Multiplexed MRM-Based Proteomics Identified Multiple Biomarkers of Disease Severity in Human Heart Failure.

Heart failure (HF) is a complex disease due to the intricate interplay of several mechanisms, which therefore implies the need for a multimarker strategy to better personalize the care of patients with HF. In this study, we developed a targeted mass spectrometry approach based on multiple reaction monitoring (MRM) to measure multiple circulating protein biomarkers, involved in cardiovascular disease, to address their relevance in the human HF, intending to assess the feasibility of the workflow in the disease monitoring and risk stratification. In this study, we analyzed a total of 60 plasma proteins in 30 plasma samples from eight control subjects and 22 age- and gender- matched HF patients. We identified a panel of four plasma proteins, namely Neuropilin-2, Beta 2 microglobulin, alpha-1-antichymotrypsin, and complement component C9, that were more abundant in HF patients in relation to disease severity and pulmonary dysfunction. Moreover, we showed the ability of the combination of these candidate proteins to discriminate, with sufficient accuracy, HF patients from healthy subjects. In conclusion, we demonstrated the feasibility and potential of a proteomic workflow based on MRM mass spectrometry for the evaluation of multiple proteins in human plasma and the identification of a panel of biomarkers of HF severity.

Neuropilin-2 expression in cancer.

AIMS: Neuropilin-2 is a coreceptor for vascular endothelial growth factor family members. Blockade of neuropilin-2 is able to suppress lymphogenous metastasis in preclinical models. The aim of this study was to validate a protocol for the evaluation of neuropilin-2 protein expression in situ, by comparison with in-situ hybridization, western blotting, and mRNA expression levels. METHODS AND RESULTS: Immunohistochemistry was performed on normal human tissues, and whole sections for 79 primary non-small-cell lung carcinomas, 65 primary breast carcinomas, 79 primary colorectal cancers, and 52 metastases. Neuropilin-2 expression was observed in lymphatic and blood vessels from all normal and malignant tissues examined. In addition, 32% of primary non-small-cell lung carcinomas, 15% of primary breast carcinomas and 22% of primary colorectal cancers showed tumour cell expression. Fifty-five primary and nine secondary malignant melanomas were also examined for neuropilin-2 expression by in-situ hybridization. All showed vascular expression, and 85% of primary malignant melanomas showed tumour cell expression. CONCLUSIONS: In the majority of lung, breast and colorectal cancers, the effects of anti-neuropilin-2 are likely to be restricted to the vasculature. These results will assist in pharmacokinetic evaluations, tolerability assessments and the choice of setting to evaluate the activity of anti-neuropilin-2 therapies.

Neuropilin-2-mediated tumor growth and angiogenesis in pancreatic adenocarcinoma.

PURPOSE: Neuropilin-2 (NRP-2) is a coreceptor for vascular endothelial growth factor (VEGF) on endothelial cells. NRP-2 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) cells relative to nonmalignant ductal epithelium. This study determined the role of NRP-2 in PDAC cells. EXPERIMENTAL DESIGN: NRP-2 expression was reduced in PDAC cells with stable short-hairpin RNA (shRNA) transfection. Western blotting was done to evaluate signaling intermediates. Migration and invasion studies were carried out in Boyden chambers. Anchorage-independent growth was assessed by soft-agar colony formation. In vivo growth was evaluated using murine subcutaneous and orthotopic xenograft models. Immunohistochemical analysis evaluated in vivo proliferation and angiogenesis. RESULTS: shRNA-NRP-2 decreased NRP-2 levels without affecting neuropilin-1 levels. Akt activation was decreased in clones with reduced NRP-2 (shRNA-NRP-2). shRNA-NRP-2 cells showed decreased migration, invasion, and anchorage-independent growth compared with control cells. In vitro proliferation rates were similar in control- and shRNA-transfected cells. Subcutaneous and orthotopic xenografts from shRNA-transfected cells were significantly smaller than those resulting from control-transfected cells (P < 0.05). Furthermore, shRNA-NRP-2 tumors exhibited less cellular proliferation and decreased microvascular area relative to control tumors (P < 0.05). Constitutive expression of the angiogenic mediator Jagged-1 was reduced in shRNA-NRP-2 cells, whereas vascular endothelial growth factor levels were unchanged. CONCLUSION: Reduction of NRP-2 expression in PDAC cells decreased survival signaling, migration, invasion, and ability to grow under anchorage-independent conditions. In vivo, reduction of NRP-2 led to decreased growth of xenograft tumors and decreased vascular area, which was associated with decreased Jagged-1 levels. NRP-2 is a potential therapeutic target on PDAC cells.

Clusterin and neuropilin-2 as potential biomarkers of tumor progression in benzo[a]pyrene-transformed 16HBE cells xenografted nude mouse model.

Benzo[a]pyrene (BaP) is a ubiquitous environment contaminant and its exposure could increase incidence of human lung cancer. In order to confirm and compare potential biomarkers of BaP-induce carcinogenesis and tumor progression, time-dependent changes of clusterin (CLU) and neuropilin-2 (NRP2) levels were evaluated in sera of BaP-transformed 16HBE cell line T-16HBE-C1 cells xenografted nude mice. Performance of CLU and NRP2 on tissue classification and tumor progression forecast was also calculated. Levels of CLU and NRP2 were significant elevated in both culture supernatant of T-16HBE-C1 cells and sera of T-16HBE-C1 cells xenografted nude mice compared with control. CLU and NRP2 were both found positively stained in tumor tissue. CLU and NRP2 alone could well predicate tumor progression in nude mice and CLU appeared to be more sensitive than NRP2. When both of them combined, performance of predication would improve. In conclusion, CLU and NRP2 could serve as potential biomarkers of tumor progression in nude mice xenografted with T-16HBE-C1 cells.

Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera.

To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis.

High diversity of non-human leukocyte antigens in transplant-associated coronary artery disease.

BACKGROUND: Antibodies to endothelial derived non-human leukocyte antigens (HLA) have been associated with transplant (Tx)-associated coronary artery disease (CAD) after cardiac transplantation; however, few have been identified. The aim of this study was to screen a human coronary artery endothelial cell cDNA library with patient sera to establish the diversity and nature of the target antigens. METHODS: A human coronary artery endothelial cell cDNA library was screened with sera from seven long-term cardiac transplant patients with angiographically diagnosed TxCAD and sera from five healthy volunteers. RESULTS: Of the seven patients' sera, five showed reactivity, as did sera from two of the five normal subjects. Eighteen positive cDNA clones were isolated by TxCAD sera; DNA sequence analysis and DNA database searching identified all but one clone; 16 were nuclear or cytoplasmic proteins and 1 of them was the cell surface protein neuropilin 2. Five clones were targeted by normal sera. A different spectrum of reactive clones was identified by the sera of each patient where reactive clones were evident. CONCLUSIONS: A high diversity of non-HLA antigens, probably autoantigens, are involved in the pathogenesis of TxCAD.

Dysregulation of vascular endothelial growth factors and their neuropilin receptors in the eutopic endometrium of women with endometriosis.

Despite the importance of neuropilins (NRPs) in a number of processes that are altered in endometriosis, such as angiogenesis and neuronal guidance, these molecules have not been previously studied in the disease. Similarly, potent lymphangiogenic factors, vascular endothelial growth factor C (VEGF-C) and VEGF-D, have not been comprehensively investigated in endometriosis. The objective of this study was to examine their expression in women with and without endometriosis. NRPs and VEGFs were quantified in 79 histologically normal uterine tissue samples (37 control and 42 endometriosis, all menstrual cycle phases) using immunohistochemistry and automated cellular imaging analysis. NRP-1 was significantly reduced in women with endometriosis (P = .004). The normal significant menstrual cyclical variations in endometrial NRP-1, NRP-2, and VEGF-C were absent in endometriosis, and VEGF-D was dysregulated. Dysregulated expression of growth factors and receptors, such as NRPs and VEGFs, likely contribute to altered angiogenesis, lymphangiogenesis, neurogenesis and immune function in endometriosis and may reflect altered hormone signals.

Maternal dietary restriction and selenium supply alters messenger ribonucleic acid expression of angiogenic factors in maternal intestine, mammary gland, and fetal jejunal tissues during late gestation in pregnant ewe lambs.

The objectives of this study were to evaluate effects of maternal dietary restriction and Se supply on angiogenic factor mRNA expression in intestinal and mammary tissues, and jejunal crypt cell proliferation and vascularity in late-term fetal intestines. In Exp. 1, pregnant ewe lambs (n = 32; initial BW = 45.6 +/- 2.3 kg) were allotted randomly to 1 of 4 treatments. Treatments (initiated d 50 +/- 5 of gestation) were control (3.5 microg of Se.kg of BW(-1).d(-1)), Se-wheat (75 microg of Se.kg of BW(-1).d(-1)), selenate (Se3; providing 75 microg of Se.kg of BW(-1).d(-1)), selenate (Se15; providing 375 microg of Se.kg of BW(-1).d(-1)). Diets (DM basis) were similar in CP (15.5%) and ME (2.68 Mcal/kg). In Exp. 2, pregnant ewe lambs (n = 36; initial BW 53.8 +/- 1.3 kg) were allotted randomly to treatments in a 2 x 2 factorial arrangement. Factors were nutrition (control, 100% of requirements vs. restricted nutrition, 60% of controls) and dietary Se (adequate Se; 6 microg of Se.kg of BW(-1).d(-1) vs. high Se; 80 microg of Se.kg of BW(-1).d(-1)). Selenium treatments were initiated 21 d before breeding, and nutritional treatments were initiated on d 64 of gestation. Diets (DM basis) were 16% CP and 2.12 Mcal/kg of ME. In Exp. 1, Se15 increased (P = 0.07) vascular endothelial growth factor (VEGF) mRNA expression, whereas Se supplementation decreased (P = 0.06) kinase insert domain receptor (KDR) mRNA in maternal mucosal scrape on d 134 of gestation. Expression of VEGF mRNA was decreased by Se (P = 0.10) in fetal jejunum. In mammary tissue, fms-related tyrosine kinase 1 and KDR mRNA were greater in Se-wheat compared with Se3, and KDR expression was increased (P = 0.10) in Se15 compared with Se3. In Exp. 2, dietary restriction increased (P < or = 0.07) expression of mRNA for VEGF, fms-related tyrosine kinase 1, KDR, neuropilin 1, neuropilin 2, and hypoxia-inducible factor 1, alpha subunit in mucosal scrapes from maternal jejunum. In fetal jejunum, soluble guanylate cyclase, was decreased (P = 0.01) by maternal dietary restriction from d 64 to 135 of gestation. Total microvascularity in fetal jejunum was reduced (P = 0.002) by maternal dietary restriction. Mammary gland expression of VEGF, neuropilin 1, angiopoietin receptor (endothelial tyrosine kinase), and endothelial nitric oxide synthase 3 increased (P < or = 0.09), whereas angiopoietin 1 decreased (P = 0.05) due to nutrient restriction. Data indicate that expression of angiogenic factors and receptors in maternal intestine, mammary gland, and fetal jejunum are responsive to maternal nutrition and likely explain observed changes in tissue vascularity.

Autocrine VEGF-A system in podocytes regulates podocin and its interaction with CD2AP.

Vascular endothelial growth factor (VEGF-A) signaling is required for endothelial cell differentiation, vasculogenesis, angiogenesis, and vascular patterning. During kidney morphogenesis, podocyte VEGF-A guides endothelial cells toward developing glomeruli. Podocyte VEGF-A expression continues throughout life but its function after completion of development remains unclear. Here, we examined the expression of VEGF-A and its receptors VEGFR1, VEGFR2, NP1, and NP2 in conditionally immortalized mouse podocytes cultured in undifferentiated and differentiated conditions using RT-PCR and Western analysis. VEGF-A secretion was assessed by ELISA and Western analysis. Upon podocyte differentiation, VEGF-A protein expression and secretion increased threefold. Differentiated podocytes expressed eightfold higher VEGFR2 mRNA levels than undifferentiated podocytes, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels were similar. We examined the regulation and function of the VEGF-A system by exposing differentiated podocytes to recombinant VEGF(165) (20 ng/ml) or control media for 24 h. VEGF(165) induced a twofold increase in VEGFR2 mRNA and protein levels, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels remained unchanged. VEGF(165) induced VEGFR2 phosphorylation. VEGF(165) reduced podocyte apoptosis approximately 40%, whereas anti-VEGFR2 neutralizing antibody enhanced it twofold. We determined that VEGF-A signaling regulates slit diaphragm proteins by inducing a dose-response podocin upregulation and increasing its interaction with CD2AP. The data indicate that podocytes in culture have a functional autocrine VEGF-A system that is regulated by differentiation and ligand availability. VEGF-A functions in podocytes include promoting survival through VEGFR2, inducing podocin upregulation and increasing podocin/CD2AP interaction.

Expression of VEGF, semaphorin SEMA3F, and their common receptors neuropilins NP1 and NP2 in preinvasive bronchial lesions, lung tumours, and cell lines.

Two receptors, neuropilin 1 (NP1) and neuropilin 2 (NP2), bind class 3 semaphorins, axon guidance molecules including SEMA3F, the gene for which was isolated from a 3p21.3 deletion in lung cancer. In addition, they bind VEGF (vascular endothelial growth factor), enhancing the effects of VEGF binding to KDR/Flk-1. Elevated VEGF levels are associated with the loss and cytoplasmic delocalization of SEMA3F in lung cancer, suggesting competition for their NP1 and NP2 receptors. To determine the timing of these events, we compared by immunohistochemistry VEGF, SEMA3F, NP1 and NP2 expression in 50 preneoplastic lesions and 112 lung tumours. In preneoplastic lesions, VEGF increased from low-grade to high-grade dysplasia (p=0.001) whereas SEMA3F levels remained low. NP1 and NP2 levels increased from dysplasia to microinvasive carcinoma (p=0.0001) and correlated with VEGF expression (p=0.04 and 0.0002, respectively). Non-small cell lung carcinoma overexpressed VEGF and NP1 and NP2 significantly more often than neuroendocrine tumours including small cell lung carcinoma. SEMA3F loss or delocalization correlated with advanced tumour stage. Migrating cells overexpressed VEGF, SEMA3F, NP1 and NP2 with cytoplasmic delocalization of NP1 as demonstrated in an in vitro wound assay. These results demonstrate early alteration of the VEGF/SEMA3F/NP pathway in lung cancer progression.