Inhibition of VEGF- and NO-dependent angiogenesis does not impair liver regeneration

Abstract

Angiogenesis occurs through a convergence of diverse signaling mechanisms with prominent pathways that include autocrine effects of endothelial nitric oxide (NO) synthase (eNOS)-derived NO and vascular endothelial growth factor (VEGF). However, the redundant and distinct roles of NO and VEGF in angiogenesis remain incompletely defined. Here, we use the partial hepatectomy model in mice genetically deficient in eNOS to ascertain the influence of eNOS-derived NO on the angiogenesis that accompanies liver regeneration. While sinusoidal endothelial cell (SEC) eNOS promotes angiogenesis in vitro, surprisingly the absence of eNOS did not influence the angiogenesis that occurs after partial hepatectomy in vivo. While this observation could not be attributed to induction of alternate NOS isoforms, it was associated with induction of VEGF signaling as evidenced by enhanced levels of VEGF ligand in regenerating livers from mice genetically deficient in eNOS. However, surprisingly, mice that were genetically heterozygous for deficiency in the VEGF receptor, fetal liver kinase-1, also maintained unimpaired capacity for liver regeneration. In summary, inhibition of VEGF- and NO-dependent angiogenesis does not impair liver regeneration, indicating signaling redundancies that allow liver regeneration to continue in the absence of this canonical vascular pathway.

Fig. 1.

Endothelial (e) nitric oxide synthase (NOS) overexpression increases proliferation and tube formation in human hepatic (HH) sinusoidal endothelial cells (SEC). A: HHSEC were transduced with adenoviral vectors for eNOS (AdeNOS) or LacZ control (AdLacZ). Following transduction, 5 × 10³ HHSEC were plated on a 96-well plate and serum starved for 10 h, and, subsequently, the proliferation index was evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. HHSEC transduced with AdeNOS showed significantly increased proliferation compared with the AdLacZ-transduced group (n = 3 separate experiments, each in triplicate *P < 0.05). B: to assess tube formation, HHSEC were transduced with AdeNOS and AdLacZ and were then plated on matrigel-coated four-chamber slides for 18 h in basal endothelial cell medium. Tubulogenesis was measured using Image-Pro Plus software. Cells transduced with AdeNOS showed a greater increase in total tube length compared with the AdLacz-transduced group (scale bar represents the tube size, n = 3 separate experiments with 15 representative images taken and analyzed from each group in each experiment; *P < 0.05).

Fig. 2.

Sinusoidal lining cell and hepatocyte proliferation kinetics after partial hepatectomy: Wild-type C57BL/6J mice (n = 6/group) underwent partial hepatectomy; mice were killed at 0, 2, 4, 6, and 8 days following the procedure. The remnant liver was weighed and embedded in optimum-cutting temperature medium for subsequent sectioning. A: left, photomicrographs along with scale bars of hematoxylin and eosin (H&E) and Ki-67 staining of remnant liver sections show the proliferation pattern at different time points. Images at bottom convey the different morphological pattern of hepatocyte and sinusoidal lining staining. Right, Ki-67-positive cells among both the hepatocyte and sinusoidal lining cells were counted and expressed as a fraction of the total cells in that respective cell population. Although proliferation of hepatocytes was significantly greater than sinusoidal lining cells at day 2, this pattern was reversed at day 4 [P < 0.05, hepatocyte vs. sinusoidal lining cell at day 2 (*) and sinusoidal lining cell vs. hepatocyte at day 4 (**)]. B: Western blot analysis of liver tissue lysates was performed to determine changes in vascular endothelial growth factor (VEGF)-A expression at day 2, day 4, day 6, and day 8 in mice posthepatectomy and in sham-operated mice; β-actin served as a loading control. VEGF-A expression was highest at day 2. C: conversion of l-[³H]arginine to l-[³H]citrulline was used to assess the NOS activity in liver samples obtained from C57BL/6J mice 2, 4, and 8 days following partial hepatectomy (n = 4 in each group) and was compared with sham-operated mice. Peak in NOS activity was observed at day 2.

Fig. 3.

Liver regeneration is not impaired following eNOS inhibition. Partial hepatectomy was conducted in eNOS wild-type (+/+) and eNOS knockout (−/−) mice; restituted liver mass was measured, and tissue was retrieved at specific time points after surgery for immunohistochemical analyses. A: immunostaining with Ki-67 was used as a proliferation marker to determine the fraction of positively staining cells in the hepatocyte population (left) or the sinusoidal lining cell (SLC) population (right) in both groups at different time points. No differences were observed between eNOS^+/+ and eNOS^+/+ mice (day 2, n = 4 for eNOS^−/− and n = 6 for eNOS^+/+; day 4, n = 6 for eNOS^−/− and n = 6 for eNOS^+/+; day 6, n = 5 for eNOS^−/− and n = 6 for eNOS^+/+; day 8, n = 4 for eNOS^−/− and n = 6 for eNOS^+/+; P > 0.05). B: the remnant liver removed at day 2, day 4, day 6, and day 8 was used to calculate the restituted liver mass according to the formula mentioned in materials and methods, and the two groups were compared for each time point. There was no significant difference in regenerating liver mass in eNOS^−/− compared with eNOS^+/+ mice (P > 0.05). C: administration of N^G-nitro-l-arginine methyl ester (l-NAME, 100 mg/kg body wt ip) did not significantly alter the proliferation pattern of hepatocytes and SLC compared with the vehicle-treated group (n = 4 mice/group, P > 0.05).

Fig. 4.

Alternative NOS isoforms do not compensate for eNOS deficiency after hepatectomy. Total RNA extracted from the harvested liver of eNOS^−/− and eNOS^+/+ mice following partial hepatectomy was used to compare the expression of neuronal NOS (nNOS; A) and inducible NOS (iNOS, B) between eNOS^−/− and wild-type control mice at different time points using qPCR. The mRNA levels of nNOS were not statistically significant at any time point under consideration between the two groups; however, there was an increase in iNOS mRNA levels in eNOS^+/+ mice compared with eNOS^−/− at day 6 (*P < 0.05; n = 3 animals/group).

Fig. 5.

VEGF mRNA levels increase in eNOS^−/− mice to a greater extent than wild-type mice posthepatectomy: VEGF mRNA levels were measured in the wild-type and eNOS^−/− mice after partial hepatectomy. eNOS^−/− animals showed a significant increase in VEGF mRNA levels compared with the wild-type mice at days 2, 4, and 8 (n = 3 animals/group; *P < 0.05).

Fig. 6.

Normal regeneration in mice genetically heterozygous for fetal liver kinase-1 (Flk-1). Flk-1^+/+ and Flk-1^+/− mice were subjected to partial hepatectomy, and the remnant liver was removed at day 2 (n = 3 for Flk-1^+/− and n = 3 for Flk-1^+/+) and day 4 (n = 3 for Flk-1^+/− and n = 3 for Flk-1^+/+) for analysis following surgery. Immunostaining of 5-μm section of the regenerated liver sections with Ki-67 was used to determine the fraction of positively staining cells in the hepatocyte population (A) or the SLC population (B) in both groups at the different time points. There were no significant differences in proliferation observed between the Flk-1^+/− and Flk-1^+/+ mice in hepatocyte or SLC populations (P > 0.05).