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Rationale: Our previous studies found that Na+/H+ exchanger (NHE) activity played an essential role in pulmonary artery smooth muscle cell (PASMC) proliferation and in the development of hypoxia-induced pulmonary hypertension and vascular remodeling. Other investigators recently observed increased expression of the NHE isoform 1 (NHE1) gene in rodents with pulmonary hypertension induced by hypoxia. However, a causal role for the NHE1 gene in pulmonary hypertension has not been determined.
Objectives: To determine the causal role of the NHE1 gene in pulmonary hypertension and vascular remodeling.
Methods: We used NHE1-null mice to define the role of the NHE1 gene in the development of pulmonary hypertension and remodeling induced by hypoxia and to delineate the NHE1 regulatory pathway.
Measurements and Main Results: After 2 weeks of exposure to hypoxia, in contrast to wild-type hypoxic littermates, there was no significant increase in right ventricular systolic pressure, in the ratio of right ventricular to left ventricular plus septal weight [RV/(LV + S)], or in medial wall thickness of the pulmonary arterioles in homozygous mice (NHE1−/−). There was a significant decrease in Rho kinase (ROCK1 and ROCK2) expression, accompanied by an increase in p27 expression in NHE1−/− mice.
Conclusions: Our study demonstrated that deficiency of the NHE1 gene prevented the development of hypoxia-induced pulmonary hypertension and vascular remodeling in mice and revealed a novel regulatory pathway associated with NHE1 signaling.
Na+/H+ exchanger (NHE) activity plays an essential role in pulmonary artery smooth muscle cell proliferation and development of hypoxia-induced pulmonary hypertension and vascular remodeling, but a causal role for the NHE gene in pulmonary hypertension has not been determined.
We demonstrated that the NHE1 gene is a key determinant in development of hypoxia-induced pulmonary hypertension and vascular remodeling and revealed a novel regulatory pathway for NHE1.
The Na+/H+ exchanger (NHE) expressed in many mammalian cell types is a protein localized to the plasma and the mitochondrial inner membrane (1). The function of NHE is to regulate intracellular pH and cell volume by extruding H+ from and taking up Na+ into cells (2). Since NHE was first analyzed in 1976 (3) and the first NHE cDNA was cloned in 1989 (4), nine NHE isoforms have been identified and cloned (2), of which NHE isoform 1 (NHE1) is ubiquitously expressed.
Pulmonary hypertension caused by many chronic lung diseases associated with prolonged hypoxia can result in right ventricular hypertrophy and heart failure (5). An important pathological feature of pulmonary hypertension is increased medial thickening of the pulmonary artery resulting from hypertrophy and hyperplasia of the pulmonary artery smooth muscle cells (PASMCs) (6, 7). In previous studies, we reported that NHE activity played a significant role in regulation of intracellular pH in PASMCs (8) and found that the inhibition of NHE activity was related to the inhibition of bovine PASMC proliferation induced by growth factors (9, 10). We also found that inhibition of NHE activity significantly reduced pulmonary hypertension and vascular remodeling induced by chronic hypoxia in rats (11). Our studies suggested that NHE activity might play an important role in development of pulmonary hypertension and vascular remodeling.
Recently, increased NHE1 gene expression was observed in the lung in hypoxia- induced pulmonary hypertension in rats (12). Rios and colleagues investigated NHE activity and NHE gene expression in hypoxic animals and found an increase in NHE activity and NHE1 mRNA and protein expression in PASMCs isolated from the intrapulmonary arteries of mice with hypoxia-induced pulmonary hypertension (13, 14). These findings suggested that the NHE1 gene might contribute to the development of hypoxia-induced pulmonary hypertension. However, the causal role of NHE1 gene in pulmonary hypertension and vascular remodeling has not been fully defined.
Pederson has shown an involvement of NHE1 in stress-induced cell signaling regulation (15). However, the signaling pathway that NHE1 uses to regulate PASMC proliferation and pulmonary hypertension is unclear. We previously have determined that p27, a cyclin-dependent kinase inhibitor, played a critical role in inhibition of PASMC proliferation and pulmonary hypertension induced by hypoxia (16). Several studies have indicated that RhoA kinase (ROCK) was an upstream factor of p27 that regulated p27 activity by degrading p27 protein (17–19), although some reports have suggested that p27 regulates RhoA/ROCK (20, 21). We therefore also investigated RhoA/ROCK expression in NHE1−/− mice to clarify the regulatory pathway between NHE1 and RhoA/ROCK in hypoxia-induced pulmonary hypertension.
Animal experiments were approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital. The NHE1 homozygous (NHE1−/−), heterozygous (NHE1+/−), and wild-type (NHE1+/+) male mice were bred from three breeding pairs of heterozygous NHE1 mice (mixed genetic background of C57BL/6 and Black Swiss strains; kind gifts of Dr. Gary Shull, University of Cincinnati College of Medicine, Cincinnati, OH). The offspring genotype of the mice was determined by polymerase chain reaction (PCR) analysis of genomic DNA from tail biopsies (22) (Figure 1).
Hypoxia exposure was performed as previously described (16). Briefly, the mice (8- to 10-week-old littermates) were weighed and placed in a tightly sealed hypoxic chamber or exposed to normoxia in the same chamber for 2 weeks. Oxygen concentration was maintained at 10% by controlling the flow rates of compressed air and N2. Cage concentration of O2 was checked daily.
The measurement for right ventricular pressure was performed as previously described (16). After 14 days in the chamber, the animals were removed and anesthetized with intraperitoneal ketamine (80 mg/kg) and diazepam (5 mg/kg). Animals were placed on a warming blanket to maintain body temperature at 37°C. Right ventricular systolic pressure (RVSP) was measured via a catheter (0.012" × 0.021" silicone tubing) passed through the right external jugular vein. Once the RVSP was obtained, the animals were killed with 200 mg/kg of pentobarbital and used immediately for the determination of right ventricular hypertrophy, hematocrit, lung pathology, and gene expression.
The ventricles and septum of the animals were collected, and the wet and dry ventricle and septal weight was obtained by drying them for 24 hours at 60°C. A ratio of right ventricle to left ventricle plus septum weight [RV/(LV+S)] was calculated for determination of right ventricular hypertrophy.
Blood samples were centrifuged in microcapillary tubes for 3 minutes, and the hematocrit was read directly.
Elastic fibers in pulmonary arteries were stained for measurement of medial wall thickness of pulmonary arteries. Percent wall thickness (%WT) and percent thick-walled vessels were used for evaluation of pulmonary artery remodeling as previously described (16). %WT was landmarked to terminal bronchial arterioles (%WT-TA) and the intraacinous arterioles (%WT-IA). It was calculated as the average diameter of the external elastic lamina minus the average diameter of internal elastic lamina divided by the average diameter of external elastic lamina. Percent thickness was calculated as percentage of thick-walled vessels as a fraction of total intraacinous vessels. A computer imaging analysis was applied for measurement of wall thickness. Images of individual pulmonary arteries were captured using a digital camera, mounted on a light microscope, and linked to a computer. The detail on measurement of wall thickness was described previously (11, 23).
Immunohistochemical staining with anti–α-smooth muscle–specific actin antibody (mouse monoclonal; Calbiochem, San Diego, CA) was performed for additional evaluation of pulmonary vascular remodeling. A modified vessel category of muscularization was used (24, 25). Muscularized vessels were defined as presence of smooth muscle cells stained brown with anti–α-smooth muscle–specific actin in 50% or more of the vessel circumference of intraacinous arterioles (24). Muscularized vessel number were quantified and expressed per 100 alveoli (25).
Immunohistochemical staining with Ki67, a cell proliferation marker, was performed to analyze cell proliferation in the medial wall of pulmonary arterioles as described previously (16). A percentage of Ki-67–positive cells expressed as Ki67 proliferative index was estimated by calculating the ratio of Ki67 expressing cell nuclei to the total number of cell nuclei in the vessel wall of cross-sections of 10 terminal bronchial arterioles per slide.
Pulmonary artery and PASMCs were used for further biological analysis. Pulmonary arteries, including main pulmonary and left and right branches, were isolated under the anatomic microscope. NHE1 mouse PASMC isolation and culture were performed according to a method described previously (14). The PASMCs were characterized by the presence of anti–α-smooth muscle–specific actin antibody.
Total RNA (2 μg), extracted from mouse lungs, pulmonary arteries, and isolated PASMCs, was used to carry out reverse transcription (RT)–PCR to measure mRNA expression with Ready-To-Go You-Prime First-strand beads for RT (GE Healthcare, Buckinghamshire, UK) and PCR Master Mix for PCR (Promega, Madson, WI). Primer pairs for RhoA, ROCK1 and ROCK2 (26), p27 (16), cyclin D1 (27), and for the housekeeping gene GAPDH were purchased from Sigma Genosys (Woodlands, TX).
Total cell lysates were obtained from mouse lungs. Antibodies included RhoA, cyclin D1, MYPT1 (myosin phosphatase target subunit 1), total MYPT1 and phospho-MYPT1 (Thr696) (Santa Cruz Biotechnology, Santa Cruz, CA), ROCK1 (ROCK-I/ROKβ), ROCK2 (ROCK-II/ROKα), p27 mouse monoclonal antibody (clone 57) (BD Biosciences Pharmingen, San Diego, CA), and GAPDH mouse monoclonal antibody (clone 6C5) (Research Diagnostics, Inc., Flanders, NJ). Western blot was performed as described previously (28).
Statistics were performed using the computer program Statview (SAS Institute, Inc., Cary, NC) with factorial analysis of variance. All values were expressed as the mean ± SEM. Significance was set at P < 0.05.
During normoxia, NHE1+/+, NHE1+/−, and NHE1−/− mice had similar hemodynamic parameters. After 2 weeks exposure to hypoxia, NHE1+/+ mice developed pulmonary hypertension with a significant increase in RVSP as compared with their normoxic littermates; in hypoxic NHE1+/− mice, the RVSP value was significantly lower than in the hypoxic NHE1+/+ mice, although the pressure was still higher than that in the hypoxic NHE1−/− mice and the normoxic littermates. There was no significant increase in RVSP observed in NHE1−/− hypoxic mice when compared with normoxic mice (Figures 2A and 2B).
Two weeks of hypoxic exposure significantly induced right ventricular hypertrophy in NHE1+/+ and NHE1+/− mice as indicated by the increase in the ratio of right ventricular weight to left ventricular plus septum weight, RV/(LV + S), but the ratio was significantly smaller in NHE1+/− mice than that in NHE1+/+ mice. There was not a significant increase in the ratio of RV/(LV + S) in NHE1−/− hypoxic mice (Figure 2C) when compared with normoxic mice. NHE1−/− mice also did not show any difference in RV/(LV + S) from NHE1+/+ mice in normoxia.
Hypoxia significantly increased hematocrit values in all NHE1 mice as compared with their normoxic littermates. No significant difference was observed between NHE1+/+, NHE1+/−, and NHE1−/− mice (Figure 2D).
In NHE1+/+ mice, the medial wall thickness of pulmonary arterioles at the level of the terminal bronchioles (%WT-TA) and at the level of the intraacinous arterioles (%WT-IA) as well as the percent thick, were significantly increased after 2 weeks exposure to hypoxia. In NHE1+/− mice, there was an increase in %WT-TA but significantly less than that in NHE1+/+ mice. No significant increase in %WT-IA was seen in NHE1+/− mice, and there was a significantly lower %WT-IA in hypoxic NHE1+/− mice than that in hypoxic NHE1+/+ mice. Consistent with the lack of rise in RVSP and RV/(LV + S) in NHE1−/− mice, there was no significant change in pulmonary vascular remodeling (Figure 3A–3C).
Hypoxia significantly increased muscularized vessel number in NHE1+/+ mouse lungs as compared with their normoxic littermates, but no significant change was seen in NHE1−/− mice. Although increased vascular muscularization was observed in hypoxic NHE1+/− mice when compared with their normoxic littermates, the increase was significantly less than that in hypoxic NHE+/+ mice (Figures 4A and 4B).
Consistent with the results we observed, exposure to hypoxia significantly increased Ki67 proliferative index in NHE1+/+ and NHE1+/− mice, but NHE1+/− mice had significant lower index than NHE1+/+ mice. We did not observe an increase in Ki67 proliferative index in hypoxic NHE1−/− mice (Figures 4C and 4D).
To determine whether RhoA/ROCK were altered along with the failure to develop pulmonary hypertension in hypoxia by NHE1 gene knockout mice, we analyzed mRNA and protein expression of RhoA, ROCK1, and ROCK2 in the mouse lungs. We found that the expression of ROCK1 and ROCK2 mRNA and protein were significantly down-regulated in the lungs from hypoxic and normoxic NHE1−/− mice (Figures 5A–5C). Hypoxia increased RhoA mRNA expression in NHE1+/+but not in NHE1−/− mice (Figure 5A).
Because a significant decrease in ROCK was found in NHE1−/− mouse lung, we isolated pulmonary artery and PASMCs from NHE1-null mice for further analysis of ROCK expression. We confirmed the significant down-regulation of ROCK1 and ROCK2 in the pulmonary arteries (Figures 5D and 5E) and PASMCs (Figures 5F and 5G) from NHE1−/− mice as compared with NHE1+/+ mice.
To examine if the reduction of ROCK expression by NHE1 gene knockout affected ROCK activity, we measured MYPT1, a direct downstream effector of ROCK. We found that ROCK activity was significantly decreased, showing an increase in phospho-MYPT1 in the PASMCs from NHE1+/+ mice (Figure 6A).
To determine if the down-regulation of ROCKs by NHE1 knockout occurred in other organs, we measured RhoA and ROCKs in heart, liver, and kidney in NHE1−/− animals. Similar to the lungs, a significant decrease in expression of ROCKs, but not of RhoA, was observed in the three tissues (Figure 6B).
Because p27 is a downstream factor for ROCK and plays an important role in regulation of cell proliferation, we analyzed p27 and its downstream factor, cyclin D1. A significant increase in p27 expression (Figures 7A–7C) and a decrease in cyclin D1 expression (Figures 7D–7F) were observed in the lungs, pulmonary arteries, and PASMCs from NHE1-null mice.
Mice with homozygous deficiency for the NHE1 gene (see Figure 1) did not respond to chronic hypoxia with a rise in RVSP (see Figures 2A and 2B), right ventricular hypertrophy (see Figure 2C), or pulmonary vascular remodeling (see Figures 3A–3C) compared with hypoxic wild-type littermates. Heterozygous NHE1-deficient mice made a modest but significant response to hypoxia, although much less than wild-type littermate hypoxic control mice. ROCK1 and 2, which have been shown to be part of the signaling cascade through p27 and cyclin D for the proliferation of normoxic vascular smooth muscle cells, had a significantly reduced expression in lung (see Figures 5A–5C) and pulmonary artery (see Figures 5D and 5E) and PASMCs (see Figures 5F and 5G), which was accompanied by a decrease in ROCK activity (see Figure 6A) and an increase in p27 (Figures 7A–7C) and a decrease in cyclin D1 (Figures 7D and 7F).
Abnormal growth of smooth muscle cells in the medial wall of pulmonary arterioles is associated with an increase in pulmonary artery pressure and pulmonary vascular remodeling. NHE1 controls intracellular pH and cell volume by extruding H+ from and taking up Na+ into cells. This function seems to play an important role in regulating vascular smooth muscle cell proliferation (29). A wide range of mitogenic factors can stimulate cell proliferation and activate NHE (30), including platelet-derived growth factor (PDGF) (9, 31) and epidermal growth factor (EGF) (9, 32, 33). PDGF is one of the growth factors released by endothelial cells exposed to hypoxia (6, 23). Our previous study found that in cultured bovine PASMCs, PDGF increased NHE activity, causing a rise in intracellular pH, which was associated with stimulation of cell proliferation. We also found in PASMCs that inhibition of NHE activity with the inhibitor dimethyl amiloride was associated with the loss of intracellular alkalization and of proliferation in response to growth factors such as PDGF (11). We found that the NHE inhibitors dimethyl amiloride and ethylisopropyl amiloride (EIPA) significantly reduced pulmonary artery pressure and total pulmonary vascular resistance index and pulmonary vascular remodeling in rats exposed to chronic hypoxia (11). Rios and colleagues observed that PASMCs isolated from chronically hypoxic mice exhibited elevated basal intracellular pH value and increased NHE activity and expression of NHE1 mRNA and protein (13). Yao and colleagues also reported elevated intracellular pH and increased expression of NHE1 mRNA in PASMCs isolated from chronically hypoxic rats (12). These studies and others showing a relationship between NHE activity and PASMC proliferation (8–10, 13, 14) support our hypothesis that NHE, and in particular NHE1, plays a major role in the development of hypoxia-induced pulmonary artery hypertension and pulmonary vascular remodeling.
In this study, we observed that knockout of the NHE1 gene not only protected mice against hypoxia-induced pulmonary hypertension and vascular remodeling but also inhibited cell proliferation in the pulmonary artery medial wall as assessed by the Ki67 proliferative index consistent with our previous work showing that heparin can inhibit Na+/H+ exchange (8, 9) and inhibit cell proliferation in the pulmonary vascular medial wall in p27+/+ mice (16). However, the exact mechanism by which loss of the NHE1 gene inhibits cell proliferation is not clear. Wu and colleagues recently reported that a new NHE inhibitor, sabiporide, inhibited human PASMC proliferation and DNA synthesis and arrested the cell at the G0/G1 phase. Their finding demonstrated that NHE activation plays a permissive role in entrance of cells into cell cycle, suggesting that the NHE1 gene might be mediating cell proliferation through cell cycle regulating genes (34).
Mitogen-induced activation of NHE1 has been reported to involve Janus kinase 2 in rat vascular smooth muscle cells (35), and the inhibitory effect of NHE1 on cardiomyocyte hypertrophy has been associated with reduction of p38 and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen-activated protein kinases (MAPKs) activation (36). An early study (37) showed that p38 and ERK1/2, but not Jun N-terminal kinase (JNK), were involved in the regulation of NHE1 activity mediated by angiotension II in vascular smooth muscle cells. A recent study observed that full expression of HIF1 was essential for hypoxic induction of NHE1 expression and change in PASMC pH homeostasis (13). In addition, the effect of NHE1 on cell proliferation regulation involved the MAPK, protein kinase B (Akt/PKB) pathway (14). The full pathway for NHE1 signaling in the regulation of cell proliferation has not been determined.
The effects of RhoA/ROCK on mediating NHE1 activity have been reported. NHE1 has been shown to act as a downstream effecter of RhoA/ROCK (38–44). Tominaga and Barber (41) found that NHE1 activity was necessary for RhoA-induced formation of actin-stress fibers. It was observed that a low concentration of serum increased NHE1 activity by decreasing RhoA and increasing Rac in human mammary epithelial cancer cell line (43). Cardone found that NHE1 activity was regulated via PKA/RhoA/ROCK/p38MAPK signaling pathway (44). In the present study, we found that deficiency of NHE1 gene in mice decreased ROCK expression not only in PASMCs and pulmonary artery but also in lung and other organs. We also found that the decrease in ROCK expression was accompanied by an increase in phospho-MYPT1. This result demonstrated that reduction of ROCK expression affected its activity because ROCK can phosphorylate MYPT1 of smooth muscle myosin phosphatase, resulting in the inhibition of MYPT1 activity (45). We also observed that ROCK down-regulation in NHE1-null mice was associated with increased p27 expression and decreased cyclin D1. This novel discovery suggested that NHE1 gene might act upstream of RhoA/ROCK in regulating PASMC proliferation and pulmonary hypertension by directly inhibiting ROCK gene expression. Putney and Barber (46) investigated gene expression in NHE1-null mouse fibroblasts by using a cDNA microarray. They observed changes in some genes, including cell proliferation regulating genes, but they did not report on RhoA/ROCK.
The best way to reflect gene expression for pulmonary hypertension is to use distal pulmonary arterioles or isolate PASMCs from the distal vessels because the most significant morphological changes in pulmonary lung during pulmonary hypertension occur in the distal arterioles. However, we have not found a way to obtain distal pulmonary arterioles or PASMCs from the arterioles from mice. Therefore, we used main and branch pulmonary arterioles and the PASMCs isolated from the vessels in this study.
We demonstrated a decrease in ROCK1 and ROCK2 but not RhoA in cardiac muscle of our NHE−/− mice (see Figure 6B). Sugiyama and colleagues have shown that Y-27632, an inhibitor of RhoA, mildly decreased myocardial contractibility in the isolated perfused dog heart (47). Inhibition of Rho/ROCK diabetic rats has been shown to enhance contractibility in the isolated working heart (48). These constitutive Rho/ROCK may influence cardiac contractibility and their cardiac output. We doubt, though, that the cardiac output in our NHE1−/− mice was an important factor in their response to 10% O2 because baseline RVSP and pulmonary architecture was similar, as were the cardiac weights in our NHE1+/+ mice and the NHE1−/− mice, suggesting baseline cardiac outputs were similar. We have previously shown in guinea pigs and rats (49, 50) that 10% O2 did not change cardiac output. We therefore suspect a low cardiac output did contribute to the failure of the NHE−/− mice to develop pulmonary hypertension in chronic hypoxia.
In conclusion, in the present study, we found that the NHE1 gene knockout resulted in the loss of chronic hypoxia–induced pulmonary hypertension and vascular remodeling in mice. This result has demonstrated that the NHE1 gene is a key determinant in the development of pulmonary hypertension and vascular remodeling induced by hypoxia. In addition, in this study, we found that the NHE1 gene knockout significantly knocked down ROCKs and increased p27. This finding suggested that the inhibition of hypoxic pulmonary hypertension by NHE1 gene deficiency was associated with direct down-regulation of ROCK, which may provide a novel regulatory pathway for NHE1.
Supported by National Institutes of Health grant HL039150 (C.A.H.).
Originally Published in Press as DOI: 10.1164/rccm.200710-1522OC on February 28, 2008
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.