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Background and aims In clinical and experimental NASH, the origin of the scar-forming myofibroblast is the HSC. We used foz/foz mice on a Western diet to characterize in detail the phenotypic changes of HSCs in a NASH model. Approach and results We examined the single-cell expression profiles (scRNA sequencing) of HSCs purified from the normal livers of foz/foz mice on a chow diet, in NASH with fibrosis of foz/foz mice on a Western diet, and in livers during regression of NASH after switching back to a chow diet. Selected genes were analyzed using immunohistochemistry, quantitative real-time PCR, and short hairpin RNA knockdown in primary mouse HSCs. Our analysis of the normal liver identified two distinct clusters of quiescent HSCs that correspond to their acinar position of either pericentral vein or periportal vein. The NASH livers had four distinct HSC clusters, including one representing the classic fibrogenic myofibroblast. The three other HSC clusters consisted of a proliferating cluster, an intermediate activated cluster, and an immune and inflammatory cluster. The livers with NASH regression had one cluster of inactivated HSCs, which was similar to, but distinct from, the quiescent HSCs. Conclusions Analysis of single-cell RNA sequencing in combination with an interrogation of previous studies revealed an unanticipated heterogeneity of HSC phenotypes under normal and injured states.

Background & Aims How benign liver steatosis progresses to nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC) remains elusive. NASH progression entails diverse pathogenic mechanisms and relies on complex cross-talk between multiple tissues such as the gut, adipose tissues, liver, and the brain. Using a hyperphagic mouse fed with a Western diet (WD), we aimed to elucidate the cross-talk and kinetics of hepatic and extrahepatic alterations during NASH–HCC progression, as well as regression. Methods Hyperphagic mice lacking a functional Alms1 gene (Foz/Foz) and wild-type littermates were fed WD or standard chow for 12 weeks for NASH/fibrosis and for 24 weeks for HCC development. NASH regression was modeled by switching back to normal chow after NASH development. Results Foz+WD mice were steatotic within 1 to 2 weeks, developed NASH by 4 weeks, and grade 3 fibrosis by 12 weeks, accompanied by chronic kidney injury. Foz+WD mice that continued on WD progressed to cirrhosis and HCC within 24 weeks and had reduced survival as a result of cardiac dysfunction. However, NASH mice that were switched to normal chow showed NASH regression, improved survival, and did not develop HCC. Transcriptomic and histologic analyses of Foz/Foz NASH liver showed strong concordance with human NASH. NASH was preceded by an early disruption of gut barrier, microbial dysbiosis, lipopolysaccharide leakage, and intestinal inflammation. This led to acute-phase liver inflammation in Foz+WD mice, characterized by neutrophil infiltration and increased levels of several chemokines/cytokines. The liver cytokine/chemokine profile evolved as NASH progressed, with subsequent predominance by monocyte recruitment. Conclusions The Foz+WD model closely mimics the pathobiology and gene signature of human NASH with fibrosis and subsequent HCC. Foz+WD mice provide a robust and relevant preclinical model of NASH, NASH-associated HCC, chronic kidney injury, and heart failure., Supplemental Graphical Summary

Introduction: Alström syndrome is a rare recessive genetic disease caused by mutations in ALMS1, which encodes a protein that is related to cilia function and intracellular endosome trafficking. The syndrome has been linked to impaired glucose metabolism and CKD. Polymorphisms in Alms1 have likewise been linked to CKD, but little is known about the modification of the phenotype by environmental factors. Methods: To gain further insights, the fat aussie (foz) mouse strain, a genetic murine model of Alström syndrome, was exposed to a normal chow (NC) or to a Western diet (WD, 20% fat, 34% sucrose by weight, and 0.2% cholesterol) and renal outcomes were measured. Results: Body weight and albuminuria were higher in foz than in wild-type (WT) mice on both diets but WD significantly increased the difference. Measurement of plasma creatinine and cystatin C indicated that glomerular filtration rate was preserved in foz versus WT independent of diet. Renal markers of injury, inflammation, and fibrosis were similar in both genotypes on NC but significantly greater in foz than in WT mice on WD. A glucose tolerance test performed in foz and WT mice on WD revealed similar basal blood glucose levels and subsequent blood glucose profiles. Conclusions: WD sensitizes a murine model of Alström syndrome to kidney injury, inflammation, and fibrosis, an effect that may not be solely due to effects on glucose metabolism. Polymorphisms in Alms1 may induce CKD in part by modulating the deleterious effects of high dietary fat and sucrose on kidney outcome.

Introduction: Alstrom syndrome is a rare recessive genetic disease caused by mutations in ALMS1, which encodes a protein that is related to cilia function and intracellular endosome trafficking. The syndrome has been linked to impaired glucose metabolism and CKD. Polymorphisms in Alms1 have likewise been linked to CKD, but little is known about the modification of the phenotype by environmental factors. Methods: To gain further insights, the fat aussie (foz) mouse strain, a genetic murine model of Alstrom syndrome, was exposed to a normal chow (NC) or to a Western diet (WD, 20% fat, 34% sucrose by weight, and 0.2% cholesterol) and renal outcomes were measured. Results: Body weight and albuminuria were higher in foz than in wild-type (WT) mice on both diets but WD significantly increased the difference. Measurement of plasma creatinine and cystatin C indicated that glomerular filtration rate was preserved in foz versus WT independent of diet. Renal markers of injury, inflammation, and fibrosis were similar in both genotypes on NC but significantly greater in foz than in WT mice on WD. A glucose tolerance test performed in foz and WT mice on WD revealed similar basal blood glucose levels and subsequent blood glucose profiles. Conclusions: WD sensitizes a murine model of Alstrom syndrome to kidney injury, inflammation, and fibrosis, an effect that may not be solely due to effects on glucose metabolism. Polymorphisms in Alms1 may induce CKD in part by modulating the deleterious effects of high dietary fat and sucrose on kidney outcome.

Background & Aims Although there are associations among oxidative stress, reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice after administration of a liver carcinogen. Methods Fourteen-day-old Nox1–/– mice, Nox4–/– mice, Nox1–/–Nox4–/– (double-knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1ΔHep), hepatic stellate cells (Nox1ΔHep), or macrophages (Nox1ΔMac). Some mice were also given injections of the NOX1-specific inhibitor ML171. To study the acute effects of DEN, 8–12-week-old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hours. Liver tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots. Results Nox4–/– mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1–/– mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1ΔHep and Nox1ΔHSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1ΔMac developed fewer and smaller tumors, similar to Nox1–/– mice. After DEN injection, levels of tumor necrosis factor, interleukin 6 (IL6), and phosphorylated signal transducer and activator of transcription 3 were increased in livers from WT, but not Nox1–/– or Nox1ΔMac, mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of tumor necrosis factor, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1ΔMac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle. Conclusions In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damage-associated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow hepatocellular carcinoma progression.

Nitric oxide (NO) acts as a signalling molecule that has direct and indirect regulatory roles in various functional processes in biology, though in plant kingdom its role is relatively unexplored. One reason for this is the fact that sensing of NO is always challenging. There are very few probes that can classify the different NO species. The present paper proposes a simple but straightforward way for sensing different NO species using chlorophyll, the source of inspiration being hemoglobin that serves as NO sink in mammalian systems. The proposed method is able to classify NO from DETA-NONOate or (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate, nitrite, nitrate and S-nitrosothiol or SNO. This discrimination is carried out by chlorophyll a (chl a) at nano molar (nM) order of sensitivity and at 293 K-310 K. Molecular docking reveals the differential binding effects of NO and SNO with chlorophyll, the predicted binding affinity matching with the experimental observation. Additional experiments with a diverse range of cyanobacteria reveal that apart from the spectroscopic approach the proposed sensing module can be used in microscopic inspection of NO species. Binding of NO is sensitive to temperature and static magnetic field. This provides additional support for the involvement of the porphyrin ring structures to the NO sensing process. This also, broadens the scope of the sensing methods as hinted in the text.

Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage.

Cigarette smoking can cause fatal pulmonary injury involving extensive structural and functional damage of the lung through a disease called emphysema followed by extensive vascular remodeling, that restricts pulmonary circulation to cause pulmonary hypertension and right ventricular dystrophy of the heart. We demonstrate that oxidant(s) present in tobacco smoke not only inflict direct oxidative damage to lung tissues to predispose them to increased proteolysis through endogenous proteases like matrix-metalloproteinase-9 (MMP-9), but are also responsible for triggering the extensive pulmonary vascular remodeling that is causally associated with pulmonary hypertension. Our present work also reveals that tobacco-smoke oxidants elicit such degenerative lung injury and lung remodeling in two different cellular environments in the lung by activating diverse classes of inflammatory cytokines. Overexpression of two key enzymes in the lung, inducible Nitric-Oxide Synthase (iNOS) and Arginase (Arginase I) also seem to play a determining role in predisposing lung tissues to emphysema and pulmonary hypertension respectively. The versatile dietary antioxidant, ascorbate or vitamin C, was found to comprehensively prevent emphysematous lung damage by inhibiting both tobacco-smoke induced direct lung protein oxidation as well as inflammatory cytokine-mediated oxido-nitrosative modification of lung proteins, that otherwise leads to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases during emphysema. Vitamin C also restricts the upregulation of MMP-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco-smoke induced emphysema. Interestingly, it also prevents the cytokines involved in triggering the subsequent peri-vascular collagen deposition and medial thickening characterizing vascular remodeling and pulmonary hypertension as well as the overexpression of the enzyme, Arginase I involved in this process. Overall, our findings implicate tobacco-smoke oxidant(s) as the key etiopathogenic factor behind both the degenerative breakdown (emphysema) and vascular remodeling (pulmonary hypertension) of the lung during smoking. It also reveals the inexpensive prospects of holistic prevention of both these major forms of tobacco-smoke induced lung damage by vitamin C particularly in those inveterate smokers who fail to bail themselves out from the addictive influence of smoking.

Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung followed by pulmonary hypertension that restricts pulmonary circulation and causes right ventricular dysfunction of the heart. Using a guinea-pig model and human lung cells we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of emphysematous lung injury, but are also responsible for the extensive pulmonary vascular remodelling that characterizes pulmonary hypertension. Tobacco-smoke oxidants activate the lung pro-inflammatory factor, Rtp801 which in turn stimulates nuclear-factor κB and consequent inducible nitric-oxide synthase (iNOS) mediated overproduction of nitric-oxide (NO) that contributes to lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-​(1-​iminoethyl)-​L-​lysine,​ dihydrochloride (L-NIL) solely restricts the observed lung protein nitration but fails to prevent or reverse the major tobacco-smoke induced oxidative lung injury, contrary to recent reports (Cell;147(2):293-305, 2011). In fact, inhibition of pulmonary iNOS also significantly accelerates lung peri-vascular collagen deposition by up-regulating the expression of arginase I, a key enzyme associated with vascular remodelling and pulmonary hypertension. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent emphysematous lung damage by inhibiting both tobacco-smoke induced lung protein oxidation as well as activation of pulmonary Rtp801 and nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, causing emphysema. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco-smoke induced emphysema as well as arginase I, to prevent the subsequent peri-vascular collagen deposition and medial thickening of the pulmonary vessels causing pulmonary hypertension. Thus, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the emphysematous and vascular pathogenesis of the lung associated with smoking. More importantly, it demonstrates the potential of vitamin C to accomplish holistic prevention of both these major forms of tobacco-smoke induced lung damage Reference Proc. Natl. Acad. Sci, USA,113(29):E4208-17, 2016