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.

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.

Endothelial Glycocalyx Integrity in Treatment-Naïve People Living with HIV before and One Year after Antiretroviral Treatment Initiation.

Endothelial glycocalyx (EG) derangement has been associated with cardiovascular disease (CVD). Studies on EG integrity among people living with HIV (PLWH), are lacking. We conducted a prospective cohort study among treatment-naïve PLWH who received emtricitabine/tenofovir alafenamide, combined with either an integrase strand transfer inhibitor (INSTI, dolutegravir, raltegravir or elvitegravir/cobicistat), or a protease inhibitor (PI, darunavir/cobicistat). We assessed EG at baseline, 24 (±4) and 48 (±4) weeks, by measuring the perfused boundary region (PBR, inversely proportional to EG thickness), in sublingual microvessels. In total, 66 consecutive PLWH (60 (90.9%) males) with a median age (interquartile range, IQR) of 37 (12) years, were enrolled. In total, 40(60.6%) received INSTI-based regimens. The mean (standard deviation) PBR decreased significantly from 2.17 (0.29) µm at baseline to 2.04 (0.26) µm (p = 0.019), and then to 1.93 (0.3) µm (p < 0.0001) at 24 (±4) and 48 (±4) weeks, respectively. PBR did not differ among treatment groups. PLWH on INSTIs had a significant PBR reduction at 48 (±4) weeks. Smokers and PLWH with low levels of viremia experienced the greatest PBR reduction. This study is the first to report the benefit of antiretroviral treatment on EG improvement in treatment-naïve PLWH and depicts a potential bedside biomarker and therapeutic target for CVD in PLWH.

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.

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.

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.

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.

Molecular Pathogenesis of Endotheliopathy and Endotheliopathic Syndromes, Leading to Inflammation and Microthrombosis, and Various Hemostatic Clinical Phenotypes Based on "Two-Activation Theory of the Endothelium" and "Two-Path Unifying Theory" of Hemostasis.

Endotheliopathy, according to the "two-activation theory of the endothelium", can be triggered by the activated complement system in critical illnesses, such as sepsis and polytrauma, leading to two distinctly different molecular dysfunctions: (1) the activation of the inflammatory pathway due to the release of inflammatory cytokines, such as interleukin 6 and tumor necrosis factor-α, and (2) the activation of the microthrombotic pathway due to the exocytosis of hemostatic factors, such as ultra-large von Willebrand factor (ULVWF) multimers and FVIII. The former promotes inflammation, including inflammatory organ syndrome (e.g., myocarditis and encephalitis) and multisystem inflammatory syndrome (e.g., cytokine storm), and the latter provokes endotheliopathy-associated vascular microthrombotic disease (VMTD), orchestrating thrombotic thrombocytopenic purpura (TTP)-like syndrome in arterial endotheliopathy, and immune thrombocytopenic purpura (ITP)-like syndrome in venous endotheliopathy, as well as multiorgan dysfunction syndrome (MODS). Because the endothelium is widely distributed in the entire vascular system, the phenotype manifestations of endotheliopathy are variable depending on the extent and location of the endothelial injury, the cause of the underlying pathology, as well as the genetic factor of the individual. To date, because the terms of many human diseases have been defined based on pathological changes in the organ and/or physiological dysfunction, endotheliopathy has not been denoted as a disease entity. In addition to inflammation, endotheliopathy is characterized by the increased activity of FVIII, overexpressed ULVWF/VWF antigen, and insufficient ADAMTS13 activity, which activates the ULVWF path of hemostasis, leading to consumptive thrombocytopenia and microthrombosis. Endothelial molecular pathogenesis produces the complex syndromes of inflammation, VMTD, and autoimmunity, provoking various endotheliopathic syndromes. The novel conceptual discovery of in vivo hemostasis has opened the door to the understanding of the pathogeneses of many endotheliopathy-associated human diseases. Reviewed are the hemostatic mechanisms, pathogenesis, and diagnostic criteria of endotheliopathy, and identified are some of the endotheliopathic syndromes that are encountered in clinical medicine.

Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature.

Endothelial cells are highly sensitive to ionizing radiation, and exposure leads to multiple adaptive changes. Remarkably, part of this response is the translocation of normally intracellular proteins to the cell surface. It is unclear whether this ectopic expression has a protective or deleterious function, but, regardless, these surface-exposed proteins may provide unique discriminatory targets for radiation-guided drug delivery to vascular malformations or tumor vasculature. We investigated the ability of an antibody-thrombin conjugate targeting mitochondrial PDCE2 (E2 subunit of pyruvate dehydrogenase) to induce precision thrombosis on irradiated endothelial cells in a parallel-plate flow system. Click-chemistry was used to create antibody-thrombin conjugates targeting PDCE2 as the vascular targeting agent (VTA). VTAs were injected into the parallel-plate flow system with whole human blood circulating over irradiated cells. The efficacy and specificity of fibrin-thrombus formation was assessed relative to non-irradiated controls. The PDCE2-targeting VTA dose-dependently increased thrombus formation: minimal thrombosis was induced in response to 5 Gy radiation; doses of 15 and 25 Gy induced significant thrombosis with equivalent efficacy. Negligible VTA binding or thrombosis was demonstrated in the absence of radiation or with non-targeted thrombin. PDCE2 represents a unique discriminatory target for radiation-guided drug delivery and precision thrombosis in pathological vasculature.

Melanoma Mediated Disruption of Brain Endothelial Barrier Integrity Is Not Prevented by the Inhibition of Matrix Metalloproteinases and Proteases.

We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated barrier disruption, which was localised to the paracellular junctions of the brain endothelial cells. Melanoma cells are known to release molecules which cleave the surrounding matrix and allow traversal within and out of their metastatic niche. Enzymatic families, such as matrix metalloproteinases (MMPs) and proteases are heavily implicated in this process and their complex nature in vivo makes them an intriguing family to assess in melanoma metastasis. Herein, we assessed the expression of MMPs and other proteases in melanoma conditioned media. Our results showed evidence of a high expression of MMP-2, but not MMP-1, -3 or -9. Other proteases including Cathepsins D and B were also detected. Recombinant MMP-2 was added to the apical face of brain endothelial cells (hCMVECs), to measure the change in barrier integrity using biosensor technology. Surprisingly, this showed no decrease in barrier strength. The addition of potent MMP inhibitors (batimastat, marimastat, ONO4817) and other protease inhibitors (such as aprotinin, Pefabloc SC and bestatin) to the brain endothelial cells, in the presence of various melanoma lines, showed no reduction in the melanoma mediated barrier disruption. The inhibitors batimastat, Pefabloc SC, antipain and bestatin alone decreased the barrier strength. These results suggest that although some MMPs and proteases are released by melanoma cells, there is no direct evidence that they are substantially involved in the initial melanoma-mediated disruption of the brain endothelium.

PET imaging of hepatocellular carcinoma by targeting tumor-associated endothelium using [68Ga]Ga-PSMA-617.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a malignant tumor associated with high morbidity and mortality rates. In many non-prostate solid tumors such as HCC, prostate-specific membrane antigens (PSMA) are overexpressed in tumor-associated endothelial cells. Therefore, the aim of this study was to evaluate the performance of [68Ga]Ga-PSMA-617 PET imaging on HCC with different animal models, including cell line-derived xenografts (CDX) and patient-derived xenografts (PDX), and to explore its mechanisms of function. METHODS: [68Ga]Ga-PSMA-617 was prepared. The expression level of PSMA in two human hepatocellular cancer cells (HepG2 and HuH-7) was evaluated, and the cellular uptakes of [68Ga]Ga-PSMA-617 were assayed. HepG2 and HuH-7 subcutaneous xenograft models, HepG2 orthotopic xenograft models, and four different groups of PDX models were prepared. Preclinical pharmacokinetics and performance of [68Ga]Ga-PSMA-617 were evaluated in different types of HCC xenografts models using small animal PET and biodistribution studies. RESULTS: Low PSMA expression level of HepG2 and HuH-7 cells was observed, and the cellular uptake and blocking study confirmed the non-specificity of the PSMA-targeted probe binding to HepG2 and HuH-7 cells. In the subcutaneous xenograft models, the tumor uptakes at 0.5 h were 0.76 ± 0.12%ID/g (HepG2 tumors) and 0.78 ± 0.08%ID/g (HuH-7 tumors), respectively, which were significantly higher than those of the blocking groups (0.23 ± 0.04%ID/g and 0.20 ± 0.04%ID/g, respectively). In the orthotopic xenograft models, PET images clearly displayed the tumor locations based on the preferential accumulation of [68Ga]Ga-PSMA-617 in tumor tissue versus normal liver tissue, suggesting the possibility of using [68Ga]Ga-PSMA-617 PET imaging to detect primary HCC lesions in deep tissue. In the four different groups of HCC PDX models, PET imaging with [68Ga]Ga-PSMA-617 provided clear tumor uptakes with prominent tumor-to-background contrast, further demonstrating its potential for the clinical imaging of PSMA-positive HCC lesions. The staining of tumor tissue sections with CD31- and PSMA-specific antibodies visualized the tumor-associated blood vessels and PSMA expression on endothelial cells in subcutaneous, orthotopic tissues, and PDX tissues, confirming the imaging with [68Ga]Ga-PSMA-617 might be mediated by targeting tumor associated endothelium. CONCLUSION: In this study, in vivo PET on different types of HCC xenograft models illustrated high uptake within tumors, which confirmed that [68Ga]Ga-PSMA-617 PET may be a promising imaging modality for HCC by targeting tumor associated endothelium.

Current Insights on the Role of Irisin in Endothelial Dysfunction.

Endothelial dysfunction is a crucial physiopathological mechanism for cardiovascular diseases that results from the harmful impact of metabolic disorders. Irisin, a recently discovered adipomyokine, has been shown to exert beneficial metabolic effects by increasing energy consumption, improving insulin sensitivity, and reducing the proinflammatory milieu. Multiple preclinical models have assessed irisin's possible role in the development of endothelial dysfunction, displaying that treatment with exogenous irisin can decrease the production of oxidative stress mediators by up-regulating Akt/mTOR/Nrf2 pathway, promote endothelial-dependent vasodilatation through the activation of AMPK-PI3K-AkteNOS pathway, and increase the endothelial cell viability by activation of ERK proliferation pathway and downregulation of Bad/Bax/Caspase 3 pro-apoptotic pathway. However, there is scarce evidence of these mechanisms in clinical studies, and available results are controversial. Some have shown negative correlations of irisin levels with the burden of coronary atherosclerosis and leukocyte adhesion molecules' expression. Others have demonstrated associations between irisin levels and increased atherosclerosis risk and higher carotid intima-media thickness. Since the role of irisin in endothelial damage remains unclear, in this review, we compare, contrast, and integrate the current knowledge from preclinical and clinical studies to elucidate the potential preventive role and the underlying mechanisms and pathways of irisin in endothelial dysfunction. This review also comprises original figures to illustrate these mechanisms.