© Borgis - Postępy Nauk Medycznych 6/2014, s. 389-394
*Brygida Adamek1, Joanna Katarzyna Strzelczyk1, Marzena Zalewska-Ziob1, Janusz Kasperczyk2, Grażyna Spausta1, Michał Kukla3, Marek Hartleb3, Andrzej Wiczkowski1
Stężenie naczyniowo-śródbłonkowego czynnika wzrostowego (VEGF) w tkance wątrobowej w późniejszych etapach regeneracji wątroby szczura poddanej działaniu interferonu α2b
Vascular endothelial growth factor (VEGF) liver tissue concentration in delayed stages of rat liver regeneration upon interferon α2b influence
1Chair and Department of General Biology, Medical University of Silesia, Katowice
Head of Department: prof. Andrzej Wiczkowski, MD, PhD
2Chair and Department of Medicine and Environmental Epidemiology, Medical University of Silesia, Katowice
Head of Department: prof. Jadwiga Jośko-Ochojska, MD, PhD
3Department of Gastroenterology and Hepatology, Medical University of Silesia, Katowice
Head of Department: prof. Marek Hartleb, MD, PhD
Streszczenie
Wstęp. Uważa się, że naczyniowo-śródbłonkowy czynnik wzrostowy (ang. vascular endothelial growth factor – VEGF) odgrywa istotną rolę w regeneracji wątroby, natomiast interferon α2b (IFN-α2b) wywiera na ten proces wpływ hamujący. VEGF wzmaga proliferację komórek śródbłonka zatok (ang. sinusoidal endothelial cells – SECc) in vitro, ale znaczenie tych oddziaływań w przebiegu regeneracji wątroby in vivo nie jest określone.
Cel pracy. Oznaczono stężenia VEGF w tkance wątrobowej szczura w późniejszej fazie regeneracji, zarówno podczas naturalnego przebiegu tego procesu, jak i po podaniu IFN-a2b.
Materiał i metody. 45 trzymiesięcznych samców szczurów rasy Wistar podzielono na trzy grupy. Pierwsza grupa otrzymała podskórnie IFN-α2b 24 godziny przed częściową hepatektomią, a następnie 24 godziny po niej (ang. partial hepatectomy – PH). Ten sam schemat zastosowano w grupie drugiej, która otrzymała dwukrotnie podskórną iniekcję 0,5 ml 0,9% NaCl. Trzecia grupa otrzymała dwie dawki IFN-α2b w odstępie 48 godzin, ale została poddana zabiegowi pozorowanemu. Szczury uśmiercano w podgrupach po pięć sztuk, kolejno w 48., 72. i 96. godzinie po przeprowadzonym zabiegu. Fragmenty tkanki wątrobowej pobierano w trakcie hepatektomii oraz podczas autopsji. Stężenia VEGF w homogenatach wątrób oznaczano metodą ELISA.
Wyniki. Stężenia VEGF przed i po PH nie różniły się, a podawany IFN-α2b nie miał na nie istotnego wpływu w analizowanych punktach czasowych.
Wnioski. Szlak VEGF nie ulega aktywacji w wątrobie szczura pomiędzy 48. a 96. godziną po częściowej hepatektomii, a oddziaływanie IFN-α2b nie ma wpływu na jego poziomy tkankowe ani w prawidłowej, ani w regenerującej wątrobie.
Summary
Introduction. Vascular endothelial growth factor (VEGF) is postulated to play an important role in liver regeneration and interferon α2b (IFN-α2b) is believed to inhibit this process. VEGF enhances proliferation of sinusoidal endothelial cells (SECc) in vitro, but its significance on liver regeneration in vivo is not well defined.
Aim. Investigation of the VEGF concentration in rat liver tissue in delayed stage of hepatic regeneration in baseline conditions and after IFNα2b administration.
Material and methods. The 45 three-months-old male Wistar rats were divided into three groups. The first group was injected subcutaneously with IFN-α2b 24 h before and 24 h after partial hepatectomy (PH). The similar schedule was realized in the second group injected with 0.5 ml of 0.9% NaCl. The third group underwent sham-operation and was given two doses of IFN-α2b with the 48 h interval. Rats were sacrificed in subgroups of five at 48, 72 and 96 h after surgery. The liver samples were obtained during surgery or autopsy. VEGF concentration was assayed in tissue homogenates with ELISA method.
Results. VEGF concentrations were not different before and after PH and IFN-α2b had no significant influence on VEGF in analyzed time points.
Conclusions. VEGF pathway is not activated in rat liver between 48 and 96 h post PH and administration of IFN-α2b has no impact on its tissue level neither in intact nor regenerating liver.
INTRODUCTION
Liver regeneration after the loss of effective hepatic mass is fundamental event in case of hepatic injury. Studies with hepatic resections showed that the regenerative response is proportional to the amount of liver removed (1, 2). The events triggered by partial loss of liver tissue involve mostly proliferation of hepatocytes, but also regeneration of all non-parenchymal mature cell populations. Hepatocyte proliferation starts in the periportal zone, continuing during next 36 to 48 hours towards pericentral areas of the lobules (3). The proliferation of non-parenchymal cells and the synthesis of new matrix are required for whole liver reconstitution (4, 5). Hepatic stellate cells (HSC), Kupffer cells and sinusoidal endothelial cells (SECs) enter cell cycle about 24 hours later than hepatocytes with a peak of DNA synthesis set at 48 h or later (6). Cellular sequential proliferations result in the formation of avascular liver tissue islands, with subsequent migration of the SECs into clusters of newly repopulated hepatocytes (7). Remodeling of regenerative liver architecture involves the formation of a complex network of sinusoids (8, 9). The stimuli to this process remain relatively unexplored.
Vascular endothelial growth factor (VEGF) is the best known angiogenic factor with documented growth-promoting effect on endothelial cells (10). VEGF is also a survival factor for SECs, as well as an inductor of their fenestrated phenotype important for microvascular permeability (11). There are several distinct isoforms of this homodimeric heparin-binding glycoprotein, which are the products of alternate splicing of the same gene (12, 13). VEGF binds to two receptor-type tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR/Flk 1 (VEGF receptor-2), interacting with a family of co-receptors and membrane proteins (neurophilins), which do not contain a tyrosine kinase domain (10, 14).
Partial hepatectomy (PH), in which two-thirds (70%) of the liver is removed, is the widely accepted experimental rat model to study mechanisms of liver regeneration (15-19). However, the knowledge on regulation of sinusoidal net rebuilding during liver regeneration in partially hepatectomized rat is largely missing (20). SECs do not initiate DNA synthesis until 48 to 72 hours after resection, starting to divide approximately 96 hours post PH, with ongoing proliferation lasting at least 8 days following PH (6, 21). Assy et al. showed that serum levels of VEGF do not change significantly and remain on physiological levels following 70% PH (22). This finding encourages investigation of local behavior of VEGF concentrations.
The interferons (IFNs) are abundantly expressed cytokines, which show antiviral, immunomodulatory, growth-inhibitory and anti-fibrogenic activities (23-26). Moreover, interferon α (IFNα) is listed among angiogenesis inhibitors (27, 28). It is postulated, that activation of more than one signaling pathway is required for the generation of different effects of IFNs as no single signaling cascade is sufficient to reach any given biological end-point (29). To date, it has been not established if IFN administration has any impact on VEGF concentration within regenerating liver.
AIM
The purpose of this study was to investigate the hepatic concentration of VEGF in partially hepatectomized rats without pharmacological intervention and under influence of IFNα.
MATERIAL AND METHODS
Animal experiment
The 45 adult male Wistar rats (300-330 g) were maintained on rat chow and water under standard conditions with a 12-hour light-dark cycle. According to the study protocol they were divided into three groups, 15 of animals each. The first group (IFN/H) was injected subcutaneously with 0.5 ml IFN-α2b (Intron A, Shering-Plough, 5 MU/100 ml 0.9% NaCl) and 24 hours later the 2/3 PH was conducted. After next 24 h the second IFN-α2b dose was administered. The second group (IFN/O) was injected with the same doses of IFN-α2b and was sham operated between the doses. Control rats (NaCl/H) underwent PH and received an identical volume (0.5 ml) of isotonic saline in the same time intervals in relation to PH. Injections and surgery (anesthesia: 50 mg/kg of ketamine given intraperitoneally) were carried out between 9.00-11.00 a.m. to minimize the influence of circadian variations. Rats were sacrificed in groups of five 48, 72 and 96 h after PH. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected by the a priori approval (no. 1/02; 19.02.2002) of the Local Ethics Commission for Animal Experiments of the Medical University of Silesia.
Samples of excised livers in IFN/H and NaCl/H groups were marked with “1”, and liver samples in IFN/O group obtained during autopsy were marked with “2”. The study design is shown in figure 1.
↓↓ →24h → PH (IFN/H/1)
↓↓ →24h → SO (IFN/O)
↓↓ →24h → PH (NaCl/H/1) | 24h → ↓↓ → 24h
24h → ↓↓ → 24h
24h → ↓↓ → 24h | → S (IFN/H/2)
→ S (IFN/O/2)
→ S (NaCl/H/2) | 48h |
| | | | |
↓↓ →24h → PH (IFN/H/1)
↓↓ →24h → SO (IFN/O)
↓↓ →24h → PH (NaCl/H/1) | 24h → ↓↓ → 48h
24h → ↓↓ → 48h
24h → ↓↓ → 48h | → S (IFN/H/2)
→ S (IFN/O/2)
→ S (NaCl/H/2) | 72h |
| | | | |
↓↓ →24h → PH (IFN/H/1)
↓↓ →24h → SO (IFN/O)
↓↓ →24h → PH (NaCl/H/1) | 24h → ↓↓ → 72h
24h → ↓↓ → 72h
24h → ↓↓ → 72h | → S (IFNH/2)
→ S (IFN/O/2)
→ S (NaCl/H/2) | 96h |
Fig. 1. The experiment schedule.
IFN/H – interferon α2b-injected/hepatectomized animals; IFN/O – interferon α2b-injected/sham-operated animals, NaCl/H – saline injected/hepatectomized animals; ? – IFN/saline injections, PH – partial hepatectomy, SO – sham operation, S – sacrification; “1” – tissue samples obtained during surgery; “2” – tissue samples obtained during autopsy; 48h, 72h, 96h – hours post surgery
Homogenization and total protein concentration
Weighed samples of rat liver (100 mg) were homogenized using a PRO 200 homogenizer (PRO Scientific Inc, USA) at 10 000 RPM in nine volumes of phosphate-buffered saline solution (PBS without Ca and Mg, BIOMED, Poland) containing 0.5% Triton X-100 (Sigma-Aldrich, USA). Then homogenates were centrifuged at 12 000 RPM for 15 minutes at 4°C, and supernatants were divided into appropriate portions and frozen at – 80°C until required for further surveys.
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