Plasmalemma Vesicle-associated Protein Research Articles

Immunohistochemical and Morphometric Assessment on the Biological Function and Vascular Endothelial Cells in the Initial Process of Cortical Porosity in Mice With PTH Administration.

To clarify the cellular mechanism of cortical porosity induced by intermittent parathyroid hormone (PTH) administration, we examined the femoral cortical bone of mice that received 40 µg/kg/day (four times a day) human PTH (hPTH) (1-34). The PTH-driven cortical porosity initiated from the metaphyseal region and chronologically expanded toward the diaphysis. Alkaline phosphatase (ALP)-positive osteoblasts in the control mice covered the cortical surface, and endomucin-positive blood vessels were distant from these osteoblasts. In PTH-administered mice, endomucin-reactive blood vessels with TRAP-positive penetrated the ALP-positive osteoblast layer, invading the cortical bone. Statistically, the distance between endomucin-positive blood vessels and the cortical bone surface abated after PTH administration. Transmission electron microscopic observation demonstrated that vascular endothelial cells often pass through the flattened osteoblast layer and accompanied osteoclasts in the deep region of the cortical bone. The cell layers covering mature osteoblasts thickened with PTH administration and exhibited ALP, α-smooth muscle actin (αSMA), vascular cell adhesion molecule-1 (VCAM1), and receptor activator of NF-κB ligand (RANKL). Within these cell layers, osteoclasts were found near endomucin-reactive blood vessels. In PTH-administered femora, osteocytes secreted Dkk1, a Wnt inhibitor that affects angiogenesis, and blood vessels exhibited plasmalemma vesicle-associated protein, an angiogenic molecule. In summary, endomucin-positive blood vessels, when accompanied by osteoclasts in the ALP/αSMA/VCAM1/RANKL-reactive osteoblastic cell layers, invade the cortical bone, potentially due to the action of osteocyte-derived molecules such as DKK1.

A sophisticated virulence repertoire and colistin resistance of Citrobacter freundii ST150 from a patient with sepsis admitted to ICU in a tertiary care hospital in Uganda, East Africa: Insight from genomic and molecular docking analyses

Sepsis and multidrug resistance comprise a complex of factors attributable to mortality among intensive care unit (ICU) patients globally. Pathogens implicated in sepsis are diverse, and their virulence and drug resistance remain elusive. From a tertiary care hospital ICU in Uganda, we isolated a Citrobacter freundii strain RSM030 from a patient with sepsis and phenotypically tested it against a panel of 16 antibiotics including imipenem levofloxacin, cotrimoxazole and colistin, among others. We sequenced the organism's genome and integrated multilocus sequencing (MLST), PathogenFinder with Virulence Factor analyzer (VFanalyzer) to establish its pathogenic relevance. Thereafter, we combined antiSMASH and PRISM genome mining with molecular docking to predict biosynthetic gene clusters (BGCs), pathways, toxin structures and their potential targets in-silico. Finally, we coupled ResFinder with comprehensive antibiotic resistance database (CARD) to scrutinize the genomic antimicrobial resistance profile of the isolate. From PathogenFinder and MLST, this organism was confirmed to be a human pathogen (p = 0.843), sequence type (ST)150, whose virulence is determined by chromosomal type III secretion system (T3SS) (the injectosome) and plasmid-encoded type IV secretion system (T4SS), the enterobactin biosynthetic gene cluster and biofilm formation through the pgaABCD operon. Pathway and molecular docking analyses revealed that the shikimate pathway can generate a toxin targeting multiple host proteins including spectrin, detector of cytokinesis protein 2 (Dock2) and plasmalemma vesicle-associated protein (PLVAP), potentially distorting the host cell integrity. From phenotypic antibiotic testing, we found indeterminate results for amoxicillin/clavulanate and levofloxacin, with resistance to cotrimoxazole and colistin. Detailed genome analysis revealed chromosomal beta lactam resistance genes, i.e. blaCMY-79, blaCMY-116 and blaTEM-1B, along with multiple mutations of the lipopolysaccharide modifying operon genes PmrA/PmrB, pmrD, mgrA/mgrB and PhoP/PhoQ, conferring colistin resistance. From these findings, we infer that Citrobacter freundii strain RSM030 is implicated in sepsis and resistance to standard antibiotics, including colistin, the last resort.

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.

From Homogeneity to Turing Pattern: Kinetically Controlled Self-Organization of Transmembrane Protein.

Understanding the spatial organization of membrane proteins is crucial for unraveling key principles in cell biology. The reaction-diffusion model is commonly used to understand biochemical patterning; however, applying reaction-diffusion models to subcellular phenomena is challenging because of the difficulty in measuring protein diffusivity and interaction kinetics in the living cell. In this work, we investigated the self-organization of the plasmalemma vesicle-associated protein (PLVAP), which creates regular arrangements of fenestrated ultrastructures, using single-molecule tracking. We demonstrated that the spatial organization of the ultrastructures is associated with a decrease in the association rate by actin destabilization. We also constructed a reaction-diffusion model that accurately generates a hexagonal array with the same 130 nm spacing as the actual scale and informs the stoichiometry of the ultrastructure, which can be discerned only through electron microscopy. Through this study, we integrated single-molecule experiments and reaction-diffusion modeling to surpass the limitations of static imaging tools and proposed emergent properties of the PLVAP ultrastructure.

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.

Downregulation of VEGF in the retinal pigment epithelium followed by choriocapillaris atrophy after NaIO3 treatment in mice

Sodium iodate (NaIO3) induces retinal pigment epithelium (RPE) dysfunction, which leads to photoreceptor degeneration. Previously, we used electron microscopy to show that the administration of NaIO3 resulted in the accumulation of cell debris in the subretinal space, which was thought to be caused by failed phagocytosis in the outer segment of the photoreceptor due to RPE dysfunction. We further analyzed the pathological changes in the retina and choroid of NaIO3-injected mice, and found that the expression of OTX2, an RPE marker, disappeared from central part of the RPE 1 day after NaIO3 administration. Furthermore, fenestrated capillaries (choriocapillaris, CC) adjacent to the RPE could not be identified only 2 days after NaIO3 administration. An examination of the expression of the CC-specific protein plasmalemma vesicle-associated protein (PLVAP), in sections and flat-mount retina/choroid specimens showed destruction of the CC, and complete disappearance of the PLVAP signal 7 days after NaIO3 administration. In contrast, CD31 flat-mount immunohistochemistry of the retina indicated no difference in retinal vessels between NaIO3-treated mice and controls. Electron microscopy showed that the fenestrated capillaries in the kidney and duodenum were morphologically indistinguishable between control and NaIO3-treated mice. We examined cytokine production in the retina and RPE, and found that the Vegfa transcript level in the RPE decreased starting 1 day after NaIO3 administration. Taken together, these observations show that NaIO3 reduces the CC in the early stages of the pathology, which is accompanied by a rapid decrease in Vegfa expression in the RPE.

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

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.

Regulation of fenestra formation via actin-dynamin2 interaction in rat pituitary endothelial cells.

Endothelial fenestrae are transcellular pores divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). They function as a channel for peptide hormones and other substances. Invagination of the plasma membrane is necessary for the fenestra formation. The actin cytoskeleton is essential for scission of endocytic vesicles from the invaginated plasma membrane. Therefore, we examined the involvement of the actin cytoskeleton in fenestra formation in cultured endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, using immunofluorescence and scanning electron microscopy. Inhibition of polymerization and depolymerization of the actin cytoskeleton by latrunculin A and jasplakinolide, respectively, remarkably increased the PLVAP-positive sieve plate area and number of fenestrae. Jasplakinolide significantly affected the arrangement of the fenestra on the cell surface, resulting in parallel serpentine furrows of the fenestra. These results suggest that the actin cytoskeleton not only induces fenestra formation but also regulates cell arrangement. Dynamin is a scission protein of the invaginated plasma membrane and interacts with the actin cytoskeleton. We found that dynamin2 is mainly expressed in the endothelial cells of the rat AL. We then investigated the function of dynamin2 by the treatment with dyngo-4a, a potent inhibitor of dynamin1 and dynamin2, on the fenestra formation. As a result, the PLVAP-positive area is significantly increased by the treatment. These results show that the actin-dynamin2 interaction is essential for the control of the fenestra formation in endothelial cells of rat AL. In conclusion, the actin cytoskeleton and dynamin2 function as regulators of endothelial fenestra formation.

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.

Analysis of Non-Relapsed and Relapsed Adult Type Granulosa Cell Tumors Suggests Stable Transcriptomes during Tumor Progression.

Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3′ mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2 (FOXL2) mutation. We compared the transcriptomic profiles of 14 non-relapsed archival primary AGCTs (follow-up time 17–26 years after diagnosis) with 13 relapsed primary AGCTs (follow-up time 1.7–18 years) and eight relapsed tumors (follow-up time 2.8–18.9 years). Non-relapsed and relapsed primary AGCTs had similar transcriptomic profiles. In relapsed tumors three genes were differentially expressed: plasmalemma vesicle associated protein (PLVAP) was upregulated (p = 0.01), whereas argininosuccinate synthase 1 (ASS1) (p = 0.01) and perilipin 4 (PLIN4) (p = 0.02) were downregulated. PLVAP upregulation was validated using tissue microarray RNA in situ hybridization. In our patient cohort with extremely long follow-up, we observed similar gene expression patterns in both primary AGCT groups, suggesting that relapse is not driven by transcriptomic changes. These results reinforce earlier findings that molecular markers do not predict AGCT behavior or risk of relapse.

Blocking of VEGF-A is not sufficient to completely revert its long-term effects on the barrier formed by retinal endothelial cells

The VEGF-A-induced functional impairment of the barrier formed by retinal endothelial cells (REC) can be prevented and even – at least temporarily – reverted by trapping the growth factor in a complex with a VEGF-binding protein or by inhibiting the activity of the VEGF receptor 2 (VEGFR2). In an approach to emulate the clinically relevant situation of constant exposure to effectors, we investigated (1) whether prolonged exposure to VEGF-A165 for up to six days results in a different type of disturbance of the barrier formed by immortalized bovine REC (iBREC) and (2) whether alterations of the barrier induced by VEGF-A165 can indeed be sustainably reverted by subsequent treatment with the VEGF-A-binding proteins ranibizumab or brolucizumab. As a measure of barrier integrity, the cell index (CI) of iBREC cultivated on gold electrodes was monitored continuously. CI values declined shortly after addition of the growth factor and then remained low for more than six days over which considerable amounts of both extra- and intracellular VEGF-A were measured. Interestingly, the specific VEGFR2 inhibitor nintedanib normalized the lowered CI when added to iBREC pre-treated with VEGF-A165 for one day, but failed to do so when cells had been exposed to the growth factor for six days. Expression of the tight junction (TJ) protein claudin-5 was unchanged early after addition of VEGF-A165 but higher after prolonged treatment, whereas decreased amounts of the TJ-protein claudin-1 remained low, and increased expression of the plasmalemma vesicle-associated protein (PLVAP) remained high during further exposure. After two days, the characteristic even plasma membrane stainings of claudin-1 or claudin-5 appeared weaker or disordered, respectively. After six days the subcellular localization of claudin-5 was similar to that of control cells again, but claudin-1 remained relocated from the plasma membrane. To counteract these effects of VEGF-A165, brolucizumab or ranibizumab was added after one day, resulting in recovery of the then lowered CI to normal values within a few hours. However, despite the VEGF antagonist being present, the CI declined again two days later to values that were just slightly higher than without VEGF inhibition during further assessment for several days. At this stage, neither the supernatants nor whole cell extracts from iBREC treated with VEGF-A165 and its antagonists contained significant amounts of free VEGF-A. Treatment of VEGF-A165-challenged iBREC with ranibizumab or brolucizumab normalized expression of claudin-1 and claudin-5, but not completely that of PLVAP. Interestingly, the characteristic VEGF-A165-induced relocalization of claudin-1 from the plasma membrane was reverted within one day by any of the VEGF antagonists, but reappeared despite their presence after further exposure for several days. Taken together, barrier dysfunction induced by VEGF-A165 results from deregulated para- and transcellular flow but the precise nature or magnitude of underlying changes on a molecular level clearly depend on the time of exposure, evolving into a stage of VEGF-A165-independent barrier impairment. These findings also provide a plausible explanation for resistance to treatment with VEGF-A antagonists frequently observed in clinical practice.

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.

Inflammatory and Oxidative Pathways Are New Drug Targets in Multiple Episode Schizophrenia and Leaky Gut, Klebsiella pneumoniae, and C1q Immune Complexes Are Additional Drug Targets in First Episode Schizophrenia.

Breakdown of paracellular and vascular pathways and activated neuroimmune and oxidative pathways was established in (deficit) schizophrenia. The aim of this study was to delineate (a) the differences in these pathways between stable-phase, first (FES) and multiple (MES) episode schizophrenia and (b) the pathways that determine the behavioral-cognitive-physical-psychosocial (BCPS) deterioration in FES/MES. This study included 21 FES and 58 FES patients and 40 healthy controls and measured indicants of serum C1q circulating immune complexes (CIC), leaky gut, immune activation, and oxidative stress toxicity (OSTOX). We constructed a BCPS-worsening index by extracting a latent vector from symptomatic, neurocognitive, and quality of life data. FES was associated with higher IgA CIC-C1q, IgA directed to cadherin, catenin, and plasmalemma vesicle-associated protein, and IgA/IgM to Gram-negative bacteria as compared with FES and controls. In FES patients, the BCPS-worsening score was predicted (48.7%) by IgA to Klebsiella pneumoniae and lowered paraoxonase 1 activity. In MES patients, the BCPS-worsening score was explained (42.7%) by increased tumor necrosis factor-α, OSTOX, and number of episodes. In schizophrenia, 34.0% of the variance in the BCPS-worsening score was explained by IgA to K. pneumoniae, OSTOX, and number of episodes. Increased IgA to K. pneumoniae was the single best predictor of residual psychotic symptoms in FES and MES. This study delineated different mechanistic processes in FES, including breakdown of adherens junctions, bacterial translocation, and IgA CIC-C1q formation, and MES, including immune and oxidative neurotoxic pathways. FES and MES comprise different staging subtypes, i.e., FES and MES with and without worsening.

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.

Fibronectin-integrin signaling regulates PLVAP localization at endothelial fenestrae by microtubule stabilization

Endothelial fenestrae are the transcellular pores existing on the capillary walls which are organized in clusters referred to as sieve plates. They are also divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). In this study, we examined the involvement of fibronectin signaling in the formation of fenestra and diaphragm in endothelial cells. Results showed that Itga5 and Itgb1 were expressed in PECAM1-positive endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, and integrin α5 was localized at the fenestrated capillaries of the rat pituitary and cultured PECAM1-positive endothelial cells isolated from AL (CECAL). Inhibition of both integrin α5β1 and FAK, a key molecule for integrin-microtubule signaling, respectively, by ATN-161 and FAK inhibitor 14, caused the delocalization of PLVAP at the sieve plates and depolymerization of microtubules in CECAL. Paclitaxel prevented the delocalization of PLVAP by the inhibition of integrin α5β1. Microtubule depolymerization induced by colcemid also caused the delocalization of PLVAP. Treatment of CECAL with ATN-161 and colcemid caused PLVAP localization at the Golgi apparatus. The localization of PLVAP at the sieve plates was inhibited by BFA treatment in a time-dependent manner and spread diffusely to the cytoplasm. These results indicate that a constant supply of PLVAP proteins by the endomembrane system via the Golgi apparatus is essential for the localization of PLVAP at sieve plates. In conclusion, the endomembrane transport pathway from the Golgi apparatus to sieve plates requires microtubule cytoskeletons, which are regulated by fibronectin-integrin α5β1 signaling.