Tumor necrosis factor receptor-2 signaling attenuates vein graft neointima formation by promoting endothelial recovery.

OBJECTIVE: Inflammation appears intricately linked to vein graft arterialization. We have previously shown that tumor necrosis factor (TNF) receptor-1 (TNFR1, p55) signaling augments vein graft neointimal hyperplasia (NH) and remodeling through its effects on vascular smooth muscle cells (SMCs). In this study we examined the role of TNFR2 (p75) signaling in vein graft arterialization. METHODS AND RESULTS: Inferior vena cava-to-carotid artery interposition grafting was performed between p75-/- and congenic (C57B1/6J) wild-type (WT) mice. Six weeks postoperatively, neointimal and medial dimensions were greater in p75-/- grafts placed into p75-/- recipients (by 42% or 60%, respectively; P<0.05), when compared with WT veins grafted into WT recipients. Relative to WT vein grafts, p75 deficiency augmented early (2-week-old) graft vascular cell adhesion molecule (VCAM)-1 expression (by 2.4-fold, P<0.05), increased endothelial cell apoptosis (2-fold), and delayed graft re-endothelialization. Both cellular proliferation in early, and collagen I content of mature (6-week-old) vein grafts were increased (by 70% and 50%, respectively) in p75-/- grafts. P75 deficiency augmented TNF-induced apoptosis of cultured endothelial cells, but did not affect TNF-stimulated SMC proliferation or migration induced by co-cultured macrophages. CONCLUSIONS: TNF signaling via p75 reduces vein graft neointimal hyperplasia through mechanisms involving reduction of adhesion molecule expression and endothelial cell apoptosis.

Expression of tumor necrosis factor receptor-1 in arterial wall cells promotes atherosclerosis.

OBJECTIVE: Mechanisms by which tumor necrosis factor-alpha (TNF) contributes to atherosclerosis remain largely obscure. We therefore sought to determine the role of the arterial wall TNF receptor-1 (TNFR1) in atherogenesis. METHODS AND RESULTS: Carotid artery-to-carotid artery interposition grafting was performed with tnfr1-/- and congenic (C57Bl/6) wild-type (WT) mice as graft donors, and congenic chow-fed apolipoprotein E-deficient mice as recipients. Advanced atherosclerotic graft lesions developed within 8 weeks, and had 2-fold greater area in WT than in tnfr1-/- grafts. While the prevalence of specific atheroma cells was equivalent in WT and tnfr1-/- grafts, the overall abundance of cells was substantially greater in WT grafts. WT grafts demonstrated greater MCP-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression at both early and late time points, and proliferating cell nuclear antigen expression at early time points. Aortic atherosclerosis was also reduced in 14-month-old apoe(-/-)/tnfr1(-/-) mice, as compared with cognate apoe-/- mice. In coculture with activated macrophages, smooth muscle cells expressing the TNFR1 demonstrated enhanced migration and reduced scavenger receptor activity. CONCLUSIONS: TNFR1 signaling, just in arterial wall cells, contributes to the pathogenesis of atherosclerosis by enhancing arterial wall chemokine and adhesion molecule expression, as well as by augmenting medial smooth muscle cell proliferation and migration.

Expression and function of lysophosphatidic acid LPA1 receptor in prostate cancer cells.

The bioactive phospholipid lysophosphatidic acid (LPA) promotes cell proliferation, survival, and migration by acting on cognate G protein-coupled receptors named LPA(1), LPA(2), and LPA(3). We profiled gene expression of LPA receptors in androgen-dependent and androgen-insensitive prostate cancer cells and found that LPA(1) gene is differentially expressed in androgen-insensitive and LPA-responsive but not androgen-dependent and LPA-resistant cells. In human prostate specimens, expression of LPA(1) gene was significantly higher in the cancer compared with the benign tissues. The androgen-dependent LNCaP cells do not express LPA(1) and do not proliferate in response to LPA stimulation, implying LPA(1) transduces cell growth signals. Accordingly, stable expression of LPA(1) in LNCaP cells rendered them responsive to LPA-induced cell proliferation and decreased their doubling time in serum. Implantation of LNCaP-LPA(1) cells resulted in increased rate of tumor growth in animals compared with those tumors that developed from the wild-type cells. Growth of LNCaP cells depends on androgen receptor activation, and we show that LPA(1) transduces Galphai-dependent signals to promote nuclear localization of androgen receptor and cell proliferation. In addition, treatment with bicalutamide inhibited LPA-induced cell cycle progression and proliferation of LNCaP-LPA(1) cells. These results suggest the possible utility of LPA(1) as a drug target to interfere with progression of prostate cancer.

Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2.

We have recently described a novel gene on human chromosome 20q 12-13.2 called Eppin (Epididymal protease inhibitor) that expresses three mRNAs encoding two isoforms of a cysteine-rich protein containing both Kunitz-type and WAP-type (four disulfide core) consensus sequences (Richardson et al., 2001). To further our studies on Eppin, we have cloned, sequenced and characterized mouse Eppin and report that it lies within a 200 Kb cluster of putative Eppin-like genes on mouse chromosome 2. Analysis of the homologies between the genes in the human and mouse Eppin clusters indicates that the first part of the cluster immediately surrounding Eppin represents a conserved linkage because the order of homologous genes is conserved. Sequencing of reverse transcription polymerase chain reaction (RT-PCR) products confirmed the expression of five of these novel Eppin-like genes in the mouse, which include the mouse homologue of HE-4. These genes are characterized by having either one or both of the Kunitz-type and WAP-type consensus sequences. Additional RT-PCR experiments revealed that expression of some of the Eppin-like genes is restricted to epididymis and testis while others are expressed in several somatic tissues. Northern blot analysis of 22 different mouse tissues identified Eppin transcripts only in the epididymis and testis. Immunostaining of Eppin with anti-recombinant mouse Eppin demonstrated Eppin predominantly on the postacrosomal region of mouse spermatozoa, in Sertoli cells, Leydig cells, and round spermatids in the testis, and in the principal cells of the cauda epididymidis epithelium. Eppin is first expressed by Sertoli cells of 12-day-old mice and subsequently in round spermatids, which is consistent with androgen regulation. Our results demonstrate that mouse chromosome 2 contains a conserved linkage of Eppin-like protease inhibitor genes that are expressed in the epididymis.

A rapid membrane potential assay to monitor CFTR function and inhibition.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important regulator of ion transport and fluid secretion in humans. Mutations to CFTR cause cystic fibrosis, which is a common recessive genetic disorder in Caucasians. Involvement of CFTR has been noted in other important diseases, such as secretory diarrhea and polycystic kidney disease. The assays to monitor CFTR function that have been described to date either are complicated or require specialized instrumentation and training for execution. In this report, we describe a rapid FlexStation-based membrane potential assay to monitor CFTR function. In this assay, agonist-mediated activation of CFTR results in membrane depolarization that can be monitored using a fluorescent membrane potential probe. Availability of a simple mix-and-read assay to monitor the function of this important protein might accelerate the discovery of CFTR ligands to study a variety of conditions.

Interleukin 6 mediates the lysophosphatidic acid-regulated cross-talk between stromal and epithelial prostate cancer cells.

The interaction between stromal and epithelial cells is critical for the initiation and progression of prostate cancer, but the molecular determinants responsible for the cross-talk between these two cell types remain largely unknown. Here, we used a co-culture cell assay to identify messengers involved in the cross-talk between human prostate stromal PS30 and epithelial LNCaP cells. Stimulation with lysophosphatidic acid (LPA) activates the mitogenic ERK signaling pathway in PS30, but not LNCaP, cells. The co-culture of PS30 and LNCaP cells results in the activation of ERK in LNCaP cells and that is further increased in response to stimulation with LPA. Physiologic relevance of the interaction between PS30 and LNCaP cells is demonstrated using LNCaP xenograft tumor assays. Animals implanted with a mixture of both cell types develop larger tumors with higher frequency compared with those injected with LNCaP cells alone. Conditioned medium transfer experiments reveal the PS30-derived inducing factor is soluble and promotes mitogenic ERK and STAT3 signaling pathways in LNCaP cells. Protein analysis demonstrates that treatment of the PS30 cells with LPA induces synthesis of interleukin 6 (IL-6). Antibody neutralization experiments reveal that IL-6 is responsible for the LPA-induced mitogenic signaling and growth of the LNCaP cells. Our findings reveal that the LPA-regulated secretion of IL-6 is an important messenger linking stromal and epithelial prostate cells, which may be exploited for the effective treatment of patients with advanced prostate cancer.

Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif.

The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.