Sildenafil improves hemodynamic changes caused by acute pulmonary embolism by inhibiting Rho kinase activity

Abstract

Objective

This study examined the effects of sildenafil on acute pulmonary embolism (APE) using a rat model.

Methods

Sprague–Dawley rats were randomly divided into the sham, pulmonary thromboembolism (PTE), and sildenafil groups. The sham and PTE groups received normal saline once daily via gavage for 14 consecutive days, whereas the sildenafil group received sildenafil (0.5 mg/kg/day) once daily via gavage for 14 consecutive days. Autologous emboli were prepared from blood samples collected from the left femoral artery of rats in each group on day 13, and autologous emboli were injected into the jugular vein cannula of rats in the PTE and sildenafil groups on day 14. Sham-treated rats received the same volume of saline. Right systolic ventricular pressure (RVSP) and mean pulmonary arterial pressure (MPAP) were used to assess pulmonary embolism, and western blotting and enzyme-linked immunosorbent assay were used to detect relevant markers.

Results

The Rho kinase signaling pathway was significantly activated in rats with APE, and sildenafil significantly inhibited this activation.

Conclusions

Sildenafil protected against APE through inhibiting Rho kinase activity, thereby reducing pulmonary vasoconstriction and decreasing elevated pulmonary arterial pressure. These findings might provide new ideas for the clinical treatment of acute pulmonary thromboembolism.

Introduction

Acute pulmonary embolism (APE) is a clinical syndrome of pulmonary circulation disorder attributable to obstruction of the pulmonary artery or its branches by endogenous or exogenous emboli, and the disease is mainly caused by thromboembolism, air embolism, and fat embolism. The most common cause of thromboembolism of the pulmonary artery and its branches in clinical practice is lower-limb venous thrombosis.^1,2 Acute right heart failure and/or circulatory shock caused by a dramatic increase in pulmonary vascular resistance are the main causes of death from APE, and drug treatment to alleviate neurohumoral factor-mediated pulmonary vasoconstriction might reduce APE-induced hemodynamic disturbances and improve prognosis.^3,4

Abnormal activation of the Rho/Rho kinase (ROCK) signaling pathway is involved in the formation of experimental pulmonary hypertension (PH), and ROCK inhibitors can prevent and reverse pulmonary vasoconstriction and pulmonary vascular remodeling during PH. ⁵ Rho is a member of the Ras superfamily of monomeric GTPases, and its isoforms are RhoA, RhoB, and RhoC3. RhoA is a molecular switch of multiple extracellular signals involved in regulating a variety of biological functions of cells, including contraction, migration, adhesion, cell cycle progression, and gene expression. Physiologically, the ROCK signaling pathway is constitutively active in the pulmonary vasculature. ⁶ Acute hypoxia can increase the activity of ROCK in pulmonary artery smooth muscle cells (PASMCs), promote the phosphorylation of myosin light chain (MLC), and enhance the contractile sensitivity of PASMCs to Ca²⁺. Y-27632 can effectively prevent and reverse hypoxic pulmonary vasoconstriction, indicating that increased ROCK activity is important in the development of hypoxic pulmonary vasoconstriction. ⁷

Sildenafil is a type 5 phosphodiesterase (PDE5) inhibitor that degrades and converts substances such as cyclic guanosine monophosphate and cyclic adenosine monophosphate into inactive substances to block cell–cell signal transmission, thereby affecting the blood vessels, structure, and function of the lungs and right ventricular myocardium.^8–10 To date, functional studies of sildenafil have mainly focused on the disease condition, hemodynamics, and exercise tolerance of patients with PH, with the drug exhibiting a certain alleviating effect. Conversely, the mechanism by which sildenafil improves the hemodynamics of PAH caused by APE is unclear.^11–13 In this study, we constructed a rat model of APE and evaluated the effect and molecular mechanism of sildenafil in the treatment of APE. Our studies will provide an important theoretical basis for APE and explore possible therapeutic targets.

Materials and methods

Animal study

All animal experiments were approved by the Ethics Committee of Shaanxi Provincial People’s Hospital (no. SP20220320). Specific pathogen-free-grade male Sprague–Dawley rats aged 42 to 56 days were purchased from Hubei Laboratory Animal Research Center (Wuhan, China). Sildenafil (0.5 mg/kg/day, MedChemExpress, Shanghai, China) was administered once daily by gavage for 14 consecutive days. Y-27632 (MedChemExpress) was administered at 5 mg/kg once every 2 days for 14 consecutive days. Animals were randomly divided into five groups: sham group (n = 6); PTE group (n = 6); PTE + sildenafil group (n = 6); PTE +Y-27632 group (n = 3); and PTE + sildenafil +inhibitor Y-27632 group (n = 3).

Autologous emboli were prepared on day 13, and rats were anesthetized and fixed in the supine position. ¹⁴ Briefly, 0.5 mL of blood were collected from the femoral artery at the left groin using a microcatheter with an inner diameter of approximately 1.1 mm. After blood collection, the microcatheter was allowed to stand at room temperature for 30 minutes and heated in a 60°C water bath for 5 minutes. The thrombus was then transferred to a sterile plate with a syringe, cut into pieces of approximately 1.1 × 3 mm², and rinsed three times with normal saline. Next, 50 emboli were extracted with a 2-mL syringe and stored in a 4°C refrigerator for future use. The aforementioned operations were performed under aseptic conditions. On day 14, the rats were anesthetized and fixed in the supine position, and their necks were shaved and disinfected. A 5-mm longitudinal incision was made in the left neck, blunt dissection was performed to expose the left external jugular vein, the distal end was ligated, and a kink was left at the proximal end for preparation. The vein was cut close to the ligation site, an 18-G arteriovenous indwelling needle was inserted, the venous cannula was ligated and fixed, and 50 autologous emboli and 1 mL of normal saline were slowly injected. The arteriovenous indwelling needle was removed, the proximal end of the external jugular vein was ligated, the subcutaneous tissue and skin were sutured layer by layer, and the rats were returned to their home cages after recovery to continue feeding. Rats in the sham group were injected with only the same amount of saline without autologous emboli, and the other procedures were the same.

Right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure (MPAP) were measured at 24 hours by echocardiography. The rats were sacrificed by carotid artery exsanguination. Blood was collected, anticoagulated, centrifuged to prepare plasma, and cryopreserved for future use. Each lung was bisected, with one half fixed and the other half cryopreserved for future use.

Hematoxylin and eosin staining

Frozen sections were removed from the refrigerator, allowed to reach room temperature, and soaked in deionized water for several minutes. The sections were stained with Mayer’s hematoxylin (the staining background was clean and differentiation was not required; Beyotime Biotechnology, Shanghai, China) for 5 minutes and rinsed with tap water. The sections were then stained with 1% water-soluble eosin staining solution (Beyotime Biotechnology) for 5 min and immersed in tap water for 30 s, 95% alcohol for 30 s, 95% alcohol for 1 minute, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. After air-drying, the sections were mounted and examined microscopically.

Western blotting

A small amount of minced tissue block was placed in a 2-mL Eppendorf tube, and cleaned steel beads were added. Then, 300 μL of detergent lysate containing phenylmethylsulfonyl fluoride were added to each tube, which was placed in an automatic homogenizer for homogenization. After incubation on ice for 30 minutes, the supernatant was collected. After determining the protein concentration, samples were denatured, and 40 µg of protein were loaded into each well and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA), and the membranes were then blocked with 5% non-fat milk in Tris-buffered saline/Tween 20 for 2 hours at room temperature. Primary antibodies (all from Affinity, Shanghai, China) against GAPDH (1:1000), MYPT1 (1:1000), p-MYPT1 (1:1000), ROCK1 (1:1000), and ROCK2 (1:1000) were diluted with blocking solution, and the membranes were incubated in the primary antibody overnight at 4°C. The membranes were thoroughly washed with Tris-buffered saline/Tween 20 five times for 5 minutes each. The membranes were soaked in secondary antibody (1:600) for 2 hours at room temperature on a shaker. The membranes were again thoroughly washed with Tris-buffered saline/Tween 20 five times for 5 minutes each. The film was developed and then scanned.

Enzyme-linked immunosorbent assay

Rat plasma was centrifuged at 1000 × g to obtain the supernatant. After washing the plate twice, 100 μL of the standard sample were added, and specimen universal diluent was added to the blank well. Subsequently, 100 μL of standards at different concentrations or the samples to be tested were added into the other wells. The plates were coated and then incubated at 37°C for 90 minutes, after which the plates were washed five times. After discarding the liquid, 100 μL of biotinylated antibody working solution were added to each well to coat the microtiter plate, and the plate was incubated at 37°C for 1 hour. Subsequently, the liquid in each well was discarded, and the plate was washed five times with PBST. Following this, 100 μL of enzyme conjugate working solution were added to each well, and the plate was covered with a covering membrane and incubated at 37°C for 30 minutes. After discarding the liquid in the wells, the plate was shaken to dryness and washed five times. Then, 100 μL of chromogenic substrate (3,3′,5,5′-tetramethylbenzidine) were added to each well, and the microtiter plate was incubated with the overlay at 37°C for 15 minutes in the dark. Finally, 100 μL of stop solution were added to each well to stop the reaction, at which time the blue color turned yellow instantly. The optical density of each well was immediately measured using a microplate reader at 450 nm.

Statistical analysis

All results are presented as the mean ± standard deviation. PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA) was used to analyze all data. An unpaired Student’s t-test was used to analyze the differences between two groups. One-way analysis of variance followed by Dunnett’s multiple comparisons test was used to analyze significance among multiple groups. P < 0.05 indicated statistical significance.

Results

Sildenafil improved hemodynamic changes in rats with pulmonary embolism.

Sprague–Dawley rats were randomly divided into the sham, PTE, and sildenafil groups. Rats in the sham and PTE groups received normal saline once daily via gavage for 14 consecutive days, and those in the sildenafil group received sildenafil (0.5 mg/kg/day) via gavage once daily for 14 consecutive days. Autologous emboli were prepared from blood samples collected from the left femoral artery of rats in each group on day 13. Autologous emboli were injected into the jugular vein cannula in the PTE and sildenafil groups on day 14, and only the same volume of saline was injected into the sham group. Our results illustrated that pulmonary embolism increased RVSP and MPAP by 1.75- and 1.52-fold, respectively, in the embolization group compared with that in the sham group. Sildenafil pretreatment effectively prevented increases in RVSP and MPAP (Figure 1a). Twenty-four hours after model establishment, the lung tissue structure was normal in the sham group, and no embolism was observed. In the PTE group, staining of lung tissue revealed emboli of different sizes in the pulmonary arteries and other small vessels accompanying the bronchi, diffuse lesions in the lung tissue, endothelial cell swelling, partial cytoplasmic lysis, marked congestion, edema and thickening of the alveolar wall, severe congestion and edema of the pulmonary interstitium, massive inflammatory cell infiltration, and massive exudates and red blood cells in the alveoli. The structure of lung tissue in the sildenafil group was similar to that in sham group, and the pathological changes were mainly manifested as mild-to-moderate alveolar wall edema, accompanied by a small amount of inflammatory cell infiltration (Figure 1b). Western blotting revealed that ROCK1, ROCK2, and p-MYPT1 expression was significantly increased in the PTE group compared with that in the sham group, whereas MYPT1 expression was unchanged. The increases in protein expression in the PTE group were blocked by sildenafil pretreatment (Figure 1c). Serum TNF-α and IL-6 levels were significantly higher in the PTE group than in the sham group, whereas their levels were significantly lower in the sildenafil group than in the PTE group (Figure 1d). These results suggested that sildenafil improved hemodynamic changes in rats with pulmonary embolism.

Figure 1.

Sildenafil improved hemodynamic changes in rats with pulmonary embolism. (a) RVSP and MPAP in the groups at 24 hours after model establishment. (b) Gross and pathological changes of lung tissue in the three groups. (c) Western blotting of ROCK1, ROCK2, p-MYPT1, and MYPT1 and (d) Enzyme-linked immunosorbent assay of TNF-α and IL-6. All experiments were duplicated three times. Data are presented as the mean ± standard deviation. ***P < 0.001, ****P < 0.0001 vs. the Sham group. Sham, sham group; PTE; pulmonary embolism model group; sildenafil, PTE + sildenafil group. scale bar = 50 μm. RVSP, right ventricular systolic pressure; MPAP, mean pulmonary arterial pressure; ROCK, Rho kinase.

Sildenafil improved hemodynamic changes during pulmonary embolism by inhibiting activation of the Rho pathway.

Our results revealed that sildenafil and the ROCK inhibitor Y-27632 reduced RVSP and MPAP in rats with pulmonary embolism (Figure 2a). The effects of sildenafil on RVSP and MPAP were enhanced by the addition of Y-27632 (Figure 2a). Coagulation thrombi, alveolar swelling and thickening, and massive inflammatory cell infiltration around the blood vessels were observed in the lung tissue of rats in the PTE group. The pathological changes in the sildenafil, Y-27632, and sildenafil + Y-27632 groups mainly manifested as mild-to-moderate alveolar wall edema accompanied by a low level of inflammatory cell infiltration (Figure 2b). Compared with the findings in the PTE group, serum TNF-α and IL-6 levels were significantly lower in the sildenafil and Y-27632 groups (Figure 2c). TNF-α and IL-6 levels further decreased in the sildenafil + Y-27632 group (Figure 2c). These results demonstrated that sildenafil improved hemodynamic changes during pulmonary embolism by inhibiting activation of the Rho pathway.

Figure 2.

Sildenafil improved hemodynamic changes during pulmonary embolism by inhibiting activation of the Rho pathway. (a) RVSP and MPAP in the five groups of rats. (b) Gross and pathological changes of lung tissue in the five groups of rats and (c) Enzyme-linked immunosorbent assay of TNF-α and IL-6 in the five groups. All experiments were duplicated three times. Data are presented as the mean ± standard deviation. **P < 0.01, ****P < 0.0001 vs. Sham group. ^&&P < 0.01, ^&&&P < 0.001, ^&&&&P < 0.0001 vs. PTE group.

Discussion

The direct causes of death following APE include severe lung injury and acute pulmonary arterial hypertension (PAH). Studies have found that PAH caused by APE is closely related to pulmonary arterial mechanical obstruction, and thrombotic mechanical obstruction after pulmonary embolism is considered the direct cause of PAH.^15,16 When APE occurs, a large number of inflammatory cells accumulate and infiltrate around the embolized vessels, and the cytokines released by these accumulated inflammatory cells further aggravate pulmonary vascular endothelial injury. Thus, these cytokines play an important role in the promotion of PAH by APE.^17–20 In this study, RVSP and MPAP were higher in the PTE group than in the sham group. Staining of lung tissue revealed emboli of different sizes in the pulmonary arteries and other small vessels accompanying the bronchi, diffuse lesions in the lung tissue, endothelial cell swelling, partial cytoplasmic lysis, marked congestion, edema, and thickening of the alveolar wall, high congestion and edema of the pulmonary interstitium, massive inflammatory cell infiltration, and massive exudates and red blood cells in the alveoli in the PTE group, Additionally, serum TNF-α and IL-6 levels were significantly higher in the PTE group than in the sham group. These results demonstrated that RVSP, MPAP and inflammation might be associated with APE.

The small GTPase RhoA and its downstream effector protein ROCK regulate various cellular functions such as vascular smooth muscle contraction, stress fiber formation, cell proliferation, and cell migration and play an important role in the pathogenesis of vascular diseases such as hypertension, atherosclerosis, stroke, and myocardial reperfusion injury. ⁵ ROCK activation leads to MYPT1 phosphorylation, which inhibits myosin light chain phosphatase (MLCP) activity suppresses its ability to catalyze MLC dephosphorylation, causing increased MLC phosphorylation and enhancing smooth muscle contractility at certain Ca²⁺ levels. In addition, increased ROCK activity induces phosphorylation of the MLCP inhibitor CPI-7, resulting in inhibition of MLCP activity. ROCK activation can directly lead to MLC phosphorylation. ²¹ Thus, ROCK activation enhances the contractility of smooth muscle cells by promoting MLC phosphorylation through multiple pathways. The exact mechanism of hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling is unknown. Studies have revealed that chronic hypoxia can increase Rho and ROCK expression in PASMCs, and Y-27632 intervention can reduce RVSP, pulmonary artery muscularization, and right ventricular hypertrophy in hypoxic PH mice, ²² indicating that the ROCK signaling pathway is involved in hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling. Our results revealed that ROCK1, ROCK2, and p-MYPT1 expression was significantly higher in the PTE group than in the sham group. Y-27632 significantly reduced RVSP and MPAP in rats with pulmonary embolism. Y-27632 also improved right heart hemodynamic disturbances and significantly reduced serum TNF-α and IL-6 levels in rats with PTE. These results indicated that the ROCK signaling pathway might be involved in APE.

PDE5 degrades and converts substances such as cyclic guanosine monophosphate and cyclic adenosine monophosphate into inactive substances to block cell–cell signal transmission, thereby affecting the blood vessels, structure, and function of the lung and right ventricular myocardium. The main pathological manifestations of PH include decreased nitric oxide production in the endothelium and significantly increased PDE5 activity and expression in the right ventricular myocardium and PASMCs. Sildenafil selectively inhibits PDE5 and enhances the nitric oxide/cyclic guanosine monophosphate signaling pathway. The drug dilates pulmonary arteries, relaxes pulmonary artery smooth muscle, reduces the proliferation of pulmonary vascular smooth muscle cells and pulmonary vascular remodeling while enhancing right ventricular contraction, and ultimately improves pulmonary circulation.^23–25 Current functional studies of sildenafil have focused on the relief of disease, hemodynamics, and exercise tolerance in patients with PH. Previous experiments illustrated that pretreatment with sildenafil effectively prevented PAH induced by APE, whereas acetylcysteine infusion enhanced this beneficial effect by inhibiting oxidative stress and lipid peroxidation. ²⁶ Our studies revealed that sildenafil inhibited the activation of ROCK signaling induced by APE in rats. Sildenafil significantly reduced RVSP and MPAP in rats with pulmonary embolism. Sildenafil also improved right heart hemodynamic disturbances and significantly reduced serum TNF-α and IL-6 levels in rats with PTE. These results indicated that the ROCK signaling pathway might be involved in APE. The effects of sildenafil on APE in rats was enhanced by the addition of Y-27632. These results indicated that sildenafil protected against APE by inhibiting ROCK activity.

Taken together, our study demonstrated that the protective effect of sildenafil against APE involved the inhibition of ROCK activity, leading to a reduction in pulmonary vasoconstriction and a decrease in pulmonary arterial pressure. Thus, our study identified sildenafil as a potential therapeutic candidate in the clinical treatment of APE. We plan to conduct an in-depth exploration of the molecular mechanisms by which sildenafil improves APE. These efforts will establish a crucial theoretical foundation for APE and the investigation of potential therapeutic targets.