In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity.

Recent success in clinical hepatocyte transplantation therapy has encouraged further investigation into bioengineering hepatic tissues in vivo. Engineering tissues in the subcutaneous space is an attractive method; however, hepatocyte survival has been transient due to insufficient vascular network formation. To establish a vascularized cavity, we created a polyethylene terephthalate mesh device coated with poly(vinylalcohol) that allowed for the gradual release of basic fibroblast growth factor (bFGF), a potent angiogenic factor. The efficacy of the bFGF-releasing device in inducing vascular network formation in the subcutaneous space was observed in mouse and rat studies. Isolated mouse hepatocytes transplanted into newly vascularized subcutaneous cavities allowed for persistent survival up to 120 days. In the absence of a vascularized compartment, the survival of the transplanted hepatocytes was markedly diminished. Functional maintenance of the engineered hepatic tissues was confirmed by high expression of liver-specific mRNAs and proteins. These engineered hepatic tissues have the ability to take up inoculated compounds and express strong induction of drug-metabolizing enzymes, demonstrating functional relevance as a metabolic tissue. In conclusion, we have created a novel technology to engineer functionally active hepatic tissues in the subcutaneous space, which will likely facilitate hepatocyte-based therapies.

Heterotopically transplanted hepatocyte survival depends on extracellular matrix components.

The novel approach of tissue engineering to treat many forms of liver diseases using hepatocytes requires sufficient numbers and sustained survival of the transplanted cells. It has been shown that providing extracellular matrix components extracted from Engelbreth-Holm-Swarm cells (EHS-ECMs) to heterotopically transplanted hepatocytes allows significantly greater hepatocyte survival. We investigated the survival and morphology of hepatocytes and EHS-ECMs transplanted under the kidney capsule compared with hepatocytes with growth factor-reduced EHS-ECMs in mice. Both the EHS-ECMs and growth factor-reduced EHS-ECMs showed a large number of surviving hepatocytes under the kidney capsule without any intergroup differences. Histologically, transplanted hepatocytes in both groups retained their characteristic morphologies and formed small liver tissues. These data indicate that extracellular matrix components are the predominant factor in EHS-ECMs required to maintain hepatocytes at heterotopic sites.

Route of feeding influences the production and expression of tumor necrosis factor alpha in burned rats.

The effect of nutritional route on tumor necrosis factor alpha (TNF-alpha) production in burned rats was examined. Scald burns covering about 30% of the whole body surface area were inflicted on 43 male Wistar rats weighing about 200g. The animals were divided into three groups: CHOW (n = 10), total parenteral nutrition (TPN) (n = 22), and total enteral nutrition (TEN) (n = 11), continuously given a chow diet, TPN solution, or an enteral diet, respectively, for 7 days after the burn injury. The rate of detection of TNF-alpha in plasma on day 7 was significantly higher in the TPN group than in the CHOW or TEN groups. The messenger RNA (mRNA) expression of TNF-alpha was significantly increased in the spleen, lungs, liver, and ileum of the rats receiving TPN compared with the CHOW and TEN rats. On the other hand, the expression of TNF-alpha mRNA was markedly decreased in the thymus of the TPN group compared with the CHOW group. The mortality rate in the TPN group (63.6%) was higher than that in the CHOW (0.0%) or TEN (27.3%) groups on day 7 after burn injury. These data suggest that TPN increases the expression of TNF-alpha mRNA in organ tissues and systemic TNF-alpha production, and reduces the survival rate of rats after thermal injury, but TEN does not.

Cap ribose methylation of c-mos mRNA stimulates translation and oocyte maturation in Xenopus laevis.

In Xenopus oocytes, progesterone stimulates the cytoplasmic polyadenylation and resulting translational activation of c-mos mRNA, which is necessary for the induction of oocyte maturation. Although details of the biochemistry of polyadenylation are beginning to emerge, the mechanism by which 3' poly(A) addition stimulates translation initiation is enigmatic. A previous report showed that polyadenylation induced cap-specific 2'-O-methylation, and suggested that this 5' end modification was important for translational activation. Here, we demonstrate that injected c-mos RNA undergoes polyadenylation and cap ribose methylation. Inhibition of this methylation by S-isobutylthioadenosine (SIBA), a methyltransferase inhibitor, has little effect on progesterone-induced c-mos mRNA polyadenylation or general protein synthesis, but prevents the synthesis of Mos protein as well as oocyte maturation. Maturation can be rescued, however, by the injection of factors that act downstream of Mos, such as cyclin A and B mRNAs. Most importantly, we show that the translational efficiency of injected mRNAs containing cap-specific 2'-O-methylation (cap I) is significantly enhanced compared to RNAs that do not contain the methylated ribose (cap 0). These results suggest that cap ribose methylation of c-mos mRNA is important for translational recruitment and for the progression of oocytes through meiosis.

Cytoplasmic 3' poly(A) addition induces 5' cap ribose methylation: implications for translational control of maternal mRNA.

During the early development of many animal species, the expression of new genetic information is governed by selective translation of stored maternal mRNAs. In many cases, this translational activation requires cytoplasmic poly(A) elongation. However, how this modification at the 3' end of an mRNA stimulates translation from the 5' end is unknown. Here we show that cytoplasmic polyadenylation stimulates cap ribose methylation during progesterone-induced oocyte maturation in Xenopus laevis. Translational recruitment of a chimeric reporter mRNA that is controlled by cytoplasmic polyadenylation coincides temporally with cap ribose methylation during this period. In addition, the inhibition of cap ribose methylation by S-isobutyladenosine significantly reduces translational activation of a reporter mRNA without affecting the increase of general protein synthesis or polyadenylation during maturation. These results provide evidence for a functional interaction between the termini of an mRNA molecule and suggest that 2'-O-ribose cap methylation mediates the translational recruitment of maternal mRNA.

Maturation of Xenopus laevis oocyte by progesterone requires poly(A) tail elongation of mRNA.

Meiotic maturation of Xenopus laevis oocytes by progesterone requires translation of stored maternal mRNAs. We investigated the role of poly(A) tail elongation of mRNAs during this process using cordycepin, which inhibits poly(A) tail elongation of mRNAs. When oocytes were treated with the buffer containing 10 mM cordycepin for 12 h, concentration of 3'-dATP in cytosol of oocytes increased to 0.7 mM, while that of ATP remained constant at around 1.2 mM. Incorporation of [32P]AMP into poly(A) mRNA was inhibited almost completely by this treatment. Progesterone-induced germinal vesicle breakdown (GVBD) was also abolished. Dose dependence of inhibition of progesterone-induced GVBD on cordycepin was similar to that of [32P]AMP incorporation into poly(A) mRNA. However, maturation-promoting factor-induced GVBD was unaffected by treatment of oocytes with cordycepin. Furthermore, the inhibition of GVBD by cordycepin was rescued by removal of cordycepin even in the presence of actinomycin D. Therefore, we concluded that poly(A) tail elongation of mRNA is required for induction of meiotic maturation of X. laevis oocytes. In addition, progesterone induced a 2.7-fold activation of [32P]AMP incorporation into the poly(A) tail of mRNA after a lag period of 3 h whereas GVBD was induced after 6-8 h from the progesterone treatment. Syntheses of most of the proteins were unaffected by treatment of oocytes with progesterone or cordycepin. However, syntheses of several proteins were increased or decreased by progesterone and cordycepin treatment.