The endothelium: gatekeeper to lung ischemia-reperfusion injury.

The success of lung transplantation is limited by the high rate of primary graft dysfunction due to ischemia-reperfusion injury (IRI). Lung IRI is characterized by a robust inflammatory response, lung dysfunction, endothelial barrier disruption, oxidative stress, vascular permeability, edema, and neutrophil infiltration. These events are dependent on the health of the endothelium, which is a primary target of IRI that results in pulmonary endothelial barrier dysfunction. Over the past 10 years, research has focused more on the endothelium, which is beginning to unravel the multi-factorial pathogenesis and immunologic mechanisms underlying IRI. Many important proteins, receptors, and signaling pathways that are involved in the pathogenesis of endothelial dysfunction after IR are starting to be identified and targeted as prospective therapies for lung IRI. In this review, we highlight the more significant mediators of IRI-induced endothelial dysfunction discovered over the past decade including the extracellular glycocalyx, endothelial ion channels, purinergic receptors, kinases, and integrins. While there are no definitive clinical therapies currently available to prevent lung IRI, we will discuss potential clinical strategies for targeting the endothelium for the treatment or prevention of IRI. The accruing evidence on the essential role the endothelium plays in lung IRI suggests that promising endothelial-directed treatments may be approaching the clinic soon. The application of therapies targeting the pulmonary endothelium may help to halt this rapid and potentially fatal injury.

The Proteome of Extracellular Vesicles Released from Pulmonary Microvascular Endothelium Reveals Impact of Oxygen Conditions on Biotrauma.

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia.

Placenta accreta spectrum disorder at single-cell resolution: a loss of boundary limits in the decidua and endothelium.

BACKGROUND: Placenta accreta spectrum disorders are associated with severe maternal morbidity and mortality. Placenta accreta spectrum disorders involve excessive adherence of the placenta preventing separation at birth. Traditionally, this condition has been attributed to excessive trophoblast invasion; however, an alternative view is a fundamental defect in decidual biology. OBJECTIVE: This study aimed to gain insights into the understanding of placenta accreta spectrum disorder by using single-cell and spatially resolved transcriptomics to characterize cellular heterogeneity at the maternal-fetal interface in placenta accreta spectrum disorders. STUDY DESIGN: To assess cellular heterogeneity and the function of cell types, single-cell RNA sequencing and spatially resolved transcriptomics were used. A total of 12 placentas were included, 6 placentas with placenta accreta spectrum disorder and 6 controls. For each placenta with placenta accreta spectrum disorder, multiple biopsies were taken at the following sites: placenta accreta spectrum adherent and nonadherent sites in the same placenta. Of note, 2 platforms were used to generate libraries: the 10× Chromium and NanoString GeoMX Digital Spatial Profiler for single-cell and spatially resolved transcriptomes, respectively. Differential gene expression analysis was performed using a suite of bioinformatic tools (Seurat and GeoMxTools R packages). Correction for multiple testing was performed using Clipper. In situ hybridization was performed with RNAscope, and immunohistochemistry was used to assess protein expression. RESULTS: In creating a placenta accreta cell atlas, there were dramatic difference in the transcriptional profile by site of biopsy between placenta accreta spectrum and controls. Most of the differences were noted at the site of adherence; however, differences existed within the placenta between the adherent and nonadherent site of the same placenta in placenta accreta. Among all cell types, the endothelial-stromal populations exhibited the greatest difference in gene expression, driven by changes in collagen genes, namely collagen type III alpha 1 chain (COL3A1), growth factors, epidermal growth factor-like protein 6 (EGFL6), and hepatocyte growth factor (HGF), and angiogenesis-related genes, namely delta-like noncanonical Notch ligand 1 (DLK1) and platelet endothelial cell adhesion molecule-1 (PECAM1). Intraplacental tropism (adherent versus non-adherent sites in the same placenta) was driven by differences in endothelial-stromal cells with notable differences in bone morphogenic protein 5 (BMP5) and osteopontin (SPP1) in the adherent vs nonadherent site of placenta accreta spectrum. CONCLUSION: Placenta accreta spectrum disorders were characterized at single-cell resolution to gain insight into the pathophysiology of the disease. An atlas of the placenta at single cell resolution in accreta allows for understanding in the biology of the intimate maternal and fetal interaction. The contributions of stromal and endothelial cells were demonstrated through alterations in the extracellular matrix, growth factors, and angiogenesis. Transcriptional and protein changes in the stroma of placenta accreta spectrum shift the etiologic explanation away from "invasive trophoblast" to "loss of boundary limits" in the decidua. Gene targets identified in this study may be used to refine diagnostic assays in early pregnancy, track disease progression over time, and inform therapeutic discoveries.

Disturbed endothelial cell signaling in tumor progression and therapy resistance.

Growth of new blood vessels is considered requisite to cancer progression. Recent findings revealed that in addition to inducing angiogenesis, tumor-derived factors alter endothelial cell gene transcription within the tumor mass but also systemically throughout the body. This subsequently contributes to immunosuppression, altered metabolism, therapy resistance and metastasis. Clinical studies demonstrated that targeting the endothelium can increase the success rate of immunotherapy. Single-cell technologies revealed remarkable organ-specific endothelial heterogeneity that becomes altered by the presence of a tumor. In metastases, endothelial transcription differs remarkably between newly formed and co-opted vessels which may provide a basis for developing new therapies to target endothelial cells and overcome therapy resistance more effectively. This review addresses how cancers impact the endothelium to facilitate tumor progression.

Discrete class I molecules on brain endothelium differentially regulate neuropathology in experimental cerebral malaria.

Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood-brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood-brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

Interleukin-30 subverts prostate cancer-endothelium crosstalk by fostering angiogenesis and activating immunoregulatory and oncogenic signaling pathways.

BACKGROUND: Cancer-endothelial interplay is crucial for tumor behavior, yet the molecular mechanisms involved are largely unknown. Interleukin(IL)-30, which is expressed as a membrane-anchored cytokine by human prostate cancer (PC) cells, promotes PC vascularization and progression, but the underlying mechanisms have yet to be fully explored. METHODS: PC-endothelial cell (EC) interactions were investigated, after coculture, by flow cytometry, transcriptional profiling, western blot, and ELISA assays. Proteome profiler phospho-kinase array unveiled the molecular pathways involved. The role of tumor-derived IL30 on the endothelium's capacity to generate autocrine circuits and vascular budding was determined following IL30 overexpression, by gene transfection, or its deletion by CRISPR/Cas9 genome editing. Clinical value of the experimental findings was determined through immunopathological study of experimental and patient-derived PC samples, and bioinformatics of gene expression profiles from PC patients. RESULTS: Contact with PC cells favors EC proliferation and production of angiogenic and angiocrine factors, which are boosted by PC expression of IL30, that feeds autocrine loops, mediated by IGF1, EDN1, ANG and CXCL10, and promotes vascular budding and inflammation, via phosphorylation of multiple signaling proteins, such as Src, Yes, STAT3, STAT6, RSK1/2, c-Jun, AKT and, primarily CREB, GSK-3α/ß, HSP60 and p53. Deletion of the IL30 gene in PC cells inhibits endothelial expression of IGF1, EDN1, ANG and CXCL10 and substantially impairs tumor angiogenesis. In its interaction with IL30-overexpressing PC cells the endothelium boosts their expression of a wide range of immunity regulatory genes, including CCL28, CCL4, CCL5, CCR2, CCR7, CXCR4, IL10, IL13, IL17A, FASLG, IDO1, KITLG, TNFA, TNFSF10 and PDCD1, and cancer driver genes, including BCL2, CCND2, EGR3, IL6, VEGFA, KLK3, PTGS1, LGALS4, GNRH1 and SHBG. Immunopathological analyses of PC xenografts and in silico investigation of 1116 PC cases, from the Prostate Cancer Transcriptome Atlas, confirmed the correlation between the expression of IL30 and that of both pro-inflammatory genes, NOS2, TNFA, CXCR5 and IL12B, and cancer driver genes, LGALS4, GNRH1 and SHBG, which was validated in a cohort of 80 PC patients. CONCLUSIONS: IL30 regulates the crosstalk between PC and EC and reshapes their transcriptional profiles, triggering angiogenic, immunoregulatory and oncogenic gene expression programs. These findings highlight the angiostatic and oncostatic efficacy of targeting IL30 to fight PC.

Myeloperoxidase Staining Identifies Endothelial Injury of Peritubular Capillaries and Glomeruli in Renal Antibody-Mediated Rejection.

OBJECTIVE: It remains unclear if C4d staining is related to any peritubular and glomerular injury during antibody mediated rejection (ABMR). The goal of this study was to determine if myeloperoxidase (MPO) staining can highlight endothelial injury in peritubular capillaries (PTC) and glomeruli. METHODS: The study included 12 native negative controls, 19 transplant biopsies with borderline changes (BC) as transplant controls, and one group of renal transplant biopsies with ABMR as the study group (acute/chronic, n=22). All three groups were stained for MPO immunohistochemically, and the MPO expressions in the endothelium of PTC and glomeruli were evaluated and correlated with serum creatinine (SCr). In addition, the ultrastructural layers of the PTC (an index for chronic allograft rejection) were correlated with MPO indices in PTC. RESULTS: The negative control group and the transplant controls showed no MPO expression in the endothelium of glomeruli and PTC. However, in the biopsies with ABMR, there were MPO-positive stains in the endothelial cells of glomeruli (15/21 cases, 71.4 %) and PTC (16/22 cases, 72.7 %). There were significant correlations between the peritubular MPO staining versus SCr (r=0.355 and p=0.0106) and glomerular MPO staining versus SCr (r=0.365 and p=0.0092). Furthermore, the layers of PTC by electron microscopy were significantly correlated with MPO scores in PTC (r=0.696, p=0.0001). CONCLUSION: Our data suggest that the MPO-positive endothelial injuries are most likely the cause leading to renal graft dysfunction following ABMR.

Engineering tumoral vascular leakiness with gold nanoparticles.

Delivering cancer therapeutics to tumors necessitates their escape from the surrounding blood vessels. Tumor vasculatures are not always sufficiently leaky. Herein, we engineer therapeutically competent leakage of therapeutics from tumor vasculature with gold nanoparticles capable of inducing endothelial leakiness (NanoEL). These NanoEL gold nanoparticles activated the loss of endothelial adherens junctions without any perceivable toxicity to the endothelial cells. Microscopically, through real time live animal intravital imaging, we show that NanoEL particles induced leakiness in the tumor vessels walls and improved infiltration into the interstitial space within the tumor. In both primary tumor and secondary micrometastases animal models, we show that pretreatment of tumor vasculature with NanoEL particles before therapeutics administration could completely regress the cancer. Engineering tumoral vasculature leakiness represents a new paradigm in our approach towards increasing tumoral accessibility of anti-cancer therapeutics instead of further increasing their anti-cancer lethality.

Injury-induced endotheliopathy: What you need to know.

ABSTRACT: The endotheliopathy of trauma involves a complex interplay between the glycocalyx, von Willebrand factor, and platelets that leads to abnormalities in coagulation, inflammation, and endothelial cell (EC) function. The current review presents a synopsis of EC function under homeostatic conditions, the structure and function of the endothelial glycocalyx; mechanisms of EC injury and activation after trauma; pathological consequences of the EoT at the cellular level; and clinical implications of the EoT. Recent evidence is presented that links the EoT to extracellular vesicles and hyperadhesive ultralarge von Willebrand factor multimers through their roles in coagulopathy. Lastly, potential therapeutics to mitigate the EoT are discussed. Most research to date has focused on blood products, primarily plasma, and its contribution to restoring postinjury EC dysfunction. Additional therapeutic adjuvants that target the glycocalyx, ultralarge von Willebrand factor, low ADAMTS-13, and pathologic extracellular vesicles are reviewed. Much of the pathobiology of EoT is known, but a better mechanistic understanding can help guide therapeutics to further repair the EoT and improve patient outcomes.

Bicuspid aortic valve aortopathy is characterized by embryonic epithelial to mesenchymal transition and endothelial instability.

Bicuspid aortic valve (BAV) is the most common congenital heart malformation frequently associated with ascending aortic aneurysm (AscAA). Epithelial to mesenchymal transition (EMT) may play a role in BAV-associated AscAA. The aim of the study was to investigate the type of EMT associated with BAV aortopathy using patients with a tricuspid aortic valve (TAV) as a reference. The state of the endothelium was further evaluated. Aortic biopsies were taken from patients undergoing open-heart surgery. Aortic intima/media miRNA and gene expression was analyzed using Affymetrix human transcriptomic array. Histological staining assessed structure, localization, and protein expression. Migration/proliferation was assessed using ORIS migration assay. We show different EMT types associated with BAV and TAV AscAA. Specifically, in BAV-associated aortopathy, EMT genes related to endocardial cushion formation were enriched. Further, BAV vascular smooth muscle cells were less proliferative and migratory. In contrast, TAV aneurysmal aortas displayed a fibrotic EMT phenotype with medial degenerative insults. Further, non-dilated BAV aortas showed a lower miRNA-200c-associated endothelial basement membrane LAMC1 expression and lower CD31 expression, accompanied by increased endothelial permeability indicated by increased albumin infiltration. Embryonic EMT is a characteristic of BAV aortopathy, associated with endothelial instability and vascular permeability of the non-dilated aortic wall. KEY MESSAGES: Embryonic EMT is a feature of BAV-associated aortopathy. Endothelial integrity is compromised in BAV aortas prior to dilatation. Non-dilated BAV ascending aortas are more permeable than aortas of tricuspid aortic valve patients.

Endothelium-dependent relaxation is impaired in Schlager hypertensive (BPH/2J) mice by region-specific mechanisms in conductance and resistance arteries.

AIMS: Endothelial dysfunction and arterial stiffness are hallmarks of hypertension, and major risk factors for cardiovascular disease. BPH/2J (Schlager) mice are a genetic model of spontaneous hypertension, but little is known about the vascular pathophysiology of these mice and the region-specific differences between vascular beds. Therefore, this study compared the vascular function and structure of large conductance (aorta and femoral) and resistance (mesenteric) arteries of BPH/2J mice with their normotensive BPN/2J counterparts. MAIN METHODS: Blood pressure was measured in BPH/2J and BPN/3J mice via pre-implanted radiotelemetry probes. At endpoint, vascular function and passive mechanical wall properties were assessed using wire and pressure myography, qPCR and histology. KEY FINDINGS: Mean arterial blood pressure was elevated in BPH/2J mice compared to BPN/3J controls. Endothelium-dependent relaxation to acetylcholine was attenuated in both the aorta and mesenteric arteries of BPH/2J mice, but through different mechanisms. In the aorta, hypertension reduced the contribution of prostanoids. Conversely, in the mesenteric arteries, hypertension reduced the contribution of both nitric oxide and endothelium-dependent hyperpolarization. Hypertension reduced volume compliance in both femoral and mesenteric arteries, but hypertrophic inward remodelling was only observed in the mesenteric arteries of BPH/2J mice. SIGNIFICANCE: This is the first comprehensive investigation of vascular function and structural remodelling in BPH/2J mice. Overall, hypertensive BPH/2J mice exhibited endothelial dysfunction and adverse vascular remodelling in the macro- and microvasculature, underpinned by distinct region-specific mechanisms. This highlights BPH/2J mice as a highly suitable model for evaluating novel therapeutics to treat hypertension-associated vascular dysfunction.

Brain endothelial cells exposure to malaria parasites links type I interferon signalling to antigen presentation, immunoproteasome activation, endothelium disruption, and cellular metabolism.

Introduction: Cerebral malaria (CM) lethality is attributable to induction of brain edema induction but the cellular mechanisms involving brain microvascular endothelium in CM pathogenesis are unexplored. Results: Activation of the STING-INFb-CXCL10 axis in brain endothelial cells (BECs) is a prominent component of the innate immune response in CM development in mouse models. Using a T cell-reporter system, we show that Type 1 IFN signaling in BECs exposed to Plasmodium berghei-infected erythrocytes (PbA-IE), functionally enhances MHC Class-I antigen presentation through gamma-interferon independent immunoproteasome activation and impacted the proteome functionally related to vesicle trafficking, protein processing/folding and antigen presentation. In vitro assays showed that Type 1 IFN signaling and immunoproteasome activation are also involved in the dysfunction of the endothelial barrier through disturbing gene expression in the Wnt/ß-catenin signaling pathway. We demonstrate that IE exposure induces a substantial increase in BECs glucose uptake while glycolysis blockade abrogates INFb secretion impairing immunoproteasome activation, antigen presentation and Wnt/ß-catenin signaling. Discussion: Metabolome analysis show that energy demand and production are markedly increased in BECs exposed to IE as revealed by enriched content in glucose and amino acid catabolites. In accordance, glycolysis blockade in vivo delayed the clinical onset of CM in mice. Together the results show that increase in glucose uptake upon IE exposure licenses Type 1 IFN signaling and subsequent immunoproteasome activation contributing to enhanced antigen presentation and impairment of endothelial barrier function. This work raises the hypothesis that Type 1 IFN signaling-immunoproteasome induction in BECs contributes to CM pathology and fatality (1) by increasing antigen presentation to cytotoxic CD8+ T cells and (2) by promoting endothelial barrier dysfunction, that likely favor brain vasogenic edema.

FGF13-Sensitive Alteration of Parkin Safeguards Mitochondrial Homeostasis in Endothelium of Diabetic Nephropathy.

Studies of diabetic glomerular injury have raised the possibility of developing useful early biomarkers and therapeutic approaches for the treatment of type 2 diabetic nephropathy (T2DN). In this study, we found that FGF13 expression is induced in glomerular endothelial cells (GECs) during T2DN progression. Endothelial-specific deletion of Fgf13 potentially alleviates T2DN damage, while Fgf13 overexpression has the opposite effect. Mechanistically, Fgf13 deficiency results in improved mitochondrial homeostasis and endothelial barrier integrity in T2DN. Moreover, FGF13-sensitive alteration of Parkin safeguards mitochondrial homeostasis in endothelium of T2DN through promotion of mitophagy and inhibition of apoptosis. Additionally, it is confirmed that the beneficial effects of Fgf13 deficiency on T2DN are abolished by endothelial-specific double deletion of Fgf13 and Prkn. The effects of Fgf13 deficiency on mitophagy and apoptosis through Parkin-dependent regulation may be distinct and separable events under diabetic conditions. These data show that the bifunctional role of Fgf13 deficiency in promoting mitophagy and inhibiting apoptosis through Parkin can shape mitochondrial homeostasis regulation in GECs and T2DN progression. As a potential therapeutic target for prevention and control of T2DN, a mechanistic understanding of the biofunction of FGF13 may also be relevant to the pathogenesis of other FGF13- and Parkin-associated diseases.

Arginase 1 Expression by Macrophages Promotes Cryptococcus neoformans Proliferation and Invasion into Brain Microvascular Endothelial Cells.

Cryptococcal meningoencephalitis caused by Cryptococcus neoformans infection is the most common cause of death in HIV/AIDS patients. Macrophages are pivotal for the regulation of immune responses to cryptococcal infection by either playing protective function or facilitating fungal dissemination. However, the mechanisms underlying macrophage responses to C. neoformans remain unclear. To analyze the transcriptomic changes and identify the pathogenic factors of macrophages, we performed a comparative transcriptomic analysis of alveolar macrophage responses during C. neoformans infection. Alveolar macrophages isolated from C. neoformans-infected mice showed dynamic gene expression patterns, with expression change from a protective M1 (classically activated)-like to a pathogenic M2 (alternatively activated)-like phenotype. Arg1, the gene encoding the enzyme arginase 1, was found as the most upregulated gene in alveolar macrophages during the chronic infection phase. The in vitro inhibition of arginase activity resulted in a reduction of cryptococcal phagocytosis, intracellular growth, and proliferation, coupled with an altered macrophage response from pathogenic M2 to a protective M1 phenotype. In an in vitro model of the blood-brain barrier, macrophage-derived arginase was found to be required for C. neoformans invasion of brain microvascular endothelium. Further analysis of the degree of virulence indicated a positive correlation between arginase 1 expression in macrophages and cryptococcal brain dissemination in vivo. Thus, our data suggest that a dynamic macrophage activation that involves arginase expression may contribute to the cryptococcal disease by promoting cryptococcal growth, proliferation, and the invasion to the brain endothelium.

A Case of a Rare Histologic Variant: Cavernous Hemangioma-Like Kaposi Sarcoma.

ABSTRACT: Kaposi sarcoma (KS) is an endothelial tumor associated with human herpesvirus 8. Cutaneous lesions can present with pink or purple patches, plaques, and nodules which can be ulcerated. The main subtypes of KS generally have similar histologic appearances, with spindle cells and expression of human herpesvirus 8 being characteristic features. However, various histologic variants have been reported. We present the case of a 55-year-old man with cutaneous KS with cavernous hemangioma-like histological features. Cavernous hemangioma-like KS is a rare morphologic type of KS, with only a handful of cases reported in the literature.

Biophysical determinants of cancer organotropism.

Metastasis remains the leading cause of cancer lethality. The 'seed/soil' hypothesis provides the framework to explain this cancer phenomenon where the concept of organotropism has been in part mechanistically explained by the properties of the tumor cells and their compatibility with the stromal environment of the distal site. The 'mechanical' hypothesis counters that non-random seeding is driven solely by the circulation patterns and vascular networks of organ systems. We incorporate concepts of mechanobiology and revisit the two hypotheses to provide additional insights into the mechanisms that regulate organ selection during metastatic outgrowth. We focus on the latter stages of the metastatic cascade and examine the role of the endothelium in regulating organ selectivity.

Steppogenin suppresses tumor growth and sprouting angiogenesis through inhibition of HIF-1α in tumors and DLL4 activity in the endothelium.

BACKGROUND: Hypoxia is a characteristic feature of many solid tumors. As an adaptive response to hypoxia, tumor cells activate hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, angiogenesis mediated by HIF-1α is involved in the growth and metastasis of tumor cells. During the angiogenic process, differentiated tip endothelial cells (ECs) characterized by high expression of DLL4 promote angiogenic germination through filopodia. Inhibitors of HIF-1α or DLL4 have been widely studied PURPOSE: We tried to find inhibitors targeting both HIF-1α and DLL4 in tumor which have not yet been developed. STUDY DESIGN: In this study, we examined a natural compound that inhibits sprouting angiogenesis and tumor growth by targeting both HIF-1α and DLL4 under hypoxic conditions. METHODS: After examining cell viability of 70 selected natural compounds, we assessed the effects of compounds on HIF-1α and DLL4 transcriptional activity using a dual-luciferase reporter assay. Western blot analysis, immunofluoresecnt assay and real-time qPCR were performed to identify expression of proteins, such as HIF-1α and DLL4, as well as HIF-1α target genes under hypoxic conditions. In vitro angiogenesis assay and in vivo allograft tumor experiment were performed to investigate inhibition of tumor growth through anti-angiogenic activity. RESULTS: Among these compounds, steppogenin, which is extracted from the root bark of Morus alba l, respectively inhibited the transcriptional activity of HIF-1α under hypoxic conditions in HEK293T cells and vascular endothelial growth factor (VEGF)-induced DLL4 expression in vascular ECs in a dose-dependent manner. In tumor cells and retinal pigment epithelial cells, steppogenin significantly suppressed HIF-1α protein levels under hypoxic conditions as well as VEGF-induced DLL4 expression in ECs. Furthermore, steppogenin suppressed hypoxia-induced vascular EC proliferation and migration as well as VEGF-induced sprouting of EC spheroids. CONCLUSION: These results suggest that the natural compound steppogenin could potentially be used to treat angiogenic diseases, such as those involving solid tumors, because of its dual inhibition of HIF-1α and DLL4.

MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes.

The trafficking of autoreactive leucocytes across the blood-brain barrier endothelium is a hallmark of multiple sclerosis pathogenesis. Although the blood-brain barrier endothelium represents one of the main CNS borders to interact with the infiltrating leucocytes, its exact contribution to neuroinflammation remains understudied. Here, we show that Mcam identifies inflammatory brain endothelial cells with pro-migratory transcriptomic signature during experimental autoimmune encephalomyelitis. In addition, MCAM was preferentially upregulated on blood-brain barrier endothelial cells in multiple sclerosis lesions in situ and at experimental autoimmune encephalomyelitis disease onset by molecular MRI. In vitro and in vivo, we demonstrate that MCAM on blood-brain barrier endothelial cells contributes to experimental autoimmune encephalomyelitis development by promoting the cellular trafficking of TH1 and TH17 lymphocytes across the blood-brain barrier. Last, we showcase ST14 as an immune ligand to brain endothelial MCAM, enriched on CD4+ T lymphocytes that cross the blood-brain barrier in vitro, in vivo and in multiple sclerosis lesions as detected by flow cytometry on rapid autopsy derived brain tissue from multiple sclerosis patients. Collectively, our findings reveal that MCAM is at the centre of a pathological pathway used by brain endothelial cells to recruit pathogenic CD4+ T lymphocyte from circulation early during neuroinflammation. The therapeutic targeting of this mechanism is a promising avenue to treat multiple sclerosis.

Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy.

PURPOSE: Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. EXPERIMENTAL DESIGN: Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. RESULTS: It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. CONCLUSION: The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.

Salvianolic Acid Ameliorates Pressure Overload-Induced Cardiac Endothelial Dysfunction via Activating HIF1[Formula: see text]/HSF1/CD31 Pathway.

Pressure overload is a major risk factor for various cardiovascular diseases. Disorders of the endothelium are involved in the pathological mechanisms of pressure, and maintaining endothelial function is a practical strategy to alleviate pressure overload-induced cardiac injury. In this study, we provided evidence that salvianolic acid, the active component of Danshen, a traditional Chinese herb medicine, preserved pressure overload-induced cardiac dysfunction via protecting endothelium. Male C57BL/6J mice were imposed with transverse aortic constriction to mimic pressure overload and treated with salvianolic acid (200[Formula: see text]mg/kg/day) or vehicle for 6 weeks. The hemodynamic and cardiac functional parameters were detected by the cardiac catheter and transthoracic echocardiography. The pathological measurements were conducted by heart hematoxylin-eosin, wheat germ agglutinin staining, Masson's trichrome staining, and immunofluorescence staining. Endothelial cell (EC) proliferation was estimated using the Cell Counting Kit-8, EC migration was evaluated by scratched assay, and EC integrity was observed by electron microscope. Salvianolic acid notably inhibited cardiac chamber enlargement, restrained cardiac contractile dysfunction, and repressed cardiac fibrosis caused by chronic pressure overload. Salvianolic acid maintained endothelial tight junction integrity by boosting the expression of CD31. Furthermore, the endothelial protective effect of salvianolic acid against pressure overload is dependent on the activation of hypoxia-inducible factor 1[Formula: see text], which consequently activated heat shock factor 1 and promoted CD31 expression. Our study uncovered that salvianolic acid protected cardiac ECs against pressure overload via a HIF1[Formula: see text]/HSF1/CD31 pathway, indicating a potential appliance of salvianolic acid in hypertensive heart disease.