Kallikrein-related peptidase 5 contributes to the remodeling and repair of bronchial epithelium.

Inflammation, oxidative stress, and protease/protease inhibitor imbalance with excessive production of proteases are factors associated with pathogenesis of the chronic obstructive pulmonary disease (COPD). In this study, we report that kallikrein-related peptidase 5 (KLK5) is a crucial protease involved in extracellular matrix (ECM) remodeling and bronchial epithelial repair after injury. First, we showed that KLK5 degrades the basal layer formed by culture of primary bronchial epithelial cells from COPD or non-COPD patients. Also, exogenous KLK5 acted differently on BEAS-2B cells already engaged in epithelial-to-mesenchymal transition (EMT) or on 16HBE 14o- cells harboring epithelial characteristics. Indeed, by inducing EMT, KLK5 reduced BEAS-2B cell adherence to the ECM. This effect, neutralized by tissue factor pathway inhibitor 2, a kunitz-type serine protease inhibitor, was due to a direct proteolytic activity of KLK5 on E-cadherin, ß-catenin, fibronectin, and α5ß1 integrin. Thus, KLK5 may strengthen EMT mechanisms and promote the migration of cells by activating the mitogen-activated protein kinase signaling pathway required for this function. In contrast, knockdown of endogenous KLK5 in 16HBE14o- cells, accelerated wound healing repair after injury, and exogenous KLK5 addition delayed the closure repair. These data suggest that among proteases, KLK5 could play a critical role in airway remodeling events associated with COPD during exposure of the pulmonary epithelium to inhaled irritants or smoking and the inflammation process.

Tissue factor pathway inhibitor 2 is a potent kallikrein-related protease 12 inhibitor.

The protease activities are tightly regulated by inhibitors and dysregulation contribute to pathological processes such as cancer and inflammatory disorders. Tissue factor pathway inhibitor 2 (TFPI-2) is a serine proteases inhibitor, that mainly inhibits plasmin. This protease activated matrix metalloproteases (MMPs) and degraded extracellular matrix. Other serine proteases are implicated in these mechanisms like kallikreins (KLKs). In this study, we identified for the first time that TFPI-2 is a potent inhibitor of KLK5 and 12. Computer modeling showed that the first Kunitz domain of TFPI-2 could interact with residues of KLK12 near the catalytic triad. Furthermore, like plasmin, KLK12 was able to activate proMMP-1 and -3, with no effect on proMMP-9. Thus, the inhibition of KLK12 by TFPI-2 greatly reduced the cascade activation of these MMPs and the cleavage of cysteine-rich 61, a matrix signaling protein. Moreover, when TFPI-2 bound to extracellular matrix, its classical localisation, the KLK12 inhibition was retained. Finally, TFPI-2 was downregulated in human non-small-cell lung tumour tissue as compared with non-affected lung tissue. These data suggest that TFPI-2 is a potent inhibitor of KLK12 and could regulate matrix remodeling and cancer progression mediated by KLK12.

Platelet Integrins in Tumor Metastasis: Do They Represent a Therapeutic Target?

Platelets are small anucleated cell fragments that ensure the arrest of bleeding after a vessel wall injury. They are also involved in non-hemostatic function such as development, immunity, inflammation, and in the hematogeneous phase of metastasis. While the role of platelets in tumor metastasis has been recognized for 60 years, the molecular mechanism underlying this process remains largely unclear. Platelets physically and functionally interact with various tumor cells through surface receptors including integrins. Platelets express five integrins at their surface, namely α2ß1, α5ß1, α6ß1, αvß3, and αIIbß3, which bind preferentially to collagen, fibronectin, laminin, vitronectin, and fibrinogen, respectively. The main role of platelet integrins is to ensure platelet adhesion and aggregation at sites of vascular injury. Two of these, α6ß1 and αIIbß3, were proposed to participate in platelet-tumor cell interaction and in tumor metastasis. It has also been reported that pharmacological agents targeting both integrins efficiently reduce experimental metastasis, suggesting that platelet integrins may represent new anti-metastatic targets. This review focuses on the role of platelet integrins in tumor metastasis and discusses whether these receptors may represent new potential targets for novel anti-metastatic approaches.

Role of Tumor Pericytes in the Recruitment of Myeloid-Derived Suppressor Cells.

BACKGROUND: Pericytes are members of the tumor stroma; however, little is known about their origin, function, or interaction with other tumor components. Emerging evidence suggest that pericytes may regulate leukocyte transmigration. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with powerful inhibitory effects on T-cell-mediated antitumor reactivity. METHODS: We generated subcutaneous tumors in a genetic mouse model of pericyte deficiency (the pdgfb (ret/ret) mouse) and littermate control mice (n = 6-25). Gene expression profiles from 253 breast cancer patients (stage I-III) were evaluated for clinic-pathological parameters and survival using Cox proportional hazard ratios (HRs) and 95% confidence intervals (CIs) based on a two-sided Wald test. RESULTS: We report that pericyte deficiency leads to increased transmigration of Gr1(+)/CD11b(+) cells in experimentally induced tumors. Pericyte deficiency produced defective tumor vasculature, resulting in a more hypoxic microenvironment promoting IL-6 upregulation in the malignant cells. Silencing IL-6 expression in tumor cells attenuated the observed differences in MDSC transmigration. Restoring the pericyte coverage in tumors abrogated the increased MDSC trafficking to pericyte-deficient tumors. MDSC accumulation in tumors led to increases in tumor growth and in circulating malignant cells. Finally, gene expression analysis from human breast cancer patients revealed increased expression of the human MDSC markers CD33 and S100A9 with concomitant decreased expression of pericyte genes and was associated with poor prognosis (HR = 1.88, 95% CI = 1.08 to 3.25, P = .03). CONCLUSIONS: Our data uncovers a novel paracrine interaction between tumor pericytes and inflammatory cells and delineates the cellular events resulting in the recruitment of MDSC to tumors. Furthermore, we propose for the first time a role for tumor pericytes in modulating the expression of immune mediators in malignant cells by promoting a hypoxic microenvironment.

Beneficial role of overexpression of TFPI-2 on tumour progression in human small cell lung cancer.

Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI-2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI-2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI-2 in tumour progression, TFPI-2 was overexpressed in NCI-H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI-2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI-H209 cells expressing TFPI-2. We also demonstrated that TFPI-2 upregulation in NCI-H209 cells decreased MMP expression, particularly by downregulating MMP-1 and MMP-3. Moreover, TFPI-2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP-1 was predominant in SCLC tissues and was inversely expressed with TFPI-2 in 35% of cases. These results suggest that downregulation of TFPI-2 expression could favour the development of SCLC.

Pro-angiogenic effect of human kallikrein-related peptidase 12 (KLK12) in lung endothelial cells does not depend on kinin-mediated activation of B2 receptor.

Kallikrein-12 (KLK12) may play an important role in angiogenesis modulating proangiogenic factor bioavailability and activating the kinin receptor B2 pathway. We studied whether KLK12 had an impact on angiogenesis and the activation of kinin receptor B2 results from the KLK12-dependent generation of kinins. KLK12 efficiently hydrolyzed high molecular weight kininogen, liberating a fragment containing the carboxy-terminal end of kinins. The kininogenase activity of KLK12 was poor, however, due to the cleavage resistance of the N-terminal side of the kinin sequence. A very low amount of kinins was accordingly released after in vitro incubation of high molecular weight kininogen with KLK12 and thus the proangiogenic activity of KLK12 in lung endothelial cells was not related to a kinin release.

Monitoring of tumour progression using bioluminescence imaging and computed tomography scanning in a nude mouse orthotopic model of human small cell lung cancer.

Human small cell lung carcinoma (SCLC) is the most aggressive type of lung cancer but no clinically relevant animal model has been developed to date. Such a model would be valuable to study the molecular aspects of tumour progression and to test the effectiveness of new treatment agents. We generated a reproducible and reliable nude mouse orthotopic model of human SCLC with NCI-H209 tumour cells genetically modified to express firefly luciferase. Cells were analysed for long-term stability of bioluminescence and a clone was passaged twice subcutaneously to enhance tumorigenicity. Cells resuspended in Matrigel and/or EDTA RPMI medium containing a (99m)Tc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea and positioned in the main bronchus using X-ray-guided imaging. Deposition of cells into the lung was then assessed by scintigraphy. The growth of the primary tumour was sensitively and non-invasively followed by bioluminescence imaging that allowed real-time monitoring of tumour progression in the same animals over a 2-12-week period. Additional 3D bioluminescence imaging and computed tomography scanning were used to document tumour location and measurements that were confirmed by histological analyses. In conclusion, this original nude mouse orthotopic model resembles various stages of human small cell lung cancer, and therefore could be used to evaluate new treatment strategies.

Purification and characterization of avian beta-defensin 11, an antimicrobial peptide of the hen egg.

Natural antimicrobial peptides are present in different compartments (eggshell, egg white, and vitelline membranes) of the hen egg and are expected to be involved in the protection of the embryo during its development and to contribute to the production of pathogen-free eggs. In the present study, we used vitelline membranes from hen (Gallus gallus) eggs as a source of avian ß-defensin 11 (AvBD11). A purification scheme using affinity chromatography and reverse-phase chromatography was developed. Purified AvBD11 was analyzed by a combination of mass spectrometry approaches to characterize its primary sequence and structure. A monoisotopic molecular species at [M + H](+) of 9,271.56 Da was obtained, and its N- and C-terminal sequences were determined. We also examined posttranslational modifications and identified the presence of 6 internal disulfide bonds. AvBD11 was found to exhibit antimicrobial activity toward both Gram-positive and Gram-negative bacteria.