Angiopoietin‐2 Promotes Pathological Angiogenesis and Is a Therapeutic Target in Murine Nonalcoholic Fatty Liver Disease

Abstract

Angiogenesis contributes to the development of nonalcoholic steatohepatitis (NASH) and promotes inflammation, fibrosis, and progression to hepatocellular carcinoma (HCC). Angiopoietin‐2 (Ang‐2) is a key regulator of angiogenesis. We aimed to investigate the role of Ang‐2 and its potential as a therapeutic target in NASH using human samples, in vivo mouse models, and in vitro assays. Serum Ang‐2 levels were determined in 104 obese patients undergoing bariatric surgery and concomitant liver biopsy. The effect of the Ang‐2/Tie2 receptor inhibiting peptibody L1‐10 was evaluated in the methionine‐choline deficient (MCD) and streptozotocin‐western diet nonalcoholic fatty liver disease mouse models, and in vitro on endothelial cells and bone marrow–derived macrophages. The hepatic vasculature was visualized with µCT scans and scanning electron microscopy of vascular casts. Serum Ang‐2 levels were increased in patients with histological NASH compared with patients with simple steatosis and correlated with hepatic CD34 immunoreactivity as a marker of hepatic angiogenesis. Serum and hepatic Ang‐2 levels were similarly increased in mice with steatohepatitis. Both preventive and therapeutic L1‐10 treatment reduced hepatocyte ballooning and fibrosis in MCD diet‐fed mice and was associated with reduced hepatic angiogenesis and normalization of the vascular micro‐architecture. Liver‐isolated endothelial cells and monocytes from MCD‐fed L1‐10–treated mice showed reduced expression of leukocyte adhesion and inflammatory markers, respectively, compared with cells from untreated MCD diet‐fed mice. In the streptozotocin‐western diet model, therapeutic Ang‐2 inhibition was able to reverse NASH and attenuate HCC progression. In vitro, L1‐10 treatment mitigated increased cytokine production in lipopolysaccharide‐stimulated endothelial cells but not in macrophages. Conclusion: Our findings provide evidence for Ang‐2 inhibition as a therapeutic strategy to target pathological angiogenesis in NASH.

Tie‐ing Up Angiogenesis to Treat Nonalcoholic Steatohepatitis

“…The authors further undertook elegant three-dimensional reconstruction imaging of the hepatic vasculature in those various groups of MCD diet-fed mice and demonstrated that L1-10 treatment, as either a preventive or a therapeutic intervention, reduced abnormal angiogenesis with decreased vascular density compared to placebo. (5) They also showed that L1-10 treatment reduced angiogenic signaling from cultured endothelial cells following lipopolysaccharide stimulation, confirming their observations that L1-10 reduces neoangiogenesis primarily through cell autonomous (i.e., endothelial) signaling. In a final series of studies, the authors returned to the streptozotocin-western diet model of NASH and asked if L1-10 would mitigate the development of HCC.…”

“…Administration of L1‐10 reduced inflammation, ballooning, and fibrosis in MCD diet–fed mice but did not change the degree of steatosis. The authors further undertook elegant three‐dimensional reconstruction imaging of the hepatic vasculature in those various groups of MCD diet–fed mice and demonstrated that L1‐10 treatment, as either a preventive or a therapeutic intervention, reduced abnormal angiogenesis with decreased vascular density compared to placebo . They also showed that L1‐10 treatment reduced angiogenic signaling from cultured endothelial cells following lipopolysaccharide stimulation, confirming their observations that L1‐10 reduces neoangiogenesis primarily through cell autonomous (i.e., endothelial) signaling.…”

“…(8) Lefere and colleagues show that serum ANG2 levels are increased in NASH patients and in proportion to the histological grade of inflammation, steatosis, and ballooning, although surprisingly not with the degree of fibrosis. (5) They further show that serum ANG2 levels correlate with CD34 immunohistochemical staining in liver sections, with reactivity confined to macrophages and endothelial cells. Those findings together strongly suggest that NASH subjects display increased circulating serum levels of ANG2, which correlates with markers of neovascularization (as indicated by CD34 staining) and that hepatic vascular endothelial cells are a plausible source of ANG2.…”

“…In addition, they observed a decreased tumor burden in the L1‐10‐treated mice with reduced expression of glypican‐3 and heat shock protein 70 as well as reduced alpha‐fetoprotein. Those findings suggest that L1‐10 treatment attenuates tumorigenesis in experimental NASH‐associated HCC, although the underlying mechanisms are still to be explored …”

“…The inflammation‐associated cleavage of Tie1 thus initiates a feed‐forward activation loop in which ANG2 promotes abnormal vascular remodeling . Lefere and colleagues show that serum ANG2 levels are increased in NASH patients and in proportion to the histological grade of inflammation, steatosis, and ballooning, although surprisingly not with the degree of fibrosis . They further show that serum ANG2 levels correlate with CD34 immunohistochemical staining in liver sections, with reactivity confined to macrophages and endothelial cells.…”