Expression Of Plasmalemma Vesicle-associated Protein Research Articles

Angiotensin-converting enzyme inhibitors provide a protective effect on hypoxia-induced injury in human coronary artery endothelial cells via Nrf2 signaling and PLVAP

BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEIs) were reported to protect from hypoxia-induced oxidative stress in coronary endothelial cells (CECs) after acute myocardial infarction (AMI). Nrf2 shows a protective effect in hypoxia-induced CECs after AMI. Plasmalemma vesicle-associated protein (PLVAP) plays a pivotal role in angiogenesis after AMI. AIM: To explore the protective effect of ACEIs and the involved mechanisms under hypoxia challenge. METHODS: Human coronary endothelial cells (HCAECs) were used to establish hypoxia-induced oxidative stress injury in vitro. Flow cytometry was used to evaluate the protective effect of ACEI on hypoxia conditions.ET-1, NO, ROS, and VEGF were detected by ELISA. HO-1, Nrf2, and Keap-1, the pivotal member in the Nrf2 signaling pathway, eNOS and PLVAP were detected in HEAECs treated with ACEI by immunofluorescence, qPCR, and western blotting. RESULTS: The hypoxia ACEI or Nrf2 agonist groups showed higher cell viability compared with the hypoxia control group at 24 (61.75±1.16 or 61.23±0.59 vs. 44.24±0.58, both P < 0.05) and 48 h (41.85±1.19 or 59.64±1.13 vs. 22.98±0.25, both P < 0.05). ACEI decreased the levels of ET-1 and ROS under hypoxia challenge at 24 and 48 h (all P < 0.05); ACEI increased the VEGF and NO levels (all P < 0.05). ACEI promoted the expression level of eNOS, HO-1, Nrf2 and PLVAP but inhibited Keap-1 expression at the mRNA and protein levels (all P < 0.05). Blockade of the Nrf2 signaling pathway significantly decreased the expression level of PLVAP. CONCLUSION: ACEI protects hypoxia-treated HEAECs by activating the Nrf2 signaling pathway and upregulating the expression of PLVAP.

Expression analysis of plvap in mouse heart development, homeostasis and injury

Plasmalemma vesicle associated protein (PLVAP) is commonly considered to be specifically expressed in endothelial cells in which it localized to diaphragms of caveolae, fenestrae, and transendothelial channels. PLVAP is reported to be an important regulator of heart development and a novel target to promote cardiac repair in the ischemic heart. However, the dynamics of plvap expression in heart development, homeostasis and pathology have not been comprehensively described. In this study, we analyzed the temporal and spatial expression of plvap in mouse heart under different conditions. We found that, during embryonic and neonatal stages, PLVAP was detected in endocardial endothelial cells, epicardial mesothelial cells, and a small amount of coronary vascular endothelial cells. In adult heart, PLVAP was also identified in endocardial cells and a few coronary vascular endothelial cells. However, epicardial expression of PLVAP was lost during postnatal heart development and cannot be detected in mouse heart by immunostaining since 3-week-old. We also analyzed the expression of plvap in a model of cardiac hypertrophy and failure induced by transverse aortic constriction surgery, and identified expression of PLVAP in endocardial cells and coronary vascular endothelial cells in the injured heart. This study provides new evidence to better understand the role of plvap in mouse heart development and injury.

The senescent secretome drives PLVAP expression in cultured human hepatic endothelial cells to promote monocyte transmigration

Liver sinusoidal endothelial cells (LSEC) undergo significant phenotypic change in chronic liver disease (CLD), and yet the factors that drive this process and the impact on their function as a vascular barrier and gatekeeper for immune cell recruitment are poorly understood. Plasmalemma-vesicle-associated protein (PLVAP) has been characterized as a marker of LSEC in CLD; notably we found that PLVAP upregulation strongly correlated with markers of tissue senescence. Furthermore, exposure of human LSEC to the senescence-associated secretory phenotype (SASP) led to a significant upregulation of PLVAP. Flow-based assays demonstrated that SASP-driven leukocyte recruitment was characterized by paracellular transmigration of monocytes while the majority of lymphocytes migrated transcellularly. Knockdown studies confirmed that PLVAP selectively supported monocyte transmigration mediated through PLVAP's impact on LSEC permeability by regulating phospho-VE-cadherin expression and endothelial gap formation. PLVAP may therefore represent an endothelial target that selectively shapes the senescence-mediated immune microenvironment in liver disease.

PLVAP protein expression correlated with microbial composition, clinicopathological features, and prognosis of patients with stomach adenocarcinoma.

Plasmalemma vesicle-associated protein (PLVAP) is involved in many immune‑related signals; however, its role in stomach adenocarcinoma (STAD) remains to be elucidated. This study investigated PLVAP expression in tumor tissues and defined the value in STAD patients. A total of 96 patient paraffin-embedded STAD specimens and 30 paraffin-embedded adjacent non-tumor specimens from the Ninth Hospital of Xi'an were consecutively recruited in analyses. All RNA‑sequence data were available from the Cancer Genome Atlas database (TCGA). PLVAP protein expression was detected using immunohistochemistry. Microbial community analysis was performed by 16S rRNA gene sequencing using Illumina MiSeq. PLVAP mRNA expression was explored with the Tumor Immune Estimation Resource (TIMER), GEPIA, and UALCAN databases. The effect of PLVAP mRNA on prognosis was analyzed via GEPIA, and Kaplan-Meier plotter database. GeneMANIA and STRING databases were used to predict gene/protein interactions and functions. The relationships between PLVAP mRNA expression and tumor-infiltrated immune cells were analyzed via the TIMER and GEPIA databases. Significantly elevated transcriptional and proteomic PLVAP expressions were found in STAD samples. Increased PLVAP protein and mRNA expression were significantly associated with advanced clinicopathological parameters and correlated with shorter disease-free survival (DFS) and overall survival (OS) in TCGA (P < 0.001). The microbiota in the PLVAP-rich (3+) group was significantly different from that in the PLVAP-poor (1+) group (P < 0.05). The results from TIMER showed that high PLVAP mRNA expression had significant positive correlations with CD4 + T cell (r = 0.42, P < 0.001). PLVAP is a potential biomarker to predict the prognosis of patients with STAD, and the high level of PLVAP protein expression was closely related to bacteria. The relative abundance of Fusobacteriia was positvely associated with the level of PLVAP. In conclusion, positive staining for PLVAP was useful for predicting the poor prognosis of STAD with Fusobacteriia infection.

PLVAP as an Early Marker of Glomerular Endothelial Damage in Mice with Diabetic Kidney Disease.

Plasmalemma vesicle-associated protein (PLVAP) is the main component of endothelial diaphragms in fenestrae, caveolae, and transendothelial channels. PLVAP is expressed in the adult kidney glomerulus upon injury. Glomerular endothelial injury is associated with progressive loss of kidney function in diabetic kidney disease (DKD). This study aimed to investigate whether PLVAP could serve as a marker for glomerular endothelial damage in DKD. Glomerular PLVAP expression was analyzed in different mouse models of DKD and their respective healthy control animals using automatic digital quantification of histological whole kidney sections. Transgenic mice expressing a dominant-negative GIP receptor (GIPRdn) in pancreatic beta-cells as a model for diabetes mellitus (DM) type 1 and black and tan brachyuric (BTBR) ob/ob mice, as a model for DM type 2, were used. Distinct PLVAP induction was observed in all diabetic models studied. Traces of glomerular PLVAP expression could be identified in the healthy control kidneys using automated quantification. Stainings for other endothelial injury markers such as CD31 or the erythroblast transformation-specific related gene (ERG) displayed no differences between diabetic and healthy groups at the time points when PLVAP was induced. The same was also true for the mesangial cells marker α8Integrin, while the podocyte marker nephrin appeared to be diminished only in BTBR ob/ob mice. Glomerular hypertrophy, which is one of the initial morphological signs of diabetic kidney damage, was observed in both diabetic models. These findings suggest that PLVAP is an early marker of glomerular endothelial injury in diabetes-induced kidney damage in mice.

PLVAP is associated with glioma-associated malignant processes and immunosuppressive cell infiltration as a promising marker for prognosis

Previous reports have confirmed the significance of plasmalemma vesicle-associated protein (PLVAP) in the progression of multiple tumors; however, there are few studies examining its immune properties in the context of gliomas. Here, we methodically investigated the pathophysiological characteristics and clinical manifestations of gliomas. A total of 699 patients diagnosed with gliomas in the cancer genome atlas along with 325 glioma patients in the Chinese glioma genome atlas were collected for the training and validation sets. We analyzed and visualized the total statistics using RStudio. PLVAP was markedly upregulated among high grade gliomas, O6-methylguanine-DNA methyltransferase promoter unmethylated subforms, isocitrate dehydrogenase wild forms, 1p19q non-codeletion subforms, and mesenchyme type gliomas. The receiver operating characteristics analysis illustrated the favorable applicability of PLVAP in regard to estimating mesenchyme subform gliomas. Subsequent Kaplan–Meier curves together with multivariable Cox analyses upon survival identified high-expression PLVAP as a distinct prognostic variable for patients with gliomas. Gene ontology analysis of PLVAP among gliomas has documented the predominant role of this protein in glioma-associated immunobiological processes and also in inflammatory responses. We consequently examined the associations of PLVAP with immune-related meta-genes, and PLVAP was positively correlated with hematopoietic cell kinase, lymphocyte-specific protein tyrosine kinase, major histocompatibility complex (MHC) I, MHC II, signal transducer and activator of transcription 1, and interferon and was negatively correlated with immunoglobulin G. Moreover, association analyses between PLVAP and glioma-infiltrating immunocytes indicated that the infiltrating degrees of most immune cells exhibited positive correlations with PLVAP expression, particularly immunosuppressive subsets such as tumor-related macrophages, myeloid-derived suppressor cells, and regulatory T lymphocytes. In summary, we originally demonstrated that PLVAP is markedly associated with immunosuppressive immune cell infiltration degrees, unfavorable survival, and adverse pathology types among gliomas, thus identifying PLVAP as a practicable marker and a promising target for glioma-based precise diagnosis and therapeutic strategies.

THU192 - Plasmalemma vesicle-associated protein expression is driven by senescent cell-endothelial crosstalk and shapes the immune landscape in chronic liver disease

THU192 - Plasmalemma vesicle-associated protein expression is driven by senescent cell-endothelial crosstalk and shapes the immune landscape in chronic liver disease

Stimulatory G-Protein α Subunit Modulates Endothelial Cell Permeability Through Regulation of Plasmalemma Vesicle-Associated Protein.

Endothelial cell leakage occurs in several diseases. Intracellular junctions and transcellular fashion are involved. The definite regulatory mechanism is complicated and not fully elucidated. The alpha subunit of the heterotrimeric G-stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of Gsα in the endothelial barrier remains unclear. In this study, mice with knockout of endothelial-specific Gsα (GsαECKO) were generated by crossbreeding Gsαflox/flox mice with Cdh5-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. GsαECKO mice displayed phenotypes of edema, anemia, hypoproteinemia and hyperlipoproteinemia, which indicates impaired microvascular permeability. Mechanistically, Gsα deficiency reduces the level of endothelial plasmalemma vesicle-associated protein (PLVAP). In addition, overexpression of Gsα increased phosphorylation of cAMP response element-binding protein (CREB) as well as the mRNA and protein levels of PLVAP. CREB could bind to the CRE site of PLVAP promoter and regulate its expression. Thus, Gsα might regulate endothelial permeability via cAMP/CREB-mediated PLVAP expression.

Plasmalemma vesicle-associated protein promotes angiogenesis in cholangiocarcinoma via the DKK1/CKAP4/PI3K signaling pathway.

Cholangiocarcinoma (CCA) is aggressive and has poor clinical outcomes because of typically delayed diagnosis and a lack of effective non-surgical therapeutic options. Recent studies have shown that plasmalemma vesicle-associated protein (PLVAP) is related to angiogenesis in various tumors, and in vivo PLVAP targeting therapy has been proven effective against hepatocellular carcinoma and pancreatic cancer. The goal of this study was to determine the potential therapeutic utility of targeting PLVAP and thus angiogenesis in CCA and explore the underlying molecular mechanisms. We found that the PLVAP expression levels were significantly higher in CCA tissues when compared with matched adjacent non-tumor tissues obtained from a total of 90 CCA patients; higher expression levels of PLVAP were associated with shorter overall survival of patients. In addition, overexpression of PLVAP was associated with higher micro-vessel density in CCA tissues. In a PLVAP overexpressing CCA patient-derived xenograft model, a novel humanized anti-PLVAP antibody in combination with Gemcitabine plus Cisplatin was significantly inhibited tumor growth. Molecular analysis of CCA cells co-cultured with human umbilical vascular endothelial cells or human hepatic sinusoidal endothelial cells showed that Dickkopf-related protein 1 (DKK1) secreted by CCA cells activated the PI3K/Akt pathway after binding to its receptor, cytoskeleton-associated protein 4 (CKAP4), resulting in the upregulation of PLVAP. Thus, CCA cells increased the angiogenic potency of endothelial cells in a paracrine fashion. Consistently, patients bearing CKAP4 and PLVAP overexpressing tumors had a poor prognosis. In conclusion, the DKK1/CKAP4/PI3K/PLVAP pathway increases angiogenesis in CCA and is therefore a potential anti-angiogenic target.

Picturing Breast Cancer Brain Metastasis Development to Unravel Molecular Players and Cellular Crosstalk

Simple SummaryBreast cancer is a devastating disorder affecting millions of women worldwide. With improved therapeutics for the primary tumor, the appearance of metastasis has been increasing. Breast cancer frequently metastasizes to the brain, constituting a major hurdle without cure and with a poor survival. It is imperative to better understand the mechanisms involved in malignant cell transposition of the brain microvasculature and parenchymal colonization by deciphering the alterations occurring in the tumor and microvascular cells, as well as the occurrence of intercellular communication during the process. We aimed to profile the process of the formation of breast cancer brain metastasis and the timeline of events governing it. We used a specific mouse model of the disease to perform extensive microscopic analyses. We identified phenotypic changes and the activation of relevant molecular players in tumorigenesis, together with vascular alterations, and the occurrence of crosstalk. Our findings unravel putative therapeutic targets to tackle breast cancer brain metastasis.With breast cancer (BC) therapy improvements, the appearance of brain metastases has been increasing, representing a life-threatening condition. Brain metastasis formation involves BC cell (BCC) extravasation across the blood–brain barrier (BBB) and brain colonization by unclear mechanisms. We aimed to disclose the actors involved in BC brain metastasis formation, focusing on BCCs’ phenotype, growth factor expression, and signaling pathway activation, correlating with BBB alterations and intercellular communication. Hippocampi of female mice inoculated with 4T1 BCCs were examined over time by hematoxylin-eosin, immunohistochemistry and immunofluorescence. Well-established metastases were observed at seven days, increasing thereafter. BCCs entering brain parenchyma presented mesenchymal, migratory, and proliferative features; however, with time, they increasingly expressed epithelial markers, reflecting a mesenchymal–epithelial transition. BCCs also expressed platelet-derived growth factor-B, β4 integrin, and focal adhesion kinase, suggesting autocrine and/or paracrine regulation with adhesion signaling activation, while balance between Rac1 and RhoA was associated with the motility status. Intercellular communication via gap junctions was clear among BCCs, and between BCCs and endothelial cells. Thrombin accumulation, junctional protein impairment, and vesicular proteins increase reflect BBB alterations related with extravasation. Expression of plasmalemma vesicle-associated protein was increased in BCCs, along with augmented vascularization, whereas pericyte contraction indicated mural cells’ activation. Our results provide further understanding of BC brain metastasis formation, disclosing potential therapeutic targets.

Transcriptional Dynamics of Hepatic Sinusoid‐Associated Cells After Liver Injury

Background and AimsHepatic sinusoidal cells are known actors in the fibrogenic response to injury. Activated hepatic stellate cells (HSCs), liver sinusoidal endothelial cells, and Kupffer cells are responsible for sinusoidal capillarization and perisinusoidal matrix deposition, impairing vascular exchange and heightening the risk of advanced fibrosis. While the overall pathogenesis is well understood, functional relations between cellular transitions during fibrogenesis are only beginning to be resolved. At single‐cell resolution, we here explored the heterogeneity of individual cell types and dissected their transitions and crosstalk during fibrogenesis.Approach and ResultsWe applied single‐cell transcriptomics to map the heterogeneity of sinusoid‐associated cells in healthy and injured livers and reconstructed the single‐lineage HSC trajectory from pericyte to myofibroblast. Stratifying each sinusoidal cell population by activation state, we projected shifts in sinusoidal communication upon injury. Weighted gene correlation network analysis of the HSC trajectory led to the identification of core genes whose expression proved highly predictive of advanced fibrosis in patients with nonalcoholic steatohepatitis (NASH). Among the core members of the injury‐repressed gene module, we identified plasmalemma vesicle–associated protein (PLVAP) as a protein amply expressed by mouse and human HSCs. PLVAP expression was suppressed in activated HSCs upon injury and may hence define hitherto unknown roles for HSCs in the regulation of microcirculatory exchange and its breakdown in chronic liver disease.ConclusionsOur study offers a single‐cell resolved account of drug‐induced injury of the mammalian liver and identifies key genes that may serve important roles in sinusoidal integrity and as markers of advanced fibrosis in human NASH.

Podocyte and endothelial-specific elimination of BAMBI identifies differential transforming growth factor-β pathways contributing to diabetic glomerulopathy

Transforming growth factor-β (TGF-β) is a central mediator of diabetic nephropathy. The effect of TGF-β, mediated by the type I TGF-β receptor, ALK5, and subsequent Smad2/3 activation results in podocyte apoptosis and loss. Previously, we demonstrated that the genetic deletion of the BMP and Activin Membrane-Bound Inhibitor (BAMBI), a negative modulator TGF-β signaling, accelerates diabetic nephropathy in mice. This was associated with heightened ALK1-mediated activation of Smad1/5 in the glomerular endothelial cells (ECs). Therefore, to evaluate the glomerular cell-specific effects of TGF-β in diabetic nephropathy we examined the effects of the podocyte- or EC-specific loss of Bambi (Pod-Bambi-/- or EC-Bambi-/-) in streptozotocin-induced diabetic mice with endothelial nitric oxide synthase deficiency. Interestingly, although hyperglycemia and body weight loss were similar in all groups of diabetic mice, significant hypertension was present only in the diabetic EC-Bambi-/- mice. While the podocyte or EC-specific loss of BAMBI both accelerated the progression of diabetic nephropathy, the worsened podocyte injury and loss observed in the diabetic Pod-Bambi-/- mice were associated with enhanced Smad3 activation. Increased Smad1/5 activation and EC proliferation were apparent only in the glomeruli of diabetic EC-Bambi-/- mice. The enhanced Smad1/5 activation in diabetic EC-Bambi-/- mice was associated with increased glomerular expression of plasmalemma vesicle-associated protein, pointing to the involvement of immature or dedifferentiated glomerular ECs in diabetic nephropathy. Notably, diabetic EC-Bambi-/- mice displayed podocyte injury and loss that were comparable to diabetic Pod-Bambi-/- mice. Thus, our results highlight the glomerular cell-specific contribution of TGF-β signaling and the intricate cross-talk between injured glomerular cells in the progression of diabetic nephropathy.

Retinal VEGFA maintains the ultrastructure and function of choriocapillaris by preserving the endothelial PLVAP

Fenestrations in choriocapillaris act as a window for molecular transports between the retina and choroid, and is crucial for maintaining visual function. Plasmalemma vesicle-associated protein (PLVAP) is essential for the development of endothelial fenestrations. There is little knowledge about how the choriocapillaris maintains the fenestrated endothelium. This study aimed to evaluate the role of vascular endothelial growth factor-A (VEGFA)-PLVAP axis in the maintenance of choroidal fenestrations using oxygen-induced retinopathy (OIR) model. In C57BL/6 J mice, the mice with OIR on postnatal day 12 (P12) presented thicker endothelium and less fenestration compared to the non-OIR mice. However, the OIR on 17 mice showed thinner endothelium with more fenestration compared to OIR on P12. In vivo angiography demonstrated the presence of hyperpermeable choroidal vessels on P17 in OIR mice. These dramatic changes in choriocapillaris were not observed in the BALB/cJ OIR model. The ultrastructural changes in the choriocapillaris were correlated with temporal variations in the expression of VEGFA and PLVAP. VEGFA stimulated expression of PLVAP in the choroidal endothelial cells. Loss of PLVAP disrupts the polarized structure of the choriocapillaris leading to retinal degeneration. These results indicate that the expression of retinal VEGFA is essential for maintaining the structure and function of choriocapillaris by preserving the endothelial PLVAP.

Detection of Intragraft Anti-Blood Group A and B Antibodies Following Renal Transplantation

BackgroundGraft immunocomplex capture fluorescence analysis is an attractive method to detect intragraft donor-specific anti-HLA antibodies. In ABO-incompatible transplantation, anti-A and B antibodies are also considered to be important donor specific antibodies (ABO-DSA). Therefore, it is useful to monitor intragraft ABO-DSAs to assess antibody-mediated rejection. MethodsTo capture A and B antigens, anti-Band III, von Willebrand factor (VW), and plasmalemma vesicle-associated protein (PLVAP) beads were produced. The allograft specimen was homogenized in a lysis buffer. Subsequently, A and B antigens were captured by anti-Band III, VW, or PLVAP beads. The immune complexes were then detected by phycoerythrin-conjugated anti-human IgG antibodies and analyzed using a Luminex system. ResultsAlthough Band III and VW beads yielded false positives and false negatives, PLVAP beads captured A and B antigens with high sensitivity (91.7%) and specificity (100%) when an index > 1.5 was considered positive. The proximity in A and B antigens and PLVAP expression was confirmed using immunohistochemical evaluation. Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis supported that PLVAP is an A and B antigen carrier protein. Case ReportBiopsies were conducted following an ABO-incompatible renal transplant (type A to O) and evaluated for ABO-DSA. Graft immunocomplex capture fluorescence analysis was demonstrated as follows: 3.19 (1 h, serum creatinine [s-Cr] 3.95 mg/dL, titer IgG 1:512, glomerulitis [g] 0, peritubular capillaritis [ptc] 0, complement 4d [C4d] 1); 1.8 (4 d, s-Cr 2.29 mg/dL, titer 1:256, g 0, ptc 0, C4d 3); 1.2 (22 d, s-Cr 1.58 mg/dL, titer 1:128, g 0, ptc 2, C4d 3). This result indicated that the remnant ABO-DSA were adsorbed and subsequently removed from the allograft successfully. ConclusionsThis novel application could be used to detect intragraft ABO-DSAs, which could lead to a correct diagnosis and shed light on the ABO-DSA kinetics following ABO-incompatible transplantation.

Phorbol esters induce PLVAP expression via VEGF and additional secreted molecules in MEK1-dependent and p38, JNK and PI3K/Akt-independent manner.

Endothelial diaphragms are subcellular structures critical for mammalian survival with poorly understood biogenesis. Plasmalemma vesicle associated protein (PLVAP) is the only known diaphragm component and is necessary for diaphragm formation. Very little is known about PLVAP regulation. Phorbol esters (PMA) are known to induce de novo PLVAP expression and diaphragm formation. We show that this induction relies on the de novo production of soluble factors that will act in an autocrine manner to induce PLVAP transcription and protein expression. We identified vascular endothelial growth factor‐A (VEGF‐A) signalling through VEGFR2 as a necessary but not sufficient downstream event as VEGF‐A inhibition with antibodies and siRNA or pharmacological inhibition of VEGFR2 only partially inhibit PLVAP upregulation. In terms of downstream pathways, inhibition of MEK1/Erk1/2 MAP kinase blocked PLVAP upregulation, whereas inhibition of p38 and JNK MAP kinases or PI3K and Akt had no effect on PMA‐induced PLVAP expression. In conclusion, we show that VEGF‐A along with other secreted proteins act synergistically to up‐regulate PLVAP in MEK1/Erk1/2 dependent manner, bringing us one step further into understanding the genesis of the essential structures that are endothelial diaphragms.

Glucocorticoids exert differential effects on the endothelium in an invitro model of the blood-retinal barrier.

Glucocorticoids (GCs) are used as treatment in diabetic macular oedema, a condition caused by blood-retinal barrier (BRB) disruption. The proposed mechanisms by which GCs reduce macular oedema are indirect anti-inflammatory effects and inhibition of VEGF production, but direct effects on the BRB endothelium may be equally important. Here, we investigated direct effects of GCs on the endothelium to understand the specific pathways of GC action, to enable development of novel therapeutics lacking the adverse side-effects of the presently used GCs. Primary bovine retinal endothelial cells (BRECs) were grown on Transwell inserts and treated with hydrocortisone (HC), dexamethasone (Dex) or triamcinolone acetonide (TA). Molecular barrier integrity of the BRB was determined by mRNA and protein expression, and barrier function was assessed using permeability assays. In addition, we investigated whether TA was able to prevent barrier disruption after stimulation with VEGF or cytokines. Treatment of BRECs with GCs resulted in upregulation of tight junction mRNA (claudin-5, occludin, ZO-1) and protein (claudin-5 and ZO-1). In functional assays, only TA strengthened the barrier function by reducing endothelial permeability. Moreover, TA was able to prevent cytokine-induced permeability in human retinal endothelial cells and VEGF-induced expression of plasmalemma vesicle-associated protein (PLVAP), a key player in VEGF-induced retinal vascular leakage. Glucocorticoids have differential effects in an experimental invitro BRB model. TA is the most potent in improving barrier function, both at the molecular and functional levels, and TA prevents VEGF-induced expression of PLVAP.

PLVAP and GKN3 Are Two Critical Host Cell Receptors Which Facilitate Japanese Encephalitis Virus Entry Into Neurons

Japanese Encephalitis Virus (JEV), a globally important pathogen, belongs to the family Flaviviridae, is transmitted between vertebrate hosts by mosquitoes, principally by Culex tritaeniorhynchus. The E-glycoprotein of the virus mediates its attachment to the host cell receptors. In this study, we cloned and purified JEV E-glycoprotein in pET28a vector using E. coli BL21 (DE3) cells. A pull down assay was performed using plasma membrane fraction of BALB/c mouse brain and E-glycoprotein as a bait protein. 2-Dimensional Gel Electrophoresis based separation of the interacting proteins was analyzed by mass spectrometry. Among all the identified partners of E-glycoprotein, PLVAP (Plasmalemma vesicle associated protein) and GKN3 (Gastrokine3) showed significant up-regulation in both JEV infected mouse brain and neuro2a cells. In-silico studies also predicted significant interaction of these receptors with E-glycoprotein. Additionally, overexperssion and silencing of these receptors resulted in increase and reduction in viral load respectively, suggesting them as two critical cellular receptors governing JEV entry and propagation in neurons. In support, we observed significant expression of PLVAP but not GKN3 in post-mortem autopsied human brain tissue. Our results establish two novel receptor proteins in neurons in case of JEV infection, thus providing potential targets for antiviral research.

A Novel Choroidal Endothelial Cell Line Has a Decreased Affinity for the Age-Related Macular Degeneration–Associated Complement Factor H Variant 402H

Choroidal endothelial cells play a central role in the pathogenesis of age-related macular degeneration (AMD). Protocols for isolating primary choroidal endothelial cells have been described but require access to human donor eyes, which is a limiting factor. Therefore, a conditionally immortalized choroidal endothelial cell (ciChEnC) line has been established. Choroidal endothelial cells were selected by magnetic-activated cell sorting and conditionally immortalized using temperature-sensitive simian virus 40 large T antigen and human telomerase. The cell line obtained was characterized based on expression of endothelial marker proteins and endothelial cell-specific responses to various stimuli. Binding of AMD-associated and non-AMD variants of complement factor H in the context of a recombinant CCP6-8 (complement control protein domains 6-8) construct was determined using ELISA. ciChEnCs maintained morphology and von Willebrand factor and vascular endothelial cadherin expression for up to 27 passages. The cells internalized acetylated low-density lipoprotein, formed tubes on Matrigel, and increased intercellular adhesion molecule-1 expression in response to tumor necrosis factor-α. Cells grew into dense monolayers with barrier function and showed characteristics of choriocapillary cells, such as expression of plasmalemma vesicle-associated protein, human leukocyte antigen ABC, carbonic anhydrase IV, and membrane indentations reflecting fenestrations. ciChEnCs synthesized glycosaminoglycans chondroitin sulfate and the complement factor H ligand heparan sulfate. Interestingly, binding of the AMD-associated 402H variant of factor H to ciChEnC was significantly decreased compared to the 402Y variant. A novel ciChEnC cell line with choriocapillary characteristics has been established and should greatly facilitate investigation of the pathogenesis of AMD in the context of the choriocapillary microenvironment.

Plasmalemma vesicle-associated protein: A crucial component of vascular homeostasis.

Endothelial subcellular structures, including caveolae, fenestrae and transendothelial channels, are crucial for regulating microvascular function. Plasmalemma vesicle-associated protein (PLVAP) is an endothelial cell-specific protein that forms the stomatal and fenestral diaphragms of blood vessels and regulates basal permeability, leukocyte migration and angiogenesis. Loss of PLVAP in mice leads to premature mortality due to disrupted homeostasis. Evidence from previous studies suggested that PLVAP is involved in cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Specifically, PLVAP expression has been demonstrated to be upregulated in these diseases, accompanied by pro-angiogenic or pro-inflammatory responses. Therefore, PLVAP is considered a novel therapeutic target, in addition to an endothelial cell marker. The present review summarizes the structure and functions of PLVAP, and its roles in pathophysiological processes.

Plasmalemma Vesicle–Associated Protein Has a Key Role in Blood-Retinal Barrier Loss

Loss of blood-retinal barrier (BRB) properties induced by vascular endothelial growth factor (VEGF) and other factors is an important cause of diabetic macular edema. Previously, we found that the presence of plasmalemma vesicle-associated protein (PLVAP) in retinal capillaries associates with loss of BRB properties and correlates with increased vascular permeability in diabetic macular edema. In this study, we investigated whether absence of PLVAP protects the BRB from VEGF-induced permeability. We used lentiviral-delivered shRNA or siRNA to inhibit PLVAP expression. The barrier properties of in vitro BRB models were assessed by measuring transendothelial electrical resistance, permeability of differently sized tracers, and the presence of endothelial junction complexes. The effect of VEGF on caveolae formation was studied in human retinal explants. BRB loss in vivo was studied in the mouse oxygen-induced retinopathy model. The inhibition of PLVAP expression resulted in decreased VEGF-induced BRB permeability of fluorescent tracers, both in vivo and in vitro. PLVAP inhibition attenuated transendothelial electrical resistance reduction induced by VEGF in BRB models in vitro and significantly increased transendothelial electrical resistance of the nonbarrier human umbilical vein endothelial cells. Furthermore, PLVAP knockdown prevented VEGF-induced caveolae formation in retinal explants but did not rescue VEGF-induced alterations in endothelial junction complexes. In conclusion, PLVAP is an essential cofactor in VEGF-induced BRB permeability and may become an interesting novel target for diabetic macular edema therapy.