Reduced Collagen Accumulation Research Articles

FGF21 protects against hepatic lipotoxicity and macrophage activation to attenuate fibrogenesis in nonalcoholic steatohepatitis.

Analogues of the hepatokine fibroblast growth factor 21 (FGF21) are in clinical development for type 2 diabetes and nonalcoholic steatohepatitis (NASH) treatment. Although their glucose-lowering and insulin-sensitizing effects have been largely unraveled, the mechanisms by which they alleviate liver injury have only been scarcely addressed. Here, we aimed to unveil the mechanisms underlying the protective effects of FGF21 on NASH using APOE*3-Leiden.CETP mice, a well-established model for human-like metabolic diseases. Liver-specific FGF21 overexpression was achieved in mice, followed by administration of a high-fat high-cholesterol diet for 23 weeks. FGF21 prevented hepatic lipotoxicity, accompanied by activation of thermogenic tissues and attenuation of adipose tissue inflammation, improvement of hyperglycemia and hypertriglyceridemia, and upregulation of hepatic programs involved in fatty acid oxidation and cholesterol removal. Furthermore, FGF21 inhibited hepatic inflammation, as evidenced by reduced Kupffer cell (KC) activation, diminished monocyte infiltration, and lowered accumulation of monocyte-derived macrophages. Moreover, FGF21 decreased lipid- and scar-associated macrophages, which correlated with less hepatic fibrosis as demonstrated by reduced collagen accumulation. Collectively, hepatic FGF21 overexpression limits hepatic lipotoxicity, inflammation, and fibrogenesis. Mechanistically, FGF21 blocks hepatic lipid influx and accumulation through combined endocrine and autocrine signaling, respectively, which prevents KC activation and lowers the presence of lipid- and scar-associated macrophages to inhibit fibrogenesis.

Reactive Oxygen Species-Responsive Polypeptide Drug Delivery System Targeted Activated Hepatic Stellate Cells to Ameliorate Liver Fibrosis.

Hepatic fibrosis is a chronic liver disease that lacks effective pharmacotherapeutic treatments. As part of the disease's mechanism, hepatic stellate cells (HSCs) are activated by damage-related stimuli to secrete excessive extracellular matrix, leading to collagen deposition. Currently, the drug delivery system that targets HSCs in the treatment of liver fibrosis remains an urgent challenge due to the poor controllability of drug release. Since the level of reactive oxygen species (ROS) increases sharply in activated HSCs (aHSCs), we designed ROS-responsive micelles for the HSC-specific delivery of a traditional Chinese medicine, resveratrol (RES), for treatment of liver fibrosis. The micelles were prepared by the ROS-responsive amphiphilic block copolymer poly(l-methionine-block-Nε-trifluoro-acetyl-l-lysine) (PMK) and a PEG shell modified with a CRGD peptide insertion. The CRGD-targeted and ROS-responsive micelles (CRGD-PMK-MCs) could target aHSCs and control the release of RES under conditions of high intracellular ROS in aHSCs. The CRGD-PMK-MCs treatment specifically enhanced the targeted delivery of RES to aHSCs both in vitro and in vivo. In vitro experiments show that CRGD-PMK-MCs could significantly promote ROS consumption, reduce collagen accumulation, and avert activation of aHSCs. In vivo results demonstrate that CRGD-PMK-MCs could alleviate inflammatory infiltration, prevent fibrosis, and protect hepatocytes from damage in fibrotic mice. In conclusion, CRGD-PMK-MCs show great potential for targeted and ROS-responsive controlled drug release in the aHSCs of liver fibrosis.

Rosemary Extract-Induced Autophagy and Decrease in Accumulation of Collagen Type I in Osteogenesis Imperfecta Skin Fibroblasts.

Osteogenesis imperfecta (OI) is a heterogeneous connective tissue disease mainly caused by structural mutations in type I collagen. Mutant collagen accumulates intracellularly, causing cellular stress that has recently been shown to be phenotype-related. Therefore, the aim of the study was to search for potential drugs reducing collagen accumulation and improving OI fibroblast homeostasis. We found that rosemary extract (RE), which is of great interest to researchers due to its high therapeutic potential, at concentrations of 50 and 100 µg/mL significantly reduced the level of accumulated collagen in the fibroblasts of four patients with severe and lethal OI. The decrease in collagen accumulation was associated with RE-induced autophagy as was evidenced by an increase in the LC3-II/LC3-I ratio, a decrease in p62, and co-localization of type I collagen with LC3-II and LAMP2A by confocal microscopy. The unfolded protein response, activated in three of the four tested cells, and the level of pro-apoptotic markers (Bax, CHOP and cleaved caspase 3) were attenuated by RE. In addition, the role of RE-modulated proteasome in the degradation of unfolded procollagen chains was investigated. This study provides new insight into the beneficial effects of RE that may have some implications in OI therapy targeting cellular stress.

Antifibrotic pyridine-containing monoterpene alkaloids from Caryopteris glutinosa

Three undescribed dimeric pyridine-containing alkaloids, caryopterisines C – E, and four unreported cyclopenta[c]pyridine-derived alkaloids, caryopterisines F – I, were identified from Caryopteris glutinosa Rehder (Lamiaceae), together with two known monoterpene alkaloids. Caryopterisine C, featuring with an unprecedented 6/5/6/6/5 pentacyclic rings scaffold, may biosynthetically stem from a Diels-Alder reaction of two cyclopenta[c]pyridine-containing monomers and a following aromatization rearrangement reaction. Caryopterisines D and E, possessing an unprecedented 6/6/6/6/5 fused rings framework, may originate from a same Diels-Alder reaction of two monomers and subsequent aromatization arrangement, Baeyer-Villiger oxidation, and a set of tailoring reactions. Caryopterisine C showed strong inhibition on collagen accumulation in NIH3T3 cells (IC50 = 14.26 ± 1.46 μM). Caryopterisines G and I reduce collagen accumulation with IC50 values 88.91 ± 0.95 μM and 33.09 ± 1.38 μM, respectively. The Western blotting of the transforming growth factor-β-activated signaling pathways revealed that caryopterisine C inhibits collagen expression and accumulation via suppression of the phosphorylation of ERK1/2, P38, and SMAD2/3. The present works indicate caryopterisine C is a potential lead compound for the development of antifibrotic drugs.

Berberine inhibits glioma cell migration and invasion by suppressing TGF-β1/COL11A1 pathway

Glioma is a clinically heterogeneous disease with a poor prognosis. Berberine (BBR), as a multi-target anti-tumor alkaloid, has the ability to penetrate the blood-brain barrier and shows cytotoxicity to glioma cells. In previous studies, we demonstrated that berberine inhibits glioma cell proliferation by inhibiting mutant p53 protein and promoting mitochondrial damage. In addition, berberine has been shown to reduce collagen accumulation in pulmonary fibrosis, diabetic nephropathy and arthritis. However, its effect on collagen in cancer needs to be further elucidated. In this study, we proved that the collagen XI alpha 1 chain (COL11A1) is highly expressed in glioma cell lines and associated with migration and invasion of glioma cells. Knocking down COL11A1 caused decreased expression of MMPs. Berberine could inhibit the migration and invasion of glioma cells by suppressing the TGF-β1/COL11A1 pathway and changes actin cytoskeleton arrangement. Nude mouse subcutaneous xenografts model also showed that berberine inhibited the expression of COL11A1 in vivo. Collectively, berberine that targets COL11A1 to inhibit glioma migration and invasion, may serve as a promising candidate for the development of anti-glioma drugs in the future.

Comprehensive analysis of transcriptomics and metabolomics to illustrate the underlying mechanism of helenalin against hepatic fibrosis

This study aimed to investigate the protective mechanisms of helenalin on hepatic fibrosis. In brief, rats were intragastrically administrated with 50% CCl4 for 9 weeks to induce liver fibrosis, followed by treatment with various agents for 6 weeks. The effects of helenalin on hepatic injury were assessed by pathological examinations. The potential targets were predicted by the “Drug-Disease” bioinformatic analysis and then verified by multiple experiments. Moreover, the underlying mechanism was investigated by transcriptomics and metabolomics as a whole. The results showed that helenalin significantly alleviated hepatocyte necrosis and hepatic injury, as proved by the pathological examinations. Also, helenalin markedly attenuated hepatocyte apoptosis by regulating the expression of caspase-3 and Bcl-2 families. Besides, helenalin could significantly reduce collagen accumulation, as evidenced by the decreased contents of collagen, hyaluronic acid and laminin. Moreover, helenalin significantly down-regulated the phosphorylation of PI3K, Akt, FAK, mTOR and P70S6K, and PTEN protein expression, suggesting that helenalin inhibited the PI3K/Akt signaling cascade. Meanwhile, helenalin inhibited the NF-κB signaling pathway by reducing the phosphorylation of IκBα, NF-κB p65 and IKKα/β, alleviating inflammation response. Interestingly, the analysis of transcriptomics and metabolomics indicated that helenalin inhibited the glycerophospholipid metabolism pathway by down-regulating the target genes (CHKA, ETNPPL, LYPLA1, PCYT2, PLD4 and PNPLA6), ultimately ameliorating hepatocyte damage. In conclusion, helenalin ameliorates hepatic fibrosis by regulating the PI3K/Akt and NF-κB signaling pathways and the glycerophospholipid metabolism pathway.

Sıçanlarda Karaciğer Fibrozu Üzerine 5-Florourasil Tedavisinin Etkinliği: Deneysel Çalışma

Objective: Liver fibrosis is a common pathological condition that can lead to liver failure. Previous studies demonstrated that 5-fluorouracil (5-FU) is an effective agent in preventing fibrosis. This experimental study aimed to evaluate the efficacy of 5-FU therapy on liver fibrosis in rats. Material and Methods: Thirty-eight rats were divided into 3 groups: Group 1 (sham-operated, n=10), the bile duct (BD) was exposed but not ligated; Group 2 [bile duct ligation (BDL)-saline, n=14], the BD was ligated and normal saline solution was given; Group 3 (BDL-5-FU, n=14), the BD was ligated and treated with 5-FU. The liver and blood samples were harvested at postoperative day 21. Liver function analyses, tissue hydroxyproline (HP) content, and serum and tissue transforming growth factor beta 1 (TGF-β1) levels were determined. The liver sections were analyzed and percentage of tissue collagen content was measured. Results: There were no significant differences between the groups in terms of tissue (p=0.748) and serum (p=0.123) levels of TGF-β1. Mean HP level in the sham-operated, BDL-saline, and BDL-5-FU groups were 253±42 ng/L, 360±32 ng/L and 305±41 ng/L, respectively. 5-FU significantly decreased HP level (p=0.036). Mean percentage of collagen content in the sham-operated, BDL-saline, and BDL-5-FU groups were 0.99±0.39%, 4.92±1.03%, and 2.62±0.64%, respectively. 5-FU significantly reduced collagen accumulation (p<0.001). Conclusion: 5-FU has a preventive effect on liver fibrosis in rats. This result suggests that 5-FU can be a potential agent for prevention of liver fibrosis in clinical practice.

Therapeutic Effect of Biejiaxiaozheng Pills on Carbon Tetrachloride-Induced Hepatic Fibrosis in Rats through the NF-κB/Nrf2 Pathway.

Aims To explore the effects of Biejiaxiaozheng pills on carbon tetrachloride-induced hepatic fibrosis in rats through the NF-κB/Nrf2 pathway and to explore the possible antifibrotic mechanisms of the drug. Material and Method. A rat model of hepatic fibrosis was established via CCl4 induction. Liver function and antioxidant indices were detected using commercial kits. Hematoxylin-eosin and Masson staining were used to detect pathological changes in hepatic tissues. ELISA was used to measure plasma TNF-α, IL-β, and IL-6 levels. RT-PCR was used to measure changes in TNF-α, IL-β, and IL-6 levels in hepatic tissues. Changes in p65, P-p65, Nrf2, and HO-1 protein expression were detected using western blotting. Results In rats with hepatic fibrosis, Biejiaxiaozheng pills effectively improved liver function, alleviated fibrosis in hepatic tissues, and significantly reduced collagen accumulation. The pills significantly downregulated inflammatory cytokine expression in hepatic tissues by suppressing p65 phosphorylation and reduced plasma inflammatory cytokine levels to some extent. The pills upregulated Nrf2 and HO-1 expression in hepatic tissues, enhanced antioxidant potential, and upregulated plasma antioxidant levels. Conclusion Biejiaxiaozheng pills improved hepatic fibrosis symptoms and lesions in rats, likely by inhibiting the NF-κB pathway and promoting the Nrf2 pathway.

MiR-150-5p affects AS plaque with ASMC proliferation and migration by STAT1.

We explore miR‐150‐5p in atherosclerosis (AS). The AS model was constructed using Apo E−/− mice with an injection of the miR-150-5p mimic or an inhibitor. Pathological characteristics were assessed using Oil red O staining and Masson staining. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot were used to analyze the expressions of microRNA-150-5p (miR-150-5p), STAT1, α-SMA (α-smooth muscle actin) and proliferating cell nuclear antigen (PCNA). Targetscan and dual-luciferase reporter assay were used to analyze the interaction between miR-150-5p and STAT1. The viability, migration, cell cycle and α-SMA and PCNA expressions in oxidized low-density lipoprotein (ox-LDL)-stimulated primary human aortic smooth muscle cells (ASMCs) were assessed using molecular experiments. miR-150-5p was reduced in both AS mice and ox-LDL-stimulated human aortic smooth muscle cells but STAT1 had the opposite effect. The miR‐150‐5p inhibitor alleviated the increase of lipid plaque and reduced collagen accumulation in the aortas during AS. Upregulation of α-SMA and PCNA was reversed by miR-150-5p upregulation. STAT1 was targeted by miR‐150‐5p, and overexpressed miR-150-5p weakened the ox-LDL-induced increase of viability and migration abilities and blocked cell cycle in ASMCs, but overexpressed STAT1 blocked the effect of the miR‐150‐5p mimic. This paper demonstrates that miR-150-5p has potential as a therapeutic target in AS, with plaque stabilization by regulating ASMC proliferation and migration via STAT1.

Metformin and leucine increase satellite cells and collagen remodeling during disuse and recovery in aged muscle.

Loss of muscle mass and strength after disuse followed by impaired muscle recovery commonly occurs with aging. Metformin (MET) and leucine (LEU) individually have shown positive effects in skeletal muscle during atrophy conditions but have not been evaluated in combination nor tested as a remedy to enhance muscle recovery following disuse atrophy in aging. The purpose of this study was to determine if a dual treatment of metformin and leucine (MET + LEU) would prevent disuse-induced atrophy and/or promote muscle recovery in aged mice and if these muscle responses correspond to changes in satellite cells and collagen remodeling. Aged mice (22-24 months) underwent 14days of hindlimb unloading (HU) followed by 7 or 14days of reloading (7 or 14days RL). MET, LEU, or MET + LEU was administered via drinking water and were compared to Vehicle (standard drinking water) and ambulatory baseline. We observed that during HU, MET + LEU resolved whole body grip strength and soleus muscle specific force decrements caused by HU. Gastrocnemius satellite cell abundance was increased with MET + LEU treatment but did not alter muscle size during disuse or recovery conditions. Moreover, MET + LEU treatment alleviated gastrocnemius collagen accumulation caused by HU and increased collagen turnover during 7 and 14days RL driven by a decrease in collagen IV content. Transcriptional pathway analysis revealed that MET + LEU altered muscle hallmark pathways related to inflammation and myogenesis during HU. Together, the dual treatment of MET and LEU was able to increase muscle function, satellite cell content, and reduce collagen accumulation, thus improving muscle quality during disuse and recovery in aging.

Eugenol modulates insulin sensitivity by upregulating insulin receptor substrate-2 in non-alcoholic fatty liver disease in rats.

The purpose of this study was to estimate the possible modulatory effect of Eugenol (EUG) on insulin resistance (IR) and liver fibrosis in high-fat diet (HFD)-induced experimental non-alcoholic fatty liver disease (NAFLD) in rats. It has been shown that EUG, a natural phenolic compound, has anti-hyperglycaemic, antioxidant and anti-inflammatory actions. For 8 consecutive weeks, standard rat chow diet (control group, EUG only treated group) or HFD (HFD group and HFD+EUG-treated group) were fed to rats daily. HFD+EUG-treated group and EUG only treated group were administered EUG (10 mg/kg) orally three times per week. Various indices of hepatotoxicity, oxidative stress, indicators of inflammation and liver fibrosis were investigated. HFD-induced liver transaminases and triglycerides (TGs) were significantly decreased and histopathological lesions were improved with EUG treatment. EUG significantly improved IR evoked by HFD, as demonstrated by Homeostasis model assessment for insulin resistance (HOMA-IR) and increased insulin receptor substrate-2 (IRS-2) sensitivity. In addition, EUG improved oxidative stress damage elicited by HFD as shown by the restoration of reduced glutathione (GSH) level and nuclear factor erythroid-2-related factor 2 (Nrf-2) expression and plummeting lipid peroxidation. Further, EUG lessened pro-inflammatory cytokines surge [tumour necrosis factor-α (TNF-α) and IL-6] via inhibiting nuclear factor-κB (NF-κB) stimulation. As markers of fibrosis, EUG reduced collagen accumulation and smooth muscle alpha actin (SMaA) and TGF-β expression. EUG may have protective effect against progression of fibrosis in NAFLD. The antifibrotic effect of EUG is probably due to EUG's antioxidant, anti-inflammatory and anti-hyperglycaemic.

Adipocyte Fatty Acid Binding Protein Promotes the Onset and Progression of Liver Fibrosis via Mediating the Crosstalk between Liver Sinusoidal Endothelial Cells and Hepatic Stellate Cells.

Development of liver fibrosis results in drastic changes in the liver microenvironment, which in turn accelerates disease progression. Although the pathological function of various hepatic cells in fibrogenesis is identified, the crosstalk between them remains obscure. The present study demonstrates that hepatic expression of adipocyte fatty acid binding protein (A‐FABP) is induced especially in the liver sinusoidal endothelial cells (LSECs) in mice after bile duct ligation (BDL). Genetic ablation and pharmacological inhibition of A‐FABP attenuate BDL‐ or carbon tetrachloride‐induced liver fibrosis in mice associating with reduced collagen accumulation, LSEC capillarization, and hepatic stellate cell (HSC) activation. Mechanistically, elevated A‐FABP promotes LSEC capillarization by activating Hedgehog signaling, thus impairs the gatekeeper function of LSEC on HSC activation. LSEC‐derived A‐FABP also acts on HSCs in paracrine manner to potentiate the transactivation of transforming growth factor β1 (TGFβ1) by activating c‐Jun N‐terminal kinase (JNK)/c‐Jun signaling. Elevated TGFβ1 subsequently exaggerates liver fibrosis. These findings uncover a novel pathological mechanism of liver fibrosis in which LSEC‐derived A‐FABP is a key regulator modulating the onset and progression of the disease. Targeting A‐FABP may represent a potential approach against liver fibrosis.

Prediction and verification of target of helenalin against hepatic stellate cell activation based on miR-200a-mediated PI3K/Akt and NF-κB pathways

Hepatic stellate cell (HSC) activation is a crucial event in the progress of liver fibrosis. In this study, the target of helenalin was firstly predicted by bioinformatics analysis, and then the prediction was verified by various experiments. HSC‐T6 cells were activated by interleukin-1 beta (IL-1β) and then treated with helenalin. Moreover, HSC‐T6 cells were transfected with miR-200a mimic or inhibitor, and the effect of helenalin on the miR-200a-mediated PI3K/Akt and NF-κB signaling pathways was investigated. The bioinformatics analysis indicated that miR-200a might regulate the PI3K/Akt pathway, NF-κB activation, Bcl-2 family and Caspases, ultimately affecting cell survival and apoptosis. Interestingly, the molecular docking demonstrated that the target of helenalin might be miR-200a-mediated the PI3K/Akt and NF-κB pathways. Moreover, the experiments showed that helenalin administration led to the inactivation of HSC-T6 cells, as evidenced by the inhibition of cell proliferation, α-SMA expression and collagen production. The mechanism studies showed that helenalin reduced collagen accumulation by restoring the balance of MMPs/TIMPs. Moreover, helenalin markedly suppressed HSC activation by inhibiting the PI3K/Akt pathway and alleviated inflammatory response by blocking the NF-κB signal transduction. Further study indicated that helenalin up-regulated miR-200a expression, thus leading to the inhibition of the PI3K/Akt and NF-κB signaling pathways. In conclusion, helenalin inhibits HSC activation via inhibiting the miR-200a-mediated PI3K/Akt and NF-κB pathways, and it may be developed as a potential medicine for the treatment of liver fibrosis.

Validation of highly selective sphingosine kinase 2 inhibitors SLM6031434 and HWG-35D as effective anti-fibrotic treatment options in a mouse model of tubulointerstitial fibrosis

Renal fibrosis is characterized by chronic inflammation and excessive accumulation of extracellular matrix and progressively leads to functional insufficiency and even total loss of kidney function. In this study we investigated the anti-fibrotic potential of two highly selective and potent SK2 inhibitors, SLM6031434 and HWG-35D, in unilateral ureter obstruction (UUO), a model for progressive renal fibrosis, in mice. In both cases, treatment with SLM6031434 or HWG-35D resulted in an attenuated fibrotic response to UUO in comparison to vehicle-treated mice as demonstrated by reduced collagen accumulation and a decreased expression of collagen-1 (Col1), fibronectin-1 (FN-1), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA). Similar to our previous study in Sphk2−/− mice, we found an increased protein expression of Smad7, a negative regulator of the pro-fibrotic TGFβ/Smad signalling cascade, accompanied by a strong accumulation of sphingosine in SK2 inhibitor-treated kidneys. Treatment of primary renal fibroblasts with SLM6031434 or HWG-35D dose-dependently increased Smad7 expression and ameliorated the expression of Col1, FN-1 and CTGF.In summary, these data prove the anti-fibrotic potential of SK2 inhibition in a mouse model of renal fibrosis, thereby validating SK2 as pharmacological target for the treatment of fibrosis in chronic kidney disease.

Antler stem cells as a novel stem cell source for reducing liver fibrosis

Liver fibrosis results from collagen fiber deposition. Antler stem cells (ASCs) naturally in vivo differentiate into cartilage, which is only made of Col II in collagen component; whereas liver fibrosis is caused by over-abundance of Col I and III. In addition, ASCs can effectively promote regenerative wound healing in which tissue contains very few collagen fibers (Col I). In this study, we investigate the therapeutic effects of ASCs in a rat model of CCl4-induced liver fibrosis. Rats were treated with ASCs for 4 weeks in vivo, then biochemical and histopathological analyses were performed. Furthermore, we established cell co-culture systems of hepatic stellate cells (HSCs) and ASCs and of M1 macrophages and ASCs in vitro. Mesenchymal stem cells (MSCs) were used as a positive control. The results showed that ASC transplantation alleviated liver fibrosis effectively as evidenced by reduced collagen accumulation, decreased fatty degeneration, increased hepatocyte regeneration, decreased inflammation and significantly enhanced liver function; moreover, ASCs decreased the expression of pro-fibrogenic factors including TGF-β and α-SMA. Additionally, our study showed that ASCs inhibit HSC activation and proliferation by controlling the expression of MMPs, TIMP1, TGF-β, α-SMA and COL1A2 involved in these processes. Our results suggested that ASCs alleviate liver fibrosis effectively and inhibit HSC activation. Thus, ASCs may serve as a novel stem cell source for the treatment of liver fibrosis in the clinic.

CCN1 expression by fibroblasts is required for bleomycin-induced skin fibrosis

The microenvironment contributes to the excessive connective tissue deposition that characterizes fibrosis. Members of the CCN family of matricellular proteins are secreted by fibroblasts into the fibrotic microenvironment; however, the role of endogenous CCN1 in skin fibrosis is unknown. Mice harboring a fibroblast-specific deletion for CCN1 were used to assess if CCN1 contributes to dermal homeostasis, wound healing, and skin fibrosis. Mice with a fibroblast-specific CCN1 deletion showed progressive skin thinning and reduced accumulation of type I collagen; however, the overall mechanical property of skin (Young's modulus) was not significantly reduced. Real time-polymerase chain reaction analysis revealed that CCN1-deficient skin displayed reduced expression of mRNAs encoding enzymes that promote collagen stability (including prolyl-4-hydroxylase and PLOD2), although expression of COL1A1 mRNA was unaltered. CCN1-deficent skin showed reduced hydroxyproline levels. Electron microscopy revealed that collagen fibers were disorganized in CCN1-deficient skin. CCN1-deficient mice were resistant to bleomycin-induced skin fibrosis, as visualized by reduced collagen accumulation and skin thickness suggesting that deposition/accumulation of collagen is impaired in the absence of CCN1. Conversely, CCN1-deficient mice showed unaltered wound closure kinetics, suggesting de novo collagen production in response to injury did not require CCN1. In response to either wounding or bleomycin, induction of α-smooth muscle actin-positive myofibroblasts was unaffected by loss of CCN1. CCN1 protein was overexpressed by dermal fibroblasts isolated from lesional (i.e., fibrotic) areas of patients with early onset diffuse scleroderma. Thus, CCN1 expression by fibroblasts, being essential for skin fibrosis, is a viable anti-fibrotic target.

Abstract 98: Stromal-targeting with quercetin in patient-derived models of head and neck squamous cell carcinoma (HNSCC)

Abstract Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a high recurrence rate. Recurrences occur in ~25% cases of early -stage patients (Stage I-II) and 50-60% cases of advanced stage (Stage II-IV) patients. Cumulative evidences suggest that tumor stroma, especially cancer-associated fibroblast (CAF) plays a protective role and contributes to drug-resistance in HNSCC, leading to recurrences after treatment. Quercetin is a flavonoid purified from natural plants, such as green tea, onions and berries. It has been reported to suppress CAFs and reduce collagen accumulation surrounding tumors, potentially facilitating drug delivery to tumors. Here, we hypothesize that quercetin targeting of CAFs may enhance the anti-tumor efficacy of cisplatin, and potentially other antitumor therapies in HNSCC. Using a primary co-culture system with primary HNSCC cells (70%) and the paired CAFs (30%) from HNSCC patients, we investigated the effect of quercetin and cisplatin combination treatment in vitro. We found that combination of quercetin and cisplatin elicited a significantly greater growth inhibition (70% of inhibition) as compared to 37% of inhibition with cisplatin and 15% of inhibition with quercetin alone. This result appeared to demonstrate some anti-CAF effects and antitumor effects exerted by this combination strategy.Using patient-derived HNSCC primary tumor cells, stromal-rich xenografts were successfully developed in vivo. The stromal-suppressive effect of quercetin was then examined upon treatment with increasing doses of quercetin (DMF, 2.5, 5 or 10 μM in 5 microliter volume, 2 times injection). Potential alteration of the tumor architecture by quercetin (vs. vehicle treatment) was examined by H&amp;E staining. A marked reduction of stromal component was found in quercetin -treated tumors vs. vehicle-treated tumors. Importantly, we also found that these quercetin-treated tumors have 80.4% reduction in α-SMA protein expression (6.0% in vehicle vs.1.2% in treatment group), strongly indicating a significant stromal suppressive action of quercetin in an HNSCC primary culture-derived xenograft model. Furthermore, quercetin (10 μM) treatment reduced collagen expression by ~80% (vs. vehicle treatment) in the HNSCC primary culture-derived xenograft model by Masson’s trichrome staining. In conclusion, quercetin can effectively suppress the activated status of CAFs and may potentially enhance anti-tumor effect of cisplatin in an HNSCC patient-relevant manner. The antitumor efficacy of quercetin combination strategies should warrant further investigations. Citation Format: Hui Li, Wenying Piao, Pik Yuk Lau, Chin Wang Lau, Jason Ying Kuen Chan, Yu Xiong Su, Leaf Huang, Kai Li Hu, Vivian Wai Yan Lui. Stromal-targeting with quercetin in patient-derived models of head and neck squamous cell carcinoma (HNSCC) [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 98.

CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis.

Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha gene (PPARGC1A, encoding PGC1α). Both TGFβ and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGFβ- and matrix stiffness-promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation, and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.

Nicotinamide riboside, an NAD+ precursor, attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis

ObjectiveLiver fibrosis is part of the non-alcoholic fatty liver disease (NAFLD) spectrum, which currently has no approved pharmacological treatment. In this study, we investigated whether supplementation of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor, can reduce the development of liver fibrosis in a diet-induced mouse model of liver fibrosis. MethodsMale C57BL/6 J mice were fed a low-fat control (LF), a high-fat/high-sucrose/high-cholesterol control (HF) or a HF diet supplemented with NR at 400 mg/kg/day (HF-NR) for 20 weeks. Features of liver fibrosis were assessed by histological and biochemical analyses. Whole-body energy metabolism was also assessed using indirect calorimetry. Primary mouse and human hepatic stellate cells were used to determine the anti-fibrogenic effects of NR in vitro. ResultsNR supplementation significantly reduced body weight of mice only 7 weeks after mice were on the supplementation, but did not attenuate serum alanine aminotransferase levels, liver steatosis, or liver inflammation. However, NR markedly reduced collagen accumulation in the liver. RNA-Seq analysis suggested that the expression of genes involved in NAD+ metabolism is altered in activated hepatic stellate cells (HSCs) compared to quiescent HSCs. NR inhibited the activation of HSCs in primary mouse and human HSCs. Indirect calorimetry showed that NR increased energy expenditure, likely by upregulation of β-oxidation in skeletal muscle and brown adipose tissue. ConclusionNR attenuated HSC activation, leading to reduced liver fibrosis in a diet-induced mouse model of liver fibrosis. The data suggest that NR may be developed as a potential preventative for human liver fibrosis.

Nitric Oxide Synthase 2 Induction Promotes Right Ventricular Fibrosis.

The ability of the right ventricle to compensate pressure overload determines survival in pulmonary arterial hypertension (PAH). Nitric oxide (NO) reduces the right ventricular afterload through pulmonary vasodilation, but excessive NO amounts cause oxidative stress. Oxidative stress drives remodeling of pulmonary arteries and the right ventricle. In the present study, we hypothesized that nitric oxide synthase 2 (NOS2) induction leads to excessive NO amounts that contribute to oxidative stress and impair right ventricular adaptation to PAH. We used a surgical pulmonary artery banding (PAB) mouse model in which right ventricular dysfunction and remodeling occur independently of changes in the pulmonary vasculature. Three weeks after PAB, NOS2 expression was increased twofold in the hypertrophied right ventricle on transcript and protein levels together with increased NO production. Histomorphology localized NOS2 in interstitial and perivascular cardiac fibroblasts after PAB, which was confirmed by cell isolation experiments. In the hypertrophied right ventricle, NOS2 induction was accompanied by an increased formation of reactive oxidants blocked by ex vivo NOS inhibition. We show that reactive oxidant formation in the hypertrophied right ventricle is in part NOS2 dependent (in NOS2-deficient mice [NOS2-/-]). Lack of NOS2 induction prevented superoxide scavenging and decreased reactive oxidant formation. Functional measures of cardiac function by noninvasive echocardiography together with intracardiac catheterization revealed no differences in heart function between both genotypes after PAB. However, reduced NO and reactive oxidant formation in the hypertrophied right ventricle of NOS2-/- mice was linked to reduced collagen accumulation through reduced collagen deposition from the cardiac fibroblast. Together, our data demonstrate a profibrotic role for NOS2 induction in the hypertrophied right ventricle.