Fibrotic Foci Research Articles

Industrial Elimination of Essential Oils from Rosmarinus Officinalis: In Support of the Synergic Antifibrotic Effect of Rosmarinic and Carnosic Acids in Bleomycin Model of Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by collagen deposition as a consequence of excessive lung fibroblasts and myofibroblasts proliferation. We aimed to investigate for the first time the effect of rosemary leaf extract rich with carnosic acid (CA) or rosmarinic acid (RA), after industrial elimination of essential oils, against bleomycin (BLM)-induced lung fibrosis in rats. Male Wistar rats were given a single dose of BLM (4 mg/kg, intratracheal), while CA rich extract, RA rich extract or the combination RA/CA rich extracts (10, 75 and 150 mg/kg, intraperitoneal) were administered 3 day later and continued for 4 weeks. We reveled by HPLC an important similar amount of phenolic compounds such as pyrogallol, vanillic, gallic and ellagic acids in both rosemary extracts. BLM induced lung fibrotic foci and disturbance in superoxide dismutase, catalase and malondialdehyde levels. At 10 mg/kg, both rosemary extracts administrated alone or in combination alleviated synergistically lung fibrosis and ameliorated oxidative changes induced by BLM. In conclusion, industrial elimination of essential oils from rosemary allowed us to obtain two extracts with potent antifibrotic activities due to the large amount of RA and CA that appear much higher and effective than wild rosemary extract.

RAPIDLY PROGRESSIVE INTERSTITIAL LUNG DISEASE IN ASSOCIATION WITH MDA5 ANTIBODY DERMATOMYOSITIS

SESSION TITLE: Medical Student/Resident Diffuse Lung Disease SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Clinically Amyopathic Dermatomyositis (CADM) is a subtype of dermatomyositis (DM) characterized by the presence of cutaneous lesions, without clinical evidence of muscle weakness. The presence of autoantibodies against Melanoma Differentiation-Associated protein 5 (MDA5) in this group is highly associated with rapidly progressive interstitial lung disease (RP-ILD) and a worse prognosis. We present a case of anti-MDA5 associated RP-ILD to illustrate the importance of screening for MDA5 in patients with CADM and DM. CASE PRESENTATION: Our patient was a 45-year-old woman with a history of hypothyroidism, seronegative inflammatory polyarthritis treated with hydroxychloroquine, and notably, a history of biopsy-proven DM with a negative myositis-specific antibodies (MSA) panel (which did not include MDA5 antibodies). She had been successfully treated for DM, and maintained in remission with oral steroids. Four years following the diagnosis of DM, she presented with insidious and gradually progressive dyspnea for over a year. Physical examination was unremarkable. Pulmonary function testing showed moderate restrictive lung disease. Chest X-ray was notable for bibasilar interstitial changes. Computed tomography (CT) scan of the chest revealed nonspecific, bilateral ground-glass opacities in the middle and lower lung zones, with associated bibasilar bronchiectasis in the affected areas (Figure 1). Bronchoscopic evaluation, including transbronchial biopsy, was performed, which was negative for malignancy or infection. Open lung biopsy was obtained via robotic-assisted thoracoscopy from the affected lobes, with histopathology revealing interstitial fibrosis with multiple fibrotic foci without clear evidence of honeycombing. The pattern was suggestive of usual interstitial pneumonia (UIP). Repeat testing for MSA revealed an elevated anti-MDA5 antibody level at 22 SI. The patient was consequently diagnosed with RP-ILD in conjunction with CADM, prompting the initiation of oral prednisone and azathioprine. DISCUSSION: Anti-MDA5 antibodies recognize the RNA helicase encoded by MDA5, a protein that is involved in innate immune processes. The presence of anti-MDA5 antibodies in patients with CADM has been linked to the presence of RP-ILD, and portends a poor prognosis. The mechanism of this autoantibody production is not fully understood. One meta-analysis found that the presence of these antibodies was 77% sensitive and 86% specific for the identification of RP-ILD. Additionally, anti-MDA5 antibodies can be utilized to predict clinical outcomes and monitor disease activity in these patients. Due to its high mortality rate from respiratory failure, immediate therapy with high-intensity combination immunosuppression is essential once the diagnosis is made. CONCLUSIONS: This case highlights the importance of screening for anti-MD5 antibodies in patients with CADM to elucidate their risk of developing RP-ILD. Reference #1: Li L, Wang Q, Yang F, et al. Anti-MDA5 antibody as a potential diagnostic and prognostic biomarker in patients with dermatomyositis. Oncotarget. 2017;8(16):26552–26564. doi:10.18632/oncotarget.15716 Reference #2: Chen, Z., Cao, M., Plana, M.N., Liang, J., Cai, H., Kuwana, M. and Sun, L. (2013), Utility of Anti–Melanoma Differentiation–Associated Gene 5 Antibody Measurement in Identifying Patients With Dermatomyositis and a High Risk for Developing Rapidly Progressive Interstitial Lung Disease: A Review of the Literature and a Meta-Analysis. Arthritis Care & Research, 65: 1316-1324. doi:10.1002/acr.21985 DISCLOSURES: No relevant relationships by Komal Ejaz, source=Web Response No relevant relationships by Dan Kazmierski, source=Web Response No relevant relationships by Bassel Noumi, source=Web Response No relevant relationships by Pius Ochieng, source=Web Response No relevant relationships by NISHANT SHARMA, source=Web Response No relevant relationships by Yichen Wang, source=Web Response

Osteopontin enhances the migration of lung fibroblasts via upregulation of interleukin-6 through the extracellular signal-regulated kinase (ERK) pathway.

Fibrosis is a phenomenon in which parenchyma is replaced with fibrous tissue. Persistent inflammation accompanied by dysregulation of cytokine production and repeated cycles of inflammation-associated tissue-repair induces fibrosis in various organs including the liver, lung, and kidney. In idiopathic pulmonary fibrosis, production of interleukin (IL)-6 and osteopontin (OPN) are dysregulated. Fibrosis leads to qualitative rather than quantitative changes of fibroblasts at the sites of tissue repair, and this leads to enlargement of fibrotic foci. These fibroblasts are immunohistochemically positive for OPN; however, the effect of overexpressed OPN in fibroblasts is not fully understood yet. In this study, we investigated the effect of OPN on IL-6 secretion and on migration and proliferation of fibroblasts. Lung fibroblasts overexpressing exogenous OPN showed that OPN was linked to the enhancement of cell migration through increased IL-6 secretion via the extracellular signal-regulated kinase (ERK) pathway. These results suggest that OPN may exert its pro-fibrotic functions, such as enhancement of fibroblasts migration by cooperating with chemoattractant IL-6, and may be involved in enlargement of fibrotic foci.

Identifying collagen VI as a target of fibrotic diseases regulated by CREBBP/EP300

Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren's disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, ACTA2 and COL1A1 Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren's nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders.

Sodium tanshinone IIA sulfonate attenuates silica-induced pulmonary fibrosis in rats via activation of the Nrf2 and thioredoxin system

Silicosis is characterized by pulmonary fibrosis due to long-term inhalation of silica particles. Although the cause of this serious disease is known, its pathogenesis remains unclear and there are currently no specific treatments. Recent studies have shown that the anti-oxidant transcription factor Nrf2 is expressed at reduced levels in fibrotic foci, which may be related to disease progression. However, the molecular mechanisms by which this might occur have yet to be elucidated. Sodium tanshinone IIA sulfonate (STS), an extract of Salvia miltiorrhiza, is used in traditional Chinese medicine in the treatment of coronary heart disease. STS has been shown to play a strong anti-oxidative role in various organs. Here, we employed a rat model to explore the effects of STS on oxidative stress and the progression of fibrosis in silicosis. STS significantly reduced collagen deposition in the lungs, thereby antagonising silicosis. Immunohistochemical and immunofluorescence staining showed that Nrf2 was differentially expressed in lung cells during silica induced fibrosis, and chromatin immunoprecipitation-sequencing experiments demonstrated that Nrf2 promoted the expression of the antioxidant proteins thioredoxin and thioredoxin reductase. Our results suggest that the anti-fibrotic effects of STS may be related to upregulation of Nrf2 nuclear expression, especially in fibrotic lesions, and the promotion of thioredoxin and thioredoxin reductase expression. Our findings may open up new avenues for the development of STS as a treatment for silicosis.

The association between MRI findings and breast cancer subtypes: focused on the combination patterns on diffusion-weighted and T2-weighted images.

To assess morphology on diffusion-weighted imaging (DWI) and intratumoral signal intensity (SI) on T2-weighted images (T2WI) of breast carcinomas, and to evaluate the association between the combined DWI and T2WI findings and breast cancer subtypes. Two hundred and eighty breast cancer patients who underwent breast MRI prior to therapy were included in this retrospective study. All had invasive carcinomas, which were classified into five subtypes: Luminal A-like (n = 149), Luminal B-like (n = 63), Hormone receptor-positive HER2 (n = 31), Hormone receptor-negative HER2 (n = 13), or Triple-negative (TN) (n = 24). Based on the morphology on DWI, the tumors were classified into two patterns: DWI-homogeneous or DWI-heterogeneous. If DWI-heterogeneous, an assessment of intratumoral SI on T2WI was performed: tumors with intratumoral high/low SI on T2WI were classified as Hete-H/Hete-L, respectively. The associations between (1) the morphological patterns on DWI and the five subtypes, and (2) the intratumoral SI patterns on T2WI and the five subtypes in DWI-heterogeneous were evaluated. There was a significant association between (1) the morphological patterns on DWI and the five subtypes (p < 0.0001), and (2) the intratumoral SI patterns on T2WI and the five subtypes in DWI-heterogeneous (p < 0.0001). DWI-homogeneous was dominant in Luminal A-like (67.1%), and Hete-H was dominant in TN type (75%). Hete-H, suggesting the presence of intratumoral necrosis, included high proliferative and/or aggressive subtypes more frequently (80%) than Hete-L, suggesting the presence of fibrotic focus. Fibrotic focus was seen more commonly in the luminal subtypes. The combined findings on DWI and T2WI revealed breast carcinomas that were associated with particular subtypes.

Sphingosine Kinase 1/S1P Signaling Contributes to Pulmonary Fibrosis by Activating Hippo/YAP Pathway and Mitochondrial Reactive Oxygen Species in Lung Fibroblasts.

The sphingosine kinase 1 (SPHK1)/sphingosine–1–phosphate (S1P) signaling axis is emerging as a key player in the development of idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM)-induced lung fibrosis in mice. Recent evidence implicates the involvement of the Hippo/Yes-associated protein (YAP) 1 pathway in lung diseases, including IPF, but its plausible link to the SPHK1/S1P signaling pathway is unclear. Herein, we demonstrate the increased co-localization of YAP1 with the fibroblast marker FSP1 in the lung fibroblasts of BLM-challenged mice, and the genetic deletion of Sphk1 in mouse lung fibroblasts (MLFs) reduced YAP1 localization in fibrotic foci. The PF543 inhibition of SPHK1 activity in mice attenuated YAP1 co-localization with FSP1 in lung fibroblasts. In vitro, TGF-β stimulated YAP1 translocation to the nucleus in primary MLFs, and the deletion of Sphk1 or inhibition with PF543 attenuated TGF-β-mediated YAP1 nuclear localization. Moreover, the PF543 inhibition of SPHK1, or the verteporfin inhibition of YAP1, decreased the TGF-β- or BLM-induced mitochondrial reactive oxygen species (mtROS) in human lung fibroblasts (HLFs) and the expression of fibronectin (FN) and alpha-smooth muscle actin (α-SMA). Furthermore, scavenging mtROS with MitoTEMPO attenuated the TGF-β-induced expression of FN and α-SMA. The addition of the S1P antibody to HLFs reduced TGF-β- or S1P-mediated YAP1 activation, mtROS, and the expression of FN and α-SMA. These results suggest a role for SPHK1/S1P signaling in TGF-β-induced YAP1 activation and mtROS generation, resulting in fibroblast activation, a critical driver of pulmonary fibrosis.

Abstract P4-07-02: Clinicopathological features and BRCA 1/2 status in a large prospective cohort of young women with breast cancer

Abstract Background: Breast cancer arising in young women is more likely to have higher risk features than in older women and more likely to be associated with germline BRCA1 or BRCA2 mutations. We previously reported a higher distribution of luminal B-like tumors among young women. Here we present the clinical and pathologic features of invasive disease in a large prospective cohort of young women with breast cancer, and associations between surrogate molecular subtype and BRCA 1/2 mutation status. Methods: Among 1,297 young women (aged ≤40 years) with breast cancer enrolled from 2006-2016, tumor histopathological features were examined by central review, and biomarker status was extracted from pathology reports. Based on histologic grade and biomarker expression, invasive tumors were categorized as luminal A-like (LA=ER/PR+ and HER2-, histologic grade 1 or 2), luminal B-like (LB=ER/PR+ and HER2+, or ER/PR+, HER2-, and grade 3), HER2 enriched (E) (ER/PR- and HER2+) or triple negative (TN=ER-, PR- and HER2-). Tumor stage, clinical characteristics, and BRCA status were retrieved by medical record review (and patient survey for BRCA status if not in medical record). Results: The majority of women presented with early stage invasive disease (8% stage 0, 32% stage 1, 40% stage 2, 15% stage 3; 5% stage 4). Among invasive tumors with evaluable molecular phenotype (n=1,136), 57% were high grade and the distribution of subtypes was: 33% LA, 42% LB, 8% HER2E, and 16% TN (Table 1). Among different age groups (≤ 30, 31-35, and 36-40 years), there were no significant differences in molecular phenotype, tumor stage, tumor grade or histopathological features including presence of tumor necrosis, lymphocytic infiltration, and central fibrotic focus. 96% of women were tested for BRCA1 and BRCA2 germline mutations, and 127 (11%) tested positive: 65% were BRCA1+, and 35% were BRCA2+ (table 2). Among BRCA1+ patients, 2% of cancers were LA, 30% LB, 5% HER2E, and 62% TN; among BRCA2+, 29% were LA, 56% LB, 2% HER2E, and 13% TN. These proportions were relatively consistent across age groups. BRCA1+ patients’ tumors were proportionately more TN and BRCA2+ patients’ tumors more likely to be LB/HER2 negative (p&amp;lt;0.0001), irrespective of age category. Conclusion: In a large cohort of women diagnosed with breast cancer at age ≤40, there was no association between age group (≤ 30, 31-35, vs. 36-40 years) and tumor stage, grade, or tumor molecular phenotype among women with invasive disease. As in older women, tumor molecular phenotype does vary by BRCA1 or BRCA2 mutation status, which has implications for prevention and treatment. Table 1. Subtype by age groupTotal (n=1136)≤30 yrs (n=144)31-35 yrs (n=314)36-40 yrs (n=678)p=0.42Luminal A-like377 (33%)48 (33%)99 (32%)230 (34%)Luminal B-like477 (42%)59 (41%)137 (44%)281 (41%)• ER/PR+, HER2-, grade 3245 (22%)27 (19%)68 (22%)150 (22%)• ER/PR+, HER2+232 (20%)32 (22%)69 (22%)131 (19%)HER2E95 (8%)13 (9%)34 (11%)48 (7%)Triple Negative187 (16%)24 (17%)44 (14%)119 (18%) Table 2. Subtype by BRCA statusTotal(n=1136)BRCA1+(n=82)BRCA2+(n=45)No mutation/not tested(n=1009)p&amp;lt;0.0001Luminal A-like377 (33%)2 (2%)13 (29%)362 (36%)Luminal B-like477 (42%)25 (30%)25 (56%)427 (42%)• ER/PR+, HER2-, grade 3245 (22%)21 (26%)18 (40%)206 (20%)• ER/PR+, HER2+232 (20%)4 (5%)7 (16%)221 (22%)HER2E95 (8%)4 (5%)1 (2%)90 (9%)Triple Negative187 (16%)51 (62%)6 (13%)130 (13%) Citation Format: Yaileen D Guzman-Arocho, Shoshana M. Rosenberg, Philip Poorvu, Kathryn J. Ruddy, Greg Kirkner, Craig Snow, Rulla M. Tamimi, Jeffrey Peppercorn, Lidia Schapira, Virginia F. Borges, Steven E. Come, Elena F. Brachtel, Ellen Warner, Ann H. Partridge, Laura C. Collins. Clinicopathological features and BRCA 1/2 status in a large prospective cohort of young women with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-07-02.

Morphological features of pulmonary fibrosis in workers occupationally exposed to alpha radiation

Purpose: The article reports on a comparative analysis of biological specimens of lung tissues collected from workers with pulmonary fibrosis induced by internal exposure to plutonium alpha-particles (plutonium-induced pulmonary fibrosis [PuPF]) and with etiologically different pulmonary fibrosis (non-PuPF) that developed as an outcome of a chronic obstructive pulmonary disease (COPD).Materials and methods: To perform histological examinations, lung tissues were sampled during autopsy. Six samples of various lung regions (the apical region, the lingula of the left lung and the inferior lobe) were collected from each donor. The resected tissue samples were fixed in 10% neutral-buffered formalin during 24 h and embedded into paraffin blocks (FFPE). FFPE blocks with lung tissue specimens collected from 56 workers with PuPF, 34 workers with non-PuPF and 35 workers without any lung disease were used in the study. To perform microscopic examination, lung tissue specimens were hematoxylin and eosin stained. To examine the connective-tissue scaffold of lung stroma and identify foci of pulmonary fibrosis, the cut sections of paraffin blocks were stained by Van Gizon’s method (to assess the total volume of fibrosis-affected tissues), Gomori’s technique (to define the reticular scaffold of lung stroma) and Weigert’s technique (to examine elastic fibers). Morphological patterns of all biological specimens were studied using immunohistochemistry. To fit the empirical data, the Weibull’s model was used.Results and conclusions: The study found qualitative and quantitative morphological features specific for PuPF compared to non-PuPF. The study demonstrated that hyper-production of collagen type V plays a key role in PuPF. The collagen type V content in fibrotic foci in lung tissue specimens from workers with PuPF was found to be increased.

Complimentary Diagnostic Tools for Endometrosis in Biopsies of Mares with Clinical Subfertility

Background: Endometrosis is a multifactorial disease and one of the main causes of infertility in mares, its etiology and pathogenesis are not completely understood. It is defined as peri glandular and/or stromal endometrial fibrosis with glandular alterations. Due to the few clinical symptoms, besides anamnesis and fertility data, endometrosis requires histological confirmation. The histo-morphology and immune histochemical characteristics of the endometrium vary among individuals according to the disease progression. The aim of this research was to combine histology with new immune and histochemical tools for a more precise detection of fibrotic changes of mares with endometrosis.Materials, Methods &amp; Results: The endometrium of forty thoroughbred mares aged 5-18 years, that did not become pregnant during the last two breeding seasons in a Chilean commercial equine breeding center were biopsied. Samples were subjected to conventional histopathology with hematoxylin-eosin as well as to specific histological staining using specific techniques such as Alcian blue and Masson Fontana, aimed to ascertain what types of mucopolysaccharides were present in those samples. In order to have a deeper picture of the progression of the pathology, immune histochemical methods for the detection of vimentin, cytokeratin, progesterone receptor and lymphocyte marker CD3 were used. Finally in order to detect fibrillar collagen we used second harmonic generation (SHG) technique with detects fibrillar collagen without staining, due to intrinsic hyperpolarization ability of this type of collagen, which can be detected by atomic force microscopy. As a result of our research samples were categorized according to the scale of Keeney and Doig into categories I, IIa, IIb and III (45, 42, 7.5 and 5% respectively). These samples also were characterized by the methods listed earlier and a result we found specific staining in 15 samples coming from higher endometrial damage using Masson-Fontana, while acid staining indicative of acid mucopolysaccharides were detected in 97% of the samples. At immunostaining, we found cytokeratin and vimentin differentially expressed as a function of the degree of the lesions. Cytokeratin was detected in glands from healthy mares, but it was almost absent as the injury was increased. Vimentin in turn, was detected in 26 samples with different degree of intensity. In nine animals, vimentin expression was anomalous, and the marker was detected in fibrotic foci concentrically surrounding glands in biopsies of grade I and IIA only. Although progesterone receptor was detected, there was no correlation with endometrosis. Finally, the lymphocyte T specific CD3 marker was positive in 100% of cases analyzed in which moderate lymphocytic infiltration was found by hematoxylin-eosin; however no staining could be detected in mares with more advanced endometrosis. By combining immunofluorescence with the detection of second harmonic it was possible to detect in the same sample two proteins and collagen deposition at the same time, this had not been reported earlier for endometrosis and mares.Discussion: Our results suggest that the combination of the different methods mentioned is useful for validation and further characterization of the routine hematoxylin-eosin, and can be used as a complementary tool for the diagnosis. Of value, it was possible to combine immunofluorescence with SHG in a single sample in situ.

Incidental Lung Cancer of Explanted Lungs from Lung Transplant Recipients: Incidence, Characteristics, and 5-Year Survival.

PurposeRecent history of malignancy without 5-year disease-free interval is an absolute contraindication for lung transplantation (LTx). However, in rare cases, lung cancer may be incidentally diagnosed in the explanted lung of recipients. We evaluated the prevalence, 5-year survival, and prognosis of incidental lung cancer after LTx.Materials and MethodsMedical records of patients who underwent LTx at Severance Hospital between January 1, 2012 and June 30, 2019 were reviewed. Patients with incidental lung cancer were included, and those with histologically proven pre-transplant lung cancer were excluded.ResultsOf the 247 patients who underwent LTx, 6 (2.4%) were diagnosed with incidental lung cancer. Interstitial lung disease (ILD) was the underlying lung disease in all patients. The median interval from the last preoperative computed tomography (CT) screening to LTx was 26 days. The most common histological type of incidental lung cancer was adenocarcinoma (n=4, 66.7%). All Stage IV cases were misdiagnosed as fibrosis on preoperative chest CT. Patients with incidental lung cancer showed lower 5-year survival than those without malignancy (median survival: 8.5 months vs. not reached, p=0.047, respectively). Patients with Stage III or IV demonstrated lower 5-year survival than those with Stage I or II and those without malignancy (median survival: 5 months, 19 months, and not reached, respectively, p=0.011).ConclusionMultidisciplinary preoperative screening and serial imaging studies within short intervals are required to differentiate lung malignancy from fibrotic foci. Furthermore, active pathologic examination of suspicious lung lesions is required in patients at high risk for lung cancer.

Loss of ELK1 has differential effects on age-dependent organ fibrosis.

ETS domain-containing protein-1 (ELK1) is a transcription factor important in regulating αvβ6 integrin expression. αvβ6 integrins activate the profibrotic cytokine Transforming Growth Factor β1 (TGFβ1) and are increased in the alveolar epithelium in idiopathic pulmonary fibrosis (IPF). IPF is a disease associated with aging and therefore we hypothesised that aged animals lacking Elk1 globally would develop spontaneous fibrosis in organs where αvβ6 mediated TGFβ activation has been implicated. Here we identify that Elk1-knockout (Elk1-/0) mice aged to one year developed spontaneous fibrosis in the absence of injury in both the lung and the liver but not in the heart or kidneys. The lungs of Elk1-/0 aged mice demonstrated increased collagen deposition, in particular collagen 3α1, located in small fibrotic foci and thickened alveolar walls. Despite the liver having relatively low global levels of ELK1 expression, Elk1-/0 animals developed hepatosteatosis and fibrosis. The loss of Elk1 also had differential effects on Itgb1, Itgb5 and Itgb6 expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1 in human disease we exposed human lung epithelial cells and murine lung slices to cigarette smoke extract, which lead to reduced ELK1 expression andmay explain the loss of ELK1 in human disease. These data support a fundamental role for ELK1 in protecting against the development of progressive fibrosis via transcriptional regulation of beta integrin subunit genes, and demonstrate that loss of ELK1 can be caused by cigarette smoke.

Modeling pulmonary fibrosis through bleomycin delivered by osmotic minipump: a new histomorphometric method of evaluation.

The systemic delivery of bleomycin (BLM) to mice through subcutaneously implanted osmotic minipumps may be used to experimentally mimic the typical features of systemic sclerosis and related interstitial lung diseases. The published studies on this model principally have focused on induced dermal modifications, probably because lung lesions are typically mild, subpleurally localized, and difficult to analyze. The use of high BLM doses to increase their severity has been proposed but is ethically questionable because of the compromising of animal welfare. We propose a tailored histomorphometric method suitable to detect and quantify this type of mild lung lesions. Using a two-step automated image analysis, a peripheral region of interest with a depth of 250 µm from the pleural edge was defined on whole slide images, and the fibrotic foci were histomorphometrically characterized. The effects of different BLM doses on lung alterations were evaluated in C57BL/6 mice and 60 U/kg resulted in a fair compromise between fibrotic lesions and animal welfare. This dose was also tested in time course experiments. The analysis revealed a peak of histological fibrotic-like alterations, cytokine expression, metalloprotease, and macrophagic activation between the 21st and 28th day after pump implant. The induced dermal fibrosis was characterized by the progressive loss of the white dermal adipose layer, an increase in dermal thickness, dermal hyperplasia, and more compacted collagen fibers. Despite the trend toward spontaneous resolution, our model allowed a double organ readout of the BLM effect and the identification of a therapeutic window for testing pharmacological compounds without using life-threatening doses.

Association between the degree of fibrosis in fibrotic focus and the unfavorable clinicopathological prognostic features of breast cancer.

ObjectiveTo explore the association between the degree of fibrosis in fibrotic focus (FF) and the unfavorable clinicopathological prognostic features of breast cancer.MethodsA total of 169 cases of breast invasive ductal carcinoma (IDC) were included in the study. Hematoxylin and eosin (H&E) staining was performed in the primary lesion of breast IDC and the degree of fibrosis in tumor-stromal FF was assessed. The association between the degree of fibrosis in FF and the well-known clinicopathologic features of breast cancer was investigated and the influence of the degree of fibrosis in FF on the survival was analyzed.ResultsTumor size >2 cm (P = 0.023), vascular invasion (P = 0.011), lymphatic vessel invasion (P < 0.001) and HER-2+ (P = 0.032) were positively correlated with the degree of fibrosis in FF in breast IDC. The result of multivariate analysis showed that lymphatic vessel invasion was the only independent correlation factor of high fibrosis in FF in breast IDC (OR = 3.82, 95% CI[1.13 ∼ 12.82], P = 0.031). The Nottingham prognostic index (NPI) of high fibrosis in FF was significantly higher than that of mild and moderate fibrosis in FF in the no vascular infiltration subgroup, the no nerve infiltration subgroup, and the Luminal A subgroup (P = 0.014, 0.039, and 0.018; respectively).ConclusionsThe high fibrosis in FF is closely associated with the strong invasiveness and the high malignancy of breast IDC. The degree of fibrosis in FF might be considered as a very practical and meaningful pathological feature of breast cancer.

Fibrotic Focus in Breast Cancer

Fibrotic Focus in Breast Cancer

Bardet-Biedl Syndrome in rhesus macaques: A nonhuman primate model of retinitis pigmentosa

The development of therapies for retinal disorders is hampered by a lack of appropriate animal models. Higher nonhuman primates are the only animals with retinal structure similar to humans, including the presence of a macula and fovea. However, few nonhuman primate models of genetic retinal disease are known. We identified a lineage of rhesus macaques with a frameshift mutation in exon 3 of the BBS7 gene c.160delG (p.Ala54fs) that is predicted to produce a non-functional protein. In humans, mutations in this and other BBS genes cause Bardet-Biedl syndrome, a ciliopathy and a syndromic form of retinitis pigmentosa generally occurring in conjunction with kidney dysfunction, polydactyly, obesity, and/or hypogonadism. Three full- or half-sibling monkeys homozygous for the BBS7 c.160delG variant, at ages 3.5, 4 and 6 years old, displayed a combination of severe photoreceptor degeneration and progressive kidney disease. In vivo retinal imaging revealed features of severe macular degeneration, including absence of photoreceptor layers, degeneration of the retinal pigment epithelium, and retinal vasculature atrophy. Electroretinography in the 3.5-year-old case demonstrated loss of scotopic and photopic a-waves and markedly reduced and delayed b-waves. Histological assessments in the 4- and 6-year-old cases confirmed profound loss of photoreceptors and inner retinal neurons across the posterior retina, with dramatic thinning and disorganization of all cell layers, abundant microglia, absent or displaced RPE cells, and significant gliosis in the subretinal space. Retinal structure, including presence of photoreceptors, was preserved only in the far periphery. Ultrasound imaging of the kidneys revealed deranged architecture, and renal histopathology identified distorted contours with depressed, fibrotic foci and firmly adhered renal capsules; renal failure occurred in the 6-year-old case. Magnetic resonance imaging obtained in one case revealed abnormally low total brain volume and unilateral ventricular enlargement. The one male had abnormally small testes at 4 years of age, but polydactyly and obesity were not observed. Thus, monkeys homozygous for the BBS7 c.160delG variant closely mirrored several key features of the human BBS syndrome. This finding represents the first identification of a naturally-occurring nonhuman primate model of BBS, and more broadly the first such model of retinitis pigmentosa and a ciliopathy with an associated genetic mutation. This important new preclinical model will provide the basis for better understanding of disease progression and for the testing of new therapeutic options, including gene and cell-based therapies, not only for BBS but also for multiple forms of photoreceptor degeneration.

P120-catenin regulates pulmonary fibrosis and TGF-β induced lung fibroblast differentiation

P120-catenin (P120) was known to function in adhesion between cells and signal transduction in many types of cells. In this study, we investigated the expression and role of P120 in pulmonary fibrosis and transforming growth factor beta (TGF-β) mediated lung fibroblast-to-myofibroblast differentiation (fibroblast differentiation). Our data indicated that P120 expression increased in lung fibrotic foci and primary lung fibroblasts isolated from bleomycin- (BLM) challenged mice, compared to controls. In vitro, TGF-β induced P120 expression in human lung fibroblasts, and siRNA-mediated SMAD3 depletion inhibited TGF-β stimulated P120 expression. Blocking nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway through chemical inhibitor or knockdown of NF-kB p65 subunit also suppressed TGF-β induced P120 expression in human lung fibroblast. Knockdown of P120 expression inhibited TGF-β induced human lung fibroblast differentiation, as well as suppressed the activation of SMAD and ERK signaling pathways. Administration of lentivirus coding mouse P120 sh-RNA into mouse lung tissue dramatically attenuated the expression of P120 in lung tissue and lung fibroblast, suppressed BLM induced increase of TGF-β, alpha smooth muscle actin (α-SMA) and fibronectin (FN) expression, and decreased the deposition of collagen and pulmonary fibrosis. Collectively, these results suggested that P120 involved in lung fibroblast differentiation and pulmonary fibrosis, and inhibition of P120 expression decreased pulmonary fibrosis in BLM challenged mice. Thus, attenuation of P120 expression might be a potential therapeutic strategy for human lung fibrosis.

Combined Activation of Guanylate Cyclase and Cyclic AMP in Lung Fibroblasts as a Novel Therapeutic Concept for Lung Fibrosis.

Remodelling of the peripheral lung tissue and fibrotic foci are the main pathologies of idiopathic pulmonary fibrosis (IPF), a disease that is difficult to treat. TGF-β activation of peripheral lung fibroblasts is indicated as the major cause of tissue remodelling in IPF and is resulting in fibroblast hyperplasia and deposition of extracellular matrix. Soluble guanylate cyclase (sGC) stimulators combined with cyclic AMP (cAMP) activators have been reported to reduce proliferation and matrix deposition in other conditions than IPF. Therefore, this drug combination may present a novel therapeutic concept for IPF. This study investigated the effect of BAY 41-2272 and forskolin on remodelling parameters in primary human lung fibroblasts. The study determined TGF-β induced proliferation by direct cell counts after 3 days; and deposition of collagen type-I, type III, and fibronectin. BAY 41-2272 significantly reduced TGF-β induced fibroblast proliferation, but did not reduce viability. This inhibitory effect was further supported by forskolin. Both BAY 41-2272 and forskolin alone reduced TGF-β induced collagen and fibronectin de novo synthesis as well as deposition. This effect was significantly stronger when the two compounds were combined. Furthermore, the TGF-β induced expression of fibrilar α-smooth muscle actin was reduced by BAY 41-2272 and this effect was strengthened by forskolin. In addition, BAY 41-2272 and forskolin reduced TGF-β induced β-catenin. All effects of BAY 41-2272 were concentration dependent. The findings suggest that BAY 41-2272 in combination with cAMP stimulation may present a novel therapeutic strategy to reduce tissue remodelling in IPF.

Delayed resolution of bleomycin-induced pulmonary fibrosis in absence of MMP13 (collagenase 3)

Matrix metalloprotease 13 (MMP13) deficiency in pulmonary fibrosis has described contradictory phenotypes on inflammatory and fibrotic responses after lung injury, and its role during lung fibrosis resolution is still undefined. MMP13 has been considered the main collagenase in rodents, and the remodeling of fibrillar collagen is widely attributed to the action of this enzyme. In this study we aimed to explore the role of MMP13 during lung fibrosis progression and resolution. Lung fibrosis was induced by intratracheal instillation, and inflammatory, fibrotic, and resolution stages were evaluated in Mmp13-null and wild-type (WT) mice. Bronchoalveolar lavage fluid was taken for cytokine array analysis and activity of gelatinases. Our results showed that MMP13 is upregulated mainly during two stages after lung injury, inflammation and resolution of fibrosis, and it is mainly expressed by alveolar and interstitial macrophages. Mmp13-null mice exhibited more extensive inflammation at 7 days after bleomycin treatment, and it was characterized by increased macrophage infiltration and significant alterations in proinflammatory cytokines. We also documented that Mmp13-deficient mice experienced more severe and prolonged lung fibrosis compared with WT mice. Delayed resolution in Mmp13-deficient lungs was characterized by a decreased overall collagenolytic activity and persistent fibrotic foci associated with emphysema-like areas. Together, our findings indicate that MMP13 plays an antifibrotic role and its activity is crucial in lung repair and restoration of tissue integrity during fibrosis resolution.

Substrate Viscosity Dictates Cellular Response

Cells respond to and are regulated by the mechanical properties of the substrate to which they are attached. Previous studies have shown that cells can generate larger forces on stiffer substrates, driving extracellular matrix assembly and increasing the substrate stiffness. In vitro models of fibrotic tissue focus on increasing the elastic modulus of the polymer substrate. However, this approach ignores the viscoelastic nature of fibrotic tissue. Recent research has suggested that as fibrotic tissue matures, the matrix transitions from one with a high viscosity to one that is more purely elastic. In this work, we investigate mechanosensitive effects of viscoelasticity using single and dual blends of poly (dimethyl siloxane) (PDMS) formulations with tunable viscous and elastic properties. We found that 3T3 fibroblast cells on a substrate with higher viscosity have larger cell area, but assemble less fibronectin. To elucidate the mechanism of altered viscosity, we simulated a modification of the Chan-Odde motor-clutch model, representing the substrate as a viscoelastic spring-dashpot system instead of a purely spring element. The viscoelastic model shows that cells are sensitive to changes in substrate viscosity, switching from a “frictional slippage” to “load and fail” regime. Our experiments and simulations demonstrate that the viscous component of underlying substrates plays a larger role in cell functionality than previously expected. This may be key in exploring the dynamics of and potential treatments for fibrosis.