Transforming Growth Factor-ß1 Induced Epithelial Mesenchymal Transition is blocked by a chemical antagonist of translation factor eIF4E.

The epithelial to mesenchymal transition (EMT) imparts disease-defining properties to epithelial cells in cancer and organ fibrosis. Prior studies identify EMT control points at the level of transcription and translation, and indicate that activation of translation initiation factor 4E (eIF4E) is involved in the mechanisms coordinating these two levels of control. Here we show that 4Ei-1, a specific chemical antagonist of the eIF4E-mRNA cap interaction, potently inhibits transforming growth factor beta 1 (TGF-ß1) mediated EMT in lung epithelial cells. Upon treatment with TGF-ß1, we observed a rapid recruitment of Snail1 mRNA into the actively translated polysome pool accompanied by accumulation of the EMT transcription factor Snail1 in the nucleus. 4Ei-1 blocks ribosome recruitment to the Snail1 transcript thereby preventing accumulation of the Snail1 protein in the nucleus. Our findings establish an obligatory role for upstream translational control of downstream Snail1-mediated transcriptional events in TGF-ß1 induced EMT, and provide proof of concept for efforts to pharmacologically modulate the eIF4E-cap interaction as a means to inhibit pathological EMT in the setting of cancer and organ fibrosis.

Cap-dependent mRNA translation and the ubiquitin-proteasome system cooperate to promote ERBB2-dependent esophageal cancer phenotype.

Pathological post-transcriptional control of the proteome composition is a central feature of malignancy. Two steps in this pathway, eIF4F-driven cap-dependent mRNA translation and the ubiquitin-proteasome system (UPS), are deregulated in most if not all cancers. We tested a hypothesis that eIF4F is aberrantly activated in human esophageal adenocarcinoma (EAC) and requires elevated rates of protein turnover and proteolysis and thereby activated UPS for its pro-neoplastic function. Here, we show that 80% of tumors and cell lines featuring amplified ERBB2 display an aberrantly activated eIF4F. Direct genetic targeting of the eIF4F in ERBB2-amplified EAC cells with a constitutively active form of the eIF4F repressor 4E-BP1 decreased colony formation and proliferation and triggered apoptosis. In contrast, suppression of m-TOR-kinase activity towards 4E-BP1with rapamycin only modestly inhibited eIF4F-driven cap-dependent translation and EAC malignant phenotype; and promoted feedback activation of other cancer pathways. Our data show that co-treatment with 2 FDA-approved agents, the m-TOR inhibitor rapamycin and the proteasome inhibitor bortezomib, leads to strong synergistic growth-inhibitory effects. Moreover, direct targeting of eIF4F with constitutively active 4E-BP1 is significantly more potent in collaboration with bortezomib than rapamycin. These data support the hypothesis that a finely tuned balance between eIF4F-driven protein synthesis and proteasome-mediated protein degradation is required for the maintenance of ERBB2-mediated EAC malignant phenotype. Altogether, our study supports the development of pharmaceuticals to directly target eIF4F as most efficient strategy; and provides a clear rationale for the clinical evaluation of combination therapy with m-TOR inhibitors and bortezomib for EAC treatment.

Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eIF4F complex in mesothelioma.

BACKGROUND: Insulin-like growth factor (IGF)-I signalling stimulates proliferation, survival, and invasion in malignant mesothelioma and other tumour types. Studies have found that tumourigenesis is linked to dysregulation of cap-dependent protein translation. METHODS: The effect of IGF stimulation on cap-mediated translation activation in mesothelioma cell lines was studied using binding assays to a synthetic 7-methyl GTP-cap analogue. In addition, cap-mediated translation was genetically repressed in these cells with a dominant active motive of 4E-BP1. RESULTS: In most mesothelioma cell lines, IGF-I stimulation resulted in a hyperphosphorylation-mediated inactivation of 4E-BP1 compared with that in normal mesothelial cells. An inhibitor of Akt diminished IGF-I-mediated phosphorylation of 4E-BP1, whereas inhibiting MAPK signalling had no such effect. IGF-I stimulation resulted in the activation of the cap-mediated translation complex as indicated by an increased eIF4G/eIF4E ratio in cap-affinity assays. Akt inhibition reversed the eIF4G/eIF4E ratio. Mesothelioma cells transfected with an activated 4E-BP1 protein (4E-BP1(A37/A46)) were resistant to IGF-I-mediated growth, motility, and colony formation. In a murine xenograft model, mesothelioma cells expressing the dominant active 4E-BP1(A37/A46) repressor protein showed abrogated tumourigenicity compared with control tumours. CONCLUSION: IGF-I signalling in mesothelioma cells drives cell proliferation, motility, and tumourigenesis through its ability to activate cap-mediated protein translation complex through PI3K/Akt/mTOR signalling.

Adenosarcoma in a patient with vaginal endometriosis.

BACKGROUND: Adenosarcoma in a patient with extraovarian endometriosis is a rare event and can be easily overlooked. CASE: A woman with a history of endometriosis underwent multiple resections of a vaginal mass and medical treatment for presumed recurrent endometriosis. Eventually, a vaginal adenosarcoma was diagnosed. CONCLUSION: The possibility of adenosarcoma should be considered if an enlarging mass occurs at the site of extraovarian endometriosis.

Inhibition of mesothelioma cell growth in vitro by doxycycline.

Malignant mesothelioma causes profound morbidity and nearly universal mortality that is often refractory to conventional treatment modalities of aggressive surgery, radiotherapy, or chemotherapy. Doxycycline, a commonly used antibiotic, has anti-tumor activity against several malignancies, but its anti-tumor effects on malignant mesothelioma have not been evaluated. We report here that concentrations of doxycycline achievable in serum with typical oral doses had cytostatic effects to varying extent on all eight of the mesothelioma cell lines studied but did not affect normal lung fibroblasts. Doxycycline inhibited the production of mitochondrial cytochrome c oxidase, especially in mesothelioma cells more sensitive to its cytostatic effects, and directly inhibited gelatinase A activity; both of these activities are putative mechanisms for the cytostatic activity of doxycycline in other tumor cells. Thus doxycycline may have a role as adjuvant therapy for malignant mesothelioma.

Postmenopausal virilization, simple ovarian cyst, and hilus cell hyperplasia--is there an association?

OBJECTIVE: To report a case of virilizing ovarian hilus cell hyperplasia detected postmenopausally in association with a simple cyst and to review the related literature, including four similar cases. METHODS: Hormonal and pathologic studies were conducted, and ovarian venous catheterization was performed during total abdominal hysterectomy. RESULTS: In our 69-year-old female patient, serum testosterone levels were 508, >3,200, and 11 ng/dL, respectively, in peripheral blood preoperatively, in ovarian venous blood obtained intraoperatively, and in peripheral blood postoperatively. The wall of the cyst contained several clusters of hilus cells, which were also found asymmetrically lateralized to the affected ovary. CONCLUSION: Hilus cell hyperplasia should be suspected in any case of postmenopausal virilization in which ultrasonography or magnetic resonance imaging suggests the presence of a simple ovarian cyst.

Translational control of the antiapoptotic function of Ras.

Activated Ras has been shown to provide powerful antiapoptotic signals to cells through well defined transcriptional and post- translational pathways, whereas translational control as a mechanism of Ras survival signaling remains unexplored. Here we show a direct relationship between assembly of the cap-dependent translation initiation apparatus and suppression of apoptosis by oncogenic Ras in vitro and in vivo. Decreasing protein synthesis with rapamycin, which is known to inhibit cap-dependent translation, increases the susceptibility of Ras-transformed fibroblasts to cytostatic drug-induced apoptosis. In contrast, suppressing global protein synthesis with equipotent concentrations of cycloheximide actually prevents apoptosis. Enforced expression of the cap-dependent translational repressor, the eukaryotic translation initiation factor (eIF) 4E-binding protein (4E-BPI), sensitizes fibroblasts to apoptosis in a manner strictly dependent on its ability to sequester eIF4E from a translationally active complex with eIF4GI and the co-expression of oncogenic Ras. Ectopic expression of 4E-BP1 also promotes apoptosis of Ras-transformed cells injected into immunodeficient mice and markedly diminishes their tumorigenicity. These results establish that eIF4E-dependent protein synthesis is essential for survival of fibroblasts bearing oncogenic Ras and support the concept that activation of cap-dependent translation by extracellular ligands or intrinsic survival signaling molecules suppresses apoptosis, whereas synthesis of proteins mediating apoptosis can occur independently of the cap.

KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT.

We investigated keratinocyte growth factor (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H2(k)) recipient mice were transplanted with C57BL/6 (H2(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th2 cytokines in situ.

Inhibition of Myc-dependent apoptosis by eukaryotic translation initiation factor 4E requires cyclin D1.

Ectopically expressed eukaryotic translation initiation factor 4E (eIF4E) stimulates cell proliferation, suppresses apoptosis in growth factor restricted cells, and induces malignant transformation in primary rodent fibroblasts when coexpressed with protooncogene myc. We report here that eIF4E rescued rat embryo fibroblasts ectopically expressing c-Myc (REF/Myc) from genotoxic and non-genotoxic cytostatic drugs and identify cyclin D1 as a downstream effector in the antiapoptotic mechanism. In clones of REF/Myc ectopically expressing eIF4E, resistance to apoptosis paralleled steady state levels of cyclin D1. Stable expression of cyclin D1 in REF/Myc inhibited apoptosis in response to a broad range of cell cycle specific cytostatic agents. Partial loss-of-cyclin D1 function in REF/Myc ectopically expressing eIF4E (REF/Myc/4E) significantly increased chemosensitivity; either soluble antisense cyclin D1 oligomers or transfection with a dominant negative cyclin D1 mutant that prevents translocation of cyclin D-dependent kinases to the nucleus, significantly blunted the antiapoptotic effect of eIF4E. These data directly link eIF4E rescue from cytostatic drugs to cyclin D1. Since overexpression of eIF4E and cyclin D1 is observed in many aggressive forms of chemoresistant cancers, these findings provide insight into possible mechanisms responsible for this biological behavior.

Interrater Variability in Diagnosis of Cervical Biopsies from Women with HIV-1: Results from the Women's Interagency HIV Study.

OBJECTIVES: To determine interrater variability in classifying cervical biopsies from women with human immunodeficiency virus (HIV). MATERIALS AND METHODS: Cervical biopsies performed on women participating in the Women's Interagency HIV Study (WIHS) were read at the six participating sites. A 10% random sample was retrieved and reviewed using standardized terminology by pathologists with a special interest in gynecologic pathology. Results were compared with kappa values and Mantel-Haentzel tests. RESULTS: Biopsies from 288 HIV-seropositive and 24 HIV-seronegative women were reviewed. The weighted kappa value of 0.67 indicated moderate to strong agreement between original and review diagnoses, with a range of 0.54 to 0.84 across sites. No cancers were identified. Significantly more specimens showing cervical intraepithelial neoplasia (CIN) grade 2 or 3 were identified by review pathologists (p = .02). CIN2 or CIN3 was graded less severely by local pathologists in 18 (51%) of 35 cases, all from HIV-seropositive women. Local pathologists' diagnoses of CIN2 or CIN3 were downgraded by reviewers in 4 of 21 cases (19%). Discrepancies were more common among women with lower CD4 lymphocyte counts. CONCLUSIONS: Although discrepancies occur, interrater correlation in the interpretation of cervical biopsies from women with HIV is moderate to strong.

Functional failure of fascia lata allografts.

OBJECTIVES: Fascia lata allografts are commonly used in urogynecologic procedures. Functional failure of several grafts has occurred, and such failure has been recognized as a materials problem in 12 patients. STUDY DESIGN: Twelve patients with failure of an initial urogynecologic procedure performed with irradiated and freeze-dried donor fascia lata grafts underwent reoperation. Portions of the implanted fascia lata grafts could be retrieved in 7 cases. Graft specimens underwent histologic processing followed by hematoxylin and eosin staining. RESULTS: Histopathologic analyses of the retrieved material demonstrated several ongoing processes in the failed grafts. A few grafts showed areas of ideal remodeling. Most grafts, however, showed areas of disorganized remodeling and areas of graft degeneration. Evidence of immune reaction to the graft was observed in some cases. CONCLUSION: The high materials failure rate associated with the use of irradiated and freeze-dried donor fascia lata grafts suggests that such tissue should not be used for urogynecologic procedures.

Verrucous carcinoma of the vulva in a patient infected with the human immunodeficiency virus.

Verrucous carcinomas of the vulva are rare and have not been reported in women infected with the human immunodeficiency virus. We present such a case in a 32-year-old woman characterized by bladder involvement that failed therapy with 13-cis-retinoic acid and interferon-alpha and required anterior exenteration.

Lovastatin induces fibroblast apoptosis in vitro and in vivo. A possible therapy for fibroproliferative disorders.

Diseases associated with pathological fibroproliferation represent a major cause of morbidity and mortality. Despite the importance of this class of disorders, current therapy is of limited value, and no therapy is available to reduce the fibroblast population size within existing fibrotic lesions. In this regard, constitutive expression of growth-promoting genes can sensitize cells to undergo apoptosis. Studies in our laboratory have demonstrated that lovastatin potently induces apoptosis in fibroblasts constitutively expressing Myc, and that lung fibroblasts isolated from fibrotic lesions constitutively express growth-promoting genes. In this study, we sought to determine if nontransformed lung fibroblasts would manifest susceptibility to lovastatin-induced apoptosis similar to that observed in fibroblasts ectopically expressing Myc. Here we show that clinically achievable concentrations of lovastatin induce apoptosis in normal and fibrotic lung fibroblasts in vitro, as evidenced by acridine orange staining, terminal transferase nick end translation (TUNEL), and DNA laddering. Apoptosis of human lung fibroblasts was dose- and time-dependent, and blocked by exogenous mevalonic acid. Furthermore, apoptosis was associated with decreased levels of mature Ras, a molecule directly implicated in fibroblast rescue from apoptosis. The ability of lovastatin to induce fibroblast apoptosis in vivo was examined using a guinea pig wound chamber model. Lovastatin (5 microM, 8 d) reduced granulation tissue formation in the wound chambers by 64.7%, with associated ultrastructural evidence of fibroblast apoptosis. These findings support further study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors as potential therapy for patients with fibroproliferative disorders.

Pancreatic masses: a multi-institutional study of 364 fine-needle aspiration biopsies with histopathologic correlation.

Only 15% of primary malignant tumors are confined to the gland at the time of presentation. Fine-needle aspiration (FNA) is valuable in confirming the malignant nature of these unresectable lesions. Benign pancreatic lesions and metastatic neoplasms can also be evaluated by fine-needle aspiration. We undertook a retrospective study to evaluate the efficacy of FNA in assessing pancreatic masses. Three hundred and sixty-four radiologically guided FNAs of the pancreas performed between 1986-1996 were reviewed. Surgical material was also evaluated when available and compared to the FNA material. There were 223 men and 141 women. Their ages ranged from 23-90, with a mean of 64 yr. Two hundred and twelve patients (58%) had primary malignant tumors, 183 had adenocarcinomas, 15 had mucinous cystadenocarcinomas, 12 had neuroendocrine tumors, and 2 had pleomorphic giant-cell carcinomas. Ninety-one (43%) had available surgical material which showed adenocarcinoma. Ninety-one patients (25%) had benign aspirates, including 53 showing benign ductal epithelium, 23 showing pancreatitis/inflammation. 10 showing pseudocysts, and 7 showing serous cystadenomas. Surgical material was available in 24 (26%) of these patients. Two of these showed adenocarcinoma. Sixteen aspirates (4%) were suspicious for malignancy, 13 (81%) of which showed adenocarcinoma on follow-up biopsies. Twenty-two aspirates (6%) showed metastatic neoplasms. Twenty-three (6%) had unsatisfactory specimens. Ten (43%) of these had follow-up biopsies, 3 of which were malignant. FNA of primary benign and malignant pancreatic masses is highly sensitive (98%) and specific (100%). Eighty-one percent of the suspicious lesions showed adenocarcinoma on follow-up biopsy. FNA of metastatic neoplasms to the pancreas is also very accurate. This technique can be useful in avoiding unnecessary surgery.

Villoglandular adenocarcinoma of the endometrium: a clinicopathologic study of 61 cases: a gynecologic oncology group study.

Papillary endometrioid or villoglandular adenocarcinoma (VGA) is a relatively common type of endometrial adenocarcinoma, but studies describing its behavior have yielded conflicting results. Patients with a component of VGA were identified in a review of 819 women entered in a Gynecology Oncology Group Study (Protocol 33) of clinical stages I and II endometrial adenocarcinoma. Cases with coexisting foci of serous or clear cell carcinoma were excluded from further consideration. Of the 61 cases that formed the study sample, there were 24 with pure villoglandular differentiation and 37 who were admixed with typical endometrioid adenocarcinoma (EA). The general clinicopathologic features of patients with pure and mixed VGA are compared with 469 patients with pure EA. The VGAs were better differentiated (grade 1 or 2--97% of VGA versus 74% EA, p = 0.001). but they were not significantly different with respect to median age, depth of invasion, or frequency of nodal spread. Six of the 61 patients with VGA died of their tumor. The disease-specific survival rate at 3 years for VGA is 94% (95% confidence interval: 0.88-0.99) compared with 88% (95% CI: 0.86-0.91) for EA. Two of the patients who died had pure villoglandular tumors and four had mixed villoglandular and endometrioid carcinoma. In view of the frequent admixture of VGA and EA and their generally similar biological characteristics, with a prognosis similar to that of typical EA, we conclude that VGA should be considered a variant of EA.

Lovastatin induces apoptosis in malignant mesothelioma cells.

Malignant mesothelioma causes profound morbidity and nearly universal mortality that is refractory to conventional treatment with aggressive surgery, radiotherapy, or chemotherapy. We report that pharmacologic concentrations of lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, induced apoptosis in human malignant mesothelioma cell lines. Mesothelioma cell viability was decreased in a dose-dependent manner by lovastatin (5 to 30 microM). These effects were not reversed by exogenous growth factors or cholesterol, but were reversed by addition of 100 microM mevalonate, confirming that lovastatin affected mesothelioma viability by inhibiting mevalonate synthesis. Lovastatin appeared to decrease mesothelioma viability by inducing apoptosis, as indicated by morphologic changes, histologic evidence of nuclear condensation and degeneration, and flow-cytometric analysis of DNA content. Lovastatin's effects on cell viability were partially reversed in the presence of farnesol, and treatment of mesothelioma cells with a specific farnesyl-protein transferase (FTP) inhibitor decreased cell viability and induced morphologic changes indistinguishable from those caused by lovastatin. In addition, lovastatin-treated cells showed translocation of ras guanosine triphosphate (GTP)-binding proteins from membrane to cytosolic fractions on Western blots, suggesting that lovastatin's effects on mesothelioma were mediated in part by disrupting acylation of GTP-binding proteins. Thus, lovastatin is a commercially available and clinically well-tolerated agent that reduces viability and induces apoptosis of mesothelioma cells, and may provide the basis for adjunctive treatments of patients with mesothelioma.

The critical early proinflammatory events associated with idiopathic pneumonia syndrome in irradiated murine allogeneic recipients are due to donor T cell infusion and potentiated by cyclophosphamide.

We have hypothesized that lung damage occurring in the peri-bone marrow transplant (BMT) period is critical for the subsequent generation of idiopathic pneumonia syndrome (IPS), a major complication following human BMT. The proinflammatory events induced by a common pre-BMT conditioning regimen, cyclophosphamide (Cytoxan(R)) (Cy) and total body irradiation, were analyzed in a murine BMT model. Electron microscopy indicated that Cy exacerbated irradiation-induced epithelial cell injury as early as day 3 after BMT. Allogenicity was an important contributing factor to lung injury as measured by lung wet and dry weights and decreased specific lung compliance. The most significant pulmonary dysfunction was seen in mice receiving both allogeneic T cells and Cy conditioning. IPS was associated with an influx of T cells, macrophages, and neutrophils early post-BMT. Hydroxyproline levels were not increased, indicating that the injury was not fibrotic early post-BMT. As early as 2 h after chemoradiation, host macrophages increased in number in the lung parenchyma. Continued increases in macrophages occurred if splenic T cells were administered with the donor graft. The expression of costimulatory B7 molecules correlated with macrophage numbers. Frequencies of cells expressing mRNA for the inflammatory proteins TNF-alpha, IL-1beta, and TGFbeta were increased. Cy accelerated the upregulation of TGFbeta and increase in host macrophages. The exacerbation of macrophage activation and severity of IPS was dependent on allogeneic T cells, implicating immune-mediated mechanisms as critical to the outcome of IPS. This demonstration of early injury after BMT indicates the need for very early therapeutic intervention before lung damage becomes profound and irreversible.

Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc.

There is increasing evidence that cell cycle transit is potentially lethal, with survival depending on the activation of metabolic pathways which block apoptosis. However, the identities of those pathways coupling cell cycle transit to survival remain undefined. Here we show that the eukaryotic translation initiation factor 4E (eIF4E) can mediate both proliferative and survival signaling. Overexpression of eIF4E completely substituted for serum or individual growth factors in preserving the viability of established NIH 3T3 fibroblasts. An eIF4E mutant (Ser-53 changed to Ala) defective in mediating its growth-factor-regulated functions was also defective in its survival signaling. Survival signaling by enforced expression of eIF4E did not result from autocrine release of survival factors, nor did it lead to increased expression of the apoptosis antagonists Bcl-2 and Bcl-XL. In addition, the execution apparatus of the apoptotic response in eIF4E-overexpressing cells was found to be intact. Increased expression of eIF4E was sufficient to inhibit apoptosis in serum-restricted primary fibroblasts with enforced expression of Myc. In contrast, activation of Ha-Ras, which is required for eIF4E proliferative signaling, did not suppress Myc-induced apoptosis. These data suggest that the eIF4E-activated pathways leading to survival and cell cycle progression are distinct. This dual signaling of proliferation and survival might be the basis for the potency of eIF4E as an inducer of neoplastic transformation.

Induction of fibroblast apoptosis by anti-CD44 antibody: implications for the treatment of fibroproliferative lung disease.

Fibroblast migration and proliferation within the alveolar wall and airspace after lung injury can lead to the development of fibrosis. Fibroblast cell surface CD44 is an adhesion receptor for provisional matrix proteins and mediates fibroblast invasion into fibrin matrices. Here we show that incubation of cultured fibroblasts with an anti-CD44 monoclonal antibody induces fibroblast detachment from the substratum and morphological changes compatible with apoptosis. In addition, we show that anti-CD44 monoclonal antibody rapidly induces fibroblast apoptosis within fibrin matrices. The effect of anti-CD44 antibody on induction of fibroblast apoptosis occurred within 8 hours and was dose dependent. Anti-CD44 antibody also induced fibroblast apoptosis in suspension. Furthermore, fibroblasts plated on anti-CD44-antibody-coated surfaces initially attached and spread on the antibody; however, after an 8-hour incubation time, many of the cells developed characteristic morphological features of apoptosis. Collectively, these data indicate that apoptosis did not result solely due to detachment from the substratum. Our results identify a new function for fibroblast cell surface CD44 related to the control of cell viability. We suggest this function may be important in fibroblast population control and could potentially be exploited in designing anti-fibrotic therapies.