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From the Departments of Surgery,* Medical Oncology, and Pathology, University Medical Center Utrecht, Utrecht, The Netherlands
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Abstract |
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. The aim of this study was to evaluate the contribution of postischemic microcirculatory disturbances, hypoxia, and HIF-1 to I/R-accelerated tumor growth. Partial hepatic I/R attributable to temporary clamping of the left liver lobe induced microcirculatory failure for up to 5 days. This was accompanied by profound and prolonged perinecrotic tissue hypoxia, stabilization of HIF-1, and massive perinecrotic outgrowth of pre-established micrometastases. Restoration of the microcirculation by treatment with Atrasentan and L-arginine minimized hypoxia and HIF-1 stabilization and reduced the accelerated outgrowth of micrometastases by 50%. Destabilization of HIF-1 by the HSP90 inhibitor 17-DMAG caused an increase in tissue necrosis but reduced I/R-stimulated tumor growth by more than 70%. In conclusion, prevention of postischemic microcirculatory disturbances and perinecrotic hypoxia reduces the accelerated outgrowth of colorectal liver metastases after I/R. This may, at least in part, be attributed to the prevention of HIF-1 stabilization. Prevention of tissue hypoxia or inhibition of HIF-1 may represent attractive approaches to limiting recurrent tumor growth after hepatic surgery.
Microcirculatory disturbances, attributable to an imbalance of vasoconstrictors (eg, endothelin-1) and vasodilators (eg, nitric oxide), play a pivotal role in the manifestation of I/R injury.17-21
This no-reflow phenomenon has been extensively studied at the microcirculatory level, but measurements have been limited to relatively short-term reperfusion periods (0 to 120 minutes). Prolonged microcirculatory failure beyond 2 hours of reperfusion may lead to sustained hypoxia in the liver, but data to support this are currently lacking. In response to tissue hypoxia, the transcription factor hypoxia-inducible factor (HIF)-1 is stabilized and acts as a cellular survival factor.22-28
HIF-1 is a strong stimulator of tumor cell proliferation, anaerobic metabolism, migration, and angiogenesis.29-33
Thus, hypoxia and consequent stabilization of HIF-1 in tissue areas containing residual micrometastases may lead to enhanced tumor cell growth.
The aim of this study was to assess the role of microcirculatory disturbances and hypoxia on the outgrowth of micrometastases after I/R. Therefore, we investigated whether improvement of the microcirculation by restoring the endothelin-1/nitric oxide balance by means of Atrasentan and L-arginine could reduce tissue hypoxia and thereby prevent the accelerated outgrowth of pre-established micrometastases after I/R. Moreover, we used the geldanamycin analogue 17-DMAG, which has been shown to induce a von Hippel-Lindau-independent degradation of HIF-134,35
and inhibit hypoxia-dependent tumor growth.36,37
We investigated whether destabilization of HIF-1 by 17-DMAG could reduce I/R-accelerated tumor growth.
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Materials and Methods |
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All experiments were performed in accordance with the guidelines of the Animal Welfare Committee of the University Medical Centre Utrecht, Utrecht, The Netherlands. Male BALB/c mice (10 to 12 weeks of age) were purchased from Charles River (Sulzfeld, Germany) and housed under standard laboratory conditions.
Drug Characteristics
Atrasentan (ABT-627), a selective endothelin-A receptor antagonist, was kindly provided by Abbott Laboratories (Abbott Park, IL). Atrasentan was dissolved in 4.2% NaHCO3 and injected intravenously as a single bolus (10 mg/kg body weight) before ischemia, followed by continuous oral administration offered in the drinking water (10 mg/kg body weight per day). The nitric oxide donor L-arginine (Sigma-Aldrich, St. Louis, MO) was administered via continuous subcutaneous infusion using Alzet osmotic minipumps (model 2001; Durect Corp., Cupertino, CA) at a dose of 30 µg/kg/minute starting before ischemia. 17-DMAG (Invivogen, San Diego, CA) was dissolved in saline and administered by intraperitoneal injections at a preoperative dose of 15 mg/kg body weight, followed by three postoperative doses of 7.5 mg/kg body weight every 12 hours after ischemia.
Standardized Murine Model of Hepatic I/R
We used an established model of partial hepatic I/R as previously described.16
In brief, after a midline incision, temporary ischemia was induced by occluding the vascular inflow of the left lateral liver lobe for 45 minutes. Sham-operated mice underwent laparotomy with exposure of the liver but without interruption of hepatic blood flow. Surgical procedures were performed under isoflurane inhalation anesthesia. Body temperature was maintained at 36.5 to 37.0°C. Nontumor-bearing animals were subjected to left lobar I/R and were randomized to receive control vehicle or Atrasentan treatment combined with L-arginine (n = 8 each group). Sham-operated mice served as controls. After 2 hours, 6 hours, 24 hours, and 5 days of reperfusion, the animals were reanesthetized for measurements of the hepatic microcirculation. At each time point, ethylenediaminetetraacetic acid plasma samples were collected via cardiac puncture for measurements of liver enzymes and livers were harvested and processed for pimonidazole and HIF-1 immunohistochemistry.
Liver Metastases Model
Colorectal liver metastases were induced in mice as described.16,38,39 In brief, routinely cultured C26 colorectal carcinoma cells were injected into the splenic parenchyma (5 x 104 cells/100 µl). After 10 minutes, the spleen was removed to prevent intrasplenic tumor growth. Five days later, animals were subjected to left lobar I/R and randomized into different treatment groups (n = 8 each group). Mice received either continuous administration of Atrasentan and L-arginine or control vehicle. In a second experiment, I/R-treated mice received either 17-DMAG or control vehicle. Sham-operated mice served as controls (n = 8 each group). Morphometric assessment of tumor growth and hepatic necrosis was performed on nonclamped and clamped liver lobes harvested 5 days later.
Intravital Fluorescence Microscopy
The hepatic microcirculation was analyzed by intravital fluorescence microscopy using a Nikon TE-300 inverted microscope (Uvikon, Bunnik, The Netherlands) equipped with a fluorescence filter for fluorescein isothiocyanate (excitation 450 to 490 nm, emission >515 nm).40 For contrast enhancement, fluorescein isothiocyanate-labeled dextran (molecular weight 446.000, 2% dextran in 0.9% NaCl; 100 µl per 20 g of body weight) was injected intravenously and excited with blue light (450 to 490 nm). The midline incision was reopened, and the left liver lobe was exteriorized and moistened. The mice were placed on an inverted microscopic stage, using a template to minimize tension and respiratory movement. Body temperature was monitored and maintained at 36.5 to 37.0°C during the entire experiment. Ten to 15 fields were randomly selected per animal and recorded for 10 seconds at a x40 magnification. Images were captured by a charge-coupled device camera (Exwave HAD; Sony, Badhoevedorp, The Netherlands) and relayed to a personal computer for off-line analysis. Sinusoidal perfusion rates were analyzed by two independent observers, blinded to treatment. Sinusoidal perfusion rates were expressed as the percentage of the number of normally perfused sinusoids divided by the total number of sinusoids observed. Perfusion rates of sham-operated animals were set at 100%. Using this method, interobserver and intraobserver variability was less than 5%.
Liver Enzymes
Plasma levels of alanine aminotransferase and aspartate aminotransferase were analyzed using commercially available diagnostic kits (Instruchemie BV, Delfzijl, The Netherlands) (n = 8 each group).
Quantification of Hepatocellular Necrosis
The percentage of hepatocellular necrosis was scored on two nonsequential hematoxylin and eosin (H&E)-stained sections using an interactive video overlay system including an automated microscope (Q-Prodit; Leica Microsystems, Rijswijk, The Netherlands) at a magnification of x40. Using a four-point grid overlay, the ratio of necrotic cells versus healthy hepatocytes plus tumor cells was determined on at least 100 fields per animal (n = 8 each group). The percentage of hepatocellular necrosis was expressed as the mean area ratio of all fields.
Pimonidazole and HIF-1 Immunohistochemistry
Liver samples were harvested, fixed in 4% buffered formalin, paraffin-embedded, and sectioned at 5 µm. Tissue sections of clamped and nonclamped liver lobes were H&E-stained for standard histology. The extent and localization of hypoxia were analyzed using the hypoxia marker pimonidazole hydrochloride (Hypoxyprobe-1, 90201; Chemicon International, Temecula, CA) (n = 4 each group).41,42
Under hypoxic conditions, pimonidazole binds to thiol groups in proteins, peptides, and amino acids and therefore serves as a valuable marker for measuring in vivo hypoxia at the cellular level. One hour before termination, pimonidazole was injected intravenously at a dose of 60 mg/kg. Pimonidazole adducts were visualized by immunohistochemistry by using fluorescein isothiocyanate-conjugated mouse anti-Hypoxyprobe-1 monoclonal antibody (1:100, TM 90529; Chemicon International), followed by anti-fluorescein isothiocyanate antibody labeled with horseradish peroxidase (1:50; DAKO, Glostrup, Denmark). For HIF-1 detection, a polyclonal rabbit antibody against mouse HIF-1 was used for incubation overnight (1:50, NB 100-449; Novus, Littleton, CO) and goat anti-rabbit PowerVision+ with 2% mouse serum was used as secondary antibody. Before immunolabeling, endogenous peroxidase activity was blocked using a solution of methanol and hydrogen peroxide. Antigen retrieval was performed by boiling sections for 20 minutes in 0.01 mol/L citrate buffer (pH 6.0) for pimonidazole staining and in ethylenediaminetetraacetic acid-buffered solution (pH 9.0) for HIF-1 staining. Reactions were developed using diaminobenzidine/H2O2 as a chromogen substrate. Primary-deleted negative controls were treated with the antibody diluent alone and were all free of nonspecific background staining. Immunostaining for HIF-1 was scored on three to four randomly selected fields at a magnification of x40 by two independent observers, blinded to treatment (n = 6 each group). The nuclear and cytoplasmic staining patterns were scored as the product of the staining intensity (weak, 1+; moderate, 2+; strong, 3+) and the percentage of positive cells (1 to 10% of cells, 1+; 11 to 50% of cells, 2+; >50% of cells, 3+).
Tumor Load
Intrahepatic tumor load was scored as the hepatic replacement area (HRA).16,38,39 For each liver lobe at least 100 fields were selected on two nonsequential H&E-stained sections using an interactive video overlay system including an automated microscope (Q-Prodit) at a magnification of x40. Using a four-point grid overlay, the ratio of tumor cells versus normal hepatocytes plus necrotic cells was determined for each field. Tumor load (HRA) was expressed as the mean area ratio of all fields. Observers were blinded to treatment. Finally, HRA ratios between clamped and nonclamped lobes were calculated for each animal to express the proportional increase in HRA in the clamped (left) lobes versus the nonclamped (right plus median) lobes.
Statistical Analysis
Statistical differences between groups were analyzed using the Mann-Whitney U-test. Data are expressed as mean ± SEM, unless otherwise stated.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Intravital fluorescence microscopy revealed normal hepatic microcirculation with >80% regularly perfused sinusoids in sham-operated animals throughout the study period. I/R induced severe sinusoidal perfusion failure, as shown by a 70% relative reduction of perfused sinusoids after 2 hours of reperfusion when compared with sham-operated mice (Figure 1a)
. Sinusoidal perfusion rates remained low during the 5-day reperfusion period demonstrating a long-term disturbance of the microcirculation. Concurrently, I/R induced severe hepatocellular injury as shown by elevated levels of plasma alanine aminotransferase (Figure 1b)
and aspartate aminotransferase (Figure 1c)
, with a maximum increase at 6 hours of reperfusion followed by normalization after 5 days. Histopathologically, areas of severe hepatocellular damage were seen at 2 and 6 hours after ischemia in all clamped liver lobes, characterized by hemorrhage, eosinophilic hepatocytes, signs of nuclear pyknosis, and loss of cell-cell contact (Figure 2a)
. After 24 hours, areas of necrosis started to develop, characterized by a massive infiltration of neutrophils. Five days after I/R, necrotic areas were observed in all animals, covering 17 ± 3% of the clamped liver tissue (Figure 1d)
. As reported previously, these necrotic areas were surrounded by a massive inflammatory infiltrate (Figure 2a)
.
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.41
Two hours after I/R, profound diffuse tissue hypoxia was observed throughout the liver parenchyma of the clamped but not of the nonclamped lobes (Figure 2b)
. During the ensuing 5 days, the liver parenchyma remained remarkably hypoxic. Whereas pimonidazole staining was diffuse after 2 hours of reperfusion, it was selectively localized around areas of hepatocellular necrosis at 5 days after clamping.
Increased Expression of HIF-1 in Hypoxic Tissue Areas
HIF-1 immunohistochemistry was performed on nontumor-bearing tissue sections of livers from sham-operated mice and of clamped and nonclamped liver lobes 2 hours, 6 hours, 24 hours, and 5 days after I/R. In sham-operated mice and in the nonclamped liver lobes, HIF-1 staining was observed around central venules, similar to the pimonidazole staining (Figure 2c)
.43
Two hours after I/R, strong cytoplasmic and nuclear HIF-1 staining were observed in hepatocytes throughout the clamped liver lobes (Figure 3, a and b
; and Figure 2c
). After 6 hours of reperfusion, staining was mainly nuclear and was localized in zones surrounding tissue necrosis (Figure 3, a and b
; and Figure 2c
). From 24 hours onwards, both cytoplasmic and nuclear HIF-1 staining could still clearly be observed in perinecrotic zones (Figure 3, a and b)
, which is consistent with the perinecrotic pimonidazole staining (Figure 2c)
. The nuclear staining at 5 days after clamping was partly attributable to nuclear staining of inflammatory cells surrounding the necrotic tissue areas.
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Expression
I/R resulted in a sixfold to sevenfold increase in micrometastasis outgrowth in occluded liver lobes when compared with nonoccluded liver lobes (Figure 4a)
. Similar to our earlier observations,16
accelerated tumor growth was predominantly located around necrotic tissue areas (Figure 4b)
. We observed a strong association of I/R-stimulated tumor growth with areas of tissue hypoxia and increased parenchymal HIF-1 expression. Strikingly, high levels of HIF-1 were detected in the nuclei of tumor cells at the tumor-necrosis margin, 5 days after clamping (Figure 4c)
. HIF-1 immunostaining in control tumor tissue in sham-operated mice is rare (Figure 4c)
.
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Treatment with Atrasentan/L-arginine markedly restored the postischemic microcirculation, with a significantly higher percentage of perfused sinusoids at all time points when compared with the control vehicle-treated I/R group (Figure 1a)
. Correspondingly, Atrasentan/L-arginine treatment effectively reduced hepatocellular injury (Figure 1, b and c)
. In addition, tissue necrosis was reduced by 70% and covered 5 ± 2% of the hepatic tissue (Figure 1d)
. Pimonidazole immunohistochemistry revealed minimal tissue hypoxia at 2 hours of reperfusion in mice treated with Atrasentan/L-arginine (Figure 2d)
. At later time points, Atrasentan/L-arginine had completely prevented detectable tissue hypoxia. HIF-1 immunohistochemistry was reminiscent of pimonidazole staining and revealed reduced immunostaining when compared with I/R at all time points (Figure 2e)
. Despite a distinct cytoplasmic immunostaining at 2 hours of reperfusion, this was not associated with exacerbated nuclear staining (Figure 3, a and b)
. Next, we investigated whether improvement of the microcirculation and reduction of tissue hypoxia by means of Atrasentan/L-arginine treatment would also reduce tumor growth after I/R. Because Atrasentan and/or L-arginine may influence tumor growth independent of I/R damage, we first evaluated the effect of Atrasentan/L-arginine treatment on tumor growth in sham-operated mice. In these animals, tumor growth was unaffected (data not shown). In mice subjected to I/R, the accelerated outgrowth of micrometastases was inhibited by 50% after Atrasentan/L-arginine treatment (Figure 4a)
. Interestingly, microscopic evaluation revealed that the few necrotic areas present were surrounded by fields of tumor cells (Figure 4b)
, which accounted for a residual 50% increase in HRA ratio when compared with sham operation. Similar to the observations in vehicle-treated animals, we found strong nuclear HIF-1 staining in cells at the tumor-necrosis margin (Figure 4c)
. These data strongly suggest that perinecrotic tissue hypoxia and, possibly, the subsequent stabilization of HIF-1 after I/R contribute to the accelerated outgrowth of micrometastases.
Inhibition of I/R-Accelerated Tumor Growth by 17-DMAG
Finally, we used the heat shock protein-90 inhibitor 17-DMAG to promote destabilization of HIF-1.34,35
In control vehicle-treated mice, tumor growth was stimulated more than sevenfold after I/R, similar to the first set of experiments (Figure 5, a and b)
. Again, tumor growth was associated with tissue necrosis (Figure 5, c and d)
and positive nuclear HIF-1 immunostaining at the tumor-necrosis margin (Figure 5e)
. 17-DMAG had no significant inhibitory effect on tumor growth in sham-operated mice (data not shown). Strikingly, 17-DMAG induced a significant reduction in tumor growth in the clamped liver lobes (Figure 5a)
, without affecting tumor growth in nonclamped liver lobes. This resulted in a 70% decrease in HRA ratio, reflecting a selective inhibitory effect on the perinecrotic stimulation of tumor growth (Figure 5b)
. Nonetheless, the percentage of necrotic tissue after 17-DMAG had increased from 31 to 56% (Figure 5c)
, indicating that the treatment had also interfered with the tissue-protecting effect of HIF-1. Despite this increase in tissue necrosis in the clamped liver lobes, the accelerated perinecrotic tumor growth was not observed (Figure 5d)
. This is the first time that we observed extensive tissue necrosis without associated tumor growth. Most importantly, microscopic lesions at the necrosis margin did not show significant HIF-1 staining, indicating that 17-DMAG indeed prevented HIF-1 stabilization (Figure 5e)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; 2) prevention of postischemic microcirculatory disturbances minimizes tissue hypoxia, avoids HIF-1 stabilization, and reduces the accelerated outgrowth of micrometastases; and 3) destabilization of HIF-1 by 17-DMAG reduces I/R-stimulated tumor growth. We conclude that prolonged tissue hypoxia and, possibly, subsequent stabilization of HIF-1 play an important role in the altered behavior of micrometastases in the liver after I/R.
Whereas prolonged periods of hypoxia are deleterious to most cells, tumor cells have adapted to survive under hypoxic conditions.44
Intratumoral hypoxia has been demonstrated in a number of human cancers, and elevated expression of HIF-1 has been related to tumor aggressiveness.33,45-49
HIF-1 activates the transcription of several genes that are implicated in cancer progression, including proliferation-promoting cytokines and growth factors; angiogenesis-promoting growth factors; glucose transporters; and serine-, aspartic-, and metalloproteases.29-33
Thus, prolonged hypoxia as it occurs after I/R may provide a protumorigenic microenvironment through up-regulation of HIF-1.
Interestingly, pimonidazole and HIF-1 immunostaining were mainly located around necrotic tissue areas. This is consistent with the HIF-1 staining patterns at the necrosis-viable margin of several tumors.47,50
In the clamped liver lobes, HIF-1 immunoreactivity in hepatocytes was primarily seen after 2 and 6 hours of reperfusion. The strong cytoplasmic staining may be the result of enhanced protein stabilization and accumulation.43,46,47
It was recently suggested that in the liver, HIF-1 targets to the peroxisome rather than the nucleus after hepatic hypoxia/reoxygenation.28
We found that HIF-1 staining was sustained up to 5 days, which is consistent with other reports.51
We used the geldanamycin analogue 17-DMAG to promote a von Hippel-Lindau-independent degradation of HIF-1.34,35
In this study a short-term treatment of 17-DMAG markedly reduced the accelerated outgrowth of micrometastases. Because 17-DMAG inhibits the function of heat shock protein-90, other mechanisms may have co-contributed to the effectiveness of 17-DMAG in reducing postischemic tumor growth. Heat shock proteins are expressed acutely in response to hypoxia and I/R, and they have numerous target genes aimed at promoting cell survival.
Several other mechanisms may have indirectly or directly contributed to I/R-stimulated tumor growth. After I/R, endothelin-1 is increased in the reperfusion period and exerts its vasoconstrictive action via the endothelin-A receptor, contributing to the microcirculatory disturbances after I/R.52-54
Endothelin-1 may also directly stimulate tumor cell proliferation,55,56
and its expression correlates with the stabilization of HIF-1.57
Finally, the inflammatory response associated with I/R, including the influx of neutrophils and activation of Kupffer cells, may also contribute to the stimulation of tumor growth. Both activated neutrophils and macrophages have been associated with increased metastatic potential, proliferation, and invasion.58-62
Interestingly, hypoxic macrophages secrete growth factors and angiogenic factors that may favor tumor progression. Furthermore, increased HIF expression has been associated with the presence of tumor-associated macrophages.63
In addition, the influx of neutrophils contributes to microcirculatory disturbances.64
Evidently, inflammation and hypoxia mutually influence each other and may co-activate tumor cell proliferation. Our observation that perinecrotic hypoxia was closely associated with the presence of inflammatory cells supports this notion. Thus, tampering the inflammatory response may also minimize hypoxia and thereby reduce tumor growth.
Taken together, although the effects of endogenous (ie, intratumoral) hypoxia on tumor growth have been well documented, this study now shows that exogenous (ie, I/R-induced) hypoxia stimulates tumor growth as well. This may possibly be, at least in part, attributed to the stabilization of HIF-1. Clinically, this is very relevant because recurrent tumor growth after an apparently complete tumor resection occurs in the majority of patients undergoing liver surgery for colorectal liver metastases. We recently found that severe ischemia as a result from prolonged vascular clamping was associated with a reduced time to develop liver recurrence and a decreased disease-free survival (unpublished data). However, these issues are not only important in case of inevitable clamping of the hepatic blood flow during liver surgery but also during other events that cause I/R injury, such as hemorrhagic shock or sepsis.65,66
Similar phenomena may occur during surgery-induced tissue hypoxia after wounding, inflammation, and organ manipulation.42,67-69
Thus, prevention of tissue hypoxia or inhibition of HIF-1 activity may represent attractive approaches to limiting recurrent tumor growth after hepatic surgery. First, therapeutic strategies that restore postischemic microcirculatory flow are potential candidates for reducing I/R-accelerated tumor growth. In the past, several compounds that aim at restoring the endothelin-1/nitric oxide imbalance have been successfully applied to reduce microcirculatory disturbances and liver tissue damage.18,19,54,70,71
Atrasentan is effective in reducing postischemic microcirculatory disturbances in models of renal,72
cardiac,73
and cerebral74
I/R and we here show that, in combination with the nitric oxide donor L-arginine, it provides maximal improvement of the microcirculation after hepatic I/R. Second, agents that target the HIF-1 pathway, including 17-DMAG, are gaining increased attention as novel anti-cancer drugs.75,76
Such compounds may ideally be administered perioperatively to prevent hypoxia-induced tumor growth stimulation. However, HIF-1 is also involved in the physiological response to tissue damage, and thus, their perioperative application may hamper wound and anastomosis healing, which may contribute to increased morbidity. Indeed, we found increased necrosis in clamped liver lobes after treatment with 17-DMAG. Further studies are needed to investigate the effectiveness and safety of such compounds in the postoperative setting.
In conclusion, long-term microcirculatory disturbances, perinecrotic hypoxia, and, possibly, the stabilization of HIF-1 play an important role in the accelerated outgrowth of colorectal liver metastases after I/R. In case of inevitable ischemic damage and hypoxia during hepatic surgery, improving microcirculatory flow or targeting the HIF-1 pathway may decrease the stimulation of microscopic tumor deposits and improve prognosis in colorectal cancer patients.
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Footnotes |
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Supported by The Netherlands Organization for Health Research and Development (grant no. 920-03-313).
Accepted for publication January 10, 2007.
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