PV-1: Leukocyte trafficking molecule and vascular marker

The distinction between lymphatic vessels and blood vessels is a crucial factor in many studies in immunology, vascular biology and cancer biology. They both share several characteristics and perform related, though different functions. They are equally important for the performance of the human immune system with the continuous recirculation of leukocytes from the tissues via lymphatics to the blood vessels and back into the tissue presenting the link between both systems. This study was undertaken to elucidate the differences in the gene expression between primary blood- and lymphatic endothelial cells as well as the two immortalized cell lines HMEC-1 (human microvascular endothelial cell line 1) and TIME (telomerase immortalized microvascular endothelial cell line). Furthermore, we wanted to investigate the mystery surrounding the identity of the antigen recognized by the prototype blood vascular marker PAL-E. In the last step we wanted to study whether the PAL-E antigen would be involved in the process of leukocyte migration from the bloodstream into the surrounding tissue. Our results clearly show that the gene expression in primary blood endothelial cells (BEC), lymphatic endothelial cells (LEC) and the cell lines HMEC-1 and TIME is plastic. Comparison of a large set of BEC- and LEC datasets allowed us to assemble a catalog of new, stable BEC- or LEC specific markers, which we verified in independent experiments. Additionally, several lines of evidence demonstrated that PAL-E recognizes plasmalemma vesicle associated protein 1 (PV-1), which can form complexes with vimentin and neuropilin-1. Finally, numerous in vitro and in vivo experiments identify the first function of the protein PV-1 during leukocyte trafficking, where it acts as regulatory molecule.

The Role of PME-1 in Cancer: Therapeutic Implication

Protein phosphatase 2A (PP2A) plays a major role in maintaining cellular signaling homeostasis in human cells by reversibly affecting the phosphorylation of a variety of proteins. Protein phosphatase methylesterase-1 (PME-1) negatively regulates PP2A activity by reversible demethylation and active site binding. Thus far, it is known that overexpression of PME-1 in human gliomas contributes to ERK pathway signaling, cell proliferation, and malignant progression. Whether PME-1-mediated PP2A inhibition promotes therapy resistance in gliomas is unknown. Specific PP2A targets regulated by PME-1 in cancers also remain elusive. Additionally, whether oncogenic function of PME-1 can be generalized to various human cancers needs to be investigated. This study demonstrated that PME-1 expression promotes kinase inhibitor resistance in glioblastoma (GBM). PME-1 silencing sensitized GBM cells to a group of clinically used indolocarbazole multikinase inhibitors (MKIs). To facilitate the quantitative evaluation of MKIs by cancer-cell specific colony formation assay, Image-J software-plugin ‘ColonyArea’ was developed. PME-1-silencing was found to reactivate specific PP2A complexes and affect PP2A-target histone deacetylase HDAC4 activity. The HDAC4 inhibition induced synthetic lethality with MKIs similar to PME-1 depletion. However, synthetic lethality by both approaches required co-expression of a pro-apoptotic protein BAD. In gliomas, PME-1 and HDAC4 expression was associated with malignant progression. Using tumor PME-1, HDAC4 and BAD expression based stratification signatures this study defined patient subgroups that are likely to respond to MKI alone or in combination with HDAC4 inhibitor therapies. In contrast to the oncogenic role of PME-1 in certain cancer types, this study established that colorectal cancer (CRC) patients with high tumor PME-1 expression display favorable prognosis. Interestingly, PME-1 regulated survival signaling did not operate in CRC cells. Summarily, this study potentiates the candidacy of PME-1 as a therapy target in gliomas, but argues against generalization of these findings to other cancers, especially CRC.

Role of Membrane Transporters, P-Glycoprotein and Mrp1, in The Placental Transfer of Drugs With Special Reference to Saquinavir and Quetiapine

Drug transporting membrane proteins are expressed in various human tissues and blood-tissue barriers, regulating the transfer of drugs, toxins and endogenous compounds into or out of the cells. Various in vitro and animal experiments suggest that P-glycoprotein (P-gp) forms a functional barrier between maternal and fetal blood circulation in the placenta thereby protecting the fetus from exposure to xenobiotics during pregnancy. The multidrug resistance-associated protein 1 (MRP1) is a relatively less studied transporter protein in the human placenta. The aim of this study series was to study the role of placental transporters, apical P-gp and basal MRP1, using saquinavir as a probe drug, and to study transfer of quetiapine and the role of P-gp in its transfer in the dually perfused human placenta/cotyledon. Furthermore, two ABCB1 (encoding P-gp) polymorphisms (c.3435C>T, p.Ile1145Ile and c.2677G>T/A, p.Ala893Ser/Thr) were studied to determine their impact on P-gp protein expression level and on the transfer of the study drugs. Also, the influence of the P-gp protein expression level on the transfer of the study drugs was addressed. Because P-gp and MRP1 are ATP-dependent drug-efflux pumps, it was studied whether exogenous ATP is needed for the function of ATP-dependent transporter in the present experimental model. The present results indicated that the addition of exogenous ATP was not necessary for transporter function in the perfused human placental cotyledon. Saquinavir and quetiapine were both found to cross the human placenta; transplacental transfer (TPTAUC %) for saquinavir was <0.5% and for quetiapine 3.7%. Pharmacologic blocking of P-gp led to disruption of the blood-placental barrier (BPB) and increased the placental transfer of P-gp substrate, saquinavir, into the fetal circulation by 6- to 8-fold. In reversed perfusions P-gp, MRP1 and possibly OATP2B1 had a negligible role in the fetal-to-maternal transfer of saquinavir. The TPTAUC % of saquinavir was about 100-fold greater from the fetal side to the maternal side compared with the maternal-to-fetal transfer. P-gp activity is not likely to modify the placental transfer of quetiapine. Higher P-gp protein expression levels were associated with the variant allele 3435T, but no correlation was found between the TPTAUC % of saquinavir and placental P-gp protein expression. The present results indicate that P-gp activity drastically affects the fetal exposure to saquinavir, and suggest that pharmacological blockade of the P-gp activity during pregnancy may pose an increased risk for adverse fetal outcome. The blockade of P-gp activity could be used in purpose to obtain higher drug concentration to the fetal side, for example, in prevention (to decrease virus transfer to fetal side) or in treating sick fetus.

Leukocyte trafficking: a special focus on VAP-1 and CLEVER-1

It is crucial that lymphocytes patrol the body against foreign intruders and that leukocytes invade inflamed tissues to ameliorate the infection or injury. The adhesion molecules in leukocytes and endothelial cells play an essential role in the immune response by directing the traffic of leukocytes. However, the same molecules that guide leukocyte traffic under physiological conditions are also involved in pathological situations, when an overly excessive or harmful inflammatory response leads to tissue destruction and organ dysfunction or tumor growth. Vascular adhesion protein-1 (VAP-1) and Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) are endothelial molecules that participate in the adhesion of leukocytes to the endothelia. This study was designed to elucidate, using different inflammation models, the role of VAP-1 and CLEVER-1 in leukocyte migration to the inflamed tissue, and to evaluate the use of antibodies against these molecules as an anti-adhesive therapy. Also, the role of CLEVER-1 during tumorigenesis was studied. Blocking the function of VAP-1 with antibodies significantly decreased the accumulation of leukocytes in the inflamed tissue. Targeting CLEVER-1 prevented cell migration via lymphatic vessels, as well as leukocyte traffic during inflammation. Following the anti-CLEVER-1 antibody treatment the number of immune regulating leukocytes in tumors was reduced, which led to a decrease in tumor growth. However, the normal immune response towards immunization or bacterial infection was not compromised. Thus, VAP-1 and CLEVER-1 are both potential targets for antiinflammatory therapies for preventing the harmful accumulation of leukocytes in inflamed areas. Targeting CLEVER-1 may also inhibit tumor growth by reducing immunosuppressive leukocytes in tumors

Islet antigen-specific T cells and regulatory T cells in type 1 diabetes

T cells are the key players in the development of type 1 diabetes (T1D), mediating autoimmune reactions leading to the destruction of insulin producing beta cells in the islets. We aimed to analyze the role of different T-cell subtypes in the autoimmunity and pathogenesis of T1D. The frequency of islet antigen-specific (GAD65-, proinsulin-, and insulin-specific) CD4+ T cells was investigated in vitro in T1D patients, at-risk individuals (diabetes-associated autoantibody positive), and in controls, using MHC class II tetramers. An overall higher frequency of CD4+ T-cells recognizing the GAD65 555−567 peptide was detected in at-risk individuals. In addition, increased CD4+ T-cell responses to the same GAD65 epitope displaying a memory phenotype were observed in at-risk and diabetic children, which demonstrate a previous encounter with the antigen in vivo. Avidity and phenotypic differences were also observed among CD4+ T-cell clones induced by distinct doses of GAD65 autoantigen. T-cell clones generated at the lowest peptide dose displayed the highest avidity and expressed more frequently the TCR Vβ5.1 chain than low-avidity T cells. These findings raise attention to the antigen dose when investigating the diversity of antigen-specific T cells. Furthermore, an increased regulatory response during the preclinical phase of T1D was also found in genetically at-risk children. Higher frequencies of regulatory T (Treg) cells (CD4+CD25_high HLA-DR-/CD69-) and natural killer T (NKT) cells (CD161+Vbeta11+) were observed in children with multiple autoantibodies compared to autoantibody-negative controls. Taken together, these data showed increased frequency of islet-specific CD4+ T-cells, especially to the GAD65 555-567 epitope, and Treg and NKT cell upregulation in children at-risk for T1D, suggesting their importance in T1D pathogenesis