Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver

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Abstract

Background/Aims: Angiogenesis is essential in liver regeneration. However, only little is known about sinusoidal endothelial cell proliferation and the role of different angiogenic growth factors and their receptors during regeneration.

Methods: Seventy percent hepatectomy was carried out on male rats. Serial changes in endothelial cell proliferation were evaluated by immunohistochemistry. The mRNA expressions of angiogenic growth factors (vascular endothelial growth factor (VEGF) and angiopoietins 1 and 2) and their receptors (flt-1, flk-1, Tie-1 and Tie-2) in the whole liver were evaluated by semi-quantitative RT-PCR.

Results: Significant elevation of endothelial cell proliferation started at 48 h and peaked at 72 h after hepatectomy. The ratio of sinusoids to liver tissue area initially decreased at 72 h, and thereafter, significantly increased at 96 h. VEGF related factors had early peaks, which coincided with the endothelial proliferation. flt-1, flk-1 and VEGF expressions peaked at 24, 48 and 72 h, respectively. angiopoietin/Tie factors peaked at 96 h, except Ang-2, which gradually increased and peaked at 168 h.

Conclusions: During liver regeneration, hepatocyte proliferation was followed by endothelial cell proliferation. The VEGF family and angiopoietin/Tie system may have distinct roles in angiogenesis, with an enhanced expression of the VEGF family in the early phase of regeneration followed by angiopoietin/Tie expression.

Introduction

The liver is the metabolic powerhouse of the body and is unique in its capability to regenerate and restore the metabolic demand following injury or resection. To reconstitute the injured liver, multiple types of cellular entities work in concert for a regenerative impulse, making the procedure a complicated one. Sinusoidal endothelial cells are the largest number of resident liver cells next to hepatocytes [1], [2], and are vital for their role in supplying nutrients and growth factors to proliferating hepatocytes by the formation of new vessels during regeneration. Extensive works have been carried out on hepatocyte regeneration, however, the details of endothelial cell proliferation and sinusoid formation during liver regeneration are yet to be delineated.

Angiogenesis is a fundamental process required for normal embryonic development, wound healing, regeneration, tumor growth and ovulation [3], [4], [5], [6], [7]. However, under physiological conditions, the rate of endothelial cell multiplication in most adult organs is comparatively slow [8], [9]. This could be attributed to the limited ability of mature adult organs to regenerate. The pioneer work on tumor angiogenesis by Folkman et al. [4] has produced much interest in this field, and has helped to evolve sophisticated techniques for the assessment of the extent of angiogenesis in situ by immunohistochemical staining and quantification by image analysis. Until now, only a few studies have addressed the growth kinetics of sinusoidal endothelial cells in regenerating liver [10], [11], [12]. Also, the quantification of sinusoidal tube formation in regenerating liver is very difficult because the structure of vessels in the hepatic lobule is different from other tissues and exhibits a sinusoidal structure. Recently, several studies [13], [14] have demonstrated that image analysis is more reliable and objective than manual counts of microvessels. Therefore, changes in the area occupied by the endothelial cells during regeneration were determined with a computer-assisted image analysis technique.

Moreover, we investigated the kinetics and expression of the members of two major angiogenic growth factor systems during hepatic regeneration. Extensive analysis has implicated the vascular endothelial growth factor (VEGF) and its two tyrosine kinase receptors, flt-1 and flk-1, as major regulators of physiological and pathological angiogenesis. In addition to the VEGF family, angiopoietin (Ang)/Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) is also an important regulator of angiogenesis. A recent study [15] has shown that VEGF and its receptors are augmented during liver regeneration. However, its correlation with sinusoid formation and the role of the Ang/Tie family during liver regeneration are still open to discussion.

Therefore, in addition to an assessment of sinusoidal endothelial cell proliferation, we investigated the kinetics and relative contribution of different angiogenic growth factors, the VEGF family and Ang/Tie family, during rat liver regeneration after 70% partial hepatectomy.

Section snippets

Animals and experimental conditions

Male Sprague–Dawley rats of 7–8 weeks of age (180–250 g; Charles River Breeding Laboratories, Kanagawa, Japan) were used for all experiments. The rats were maintained in temperature-controlled rooms under a 12 h dark/light cycle and allowed food (standard laboratory chow) and water ad libitum. Partial hepatectomy was performed by a standard two-thirds partial hepatectomy under diethylether anesthesia [16]. For the assessment of DNA synthesis and endothelial cell area (sinusoidal tube

DNA synthesis of hepatocytes and endothelial cells

The percentages of labeled hepatocytes and endothelial cells by BrdU are shown in Fig. 1. The number of labeled hepatocytes peaked at 24 h, and decreased gradually, approaching the basal preoperative level at 120 h posthepatectomy. The number of labeled endothelial cells sharply increased at 48 h, and peaked at 72 h posthepatectomy. Thereafter, the number of labeled endothelial cells decreased gradually, approaching the basal preoperative level at 120 h posthepatectomy (Fig. 1a). During liver

Acknowledgements

The authors thank Mrs Miki Asazu and Mrs Mika Nada for much technical assistance.

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