Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.

Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD. Additionally, blocking ACAT1/SOAT1 activity stimulates autophagy and lysosomal biogenesis; however, the exact molecular connection between the ACAT1/SOAT1 blockade and these observed benefits remain unknown. Here, using biochemical fractionation techniques, we observe cholesterol accumulation at the MAM which leads to ACAT1/SOAT1 enrichment in this domain. MAM proteomics data suggests that ACAT1/SOAT1 inhibition strengthens the ER-mitochondria connection. Confocal and electron microscopy confirms that ACAT1/SOAT1 inhibition increases the number of ER-mitochondria contact sites and strengthens this connection by shortening the distance between these two organelles. This work demonstrates how directly manipulating local cholesterol levels at the MAM can alter inter-organellar contact sites and suggests that cholesterol buildup at the MAM is the impetus behind the therapeutic benefits of ACAT1/SOAT1 inhibition.

Pattern of tamoxifen-induced Tie2 deletion in endothelial cells in mature blood vessels using endo SCL-Cre-ERT transgenic mice.

Tyrosine-protein kinase receptor Tie2, also known as Tunica interna Endothelial cell Kinase or TEK plays a prominent role in endothelial responses to angiogenic and inflammatory stimuli. Here we generated a novel inducible Tie2 knockout mouse model, which targets mature (micro)vascular endothelium, enabling the study of the organ-specific contribution of Tie2 to these responses. Mice with floxed Tie2 exon 9 alleles (Tie2floxed/floxed) were crossed with end-SCL-Cre-ERT transgenic mice, generating offspring in which Tie2 exon 9 is deleted in the endothelial compartment upon tamoxifen-induced activation of Cre-recombinase (Tie2ΔE9). Successful deletion of Tie2 exon 9 in kidney, lung, heart, aorta, and liver, was accompanied by a heterogeneous, organ-dependent reduction in Tie2 mRNA and protein expression. Microvascular compartment-specific reduction in Tie2 mRNA and protein occurred in arterioles of all studied organs, in renal glomeruli, and in lung capillaries. In kidney, lung, and heart, reduced Tie2 expression was accompanied by a reduction in Tie1 mRNA expression. The heterogeneous, organ- and microvascular compartment-dependent knockout pattern of Tie2 in the Tie2floxed/floxed;end-SCL-Cre-ERT mouse model suggests that future studies using similar knockout strategies should include a meticulous analysis of the knockout extent of the gene of interest, prior to studying its role in pathological conditions, so that proper conclusions can be drawn.

Primary myxoid and epithelioid mesenchymal tumor of the kidney with a novel GLI1-FOXO4 fusion.

To our knowledge, we describe the first mesenchymal tumor with a novel GLI1-FOXO4 fusion gene. This well-circumscribed kidney tumor displayed variably myxoid and epithelioid histologic features with a focally nodular growth pattern. The tumor cells showed bland, round to ovoid nuclei, with no overt high-grade features. The tumor showed focal immunopositivity for smooth muscle actin and Melan-A, which raised the possibility of a relationship with a perivascular epithelioid cell tumor. The clinical and morphologic features appear distinct from other reported neoplasms harboring GLI1 or FOXO4 gene rearrangements. The patient underwent radical nephrectomy and is without evidence of disease during a relatively short clinical follow-up period. However, the features of this tumor likely warrant long-term follow-up to monitor for the possibility of a late recurrence or metastasis. In addition to reporting this novel fusion-positive tumor, we also provide a brief review of GLI1 and FOXO4 gene functions in both normal and neoplastic contexts.

The Role of PTEN in Neurodevelopment.

PTEN is a lipid and protein phosphatase that regulates cell growth and survival. Mutations to PTEN are highly penetrant for autism spectrum disorder (ASD). Here, we briefly review the evidence linking PTEN mutations to ASD and the mouse models that have been used to study the role of PTEN in neurodevelopment. We then focus on the cellular phenotypes associated with PTEN loss in neurons, highlighting the role PTEN plays in neuronal proliferation, migration, survival, morphology, and plasticity.

EphB1 interaction with caveolin-1 in endothelial cells modulates caveolae biogenesis.

Caveolae, the cave-like structures abundant in endothelial cells (ECs), are important for multiple signaling processes such as production of nitric oxide and caveolae-mediated intracellular trafficking. Using superresolution microscopy, fluorescence resonance energy transfer, and biochemical analysis, we observed that the EphB1 receptor tyrosine kinase constitutively interacts with caveolin-1 (Cav-1), the key structural protein of caveolae. Activation of EphB1 with its ligand Ephrin B1 induced EphB1 phosphorylation and the uncoupling EphB1 from Cav-1 and thereby promoted phosphorylation of Cav-1 by Src. Deletion of Cav-1 scaffold domain binding (CSD) motif in EphB1 prevented EphB1 binding to Cav-1 as well as Src-dependent Cav-1 phosphorylation, indicating the importance of CSD in the interaction. We also observed that Cav-1 protein expression and caveolae numbers were markedly reduced in ECs from EphB1-deficient (EphB1-/-) mice. The loss of EphB1 binding to Cav-1 promoted Cav-1 ubiquitination and degradation, and hence the loss of Cav-1 was responsible for reducing the caveolae numbers. These studies identify the crucial role of EphB1/Cav-1 interaction in the biogenesis of caveolae and in coordinating the signaling function of Cav-1 in ECs.

Lymph node conduits transport virions for rapid T cell activation.

Despite intense interest in antiviral T cell priming, the routes by which virions move in lymph nodes (LNs) are imperfectly understood. Current models fail to explain how virus-infected cells rapidly appear within the LN interior after viral infection. To better understand virion trafficking in the LN, we determined the locations of virions and infected cells after administration to mice of vaccinia virus or Zika virus. Notably, many rapidly infected cells in the LN interior were adjacent to LN conduits. Through the use of confocal and electron microscopy, we clearly visualized virions within conduits. Functionally, CD8+ T cells rapidly and preferentially associated with vaccinia virus-infected cells in the LN paracortex, which led to T cell activation in the LN interior. These results reveal that it is possible for even large virions to flow through LN conduits and infect dendritic cells within the T cell zone to prime CD8+ T cells.

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.

Spatially controlled assembly of affinity ligand and enzyme cargo enables targeting ferritin nanocarriers to caveolae.

One of the goals of nanomedicine is targeted delivery of therapeutic enzymes to the sub-cellular compartments where their action is needed. Endothelial caveolae-derived endosomes represent an important yet challenging destination for targeting, in part due to smaller size of the entry aperture of caveolae (ca. 30-50 nm). Here, we designed modular, multi-molecular, ferritin-based nanocarriers with uniform size (20 nm diameter) for easy drug-loading and targeted delivery of enzymatic cargo to these specific vesicles. These nanocarriers targeted to caveolar Plasmalemmal Vesicle-Associated Protein (Plvap) deliver superoxide dismutase (SOD) into endosomes in endothelial cells, the specific site of influx of superoxide mediating by such pro-inflammatory signaling as some cytokines and lipopolysaccharide (LPS). Cell studies showed efficient internalization of Plvap-targeted SOD-loaded nanocarriers followed by dissociation from caveolin-containing vesicles and intracellular transport to endosomes. The nanocarriers had a profound protective anti-inflammatory effect in an animal model of LPS-induced inflammation, in agreement with the characteristics of their endothelial uptake and intracellular transport, indicating that these novel, targeted nanocarriers provide an advantageous platform for caveolae-dependent delivery of biotherapeutics.

Flexible Nanoparticles Reach Sterically Obscured Endothelial Targets Inaccessible to Rigid Nanoparticles.

Molecular targeting of nanoparticle drug carriers promises maximized therapeutic impact to sites of disease or injury with minimized systemic effects. Precise targeting demands addressing to subcellular features. Caveolae, invaginations in cell membranes implicated in transcytosis and inflammatory signaling, are appealing subcellular targets. Caveolar geometry has been reported to impose a ≈50 nm size cutoff on nanocarrier access to plasmalemma vesicle associated protein (PLVAP), a marker found in caveolae in the lungs. The use of deformable nanocarriers to overcome that size cutoff is explored in this study. Lysozyme-dextran nanogels (NGs) are synthesized with ≈150 or ≈300 nm mean diameter. Atomic force microscopy indicates the NGs deform on complementary surfaces. Quartz crystal microbalance data indicate that NGs form softer monolayers (≈60 kPa) than polystyrene particles (≈8 MPa). NGs deform during flow through microfluidic channels, and modeling of NG extrusion through porous filters yields sieving diameters less than 25 nm for NGs with 150 and 300 nm hydrodynamic diameters. NGs of 150 and 300 nm diameter target PLVAP in mouse lungs while counterpart rigid polystyrene particles do not. The data in this study indicate a role for mechanical deformability in targeting large high-payload drug-delivery vehicles to sterically obscured targets like PLVAP.

Targeting superoxide dismutase to endothelial caveolae profoundly alleviates inflammation caused by endotoxin.

Inflammatory mediators binding to Toll-Like receptors (TLR) induce an influx of superoxide anion in the ensuing endosomes. In endothelial cells, endosomal surplus of superoxide causes pro-inflammatory activation and TLR4 agonists act preferentially via caveolae-derived endosomes. To test the hypothesis that SOD delivery to caveolae may specifically inhibit this pathological pathway, we conjugated SOD with antibodies (Ab/SOD, size ~10nm) to plasmalemmal vesicle-associated protein (Plvap) that is specifically localized to endothelial caveolae in vivo and compared its effects to non-caveolar target CD31/PECAM-1. Plvap Ab/SOD bound to endothelial cells in culture with much lower efficacy than CD31 Ab/SOD, yet blocked the effects of LPS signaling with higher efficiency than CD31 Ab/SOD. Disruption of cholesterol-rich membrane domains by filipin inhibits Plvap Ab/SOD endocytosis and LPS signaling, implicating the caveolae-dependent pathway(s) in both processes. Both Ab/SOD conjugates targeted to Plvap and CD31 accumulated in the lungs after IV injection in mice, but the former more profoundly inhibited LPS-induced pulmonary inflammation and elevation of plasma level of interferon-beta and -gamma and interleukin-27. Taken together, these results indicate that targeted delivery of SOD to specific cellular compartments may offer effective, mechanistically precise interception of pro-inflammatory signaling mediated by reactive oxygen species.

INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

Mitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through ER-bound inverted formin 2 (INF2) stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER-mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment.

Ascending Vasa Recta Are Angiopoietin/Tie2-Dependent Lymphatic-Like Vessels.

Urinary concentrating ability is central to mammalian water balance and depends on a medullary osmotic gradient generated by a countercurrent multiplication mechanism. Medullary hyperosmolarity is protected from washout by countercurrent exchange and efficient removal of interstitial fluid resorbed from the loop of Henle and collecting ducts. In most tissues, lymphatic vessels drain excess interstitial fluid back to the venous circulation. However, the renal medulla is devoid of classic lymphatics. Studies have suggested that the fenestrated ascending vasa recta (AVRs) drain the interstitial fluid in this location, but this function has not been conclusively shown. We report that late gestational deletion of the angiopoietin receptor endothelial tyrosine kinase 2 (Tie2) or both angiopoietin-1 and angiopoietin-2 prevents AVR formation in mice. The absence of AVR associated with rapid accumulation of fluid and cysts in the medullary interstitium, loss of medullary vascular bundles, and decreased urine concentrating ability. In transgenic reporter mice with normal angiopoietin-Tie2 signaling, medullary AVR exhibited an unusual hybrid endothelial phenotype, expressing lymphatic markers (prospero homeobox protein 1 and vascular endothelial growth factor receptor 3) as well as blood endothelial markers (CD34, endomucin, platelet endothelial cell adhesion molecule 1, and plasmalemmal vesicle-associated protein). Taken together, our data redefine the AVRs as Tie2 signaling-dependent specialized hybrid vessels and provide genetic evidence of the critical role of AVR in the countercurrent exchange mechanism and the structural integrity of the renal medulla.

Growth Differentiation Factor 6 Promotes Vascular Stability by Restraining Vascular Endothelial Growth Factor Signaling.

OBJECTIVE: The assembly of a functional vascular system requires a coordinated and dynamic transition from activation to maturation. High vascular endothelial growth factor activity promotes activation, including junction destabilization and cell motility. Maturation involves junctional stabilization and formation of a functional endothelial barrier. The identity and mechanism of action of prostabilization signals are still mostly unknown. Bone morphogenetic protein receptors and their ligands have important functions during embryonic vessel assembly and maturation. Previous work has suggested a role for growth differentiation factor 6 (GDF6; bone morphogenetic protein 13) in vascular integrity although GDF6's mechanism of action was not clear. Therefore, we sought to further explore the requirement for GDF6 in vascular stabilization. APPROACH AND RESULTS: We investigated the role of GDF6 in promoting endothelial vascular integrity in vivo in zebrafish and in cultured human umbilical vein endothelial cells in vitro. We report that GDF6 promotes vascular integrity by counteracting vascular endothelial growth factor activity. GDF6-deficient endothelium has increased vascular endothelial growth factor signaling, increased vascular endothelial-cadherin Y658 phosphorylation, vascular endothelial-cadherin delocalization from cell-cell interfaces, and weakened endothelial cell adherence junctions that become prone to vascular leak. CONCLUSIONS: Our results suggest that GDF6 promotes vascular stabilization by restraining vascular endothelial growth factor signaling. Understanding how GDF6 affects vascular integrity may help to provide insights into hemorrhage and associated vascular pathologies in humans.

HS3ST1 genotype regulates antithrombin's inflammomodulatory tone and associates with atherosclerosis.

The HS3ST1 gene controls endothelial cell production of HSAT+ - a form of heparan sulfate containing a specific pentasaccharide motif that binds the anticoagulant protein antithrombin (AT). HSAT+ has long been thought to act as an endogenous anticoagulant; however, coagulation was normal in Hs3st1-/- mice that have greatly reduced HSAT+ (HajMohammadi et al., 2003). This finding indicates that HSAT+ is not essential for AT's anticoagulant activity. To determine if HSAT+ is involved in AT's poorly understood inflammomodulatory activities, Hs3st1-/- and Hs3st1+/+ mice were subjected to a model of acute septic shock. Compared with Hs3st1+/+ mice, Hs3st1-/- mice were more susceptible to LPS-induced death due to an increased sensitivity to TNF. For Hs3st1+/+ mice, AT treatment reduced LPS-lethality, reduced leukocyte firm adhesion to endothelial cells, and dilated isolated coronary arterioles. Conversely, for Hs3st1-/- mice, AT induced the opposite effects. Thus, in the context of acute inflammation, HSAT+ selectively mediates AT's anti-inflammatory activity; in the absence of HSAT+, AT's pro-inflammatory effects predominate. To explore if the anti-inflammatory action of HSAT+ also protects against a chronic vascular-inflammatory disease, atherosclerosis, we conducted a human candidate-gene association study on >2000 coronary catheterization patients. Bioinformatic analysis of the HS3ST1 gene identified an intronic SNP, rs16881446, in a putative transcriptional regulatory region. The rs16881446G/G genotype independently associated with the severity of coronary artery disease and atherosclerotic cardiovascular events. In primary endothelial cells, the rs16881446G allele associated with reduced HS3ST1 expression. Together with the mouse data, this leads us to conclude that the HS3ST1 gene is required for AT's anti-inflammatory activity that appears to protect against acute and chronic inflammatory disorders.

Epithelial cell integrin ß1 is required for developmental angiogenesis in the pituitary gland.

As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin ß1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin ß1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin ß1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin ß1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non-cell-autonomous effects on angiogenesis. We conclude that epithelial integrin ß1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development.

Endothelial Plasmalemma Vesicle-Associated Protein Regulates the Homeostasis of Splenic Immature B Cells and B-1 B Cells.

Plasmalemma vesicle-associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time, Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble Ags into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap results in a dramatic decrease of IgM+IgDlo B cells in both the spleen and the peritoneal cavity. Tissue-specific deletion of Plvap demonstrates that the defect is B cell extrinsic, because B cell and pan-hematopoietic Plvap deletion has no effect on IgM+IgDlo B cell numbers. Endothelial-specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype, whereas endothelial-specific reconstitution of Plvap under the Chd5 promoter rescues the IgM+IgDlo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM+ B cells in the spleen and peritoneal cavity.

VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread.

The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2(Y949F/Y949F) mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2(Y949F/Y949F) mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms.

Uncoupling Caveolae From Intracellular Signaling In Vivo.

RATIONALE: Caveolin-1 (Cav-1) negatively regulates endothelial nitric oxide (NO) synthase-derived NO production, and this has been mapped to several residues on Cav-1, including F92. Herein, we reasoned that endothelial expression of an F92ACav-1 transgene would let us decipher the mechanisms and relationships between caveolae structure and intracellular signaling. OBJECTIVE: This study was designed to separate caveolae formation from its downstream signaling effects. METHODS AND RESULTS: An endothelial-specific doxycycline-regulated mouse model for the expression of Cav-1-F92A was developed. Blood pressure by telemetry and nitric oxide bioavailability by electron paramagnetic resonance and phosphorylation of vasodilator-stimulated phosphoprotein were determined. Caveolae integrity in the presence of Cav-1-F92A was measured by stabilization of caveolin-2, sucrose gradient, and electron microscopy. Histological analysis of heart and lung, echocardiography, and signaling were performed. CONCLUSIONS: This study shows that mutant Cav-1-F92A forms caveolae structures similar to WT but leads to increases in NO bioavailability in vivo, thereby demonstrating that caveolae formation and downstream signaling events occur through independent mechanisms.

An updated h-index measures both the primary and total scientific output of a researcher.

The growing interest in scientometry stems from ethical concerns related to the proper evaluation of scientific contributions of an author working in a hard science. In the absence of a consensus, institutions may use arbitrary methods for evaluating scientists for employment and promotion. There are several indices in use that attempt to establish the most appropriate and suggestive position of any scientist in the field he/she works in. A scientist's Hirsch-index (h-index) quantifies their total effective published output, but h-index summarizes the total value of their published work without regard to their contribution to each publication. Consequently, articles where the author was a primary contributor carry the same weight as articles where the author played a minor role. Thus, we propose an updated h-index named Hirsch(p,t)-index that informs about both total scientific output and output where the author played a primary role. Our measure, h(p,t) = h(p),h(t), is composed of the h-index h(t) and the h-index calculated for articles where the author was a key contributor; i.e. first/shared first or senior or corresponding author. Thus, a h(p,t) = 5,10 would mean that the author has 5 articles as first, shared first, senior or corresponding author with at least 5 citations each, and 10 total articles with at least 10 citations each. This index can be applied in biomedical disciplines and in all areas where the first and last position on an article are the most important. Although other indexes, such as r- and w-indexes, were proposed for measuring the authors output based on the position of researchers within the published articles, our simpler strategy uses the already established algorithms for h-index calculation and may be more practical to implement.

Mutations in Plasmalemma Vesicle Associated Protein Result in Sieving Protein-Losing Enteropathy Characterized by Hypoproteinemia, Hypoalbuminemia, and Hypertriglyceridemia.

BACKGROUND & AIMS METHODS: Severe intestinal diseases observed in very young children are often the result of monogenic defects. We used whole exome sequencing (WES) to examine the genetic cause in a patient with a distinct severe form of protein losing enteropathy (PLE) characterized by hypoproteinemia, hypoalbuminemia, and hypertriglyceridemia. METHODS: WES was performed at the Centre for Applied Genomics, Hospital for Sick Children, Toronto, Canada. Exome library preparation was performed using the Ion Torrent AmpliSeq RDY Exome Kit. Functional studies were carried out based on the identified mutation. RESULTS: Using whole exome sequencing we identified a homozygous nonsense mutation (1072C>T; p.Arg358*) in the PLVAP (plasmalemma vesicle associated protein) gene in an infant from consanguineous parents who died at five months of age of severe protein losing enteropathy. Functional studies determined that the mutated PLVAP mRNA and protein were not expressed in the patient biopsy tissues, presumably secondary to nonsense-mediated mRNA decay. Pathological analysis showed that the loss of PLVAP resulted in disruption of endothelial fenestrated diaphragms. CONCLUSIONS: PLVAP p.Arg358* mutation resulted in loss of PLVAP expression with subsequent deletion of the diaphragms of endothelial fenestrae leading to plasma protein extravasation, protein-losing enteropathy and ultimately death.