Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats.

Enteric neurons and blood vessels form intricate networks throughout the gastrointestinal tract. To support the hypothesis of a possible interaction of both networks, we investigated whether primary mesenteric vascular cells (MVCs) and enteric nervous system (ENS)-derived cells (ENSc) depend on each other using two- and three-dimensional in vitro assays. In a confrontation assay, both cell types migrated in a target-oriented manner towards each other. The migration of MVCs was significantly increased when cultured in ENSc-conditioned medium. Co-cultures of ENSc with MVCs resulted in an improved ENSc proliferation and differentiation. Moreover, we analysed the formation of the vascular and nervous system in developing mice guts. It was found that the patterning of newly formed microvessels and neural stem cells, as confirmed by nestin and SOX2 stainings, is highly correlated in all parts of the developing gut. In particular in the distal colon, nestin/SOX2-positive cells were found in the tissues adjacent to the capillaries and in the capillaries themselves. Finally, in order to provide evidences for a mutual interaction between endothelial and neural cells, the vascular patterns of a RET((-/-)) knockout mouse model as well as human Hirschsprung's cases were analysed. In the distal colon of postnatal RET((-/-)) knockout mice, the vascular and neural networks were similarly disrupted. In aganglionic zones of Hirschsprung's patients, the microvascular density was significantly increased compared with the ganglionic zone within the submucosa. Taken together, these findings indicate a strong interaction between the enteric nervous and vascular system.

Hepatic stellate cells (HSCs) surround liver sinusoids and store retinol while they are quiescent. During fibrotic liver diseases and acute-on-chronic liver failure they change to the activated state in which they proliferate, lose their retinol content and deposit extracellular matrix molecules. The process of HSC activation is of utmost interest, but so far only insufficiently understood, because there is a lack of techniques to address the function of single HSCs in the tissue context. In this contribution, the potential of Raman micro-spectroscopy for the label-free detection of HSCs in mouse liver samples is demonstrated. First, culture-induced activation of primary mouse HSCs is followed in vitro and characterized by means of Raman spectroscopy. The HSC activation state is confirmed by immunofluorescence labeling of glial fibrillary acidic protein (GFAP) and α-smooth muscle actin (ASMA). As expected, the unique Raman spectrum of retinol in quiescent HSCs is lost during activation. Nevertheless, successful discrimination of HSCs from primary hepatocytes is possible during all states of activation. A classification model based on principal component analysis followed by linear discriminant analysis (PCA-LDA) of the lipid droplet Raman data yields a prediction accuracy of 99%. The in vitro results are transferred to fresh liver slices and freshly sampled livers. Quiescent HSCs and a HSC transforming from quiescent to activated state are identified based on their Raman signature. This provides valuable information on HSC activation state in the liver.

Hydrogen sulfide (H2S) is an endogenous gas with important physiological functions. The role of H2S in liver injury remains controversial. Exogenous H2S is protective in ischemia/reperfusion and burns, whereas the inhibition of endogenous H2S is protective in sepsis models. We have demonstrated increased oxygen consumption in the liver with low levels of exogenous H2S (<200ìM) via mitochondrial sulfide oxidation but inhibition at higher levels. In vivo H2S infused into the portal vein decreased hepatic tissue PO2. Thus, we hypothesized that H2S contributes to liver injury via exacerbation of cellular hypoxia. To test this, primary rat hepatocytes were subjected to one hour of hypoxia (PO2 < 25 mmHg) in the presence of the slow releasing H2S donor, GYY 4137 (GYY). Hypoxia caused significant cell death in hepatocytes plated at >75% confluence. Contrary to our hypothesis, GYY significantly improved cell viability following hypoxia (84% survival vs. 30% in control, P= 0.0017). Since mitochondrial stress contributes to cell injury, we tested whether GYY decreased mitochondrial reactive oxygen during hypoxia. GYY significantly reduced mitochondrial ROS in hypoxia as visualized by the mitochondrial ROS specific dye Mitosox (TM) (50.3 vs 31.3 units, P=0.006). Thus, H2S protects against hepatocellular injury through a reduction in mitochondrial‐derived reactive oxygen production. Supported by DK38201