C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement.

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.

A Phase IIb Randomized Clinical Study of an Anti-αvß6 Monoclonal Antibody in Idiopathic Pulmonary Fibrosis.

Rationale: Treatment options for idiopathic pulmonary fibrosis (IPF) are limited. Objectives: To evaluate the efficacy and safety of BG00011, an anti-αvß6 IgG1 monoclonal antibody, in the treatment of patients with IPF. Methods: In a phase IIb randomized, double-blind, placebo-controlled trial, patients with IPF (FVC ⩾50% predicted, on or off background therapy) were randomized 1:1 to once-weekly subcutaneous BG00011 56 mg or placebo. The primary endpoint was FVC change from baseline at Week 52. Because of early trial termination (imbalance in adverse events and lack of clinical benefit), endpoints were evaluated at Week 26 as an exploratory analysis. Measurements and Main Results: One hundred six patients were randomized and received at least one dose of BG00011 (n = 54) or placebo (n = 52). At Week 26, there was no significant difference in FVC change from baseline between patients who received BG00011 (n = 20) or placebo (n = 23), least squares mean (SE) -0.097 L (0.0600) versus -0.056 L (0.0593), respectively (P = 0.268). However, after Week 26, patients in the BG00011 group showed a worsening trend. Eight (44.4%) of 18 who received BG00011 and 4 (18.2%) of 22 who received placebo showed worsening of fibrosis on high-resolution computed tomography at the end of treatment. IPF exacerbation/or progression was reported in 13 patients (all in the BG00011 group). Serious adverse events occurred more frequently in BG00011 patients, including four deaths. Conclusions: The results do not support the continued clinical development of BG00011. Further research is warranted to identify new treatment strategies that modify inflammatory and fibrotic pathways in IPF. Clinical trial registered with www.clinicaltrials.gov (NCT03573505).

Inhibition of integrin αVß6 changes fibril thickness of stromal collagen in experimental carcinomas.

BACKGROUND: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-ß1 and -ß3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma. METHODS: The potential role of αVß6 integrin-mediated activation of latent TGF-ß was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVß6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVß6 integrin activity. RESULTS: Both KAT-4 and Capan-2 cells expressed the αVß6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-ß in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-ß1 and -ß3 reduced collagen fibril thickness in both tumor models. CONCLUSION: Our data demonstrate that the αVß6-directed activation of latent TGF-ß plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVß6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-ß activation and the extent of desmoplasia.

Inhibition of αvß5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvß5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvß5 in a rat model of renal IRI. Pretreatment with this anti-αvß5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvß5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvß5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvß5 antibody treatment. Immunostaining for αvß5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvß5 in modulating vascular leak. Additional studies showed αvß5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvß5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvß5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvß5 inhibition as a promising therapeutic strategy for AKI.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvß6 Expression.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFß1 is considered central to the pathogenesis of IPF. A major mechanism of TGFß1 activation in the lung involves the epithelially restricted αvß6 integrin. Expression of the αvß6 integrin is dramatically increased in IPF. How αvß6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the ß6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvß6 integrin expression in vitro We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvß6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

Connective tissue growth factor and integrin αvß6: a new pair of regulators critical for ductular reaction and biliary fibrosis in mice.

UNLABELLED: Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvß6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvß6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvß6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvß6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvß6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvß6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-ß1 activation in vitro. CONCLUSIONS: CTGF and integrin αvß6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-ß1. CTGF and integrin αvß6 are potential therapeutic targets to control DRs and fibrosis in related liver disease.

αvß6 integrin is required for TGFß1-mediated matrix metalloproteinase2 expression.

Transforming growth factor (TGF) ß1 activity depends on a complex signalling cascade that controls expression of several genes. Among others, TGFß1 regulates expression of matrix metalloproteinases (MMPs) through activation of Smads. In the present study, we demonstrate for the first time that the αvß6 integrin interacts with TGFß receptor II (TßRII) through the ß6 cytoplasmic domain and promotes Smad3 activation in prostate cancer (PrCa) cells. Another related αv integrin, αvß5, as well as the αvß6/3 integrin, which contains a chimeric form of ß6 with a ß3 cytoplasmic domain, do not associate with TßRII and fail to show similar responses. We provide evidence that αvß6 is required for up-regulation of MMP2 by TGFß1 through a Smad3-mediated transcriptional programme in PrCa cells. The functional relevance of these results is underscored by the finding that αvß6 modulates cell migration in an MMP2-dependent manner on an αvß6-specific ligand, latency-associated peptide (LAP)-TGFß. Overall, these mechanistic studies establish that expression of a single integrin, αvß6, is sufficient to promote activation of Smad3, regulation of MMP2 levels and consequent catalytic activity, as well as cell migration. Our study describes a new TGFß1-αvß6-MMP2 signalling pathway that, given TGFß1 pro-metastatic activity, may have profound implications for PrCa therapy.

αvß6 integrin may be a potential prognostic biomarker in interstitial lung disease.

Idiopathic pulmonary fibrosis (IPF) and fibrotic nonspecific interstitial pneumonitis are progressive interstitial lung diseases (ILDs) with limited treatment options and poor survival. However, the rate of disease progression is variable, implying there may be different endotypes of disease. We hypothesised that immunophenotyping biopsies from ILD patients might reveal distinct endotypes of progressive fibrotic disease, which may facilitate stratification when undertaking clinical trials of novel therapies for IPF.43 paraffin-embedded, formalin-fixed lung tissue sections were immunostained for five molecules implicated in the pathogenesis of the fibrosis: α-smooth muscle actin (αSMA), αvß6 integrin, pro-surfactant protein C (SP-C), hepatocyte growth factor (HGF) and tenascin-C (TenC). Levels of immunostaining and numbers of fibroblastic foci were quantified using operator-dependent and -independent methods. The relationship of all these markers to overall survival was analysed.Staining revealed high levels of αSMA, αvß6 integrin, pro-SP-C, HGF and TenC, and fibroblastic foci. Immunostaining varied across samples for all molecules but only the extent of αvß6 integrin immunostaining was associated with increased mortality. There was no association with the other markers measured.Our data suggest high levels of αvß6 integrin may identify a specific endotype of progressive fibrotic lung disease.

Future directions in idiopathic pulmonary fibrosis research. An NHLBI workshop report.

The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.

Inhibition of the αvß6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis.

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-ß (TGF-ß) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-ß induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-ß pathway in the lung is unknown. The αvß6 integrin is an important in vivo activator of TGF-ß activation in the lung. Immunohistochemical analysis of αvß6 protein expression and bronchoalveolar analysis of TGF-ß pathway signaling indicates activation of the αvß6/TGF-ß pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvß6/TGF-ß pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvß6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvß6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the ß6 integrin, TGF-α transgenic mice were mated with ß6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the ß6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the ß6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvß6/TGF-ß pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

Lysophosphatidic acid increases proximal tubule cell secretion of profibrotic cytokines PDGF-B and CTGF through LPA2- and Gαq-mediated Rho and αvß6 integrin-dependent activation of TGF-ß.

After ischemia-reperfusion injury (IRI), kidney tubules show activated transforming growth factor ß (TGF-ß) signaling and increased expression of profibrotic peptides, platelet-derived growth factor-B (PDGF-B) and connective tissue growth factor (CTGF). If tubule repair after IRI is incomplete, sustained paracrine activity of these peptides can activate interstitial fibroblast progenitors and cause fibrosis. We show that lysophosphatidic acid (LPA), a ubiquitous phospholipid that is increased at sites of injury and inflammation, signals through LPA2 receptors and Gαq proteins of cultured proximal tubule cells to transactivate latent TGF-ß in a Rho/Rho-kinase and αvß6 integrin-dependent manner. Active TGF-ß peptide then initiates signaling to increase the production and secretion of PDGF-B and CTGF. In a rat model of IRI, increased TGF-ß signaling that was initiated early during reperfusion did not subside during recovery, but progressively increased, causing tubulointerstitial fibrosis. This was accompanied by correspondingly increased LPA2 and ß6 integrin proteins and elevated tubule expression of TGF-ß1, together with PDGF-B and CTGF. Treatment with a pharmacological TGF-ß type I receptor antagonist suppressed TGF-ß signaling, decreased the expression of ß6 integrin, PDGF-B, and CTGF, and ameliorated fibrosis. We suggest that LPA-initiated autocrine signaling is a potentially important mechanism that gives rise to paracrine profibrotic signaling in injured kidney tubule cells.

Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7-13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As αvß6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. αvß6 was markedly up-regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of αvß6 using oral keratinocyte-derived cells genetically modified to express high αvß6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low αvß6 (OKF6/TERT-1). VB6 cells showed significant αvß6-dependent activation of TGF-ß1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF-ß1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte αvß6 expression. This was modulated through the M(4) muscarinic acetylcholine receptor and was suppressed by the M(4) antagonist, tropicamide. Arecoline-dependent αvß6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF-related oral cancers examined had moderate/high αvß6 expression. These data suggest that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of αvß6 integrin.

Stromal features are predictive of disease mortality in oral cancer patients.

Worldwide, approximately 405 000 cases of oral cancer (OSCC) are diagnosed each year, with a rising incidence in many countries. Despite advances in surgery and radiotherapy, which remain the standard treatment options, the mortality rate has remained largely unchanged for decades, with a 5-year survival rate of around 50%. OSCC is a heterogeneous disease, staged currently using the TNM classification, supplemented with pathological information from the primary tumour and loco-regional lymph nodes. Although patients with advanced disease show reduced survival, there is no single pathological or molecular feature that identifies aggressive, early-stage tumours. We retrospectively analysed 282 OSCC patients for disease mortality, related to clinical, pathological, and molecular features based on our previous functional studies [EGFR, αvß6 integrin, smooth muscle actin (SMA), p53, p16, EP4]. We found that the strongest independent risk factor of early OSCC death was a feature of stroma rather than tumour cells. After adjusting for all factors, high stromal SMA expression, indicating myofibroblast transdifferentiation, produced the highest hazard ratio (3.06, 95% CI 1.65-5.66) and likelihood ratio (3.6; detection rate: false positive rate) of any feature examined, and was strongly associated with mortality, regardless of disease stage. Functional assays showed that OSCC cells can modulate myofibroblast transdifferentiation through αvß6-dependent TGF-ß1 activation and that myofibroblasts promote OSCC invasion. Finally, we developed a prognostic model using Cox regression with backward elimination; only SMA expression, metastasis, cohesion, and age were significant. This model was independently validated on a patient subset (detection rate 70%; false positive rate 20%; ROC analysis 77%, p < 0.001). Our study highlights the limited prognostic value of TNM staging and suggests that an SMA-positive, myofibroblastic stroma is the strongest predictor of OSCC mortality. Whether used independently or as part of a prognostic model, SMA identifies a significant group of patients with aggressive tumours, regardless of disease stage.

Development and Optimization of Bifunctional Fusion Proteins to Locally Modulate Complement Activation in Diseased Tissue.

Sustained complement activation is an underlying pathologic driver in many inflammatory and autoimmune diseases. Currently approved anti-complement therapies are directed at the systemic blockade of complement. Consequently, these therapies provide widespread inhibition of complement pathway activity, beyond the site of ongoing activation and the intended pharmacodynamic (PD) effects. Given the essential role for complement in both innate and adaptive immunity, there is a need for therapies that inhibit complement in diseased tissue while limiting systemic blockade. One potential approach focuses on the development of novel fusion proteins that enable tissue-targeted delivery of complement negative regulatory proteins. These therapies are expected to provide increased potency and prolonged tissue PD, decreased dosing frequency, and the potential for improved safety profiles. We created a library of bifunctional fusion proteins that direct a fragment of the complement negative regulator, complement receptor type 1 (CR1) to sites of tissue injury. Tissue targeting is accomplished through the binding of the fusion protein to complement C3 fragments that contain a surface-exposed C3d domain and which are covalently deposited on tissues where complement is being activated. To that end, we generated a fusion protein that contains an anti-C3d monoclonal antibody recombinantly linked to the first 10 consensus repeats of CR1 (CR11-10) with the intention of delivering high local concentrations of this complement negative regulatory domain to tissue-bound complement C3 fragments iC3b, C3dg and C3d. Biochemical and in vitro characterization identified several fusion proteins that inhibit complement while maintaining the C3d domain binding properties of the parent monoclonal antibody. Preclinical in vivo studies further demonstrate that anti-C3d fusion proteins effectively distribute to injured tissue and reduce C3 fragment deposition for periods beyond 14 days. The in vitro and in vivo profiles support the further evaluation of C3d mAb-CR11-10 as a novel approach to restore proper complement activation in diseased tissue in the absence of continuous systemic complement blockade.

The coagulation system contributes to alphaVbeta6 integrin expression and liver fibrosis induced by cholestasis.

Chronic injury to intrahepatic bile duct epithelial cells (BDECs) elicits expression of various mediators, including the αVß6 integrin, promoting liver fibrosis. We tested the hypothesis that tissue factor (TF)-dependent thrombin generation and protease activated receptor-1 (PAR-1) activation contribute to liver fibrosis induced by cholestasis via induction of αVß6 expression. To test this hypothesis, mice deficient in either TF or PAR-1 were fed a diet containing 0.025% α-naphthylisothiocyanate (ANIT), a BDEC-selective toxicant. In genetically modified mice with a 50% reduction in liver TF activity fed an ANIT diet, coagulation cascade activation and liver fibrosis were reduced. Similarly, liver fibrosis was significantly reduced in PAR-1(-/-) mice fed an ANIT diet. Hepatic integrin ß6 mRNA induction, expression of αVß6 protein by intrahepatic BDECs, and SMAD2 phosphorylation were reduced by TF deficiency and PAR-1 deficiency in mice fed the ANIT diet. Treatment with either an anti-αVß6 blocking antibody or soluble transforming growth factor-ß receptor type II reduced liver fibrosis in mice fed the ANIT diet. PAR-1 activation enhanced transforming growth factor-ß1-induced integrin ß6 mRNA expression in both transformed human BDECs and primary rat BDECs. Interestingly, TF and PAR-1 mRNA levels were increased in livers from patients with cholestatic liver disease. These results indicate that a TF-PAR-1 pathway contributes to liver fibrosis induced by chronic cholestasis by increasing expression of the αVß6 integrin, an important regulator of transforming growth factor-ß1 activation.

Lysophosphatidic acid induces alphavbeta6 integrin-mediated TGF-beta activation via the LPA2 receptor and the small G protein G alpha(q).

Activation of latent transforming growth factor beta (TGF-beta) by alphavbeta6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes alphavbeta6 integrin-mediated TGF-beta activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce alphavbeta6 integrin-mediated TGF-beta activation is unknown; in addition, the alphavbeta6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces alphavbeta6-mediated TGF-beta activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced alphavbeta6 integrin-mediated TGF-beta activity is mediated via the LPA2 receptor, which signals via G alpha(q). Finally, we show that the expression levels of both the LPA2 receptor and alphavbeta6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and alphavbeta6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces alphavbeta6 integrin-mediated TGF-beta activation in epithelial cells via LPA2, G alpha(q), RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.

Interleukin-1beta causes acute lung injury via alphavbeta5 and alphavbeta6 integrin-dependent mechanisms.

Interleukin (IL)-1beta has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1beta-mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1beta production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta attenuated the IL-1beta-induced ALI. The results of in vitro studies indicated that IL-1beta caused the activation of transforming growth factor-beta via RhoA/alphavbeta6 integrin-dependent mechanisms and the inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta signaling completely blocked the IL-1beta-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1beta increased protein permeability across lung endothelial cell monolayers via RhoA- and alphavbeta5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the alphavbeta5 and alphavbeta6 integrins had an additive protective effect against IL-1beta-induced ALI. In summary, these results demonstrate a critical role for the alphavbeta5/beta6 integrins in mediating the IL-1beta-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI.

Constitutively activated Stat3 induces tumorigenesis and enhances cell motility of prostate epithelial cells through integrin beta 6.

The persistent activation of signal transducer and activator of transcription 3 (Stat3) is a common feature of prostate cancer. However, little is known about the Stat3 targets that may mediate prostate tumorigenesis. The introduction of an activating mutant form of Stat3 (Stat3-C) into immortalized prostate epithelial cells resulted in tumorigenesis. Stat3-C-expressing cells had decreased E-cadherin levels, increased numbers of lamellipodia and stress fibers, and enhanced migratory capacities compared to vector control-expressing cells, with a concomitant increase in the expression of integrin beta6 and its ligand, fibronectin (FN). Exogenously added FN increased cellular migration, with a concomitant loss of E-cadherin expression. The blockade of integrin alphavbeta6 in Stat3-C-expressing cells inhibited migration, increased E-cadherin levels, and reduced colony formation in soft agar. These results demonstrate the sufficiency of constitutively activated Stat3 in mediating prostate tumorigenesis and identify novel Stat3 targets that are involved in promoting cell migration and transformation.

Overexpression of fibroblast growth factor-10 during both inflammatory and fibrotic phases attenuates bleomycin-induced pulmonary fibrosis in mice.

RATIONALE: Fibroblast growth factor-10 (FGF10) controls survival, proliferation, and differentiation of distal-alveolar epithelial progenitor cells during lung development. OBJECTIVES: To test for the protective and regenerative effect of Fgf10 overexpression in a bleomycin-induced mouse model of pulmonary inflammation and fibrosis. METHODS: In SP-C-rtTA; tet(O)Fgf10 double-transgenic mice, lung fibrosis was induced in 2-month-old transgenic mice by subcutaneous delivery of bleomycin (BLM), using an osmotic minipump for 1 week. Exogenous Fgf10 expression in the alveolar epithelium was induced for 7 days with doxycycline during the first, second, and third weeks after bleomycin pump implantation, and lungs were examined at 28 days. MEASUREMENTS AND MAIN RESULTS: Fgf10 overexpression during Week 1 (inflammatory phase) resulted in increased survival and attenuated lung fibrosis score and collagen deposition. In these Fgf10-overexpressing mice, an increase in regulatory T cells and a reduction in both transforming growth factor-beta(1) and matrix metalloproteinase-2 activity were observed in bronchoalveolar lavage fluids whereas the number of surfactant protein C (SP-C)-positive, alveolar epithelial type II cells (AEC2) was markedly elevated. Analysis of SP-C and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end labeling) double-positive cells and isolation of AEC2 from lungs overexpressing Fgf10 demonstrated increased AEC2 survival. Expression of Fgf10 during Weeks 2 and 3 (fibrotic phase) showed significant attenuation of the lung fibrosis score and collagen deposition. CONCLUSIONS: In the bleomycin model of lung inflammation and fibrosis, Fgf10 overexpression during both the inflammatory and fibrotic phases results in a greatly reduced extent of lung fibrosis, suggesting that FGF10 may be useful as a novel approach to the treatment of pulmonary fibrosis.