Philip Gotwals

Blockade of TGF-β inhibits mammary tumor cell viability, migration, and metastases

TGF-βs are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-β interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-β type II receptor fusion protein (Fc:TβRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TβRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TβRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TβRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TβRII. Therefore, blockade of TGF-β signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.

Regulation of inflammation by collagen-binding integrins α1β1 and α2β1 in models of hypersensitivity and arthritis

Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAbs against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.

Reduction of bleomycin induced lung fibrosis by transforming growth factor β soluble receptor in hamsters

BACKGROUND Transforming growth factor β (TGF-β) is a key mediator of collagen synthesis in the development of lung fibrosis. It has previously been shown that the administration of TGF-β antibody and TGF-β binding proteoglycan, decorin, reduced bleomycin (BL) induced lung fibrosis in animals. The present study was carried out to investigate whether intratracheal instillation of TGF-β soluble receptor (TR) would minimise the BL induced lung fibrosis in hamsters. METHODS The effect of a recombinant TR (TGFβRII) on the lung collagen accumulation was evaluated in a BL hamster model of pulmonary fibrosis. Animals were divided into four groups and intratracheally injected with saline or BL at 6.5 U/4 ml/kg followed by intratracheal instillation of phosphate buffered saline (PBS) or 4 nmol TR in 0.3 ml twice a week. Twenty days after the first intratracheal instillation the hamsters were killed for bronchoalveolar lavage (BAL) fluid, biochemical, and histopathological analyses. RESULTS Treatment of hamsters with TR after intratracheal instillation of BL significantly reduced BL induced lung fibrosis as shown by decreases in the lung hydroxyproline level and prolyl hydroxylase activity, although they were still significantly higher than those of the saline control. Histopathological examination showed a considerable decrease in BL induced fibrotic lesions by TR treatment. However, TR did not prevent the BL induced increases in total cells and protein in the BAL fluid. CONCLUSIONS These results suggest that TR has antifibrotic potential in vivo and may be useful in the treatment of fibrotic diseases where increased TGF-β is associated with excess collagen accumulation.

The alpha1beta1 integrin is expressed during neointima formation in rat arteries and mediates collagen matrix reorganization.

Remodeling of the extracellular matrix by activated mesenchymal cells (myofibroblasts) is a critical aspect of wound repair in all adult organs. Collagen-dependent gel contraction, a process requiring integrin function, is an established in vitro assay thought to mimic in vivo matrix remodeling. Numerous data have implicated the alpha2beta1 integrin in various cell types as the primary collagen receptor responsible for collagen gel contraction. However, evidence from the literature suggests that the major collagen binding integrin expressed on mesenchymally derived cells in situ is the alpha1beta1 integrin, not the alpha2beta1 integrin. In this report, we use a rat vascular injury model to illustrate that the alpha1beta1 integrin is the major collagen receptor expressed on vascular smooth muscle cells after injury. Using two smooth muscle cell lines, expressing either the alpha1beta1 integrin alone or both the alpha1beta1 and alpha2beta1 integrins, along with Chinese hamster ovary cells transfected with the alpha1 integrin, we demonstrate that alpha1beta1 supports not only collagen-dependent adhesion and migration, but also gel contraction. These data suggest that in vivo the alpha1beta1 integrin is a critical collagen receptor on mesenchymally derived cells potentially involved in matrix remodeling after injury.

Transforming Growth Factor-Beta-Dependent Events in Vascular Remodeling following Arterial Injury

Constrictive remodeling has been identified as a major contributor to restenosis following angioplasty. Characterization of transforming growth factor-β (TGF-β)-mediated cellular events in the adventitia and their contribution to vascular remodeling, however, has not previously been studied in detail. The balloon catheter denudation model was performed on rat carotid artery, and groups of rats were treated with vehicle or a TGF-β inhibitor, a soluble TGF-β receptor type II (TGF-βR:Fc). Adventitial cell proliferation, which peaked 4 days after injury, was characterized by the de novo formation of several cell layers surrounding the outer adventitia and this process was not dependent upon TGF-β activity. These neoadventitial cells expressed an abundance of collagen type I and a fetal isoform of fibronectin containing the EIIIA domain, and the expression of both proteins was suppressed in the presence of TGF-βR:Fc. Lumenal narrowing was apparent 14 days after injury. Inhibition of TGF-β signaling promoted vessel enlargement. As a result, lumen size did not change despite neointima formation. In conclusion, adventitial fibrosis with abundant collagen matrix deposition but not adventitial cell proliferation is dependent upon endogenous TGF-β activity. Furthermore, inhibition of TGF-β signaling prevents injury-induced reduction in lumen area by promoting vessel enlargement.

Global expression analysis of extracellular matrix-integrin interactions in monocytes

Central to immune and inflammatory responses are the integrin-mediated adhesive interactions of cells with their extracellular matrix (ECM)-rich environment. Using a comprehensive and quantitative mRNA profiling technique, we analyzed the effect of ECM-induced attachment on monocyte gene expression, its regulation by growth factors, and the integrin specificity of this event. Adhesion of cells to fibronectin resulted in increased expression of a large number of genes, which was strongly potentiated by the presence of growth factors. Adhesion activated both the NF-kappaB and Jak/STAT pathways of gene transcription and increased expression of genes involved in inflammatory and immune responses, revealing the importance of ECM-integrin interactions in these processes.

Crystal structure of the α1β1 integrin I-domain: insights into integrin I-domain function

The α1β1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a ∼200 amino acid inserted ‘I’‐domain contained in the extracellular part of the integrin α chain. Integrin I‐domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I‐domain from the rat α1β1 integrin at 2.2 Å resolution in the absence of divalent cations. The α1 I‐domain adopts the dinucleotide binding fold that is characteristic of all I‐domain structures that have been solved to date and has a structure very similar to that of the closely related α2β1 I‐domain which also mediates collagen binding. A unique feature of the α1 I‐domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I‐domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand‐induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I‐domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.

Interference with TGF‐β1 and ‐β3 in tumor stroma lowers tumor interstitial fluid pressure independently of growth in experimental carcinoma

A high tumor interstitial fluid pressure (TIFP) is a pathologic characteristic distinguishing the stroma of carcinomas from normal interstitial loose connective tissues. The role of TGF‐β1 and ‐β3 in generating a high TIFP was investigated in xenografted experimental anaplastic thyroid carcinoma (ATC) derived from the human ATC cell line KAT‐4. A single intravenous injection of a soluble recombinant TGF‐β receptor type II‐murine Fc:IgG2A chimeric protein that specifically inhibits TGF‐β1 and ‐β3, significantly lowered TIFP in a time and concentration dependent manner but did not change total tissue water content in the tumors. Tumor growth rate was higher in tumors treated with the TGF‐β1 and ‐β3 inhibitor compared to control tumors during the first 10 days after administration of the inhibitor. The apoptotic index of carcinoma cells, and expression of the cell cycle inhibitor p27Kip1, were, however, increased in TGF‐β1 and ‐β3 inhibitor‐treated tumors. Prolonged treatment periods and administration of a second dose of the inhibitor decreased tumor growth rate. The TGF‐β1 and ‐β3 inhibitor did not affect proliferation or expression of phosphorylated Smad2 protein in KAT‐4 cells cultured in vitro. Our results indicate that members of the TGF‐β family are potential targets for novel anti‐cancer treatment directed to the stroma. First by controlling TIFP and by that potentially the uptake of anticancer drugs into tumors and second by their suggested role in maintaining a supportive tumor stroma.

Fibronectin Type III Repeats Mediate RGD-independent Adhesion and Signaling through Activated β1 Integrins

Many cell-surface and extracellular matrix proteins contain multiple modular domains known as fibronectin type III (FNIII) repeats. Cells adhere to the extracellular matrix proteins fibronectin and tenascin in part by the interaction of certain integrins with the Arg-Gly-Asp (RGD) sequence, displayed on specific FNIII repeats. We have found that, after experimental activation of β1 integrins, a number of cell types adhere and spread on FNIII repeats lacking RGD, derived from extracellular matrix proteins and cytokine receptors. Interaction between individual FNIII domains and β1 integrins mediates focal adhesion kinase phosphorylation and subsequent stress fiber and focal contact formation. These data suggest that many FNIII-containing proteins may bind and signal through activated β1 integrins, dramatically expanding the potential for integrin-dependent intercellular and cell-matrix communication.

Effect of antibody against integrin α4 on bleomycin-induced pulmonary fibrosis in mice

Integrins are a family of transmembrane glycoproteins that can interact with components of the extracellular matrix. The alpha4beta1 and alpha4beta7 integrins are heterodimeric leukocyte cell surface molecules critical to their cell and matrix adhesive interactions. Evidence for a central role for the alpha4 integrins in leukocyte pathophysiology in the lung is well documented. In this study, we tested the hypothesis that neutralizing antibody for integrin alpha4 (PS2) may reduce bleomycin (BL)-induced lung fibrosis in vivo. Male C57BL/6 mice were injected intratracheally with saline (SA) or BL (0.08 U/mouse) followed by intraperitoneal injection of SA, isotype control antibody (1E6), or PS2 (100 microg) three times a week. Twenty-one days after the intratracheal instillation, mice were killed for bronchoalveolar lavage (BAL), biochemical, histopathological, and immunohistological analyses. Treatment with PS2 significantly reduced BL-induced increases in lung lipid peroxidation and hydroxyproline content. Lung histopathology also showed reduced fibrotic lesions in the BL-treated lungs by treatment with PS2. BL-treated mouse lungs also showed induction of cells with the myofibroblast phenotype, as indicated by the increased expression of alpha-smooth muscle actin (alphaSMA), whereas treatment with PS2 minimized the BL-induced alphaSMA expression. Furthermore, treatment with PS2 reduced the BL-induced increase in the BAL total cell number, and attenuated the BL-induced increase in the BAL protein level. It is concluded that integrin alpha4 may play an important role in BL-induced pulmonary fibrosis, and the use of anti-alpha4 antibody offers therapeutic antifibrotic potential in vivo.