Fibroblast activation protein augments progression and metastasis of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinomas (PDAs) are desmoplastic and can undergo epithelial-to-mesenchymal transition to confer metastasis and chemoresistance. Studies have demonstrated that phenotypically and functionally distinct stromal cell populations exist in PDAs. Fibroblast activation protein-expressing (FAP-expressing) cells act to enhance PDA progression, while α-smooth muscle actin myofibroblasts can restrain PDA. Thus, identification of precise molecular targets that mediate the protumorigenic activity of FAP+ cells will guide development of therapy for PDA. Herein, we demonstrate that FAP overexpression in the tumor microenvironment correlates with poor overall and disease-free survival of PDA patients. Genetic deletion of FAP delayed onset of primary tumor and prolonged survival of mice in the KPC mouse model of PDA. While genetic deletion of FAP did not affect primary tumor weight in advanced disease, FAP deficiency increased tumor necrosis and impeded metastasis to multiple organs. Lineage-tracing studies unexpectedly showed that FAP is not only expressed by stromal cells, but can also be detected in a subset of CD90+ mesenchymal PDA cells, representing up to 20% of total intratumoral FAP+ cells. These data suggest that FAP may regulate PDA progression and metastasis in cell-autonomous and/or non-cell-autonomous fashions. Together, these data support pursuing FAP as a therapeutic target in PDA.

Level of serum neutrophil gelatinase-associated lipocalin in childhood asthma

BACKGROUND: The role of neutrophil gelatinase-associated lipocalin (NGAL) in childhood asthma remains unknown. This study aimed to measure the serum levels of NGAL in children with asthma and to investigate the correlation between NGAL and transforming growth factor beta 1 (TGF-β1), a good indicator of airway remodeling in children with asthma. METHODS: This prospective, cross-sectional study was conducted on 75 children. Serum NGAL and TGF-β1 concentrations were measured by the ELISA method. Complete blood count, high sensitive C reactive protein (hsCRP), eosinophil cationic protein (ECP), and total serum IgE were investigated in the study population. Atopy in the asthma group was investigated using a skin prick test and specific IgE measurements. RESULTS: Forty-three asthmatic children and 32 healthy children were enrolled in the study. Total eosinophil numbers, white blood cell count, total serum IgE levels and ECP levels were significantly higher in the asthma group than in the control group (p < 0.05). Similarly, serum TGF-β1 levels were significantly higher in children with asthma (p = 0.012). The difference in NGAL levels between the groups was insignificant (p = 0.268). NGAL levels did not show a significant correlation with total IgE, ECP, eosinophil numbers and TGF-β1 levels (p > 0.05). CONCLUSION: As a conclusion, while elevated TGF-β1 levels in children with asthma might be regarded as an indicator of airway remodeling, we did not find a similar prediction strength for NGAL. Further studies are required to better identify the role of NGAL in childhood asthma and to determine its potential use as a clinical marker

No disponible

Deficiency of fibroblast activation protein alpha ameliorates cartilage destruction in inflammatory destructive arthritis.

INTRODUCTION: Inflammatory destructive arthritis, like rheumatoid arthritis (RA), is characterized by invasion of synovial fibroblasts (SF) into the articular cartilage and erosion of the underlying bone, leading to progressive joint destruction. Because fibroblast activation protein alpha (FAP) has been associated with cell migration and cell invasiveness, we studied the function of FAP in joint destruction in RA. METHODS: Expression of FAP in synovial tissues and fibroblasts from patients with osteoarthritis (OA) and RA as well as from wild-type and arthritic mice was evaluated by immunohistochemistry, fluorescence microscopy and polymerase chain reaction (PCR). Fibroblast adhesion and migration capacity was assessed using cartilage attachment assays and wound-healing assays, respectively. For in vivo studies, FAP-deficient mice were crossed into the human tumor necrosis factor transgenic mice (hTNFtg), which develop a chronic inflammatory arthritis. Beside clinical assessment, inflammation, cartilage damage, and bone erosion were evaluated by histomorphometric analyses. RESULTS: RA synovial tissues demonstrated high expression of FAP whereas in OA samples only marginal expression was detectable. Consistently, a higher expression was detected in arthritis SF compared to non-arthritis OA SF in vitro. FAP-deficiency in hTNFtg mice led to less cartilage degradation despite unaltered inflammation and bone erosion. Accordingly, FAP(-/-) hTNFtg SF demonstrated a lower cartilage adhesion capacity compared to hTNFtg SF in vitro. CONCLUSIONS: These data point to a so far unknown role of FAP in the attachment of SF to cartilage, promoting proteoglycan loss and subsequently cartilage degradation in chronic inflammatory arthritis.

Fibroblast activation protein (FAP) is essential for the migration of bone marrow mesenchymal stem cells through RhoA activation.

BACKGROUND: The ability of human bone marrow mesenchymal stem cells (BM-MSCs) to migrate and localize specifically to injured tissues is central in developing therapeutic strategies for tissue repair and regeneration. Fibroblast activation protein (FAP) is a cell surface serine protease expressed at sites of tissue remodeling during embryonic development. It is also expressed in BM-MSCs, but not in normal tissues or cells. The function of FAP in BM-MSCs is not known. PRINCIPAL FINDINGS: We found that depletion of FAP proteins significantly inhibited the migration of BM-MSCs in a transwell chemotaxis assay. Such impaired migration ability of BM-MSCs could be rescued by re-expressing FAP in these cells. We then demonstrated that depletion of FAP activated intracellular RhoA GTPase. Consistently, inhibition of RhoA activity using a RhoA inhibitor rescued its migration ability. Inhibition of FAP activity with an FAP-specific inhibitor did not affect the activation of RhoA or the migration of BM-MSCs. Furthermore, the inflammatory cytokines interleukin-1beta (IL-1ß) and transforming growth factor-beta (TGF-ß) upregulated FAP expression, which coincided with better BM-MSC migration. CONCLUSIONS: Our results indicate FAP plays an important role in the migration of BM-MSCs through modulation of RhoA GTPase activity. The peptidase activity of FAP is not essential for such migration. Cytokines IL-1ß and TGF-ß upregulate the expression level of FAP and thus enhance BM-MSC migration.

Regulation of dipeptidyl peptidase 8 and 9 expression in activated lymphocytes and injured liver.

AIM: To investigate the expression of dipeptidyl peptidase (DPP) 8 and DPP9 in lymphocytes and various models of liver fibrosis. METHODS: DPP8 and DPP9 expression were measured in mouse splenic CD4⁺ T-cells, CD8⁺ T-cells and B-cells (B220⁺), human lymphoma cell lines and mouse splenocytes stimulated with pokeweed mitogen (PWM) or lipopolysaccharide (LPS), and in dithiothreitol (DTT) and mitomycin-C treated Raji cells. DPP8 and DPP9 expression were measured in epidermal growth factor (EGF) treated Huh7 hepatoma cells, in fibrotic liver samples from mice treated with carbon tetrachloride (CCl4) and from multidrug resistance gene 2 (Mdr2/Abcb4) gene knockout (gko) mice with biliary fibrosis, and in human end stage primary biliary cirrhosis (PBC). RESULTS: All three lymphocyte subsets expressed DPP8 and DPP9 mRNA. DPP8 and DPP9 expression were upregulated in both PWM and LPS stimulated mouse splenocytes and in both Jurkat T- and Raji B-cell lines. DPP8 and DPP9 were downregulated in DTT treated and upregulated in mitomycin-C treated Raji cells. DPP9-transfected Raji cells exhibited more annexin V⁺ cells and associated apoptosis. DPP8 and DPP9 mRNA were upregulated in CCl4 induced fibrotic livers but not in the lymphocytes isolated from such livers, while DPP9 was upregulated in EGF stimulated Huh7 cells. In contrast, intrahepatic DPP8 and DPP9 mRNA expression levels were low in the Mdr2 gko mouse and in human PBC compared to non-diseased livers. CONCLUSION: These expression patterns point to biological roles for DPP8 and DPP9 in lymphocyte activation and apoptosis and in hepatocytes during liver disease pathogenesis.

Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice.

Membrane-bound proteases have recently emerged as critical mediators of tumorigenesis, angiogenesis, and metastasis. However, the mechanisms by which they regulate these processes remain unknown. As the cell surface serine protease fibroblast activation protein (FAP) is selectively expressed on tumor-associated fibroblasts and pericytes in epithelial tumors, we set out to investigate the role of FAP in mouse models of epithelial-derived solid tumors. In this study, we demonstrate that genetic deletion and pharmacologic inhibition of FAP inhibited tumor growth in both an endogenous mouse model of lung cancer driven by the K-rasG12D mutant and a mouse model of colon cancer, in which CT26 mouse colon cancer cells were transplanted into immune competent syngeneic mice. Interestingly, growth of only the K-rasG12D-driven lung tumors was also attenuated by inhibition of the closely related protease dipeptidyl peptidase IV (DPPIV). Our results indicate that FAP depletion inhibits tumor cell proliferation indirectly, increases accumulation of collagen, decreases myofibroblast content, and decreases blood vessel density in tumors. These data provide proof of principle that targeting stromal cell-mediated modifications of the tumor microenvironment may be an effective approach to treating epithelial-derived solid tumors.

Description of a 23.9-kilobase chromosomal deletion containing a region encoding fsr genes which mainly determines the gelatinase-negative phenotype of clinical isolates of Enterococcus faecalis in urine.

Expression of virulence-related extracellular proteases, gelatinase, and serine protease of Enterococcus faecalis is regulated by a quorum-sensing system encoded by the fsr gene cluster. In this study, a 23.9-kb chromosomal deletion containing the fsr gene cluster region was found to be present in the majority (79%) of gelatinase-negative clinical isolates of E. faecalis from urine.

Reduced angiogenesis and tumor progression in gelatinase A-deficient mice.

Matrix proteolysis is thought to play a crucial role in several stages of tumor progression, including angiogenesis, and the invasion and metastasis of tumor cells. We investigated the specific role of gelatinase A (matrix metalloproteinase 2) on these events using gelatinase A-deficient mice. In these mice, tumor-induced angiogenesis was suppressed according to dorsal air sac assay. When B16-BL6 melanoma cells or Lewis lung carcinoma cells were implanted intradermally, the tumor volumes at 3 weeks after implantation in the gelatinase A-deficient mice decreased by 39% for B16-BL6 melanoma and by 24% for Lewis lung carcinoma (P < 0.03 for each tumor). The number of lung colonies of i.v. injections fell by 54% for B16-BL6 melanoma and 77% for Lewis lung carcinoma (P < 0.014 and P < 0.0015, respectively). These results indicated that host-derived gelatinase A plays an important role in angiogenesis and tumor progression, suggesting the usefulness of gelatinase A inhibitors for anticancer chemotherapy.

Unaltered secretion of beta-amyloid precursor protein in gelatinase A (matrix metalloproteinase 2)-deficient mice.

The beta-amyloid peptide, which forms extracellular cerebral deposits in Alzheimer's disease, is derived from a large membrane-spanning glycoprotein referred to as the beta-amyloid precursor protein (APP). The APP is normally cleaved within the beta-amyloid region by a putative proteinase (alpha-secretase) to generate large soluble amino-terminal derivatives of APP, and this event prevents the beta-amyloid peptide formation. It has been suggested that the gelatinase A (matrix metalloproteinase 2, a 72-kDa type IV collagenase) may act either as alpha-secretase or as beta-secretase. Mice devoid of gelatinase A generated by gene targeting develop normally, except for a subtle delay in their growth, thus providing a useful system to examine the role of gelatinase A in the cleavage and secretion of APP in vivo. We show here that APP is cleaved within the beta-amyloid region and secreted into the extracellular milieu of brain and cultured fibroblasts without gelatinase A activity. The data suggest that gelatinase A does not play an essential role in the generation and release of soluble derivatives of APP at physiological conditions.