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Assessment of a dual regulatory role for NO in liver regeneration after partial hepatectomy: protection against apoptosis and retardation of hepatocyte proliferation

Miriam Zeini, Sonsoles Hortelano, Paqui G. Través, Alicia G. Gómez-Valadés^*, Anna Pujol, José C. Perales^*, Ramón Bartrons^* and Lisardo Boscá¹

Instituto de Bioquímica (Centro Mixto CSIC-UCM) and Centro Nacional de Investigaciones Cardiovasculares, Facultad de Farmacia, Universidad, Complutense, Madrid;
^* Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Barcelona; and
Unidad de Animales transgénicos, Centro de Biotecnología Animal y Terapia Génica de la UAB, Bellaterra, Barcelona, Spain

¹ Correspondence: Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid. Spain. E-mail: lbosca{at}cnic.es

SPECIFIC AIMS

The role of hepatic nitric oxide (NO) in liver regeneration after partial hepatectomy (PH) was studied in animals expressing a nitric oxide synthase-2 (NOS-2) transgene under the control of the phospho(enol)pyruvate carboxykinase (PEPCK-Tg) promoter after hydrodynamic transfection of murine NOS-2, which targets liver as the main tissue expressing the exogenous gene, and pharmacological administration of NO donors with low decomposition rates (DETA-NO). PEPCK-Tg mice expressed NOS-2 in liver cells under fasting conditions. Early signaling, liver mass recovery, and molecular parameters related to cell proliferation and apoptosis were determined after PH. Preexisting hepatic NO synthesis as well as NO delivery by NO donors impaired early signaling involved in the regenerative response, such as attenuated NF-B and Stat-3 activation as well as TNF- and IL-6 release. Under these conditions an insufficient proliferative response was observed, but mouse survival after PH was not compromised. However, the early presence of NO exerted a protective role against apoptosis in these models of hepatectomized mice. These data suggest that the sustained presence of NO after PH exerts a dual role: attenuating liver regeneration while efficiently protecting against liver apoptosis, allowing the tissue to reach functional size. These results open the possibility of the use of pharmacological NO delivery in the liver to treat diseases in which massive apoptosis of hepatocytes occurs.

PRINCIPAL FINDINGS

After surgical resection of two-thirds of the liver mass by PH, the remnant tissue initiates a process of regeneration. This process requires injury-related cytokines, such as TNF- and IL-6, that mediate the activation of cytokine-regulated transcription factors such as NF-B and Stat-3, which govern the expression of genes controlling hepatocyte viability and proliferation. NOS-2, the high-output, cytokine-inducible NO releasing enzyme, is expressed after PH by hepatocytes and Kupffer cells. The NO released plays a regulatory role in liver regeneration, since liver regeneration in NOS-2 KO mice is impaired due to an increase in hepatocyte apoptosis. These data suggest that NO might be an important hepatoprotective factor in the regenerating liver. Similar conclusions were obtained after pharmacological inhibition of NOS-2.

It has been shown that NO prevents TNF--mediated activation of the proapoptotic caspase 3 and protects hepatocytes from cytokine-mediated death by S-nitrosylating procaspases and active caspase enzymes. The available data thus indicate that NO protects the liver by inhibiting synthesis of proinflammatory mediators and prevents apoptosis of injured cells by inhibiting caspase 3.

1. Preexisting hepatic NO synthesis delays cell proliferation and liver mass recovery after PH
The expression of NOS-2 prior to PH was undetectable in wild-type (Wt) fed or fasted animals but clearly detectable in overnight fasted PEPCK-Tg mice. Moreover, the ability of the transgene to express a functional NOS-2 enzyme was confirmed by the increase in the plasma levels of nitrates plus nitrites after starvation, a response that was inhibited by NOS-2 selective inhibitors. The role of preexisting NO on liver regeneration after PH was investigated using this transgenic animal model. Both Wt and Tg mice survived after PH whether fed or overnight fasted. In the presence of hepatic NOS-2 after overnight starvation of Tg mice, delayed mass recovery was observed at day 5 after PH; liver mass was completely restored at day 10 post-PH.

Analysis of the mechanisms responsible of the delay in liver mass recovery in Tg mice showed deficient early activation of NF-B and Stat-3. As Fig. 1 shows, increases in the levels of cyclin E, cyclin D₁, and PCNA, and incorporation of bromodeoxyuridine (BrdU) as an index of cell proliferation, were delayed in fasted Tg animals compared with their Wt counterparts. The same situation was observed for the kinetics of the decrease and resynthesis of p27 and the transcription factor C/EBP. The characteristic rise in C/EBPß levels in the early stages of hepatocytes proliferation was attenuated in Tg animals. These parameters are all linked to delayed proliferation. However, analysis of liver sections prepared from animals after PH showed an almost complete absence of apoptotic cells in the remnant livers from Tg animals, confirming the relevance of NO as an inhibitor of apoptosis in regenerating liver.

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of NO on cell cycle progression after PH. Wt and Tg animals were subjected to PH and samples of the remnant liver were processed to determine protein levels. Results show representative blots (A) and quantitative analysis (mean ± SE; n=5) (B). Incorporation of BrdU was determined after in vivo administration of 10 mg/kg (body weight) 3 h before sacrifice (C). *P < 0.01 vs. the corresponding Wt condition at the indicated time.

2. Transient expression of NOS-2 delays liver regeneration
In addition to the PEPCK-NOS-2 Tg model, which requires starvation of the animals for the hepatic expression of NOS-2, we have developed a method to deliver the NOS-2 gene into the liver via hydrodynamics-based transfection. As Fig. 2 shows, NOS-2 was expressed in the liver in 40% of the cells (as indicated by the associated GFP fluorescence). Under these conditions, the levels of ALT determined 24 h after transfection were similar among animals transfected with GFP or GFP-NOS2 and were in the range of those receiving saline (Fig. 2B ). The transfected NOS-2 was active; serum levels of NOx increased significantly in GFP-NOS2 mice but not in GFP transfected counterparts (Fig. 2C ). However, PCNA levels, incorporation of BrdU, and the shift in C/EBP levels were delayed when NO was released prior to PH compared with the GFP-transfected counterparts (Fig. 2D ). Moreover, recovery of liver mass was delayed in animals carrying the NOS-2 transgene (Fig. 2E ).

View larger version (30K): [in this window] [in a new window]   Figure 2. Effect of hydrodynamic transfection with NOS-2 on liver regeneration after PH and apoptosis after Fas challenge. Animals (n=5 for each condition) were transfected with plasmids encoding GFP or GFP-NOS2. Liver sections were analyzed 24 h after transfection to verify expression of GFP or GFP-NOS2 fusion protein, respectively (A). Liver injury caused by the injection was detected as ALT serum levels (B); NOS-2 activity was evaluated by measuring serum NOx levels (C). Cell cycle protein levels were determined by Western blot at the indicated times after PH (D). BrdU incorporation was evaluated by ELISAs (D). Liver mass recovery after PH was determined 5 and 10 days post-PH (n=5) (E). Transfected non-operated animals were injected i.p. with Jo2 anti-Fas receptor antibody (0.30 µg/g body weight). 5 h after challenge, animals were killed and caspase activities were measured in liver extracts (F). Data show the means ± SE of 5 animals per condition. *P < 0.001 vs. the GFP condition.

Finally, to confirm the view of NO as an inhibitor of caspase activation and apoptosis, animals transfected with the GFP or GFP-NOS2 plasmids were challenged with a Fas antibody and the activation of caspases was measured. Figure 2F shows that activation of caspases 8, 9, and 3 was significantly impaired in liver from animals releasing NO.

CONCLUSIONS AND SIGNIFICANCE

The incidence of liver disease has increased over the past few years, but liver transplant does not offer an effective treatment in most cases. For this reason, the molecular mechanisms governing hepatocyte growth and viability, particularly during regeneration after partial liver resection, warrant further study. NO, whose local release can be controlled, is a candidate with therapeutic potential. Indeed, release of NO has been implicated in the etiology and progression of various liver pathologies, although both beneficial and detrimental effects have been reported, depending on the disease and the time of the synthesis or pharmacological delivery.

In adult vertebrates, the capacity of regeneration is limited to a few tissues, one of which is the liver. There are a great many factors and early signals involved in the cell cycle progression of hepatocytes after PH, and only a few conditions have been described in which liver regeneration is a regressive process leading to animal death. NO seems to play an important regulatory role; indeed, NOS-2 is effectively induced in the remnant liver after PH. The rapid activation of NF-B that occurs after PH was inhibited by preexisting NO, and data from microarrays have shown that this rapid activation of NF-B is required for the expression of immediate early genes, including anti-apoptotic and proliferation genes. Furthermore, release of cytokines involved in hepatocyte priming, such as TNF- and IL-6, were significantly lower in the presence of NO. In summary, activation of NOS-2 before PH results in delay and attenuation of cell cycle progression. Interestingly enough, the same type of response has been reported with hyperstimulation with IL-6.

In contrast to its anti-proliferative and cytostatic effects, NO efficiently protected hepatocytes from apoptosis in Tg animals, an effect mimicked by the NO donor DETA-NO in Wt mice and inhibited by selective NOS-2 inhibitors. In agreement, it has been shown that NO exerts hepatoprotective and anti-apoptotic effects in several pathological situations. For example, carbon tetrachloride-induced hepatic injury is increased in animals lacking the NOS-2 gene. Fas-dependent apoptosis, which plays a major role in the pathogenesis of immuno-mediated liver diseases such as viral hepatitis and acute liver failure, can be impaired by NO, providing an additional rationale for the therapeutic treatment of these hepatic diseases.

In summary, the data presented in this study indicate that preexisting NO liver synthesis can protect hepatocytes from apoptosis after PH or challenge with Fas mAb by inhibiting caspase activation (Fig. 3 ). However, the cytostatic and anti-proliferative effects of NO might prove harmful when rapid regeneration is required. This knowledge is relevant to the specific management of several hepatic pathologies, such as fulminant hepatitis, in which inhibition of hepatocyte apoptosis can exert beneficial effects. Therefore, the development of strategies for local delivery of NO to the liver is an area of interest for the therapeutic treatment of several hepatopathies.

View larger version (14K): [in this window] [in a new window]   Figure 3. Schematic diagram showing the different targets of NO during liver regeneration after PH. NO plays anti-apoptotic and anti-proliferative roles depending on the time of administration.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3233fje; doi: 10.1096/fj.04-3233fje

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This Article Full Text (PDF) All Versions of this Article: 19/8/995 04-3233fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zeini, M. Articles by Boscá, L. Search for Related Content PubMed PubMed Citation Articles by Zeini, M. Articles by Boscá, L.