Peripheral Endothelial Cells Research Articles

Thrombomodulin (p.Cys537Stop) is released from cells by an unusual membrane insertion/leakage mechanism

AbstractExpression of the thrombomodulin (TM) variant c.1611C>A (p.Cys537Stop) leads to the synthesis of a protein with no cytoplasmic tail and a transmembrane domain shortened by 3 amino acids (TM536). However, little is known regarding the release mechanism and properties of TM536. Using umbilical vein endothelial cells and peripheral blood-derived endothelial colony-forming cells from a heterozygous carrier of the TM536 variant as well as overexpression cell models, we demonstrated that TM536 is released from cells by an unusual mechanism. First, TM536 is inserted into the endoplasmic reticulum (ER) membrane, then, because of the low hydrophobicity of its intramembrane domain, it escapes from it and follows the conventional secretory pathway to be released into the extracellular compartment without the involvement of proteolysis. This particular secretion mechanism yields a soluble TM536, which is poorly modified by chondroitin sulfate glycosaminoglycan compared with conventionally secreted soluble forms of TM, and therefore has a suboptimal capacity to mediate thrombin-dependent activation of protein C (PC). We also showed that TM536 cellular trafficking was altered, with retention in the early secretory pathway and increased sensitivity to ER-associated degradation. As expected, activation of ER-associated degradation increased TM536 degradation and reduced its release. The expression of TM536 at the cell surface was low, and its distribution in lipid raft-like membrane microdomains was altered, resulting in low thrombin-dependent PC activation on the cell surface.

CD163+ Macrophages Induce Endothelial-to-Mesenchymal Transition in Atheroma.

Cell phenotype switching is increasingly being recognized in atherosclerosis. However, our understanding of the exact stimuli for such cellular transformations and their significance for human atherosclerosis is still evolving. Intraplaque hemorrhage is thought to be a major contributor to plaque progression in part by stimulating the influx of CD163+ macrophages. Here, we explored the hypothesis that CD163+ macrophages cause plaque progression through the induction of proapoptotic endothelial-to-mesenchymal transition (EndMT) within the fibrous cap. Human coronary artery sections from CVPath's autopsy registry were selected for pathological analysis. Athero-prone ApoE-/- and ApoE-/-/CD163-/- mice were used for in vivo studies. Human peripheral blood mononuclear cell-induced macrophages and human aortic endothelial cells were used for in vitro experiments. In 107 lesions with acute coronary plaque rupture, 55% had pathological evidence of intraplaque hemorrhage in nonculprit vessels/lesions. Thinner fibrous cap, greater CD163+ macrophage accumulation, and a larger number of CD31/FSP-1 (fibroblast specific protein-1) double-positive cells and TUNEL (terminal deoxynucleotidyl transferase-dUTP nick end labeling) positive cells in the fibrous cap were observed in nonculprit intraplaque hemorrhage lesions, as well as in culprit rupture sections versus nonculprit fibroatheroma sections. Human aortic endothelial cells cultured with supernatants from hemoglobin/haptoglobin-exposed macrophages showed that increased mesenchymal marker proteins (transgelin and FSP-1) while endothelial markers (VE-cadherin and CD31) were reduced, suggesting EndMT induction. Activation of NF-κB (nuclear factor kappa β) signaling by proinflammatory cytokines released from CD163+ macrophages directly regulated the expression of Snail, a critical transcription factor during EndMT induction. Western blot analysis for cleaved caspase-3 and microarray analysis of human aortic endothelial cells indicated that apoptosis was stimulated during CD163+ macrophage-induced EndMT. Additionally, CD163 deletion in athero-prone mice suggested that CD163 is required for EndMT and plaque progression. Using single-cell RNA sequencing from human carotid endarterectomy lesions, a population of EndMT was detected, which demonstrated significant upregulation of apoptosis-related genes. CD163+ macrophages provoke EndMT, which may promote plaque progression through fibrous cap thinning.

Retracted: The Treatment Combining Antiangiogenesis with Chemoradiotherapy Impinges on the Peripheral Circulation Vascular Endothelial Cells and Therapeutic Effect in the Patients with Locally Advanced Nasopharyngeal Carcinoma.

[This retracts the article DOI: 10.1155/2022/1787854.].

Effect of antiglaucomatous eye drops on corneal endothelium

First choice therapy for reducing the intraocular pressure in context of glaucoma remains the applications of eye drops. Longterm exposure to topical therapy can clearly have an impact on the ocular surface, but as the medications penetrate through the cornea and are present in humour aqueous, also on all other layers of cornea, including corneal endothelium. In general, none of the medications from the standard pharmacological groups influences significantly an otherwise healthy corneal endothelium in an eye with glaucoma or ocular hypertension – be it in terms of central corneal thickness, be it in terms of endothelial cell density and morphology, be it for beta blockers, carbonic anhydrase inhibitors, pilocarpine, alpha2 agonists or prostaglandin analogues. The situation changes in diseased corneas, such as in Fuchs endothelial dystrophy, where topically applied carbonic anhydrase inhibitors can lead to increase in corneal thickness. Benzalkonium chloride has been proven toxic for corneal endothelial cells in vitro. However, in the daily practice and under real life circumstances, it is from the specific point of view on the corneal endothelium still not clear whether using non‐preserved topical glaucoma medications is an absolute imperative. One separate topic here is also a group of Rho‐kinase inhibiting topical medications. Apart from new mechanisms of action for reducing the intraocular pressure, this group of medications has also a direct influence on the corneal structures – from cornea verticillata on the epithelium corneal level, to mobilizing peripheral endothelial corneal cells as a possible therapeutic path for Fuchs endothelial dystrophy. One part of the lecture will address also these issues.

Long-Term Follow-Up of Descemet Stripping Only: Data Up to 7 Years Postoperatively.

The aim of this study was to report long-term follow-up of eyes undergoing Descemet stripping only (DSO). This was a retrospective study including 26 eyes of 20 patients undergoing DSO between December 2015 and November 2022. Eligibility criteria included peripheral endothelial cell count (ECC) >1000 cells/mm 2 and symptoms caused by central guttata. Patients underwent a central circular 4-mm descemetorhexis using a reverse Sinskey hook and a pair of descemetorhexis forceps using a peeling technique. Three parameters were measured before surgery and at last follow-up: best-corrected visual acuity (BCVA), central corneal thickness (CCT), and ECC measured centrally and at the periphery. The mean age was 73 ± 9 years [52-90 years]. The average follow-up period was 23.7 ± 24.8 months [3-84]. Twenty-two eyes responded to DSO with 20 female eyes (91%) and 2 male eyes (9%). The mean postoperative BCVA improved from 0.3 ± 0.17 logMAR to 0.09 ± 0.13 logMAR ( P value <0.05). The mean postoperative CCT decreased from 588 ± 41 μm to 546 ± 50 μm ( P -value <0.05). The mean postoperative central ECC was 780 ± 257 cells/mm 2 [484-1500]. Peripheral ECC decreased postoperatively (1837 ± 407 cells/mm 2 preoperatively to 864 ± 340 cells/mm 2 postoperatively, P value >0.05). Peripheral endothelial cell polymegathism was stable (average of 26.8% ± 6.8% preoperatively and 30.2% ± 14% postoperatively). Average peripheral endothelial cells polymorphism decreased postoperatively (63.1 ± 20.5% preoperatively to 33% ± 25% postoperatively, P value >0.05). Four eyes did not show improvement after DSO and underwent Descemet membrane endothelial keratoplasty surgery. There were 3 men (75%) and 1 women (25%). The preoperative trend was for nonresponders to have lower BCVA, higher CCT, more abnormal peripheral polymorphism, and polymegathism. The results of this study, with up to 7 years follow up, demonstrate the durability of DSO.

Identifying molecular tags selectively retained on the surface of brain endothelial cells to generate artificial targets for therapy delivery

Current strategies to identify ligands for brain delivery select candidates based on preferential binding to cell-membrane components (CMC) on brain endothelial cells (EC). However, such strategies generate ligands with inherent brain specificity limitations, as the CMC (e.g., the transferrin receptor TfR1) are also significantly expressed on peripheral EC. Therefore, novel strategies are required to identify molecules allowing increased specificity of therapy brain delivery. Here, we demonstrate that, while individual CMC are shared between brain EC and peripheral EC, their endocytic internalization rate is markedly different. Such differential endocytic rate may be harnessed to identify molecular tags for brain targeting based on their selective retention on the surface of brain EC, thereby generating ‘artificial’ targets specifically on the brain vasculature. By quantifying the retention of labelled proteins on the cell membrane, we measured the general endocytic rate of primary brain EC to be less than half that of primary peripheral (liver and lung) EC. In addition, through bio-panning of phage-displayed peptide libraries, we unbiasedly probed the endocytic rate of individual CMC of liver, lung and brain endothelial cells. We identified phage-displayed peptides which bind to CMC common to all three endothelia phenotypes, but which are preferentially endocytosed into peripheral EC, resulting in selective retention on the surface of brain EC. Furthermore, we demonstrate that the synthesized free-form peptides are capable of generating artificial cell-surface targets for the intracellular delivery of model proteins into brain EC with increasing specificity over time. The developed identification paradigm, therefore, demonstrates that the lower endocytic rate of individual CMC on brain EC can be harnessed to identify peptides capable of generating ‘artificial’ targets for the selective delivery of proteins into the brain vasculature. In addition, our approach identifies brain-targeting peptides which would have been overlooked by conventional identification strategies, thereby increasing the repertoire of candidates to achieve specific therapy brain delivery.

IL-33 Suppresses the Progression of Atherosclerosis via the ERK1/2-IRF1-VCAM-1 Pathway.

This study was designed to explore the effects of interleukin 33 (IL-33) on the progression of atherosclerosis and the possible mechanism. The adhesion assay was performed on isolated peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC). The expression of proteins and messenger RNA (mRNA) were detected by western blot and quantitative real-time polymerase chain reaction (PCR), including intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and P-selectin. The effect of IL-33 on the interaction of growth stimulation expressed gene 2 (ST2) with myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase (IRAK) 1/4 were investigated using co-immunoprecipitation assay. An apolipoprotein (Apo) E-/- mice model was used to confirm the effect of IL-33 on atherosclerosis progression. Area of plaques was recorded by hematoxylin-eosin (H&E) staining. The severity of atherosclerosis plaque was evaluated using immunohistochemistry assay, and lipid accumulation was measured by an oil red O staining. In contrast, western blot was performed to detect the expression levels of VCAM-1, extracellular signal-regulated kinase (ERK) 1/2, and interferon regulatory factor 1 (IRF1). Our study observed that IL-33 suppressed cell adhesion and the expression of VCAM-1 in tumor necrosis factor-α (TNF-α) exposed HUVEC. Moreover, the addition of IL-33 significantly inhibited the expression of IRF1 and the binding level of IRF1 to VCAM-1 and also promoted the phosphorylation level of IRAK1/4 and ERK1/2 compared to TNF-α-stimulated HUVEC. The ST2 neutralizing antibody or ERK pathway inhibitor SCH772984 reversed the regulatory effects of IL-33 on HUVEC, suggesting that IL-33 suppressed IRF1 and VCAM-1 dependent on binding to ST2 and activating the ERK1/2 signaling pathway. Further investigation in vivo confirmed that IL-33 decreased the expressions of IRF1 and VCAM-1 by activating the phosphorylation of ERK1/2 in the thoracic aorta of Apo E-/- mice. In conclusion, our results demonstrated that IL-33 plays a protective role in the progression of atherosclerosis by inhibiting cell adhesion via the ERK1/2-IRF1-VCAM-1 pathway. This study may provide a potential therapeutic way to prevent the development of atherosclerosis.

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells.

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro. This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Host CD34+ cells are replacing donor endothelium of transplanted heart

Endothelium dysfunction is a central problem for early rejection due to the host alloimmune response and the late status of arteriosclerosis in heart transplantation. However, reliable pieces of evidence are still limited concerning the source of the regenerated endothelium within the transplanted heart. We analyzed single-cell RNA sequencing data and constructed an inducible lineage tracing mouse, combined heart transplantation with bone marrow transplantation and a parabiosis model, cellular components, and endothelial cell populations in cardiac graft lesions. Our single-cell RNA sequencing analysis of a transplanted heart allowed for the establishment of an endothelial cell atlas with a heterogeneous population, including arterial, venous, capillary, and lymphatic endothelial cells. Along with genetic cell lineage tracing, we demonstrated that the donor cells were mostly replaced by recipient cells in the cardiac allograft, up to 83.29% 2weeks after transplantation. Furthermore, recipient nonbone marrow CD34+ endothelial progenitors contributed significantly to extracellular matrix organization and immune regulation, with higher apoptotic ability in the transplanted hearts. Mechanistically, peripheral blood-derived human endothelial progenitor cells differentiate into endocardial cells via Vascular endothelial growth factor receptor-mediated pathways. Host circulating CD34+ endothelial progenitors could repair the damaged donor endothelium presumably through CCL3-CCR5 chemotaxis. Partial depletion of host CD34+ cells resulted in delayed endothelial regeneration. We created an annotated fate map of endothelial cells in cardiac allografts, indicating how recipient CD34+ cells could replace the donor endothelium via chemokine CCL3-CCR5 interactions. The mechanisms we discovered could have a potential therapeutic effect on the long-term outcomes of heart transplantation.

LncRNA NEAT-2 regulate the function of endothelial progenitor cells in experimental Sepsis model.

Sepsis is a life-threatening disease with a limited effectiveness and the potential mechanism remains unclear. LncRNA NEAT-2 is reported to be involved in the regulation of cardiovascular disease. This study aimed to investigate the function of NEAT-2 in sepsis. We built sepsis animal model with Male Balb/C mice induced by cecal ligation and puncture (CLP). A total of 54 mice were randomly assigned into eight groups: sham operation group (n = 18), CLP group (n = 18), CLP plus si-control group (n = 3), CLP plus si-NEAT2 group (n = 3), CLP plus mimic control group (n = 3), CLP plus miR-320 group (n = 3), CLP plus normal saline group (n = 3), and normal control group (n = 3). The number of peripheral endothelial progenitor cells (EPCs), the expression level of NEAT-2 and miR-320 were detected during progression of sepsis, as well as the number of peripheral EPCs and level of TNF-α, IL-6, VEGF, ALT, AST and Cr. In addition, the function of EPCs was evaluated after NEAT-2 knockdown and miR-320 overexpression in vitro. The number of circulating EPCs increased significantly in sepsis. NEAT-2 expression was significantly increased in the progress of sepsis, accompanied with miR-320 downregulated. NEAT-2 knockdown and miR-320 overexpression attenuated hepatorenal function and increased cytokines in sepsis. Moreover, NEAT-2 knockdown and miR-320 overexpression decreased the proliferation, migration and angiogenesis of endothelial progenitor cells in vitro. LncRNA-NEAT2 regulated the number and function of endothelial progenitor cells via miR-320 in sepsis, which may contribute to the development of novel potential clinical therapy for sepsis.

Hereditary angioedema with an acute attack resolved after bone marrow transplantation for acute myeloid leukemia: a case report

BackgroundHereditary angioedema (HAE), which is caused by C1-inhibitor (C1-INH) deficiency or dysfunction, is a rare and potentially life-threatening disease. In patients with HAE, excess production of bradykinin causes acute unpredictable recurrent attacks of angioedema in localized regions, including the larynx and intestines. Given the fact that HAE is an autosomal dominant disease, C1-INH produced in patients with HAE is 50% of that produced in healthy individuals. However, most patients with HAE present plasma C1-INH function of < 25% owing to the chronic consumption of C1-INH by kallikrein–kinin, contact, complement, coagulation, and fibrinolysis cascades. Recently, several therapeutic options have been developed for acute attacks and prophylaxis in the treatment of HAE; however, currently, there is no curative therapy for HAE.Case presentationHere we report the case of a 48-year-old male patient who presented with a long-standing history of HAE and underwent bone marrow transplantation (BMT) for acute myeloid leukemia (AML) at the age of 39 years and has been in complete remission of AML and HAE thereafter. Notably, after BMT, his C1-INH function gradually increased as follows: < 25%, 29%, 37%, and 45.6%. Since his 20 s, he intermittently presented with an acute attack of HAE once every 3 months from the initial attack. Further, after undergoing BMT, the number of acute attacks decreased to twice within 4 years until the age of 45 years, and subsequently, the patient has been free of acute attacks. C1-INH is mainly synthesized by hepatocytes, but it is known to be partially produced and secreted from peripheral blood monocytes, macrophages, endothelial cells, and fibroblasts. We speculate that the C1-INH function may be increased by extrahepatic production of C1-INH, possibly synthesized by differentiated cells derived from hematopoietic and mesenchymal stem cells after BMT.ConclusionsThis case report supports efforts to focus on extrahepatic production of C1-INH in the next strategy of new treatment development for HAE.

O006 Hepatitis A virus cellular receptor 1 (HAVCR1) influences the integrity of blood brain barrier (BBB) of cerebral endothelial cells

Abstract Introduction Hepatitis A virus cellular receptor 1 (HAVcR1) has been established to play an important role in tight junction (TJ) in cancer cells and in peripheral endothelial cells. Its role in blood brain barrier (BBB) of cerebral endothelium remains unknown. This study aimed to investigate the association and functionality of HAVcR1 in BBB. Methods HAVcR1 was knocked down by siRNA in human cerebral microvessel endothelial cell/D3 (hCMEC/D3) cell lines. Changes in tight junction (TJ) behaviour were assessed using electric cell impedance sensing (ECIS), transendothelial resistance (TER), and paracellular permeability (PCP). The expression of HAVcR1 and the binding partners of HAVcR1 protein was assessed using quantitative polymerase chain reaction (qPCR). Results The HAVcR1 gene transcript, highly expressed in hCMEC/D3 cells, was successfully knocked down from the cell by anti-HAVcR1 siRNA. HAVcR1 knockdown resulted in an increase in four TJ molecules and a decrease in eight TJ molecules as demonstrated by transcript analysis. HAVcR1 knockdown cells displayed lower electric resistance compared with wild type cells during initial attachment and spreading in hCMEC/D3 cells shown by ECIS. Similarly, TER in HAVcR1 knockdown cells were also reduced. Higher PCP fluorescence signals were detected in HAVcR1 knockdown cell lines compared with wild type. Conclusion HAVcR1 regulates paracellular leakage of cerebral endothelial cells and loss of HAVcR1 renders the cells with weaker tight junction by influencing TJ protein expression. HAVcR1 have a function as part of the regulatory apparatus for TJ in BBB.

Bilateral Simultaneous Penetrating Keratoplasty: A 40-Year Odyssey

Purpose: The purpose of this study was to report 40-year follow-up on a case of bilateral simultaneous penetrating keratoplasty (PK) involving a contralateral autograft. Methods: This was a case report. Results: A 39-year-old man developed right corneal scarring and lipid keratopathy from previous herpes zoster keratouveitis with visual acuity of 20/200. The left eye had dense amblyopia with visual acuity of 20/300. In 1980, he underwent right PK using a contralateral autograft and simultaneous left PK with an allograft. The right autograft provided excellent visual acuity until the graft failed 40 years later. The left allograft underwent severe endothelial graft rejection 2 months after surgery with gradual corneal clearance over the next 21 months. The left allograft remained clear through 40 years without graft failure. Conclusions: Long-term useful vision and graft survival were achieved with a penetrating autograft by avoiding endothelial graft rejection in the setting of previous herpes zoster keratitis and the patient being functionally monocular. Despite almost failing from severe endothelial graft rejection in the absence of ocular risk factors, the left allograft recovered and remained clear through 40 years possibly because peripheral host endothelial cells repopulated the donor.

Construction of Vascularized Tissue Engineered Bone with nHA-Coated BCP Bioceramics Loaded with Peripheral Blood-Derived MSC and EPC to Repair Large Segmental Femoral Bone Defect

The regenerative repair of segmental bone defect (SBD) is an urgent problem in the field of orthopedics. Rapid induction of angiogenesis and osteoinductivity after implantation of scaffold is critical. In this study, a unique tissue engineering strategy with mixture of peripheral blood-derived mesenchymal stem cells (PBMSC) and endothelial progenitor cells (PBEPC) was applied in a 3D-printed biphasic calcium phosphate (BCP) scaffold with highly bioactive nano hydroxyapatite (nHA) coating (nHA/BCP) to construct a novel vascularized tissue engineered bone (VTEB) for rabbit femoral SBD repair. The 2D coculture of PBMSC and PBEPC showed that they could promote the osteogenic or angiogenic differentiation of the cells from each other, especially in the group of PBEPC/PBMSC = 75:25. Besides, the 3D coculture results exhibited that the nHA coating could further promote PBEPC/PBMSC adhesion, proliferation, and osteogenic and angiogenic differentiation on the BCP scaffold. In vivo experiments showed that among the four groups (BCP, BCP-PBEPC/PBMSC, nHA/BCP, and nHA/BCP-PBEPC/PBMSC), the nHA/BCP-PBEPC/PBMSC group induced the best formation of blood vessels and new bone and, thus, the good repair of SBD. It revealed the synergistic effect of nHA and PBEPC/PBMSC on the angiogenesis and osteogenesis of the BCP scaffold. Therefore, the construction of VTEB in this study could provide a possibility for the regenerative repair of SBD.

ЭНДОТЕЛИАЛЬНАЯ ДИСФУНКЦИЯ В ОЦЕНКЕ СТЕПЕНИ ТЯЖЕСТИ ОСТРОГО ПАНКРЕАТИТА

Abstract. Introduction. The questions of the efficacy estimation of the treatment carried out at various forms of acute pancreatitis and the choice of the optimal terms of surgical treatment are still debatable. The given work is an attempt to find effective indications for the adequate treatment of patients with this pathology. Aim. Aim is to estimate the number of desquamated peripheral blood endothelial cells and degree of their coloring in patients with acute pancreatitis in the dynamics of the disease. Material and methods. We studied desquamated endothelial cells in 22 patients divided into 2 groups: group A – with acute edematous pancreatitis, and group B – with acute destructive pancreatitis with surgical treatment. We were studied the dynamics of the number of desquamated peripheral blood endothelial cells and degree of their coloring in both ways of treatment and their compliance with the scales that shows the severity of the disease. Results and discussion. In group A patients on day 7–9 while clinical and laboratory parameters of acute pancreatitis was decreased we noticed that the number of desquamated endothelial cells was decreased in the field of vision from 8 to 4,7. In group B the increasement of the parameters of the scales of criteria for acute pancreatitis and leukocyte intoxication has the same result – number of cells in the field of vision were decreased from 195 to 50. This patterns of the dynamics of the intensity of coloring of desquamated endothelial cells in both groups are reflected in the coloring coefficient, that was made by our study group. We found that in group A the color coefficient decreases from 1,5 to 0,2 (hyperchromically colored cells prevail), and in group B the coefficient increases from 0,8 to 1,2 (hypochromically colored cells increased). Conclusion. The study of the degree of endotheliocyte staining, reflecting the compensatory possibilities of the organism and treatment efficacy, is of great importance in the assessment of the severity and prognosis of acute pancreatitis treatment. An increasment of the color coefficient of desquamated endothelial cells by 0,4 may indicate that it’s time for surgical methods of treatment.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of Tcell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTβR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 Tcell progenitors that differentiate into GrzB+PD1+ CD8T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

Peripheral Blood Brain Microvascular Endothelial Cells As a Novel Marker in the Detection of Blood-Brain Barrier Injury in EV71 Severe Encephalopathy

Enterovirus 71 (EV71), the main causative agent of hand, foot, and mouth disease (HFMD), is also considered a neuroinvasive virus. EV71 causes sporadic epidemics and outbreaks with more than 13 million people worldwide, predominantly occurring in the Asia-Pacific, and elicited more than 3000 deaths, including 2457 deaths in China from 2008 to 2014. However, there are no available noninvasive biomarkers in peripheral blood for neuro-EV71 infection and optimizing therapeutic options. Brain microvascular endothelial cells (BMECs) are a special type of cells that constitute the blood-brain barrier (BBB), which dynamically shed associated with BBB injury. A mouse model of BBB injury induced by microbial and non-microbial agents was performed, using magnetic bead extraction (MBE) to identify circulating endothelial cells (CECs,CD146+/DAPI+), circulating BMECs (cBMECs, CD146+/S100B+/DAPI+) and endothelial progenitor cells (EPCs, CD146+/CD133+/DAPI+), which are identified based on their S100B (brain marker)+/ CD146 (EC marker) +/CD133+ (PC marker) /DAPI (nuclei)+phenotype with significantly higher levels in the experimental group than in the control group. These results indicate that it is feasible to evaluate BBB injury by quantifying BMECs and EPCs with MBE. The whole cell proteins were prepared by us from EV71 infected BMECs, and among the samples infected with EV71, MALDI TOF / TOF mass spectrometry successfully identified 28 proteins, 4 proteins including vimentin (VIM) were significantly up-regulated, 6 proteins were down regulated, and the other 18 proteins were expressed to various degrees at different incubation times. Furthermore, we performed a glioblastoma model with low vimentin expression and found that EV71 may activate the NLRP3 inflammasome via the VIM-ERK-NFκB pathway during EV71 CNS infection, which promotes caspase-1 activation and the inflammatory factor IL-1β release, causing an inflammatory response that ultimately leads to BBB/CNS disorders and neuronal death. The same results for those experiments with neonatal mice of EV71 infection were also observed. The results showed that cBMECs may be used as potential cell-based biomarkers for indexing of BBB injury. Then peripheral blood samples were obtained from the children with severe HFMD infection from a medical center for women and children, and examination showed that the counts of cBMECs were significantly higher in severe HFMD children than in the control children, indicating that the detection of cBMECs may be used for the early diagnosis of severe HFMD. In addition, Mfsd2 antibody specifically on BMECs were attached to magnetic beads and used directly to isolate BMECs from blood, followed by immunofluorescence staining with CD146 antibody, and the two corresponding fluorophores, CD146 and DPAI, appeared microscopically to preliminarily determine BMECs. The use of mfsd2 antibody to isolate BMECs both simplifies experimental procedures and improves detection precision and efficiency. In conclusion, in this study, cBMECs are demonstrated to serve as cellular biomarkers for BBB injury caused by microbial and non-microbial factors. Proteomics combined with mechanistic studies indicate that cBMECs are important indicators of BBB/CNS disorders in severe HFMD. Ultimately, this study successfully established a method to detect EV71 severe encephalopathy in human peripheral blood, which is safer and noninvasive than conventional cerebrospinal fluid puncture and pathological collection methods, causes minimal physical trauma to patients, and is more intuitive and accurate than conventional imaging examination. Therefore, this assay has good clinical application and may provide a new means for noninvasive and visual detection of EV71 severe encephalopathy. However, further cases were also collected to verify the accuracy of peripheral blood BMEC count for diagnosing severe HFMD, while establishing suitable cut-off values for application in early diagnosis. (Acknowledgements: Corresponding author: Hong Cao, gzhcao@smu.edu.cn; The Basic Research Project of Key Laboratory of Guangzhou, China (No.202102100001) and Grant from School of Public Health of Southern Medical University, China, No.GW202222 to H.C.)

P17.10: Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation. This work was carried out with the support of the “Cooperative Research Program for Agriculture Science and Technology Development [Project No. PJ015607 (HK and KO)], Rural Development Administration, Republic of Korea. This research was partly supported by Hallym University Research Fund (HL).

Exposure of human cerebral microvascular endothelial cells hCMEC/D3 to laminar shear stress induces vascular protective responses

Endothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). ECs are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral ECs, less is known about the molecular responses of microvascular brain ECs which constitute the blood–brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm−2 SS for 3 days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong protective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins and did not afect either the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain ECs to SS and on the importance of SS for optimizing in vitro BBB models.

1100-P: Endothelial Protein Expression and Its Association with Type 1 Diabetes

Introduction: People with type 1 diabetes (T1D) have an increased risk of developing cardiovascular disease. Endothelial cell dysfunction is thought to be a factor in this increased risk but underlying cellular mechanisms related to vasodilation, inflammation and oxidative stress are unknown. Methods: Participants with T1D (n= 25) and non-T1D (n=20) were categorized into groups of high (≥80) or low (&amp;lt;15 AU) coronary artery calcium (CAC) , measured by computed tomography. Protein expression was assessed for endothelial Nitric Oxide Synthase [eNOS], estrogen receptors (ER) α and ER-β (women only) , nuclear factor [NF]κβ and nicotinamide adenine dinucleotide phosphate [NADPH] using quantitative immunofluorescence in peripheral venous endothelial cells harvested by endovascular biopsy. SphygmoCor Pulse Wave Velocity (PWV) measured arterial stiffness. Linear regression was used to test associations of high vs. low CAC and T1D with proteins of interest. All models included age, sex, CAC and diabetes. Results: As shown in table 1, eNOS and NADPH were lower and NFκβ and Nitrotyrosine [NT] were higher in T1D compared to non-T1D participants with low CAC. Among T1D, PWV was higher in those with high vs. low CAC. Among non-T1D participants, eNOS was lower and NT and ER-α were lower in those with high vs. low CAC. Conclusions: T1D is associated with adverse endothelial proteins that may contribute to endothelial dysfunction and vascular disease. Disclosure H.K.Wise: None. A.Keshawarz: None. K.Moreau: None. J.K.Snell-bergeon: Stock/Shareholder; GlaxoSmithKline plc.