The slow releasing hydrogen sulfide donor GYY4137 reduces neointima formation upon FeCl 3 injury of the carotid artery in mice

Abstract

INTRODUCTION:

Neointima formation is closely linked to vascular stenosis and occurs after endothelial damage. Hydrogen sulfide is an endogenous pleiotropic mediator with numerous positive effects on the cardio vascular system.

OBJECTIVE:

This study evaluates the effect of the slow releasing hydrogen sulfide donor GYY4137 (GYY) on neointimal formation in vivo.

METHODS:

The effect of GYY on neointimal formation in the carotid artery was studied in the FeCl₃ injury model in GYY- or vehicle-treated mice. The carotid arteries were studied at days 7 and 21 after treatment by means of histology and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and alpha smooth muscle actin (α-SMA).

RESULTS:

GYY treatment significantly reduced the maximal diameter and the area of the newly formed neointima on both days 7 and 21 when compared to vehicle treatment. GYY additionally reduced the number of PCNA- and α-SMA-positive cells within the neointima on day 21 after FeCl₃ injury of the carotid artery.

CONCLUSIONS:

Summarizing, single treatment with the slow releasing hydrogen sulfide donor GYY reduced the extent of the newly formed neointima by affecting the cellular proliferation at the site of vascular injury.

Keywords

Pleiotropic mediator gaseous transmitter arterial stenosis basic science vascular disease

1 Introduction

Neointima hyperplasia, also known as neointima formation, occurs after endothelial damage [1]. Hyperplasia starts with an influx of inflammatory cells at the site of injury followed by appearance of smooth muscle cells and myofibroblasts that produce an abundant extra-cellular matrix. This happens after endovascular angioplasty in stenosed vessels, i.e. after percutaneous coronary interventions, carotid artery stenting or percutaneous transluminal angioplasty in other peripheral vessels as well as in both synthetic and autologous bypass grafts and in arterio-venous shunts for haemodialysis. Due to the formation of this obstructive neointima patients are threatened by restenosis after angioplasty, by stenosis and occlusion of bypasses or by arterio-venous fistulas [2, 3]. Cardiovascular disorders, like peripheral artery disease, ischemic heart attack or stroke are frequently associated with renal insufficiency and are the leading cause of death in today’s society. Therefore, new therapeutic approaches are needed to reduce neointima formation and in turn to prevent vascular stenosis and subsequent ischemia of the respective tissue.

Due to the complexity of neointima hyperplasia, that involves numeral types of cells as well as paracrine and endocrine factors, endogenous mediators with a pleiotropic character are of great interest to prevent pathologic neointima formation. The gaseous mediator hydrogen sulfide (H₂S) has such a multifaceted activity spectrum. It is known as the third endogenous gaseous transmitter next to nitric oxide and carbon monoxide and is enzymatically synthetized from homocysteine metabolism by cystathione-ß-synthase, cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase [4]. H₂S affects the cardiovascular system as an endothelial derived relaxing factor, achieving vasodilatation via depolarisation of vascular smooth muscle cells by activation of subunits from adenosinetriphosphate-depending potassium channels and therefore possesses important physiological and pharmacological functions in regulation of blood pressure [5, 6]. Exogenously supplied H₂S further exerts anti-thrombotic and pro-thrombolytic actions that are based on an impaired platelet activation and platelet-leukocyte interaction [7, 8]. Vice versa a lack of H₂S due to a CSE knockout leads to development of hypertension [9] and atherosclerosis in both mice [10] and humans [11, 12]. Hyperhomocysteinemia further enhances vascular neointima formation and accelerates atherosclerosis [13, 14]. It was recently shown that the repetitively injected salty H₂S donor NaHS has the potential to reduce neointima formation in different in vivo models [15 –20].

This study aimed to test whether a single treatment with the established H₂S donor GYY4137 (GYY, Enzo Life Sciences GmbH, Lörrach, Germany) [21], characterized by a continuous liberation of the volatile mediator over time, could affect neointima formation in the murine carotid artery after FeCl₃ damage.

2 Materials and methods

2.1 Vascular injury model

All experiments were conducted in in accordance with the European Directive (2010/63/EU), and this procedure was approved by the local animal care committee (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, reference number: 7221.3-1.1-029/17). Male C57BL/6J Tyr mice were used at an age of 8-24 weeks and a body weight (bw) of 20-30 g. The animals were housed in standard laboratories with a 12 h light-dark cycle and had free ad libitum access to standard laboratory food and water.

Mice were anaesthetized by intraperitoneal (ip) injection of ketamine (90 mg/kg bw) and xylazine (25 mg/kg bw) and subjected to carotid artery injury using 10% ferric chloride as previously described [22 –25]. Briefly, the right carotid artery was carefully separated from the accompanying nerve and vein and any adventitial tissue, which might prevent diffusion of the ferric chloride solution, was removed by forceps. The carotid artery was injured by placing a 0.5–1.0 mm strip of filter paper saturated with 10% ferric chloride solution onto the adventitia for 3 min.

2.2 Treatment

After carotid injury the animals were separated in two groups that were either treated intravenously (iv) with GYY or its vehicle. Therefore, the respective right jugular vein was punctured for treatment with either GYY (133 μmol/kg bw, dissolved in 30% DMSO, n = 10) or the vehicle in an equimolar volume (n = 10). GYY, morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate, releases H₂S depending on pH and temperature over a sustained period. While ice limits H₂S release, acidic conditions promote H₂S release due to an electrophilic attack directed against the thione ring structure of GYY [21].

Then the wound was carefully sutured with prolene 6-0 (Ethicon Johnson & Johnson Medical GmbH, Norderstedt, Germany) and the mice returned to their cages.

2.3 Histology and immunohistochemistry

One or three weeks after injury, mice were anesthetized as described above and carefully perfused with physiological saline and fixed with phosphate buffered formalin (4%) through the left ventricle. Several 4 μm thick cross sections of the carotid artery were done in 200 μm intervals.

Neointima formation was quantified per specimen in hematoxylin-eosin (HE) stained sections, in particular by assessing neointima area and thickness using computerized image analysis software (cellSens, Olympus, Shinjuku, prefacture Tokyo, Japan), as previously described [23, 24]. The results were averaged for each animal. The area of neointima was measured from the highest point of the area to the internal elastic lamina.

Paraffin sections of carotid arteries at one and three weeks after arterial injury were analysed for the presence of α-actin-positive smooth muscle cells in the neointima lesion (α-SMA; ab5694, abcam, Cambridge, UK). Proliferating cells were detected using anti-proliferating cell nuclear antigen (PCNA; ab29, abcam, Cambridge, UK) antibody. α-SMA- and PCNA-positive cells were manually counted and expressed as the percentage of total cell nuclei within neointima lesion.

2.4 Statistical analysis

Differences between groups were assessed using one-way ANOVA, followed by the appropriate post hoc comparison test. For post hoc comparison of the maximal diameter of the neointima the Dunn’s Method was used while the area of the neointima and the number of PCNA-positive cells were compared by the Holm-Sidak method. For comparison of α-SMA-positive cells a t-Test followed by Bonferroni correction was applied. All data were expressed as mean and standard error of mean (SEM, in case of normality) or median and interquartile range (i.e. 25% and 75% percentile median and interquartile range (i.e. 25% and 75% percentile, in case of failing normality) and overall statistical significance was set at p < 0.05.

3 Results

3.1 Histological analysis of neointima formation

Focal arterial inflammation that results in remodelling of the vascular wall is an initial step leading to arterial disease. Using the neointima injury model the developed lesions were morphometrically analysed (Fig. 1). In vehicle treated animals the median area of neointima (Table 1) significantly increased from day 7 to day 21 (p < 0.001). Compared to the vehicle, GYY treatment significantly reduced neointima formation significantly on both day 7 and day 21 (p < 0.05 and p < 0.001 respectively).

Fig.1

Morphometric analysis of neointima formation after FeCl₃ treatment of the left carotid artery. Quantification of neointima area (A) and maximal diameter of neointima (B) in vehicle- or GYY-treated mice after carotid artery injury using 10% FeCl₃. Representative images of HE-stained cross sections from carotids of vehicle- or GYY-treated animals (C-F). Data are given as box plots indicating the median with the 25 th and 75 th percentiles. ANOVA followed by appropriate post hoc comparison test. ^∗p < 0.05 vs day 7 ^∗∗p < 0.001 vs day 7, ^#p < 0.05 vs vehicle, ^##p < 0.001 vs vehicle, n = 6-7 independent experiments. Bars represent 100 μm. Arrows indicate the location of the neointima formation upon FeCl₃ injury.

Table 1

Morphometrical analysis of neointima formation upon FeCl₃ injury of the common carotid artery. Data of neointimal area and thickness are displayed as median and interquartile range (25% and 75% percentile). *p < 0.05 vs day 7, **p < 0.001 vs day 7, ^#p < 0.05 vs vehicle, ^##p < 0.001 vs vehicle, n = 6-7 independent experiments

	Neointimal area (μm²)		Neointimal thickness (μm)
	Vehicle	GYY	Vehicle	GYY
Day 7	26074 (16996–28214)	8590 (7878–10132)^#	128.3 (93.7–163.8)	43.3 (36.9–50.3)^#
Day 21	73500 (48545–75705)^**	14071 (9752–22829)^##	154 (100.5–250.1)^*	56.3 (40.5–71.3)^#

Analysis of maximal neointima thickness (Table 1) revealed an increase over the course of time from day 7 to day 21 in vehicle treated animals (p < 0.05). In GYY-treated animals maximal neointima thickness increased as well over the course of time but significantly less compared to vehicle treatment (p < 0.05 at both timepoints).

3.2 Immunohistochemical analysis of α-SMA and PCNA expression of neointima formation

Assessment of α-SMA expression (Fig. 2A) in the area of neointima formation at day 7 and 21 after ferric chloride injury of the carotid artery showed a significant increase over the course of time from day 7 to day 21 in vehicle-treated animals (p < 0.01, Table 2). In contrast to vehicle treatment GYY-treated animals showed a delayed and attenuated increase of α-SMA-positive cells in the neointima formation on day 21 (p < 0.05).

Fig.2

Cellular composition of neointima formation. Quantification of α-SMA-positive (A) and PCNA-positive cells (B) in neointimal lesions after carotid artery injury using 10% FeCl₃. Representative images of immunohistochemical detection of α-SMA (C and D) and PCNA (E and F). Arrow heads indicating PCNA-positive cells. Data are given as mean±SEM. ANOVA followed by appropriate post hoc comparison test. ^∗p < 0.05 vs day 7, ^∗∗p < 0.01 vs day 7, ^#p < 0.05 vs vehicle, ^##p < 0.01 vs vehicle. n = 6–8 independent experiments. Bars represent 20 μm.

Table 2

Immunohistochemical analysis of proliferative activity within the neointima. Data of α-SMA- and PCNA-positive cells are given as mean±SEM. *p < 0.05 vs day 7, **p < 0.01 vs day 7, ^#p < 0.05 vs vehicle, ^##p < 0.01 vs vehicle, n = 6-8 independent experiments

	α-SMA-positive cells		PCNA-positive cells
	Vehicle	GYY	Vehicle	GYY
Day 7	17±3	11±1	6±1	4±2
Day 21	35±4^**	18±2^*#	13±1^**	5±2^##

The PCNA expression in the neointima formation (Fig. 2B) also increased over the course of time in vehicle-treated animals from day 7 to day 21 (p < 0.01, Table 2). However, GYY treatment showed no relevant effect on PCNA expression on day 7 (p = 0.79) but significantly reduced the number of PCNA-positive cells in the neointima on day 21 when compared to vehicle-treated animals (p < 0.01).

4 Discussion

In this study it was shown that the slow releasing H₂S donor GYY reduces neointima formation in vivo and decreases the number of α-SMA- and PCNA-positive cells within the neointima reflecting an anti-proliferative effect at the site of FeCl₃-induced endothelial damage.

In the last decade beneficial effect of H₂S on neointima formation were described by other groups using NaHS for donation of H₂S [15 –20].

Hu et al. treated mice ip with 50 μmol/kg bw before and once daily for 28 days after wire injury of the carotid artery. In this setting NaHS significantly reduced intima/media ratio as an expression of reduced intima formation by over 50% when compared to saline treatment [20].

Lin et al. also used NaHS for H₂S donation at 17.86 μmol/kg bw, given once daily after ligation of the carotid artery. After 28 days the neointimal hyperplasia and the atherosclerotic lesions were found significantly reduced when compared to saline treatment [19].

Qin et al. showed that ip pretreatment with 0.056, 0.56, 2.8 or 5.6 mg/kg bw NaHS once daily for three days dose-dependently enhances blood flow in the carotid artery. Thereby the maximal effect was observed at 0.56 mg/kg bw being equivalent to 10 μmol/kg bw. It was further shown that also the thrombus formation was reduced most effectively by this NaHS dosage 24 h after FeCl₃ injury. NaHS pretreatement before vascular injury additionally reduced the number of apoptotic endothelial cells upon FeCl₃ injury [18].

In parallel Yang et al. showed that daily ip administration of a markedly higher NaHS dosage of 60 μmol/kg bw significantly reduced neointima formation compared to controls in both wildtype and cystathionine gamma-lyase deficient mice [16].

Meng et al. treated rats ip with 30 μmol NaHS per kg bw before and once daily after balloon injury of the carotid artery and showed that NaHS reduced neointima formation 4 weeks after vascular injury [15].

It was further shown that daily ip injection of 3.14 mg/kg NaHS, equivalent to 56.07 μmol/kg bw, if started immediately before balloon angioplasty significantly reduces both intimal area and intima/media ratio 28 days after angioplasty in the femoral artery of rabbits [17].

In summary the used concentration of NaHS in these studies range from about 10 to 60 μmol/l. With respect to Li et al. 20 μmol/l NaHS lead to H₂S plasma concentration of about 50 μmol/l [20]. Therefore, it can be assumed that most of the previously published studies used higher H₂S concentrations [15–17 , 20] when compared to the GYY concentration of 133 μmol/l in this study, liberating about 50 μmol/l H₂S in the steady state [21]. So, it might be concluded that GYY is the more potent H₂S donor than NaHS as single GYY treatment after vascular injury was sufficient to reduce neointima formation and proliferative activity in the neointima, whereas NaHS has to be applied daily. This further accounts for the suitability of GYY with its continuously H₂S releasing kinetics. Until now no study analysed when H₂S plasma concentration reaches baseline levels after GYY administration. Li et al. showed that H₂S plasma concentrations are still increased 180 minutes after iv or ip treatment of rats [21]. In addition, Lee et al. described H₂S levels maintained above baseline in vitro over 7 days. This study was on different cancer cell lines and it was further concluded that GYY is more effective in killing of cancer cells than NaHS [26]. Although it is not known up to now how long GYY elevates H₂S plasma levels in vivo it can be assumed that an early treatment of vascular lesions with GYY affects proliferative processes on the long term and accounts for a significant reduction of neointima formation even 21 days after vascular injury.

As the vehicle of GYY contains 30% DMSO it might be discussed whether DMSO itself affects neointima formation. However, since both GYY and the vehicle contain the same DMSO concentration the described results can be attributed to the action of H₂S.

Due to the already comprehensively described mechanisms behind the effects of H₂S on neointima formation this study only addressed the intimal α-SMA and PCNA, expression that were both reduced upon GYY treatment, accounting for impaired cellular proliferation upon H₂S exposure. This is in line with the findings of Ma et al. that also described a reduction of PCNA-positive cells beside anti-apoptotic effects on vascular smooth muscle cells in neointima lesions after NaHS treatment [17]. Other beneficial effects of H₂S on vascular repair include reduction of β-1integrin and MMP-2 expression at the side of vascular injury [16], reduction of thrombus formation upon FeCl₃ injury and preservation of endothelial integrity by inhibition of endothelial cell apoptosis [18]. H₂S further enhances vasorelaxation in balloon-injured carotids, reduces cellular proliferation in the neointima [15] and accelerates re-endothelialization by induction of endothelial nitric oxide synthase and therefore mobilization of bone marrow-derived endothelial progenitor cells [20]. In addition, H₂S inhibits cytokine and chemokine production in endothelial cells by induction of angiotensin converting enzyme-2 and subsequent increases in the anti-atherosclerotic angiotensin (1–7) [19].

From all these data it can be concluded that both pre-treatment with H₂S donors or treatment after vascular damage are effective to reduce vascular injury and neointima formation at early and later time points after the injury due to the multifaceted actions of H₂S.

Recently published studies demonstrate the role of H₂S synthesis and plasma level for cardiovascular disease in human as on one hand decreased endogenous H₂S production may predispose patients with stable coronary artery disease to rupture of vulnerable plaque and thus to acute coronary syndrome, possibly in relation to circulating monocyte phenotype transformation from with differential expressions of chemokine receptors CCR2 and CX3CR1 [27]. On the other hand, chronic haemodialysis is associated with increased cardiovascular risk factors and mortality which might be due to reduced H₂S plasma levels in these patients [28]. H₂S can further inhibit the progression of atherosclerosis by a reduced haemoglobin oxidation in atherosclerotic lesions. In this context in was further shown that GYY reduces aortic atherosclerotic plaques in mice by s-sulfhydration-directed reduction of sirtuin-1 degradation [29] and improves aortic endothelium-dependent relaxation [30]. In parallel H₂S abrogates heme- and haemoglobin-mediated oxidation of human atheroma-derived lipids in an atheroprotective fashion [31].

On one hand this illustrates the potential of the endogenous mediator H₂S for prevention and treatment of atherosclerosis. On the other hand, it shows its feasibility to improve long term patency after endothelial damage or angioplasty. Treatment of reduced H₂S synthesis and plasma levels in patients with cardiovascular disease could be realised by an induction of endogenous H₂S synthesis or by exogenous H₂S supply using respective donors [32, 33]. Thereby, H₂S donors with continuous release kinetics like GYY seem to have advantage over sulfide salts like NaHS.

5 Acknowledgments

The authors kindly thank Antje Ladwig (Centogene, Rostock, Germany) and the technicians Berit Blendow and Dorothea Frenz (Institute for Experimental Surgery, Rostock University Medical Center) for their excellent technical assistance.

6 Funding

This work was supported by the FORUN program, Rostock University Medical Center, Rostock, Germany (889012).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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