Development of occlusive neointimal lesions in distal pulmonary arteries of endothelin B receptor-deficient rats: a new model of severe pulmonary arterial hypertension

Abstract

Background: Human pulmonary arterial hypertension (PAH) is characterized by proliferation of vascular smooth muscle and, in its more severe form, by the development of occlusive neointimal lesions. However, few animal models of pulmonary neointimal proliferation exist, thereby limiting a complete understanding of the pathobiology of PAH. Recent studies of the endothelin (ET) system demonstrate that deficiency of the ET(B) receptor predisposes adult rats to acute and chronic hypoxic PAH, yet these animals fail to develop neointimal lesions. Herein, we determined and thereafter showed that exposure of ET(B) receptor-deficient rats to the endothelial toxin monocrotaline (MCT) leads to the development of neointimal lesions that share hallmarks of human PAH.

Methods and results: The pulmonary hemodynamic and morphometric effects of 60 mg/kg MCT in control (MCT(+/+)) and ET(B) receptor-deficient (MCT(sl/sl)) rats at 6 weeks of age were assessed. MCT(sl/sl) rats developed more severe PAH, characterized by elevated pulmonary artery pressure, diminished cardiac output, and right ventricular hypertrophy. In MCT(sl/sl) rats, morphometric evaluation revealed the presence of neointimal lesions within small distal pulmonary arteries, increased medial wall thickness, and decreased arterial-to-alveolar ratio. In keeping with this, barium angiography revealed diminished distal pulmonary vasculature of MCT(sl/sl) rat lungs. Cells within neointimal lesions expressed smooth muscle and endothelial cell markers. Moreover, cells within neointimal lesions exhibited increased levels of proliferation and were located in a tissue microenvironment enriched with vascular endothelial growth factor, tenascin-C, and activated matrix metalloproteinase-9, factors already implicated in human PAH. Finally, assessment of steady state mRNA showed that whereas expression of ET(B) receptors was decreased in MCT(sl/sl) rat lungs, ET(A) receptor expression increased.

Conclusions: Deficiency of the ET(B) receptor markedly accelerates the progression of PAH in rats treated with MCT and enhances the appearance of cellular and molecular markers associated with the pathobiology of PAH. Collectively, these results suggest an overall antiproliferative effect of the ET(B) receptor in pulmonary vascular homeostasis.

Figure 1

Hematoxylin and eosin (H&E) staining of MCT^+/+ rat lung revealed medial hypertrophy of small pulmonary arteries. VVG staining revealed a well-defined internal elastic lamina. In contrast, near occlusion of small pulmonary arteries in the MCT^sl/sl rat lung is shown. VVG staining of MCT^sl/sl rat lung of plexogenic lesions failed to reveal a well-defined internal elastic lamina. Bar=50 μm.

Figure 2

Barium angiograms of control rat lung (+/+), MCT^+/+ rat lung, and MCT^sl/sl rat lung. Note progressive decrease in filling of distal pulmonary vasculature in MCT^+/+ and MCT^sl/sl lung.

Figure 3

Immunohistochemical staining of MCT^+/+ and MCT^sl/sl rat lung. Factor VIII (FVIII) staining of MCT^+/+ rat lung revealed a well-defined endothelial layer. Smooth muscle α-actin (SMA) staining of MCT^+/+ lungs identified VSMCs in vessel media. In MCT^sl/sl rat lung, FVIII and SMA expression was detected within occlusive plexogenic lesions, indicating the presence of EC and VSMC markers. Bar=50 μm.

Figure 4

VEGF is expressed in alveoli and blood vessel wall of MCT^+/+ rat lung. In plexogenic lesions in MCT^sl/sl lungs, staining of the proangiogenic factor VEGF is noted. Immunohistochemical staining of MCT^+/+ and MCT^sl/sl rat lung for PCNA revealed PCNA-positive cells in EC layer of MCT^+/+ group, whereas distribution of PCNA-positive cells in MCT^sl/sl group was more widespread, being detected in the adventitial cell, medial cell, and EC layers. Bar=50 μm.

Figure 5

A, In control (+/+) rats, extracellular TN-C protein was deposited throughout the lung parenchyma, as well as in smooth muscle of large and small vessels and in smooth muscle layer of airways. In untreated sl/sl lung, smooth muscle TN-C positivity increased in all tissue compartments and was especially prominent in the large airway smooth muscle layer. In addition, TN-C positivity was apparent beneath the endothelium and in the thickened parenchyma, which showed highly heterogenous TN-C staining. In MCT-treated control rats, TN-C was deposited throughout the parenchyma, beneath the airways, and in smooth layers of pulmonary- and bronchiole-associated blood vessels. Submucosal positivity was also apparent. In sl/sl rats treated with MCT, expression of TN-C was greatly increased in blood vessels, including occluded small-resistance vessels (inset). B, Whole lung TN-C was increased in sl/sl lungs without MCT, suggesting a predisposition to pulmonary arterial hypertension, but was not significantly increased after MCT. NORM indicates normoxic. Bar=50 μm.

Figure 6

Gelatin substrate zymography reveals that MMP-9 activity was increased >4-fold in MCT^sl/sl lungs compared with MCT^+/+ lungs, whereas MMP-2 activity was not different between groups. Note that MMP-2 and MMP-9 are detected as both a higher-molecular-weight proform and a lower-molecular-weight active form. STD indicates recombinant MMP-9.

Figure 7

Northern blot analysis shows that MCT^sl/sl animals have a 65±10% decrease in steady state levels of ET_B receptor mRNA and a 50±6% increase in ET_A receptor mRNA levels. In contrast, expression of mRNAs for pre-proET-1 (ppET-1) and ECE-1 did not change.