Volume 129, Issue 5, Pages (November 2005)

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Volume 129, Issue 5, Pages 1686-1695 (November 2005) Microarray Analysis of Endothelial Differentially Expressed Genes in Liver of Cirrhotic Rats Sònia Tugues, Manuel Morales–Ruiz, Guillermo Fernandez–Varo, Josefa Ros, David Arteta, Javier Muñoz–Luque, Vicente Arroyo, Juan Rodés, Wladimiro Jiménez Gastroenterology Volume 129, Issue 5, Pages 1686-1695 (November 2005) DOI: 10.1053/j.gastro.2005.09.006 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 eNOS, RECA-1, and PECAM-1 expression in LECs. (A) Representative immunofluorescent images of eNOS and RECA-1 in LECs are shown with their corresponding phase-contrast images (n = 4, original magnification ×200). (B) LECs were analyzed by FACScan for PECAM-1 or incubated with IgG isotype control antibody. The percentage of cells expressing PECAM-1 was calculated as the percentage of cells that was located under the M1 window (n = 4). Gastroenterology 2005 129, 1686-1695DOI: (10.1053/j.gastro.2005.09.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Hierarchical clustering analysis of differentially regulated transcripts in cirrhosis. Gene regulation results are explained in the red-green code bar shown on the right. (A) Unsupervised hierarchical clustering of control and cirrhotic LECs samples. (B) Supervised 2-way hierarchical clustering was performed by using the 489 most significantly differentially expressed genes found in LECs from controls and LECs from cirrhotic rats. Gastroenterology 2005 129, 1686-1695DOI: (10.1053/j.gastro.2005.09.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Validation of differentially expressed genes. (A) Real-time RT-PCR for Mmp2, Selp, Bmp2, or Cnr2 were performed in LECs from control or cirrhotic rats. Each column represents mean values ± SEM (n = 3). *P < .05 compared with LECs from control rats. (B) Total RNA extracted from LECs was amplified by RT-PCR using VEGF-D primers. MW, molecular weight; RT, samples without retrotranscriptase enzyme. (C) LECs lysates (30 μg) from control (ct) and cirrhotic (ch) rats were analyzed by Western blotting with anti-eNOS antibody. Ponceau S Red staining is shown in the lower panel. Gastroenterology 2005 129, 1686-1695DOI: (10.1053/j.gastro.2005.09.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Functional validation of microarrays. (A) Extensive positive immunohistochemical staining for podoplanin was present in the vasculature of cirrhotic livers (arrows in b) as compared with control animals (a). Some vessels did not stain for podoplanin (arrowheads in a and b). Negative control staining is also shown (panels c and d for control and cirrhotic rats, respectively) (n = 3; original magnification, ×200). (B) Time-course analysis of VEGF-D and HPRT mRNA expression in livers from control and cirrhotic rats (n = 3). Gastroenterology 2005 129, 1686-1695DOI: (10.1053/j.gastro.2005.09.006) Copyright © 2005 American Gastroenterological Association Terms and Conditions