Michael B. Duncan

Fibroblasts Derive from Hepatocytes in Liver Fibrosis via Epithelial to Mesenchymal Transition

Activated fibroblasts are key contributors to the fibrotic extracellular matrix accumulation during liver fibrosis. The origin of such fibroblasts is still debated, although several studies point to stellate cells as the principal source. The role of adult hepatocytes as contributors to the accumulation of fibroblasts in the fibrotic liver is yet undetermined. Here, we provide evidence that the pro-fibrotic growth factor, TGF-β1, induces adult mouse hepatocytes to undergo phenotypic and functional changes typical of epithelial to mesenchymal transition (EMT). We perform lineage-tracing experiments using AlbCre. R26RstoplacZ double transgenic mice to demonstrate that hepatocytes which undergo EMT contribute substantially to the population of FSP1-positive fibroblasts in CCL4-induced liver fibrosis. Furthermore, we demonstrate that bone morphogenic protein-7 (BMP7), a member of the TGFβ superfamily, which is known to antagonize TGFβ signaling, significantly inhibits progression of liver fibrosis in these mice. BMP7 treatment abolishes EMT-derived fibroblasts, suggesting that the therapeutic effect of BMP7 was at least partially due to the inhibition of EMT. These results provide direct evidence for the functional involvement of adult hepatocytes in the accumulation of activated fibroblasts in the fibrotic liver. Furthermore, our findings suggest that EMT is a promising therapeutic target for the attenuation of liver fibrosis.

Biomolecular characterization and protein sequences of the Campanian hadrosaur B. canadensis.

The Birds and the Dinosaurs The extent to which primary tissues are preserved in ancient fossils remains controversial. Schweitzer et al. (p. 626; see the news story by Service) describe well-preserved tissues and primary collagen sequences from the femur of an 80-million-year-old hadrosaur. The fossil preserved structures resembling primary bone tissues and vessels. Both extracts and tissue pieces were analyzed in multiple laboratories by mass spectrometry, which revealed ancient collagen sequences that support a close relation between birds and dinosaurs. Analysis of well-preserved tissues from an 80-million-year-old hadrosaur supports the dinosaur-bird relationship. Molecular preservation in non-avian dinosaurs is controversial. We present multiple lines of evidence that endogenous proteinaceous material is preserved in bone fragments and soft tissues from an 80-million-year-old Campanian hadrosaur, Brachylophosaurus canadensis [Museum of the Rockies (MOR) 2598]. Microstructural and immunological data are consistent with preservation of multiple bone matrix and vessel proteins, and phylogenetic analyses of Brachylophosaurus collagen sequenced by mass spectrometry robustly support the bird-dinosaur clade, consistent with an endogenous source for these collagen peptides. These data complement earlier results from Tyrannosaurus rex (MOR 1125) and confirm that molecular preservation in Cretaceous dinosaurs is not a unique event.

Counterbalancing angiogenic regulatory factors control the rate of cancer progression and survival in a stage-specific manner

Whereas the roles of proangiogenic factors in carcinogenesis are well established, those of endogenous angiogenesis inhibitors (EAIs) remain to be fully elaborated. We investigated the roles of three EAIs during de novo tumorigenesis to further test the angiogenic balance hypothesis, which suggests that blood vessel development in the tumor microenvironment can be governed by a net loss of negative regulators of angiogenesis in addition to the well-established principle of up-regulated angiogenesis inducers. In a mouse model of pancreatic neuroendocrine cancer, administration of endostatin, thrombospondin-1, and tumstatin peptides, as well as deletion of their genes, reveal neoplastic stage-specific effects on angiogenesis, tumor progression, and survival, correlating with endothelial expression of their receptors. Deletion of tumstatin and thrombospondin-1 in mice lacking the p53 tumor suppressor gene leads to increased incidence and reduced latency of angiogenic lymphomas associated with diminished overall survival. The results demonstrate that EAIs are part of a balance mechanism regulating tumor angiogenesis, serving as intrinsic microenvironmental barriers to tumorigenesis.

Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye

The ciliary body (CB) of the mammalian eye is responsible for secreting aqueous humor to maintain intraocular pressure, which is elevated in the eyes of glaucoma patients. It contains a folded two-layered epithelial structure comprising the nonpigmented inner ciliary epithelium (ICE), the pigmented outer ciliary epithelium (OCE), and the underlying stroma. Although the CB has an important function in the eye, its morphogenesis remains poorly studied. In this study, we show that conditional inactivation of the Jagged 1 (Jag1)-Notch2 signaling pathway in the developing CB abolishes its morphogenesis. Notch2 is expressed in the OCE of the CB, whereas Jag1 is expressed in the ICE. Conditional inactivation of Jag1 in the ICE or Notch2 in the OCE disrupts CB morphogenesis, but neither affects the specification of the CB region. Notch2 signaling in the OCE is required for promoting cell proliferation and maintaining bone morphogenetic protein (BMP) signaling, both of which have been suggested to be important for CB morphogenesis. Although Notch and BMP signaling pathways are known to cross-talk via the interaction between their downstream transcriptional factors, this study suggests that Notch2 maintains BMP signaling in the OCE possibly by repressing expression of secreted BMP inhibitors. Based on our findings, we propose that Jag1-Notch2 signaling controls CB morphogenesis at least in part by regulating cell proliferation and BMP signaling.

Mass Spectrometry and Antibody-Based Characterization of Blood Vessels from Brachylophosaurus canadensis

Structures similar to blood vessels in location, morphology, flexibility, and transparency have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty osteonal network after degradation of original organic components. Here, we test the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence. Two lines of evidence support this hypothesis. First, peptide sequencing of Brachylophosaurus canadensis blood vessel extracts is consistent with peptides comprising extant archosaurian blood vessels and is not consistent with a bacterial, cellular slime mold, or fungal origin. Second, proteins identified by mass spectrometry can be localized to the tissues using antibodies specific to these proteins, validating their identity. Data are available via ProteomeXchange with identifier PXD001738.

Type XVIII collagen is essential for survival during acute liver injury in mice

SUMMARY The regenerative response to drug- and toxin-induced liver injury induces changes to the hepatic stroma, including the extracellular matrix. Although the extracellular matrix is known to undergo changes during the injury response, its impact on maintaining hepatocyte function and viability in this process remains largely unknown. We demonstrate that recovery from toxin-mediated injury is impaired in mice deficient in a key liver extracellular matrix molecule, type XVIII collagen, and results in rapid death. The type-XVIII-collagen-dependent response to liver injury is mediated by survival signals induced by α1β1 integrin, integrin linked kinase and the Akt pathway, and mice deficient in either α1β1 integrin or hepatocyte integrin linked kinase also succumb to toxic liver injury. These findings demonstrate that type XVIII collagen is an important functional component of the liver matrix microenvironment and is crucial for hepatocyte survival during injury and stress.

Parstatin, a novel protease-activated receptor 1-derived inhibitor of angiogenesis

Angiogenesis, the process of forming new blood vessels, is a well established and clinically relevant feature of a variety of disease states. Whether blood vessels sprout in a given tissue environment depends on the balance between factors that stimulate angiogenesis and those that impede it. Potent pro-angiogenic factors such as vascular endothelial growth factor (VEGF) have been identified, validated, and successfully used in the clinic. Likewise, anti-angiogenic factors are also emerging as biologically relevant and therapeutically useful entities. PAR1 is a G protein-coupled receptor (GPCR) that participates in hemostasis and vascular development and that mediates the angiogenic activity of thrombin. PAR1 is activated through proteolytic cleavage of its first forty-one extracellular residues by a variety of proteases, most notably thrombin. However, little effort has focused on the forty-one-residue peptide fragment liberated during PAR1 activation. Tsopanoglou and colleagues have now demonstrated that this peptide, parstatin, has intriguing antiangiogenic activity, and, in a follow-up study, they demonstrate its potential pharmacological utility using a rat model of ischemic heart disease.

929 Type XVIII Collagen Plays a Critical Role in Liver Regeneration and is Regulated by Transforming Growth Factor Beta 1

Background: The intestinal mucus layer protects the epithelium against noxious agents and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin-2 (Muc2), secreted by goblet cells of the intestine. Experimental alcoholic liver disease is dependent on the translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. The aim of our study was to investigate the impact of the intestinal mucus layer and in particular Muc2 on alcoholic liver disease. Methods and Results: We used the Tsukamoto-French mouse model which involves continuous intragastric feeding of isocaloric diet (n=4-5) or alcohol for 1 week in Muc2-/(n=9) and wildtype (WT) mice (n=10). Muc2 was abundantly expressed in the small and large intestine of WT mice, but undetectable in the liver. The intestinal mucus layer was considerably thinner in Muc2 deficient mice as shown by PAS staining. Alcohol feeding did not result in a compensatory upregulation of other intestinal mucins in Muc2-/mice. After alcohol feeding mice deficient in Muc2 were protected from steatohepatitis as evidenced by significantly lower ALT levels and hepatic triglyceride concentrations. In addition, hepatic oxidative stress was significantly reduced in Muc2-/mice following intragastric alcohol feeding as shown by immunohistochemistry for 4-hydroxynonenal (4-HNE) and by TBARS assay. There was no significant difference in plasma alcohol levels or hepatic alcohol metabolizing enzymes alcohol dehydrogenase 1 (ADH1) and cytochrome p450 enzyme 2E1 (Cyp2E1) in alcohol fed Muc2-/as compared to WT mice. Most notably, Muc2-/mice had significantly lower systemic plasma LPS levels after alcohol feeding. In contrast to WT mice, Muc2-/mice did not exhibit intestinal bacterial overgrowth, but a higher amount of intestinal probiotic Lactobacillus after alcohol administration as shown by qPCR for 16S rRNA and Lactobacillus spp. The antimicrobial proteins Reg3b and Reg3g were found to be expressed at significantly higher levels in the proximal small intestine in isocaloric diet and alcohol fed Muc2-/mice relative to WT mice as assessed by qPCR and Western blotting. As Reg3b and Reg3g are bactericidal c-type lectins, an increase in their expression might contribute to the observed reduction of the bacterial burden and suppression of bacterial overgrowth in the intestine. Conclusion: Intestinal mucin-2 deficiency protects from alcoholic steatohepatitis. We suggest a pathway that involves higher expression of enteric antimicrobial molecules which suppresses alcohol-associated intestinal bacterial overgrowth. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation and cause less alcoholic liver disease.

Basement membrane derived inhibitors of angiogenesis

Blood vessel growth during development and disease is likely governed by the balance between proand antiangiogenic factors. Numerous reports have focused on the important role of various growth factors during angiogenesis. Peptide fragments derived from basement membranes constitute a relatively new and expanding class of antiangiogenic factors with a potential for clinical relevance. These factors have been studied in a variety of disease models, and genetic evidence for their role in controlling angiogenesis is beginning to be realized. This chapter highlights several of these factors and their mechanism of action as we understand them to date. Gaining additional insight into the full compliment of these antiangiogenic fragments from basement membranes, how they are derived, and their full mechanism of action represents an important challenge in vascular biology today. The vascular basement membrane (VBM) is a complex structure composed of a variety of functionally diverse glycoproteins and proteoglycans (Fig. 11.1) [1]. The primary molecules detected in the VBM are the heparan sulfate proteoglycans (such as perlecan and type XVIII collagen), laminin, niodogen, entactin, fibulin, as well as type IV collagen. While providing structural support to the vessel, these molecules also serve important functional roles in endothelial cell (EC) signaling and adhesion. Therefore, these basement membrane molecules play a critical role in EC proliferation, migration, morphogenesis, survival, and vessel stability [2]. Much of the interactions and subsequent biological activity of VBM macromolecules are due to their ability to engage endothelial cell surface receptors, namely the integrins [3]. In some instances, evidence suggests that the antiangiogenic functionality of basement membrane molecules is elicited only when cryptic fragments of these molecules are revealed through proteolytic processing [4]. These findings highlight an essential role for (1) understanding the structural organization of the VBM in normal and disease states, (2) identifying the full complement of antiangiogenic entities derived from the VBM and other basement membranes, and (3) defining the mechanisms by which changes in the vascular microenvironment, and subsequent changes in the balance of angiogenic mediators, occur. This chapter highlights the origin and biological activity of some recently discovered basement membrane derived antiangiogenic molecules.