Hydrostatic pressure mediates epithelial-mesenchymal transition of cholangiocytes through RhoA/ROCK and TGF-ß/smad pathways.

Biomechanical cue within the tissue microenvironment is known to play a critical role in regulating cell behaviors and maintaining tissue homeostasis. As hydrostatic pressure often increases in biliary system under pathological states, we investigated the effect of the moderate elevation of the hydrostatic pressure on biliary epithelial cells, especially on the epithelial-mesenchymal transition (EMT). Human intrahepatic biliary epithelial cells were loaded to hydrostatic pressure using a commercial device. We found that loading the cells to 50 mmHg hydrostatic pressure induced obvious morphological changes and significantly upregulated vimentin, ZEB1, and pSmad2/3, fibronectin, and collagen 1α. All changes induced by hydrostatic pressure loading were effectively mitigated by either ROCK inhibitor (Y-27632) or ALK5 inhibitor (SB-431542). Our in vitro experimental data suggests that hydrostatic pressure loading induces EMT of cholangiocytes through RhoA/ROCK and TGF-ß/Smad pathways. Elevated hydrostatic pressure in biliary duct system under pathological states may promote the biliary epithelial cells shifting to profibrotic and mesenchymal characteristics.

Chronic replication stress invokes mitochondria dysfunction via impaired parkin activity.

Replication stress is a major contributor to tumorigenesis because it provides a source of chromosomal rearrangements via recombination events. PARK2, which encodes parkin, a regulator of mitochondrial homeostasis, is located on one of the common fragile sites that are prone to rearrangement by replication stress, indicating that replication stress may potentially impact mitochondrial homeostasis. Here, we show that chronic low-dose replication stress causes a fixed reduction in parkin expression, which is associated with mitochondrial dysfunction, indicated by an increase in mtROS. Consistent with the major role of parkin in mitophagy, reduction in parkin protein expression was associated with a slight decrease in mitophagy and changes in mitochondrial morphology. In contrast, cells expressing ectopic PARK2 gene does not show mtROS increases and changes in mitochondrial morphology even after exposure to chronic replication stress, suggesting that intrinsic fragility at PARK2 loci associated with parkin reduction is responsible for mitochondrial dysfunction caused by chronic replication stress. As endogenous replication stress and mitochondrial dysfunction are both involved in multiple pathophysiology, our data support the therapeutic development of recovery of parkin expression in human healthcare.

Nicaraven attenuates the acquired radioresistance of established tumors in mouse models via PARP inhibition.

Nicaraven has been reported to inhibit the activity of poly (ADP-ribose) polymerase (PARP). In this study, we investigated the probable ability of nicaraven to attenuate cancer radioresistance during fractionated radiotherapy. Tumor models were established in C57BL/6 mice and BALB/c nude mice by subcutaneous injection of Lewis mouse lung carcinoma cancer cells and A549 human lung cancer cells, respectively. When the tumors had grown to approximately 100 mm3, we initiated fractionated radiotherapy. Nicaraven or saline was administered immediately after each irradiation exposure. Compared to saline treatment, nicaraven administration significantly induced gamma-H2AX foci formation and cell apoptosis in tumors at 1 or 3 days after an additional challenge exposure to 10 Gy and inhibited tumor growth during the short-term follow-up period, suggesting increased radiosensitivity of cancer cells. Moreover, the expression of PARP in tumor tissue was decreased by nicaraven administration. Our data suggest that nicaraven likely attenuates the acquired radioresistance of cancers through PARP inhibition.

The angiogenesis-modulating effects of coumarin-derivatives.

Coumarin is a natural compound that is rich in plants. Coumarin and its derivates were reported to have many biological activities, such as anti-bacterial, anti-tumor, and anti-coagulation. In this study, we examined the angiogenic modulating activities of six previously synthesized coumarin derivatives (Compound #1-#6) in zebrafish embryos and further confirmed them in a chick model. According to the survival rate in a zebrafish model, Compound #1 (100 %), #2 (82.5-100 %), and #4 (100 %) showed much less toxicity than Compound #3 (19.2-100 %), #5 (0-100 %), and #6 (0-100 %). Using a green blood vessel fluorescent transgenic fish Tg(fli1:egfp) to record the angiogenesis-modulating effects of Compound #1, #2, and #4, we found that Compound #2 had the highest effects in interfering intersegmental vessel growth, subintestinal vein growth, and caudal vein plexus remodeling. Chick chorioallantoic membrane (CAM) assay also showed that Compound #2 exposure led to a reduction of blood vessel growth. Real-time PCR experiments revealed that Compound #2 significantly changed the expression of vascular growth-related genes flt1, cdh5, and nrp1a in zebrafish. Based on our data from zebrafish and chick models, a new coumarin-derivative (Compound #2) possesses anti-angiogenic activity with low toxicity, but further investigation in mammal models is asked to confirm our findings.

Comparison of hypoxia- and hyperoxia-induced alteration of epigene expression pattern in lungs of Pleurodeles waltl and Mus musculus.

Epigenetics is an emerging field of research because of its involvement in susceptibility to diseases and aging. Hypoxia and hyperoxia are known to be involved widely in various pathophysiologies. Here, we compared the differential epigene expression pattern between Pleurodeles waltl and Mus musculus (commonly known as Iberian ribbed newt and mouse, respectively) exposed to hypoxia and hyperoxia. Adult healthy newts and mice were exposed to normobaric hypoxia (8% O2) and hyperoxia (80% O2) for 2 hours. We collected the lungs and analyzed the expression of hypoxia-inducible factor 1 alpha (Hif1α) and several key epigenes from DNA methyltransferase (DNMT) family, histone deacetylase (HDAC) family, and methyl-CpG binding domain (MBD) family. The exposure to hypoxia significantly increased the mRNA levels of DNA methyltransferase 3 alpha (Dnmt3α), methyl-CpG binding domain protein 2 (Mbd2), Mbd3, and histone deacetylase 2 (Hdac2) in lungs of newts, but decreased the mRNA levels of DNA methyltransferase 1 (Dnmt1) and Dnmt3α in lungs of mice. The exposure to hyperoxia did not significantly change the expression of any gene in either newts or mice. The differential epigene expression pattern in response to hypoxia between newts and mice may provide novel insights into the prevention and treatment of disorders developed due to hypoxia exposure.

Losartan alleviates renal fibrosis by inhibiting the biomechanical stress-induced epithelial-mesenchymal transition of renal epithelial cells.

Angiotensin receptor blockers (ARBs) have been reported to be beneficial of renal fibrosis, but the molecular and cellular mechanisms are still unclear. In this study, we investigated the effectiveness and relevant mechanism of ARBs in alleviating renal fibrosis, especially by focusing on biomechanical stress-induced epithelial to mesenchymal transition (EMT) of renal epithelial cells. Unilateral ureteral obstruction (UUO) renal fibrosis model was established in mice by ligating the left ureter, and then randomly received losartan at a low dose (1 mg/kg) or a regular dose (3 mg/kg) for 2 weeks. Compared to the control, histological analysis showed that losartan treatment at either a low dose or a regular dose effectively attenuated renal fibrosis in the UUO model. To further understand the mechanism, we ex vivo loaded primary human renal epithelial cells to 50 mmHg hydrostatic pressure. Western blot and immunostaining analyses indicated that the loading to 50 mmHg hydrostatic pressure for 24 h significantly upregulated vimentin, ß-catenin and α-SMA, but downregulated E-cadherin in renal epithelial cells, suggesting the EMT. The addition of 10 or 100 nM losartan in medium effectively attenuated the EMT of renal epithelial cells induced by 50 mmHg hydrostatic pressure loading. Our in vivo and ex vivo experimental data suggest that losartan treatment, even at a low dose can effectively alleviate renal fibrosis in mouse UUO model, at least partly by inhibiting the biomechanical stress-induced EMT of renal epithelial cells. A low dose of ARBs may repurpose for renal fibrosis treatment.

Nicaraven Exerts a Limited Effect on Radiation-Induced Inhibition of Tumor Growth in a Subcutaneous Murine Tumor Model.

Nicaraven selectively protects normal tissue from radiation-induced injury. To further develop the clinical application of nicaraven for mitigating the side effects of cancer radiotherapy, we investigated the potential effect of nicaraven administration in radiation-induced inhibition of tumor growth. A subcutaneous tumor model was established in mice by the injection of Lewis lung cancer cells at the back of the chest. X-ray radiation was delivered to the thoracic area and different doses of nicaraven (0, 20, 50, 100 mg/kg) were administrated intraperitoneally pre- or post-irradiation. The tumor size was measured every other day. Mice were euthanized on day 30, and the tumor weight and the levels of cytokines in tumor tissue were measured. Pre- or post-irradiation administration of nicaraven up to a dose of 100 mg/kg did not significantly diminish the radiation-induced inhibition of tumor growth, but post-irradiation administration of 20 and 50 mg/kg nicaraven resulted in relatively lower tumor weight. The levels of IL-1ß, IL-6, IL-10, MCP-1, MIP-2a, TGF-ß1, VEGF, p53, p21, cyclin D1 and caspase-3 in tumor tissue did not change by nicaraven administration and were not significantly associated with the tumor weights. According to our experimental data, nicaraven will not significantly diminish the radiation-induced inhibition of tumor growth, even with pre-irradiation administration at a high dose.

Hyperoxia but not high tidal volume contributes to ventilator-induced lung injury in healthy mice.

BACKGROUND: Mechanical ventilation is a supportive therapy used to maintain respiratory function in several clinical and surgical cases but is always accompanied by lung injury risk due to improper treatment. We investigated how tidal volume and oxygen delivery would contribute independently or synergistically to ventilator-induced lung injury (VILI). METHODS: Under general anesthesia and tracheal intubation, healthy female C57BL/6 N mice (9 weeks old) were randomly ventilated for 2 h by standard (7 ml/kg) or high (14 ml/kg) tidal volume at positive end-expiratory pressure (PEEP) of 2 cmH2O, with room air, 50% O2 (moderate hyperoxia), or 100% O2 (severe hyperoxia); respectively. Mice were sacrificed 4 h after mechanical ventilation, and lung tissues were collected for experimental assessments on lung injury. RESULTS: Compared with the healthy control, severe hyperoxia ventilation by either standard or high tidal volume resulted in significantly higher wet-to-dry lung weight ratio and higher levels of IL-1ß and 8-OHdG in the lungs. However, moderate hyperoxia ventilation, even by high tidal volume did not significantly increase the levels of IL-1ß and 8-OHdG in the lungs. Western blot analysis showed that the expression of RhoA, ROCK1, MLC2, and p-MLC2 was not significantly induced in the ventilated lungs, even by high tidal volume at 2 cmH2O PEEP. CONCLUSION: Severe hyperoxia ventilation causes inflammatory response and oxidative damage in mechanically ventilated lungs, while high tidal volume ventilation at a reasonable PEEP possibly does not cause VILI.

A novel ex vivo lung cancer model based on bioengineered rat lungs.

Introduction: Two-dimensional cell cultures have contributed substantially to lung cancer research, but 3D cultures are gaining attention as a new, more efficient, and effective research model. A model reproducing the 3D characteristics and tumor microenvironment of the lungs in vivo, including the co-existence of healthy alveolar cells with lung cancer cells, is ideal. Here, we describe the creation of a successful ex vivo lung cancer model based on bioengineered lungs formed by decellularization and recellularization. Methods: Human cancer cells were directly implanted into a bioengineered rat lung, which was created with a decellularized rat lung scaffold reseeded with epithelial cells, endothelial cells and adipose-derived stem cells. Four human lung cancer cell lines (A549, PC-9, H1299, and PC-6) were applied to demonstrate forming cancer nodules on recellularized lungs and histopathological assessment were made among these models. MUC-1 expression analysis, RNA-seq analysis and drug response test were performed to demonstrate the superiority of this cancer model. Results: The morphology and MUC-1 expression of the model were like those of lung cancer in vivo. RNA sequencing revealed an elevated expression of genes related to epithelial-mesenchymal transition, hypoxia, and TNF-α signaling via NF-κB; but suppression of cell cycle-related genes including E2F. Drug response assays showed that gefitinib suppressed PC-9 cell proliferation equally well in the 3D lung cancer model as in 2D culture dishes, albeit over a smaller volume of cells, suggesting that fluctuations in gefitinib resistance genes such as JUN may affect drug sensitivity. Conclusions: A novel ex vivo lung cancer model was closely reproduced the 3D structure and microenvironment of the actual lungs, highlighting its possible use as a platform for lung cancer research and pathophysiological studies.

Negative pressure induces dedifferentiation of hepatocytes via RhoA/ROCK pathway.

Biomechanical forces are known to regulate the biological behaviors of cells. Although negative pressure has been used for wound healing, it is still unknown about its role in regulating cell plasticity. We investigated whether negative pressure could induce the dedifferentiation of hepatocytes. Using a commercial device, we found that the exposure of primary human hepatocytes to -50 mmHg quickly induced the formation of stress fibers and obviously changed cell morphology in 72 h. Moreover, the exposure of hepatocytes to -50 mmHg significantly upregulated RhoA, ROCK1, and ROCK2 in 1-6 h, and dramatically enhanced the expression of marker molecules on "stemness", such as OCT4, SOX2, KLF4, MYC, NANOG, and CD133 in 6-72 h. However, all these changes in hepatocytes induced by -50 mmHg stimulation were almost abrogated by ROCK inhibitor Y27623. Our data suggest that an appropriate force of negative pressure stimulation can effectively induce the dedifferentiation of hepatocytes via RhoA/ROCK pathway activation.

Ex vivo expansion of primary cells from limb tissue of Pleurodeles waltl.

Pleurodeles waltl is coming to light as a model animal, especially in regeneration studies, but deep studies on the molecular mechanisms have been limited due to the absence of primary tissue cells for wide usage. Therefore, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explants" on culture dishes coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin supported quicker outgrowth of cells from explants and faster cell adhesion, and fibronectin showed significantly better performance than gelatin. Interestingly, the doubling time of cells on fibronectin- and gelatin-coated surfaces was almost the same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly different from that on non-coated plates (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed a multiplication capacity that was similar to that of fresh cells. Senescent cells were barely detected even after long-term sub-culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H2 O2 exposure confirmed the respondence to chemical stimuli. Collectively, our results show that we are able to grow enough good-quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrowth and attachment.

Nicaraven protects against endotoxemia-induced inflammation and organ injury through modulation of AMPK/Sirt1 signaling in macrophages.

Endotoxemia is a disease characterized by systemic inflammatory responses and organ injury caused by lipopolysaccharide (LPS) infection, with high mortality. Nicaraven (AVS), a potent hydroxyl radical scavenger, has been proven to regulate the inflammatory response in tumors. To investigate the protective effects and mechanisms of AVS in endotoxemia, mice were injected intraperitoneally with LPS to induce endotoxemia. AVS treatment significantly decreased the levels of pro-inflammatory cytokines in the serum, reduced neutrophil infiltration, attenuated multiple organ injury, and increased the survival rate in LPS-challenged mice. In the LPS-induced inflammatory model of macrophages, AVS inhibited macrophage activation, suppressed nitric oxide (NO) production, and inhibited the expression and secretion of pro-inflammatory cytokines. Mechanistically, AVS treatment up-regulated silence information regulator transcript-1 (Sirt1) expression in a time- and dose-dependent manner. AVS treatment activated the AMP-dependent protein kinase (AMPK)/Sirt1 signaling pathway and suppressed the activation of nuclear factor kappa B (NF-κB) in macrophages exposed to LPS. However, the anti-inflammatory effects of AVS could be reversed by the AMPK, the Sirt1 inhibitor, or the histone deacetylase inhibitor. We confirmed that the AMPK inhibitor inhibited AVS-mediated AMPK/Sirt1 activation and NF-κB p65 acetylation. These results suggested that AVS alleviated endotoxemia by activating the AMPK/Sirt1 signaling pathway in macrophages.

Smooth muscle cell fate decisions decipher a high-resolution heterogeneity within atherosclerosis molecular subtypes.

BACKGROUND: Mounting evidence has revealed the dynamic variations in the cellular status and phenotype of the smooth muscle cell (SMC) are vital for shaping the atherosclerotic plaque microenvironment and ultimately mapping onto heterogeneous clinical outcomes in coronary artery disease. Currently, the underlying clinical significance of SMC evolutions remains unexplored in atherosclerosis. METHODS: The dissociated cells from diseased segments within the right coronary artery of four cardiac transplant recipients and 1070 bulk samples with atherosclerosis from six bulk cohorts were retrieved. Following the SMC fate trajectory reconstruction, the MOVICS algorithm integrating the nearest template prediction was used to develop a stable and robust molecular classification. Subsequently, multi-dimensional potential biological implications, molecular features, and cell landscape heterogeneity among distinct clusters were decoded. RESULTS: We proposed an SMC cell fate decision signature (SCFDS)-based atherosclerosis stratification system and identified three SCFDS subtypes (C1-C3) with distinguishing features: (i) C1 (DNA-damage repair type), elevated base excision repair (BER), DNA replication, as well as oxidative phosphorylation status. (ii) C2 (immune-activated type), stronger immune activation, hyper-inflammatory state, the complex as well as varied lesion microenvironment, advanced stage, the most severe degree of coronary stenosis severity. (iii) C3 (stromal-rich type), abundant fibrous content, stronger ECM metabolism, immune-suppressed microenvironment. CONCLUSIONS: This study uncovered atherosclerosis complex cellular heterogeneity and a differentiated hierarchy of cell populations underlying SMC. The novel high-resolution stratification system could improve clinical outcomes and facilitate individualized management.

Optimization on the dose and time of nicaraven administration for mitigating the side effects of radiotherapy in a preclinical tumor-bearing mouse model.

OBJECTIVE: Radiation-induced lung injury (RILI) is one of the serious complications of radiotherapy. We have recently demonstrated that nicaraven can effectively mitigate RILI in healthy mice. Here, we further tried to optimize the dose and time of nicaraven administration for alleviating the side effects of radiotherapy in tumor-bearing mice. METHODS AND RESULTS: A subcutaneous tumor model was established in the back of the chest in C57BL/6N mice by injecting Lewis lung cancer cells. Therapeutic thoracic irradiations were done, and placebo or different doses of nicaraven (20, 50, 100 mg/kg) were administrated intraperitoneally pre-irradiation (at almost 5-10 min before irradiation) or post-irradiation (within 5 min after irradiation). Mice that received radiotherapy and nicaraven were sacrificed on the 30th day, but control mice were sacrificed on the 15th day. Serum and lung tissues were collected for evaluation. Nicaraven significantly decreased the level of CCL8, but did not clearly change the levels of 8-OHdG, TGF-ß, IL-1ß, and IL-6 in serum. Besides these, nicaraven effectively decreased the levels of TGF-ß, IL-1ß, and SOD2 in the lungs, especially by post-irradiation administration with the dose of 20 mg/kg. Although there was no significant difference, the expression of SOD1, 53BP1, and caspase 3 was detected lower in the lungs of mice received nicaraven post-irradiation than that of pre-irradiation. CONCLUSION: According to our data, the administration of nicaraven at a relatively low dose soon after radiotherapy will be recommended for attenuating the side effects of radiotherapy.

Nicaraven induces programmed cell death by distinct mechanisms according to the expression levels of Bcl-2 and poly (ADP-ribose) glycohydrolase in cancer cells.

The PARP-1 expression level and poly (ADP-ribosyl)ation activity in cancer markedly affect the therapeutic outcome. Nicaraven, a free radical scavenger has been found to inhibit PARP, but the effect on cancer cells is still unclear. In this study, we investigated the potential role and molecular mechanism of nicaraven on cancer cells. Using U937 lymphoma cells and HCT-8 colorectal cancer cells, we found that nicaraven moderately reduced the cell viability of both cells in a dose-dependent manner. Interestingly, nicaraven significantly induced apoptosis of U937 cells that are dominantly expressing Bcl-2 but induced PAR-dependent cell death (parthanatos) of HCT-8 cells that are highly expressing poly (ADP-ribose) glycohydrolase (PARG). Based on our data, nicaraven seems to induce programmed cell death through distinct mechanisms, according to the expression levels of Bcl-2 and PARG in cancer cells.

Analysis of Immune and Inflammation Characteristics of Atherosclerosis from Different Sample Sources.

Background: Atherosclerosis is the predominant cause of cardiovascular diseases. Existing studies suggest that the development of atherosclerosis is closely related to inflammation and immunity, but whether there are differences and similarities between atherosclerosis occurring at different sites is still unknown. We elucidated the pathological characteristics of peripheral vascular diseases by using bioinformatic analyses on immune cells and inflammation-related gene expression in atherosclerotic arteries and plaques. Methods: Eight data sets regarding atherosclerosis were downloaded from the Gene Expression Omnibus database. Human immune genes were obtained from the IMMPORT website. The samples were scored and divided into high- and low-immune groups. Then the samples were analysed using weighted gene co-expression network analysis, while the modules were analysed using functional enrichment. The protein-protein interaction network was constructed using the STRING and Cytoscape databases. The hub immune genes were screened, and the correlation between hub immune genes and immune cells was analysed. Results: Immune cells and their functions were significantly different during atherosclerosis development. The infiltration proportion of immune cells was approximately similar in samples from different sources of patients with carotid atherosclerosis. However, the sensitivity of lower extremity atherosclerosis samples to immune cells is lower than that of carotid atherosclerosis samples.The samples from the plaque and artery were mainly infiltrated by macrophages, T cells and mast cells. After immune cells were assessed, resting NK cells, activated mast cells and M0 macrophages were found to be key immune cells in atherosclerosis and plaque formation. In addition, CCL4, TLR2, IL1B and PTPRC were considered to be immune marker genes in atherosclerosis development. Conclusion. Bioinformatic data analysis confirms the essential role of immune cells in cardiovascular diseases, and also indicates some differences of immune and inflammation characteristics of atherosclerosis between carotid and lower extremity arteries.

Ex Vivo Hydrostatic Pressure Loading of Atrial Tissues Activates Profibrotic Transcription via TGF-ß Signal Pathway.

Excessive mechanical stress causes fibrosis-related atrial arrhythmia. Herein, we tried to investigate the mechanism of atrial fibrogenesis in response to mechanical stress by ex vivo approach. We collected atrial tissues from mice and then cultured them as "explants" under atmospheric pressure (AP group) or 50 mmHg hydrostatic pressure loading (HP group) conditions. Pathway-specific PCR array analysis on the expression of fibrosis-related genes indicated that the loading of atrial tissues to 50 mmHg for 24 hours extensively upregulated a series of profibrotic genes. qRT-PCR data also showed that loading atrial tissues to 50 mmHg enhanced Rhoa, Rock2, and Thbs1 expression at different time points. Interestingly, the enhanced expression of Thbs1 at 1 hour declined at 6-24 hours and then increased again at 72 hours. In contrast, an enhanced expression of Tgfb1 was observed at 72 hours. In contrast, daily loading to 50 mmHg for 3 hours significantly accelerated the outgrowth of mesenchymal stem-like stromal cells from atrial tissues; however, we did not observe significant phenotypic changes in these outgrowing cells. Our ex vivo experimental data clearly show the induction of profibrotic transcription of atrial tissues by HP loading, which confirms the common pathological feature of atrial fibrosis following pressure overload.

Biological Differences Between Ovarian Cancer-associated Fibroblasts and Contralateral Normal Ovary-derived Mesenchymal Stem Cells.

BACKGROUND/AIM: The aim of this study was to clarify the biological differences between ovarian cancer-associated fibroblasts (OCa-CAFs) and normal ovary-derived mesenchymal stem cells (NO-MSCs). MATERIALS AND METHODS: Surgically resected ovarian cancer and contralateral normal ovarian tissue samples were cut into small pieces for culture as "explants". The number of outgrown cells, their proliferative kinetics, and expression levels of cell surface markers of CAFs, as well as three miRNAs in OCa-CAFs and NO-MSCs were compared directly. Differentially expressed genes between both groups were also investigated. RESULTS: Comparable numbers of outgrown cells were harvested from both groups. Significantly higher expression of α-smooth muscle actin and miR-142 was found in OCa-CAFs, which decreased significantly during ex vivo cell expansion. A total of 21 differentially expressed genes were identified between both groups. CONCLUSION: OCa-CAFs showed different biological properties in direct comparison with NO-MSCs, which might play major roles in the pathogenesis of ovarian cancer.

Hydrostatic pressure induces profibrotic properties in hepatic stellate cells via the RhoA/ROCK signaling pathway.

Elevated interstitial fluid hydrostatic pressure is commonly observed in diseased livers. We herein examined the hypothesis that hydrostatic pressure induces hepatic stellate cells to acquire profibrotic properties under pathological conditions. Human hepatic stellate cells were exposed to 50 mmHg pressure for 24 h. Although we observed few changes of cell growth and morphology, PCR array data on the expression of fibrosis-associated genes suggested the acquisition of profibrotic properties. The exposure of hepatic stellate cells to 50 mmHg pressure for 24 h also significantly enhanced the expression of RhoA, ROCK1, α-SMA, TGF-ß1 , p-MLC, and p-Smad2, and this was effectively attenuated by the ROCK inhibitor Y-27632. Our ex vivo experimental data suggest that elevated interstitial fluid hydrostatic pressure under pathological conditions may promote liver fibrosis by inducing acquisition of profibrotic properties of hepatic stellate cells through the RhoA/ROCK signaling pathway.

Angiotensin receptor blocker alleviates liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.

Angiotensin receptor blockers have been reported to be beneficial to liver fibrosis, but the relevant molecular and cellular mechanisms remain unclear. We herein investigated whether low-dose angiotensin receptor blocker alleviated liver fibrosis through mechanotransduction regulation. Hydrostatic pressure-induced liver fibrosis model was established in mice by ligating partially the inferior vena cava, and then randomly received a very low dose of losartan (0.5 mg/kg) or placebo treatment for 8 weeks. We found that losartan administration interfered the expression of several mechanotransductive molecules, and effectively alleviated liver fibrosis. Using a commercial device, we further confirmed that ex vivo loading of hepatic stellate cells to 50 mmHg hydrostatic pressure for 24 h significantly upregulated RhoA, ROCK, AT1R, and p-MLC2, which was effectively attenuated by adding 10 nM losartan in medium. Our in vivo and ex vivo experimental data suggest that low-dose angiotensin receptor blockers may alleviate hydrostatic pressure-induced liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.NEW & NOTEWORTHY Our ex vivo and in vivo experiments clearly indicated that low-dose losartan alleviated liver fibrosis, likely by modulating the mechanotransduction properties of HSCs. Uncovering the biomechanical signaling pathway of ARB treatment on liver fibrosis will be helpful to develop novel molecular targeting therapy for liver diseases.