Placental growth factor modulates endothelial NO production and exacerbates experimental hepatopulmonary syndrome

Abstract

Background & aims: Hepatopulmonary syndrome (HPS) results from portal hypertension, with or without cirrhosis, and is marked by pulmonary vascular dilations leading to severe hypoxemia. Although placental growth factor (PlGF) is important for vascular growth and endothelial function, its role in HPS is unclear. This study investigated the involvement of PlGF in experimental models of HPS and in patients.

Methods: Circulating PlGF levels were measured in 64 controls and 137 patients with liver disease, with or without HPS. Two rat models, common bile duct ligation (CBDL) and long-term partial portal vein ligation (PPVL), were used. Plgf-knockout (Plgf ^-/-) rats were generated using CRISPR-Cas9. Lung RNA-sequencing analysis was performed in the CBDL model. The effects of PlGF on endothelial nitric oxide synthase (eNOS) activity in human pulmonary microvascular endothelial cells were also investigated.

Results: Circulating PlGF levels were significantly higher in patients with cirrhosis compared with healthy controls (29.4 ± 1.2 vs. 20.2 ± 0.8 pg/ml, p <0.0001), but no difference were found between patients with and without HPS. PlGF levels were not elevated in patients with extrahepatic portal hypertension. In Plgf ^-/- rats, there was a protective effect against CBDL-induced HPS, whereas PPVL-induced HPS severity remained unchanged. RNA sequencing coupled with ingenuity pathway analysis identified significant interactions between PlGF and pulmonary eNOS activity. Following CBDL, Plgf ^-/- rats showed decreased pulmonary eNOS activity and reduced circulating nitric oxide metabolites. In vitro, PlGF stimulation enhanced eNOS activity in human pulmonary microvascular endothelial cells, whereas PlGF knockdown led to a decrease.

Conclusions: These findings indicate that PlGF aggravates cirrhosis-induced HPS through modulation of pulmonary eNOS activity, and is not involved in HPS from extrahepatic portal hypertension.

Impact and implications: This study identified PlGF as a significant contributor to the exacerbation of HPS associated with cirrhosis, through its regulation of pulmonary nitric oxide production. Our findings demonstrated that PlGF deficiency mitigates the severity of both cirrhosis and HPS in the CBDL model, highlighting its potential as a therapeutic target in cirrhosis-induced HPS. Notably, this protective effect was absent in the PPVL model, which induces HPS associated with portal hypertension without cirrhosis. These results open avenues for novel pharmacological interventions aiming to improve outcomes for patients with cirrhosis-induced HPS.

Keywords: Common bile duct ligation; Hypoxemia; Intrapulmonary vascular dilations; Liver cirrhosis; Partial portal vein ligation; Portal hypertension; Pulmonary endothelial dysfunction; VEGF.

Graphical abstract

Fig. 1

PlGF levels in humans. (A) Circulating PlGF levels in healthy control subjects, patients with cirrhosis, and patients with portal cavernoma. (B) Among patients with cirrhosis, circulating PlGF levels according to the Child-Pugh class. (C). Among patients with cirrhosis, circulating PlGF levels in patients without and with HPS. (D) Among patients with HPS, correlation between circulating PlGF levels and A-aO₂ assessed by Pearson correlation. Data are mean ± SEM; comparisons were made using one-way ANOVA followed by the Tukey test or by unpaired t test. n.s., not significant (p >0.05); ∗p <0.05, ∗∗∗∗p <0.0001. A-aO₂, alveolar–arterial oxygen gradient; HPS, hepatopulmonary syndrome; PIGF, placental growth factor.

Fig. 2

HPS development in Plgf^–/– rats after CBDL and PPVL surgery. (A) Experimental procedures. (B) Circulating PlGF levels in serum determined using ELISA. (C) Detection of Plgf mRNA levels in liver and lung using RT-qPCR. (D) A-aO₂ in arterial blood. (E) Quantification of contrast-enhanced transthoracic echocardiogram: ratio of the difference in acoustic signal between baseline and after injection in the left ventricle to the right ventricle. (F) Quantification of total microspheres in one kidney by fluorescence-activated cell sorting after jugular vein injection. Data are mean ± SEM; comparisons were made using one-way ANOVA followed by the Tukey test: n.s., not significant (p >0.05); ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001, vs. Sham WT group; ^&&&p <0.001, ^&&&&p <0.0001 vs. WT CBDL. A-aO₂, alveolar–arterial oxygen gradient; CBDL, common bile duct ligation; HPS, hepatopulmonary syndrome; PIGF, placental growth factor; PPVL, partial portal vein ligation; real-time quantitative PCR (RT-qPCR); WT, wild-type.

Fig. 3

Portal hypertension and cirrhosis development in Plgf^–/– rats after CBDL and PPVL surgery. (A) Quantification of portal venous flow by liver Doppler ultrasound. (B) Spleen and liver weight. (C) Picro-Sirius Red staining of liver sections. (D) Quantification of liver fibrotic area by measuring the percentage of Picro-Sirius Red staining. Data are mean ± SEM; Comparisons were made using one-way ANOVA followed by the Tukey test: n.s., not significant (p >0.05); ∗p <0.05, ∗∗∗p <0.001, ∗∗∗∗p <0.0001 vs. Sham WT group; ^&&p <0.01, ^&&&p <0.001, ^&&&&p <0.0001 vs. WT CBDL. CBDL, common bile duct ligation; PIGF, placental growth factor; PPVL, partial portal vein ligation; real-time quantitative PCR (RT-qPCR); WT, wild-type.

Fig. 4

eNOS activity modulation by PlGF abundance in CBDL rats and in human PMECs. (A) eNOS and p-eNOS expression in WT and Plgf^–/– rats after CBDL surgery analyzed by western blot. (B) NO metabolites in WT and Plgf^–/– rats after CBDL surgery. (C) eNOS and p-eNOS expression after 30 min or 24 h of PlGF stimulation (0, 50, or 200 ng/ml). (D) eNOS and p-eNOS expression after transfection with siPlGF or scrambled sequence. Data are mean ± SEM; comparisons were made using one-way ANOVA followed by the Tukey test or by unpaired t test: n.s., not significant (p >0.05); ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. CBDL, common bile duct ligation; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; p-eNOS, phosphorylated eNOS; PlGF, placental growth factor; PMECs, pulmonary microvascular endothelial cells; siPlGF, siRNA-mediated knockdown of PlGF; WT, wild-type.