1. Volume 125, Issue 2, Pages 522-531 (August 2003)
Transduction of the liver with activated Akt normalizes portal pressure in cirrhotic rats 
Manuel Morales-ruiz, Pilar Cejudo-martı́n, Guillermo Fernández-varo, Sònia Tugues, Josefa Ros, Paolo Angeli, Francisca Rivera, Vicente Arroyo, Juan Rodés, William C Sessa, Wladimiro Jiménez 
Gastroenterology 
Volume 125, Issue 2, Pages (August 2003)
DOI: /S (03)

2. Figure 1
Impaired Akt and eNOS activation in the liver of cirrhotic animals. (A ) Liver tissue from control rats and cirrhotic rats were homogenated in lysis buffer and subjected to electrophoresis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with antibodies for P-Akt, Akt, P-eNOS, and total eNOS, as described in the Materials and Methods section. Representative Western blot analyses are shown. (B) Histograms reflect the densitometric ratios of P-Akt to Akt (left) and P-eNOS to eNOS (right) measured in 10 control and 10 cirrhotic livers. Data are shown as mean ± SEM. ∗P ≤ 0.01 compared with control rats.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
myr-Akt transduction of BAEC-induced eNOS phosphorylation and NO production. (A ) The myr-Akt mutant is based on the wild-type sequence of Akt with the exception that it is HA tagged and it incorporates an amino terminus containing a src myristoylation (myr) signal generated by PCR. This mutation allows the permanent phosphorylation of the Akt mutant in its amino acids threonine (Thr) 308 and serine (Ser) 473. These 2 amino acids have been substituted by alanine in the wild-type sequence to synthesize the HA-tagged inactive phosphorylation mutant Akt (AA-Akt). (B) BAECs were NO treated or infected with β-gal, AA-Akt, or myr-Akt adenoviruses as described in the Materials and Methods section. Cells were serum starved and NO production was quantified in the media by chemiluminescence. Finally, cells were harvested and cell lysates were subjected to immunoblot analysis with antibodies specific for P-eNOS, total eNOS, P-Akt, and total Akt (n = 3).
Gastroenterology  , DOI: ( /S (03) )

4. Figure 3
Adenovirus-mediated β-galactosidase gene expression in the liver. (A ) Control rats were killed after 3 days of infection with 5 × 1010 pfu of β-gal adenovirus. The liver, aorta, mesentery, lung, kidney, and heart tissues were collected and homogenized in lysis buffer to measure β-gal activity by using a chemiluminescent assay. Data are shown as mean ± SEM (n = 3). (B) Representative sections of livers from control rats treated with vehicle (left panel) or control rats infected with 5 × 1010 pfu of adenovirus expressing β-gal (right panel) are shown. The paraformaldehyde-fixed sections were embedded in OCT and stained histochemically by using X-gal as described in the Materials and Methods section. Original magnification ×200.
Gastroenterology  , DOI: ( /S (03) )

5. Figure 4
myr-Akt transduction in cirrhotic livers activates eNOS. (A ) Hepatocytes (Hep) and sinusoidal endothelial cells (SEC) were purified, as described in the Materials and Methods section, from liver of cirrhotic rats that were intravenously injected with myr-Akt adenoviruses (5 × 1010 pfu) 3 days earlier. Cells were lysated in lysis buffer and 30 μg of protein was subjected to electrophoresis and immunoblot analysis with specific antibodies for HA and eNOS (n = 2). (B) After cirrhotic rats were infected with adenovirus expressing β-gal or myr-Akt at a dose of 5 × 1010 pfu, immunoblot analyses were performed with P-eNOS, total eNOS, P-Akt, or HA antibodies, as described earlier. Representative Western blot analyses are shown. (C ) The intrahepatic concentration of cGMP from 10 control rats (ct), 10 cirrhotic rats (ch), 10 cirrhotic rats infected with β-gal (β-gal), and 10 cirrhotic rats infected with myr-Akt (myr-Akt) adenoviruses were determined by radioimmunoassay. Data are expressed as mean ± SEM. aP ≤ 0.01 compared with ch and bP ≤ 0.05 compared with β-gal.
Gastroenterology  , DOI: ( /S (03) )

6. Figure 5
myr-Akt transduction reduces PP and increases MAP in cirrhotic rats. After 3 days of adenoviral infection (5 × 1010 pfu), PP, MAP, and cardiac output were measured in cirrhotic rats. Data are shown as mean ± SEM (n = 10).
Gastroenterology  , DOI: ( /S (03) )

7. Figure 6
Histopathologic analysis after adenoviral treatment. Representative sections of livers from (A, B) control rats, (C, D) cirrhotic rats, (E, F ) cirrhotic rats infected with adenovirus expressing β-gal, and (G, H ) cirrhotic rats infected with adenovirus expressing myr-Akt are shown. Animals were killed 3 days after the infection and liver samples were collected for paraffin sections followed by H&E staining. Arrows denote fibrotic bands. Original magnification (A, C, E, G) 40×, and (B, D, F, H) 200×.
Gastroenterology  , DOI: ( /S (03) )