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

EGFR knockdown and sTβRII expression modulate colony formation in pancreatic cancer cells.

<p>(A) ASPC-1 and T3M4 human pancreatic cancer cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTβRII (sTβRII), or both shEGFR and sTβRII. Cell lysates and conditioned media were then subjected to immunoblotting with anti-EGFR and anti-HA-tag antibodies, respectively, the latter serving to confirm sTβRII release by the cancer cells. An anti-ERK antibody served to assess lane loading. (B) The consequences of EGFR silencing with shEGFR and TGF-β sequestration with sTβRII were assessed by monitoring colony formation in 3-D culture (B). Data are the means ± SE of triplicate determinations from three independent experiments. *p<0.05, **p<0.01, when compared with respective controls.</p

Effects of targeting EGFR and TGF-β pathways on phosphorylation status of src family members.

<p>T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), and/or WPT-sTβRII (sTβRII) as indicated. Cell lysates were then analyzed with a phospho-kinase antibody array to assess the phosphorylation status of the indicated src family members. Results were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039684#s4" target="_blank">Methods</a>. Data are the means ± SEM of triplicate determinations from three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 when compared with control.</p

Effects of EGFR knockdown and sTβRII expression on receptor phosphorylation and downstream signaling.

<p>(A) Effects on receptor phosphorylation. ASPC-1 and T3M4 cells were infected as indicated with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTβRII (sTβRII), or both shEGFR and sTβRII. Cell lysates were subjected to immunoblotting with antibodies directed against the indicated receptors and phospho-receptors. (B) Cells were infected as indicated in A, and cell lysates were subjected to immunoblotting with antibodies directed against the indicated proteins and phospho-proteins. Each panel shows data from a representative of at least two independent experiments. In both panels A and B, immnoblotting with an anti-ERK antibody confirmed equivalent lane loading, but not all ERK blots are shown.</p

Targeting EGFR and TGF-β pathways exerts different effects on the formation and growth of tumors formed by ASPC-1 and T3M4 cells.

<p>ASPC-1 (A) and T3M4 (B) cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), sTβRII, or both EGFR-LV and sTβRII, and injected subcutaneously (one injection per mouse) into the flank region of nude mice. Tumor volumes were monitored for the indicated number of days. Values are the means ± SEM of 8 mice per group, indicated in the denominator to the right of each curve. The number of tumors that formed in each group is indicated in the numerator. *p<0.05, and **p<0.01, when compared with respective controls.</p