Kimberly A. Broadhurst

Expression of HSP47, a collagen-specific chaperone, in normal and diseased human liver

HSP47 is a collagen-specific chaperone that is required for normal collagen synthesis. In animal models of liver injury, hepatic stellate cells (HSC) have been identified as a source of HSP47. Because expression of HSP47 has not been investigated in human liver, the aim of these studies was to characterize expression of HSP47 in human liver and to investigate its regulation in human HSC in vitro. Immunohistochemistry demonstrated staining for HSP47 along the sinusoids of normal and cirrhotic human livers and in fibrous septa. Dual fluorescence confocal microscopy showed colocalization of HSP47 with synaptophysin, a marker for HSC. Levels of immunoreactive HSP47 and its transcript tended to be higher in cirrhotic livers than in normal livers. The abundance of HSP47 protein was unchanged by treatment of cultured human HSC with TGF-β1, angiotensin II, hypoxia and a number of other treatments intended to increase collagen synthesis. A modest reduction in HSP47 was achieved by transfection with antisense oligonucleotides and was associated with a significant decrease in procollagen synthesis. These observations suggest that HSP47 is constitutively expressed in human HSC and that HSP47 may be a target for antifibrotic therapy.

Captopril decreases stress ulceration without affecting gastric perfusion during canine hemorrhagic shock

The renin-angiotensin axis has recently been called the source of disproportionate splanchnic vasoconstriction during shock, and blocking this axis decreased gastric stress ulceration during swine cardiogenic shock. The present study tested whether the angiotensin converting enzyme inhibitor captopril would prevent stress ulceration when given after the onset of canine hemorrhagic shock, and whether any detrimental effects would result from enhancing splanchnic perfusion with captopril during hemorrhagic shock. We found that captopril treatment was associated with a decrease in gastric mucosal injury and with a marked decrease in systemic acidosis. Captopril enhanced blood flow to the small intestine, pancreas, liver, and spleen, but not flow to the stomach, during shock. Following the reinfusion of shed blood, the captopril-treated animals had decreased mean blood pressures and increased heart rates compared with untreated animals. We found captopril alleviated the stress ulceration produced by canine hemorrhagic shock, but concluded that the likely mechanism was alleviating systemic acidosis through enhanced perfusion of other viscera rather than a specific enhancement of gastric perfusion.

Altered expression of iron regulatory genes in cirrhotic human livers : clues to the cause of hemosiderosis?

Hepatic iron deposition unrelated to hereditary hemochromatosis occurs commonly in cirrhosis but the pathogenesis of this condition is unknown. The aim of this study was to compare the expression of genes involved in the regulation of iron metabolism in cirrhotic (n=22) and control human livers (n=5). Transcripts were quantitated by real-time RT-PCR and protein levels were assessed by western blot. Hepatic iron concentrations (HICs) were measured by a spectrophotometric method. Levels of hepcidin mRNA did not differ between controls and cirrhotic livers; there was a highly significant correlation between hepcidin transcript levels and HIC in the latter group. Ferroportin, divalent metal transporter-1 (DMT1), and ferritin heavy chain mRNA levels were significantly higher in cirrhotic human livers than in controls (P=0.007, 0.039, and 0.025, respectively). By western blot, ferroportin and DMT1 levels were generally diminished in the cirrhotic livers compared to controls; neither correlated with HIC. In contrast, the abundance of ferritin increased with increasing HIC in the cirrhotic livers, whereas transferrin receptor decreased, indicating physiologically appropriate regulation. In conclusion, hepcidin expression appears to be appropriately responsive to iron status in cirrhosis. However, there are complex alterations in DMT1 and ferroportin expression in cirrhotic liver, including decreases in ferroportin and DMT1 at the protein level that may play a role in aberrant regulation of iron metabolism in cirrhosis.

Gastric contractions, secretions and injury in cold restraint

The clinical syndrome of stress ulceration has been studied for years using rodent cold restraint stress models, although the pathogenesis of the characteristic focal gastric mucosal lesions produced in these models has been controversial. We used gastric strain gauges to characterize fully the gastric motility effects of a 4‐h cold restraint protocol, and we determined the relationship of variations in gastric contents and in gastric contractions to the amount of gastric mucosal injury. Additionally, we examined rat stomachs histologically, and determined the location of focal haemorrhagic mucosal lesions on the mucosal rugae. We found a consistent relationship between force of gastric contractions and gastric mucosal injury, and also a relationship between the initial duration of contractions during restraint and ultimate mucosal injury. Volume, acidity and mucus in the gastric contents were unrelated to mucosal injury. The majority (91%) of the mucosal lesions had some relationship to a rugal fold, with 59% of all lesions at the base of a rugal fold. Thus, the mechanical forces of gastric hypercontractility may contribute to the gastric mucosal injury of rodent cold restraint models.

Gastric Mucosal Protection from Enteral Nutrients: Role of Motility

BACKGROUND Cold restraint stress increases the force of gastric contractions and produces gastric mucosal injury in rats. The aim of our study was to determine whether enteral glucose or hyperglycemia alone would alter the stress-induced gastric motility pattern and ameliorate the associated gastric mucosal injury. METHODS Adult male rats underwent surgical placement of gastric catheters, jugular venous catheters, and gastric strain gauge transducers 5 days before cold restraint. Three groups of rats received different substances during the same cold restraint stress protocol. Group 1 received 0.9% NaCl, 2 mL/h infused both intravenously (i.v.) and intragastrically (i.g.); group 2 received 0.9% NaCl, 2 mL/h i.g. plus 25% glucose, 2 mL/h i.v.; and group 3 received 0.9% NaCl, 2 mL/h i.v. plus 25% glucose i.g. Following baseline gastric motility measurements, all rats were restrained for 2 hours at 20 degrees C followed by 2 hours at 4 degrees C. RESULTS Restraint even at room temperature increased the force of gastric contractions; the cold environment gradually prolonged gastric contractions. Enteral glucose blunted the effects of stress on gastric motility, increased gastric residual volume, decreased gastric acidity, and prevented gastric mucosal injury. Parenteral glucose had little effect on any gastric parameters. CONCLUSIONS Enteral glucose prevents the abnormal gastric motility pattern that is necessary to produce the gastric mucosal injury associated with cold restraint stress, but hyperglycemia alone has little effect on the pathophysiology of cold restraint.

Strain- and time-dependent alterations in hepatic iron metabolism in a murine model of nonalcoholic steatohepatitis: Iron metabolism in murine NASH

Nonalcoholic steatohepatitis is a common liver disease that is often accompanied by dysregulated iron metabolism. The aim of the study was to test the hypothesis that aberrant iron metabolism in nonalcoholic steatohepatitis is modulated by genetic susceptibility to inflammation and oxidative stress. Hepatic histology and iron content were assessed in 3 inbred strains of mice (C57BL/6, BALB/c, and C3H/HeJ) fed an atherogenic diet (AD). Hepatic expression of genes relevant to iron metabolism, inflammation, and oxidative stress were quantitated by real‐time reverse transcription‐polymerase chain reaction. At 6 weeks on the AD, histologic injury and induction of inflammatory and oxidative stress‐associated gene expression were most pronounced in C57BL/6. At 18 weeks on the AD, these parameters were similar in C57BL/6 and BALB/c. Atherogenic diet–fed C3H/HeJ showed milder responses at both time points. The AD was associated with decreased hepatic iron concentrations in all strains at 6 and 18 weeks. The decrease in hepatic iron concentrations did not correlate with changes in hepcidin expression and was not associated with altered expression of iron transporters. These findings are similar to those observed in models of obesity‐induced steatosis and indicate that hepatic steatosis can be associated with depletion of iron stores that is not explained by upregulation of hepcidin expression by inflammation.