Inhibitory effect of adenosine on intimal hyperplasia and proliferation of smooth muscle cells in a carotid arterial anastomosis animal model

Abstract

Purpose

The effect of adenosine (9-β-0-ribifuranosyladenine) on the endothelial cell proliferation and neointimal hyperplasia is investigated in the rabbit carotid artery anastomosis model.

Methods

Twenty-eight New Zealand white rabbits were arranged in four groups of seven animals each. The right carotid arteries of each animal were transsected and re-anastomosed. The left sides remained as control. In Group A, no medication was used. In Group B, subcutaneous Adenosine was applied for 3 days. In Group C, the same dose was applied for 7 days, and in Group D for 21 days. After 28 days, the luminal diameters, luminal areas, intima/media ratios were all measured by using histopathological evaluation.

Findings

The mean luminal diameters and areas of the four groups were smaller than the control ones. Massive thickening of smooth muscle cell proliferation and dense intensifying in the connecting tissues were observed most prominently in Group A, in decreasing degrees within other groups. Intima/media ratio was highest in Group A. Scoring the quantity of e-NOS positive staining also revealed a significant difference between the experimental groups and their control associates.

Conclusion

The process of endothelial cell proliferation and neointimal hyperplasia can be significantly reduced by the use of adenosine.

Keywords

Adenosine endothelial cell proliferation neointimal hyperplasia vasculary anastomosis

Introduction

Autologous venous or arterial grafts are widely used in the area of cardiovascular surgery. These grafts are anastomized in various positions either for the purpose of interposition or bypassing. Whether the anastomosis would be successful and a normal flow pattern would be achieved or not, two different processes in the early and late period can reduce the size of the anastomotic area, leading to stenotic results.¹ These are endothelial cell proliferation and neo-intimal hyperplasia, which can also be defined as smooth muscle cell (SMC) proliferation and extracellular matrix accumulation.^2–4 The process generally begins to develop within the 24 hours.

Adenosine (9-β-0-ribifuranosyladenine) is a molecule having a role in the activation of adenylate cyclase and production of cAMP. By using the A2a and A2b receptors located on the surface of endothelial cells and SMCs, this molecule can lead to inhibition of vasculary SMC proliferation and collagen synthesis, inhibition of thrombocyte aggregation, neutrophilsadhesion and diminishing of extracellular matryx synthesis.^5,6 These processes can impede endothelial cell proliferation and migration. Previous studies had shown the effectiveness of adenosine in this area.⁷ This is the first research study planned for the purpose of investigating the effects of this molecule on intimal hyperplasia and vascular SMC proliferation in vivo conditions. The side effects of adenosine include hyperemia, headaches, sweating, palpitations, chest pain, hypotension, respiratory problems (shortness of breath/dypsnea), chest tightness, and hyperventilation.

Methods

Experimental design and operative procedure

The experiment was performed in compliance with the “Principles of Laboratory Animal Care” formulated by the National Institutes of Health (National Institutes of Health publication no. 96 to 23, revised, 1996). The experiment and animal care protocol was approved by the Ethic Committee for Animal Care, which had been established in Dokuz Eylul University School of Medicine. Twenty-eight New Zealand white rabbits weighing 2.650 ± 0.1 kg were used in the study. Four groups were arranged of seven animals each, by randomisation. After a premedication of 5 mg/kg xylasine, 50 mg/kg intramuscular ketamine administration was used. Additional ketamine doses were done if required, in order to maintain an enough depth of anesthesia, allowing spontaneous respiration, and without endotracheal intubation. The right carotid artery of each animal was explored. Surgery was performed with the use of an operating microscope. After a heparinization with a dose of 100 IU/kg, the artery was clamped 15 mm away from the anastomosis site. The artery was transected and re-anastomosed by using 8/0 polypropylene sutures. Interrupted suture technique was used The patency of the artery was controlled with hand-held doppler ultrasound. The left sides remained as control.

In Group A, no medication or additional material was used. In group B, subcutaneous Adenosine was applied for 3 days in the dose of 1 mg/kg-day. In Group C, the same dose was applied for 7 days, and in Group D for 21 days. Standard diet and water were provided ad libitum.

After 28 days, the animals were sacrificed. Euthanasia was performed with an overdose of ketamine and xylazine. The carotid arteries of both sides were excised, after being fixed in 10% formaldehyde with pressure perfusion technique at 100 mmHg.The specimens included the anastomosis side and the vascular tissue near anastomosis (approximately 5 mm).

Histopathological evaluation

After fixation of the vessels in the 10% formaldehide solution, they were buried in the paraffin wax. Paraffin blocks were placed in a rotary microtome (Leica RM 2135, Leica Instruments, Nussloch, Germany) with disposable metal microtome blades (Type S35, Feather Company, Osaka, Japan) to obtain serial coronal sections of 5-µm thickness. Three chosen transverse sections from each sample were stained with two different stains: Haematoxylin-eosin (H&E) and Masson’s trichrome (MT). The specimens were examined in the light microscope (Olympus BH-2, Tokyo, Japan). The H&E stained sections were used to evaluate the general morphology of the vessels and MT stain was used to evaluate the collagen content of the matrix. With this technique, increase in blue color in the matrix assesses the increase in connective tissue. With the help of the digital image analysis program (JVC TK-890E, Japan and UTSCSA, Image tool version 3.0), the luminal diameters, luminal areas, intima/media ratios were all measured and the groups were compared in terms of these parameters.

Immunohistochemical analysis

Serial coronal sections of 4–5 µm thickness from paraffin blocks were collected on lysine-coated slides and baked at 60℃ overnight. Tissue sections were deparaffinized and hydrated through xylenes and graded alcohol series and endogenous peroxidase was inactivated in 3% hydrogen peroxide in methanol for 10 minutes at room temperature. Sections were washed with phosphate-buffered saline (PBS) followed by antigen retrieval with 10 mmol/L sodium citrate buffer (pH 6) in a microwave oven for 20 minutes. Vectastain ABC Elite Kit P-6102 was used according to manufacturer’s instructions (Vector Laboratories İnc. Burlingame, USA). After the sections were incubated with diluted normal blocking serum for 30 minutes at room temperature, excess serum from sections were blotted. Then the sections were incubated overnight at 4℃ with the specific primary antibody; anti-eNOS monoclonal antibody 1:100 diluted in buffer (anti-eNOS mouse monoclonal Ab, Genetex, USA). On the next day, appropriate biotinylated IgG secondary antibodies (Vector, Burlingame, CA) were applied for 30 minutes at room temperature. Sections were washed with PBS followed by incubation with VECSTATİN Elite ABC® Reagent for 30 minutes. Sections were washed with PBS followed by chromogenic detection with stable diaminobenzidine (DAB) substrate (Roche Diagnostics GmbH, Mannheim, Germany) for 5 minutes at room temperature and counterstained with Harris haematoxylin, cleared and mounted. Negative control samples in which an equal amount of IgG was substituted for the primary Ab were included in each assay, and were uniformly negative.

Image analysis methods

A computerized video camera based image analysis system (Image Tool, UTHSC Image software for Windows 3.0, Texas University, USA) was used. All available sections (at least three sections per vessel) were analyzed; only sections with obvious technical artifacts related to the staining procedure were excluded. After the staining process completed, sections were examined with a light microscope (Olympus BX-50, Tokyo, Japan) and images transferred to computer using a high-resolution camera (Olympus DP-70, Japan). All sections were digitally photographed.

Semi-quantification of immunostaining data

A grade system was used to score the quantity of e-NOS positive staining. The score was defined as the following: 1, very few positive staining was observed in an image and the staining was mild; 2, positive staining was moderate and between grade 1 and grade 3; 3, strong positive staining was evenly distributed in the whole image; and 0, no immunoreactivity. To maintain consistency of scoring, each section was graded by two person blind to treatments and the average was taken. Digital microscopic images were taken at the area where the positive cells were observed for each vessel section. The average of the scores was used to represent the grade of e-NOS staining for each vessel. All histomorphological analyses described above were performed by two investigators blind to rat’s treatment.

Statistical analysis

The statistical calculations were made with the program of SPSS for Windows, Release 10.0.1 Student Version (SPSS Inc., Chicago, IL, USA). The probability (p) less than 0.05 was considered significant. The data were analysed by using One-way ANOVA, Posthoc LSD, and Mann Whitney U tests. The results in the text and in the tables are expressed as arythmetical mean ± standard deviation.

Results

All the animals survived throughout the study, none of them exhibited neurological deficit or wound infection. All grafts were patent at the time of sacrifice. Figure 1(a) shows a non-transected and non-anastomized vessel segment on the contralateral side of an animal. In the cross-sections of the Group A specimens, massive thickening of the intimal layer was noticed, almost occluding the lumen in some cases and showing some recanalization. SMC proliferation and dense intensification in the connecting tissues can be easily observed in the intimal layer. Despite a slight increase in the thickening of medial layer, statistical significance could not be detected when compared with other study groups. Besides, a prominent layer of fibrous tissue could be noticed encircling the adventitia in this group (Figure 1b). Group B specimens also showed note-worthy thickening of the intimal layer, in addition to significant SMC proliferation; but the findings were less prominent. Though less obvious, the roughness of the intimal layer and protruding pieces into the lumen could be observed similar to the samples in Group A (Figure 1c). Figure 1(d) and (e) shows significantly less prominent findings of intimal hyperplasia and SMC proliferation within Groups C and D.

Figure 1.

(a) A non-transsected and non-anastomosed vessel segment of the contrlateral side. (b) Anastomosed specimen of Group A (without Adenosine) after 28 days. (c) A specimen of Group B, in which 3 days of adenosine had been used. (d) Group C: 7 days of adenosine application. (e) Group D: 21 days of adenosine application.

Luminal diameters

The mean luminal diameters of the four experimental groups were all smaller than the control ones, and significant difference was noticed between them. The mean values and significant differences can be seen in Figure 2. The least luminal diameter was observed in the Group A. The mean diameter of the Group B was significantly higher than that of A (p < 0.05), but lesser than that of Group C. There was also a significant difference between B and C (p < 0.05). An non-significant ascend was noticed between the Groups of C and D (p > 0.05). The widest mean diameter was obtained in the measurements in Group D among the experimental groups, on the other hand, the mean value of D was still significantly smaller than the control values. The numeric data with their standard error values can be seen in the Table 1.

Figure 2.

The differences for the mean luminal diameters between the groups can be seen. The difference is significant between A and B, additionally B and C. While the biggest diameter was observed in Group D, this was significantly smaller than the control values.

Table 1.

Measurement parameters and e-Nos staining scoring values.

	Mean luminal diameter (µm)	Mean luminal area (µm²)	Mean intima/media ratio	e-Nos staining scoring
Group A	407.77 ± 44.031	82.297 ± 13.320	0.64 ± 0.14	2.14
Group B	527.11 ± 14.53	257.525 ± 24.169	0.49 ± 0.05	2.0
Group C	603.26 ± 22.5	257.586 ± 17.480	0.31 ± 0.03	1.57
Group D	673.37 ± 10.72	333.033 ± 12.661	0.27 ± ± 0.04	1.42
Control A	808.61 ± 5.39	420.903 ± 27.494	0.22 ± 0.04	0.85
Control B	809.13 ± 6.78	514.424 ± 32.189	0.22 ± 0.04	1.0
Control C	819.08 ± 6.94	453.180 ± 69.939	0.23 ± 0.04	0.85
Control D	809.42 ± 7.16	498.430 ± 19.280	0.21 ± 0.05	1.0

The values are expressed as the arythmethical mean ± standard error. Groups A, B, C, D take place as the experimental groups, whereas the control ones define the values of the contralateral sides of the same animals.

Luminal areas

A significant shrinkage was observed in the experimental Group A, in which no medication was administered. In Group B, the mean area was significantly wider (p < 0.05). The mean area values were nearly the same for the Group C (p > 0.05), and there was no difference. An upward stepping was noticed as in the Group D and the difference was again significant (p < 0.05) The data can be seen in Figure 3. Also the mean value of D is significantly smaller than the values of the control groups. The numeric data with their standard error values are demonstrated in Table 1.

Figure 3.

This figure shows the mean luminal areas of the groups, with the most significant shrinkage within the Group A. Differences were significant between A and B, C and D, D and control values.

Intima/media ratio

The mean value of the Group A was highest amongst other groups, followed by B, C, and D, consecutively. The differences between A and B and also B and C were significant (p < 0.05). Though findings in Groups C, D, and the control group decreased consecutively, the statistical differences were insignificant (p > 0.05). Figure 4 shows the gradual decline in the mean values of intima/media values corresponding to the groups.

Figure 4.

The biggest intima/media ratio was obtained in Group A, whereas the others were decreasing consecutively. The differences were significant only for the first two groups.

Figure 5 shows the intimal hyperplasia rates in four different experimental groups. Typical increased hyperplasia rate is noticeable especially in Group A.

Figure 5.

Intimal hyperplasia rates in different groups.

Immunohistochemical analysis

Scoring the quantity of e-NOS positive staining revealed a significant difference between the experimental groups (in terms of Kruskal Wallis p = 0.000). Also significant differences were observed between either experimental group and their control associates (in terms of Mann Whitney U test).

Discussion

All types of vascular interventions lead to different types of injuries in the vessel wall. Even in the ideal conditions, including the qualified suture materials and successfull vascular anastomosis in obtaining a good flow pattern, the process of neo-intimal hyperplasia begins to develop within the first 24 hours. This process can decrease the surgical success rates around the middle and late postoperative periods, by reducing the size of anastomotic area, thus leading to re-stenotic results. Unfavorable results with a ratio of 30% have been reported after 2 years following femoro-popliteal bypasses, in which saphenous graft was used. Similar results have been reported in the ratio of 40–50% with the synthetic grafts, and in the ratio of 20–50% following percutaneous peripheric arterial angioplastic procedures.⁸ SMC proliferation and their migration to the intimal layer, extracellular matrix protein synthesis, and their accumulation in the intima are the main culprits of this process.²

Till date several studies have been done concerning the inhibitory effects of various substances on the processes of intimal hyperplasia and SMC proliferation, following surgical anastomosis or angioplastic procedures. Different growth factor inhibitors, selective A2a adenosine receptor agonists, immunosupressives, Ca⁺⁺ channel blocking agents, statins, acetyl salicilic acid, zoledronic acid, rosiglitazone, iloprost, forms of heparin, angiotensin converting enzyme inhibitors can all be reckoned as some of the frequently studied agents.^9–13 Previous in vitro and cell culture studies has shown that adenosine and its receptor agonist can obviously limit the endothelial cell proliferation, adhesion and migration.^7,14–16 Adenosine shows its inhibitory effects on vasculary SMC proliferation by inhibiting thrombocyte aggregation, diminishing neutrophyle adhesion, inhibiting renin-angiotensin system, increasing NO production, increasing cAMP levels via sympathetic nervous system, and preventing neutrophil-mediated cell injury.¹⁵ Adenylate cyclase activation takes place as the result of adenosine A2 receptor stimulation. This leads to increase in cAMP levels, which is known to inhibit vascular SMC proliferation. Selective adenosine A2a receptor agonist (YT-146e) had been used in a study done by Takiguchi et al.⁷ after creating an injury in the femoral artery of rats by photochemical reaction, the agent was used for 3, 7, and 28 days in subcutaneous doses in order to observe its effects on the intima/media areas ratios. Significant decrease in neo-intimal hyperplasia was noticed at the end of 28 days. But there was no difference between the groups after different administration periods. They had linked their findings to the inhibitory effect of this receptor agonist on the conversion of the cells from G1 phase to the S phase in the early period. Our study had been planned for the purpose of investigating the effects of adenosine on intimal hyperplasia and endothelial cell proliferation in vivo conditions.

It has been shown that stimulation of adenosine A2a receptors lead to an increase of NO production from the arterial endothelial cells.¹⁷ Excess NO formation decreases SMC proliferation, extracellular matrix synthesis, and accumulation. One of the causes of significant decrease in neointimal hyperplasia can be linked to increased NO formation in our study. cAMP activation and NO formation both are effective in apoptotic processes of the SMCs.¹⁴ Increase in nitric oxide contributes to a decrease in SMC proliferation, extracellular matrix synthesis and deposition. In a study including baboons, Mattsson and collegues¹⁸ observed a decrease in intimal thickness in PTFE grafts secondary to an increase in blood flow. They related the increase in nitric oxide levels to increased blood flow. For this reason, nitric oxide decreased SMC proliferation and matrix deposition resulting in decrease in intimal hyperplasia. We thought at the end of our study that increase in nitric oxide is one of the effects of adenosine that decreases intimal hyperplasia.

By increasing cAMP, A2b receptor is responsible for apoptosis and inhibition of SMC proliferation. In addition, adenosine increases nitric oxide synthesis. In the same way, nitrix oxide is also shown to be responsible for apoptosis and inhibition of SMC proliferation. It is observed in a study by Peyot and collegues¹⁹ that cAMP increases secondary to the stimulation of A2b receptors by adenosine resultng in smooth musce cell apoptosis and increases adenosine secretion through pro-apopitotic effect.

Most experimental studies of balloon angioplasty traumatization reported lesser differences regarding different administration periods, possibly due to lesser traumatization of the vascular wall limited to intimal layer only. On the other hand, our study revealed more significant differences among groups with different administration periods. This can be attributed to extensive surgical injury to the three layers of the vessel wall. The role of the adventitial layer on the hyperplastic process has also been stated in different rabbit models.²⁰ In a study by More et al.,²¹ they detected that re-endothelialization began 3 days after balloon injury in rats and intimal thickening reached maximum within 1 month due to extracellular matrix accumulation and decreased within 3 months.²¹ All veins implanted to arterial system showed intimal thickening within four-six weeks after implantation and narrows the lumen by approximately 25%. This rarely leads to significant stenosis. This thickening may only be shown with histopathologic studies in the early period.²² We also obtained specimens after 28th day, in which the proliferation processes were at their peak levels. The degree of the proliferation reached such an extreme point that some specimens from Group A were revealing a neo-intimal thickening which nearly obstruct the lumen, or containing some protrusions kissing each other at the centrum of the lumen.

In our study, we administered adenosine for 3, 7, and 28 days after anastomosis. At the end of the study of the luminal area, we observed better results in Group D compared to Groups A, B, and C. The result was statistically suggestive. There was no statistical differences between Groups B and C. When the ratio of intima-media areas are compared, the intimal hyperplasia was significantly lesser in Group D than in Groups A and B but no difference was observed between Groups D and C. There was no statistical difference between Groups C and D. It can be observed that intimal hyperplasia is significantly decreased with adenosine administration for 7 and 21 days, when compared with 3 days. Similarly, more significant results were obtained with the technique of scoring the quantity of e-NOS staining. The method revealed a significant difference between the experimental groups. eNOS expression was highest in the Group A treated animals. Because endothelium overproduces NOS to prevent vascular lesions. Although the results of 3 days adenosine usage is significantly unfavorable than other administration days, it significantly improves when compared with patients not administered with adenosine. The equal results of adenosine usage for 3, 7, and 28 days may be explained as havıng a lesser intimal injury with photochemical reaction than surgical (anastomotic) trauma.

In our study, we did not use a selective adenosine agonist. As seen in the previous two studies, it depicted the effect of adenosine through A2a and A2b receptors. Inhibition of SMC proliferation occured in both studies. Adenosine found to be effective in our study may be secondary to the effect of both A2a and A2b receptor stimulation resulting in inhibition of SMC proliferation.

Planing a study that will include the use of a selective A2a adenosine receptor agonist with nonselective adenosine may better supply the understanding of the results clearly.

Beside all these reasons and results in nonselective adenosine usage in our study, we think that adenosine can be used effectively for the purpose of inhibiting intimal hyperplasia.

Footnotes

Conflict of interest

The authors declare that there is no conflict of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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