Annexin A1 protein regulates the expression of PMVEC cytoskeletal proteins in CBDL rat serum-induced pulmonary microvascular remodeling

Abstract

Background: Hepatopulmonary syndrome (HPS) is characterized by advanced liver disease, hypoxemia and intrapulmonary vascular dilatation (IPVD). The pathogenesis of HPS is not completely understood. Recent findings have established the role of proliferation and phenotype differentiation of pulmonary microvascular endothelial cells (PMVECs) in IPVD of HPS; the change in cytoskeletal proteins and their molecular mechanism play an essential role in the proliferation, phenotype modulation and differentiation of PMVECs. However, little is known about the relevance of cytoskeletal protein expression and its molecular mechanism in IPVD of HPS. In addition, ANX A1 protein has been identified as a key regulator in some important signaling pathways, which influences cytoskeletal remodeling in many diseases, such as lung cancer, liver cancer, etc.

Methods: PMVECs were cultured from the normal rats and then divided into three groups(Ad-ANXA1-transfected group, a non-transfected group, and an adenovirus empty vector group) and incubated by nomal rat serum or hepatopulmonary syndrome rat serum respectively. mRNA level was evaluated by real time reverse transcription polymerase chain reaction, and protein expression was detected by western blot. Cell proliferation was determined by the MTT and thymidine incorporation assay.

Results: In this study, we found that the serum from a common bile duct ligation(CBDL) Rat model decreased the expression levels of the ANX A1 mRNA and protein by at least two-fold in human PMVECs. We also found the expression of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin) in PMVECs significantly increased. After stimulating ANX A1 over-expression in PMVECs by adenovirus-mediated ANX A1 (Ad-ANXA1) transfection, we found the expression levels of cytoskeletal proteins were significantly suppressed in PMVECs at all time points. Additionally, we report here that serum from a CBDL Rat model increases the proliferation of PMVECs by nearly two-fold and that over-expression of Ad-ANXA1 significantly inhibits HPS-rat-serum-induced PMVEC proliferation (p <0.05). These findings suggest that the ANX A1 down-regulation of PMVEC proliferation in the presence of HPS-rat-serum may be the major cause of aberrant dysregulation of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin) and may, therefore, play a fundamental role in the proliferation and phenotype differentiation of PMVECs in the PVD of HPS.

Conclusion: Finally, the fact that transfection with Ad-ANXA1 results in inhibition of the aberrant dysregulation of cytoskeletal proteins and proliferation of PMVECs suggests a potential therapeutic effect on PVD of HPS.

Figure 1

Levels of ANX A1 mRNA and protein in PMVECs induced by CBDL-Rat serum at each time point. (A.B) Exposure to CBDL-Rat serum significantly decreased expression of the ANX A1 gene, including the transcript and protein levels in PMVECs (p < 0.05). (C) The mRNA and protein density of the ANX A1 gene for each group is displayed. Each data point represents the mean ± S.E.M. of four independent experiments. * p < 0.05 vs. T1 or T2; # p < 0.05 vs. Group N. Group N: normal rat serum; Group C: CBDL-Rat serum.

Figure 2

Ad-ANXA1 transfected into PMVECs was strongly expressed and the protein was found in both the cytoplasm and nuclei. (A) At 24 h after transfection with Ad-ANXA1, PMVECs had a normal structure with no observable pathological characteristics (magnification 100×). (B) Bright green fluorescence was observed in nearly all cells using an inverted fluorescence microscope following transfection with Ad-ANXA1 containing the green fluorescent protein (GFP) gene (magnification 100×). (C.D) The expression of the ANX A1 mRNA and protein in PMVECs 24 h after either the mock transfection or transfection with Ad-ANXA1 was determined by RT-PCR and western blotting. GAPDH was used as an internal control. (E) The mRNA and protein density of the ANX A1 gene for each group is displayed. Each data point represents the mean ± S.E.M. of four independent experiments. * p < 0.05; N: Non-transfected group; T: transfected group; E: empty vector group.

Figure 3

Over-expression of ANX A1 suppressed CBDL-rat-serum-induced increase in the a1-actin, a1-tubulin and destrin protein expression levels in PMVECs. (A) The change in expression levels of a1-actin, a1-tubulin and destrin proteins in PMVECs 24 h following either mock transfection or transfection with Ad-ANXA1 was determined by western blot. GAPDH was used as an internal control. (B-D) The protein density of a1-actin, a1-tubulin and destrin for each group is displayed. Each data point represents the mean ± S.E.M. of four independent experiments. * p < 0.05 vs. group N; ^#p < 0.05 vs. the non-transfected group; Δp < 0.05 vs. T1. N: Non-transfected; T: transfected with Ad-ANXA1.

Figure 4

Over-expression of the ANXA1 gene inhibited CBDL-rat-serum-induced PMVEC proliferation as determined by the ³H-TdR and MTT assay. (A.B) The change of the value of the incorporation of ³H-TdR and the absorbance of MTT assay is displayed. The inhibitory effect was investigated at T2 and T3. Each data point represents the mean ± S.E.M. of four independent experiments. * P < 0.05 vs. group N; # P < 0.05 vs. Non-transfected group and empty vector group. Non-N: Group N without Ad-ANXA1 transfection; Non-C: Group C without Ad-ANXA1 transfection; Empty-N: Group N with empty vector transfection; Empty-C: Group C with empty vector transfection; Trans-N: Group N with Ad-ANXA1 transfection; Trans-C: Group C with Ad-ANXA1 transfection.