The pathophysiology of hepatic steatosis, a prerequisite of nonalcoholic fatty liver disease, is poorly understood. Because very-low-density lipoprotein (VLDL) formation is the chief route of hepatic lipid export, we hypothesized that the synthesis of apoB-100, a rate-determining step in hepatic VLDL formation, may be altered in patients with nonalcoholic steatohepatitis (NASH). This study evaluated the relative synthesis rates of apolipoprotein B-100 (apoB-100) in patients with NASH and in lean and body mass index (BMI)-matched (obese) controls without NASH. A primed continuous infusion of L-[1-13 C] leucine was used to measure the absolute synthesis rates (ASR) of apoB-100 and fibrinogen in 7 patients with NASH and compared them with 7 lean and 7 obese (BMI-matched) controls without NASH. The ASRs of fibrinogen and albumin also were measured. The mean ASR of apoB-100 in patients with NASH was lower (31.5 ؎ 3.4 mg/kg/d) than that of obese (115.2 ؎ 7.2 mg/kg/d, P < .001) and lean controls (82.4 ؎ 4.1 mg/kg/d, P ‫؍‬ .002). In contrast, the mean ASR of fibrinogen was greater in subjects with NASH than in both control groups. These data indicate that NASH is associated with markedly altered hepatic synthesis of apoB-100. The finding that albumin synthesis was not similarly decreased in patients with NASH shows that the attenuation of apoB-100 synthesis is not on the basis of globally impaired hepatic protein synthesis. In conclusion, because apoB-100 synthesis is a rate-determining step in hepatocyte lipid export, decreased synthesis of this protein may be an important factor in the development of hepatic steatosis, a prerequisite for NASH. (HEPATOLOGY 2002;35:898-904.) N on-alcoholic steatohepatitis (NASH; part of the spectrum of nonalcoholic fatty liver disease) is characterized by macrovesicular steatosis, nuclear glycogenation, lobular and portal inflammation, and, occasionally, Mallory's hyaline, in the absence of excessive alcohol ingestion. 1 NASH is most frequently associated with obesity 2-9 and type 2 diabetes mellitus. 3,[7][8][9] Despite the high prevalence and the increasingly recognized morbidity associated with NASH, little is known about the pathophysiology of this condition. The liver is central to the regulation of fat metabolism, and the net accumulation of fat within hepatocytes, a prerequisite for steatosis and steatohepatitis, could potentially result from alterations in the uptake, synthesis, degradation, or secretory pathways of hepatic lipid metabolism.Apolipoprotein B-100 (apoB-100) is a large protein (512 kd) involved in the transport of triglycerides and cholesterol from the liver to peripheral tissues. Lipids are chiefly exported from the liver in the form of very-lowdensity lipoproteins (VLDL), a complex of protein (apoB-100), lipids (triglycerides or cholesteryl esters), and phospholipids. Although the mechanisms regulating the synthesis of apoB-100 are incompletely understood, the production of apoB-100 messenger RNA (mRNA) has been shown to be altered by insulin and unaffected by...

Although the molecular basis for the pathophysiology of nonalcoholic steatohepatitis (NASH) is poorly understood, insulin resistance and mitochondrial dysfunction are physiologic hallmarks of this condition. We sought evidence of a transcriptional or pretranscriptional basis for insulin resistance and mitochondrial dysfunction through measurement of hepatic gene expression (messenger RNA [mRNA]) using high-density synthetic oligonucleotide microarray analysis (Hu6800 GeneChip, Affymetrix, CA). Global hepatic gene expression was determined in snap-frozen liver biopsy specimens from 4 groups: (1) patients with cirrhotic-stage NASH (n ‫؍‬ 6), (2) patients with cirrhosis caused by hepatitis C virus (HCV) (n ‫؍‬ 6), (3) patients with cirrhosis secondary to primary biliary cirrhosis (PBC) (n ‫؍‬ 6), and (4) healthy controls (n ‫؍‬ 6). Genes were considered to be expressed differentially in NASH only if there was a greater than 2-fold difference in abundance of mRNA when compared with each of the control groups. Sixteen genes were uniquely differentially expressed (4 overexpressed and 12 underexpressed) in patients with cirrhotic-stage NASH. Genes that were significantly underexpressed included genes important for maintaining mitochondrial function (copper/zinc superoxide dismutase, aldehyde oxidase, and catalase). Glucose 6-phospatase, alcohol dehydrogenase, elongation factor-TU, methylglutaryl coenzyme A (CoA), acyl CoA synthetase, oxoacyl CoA thiolase, and ubiquitin also were underexpressed in NASH. Genes that were overexpressed in NASH included complement component C3 and hepatocyte-derived fibrinogen-related protein, potentially contributing to impaired insulin sensitivity. In conclusion, these studies provide evidence for a transcriptional or pretranscriptional basis for impaired mitochondrial function (attenuated capacity for the dismutation of reactive oxygen species) and diminished insulin sensitivity (increased acute phase reactants) in patients with histologically progressive NASH. Further studies are required to determine the mechanism and the physiologic significance of these findings. (HEPATOLOGY 2003;38:244-251.)

Once excess liquid gains access to air spaces of an injured lung, the act of breathing creates and destroys foam and thereby contributes to the wounding of epithelial cells by interfacial stress. Since cells are not elastic continua, but rather complex network structures composed of solid as well as liquid elements, we hypothesize that plasma membrane (PM) wounding is preceded by a phase separation, which results in blebbing. We postulate that interventions such as a hypertonic treatment increase adhesive PM-cytoskeletal (CSK) interactions, thereby preventing blebbing as well as PM wounds. We formed PM tethers in alveolar epithelial cells and fibroblasts and measured their retractive force as readout of PM-CSK adhesive interactions using optical tweezers. A 50-mOsm increase in media osmolarity consistently increased the tether retractive force in epithelial cells but lowered it in fibroblasts. The osmo-response was abolished by pretreatment with latrunculin, cytochalasin D, and calcium chelation. Epithelial cells and fibroblasts were exposed to interfacial stress in a microchannel, and the fraction of wounded cells were measured. Interventions that increased PM-CSK adhesive interactions prevented blebbing and were cytoprotective regardless of cell type. Finally, we exposed ex vivo perfused rat lungs to injurious mechanical ventilation and showed that hypertonic conditioning reduced the number of wounded subpleural alveolus resident cells to baseline levels. Our observations support the hypothesis that PM-CSK adhesive interactions are important determinants of the cellular response to deforming stress and pave the way for preclinical efficacy trials of hypertonic treatment in experimental models of acute lung injury. alveolar epithelial cell; acute lung injury; interfacial stress; osmotic response; cytoprotection THE SYNDROME OF ventilator-induced lung injury (VILI) contributes to the morbidity and mortality of critically ill patients (25). The clinical manifestations of the syndrome are indistinguishable from those of all-cause acute lung injury and at their core reflect a mechanotransduction event, i.e., the effects of deforming stress on cell and tissue injury, remodeling, and repair. The complex topographical distributions of lung mechanical properties, and hence of parenchymal stress and strain together with the numerous and nuanced injury manifestations, make it very difficult to establish mechanistic cause and effect relationships on the scale of interest, e.g., that of individual cells. Our research, therefore, focuses on a very specific mechanotransduction event, namely physical stress-related epithelial cell wounding and repair, which we believe contributes to the pathogenesis VILI.In the current study, we present a body of work in which we have explored the effects of hypertonic exposure on cell wounding in experimental models ranging from individual cells to rodent VILI preparations. The experiments were not designed to test the preclinical efficacy of an intervention, but rather to explore a cell bio...

P188 facilitates plasma membrane repair in alveolus resident cells, but has no salutary effects on lung mechanics or vascular barrier properties in live animals. This discordance may have pathophysiological significance for the interdependence of different injury mechanisms and therapeutic implications regarding the benefits of prolonging the life of stress-activated cells.

Caples SM, Rasmussen DL, Lee WY, Wolfert MZ, Hubmayr RD. Impact of buffering hypercapnic acidosis on cell wounding in ventilator-injured rat lungs.

Rationale: Wounded alveolus resident cells are identified in human and experimental acute respiratory distress syndrome models. Poloxamer 188 (P188) is an amphiphilic macromolecule shown to have plasma membrane-sealing properties in various cell types. Objectives: To investigate whether P188 (1) protects alveolus resident cells from necrosis and (2) is associated with reduced ventilatorinduced lung injury in live rats, isolated perfused rat lungs, and scratch and stretch-wounded alveolar epithelial cells. Methods: Seventy-four live rats and 18 isolated perfused rat lungs were ventilated with injurious or protective strategies while infused with P188 or control solution. Alveolar epithelial cell monolayers were subjected to scratch or stretch wounding in the presence or absence of P188. Measurements and Main Results: P188 was associated with fewer mortally wounded alveolar cells in live rats and isolated perfused lungs. In vitro, P188 reduced the number of injured and necrotic cells, suggesting that P188 promotes cell repair and renders plasma membranes more resilient to deforming stress. The enhanced cell survival was accompanied by improvement in conventional measures of lung injury (peak airway pressure, wet-to-dry weight ratio) only in the ex vivo-perfused lung preparation and not in the live animal model. Conclusions: P188 facilitates plasma membrane repair in alveolus resident cells, but has no salutary effects on lung mechanics or vascular barrier properties in live animals. This discordance may have pathophysiological significance for the interdependence of different injury mechanisms and therapeutic implications regarding the benefits of prolonging the life of stress-activated cells.

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