Impact of hyperglycemia and acute pancreatitis on the receptor for advanced glycation endproducts.

Since hyperglycemia aggravates acute pancreatitis and also activates the receptor for advanced glycation endproducts (RAGE) in other organs, we explored if RAGE is expressed in the pancreas and if its expression is regulated during acute pancreatitis and hyperglycemia. Acute pancreatitis was induced by cerulein in untreated and streptozotocin treated diabetic mice. Expression of RAGE was analyzed by Western blot and immunohistochemistry. To evaluate signal transduction the phosphorylation of ERK1/ERK2 was assessed by Western blot and the progression of acute pancreatitis was monitored by evaluation of lipase activity and the pancreas wet to dry weight ratio. RAGE is mainly expressed by acinar as well as interstitial cells in the pancreas. During acute pancreatitis infiltrating inflammatory cells also express RAGE. Using two distinct anti-RAGE antibodies six RAGE proteins with diverse molecular weight are detected in the pancreas, whereas just three distinct RAGE proteins are detected in the lung. Hyperglycemia, which aggravates acute pancreatitis, significantly reduces the production of two RAGE proteins in the inflamed pancreas.

Development of Adenoviral Delivery Systems to Target Hepatic Stellate Cells In Vivo.

Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGFp) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGFp to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGFp to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGFp transduce activated HSCs better than Ad.GFP-PEG-NGFp. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGFp.

Heterogeneity of the intrahepatic portal venous blood flow: impact on hepatocyte transplantation.

BACKGROUND: The poor repopulation rate of the liver by transplanted hepatocytes markedly hampers liver cell therapy, which might be due to a limited sequestration of cells within the hepatic microvasculature. We therefore present intravital fluorescence microscopic data of transplanted hepatocytes immediately after portal venous injection demonstrating their intrahepatic distribution. METHODS: Male Wistar rats were transplanted with freshly isolated, rhodamine 123 labelled, primary rat hepatocytes. Cells (10(6) in 0.5 ml) were slowly injected via a catheter in the V. lienalis over 2 min. Their distribution in the left lateral liver lobe was visualized simultaneously as well as over the following 30 min by intravital fluorescence microscopy. In a second set of animals green fluorescent microspheres exhibiting a size of 15 µm were injected and observed identically. For further analyses of portal venous blood flow distribution sodium fluorescein was injected via the V. lienalis as well as via the V. jugularis. RESULTS: In vivo imaging allowed the clear detection and observation of hepatocytes flowing into the liver and forming microemboli, which are trapped particularly in small distal portal branches. To a minor extent they were trapped as solitary cells in the periportal zone of sinusoids. Most interestingly, the distribution of cells within the liver was highly heterogeneous, as wide areas of acini were found free of transplanted cells after portal venous injection, while neighbouring areas showed disproportionately high hepatocyte occurrence. To further investigate this phenomenon sodium fluorescein was injected via the V. lienalis instead and an identical heterogeneous distribution pattern with clear anatomical borders defining highly, semi, and non-portal venous perfused liver acini could be observed. In contrast, systemic injection of sodium fluorescein via the V. jugularis in the same animals resulted in a homogenous dispersion within the liver. CONCLUSION: Using in vivo fluorescence microscopy and exclusive portal venous injection of a fluorescent dye, we provide evidence for the existence of liver areas, differentially supplied by portal venous blood. As a consequence, hepatocytes transplanted via the portal tract are very heterogeneously distributed within the liver. This observation forces us to reconsider our current knowledge on (i) monitoring engrafted cells, (ii) the optimal hepatocyte number to be transplanted, (iii) portal hypertension after cell injection, and last but not least (iv) the optimal transplantation route. Moreover, the established model for in vivo visualization of transplanted hepatocytes allows development of new therapeutic strategies facilitating an improved engraftment of cells.