Pharmacological activation of soluble guanylate cyclase improves vascular graft function

Abstract

 

OBJECTIVES

Ischaemia–reperfusion injury impairs the nitric oxide/soluble guanylate cyclase/cyclic guanosine monophosphate (cGMP) signalling pathway and leads to vascular dysfunction. We assessed the hypothesis that the soluble guanylate cyclase activator cinaciguat would protect the vascular graft against ischaemia–reperfusion injury.

METHODS

In the treatment groups, rats (n = 8/group) were pretreated with either intravenous saline or intravenous cinaciguat (10 mg/kg) 2 h before an aortic transplant. Aortic grafts were stored for 2 h in saline and transplanted into the abdominal aorta of the recipients. Two hours after the transplant, the grafts were harvested and mounted in an organ bath. Vascular function of the grafts was investigated in the organ bath. Terminal deoxynucleotidyl transferase dUTP nick end labelling, cluster of differentiation 31, caspase-3, endothelial nitric oxide synthase, cGMP, nitrotyrosine and vascular cell adhesion molecule 1 immunochemical reactions were also investigated.

RESULTS

Pretreatment with cinaciguat significantly improved endothelium-dependent maximal relaxation 2 h after reperfusion compared with the saline group (maximal relaxation control: 96.5 ± 1%, saline: 40.4 ± 3% vs cinaciguat: 54.7 ± 2%; P < 0.05). Pretreatment with cinaciguat significantly reduced DNA fragmentation and nitro-oxidative stress; decreased the caspase-3 and vascular cell adhesion molecule 1 scores; and increased endothelial nitric oxide synthase, cGMP and cluster of differentiation 31 scores.

CONCLUSIONS

Our results demonstrated that enhancement of cGMP signalling by pharmacological activation of the soluble guanylate cyclase activator cinaciguat might represent a beneficial therapy for treating endothelial dysfunction of arterial bypass graft during cardiac surgery.

INTRODUCTION

Although it is only a simple monolayer, the healthy endothelium is able to respond to physical and chemical signals by producing a wide range of factors that regulate tone, cellular adhesion, smooth muscle cell proliferation and vessel wall inflammation [1]. Alteration of the endothelial function of the bypass graft precedes the development of morphological changes and contributes to early and late vascular graft dysfunction [2].

The nitric oxide (NO)–cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase pathway was demonstrated to have a pivotal role in vascular- and cardioprotection [3–5]. Furthermore, elevated intracellular cGMP levels reduced oxidative stress and free radical release [6]. The soluble guanylate cyclase (sGC) activator cinaciguat modulates the sGC-cGMP signal transduction pathway in an NO-independent manner; thus, the intracellular production of cGMP is increased, even when the sGC enzyme is unresponsive to NO [7]. Cinaciguat produces potent cardioprotection effects in different pathophysiological conditions associated with impaired NO-sGC-cGMP signalling, such as heart failure [8], diabetes or atherosclerosis [9]. The first clinical study of cinaciguat in patients with acute decompensated heart failure demonstrated strong reductions in preload and afterload, with an increase in cardiac output and preservation of renal function [10].

Based on the beneficial effect of the sGC activator cinaciguat on endothelial function, we designed our study to investigate the hypothesis that pretreatment with cinaciguat could reduce the detrimental effect of oxidative stress on endothelial cells and thereby provide vascular protection of arterial grafts in a bypass model.

MATERIALS AND METHODS

Ethical statement

The experimental study was reviewed and approved by our ethical committee for animal experimentation (35-9185.81/G-297/16; approval date: 02 August 2017).

Animals

Lewis rats (weight: 250–350 g; n = 8/group) (Charles River Laboratories, Sulzfeld, Germany) received either saline or cinaciguat (10 mg/kg) intravenously 2 h before the aortic transplant. In the treatment groups, the ischaemic period was standardized to 2 h. The third group served as a non-ischaemia–reperfusion control (Table 1). Procedures concerning animals conformed to the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996).

Table 1:

Treatment protocol

Groups (n = number of animals, N = aortic rings)	Pretreatment 2 h before explantation	Ischaemia (80 min storage and 40 min warm ischaemia)	Reperfusion
(1) Control (n = 8, N = 16)	No pretreatment	No storage	No reperfusion
(2) Saline (n = 8, N = 16)	With saline	Storage in saline	2 h in vivo reperfusion
(2b) Cinaciguat (n = 8, N = 16)	With cinaciguat	Storage in saline	2 h in vivo reperfusion

Aortic transplantation

The experimental model was established according to the reported method [5, 11]. Before injecting the solution (saline or cinaciguat solution, a dose comparable to that used in previous experiments [12]) in the tail vein, we anaesthetized the Lewis rats with isofluran (3% to initiate anaesthesia, 1.75–2.5% to keep the rats anaesthetized). We also used subcutaneous buprenorphine (0.05–0.1 mg/kg) 45 min before the operation. To excise the aortic grafts, the rats were anaesthetized again with isofluran.

The donor aortic arch was explanted and flushed using cold physiological saline solution and stored for 80 min in cold saline (Table 1). After ischaemia, the arterial graft was heterotopically transplanted by 2 end-to-end anastomoses into the abdominal aorta of the recipient (∼40 min warm ischaemia). After 120 min, recipient rats were sacrificed with an overdose of sodium pentobarbital (150 mg/kg, intraperitoneally). The aortic graft segment was explanted from the abdomen, cut into 4-mm wide rings and mounted in organ baths (Radnoti Glass Technology, Monrovia, CA, USA), as previously reported [5, 11].

Graft segments (control group: donor aortic segments; cinaciguat and saline group: the implanted aortic segments after 2 h reperfusion) were fixed in buffered paraformaldehyde solution (4%) and embedded in paraffin. Then, 4-μm-thick sections were placed on adhesive slides as previously reported [5, 11].

In vitro organ bath experiments

The explanted aortic rings were used for functional vascular measurements as previously described [5, 13]. Briefly, the aortic rings (2 aortic rings/animal in each group) were mounted on stainless steel hooks under a tension of 2 g and equilibrated for 60 min [5, 12]. At the beginning of each investigation, potassium chloride (80 mM) was used to prepare the rings for stable contractions. Aortic rings were rinsed and preconstricted with phenylephrine (PE, 10⁻⁶ M) until a stable plateau was reached; relaxation responses were investigated by adding cumulative concentrations of acetylcholine (10⁻⁹–10⁻⁴ M). To test the response of smooth muscle cells, sodium nitroprusside (10⁻¹⁰–10⁻⁵ M) was also used. The sensitivity of the aortic rings to vasorelaxants was also assessed. Half-maximum response values were obtained from individual concentration responses by fitting experimental data to a sigmoidal equation using Origin 7.0 (Microcal Software, Northampton, MA, USA).

Immunohistochemical staining

We performed terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), cluster of differentiation 31 (CD-31), nitrotyrosine, cGMP, caspase-3, endothelial nitric oxide synthase (eNOS) and vascular cell adhesion molecule 1 (VCAM-1) staining as previously described [4, 5, 13].

TUNEL staining: briefly, the sections were incubated with 50 µl of terminal deoxynucleotidyl transferase enzyme and TUNEL reaction mixture for 1 h at 37°C in the dark and then washed with phosphate-buffered saline (PBS). The slides were mounted using 4′,6-diamidino-2-phenylindole-Fluoromount-G™ (SouthernBiotech, Birmingham, AL, USA), topped with a cover glass and analysed under a fluorescence microscope (Fig. 2).

Figure 2:

Aortic histomorphometry and scoring of immunohistochemical stains for nitrotyrosine, TUNEL and caspase-3. *P < 0.05 versus control; ^#P < 0.05 versus cinaciguat. TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.

CD-31 staining: As reported, we used anti-CD-31 mouse IgG (Santa Cruz Biotechnology Inc, Heidelberg, Germany) according to the manufacturer’s protocol. Endothelium-covered areas of the aortic arches were assessed microscopically (Fig. 3).

Figure 3:

Aortic histomorphometry and scoring of immunohistochemical stains for eNOS, CD-31 and VCAM-1. *P < 0.05 versus control; ^#P < 0.05 versus cinaciguat. CD-31: cluster of differentiation 31; eNOS: endothelial nitric oxide synthase; VCAM-1: vascular cell adhesion molecule 1.

Additional staining: briefly, we immersed the slices with (i) xylene, 3 washes for 5 min each; (ii) 100% ethanol, 2 washes for 10 min each; (iii) 95% ethanol, 2 washes for 10 min each; (iv) 70% ethanol, 2 washes for 10 min each; (v) 50% ethanol, 2 washes for 10 min each; (vi) deionized water, 2 washes for 5 min each. Endogenous peroxidase activity was blocked by quenching the tissue sections with 3.0% hydrogen peroxide in PBS for 20 min. Afterwards, the sections were washed by immersing them in distilled water for 5 min. After that, we placed slides in the staining dish with citrate buffer and put them into the microwave (700 W, 20 min) to achieve antigen retrieval. Afterwards, we placed the staining dish on the laboratory bench for 20–30 min before proceeding with the staining procedure. A hydrophobic barrier pen was used to draw a circle on the slide around the tissue. We blocked any non-specific binding by incubating the tissue sections with 2% horse serum in PBS for 30 min. Later, the blocking serum was removed completely. Afterwards, we added the primary antibody (VCAM-1, caspase-3: Novus Biologicals, Littleton, CO, USA; nitrotyrosine: MilliporeSigma, Burlington, MA, USA), which was diluted in 2% horse serum in PBS and was incubated for 2 h. Then, the sections were washed twice with PBS for 10 min each.

After that, we added a biotin conjugated secondary antibody and incubated the mixture for 30 min. Then, the sections were washed twice with PBS for 10 min each. Later, we added ABC-HRP reagent (VECTASTAIN universal elite ABC kit, Vector Laboratories, Burlingame, MA, USA) and incubated them for 30 min. Afterwards, expression of the target protein was visualized using 3, 3-diaminobenzidine (VECTOR DAB kit, Vector Laboratories). We monitored the reaction as the chromogenic reaction turned the epitope sites brown. Then, the sections were washed twice in PBS for 10 min each. Haematoxylin was applied according to the manufacturer’s instructions to counterstain the nuclei. Tissue sections were dehydrated by moving slides through the following solutions twice for 2 min each: (i) 95% ethanol; (ii) 100% ethanol; and (iii) xylene. Finally, we added mounting media to the slides and topped them with coverslips.

Drugs

Cinaciguat was bought from Bayer HealthCare (Wuppertal, Germany). PE, acetylcholine and sodium nitroprusside were bought from Sigma-Aldrich, Steinheim, Germany.

Statistical analyses

All data are expressed as means ± standard error of mean. Intergroup comparisons were performed using one-way analysis of variance followed by a ‘Student’s t-test’ with a Bonferroni correction for multiple comparisons. A value of P < 0.05 was considered statistically significant.

RESULTS

Vascular function of aortic rings

In arterial rings precontracted with PE, acetylcholine-induced concentration-dependent relaxation (Fig. 1). After reperfusion, the maximum endothelium-dependent vasorelaxation was significant in the saline and cinaciguat groups compared to the control. However, pretreatment with cinaciguat improved the endothelium-dependent function of the arterial rings compared to the saline group (Fig. 1; Table 2). There was no significant difference in endothelium-dependent vasorelaxation for vasorelaxation compared to sodium nitroprusside between the groups (Table 2). PE led to concentration-dependent contraction of rings in all groups. The value of maximum contraction for PE is increased in the saline and cinaciguat groups compared to the control group.

Figure 1:

Effect of cinaciguat on the vascular function of arterial graft. (A) Acetylcholine-induced endothelium-dependent and (B) sodium nitroprusside-induced endothelium independent vasorelaxation in the control, saline and cinaciguat groups. Values represent mean ± standard error of mean; *P < 0.05 versus control; ^#P < 0.05 versus cinaciguat.

Table 2:

Quantitative analysis of vascular function

	Control	Saline	Cinaciguat
PE (g)	3.3 ± 0.2	3.9 ± 0.3	3.4 ± 0.4
KCl (g)	4.0 ± 0.1	4.4 ± 0.3	3.7 ± 0.4
Rmax to acetylcholine (%)	96.5 ± 0.8	40.5 ± 2.6a	54.7 ± 1.6a,b
pD2 to acetylcholine	7.4 ± 0.1	6.5 ± 0.2a	6.8 ± 0.1a
Rmax to SNP (%)	100.5 ± 0.3	100.1 ± 0.1	100.2 ± 0.1
pD2 to SNP	8.3 ± 0.1	8.6 ± 0.1	8.5 ± 0.1
Phenylephrine (% of KCl)	81.6 ± 3.6	88.5 ± 5.4	92.1 ± 4.9

Values represent mean ± SEM.

^a P < 0.05 versus control.

^b P < 0.05 versus saline.

KCl: potassium chloride; pD₂: sensitivity to vasorelaxant; PE: phenylephrine; R_max: maximal relaxation; SEM: standard error of mean; SNP: sodium nitroprusside.

The effect of ischaemia–reperfusion injury on arterial graft (rate of oxidative stress and apoptosis)

To investigate the levels of oxidative stress in the vascular wall of the arterial graft, we assessed nitrotyrosine immunoreactivity. The intensity of nitrotyrosine staining in the saline group was higher compared to that in the control and was significantly reduced in the cinaciguat group (Fig. 2).

Furthermore, there was an increase in intensity of the TUNEL-positive nuclei of the arterial graft in the saline group, indicating a high level of DNA fragmentation (Fig. 2). However, we observed significantly decreased DNA strand breaks in the cinaciguat group compared to the saline group.

A severe apoptosis rate (higher caspase-3 positive cells) was observed in the saline and cinaciguat groups (Fig. 2). Pretreatment with cinaciguat significantly reduced the apoptosis rate compared to that in the saline group.

We performed immunohistochemical staining for eNOS to identify the amount of eNOS in the wall of the arterial graft. An enhanced reactivity for eNOS was observed in the arterial rings of the control and cinaciguat groups and was significantly lower in the saline group (Fig. 3).

The inner walls of the aortic segment in the control group showed a 93 ± 8% positive reaction for the CD-31 antigen (Fig. 3). A significantly higher CD-31 positive reaction was demonstrated in the cinaciguat group compared with that in the saline group.

Furthermore, cGMP levels, evaluated by cGMP immunohistochemical analysis, showed a high score for cGMP rings in the control group. However, a significantly lower cGMP score was detected in the saline group. Pretreatment with the cGMP-activator cinaciguat increased the cGMP score in the vascular wall of the arterial graft (Fig. 4).

Figure 4:

Aortic histomorphometry and scoring of immunohistochemical staining for cGMP. *P < 0.05 versus control; ^#P < 0.05 versus cinaciguat. cGMP: cyclic guanosine monophosphate.

Immunohistochemical staining revealed a high expression of VCAM-1 in the saline and cinaciguat groups (Fig. 3); however, VCAM-1 scores decreased significantly in the cinaciguat group compared to that in the saline group.

DISCUSSION

In this study, we presented for the first time the efficacy of the NO and haem-independent sGC activator cinaciguat in the endothelial protection of an artery during surgery. The beneficial effect of the sGC activator cinaciguat was demonstrated by the improved endothelial function, reduced apoptosis rate, lower oxidative stress, decreased VCAM-1 score and higher intracellular cGMP level. Because endothelial function is one of the major factors influencing the early- and late-term patency of vascular grafts after cardiac surgery [14], these findings provide new insights in the protection of the endothelium of bypass grafts to improve the patency of arterial grafts (Fig. 5).

Figure 5:

The role of the soluble guanylate cyclase activator cinaciguat in endothelial protection. Red line: oxidative stress; blue line: normal conditions. cGMP: cyclic guanosine monophosphate; eNOS: cyclic guanosine monophosphate; I/R: ischaemia–reperfusion; NADPH: nicotinamide adenine dinucleotide phosphate; NO: nitrous oxide; ONOO⁻⁻: peroxynitrite; sGC: soluble guanylate cyclase.

During the coronary artery bypass grafting procedure, arterial/venous grafts undergo a period of ischaemia followed by reperfusion, resulting in the generation of reactive oxygen species that cause cytotoxicity of the endothelium and smooth muscle cells [15]. The relative resistance of endothelial cells to ischaemia compared to that of cardiomyocytes was demonstrated by electron and light microscopic examination in dogs [16]. However, another electron microscopy study suggested that endothelial cells are more sensitive than cardiomyocytes to reperfusion injury after cardioplegia [17]. A reduced endothelial responsiveness (impaired endothelium-dependent vasorelaxation) has been described in enhanced nitro-oxidative stress, such as after reperfusion [18], which limits coronary blood flow and triggers a range of problems including platelet and leucocyte adhesion and aggregation. Indeed, in agreement with our previous results on endothelial dysfunction of arterial grafts [5, 19, 20], we also reported that the viability and function of the endothelium of the arterial graft were severely impaired during the early phase of ischaemia–reperfusion injury. These findings correlate with the clinical situation after cardiac surgery, where unfavourable complications (occlusion of the graft) occurred during the first 24 h postoperatively [21].

The potent protective effects of the sGC activator cinaciguat against ischaemia–reperfusion injury was demonstrated in several experimental studies involving myocardial infarction [4, 22], vascular neointima formation [23] or prevention of cardiomyocyte hypertrophy [24]. In addition, pharmacological inhibition of the phosphodiesterase-5 enzyme has been reported to decrease the harmful effect of nitro-oxidative stress and ischaemia–reperfusion injury, subsequently improving vascular graft function in vitro and in vivo [20, 25].

Whether the sGC activator cinaciguat would also be capable of preventing oxidative stress of arterial graft has not been investigated. We report here for the first time reduced nitro-oxidative stress (as evidenced by lower nitrotyrosine, caspase-3 and TUNEL scores), preserved endothelial structure (higher CD-31 score) and significantly improved vascular function of an arterial bypass graft (improved endothelium-dependent vasorelaxation) by pharmacological sGC activation in an in vivo model of surgical revascularisation. Our present data for preconditioning with cinaciguat corresponds to data from previous experimental works.

Costell et al. [26] reported that the binding of NO to the Fe²⁺ of its prosthetic haem group activates the enzyme and leads to conversion of guanosine triphosphate to the intracellular second messenger, cGMP, and causes cell membrane hyperpolarisation and smooth muscle relaxation. During the coronary artery bypass grafting operation, oxidative stress directly deteriorates the structure of sGC resulting in a haem-deficient, NO-insensitive and inactive form of the enzyme. However, the novel sGC activator cinaciguat can activate the sGC enzyme in the inactive form, because it binds to the new regulatory site of the enzyme, thus increasing its cGMP-producing activity (Fig. 5).

The endothelial protective effect of the sGC activator cinaciguat could be mediated by several mechanisms. Evgenov et al. [27] showed that cinaciguat induced the generation of cGMP and evoked vasodilation preferentially in diseased vessels. The markedly increased nitro-oxidative stress and the high level of apoptosis in the vessel wall in the saline group were significantly decreased with pretreatment with cinaciguat, suggesting the inhibition of inflammation and reactive oxygen species-induced injury. As a consequence of lower oxidative stress, the structural integrity of the endothelium (CD-31 expression on the endothelium of the arterial bypass graft) remained intact after pretreatment with cinaciguat.

Endothelial dysfunction takes place within a few min after reperfusion and is also characterized by decreased release/synthesis and/or NO. Xuan et al. [28] demonstrated that the eNOS synthase enhancer restored endothelium-dependent vasorelaxation through up-regulation of eNOS synthase expression and inhibition of production of reactive oxygen species in the human internal thoracic artery. It has also been demonstrated that NO donors could inhibit intimal hyperplasia, which is the major cause of restenosis/occlusion of venous grafts [29]. Based on these findings, we analysed the level of eNOS in the vascular wall by immunohistochemical analysis. As shown in Fig. 3, we demonstrated an elevated level of eNOS content in the cinaciguat group compared with that in the saline group. These findings show that the impaired responsiveness of the endothelium may theoretically be associated with the down-regulation of eNOS and the reduction in the living endothelial cells on the arterial wall (Fig. 3).

Recruitment of leucocytes, the principal effector cells of inflammation, is promoted by the expression of E-selectin, P-selectin and intracellular adhesion molecules (VCAM-1) on the surface of activated endothelial cells [30]. Indeed, in this study, we found a significant up-regulation of the adhesion molecule VCAM-1 after short-term reperfusion injury. However, VCAM-1 expression was lower in the cinaciguat group compared with that in the saline group, which is consistent with previously published results in experiments on vascular graft storage [19].

Limitations

Pretreatment with the sGC activator cinaciguat may have the beneficial effect of reducing the oxidative stress of a vascular graft, thereby improving the vascular function of the bypass graft and consequently the clinical outcome after surgery. However, a limitation of our experimental study is that the endothelial structure of the rat aorta differs from that of human grafts, which may limit the transferability of the results. Nevertheless, our findings (endothelial dysfunction) correlate closely with the results of free arterial tissues (internal thoracic artery) in the pig model.

CONCLUSIONS

Our results demonstrate that enhancement of cGMP signalling by pharmacological activation of the sGC activator cinaciguat may be beneficial in the prevention of endothelial dysfunction of the arterial bypass graft during cardiac surgery.

Conflict of interest: none declared.

ABBREVIATIONS

CD-31

Cluster of differentiation 31

cGMP

Cyclic guanosine monophosphate

eNOS

Endothelial nitric oxide synthase

NO

Nitric oxide

PBS

Phosphate-buffered saline

PE

Phenylephrine

sGC

Soluble guanylate cyclase

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labelling

VCAM-1

Vascular cell adhesion molecule 1

Author contributions

Gábor Veres: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing—original draft; Writing—review & editing. Yang Bai: Data curation; Formal analysis; Investigation; Methodology; Project administration; Software; Validation; Writing—review & editing. Klára Aliz Stark: Investigation; Methodology; Validation; Visualization; Writing—review & editing. Harald Schmidt: Conceptualization; Formal analysis; Investigation; Methodology; Software; Supervision; Writing—review & editing. Tamás Radovits: Conceptualization; Formal analysis; Supervision; Writing—review & editing. Sivakkanan Loganathan: Conceptualization; Investigation; Supervision; Writing—review & editing. Sevil Korkmaz-Icöz: Conceptualization; Investigation; Supervision; Writing—review & editing. Gábor Szabó: Conceptualization; Funding acquisition; Methodology; Resources; Supervision; Writing—review & editing.

Reviewer information

Interactive CardioVascular and Thoracic Surgery thanks Attila Kiss, Pradeep Narayan and the other, anonymous reviewer(s) for their contribution to the peer review process of this article.