Endothelial Cell Phenotype Research Articles

SATB2, coordinated with CUX1, regulates IL-1β-induced senescence-like phenotype in endothelial cells by fine-tuning the atherosclerosis-associated p16INK4a expression.

Genome-wide association studies (GWAS) have validated a strong association of atherosclerosis with the CDKN2A/B locus, a locus harboring three tumor suppressor genes: p14ARF , p15INK4b , and p16INK4a . Post-GWAS functional analysis reveals that CUX is a transcriptional activator of p16INK4a via its specific binding to a functional SNP (fSNP) rs1537371 on the atherosclerosis-associated CDKN2A/B locus, regulating endothelial senescence. In this work, we characterize SATB2, another transcription factor that specifically binds to rs1537371. We demonstrate that even though both CUX1 and SATB2 are the homeodomain transcription factors, unlike CUX1, SATB2 is a transcriptional suppressor of p16INK4a and overexpression of SATB2 competes with CUX1 for its binding to rs1537371, which inhibits p16INK4a and p16INK4a -dependent cellular senescence in human endothelial cells (ECs). Surprisingly, we discovered that SATB2 expression is transcriptionally repressed by CUX1. Therefore, upregulation of CUX1 inhibits SATB2 expression, which enhances the binding of CUX1 to rs1537371 and subsequently fine-tunes p16INK4a expression. Remarkably, we also demonstrate that IL-1β, a senescence-associated secretory phenotype (SASP) gene itself and a biomarker for atherosclerosis, induces cellular senescence also by upregulating CUX1 and/or downregulating SATB2 in human ECs. A model is proposed to reconcile our findings showing how both primary and secondary senescence are activated via the atherosclerosis-associated p16INK4a expression.

KIAA1429 promotes infantile hemangioma regression by facilitating the stemness of hemangioma endothelial cells.

Infantile hemangiomas are common vascular tumors with a specific natural history. The proliferation and regression mechanism of infantile hemangiomas may be related to the multilineage differentiation ability of hemangioma stem cells, but the specific mechanism is not well elucidated. KIAA1429 is an N6 -methyladenosine methylation-related protein that can also exert its role in a methylation-independent manner. This study aims to explore the function of KIAA1429 in infantile hemangiomas. qRT-PCR, western blotting, and immunostaining were performed to verify the expression of KIAA1429. The endothelial and fibroblast-like phenotypes of hemangioma endothelial cells were detected after KIAA1429 knockdown and overexpression. The stemness properties of hemangioma endothelial cells and the underlying mechanism of KIAA1429 in hemangiomas were also investigated. Nude mouse models of infantile hemangiomas were conducted to ascertain the effects of KIAA1429 in vivo. The results showed that KIAA1429 was highly expressed in infantile hemangiomas, particularly in involuting hemangiomas. In vitro experiments confirmed that KIAA1429 inhibited the endothelial phenotype, enhanced the differentiation ability, and promoted the fibroblast-like phenotype of hemangioma endothelial cells by inducing endothelial cell transition to facultative stem cells. However, the effect of KIAA1429 on the potential target was shown to be independent of N6 -methyladenosine methylation modification. Mouse models further revealed that KIAA1429 could inhibit the proliferation and promote the regression of hemangiomas. In conclusion, this study found that KIAA1429 played an important role in the regression of infantile hemangiomas by enhancing the stemness of hemangioma endothelial cells and could be a potential treatment target for infantile hemangiomas.

Effects of shear stress on the properties of colonyforming endothelial cells in comparison with coronary artery endothelial cells

Highlights. It is assumed that pre-colonization by endothelial cells of the inner surface of tissue-engineered vessels of small diameter can serve as an effective way to prevent thrombosis. The question of choosing the optimal source of endothelial cells for use in tissue engineering remains debatable. The paper considers the features of the culture of colony-forming endothelial cells obtained from the peripheral blood of patients with coronary heart disease, in comparison with mature endothelial cells from the coronary artery.Aim. To study the effect of laminar flow on the morphological and functional characteristics of mature endothelial cells and peripheral blood-derived endothelial colony-forming cells.Methods. Coronary artery endothelial cells were purchased from the Cell Applications, Inc. Colony-forming endothelial cells were obtained from the peripheral blood of patients with coronary artery disease who underwent percutaneous coronary intervention. The cells were isolated using a Ficoll gradient and cultured in EGM-2MV culture medium containing 5% fetal bovine serum. The cells of the experimental group were cultured in µ-Luer plates in a perfusion system with a shear stress of 3 dyn/cm2 . The cultivation time was 2 days. The cells of the control group were cultured under static conditions. At the end of the cultivation we performed immunofluorescent staining for CD31, KDR/CD309, CD144, vWF, type IV collagen, F-actin.Results. Colony-forming endothelial cells and coronary artery endothelial cells retained high density and viability both under static and laminar flow conditions. Shear stress stimulated a change in the phenotype of colony-forming endothelial cells towards a mature endothelial cells, in particular, a significant increased the expression of KDR/CD309 and CD31. The action of laminar flow reduced the synthesis of von Willebrand factor, stimulated the synthesis of type IV collagen. Shear stress promoted the development of structural rearrangements in cells in response to transduction, which manifested in a change in F-actin fibrils orientation on the flow direction.Conclusion. Colony-forming endothelial cells showed a characteristic response to the action of shear stress, consisting in a change in morphology, phenotype, and secretory activity of cells, comparable to that of coronary artery endothelial cells.

Colchicine Inhibited Senescence Associated Secretory Phenotype in Endothelial Cells Exposed to Smoke Condensate

Colchicine Inhibited Senescence Associated Secretory Phenotype in Endothelial Cells Exposed to Smoke Condensate

Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thrombotic events and/or pregnancy morbidity in the presence of antiphospholipid antibodies detected either as anti-cardiolipin, anti-β2 Glycoprotein I (anti-β2GPI) or Lupus anticoagulant (LA). Endothelial deregulation characterizes the syndrome. To address gene expression changes accompanying the development of autoimmune phenotype in endothelial cells in the context of APS, we performed transcriptomics analysis in Human Umbilical Vein Endothelial Cells (HUVECs) stimulated with IgG from APS patients and β2GPI, followed by intersection of RNA-seq data with published microarray and ChIP-seq results (Chromatin Immunoprecipitation). Our strategy revealed that during HUVEC activation diverse signaling pathways such as TNF-α, TGF-β, MAPK38, and Hippo are triggered as indicated by Gene Ontology (GO) classification and pathway analysis. Finally, cell biology approaches performed side-by-side in naïve and stimulated cultured HUVECs, as well as, in placenta specimens derived from Healthy donors (HDs) and APS-patients verified the evolution of an APS-characteristic gene expression program in endothelial cells during the initial stages of the disease's development.

Synthetic hyaluronic acid coating preserves the phenotypes of lymphatic endothelial cells.

Lymphatic endothelial cells (LECs) play a critical role in the formation and maintenance of the lymphatic vasculature, which is essential for the immune system, fluid balance, and tissue repair. However, LECs are often difficult to study in vivo and in vitro models that accurately mimic their behaviors and phenotypes are limited. In particular, LECs have been shown to lose their lymphatic markers over time while being cultured in vitro, which reflect their plasticity and heterogeneity in vivo. Since LECs uniquely express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), we hypothesized that surface coating with hyaluronic acid (HA) can preserve LEC phenotypes and functionalities. Dopamine conjugated hyaluronic acid (HA-DP) was synthesized with 42% degree of substitution to enable surface modification and conjugation onto standard tissue culture plates. Compared to fibronectin coating and tissue culture plate controls, surface coating with HA-DP was able to preserve lymphatic markers, such as prospero homeobox protein 1 (Prox1), podoplanin (PDPN), and LYVE-1 over several passages in vitro. LECs cultured on HA-DP expressed lower levels of focal adhesion kinase (FAK) and YAP/TAZ, which may be responsible for the maintenance of the lymphatic characteristics. Collectively, the HA-DP coating may provide a novel method for culturing human LECs in vitro toward more representative studies in basic lymphatic biology and lymphatic regeneration.

Study of the Phenotype of Endothelial Cells in the Vessels of Uveal Melanoma

In tumor angiogenesis, two main mechanisms are officially recognized. They are: the formation of vascular structures by associations of endothelial cell precursors (PEK) or angioblasts from circulating peripheral blood (1) and vascular co-optation (2) using preexistent vessels. Determining the dominant phenotype of endothelial cells (EC) involved in the neoangiogenesis of uveal melanoma (UM) is very important due to the different sensitivity of different EC to antiangiogenic therapy and their biological differences. Circulating hemangioblasts of bone marrow origin (synonym: endothelial cell precursors, PEC) involved in the formation of vessels in the tumor can also originate from hematopoietic stem cells and, in conditions of an excess of proangiogenic growth factors, undergo “reverse differentiation” or “dedifferentiation”. This situation is extremely dangerous due to changes in the biological properties of EC: the rate of proliferation increases and high resistance to any type of interventional antiangiogenic therapy, including targeted antiangiogenic drugs, develops. In combination with high resistance to radiation therapy and chemotherapy of tumor cells, such a tumor becomes uncontrollable and quickly leads to death... The purpose of this study: to identify the phenotype of endothelial cells involved in UM angiogenesis by immunohistochemical analysis. The authors conducted immunohistochemical studies on paraffin blocks (n = 98) of enucleated eyes with UM. One of the recommended PEC markers was used: a monoclonal antibody to the VEGFR2-KDR/Flk-1 receptor. It was found that the expression of the VEGFR- 2/Flk-KDR-1 receptor took place only in 22.1 % of the UM. The receptor was expressed exclusively by endothelial cells (EC) of tumor vessels and was not expressed by UM cells. There was no basic expression of the receptor (at stage T1). Peak expression was observed at stage T2. At the next stages of the oncological process, there was a decline in expression. The mean IHC gradation of expression intensity also decreased from stage T2 to stage T4. Flk-positive and negative endothelial cells differed in size, shape, nature of communication with the vascular wall and the presence or absence of a nucleus in the cell. The authors concluded that both mechanisms of angiogenesis take place in UM. A small percentage (22.1 %) Flk+endothelial cells indicate that the dominant mechanism of angiogenesis in UM is co-optation of vessels based on preexistent, and the dominant population of EC is Flk-negative EC.

Endothelial dysfunction in Marfan syndrome mice is restored by resveratrol

Patients with Marfan syndrome (MFS) develop thoracic aortic aneurysms as the aorta presents excessive elastin breaks, fibrosis, and vascular smooth muscle cell (vSMC) death due to mutations in the FBN1 gene. Despite elaborate vSMC to aortic endothelial cell (EC) signaling, the contribution of ECs to the development of aortic pathology remains largely unresolved. The aim of this study is to investigate the EC properties in Fbn1C1041G/+ MFS mice. Using en face immunofluorescence confocal microscopy, we showed that EC alignment with blood flow was reduced, EC roundness was increased, individual EC surface area was larger, and EC junctional linearity was decreased in aortae of Fbn1C1041G/+ MFS mice. This modified EC phenotype was most prominent in the ascending aorta and occurred before aortic dilatation. To reverse EC morphology, we performed treatment with resveratrol. This restored EC blood flow alignment, junctional linearity, phospho-eNOS expression, and improved the structural integrity of the internal elastic lamina of Fbn1C1041G/+ mice. In conclusion, these experiments identify the involvement of ECs and underlying internal elastic lamina in MFS aortic pathology, which could act as potential target for future MFS pharmacotherapies.

Transcriptomic profiling reveals sex-specific molecular signatures of adipose endothelial cells under obesogenic conditions

Female mice display greater adipose angiogenesis and maintain healthier adipose tissue than do males upon high-fat diet feeding. Through transcriptome analysis of endothelial cells (EC) from the white adipose tissue of male and female mice high-fat-fed for 7weeks, we found that adipose EC exhibited pronouncedly sex-distinct transcriptomes. Genes upregulated in female adipose EC were associated with proliferation, oxidative phosphorylation, and chromatin remodeling contrasting the dominant enrichment for genes related to inflammation and a senescence-associated secretory of male EC. Similar sex-biased phenotypes of adipose EC were detectable in a dataset of aged EC. The highly proliferative phenotype of female EC was observed also in culture conditions. In turn, male EC displayed greater inflammatory potential than female EC in culture, based on basal and tumor necrosis factor alpha-stimulated patterns of gene expression. Our study provides insights into molecular programs that distinguish male and female EC responses to pathophysiological conditions.

Uric Acid Induces a Proatherothrombotic Phenotype in Human Endothelial Cells by Imbalancing the Tissue Factor/Tissue Factor Pathway Inhibitor Pathway.

Several evidence show that elevated plasma levels of uric acid (UA) are associated with the increased risk of developing atherothrombotic cardiovascular events. Hyperuricemia is a risk factor for endothelial dysfunction (ED). ED is involved in the pathophysiology of atherothrombosis since dysfunctional cells lose their physiological, antithrombotic properties. We have investigated whether UA might promote ED by modulating the tissue factor (TF)/TF pathway inhibitor (TFPI) balance by finally changing the antithrombotic characteristics of endothelial cells. Human umbilical vein endothelial cells were incubated with increasing doses of UA (up to 9 mg/dL). TF gene and protein expressions were evaluated by real-time polymerase chain reaction (PCR) and Western blot. Surface expression and procoagulant activity were assessed by FACS (fluorescence activated cell sorting) analysis and coagulation assay. The mRNA and protein levels of TFPI were measured by real-time PCR and Western blot. The roles of inflammasome and nuclear factor-κB (NF-κB) as possible mechanism(s) of action of the UA on TF/TFPI balance were also investigated. UA significantly increased TF gene and protein levels, surface expression, and procoagulant activity. In parallel, TFPI levels were significantly reduced. The NF-κB pathways appeared to be involved in modulating these phenomena. Additionally, inflammasome might also play a role. The present in vitro study shows that one of the mechanisms by which high levels of UA contribute to ED might be the imbalance between TF/TFPI levels in endothelial cells, shifting them to a nonphysiological, prothrombotic phenotype. These UA effects might hypothetically explain, at least in part, the relationship observed between elevated plasma levels of UA and cardiovascular events.

Protein Fractions of Natural Killer Cell Lysates Affect the Phenotype, Proliferation and Migration of Endothelial Cells in vitro

Protein Fractions of Natural Killer Cell Lysates Affect the Phenotype, Proliferation and Migration of Endothelial Cells in vitro

Extracellular Vesicles from Ocular Melanoma Have Pro-Fibrotic and Pro-Angiogenic Properties on the Tumor Microenvironment

Uveal melanoma (UM) is the most common primary intraocular tumor and often spreads to the liver. Intercellular communication though extracellular vesicles (EVs) plays an important role in several oncogenic processes, including metastasis, therapeutic resistance, and immune escape. This study examines how EVs released by UM cells modify stellate and endothelial cells in the tumor microenvironment. The surface markers, and the concentration and size of EVs derived from UM cells or choroidal melanocytes were characterized by high-resolution flow cytometry, electron microscopy, and Western blotting. The selective biodistribution of EVs was studied in mice by fluorescence imaging. The activation/contractility of stellate cells and the tubular organization of endothelial cells after exposure to melanomic EVs were determined by traction force microscopy, collagen gel contraction, or endothelial tube formation assays. We showed that large EVs from UM cells and healthy melanocytes are heterogenous in size, as well as their expression of phosphatidylserine, tetraspanins, and Tsg101. Melanomic EVs mainly accumulated in the liver and lungs of mice. Hepatic stellate cells with internalized melanomic EVs had increased contractility, whereas EV-treated endothelial cells developed more capillary-like networks. Our study demonstrates that the transfer of EVs from UM cells leads to a pro-fibrotic and pro-angiogenic phenotype in hepatic stellate and endothelial cells.

Organotypic stromal cells impact endothelial cell transcriptome in 3D microvessel networks

Endothelial cells line all major blood vessels and serve as integral regulators of many functions including vessel diameter, cellular trafficking, and transport of soluble mediators. Despite similar functions, the phenotype of endothelial cells is highly organ-specific, yet our understanding of the mechanisms leading to organ-level differentiation is incomplete. We generated 3D microvessel networks by combining a common naïve endothelial cell with six different stromal cells derived from the lung, skin, heart, bone marrow, pancreas, and pancreatic cancer. Single cell RNA-Seq analysis of the microvessel networks reveals five distinct endothelial cell populations, for which the relative proportion depends on the stromal cell population. Morphologic features of the organotypic vessel networks inversely correlate with a cluster of endothelial cells associated with protein synthesis. The organotypic stromal cells were each characterized by a unique subpopulation of cells dedicated to extracellular matrix organization and assembly. Finally, compared to cells in 2D monolayer, the endothelial cell transcriptome from the 3D in vitro heart, skin, lung, and pancreas microvessel networks are more similar to the in vivo endothelial cells from the respective organs. We conclude that stromal cells contribute to endothelial cell and microvessel network organ tropism, and create an endothelial cell phenotype that more closely resembles that present in vivo.

ApoA-I Nanotherapy Rescues Postischemic Vascular Maladaptation by Modulating Endothelial Cell and Macrophage Phenotypes in Type 2 Diabetic Mice.

Diabetes is a major risk factor for peripheral arterial disease. Clinical and preclinical studies suggest an impaired collateral remodeling and angiogenesis in response to atherosclerotic arterial occlusion in diabetic conditions, although the underlying mechanisms are poorly understood. To clarify the cellular and molecular mechanisms underlying impaired postischemic adaptive vascular responses and to evaluate rHDL (reconstituted HDL)-ApoA-I nanotherapy to rescue the defect in type 2 diabetic mouse model of hindlimb ischemia. Hindlimb ischemia was induced by unilateral femoral artery ligation. Collateral and capillary parameters together with blood flow recovery were analyzed from normoxic adductor and ischemic gastrocnemius muscles, respectively, at day 3 and 7 post-ligation. In response to femoral artery ligation, collateral lumen area was significantly reduced in normoxic adductor muscles. Distally, ischemic gastrocnemius muscles displayed impaired perfusion recovery and angiogenesis paralleled with persistent inflammation. Muscle-specific mRNA sequencing revealed differential expression of genes critical for smooth muscle proliferation and sprouting angiogenesis in normoxic adductor and ischemic gastrocnemius, respectively, at day 7 post-ligation. Genes typical for macrophage (Mϕ) subsets were differentially expressed across both muscle types. Cell-specific gene expression, flow cytometry, and immunohistochemistry revealed persistent IFN-I response gene upregulation in arterial endothelial cells, ECs and Mϕs from T2DM mice associated with impaired collateral remodeling, angiogenesis and perfusion recovery. Furthermore, rHDL nanotherapy rescued impaired collateral remodeling and angiogenesis through dampening EC and Mϕ inflammation in T2DM mice. Our results suggest that an impaired collateral remodeling and sprouting angiogenesis in T2DM mice is associated with persistent IFN-I response in ECs and Mϕs. Dampening persistent inflammation and skewing ECs and Mϕ phenotype toward less inflammatory ones using rHDL nanotherapy may serve as a potential therapeutic target for T2DM peripheral arterial disease.

Neutrophil Extracellular Traps (NETs) Thrombogenicity and Endothelial Cell Toxicity Are Ameliorated By Platelet Factor 4 (PF4): Implications to the Treatment of Sepsis

Sepsis, the dysregulated response to infection, is a leading cause of mortality worldwide for which there are no targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens. However, when NETs are degraded by circulating nucleases, they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger thrombosis, induce complement activation and cause oxidative vascular damage. We propose a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4 (CXCL4), which enhances NET resistance to DNase degradation, promoting NDP sequestration and bacterial capture, improving sepsis outcomes. As NETs are traditionally considered to be prothrombotic, we investigated whether PF4-stabilization alters the thrombogenicity of NETs. We examined the effect of PF4 on both NET- and DNA-mediated thrombus formation and endothelial cell injury using NET and DNA fragments of different lengths. Human neutrophils were stimulated to release NETs and treated with mild DNase I (4U/mL) to liberate high-molecular-weight (hmw) NETs (>50kb), and then digested with restriction enzymes to generate low-molecular-weight (lmw) fragments of ~4kb and ~250bp. We also studied single-stranded (ss) DNA, generated by heating double stranded (ds) DNA fragments to 100°C. We assessed the effects of PF4 on NETs/dsDNA/ssDNA-induced thrombin and fibrin generation in normal and in factor (F) XII- and FXI-depleted plasma. PF4 significantly reduced plasma thrombin and fibrin generation induced by digested NET and DNA fragments and by ssDNA without impairing thrombolysis. PF4 did not exert any effects on DNA-induced fibrin generation in FXII- or FXI-depleted plasma, while supplementation with purified FXII or FXI restored the anticoagulant effects of PF4, suggesting that PF4 binding reduces the ability of cfDNA and NETs to activate the contact pathway of coagulation. We next studied how PF4 alters the ability of DNA to induce a prothrombotic phenotype in human umbilical vein endothelial cells (HUVECs) incubated with dsDNA or ssDNA fragments by measuring von Willebrand factor (VWF) release with immunofluorescence and by quantifying tissue factor (TF) expression with a chromogenic substrate assay. To study the mechanism of endothelial activation, HUVECs were treated with inhibitors of toll-like receptor (TLR) 9, a pattern recognition receptor that binds to dsDNA. Unlike intact NETs or hmwDNA, both digested dsDNA and ssDNA upregulated VWF release and TF expression by endothelial cells via a TLR9-dependent mechanism, which was prevented by the addition of PF4. To study these observations in vivo, hmwDNA was administered intravenously to wildtype (WT) and Cxcl4-/- mice and plasma thrombin-anti-thrombin (TAT) levels were measured 4h post infusion. Cxcl4-/- mice exhibited higher TAT levels as compared to WT mice. We also studied two distinct sepsis-like states: (1) WT mice exposed to lipopolysaccharide (LPS, IP; 35mg/kg) ± human (h)PF4 (IV; 40mg/kg), where plasma cfDNA and TAT levels were measured at 6h, and (2) the cecal slurry model of polymicrobial infection in endothelial-specific TLR9-/- mice, where plasma TAT levels were measured at 4h. In the LPS endotoxemia model (1), TAT and cfDNA levels were elevated at 6h post LPS challenge, which were prevented with the administration of hPF4. In the cecal slurry model (2), endothelial TLR9-/- mice exhibited lower TAT levels at 4h post challenge as compared to WT controls. In summary, while intact NETs help to contain infection, NET degradation liberates lmwDNA and ssDNA that activate the contact pathway and induce a prothrombotic phenotype in endothelial cells via activation of TLR9. PF4-stabilization of NETs decreases the risk of NET-mediated toxicity by (1) inhibiting DNase cleavage of intact NETs and (2) neutralizing released lmwDNA and ssDNA. NET stabilization by PF4 tips the balance, limiting NET- and NDP-induced thrombosis and vascular toxicity, while enhancing NET's ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis. Strategies that augment the beneficial effects of PF4 merit further investigation.

Spatial Transcriptomics of Tissue-Based Immune Response to Acute Graft-Versus-Host Disease of the Lower Gastrointestinal Tract

Background: Corticosteroid resistant acute graft-versus-host disease (GVHD) of the lower gastrointestinal (GI) tract is a leading cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. We hypothesized that distinct gene expression profiles exist within affected GI tissue at GVHD diagnosis and may be associated with treatment outcomes. Methods: Unstained tissue sections from 33 patients with biopsy-confirmed acute lower GI GVHD were used for spatial transcriptomics using the NanoString GeoMxTM Digital Spatial Profiler (DSP) platform. Fluorescent morphology markers (PanCK, CD45 and CD31) were used to visualize the epithelial, immune and endothelial compartments and guide capture of RNA expression profiles using the GeoMxTM Human Whole Transcriptome Atlas (18,676 genes). A total of 739 areas of interest (AOIs) were sampled across the 33 patients with a total of 57,960 immune, 37,610 epithelial, and 2298 vascular endothelial cells analyzed (average of 1756.4 immune, 1139.7 epithelial and 69.6 endothelial cells per case). The gene specific oligos were quantified using Illumina's i5 x i7 dual-indexing system and sequenced on the Illumina NextSeq 500 using a 2 x 75 MidOutput v2.5 flow cell. The FASTQ files were processed to generate target probe-specific count files and saved in DCC format. Gene expression files were normalized per NanoString protocol. All unsupervised clustering and differential expression were done in Bioconductor in the R statistical computing space. Results: The gene expression data for each AOI of immune, epithelial, and endothelial cells were normalized and the top 500 most variable genes were used for unsupervised hierarchical clustering to obtain an unbiased classification of different subtypes. There were two clear major AOI clusters for each of the cellular compartments with immune clustering shown in Figure 1A. The left immune cluster (IC1) and the right immune cluster (IC2) were then evaluated for association with clinical GVHD outcomes. IC1 had an over-representation of patients with steroid sensitive GVHD (Fisher exact p value = <0.0001) and AOIs with low grade pathology (Fisher exact p value = 0.031). To obtain gene level differences between IC1 and IC2, differential expression was performed using a Wilcoxon Rank Sum test and volcano plots were generated to illustrate the most differentially expressed genes across the visual AOI-based clusters (Figure 1B). Genes with an adjusted p-value of 0.05 for association with the particular arm of the volcano plot and at least a 2-fold change over the other cluster were considered significant. Gene set enrichment analysis (GSEA) using curated datasets from MSigDB was performed and identified that IC1 genes were enriched for IFNγ response (q value = 2.99e-5), TNFα signaling (q value = 2.99e-5) and inflammatory response (q value = 0.0063). These IC1 differentially expressed genes included CD74 (835 vs. 361.6 mean normalized gene transcripts), LYZ, β2-microglobulin, C1QB and CD68. IC2, on the other hand, had a prominent exhausted/immunosuppressive signature significantly enriched for Th17 cell genes (q value = 0.027) by GSEA. Notable genes that were statistically significant were FCF1 (152.3 vs. 74 mean normalized transcripts) and USP17L11 (78.3 vs. 36.4 mean normalized transcripts). Extension of this analysis to the epithelial (EC) and vascular endothelial (VC) cell compartments also identified distinct clustering with both having an inflamed (EC1 and VC1) and a basal-like state (EC2 and VC2). Interestingly, the inflamed epithelial cells (EC2) associated with immunosuppressive IC2 immune cells (Fisher exact p-value = 0.006), but inflamed vascular endothelium (VC1) associated with the pro-inflammatory IC1 immune cells (Fisher exact p-value = 0.0005). Conclusion: Spatial transcriptomics provides high resolution gene expression profiles in distinct cellular compartments in human acute GI GVHD samples and provides a better understanding of the relationship of the immune response with epithelial and vascular endothelial cell phenotypes. Initial analysis demonstrates that an inflammatory immune signature is associated with steroid responsive acute GI GVHD and an exhausted phenotype with steroid resistance. Future studies are needed to confirm the predictive value of specific gene expression profiles as biomarkers of corticosteroid response at diagnostic biopsy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 15273: Impact of Lipid Lowering Therapy on Progenitor Endothelial Cell Phenotype and Function in Coronary Artery Disease

Introduction: Early revascularization of infarcted zone is crucial to improve patient′s outcomes. Despite their primary actions, major lipid-lowering drugs may also reduce cardiovascular events by pleiotropic mechanisms. Hypothesis: Conventional (statin) and novel lipid-lowering therapies [(PSCK9-inhibitor (PCSK9i) and eicosatetraenoic acid (EPA)] can improve endothelial progenitor cells (EPC) phenotype and favor neovascularization. Methods: EPC were isolated from peripheral blood of patients with acute coronary syndrome (n=62) who were statin-naïve (n=34) or chronic statin treatment (n=28) at time of inclusion. A set of statin-naïve EPC and mature endothelial cells (HUVEC) were treated in vitro with statin (20nM), PCSKi (1μM) and EPA (3μM). EPC phenotype was determined as EPC colony number and time of appearance, proliferative rate, and ability to form capillary-like structures. Serum PCSK9 and markers of angiogenesis and endothelial function were determined by multiplexed protein assay. Results: Frequency of EPC appearance was higher in samples of statin group (78%) compared to naïve group (66%) (Fig A). They also appeared much earlier (Fig B), in greater quantity (4.7 vs 2.1 colony, p = 0.0009) and had higher proliferative rate than EPCs of naïve group. Besides they had increased potential of tube formation and shaped more branched vessel-like structures (Fig C). No differences in serum levels of biomarkers were found. In vitro treatment of EPC with statin, PCSK9i and EPA did not modify colonies behavior (Fig D). PCSK9 levels showed negative correlation with EPC proliferation (r -0.4150, p=0.004), and PCSK9i treatment increased angiogenic potential of HUVEC (Fig E). Conclusions: Statin and PCSK9i have pleiotropic effects on EPC, boosting their potential to form endothelial cells colonies and to induce angiogenesis. These effects could contribute to the progression and outcomes of coronary artery disease. Funding: ISCIII - FEDER-ERDF (PI19/00264)

Abstract 15005: Tlr4/py14-Caveolin-1-Mediated Endothelial C-iap2 Expression During Schistosomiasis-Associated Pulmonary Arterial Hypertension

Introduction: During schistosomiasis-associated pulmonary arterial hypertension (Sch-PAH), hyperproliferation of abnormal endothelial cells (ECs) contributes to development of severe vascular lesions within the lungs; a life-threatening phenomenon that remains with no target therapy. Hypothesis: Here, we found that antigenic molecules from the major etiological agent of Sch-PAH, i.e., S. mansoni , amplify the secretion of the antiapoptotic protein c-IAP2 via TLR4-pY14Cav-1 signaling pathway, which we hypothesized to contribute to generation of an EC memory of survival and severity of PAH. Methods/Results: Cav-1 deficiency via shedding of extracellular vesicles (EVs) leads to survival of an apoptosis-resistant EC phenotype during idiopathic PAH. Similarly, S. mansoni egg antigens also reduced lung EC-Cav-1 expression, suggesting that Cav-1 also plays a role during pathogen-associated PAH. Cav-1 depletion via EVs precedes its phosphorylation at residue Tyr14 (pY14Cav-1), which in turn, contributes to EV loading. Among apoptotic regulators, IAPs have been found as a cargo of EVs in different conditions, including in TLR4-activated cells. Accordingly, our data indicates that S. mansoni egg antigen p40 also activated TLR4/NFkB-mediated signaling, inducing c-IAP2 expression and a time-dependent phosphorylation of Cav-1 in human lung microvascular EC (HMVEC-L: 228.2 +/- 38.28% of control; p&lt;0.05; n=4). Moreover, although Sm-p40 alone was unable to induce shedding of EVs, combination with NFkB-associated proinflammatory mediators (TNF-α, IL-6, and ATP), significantly increased EV shedding by HMVEC-L, suggesting that S. mansoni may epigenetically prime ECs, but co-factors are required to induce EV shedding. Finally, Sch-PAH model using EC- Cav1 -/- mice ( End-scl.Cre ERT2 ;Rosa mt/mg ;Cav1 fl/fl ) revealed that Cav-1 absence led to accumulation of eggs within the lungs and loss of eGFP+ cells, indicating that EC-Cav-1 phosphorylation may be required to development of Sch-PAH. Conclusions: Our data suggest that S. mansoni -induced pY14Cav-1 is associated to expression of the anti-apoptotic c-IAP2 protein, which may be critical for prolonged survival of a pathogenic EC phenotype during the development of Sch-PAH.

Abstract 10096: Genetic Control of Lysosomal Dysfunction Reprograms Sterol Metabolism in Pulmonary Arterial Hypertension

Background : Vascular inflammation critically regulates endothelial pathophenotypes, yet causative mechanisms remain incompletely defined, particularly in pulmonary arterial hypertension (PAH). Immune dysregulation and metabolic reprogramming are recognized tenets of PAH pathogenesis, but a unifying theory connecting the two has not been established. Methods &amp; Results: Guided by an unbiased, metabolome-wide association study from the multicenter PAH Biobank cohort (N=2,666) that identified a proinflammatory sterol and bile acid metabolite plasma signature significantly associated with PAH mortality (adjusted P values&lt;1.1x10 -6 ), we found in pulmonary arterial endothelial cells (ECs) that induction of the nuclear receptor coactivator 7 (NCOA7) tempered the generation of proinflammatory sterols by bolstering lysosomal acidification and constraining EC immunoactivation. Conversely, reduced NCOA7 promoted lysosomal dysfunction, resulting in sterol- and bile acid-driven inflammation and EC phenotypes consistent with PAH. Notably, the common variant intronic SNP rs11154337 in NCOA7 was found to control NCOA7 levels, lysosome activity, sterol and bile acid production, and EC immunoactivation in isogenic, CRISPR-Cas9, SNP-edited, iPSC-derived ECs, indicating a potentially widespread genetic predisposition to NCOA7 deficiency. Correspondingly, SNP rs11154337 was associated with PAH severity as noted by six-minute walk distance ( P =0.0130, β=58.15, 95% CI [14.45-119.36]) and mortality ( P =0.0250; hazard ratio=0.4368, 95% CI [0.21-0.90]) in an independent, single-center PAH cohort (N=93). In vivo , mice deficient for Ncoa7 or treated with exogenous 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA)—chosen as a representative metabolite from the sterol signature associated with mortality in the PAH Biobank—demonstrated worsened hemodynamic and histological indices of PAH. Conclusions: Our work establishes a genetic and metabolic paradigm that links lysosomal biology and sterol and bile acid processes with EC inflammation. This paradigm carries broad implications not only on molecular diagnostic and therapeutic development in PAH but also in other vascular disorders dependent upon acquired and innate immune regulation.

Tripeptide Leu-Ser-Trp Regulates the Vascular Endothelial Cells Phenotype Switching by Mediating the Vascular Smooth Muscle Cells-Derived Small Extracellular Vesicles Packaging of miR-145.

Tripeptide LSW, initially identified as a potent ACE inhibitory peptide from soybean protein, was recently reported to exert a protective effect against angiotensin II-induced endothelial dysfunction via extracellular vesicles (EVs). However, the molecular mechanisms, especially in lipid accumulation-induced atherosclerosis, still remain unclear. The study aimed to investigate whether the protective effects of LSW against endothelial dysfunction on vascular endothelial cells (VECs) was via vascular smooth muscle cells (VSMCs)-derived miRNA-145 packaged in EVs. The miRNA-145 was concentrated in EVs from LSW-treated VSMCs (LEVs), internalized into the HVUECs, and targeted the programmed cell death protein 4 (PDCD4) expression of HUVECs. Oxidized low-density lipoprotein (oxLDL) was applied to induce endothelial dysfunction in HUVECs; oxLDL-induced endothelial dysfunction in HUVECs was attenuated by PDCD4 knockout or LEVs incubation. The results of this study suggested a novel function of LSW as a regulator on the functional EVs from vascular cells in the oxLDL-induced atherosclerotic model.