Published online May 10, 2019. doi: 10.5494/wjh.v9.i2.17
Peer-review started: October 12, 2018
First decision: December 18, 2019
Revised: January 8, 2019
Accepted: March 12, 2019
Article in press: March 12, 2019
Published online: May 10, 2019
A number of cardiopulmonary and other systemic diseases are known to lead to pulmonary arterial hypertension (PAH), a serious sequel with a high mortality rate. The features of PAH are: impaired vascular relaxation, activation of proliferative pathways; smooth muscle cells (SMC) proliferation, medial hypertrophy, narrowing of the lumen, elevated pulmonary artery pressure and right ventricular hypertrophy (RVH). As the disease progresses, neointima is formed which leads to the irreversibility of PAH. Despite major advances in the field, the cure is not yet available. The diagnosis of PAH is often delayed because of the vague symptoms. This is not surprising because we have previously shown in the monocrotaline (MCT) model of pulmonary hypertension (PH), endothelial damage, progressive loss of endothelial caveolin-1 (a membrane protein) and the activation of proliferative pathways occur before the onset of PH.
By exposing the MCT-treated rats to hypoxia, the disease process is accelerated and by 4 weeks, neointimal lesions are seen, which gives us an excellent opportunity to examine the mechanism of neointima formation. If we can unravel the mechanism of neointima formation, it will allow us to expand the experimental studies to further the knowledge which will aid us in designing curative treatment.
The main objective of the study was to examine the mechanism of neointima formation which renders the diseases irreversible. Our most important observation is that the significant endothelial damage and loss of endothelial caveolin-1 is accompanied by loss of cavin-1 and caveolae, and “enhanced” expression of caveolin-1 in SMC, that is tyrosine phosphorylated. It is possible that these alterations are at least in part responsible for neointima formation.
We divided male Sprague-Dawley rats (age 6-8 wks) in 4 groups (6-8/per gr.): Gr. 1 Controls, Gr. 2 (received a single sc injection of MCT 40 mg/kg), Gr.3 (rats in the group were exposed to hypobaric hypoxia) and Gr. 4 (MCT-injected rats were exposed to hypobaric hypoxia starting on day 1). These rats were studied 4 weeks later. Hemodynamic data and RVH were assessed. In addition, we evaluated the histological changes in the pulmonary arteries and the expression of caveolin-1 using immunofluorescence technique. The expressions of caveolin-1, p-caveolin-1, caveolin-2, VE-cadherin (VE-Cad), Cavin-1 and p-Erk1/2 in the lungs were examined using western blot technique.
Significant loss of caveolin-2, VE-Cad, Cavin-1, indicative of significant endothelial damage was observed in MCT groups. In the MCT group, there was a significant loss of endothelial caveolin-1, and enhanced caveolin-1 expression in SMC in a few arteries; however, the total cav-1 expression in the lungs remained low. In MCT + hypoxia group, a significant loss of endothelial caveolin-1 was accompanied by enhanced expression of caveolin-1 in SMC in a greater number of arteries, which almost normalized the lung caveolin-1 levels. The loss of cavin-1 suggests that the caveolin-1 is not in caveolae, because cavin-1 is essential for the maintenance of caveolar curvature, and in addition, it stabilizes caveolin-1 in caveolae. Furthermore, caveolin-1 is tyrosine phosphorylated in the MCT and MCT + hypoxia groups. ERK activation is observed in all PH models including hypoxia-induced PH. Importantly, there is no loss of caveolin-1, caveolin-2, cavin-1 or VE-Cad in the hypoxia-induce PH.
Disruption of endothelial cells (EC) and progressive loss of endothelial caveolin-1 results in reciprocal activation of proliferative pathways leading to PH. Extensive damage/loss of EC exposes SMC to increased pressure and shear stress, leading to flattening of caveolae. In addition, caveolin-1 is tyrosine phosphorylated in the MCT and MCT + hypoxia groups. In cancer, loss of cavin-1 and tyrosine phosphorylation of caveolin-1 results in cell migration and metastasis. Thus, the alterations in the caveolin-1 expression in the MCT + hypoxia group may in part facilitate cell proliferation, cell migration and neointima formation. It is important to note, that hypoxia does not alter the expression of caveolin-1 and other membrane proteins; and there is no enhanced expression of caveolin-1 in SMC. However, despite the presence of endothelial caveolin-1, proliferative pathway is activated indicating that the endothelial caveolin-1 is dysfunctional in hypoxia-induced PH. There is no evidence for endothelial membrane disruption in the hypoxia model, which may explain the reported recovery of animals after removal from hypoxia.
The important observation in our study is that the disruption of EC coupled with endothelial caveolin-1 loss initiates pulmonary hypertensive changes and facilitates progression. Further studies are required to determine other factors that might facilitate the “enhanced” caveolin-1 expression in SMC in cell proliferation and migration. We will examine whether cavin-1 and caveolae can be restituted in SMC and normalize caveolin-1 expression, which may abrogate neointima formation.