The role of CX₃CL1/CX₃CR1 in pulmonary angiogenesis and intravascular monocyte accumulation in rat experimental hepatopulmonary syndrome

Abstract

Background & aims: Hepatopulmonary syndrome (HPS), classically attributed to intrapulmonary vascular dilatation, occurs in 15-30% of cirrhotics and causes hypoxemia and increases mortality. In experimental HPS after common bile duct ligation (CBDL), monocytes adhere in the lung vasculature and produce vascular endothelial growth factor (VEGF)-A and angiogenesis ensues and contribute to abnormal gas exchange. However, the mechanisms for these events are unknown. The chemokine fractalkine (CX(3)CL1) can directly mediate monocyte adhesion and activate VEGF-A and angiogenesis via its receptor CX(3)CR1 on monocytes and endothelium during inflammatory angiogenesis. We explored whether pulmonary CX(3)CL1/CX(3)CR1 alterations occur after CBDL and influence pulmonary angiogenesis and HPS.

Methods: Pulmonary CX(3)CL1/CX(3)CR1 expression and localization, CX(3)CL1 signaling pathway activation, monocyte accumulation, and development of angiogenesis and HPS were assessed in 2- and 4-week CBDL animals. The effects of a neutralizing antibody to CX(3)CR1 (anti-CX(3)CR1 Ab) on HPS after CBDL were evaluated.

Results: Circulating CX(3)CL1 levels and lung expression of CX(3)CL1 and CX(3)CR1 in intravascular monocytes and microvascular endothelium increased in 2- and 4-week CBDL animals as HPS developed. These events were accompanied by pulmonary angiogenesis, monocyte accumulation, activation of CX(3)CL1 mediated signaling pathways (Akt, ERK) and increased VEGF-A expression and signaling. Anti-CX(3)CR1 Ab treatment reduced monocyte accumulation, decreased lung angiogenesis and improved HPS. These events were accompanied by inhibition of CX(3)CL1 signaling pathways and a reduction in VEGF-A expression and signaling.

Conclusions: Circulating CX(3)CL1 levels and pulmonary CX(3)CL1/CX(3)CR1 expression and signaling increase after CBDL and contribute to pulmonary intravascular monocyte accumulation, angiogenesis and development of experimental HPS.

Fig. 1. Pulmonary fractalkine/CX₃CL1 expression and immunofluorescent localization, and plasma levels after CBDL

(A), representative double-labeling images of CX₃CL1 (green) and ED1 (red, a specific monocyte marker) and superimposition with DAPI (blue) in control and 2wk CBDL animals (shown by arrows, original magnification, 40×). The graphical summaries are shown of (B) CX₃CL1 mRNA levels in lungs and (C) circulating levels in plasma after 2wk (n=5) and 4wk CBDL (n=6) relative to control (n=6). Values are expressed as means ± SE. *P < 0.05 compared with control.

Fig. 2. Pulmonary CX₃CR1 expression and immunofluorescent localization after CBDL

(A), Representative double-labeling images of CX₃CR1 (green) and ED1 (red, a specific monocyte marker) and superimposition with DAPI (blue) in control and 2wk CBDL animals (show by arrows, original magnification 40×). (B), the graphical summary of CX₃CR1 mRNA levels in lungs after 2wk (n=5) and 4wk CBDL (n=6) relative to control (n=6). Values are expressed as means ± SE. *P < 0.05 compared with control.

Fig. 3. Effect of CX₃CR1 neutralization on pulmonary FVIII immunostaining, microvessel counts and von Willebrand factor (vWf) and PCNA levels in 2wk CBDL animals

(A), immunostaining of FVIII (red) with blue DAPI nuclear stain (blue) in control, CBDLand anti-CX₃CR1-neutralizing antibody (anti-CX₃CR1 Ab) administered CBDL animals (original magnification, 40×) and the graphical summary of lung microvessel counts in all animal groups. The representative immunoblots and graphical summaries of (B) vWf and (C) PCNA levels in control (n=6), CBDL (n=5) and anti-CX₃CR1 Ab treated CBDL (n=8) animals are shown. Values are expressed as means ± SE. *P < 0.05 compared with control. ^‡P < 0.05 compared with CBDL.

Fig. 4. Effect of CX₃CR1 neutralization on immunofluorescent localization and protein levels of lung VEGF-A, VEGFR-2 phosphorylation, monocyte accumulation and phosphorylation of Akt and ERK in 2wk CBDL animals

(A), representative double-labeling images of VEGF-A (green) and ED1 (red) with blue DAPI nuclear stain (blue) in control, CBDL and anti-CX₃CR1 Ab administered CBDL animals (shown by arrows, original magnification 40×). The representative immunoblots and graphical summaries of protein levels for (B) VEGF-A, (C) p-VEGFR-2, (D) ED1, (E) p-Akt/Akt and (F) p-ERK/ERK in control (n=6), CBDL (n=5) and anti-CX₃CR1 Ab treated CBDL (n=8) animals are shown. Values are expressed as means ± SE. *P < 0.05 compared with control. ^‡P < 0.05 compared with CBDL.

Fig. 5. Effect of CX₃CR1 neutralization on alveolar–arterial oxygen gradients, lung microvessel counts, monocytes accumulation and phosphorylation of Akt and ERK in 4wk CBDL animals

(A), the summaries of AaPO₂ in control, CBDL and anti-CX₃CR1 Ab administered CBDL animals. (B), immunostaining of FVIII (green) with DAPI nuclear stain (blue) in CBDL and anti-CX₃CR1 Ab administered CBDL animals (original magnification, 20×) and the graphical summary of lung microvessel counts in all animal groups. (C), the representative immunoblots and graphical summaries of protein levels for ED1, p-Akt/Akt and p-ERK/ERK in control (n=5), CBDL (n=6) and anti-CX₃CR1 Ab treated CBDL (n=6) animals. Values are expressed as means ± SE. *P < 0.05 compared with control. ^‡P < 0.05 compared with CBDL.