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Vascular endothelial growth factor and nitric oxide in rat liver regeneration
Life Sciences 81 (2007) 750 – 755 www.elsevier.com/locate/lifescie
Vascular endothelial growth factor and nitric oxide in rat liver regeneration Maria Teresa Ronco, Daniel Francés, Maria de Luján Alvarez, Ariel Quiroga, Juan Monti, Juan Pablo Parody, Gerardo Pisani, Maria Cristina Carrillo, Cristina Ester Carnovale ⁎ Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina Received 23 April 2007; accepted 6 July 2007
Abstract In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65 ± 10.70; PH-Control: 88.13 ± 1.60⁎; Sh-SNP: 90.35 ± 3.11⁎; PH-SNP: 119.5 ± 12.10⁎#; Sh-AG: 33.27 ± 5.23, PH-AG: 36.80 ± 3.40#) (p b 0.05 vs Sh-Control; #p b 0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats. © 2007 Elsevier Inc. All rights reserved. Keywords: Liver regeneration; Nitric oxide; Vascular endothelial growth factor; Partial hepatectomy
Introduction The liver has the unique and remarkable ability to restore itself following cell loss (toxic, viral or surgical). Despite being specialized and highly differentiated cells, adult hepatocytes remain capable of rapid proliferation when appropriately stimulated. Following a two-thirds partial hepatectomy (PH), liver cells switch from a quiescent state into a proliferative state and re-enter cell cycle, beginning DNA synthesis as a semisynchronised cohort about 12–15 h after surgery (Fausto, 2000; Mangnall et al., 2003; Li et al., 2001). The mechanisms regulating regenerative processes are complex and incompletely understood. A large number of immediate and delayed early genes, which are not normally ⁎ Corresponding author. Instituto de Fisiología Experimental, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570-2000 Rosario, Argentina. Fax: +54 341 4399473. E-mail address: [email protected]. (C.E. Carnovale). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.07.009
expressed in the quiescent liver, are activated (Li et al., 2001). Immediately after PH (1–6 h), nitric oxide (NO) is synthesized by liver parenchymal and non-parenchymal cells from Larginine, via induction of the inducible form of nitric oxide synthase (iNOS) (Obolenskaya et al., 1994; Carnovale et al., 2000; Ronco et al., 2004). NO is a highly reactive molecule, known to be involved in diverse biological processes in nearly all aspects of life. The precise role of NO in the regenerative process is still unidentified, although the importance of NO in this process is suggested by the finding that liver regeneration is impaired in iNOS-deficient mice (Rai et al., 1998). One of the well-characterized functions of NO is as a mediator of vascular dilatation and permeability and it is involved in vascular remodeling (Hortelano et al., 1995; Carnovale et al., 2000). Angiogenesis, the formation of new blood vessels, is a complicated process that implies proliferation and migration of endothelial cells. This phenomenon is required for remodeling liver architecture following liver resection
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(Kraizer et al., 2001; Assy et al., 1999). The initial wave of hepatocyte proliferation is followed by endothelial cell proliferation and penetration of vascular hepatocellular islands leading to the formation of new sinusoids (Sato et al., 2001). In liver regeneration after PH, both hepatocytes and nonparenchymal cells express vascular endothelial growth factor (VEGF) mRNA, suggesting that VEGF plays a significant role in this process. Hepatocellular production of VEGF shows the maximal levels between 48 and 72 h after PH (Mochida et al., 1996; Taniguchi et al., 2001). Moreover, recent studies have shown that inhibition of angiogenesis with angiostatin impairs liver regeneration (Redalli et al., 2004). VEGF also increases dilatation and permeability of blood vessels, and it has been suggested that NO may be involved in VEGF signalling pathways. Although there is a growing body of evidence that NO has angiogenic effects, partly mediated by VEGF induction in different cellular types (Jozkowicz et al., 2001), there is not enough evidence about the role of NO in VEGF induction during rat liver regeneration. In the present work, we investigated the role of NO in VEGF induction in liver tissue after PH in rats. Experimental procedures Animals and surgical procedures Male Wistar rats weighing 300 to 360 g were housed two per cage and maintained under a 12-hour light/dark period. Rats were fed ad libitum with a normal standard diet and water. Animals received humane care according to criteria the “Guide for the Care and Use of Laboratory Animals”. The animals were anesthetized with pentobarbital solution (50 mg/kg body weight) injected i.p. The peritoneal cavity was opened by a median incision, and the liver was freed from its ligaments with minimal manipulation. Two-thirds hepatectomy (PH) was performed following the standard Higgins and Anderson technique in which the median and left lateral lobes were ligated at their roots and were then removed (Higgins and Anderson, 1931). Surgical sham control rats (Sh-Control) underwent midline laparotomy with liver manipulation. To avoid variations due to circadian rhythms, the time to perform the surgery had to be fixed in order to sacrifice the animals always at the same time of the day (between 10:00 and 12:00 h). Increase of nitric oxide by nitroprusside sodium administration To obtain maximal NO levels at 5 h post-surgery, sodium nitroprusside (SNP; Salom et al., 2000) was administered intravenously for 30 min at a rate of 1 ml/h, beginning at 4.5 h after surgical procedures. SNP (Merck) was dissolved and diluted in 0.9% isotonic saline. Care was taken to protect SNP solutions from light due to its light sensitivity. Both Sh and PH animals were randomized in two sub-groups that received isotonic saline (Control) and SNP (0.25 mg/kg body weight). Four rats of each group (Sh-Control, Sh-SNP, PH-Control, PHSNP) were sacrificed at each time after surgery: 5, 24 and 72 h.
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Inhibition of nitric oxide synthesis A group of rats received isotonic saline (control) and another group of animals received aminoguanidine (AG), a preferential iNOS inhibitor. AG solution was prepared in isotonic saline and administered intraperitoneally (100 mg/kg body weight) once a day, beginning 3 days before surgery (Carnovale et al., 2000). Six animals from each group (Sh-Control, Sh-AG, PH-Control and PH-AG) were killed at each time after surgery: 5 and 72 hours. Analytical assays Nitrate determination NO formation was indirectly assessed in cytosolic fractions by determining the concentration of nitrate, one of the stable end products of NO oxidation. An assay based on the conversion of nitrate to nitrite by reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in the presence of nitrate reductase enzyme was used (Bories and Bories, 1995). The amount of NADPH oxidized during the reaction was stoichiometrically equivalent to the amount of nitrate present in the samples. The decrease in NADPH was followed at 340 nm. Cytosolic fractions were prepared as described by de Duve et al. Briefly, frozen liver tissues were homogenized in 3 volumes of 0.3 M sucrose containing 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1 μg/ml aprotinin. Homogenates were centrifuged at 1000 g to remove non-lysed cells, nuclei and heavy membranes. Supernatants were centrifuged at 3000 g at 4 °C for 15 min and pellets were discarded. Cytosolic fractions were obtained by centrifugation of supernatants at 56,000 rpm at 4°C for 60 min. Electron Paramagnetic Resonance (EPR) spectroscopy Sh and PH male Wistar rats, as described in “Animals and Surgical Procedures”, were used throughout. Thirty minutes before sacrifice, all animals were given ferrous sulfate (37.5 mg/ kg body weight, Cicarelli) and disodium citrate dihydrate (187.5 mg/kg body weight, Cicarelli), subcutaneously, and afterwards, diethyldithiocarbamate (DETC, 500 mg/kg w.t., Anedra), intraperitoneally. At 5 h after surgery, animals were sacrified and livers were promptly removed. After a rapid washing in cold saline solution, samples were placed in 1 ml syringes, frozen and kept in liquid nitrogen until they were subjected to EPR. Samples were transferred to a Dewar and EPR spectra were recorded at liquid nitrogen temperature on a Bruker ECS 106 ESR, operating at the microwave frecuency 9.41 GHz, microwave power 10 mW, modulation amplitude 4.75 G and central field 3350 G and were measured at RG: 1 × 103 and 1 scan (Mikoyan et al., 1997; Obolenskaya et al., 1994). Western blot analysis: vascular endothelial growth factor (VEGF) For VEGF detection, liver tissue lysates were prepared by homogenization of frozen tissues in 3 volumes of lysating RIPA buffer containing PBS, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1 μg/ml aprotinin. After 30 min of incubation at
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0 °C and three freeze–thaw cycles, lysates were cleared by centrifugation at 15,000 rpm for 30 min, and supernatants were kept at − 70 °C (Ronco et al., 2004). Protein content was quantified according to Lowry et al. Fifty micrograms of protein was subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvynil difluoride membranes (PVDF, Perkin Elmer Life Sciences, Boston, MA, USA). After blocking, blots were incubated overnight at 4 °C with an antimouse VEGF antibody (1:500, Santa Cruz, Biotechnology, USA). Membranes were then incubated with anti-mouse IgGperoxidase conjugates (1:5000, Amersham Life Science) and the bands were detected by an enhanced chemiluminescence method (ECL; Amersham Pharmacia Biotech). Autoradiographs were obtained by exposing membranes to Kodak XAR film, and the bands were quantified by densitometry (Shimadzu CS-9000). Statistical analysis Results were expressed as mean ± S.E. Statistical analysis was performed by using one-way ANOVA, followed by Tukey's test. Differences were considered to be statistically significant when the p value was b0.05. Results NO detection We determined nitrate concentration in hepatic cytosol to evaluate NO production. Fig. 1 clearly shows an increase (80%) in hepatic cytosol nitrate content at 5 h post-hepatectomy. SNP treatment, a tool for increasing NO levels, produced no changes in nitrate levels as compared to PH-Control. NO generation after PH was inhibited by AG treatment (Fig. 1), which decreased by 34% the hepatic cytosol nitrate content with respect to PH-Control.
Fig. 1. Production of nitrate, a metabolic product of NO, measured in hepatic cytosol, expressed as nmol per mg of protein at 5 h post-surgery. Sh, sham operated; Sh-SNP, Sh-treated with SNP 0.25 mg/kg body weight. PH-Control, partial hepatectomy control; PH-SNP, PH-treated with SNP 0.25 mg/kg body weight; PH-AG, PH-treated with AG 100 mg/kg body weight. Data are expressed as mean ± SE of six rats. ⁎Significantly different from Sh-Control. # Significantly different from PH-Control. (p b 0.05).
Fig. 2. EPR spectra (9.41 GHz) of NO–Fe+2–DETC complex in rat liver homogenates. A) Sh-Control; B) PH-Control; C) Sh-SNP (SNP: 0.25 mg/kg body weight); D) PH-SNP (SNP: 0.25 mg/body weight); E) Sh-AG (AG: 100 mg/kg body weight); F) PH-AG (AG: 100 mg/kg body weight). Records were performed at nitrogen liquid temperature, microwave power 10 mW, modulation amplitude 4.75 G, and central field 3350 G. The samples were determined at RG: 1 × 103 and 1 scan.
NO production was also estimated from the intensity of the EPR signal of the mononitrosyl iron complex with DETC. Fe+2–DETC complexes preformed in-vivo served as a trap for the endogenously produced NO (Quaresima et al., 1996). The typical EPR spectra corresponding to NO–Fe+2–DETC complex of rats liver producing NO were observed and a three line EPR spectrum (G = 2.038) was obtained from every tissue homogenate (Fig. 2) at 5 h after surgery. Table 1 shows the values related to the intensity of each signal of the NO–Fe+2– DETC complex. As expected, the signal intensity for PHControl animals was higher than that for Sh-Control animals. Treatment with SNP resulted in the highest EPR signal intensity of the NO trapped in liver homogenates. On the other hand, AG treatment decreased the intensity of the signal of the NO–Fe+2– DETC complex (Table 1). In-vivo EPR spectroscopy is a useful tool for the study of in-situ, real time NO generation (Quaresima et al., 1996). Its high sensitivity allows us to detect differences between PH-SNP animals and PH-Control animals, that were not observed by means of nitrate measurement. Probably, this could be due to the lack of sensitivity of the activity assay. NADPH can act as a cofactor for a variety of enzymes such as NADPH oxidase, among others. Moreover, some ions (manganese, chloride, cyanide and sulfite) are able to inactivate o retard nitrate conversion.
M.T. Ronco et al. / Life Sciences 81 (2007) 750–755 Table 1 Intensity of NO–Fe+2–DETC complex signal in liver homogenates Samples
NO–Fe+2–DETC arbitrary units
Sh-Control PH-Control Sh-SNP PH-SNP Sh-AG PH-AG
37.65 ± 10.70 88.13 ± 1.60⁎ 90.35 ± 3.11⁎ 119.5 ± 12.10⁎# 33.27 ± 5.23# 36,80 ± 3.40#
⁎Significant difference vs Sh-Control. #Significant difference vs PH-Control.
VEGF detection NO acts as a mediator of vessels dilatation and permeability and it is known that angiogenesis is a fundamental process involved in the remodeling of hepatic architecture following liver resection, playing an important role in hepatocyte blood supply. Thus, we determined the levels of VEGF protein by western blot in total liver lysates at 24 and 72 h after PH (Fig. 3). At 24 h post-hepatectomy, PH-Control and PH-SNP animals showed an increase of VEGF levels as compared to Sh-Control. At this time after PH, VEGF levels showed no differences between PH-Control and PH-SNP animals. In concordance with other authors, PH-Control animals showed an increase in VEGF levels with respect to Sh-Control at 24 and 72 h after PH. SNP treatment increases by above 300% the levels of VEGF at 72 h after PH (Fig. 3B). Other authors have previously shown that NO positively regulates VEGF expression in different tissues (Jozkowicz et al., 2001). To better understand the relationship between NO and VEGF, we inhibited NO production by administration of AG (a specific iNOS inhibitor). This study was performed only at 72 h after PH because it has been reported that VEGF expression is maximal at this time
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(Taniguchi et al., 2001). We observed that the pre-treatment with AG resulted in a diminution of VEGF protein levels of 60% at 72 h post-PH (Fig. 4). Discussion The liver ability to restore major tissue loss is a unique process that involves numerous cell interactions and a complex network of mediators. PH in rats results in the induction of gene expression and release of biologically active substances which regulate hepatic regeneration. There is a fine balance between stimulators and inhibitors, an equilibrium that makes quiescent hepatocytes proliferate after tissue resection in order to restore the functional capacity of the liver as well as its mass (Fausto, 2000; Mangnall et al., 2003; Li et al., 2001). Liver regeneration requires the formation of new blood vessels (angiogenesis) for reconstruction of hepatic sinusoids. Various angiogenic factors have been identified, but VEGF is the most potent and specific growth factor for both angiogenesis and vasculogenesis (Kraizer et al., 2001; Sato et al., 2001; Yancopoulos et al., 2000). VEGF have been demonstrated to play a central role in division, migration, and tubule formation of vascular endothelial cells. Although a variety of growth factors and cytokines have been implicated in liver regeneration, the contribution of VEGF is unclear in details. After PH, both hepatocytes and non-parenchymal cells express VEGF mRNA, suggesting that VEGF plays a significant role in this process (Taniguchi et al., 2001; Mochida et al., 1996). In agreement, we observed that liver homogenates from hepatectomized rats show increased VEGF levels compared with ShControl animals at 24 and 48 h post-PH. Previous studies of our group, in agreement with other authors, demonstrated that iNOS is up-regulated in the liver
Fig. 3. Immunoblotting analysis of VEGF protein expression in liver total lysates fraction at 24 and 72 h post-surgery: A) after 24 h: Line 1: Sh-Control; Line 2: PHControl; Line 3: Sh-SNP (SNP: 0.25 mg/kg body weight); Line 4: PH-SNP (SNP: 0.25 mg/kg body weight); B) after 72 h: Line 1: Sh-Control; Line 2: PH-Control; Line 3: Sh-SNP (SNP: 0.25 mg/kg body weight); Line 4: PH-SNP (SNP: 0.25 mg/kg body weight). The accompanying bars represent the densitometry expressed in percentage from five separate animal sets. ⁎Significantly different from Sh-Control. #Significantly different from PH-Control. (p b 0.05).
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Fig. 4. Immunoblotting analysis of VEGF protein expression in total liver lysates fraction at 72 h post-surgery. A) Line 1: Sh-Control; Line 2: PH-Control; Line 3: Sh-AG (AG: 100 mg/kg body weight); Line 4: PH-AG (AG: 100 mg/kg body weight). The accompanying bars represent the densitometry expressed in percentage from five separate animal sets. ⁎Significantly different from ShControl. #Significantly different from PH-Control. (p b 0.05).
with maximal levels of expression at 5 h post-PH; a large amount of NO is generated, returning to basal levels at 18 h after PH (Carnovale et al., 2000, Hortelano et al., 1995). In this work, we used a direct NO donor (SNP) in order to obtain the maximal levels of NO at 5 h post-surgery, time of endogenous peak. The importance of NO in the early stages of liver regeneration remains unclear. It is known that NO plays an important role in the process of vascularization, angiogenesis and permeabilization of tissue (Jozkowicz et al., 2001). Although there is a growing body of evidence that NO has angiogenic effects, partly mediated by VEGF, there is no unanimity of opinion in this regard (Jozkowicz et al., 2001; Frank et al., 1999a,b; Xiong et al., 1998). The effects of NO donors are greatly dependent upon cell type, cellular redox status, and the amount and chemical nature of NO generators. As a consequence, contradictory data have sometimes been reported (Kimura et al., 2000). Our results show that SNP treatment increases VEGF levels compared with PH-Control animals, suggesting that NO enhances VEGF expression. Accordingly, other authors have reported that the exogenous addition of NO donors or increased levels of endogenous NO enhanced VEGF synthesis in rat vascular smooth muscle cells (Dulak et al., 2000; Ramanthan et al., 2003). Furthermore, in the rabbit cornea model of angiogenesis, VEGF-induced angiogenesis is blocked by LNAME (non-selective NOS inhibitor), demonstrating that neovascularization is suppressed by the blockade of NO production (Ziche et al., 1997). However, studies about the relationship between VEGF expression and NO production carried out in the liver are lacking so far. In the present work, we demonstrated that the inhibition of NO synthesis resulted in decreased VEGF protein levels, suggesting that NO is implicated in VEGF expression. On the
other hand, Taniguchi et al. have shown that VEGF expression in regenerating rat liver occurs predominantly in periportal hepatocytes and they also demonstrated that VEGF is involved in proliferation of hepatocytes associated with proliferation of sinusoidal endothelial cells after PH in rats. Histological studies revealed that SNP treatment increases the number of vascular structures in portal areas in PH animals and that AG treatment reduces this rate (data not shown), suggesting that the augmentation of NO levels increases periportal vascularization; probably mediated by VEGF. In agreement with other authors, these studies provide further evidence that VEGF production is regulated by NO and that it plays a central role in rat liver regeneration after PH (Frank et al., 1999a,b; Chin et al., 1997). In this work, we found out that the modification of NO levels at 5 h post-PH produces changes in VEGF protein levels at 72 h after PH. It is clear that NO increase during early steps of liver regeneration might initiate a cell responsive mechanism and even activate transcriptional factors that act as signal transducers between cytoplasm and nucleus, which results in the regulation of VEGF expression. Finally, additional studies are needed to elucidate the precise mechanisms through which NO exerts its effects on VEGF expression. Our results show that NO enhances VEGF expression and it is suggested that NO is involved in the process of vascularization of the remnant liver after partial hepatectomy. We propose that NO plays a key role in the vascularization process due to its ability to control VEGF expression. Acknowledgements This work was supported by research grants from “Consejo Nacional de Investigaciones Científicas y Tecnológicas” (CONICET). The authors wish to acknowledge Cecilia Basiglio for language revision. References Assy, N., Spira, G., Paizi, M., Shenkar, L., Kraizer, Y., Cohen, T., Neufeld, G., Dabbah, B., Enat, R., Baruch, Y., 1999. Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats. Journal of Hepatology 30, 911–915. Bories, P.N., Bories, C., 1995. Nitrate determination in biological fluids by an enzymatic one step assay with nitrate reductase. Clinical Chemistry 41, 904–907. Carnovale, C., Scapini, C., Alvarez, L., Favre, C., Monti, J., Carrillo, M., 2000. Nitric oxide release and enhancement of lipid peroxidation in regeneration rat liver. Journal of Hepatology 32, 798–804. Chin, K., Kurashima, Y., Ogura, T., Tajiri, H., Yoshida, S., Esumi, H., 1997. Induction of endothelial growth factor by nitric oxide in human glioblastoma and hepatocellular carcinoma cells. Oncogene 15, 437–442. Dulak, J., Jazkowickz, A., Dembinska-Kiec, A., Guevara, I., Zdzienicka, A., Zmudzinska-Grochot, D., Florek, I., Wojtowicz, A., Suba, A., Cooke, J., 2000. Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells. Arteriosclerosis, Thrombosis, and Vascular Biology 20 (6), 59–666. Fausto, N., 2000. Liver regeneration. Journal of Hepatology 32 (suppl. 1), 19–31. Frank, S., Stallmeyer, B., Kampfer, H., Kolb, N., Pfeilschifter, J., 1999a. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair. FASEB Journal 13, 2002–2014.
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Vascular endothelial growth factor and nitric oxide in rat liver regeneration Maria Teresa Ronco, Daniel Francés, Maria de Luján Alvarez, Ariel Quiroga, Juan Monti, Juan Pablo Parody, Gerardo Pisani, Maria Cristina Carrillo, Cristina Ester Carnovale ⁎ Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina Received 23 April 2007; accepted 6 July 2007
Abstract In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65 ± 10.70; PH-Control: 88.13 ± 1.60⁎; Sh-SNP: 90.35 ± 3.11⁎; PH-SNP: 119.5 ± 12.10⁎#; Sh-AG: 33.27 ± 5.23, PH-AG: 36.80 ± 3.40#) (p b 0.05 vs Sh-Control; #p b 0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats. © 2007 Elsevier Inc. All rights reserved. Keywords: Liver regeneration; Nitric oxide; Vascular endothelial growth factor; Partial hepatectomy
Introduction The liver has the unique and remarkable ability to restore itself following cell loss (toxic, viral or surgical). Despite being specialized and highly differentiated cells, adult hepatocytes remain capable of rapid proliferation when appropriately stimulated. Following a two-thirds partial hepatectomy (PH), liver cells switch from a quiescent state into a proliferative state and re-enter cell cycle, beginning DNA synthesis as a semisynchronised cohort about 12–15 h after surgery (Fausto, 2000; Mangnall et al., 2003; Li et al., 2001). The mechanisms regulating regenerative processes are complex and incompletely understood. A large number of immediate and delayed early genes, which are not normally ⁎ Corresponding author. Instituto de Fisiología Experimental, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570-2000 Rosario, Argentina. Fax: +54 341 4399473. E-mail address: [email protected]. (C.E. Carnovale). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.07.009
expressed in the quiescent liver, are activated (Li et al., 2001). Immediately after PH (1–6 h), nitric oxide (NO) is synthesized by liver parenchymal and non-parenchymal cells from Larginine, via induction of the inducible form of nitric oxide synthase (iNOS) (Obolenskaya et al., 1994; Carnovale et al., 2000; Ronco et al., 2004). NO is a highly reactive molecule, known to be involved in diverse biological processes in nearly all aspects of life. The precise role of NO in the regenerative process is still unidentified, although the importance of NO in this process is suggested by the finding that liver regeneration is impaired in iNOS-deficient mice (Rai et al., 1998). One of the well-characterized functions of NO is as a mediator of vascular dilatation and permeability and it is involved in vascular remodeling (Hortelano et al., 1995; Carnovale et al., 2000). Angiogenesis, the formation of new blood vessels, is a complicated process that implies proliferation and migration of endothelial cells. This phenomenon is required for remodeling liver architecture following liver resection
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(Kraizer et al., 2001; Assy et al., 1999). The initial wave of hepatocyte proliferation is followed by endothelial cell proliferation and penetration of vascular hepatocellular islands leading to the formation of new sinusoids (Sato et al., 2001). In liver regeneration after PH, both hepatocytes and nonparenchymal cells express vascular endothelial growth factor (VEGF) mRNA, suggesting that VEGF plays a significant role in this process. Hepatocellular production of VEGF shows the maximal levels between 48 and 72 h after PH (Mochida et al., 1996; Taniguchi et al., 2001). Moreover, recent studies have shown that inhibition of angiogenesis with angiostatin impairs liver regeneration (Redalli et al., 2004). VEGF also increases dilatation and permeability of blood vessels, and it has been suggested that NO may be involved in VEGF signalling pathways. Although there is a growing body of evidence that NO has angiogenic effects, partly mediated by VEGF induction in different cellular types (Jozkowicz et al., 2001), there is not enough evidence about the role of NO in VEGF induction during rat liver regeneration. In the present work, we investigated the role of NO in VEGF induction in liver tissue after PH in rats. Experimental procedures Animals and surgical procedures Male Wistar rats weighing 300 to 360 g were housed two per cage and maintained under a 12-hour light/dark period. Rats were fed ad libitum with a normal standard diet and water. Animals received humane care according to criteria the “Guide for the Care and Use of Laboratory Animals”. The animals were anesthetized with pentobarbital solution (50 mg/kg body weight) injected i.p. The peritoneal cavity was opened by a median incision, and the liver was freed from its ligaments with minimal manipulation. Two-thirds hepatectomy (PH) was performed following the standard Higgins and Anderson technique in which the median and left lateral lobes were ligated at their roots and were then removed (Higgins and Anderson, 1931). Surgical sham control rats (Sh-Control) underwent midline laparotomy with liver manipulation. To avoid variations due to circadian rhythms, the time to perform the surgery had to be fixed in order to sacrifice the animals always at the same time of the day (between 10:00 and 12:00 h). Increase of nitric oxide by nitroprusside sodium administration To obtain maximal NO levels at 5 h post-surgery, sodium nitroprusside (SNP; Salom et al., 2000) was administered intravenously for 30 min at a rate of 1 ml/h, beginning at 4.5 h after surgical procedures. SNP (Merck) was dissolved and diluted in 0.9% isotonic saline. Care was taken to protect SNP solutions from light due to its light sensitivity. Both Sh and PH animals were randomized in two sub-groups that received isotonic saline (Control) and SNP (0.25 mg/kg body weight). Four rats of each group (Sh-Control, Sh-SNP, PH-Control, PHSNP) were sacrificed at each time after surgery: 5, 24 and 72 h.
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Inhibition of nitric oxide synthesis A group of rats received isotonic saline (control) and another group of animals received aminoguanidine (AG), a preferential iNOS inhibitor. AG solution was prepared in isotonic saline and administered intraperitoneally (100 mg/kg body weight) once a day, beginning 3 days before surgery (Carnovale et al., 2000). Six animals from each group (Sh-Control, Sh-AG, PH-Control and PH-AG) were killed at each time after surgery: 5 and 72 hours. Analytical assays Nitrate determination NO formation was indirectly assessed in cytosolic fractions by determining the concentration of nitrate, one of the stable end products of NO oxidation. An assay based on the conversion of nitrate to nitrite by reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in the presence of nitrate reductase enzyme was used (Bories and Bories, 1995). The amount of NADPH oxidized during the reaction was stoichiometrically equivalent to the amount of nitrate present in the samples. The decrease in NADPH was followed at 340 nm. Cytosolic fractions were prepared as described by de Duve et al. Briefly, frozen liver tissues were homogenized in 3 volumes of 0.3 M sucrose containing 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1 μg/ml aprotinin. Homogenates were centrifuged at 1000 g to remove non-lysed cells, nuclei and heavy membranes. Supernatants were centrifuged at 3000 g at 4 °C for 15 min and pellets were discarded. Cytosolic fractions were obtained by centrifugation of supernatants at 56,000 rpm at 4°C for 60 min. Electron Paramagnetic Resonance (EPR) spectroscopy Sh and PH male Wistar rats, as described in “Animals and Surgical Procedures”, were used throughout. Thirty minutes before sacrifice, all animals were given ferrous sulfate (37.5 mg/ kg body weight, Cicarelli) and disodium citrate dihydrate (187.5 mg/kg body weight, Cicarelli), subcutaneously, and afterwards, diethyldithiocarbamate (DETC, 500 mg/kg w.t., Anedra), intraperitoneally. At 5 h after surgery, animals were sacrified and livers were promptly removed. After a rapid washing in cold saline solution, samples were placed in 1 ml syringes, frozen and kept in liquid nitrogen until they were subjected to EPR. Samples were transferred to a Dewar and EPR spectra were recorded at liquid nitrogen temperature on a Bruker ECS 106 ESR, operating at the microwave frecuency 9.41 GHz, microwave power 10 mW, modulation amplitude 4.75 G and central field 3350 G and were measured at RG: 1 × 103 and 1 scan (Mikoyan et al., 1997; Obolenskaya et al., 1994). Western blot analysis: vascular endothelial growth factor (VEGF) For VEGF detection, liver tissue lysates were prepared by homogenization of frozen tissues in 3 volumes of lysating RIPA buffer containing PBS, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1 μg/ml aprotinin. After 30 min of incubation at
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0 °C and three freeze–thaw cycles, lysates were cleared by centrifugation at 15,000 rpm for 30 min, and supernatants were kept at − 70 °C (Ronco et al., 2004). Protein content was quantified according to Lowry et al. Fifty micrograms of protein was subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvynil difluoride membranes (PVDF, Perkin Elmer Life Sciences, Boston, MA, USA). After blocking, blots were incubated overnight at 4 °C with an antimouse VEGF antibody (1:500, Santa Cruz, Biotechnology, USA). Membranes were then incubated with anti-mouse IgGperoxidase conjugates (1:5000, Amersham Life Science) and the bands were detected by an enhanced chemiluminescence method (ECL; Amersham Pharmacia Biotech). Autoradiographs were obtained by exposing membranes to Kodak XAR film, and the bands were quantified by densitometry (Shimadzu CS-9000). Statistical analysis Results were expressed as mean ± S.E. Statistical analysis was performed by using one-way ANOVA, followed by Tukey's test. Differences were considered to be statistically significant when the p value was b0.05. Results NO detection We determined nitrate concentration in hepatic cytosol to evaluate NO production. Fig. 1 clearly shows an increase (80%) in hepatic cytosol nitrate content at 5 h post-hepatectomy. SNP treatment, a tool for increasing NO levels, produced no changes in nitrate levels as compared to PH-Control. NO generation after PH was inhibited by AG treatment (Fig. 1), which decreased by 34% the hepatic cytosol nitrate content with respect to PH-Control.
Fig. 1. Production of nitrate, a metabolic product of NO, measured in hepatic cytosol, expressed as nmol per mg of protein at 5 h post-surgery. Sh, sham operated; Sh-SNP, Sh-treated with SNP 0.25 mg/kg body weight. PH-Control, partial hepatectomy control; PH-SNP, PH-treated with SNP 0.25 mg/kg body weight; PH-AG, PH-treated with AG 100 mg/kg body weight. Data are expressed as mean ± SE of six rats. ⁎Significantly different from Sh-Control. # Significantly different from PH-Control. (p b 0.05).
Fig. 2. EPR spectra (9.41 GHz) of NO–Fe+2–DETC complex in rat liver homogenates. A) Sh-Control; B) PH-Control; C) Sh-SNP (SNP: 0.25 mg/kg body weight); D) PH-SNP (SNP: 0.25 mg/body weight); E) Sh-AG (AG: 100 mg/kg body weight); F) PH-AG (AG: 100 mg/kg body weight). Records were performed at nitrogen liquid temperature, microwave power 10 mW, modulation amplitude 4.75 G, and central field 3350 G. The samples were determined at RG: 1 × 103 and 1 scan.
NO production was also estimated from the intensity of the EPR signal of the mononitrosyl iron complex with DETC. Fe+2–DETC complexes preformed in-vivo served as a trap for the endogenously produced NO (Quaresima et al., 1996). The typical EPR spectra corresponding to NO–Fe+2–DETC complex of rats liver producing NO were observed and a three line EPR spectrum (G = 2.038) was obtained from every tissue homogenate (Fig. 2) at 5 h after surgery. Table 1 shows the values related to the intensity of each signal of the NO–Fe+2– DETC complex. As expected, the signal intensity for PHControl animals was higher than that for Sh-Control animals. Treatment with SNP resulted in the highest EPR signal intensity of the NO trapped in liver homogenates. On the other hand, AG treatment decreased the intensity of the signal of the NO–Fe+2– DETC complex (Table 1). In-vivo EPR spectroscopy is a useful tool for the study of in-situ, real time NO generation (Quaresima et al., 1996). Its high sensitivity allows us to detect differences between PH-SNP animals and PH-Control animals, that were not observed by means of nitrate measurement. Probably, this could be due to the lack of sensitivity of the activity assay. NADPH can act as a cofactor for a variety of enzymes such as NADPH oxidase, among others. Moreover, some ions (manganese, chloride, cyanide and sulfite) are able to inactivate o retard nitrate conversion.
M.T. Ronco et al. / Life Sciences 81 (2007) 750–755 Table 1 Intensity of NO–Fe+2–DETC complex signal in liver homogenates Samples
NO–Fe+2–DETC arbitrary units
Sh-Control PH-Control Sh-SNP PH-SNP Sh-AG PH-AG
37.65 ± 10.70 88.13 ± 1.60⁎ 90.35 ± 3.11⁎ 119.5 ± 12.10⁎# 33.27 ± 5.23# 36,80 ± 3.40#
⁎Significant difference vs Sh-Control. #Significant difference vs PH-Control.
VEGF detection NO acts as a mediator of vessels dilatation and permeability and it is known that angiogenesis is a fundamental process involved in the remodeling of hepatic architecture following liver resection, playing an important role in hepatocyte blood supply. Thus, we determined the levels of VEGF protein by western blot in total liver lysates at 24 and 72 h after PH (Fig. 3). At 24 h post-hepatectomy, PH-Control and PH-SNP animals showed an increase of VEGF levels as compared to Sh-Control. At this time after PH, VEGF levels showed no differences between PH-Control and PH-SNP animals. In concordance with other authors, PH-Control animals showed an increase in VEGF levels with respect to Sh-Control at 24 and 72 h after PH. SNP treatment increases by above 300% the levels of VEGF at 72 h after PH (Fig. 3B). Other authors have previously shown that NO positively regulates VEGF expression in different tissues (Jozkowicz et al., 2001). To better understand the relationship between NO and VEGF, we inhibited NO production by administration of AG (a specific iNOS inhibitor). This study was performed only at 72 h after PH because it has been reported that VEGF expression is maximal at this time
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(Taniguchi et al., 2001). We observed that the pre-treatment with AG resulted in a diminution of VEGF protein levels of 60% at 72 h post-PH (Fig. 4). Discussion The liver ability to restore major tissue loss is a unique process that involves numerous cell interactions and a complex network of mediators. PH in rats results in the induction of gene expression and release of biologically active substances which regulate hepatic regeneration. There is a fine balance between stimulators and inhibitors, an equilibrium that makes quiescent hepatocytes proliferate after tissue resection in order to restore the functional capacity of the liver as well as its mass (Fausto, 2000; Mangnall et al., 2003; Li et al., 2001). Liver regeneration requires the formation of new blood vessels (angiogenesis) for reconstruction of hepatic sinusoids. Various angiogenic factors have been identified, but VEGF is the most potent and specific growth factor for both angiogenesis and vasculogenesis (Kraizer et al., 2001; Sato et al., 2001; Yancopoulos et al., 2000). VEGF have been demonstrated to play a central role in division, migration, and tubule formation of vascular endothelial cells. Although a variety of growth factors and cytokines have been implicated in liver regeneration, the contribution of VEGF is unclear in details. After PH, both hepatocytes and non-parenchymal cells express VEGF mRNA, suggesting that VEGF plays a significant role in this process (Taniguchi et al., 2001; Mochida et al., 1996). In agreement, we observed that liver homogenates from hepatectomized rats show increased VEGF levels compared with ShControl animals at 24 and 48 h post-PH. Previous studies of our group, in agreement with other authors, demonstrated that iNOS is up-regulated in the liver
Fig. 3. Immunoblotting analysis of VEGF protein expression in liver total lysates fraction at 24 and 72 h post-surgery: A) after 24 h: Line 1: Sh-Control; Line 2: PHControl; Line 3: Sh-SNP (SNP: 0.25 mg/kg body weight); Line 4: PH-SNP (SNP: 0.25 mg/kg body weight); B) after 72 h: Line 1: Sh-Control; Line 2: PH-Control; Line 3: Sh-SNP (SNP: 0.25 mg/kg body weight); Line 4: PH-SNP (SNP: 0.25 mg/kg body weight). The accompanying bars represent the densitometry expressed in percentage from five separate animal sets. ⁎Significantly different from Sh-Control. #Significantly different from PH-Control. (p b 0.05).
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Fig. 4. Immunoblotting analysis of VEGF protein expression in total liver lysates fraction at 72 h post-surgery. A) Line 1: Sh-Control; Line 2: PH-Control; Line 3: Sh-AG (AG: 100 mg/kg body weight); Line 4: PH-AG (AG: 100 mg/kg body weight). The accompanying bars represent the densitometry expressed in percentage from five separate animal sets. ⁎Significantly different from ShControl. #Significantly different from PH-Control. (p b 0.05).
with maximal levels of expression at 5 h post-PH; a large amount of NO is generated, returning to basal levels at 18 h after PH (Carnovale et al., 2000, Hortelano et al., 1995). In this work, we used a direct NO donor (SNP) in order to obtain the maximal levels of NO at 5 h post-surgery, time of endogenous peak. The importance of NO in the early stages of liver regeneration remains unclear. It is known that NO plays an important role in the process of vascularization, angiogenesis and permeabilization of tissue (Jozkowicz et al., 2001). Although there is a growing body of evidence that NO has angiogenic effects, partly mediated by VEGF, there is no unanimity of opinion in this regard (Jozkowicz et al., 2001; Frank et al., 1999a,b; Xiong et al., 1998). The effects of NO donors are greatly dependent upon cell type, cellular redox status, and the amount and chemical nature of NO generators. As a consequence, contradictory data have sometimes been reported (Kimura et al., 2000). Our results show that SNP treatment increases VEGF levels compared with PH-Control animals, suggesting that NO enhances VEGF expression. Accordingly, other authors have reported that the exogenous addition of NO donors or increased levels of endogenous NO enhanced VEGF synthesis in rat vascular smooth muscle cells (Dulak et al., 2000; Ramanthan et al., 2003). Furthermore, in the rabbit cornea model of angiogenesis, VEGF-induced angiogenesis is blocked by LNAME (non-selective NOS inhibitor), demonstrating that neovascularization is suppressed by the blockade of NO production (Ziche et al., 1997). However, studies about the relationship between VEGF expression and NO production carried out in the liver are lacking so far. In the present work, we demonstrated that the inhibition of NO synthesis resulted in decreased VEGF protein levels, suggesting that NO is implicated in VEGF expression. On the
other hand, Taniguchi et al. have shown that VEGF expression in regenerating rat liver occurs predominantly in periportal hepatocytes and they also demonstrated that VEGF is involved in proliferation of hepatocytes associated with proliferation of sinusoidal endothelial cells after PH in rats. Histological studies revealed that SNP treatment increases the number of vascular structures in portal areas in PH animals and that AG treatment reduces this rate (data not shown), suggesting that the augmentation of NO levels increases periportal vascularization; probably mediated by VEGF. In agreement with other authors, these studies provide further evidence that VEGF production is regulated by NO and that it plays a central role in rat liver regeneration after PH (Frank et al., 1999a,b; Chin et al., 1997). In this work, we found out that the modification of NO levels at 5 h post-PH produces changes in VEGF protein levels at 72 h after PH. It is clear that NO increase during early steps of liver regeneration might initiate a cell responsive mechanism and even activate transcriptional factors that act as signal transducers between cytoplasm and nucleus, which results in the regulation of VEGF expression. Finally, additional studies are needed to elucidate the precise mechanisms through which NO exerts its effects on VEGF expression. Our results show that NO enhances VEGF expression and it is suggested that NO is involved in the process of vascularization of the remnant liver after partial hepatectomy. We propose that NO plays a key role in the vascularization process due to its ability to control VEGF expression. Acknowledgements This work was supported by research grants from “Consejo Nacional de Investigaciones Científicas y Tecnológicas” (CONICET). The authors wish to acknowledge Cecilia Basiglio for language revision. References Assy, N., Spira, G., Paizi, M., Shenkar, L., Kraizer, Y., Cohen, T., Neufeld, G., Dabbah, B., Enat, R., Baruch, Y., 1999. Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats. Journal of Hepatology 30, 911–915. Bories, P.N., Bories, C., 1995. Nitrate determination in biological fluids by an enzymatic one step assay with nitrate reductase. Clinical Chemistry 41, 904–907. Carnovale, C., Scapini, C., Alvarez, L., Favre, C., Monti, J., Carrillo, M., 2000. Nitric oxide release and enhancement of lipid peroxidation in regeneration rat liver. Journal of Hepatology 32, 798–804. Chin, K., Kurashima, Y., Ogura, T., Tajiri, H., Yoshida, S., Esumi, H., 1997. Induction of endothelial growth factor by nitric oxide in human glioblastoma and hepatocellular carcinoma cells. Oncogene 15, 437–442. Dulak, J., Jazkowickz, A., Dembinska-Kiec, A., Guevara, I., Zdzienicka, A., Zmudzinska-Grochot, D., Florek, I., Wojtowicz, A., Suba, A., Cooke, J., 2000. Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells. Arteriosclerosis, Thrombosis, and Vascular Biology 20 (6), 59–666. Fausto, N., 2000. Liver regeneration. Journal of Hepatology 32 (suppl. 1), 19–31. Frank, S., Stallmeyer, B., Kampfer, H., Kolb, N., Pfeilschifter, J., 1999a. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair. FASEB Journal 13, 2002–2014.
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