Cathy Culberson

Differential mechanisms of hepatic vascular dysregulation with mild vs. moderate ischemia-reperfusion

Endotoxemia produces hepatic vascular dysregulation resulting from inhibition of endothelin (ET)-stimulated NO production. Mechanisms include overexpression of caveolin-1 (Cav-1) and altered phosphorylation of endothelial nitric oxide (NO) synthase (NOS; eNOS) in sinusoidal endothelial cells. Since ischemia-reperfusion (I/R) also causes vascular dysregulation, we tested whether the mechanisms are the same. Rats were exposed to either mild (30 min) or moderate (60 min) hepatic ischemia in vivo followed by reperfusion (6 h). Livers were harvested and prepared into precision-cut liver slices for in vitro analysis of NOS activity and regulation. Both I/R injuries significantly abrogated both the ET-1 (1 microM) and the ET(B) receptor agonist (IRL-1620, 0.5 microM)-mediated stimulation of NOS activity. 30 min I/R resulted in overexpression of Cav-1 and loss of ET-stimulated phosphorylation of Ser1177 on eNOS, consistent with an inflammatory response. Sixty-minute I/R also resulted in loss of ET-stimulated Ser1177 phosphorylation, but Cav-1 expression was not altered. Moreover, expression of ET(B) receptors was significantly decreased. This suggests that the failure of ET to activate eNOS following 60-min I/R is associated with decreased protein expression consistent with ischemic injury. Thus hepatic vascular dysregulation following I/R is mediated by inflammatory mechanisms with mild I/R whereas ischemic mechanisms dominate following more severe I/R stress.

HYDROGEN SULFIDE MODULATES SINUSOIDAL CONSTRICTION AND CONTRIBUTES TO HEPATIC MICORCIRCULATORY DYSFUNCTION DURING ENDOTOXEMIA

Hydrogen sulfide (H₂S) affects vascular resistance; however, its effect on the hepatic microcirculation has not been investigated. Hepatic sinusoidal perfusion is dysregulated during sepsis, contributing to liver injury. Therefore, the present study determined the effect of H₂S on the hepatic microcirculation and the contribution of endogenous H₂S to hepatic microcirculatory dysfunction in an endotoxin model of sepsis. Portal infusion of H₂S increased portal pressure in vivo (6.8 ± 0.2 mmHg before H₂S vs. 8.6 ± 0.8 mmHg peak during H₂S infusion, P < 0.05). Using intravital microscopy, we observed decreased sinusoidal diameter (6.2 ± 0.27 μm before H₂S vs. 5.7 ± 0.3 μm after H₂S, P < 0.05) and increased sinusoidal heterogeneity during H₂S infusion (P < 0.05) and net constriction. Since hepatic H₂S levels are elevated during sepsis, we used the cystathionine γ lyase inhibitor DL-propargylglycine (PAG) to determine the contribution of H₂S to the hypersensitization of the sinusoid to the vasoconstrictor effect of endothelin-1 (ET-1). PAG treatment significantly attenuated the sinusoidal sensitization to ET-1 in endotoxin-treated animals. ET-1 infusion increased portal pressure to 175% of baseline in endotoxemic animals, which was reduced to 143% following PAG treatment (P < 0.05). PAG abrogated the increase in sinusoidal constriction after ET-1 infusion in LPS-treated rats (30.9% reduction in LPS rats vs. 11.6% in PAG/LPS rats, P < 0.05). Moreover, PAG treatment significantly attenuated the increase in NADH fluorescence following ET-1 exposure during endotoxemia (61 grayscale units LPS vs. 21 units in PAG/LPS, P < 0.05), suggesting an improvement in hepatic oxygen availability. This study is the first to demonstrate a vasoconstrictor action of H₂S on the hepatic sinusoid and provides a possible mechanism for the protective effect of PAG treatment during sepsis.

Evidence for a Novel Serum Factor Distinct From Zinc Alpha-2 Glycoprotein That Promotes Body Fat Loss Early in the Development of Cachexia

We provide evidence that a factor other than the previously identified lipid mobilizing factor, zinc alpha-2 glycoprotein, promotes lipolysis in the MCA-induced sarcoma-bearing cachexia model. Cachexia is characterized by progressive loss of adipose tissue and skeletal muscle without a concurrent increase in food intake to restore lost tissue stores. We compared tumor-bearing ad lib fed (TB) animals to nontumor bearing ad lib fed (NTB) animals or nontumor-bearing pair-fed (PF) animals at various time points throughout development of tumor derived cachexia. Prior to cachexia, the TB animals lost more than 10 ± 0.7% of their body fat before losing protein mass and decreasing their food intake. Fat loss occurred because adipocyte size, not number, was reduced. Increased turnover of palmitate and significantly higher serum triglyceride levels prior to cachexia were further indicators of an early loss of lipid from the adipocytes. Yet, circulating levels of norepinephrine, epinephrine, TNF-α, and zinc α-2 glycoprotein were not increased prior to the loss of fat mass. We provide evidence for a serum factor(s), other than zinc alpha-2 glycoprotein, that stimulates release of glycerol from 3T3-L1 adipocytes and promotes the loss of stored adipose lipid prior to the loss of lean body mass in this model.

Inhibitory effects of fusarochromanone on melanoma growth

Fusarochromanone is a toxic metabolite produced by Fusarium equiseti, a fungus present in decaying cereal plants in northern latitudes; it has been detected in various food grains. Fusarochromanone has been shown to have both stimulatory and inhibitory effects on various mammalian cells, depending on the concentration used. Whether these cytotoxic effects can be used in the clinical treatment of tumors remains to be established. Here, we evaluated the effects of fusarochromanone on the growth of human melanoma cells both in vitro and in vivo. In vitro, low concentrations (0.1–1 nmol/l) of fusarochromanone were found to be cytotoxic to many melanoma cell lines. In contrast, growth of normal melanocytes was inhibited only at much higher fusarochromanone concentrations (100–200 nmol/l). In vivo, the growth of melanoma cells implanted subcutaneously in immuno-compromised mice was significantly (P<0.05) reduced by daily administration of fusarochromanone. Immunohistological analyses indicated a significant (P<0.05) increase in the expression of active caspase-3 in tumor masses of mice treated with fusarochromanone, compared with controls. Together, these observations show that fusarochromanone increased apoptosis of tumor cells and reduced tumor growth in vivo. Therefore, the effects of fusarochromanone warrant further investigation as an adjuvant molecule to prevent growth and recurrence of melanomas.