1 suppressed the expression and secretion of the MCP-1 chemokine, induced by IL-6 in combination with sIL-6-Ra, but not the expression of ICAM-1 adhesion molecules in EA.hy926 human endothelial cells. Using specific inhibitors, the authors have shown that, in EA.hy926 cells, IL-6-induced expression of MCP-1 and ICAM-1 depends on the signaling protein and transcription activator STAT3 and, in some cases, on JNK, PI3K, and MEK1/2 kinases and is independent of p38 kinase. In this model, IL-6 induced rapid STAT3 activation, while ERK1/2 activation was less pronounced, and there was no IL-6 effect on Akt and JNK activation. SkQ1 partially suppressed STAT3 and ERK1/2 activation. Thus, we have shown that SkQ1 suppresses not only NF-kB-dependent expression of IL-6 and other proinflammatory genes but also IL-6-induced activation of JAK/STAT3 and STAT3-dependent expression of MCP-1, which probably contributes to the overall therapeutic effect of SkQ1.

Abstract P3128: Obesogenic Diet Promotes Endothelial-to-Mesenchymal Transition In Adipose Tissue

Vascular dysfunction and chronic inflammation are characteristics of obesity-induced adipose tissue dysfunction. Proinflammatory cytokines can drive Endothelial-to-Mesenchymal Transition (EndoMT), where endothelial cells transition into mesenchymal-like interstitial cells that leave the vascular wall and promote tissue inflammation and fibrosis. In this study, we aimed to determine whether obesogenic diet can promote EndoMT in adipose tissue. EndoMT was assessed in 1) transgenic endothelial cell lineage-tracing mice subjected to High-Fat/High-Sucrose (HF/HS) diet, 2) available single cell RNA sequencing datasets from human obese adipose tissue, and 3) cell culture models using primary human adipose-derived endothelial cells. In lineage-tracing mice, obesogenic diet induced EndoMT in a time-dependent manner at 13, 26, and 52 weeks on HF/HS diet in both subcutaneous and visceral adipose tissue. Interestingly, rate of EndoMT was greater in visceral compared to subcutaneous adipose tissue. After 52-weeks on HF/HS diet, EndoMT cells in visceral adipose tissue upregulate inflammatory response and TGF-Beta signaling transcriptional pathways compared to endothelial cells in the same adipose tissue depot. Initiation of EndoMT was also found in single cell RNA sequencing datasets of stromal cells from human obese adipose tissue, indicated by endothelial cells in these datasets containing sub-clusters with reduced Pecam1 and increased Acta2 expression. Finally, the capacity for EndoMT in primary human adipose-derived endothelial cells was validated by bulk RNA sequencing after treatment with proinflammatory cytokines (TNF-Alpha, IFN-Gamma, TGF-Beta1), confirming decreased expression of endothelial cell genes and increased expression of genes associated with EndoMT, inflammatory response, and TGF-Beta signaling. Together, these clinical and experimental findings indicate that chronic obesity can promote EndoMT in adipose endothelial cells.

Abstract GS104: Stress-induced Uhrf1 Epigenetically Modulates Angiogenesis And Cardiac Function In Ventricular Hypertrophy

Heart failure (HF) is accompanied by cardiac hypertrophy (CH) and myocardial tissue remodeling. Epigenetic mechanisms regulate cardiomyocyte (CM) gene expression during CH: the DNA methylation profile and several histone modification marks are profoundly modulated in mouse models of HF and in the human disease. UHRF1 is an epigenetic cofactor connecting DNA methylation to histone methylation. By screening for epigenetic genes upregulated during the early phase of HF, Uhrf1 emerged as a “hit”. We found that UHRF1 is highly expressed in the endothelial cell (EC) compartment under basal conditions in comparison to other myocardial cellular components, and its expression surges in the acute phase of CH induced through transverse aortic constriction (TAC). In vitro studies on HUVECs and H5V cells revealed that silencing of Uhrf1 is associated with defects in cell cycle progression, migration and tube formation. To gain further insight on the role of UHRF1, we generated an EC-specific conditional knockout (cKO) mouse line, Cdh5-CreERT2; Uhrf 1 fl/fl . Compared to control, Uhrf1 -cKO mice subjected to TAC exhibit a worsening of cardiac function associated with increased left ventricular dimension, suggesting a more rapid transition toward HF. Transcriptomic analysis of ECs revealed a robust correlation between UHRF1 depletion and alteration of cell cycle and angiogenesis, in agreement with in vitro results. These results were further corroborated in vivo through EdU injection, matrigel plug and retinal angiogenesis assays, and provided robust evidence for a key role for UHRF1 in the EC response to stress, linking the latter to neo-angiogenesis. We then defined whether the crosstalk between CMs and ECs during stress was altered by the absence of UHRF1. We found that, among other angiocrines, neuregulin-1 (Nrg-1) was strongly downregulated in ECs of Uhrf1 fl/fl mice subjected to TAC. In line with this evidence, ErbB2 and ERK pathways were significantly downregulated in the left ventricle of Uhrf1 fl/fl TAC mice, leading to increased tissue apoptosis.Our study identifies UHRF1 as a critical epigenetic regulator of stress-induced angiogenesis and, more broadly, underlines the relevance of epigenetics in modulating EC gene expression during myocardial stress.

Late phase endothelial cell inflammation is characterized by interferon response genes and driven by JAK/STAT, not NFκB

Chronic vascular inflammation underlies many diseases, including atherosclerosis, autoimmune vasculitides and transplant rejection. The resolution of inflammation is critical for proper healing and restoration of homeostasis, but the timing and signaling mechanisms involved in the return to a non-inflamed state are not well understood. Pro-adhesive gene expression, phenotype and secretome of human endothelial cells was measured in primary human aortic endothelium under chronic TNFα stimulation, and after short-term TNFα priming followed by withdrawal. The effects of NFκB, MAPK and JAK1/2 inhibitors on TNFα-induced gene expression were tested. The majority of inducible TNFα effectors, such as E-selectin, VCAM-1 and most chemokines, required continuous exposure for reinforcement of the altered phenotype, and were NFκB dependent. However, 3 h priming with TNFα induced late phase STAT activation and interferon response genes after 18 h, as well as enhanced ICAM-1, BST2 and CXCR3 ligand expression. Chronic activation was autonomous of continuous TNFα, and could be blocked by the JAK1/2 inhibitor ruxolitinib. The results demonstrate that NFκB is not a significant driver of the later phase of endothelial cell activation by TNFα, but that sustained inflammation is JAK1/2-dependent and characterized by adaptive chemokines.

Combining TMZ and SB225002 induces changes of CXCR2 and VEGFR signalling in primary human endothelial cells invitro.

Standard of care therapy for glioblastoma (GBM) consisting of surgical removal, temozolomide (TMZ) and radiotherapy fails to cure the disease and median survival is limited to 15 months. Therapeutic approaches targeting vascular endothelial growth factor (VEGF)-mediated angiogenesis, one of the major drivers of tumour growth, have not prolonged patient survival as reported in clinical studies. Apart from VEGFR signalling, proangiogenic C-X-C motif chemokine receptor 2 (CXCR2) is of special interest as its ligands C-X-C motif chemokine ligand 2 (CXCL2) and interleukin-8 (IL8) are upregulated and associated with reduced survival in GBM patients. As CXCR2 is also expressed by endothelial cells, the aim of the present study was to elucidate the effect of combination therapy on gene and protein expression of primary human endothelial cells (HUVECs). To mimic the GBM specific CXCL2/IL8 oversupply environment [referred to as stimulation (STIM)], HUVECs were treated with a cocktail of CXCL2/IL8 and/or TMZ and/or CXCR2-antagonist SB225002 (SB). In brief, six treatment conditions were utilized: i) Control, ii) STIM (CXCL2/IL8), iii) TMZ + SB, iv) STIM + TMZ, v) STIM + SB, vi) STIM + TMZ + SB followed by either RNA-isolation and RT-qPCR for BAX, BCL2, vascular endothelial growth receptor (VEGFR)1/2, VEGF, CXCR1/2, CXCL2 and IL8 or immunofluorescence staining for VEGFR2 and CXCR2. SB and TMZ led to morphological changes of HUVECs and downregulated antiapoptotic BCL2 in vitro. In addition, gene expression of the alternative proangiogenic CXCL2/IL8/CXCR2 signalling pathway was significantly altered by the combination therapy, while the VEGF/VEGFR1/2 axis was only mildly affected. Furthermore, VEGFR2 and CXCR2 gene and protein expression regulation differed. VEGFR2 was not altered at the gene expression level, while combination therapy with TMZ and SB led to a 74% upregulation of VEGFR2 at the protein level. By contrast, CXCR2 was upregulated 5-fold by the combination therapy at the gene expression level and downregulated by 72.5% at the protein expression level. The present study provided first insights into the molecular changes of two major proangiogenic pathways in primary endothelial cells during treatment with TMZ and SB. Different gene and protein expression levels of the proangiogenic receptors CXCR2 and VEGFR2 in vitro must be taken into consideration in future studies.

Context dependent role of p53 during the interaction of hepatocellular carcinoma and endothelial cells.

During the progression of hepatocellular carcinoma (HCC), several angiogenic factors are overexpressed in the hepatic microenvironment, which play a critical role in governing the phenotype of the endothelial cells. Mutation in the p53 gene (TP53) is a common event in HCC that may dysregulate the angiogenic signals. However, their functional messages remain largely unexplored at the onset of metastasis. Role of p53 was studied by siRNA mediated silencing of p53 in HepG2 cells (WTp53), collecting and analyzing their conditioned medium, followed by indirect co-culture with endothelial cells (HUVECs). Gene and protein expression in HCC cells and endothelial cells was studied by RT-qPCR and western blotting respectively. β-catenin protein expression and localization were analyzed by immunocytochemistry. We have studied a cell-to-cell interaction model to investigate the crosstalk of endothelial and hepatoma cells by either knocking down p53 or by using p53 null low metastatic HCC cell line. In the absence of p53, the HCC cells influence the migration and vascular network formation of endothelial cells through paracrine signaling of VEGF. Secretory VEGF activated the VEGF receptor-2 along with the survival signaling in endothelial cells. However, the β-catenin signal is upregulated in endothelial cells only during interaction with metastatic set up irrespective of absence and presence of p53, indicating context-dependent participation of p53 during communication between hepatoma cells and endothelial cells. This study highlights that the role of p53 on cellular responses during interaction of hepatocellular carcinoma and endothelial cells is distinct to cell types and context.