AJP - GI Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 294: G819-G830, 2008. First published January 10, 2008; doi:10.1152/ajpgi.00179.2007
0193-1857/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables and Figures
Right arrow All Versions of this Article:
294/3/G819    most recent
00179.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mortensen, K. E.
Right arrow Articles by Revhaug, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mortensen, K. E.
Right arrow Articles by Revhaug, A.

LIVER AND BILIARY TRACT

Regenerative response in the pig liver remnant varies with the degree of resection and rise in portal pressure

Kim Erlend Mortensen,1 Lene Nagstrup Conley,2 Jakob Hedegaard,2 Trine Kalstad,3 Peter Sorensen,2 Christian Bendixen,2 and Arthur Revhaug3

1Department of Digestive Surgery, University Hospital of Northern-Norway, Tromsø, Norway; 2Department of Genetics and Biotechnology, Research Centre Foulum, Faculty of Agricultural Sciences, University of Aarhus, Denmark; and 3Laboratory of Surgical Research, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway

Submitted 23 April 2007 ; accepted in final form 26 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
After parenchymal loss, the liver regenerates restoring normal mass and metabolic function. Prevailing theories on triggering events leading to regeneration include humoral, metabolic, and flow-mediated mechanisms, the latter emphasizing the importance of shear stress mediated nitric oxide regulation. We aimed to investigate whether the grade of resection and hence the portal venous pressure and sinusoidal shear stress increase would be reflected in the gene expression profiles in the liver remnant by using a global porcine cDNA microarray chip with ~23,000 genes represented. Six pig livers were resected with 62% (low portal pressure resection) and 75% (high portal pressure resection), resulting in a portal venous pressure increase from a baseline of 6.1–8.2 and 12 mmHg, respectively. By sampling consecutive biopsies from the liver remnants, we found differentially expressed genes in the high portal pressure resection group to have functions related primarily to apoptosis, nitric oxide metabolism and oxidative stress, whereas differentially expressed genes in the low portal pressure resection group potentially regulate the cell cycle. Common to both groups was the upregulation of genes regulating inflammation, transport, cell proliferation, development, and protein metabolism. Also common to both groups was both up- and downregulation of genes regulating cell-cell signaling, signal transduction, cell adhesion, and translation. Genes regulating the metabolism of lipids, hormones, amines, and alcohol were downregulated in both groups. In conclusion, the genetic regenerative response in the liver remnant to varies according to the level of resection.

microarray; shear stress; partial hepatectomy; regeneration

AFTER PARENCHYMAL LOSS, the liver regenerates restoring normal mass and metabolic function. Since Higgins and Anderson (33) formalized the study of liver regeneration in 1931, most studies on liver regeneration have been conducted in a model of 70% partial hepatectomy (PHx) in rodents. After PHx, several proapoptotic [transforming growth factor (TGF)-β and Fas ligand] and promitotic factors [IL-1, IL-6, EGF, hepatocyte growth factor (HGF), and TNF-{alpha}; Refs. 22, 72] are known to be important humoral factors regulating induction, propagation, and termination of liver regeneration. Knockout studies in mice have demonstrated impaired regeneration in mice receptor negative for promitotic mediators (12) and hepatomegaly in mice receptor negative for proapoptotic mediators (16). Many of these humoral factors are detectable several hours after PHx (Refs. 50, 53, 76). However, later studies (65) have shown that liver regeneration commences already after 15 min post-PHx (via the detection of c-fos mRNA expression), suggesting more immediate triggering events. There is evidence indicating that the increased portal pressure and flow per gram of remaining liver tissue and hence sinusoidal shear stress after PHx may be a primary stimulus (53, 63, 64). Endothelial shear stress results in the production of nitric oxide (NO; Refs. 49, 66, 75), and several studies (28, 65, 66) have illustrated that liver regeneration is inhibited by administration of the NO synthase antagonist NG-nitro-L-arginine methyl ester and restored by the NO donor 3-morpholinosydnonimine-1 (SIN-1).

Recent studies (5, 26, 48, 71, 77) have used microarray analysis to study liver regeneration. These experiments have been conducted on a 70% PHx model in rodents using liver-specific chips, and they collectively describe gene expression profiles in the regenerating liver over a time span of 1 min to 1 wk post-PHx. Common to all is the upregulation of genes associated with transport control; inflammatory/acute phase response; cytoskeletal and extracellular remodeling; regulation of cell cycle entry and progression, including cell cycle checkpoint genes; synthesis of constitutive plasma proteins; biosynthesis of critical cell components; and signal transduction. Genes regulating intermediary metabolism of drugs, lipids, sugars, amino acids, nucleotides, and steroids were commonly downregulated. Notably, several of these studies (5, 71) report an upregulation of genes inducing antiproliferative effects (RhoB, PAI-1, and Quiescin Q6) as early as 90 min after PHx, illustrating the dual nature of the regenerative response.

The aspects of changes in portal pressure and sinusoidal flow in the liver remnant have not been addressed in previous microarray experiments. Based on the shear stress theory (49, 53, 63, 64, 66, 73), we performed an analysis of gene expression profiles in consecutive biopsies from liver remnants after two different grades of resections: 62% resection [low portal venous pressure resection (LPPR)] and 75% resection [high portal venous pressure resection (HPPR)] with the primary aim of investigating whether the pressure and flow differences in these two sets of liver remnants would generate activation patterns corroborating the theory of shear stress induced initiation of the regenerative response. To this end, we utilized a novel porcine cDNA microarray chip with ~23,000 genes represented, encompassing the entire pig genome and enabling a more comprehensive investigation of the regenerative process compared with previous studies based on limited microarray platforms. In addition, as most previous regeneration models have been based on rodents, we chose to examine the process in the pig, as this species bears closer genetic and physiological resemblance to man.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Six castrate sus scrofa domesticus were used for all experiments. The committee of the Norwegian Experimental Animal Board approved all protocols, which were conducted in compliance with the institutional animal care guidelines and the National Institute of Health's Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985]. Anesthesia was induced and maintained as described previously from our laboratory (44). Operative procedures: after a midline laparotomy and placement of all catheters, the hepatic artery supplying segments II and III (left lateral lobe) together with the portal branch of these segments was ligated using an absorbable polyfilament suture on a large needle. Thereafter, the lobe was strangulated with a 0.5-cm wide cotton ribbon, removed, and weighed. Segments IV, V, and VIII were removed in a similar manner in the LPPR series leaving segments VI, VIII, and I in place (62% resection). In the HPPR series, the resection was continued and segments VI and VII were also removed (75% resection).

A 16-G central venous catheter (CVK, Secalon T) was placed in the left external jugular vein for administration of anesthesia and infusions. A 16-G CVK was placed in the left femoral artery for continuous arterial blood sampling and pressure monitoring. A 7-Fr 110-cm angiographic catheter (Cordis, Johnson&Johnson) was placed in the right hepatic vein just before entering the inferior caval vein for blood sampling. Correct placement was ensured by direct palpation and later controlled by postmortem dissection. A 4-Fr pediatric central venous catheter (Arrow International) was placed in the portal vein with the tip ~5 cm from the liver hilus for pressure monitoring and blood sampling.

A 12-mm flow probe (Medistim, Oslo, Norway) was placed around the portal vein. Calibrated transducers (Transpac 3, Abbott Critical Care Systems, Chicago, IL) were used for continuous pressure registration. The transducers were connected to an amplifier (Gould, 2800S). Pulsatile signals were displayed on a monitor, digitalized, and stored electronically (Advantech, Industrial Computer).

Biopsies were taken from the remaining segments VI and VII in the LPPR series and from segment I in the HPPR series and placed immediately in RNALater (Ambion). The consecutive samples were taken some distance apart to avoid disturbance from the sampling procedure. Sampling time points were 1, 30, and 90 min and 3, 4, and 6 h after resection.

Blood extraction was performed before biopsy sampling. Samples were taken from the portal vein, femoral artery, and hepatic vein. IL-1, IL-6, TNF-{alpha}, TGF-{alpha}, TGF-β, and EGF were analyzed using ELISA (Quantikine, R&D Systems, and Searchlight Pierce Biotechnology), and aspartate aminotransferase, alanine aminotransferase, glutamyl transpeptidase, pyruvate, glucose, lactate, and alkaline phosphatase levels were quantified by calorimetric, ultraviolet-photometric, and HPLC analysis (Roche, PerkinElmer). Biochemical flux across the liver remnant was analyzed using linear mixed models in SPSS version 13, testing time (T), and group x time (GT) interaction. P values ≤0.05 were considered significant.

The microarray experiments were conducted as a common reference design using liver total RNA purified from an unrelated animal as the reference, allowing comparison of all the different samples to each other (the acute phase response due to the operation in itself could in theory affect gene expression in both groups of liver remnants; however, as the perioperative conditions were almost identical, we considered this effect to be equal for the two resection groups and chose not to use a sham as the reference). Total RNA was purified and aminoallyl-cDNA (aa-cDNA) was synthesized from 20 µg of total RNA. The reference sample was labeled with Alexa 488, each individual sample was labeled with Alexa 594, and the samples were combined pair wise and hybridized to the pig array DIAS_PIG_55K2, which consists of 26,879 PCR products amplified from unique cDNA clones. After hybridization, washing, and drying, the slides were scanned, and the median intensities were computed. Statistical analysis was carried out in the R computing environment using the Bioconductor package Linear Models for Microarray Analysis (Limma; Refs. 29, 69). The log2-transformed ratios of Alexa-594 to Alexa-488 were normalized within slide using printtip-loess and were analyzed to identify genes being significantly differentially expressed by time within treatment as well as between treatments. Time contrasts were formed referring to the sample taken at the 1-min time point. The genes found significantly expressed between at least two time points were further analyzed referring to Online Mendelian Inheritance in Man (OMIM; Ref. 2) and Gene Ontology (GO) to group the genes by function. More detailed descriptions of the microarray experiments are available at the National Center for Biotechnology Information's Gene Expression Omnibus (GEO; Refs. 1, 7, 20) through the GEO series accession number GSE6860.

The data were analyzed within each resection group (LPPR and HPPR) in two ways. First, using top tables for each of the five time intervals (1 to 30 min, 1–90 min, 1 min to 3 h, 1 min to 4 h, and 1 min to 6 h post-PHx), we classified all genes into 14 functional groups by molecular function and biological process according to the GO and OMIM. Second, using K-means clustering, we defined 20 clusters in each group and selected from these 8 clusters of special interest on the basis of their nonuniform profiles for closer analysis with GO and OMIM. For direct comparison of the two resection groups, we investigated the genes that were expressed in both groups at each time point, selecting those that had a statistically different expression level in the two groups (herein termed "within time point contrasts"). We avoided using fold change cutoff values because this approach fails to take the uncertainty of variability into account (i.e., a gene may exhibit a 10-fold change and yet not be significant because of its variability) and has the potential of excluding genes of biological importance whose expression values are below the cutoff value.

For RT-PCR validation, sets of two primers and a probe for eight chosen target genes and one control gene (18S) were designed using the Primer Express software package (version 2.0; Applied Biosystems). To avoid genomic DNA contamination, the primers were designed to span exon boundaries. The probes for the target genes were labeled with either the fluorescent reporter VIC or SYBR Green. Five micrograms of total RNA of each sample were reverse transcribed using random hexamer primers and SuperScript II Reverse Transcriptase (Invitrogen). Real-time quantitative RT-PCR was performed on the ABI Prism 7900HT sequence detection system (Applied Biosystems) by monitoring the increase in fluorescence due to the binding of SYBR green or VIC to double-stranded DNA. The amplification conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles each of 95°C for 15 s and 60°C for 1 min. Each cDNA sample was run as technical triplicates. The standard curve method was used to calculate the relative mRNA levels. The quantity mean of the triplicate measurements was normalized against the 18S gene, and the mean of the biological replicates was calculated as well, yielding a single value for each timepoint. The profiles of these values were compared with the profiles of the microarray data.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General Results

Hemodynamics. Liver resection resulted in a gradual rise in portal venous pressure, reflecting an increase in resistance to blood flow through the liver sinusoids as the blood flow per gram remaining liver tissue increases. Upon additional resection of segments VI and VII (from a 62 to 75% resection), the pressure rose precipitously; from a baseline average of 6.1 mmHg, the venous portal pressure rose to 8.2 mmHg in the low-pressure group (35% increase) and to 12 mmHg in the high-pressure resection group (110% increase), remaining relatively stable until experiment termination (Fig. 1). Similarly, the average portal blood flow per gram remaining liver tissue rose from ~1–2 ml/g in the LPPR group and to 5 ml/g in the HPPR group.


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 1. Portal venous pressure in the high portal venous pressure resection (HPPR) and low portal venous pressure resection (LPPR) series (mean pressure ±SD).

 
Serum analysis. We found a significant net absorption of alanine aminotransferase, glutamyl transpeptidase, and glucose across the liver remnant after resection in both LPPR and HPPR resection groups. This uptake increased over time, but there was no difference between the groups. A net discharge of aspartate aminotransferase and alkaline phosphatase was seen. This net discharge increased over time, and there were significant group x time interactions with a larger discharge seen in the HPPR group. No changes in pyruvate flux were observed.

No significant changes in IL-1, IL-6, TNF-{alpha}, and EGF were found. Beginning at 2 h post-PHx, an increase in serum levels of TGF-{alpha} from an average of 8.9 pg/ml (±4.6 SD) to 75.85 pg/ml (±14.9 SD) was observed in the LPPR group but not in the HPPR group (P = 0.011). Serum TGF-β rose significantly in both groups (P = 0.017), but there was no group difference.

Microarray Results

A total of 1,095 genes was differentially expressed at least once between two time points in the LPPR group and 1,010 genes in the HPPR group (the online version of this article contains supplemental data; see Tables S1 and S2). Of these, 174 genes were common to both groups. Approximately 50% of the common genes regulate intermediate metabolism or have an unknown function. Approximately 35% regulate transcription, signal transduction, translation, transport, protein metabolism, and oxidoreductase activity. The remaining 15% regulate cell cycle, apoptosis, cytoskeleton, acute-phase response, nucleic acid metabolism, and cell proliferation.

Top-table analysis LPPR group. Analysis of time trends among upregulated genes in the LPPR group revealed a predominance of expression of genes governing cell proliferation in the earlier time periods, whereas cell cycle and apoptosis genes and genes regulating translation tended to be expressed in later phases. Inflammation/acute-phase response genes were evenly expressed throughout the experiment. Analysis of time trends among the downregulated genes in the LPPR group revealed a relative increase in the downregulation of genes controlling lipid/hormone/amine/alcohol metabolism towards the end of the experiment. The expression of genes regulating transport and cell-cell signaling/signal transduction and cell adhesion remained relatively stable (Fig. 2).


Figure 2
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 2. Functional classification of differentially expressed genes in top tables according to Gene Ontology and Online Mendelian Inheritance in Man.

 
Top-table analysis HPPR group. We observed a gradual increase over time in the upregulation of the genes controlling protein metabolism, cell cycle and/or apoptosis, and translation. Genes regulating cell-cell signaling/signal transduction/cell adhesion and transport were gradually less upregulated throughout the experiment. Genes regulating lipid/hormone/amine/alcohol metabolism and oxidoreductase function were increasingly downregulated over time (Fig. 2).

K-means clustering. All genes differentially expressed at least once between two time points were clustered into 20 groups (see Supplemental Figs. S3 and S4). When analyzing the chosen eight clusters in the HPPR and LPPR groups with GO and OMIM, we found the following similarities between the two resection groups (see Supplemental Figs. S1 and S2): 1) Genes regulating cell-cell signaling/signal transduction/cell adhesion and translation were up- and downregulated to similar degrees in both series. 2) With the exception the genes in cluster 13 in the LPPR series, we found genes regulating inflammation/acute-phase response to be marginally upregulated in both resection groups. 3) Genes regulating transport and cell proliferation/development were largely upregulated in both series. 4) Genes regulating lipid/hormone/amine/alcohol metabolism were generally downregulated in both series. 5) In general, more genes regulating protein metabolism were upregulated than downregulated in both series throughout the experiment. Genes regulating transcription, cell cycle and/or apoptosis, extracellular matrix/cytoskeleton, and nucleic acid metabolism were upregulated to a larger degree in the HPPR series.

Genes Regulating Cell Cycle and Apoptosis

The 26 differentially expressed genes regulating cell cycle and apoptosis are schematically presented in Table 1 and Fig. 3.


View this table:
[in this window]
[in a new window]

  		Table 1. Differentially expressed genes regulating cell cycle and apoptosis according to time intervals in the LPPR and HPPR series

 

Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 3. Schematic presentation and suggested interaction of differentially expressed genes regulating cell cycle and apoptosis in both resection series. See text for definitions.

 
A dual response was seen during the first 30 min after PHx in the HPPR, series as the upregulation of PACS-2 would in theory result in caspase activation (68) and downregulation of CASP8 would modify caspase-dependent apoptosis (57).

Potential products of the upregulated genes in the LPPR series in the time interval 1–90 min organize the microtubuli apparatus (involved in cell division; KIF20A; Ref. 34), are involved in p-53-dependent apoptosis and TGF-β-dependent apoptosis through interaction with Dapk1 (MIB1; Ref. 38, 40) and enhance cell cycle transition (CDK6; Ref. 51, 58) and protection from serum starvation-induced apoptosis (MCTS1; Ref. 67).

Products of the five upregulated genes in the HPPR group in the same time interval could potentially inhibit DNA damage and cytolysis (NME1; Refs. 21, 43), counteract apoptosis (Bcl2; Ref. 35) and MAPK8IP2 (52) facilitate entrance into the G1-phase (UBE2C; Ref. 59), and mediate p53 dependent activation of NF{kappa}-B through the RAF/MEK1(MAPK2K1)/p90(rsk) pathway (MAPK2K1; Ref. 62), which in turn could both protect or contribute to apoptosis (24). [Studying liver regeneration, Su et al. (71) found BCL-X, BCL-2-related protein A1 and bcl-3 upregulated after a PHx in mice and Locker et al. (48) found Bcl2 upregulated after PHx]. Downregulated in the HPPR group in this time period were two genes controlling apoptosis (CYCS; Ref. 47, and Casp7; Ref. 54) and one gene associated with cell cycle progression (HNRPK; Ref. 15).

Within 3 h, four genes were found upregulated in the HPPR group, whose products inhibit caspase-9-dependent apoptosis (PTMA; Ref. 39), regulate centrosome duplication in the G1 phase of the cell cycle (MPM1; Ref. 55), possibly function as a tumor suppressor (BCLAF; Ref. 41), and are found localized in the nucleoli G1 and G2 phase of the cell cycle (SBDSP; Ref. 6).

Within 4 h in the LPPR group, we found differentially expressed genes whose products regulate centrosome duplication in the G1 phase of the cell cycle (MPM1; Ref. 55) are involved in centrosome formation and mitosis (SCYL1; Ref. 42), and are found localized in the nucleoli G1 and G2 phase of the cell cycle (SBDSP; Ref. 6). In the HPPR group, we found NPM1 upregulated and other genes whose products regulate the centriole cycle and mitosis (CETN2; Ref. 46); promote the expression of the c-myc gene, abundantly found in hepatocellular carcinomas (NME2; Ref. 37); and potentially inhibit DNA damage and cytolysis (NME1; Ref. 21, 43).

Downregulated genes in this time period were only found in the HPPR group. The products of these genes are associated with complex formation with p53 in G0/G1 and S/G2/M phases of the cell cycle (MDM2; Ref. 25), regulate the homeostasis of liver cell number, and inhibit Fas-mediated cell death (FAIM2; Refs. 3, 70), and lead to G2/M cell cycle arrest and apoptosis (IGFBP5; Ref. 11). [Specifically addressing the liver regeneration, Desbarats and Newell (16) found that Fas engagement accelerates liver regeneration after PHx.]

Within 6 h post-PHx, the previously described genes NPM1 and PTMA were upregulated in the LPPR and HPPR series, respectively, whereas BTG2 was downregulated in the LPPR series. BTG2 belongs to a family of structurally related proteins that appear to have antiproliferative properties (61).

Genes Regulating NO Metabolism and Oxidative Stress

Differentially expressed genes regulating NO metabolism and oxidative stress are presented schematically in Table 2 and Fig. 4.


View this table:
[in this window]
[in a new window]

  		Table 2. Differentially expressed genes regulating NO metabolism and oxidative stress according to time intervals in the LPPR and HPPR series

 

Figure 4
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 4. Schematic presentation of differentially expressed genes regulating cell redox status and nitric oxide metabolism. See text for definitions.

 
Heat shock factor-1 (HSF-1) expression was gradually upregulated in the HPPR series with a maximum value at 3 h post-PHx. HSF-1, a major heat stress-response factor, upregulates many other heat shock protein (HSP) genes, which in turn are cytoprotective and probably play a role in ischemic preconditioning due to their ATP-sparing effects. HSPs in turn have been found to inhibit the formation of the apoptosome complex through forming complexes with Apaf1 and cytochrome c, inhibiting the final activation of caspase-3 (78). HSP90 has also been found to play a central role in statin-induced angiogenesis (9) and activates endothelial nitric oxide synthase (eNOS) upon shear stress stimulation (27). HSF-1 also activates PKB/AKT, which in turn phosporylates and activates eNOS (17).

Activation of eNOS is partially dependent on Ca2+ calmodulin that binds eNOS upon an increase in intracellular Ca2+ concentration. We found that this gene was upregulated in both resection series within 3 h post-PHx and that it stayed so until experiment termination.

In the HPPR series, we found the NOSIP transcript elevated at 3 h post-PHx. The activity of eNOS is regulated by its intracellular location and association with NOSIP. Dedio et al. (14) found possible eNOS activity inhibition by this protein and cellular translocation of eNOS from the caveolin-bound plasmalemmal location to the intracellular golgi apparatus.

Dimethylaminohydrolase (DDAH) reduces tumor asymmetric dimethylarginine in turn stimulating tumor growth and angiogenesis through increased NO levels and VEGF expression (45). In the present investigation, DDAH2 remained downregulated throughout the 6 h of observation post-PHx in both series. However, at 3 h, we observed a significant rise in the mRNA level in the HPPR series.

Oxygen radicals such as H2O2 and O2– are established activators of the caspase cascade. Dimmeler et al. (18) found that shear stress mediated upregulation of SOD1 mRNA and protein levels and eNOS activity abrogated the activation of the TNF-{alpha} induced and oxidized low-density lipoprotein induced caspase activation in human umbilical vein endothelial cells. We found SOD1 consistently downregulated in both resection series, with the exception of an increased level at 3 h in the HPPR series.

The antioxidant catalase is well established as an apoptotic inhibitor (4) being a scavenger of hydrogen peroxide. Catalase was downregulated in both resection series within 3 h in the HPPR series and within 4 h in the LPPR series.

GSTP1 was downregulated within 6 h in the HPPR series. Henderson et al. (31) reported that in GSTP1-null mice the absence of the enzyme significantly enhanced the ability of the liver to maintain GSH homeostasis in the face of oxidative stress, suggesting that GSTP1 causes a higher level of oxidative stress in the hepatocytes by redox cycling. In the absence of GSTP1, the constitutive activation Jun N-terminal kinase results in activation of c-jun rendering both cytoprotective effects and apoptosis. Interestingly, Hojo et al. (36) found that laminar fluid shear stress inhibited H2O2-induced JNK activation and increased the GSH-to-GSSG ratio, and Hermann et al. (32) found that the combined inhibition of NO synthase and GSH biosynthesis completely reversed the protective effect of shear stress, suggesting that both NO synthase and the GSH redox cycle system control the apoptosis-suppressing effect of shear stress.

Src-associated protein 2 (SCAP2) was upregulated in both series after PHx but significantly more in the LPPR group at 3 h. In a bovine aortic endothelial cell culture, Davis et al. (13) found that shear stress induced a transient expression of eNOS and prolonged half-life of eNOS mRNA through interaction with c.Src. SCAP2 is phosphorylated by Src family kinases.

RT-PCR validation. A comparison of the RT-PCR and microarray profiles of eight selected genes is illustrated in Fig. 5. The profiles of were congruent for NOSIP, SOD1, NOS3, and cFOS. The remaining gene proflies were only comparable over certain time points.


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. Comparison of average relative mRNA levels (±SD) for 8 genes as measured by RT-PCR (bullet; Qty mean target/18S) and microarray ({blacksquare}; M-Log base 2).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study shows that the immediate regenerative response in the liver remnant, as quantified by gene expression, is influenced by the grade of resection and increase in portal pressure; 62% resection affects genes primarily regulating the cell cycle, whereas 75% resection affects genes primarily regulating apoptosis, NO metabolism, and oxidative stress.

With respect to genes controlling the cell cycle and apoptosis, we observed quantitative and qualitative differences between resections yielding LPPR (62% resection) and HPPR (75% resection). Of all the 26 genes differentially expressed, a major part (18 genes) was found in the HPPR group. Qualitatively, the genetic response after a high-grade resection seems to center around the regulation of apoptosis, inhibiting death-promoting pathways, particularly the caspase system, whereas after a lower grade resection we observe the differential regulation primarily of genes regulating the cell cycle and cytoskeletal framework. Specifically, we observed in the LPPR group that genes promoting progression through the G1 phase were upregulated and genes inhibiting progression were downregulated. In addition, genes associated with the microtubuli apparatus and centrosomes were found to be upregulated. Over time, it seems that apoptosis is downregulated during the earlier time points (within 90 min), whereas cell cycle progression and microtubuli/centrosome regulation are regulated somewhat later (90 min to 4 h post-PHx). From these results, it would seem that a higher grade of resection primarily results in an inhibition of the apoptotic apparatus, whereas a lower grade resection stimulates primarily G1-phase cell cycle progression.

With regard to genes controlling NO metabolism and oxidative stress, we also observed several patterns distinguishing the two resection groups. However, the potential role of NO in liver regeneration appears to be quite complex and necessitates more discussion here. NO has been shown to inhibit apoptosis of endothelial cells through the regulation of Bcl2 (8) and the caspase cascade (18, 19, 60). On the other hand, NO has shown both antiproliferative properties through the induction of p21 (30) and potential proliferative properties through the activation of activator protein-1 (56). Furthermore, other studies (65, 66) have illustrated that NOS inhibitors (NG-nitro-L-arginine methyl ester) have blocked liver regeneration after PHx, while the addition of NO donors (SIN-1) has restored the process. NO may also have direct apoptotic effects by combining with superoxide O– forming peroxynitrite ONOO– leading to DNA damage and apoptosis (10). In the present study, a major part of genes regulating NO metabolism is found in the HPPR group (Table 2). Several genes regulating the activity of eNOS were found differentially expressed. NOSIP competes with caveolin-1 (recently found essential for liver regeneration; Ref. 23) in the binding of eNOS. Upon binding eNOS, NOSIP (upregulated in the HPPR group) translocates the enzyme from the plasmalemma to the Golgi apparatus and possibly the mitochondria, reflecting functional regulation by cellular compartmentalization after PHx. DDAH2 (upregulated in the HPPR group) regulates eNOS activity indirectly by its degradation of asymmetric dimethylarginine, which in turn converts eNOS to methylamine and citrulline. This could possibly reflect a very early physiological response in NO regulation and neovascularization in the liver remnant, as angiogenesis is central to neovascularization of regenerated hepatocyte islands in the regenerating liver (74). Genes encoding calcium calmodulin (essential for eNOS activity) were upregulated in both resection series, and HSF-1 (activating eNOS via PKB/AKT) was upregulated in the HPPR series, reflecting activation of eNOS in the liver remnants.

Interestingly, several genes, previously found activated in response to various cellular stresses, were found to be downregulated in our liver remnants (SOD1, catalase, and GSTP1). Taken together, this suggests that the cells in the liver remnants were under reduced oxidative stress over time. This is in contrast to Fausto's metabolic theory of reactive oxygen species triggering the regenerative response after PHx (22). Dimmeler et al. (18) showed that laminar flow shear stress protects against oxidative stress by the upregulation of SO, and Hojo et al. (36) found that laminar fluid shear stress inhibited H2O2-induced JNK activation and increased GSH-to-GSSG ratio. Of the genes associated with redox cycling, all were differentially expressed in the HPPR, whereas only one (catalase) was found in the LPPR group. Taken together, this may suggest that the increased shear stress in the HPPR group decreases the level of oxidative stress within the endothelial and juxtaposed hepatocytes.

Functional classification of the differentially expressed genes with GO demonstrates the complexity of the immediate genetic response in the liver remnant after PHx, as genes representing almost all functional groups are differentially expressed at one time or another. This is not surprising, as the process of liver regeneration necessarily involves multiple metabolic pathways. However, we find some differences between the two resection groups when it comes to functional classification, as genes regulating transcription, cell cycle and/or apoptosis, extracellular matrix and/or cytoskeleton, and nucleic acid metabolism seem to be upregulated to a larger degree after a larger resection, tentatively due to a greater increase in portal pressure. Apart from this, our porcine study corroborates the findings (5, 26, 48, 71, 77) in previous microarray studies on liver regeneration in rodents that report the differential expression of genes in various functional groups some of which are upregulated and some of which are downregulated (see Table 3).


View this table:
[in this window]
[in a new window]

  		Table 3. Overview of functional classification of genes according to Gene Ontology and their expression at various time points in 5 reference studies using microarray analysis of gene expression in liver remnants after partial hepatectomy. Gray squares denote time points of mRNA extraction in the respective studies. Black lines denote time points in which genes in the different functional groups were collectively differentially expressed.

 
There are several important differences between the referred studies in Table 3 and the present study: our microarray analysis is based on a cDNA chip containing ~23,000 gene elements (vs. 2,500–6,000 gene elements), the biopsies are taken consecutively from the same animal (vs. different animals killed at different times), and the species studied is different (pig vs. rodent). In our opinion, using a global chip containing more gene elements gives a superior picture of the genome-wide expression in the liver remnant after PHx. Using a large animal model allowed us to sample consecutively from the same individual animal, which reduces the likelihood of individual animals distorting the overall picture should their profiles vary greatly from the mean. Furthermore, 7 of the 26 genes regulating cell cycle and/or apoptosis that we report in the present study have been previously reported in microarray studies of liver regeneration in rodents (5, 26, 48, 71, 77). The remaining 19 genes are mainly described in models of cell culture. When studying a highly coordinated process such as liver regeneration, we maintain that it is better to do so in an integrated biological system rather than in an isolated cell culture. Finally, as porcine genetics are closer to humans than rodents are, studying liver regeneration in a porcine model ought to be advantageous as far as translational science is concerned.

The RT-PCR validation of the microarray analysis was not entirely satisfactory. We suggest two explanations for this. First, the partial incompatibility may be explained by the fact that RT-PCR measures the abundance of a single transcript or transcript segment, whereas microarray data may result from hybridization of multiple transcript isoforms to a probe or probes on an array. Second, with comparisons between time-course experiments such as the present one, there will be noise not only due to biological variation in the individual animals as a response to the surgery itself but also due to the time perspective of when it happens, that is, the genetic response of some animals to an intervention may be slower or faster than others. These factors may increase the variability not only of the microarray data but also of the RT-PCR data. We believe that, although the microarray data are not fully substantiated by the RT-PCR validation, the genes found differentially expressed by microarray analysis in our study are nonetheless found coassociated within the biological context of the cell cycle and/or apoptosis, NO metabolism, and the redox state. In our opinion, this constellation is an argument advocating that the genes in fact are differentially expressed because it infers biological meaning and thus adds credibility to our conclusions.

A microarray experiment cannot specifically falsify or confirm a null hypothesis, as it is, in essence, a screening technique. However, we applied the method in this study to shed light on present regeneration theories of portal pressure and sinusoidal shear stress and ventured to hypothesize that the immediate regenerative response in the liver remnant after a liver resection would vary according to the level of resection because of the differences in portal pressure incurred by varying levels of resection. We believe that this study presents data suggesting that there are qualitative and quantitative differences in the regenerative response depending on the level of resection and that these differences may be caused by differences in portal and hence sinusoidal pressure and shear stress in the respective liver remnants.

Graded liver resection in the pig may serve as a model to investigate shear stress in the liver remnant. Refined in vivo models of shear stress in the liver need to be explored to investigate increased sinusoidal flow per se (without prior resection) to isolate the phenomenon of increased flow. In addition, further studies with a longer time frame need to be conducted investigating the possible physiological consequences (such as differences in mitosis, apoptosis, and liver mass restoration) of the differences in the regenerative response between high- and low-pressure resections observed in the present study.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was financed by the Norwegian Research Council and Northern Norway Regional Health Authority.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. E. Mortensen, Dept. of Digestive Surgery, Univ. Hospital of Northern-Norway, 9038 Tromsoe, Norway (e-mail: kimem{at}fagmed.uit.no)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anonymous. Gene Expression Omnibus (Online). http://www.ncbi.nlm.nih.gov/geo/ [2006].
  2. Anonymous. Online Mendelian Inheritance in Man (O.M.I.M.) (Online). http://www.nslij-genetics.org/search_omim.html [2006].
  3. Adachi M, Suematsu S, Kondo T, Ogasawara J, Tanaka T, Yoshida N, Nagata S. Targeted mutation in the fas gene causes hyperplasia in peripheral lymphoid organs and liver. Nat Genet 11: 294–300, 1995.[CrossRef][Web of Science][Medline]
  4. Ande SR, Kommoju PR, Draxl S, Murkovic M, Macheroux P, Ghisla S, Ferrando-May E. Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom. Apoptosis 11: 1439–1451, 2006.[CrossRef][Web of Science][Medline]
  5. Arai M, Yokosuka O, Chiba T, Imazeki F, Kato M, Hashida J, Ueda Y, Sugano S, Hashimoto K, Saisho H, Takiguchi M, Seki N. Gene expression profiling reveals the mechanism and pathophysiology of mouse liver regeneration. J Biol Chem 278: 29813–29818, 2003.[Abstract/Free Full Text]
  6. Austin KM, Leary RJ, Shimamura A. The Shwachman-Diamond SBDS protein localizes to the nucleolus. Blood 106: 1253–1258, 2005.[Abstract/Free Full Text]
  7. Barrett T, Suzek TO, Troup DB, Wilhite SE, Ngau WC, Ledoux P, Rudnev D, Lash AE, Fujibuchi W, Edgar R. NCBI GEO: mining millions of expression profiles-database and tools. Nucleic Acids Res 33: D562–D566, 2005.[Abstract/Free Full Text]
  8. Boo YC, Jo H. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases. Am J Physiol Cell Physiol 285: C499–C508, 2003.[Abstract/Free Full Text]
  9. Brouet A, Sonveaux P, Dessy C, Moniotte S, Balligand JL, Feron O. Hsp90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circulation Res 89: 866–873, 2001.[Abstract/Free Full Text]
  10. Burney S, Tamir S, Gal A, Tannenbaum SR. A mechanistic analysis of nitric oxide-induced cellular toxicity. Nitric Oxide 1: 130–144, 1997.[CrossRef][Web of Science][Medline]
  11. Butt AJ, Dickson KA, McDougall F, Baxter RC. Insulin-like growth factor-binding protein-5 inhibits the growth of human breast cancer cells in vitro and in vivo. J Biol Chem 278: 29676–29685, 2003.[Abstract/Free Full Text]
  12. Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V, Taub R. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 274: 1379–1383, 1996.[Abstract/Free Full Text]
  13. Davis ME, Cai H, Drummond GR, Harrison DG. Shear stress regulates endothelial nitric oxide synthase expression through c-Src by divergent signaling pathways. Circ Res 89: 1073–1080, 2001.[Abstract/Free Full Text]
  14. Dedio J, Konig P, Wohlfart P, Schroeder C, Kummer W, Muller-Esterl W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J 15: 79–89, 2001.[Abstract/Free Full Text]
  15. Dejgaard K, Leffers H, Rasmussen HH, Madsen P, Kruse TA, Gesser B, Nielsen H, Celis JE. Identification, molecular-cloning, expression and chromosome mapping of a family of transformation up-regulated Hnrnp-K proteins derived by alternative splicing. J Mol Biol 236: 33–48, 1994.[CrossRef][Web of Science][Medline]
  16. Desbarats J, Newell MK. Fas engagement accelerates liver regeneration after partial hepatectomy. Nat Med 6: 920–923, 2000.[CrossRef][Web of Science][Medline]
  17. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399: 601–605, 1999.[CrossRef][Medline]
  18. Dimmeler S, Hermann C, Galle J, Zeiher AM. Upregulation of superoxide dismutase and nitric oxide synthase mediates the apoptosis-suppressive effects of shear stress on endothelial cells. Arterioscler Thromb Vasc Biol 19: 656–664, 1999.[Abstract/Free Full Text]
  19. Dimmeler S, Zeiher AM. Nitric oxide–an endothelial cell survival factor. Cell Death Differ 6: 964–968, 1999.[CrossRef][Web of Science][Medline]
  20. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30: 207–210, 2002.[Abstract/Free Full Text]
  21. Fan Z, Beresford PJ, Oh DY, Zhang D, Lieberman J. Tumor suppressor NM23–H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor. Cell 112: 659–672, 2003.[CrossRef][Web of Science][Medline]
  22. Fausto N. Liver regeneration. J Hepatol 32: 19–31, 2000.[Web of Science][Medline]
  23. Fernandez MA, Albor C, Ingelmo-Torres M, Nixon SJ, Ferguson C, Kurzchalia T, Tebar F, Enrich C, Parton RG, Pol A. Caveolin-1 is essential for liver regeneration. Science 313: 1628–1632, 2006.[Abstract/Free Full Text]
  24. Foo SY, Nolan GP. NF-kappa B to the rescue–RELs, apoptosis and cellular transformation. Trends Genet 15: 229–235, 1999.[CrossRef][Web of Science][Medline]
  25. Fuchs SY, Adler V, Buschmann T, Yin ZM, Wu XW, Jones SN, Ronai Z. JNK targets p53 ubiquitination and degradation in nonstressed cells. Genes Dev 12: 2658–2663, 1998.[Abstract/Free Full Text]
  26. Fukuhara Y, Hirasawa A, Li XK, Kawasaki M, Fujino M, Funeshima N, Katsuma S, Shiojima S, Yamada M, Okuyama T, Suzuki S, Tsujimoto G. Gene expression profile in the regenerating rat liver after partial hepatectomy. J Hepatol 38: 784–792, 2003.[CrossRef][Web of Science][Medline]
  27. Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, Sessa WC. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 392: 821–824, 1998.[CrossRef][Medline]
  28. Garcia-Trevijano ER, Martinez-Chantar ML, Latasa MU, Mato JM, Avila MA. NO sensitizes rat hepatocytes to proliferation by modifying S-adenosylmethionine levels. Gastroenterology 122: 1355–1363, 2002.[CrossRef][Web of Science]
  29. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80, 2004.[CrossRef][Medline]
  30. Gu MF, Brecher P. Nitric oxide-induced increase in p21(Sdi1/Cip1/Waf1) expression during the cell cycle in aortic adventitial fibroblasts. Arterioscler Thromb Vasc Biol 20: 27–34, 2000.[Abstract/Free Full Text]
  31. Henderson CJ, Wolf CR, Kitteringham N, Powell H, Otto D, Park BK. Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi. Proc Natl Acad Sci USA 97: 12741–12745, 2000.[Abstract/Free Full Text]
  32. Hermann C, Zeiher AM, Dimmeler S. Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. Arterioscler Thromb Vasc Biol 17: 3588–3592, 1997.[Abstract/Free Full Text]
  33. Higgins G, Anderson GM. Experimental pathology of the liver. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol 12: 186–202, 1931.[Web of Science]
  34. Hirokawa N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279: 519–526, 1998.[Abstract/Free Full Text]
  35. Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondrial-membrane protein that blocks programmed cell-eeath. Nature 348: 334–336, 1990.[CrossRef][Medline]
  36. Hojo Y, Saito Y, Tanimoto T, Hoefen RJ, Baines CP, Yamamoto K, Haendeler J, Asmis R, Berk BC. Fluid shear stress attenuates hydrogen peroxide-induced c-jun NH2-terminal kinase activation via a glutathione reductase-mediated mechanism. Circ Res 91: 712–718, 2002.[Abstract/Free Full Text]
  37. Iizuka N, Mori N, Tamesa T, Tangoku A, Oka M. Telomerase activity and nm23–H2 protein expression in hepatocellular carcinoma. Anticancer Res 23: 43–47, 2003.[Web of Science][Medline]
  38. Jang. TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nature Cell Biol 4: 328, 2002.[CrossRef]
  39. Jiang XJ, Kim HE, Shu HJ, Zhao YM, Zhang HC, Kofron J, Donnelly J, Burns D, Ng SC, Rosenberg S, Wang XD. Distinctive roles of PHAP proteins and prothymosin-alpha in a death regulatory pathway. Science 299: 223–226, 2003.[Abstract/Free Full Text]
  40. Jin Y, Blue EK, Dixon S, Shao Z, Gallagher PJ. A death-associated protein kinase (DAPK)-interacting protein, DIP-1, is an E3 ubiquitin ligase that promotes tumor necrosis factor-induced apoptosis and regulates the cellular levels of DAPK. J Biol Chem 277: 46980–46986, 2002.[Abstract/Free Full Text]
  41. Kasof GM, Goyal L, White E. Btf, a novel death-promoting transcriptional repressor that interacts with Bcl-2-related proteins. Mol Cell Biol 19: 4390–4404, 1999.[Abstract/Free Full Text]
  42. Kato M, Yano K, Morotomi-Yano K, Saito H, Miki Y. Identification and characterization of the human protein kinase-like gene NTKL: mitosis-specific centrosomal localization of an alternatively spliced isoform. Genomics 79: 760–767, 2002.[CrossRef][Web of Science][Medline]
  43. Keim D, Hailat N, Melhem R, Zhu XX, Lascu I, Veron M, Strahler J, Hanash SM. Proliferation-related expression of P19/Nm23 nucleoside diphosphate kinase. J Clin Invest 89: 919–924, 1992.[Web of Science][Medline]
  44. Korvald C, Elvenes OP, Myrmel T. Myocardial substrate metabolism influences left ventricular energetics in vivo. Am J Physiol Heart Circ Physiol 278: H1345–H1351, 2000.[Abstract/Free Full Text]
  45. Kostourou V, Robinson SP, Cartwright JE, Whitley GS. Dimethylarginine dimethylaminohydrolase I enhances tumour growth and angiogenesis. Br J Cancer 87: 673–680, 2002.[CrossRef][Web of Science][Medline]
  46. Laoukili J, Perret E, Middendorp S, Houcine O, Guennou C, Marano F, Bornens M, Tournier F. Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro. J Cell Sci 113: 1355–1364, 2000.[Abstract]
  47. Liu XS, Kim CN, Yang J, Jemmerson R, Wang XD. Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 86: 147–157, 1996.[CrossRef][Web of Science][Medline]
  48. Locker J, Tian JM, Carver R, Concas D, Cossu C, Ledda-Columbano GM, Columbano A. A common set of immediate-early response genes in liver regeneration and hyperplasia. Hepatology 38: 314–325, 2003.[Web of Science][Medline]
  49. Macedo MP, Lautt WW. Shear-induced modulation of vasoconstriction in the hepatic artery and portal vein by nitric oxide. Am J Physiol Gastrointest Liver Physiol 274: G253–G260, 1998.[Abstract/Free Full Text]
  50. Mars WM, Liu ML, Kitson RP, Goldfarb RH, Gabauer MK, Michalopoulos GK. Immediate-early detection of urokinase receptor after partial-hepatectomy and its implications for initiation of liver-regeneration. Hepatology 21: 1695–1701, 1995.[CrossRef][Web of Science][Medline]
  51. Meyerson M, Harlow E. Identification of G(1) kinase-activity for Cdk6, a novel cyclin-D partner. Mol Cell Biol 14: 2077–2086, 1994.[Abstract/Free Full Text]
  52. Negri S, Oberson A, Steinmann M, Sauser C, Nicod P, Waeber G, Schorderet DF, Bonny C. cDNA cloning and mapping of a novel islet-brain/JNK-interacting protein. Genomics 64: 324–330, 2000.[CrossRef][Web of Science][Medline]
  53. Niiya T, Murakami M, Aoki T, Murai N, Shimizu Y, Kusano M. Immediate increase of portal pressure, reflecting sinusoidal shear stress, induced liver regeneration after partial hepatectomy. J Hepatobiliary Pancreat Surg 6: 275–280, 1999.[CrossRef][Medline]
  54. Nunez G, Benedict MA, Hu YM, Inohara N. Caspases: the proteases of the apoptotic pathway. Oncogene 17: 3237–3245, 1998.[CrossRef][Web of Science][Medline]
  55. Okuda M, Horn HF, Tarapore P, Tokuyama Y, Smulian AG, Chan PK, Knudsen ES, Hofmann IA, Snyder JD, Bove KE, Fukasawa K. Nucleophosmin/B23 is a target of CDK2/Cyclin E in centrosome duplication. Cell 103: 127–140, 2000.[CrossRef][Web of Science][Medline]
  56. Pilz RB, Suhasini M, Idriss S, Meinkoth JL, Boss GR. Nitric-oxide and Cgmp analogs activate transcription from Ap-1-responsive promoters in mammalian-cells. FASEB J 9: 552–558, 1995.[Abstract]
  57. Poulaki V, Mitsiades CS, McMullan C, Fanourakis G, Negri J, Goudopoulou A, Halikias IX, Voutsinas G, Tseleni-Balafouta S, Miller JW, Mitsiades N. Human retinoblastoma cells are resistant to apoptosis induced by death receptors: role of caspase-8 gene silencing. Invest Ophthalmol Vis Sci 46: 358–366, 2005.[Abstract/Free Full Text]
  58. Prosniak M, Dierov J, Okami K, Tilton B, Jameson B, Sawaya BE, Gartenhaus RB. A novel candidate oncogene, MCT-1, is involved in cell cycle progression. Circ Res 58: 4233–4237, 1998.
  59. Rape M, Kirschner MW. Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature 432: 588–595, 2004.[CrossRef][Medline]
  60. Rossig L, Fichtlscherer B, Breitschopf K, Haendeler J, Zeiher AM, Mulsch A, Dimmeler S. Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. J Biol Chem 274: 6823–6826, 1999.[Abstract/Free Full Text]
  61. Rouault JP, Falette N, Guehenneux F, Guillot C, Rimokh R, Wang Q, Berthet C, MoyretLalle C, Savatier P, Pain B, Shaw P, Berger R, Samarut J, Magaud JP, Ozturk M, Samarut C, Puisieux A. Identification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway. Nat Genet 14: 482–486, 1996.[CrossRef][Web of Science][Medline]
  62. Ryan KM, Ernst MK, Rice NR, Vousden KH. Role of NF-kappa B in p53-mediated programmed cell death. Nature 404: 892–897, 2000.[CrossRef][Medline]
  63. Sato Y, Tsukada K, Hatakeyama K. Role of shear stress and immune responses in liver regeneration after a partial hepatectomy. Surg Today 29: 1–9, 1999.[CrossRef][Web of Science][Medline]
  64. Satoh Y, Koyama S, Tsukada K, Hatakeyama K. Acute portal hypertension reflecting shear stress as a trigger of liver regeneration after partial hepatectomy. Surg Today 27: 518–526, 1997.[CrossRef][Web of Science][Medline]
  65. Schoen JM, Lautt WW. Nitric oxide potentiates c-fos mRNA expression after 2/3 hepatectomy. Proc West Pharmacol Soc 45: 47–48, 2002.[Medline]
  66. Schoen JM, Wang HH, Minuk GY, Lautt WW. Shear stress-induced nitric oxide release triggers the liver regeneration cascade. Nitric Oxide 5: 453–464, 2001.[CrossRef][Web of Science][Medline]
  67. Shi B, Levenson V, Gartenhaus RB. Identification and characterization of a novel enhancer for the human MCT-1 oncogene promoter. J Cell Biochem 90: 68–79, 2003.[CrossRef][Web of Science][Medline]
  68. Simmen T, Aslan JE, Blagoveshchenskaya AD, Thomas L, Wan L, Xiang Y, Feliciangeli SF, Hung CH, Crump CM, Thomas G. PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis. EMBO J 24: 717–729, 2005.[CrossRef][Web of Science][Medline]
  69. Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article 3, 2004.
  70. Somia NV, Schmitt MJ, Vetter DE, Van Antwerp D, Heinemann SF, Verma IM. LFG: An anti-apoptotic gene that provides protection from Fas-mediated cell death. Proc Natl Acad Sci USA 96: 12667–12672, 1999.[Abstract/Free Full Text]
  71. Su AI, Guidotti LG, Pezacki JP, Chisari FV, Schultz PG. Gene expression during the priming phase of liver regeneration after partial hepatectomy in mice. Proc Natl Acad Sci USA 99: 11181–11186, 2002.[Abstract/Free Full Text]
  72. Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 5: 836–847, 2004.[CrossRef][Web of Science][Medline]
  73. Uematsu M, Ohara Y, Navas JPNK, Murphy TJ, Alexander RW, Nerem RM, Harrison, DG. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol Cell Physiol 269: C1371–C1378, 1995.[Abstract/Free Full Text]
  74. Wack KE, Ross MA, Zegarra V, Sysko LR, Watkins SC, Stolz DB. Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver. Hepatology 33: 363–378, 2001.[CrossRef][Web of Science][Medline]
  75. Wang HH, Lautt WW. Evidence of nitric oxide, a flow-dependent factor, being a trigger of liver regeneration in rats. Can J Physiol Pharmacol 76: 1072–1079, 1998.[CrossRef][Web of Science][Medline]
  76. Wang HH, Lautt WW. Hepatocyte primary culture bioassay: a simplified tool to assess the initiation of the liver regeneration cascade. J Pharmacol Toxicol Methods 38: 141–150, 1997.[CrossRef][Web of Science][Medline]
  77. White P, Brestelli JE, Kaestner KH, Greenbaum LE. Identification of transcriptional networks during liver regeneration. J Biol Chem 280: 3715–3722, 2005.[Abstract/Free Full Text]
  78. Zou Y, Zhu W, Sakamoto M, Qin Y, Akazawa H, Toko H, Mizukami M, Takeda N, Minamino T, Takano H, Nagai T, Nakai A, Komuro I. Heat shock transcription factor 1 protects cardiomyocytes from ischemia/reperfusion injury. Circulation 108: 3024–3030, 2003.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
B. Vollmar and M. D. Menger
The Hepatic Microcirculation: Mechanistic Contributions and Therapeutic Targets in Liver Injury and Repair
Physiol Rev, October 1, 2009; 89(4): 1269 - 1339.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables and Figures
Right arrow All Versions of this Article:
294/3/G819    most recent
00179.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mortensen, K. E.
Right arrow Articles by Revhaug, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mortensen, K. E.
Right arrow Articles by Revhaug, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.