Onco-fetal Reprogramming of Endothelial Cells Drives Immunosuppressive Macrophages in Hepatocellular Carcinoma.

We employed scRNA sequencing to extensively characterize the cellular landscape of human liver from development to disease. Analysis of ∼212,000 cells representing human fetal, hepatocellular carcinoma (HCC), and mouse liver revealed remarkable fetal-like reprogramming of the tumor microenvironment. Specifically, the HCC ecosystem displayed features reminiscent of fetal development, including re-emergence of fetal-associated endothelial cells (PLVAP/VEGFR2) and fetal-like (FOLR2) tumor-associated macrophages. In a cross-species comparative analysis, we discovered remarkable similarity between mouse embryonic, fetal-liver, and tumor macrophages. Spatial transcriptomics further revealed a shared onco-fetal ecosystem between fetal liver and HCC. Furthermore, gene regulatory analysis, spatial transcriptomics, and in vitro functional assays implicated VEGF and NOTCH signaling in maintaining onco-fetal ecosystem. Taken together, we report a shared immunosuppressive onco-fetal ecosystem in fetal liver and HCC. Our results unravel a previously unexplored onco-fetal reprogramming of the tumor ecosystem, provide novel targets for therapeutic interventions in HCC, and open avenues for identifying similar paradigms in other cancers and disease.

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.

Caveolae-mediated endocytosis pathway regulates endothelial fenestra homeostasis in the rat pituitary.

Endothelial fenestrae are transcellular pores separated by diaphragms formed by plasmalemma vesicle-associated proteins (PLVAP) and function as channels for peptide hormones and other substances. Caveola, a key regulator of clathrin-independent endocytosis, may be involved in the invagination and fusion of plasma membranes, which are essential for fenestra formation. In this study, we first found that caveolin-1 and -2, the major components of caveolae, was localized in fenestrated endothelial cells in the anterior lobe of the rat pituitary by immunohistochemistry. As we also observed caveolae in the endothelial cells of the anterior lobe of the rat pituitary by transmission electron microscopy, we studied the relationship between the caveolae-mediated endocytosis pathway and fenestrae structure in cultured endothelial cells isolated from the anterior lobe of the rat pituitary (CECAL) by immunofluorescence staining and scanning electron microscopy. The inhibition of caveolae-mediated endocytosis by genistein enlarged the PLVAP-positive oval-shaped structure that represented the sieve plate and induced the formation of a doughnut-shaped bulge around the fenestra in CECAL. In contrast, the acceleration of caveolae-mediated endocytosis by okadaic acid induced the diffusion of PLVAP-positive signals in the cytoplasm and reduced the number of fenestrae in CECAL. These results indicate that the caveolae-mediated endocytosis pathway is involved in the fenestra homeostasis in the fenestrated endothelial cells of the rat pituitary.

The role of PLVAP in endothelial cells.

Endothelial cells play a major part in the regulation of vascular permeability and angiogenesis. According to their duty to fit the needs of the underlying tissue, endothelial cells developed different subtypes with specific endothelial microdomains as caveolae, fenestrae and transendothelial channels which regulate nutrient exchange, leukocyte migration, and permeability. These microdomains can exhibit diaphragms that are formed by the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), the only known protein component of these diaphragms. Several studies displayed an involvement of PLVAP in diseases as cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Besides an upregulation of PLVAP expression within these diseases, pro-angiogenic or pro-inflammatory responses were observed. On the other hand, loss of PLVAP in knockout mice leads to premature mortality due to disrupted homeostasis. Generally, PLVAP is considered as a major factor influencing the permeability of endothelial cells and, finally, to be involved in the regulation of vascular permeability. Following these observations, PLVAP is debated as a novel therapeutic target with respect to the different vascular beds and tissues. In this review, we highlight the structure and functions of PLVAP in different endothelial types in health and disease.

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.

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 α5ß1 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 α5ß1. 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 α5ß1 signaling.

Fibronectin is essential for formation of fenestrae in endothelial cells of the fenestrated capillary.

Endothelial fenestrae are transcellular pores that pierce the capillary walls in endocrine glands such as the pituitary. The fenestrae are covered with a thin fibrous diaphragm consisting of the plasmalemma vesicle-associated protein (PLVAP) that clusters to form sieve plates. The basal surface of the vascular wall is lined by basement membrane (BM) composed of various extracellular matrices (ECMs). However, the relationship between the ECMs and the endothelial fenestrae is still unknown. In this study, we isolated fenestrated endothelial cells from the anterior lobe of the rat pituitary, using a dynabeads-labeled antibody against platelet endothelial cell adhesion molecule 1 (PECAM1). We then analyzed the gene expression levels of several endothelial marker genes and genes for integrin α subunits, which function as the receptors for ECMs, by real-time polymerase chain reaction (PCR). The results showed that the genes for the integrin α subunit, which binds to collagen IV, fibronectin, laminin-411, or laminin-511, were highly expressed. When the PECAM1-positive cells were cultured for 7 days on collagen IV-, fibronectin-, laminins-411-, or laminins-511-coated coverslips, the sieve plate structures equipped with probably functional fenestrae were maintained only when the cells were cultured on fibronectin. Additionally, real-time PCR analysis showed that the fibronectin coating was effective in maintaining the expression pattern of several endothelial marker genes that were preferentially expressed in the endothelial cells of the fenestrated capillaries. These results indicate that fibronectin functions as the principal factor in the maintenance of the sieve plate structures in the endothelial cells of the fenestrated capillary.

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

Transforming growth factor-ß (TGF-ß) is a central mediator of diabetic nephropathy. The effect of TGF-ß, mediated by the type I TGF-ß 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-ß 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-ß 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-ß 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.

Mutations in plasmalemma vesicle-associated protein cause severe syndromic protein-losing enteropathy.

BACKGROUND: Protein-losing enteropathy (PLE) is characterised by gastrointestinal protein leakage due to loss of mucosal integrity or lymphatic abnormalities. PLE can manifest as congenital diarrhoea and should be differentiated from other congenital diarrhoeal disorders. Primary PLEs are genetically heterogeneous and the underlying genetic defects are currently emerging. OBJECTIVES: We report an infant with fatal PLE for whom we aimed to uncover the underlying pathogenic mutation. METHODS: We performed whole exome sequencing (WES) for the index patient. Variants were classified based on the American College of Medical Genetics and Genomics guidelines. WES results and our detailed clinical description of the patient were compared with the literature. RESULTS: We discovered a novel homozygous stop mutation (c.988C>T, p.Q330*) in the Plasmalemma Vesicle-Associated Protein (PLVAP) gene in a newborn with fatal PLE, facial dysmorphism, and renal, ocular and cardiac anomalies. The Q330* mutation is predicted to result in complete loss of PLVAP protein expression leading to deletion of the diaphragms of endothelial fenestrae, resulting in plasma protein extravasation and PLE. Recently, another single homozygous stop mutation in PLVAP causing lethal PLE in an infant was reported. CONCLUSIONS: Our findings validate PLVAP mutations as a cause of syndromic PLE. Prenatal anomalies, severe PLE and syndromic features may guide the diagnosis of this rare disease.

Establishing the role of PLVAP in protein-losing enteropathy: a homozygous missense variant leads to an attenuated phenotype.

BACKGROUND: Intestinal integrity is essential for proper nutrient absorption and tissue homeostasis, with damage leading to enteric protein loss, that is, protein-losing enteropathy (PLE). Recently, homozygous nonsense variants in the plasmalemma vesicle-associated protein gene (PLVAP) were reported in two patients with severe congenital PLE. PLVAP is the building block of endothelial cell (EC) fenestral diaphragms; its importance in barrier function is supported by mouse models of Plvap deficiency. OBJECTIVE: To genetically diagnose two first-degree cousins once removed, who presented with PLE at ages 22 and 2.5 years. METHODS: Family-based whole exome sequencing was performed based on an autosomal recessive inheritance model. In silico analyses were used to predict variant impact on protein structure and function. RESULTS: We identified a rare homozygous variant (NM_031310.2:c.101T>C;p.Leu34Pro) in PLVAP, which co-segregated with the disease. Leu34 is predicted to be located in a highly conserved, hydrophobic, α-helical region within the protein's transmembrane domain, suggesting Leu34Pro is likely to disrupt protein function and/or structure. Electron microscopy and PLVAP immunohistochemistry demonstrated apparently normal diaphragm morphology, predicted to be functionally affected. CONCLUSIONS: Biallelic missense variants in PLVAP can cause an attenuated form of the PLE and hypertriglyceridaemia syndrome. Our findings support the role of PLVAP in the pathophysiology of PLE, expand the phenotypic and mutation spectrums and underscore PLVAP's importance in EC barrier function in the gut.

Novel PLVAP Mutation in Protein Losing Enteropathy.

Introduction: A genetic cause of the protein-losing enteropathy (PLE) disease Diarrhea 10 (DIAR10) are mutations in the recently described PLVAP (plasmalemma vesicle protein). Case report: An infant with fatal PLE had a novel homozygous frameshift mutation (c.339dupT; p.Ala114Cysfs*9) leading to a premature stop codon in exon 1 of the PLVAP (NM_031310) gene detected by Whole Exome Sequencing (WES). Conclusion: The frameshift mutation (PLVAP; c.339dupT; p.Ala114Cysfs*9) we have described in our patient has not been previously reported. This is the fifth case reported with a mutation in PLVAP associated with PLE and DIAR10.

CNS angiogenesis and barriergenesis occur simultaneously.

The blood-brain barrier (BBB) plays a vital role in the central nervous system (CNS). A comprehensive understanding of BBB development has been hampered by difficulties in observing the differentiation of brain endothelial cells (BECs) in real-time. Here, we generated two transgenic zebrafish line, Tg(glut1b:mCherry) and Tg(plvap:EGFP), to serve as in vivo reporters of BBB development. We showed that barriergenesis (i.e. the induction of BEC differentiation) occurs immediately as endothelial tips cells migrate into the brain parenchyma. Using the Tg(glut1b:mCherry) transgenic line, we performed a genetic screen and identified a zebrafish mutant with a nonsense mutation in gpr124, a gene known to play a role in CNS angiogenesis and BBB development. We also showed that our transgenic plvap:EGFP line, a reporter of immature brain endothelium, is initially expressed in newly formed brain endothelial cells, but subsides during BBB maturation. Our results demonstrate the ability to visualize the in vivo differentiation of brain endothelial cells into the BBB phenotype and establish that CNS angiogenesis and barriergenesis occur simultaneously.

Molecular analysis of blood-retinal barrier loss in the Akimba mouse, a model of advanced diabetic retinopathy.

The molecular mechanisms of vascular leakage in diabetic macular edema and proliferative retinopathy are poorly understood, mainly due to the lack of reliable in vivo models. The Akimba (Ins2(Akita)VEGF(+/-)) mouse model combines retinal neovascularization with hyperglycemia, and in contrast to other models, displays the majority of signs of advanced clinical diabetic retinopathy (DR). To study the molecular mechanism that underlies the breakdown of the blood-retinal barrier (BRB) in diabetic macular edema and proliferative diabetic retinopathy, we investigated the retinal vasculature of Akimba and its parental mice Kimba (trVEGF029) and Akita (Ins2(Akita)). Quantitative PCR, immunohistochemistry and fluorescein angiography were used to characterize the retinal vasculature with special reference to the inner BRB. Correlations between the degree of fluorescein leakage and retinal gene expression were tested by calculating the Spearman's correlation coefficient. Fluorescein leakage demonstrating BRB loss was observed in Kimba and Akimba, but not in Akita or wild type mice. In Kimba and Akimba mice fluorescein leakage was associated with focal angiogenesis and correlated significantly with Plvap gene expression. PLVAP is an endothelial cell-specific protein that is absent in intact blood-retinal barrier, but its expression significantly increases in pathological conditions such as DR. Furthermore, in Akimba mice BRB disruption was linked to decreased expression of endothelial junction proteins, pericyte dropout and vessel loss. Despite fluorescein leakage, no alteration in BRB protein levels or pericyte coverage was detected in retinas of Kimba mice. In summary, our data not only demonstrate that hyperglycemia sensitizes retinal vasculature to the effects of VEGF, leading to more severe microvascular changes, but also confirm an important role of PLVAP in the regulation of BRB permeability.

Screening immune-related blood biomarkers for DKD-related HCC using machine learning.

Background: Diabetes mellitus is a significant health problem worldwide, often leading to diabetic kidney disease (DKD), which may also influence the occurrence of hepatocellular carcinoma (HCC). However, the relationship and diagnostic biomarkers between DKD and HCC are unclear. Methods: Using public database data, we screened DKD secretory RNAs and HCC essential genes by limma and WGCNA. Potential mechanisms, drugs, and biomarkers for DKD-associated HCC were identified using PPI, functional enrichment, cMAP, and machine learning algorithms, and a diagnostic nomogram was constructed. Then, ROC, calibration, and decision curves were used to evaluate the diagnostic performance of the nomograms. In addition, immune cell infiltration in HCC was explored using CIBERSORT. Finally, the detectability of critical genes in blood was verified by qPCR. Results: 104 DEGs associated with HCC using WGCNA were identified. 101 DEGs from DKD were predicated on secreting into the bloodstream with Exorbase datasets. PPI analysis identified three critical modules considered causative genes for DKD-associated HCC, primarily involved in inflammation and immune regulation. Using lasso and RM, four hub genes associated with DKD-associated HCC were identified, and a diagnostic nomogram confirmed by DCA curves was established. The results of immune cell infiltration showed immune dysregulation in HCC, which was associated with the expression of four essential genes. PLVAP was validated by qPCR as a possible blood-based diagnostic marker for DKD-related HCC. Conclusion: We revealed the inflammatory immune pathways of DKD-related HCC and developed a diagnostic nomogram for HCC based on PLVAP, C7, COL15A1, and MS4A6A. We confirmed with qPCR that PLVAP can be used as a blood marker to assess the risk of HCC in DKD patients.

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

PURPOSE: Plasmalemma vesicle-associated protein (PLVAP) is involved in many immune­related 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. METHODS: 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 RNA­sequence 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. RESULTS: 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). CONCLUSION: 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 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.

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.

A multistate stem cell dynamics maintains homeostasis in mouse spermatogenesis.

In mouse testis, a heterogeneous population of undifferentiated spermatogonia (Aundiff) harbors spermatogenic stem cell (SSC) potential. Although GFRα1+ Aundiff maintains the self-renewing pool in homeostasis, the functional basis of heterogeneity and the implications for their dynamics remain unresolved. Here, through quantitative lineage tracing of SSC subpopulations, we show that an ensemble of heterogeneous states of SSCs supports homeostatic, persistent spermatogenesis. Such heterogeneity is maintained robustly through stochastic interconversion of SSCs between a renewal-biased Plvap+/GFRα1+ state and a differentiation-primed Sox3+/GFRα1+ state. In this framework, stem cell commitment occurs not directly but gradually through entry into licensed but uncommitted states. Further, Plvap+/GFRα1+ cells divide slowly, in synchrony with the seminiferous epithelial cycle, while Sox3+/GFRα1+ cells divide much faster. Such differential cell-cycle dynamics reduces mitotic load, and thereby the potential to acquire harmful de novo mutations of the self-renewing pool, while keeping the SSC density high over the testicular open niche.

An anti-PLVAP antibody suppresses laser-induced choroidal neovascularization in monkeys.

Plasmalemma vesicle-associated protein (PLVAP, also called PV-1) is the only protein that forms endothelial diaphragms. PLVAP expression is very low in the normal blood-retinal barrier; however, pathological factors such as high glucose and vascular endothelial growth factor (VEGF) induce its expression, leading to the exacerbation of cellular permeability. Because the new blood vessels are fragile and leaky, PLVAP could possibly be considered a therapeutic target against retinovascular diseases. VEGF inhibitors are commonly used for the treatment of such diseases; however, there are several concerns associated with their use, especially in the case of chronic suppression of VEGF. In this study, we investigated the expressional level of PLVAP mRNA in VEGF-treated endothelial cells and the retinas of 2 animal models: streptozotocin-induced diabetic Brown Norway rats and Sprague-Dawley rats with oxygen-induced retinopathy. Among transcellular transport-related genes, the induction of PLVAP mRNA is the most apparent; the increase of PLVAP mRNA levels in the retina is evident during pathological progression. Furthermore, anti-PLVAP antibodies were generated, and their efficacy against laser-induced choroidal neovascularization was tested in cynomolgus monkeys. Although the leakage was exacerbated in the saline-injected group during the progression of neovascularization, the intravitreal injection of anti-PLVAP antibodies significantly ameliorated the exudation. These data imply that the PLVAP inhibition is a promising therapeutic approach against retinal diseases such as diabetic macular edema, retinopathy of prematurity, and wet age-related macular degeneration.