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 antigens 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(+)IgD(lo) B cells in both the spleen and peritoneal cavity. Tissue specific deletion of Plvap demonstrates that the defect is B cell extrinsic, as B cell and pan hematopoietic Plvap deletion has no effect on IgM(+)IgD(lo) 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(+)IgD(lo) 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

PV1 Down-Regulation via shRNA Inhibits the Growth of Pancreatic Adenocarcinoma Xenografts

PV1 is an endothelial-specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour-bearing mice by single-dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down-regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC-1 and BxPC-3). The effect observed is because of down-regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down-regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis

Dynamic Dual-Tracer MRI-Guided Fluorescence Tomography to Quantify Receptor Density In Vivo

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies

Caveolae, Fenestrae and Transendothelial Channels Retain PV1 on the Surface of Endothelial Cells

PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin- and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1

Concomitant Targeting of EGF Receptor, TGF-beta and Src Points to a Novel Therapeutic Approach in Pancreatic Cancer

To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-β) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-β sequestration by soluble TGF-β receptor II (sTβRII). Effects on colony formation in 3-dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTβRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTβRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTβRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTβRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTβRII due to increased cancer cell proliferation. The combination of shEGFR and sTβRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTβRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-β, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-β-dependent pathways

Implementation of Multicolor Melt Curve Analysis for High-Risk Human Papilloma Virus Detection in Low- and Middle-Income Countries: A Pilot Study for Expanded Cervical Cancer Screening in Honduras

Purpose: Cervical cancer is a leading cause of cancer-related mortality in low- and middle-income countries (LMICs) and screening in LMICs is extremely limited. We aimed to implement on-site high-risk human papillomavirus (hrHPV) DNA testing in cohorts of women from an urban factory and from a rural village. Methods: A total of 802 women were recruited for this study in partnership with La Liga Contra el Cancer through the establishment of women’s health resource fairs at two locations in Honduras: a textile factory (n = 401) in the city of San Pedro Sula and the rural village of El Rosario (n = 401) in Yoro. Participants received a routine cervical examination during which three sterile cytobrushes were used to collect cervical samples for testing. hrHPV genotyping was performed using a hrHPV genotyping assay and a real-time polymerase chain reaction instrument. Results: hrHPV status across all participants at both sites was 13% hrHPV positive and 67% hrHPV negative. When hrHPV status was compared across all three testing sites, hrHPV-positive rates were approximately equal among the factory (13%), village (12%), and confirmatory testing at Dartmouth-Hitchcock Medical Center (Lebanon, NH; 14%). hrHPV genotype was compared across sites, with HPV16 showing the highest infection rate (15%), followed by HPV59 (12%), and HPV68 (11%). There was a low prevalence of HPV18 observed in both populations compared with the hrHPV-positive population in the United States. Conclusion: In collaboration with oncologists and pathologists from La Liga Contra el Cancer, we were able to provide a continuum of care once health-fair testing was performed. We established a method and implementation plan for hrHPV testing that is sustainable in LMICs

The diaphragms of fenestrated endothelia:gatekeepers of vascular permeability and blood composition

Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition

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

Background & Aims: Severe intestinal diseases observed in very young children are often the result of monogenic defects. We used whole-exome sequencing (WES) to examine genetics 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, and exome library preparation was performed with the Ion Torrent AmpliSeq RDY Exome Kit. Functional studies were based on the identified mutation. Results: Using WES 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 5 months of age of severe PLE. 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: The PLVAP p.Arg358* mutation resulted in the loss of PLVAP expression with subsequent deletion of the diaphragms of endothelial fenestrae, which led to plasma protein extravasation, PLE, and ultimately death. Keywords: Endothelium, Fenestrae, Hypertriglyceridemia, Hypoalbuminemia, Hypoproteinemia, Very Early Onset Inflammatory Bowel Disease, Monogenic Diseases, Protein-Losing Enteropathy, Whole-Exome Sequencin

Protein level of PV1 correlates with the number of structures capable of forming diaphragms <i>in vivo</i>.

<p>A) Electron micrographs of capillary ECs of the kidneys (<i>top panels</i>) and lungs (<i>middle and bottom panels</i>) of WT, Cav1−/− and cavin-1−/− mice, as indicated. TEC and fenestrae are present in the kidneys of WT, Cav1−/− and cavin-1−/− mice (<i>top panels</i>). (<i>Middle and bottom panels</i>) Caveolae with stomatal diaphragms are present in the lungs of WT and absent in Cav1−/− and cavin-1−/− mice (<i>middle panel</i>). Insets in <i>middle panels</i> are a 2-fold magnification of the noted stretches of ECs. Bottom panels are a 3-fold magnification of ECs of Cav1−/− (<i>left</i>) and cavin-1−/− (<i>right</i>). Bars −200 nm. B) Morphometric analysis of the number of lung endothelial caveolae in WT and Cav1−/− mice demonstrating the absence of caveolae in the latter. C) Morphometric analysis of the numbers of kidney endothelial TEC, fenestrae and caveolae in WT and Cav1−/− mice.</p