The role of canonical WNT/β-catenin signaling pathway on pulmonary arterial hypertension induced by Monocrotaline in rats

Abstract

Background: Pulmonary vascular remodeling seems to play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). The pulmonary vascular remodeling is closely associated with migration and proliferation of pulmonary arterial smooth muscle cells (PASMCs). The canonical WNT/β-catenin pathway has been associated with formation and remodeling of the vasculature. However, the association between the canonical WNT/β-catenin pathway and PAH, especially PASMC proliferation and vascular remodeling remains unclear. This study evaluated a potential role of canonical WNT/β-catenin pathway on PASMC proliferation and vascular remodeling of PAH and its possible mechanism in Monocrotaline (MCT)-induced PAH rat model.

Methods: Male Sprague-Dawley rats were randomized to administer a single subcutaneous injection of MCT (70 mg/kg) and phosphate-buffered saline (PBS) of an equal volume. All rats with MCT were randomly received β-catenin-targeted small interfering RNA (siRNA) or PBS via intratracheal route three times a week. To measure right ventricle (RV) function, echocardiography was performed at 28 days after MCT injection. Assessment of RV hypertrophy and histological examination were carried out at 14, 28 days after MCT injection. Samples of rat lungs and pulmonary arteries were obtained for immunohistochemistry and quantitative real-time PCR of β-catenin, Tenacin-C (TN-C), and PDGFR-β.

Results: MCT induced PAH, RV hypertrophy, and pulmonary vascular remodeling at 28 days after MCT injection, as judged by echocardiography and histology. There were markedly increased expression of β-catenin in alveolar epithelial cells and mild increased expression in neointima of pulmonary artery (PA), accompanied with a significant upregulation in mRNA expression of β-catenin at 14 days of MCT-induced PAH rat model (P < 0.05). The mRNA expression of Tenascin-C was significantly increased at 28 days in MCT-induced rat lung homogenates (P < 0.05). However, no significant difference was found between controls and MCT-induced rats in mRNA expression of PDGFR-β. The blockade of the WNT/β-catenin pathway by β-catenin-targeted siRNA attenuated MCT-induced PAH, RV hypertrophy, and pulmonary vascular remodeling. Also, the siRNA-mediated inhibition of β-catenin in vivo led to reduce TN-C expression. However, expression of PDGFR-β was unaffected by β-catenin blockade.

Conclusion: Inhibition of the canonical WNT/β-catenin pathway by β-catenin-targeted siRNA attenuated MCT-induced PAH, RV hypertrophy, and pulmonary vascular remodeling. The canonical WNT/β-catenin pathway appears to contribute to the pathogenesis of PAH and pulmonary vascular remodeling in MCT-induced PAH rat model. |배경: 폐동맥 고혈압은 폐혈관 저항을 증가시키는 심각한 질환으로 광범위한 폐혈관의 리모델링을 특징으로 한다. 폐동맥 고혈압의 원인은 아직까지 구체적으로 밝혀지지 않고 있으나, 폐혈관의 리모델링은 중요한 원인 중에 하나로 알려져 있다. 폐혈관의 리모델링은 폐동맥 근육세포의 이주와 증식과 관련이 많다고 알려져 있다. 폐동맥 고혈압과 폐동맥 근육세포의 증식에 정규 WNT/β-catenin 신호경로가 관여한다는 보고가 있었다. 이에 본 저자는 Monocrotaline (MCT)으로 유도된 폐동맥 고혈압 백서 모델을 통하여 폐혈관 리모델링과 폐동맥 고혈압에 대한 정규 WNT/β-catenin 신호경로의 역할을 알아보고자 하였다.

방법: 6주 령의 Sprague Dawley 쥐를 대조군과 MCT 군으로 나누었다. MCT 군에는 70 mg/kg의 MCT 를 배부에 피하주사하였고, 대조군에는 동량의 생리식염수를 피하주사하였다. 폐동맥 고혈압의 유도를 확인하기 위해 MCT 주사 4주 후 심초음파로 측정하였고, MCT 주사 2주, 4주 후 우심실 비대(우심실/좌심실+심실벽 무게 비), 폐동맥의 리모델링(근육성 폐세동맥의 비율, 근육성 폐세동맥에서의 중막 비후율) 지표를 측정하였다. 얻어진 폐, 폐동맥 조직에서 β-catenin, Tenascin-C 그리고 PDGFR-β의 면역염색과 중합효소연쇄반응 검사를 시행하였다.

결과: MCT 군에서 MCT 투여 4주 후 조직검사와 심초음파 검사로 폐동맥 고혈압 발생을 확인하였다. β-catenin 면역 염색 및 중합효소연쇄반응에서 대조군에 비하여 MCT로 유도된 폐동맥 고혈압 모델의 β-catenin 발현이 MCT 주사 2주 후 폐조직에서 유의하게 증가하였다 (p < 0.05). 면역 염색 및 중합효소연쇄반응에서 Tenascin-C 발현은 MCT 투여 4주 후 유의하게 증가하였으나 (p < 0.05), PDGFR-β 발현은 유의하게 증가하지 않았다. β-catenin 표적 siRNA를 통한 정규 WNT 신호경로의 차단은 생체 내에서 β-catenin 발현을 억제하였고, 폐동맥 고혈압, 우심실 비대 및 폐동맥 리모델링을 완화하였다. 이러한 정규 WNT/β-catenin 신호경로의 차단은 생체 내에서 TN-C 발현을 억제하였으나, PDGFR-β발현은 억제하지 못하였다.

결론: β-catenin 표적 siRNA 를 통한 정규 WNT/β-catenin 신호경로의 차단은 MCT 로 유도된 폐동맥 고혈압 백서 모델에서 TN-C을 억제함으로서 폐동맥 고혈압과 폐혈관 리모델링을 완화하였다. 정규 Wnt/β-catenin 신호경로는 MCT 로 유도된 폐동맥 고혈압 백서 모델에서 폐동맥 고혈압과 폐동맥 리모델링의 발생에 의미있는 역할을 하였다.; Background: Pulmonary vascular remodeling seems to play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). The pulmonary vascular remodeling is closely associated with migration and proliferation of pulmonary arterial smooth muscle cells (PASMCs). The canonical WNT/β-catenin pathway has been associated with formation and remodeling of the vasculature. However, the association between the canonical WNT/β-catenin pathway and PAH, especially PASMC proliferation and vascular remodeling remains unclear. This study evaluated a potential role of canonical WNT/β-catenin pathway on PASMC proliferation and vascular remodeling of PAH and its possible mechanism in Monocrotaline (MCT)-induced PAH rat model.

Methods: Male Sprague-Dawley rats were randomized to administer a single subcutaneous injection of MCT (70 mg/kg) and phosphate-buffered saline (PBS) of an equal volume. All rats with MCT were randomly received β-catenin-targeted small interfering RNA (siRNA) or PBS via intratracheal route three times a week. To measure right ventricle (RV) function, echocardiography was performed at 28 days after MCT injection. Assessment of RV hypertrophy and histological examination were carried out at 14, 28 days after MCT injection. Samples of rat lungs and pulmonary arteries were obtained for immunohistochemistry and quantitative real-time PCR of β-catenin, Tenacin-C (TN-C), and PDGFR-β.

Results: MCT induced PAH, RV hypertrophy, and pulmonary vascular remodeling at 28 days after MCT injection, as judged by echocardiography and histology. There were markedly increased expression of β-catenin in alveolar epithelial cells and mild increased expression in neointima of pulmonary artery (PA), accompanied with a significant upregulation in mRNA expression of β-catenin at 14 days of MCT-induced PAH rat model (P < 0.05). The mRNA expression of Tenascin-C was significantly increased at 28 days in MCT-induced rat lung homogenates (P < 0.05). However, no significant difference was found between controls and MCT-induced rats in mRNA expression of PDGFR-β. The blockade of the WNT/β-catenin pathway by β-catenin-targeted siRNA attenuated MCT-induced PAH, RV hypertrophy, and pulmonary vascular remodeling. Also, the siRNA-mediated inhibition of β-catenin in vivo led to reduce TN-C expression. However, expression of PDGFR-β was unaffected by β-catenin blockade.

Conclusion: Inhibition of the canonical WNT/β-catenin pathway by β-catenin-targeted siRNA attenuated MCT-induced PAH, RV hypertrophy, and pulmonary vascular remodeling. The canonical WNT/β-catenin pathway appears to contribute to the pathogenesis of PAH and pulmonary vascular remodeling in MCT-induced PAH rat model.