Immunohistochemistry For α-SMA Research Articles

A novel pulmonary fibrosis NOD/SCID murine model with natural aging

BackgroundIdiopathic pulmonary fibrosis (IPF) is an age-related disease severely affecting life quality with its prevalence rising as the population ages, yet there is still no effective treatment available. Cell therapy has emerged as a promising option for IPF, however, the absence of mature and stable animal models for IPF immunodeficiency hampers preclinical evaluations of human cell therapies, primarily due to rapid immune clearance of administered cells. This study aims to establish a reliable pulmonary fibrosis (PF) model in immunodeficient mice that supports autologous cell therapy and to investigate underlying mechanism.MethodsWe utilized thirty 5-week-old male NOD/SCID mice, categorizing them into three age groups: 12weeks, 32 weeks and 43 weeks, with 6 mice euthanized randomly from each cohort for lung tissue analysis. We assessed fibrosis using HE staining, Masson’s trichrome staining, α-SMA immunohistochemistry and hydroxyproline content measurement. Further, β-galactosidase staining and gene expression analysis of MMP9, TGF-β1, TNF-α, IL-1β, IL-6, IL-8, SOD1, SOD2, NRF2, SIRT1, and SIRT3 were performed. ELISA was employed to quantify protein levels of TNF-α, TGF-β1, and IL-8.ResultsWhen comparing lung tissues from 32-week-old and 43-week-old mice to those from 12-week-old mice, we noted a marked increase in inflammatory infiltration, fibrosis severity, and hydroxyproline content, alongside elevated expression levels of α-SMA and MMP9. Notably, the degree of fibrosis intensified with age. Additionally, β-galactosidase staining became more pronounced in older mice. Quantitative PCR analyses revealed age-related, increases in the expression of senescence markers (GLB1, P16, P21), and proinflammatory genes (TGF-β1, TNF-α, IL-1β, IL-6, and IL-8). Conversely, the expression of anti-oxidative stress-related genes (SOD1, SOD2, NRF2, SIRT1, and SIRT3) declined, showing statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001). ELISA results corroborated these findings, indicating a progressive rise in the protein levels of TGF-β1, TNF-α, and IL-8 as the mice aged.ConclusionsThe findings suggest that NOD/SCID mice aged 32 weeks and 43 weeks effectively model pulmonary fibrosis in an elderly context, with the disease pathogenesis likely driven by age-associated inflammation and oxidative stress.

Pdgfrβ+ Lineage Cells Transiently Increase at the Site of Achilles Tendon Healing.

The purpose of this study was to track platelet-derived growth factor receptor-beta (Pdgfr-β) lineage cells at the site of Achilles tendon injury over time. Pdgfr-β-CreERT2 :Ai9 mice were generated in order to track Pdgfr-β lineage cells in adult mice. A surgical Achilles transection injury model was employed to examine the presence of Pdgfr-β lineage cells in the healing tendon over time, with 5 mice per time point at 3, 7, 14, 28, and 56 days postoperatively. Histology and immunohistochemistry for tdTomato (Pdgfr-β lineage cells), PCNA (proliferating cell nuclear antigen, cell proliferation), and α-SMA (alpha smooth muscle actin, myofibroblasts) were performed. The percentage of cells at the healing tendon site staining positive for tdTomato and PCNA were quantified. Over 75% of cells at the injury site were Pdgfr-β lineage cells at days 3, 7, and 14, and this percentage decreased significantly by days 28 and 56 post-injury. Cell proliferation at the injury site peaked on day 7 and decreased thereafter. Immunohistochemistry for α-SMA demonstrated minimal co-localization of myofibroblasts with Pdgfr-β lineage cells. This study demonstrates that in a mouse model of Achilles tendon injury, Pdgfr-β lineage cells' presence at the injury site is transient. Thus, we conclude that they are unlikely to be the cells that differentiate into myofibroblasts and directly contribute to tendon fibrous scar formation. This article is protected by copyright. All rights reserved.

Edaravone protects against liver fibrosis progression via decreasing the IL-1β secretion of macrophages

Edaravone (EDA), a strong novel free radical scavenger, have been demonstrated to exert neurovascular protective effects clinically. Furthermore, EDA can suppress the lung injury, pulmonary fibrosis and skin fibrosis, while the precise effects and mechanisms of EDA on liver injury and fibrosis remain unclear. The effects of EDA on the Thioacetamide (TAA)-induced liver fibrosis were evaluated by sirius red staining, α-SMA immunohistochemistry. The percentages of immune cell subsets were analyzed by flow cytometry. Immunofluorescence assay was performed to identify the fibrotic properties of hepatic stellate cells (HSCs). Western blot and qPCR were used to detect the levels of liver fibrosis-related molecules and IL-1β. EDA displayed a hepatic protective role in TAA-induced chronic liver fibrosis via inhibiting monocyte/macrophages recruitment and IL-1β production of macrophages. Mechanically, EDA inhibited of NF-κB signal pathway and reactive oxygen species (ROS) production in macrophages. Moreover, EDA treatment indirectly suppressed the activation of HSCs by decreasing the IL-1β secretion of macrophages. Together, EDA protects against TAA-induced liver fibrosis via decreasing the IL-1β production of macrophages, thereby providing a feasible solution for clinical treatment of liver fibrosis.

Myofibroblastic CAF Density, Not Activated Stroma Index, Indicates Prognosis after Neoadjuvant Therapy of Pancreatic Carcinoma

Simple SummaryPancreatic cancer is increasingly treated with chemotherapy before surgery, but this does not improve prognosis for every patient. On a cellular level, pancreatic cancer tissue is usually mixed with a dense matrix called the stroma which interacts with the tumor and contains fibers, large molecules and cells. The role of this stroma in chemotherapy and subsequent surgical tumor resection is unclear, especially for current first-line chemotherapy regimens. We analyzed two of the main stromal components, activated fibroblasts and collagen, and found that treatment with gemcitabine + nab-paclitaxel reduced collagen content while FOLFIRINOX had no quantitative effect. Meanwhile, a higher number of activated fibroblasts was beneficial for prognosis after chemotherapy. These findings will serve to further elucidate mechanisms of response and chemoresistance in pancreatic cancer and potentially to stratify patients for different treatment pathways.Neoadjuvant therapy (NT) for advanced PDAC is an emerging concept, affecting both stroma and tumor. The Activated Stroma Index (ASI; ratio of activated cancer-associated fibroblasts (CAF) to collagen deposition) is a prognostic marker in upfront resected pancreatic adenocarcinoma (PDAC). We assessed ASI and its prognostic relevance after NT. Tissue from resection specimens of n = 48 PDAC patients after neoadjuvant chemotherapy with FOLFIRINOX (FOL; n = 31), gemcitabine + nab-paclitaxel (GEM; 7) or combination treatment (COMB; 10) was compared with upfront resected matched controls (RES; 69). Activated CAFs were assessed by immunohistochemistry for α-SMA, and collagen was stained with aniline blue; the stained area was then determined by computational imaging analysis and ASI was calculated. In GEM, ASI was significantly higher and collagen deposition lower than in controls and FOL. The lowest quartile of ASI values had significantly longer overall survival (OS) in RES, whereas in FOL, the highest quartile had the best prognosis. After NT, OS was significantly improved in the α-SMA-high group; in RES, however, survival was independent of α-SMA. Reversed prognostic association of ASI thus points to the differing significance of stromal composition after FOL, while improved prognosis with high CAF abundance suggests a synergistic effect of myofibroblasts with chemotherapy. These divergences impede usability of ASI after NT.

Baoganning formula alleviates liver fibrosis in mice by inhibiting hepatic IDO1 expression and promoting phenotypic maturation of dendritic cells

To investigate the protective effect of Bao Gan Ning (BGN), a traditional Chinese medicinal formula, against CCl4-induced liver fibrosis in mice and explore the mechanism. C57BL/6 mice were randomly divided into control group, liver fibrosis model group, positive control group and the low-, middle-, and high-dose BGN groups (n=10). In all but the control group, the mice were subjected to daily intraperitoneal injection of 25% CCl4 (in olive oil) to induce liver fibrosis and intragastric gavage of corresponding drugs. After 8 weeks, serum levels of ALT and AST were detected. Pathological examination of the liver was performed using HE and Sirius Red staining and α-SMA immunohistochemistry. The expression level of indoleamine 2, 3-dioxygenase 1 (IDO1) and phenotypic changes of hepatic DCs in the liver were measured. Another 18 mice were randomly divided into AAV9-NC, AAV9-IDO1 and high-dose BGN + AAV-IDO1 groups (n=6) for corresponding treatment, and 4 weeks later the deposit of hepatic IDO1 and phenotypic changes of the hepatic DCs were analyzed. Compared with those in the model group, serum AST and ALT levels decreased significantly in BGN group (P < 0.01). Obvious liver fibrosis was observed in the model group, while the mice treated with BGN showed obviously reduced cell necrosis and collagen proliferation in the liver with significantly lowered the expression levels of hepatic α-SMA and IDO1 (P < 0.05). The percentages of CD11C + DCs, CD11C +CD80 + DCs, CD11C +CD86 + DCs, CD11C +CD40 + DCs, CD11C +MHCII + DCs, CD3 + T cells, and CD3 +CD4 + T cells all increased significantly in BGN group as compared with the model group (P < 0.05). In mice with adenovirus-mediated IDO1 overexpression in the liver, BGN treatment significantly lowered the expression level of IDO1 (P=0.000) and increased the percentages of hepatic CD11C +CD40 + DCs, CD11C +MHCII + DCs and CD3 +CD4 + T cells (P < 0.05). BGN can effectively inhibit liver fibrosis in mice possibly by lowering the expression level of IDO1 in the liver, thus improving the function of hepatic DCs and subsequently promoting proliferation of T cells.

Decreased ferroportin in hepatocytes promotes macrophages polarize towards an M2-like phenotype and liver fibrosis

Iron release from macrophages is closely regulated by the interaction of hepcidin, a peptide hormone produced by hepatocytes, with the macrophage iron exporter ferroportin (FPN1). However, the functions of FPN1 in hepatocyte secretion and macrophage polarization remain unknown. CD68 immunohistochemical staining and double immunofluorescence staining for F4/80 and Ki67 in transgenic mouse livers showed that the number of macrophages in FPN1−/+ and FPN1−/− mouse livers was significantly increased compared to that in WT (FPN+/+) mice. FPN1 downregulation in hepatic cells increased the levels of the M2 markers CD206, TGF- β, VEGF, MMP-9, Laminin, Collagen, IL-4 and IL-10. Furthermore, the expression of CD16/32 and iNOS, as M1 markers, exhibited the opposite trend. Meanwhile, α-SMA immunohistochemistry and Sirius red staining showed that the trend of liver fibrosis in FPN1−/− mice was more significant than that in control mice. Similarly, in vitro FPN1 knockdown in L02-Sh/L02-SCR liver cell lines yielded similar results. Taken together, we demonstrated that downregulated FPN1 expression in hepatocytes can promote the proliferation and polarization of macrophages, leading to hepatic fibrosis. Above all, the FPN1 axis might provide a potential target for hepatic fibrosis.

The Propensity of the Human Liver to Form Large Lipid Droplets Is Associated with PNPLA3 Polymorphism, Reduced INSIG1 and NPC1L1 Expression and Increased Fibrogenetic Capacity.

In nonalcoholic steatohepatitis animal models, an increased lipid droplet size in hepatocytes is associated with fibrogenesis. Hepatocytes with large droplet (Ld-MaS) or small droplet (Sd-MaS) macrovesicular steatosis may coexist in the human liver, but the factors associated with the predominance of one type over the other, including hepatic fibrogenic capacity, are unknown. In pre-ischemic liver biopsies from 225 consecutive liver transplant donors, we retrospectively counted hepatocytes with Ld-MaS and Sd-MaS and defined the predominant type of steatosis as involving ≥50% of steatotic hepatocytes. We analyzed a donor Patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphism, hepatic expression of proteins involved in lipid metabolism by RT-PCR, hepatic stellate cell (HSC) activation by α-SMA immunohistochemistry and, one year after transplantation, histological progression of fibrosis due to Hepatitis C Virus (HCV) recurrence. Seventy-four livers had no steatosis, and there were 98 and 53 with predominant Ld-MaS and Sd-MaS, respectively. In linear regression models, adjusted for many donor variables, the percentage of steatotic hepatocytes affected by Ld-MaS was inversely associated with hepatic expression of Insulin Induced Gene 1 (INSIG-1) and Niemann-Pick C1-Like 1 gene (NPC1L1) and directly with donor PNPLA3 variant M, HSC activation and progression of post-transplant fibrosis. In humans, Ld-MaS formation by hepatocytes is associated with abnormal PNPLA3-mediated lipolysis, downregulation of both the intracellular cholesterol sensor and cholesterol reabsorption from bile and increased hepatic fibrogenesis.

The Optimal Application of Medium Potency Topical Corticosteroids in Preventing Laser-Induced Inflammatory Responses-An Animal Study.

Background: During ablative fractional resurfacing (AFR) laser therapy, thermal damage to the skin is inevitable, resulting in inflammatory responses and small wounds. Corticosteroids are known for their anti-inflammatory effect. However, inappropriate application of corticosteroids carries the risk of delayed wound healing. Therefore, we aimed to find the optimal administration route, timing, and duration of medium potency corticosteroid treatment to prevent AFR laser-induced inflammatory responses and to minimize the risk of delayed wound healing. Methods: We determined the anti-inflammatory efficacy of corticosteroids by skin erythema and tissue biopsies on C57BL/6 mice. Wound healing was evaluated by crust area and epithelial gap. Finally, Masson’s trichrome stain and α-SMA immunohistochemistry stain were used to analyze scar contracture. Results: Our results demonstrated that one dose of medium-potency topical corticosteroid applied immediately after AFR laser treatment could prevent erythema effectively with minimal disruption to wound healing. Notably, when more than one dose was administered, wound healing was delayed and scar contracture was aggravated by the application of medium-potency topical corticosteroids in a dosage-dependent manner. Conclusion: Our findings suggested that single-dose medium-potency topical corticosteroids could potentially improve AFR laser-induced acute inflammatory responses in clinical applications.

Abstract 5110: Cancer-associated fibroblast phenotypes vary across colorectal cancers and correlate with CD8+ T-cell infiltration

Abstract BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment (TME). This has been correlated with changes in cancer biology, including tumor progression and treatment response. CAFs exhibit divergent phenotypes differentiated by their expression profiles and function. Across cancer types, two CAF subsets have been consistently identified: myofibroblastic and non-myofibroblastic. Here we examine podoplanin (PDPN), α-smooth muscle actin (αSMA), and collagen as potential CAF subtype markers in the stromal compartment of colorectal cancers (CRCs). These markers were compared with versican (VCAN), an immunoregulatory proteoglycan associated with an immune excluded TME. METHODS: A tissue microarray containing representative tumor and normal tissue from 122 CRC patients was stained using Masson's Trichrome (collagen) or immunohistochemistry for αSMA, PDPN, VCAN and versikine (Vkine, a proteolytic fragment of VCAN). Quantification of the stains (0 to 3+) was performed by at least two observers in consultation with a surgical pathologist. Smooth muscle from the muscularis propria was excluded from quantification in the αSMA stain. CD8 staining was quantified as the number of tumor infiltrating CD8+ lymphocytes (TILs) per high power field (HPF). Separate tissue cores were sequenced for 107 of these patients using the Qiagen Comprehensive Cancer Panel. Mutations were identified using Strelka; SNPs and indels were cross-referenced to ClinVar. RESULTS: Stromal staining for αSMA, PDPN, and collagen varied across CRC sections. The abundance and intensity of staining was independent of disease stage. Those cancers with 3+ collagen or αSMA had reduced TILs (collagen 0-1+: 8.0 TILs/HPF vs 2-3+: 3.0, p=0.02; αSMA 0-1+: 7.4 vs 2-3+: 3.6, p=0.04). There was a similar trend observed for PDPN (0-1+: 6.5 vs 2-3+: 3.5, p=0.1). VCAN proteolysis predominant (VPP; VCAN 0-1+ and Vkine 2-3+) samples, previously reported to associate with higher CD8+ TILs, had lower collagen vs VCAN proteolytic weak (VPW; mean collagen score 1.6 vs 2.2, p=0.002) and lower αSMA (VPP: 1.7 vs VPW: 2.1, p=0.02). PDPN was not associated with proteolysis (p=0.4). In VPP tumors, stromal marker abundance did not correlate with CD8+ TILs; however, in VPW tumors, CD8+ TILs were higher in low PDPN (4.1 vs 1.5 TILs/HPF, p = 0.02) and low αSMA tumors (4.8 vs 1.6; p = 0.009). The trend was similar for low collagen tumors (3.7 vs 1.9, p = 0.1). Collagen was more abundant in TP53-mutant cancers (p=0.03, Chi-Square Test). CONCLUSIONS: The stromal microenvironment of CRC is highly variable across patients with diverse CAF densities and phenotypes. The makeup of this microenvironment has potential clinical impacts, as it correlates with immune cell infiltration. Further investigation into CAF biology in the TME could reveal therapeutic targets to alter immune surveillance of cancers. Citation Format: Katherine A. Johnson, Philip B. Emmerich, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. Cancer-associated fibroblast phenotypes vary across colorectal cancers and correlate with CD8+ T-cell infiltration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5110.

Comparative Study on Effect of Mesenchymal Stem Cells and Endothelial Progenitor Cells on Treatment of Experimental CCL4 Induced Liver Fibrosis

Abstract Background Bone marrow mesenchymal stem cells (BM-MSCs) and human umbilical cord endothelial progenitor cells (UC-EPCs) have several benefits for liver regeneration. We speculate huge impacts of rat BM-MSCs and UC-EPCs on reversal of hepatic injury and improvement of liver function in liver fibrosis induced by carbon tetrachloride (CCl4). Methods Forty adult rats were divided into 4 groups; control group, CCl4 group, CCl4/BM-MSCs group and CCl4/UCEPCs group. Blood samples were driven from rats at 1, 2 and 3months to measure serum concentration of albumin and alanine aminotransferase (ALT). Quantitative expression of HGF,TGF-β, MMP-2, and VEGF were assessed by polymerase chain reaction. Histological examination of the liver tissue was performed. α-SMA immunohistochemistry to identify the myoepithelial cells (MECs) and Ki-67 to identify cell prolifration immunohistochemistry are detected in groups injected with cells to confirm cells regeneration. Results Regarding liver function, there was elevating albumin (P &amp;lt; 0.05) and reducing ALT (P &amp;lt; 0.05) concentrations in groups treated with BM-MSCs and UC-EPCs effect compared to untreated CCL4 group. Concerning gene expression, UC-EPCs treated group have significantly higher MMP-2 and VEGF (P &amp;lt; 0.01) genes expression than BM-MSCs treated group. Furthermore, UC-EPCs were more valuable than BM-MSCs in increasing gene expression of HGF (P &amp;lt; 0.05) and immunohistochemistry of α-SMA and Ki-67 (P &amp;lt; 0.01). BM-MSCs have significantly lower TGF- β (P &amp;lt; 0.00) compared to UC-EPCs. Conclusion This study highlighted on liver regeneration role of both human UC-EPCs and BM-MSCs in liver fibrosis by different signaling mechanistic.

Lats2-Underexpressing Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate LPS-Induced Acute Lung Injury in Mice

The pathophysiology of the acute lung injury (ALI) is characterized by the damage of alveolar epithelial cells, which can be repaired by exogenous bone marrow-derived mesenchymal stem cells (BMSCs). However, the migration and differentiation abilities of BMSCs are not sufficient for the purpose, and a new approach that could strengthen the repair effects of BMSCs in ALI still needs to be clarified. We have previously proved that in vitro large tumor suppressor kinase 2- (Lats2-) underexpressing BMSCs may enhance their tissue repair effects in ALI; thus, in the present study, we tried to explore whether Lats2-underexpressing BMSCs could rescue lipopolysaccharide- (LPS-) induced ALI in vivo. BMSCs from C57BL/6 mice transfected with Lats2-interfering lentivirus vector or lentivirus blank controls were transplanted intratracheally into LPS-induced ALI mice. The retention and differentiation of BMSCs in the lung were evaluated by in vivo imaging, immunofluorescence staining, and Western blotting. The lung edema and permeability were assessed by lung wet weight/body weight ratio (LWW/BW) and measurements of proteins in bronchoalveolar lavage fluid (BALF) using ELISA. Acute lung inflammation was measured by the cytokines in the lung homogenate and BALF using RT-qPCR and ELISA, respectively. Lung injury was evaluated by HE staining and lung injury scoring. Pulmonary fibrosis was evaluated by Picrosirius red staining, immunohistochemistry for α-SMA and TGF-β1, and hydroxyproline assay and RT-qPCR for Col1α1 and Col3α1. Lats2-mediated inhibition of the Hippo pathway increased the retention of BMSCs and their differentiation toward type II alveolar epithelial cells in the lung. Furthermore, Lats2-underexpressing BMSCs improved lung edema, permeability of the lung epithelium, and lung inflammation. Finally, Lats2-underexpressing BMSCs alleviated lung injury and early pulmonary fibrosis. Our studies suggest that underexpression of Lats2 could further enhance the repair effects of BMSCs against epithelial impair and the therapeutic potential of BMSCs in ALI mice.

A novel model of reno-cardiac syndrome in the C57BL/ 6 mouse strain

BackgroundThe end stage renal disease population has a 20 fold higher incidence of cardiovascular mortality compared to the overall population. The development of reno-cardiac syndrome in these patients will result in cardiovascular events to be the cause of 50% of fatalities. There is therefore a need to research improved therapeutic strategies to combat renal cardiac pathologies. Murine in vivo models contribute greatly to such research allowing for specific genetic modification and reduced miscellany, however there is currently no reliable model of reno-cardiac syndrome in the most common genetically modified mouse strain, the C57BL/6. In this study we have manipulated an established model of chronic renal disease using adenine infused diet and prolonged the course of its pathology achieving chronic renal failure and subsequent reno-cardiac syndrome in the C57BL/6 mouse.MethodsEight week-old male C57BL/ 6 mice were acclimatised for 7 days before administration of a 0.15% adenine diet or control diet for 20 weeks.Cardiac function was assessed in mice at week 20 by echocardiography. At experiment termination blood and urine samples were analysed biochemically and organ dysfunction/injury was determined using immunoblotting and immunohistochemistry.ResultsAdministration of 0.15% adenine diet caused progressive renal failure resulting in reno-cardiac syndrome. At endpoint uraemia was confirmed by blood biochemistry which in the adenine fed mice showed significant increases in serum creatinine, urea, calcium (P < 0.0001) potassium (P < 0.05), and a significantly reduced glomerular filtration rate (P < 0.05). Reno-cardiac syndrome was confirmed by a significantly increased heart to body weight ratio (P < 0.0001) and echocardiography which showed significant reductions in percentage of ejection fraction, fractional shortening, fractional area change, (P < 0.0001) and an increase in left ventricular end diastolic volume (P < 0.05). Immunoblotting of kidney and heart tissue showed increased apoptosis (caspase 3) and fibrosis (fibronectin) and increases in the cardiac levels of phosphorylated Akt, and renal total Akt. Immunohistochemistry for α-SMA, collagen 1 and collagen 3 further confirmed fibrosis.ConclusionsWe present a novel regimen of adenine diet which induces both chronic kidney disease and reno-cardiac syndrome in the C57/BL6 mouse strain. The non-surgical nature of this model makes it highly reproducible compared to other models currently available.

Elimination of Wnt Secretion From Stellate Cells Is Dispensable for Zonation and Development of Liver Fibrosis Following Hepatobiliary Injury.

Alterations in the Wnt signaling pathway including those impacting hepatic stellate cells (HSCs) have been implicated in liver fibrosis. In the current study, we first examined the expression of Wnt genes in human HSC (HHSCs) after treatment with a profibrogenic factor TGF-β1. Next, we generated HSC-specific Wntless (Wls) knockout (KO) using the Lrat-cre and Wls-floxed mice. KO and littermate controls (CON) were characterized for any basal phenotype and subjected to two liver fibrosis protocols. In vitro, TGF-β1 induced expression of Wnt2, 5a and 9a while decreasing Wnt2b, 3a, 4, and 11 in HHSC. In vivo, KO and CON mice were born at normal Mendelian ratio and lacked any overt phenotype. Loss of Wnt secretion from HSCs had no effect on liver weight and did not impact β-catenin activation in the pericentral hepatocytes. After 7 days of bile duct ligation (BDL), KO and CON showed comparable levels of serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total and direct bilirubin. Comparable histology, Sirius red staining, and immunohistochemistry for α-SMA, desmin, Ki-67, F4/80, and CD45 indicated similar proliferation, inflammation, and portal fibrosis in both groups. Biweekly administration of carbon tetrachloride for 4 or 8 weeks also led to comparable serum biochemistry, inflammation, and fibrosis in KO and CON. Specific Wnt genes were altered in HHSCs in response to TGF-β1; however, eliminating Wnt secretion from HSC did not impact basal β-catenin activation in normal liver nor did it alter the injury-repair response during development of liver fibrosis.

Protective effects of combined Losartan and Nilotinib on carbon tetrachloride (CCl4)-induced liver fibrosis in rats

Tyrosine kinase inhibitors (TKIs) have been developed as therapeutic compounds for inhibiting the progression of liver fibrosis. In the present study, the simultaneous treatment of Nilotinib (TKIs) and Losartan was studied. Forty rats were divided into eight groups of fibrosis induced by carbon tetrachloride (CCl4) and therapeutics (Nilotinib, Losartan, and combination therapy). In the end, serum parameters of the liver and gene expression analysis of transforming growth factor-β1, its receptors (TβRII), platelet-derived growth factor, its receptors (PDGFRβ), matrix metalloproteinases (MMP-2 and MMP-9), tumor necrosis factor-α, cytochrome P450 2E1, and collagen1 type 1 were performed. The oxidant/antioxidant factors were also analyzed. Histopathology analysis along with α-SMA immunohistochemistry and hydroxyproline evaluation was also conducted for a more in-depth study. The overall results indicated a better therapeutic effect of co-treatment of Nilotinib–Losartan in comparison with the treatment of each of them alone. Interestingly, some gene and protein factors and fibrotic indices were reduced even to the normal levels of the control group. The results of this study suggest that co-administration of these two combinations, strengthens their anti-fibrotic properties and, due to the routine use of these compounds against AML and blood pressure, these compounds can be used with caution against human liver fibrosis.

Arginase and α-smooth muscle actin induction after hyperoxic exposure in a mouse model of bronchopulmonary dysplasia.

The L-arginine/NO pathway is an important regulator of pulmonary hypertension, the leading cause of mortality in patients with the chronic lung disease of prematurity, bronchopulmonary dysplasia. L-arginine can be metabolized by NO synthase (NOS) to form L-citrulline and NO, a potent vasodilator. Alternatively, L-arginine can be metabolized by arginase to form urea and L-ornithine, a precursor to collagen and proline formation important in vascular remodelling. In the current study, we hypothesized that C3H/HeN mice exposed to prolonged hyperoxia would have increased arginase expression and pulmonary vascular wall cell proliferation. C3H/HeN mice were exposed to 14days of 85% O2 or room air and lung homogenates analyzed by western blot for protein levels of arginase I, arginase II, endothelial NOS (eNOS), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT), and α-smooth muscle actin (α-SMA). Hyperoxia did not change arginase I or eNOS protein levels. However, arginase II protein levels were 15-fold greater after hyperoxia exposure than in lungs exposed to room air. Greater protein levels of ODC and OAT were found in lungs following hyperoxic exposure than in room air animals. α-SMA protein levels were found to be 7-fold greater in the hyperoxia exposed lungs than in room air lungs. In the hyperoxia exposed lungs there was evidence of greater pulmonary vascular wall cell proliferation by α-SMA immunohistochemistry than in room air lungs. Taken together, these data are consistent with a more proliferative vascular phenotype, and may explain the propensity of patients with bronchopulmonary dysplasia to develop pulmonary hypertension.

The 50/10 Oxygen-Induced Retinopathy Model Serves as a Hyperoxia and Hypoxia Model of Bronchopulmonary Dysplasia

BackgroundAnimal models of bronchopulmonary dysplasia (BPD) are mainly created by hyperoxia exposure. However, these models do not fully recapitulate BPD pathophysiology as observed in clinical practice. To find a better BPD model, we established a rat 50/10 oxygen-induced retinopathy (OIR) model and analyzed the pathologic features of the lungs. MethodsThe rat OIR model was established by exposing newborn rats (P0) to 50% and 10% oxygen (hyperoxia and hypoxia) on alternating days for 14 days. Lungs were harvested immediately on postnatal day 14 (P14) and on P18 after 4 days of normoxia exposure for hematoxylin and eosin staining, antialpha smooth muscle actin (α-SMA) immunohistochemistry and Picrosirius red staining of collagen. Retinas were obtained to confirm successful model establishment by isolectin B4 staining of retinal vasculature. ResultsOIR rats presented with fewer and enlarged alveoli, and the septal walls were thicker than those in age-matched controls. α-SMA immunohistochemistry indicated increased abundance of myofibroblasts in OIR rats. At P18, α-SMA-positive myofibroblasts were present at extremely low levels from the alveolar walls of control rats, while OIR rats showed myofibroblast persistence. The amount of collagen in OIR rats was also higher than that in control rats at both P14 and P18 as evidenced by Picrosirius red staining. ConclusionsAlveolar changes observed by hematoxylin and eosin staining, prolonged and stronger α-SMA expression and augmented collagen accumulation resemble the histopathology of BPD, suggesting that the rat 50/10 OIR model is suitable for use in BPD research.

Comparison of renal interstitial fibrosis induced by different animal models of urinary tract infection

Objectives: To identify a better animal model that more closely mimics early renal interstitial fibrosis induced by long-term recurrent urinary tract infection (UTI), as occurs in humans, three different murine models were compared. Methods: Three different murine models of upper UTI were established, including direct injection of bacteria into renal tissues, ascending urinary infection with partial unilateral ureteric obstruction, and repeated infusion of bacteria into the bladder. The histopathology of the kidneys was assessed by hematoxylin and eosin staining. Masson's trichrome staining and immunohistochemistry for α-smooth muscle actin (α-SMA) expression were used for the detection of fibrosis. Results: All three models developed different levels of inflammation in the kidney. However, in contrast to the severe renal interstitial fibrosis in the other two models, the model of repeated infusion of bacteria into the bladder demonstrated early renal interstitial fibrosis by Masson's trichrome staining and immunohistochemistry for α-SMA. Conclusions: The model of repeated infusion of bacteria into the bladder developed low levels of renal interstitial fibrosis, which resembles the early tissue damage in the kidney induced by recurrent UTI s in humans. This model may therefore offer a better way to study the early therapeutic intervention of renal interstitial fibrosis caused by inflammation.

Porphyromonas gingivalis oral infection promote T helper 17/Treg imbalance in the development of atherosclerosis

Background/purposeIncreasing studies have indicated the involvement of Porphyromonas gingivalis in atherosclerosis. T helper 17 (Th17)/Treg balance is critical during atherosclerosis. However, whether P. gingivalis oral infection is associated with Th17/Treg imbalance is unclear. The aim of the present study was to investigate the effect of P. gingivalis on Th17/Treg balance during atherosclerosis. Materials and methodsApoE–/– and C57BL/6 mice were inoculated orally with P. gingivalis ATCC 33277 for 9 weeks. The alveolar bone loss was assessed by microcomputerized tomography. The area of atherosclerosis plaque was identified by oil red O staining. Plaque stability was analyzed by CD68 and αSMA immunohistochemistry staining and Masson staining. The frequency of Th17 and Treg in spleen was detected by flow cytometry. The mRNA expression of Th17- and Treg-related factors was determined by quantitative polymerase chain reaction. Interleukin (IL)-6, a critical factor in modulating T-cell differentiation, was determined from spleen cells and mouse dendritic cells by enzyme-linked immunosorbent assay. ResultsLong-term P. gingivalis oral infection induced alveolar bone resorption. In ApoE–/– mice, P. gingivalis enhanced atherosclerotic lesion formation and plaque instability accompanied with a decreased Treg frequency and an increased Th17 cell frequency. In addition, mRNA expression of retinoic acid receptor-related orphan receptor γt and IL-17 was increased, and that of transforming growth factor (TGF) β and IL-10 was decreased in P. gingivalis-infected ApoE–/– mice. Furthermore, secretion of IL-6 was elevated in the spleen of P. gingivalis-infected ApoE–/– mice, as well as in mouse dendritic cells after P. gingivalis infection. ConclusionP. gingivalis oral infection may promote Th17/Treg imbalance by influencing T-cell differentiation during the process of atherosclerosis, with a larger lesion area and decreasing plaque instability.

Thyroid Hormone Receptor (TR): a regulator in Liver Fibrogenesis

Thyroid hormone (TH) signaling is critical for tissue-organ development, growth, differentiation and metabolism, and in the liver, the most widely expressed TH receptor is TRβ. In a recent study of patients with nonalcoholic steatohepatitis (NASH), progressive liver fibrosis was associated with disrupted TH signaling. In a separate study, low serum triiodothyronine (T3) was associated with advanced NASH-fibrosis. These findings suggest that TH signaling may be a novel regulator of adult liver fibrogenesis. Herein, we hypothesized that the TH-TR axis modulates HSC phenotype and perturbations in TH-TR axis occur during liver injury. Methods: In vivo: Two murine liver injury models were used (6 weeks of methionine choline deficient (MCD) diet and 6 weeks carbon tetrachloride (CCL4) injection). Human tissues were obtained from explanted NASH and healthy donor livers. Liver fibrosis was assessed by Sirius Red (SR) staining, αSMA immunohistochemistry, and the hepatic hydroxyproline assay. Total liver expression of TRα and TRβ, αSMA and Collagen 1α1 mRNA were determined by qRT-PCR. In vitro: To determine whether TH modulates HSC activation, primary HSC and a HSC line were treated with transforming growth factor (TGF)-β (5 ng/ml), in the presence or absence of T3 (10 ng/ml). TRα knockdown in HSC was achieved using lentiviral-mediated shRNA (shTRα). Results were confirmed using TRα knock-out (TRαKO) mouse embryonic fibroblasts (MEF). HSC migration was assessed using the wound healing assay, western blot and qRT-PCR (αSMA, collagen 1α1, TRα and β). Hepatocytes were used as positive control for TRβ expression. Results: Total liver TRα and TRβ mRNA were downregulated by 4-fold (p < 0.05) during liver fibrogenesis in mice and humans. In HSC, TRα is the predominant TR (4 fold higher than TRβ; p < 0.05). TGF-β stimulation of HSC repressed TRα and TRβ mRNA expression, but this effect was blunted by T3. shTRα-HSC exhibited greater activation at baseline, with increased fibrogenesis markers αSMA and collagen 1α1, and enhanced responses to TGF-β (increased p-Smad2/3). shTRα-HSC also had greater response in wound-healing assay (1.3-fold, p < 0.05), which was consistent with enriched contractility/migration pathways obtained from RNA sequencing analysis. Conclusions: Liver expression of TRα and β is repressed during fibrogenesis following chronic liver injury. TRα is the predominant TR in HSC and perturbations in TH-TR levels regulate HSC phenotype via the TGF-β pathway. Future studies will be needed to determine if whether TH treatment could inhibit liver fibrosis progression.

Abstract B15: Targeting NOX4 in the tumor microenvironment inhibits myofibroblast differentiation and suppresses tumor progression

Abstract Purpose: Smooth muscle actin (SMA)-positive myofibroblasts (cancer-associated fibroblasts), within the tumor microenvironment, promote many of the hallmarks of malignancy and correlate with poor prognosis in multiple cancer types. The purpose of this study was to investigate the mechanisms regulating myofibroblast differentiation in solid tumors in order to therapeutically target this cell population. Experimental procedures: Clinical Samples: Human tumor samples were analyzed by immunohistochemistry and RNA sequencing followed by bioinformatics analyses. In vitro: Myofibroblast differentiation was analyzed in Human fetal foreskin fibroblasts (HFFF2), adult primary fibroblasts and cancer associated fibroblasts (CAFs) by Western blotting for α-SMA; immunofluorescence for α-SMA stress fiber formation; collagen gel contraction assays and analysis of gene expression by RNA sequencing/PCR. Intracellular Reactive Oxygen Species (ROS) generation was measured using FACS analysis of DCFH-DA fluorescence. In vivo: To monitor tumor growth in vivo, a xenograft model of Head &amp; Neck squamous cell carcinoma (HNSCC; 5PT) co-injected with HFFF2s in B6.129S7-Rag1tm1Mom/J mice; and an isograft model (K2) in C57BL/6-Tg(TRAMP)8247Ng/J mice was used. Lymph node (LN) metastases were assayed in the 5PT model using the LI-COR IRDye® 800CW 2-DG Optical Probe. Myofibroblast accumulation was assessed by immunohistochemistry for α-SMA and collagen deposition by Masson's trichrome staining. Results: Using TGF-β1 treatment to model fibroblast-to-myofibroblast differentiation in vitro, we found that a delayed phase of intracellular ROS generation, beginning 24/48 hours post-treatment was required to induce differentiation in HFFF2s and adult primary fibroblasts. RNA-Sequencing of fibroblasts treated with TGF-β1 showed that NOX4 was the most significantly up-regulated ROS-producing enzyme. NOX4 was also found to be up-regulated in the stroma of multiple solid tumors, co-localized with α-SMA, and correlating with a myofibroblast gene signature. Genetic (si/shRNA) and pharmacological (GKT137831) inhibition of NOX4 abrogated multiple processes associated with myofibroblast differentiation, down-stream of TGF-β1: suppressing ROS generation; α-SMA expression; collagen gel contraction and expression of genes associated with ECM deposition (COL1A1, COL3A1, FN1 and CTGF). Furthermore, NOX4 inhibition reversed the myofibroblast phenotype in Cancer Associated Fibroblasts (CAFs) isolated from HNSCC and esophageal adenocarcinoma. Genetic and pharmacological inhibition of NOX4 in fibroblasts significantly slowed tumor growth and myofibroblast accumulation in the 5PT and K2, in vivo, model systems. Notably, LN metastases in the 5PT model were significantly reduced following shRNA mediated NOX4 knockdown in HFFF2s. Conclusions: NOX4 is a critical regulator of myofibroblast accumulation in multiple tumor types. Fibroblast-specific inhibition of this enzyme suppresses tumor growth and metastatic spread, suggesting that NOX4 is a promising therapeutic target for preventing myofibroblast differentiation within the tumor microenvironment. Citation Format: Christopher J. Hanley, Massimiliano Mellone, Gareth J. Thomas. Targeting NOX4 in the tumor microenvironment inhibits myofibroblast differentiation and suppresses tumor progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B15.