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Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding α(v) integrin subunit because various α(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of β₃, β₅ or β₆ integrins or conditional loss of β₈ integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature., Author(s): Michael Vanlandewijck [1, 3]; Liqun He [2]; Maarja Andaloussi Me [3]; Johanna Andrae [3]; Koji Ando [3]; Francesca Del Gaudio [4]; Khayrun Nahar [3]; Thibaud Lebouvier [3, 5]; Brbara [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.10.049 Byline: Elisabeth Raschperger (a), Etienne P.A. Neve (b), Annika Wernerson (c), Kjell Hultenby (d), Ralf F. Pettersson (a), Arindam Majumdar (e) Keywords: CXADR; CAR; CTX; Zebrafish; Pronephros; Podocyte; Tubulogenesis; Microvillus; Tight junction Abstract: The coxsackie and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily and a component of vertebrate tight junctions. CAR protein is widely expressed in fish and mammals in organs of epithelial origin suggesting possible functions in epithelial biology. In order to gain insight into its function, we knocked the CAR gene down in zebrafish using antisense morpholinos. We identified a requirement for CAR in the terminal differentiation of glomerular podocytes and pronephric tubular epithelia. Podocytes differentiate in CAR morphants but are not able to elaborate a regularly patterned architecture of foot processes. In the tubules, CAR was required for the apposition of plasma membranes from adjacent epithelial cells but did not appear to be necessary for the formation of tight junctions. Additionally, tubular epithelia lacking CAR were not able to elaborate apical brush border microvilli. These results establish a requirement for CAR in the terminal differentiation of renal glomerular and tubular cell types. Author Affiliation: (a) Ludwig Institute for Cancer Research, Karolinska Institutet, Stockholm, Sweden (b) Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden (c) Department of Laboratory Medicine, Division of Pathology, Karolinska Institute Karolinska University Hospital, Huddinge, Sweden (d) Department of Pathology, Karolinska University Hospital/Huddinge, Karolinska Institutet, Stockholm, Sweden (e) Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Article History: Received 24 May 2007; Revised 26 October 2007; Accepted 30 October 2007

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding αv integrin subunit because various αv-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of β3, β5 or β6 integrins or conditional loss of β8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the αv integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of αv-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases. [ABSTRACT FROM AUTHOR]

The expression of the cyclin-dependent kinase inhibitor p15INK4B in normal cells after induction with TGF-β1, or following overexpression from an adenovirus-encoded cDNA, appears on an SDS-polyacrylamide gel as a doublet. Here, the underlying mechanism behind the synthesis of the two species has been studied. By expressing cDNAs truncated at their 5’ end, we found that the synthesis of the more slowly migrating form, called p15.5INK4B, is dependent on a sequence upstream of the first AUG codon thought to initiate translation of p15INK4B. Two potential, in frame, alternative upstream initiation codons, ACG and GUG, were individually changed to GCA encoding alanine. Analysis by in vitro translation, or immunoblotting of lysates from transfected 293 cells, showed that translation of p15.5INK4B is initiated at the GUG located 13 codons upstream of the first AUG. When this AUG was mutated, p15INK4B was no longer made. Instead, a shorter form, initiated at an in frame AUG located seven codons downstream, was synthesized. Finally, when both these AUGs were mutated, only p15.5INK4B was generated. Both p15INK4B and p15.5INK4B bound to CDK4 and CDK6, inhibited DNA synthesis, and caused replicative senescence of a human glioma cell line. We thus conclude that p15INK4B and p15.5INK4B, encoded by the CDKN2B gene, are functionally indistinguishable as based on these assays. Oncogene (2000) 19, 1724–1728 [ABSTRACT FROM AUTHOR]