Volume 117, Issue 5, Pages 1222-1228 (November 1999) Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat  Vijay Shah*,‡,§, Murat Toruner§, Faddi Haddad§,∥, Gregory Cadelina§, Andreas Papapetropoulos¶, Kenneth Choo*,¶, William C. Sessa‡,¶, Roberto J. Groszmann*,‡,§  Gastroenterology  Volume 117, Issue 5, Pages 1222-1228 (November 1999) DOI: 10.1016/S0016-5085(99)70408-7 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Impaired NOx production, pressure regulation, and NOS activity in cirrhotic liver. (A) NOx in the perfused liver was significantly less in cirrhotic than in control animals (*P < 0.05). Addition of L-NMMA to the perfusate significantly inhibited NOx production in both groups (**P < 0.05; n = 16 control, n = 8 cirrhosis). ■, Before L-NMMA; □, after L-NMMA). (B) Flow-induced increases in perfusion pressure in both experimental groups. The increase in perfusion pressure was greater in cirrhotic animals as demonstrated by a significantly greater perfusion pressure at each flow rate (*P < 0.05) and a significantly greater pressure-flow slope (k) (**P < 0.05; n = 4 control [○], n = 4 cirrhosis [●]). (C) NOS activity, measured by 3H-labeled L-citrulline generation, was assessed in homogenized liver tissue from pairs of control (n = 5) and cirrhotic (n = 5) rats studied in parallel, as described in Materials and Methods. NOS activity was significantly reduced in liver tissue from cirrhotic animals compared with parallel control animals when normalized per milligram of protein (left panel) and per milligram of liver tissue (right panel, *P < 0.05, Wilcoxon signed-rank test). Gastroenterology 1999 117, 1222-1228DOI: (10.1016/S0016-5085(99)70408-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 eNOS protein levels and cellular localization are not changed in cirrhotic rat liver compared with control rat liver. (A) Left panel, a representative blot showing a lack of reduction in eNOS protein levels in cirrhotic liver tissue compared with control tissue in immunoprecipitated samples (IP). Quantitation of the immunoprecipitation is demonstrated by the lack of eNOS signal after reprecipitation of eNOS from postimmunoprecipitated supernatants (PIP). Right panel, densitometric analysis of blots (n = 6, each group). (B) Left, in control tissue, peroxidase staining for eNOS is visualized predominantly in hepatic sinusoidal and venular ECs (original magnification 200×). Right, in cirrhotic liver, regenerative nodules and fibrosis are evident. A similar pattern of staining is observed in sinusoidal and venular endothelium. Gastroenterology 1999 117, 1222-1228DOI: (10.1016/S0016-5085(99)70408-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Caveolin-1 association with eNOS is enhanced in cirrhotic liver. Protein lysates were prepared from control and cirrhotic liver and prepared for gel electrophoresis or, alternatively, first for eNOS immunoprecipitation. (A) Left, representative blot showing enhanced caveolin-1 association and diminished calmodulin association with eNOS in cirrhotic liver tissue compared with control (n = 3 control, n = 3 cirrhosis; performed in duplicate). Higher-molecular-weight bands detected on caveolin-1 Western blots represent SDS-resistant heteromeric and homomeric caveolin complexes, as described previously.15,25 Right, densitometric analysis showing a severalfold increase in caveolin-1 bound to eNOS. (B) Left, representative blot showing increased caveolin-1 protein levels in cirrhotic liver tissue compared with control (n = 3 control, n = 3 cirrhosis; performed in duplicate). Calmodulin protein levels are similar in control and cirrhotic liver tissue. Right, by densitometric analysis, a severalfold increase in caveolin-1 protein levels is detected in total liver lysates. Gastroenterology 1999 117, 1222-1228DOI: (10.1016/S0016-5085(99)70408-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Caveolin-1 expression is enhanced in ECs in cirrhotic liver. Immunoperoxidase staining for caveolin-1 was performed in control and cirrhotic liver fixed in formalin and processed in parallel. (A) Negative control sections were incubated with appropriate serum substituted for the primary antibody (original magnification 500×). Left panel, in control liver tissue incubated with caveolin-1 PAb, peroxidase staining for caveolin-1 is shown in (B) portal venule (arrow), (C) central venule (arrow), and (D) lobule. Note the low levels of detection in the control liver. (A) Right panel, in cirrhotic liver tissue, negative control sections were incubated with appropriate serum substituted for the primary antibody. In cirrhotic liver tissue incubated with caveolin-1 PAb, enhanced peroxidase staining for caveolin-1 is detected in (B) portal venule (arrow), (C) central venule (arrow), and (D) lobule (original magnification 500×). Gastroenterology 1999 117, 1222-1228DOI: (10.1016/S0016-5085(99)70408-7) Copyright © 1999 American Gastroenterological Association Terms and Conditions