Gastroenterology

Gastroenterology

Volume 136, Issue 3, March 2009, Pages 1070-1080
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
Pulmonary Angiogenesis in a Rat Model of Hepatopulmonary Syndrome

https://doi.org/10.1053/j.gastro.2008.12.001Get rights and content

Background & Aims

Hepatopulmonary syndrome (HPS), defined as intrapulmonary vasodilation, occurs in 10%–30% of cirrhotics and increases mortality. In a rat model of HPS induced by common bile duct ligation (CBDL), but not thioacetamide (TAA)-induced nonbiliary cirrhosis, lung capillary density increases, monocytes accumulate in the microvasculature, and signaling factors in the angiogenesis pathway (Akt and endothelial nitric oxide synthase [eNOS]) are activated. Pentoxifylline (PTX) directly decreases lung endothelial Akt and eNOS activation, blocks intravascular monocyte accumulation, and improves experimental HPS; we evaluated whether pulmonary angiogenesis develops in this model.

Methods

TAA- and PTX-treated animals were evaluated following CBDL. Lung angiogenesis was assessed by quantifying factor VIII-positive microvessels and levels of von Willebrand factor (vWf), vascular endothelial cadherin (VE-cadherin), and proliferating cell nuclear antigen (PCNA). Angiogenic factors including phospho-Akt, phospho-eNOS, vascular endothelial growth factor (VEGF)-A, and phospho-VEGF receptor-2 (p-VEGFR-2) were compared and monocyte accumulation was assessed.

Results

Following CBDL, but not TAA exposure, rats developed HPS that was temporally correlated with increased numbers of lung microvessel; increased levels of vWf, VE-cadherin and PCNA; and activation of Akt and eNOS. Angiogenesis was accompanied by increased pulmonary VEGF-A and p-VEGFR-2 levels, with VEGF-A staining in accumulated intravascular monocytes and alveolar endothelial cells. Following CBDL, PTX-treated rats had reduced numbers of microvessels, reduced lung monocyte accumulation, downregulation of pulmonary angiogenic factors, and reduced symptoms of HPS.

Conclusions

A specific increase in pulmonary angiogenesis occurs as experimental HPS develops, accompanied by activation of VEGF-A-associated angiogenic pathways. PTX decreases the angiogenesis, reduces the symptoms of HPS, and downregulates VEGF-A mediated pathways.

Section snippets

Animals

Male Sprague–Dawley rats (200–250 g; Charles River, Wilmington, MA) were used in all experiments. CBDL was performed as described.22 Normal control animals underwent mobilization of the common bile duct without ligation. Some rats were intraperitoneally injected with TAA (Sigma–Aldrich, St Louis, MO) 200 mg/kg body weight or saline 3 times each week for 2 or 8 weeks as described.7 PTX (Sigma–Aldrich) 50 mg/kg body weight per day was given orally in drinking water for 2 weeks beginning 1 week

Physiologic Assessment for Hepatopulmonary Syndrome After CBDL and TAA Administration

To evaluate the development of HPS in relation to hepatic and pulmonary alterations in models of biliary and nonbiliary cirrhosis, we measured arterial blood gases and microsphere shunting through the pulmonary microcirculation, portal venous pressure, spleen weight, and liver histology at serial time points after CBDL and TAA administration (Table 1). HPS developed only after CBDL, as reflected by the development of a widened alveolar arterial oxygen gradient and increased shunting of

Discussion

The pathogenesis of HPS is poorly understood and has hampered the development of effective treatments. The current study was undertaken to define whether pulmonary angiogenesis is a unique feature that contributes to experimental HPS. We find that HPS induced by CBDL is associated with pulmonary angiogenesis, activation of Akt and eNOS in the pulmonary endothelium, and VEGF-A production in intravascular monocytes. In contrast, angiogenesis was absent; monocyte accumulation was minimal; and Akt,

Acknowledgments

J.H. and B.L. contributed equally to this paper.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by DK02030 and a VA Merit Review grant (to M.B.F.) and AHA 0735468N (to J.Z.).

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