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In JoVE (1)

Other Publications (5)

Articles by Xiaogang Hou in JoVE

 JoVE Bioengineering

Tissue Engineering of the Intestine in a Murine Model

1Children's Hospital Los Angeles, Division of Pediatric Surgery, Saban Research Institute, Keck School of Medicine of the University of Southern California


JoVE 4279

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Other articles by Xiaogang Hou on PubMed

Angiogenic Gene Therapy for Experimental Critical Limb Ischemia: Acceleration of Limb Loss by Overexpression of Vascular Endothelial Growth Factor 165 but Not of Fibroblast Growth Factor-2

Recent studies suggest the possible therapeutic effect of intramuscular vascular endothelial growth factor (VEGF) gene transfer in individuals with critical limb ischemia. Little information, however, is available regarding (1) the required expression level of VEGF for therapeutic effect, (2) the related expression of endogenous angiogenic factors, including fibroblast growth factor-2 (FGF-2), and (3) the related adverse effects due to overexpression of VEGF. To address these issues, we tested effects of overexpression of VEGF165 using recombinant Sendai virus (SeV), as directly compared with FGF-2 gene transfer. Intramuscular injection of SeV strongly boosted FGF-2, resulting in significant therapeutic effects for limb salvage with increased blood perfusion associated with enhanced endogenous VEGF expression in murine models of critical limb ischemia. In contrast, VEGF165 overexpression, 5-times higher than that of baseline on day 1, also strongly evoked endogenous VEGF in muscles, resulting in an accelerated limb amputation without recovery of blood perfusion. Interestingly, viable skeletal muscles of either VEGF165- or FGF-2-treated ischemic limbs showed similar platelet-endothelial cell adhesion molecule-1-positive vessel densities. Maturation of newly formed vessels suggested by smooth muscle cell actin-positive cell lining, however, was significantly disturbed in muscles with VEGF. Further, therapeutic effects of FGF-2 were completely diminished by anti-VEGF neutralizing antibody in vivo, thus indicating that endogenous VEGF does contribute to the effect of FGF-2. These results suggest that VEGF is necessary, but should be delicately regulated to lower expression to treat ischemic limb. The therapeutic effect of FGF-2, associated with the harmonized angiogenic effects seen with endogenous VEGF, provides important insights into therapeutic angiogenesis.

Mutations in Sendai Virus Variant F1-R That Correlate with Plaque Formation in the Absence of Trypsin

With the emergence of new viruses, such as the SARS virus and the avian influenza virus, the importance of investigations on the genetic basis of viral infections becomes clear. Sendai virus causes a localized respiratory tract infection in rodents, while a mutant, F1-R, causes a systemic infection. It has been suggested that two determinants are responsible for the systemic infection caused by F1-R [Okada et al (1998) Arch Virol 143:2343-2352]. The primary determinant of the pantropism is the enhanced proteolytic cleavability of the fusion (F) protein of F1-R, which allows the virus to undergo multiple rounds of replication in many different organs, whereas wild-type virus can only undergo multiple rounds of replication in the lungs. The enhanced cleavability of F1-R F was previously attributed to an amino acid change at F115 that is adjacent to the cleavage site at amino acid 116. Secondly, wild-type virus buds only from the apical domain of bronchial epithelium, releasing virus into the lumen of the respiratory tract, whereas F1-R buds from both apical and basolateral domains. Thus, virus is released into the basement membrane where it can easily gain access to the bloodstream for dissemination. The microtubule disruption is attributed to two amino acid differences in M protein. To confirm that the F and M gene mutations described above are solely responsible for the phenotypic differences seen in wild-type versus F1-R infections, reverse genetics was used to construct recombinant Sendai viruses with various combinations of the mutations found in the M and F genes of F1-R. Plaque assays were performed with or without trypsin addition. A recombinant virus containing all F1-R M and F mutations formed plaques in LLC-MK2 cells and underwent multiple cycles of replication without trypsin addition. To clarify which mutation(s) are necessary for plaque formation, plaque assays were done using other recombinant viruses. A virus with only the F115 change, which was previously thought to be the only change important for plaque formation of F 1-R F, did not confer upon the virus the ability to form plaques without the addition of trypsin. Another virus with the F115 and both M changes gave the same result. Therefore, more than one mutation in the F gene contributes to the ability of F1-R to form plaques without trypsin addition.

The Critical Role of AKT2 in Hepatic Steatosis Induced by PTEN Loss

Insulin signaling in the liver leads to accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Deletion of the phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) reduces PIP3 levels and leads to fatty liver development. The purpose of this study was to investigate the mechanisms underlying lipogenesis that result from PIP3 accumulation using liver Pten-deletion mice. To explore the role of AKT2, the major liver AKT isoform in steatosis induced by deletion of Pten, we created mice lacking both Pten and Akt2 in hepatocytes and compared the effect of deleting Akt2 and Pten in the double mutants to the Pten deletion mice alone. Hepatic lipid accumulation was significantly reduced in mice lacking both PTEN and AKT2, as compared with Pten mutant mice alone. This effect was due to the role of AKT2 in maintaining expression of genes involved in de novo lipogenesis. We showed that lipid accumulation in the double mutant hepatocytes was partially reversed by expression of constitutive active FOXO1, a transcription factor downstream of AKT not dependent on inhibition of atypical protein kinase C. In summary, this study delineated regulation of lipid metabolism by PI3K signaling pathway by showing that AKT mediates PIP3 accumulation (mimicked by PTEN loss) induced lipid deposition in the liver and provided an important molecular mechanism for insulin-regulated hepatic lipogenesis.

Age-changes in Gene Expression in Primary Mixed Glia Cultures from Young Vs. Old Rat Cerebral Cortex Are Modified by Interactions with Neurons

Astrocytic GFAP expression increases during normal aging in many brain regions and in primary astrocyte cultures derived from aging rodent brains. As shown below, we unexpectedly found that the age-related increase of GFAP expression was suppressed in mixed glia (astrocytes+microglia). However, the age-related increase of GFAP was observed when E18 neurons were co-cultured with mixed glia. Thus, the presence of microglia can suppress the age-related increase of GFAP, in primary cultures of astrocytes. To more broadly characterize how aging and co-culture with neurons alters glial gene expression, we profiled gene expression in mixed glia from young (3 mo) and old (24 mo) male rat cerebral cortex by Affymetrix microarray (Rat230 2.0). The majority of age changes were independent of the presence of neurons. Overall, the expression of twofold more genes increased with age than decreased with age. The minority of age changes that were either suppressed or revealed by the presence of neurons may be useful to analyze glial-neuron interaction during aging. Some in vitro changes are shared with those of aging rat hippocampus in studies from the Landfield group (Rowe et al., 2007; Kadish et al., 2009).

Human Tissue-engineered Colon Forms from Postnatal Progenitor Cells: an in Vivo Murine Model

Aim: Loss of colon reservoir function after colectomy can adversely affect patient outcomes. In previous work, human fetal intestinal cells developed epithelium without mesenchyme following implantation in mice. However, for humans, postnatal tissue would be the preferred donor source. We generated tissue-engineered colon (TEC) from postnatal human organoid units. Materials & methods: Organoid units were prepared from human colon waste specimens, loaded onto biodegradable scaffolds and implanted into immunocompromised mice. After 4 weeks, human TEC was harvested. Immunofluorescence staining confirmed human origin, identified differentiated epithelial cell types and verified the presence of supporting mesenchyme. Results: Human TEC demonstrated a simple columnar epithelium. Immunofluorescence staining demonstrated human origin and the three differentiated cell types of mature colon epithelium. Key mesenchymal components (smooth muscle, intestinal subepithelial myofibroblasts and ganglion cells) were seen. Conclusion: Colon can form from human progenitor cells on a scaffold in a mouse host. This proof-of-concept experiment is an important step in transitioning TEC to human therapy.

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