Skip to main content
NCBI home page
Pulmonary Circulation logoLink to Pulmonary Circulation
. 2016 Sep;6(3):369–380. doi: 10.1086/687766

Sphingosine-1-phosphate is involved in the occlusive arteriopathy of pulmonary arterial hypertension

Salina Gairhe 1,2,✉, Sachindra R Joshi 2,3, Mrigendra M Bastola 2, Jared M McLendon 2,3, Masahiko Oka 1,2, Karen A Fagan 1,2, Ivan F McMurtry 1,2
PMCID: PMC5019090  PMID: 27683614

Abstract Abstract

Despite several advances in the pathobiology of pulmonary arterial hypertension (PAH), its pathogenesis is not completely understood. Current therapy improves symptoms but has disappointing effects on survival. Sphingosine-1-phosphate (S1P) is a lysophospholipid synthesized by sphingosine kinase 1 (SphK1) and SphK2. Considering the regulatory roles of S1P in several tissues leading to vasoconstriction, inflammation, proliferation, and fibrosis, we investigated whether S1P plays a role in the pathogenesis of PAH. To test this hypothesis, we used plasma samples and lung tissue from patients with idiopathic PAH (IPAH) and the Sugen5416/hypoxia/normoxia rat model of occlusive PAH. Our study revealed an increase in the plasma concentration of S1P in patients with IPAH and in early and late stages of PAH in rats. We observed increased expression of both SphK1 and SphK2 in the remodeled pulmonary arteries of patients with IPAH and PAH rats. Exogenous S1P stimulated the proliferation of cultured rat pulmonary arterial endothelial and smooth-muscle cells. We also found that 3 weeks of treatment of late-stage PAH rats with an SphK1 inhibitor reduced the increased plasma levels of S1P and the occlusive pulmonary arteriopathy. Although inhibition of SphK1 improved cardiac index and the total pulmonary artery resistance index, it did not reduce right ventricular systolic pressure or right ventricular hypertrophy. Our study supports that S1P is involved in the pathogenesis of occlusive arteriopathy in PAH and provides further evidence that S1P signaling may be a novel therapeutic target.

Keywords: occlusive lesions, S1P, pulmonary, cardiac

Pulmonary arterial hypertension (PAH) remains debilitating and deadly despite advanced and expensive medical treatment.1 Its pathogenic mechanisms have not been fully identified and therapeutically targeted. A limitation in the current treatment of patients with PAH is the inability of prostanoids, endothelin receptor blockers, phosphodiesterase inhibitors, or their various combinations to reverse the pulmonary arteriopathy.2,3 An effective strategy in the development of more efficacious therapy would be to identify the molecular determinants of the arterial wall remodeling.

Sphingosine-1-phosphate (S1P) is a biologically active lipid synthesized intracellularly by sphingosine kinase 1 (SphK1) and SphK2 and degraded by S1P lyase and various phosphatases. It is released by endothelial cells, red blood cells, activated platelets, and monocytes and has regulatory roles in several physiological and pathological processes.4,5 Physiological levels of circulating S1P are vasculoprotective, whereas abnormal activation of SphK/S1P signaling is associated with diseases such as cancer, fibrosis, diabetes, and hypertension.6-10 At high cellular or tissue levels, S1P is a proproliferative, antiapoptotic, promigratory, profibrotic, and proinflammatory signaling molecule.11 SphK activity and S1P production are stimulated by numerous signals, including growth factors, cytokines, mitogens, and G protein–coupled receptor agonists.12 S1P mediates diverse cellular effects by acting on 1 or more of its 5 G protein–coupled receptors, S1PR1 through S1PR5, and on certain intracellular targets.13

Exogenous S1P elicits Ca2+- and Rho kinase–dependent vasoconstriction in mouse and rat lungs via S1PR2 and S1PR4, respectively.14,15 There is also evidence that hypoxic pulmonary vasoconstriction in mice is mediated, at least partly, by SphK1-derived S1P acting on both S1PR2 and S1PR4.15 Chen et al.10 have recently found that the SphK1/S1P axis is activated in the lungs and pulmonary artery smooth-muscle cells (PASMCs) of patients with idiopathic PAH (IPAH) and in the lungs and pulmonary arteries of chronically hypoxic mice and rats. They further observed that S1P causes proliferation of IPAH PASMCs and is involved in the muscularization of pulmonary arteries in chronically hypoxic mice via ligation of S1PR2. In contrast to this evidence that S1P mediates the development of PAH, Haberberger et al.16 found that long-term exposure to an allergen exaggerated pulmonary arterial responsiveness and wall thickening in SphK1−/− mice compared with that in wild-type mice. Similarly, Tian et al.17 have recently proposed that pulmonary artery endothelial cell (PAEC) apoptosis and formation of occlusive pulmonary arterial lesions in the Sugen5416/athymic rat model of PAH is due to leukotriene B4–induced inhibition of SphK1/S1P/nitric oxide signaling.

Therefore, to gain more insight into the role of S1P in the pathogenesis of PAH, we investigated whether the plasma levels of S1P were increased in patients with IPAH and in early and late stages of occlusive PAH in Sugen5416/hypoxia/normoxia (Su/Hx/Nx)–exposed rats.18 We also examined the expression of SphK1 and SphK2 in the remodeled pulmonary arteries of human and rat PAH lungs and the effects of S1P on the proliferation of cultured rat PAECs and PASMCs. Additionally, we determined the effects of chronic treatment of late-stage PAH rats with an SphK1 inhibitor on the occlusive arteriopathy and cardiovascular function. Collectively, we report that SphK/S1P signaling contributes to the formation and maintenance of occlusive neointimal lesions and may be an additional molecular target for the treatment of IPAH.

Methods

Human blood and lung samples

All studies involving human samples were conducted after obtaining informed consent with protocol approval by the institutional review board of the Office of Regulatory Compliance, University of South Alabama. Whole-blood samples were collected from patients with IPAH and healthy controls (without any pulmonary or cardiovascular diseases) who were attending the 2012 Meeting of the Pulmonary Hypertension Association. Formalin-fixed, paraffin-embedded (FFPE) sections of lung tissues from patients with idiopathic PAH and healthy controls were obtained from Sébastien Bonnet and Steeve Provencher of the Pulmonary Hypertension Research Group of the Quebec Heart and Lung Institute, Quebec Research Center, Laval University, Quebec, Canada.

Animal model of severe occlusive PAH

All experimental procedures were conducted in accordance with the Animal Welfare Act and approved by the Institutional Animal Care and Use Committee of the University of South Alabama. Adult male Sprague-Dawley rats (∼250 g) were used. Severe occlusive PAH was induced in rats by a single subcutaneous injection (20 mg/kg) of the vascular endothelial growth factor (VEGF) receptor blocker Sugen-5416 (Cayman Chemical) and exposure to 3 weeks of normobaric hypoxia (10% O2) followed by reexposure to normoxia (21% O2) for an additional 10 weeks (Su/Hx/Nx PAH rats).18,19 Age-matched control rats were given vehicle alone (0.5% carboxy-methylcellulose sodium, 0.9% NaCl, 0.4% polysorbate, 0.9% benzyl alcohol in deionized water) and exposed to normoxia (21%) for the entire 13-week study. Peripheral venous blood was drawn from the tail vein at 4, 8, 10, and 12 weeks. The time points were chosen according to the progression of occlusive lesions during the development of severe occlusive PAH in Su/Hx/Nx rats as described by Toba et al.20

SphK1 inhibitor treatment

For chronic treatment, 10-week Su/Hx/Nx PAH rats were given the SphK1 inhibitor, SLP711122821 (SphynKx Therapeutics, Charlottesville, VA), at a dosage of 10 mg/kg administered intraperitoneally once daily for 3 weeks or vehicle 2% hydroxypropyl-β-cyclodextrin (HPCD). SphK1 inhibitor SLP7111228 was chosen because of its specificity (nanomolar range), selectivity (>200-fold), and in vivo stability (half-life of 4 hours).21 Echocardiography (see below) was performed to determine cardiac function in 10- and 12-week Su/Hx/Nx PAH rats before and after SphK1 inhibition. Twenty-four hours after the last inhibitor treatment, the rats were anesthetized and catheterized for right and left ventricular hemodynamic measurements (see below). After completion of hemodynamic measurements, blood samples were collected, and the animals were euthanized by an overdose of pentobarbital (150 mg/kg administered intravenously). The heart and lungs were collected. Histological changes in the pulmonary arteries were examined with hematoxylin and eosin staining. The degree of right ventricular hypertrophy was determined by calculating the right ventricular weight divided by the weight of the left ventricle plus septum (RV/LV+S).

Enzyme-linked immunosorbent assay (ELISA) for S1P

Plasma samples isolated from peripheral venous blood of healthy control patients and patients with IPAH were analyzed for S1P levels by an ELISA kit according to the manufacturer’s protocol (K-1900; Echelon Bioscience). Data were quantified using a standard curve of known concentrations of human S1P standards.

Liquid chromatography–mass spectrometry (LCMS)

Plasma levels of S1P in control rats; PAH rats at 4, 8, 10, 12, and 13 weeks; and PAH rats treated with SphK1 inhibitor for 3 weeks were measured by LCMS as described elsewhere.22

Histopathological and immunohistochemical studies

Histopathological and immunohistochemical procedures were performed as described elsewhere.23 Briefly, rat lungs were inflated at 20 cm H2O with 0.05% (w/v) agarose in 1% neutral buffered formalin and fixed in 10% neutral buffered formalin. Formalin-fixed lungs were paraffin embedded, and 5-µm-thick tissue sections were prepared. For histopathological analysis, the tissue was stained with hematoxylin and eosin.

For immunohistochemical staining of human and rat lung sections, FFPE sections were deparaffinized and rehydrated. Endogenous peroxide activity was blocked with hydrogen peroxide for 10 minutes. For antibody epitope retrieval, heated citrate buffer was applied for 30 minutes. The sections were then blocked with normal horse serum for 1 hour. Tissue sections were incubated overnight at 4°C with primary antibodies against SphK1 and SphK2 (Abgent; 1∶500). Sections were incubated with biotinylated secondary antibodies for 30 minutes, washed with phosphate-buffered saline, and incubated in ABC Reagent (Vector Labs) for another 30 minutes. Western blot analysis was performed to determine antibody selectivity. Rabbit immunoglobulin G isotype was used as a negative control. Diaminobenzidine, as a substrate for horseradish peroxidase, was used to identify antibody-labeled cells. Hematoxylin was used as a counterstain. Lung sections were analyzed by light microscopy.

Western blot analysis

Lung tissues were harvested from normal and Su/Hx/Nx PAH rats. Thirty micrograms of lung tissue protein was used to perform Western blot analysis for SphK1 and SphK2 (Abgent; 1∶1,000) as described elsewhere.24

Measurements of pulmonary and cardiac function in PAH rats

Echocardiography

Symptoms of PAH in rats were monitored by echocardiography at 4, 8, 10, and 12 weeks after induction of PAH. Echocardiography was performed using a Vevo770 imaging system (Visual Sonics, Toronto, Canada) as described elsewhere.20,25 Rats were anesthetized with <3% sevoflurane in O2. Adequate anesthesia was determined by checking for the lack of withdrawal reflex (toe pinch). Temperature, heart rate, and respiratory rate were continuously monitored. A measurement of the right ventricular inner diameter (RVID) in diastole was acquired for the assessment of right ventricular function as described elsewhere.20,25

Hemodynamic measurements

Rats were anesthetized with pentobarbital (30 mg/kg administered intraperitoneally), and anesthesia during surgery and catheterization was maintained by sevoflurane inhalation (1.0%–2.0%). Physiological body temperature was maintained with a heating pad. The level of anesthesia was monitored by toe pinch reflex. After an appropriate level of anesthesia, the right common carotid artery was catheterized with a 2.0F Millar Mikro-Tip pressure-volume catheter (SPR-838, Millar Instruments) for measurement of systemic arterial pressure. The catheter was then advanced into the left ventricle for measurement of cardiac output. Another catheter (polyvinyl-1, internal diameter of 0.28 mm) was threaded into the right ventricle through the right jugular vein and atrium to measure right ventricular systolic pressure (RVSP). The signals were continuously recorded by the Millar Pressure-Volume Systems–300 system with PowerLab/4SP, A/D converter (AD Instruments), and a personal computer. Cardiac index (CI) was calculated by dividing cardiac output by body weight, and total pulmonary artery resistance index (TPRI) was calculated by dividing RVSP by CI.

Morphometric assessment

Pulmonary arterial histology was quantified as described elsewhere.20,24 After hematoxylin and eosin staining, lung sections were assessed microscopically for occluded vessels. The quantitative analysis of luminal obstruction was measured in at least 30 randomly selected pulmonary arteries with outer diameter (OD) of <50 mm, 50–100 mm, and >100 mm per lung section from each rat. Vessels were graded as grade 0 for no evidence of luminal occlusion, grade 1 for partial luminal occlusion (<50%), and grade 2 for ≥50% luminal occlusion. The quantitative analysis of the luminal occlusion was performed by personnel unaware of the treatment groups.

Cell culture and treatment

To determine whether S1P induces proliferation of cultured rat PAECs and PASMCs, the cells were harvested from normal adult male Sprague-Dawley rats and cultured as described elsewhere.26 Both cell types were cultured in low-serum medium (Dulbecco’s modified Eagle medium [DMEM] plus 2% fetal bovine serum [FBS]) for 24 hours prior to drug treatment. Low-serum medium was replaced with growth-arrest medium (DMEM plus 0.1% FBS for PAECs and DMEM-F12 50∶50 for PASMCs) for 24 hours. PAECs and PASMCs were then treated with S1P (5 μM, Sigma), ethanol (0.1%) as a vehicle control, or left untreated for the next 24 and 48 hours of incubation at 37°C in air plus 5% CO2. Media with 10% serum was used as a positive control. The concentration of live cells and cell viability were determined using Countess Automated Cell Counter. Each experiment was independently repeated 3 times.

Data and statistical analyses

Data are reported as mean values (±SEM) and analyzed by paired or unpaired t test or one-way ANOVA with or without replication. Bonferroni post hoc test was used for simultaneous multiple comparisons. Pearson correlation coefficient (r) was used for the linear correlation analysis. Differences were considered significant at P < 0.05.

Results

Plasma S1P levels are elevated in patients with IPAH and Su/Hx/Nx PAH rats

To determine whether circulating levels of S1P were elevated in PAH, plasma S1P levels were measured in peripheral venous blood samples collected from patients with IPAH and healthy controls. Circulating levels of S1P were higher in the patients with IPAH (Fig. 1A). We next measured levels of S1P in the peripheral venous blood at 4, 8, 10, and 12 weeks of the disease progression in the Su/Hx/Nx PAH rats. Plasma S1P levels were elevated in the PAH rats from 4 to 12 weeks in comparison with normal controls (Fig. 1B). Similarly, levels of plasma sphingosine, a precursor of S1P, were also elevated at all time points in Su/Hx/Nx PAH rats (Fig. 1C).

Figure 1.

Figure 1

Open in a new tab

Plasma sphingosine-1-phosphate (S1P) level is elevated in pulmonary arterial hypertension (PAH). A, Plasma levels of S1P in human patients with idiopathic PAH (IPAH) and in healthy controls. B, Plasma levels of S1P in severe occlusive (Sugen5416/hypoxia/normoxia [Su/Hx/Nx]) PAH and normal control rats. C, Plasma levels of sphingosine, an S1P precursor, in Su/Hx/Nx PAH and control rats. D, Quantitative assessment of right ventricular internal diameter at diastole (RVID) from B-mode image in short-axis view of Su/Hx/Nx PAH and control rat hearts at the level of papillary muscles. E, Correlation analysis of RVID with the plasma levels of S1P at various stages of Su/Hx/Nx PAH. Data are mean ± SEM (n = 3–7). Differences are significant (P < 0.05) compared with normal control. Wk: week.

Next, we used echocardiography to evaluate the RVID at diastole to monitor the severity of PAH in Su/Hx/Nx rats at 4, 8, 10, and 12 weeks. RVID was increased at all 4 time points (Fig. 1D), suggesting dilatation of the right ventricle. We have previously found in the Su/Hx/Nx PAH model that RVSP levels off and cardiac output drops as disease progresses.20 Interestingly, the increase in RVID with the progression of PAH was positively correlated with the increase in plasma S1P levels (Fig. 1E). Taken together, these data suggest that an increase in circulating levels of S1P might be an indicator of the progression of PAH.

Pulmonary arterial expression of SphK1 and SphK2 increases with the progression of PAH

Because both SphK1 and SphK2 synthesize S1P, we next determined whether the expression of these enzymes was increased in PAH. Western blot analysis of whole-lung homogenates showed increased expression of SphK1 and SphK2 in 5-, 8-, and 13-week Su/Hx/Nx PAH rats compared with controls (Fig. 2A). We then immunostained lung sections of normal control and 5-, 8-, and 13-week Su/Hx/Nx PAH rats and patients with IPAH. Immunostaining of both SphKs was observed in the occlusive lesions of Su/Hx/Nx PAH rats, and the intensity of staining appeared to increase with the progression of PAH (Fig. 2B). The immunostaining of both SphKs was also elevated in lung sections from patients with IPAH compared with sections from human lungs used as a control (Fig. 2C).

Figure 2.

Figure 2

Open in a new tab

Expression of sphingosine kinase 1 (SphK1) and SphK2 is increased in pulmonary arterial hypertension (PAH). A, Western blot analysis of SphK1 and SphK2 expression in whole-lung homogenates of normal control (NC) and Sugen5416/hypoxia/normoxia (Su/Hx/Nx) PAH rats at various stages (5, 8, and 13 weeks [Wk]) of PAH. B, Images of SphK1 and SphK2 immunohistochemistry in distal pulmonary arteries of normal control and 5-, 8-, and 13-Wk Su/Hx/Nx PAH rats. Images are representative of 5 independent experiments. C, Images of SphK1 and SphK2 immunohistochemistry in pulmonary arteries of control and idiopathic PAH human lungs. Images are representative of 3 different donors. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Exogenous S1P induces proliferation of rat PAECs and PASMCs

Proliferation of PAECs and PASMCs is associated with occlusive arteriopathy in PAH.27 Because S1P has proproliferative effects on numerous cell types,28-30 including human PASMCs,10 we investigated whether S1P induces proliferation of rat PAECs and PASMCs. Cultured PAECs and PASMCs were serum-deprived for 24 hour and then treated with 5 µM S1P or vehicle (0.1% ethanol) for 24 and 48 hours. Media with 10% serum was used as a positive control. When compared with vehicle-treated and serum-starved cells, the number of S1P-treated PAECs (Fig. 3A) and PASMCs (Fig. 3B) was higher at both the 24- and 48-hour time points. This observation supports that S1P may promote the PAEC and PASMC proliferation of PAH.

Figure 3.

Figure 3

Open in a new tab

Exogenous sphingosine-1-phosphate (S1P) induces pulmonary artery endothelial cell (PAEC) and pulmonary artery smooth-muscle cell (PASMC) proliferation. The number of growth synchronized PAECs (A) and PASMCs (B) over 48 hours (Hrs) in the presence of S1P (5 µM), dimethyl sulfoxide (vehicle control), 10% fetal bovine serum (serum), and without 10% fetal bovine serum (serum free). Asterisk indicates P < 0.05 S1P versus vehicle control or serum free (24 Hrs), and pound sign indicates P < 0.05 S1P versus vehicle control or serum free (48 Hrs). Data are mean ± SEM (n = 3 independent experiments with triplicates in each experiment).

Inhibition of SphK1 reduces plasma S1P levels

To examine whether chronic inhibition of SphK1 at the late stage of severe PAH has therapeutic benefits, we delivered the SphK1 inhibitor, SLP7111228 (10 mg/kg administered intraperitoneally), to 10-week Su/Hx/Nx PAH rats every day for 3 weeks. After the 3 weeks of treatment, we observed a significant decrease in plasma levels of S1P (Fig. 4A) but not of the S1P precursor sphingosine (Fig. 4B). These findings show the effectiveness of SLP7111228 in reducing SphK1 activity.

Figure 4.

Figure 4

Open in a new tab

Inhibition of sphingosine kinase 1 (SphK1) reduces plasma levels of sphingosine-1-phosphate (S1P) in Sugen5416/hypoxia/normoxia (Su/Hx/Nx) pulmonary arterial hypertension (PAH) rats. Plasma levels of S1P (A) and its precursor sphingosine (B) in PAH rats after intraperitoneal delivery of SphK1 inhibitor (10 mg/kg, SLP7111228 dissolved in 2% 2-hydroxypropyl-beta-cyclodextrin [HPCD]) or vehicle (HPCD) for 3 weeks beginning at 10 weeks. Data are mean ± SEM (n = 3 in each group). Difference is significant (P < 0.05) compared with vehicle control. NS: not significant.

Inhibition of SphK1 reduces the occlusive pulmonary arteriopathy

Occlusive lesions are the hallmark of severe PAH. To examine whether chronic inhibition of SphK1 reduces the occlusive pulmonary arteriopathy, we performed hematoxylin and eosin staining of the FFPE lung sections (Fig. 5A) of Su/Hx/Nx PAH rats treated with SphK1 inhibitor or vehicle, and occluded pulmonary arteries were assessed quantitatively. The percentage of occluded pulmonary arteries (grade 1 and grade 2) with OD of <50 µm was decreased in the SphK1 inhibitor–treated group compared with the vehicle control group (Fig. 5A, 5B), whereas the percentage of nonoccluded (grade 0) pulmonary arteries was increased (Fig. 5B). Similarly, the percentage of occluded pulmonary arteries (grade 1) with OD between 50 and 100 µm was also decreased in the SphK1 inhibitor–treated group compared with the vehicle control group (Fig. 5C). This was accompanied by an increase in the percentage of nonoccluded (grade 0) pulmonary arteries (Fig. 5C). Also, the percentage of occluded pulmonary arteries (grade 2) with OD > 100 µm was decreased in the SphK1 inhibitor treated group compared with the vehicle control group (Fig. 5D). These results suggest that increased SphK1 leading to increased S1P levels contributes to the pulmonary arteriopathy in severe PAH.

Figure 5.

Figure 5

Open in a new tab

Inhibition of sphingosine kinase 1 (SphK1) reverses occlusive pulmonary arteriopathy in Sugen5416/hypoxia/normoxia (Su/Hx/Nx) pulmonary arterial hypertension (PAH) rats. A, Images of hematoxylin and eosin–stained distal pulmonary arteries of Su/Hx/Nx PAH rats showing occluded arteries in comparison with normal control rats. Right panel shows reversal of occluded arteries in the SphK1 inhibitor–treated Su/Hx/Nx PAH rats. Images are representative of 3 independent experiments. B, C, and D show quantitative determination of occluded arteries in normal control rats, vehicle-treated Su/Hx/Nx PAH rats, and SphK1 inhibitor–treated Su/Hx/Nx PAH rats. Percentage (mean) of grade 0 for no evidence of luminal occlusion, grade 1 for partial luminal occlusion (<50%), and grade 2 for full luminal occlusion (≥50%). B, Pulmonary arteries with outer diameter (OD) of <50 μm. C, Pulmonary arteries with OD of 50–100 μm. D, Pulmonary arteries with OD of >100 μm. Asterisk indicates P < 0.05 grade 0 vehicle control versus normal control or SphK1 inhibitor, pound sign indicates P < 0.05 grade 1 vehicle control versus normal control or SphK1 inhibitor, and @ indicates P < 0.05 grade 2 vehicle control versus normal control or SphK1 inhibitor.

Inhibition of SphK1 improves cardiovascular function

RVID was increased in the PAH rats (Fig. 6A, 6B). Moreover, we showed earlier that the increase in S1P levels was positively correlated with deteriorating RV function, indicated by increasing RVID (Fig. 1E); therefore, we measured the RVID after 3 weeks of SphK1 inhibitor treatment in Su/Hx/Nx PAH rats. The increase in RVID in the severe PAH rats was reduced by chronic SphK1 inhibition (Fig. 6A, 6B). Importantly, the decrease in RVID was positively correlated with the decrease in plasma S1P levels (Fig. 6C). In addition to the decrease in RVID as an indicator of improvement in cardiovascular function, we also assessed CI and TPRI by cardiac catheterization. Decreased CI (Fig. 6D) and increased TPRI (Fig. 6E) in the PAH rats were reversed in the SphK1 inhibitor–treated group (Fig. 6D). However, the improvement in cardiac function was not accompanied by a decrease in either RVSP (vehicle control: 85.7 ± 17.6 mmHg; SphK1 inhibitor Su/Hx/Nx: 91.9 ± 9.5 mmHg; n = 3) or RV/LV+S (vehicle control: 0.50 ± 0.08; SphK1 inhibitor Su/Hx/Nx: 0.53 ± 0.02; n = 3). Chronic SphK1 inhibition had no effects on systemic arterial pressure and heart rate (Fig. 6F, 6G). Altogether, these data indicate that inhibition of SphK1 reduced circulating S1P levels and the degree of occlusive arteriopathy and improved cardiac function in PAH rats.

Figure 6.

Figure 6

Open in a new tab

Inhibition of sphingosine kinase 1 (SphK1) improves cardiac function in Sugen5416/hypoxia/normoxia (Su/Hx/Nx) pulmonary arterial hypertension (PAH) rats. A, Representative echocardiogram of rat heart in short-axis B-mode view of a normal control and an Su/Hx/Nx PAH rat treated with vehicle or SphK1 inhibitor showing the restoration of normal position and orientation of the interventricular septum (dotted red lines) in SphK1 inhibitor–treated rat. B, Quantitative assessment of right ventricular internal diameter during diastole (RVID) from the short-axis B-mode echocardiogram of the hearts of normal control rats and Su/Hx/Nx PAH rats treated with vehicle or SphK1 inhibitor. C, Pearson correlation between RVID and plasma sphingosine-1-phosphate (S1P) level in Su/Hx/Nx rats treated with vehicle or SphK1 inhibitor. Measurement of cardiovascular function showing cardiac index (CI; D), total pulmonary artery resistance index (TPRI; E), mean systemic arterial pressure (mAP; F), and heart rate (HR; G) of control rats and Su/Hx/Nx PAH rats treated with vehicle or SphK1 inhibitor. Data are mean ± SEM (n = 3). Difference is significant (P < 0.05) compared with vehicle control Su/Hx/Nx PAH rats. NS: not significant.

Discussion

We found that the plasma levels of S1P were elevated in both patients with IPAH and PAH rats. Only 1 time point was measured in patients, but repeated measurements in rats showed that the plasma levels of S1P were increased at 4 weeks and sustained to 12 weeks of PAH. In a similar context, the serum level of S1P in patients with obstructive coronary artery disease was found to be a better predictor of occurrence and severity of disease than traditional risk factors.31 These observations suggest that increased plasma levels of S1P might serve as an early indicator of PAH pathology.

Chen et al.10 have recently found that the expression of SphK1, but not of SphK2, is increased in the lungs and PASMCs of patients with IPAH and in the lungs of chronically hypoxic mice and rats. The increased expression of SphK1 was accompanied by increased levels of S1P in the IPAH lungs and in pulmonary arteries of the hypoxic rodents. In contrast, we found elevated levels of both SphK1 and SphK2 in the total lung homogenates of Su/Hx/Nx PAH rats. Therefore, we analyzed by immunostaining the expression of SphK1 and SphK2 in lungs of patients with IPAH and PAH rats. We observed that the expression of both SphKs was increased predominantly in the pulmonary arterial wall, suggesting the involvement of pulmonary vascular cells in an SphK/S1P-mediated pathogenesis of PAH. These findings are supported by an earlier report that the messenger RNA of acid ceramidase, which hydrolyzes ceramide to the SphK substrate sphingosine, is upregulated in IPAH lungs.32 In addition, it has recently been reported that sphingolipid metabolites are increased in human PAH lungs.33 Our finding that the expression of both kinases is increased raises the possibility of additional studies detailing the relative roles of SphK1 and SphK2 in the pathogenesis of PAH. Although there is considerable evidence for the role of SphK1 in promoting cell survival, proliferation, and neoplastic transformation, emerging evidence also implicates SphK2 in a variety of diseases.34

Occlusive lesions in PAH are considered to be cancer-like.35,36 The increased expression of SphK1 in PAH is consistent with literature suggesting that it is upregulated in many types of cancer and that the S1P-generating enzyme has many features of an oncogene.37-40 SphK1 can be activated by several mediators, and S1P produced and released into the extracellular milieu promotes cell proliferation and survival by acting on its G protein–coupled receptors and by transactivating a spectrum of receptor tyrosine kinases, including those of platelet-derived growth factor (PDGF), transforming growth factor β, epidermal growth factor, hepatocyte growth factor, and VEGF.41 Importantly, constitutive activation of SphK1 in endothelial cells induces a proinflammatory and proangiogenic phenotype,42 and SphK inhibitors block pathologic neovascularization in animal models.43 Given that many of the aforementioned growth factor receptors and pathogenic processes have been implicated in the pathogenesis of occlusive lesions in PAH,44 we examined the effect of exogenous S1P on rat PAECs and found that it stimulated cell proliferation. This finding agrees with other reports that S1P of >5 μM induces endothelial dysfunction.45-47 Therefore, our finding raises the possibility that increased S1P levels are an early insult signal, which induces endothelial dysfunction leading to pulmonary arteriopathy. In PAH, endothelial dysfunction triggers the proliferation of apoptosis-resistant PAECs and the secretion of growth factors that stimulate PASMC proliferation. Independent of PAEC dysfunction–mediated PASMC proliferation, we also observed that S1P directly induced PASMC proliferation, which is consistent with the report by Chen et al.10 and the recent finding that early growth response protein 1 mediates PDGF-induced SphK1 expression and cell proliferation in human PASMCs.48

Although the study by Chen et al.10 demonstrates that pharmacological inhibition or gene knock down of SphK1 prevents hypoxia-induced pulmonary hypertension, it is not clear whether pharmacological inhibition of SphK1 can reverse the pulmonary vascular remodeling and improve cardiovascular function in severe occlusive PAH. Therefore, to assess the effect of pharmacological inhibition of SphK1 in PAH pathology, we treated late-stage (10-week) Su/Hx/Nx PAH rats with the inhibitor SLP7111228 daily for 3 weeks. Chronic inhibition of SphK1 in PAH rats decreased the elevated levels of plasma S1P and reduced the occlusive pulmonary arteriopathy. Given that physiological levels of S1P enhance endothelial barrier function, increased vascular permeability was a possible adverse effect of chronic inhibition of S1P synthesis.49,50 However, because SphK1 inhibitors reduce circulating S1P levels by only ∼50%, we did not observe lung edema formation in the rats.22

In contrast to our study and others10,33,51 suggesting a role for S1P in the pathogenesis of PH, long-term exposure to an allergen exaggerated pulmonary arterial responsiveness and wall thickening in SphK1-deficient mice.16 Similarly, Tian et al.17 have recently proposed that PAEC apoptosis and formation of occlusive pulmonary arterial lesions in the Sugen5416/athymic rat model of PAH is due to leukotriene B4 (LTB4)–induced inhibition of SphK1/S1P/nitric oxide signaling. However, they also found that LTB4 signaling was not activated in the Su/Hx/Nx PAH model. It is possible that the Sugen/athymic model recapitulates the pathology associated with connective tissue/immune disorder and that the Su/Hx/Nx model more closely mimics that associated with IPAH, and the signaling mechanisms of S1P might be different in the 2 models.

Chronic inhibition of SphK1 in the late-stage PAH rats improved CI and reduced TPRI, right ventricular diastolic dilation, and flattening of the ventricular septum. Despite these improvements, there was no decrease in RVSP or RV/LV+S. In contrast to our observation, pharmacological inhibition of SphK1 in chronically hypoxic rodents prevented the increases in RVSP and RV/LV+S.10 This difference in outcome can possibly be attributed to the differences in the 2 models of pulmonary hypertension. Chronic hypoxia–induced pulmonary hypertension does not develop occlusive arteriopathy, and the RV does not undergo transition from adaptive hypertrophy to maladaptive hypertrophy resulting in RV failure. The Su/Hx/Nx model of PAH develops occlusive lesions, and the RV transitions from adaptive hypertrophy to maladaptive hypertrophy with clear evidence of cardiac fibrosis and dysfunction.52,53 It has recently been shown that inhibition of SphK1/S1P signaling ameliorates infarction-induced cardiac remodeling and dysfunction,54 and our finding of improvement in cardiac function after SphK1 inhibition without decreases in RVSP and RV hypertrophy might be attributable to a direct therapeutic benefit in maladaptive RV hypertrophy. Another factor is the short therapeutic window (3 weeks). We speculate that an increase in the therapeutic window from 3 to 5 weeks might show improvement in the pressure and hypertrophy. Additionally, the differences in the treatment protocols may also account for the differences in the therapeutic outcome. In our study, SphK1 inhibition was started at 10 weeks of Su/Hx/Nx exposure, at which time the animals have developed severe pulmonary hypertension,18,20 whereas in the study by Chen et al.,10 inhibition of SphK1 was started at day 1 before the manifestation of pulmonary hypertension. Even though our study supports the hypothesis that inhibition of S1P synthesis in PAH reverses pulmonary and cardiac dysfunction, the mechanisms of the improvement are unknown and require additional studies.

Our study has limitations. We have not determined the source of increased circulating S1P. However, on the basis of the immunohistochemical analysis, we can infer that hypertensive pulmonary arteries might be one source of the S1P. Furthermore, use of pharmacologic inhibitors always raises concern about selectivity and specificity, but the established selectivity of the inhibitor used in this study minimizes this problem.21 However, additional studies using SphK-null mice (SphK1−/− and SphK2−/− mice) are needed to determine whether either of these genotypes is protected from Su/Hx- or prolyl-4 hydroxylase 2 knockdown–induced PAH.55,56

In summary, we found that circulating levels of S1P were elevated in patients with IPAH and in a rat model of severe occlusive PAH. Importantly, inhibition of S1P synthesis improved the pulmonary arterial structure and cardiac function of the PAH rats (Fig. 7). We further identified S1P as a proliferative mediator of rat PAECs and PASMCs. Collectively, these observations suggest that S1P is a molecular signal in the pathogenesis of PAH and is involved in increased proliferation and survival of the vascular cells, key events in the occlusive vascular remodeling. This study extends the role of endogenous S1P in the pathobiology of PAH and will hopefully lead to additional translational studies.

Figure 7.

Figure 7

Open in a new tab

Increase in plasma sphingosine-1-phosphate (S1P) levels and its inhibition in pulmonary arterial hypertension (PAH). In PAH, a normal lung with compliant thin-walled pulmonary arteries undergoes remodeling leading to the narrowing of the lumen with occlusive lesions. The decrease in lumen diameter increases pulmonary arterial stiffness and resistance leading to increased afterload in the right ventricle (RV). Increased afterload in RV changes the orientation of the normal interventricular septum leading to dilatation and ultimately failure. Plasma levels of S1P were increased in Su/Hx/Nx PAH rats as the disease progressed. Inhibition of sphingosine kinase 1 (SphK1) decreased plasma S1P levels, reduced pulmonary arteriopathy, and improved cardiovascular function. LV: left ventricle.

Acknowledgments

Dr. Kevin Lynch and Andrew Blot from SphynKx Therapeutics, Charlottesville, Virginia, provided the SphK1 inhibitor and LCMS measurement of S1P in rat plasma samples.

Source of Support: Departments of Internal Medicine and Pharmacology and the Center for Lung Biology, University of South Alabama, American Heart Association grant 11POST7720023.

Conflict of Interest: None declared.

References

  • 1.Macchia A, Marchioli R, Marfisi R, Scarano M, Levantesi G, Tavazzi L, Tognoni G. A meta-analysis of trials of pulmonary hypertension: a clinical condition looking for drugs and research methodology. Am Heart J 2007;153:1037–1047. [DOI] [PubMed]
  • 2.Pogoriler JE, Rich S, Archer SL, Husain AN. Persistence of complex vascular lesions despite prolonged prostacyclin therapy of pulmonary arterial hypertension. Histopathology 2012;61:597–609. [DOI] [PMC free article] [PubMed]
  • 3.Stacher E, Graham BB, Hunt JM, Gandjeva A, Groshong SD, McLaughlin VV, Jessup M, et al. Modern age pathology of pulmonary arterial hypertension. Am J Resp Crit Care Med 2012;186:261–272. [DOI] [PMC free article] [PubMed]
  • 4.Kawamori T, Osta W, Johnson KR, Pettus BJ, Bielawski J, Tanaka T, Wargovich MJ, et al. Sphingosine kinase 1 is up-regulated in colon carcinogenesis. FASEB J 2006;20:386–388. [DOI] [PubMed]
  • 5.Hla T. Physiological and pathological actions of sphingosine 1-phosphate. Semin Cell Dev Biol 2004;15:513–520. [DOI] [PubMed]
  • 6.Maceyka M, Milstien S, Spiegel S. Sphingosine-1-phosphate: the Swiss army knife of sphingolipid signaling. J Lipid Res 2009;50(suppl):S272–S276. [DOI] [PMC free article] [PubMed]
  • 7.Ryland LK, Fox TE, Liu X, Loughran TP, Kester M. Dysregulation of sphingolipid metabolism in cancer. Cancer Biol Ther 2011;11:138–149. [DOI] [PubMed]
  • 8.Delgado A, Fabrias G, Bedia C, Casas J, Abad JL. Sphingolipid modulation: a strategy for cancer therapy. Anticancer Agents Med Chem 2012;12:285–302. [DOI] [PubMed]
  • 9.Russo SB, Ross JS, Cowart LA. Sphingolipids in obesity, type 2 diabetes, and metabolic disease. Handb Exp Pharmacol 2013:373–401. [DOI] [PMC free article] [PubMed]
  • 10.Chen J, Tang H, Sysol JR, Moreno-Vinasco L, Shioura KM, Chen T, Gorshkova I, et al. The sphingosine kinase 1/sphingosine-1-phosphate pathway in pulmonary arterial hypertension. Am J Resp Crit Care Med 2014;190:1032–1043. [DOI] [PMC free article] [PubMed]
  • 11.Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 2003;4:397–407. [DOI] [PubMed]
  • 12.Young N, Van Brocklyn JR. Signal transduction of sphingosine-1-phosphate G protein-coupled receptors. ScientificWorldJournal 2006;6:946–966. [DOI] [PMC free article] [PubMed]
  • 13.Brinkmann V. Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology. Pharmacol Ther 2007;115:84–105. [DOI] [PubMed]
  • 14.Szczepaniak WS, Pitt BR, McVerry BJ. S1P2 receptor-dependent Rho-kinase activation mediates vasoconstriction in the murine pulmonary circulation induced by sphingosine 1-phosphate. Am J Physiol Lung Cell Mol Physiol 2010;299:L137–L145. [DOI] [PMC free article] [PubMed]
  • 15.Ota H, Beutz MA, Ito M, Abe K, Oka M, McMurtry IF. S1P(4) receptor mediates S1P-induced vasoconstriction in normotensive and hypertensive rat lungs. Pulm Circ 2011;1:399–404. [DOI] [PMC free article] [PubMed]
  • 16.Haberberger RV, Tabeling C, Runciman S, Gutbier B, Konig P, Andratsch M, Schutte H, Suttorp N, Gibbins I, Witzenrath M. Role of sphingosine kinase 1 in allergen-induced pulmonary vascular remodeling and hyperresponsiveness. J Allergy Clin Immunol 2009;124:933–941.e1–9. [DOI] [PubMed]
  • 17.Tian W, Jiang X, Tamosiuniene R, Sung YK, Qian J, Dhillon G, Gera L, et al. Blocking macrophage leukotriene B4 prevents endothelial injury and reverses pulmonary hypertension. Sci Transl Med 2013;5:200ra117. [DOI] [PMC free article] [PubMed]
  • 18.Abe K, Toba M, Alzoubi A, Ito M, Fagan KA, Cool CD, Voelkel NF, McMurtry IF, Oka M. Formation of plexiform lesions in experimental severe pulmonary arterial hypertension. Circulation 2010;121:2747–2754. [DOI] [PubMed]
  • 19.Taraseviciene-Stewart L, Kasahara Y, Alger L, Hirth P, McMahon G, Waltenberger J, Voelkel NF, Tuder RM. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J 2001;15:427–438. [DOI] [PubMed]
  • 20.Toba M, Alzoubi A, O’Neill KD, Gairhe S, Matsumoto Y, Oshima K, Abe K, Oka M, McMurtry IF. Temporal hemodynamic and histological progression in Sugen5416/hypoxia/normoxia-exposed pulmonary arterial hypertensive rats. Am J Physiol Heart Circ Physiol 2014;306:H243–H250. [DOI] [PMC free article] [PubMed]
  • 21.Patwardhan NN, Morris EA, Kharel Y, Raje MR, Gao M, Tomsig JL, Lynch KR, Santos WL. Structure-activity relationship studies and in vivo activity of guanidine-based sphingosine kinase inhibitors: discovery of SphK1- and SphK2-selective inhibitors. J Med Chem 2015;58:1879–1899. [DOI] [PMC free article] [PubMed]
  • 22.Kharel Y, Mathews TP, Gellett AM, Tomsig JL, Kennedy PC, Moyer ML, Macdonald TL, Lynch KR. Sphingosine kinase type 1 inhibition reveals rapid turnover of circulating sphingosine 1-phosphate. Biochem J 2011;440:345–353. [DOI] [PMC free article] [PubMed]
  • 23.Gairhe S, Bauer NN, Gebb SA, McMurtry IF. Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation. Am J Physiol Lung Cell Mol Physiol 2012;303:L767–L777. [DOI] [PubMed]
  • 24.Oka M, Homma N, Taraseviciene-Stewart L, Morris KG, Kraskauskas D, Burns N, Voelkel NF, McMurtry IF. Rho kinase-mediated vasoconstriction is important in severe occlusive pulmonary arterial hypertension in rats. Circ Res 2007;100:923–929. [DOI] [PubMed]
  • 25.McLendon JM, Joshi SR, Sparks J, Matar M, Fewell JG, Abe K, Oka M, McMurtry IF, Gerthoffer WT. Lipid nanoparticle delivery of a microRNA-145 inhibitor improves experimental pulmonary hypertension. J Control Release 2015;210:67–75. [DOI] [PMC free article] [PubMed]
  • 26.Stevens T, Creighton J, Thompson WJ. Control of cAMP in lung endothelial cell phenotypes: implications for control of barrier function. Am J Physiol 1999;277:L119–L126. [DOI] [PubMed]
  • 27.Sakao S, Tatsumi K, Voelkel NF. Endothelial cells and pulmonary arterial hypertension: apoptosis, proliferation, interaction and transdifferentiation. Respir Res 2009;10:95. [DOI] [PMC free article] [PubMed]
  • 28.Pyne NJ, Pyne S. Sphingosine 1-phosphate and cancer. Nat Rev Cancer 2010;10:489–503. [DOI] [PubMed]
  • 29.Calise S, Blescia S, Cencetti F, Bernacchioni C, Donati C, Bruni P. Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells: role of S1P receptors. Biochim Biophys Acta 2012;1823:439–450. [DOI] [PubMed]
  • 30.Proia RL, Hla T. Emerging biology of sphingosine-1-phosphate: its role in pathogenesis and therapy. J Clin Invest 2015;125:1379–1387. [DOI] [PMC free article] [PubMed]
  • 31.Deutschman DH, Carstens JS, Klepper RL, Smith WS, Page MT, Young TR, Gleason LA, Nakajima N, Sabbadini RA. Predicting obstructive coronary artery disease with serum sphingosine-1-phosphate. Am Heart J 2003;146:62–68. [DOI] [PubMed]
  • 32.Edgar AJ, Chacon MR, Bishop AE, Yacoub MH, Polak JM. Upregulated genes in sporadic, idiopathic pulmonary arterial hypertension. Respir Res 2006;7:1. [DOI] [PMC free article] [PubMed]
  • 33.Zhao YD, Chu L, Lin K, Granton E, Yin L, Peng J, Hsin M, et al. A biochemical approach to understand the pathogenesis of advanced pulmonary arterial hypertension: metabolomic profiles of arginine, sphingosine-1-phosphate, and heme of human lung. PLoS One 2015;10:e0134958. [DOI] [PMC free article] [PubMed]
  • 34.Neubauer HA, Pitson SM. Roles, regulation and inhibitors of sphingosine kinase 2. FEBS J 2013;280:5317–5336. [DOI] [PubMed]
  • 35.Lee SD, Shroyer KR, Markham NE, Cool CD, Voelkel NF, Tuder RM. Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension. J Clin Invest 1998;101:927–934. [DOI] [PMC free article] [PubMed]
  • 36.Voelkel NF, Cool C, Lee SD, Wright L, Geraci MW, Tuder RM. Primary pulmonary hypertension between inflammation and cancer. Chest 1998;114:225S–230S. [DOI] [PubMed]
  • 37.Sabbadini RA. Targeting sphingosine-1-phosphate for cancer therapy. Br J Cancer 2006;95:1131–1135. [DOI] [PMC free article] [PubMed]
  • 38.Olivera A, Kohama T, Edsall L, Nava V, Cuvillier O, Poulton S, Spiegel S. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival. J Cell Biol 1999;147:545–558. [DOI] [PMC free article] [PubMed]
  • 39.Nagahashi M, Ramachandran S, Kim EY, Allegood JC, Rashid OM, Yamada A, Zhao R, et al. Sphingosine-1-phosphate produced by sphingosine kinase 1 promotes breast cancer progression by stimulating angiogenesis and lymphangiogenesis. Cancer Res 2012;72:726–735. [DOI] [PMC free article] [PubMed]
  • 40.Zhang H, Li W, Sun S, Yu S, Zhang M, Zou F. Inhibition of sphingosine kinase 1 suppresses proliferation of glioma cells under hypoxia by attenuating activity of extracellular signal-regulated kinase. Cell Prolif 2012;45:167–175. [DOI] [PMC free article] [PubMed]
  • 41.Lebman DA, Spiegel S. Cross-talk at the crossroads of sphingosine-1-phosphate, growth factors, and cytokine signaling. J Lipid Res 2008;49:1388–1394. [DOI] [PMC free article] [PubMed]
  • 42.Limaye V, Xia P, Hahn C, Smith M, Vadas MA, Pitson SM, Gamble JR. Chronic increases in sphingosine kinase-1 activity induce a pro-inflammatory, pro-angiogenic phenotype in endothelial cells. Cell Mol Biol Lett 2009;14:424–441. [DOI] [PMC free article] [PubMed]
  • 43.Maines LW, Fitzpatrick LR, French KJ, Zhuang Y, Xia Z, Keller SN, Upson JJ, Smith CD. Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase. Dig Dis Sci 2008;53:997–1012. [DOI] [PMC free article] [PubMed]
  • 44.Schermuly RT, Ghofrani HA, Wilkins MR, Grimminger F. Mechanisms of disease: pulmonary arterial hypertension. Nat Rev Cardiol 2011;8:443–455. [DOI] [PMC free article] [PubMed]
  • 45.McVerry BJ, Garcia JG. Endothelial cell barrier regulation by sphingosine 1-phosphate. J Cell Biochem 2004;92:1075–1085. [DOI] [PubMed]
  • 46.Singleton PA, Dudek SM, Chiang ET, Garcia JG. Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin. FASEB J 2005;19:1646–1656. [DOI] [PubMed]
  • 47.Li Q, Chen B, Zeng C, Fan A, Yuan Y, Guo X, Huang X, Huang Q. Differential activation of receptors and signal pathways upon stimulation by different doses of sphingosine-1-phosphate in endothelial cells. Exp Physiol 2015;100:95–107. [DOI] [PubMed]
  • 48.Sysol JR, Natarajan V, Machado RF. PDGF induces SphK1 expression via Egr-1 to promote pulmonary artery smooth muscle cell proliferation. Am J Physiol Cell Physiol 2016;310:C983–C992. [DOI] [PMC free article] [PubMed]
  • 49.Abbasi T, Garcia JG. Sphingolipids in lung endothelial biology and regulation of vascular integrity. Handb Exp Pharmacol 2013;216:201–226. [DOI] [PMC free article] [PubMed]
  • 50.Natarajan V, Dudek SM, Jacobson JR, Moreno-Vinasco L, Huang LS, Abassi T, Mathew B, et al. Sphingosine-1-phosphate, FTY720, and sphingosine-1-phosphate receptors in the pathobiology of acute lung injury. Am J Respir Cell Mol Biol 2013;49:6–17. [DOI] [PMC free article] [PubMed]
  • 51.Tabeling C, Yu H, Wang L, Ranke H, Goldenberg NM, Zabini D, Noe E, et al. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction. Proc Natl Acad Sci U S A 2015;112:E1614–E1623. [DOI] [PMC free article] [PubMed]
  • 52.Alzoubi A, Toba M, Abe K, O’Neill KD, Rocic P, Fagan KA, McMurtry IF, Oka M. Dehydroepiandrosterone restores right ventricular structure and function in rats with severe pulmonary arterial hypertension. Am J Physiol Heart Circ Physiol 2013;304:H1708–H1718. [DOI] [PubMed]
  • 53.Gomez-Arroyo J, Mizuno S, Szczepanek K, Van Tassell B, Natarajan R, dos Remedios CG, Drake JI, et al. Metabolic gene remodeling and mitochondrial dysfunction in failing right ventricular hypertrophy secondary to pulmonary arterial hypertension. Circ Heart Fail 2013;6:136–144. [DOI] [PMC free article] [PubMed]
  • 54.Zhang F, Xia Y, Yan W, Zhang H, Zhou F, Zhao S, Wang W, et al. Sphingosine 1-phosphate signaling contributes to cardiac inflammation, dysfunction, and remodeling following myocardial infarction. Am J Physiol Heart Circ Physiol 2015;310:H250–H261. [DOI] [PubMed]
  • 55.Ciuclan L, Bonneau O, Hussey M, Duggan N, Holmes AM, Good R, Stringer R, et al. A novel murine model of severe pulmonary arterial hypertension. Am J Respir Crit Care Med 2011;184:1171–1182. [DOI] [PubMed]
  • 56.Dai Z, Li M, Wharton J, Zhu MM, Zhao YY. PHD2 deficiency in endothelial cells and hematopoietic cells induces obliterative vascular remodeling and severe pulmonary arterial hypertension in mice and humans through HIF-2alpha. Circulation 2016;133:2447–2458. [DOI] [PMC free article] [PubMed]

Articles from Pulmonary Circulation are provided here courtesy of Wiley

RESOURCES