Tissue Fibroblasts Research Articles

Biallelic Variants in ASNA1 , Encoding a Cytosolic Targeting Factor of Tail-Anchored Proteins, Cause Rapidly Progressive Pediatric Cardiomyopathy

Pediatric cardiomyopathies are a clinically and genetically heterogeneous group of heart muscle disorders associated with high morbidity and mortality. Although knowledge of the genetic basis of pediatric cardiomyopathy has improved considerably, the underlying cause remains elusive in a substantial proportion of cases. Exome sequencing was used to screen for the causative genetic defect in a pair of siblings with rapidly progressive dilated cardiomyopathy and death in early infancy. Protein expression was assessed in patient samples, followed by an in vitro tail-anchored protein insertion assay and functional analyses in zebrafish. We identified compound heterozygous variants in the highly conserved ASNA1 gene (arsA arsenite transporter, ATP-binding, homolog), which encodes an ATPase required for post-translational membrane insertion of tail-anchored proteins. The c.913C>T variant on the paternal allele is predicted to result in a premature stop codon p.(Gln305*), and likely explains the decreased protein expression observed in myocardial tissue and skin fibroblasts. The c.488T>C variant on the maternal allele results in a valine to alanine substitution at residue 163 (p.Val163Ala). Functional studies showed that this variant leads to protein misfolding as well as less effective tail-anchored protein insertion. Loss of asna1 in zebrafish resulted in reduced cardiac contractility and early lethality. In contrast to wild-type mRNA, injection of either mutant mRNA failed to rescue this phenotype. Biallelic variants in ASNA1 cause severe pediatric cardiomyopathy and early death. Our findings point toward a critical role of the tail-anchored membrane protein insertion pathway in vertebrate cardiac function and disease.

109. An investigational study of a dual-layer, chorion-free amnion patch as a protective barrier following lumbar laminectomy in a sheep model

BACKGROUND CONTEXT A decompressive laminectomy is the most common surgery performed to relieve symptoms of lumbar spinal stenosis. While this surgery has been shown to be successful in relieving patient symptoms, partially removing the lamina exposes the dura to elements and potential damage, from the surrounding environment. While a number of different biomaterials have been investigated for use as a protective barrier in spine surgery, the inherent properties of the human amniotic membrane (HAM) may make it favorable to protect neural elements and anterior vessels from the surrounding environment. The effect of a dual-layer HAM patch used as a physical barrier following a laminectomy in an animal model has yet to be investigated. PURPOSE The objective of this study was to evaluate the effect of a dual-layer, chorion-free amnion patch (DLAM) processed from HAM as a protective barrier following lumbar laminectomy in a sheep model. STUDY DESIGN/SETTING Prospective animal study. PATIENT SAMPLE Twelve skeletally mature female crossbred Suffolk sheep randomly divided into two equal postoperative periods (4 and 10 weeks). OUTCOME MEASURES A multiplex immunoassay was performed to quantify the inherent cytokines present in the amnion after processing. Additionally, at each time point extent of epidural fibrosis and neurohistopathological responses at the laminectomy sites was assessed based on epidural fibrosis-dura tenacity scores and decalcified histology, respectively. METHODS Each sheep underwent a laminectomy at L3 and L5, and one of the surgical sites randomly received the DLAM treatment. At each post-surgical time point, the extent of epidural fibrosis and neurohistopathological responses at the laminectomy sites was assessed based on epidural fibrosis-dura tenacity scores and decalcified histology, respectively. RESULTS Immunoassay results showed that inflammatory mediators and immunomodulatory cytokines were present in the amnion after processing, but no pro-angiogenic cytokines were detected. At 10 weeks, tissue tenacity was significantly less in the DLAM treatment group when compared to the operative control (1.2 ± 0.4 vs 2.8 ± 0.4, p CONCLUSIONS This study found that DLAM reduced fibroblast infiltration and tissue tenacity following lumbar laminectomy in a sheep animal model. These findings support the potential use of DLAM in clinical practice as a protective barrier for neural elements and anterior vessels. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.

Acyltransferase skinny hedgehog regulates TGFβ-dependent fibroblast activation in SSc

ObjectivesSystemic sclerosis (SSc) is characterised by aberrant hedgehog signalling in fibrotic tissues. The hedgehog acyltransferase (HHAT) skinny hedgehog catalyses the attachment of palmitate onto sonic hedgehog (SHH). Palmitoylation of SHH...

Elevated Thrombospondin 2 Contributes to Delayed Wound Healing in Diabetes.

Impaired wound healing is a major complication of diabetes, and despite the associated risks, treatment strategies for diabetic wounds remain limited. This is due, in part, to an incomplete understanding of the underlying pathological mechanisms, including the effects of hyperglycemia on components of the extracellular matrix (ECM). In the current study, we explored whether the expression of thrombospondin 2 (TSP2), a matricellular protein with a demonstrated role in response to injury, was associated with delayed healing in diabetes. First, we found that TSP2 expression was elevated in diabetic mice and skin from patients with diabetes. Then, to determine the contribution of TSP2 to impaired healing in diabetes, we developed a novel diabetic TSP2-deficient model. Though the TSP2-deficient mice developed obesity and hyperglycemia comparable with diabetic control mice, they exhibited significantly improved healing, characterized by accelerated reepithelialization and increased granulation tissue formation, fibroblast migration, and blood vessel maturation. We further found that hyperglycemia increased TSP2 expression in fibroblasts, the major cellular source of TSP2 in wounds. Mechanistically, high glucose increased activation of the hexosamine pathway and nuclear factor-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes.

Senescence-associated tissue microenvironment promotes colon cancer formation through the secretory factor GDF15.

The risk of colorectal cancer (CRC) varies between people, and the cellular mechanisms mediating the differences in risk are largely unknown. Senescence has been implicated as a causative cellular mechanism for many diseases, including cancer, and may affect the risk for CRC. Senescent fibroblasts that accumulate in tissues secondary to aging and oxidative stress have been shown to promote cancer formation via a senescence‐associated secretory phenotype (SASP). In this study, we assessed the role of senescence and the SASP in CRC formation. Using primary human colon tissue, we found an accumulation of senescent fibroblasts in normal tissues from individuals with advanced adenomas or carcinomas in comparison with individuals with no polyps or CRC. In in vitro and ex vivo model systems, we induced senescence using oxidative stress in colon fibroblasts and demonstrated that the senescent fibroblasts secrete GDF15 as an essential SASP factor that promotes cell proliferation, migration, and invasion in colon adenoma and CRC cell lines as well as primary colon organoids via the MAPK and PI3K signaling pathways. In addition, we observed increased mRNA expression of GDF15 in primary normal colon tissue from people at increased risk for CRC in comparison with average risk individuals. These findings implicate the importance of a senescence‐associated tissue microenvironment and the secretory factor GDF15 in promoting CRC formation.

Cancer‑associated fibroblast‑derived exosomal miR‑382‑5p promotes the migration and invasion of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC), with high potential for metastasis, is the most common malignant tumor of the head and neck. Cancer-associated fibroblasts (CAFs) are the main stromal cells in the microenvironment and aggravate tumor progression. However, whether CAFs are associated with the progression of OSCC remains unknown and the underlying mechanism remains unclear. In the present study, the role of CAFs in mediating OSCC cell migration and invasion was investigated, and the participation of exosomal miR-382-5p in this process was elucidated. In this study, according to the α-SMA staining with immunohistochemistry, 47 OSCC patients were divided into CAFs-rich and CAFs poor groups, and association of CAF density and clinicopathologic features of the OSCC patients were analyzed with Pearson χ2 test. Transwell assay was used for evaluating cell migration and invasion ability of OSCC cells after being co-cultured with NFs or CAFs, or after added exosomes. qPCR was used to detect the expression of miR-382-5p. Western blot analysis was used to measure the expression of migration and invasion-associated proteins. In the present study, the CAF density in tumor tissues was found to be relevant to OSCC lymph node metastasis and TNM stage. Furthermore, we revealed that miR-382-5p was overexpressed in CAFs compared with that in fibroblasts of adjacent normal tissue and miR-382-5p overexpression was responsible for OSCC cell migration and invasion. Finally, we demonstrated that CAF-derived exosomes transported miR-382-5p to OSCC cells. The present study confirmed a new mechanism of CAF-facilitated OSCC progression and may be beneficial for identifying new cancer therapeutic targets.

An investigational study of a dual-layer, chorion-free amnion patch as a protective barrier following lumbar laminectomy in a sheep model.

The inherent properties of the human amniotic membrane (HAM) suggest its potential for use as a physical barrier during surgery to protect neural elements and vessels from the surrounding environment. The objective of this study was to evaluate the effect of a dual-layer, chorion-free amnion patch (DLAM; ViaShield®, Globus Medical Inc., Audubon, PA, USA) processed from HAM as a protective barrier following lumbar laminectomy in a sheep model. A multiplex immunoassay was performed to quantify the inherent cytokines present in the amnion after processing. Twelve skeletally mature female crossbred Suffolk sheep were randomly divided into two equal post-operative periods (4 and 10 weeks). Each sheep underwent a laminectomy at L3 and L5, and one of the surgical sites randomly received the DLAM treatment. At each postsurgical time point, the extent of epidural fibrosis and neurohistopathological responses at the laminectomy sites was assessed based on epidural fibrosis-dura tenacity scores and decalcified histology, respectively. Immunoassay results showed that inflammatory mediators and immunomodulatory cytokines were present in the amnion after processing, but no proangiogenic cytokines were detected. At 10 weeks, tissue tenacity was significantly less in the DLAM treatment group when compared with the operative control (1.2 ± 0.4 vs. 2.8 ± 0.4, p < 0.05), demonstrating the ability of DLAM to act as a barrier and cover the dura. Gross observations showed fewer fibroblasts in the DLAM group in comparison with the control at both post-operative time points. Fibroblast infiltration analysis indicated that at both 4 and 10 weeks, there were significantly more infiltrated fibroblasts in the operative control sites than in the DLAM-treated sites, expressed as a percentage of the total number of fibroblasts present (4 weeks: 72.3 ± 10.2% vs. 10.8 ± 10.1%, p < .05; 10 weeks: 84.9 ± 15.8% vs. 43.1 ± 11.6%, p < .05). Additionally, fibroblasts travelled further into the dura in the operative control group compared with the DLAM-treated group at both time points. In conclusion, this study found that DLAM reduced fibroblast infiltration and tissue tenacity following lumbar laminectomy in a sheep animal model. These findings support the potential use of DLAM in clinical practice as a protective barrier for neural elements and anterior vessels.

Global DNA Methylation Pattern of Fibroblasts in Idiopathic Pulmonary Fibrosis.

Our previous transcriptome study of cultured fibroblasts identified 178 genes that were differentially expressed by 8 idiopathic pulmonary fibrosis (IPF) fibroblasts compared with 4 controls. Here, we performed genome-wide DNA methylation analysis to evaluate the relationship of CpG methylation to differential gene expression. Among 485,577 loci, 5850 loci on 2282 genes showed significant differences between the 2 groups (delta-beta >10.21 and p-value <0.05). Among these, beta values of 80 CpGs (30 hypermethylated and 50 hypomethylated) were significantly correlated with mRNA expression of 34 genes (19.1%) of the 178 differentially expressed genes between the 2 groups (13 downregulated and 21 upregulated). Gene ontology enrichment of these genes included cell adhesion, molecule binding, chemical homeostasis, surfactant homeostasis, and receptor binding. One-third of them are involved in the known process of fibrosis; the others are novel genes with respect to pulmonary fibrosis. We identified relationships between the altered DNA methylation levels and about one-fifth of the corresponding changes in gene expression by lung tissue fibroblasts. Findings from this study provide new information on novel genes responsible for the pathogenesis of IPF under the control of CpG methylation changes in IPF lungs.

Sphingosine-1-Phosphate Facilitates Skin Wound Healing by Increasing Angiogenesis and Inflammatory Cell Recruitment with Less Scar Formation.

Wound healing starts with the recruitment of inflammatory cells that secrete wound-related factors. This step is followed by fibroblast activation and tissue construction. Sphingosine-1-phosphate (S1P) is a lipid mediator that promotes angiogenesis, cell proliferation, and attracts immune cells. We investigated the roles of S1P in skin wound healing by altering the expression of its biogenic enzyme, sphingosine kinase-1 (SphK1). The murine excisional wound splinting model was used. Sphingosine kinase-1 (SphK1) was highly expressed in murine wounds and that SphK1−/− mice exhibit delayed wound closure along with less angiogenesis and inflammatory cell recruitment. Nanoparticle-mediated topical SphK1 overexpression accelerated wound closure, which associated with increased angiogenesis, inflammatory cell recruitment, and various wound-related factors. The SphK1 overexpression also led to less scarring, and the interaction between transforming growth factor (TGF)-β1 and S1P receptor-2 (S1PR2) signaling is likely to play a key role. In summary, SphK1 play important roles to strengthen immunity, and contributes early wound healing with suppressed scarring. S1P can be a novel therapeutic molecule with anti-scarring effect in surgical, trauma, and chronic wound management.

The impact of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells for model rats with diabetic foot ulcer

Objective To study effect of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells (HUCMSCs) for model rats with diabetic foot ulcer (DFU), and analyzed the possible action mechanism of HUCMSCs therapy DFU. Methods 50 specific pathogen free (SPF)-level male rats were fed with high-fat beverage for 4 weeks. The DFU models were established by means of intraperitoneal injection of 1% streptozotocin (40 mg/kg) and steam fumigation. 30 successful model rats were divided into model group, high-dose HUCMSCs group (high-dose group: femoral artery injection with HUCMSCs 2.0×106/0.2 ml) and low-dose HUCMSCs group (low-dose group: femoral artery injection with HUCMSCs 1.0×106/0.2 ml), 10 rats/each group. Another 10 SPF-level male rats were selected and set as blank control group (blank group). Then wound healing rate, serum inflammatory factors, related cell growth factors, granulation tissue fibroblasts and newly born capillaries were compared between the groups. Results (1) Wound healing rate: The model group, low-dose group, high-dose group respectively were (26.31±4.32)%, (66.92±7.24)%, (74.74±8.32)%. low-dose group and high-dose group wound healing rate were significantly higher than model group (t=8.424, 11.201, P<0.01), high-dose group wound healing rate were significantly higher than low-dose group (t=3.148, P<0.05). (2) Serum inflammatory factors: model group, low-dose group and high-dose group interleukin (IL)-1, tumor necrosis factor-α (TNF-α), hs-CRP respectively were (6.12±0.25), (4.54±0.36), (4.20±0.33) ng/L; (29.45±3.12), (21.13±3.24), (18.20±2.65) ng/L; (0.61±0.08), (0.45±0.07), (0.37±0.06) ng/L. low-dose group and high-dose group IL-1, TNF-α, hs-CRP were significantly lower than model group (t=11.400, 5.849, 5.111, 14.666, 8.691, 8.222, P<0.05, P<0.01), high-dose group were significantly lower low-dose group (t=2.202, 2.214, 2.744, P<0.05); (3) Related cell growth factors: model group, low-dose group and high-dose group basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) respectively were (46.42±6.36), (61.35±7.20), (68.24±8.12) ng/L; (546.32±30.24), (722.36±32.17), (772.45±34.26) μg/L; (2.24±0.32), (3.65±0.41), (4.26±0.55) μg/L. Low-dose group and high-dose group bFGF, EGF, VEGF were significantly higher than model group (t=4.915, 12.609, 8.573, 6.690, 15.648, 10.039, P<0.05, P<0.01). high-dose group bFGF, EGF and VEGF were significantly higher than low-dose group (t=2.031, 3.740, 2.812, P<0.05); (4) Granulation tissue fibroblasts and newly born capillaries: model group, low-dose group and high-dose group granulation tissue fibroblasts and newly born capillaries were respectively (155.24±8.45), (278.65±9.12), (305.12±10.25) cells/visual field; (4.36±0.45), (6.45±0.62), (7.24±0.60)×10/μm2. The low-dose group and high-dose group were significantly higher than model group (t=31.389, 8.627, 35.679, 12.143, P<0.01), high-dose group were significantly higher than low-dose group (t=6.101, 2.896, P<0.05). Conclusion HUMSCs help to promote the ulcerative healing of DFU rats. Key words: Diabetic foot ulcer; Human umbilical cord mesenchymal stem cells; Inflammatory factor; Growth factor

Abstract 2645: Quantitative measurement of multiple signal transduction pathway activities in cell and tissue culture, including cancer, fibroblast, and immune cell types

Abstract To improve pathophysiology research, biomarker discovery and drug development, cell culture models should adequately mimic human (patho)physiology and provide reproducible results. This requires comparison between cultured cells/tissue and actual histopathology in the patient, as well as standardization of culture experiments to ensure experimental reproducibility, preferably in a quantitative manner1. 10-15 signal transduction pathways govern major cellular processes, e.g. cell division, differentiation and migration. The past decade we developed tests to quantitatively measure functional activity of signal transduction pathways in individual cell/tissue samples, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the transcription factor associated with the respective signalling pathway. Tests provide quantitative pathway activity scores and are intended to be used for diagnostics and life sciences research2-4. Method: Tests have been developed for androgen (AR) and estrogen receptor (ER), Hedgehog (HH), Wnt, TGFβ, Notch, NFκB, PI3K, JAK-STAT 1/2 and 3, and MAPK pathways. After calibration and freezing of the models, extensive biological test validation was performed on healthy/diseased cell and tissue types, including multiple cancer, fibroblast and immune cell types. Using Affymetrix expression microarray data (GEO database) &amp;gt;900 cell lines of most cell and cancer types were analyzed, as well as primary cultures of most immune cell types. LnCaP (prostate), MCF7, BT474 (breast), HCC827 (lung) and A2780 (ovarian) cancer cell lines were compared across laboratories. Results: Typical expected single or combined pathway activities were confirmed, e.g. ER activity in breast, AR activity in prostate, and Wnt activity in colon cancer; HH activity in soft tissue tumor, NFκB activity in lymphoma, frequently combined with PI3K and/or MAPK and/or JAK-STAT growth factor pathways. Quantitative pathway activities were reproducible within studies, but highly variable between labs, and dependent on culture conditions. Conclusion: Our pathway tests measure signaling pathway activity in many cell and tissue types, and can be used as quantitative readout for cell/tissue culture. Applications are: standardization of cell/tissue culture to ensure reproducibility; comparison between culture-based disease model and patient histopathology; quantitative assessment of drug efficacy on disease models; assessment of toxicity on healthy cell/tissue models. A number of tests have been adapted to qPCR, enabling use on FFPE tissue and small samples. 1 Ben-David U, et al. Nature, 2018;560(7718):325; 2 Verhaegh W, et al. Cancer Res 2014;74(11):2936-45; 3 Verhaegh W, Stolpe A van de. Oncotarget, 2014:5(14):5196-7; 4 Ooijen H. van, et al. Am J Pathol 2018;188(9):1956-1972 Citation Format: Anja Van De Stolpe, Marcia Alves de Inda, Eveline den Biezen-Timmermans, Laurent Holtzer, Henk van Ooijen, Wim Verhaegh. Quantitative measurement of multiple signal transduction pathway activities in cell and tissue culture, including cancer, fibroblast, and immune cell types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2645.

Abstract 2699: Targeting an ubiquitin-activating enzyme in small-cell lung cancer (SCLC)

Abstract Background: Despite valiant efforts to improve patient outcomes, small cell lung cancer (SCLC) (~15-17% of lung cancer) remains an aggressive lung cancer, with an overall five-year survival of 7%. Current first-line therapy (chemotherapy+/-radiation) has not changed in 30+ years. TAK-243, an inhibitor of the ubiquitin activating enzyme (UAE), E1 (encoded by UBA1), poses a novel approach for cancer therapy. TAK-243 limits the formation of ubiquitin conjugates that ultimately mediate many cellular processes, including DNA repair signalling and protein degradation. Consequently, TAK-243 may disrupt cancer-specific dependencies of UAE to induce malignant cell death, and potentiate radiation-induced DNA damage. The discovery publication revealed that TAK-243 is especially potent in 2 SCLC cell-lines compared to healthy breast and fibroblastic tissues. With these limited data, the potential benefit of this novel agent as monotherapy and in combination with chemotherapy for SCLC has yet to be elucidated. Methods: We assessed the anti-proliferative effect of TAK-243 monotherapy in a large panel (n = 10) of variant and classical SCLC cell-lines, including assorted MYC family member amplifications and different UBA1 mRNA expression levels. TAK-243 EC50 single agent doses were determined by treating cell-lines with incremental concentrations of TAK-243 (0-1μM). For combinatorial analysis, TAK-243 (0-1μM) was administered to cell-lines alongside cisplatin/etoposide (CE) at cell-line specific optimized doses yielding 30-35% kill by CE. Short-term efficacy was determined by a cell viability resazurin assay, 48 hours after treatment initiation. ΔEC50 was measured as the single agent EC50 subtracted by the EC50 of TAK-243+CE. Positive ΔEC50 values show potential synergy between TAK-243 and CE. Results: Single-agent therapy. SCLC cell-lines demonstrated a range of sensitives to TAK-243 monotherapy after 48 hours. The cell-lines tested are ranked from highest to lowest sensitivity: NCI-H82, NCI-H69, NCI-H1618, LX22 (ex-vivo cultured PDX), NCI-H1092, NCI-H446, SHP77 and NCI-H196; with EC50 values ranging from 18nM to 80nM. However, NCI-H526, NCI-H889, and SBC-5 were non-responsive to therapy at 1μM. Combination therapy. TAK-243 upon combination treatment with CE revealed ΔEC50 values ranging from 5.23-14.54nM. NCI-H82, NCI-H69, SHP77 and NCI-H1092 demonstrated potential chemosensitivity to combination treatment. Conclusions: These data begin to hint at synergy between TAK-243 and CE, where lower doses of TAK-243 may be required for certain SCLC cell-lines when used in combination. These preliminary results suggest that TAK-243 may have the potential to improve the current SCLC standard therapies in molecular defined subgroups. These results are being confirmed in additional cell-lines and through in vivo experiments using patient-derived xenograft models of SCLC. *S.M and M.K.A contributed equally to this work Citation Format: Safa Majeed, Mansi K. Aparnathi, Lifang Song, Aaron D. Schimmer, Ming S. Tsao, Geoffrey Liu, Benjamin H. Lok. Targeting an ubiquitin-activating enzyme in small-cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2699.

Roles of the Mesenchymal Stromal/Stem Cell Marker Meflin in Cardiac Tissue Repair and the Development of Diastolic Dysfunction.

Myofibroblasts have roles in tissue repair following damage associated with ischemia, aging, and inflammation and also promote fibrosis and tissue stiffening, causing organ dysfunction. One source of myofibroblasts is mesenchymal stromal/stem cells that exist as resident fibroblasts in multiple tissues. We previously identified meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue), a glycosylphosphatidylinositol-anchored membrane protein, as a specific marker of mesenchymal stromal/stem cells and a regulator of their undifferentiated state. The roles of meflin in the development of heart disease, however, have not been investigated. We examined the expression of meflin in the heart and its involvement in cardiac repair after ischemia, fibrosis, and the development of heart failure. We found that meflin has an inhibitory role in myofibroblast differentiation of cultured mesenchymal stromal/stem cells. Meflin expression was downregulated by stimulation with TGF (transforming growth factor)-β, substrate stiffness, hypoxia, and aging. Histological analysis revealed that meflin-positive fibroblastic cells and their lineage cells proliferated in the hearts after acute myocardial infarction and pressure-overload heart failure mouse models. Analysis of meflin knockout mice revealed that meflin is essential for the increase in the number of cells that highly express type I collagen in the heart walls after myocardial infarction induction. When subjected to pressure overload by transverse aortic constriction, meflin knockout mice developed marked cardiac interstitial fibrosis with defective compensation mechanisms. Analysis with atomic force microscopy and hemodynamic catheterization revealed that meflin knockout mice developed stiff failing hearts with diastolic dysfunction. Mechanistically, we found that meflin interacts with bone morphogenetic protein 7, an antifibrotic cytokine that counteracts the action of TGF-β and augments its intracellular signaling. These data suggested that meflin is involved in cardiac tissue repair after injury and has an inhibitory role in myofibroblast differentiation of cardiac fibroblastic cells and the development of cardiac fibrosis.

Recovery Processes in the Adrenal Glands of Dogs

The aim of this research was a comparative study of cyclic changes in secretory, destructive and proliferative processes in the adrenal cortex in dogs. Material and methods . Cyclic changes of morphofunctional indices of adrenal cortex in dogs (Canis familiaris) were studied. Healthy mature males were used. The material was collected in the period from December to April with a monthly interval. For light microscopy the fragments of the adrenal glands were fixed in Steve, Bouin and Zenker liquids, 10% neutral formalin solution. PAS-reaction was used. RNA was detected by the Brachet reaction and gallocyanine according to Einarsson. The material fixed in a 10% solution of neutral formalin was used to study lipids. Results. In January, in the adrenal cortex clearly expressed glomerular, fasciculate and reticulae zones. In the glomerular zone elongated cells with oval nuclei are situated, in the fascicular - polygonal cells with rounded nuclei. In February, a decrease in the functional activity of the cortical parenchyma and the development of extensive destructive changes in glandular cells, especially in the glomerular zone, were found in the adrenal cortex. In March, the development of recovery processes and increased secretory activity of the adrenal cortex were noted. Proliferative processes are accompanied by loosening of the capsule, the formation of connective tissue strands going into the fascicular zone. Among the fibers of connective tissue fibroblasts, fibrocytes and small poorly differentiated cells with rounded nuclei are disposed. These cells are converted into elongated cells of the glomerular zone and polygonal cells of the fascicular zone.

Role of the Notch Signaling Pathway in Fibrosis of Denervated Skeletal Muscle.

In order to investigate the role of the Notch signaling pathway in skeletal muscle fibrosis after nerve injury, 60 Sprague-Dawley rats were selected and divided randomly into a control and two experimental groups. Group A served as controls without any treatment. Rats in groups B were injected intraperitoneally with 0.2 mL PBS and those in group C were injected intraperitoneally with 0.2 mL PBS+100 μmol/L, 0.2 mL N-[N-(3,5-difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT, a gamma-secretase inhibitor that suppresses Notch signaling) respectively, on postoperative days 1, 3, 7, 10, and 14 in a model of denervation-induced skeletal muscle fibrosis by right sciatic nerve transection. Five rats from each group were euthanized on postoperative days 1, 7, 14, and 28 to collect the right gastrocnemii, and hematoxylin and eosin (HE) staining, immunohistochemistry test, real-time PCR, and Western blotting were performed to assess connective tissue hyperplasia and fibroblast density as well as expression of Notch 1, Jagged 1, and Notch downstream molecules Hes 1 and collagen I (COL I) on day 28. There was no significant difference in HE-stained fibroblast density between group B and C on postoperative day 1. However, fibroblast density was significantly higher in group B than in group C on postoperative days 7, 14, and 28. Notch 1, Jagged 1, Hes 1, and COL I proteins in the gastrocnemius were expressed at very low levels in group A but at high levels in group B. Expression levels of these proteins were significantly lower in group C than in group B (P<0.05), but they were higher in group C than in group A (P<0.05) on postoperative day 28. We are led to conclude that locking the Notch signaling pathway inhibits fibrosis progression of denervated skeletal muscle. Thus, it may be a new approach for treatment of fibrosis of denervated skeletal muscle.

Prognostic significance of combining immunohistochemical markers for cancer-associated fibroblasts in lung adenocarcinoma tissue.

Cancer-associated fibroblasts (CAFs), activated fibroblasts in a cancer microenvironment, exert various effects upon carcinoma cells including lung adenocarcinoma cells. Various markers identifying CAFs have been proposed, but the correlations among these markers proposed and their clinicopathological significance have remained largely unknown. Therefore, in this study, we immunohistochemically evaluated the expression of alpha-smooth muscle actin (α-SMA), podoplanin, and periostin among these proposed markers in 92 cases of lung adenocarcinoma. These three markers were weakly correlated, but the relative abundance of α-SMA was significantly associated with high Ki-67 labelling index (LI), lymph node metastasis, and low 5-year overall survival (OS) rate of the patients. That of podoplanin was significantly associated with high pT and Ki-67 LI, distant metastasis, and low 5-year OS rate and that of periostin with high pT and Ki-67 LI. We then tentatively subclassified these cases into four groups according to high or low status of each of paired markers: α-SMA/podoplanin, α-SMA/periostin, and periostin/podoplanin. The α-SMA high/podoplanin high group was associated with the lowest survival rate (53.3%) among the four groups with significance. However, there were no significant differences in overall survival when the patients were classified according to the combinations of α-SMA/periostin or periostin/podoplanin. Results of our study firstly revealed the heterogeneity of CAFs in human lung adenocarcinoma tissue, and the analysis employing multiple markers of CAFs is generally required to study the clinical significance of CAFs in clinical materials.

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.

Changes in the functional state of the vascular endothelium in young athletes of varying skill levels

Recent studies indicate to the special importance of endothelial function (EF) in processes of the regulation of blood circulation. There are presented data on the influence of physical loads on changes in EF on the basis of analysis of the changes of serum content of angiogenin, vascular endothelial growth factor, fibroblast growth factor, matrix metalloproteinase-9 and tissue inhibitor of MMP, thrombospondin and endothelin in young swimmers of various sports skill levels. During training activities and with gain in sports skill levels in young athletes there was found that a significant increment of the concentration of mentioned regulators may be a response to intense exercises indicating to active participation of endothelial vasculature of the growing body in a physiological mechanisms of adaptation to physical loads in children.

Novel Silver-NHC complexes: Synthesis and anticancer properties

The aim of this study was to present the synthesis, characterization and anticancer activities of two novel benzimidazole-based NHC salts (1a-b) and their silver(I) complexes (2a-b) with methylbenzyl and isopropylbenzyl chains. All compounds were prepared using Schlenk techniques in an inert atmosphere. The carbene complexes were prepared with the interaction of carbene precursors and Ag2O. All new compounds were characterized by elemental analysis, LC-MS, FT-IR, 1H NMR, and 13C NMR spectroscopic techniques. The anticancer activities of the salts and complexes were determined by cell proliferation analysis using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human prostate cancer cells (DU-145) and human breast cancer cells (MCF-7, MDA-MB-231). L-929, mouse adipose tissue fibroblasts were used as model normal cells. The cells were plated at a cell density of 1 × 105 cells in 96-well plates and treated with different concentrations (1–20 μM) of salts and complexes during 24, 48 and 72 h. The MTT assay results indicated that the salts (1a-b) have lower anticancer activity on cancer cells than their silver(I) complexes (2a-b). It was also observed that benzimidazole salts (1a-b) and their Ag-NHC complexes (2a-b) displayed lower anticancer activities on L-929 normal cells than on cancer cells. These results highlight that the anticancer activities of N-heterocyclic carbene-silver complexes vary according to the structure of the silver complex and cell line type.

Adherent muscle connective tissue fibroblasts are phenotypically and biochemically equivalent to stromal fibro/adipogenic progenitors

Extracellular matrix (ECM) gives structure, support, and is the niche for several cells found in skeletal muscle. ECM is mainly produced by muscle connective tissue (CT) fibroblasts during development and regeneration. Stromal fibroadipogenic progenitors (FAPs) are CT fibroblasts-like mesenchymal progenitors (MPs) with important roles in regeneration and degeneration. Chronic damage restrains the normal regenerative behavior of muscle fibroblasts/FAPs. Thus, the isolation and study of these mesenchymal progenitors are of crucial importance for understanding their behavior and biology. We investigated whether adult muscle CT fibroblasts (hereafter referred to as adherent fibroblasts [aFbs]) cultured via pre-plating strategy belong to a heterogeneous population of FAPs. By combining microscopy, western blot analyses, flow cytometry, and FACS we determined that aFbs isolated from skeletal muscle largely overlap with FAPs. In addition, we used the PDGFRαEGFP mice in order to corroborate our results with EGFP+ FAPs. Moreover, our strategy allows the isolation of activated EGFP+ FAPs from the murine DMD model PDGFRαEGFP; mdx and PDGFRαEGFP denervated mice. Here we report that 1 h 30 min of pre-plating strategy allows the isolation and culture of a highly enriched population of aFbs. These cells are phenotypically and biochemically a FAPs-like population of adherent cells. In addition, aFbs respond in the same fashion as FAPs to Nilotinib, an inducer of FAPs apoptosis. Moreover, flow cytometry characterization of these aFbs suggests that 85% of them express the MP marker PDGFRα, and isolation of aFbs from the PDGFRαEGFP mice suggests that 75% of them show high EGFP expression. Furthermore, TGF-β1 induces aFbs proliferation, myofibroblast differentiation, and ECM production. We were also able to isolate activated aFbs from skeletal muscle of the DMD mice and from the PDGFRαEGFP mice 2-days after denervation. Our findings suggest that the in vitro pre-plating strategy allows the isolation and culture of a relatively pure aFbs population, which resembles FAPs in vitro.