In this study, we found that the silencing of NHE1 by siRNA significantly inhibited the proliferation of human PASMCs () and the cell-cycle progression induced by hypoxia (). The inhibitory effect of NHE1 siRNA was blocked by overexpressing E2F1 (). We found that NHE1 siRNA attenuated the hypertrophy of human PASMCs () and the migration induced by hypoxia (), and that the overexpression of E2F1 prevented these attenuations. NHE1 siRNA also decreased the hypoxia-induced proliferation of human PAECs and PAFs ().
The proliferation of PASMCs is considered an important pathological change in the development of the pulmonary hypertension and vascular remodeling induced by hypoxia. In previous studies, we reported that the NHE inhibitors dimethyl amiloride and 5-ethylisopropyl amiloride significantly inhibited the growth of bovine PASMCs (6
) in vitro
, and inhibited hypoxia-induced pulmonary hypertension in animals (7
). However, those NHE inhibitors were not NHE1-specific, but also affected NHE2 and NHE3 (5
). Wu and colleagues reported that a new NHE1 inhibitor, sabiporide, significantly inhibited the proliferation of human PASMCs and migration under normoxia (37
). However, that inhibitor also affected other NHEs (5
). Therefore, the role of NHE1 in the regulation of PASMC proliferation has not been specifically investigated. We recently found that NHE1 gene knockout prevented hypoxia-induced pulmonary hypertension and vascular remodeling in mice, accompanied by a significant decrease in the proliferation of PASMCs (11
) and indicating specific effects of the NHE1 gene in regulating the proliferation of PASMCs. In the present study, we used NHE1-specific siRNA to silence NHE1 in human PASMCs under normoxia and hypoxia, and found that silencing NHE1 not only inhibited the proliferation of PASMCs, but also reduced cell-cycle progression. These results demonstrate that NHE1 is a critical regulator of the hypoxia-induced proliferation of PASMCs.
Zhang and colleagues reported a significant increase in the inhibition of NHE1 expression by using two sets of NHE1 siRNA for one transfection (38
). To obtain the maximal silencing of the NHE1 gene in PASMCs, we simultaneously transfected two sets of NHE1 siRNA into human PASMCs. However, we did not observe a significantly increased inhibition of PASMC proliferation under normoxia or hypoxia, although a slightly greater inhibition of cell growth was evident in the group with two sets of NHE1 siRNA.
NHE1 has been studied for years, but the mechanism by which NHE1 mediates the proliferation of PASMCs is not well-understood. We previously found that NHE1 gene knockout significantly decreased the expression of Rho-associated, coiled-coil containing protein kinase 1 or Rho-associated protein kinase 1 (ROCK1) and ROCK2, which was accompanied by the significantly increased expression of p27 and decreased expression of cyclin D1 (11
). Because E2F1 is a downstream factor of p27 and cyclin D1, we hypothesized that E2F1 plays an important role in the inhibition by NHE1 siRNA of human PASMC proliferation. The E2F family of transcription factors, composed of eight different members, regulates cell-cycle progression (12
). E2F1 is one of these members and is essential for cellular proliferation. E2F1 functions to activate nuclear transcription factors and promote cell-cycle progression, after its release from the Rb/E2F1 complex (12
). Bindra and colleagues (40
) reported that E2Fs may be linked to the transcriptional response to hypoxia. Previous studies demonstrated the involvement of E2F1 in cell proliferation (13
). Tammali and colleagues (13
) found that the inhibition of high glucose–induced and TNF-α–induced rat aorta vascular smooth muscle cell proliferation by aldose reductase was accompanied by the decreased expression of E2F1. Grouwels and colleagues (14
) found that the overexpression of E2F1 by an adenovirus E2F1 promoter increased the proliferation of primary β-cells isolated from the pancreas of rats. Amrani and colleaues (15
) reported that interferon-γ inhibited the proliferation of airway smooth muscle cells by inhibiting the expression of the E2F1 gene. Goukassian and colleagues (16
) reported that the overexpression of E2F1 negated the inhibitory effects of a potent repressor of cellular proliferation, PCA-4230, on the proliferation of human vascular smooth muscle cells. In this study, we found that the expression of E2F1 was induced in PASMCs under hypoxia, and that NHE1 siRNA significantly decreased the induction of EF21 in PASMCs. We also found that the overexpression of E2F1 completely blocked the inhibitory effects of NHE1 siRNA on the hypoxia-induced proliferation of PASMCs. These results indicate that E2F1 signaling is essential for the regulation by NHE1 of PASMC proliferation.
In addition to proliferation, hypertrophy is another pathological characteristic of PASMCs in pulmonary hypertension (41
). Studies indicated the relationship between NHE1 and myocardial hypertrophy, showing that the inhibition of NHE1 activity by inhibitors reduced cardiac hypertrophy in animals and cultured cardiomyocytes (23
). The increased medial wall thickness of pulmonary arteries in pulmonary hypertension and vascular remodeling is involved not only in the proliferation of PASMCs, but also in the hypertrophy of PASMCs. In our previous study, we found a decrease in medial wall thickness of the pulmonary arteries in NHE1-deficient mice. We therefore investigated the effects of NHE1 on the hypertrophy of PASMCs in this study. We found that silencing the NHE1 gene significantly decreased the hypoxia-increased size of human PASMCs. In addition to cell size, cell volume, established by calculating the protein/DNA ratio, was used for the analysis of cell hypertrophy (39
), because research indicates that the stimulation of protein synthesis without DNA replication is a feature of hypertrophy (33
). We found that silencing NHE1 significantly decreased the protein/DNA ratio in human PASMCs cultured under hypoxia. E2F1 was shown to be involved in the pathogenesis of cardiac hypertrophy. Wohlschlaeger and colleagues (20
) reported a significant increase in the expression of E2F1 in myocardial tissues from patients with chronic hear failure–associated cardiomyocyte hypertrophy. Hlaing and colleagues (21
) found that E2F1 regulated the hypertrophy of skeletal myoblasts induced by angiotensin II. Vara and colleagues (22
) showed that the inhibition of E2F1 function prevented the development of myocyte hypertrophy in rats. In this study, we found that the overexpression of E2F1 also blocked the inhibition by NHE1 siRNA of PASMC hypertrophy, indicating the involvement of E2F1 in the regulation by NHE1 of PASMC hypertrophy.
In addition to proliferation and hypertrophy, the migration of PASMCs is also involved in the vascular remodeling underlying pulmonary hypertension. The extension of PASMCs into nonmuscularized vessels suggests the involvement of smooth muscle cell migration in the pathogenesis of pulmonary hypertension (28
). The migration of PASMCs was reportedly induced by sustained hypoxia (29
), endothelin-1 (30
), serotonin (28
), and other factors (31
). NHE1 is reportedly involved in regulating the migration of different types of cells (40
), and the NHE1 inhibitor sabiporide also inhibited the migration of human PASMCs under normoxia (38
). In this study, we found that silencing NHE1 siRNA significantly diminished the migration of PASMCs induced by hypoxia. Interestingly, we also found that the overexpression of the E2F1 gene prevented the inhibition by NHE1 siRNA of human PASMC migration.
The effects of hypoxia on the proliferation of PASMCs were studied in different laboratories, and inconsistent results were reported (44
). To determine the best concentration of oxygen that stimulates the proliferation of human PASMCs, we investigated the effects of different levels of oxygen (1, 2, 3, 5, and 10% O2
) on the proliferation of PASMCs. We found that 1, 2, and 3% oxygen significantly stimulated the proliferation of PASMCs. Although some investigators obtained controversial results (44
), we did not find a significantly increased growth of PASMCs under 5 and 10% oxygen. This result was consistent with the data we obtained in another study (32
). The mechanisms underlying the reactions of PASMCs to different oxygen concentrations have not been fully clarified. One report stated that cells in the medial wall of the artery normally exist under conditions of low oxygen (45
). Santilli and colleagues (45
) measured the concentration of transarterial wall oxygen in rabbit aortas by using an oxygen microelectrode, and found the lowest oxygen concentrations in the medial wall of the artery, compared with the adventitia and lumen. The oxygen concentration was only approximately 4% in the center of the media, but at the same time, oxygen concentrations were around 12% in the lumen and 8% in the adventitia. Because 10% oxygen was widely used to induce pulmonary hypertension and vascular remodeling in rodents, then based on the findings of Santilli and colleagues (45
), the oxygen concentration would be lower than 5 or 10% in the media of the pulmonary artery when animals were placed in a hypoxia chamber (10% O2
), which resulted in the proliferation and hypertrophy of smooth muscle cells in the medial walls of pulmonary arteries. Thus, a slight reduction of oxygen concentrations to 5 or 10% may not be enough to stimulate the proliferation of PASMCs, because cells in the medial walls of the artery were already under a low oxygen condition. This may explain why investigators generally use 1–3% oxygen for studies related to the proliferation of PASMCs (44
Moreover, we investigated whether the regulation by E2F1 of NHE is NHE1-specific. We did not detect the expression of NHE2 in human PASMCs. Although the expression of NHE3 was evident in PASMCs, the proliferation of cells was not significantly affected by either the NHE3 inhibitor S-3226 or NHE3 siRNA. Previous reports showed that no expression of NHE2 and NHE3 was evident in human lung tissue (48
) and pulmonary endothelial cells (49
). Therefore, we did not investigate the effects of E2F1 on NHE2 and NHE3 in this study.
In conclusion, we demonstrated that the specific inhibition of the NHE1 gene by siRNA significantly inhibited the hypoxia-induced proliferation, hypertrophy, and migration of human PASMCs via the repression of nuclear transcription factor E2F1. This observation revealed a novel mechanism underlying the regulation by NHE1 of hypoxic pulmonary hypertension and vascular remodeling.