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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.

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.

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.

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.

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.

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.

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 others^10,33,51 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.

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.

Footnotes

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.

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Sphingosine-1-Phosphate is Involved in the Occlusive Arteriopathy of Pulmonary Arterial Hypertension

Abstract

Keywords

METHODS

Human blood and lung samples

Animal model of severe occlusive PAH

SphK1 inhibitor treatment

Enzyme-linked immunosorbent assay (ELISA) for S1P

Liquid chromatography–mass spectrometry (LCMS)

Histopathological and immunohistochemical studies

Western blot analysis

Measurements of pulmonary and cardiac function in PAH rats

Morphometric assessment

Cell culture and treatment

Data and statistical analyses

RESULTS

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

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

Exogenous S1P induces proliferation of rat PAECs and PASMCs

Inhibition of SphK1 reduces plasma S1P levels

Inhibition of SphK1 reduces the occlusive pulmonary arteriopathy

Inhibition of SphK1 improves cardiovascular function

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References