Arq Bras CardiolabcArquivos Brasileiros de CardiologiaArq. Bras. Cardiol.0066-782X1678-4170Sociedade Brasileira de Cardiologia - SBCWmSCjxTk5dfNFd4Frq9j4jtS0066-782X2019000100003S0066-782X201800502010110.5935/abc.20180247Original ArticleRapamycin Combined with α-Cyanoacrylate Contributes to Inhibiting Intimal Hyperplasia in Rat ModelsTianshu-ChuCongrong-GaoZhiwei-zhaoFei-LingAyu-SunYuanbiao-ZhengJing-Cao0000-0002-9424-6049GeJianjunAnhui Medical UniversityHefeiChinaAnhui Medical University, Hefei - ChinaMailing Address: Jianjun Ge, 17 # Lujiang Road, Hefei 230001, P.R. - China. E-mail: SLGejianjun@outlook.com
Author contributions
Conception and design of the research: Tianshu-Chu, Congrong-Gao, Zhiwei-zhao; acquisition of data: Tianshu-Chu, Fei-Ling, Ayu-Sun; analysis and interpretation of the data: Tianshu-Chu, Jing-Cao, Yuanbiao-Zheng, Jianjun Ge; statistical analysis: Tianshu-Chu, Congrong-Gao, Zhiwei-zhao; writing of the manuscript: Tianshu-Chu, Congrong-Gao, Fei-Ling, Ayu-Sun; critical revision of the manuscript for intellectual content: Tianshu-Chu, Congrong-Gao, Zhiwei-zhao, Fei-Ling, Ayu-Sun, Jianjun Ge.
Potential Conflict of Interest
No potential conflict of interest relevant to this article was reported.
17122018120191121310061120172606201823072018This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly citedAbstractBackground:
Vein graft restenosis has an adverse impact on bridge vessel circulation and patient prognosis after coronary artery bypass grafting.
Objectives:
We used the extravascular supporter α-cyanoacrylate (α-CA), the local application rapamycin/sirolimus (RPM), and a combination of the two (α-CA-RPM) in rat models of autogenous vein graft to stimulate vein graft change. The aim of our study was to observe the effect of α-CA, RPM, and α-CA-RPM on vein hyperplasia.
Methods:
Fifty healthy Sprague Dawley (SD) rats were randomized into the following 5 groups: sham, control, α-CA, RPM, and α-CA-RPM. Operating procedure as subsequently described was used to build models of grafted rat jugular vein on carotid artery on one side. The level of endothelin-1 (ET-1) was determined by enzyme-linked immunosorbent assay (ELISA). Grafted veins were observed via naked eye 4 weeks later; fresh veins were observed via microscope and image-processing software in hematoxylin-eosin (HE) staining and immunohistochemistry after having been fixed and stored” (i.e. First they were fixed and stored, and second they were observed); α-Smooth Muscle Actin (αSMA) and von Willebrand factor (vWF) were measured with reverse transcription-polymerase chain reaction (RT-PCR). Comparisons were made with single-factor analysis of variance and Fisher’s least significant difference test, with p < 0.05 considered significant.
Results:
We found that intimal thickness of the α-CA, RPM, and α-CA-RPM groups was lower than that of the control group (p < 0.01), and the thickness of the α-CA-RPM group was notably lower than that of the α-CA and RPM groups (p < 0.05).
Conclusion:
RPM combined with α-CA contributes to inhibiting intimal hyperplasia in rat models and is more effective for vascular patency than individual use of either α-CA or RPM.
Coronary artery bypass grafting (CABG) is one of the main therapies for coronary heart disease. However, 40% of bridge vessels are totally obstructed and 30% of bridge vessel blood flow is reduced after CABG, which seriously affects patient survival and prognosis.1,2 Mechanisms of restenosis include thrombosis, intimal hyperplasia, and atherosclerosis. Immigration of endothelial cells and vascular smooth muscle cells is vital for intimal hyperplasia, which is the main cause of restenosis.3
Although drugs for inhibiting cytokinin and cell cycle regulation contribute to inhibiting intimal hyperplasia, the systemic side effects are harmful for patients. Therefore, local application is very important. Rapamycin (sirolimus) is widely used for anti-rejection after transplant operations, and drug-eluting stents are widely used in coronary arteries. Researchers have found that applying rapamycin to grafted veins is effective in inhibiting intimal hyperplasia by inhibiting proliferation and promoting apoptosis of smooth muscle cells.4
In 1963, Parsonnet et al. observed that perivenous supporters were effective for vascular patency.5 Subsequently, basic and clinical researchers found that perivenous supporters could enhance patency rates by reducing intimal hyperplasia in grafted veins. α-CA, which is liquid at room temperature, is harmless to the human body. Degradation time is 1-3 months, depending on the dosage. α-CA is used in surgery for bleeding closure and wound binding.6
α-CA and RPM are usually used as perivenous supporters and local applications, respectively. We innovatively investigated the pathophysiological process of neointima hyperplasia in grafted veins after CABG via rat models of autogenous vein graft. We are interested in finding new methods to inhibit intimal hyperplasia.
MethodsReagent and method
α-CA (99% n-octyl-α-cyanoacrylate + n-butyl-α-cyanoacrylate) was purchased from Beijing Fuaile Science and Technology Development Co. (Beijing, China). RPM was purchased from Selleck Company. We dissolved 8 mg of RPM in 1 ml of α-CA (taken by pipette) in a sterile EP tube. A magnetic stirrer was then used to mix them to α-CA-RPM of 8 mg/ml, stored in a refrigerator between 2-8°C. RPM was hydrosolvent, prepared by the same method.7
Models and groups
Fifty SD rats (provided by Anhui Lab Animal Research Center and identified by the medical ethics committee of Anhui Medical University), male and female, aged 10-12 weeks, weighing 220-280 g, were randomized (completely randomized design) into 5 groups, each group containing 10 rats, and fed for 4 weeks after operation. Operating procedure and sample size were determined according to pilot experiments and previous studies, as subsequently described.
Operating procedure: an intraperitoneal injection of 10% chloralic hydras was used to anaesthetize rats. Heparin (700 IU/Kg) was injected through the caudal vein to induce heparinization. A vertical incision of approximately 1 cm was made in the middle of the neck (deflected to the operation side), and veins were dissociated on one side. Epitheca of 1-2 mm were taken from 20G red arterial puncture needle (BD Company), used as cannula. The carotid artery was isolated until the branches. Then two suture traction lines and hemoclips were placed at both ends of the artery to block blood flow. The middle of the artery was isolated and turned carefully to 1-1.2 mm above the cannula. A 6/0 silk suture was used to knot and fix in order to isolate the vein from arteries; we were then able to open vascular clamps. The incision was sutured after we verified that the pulse of the grafted vein was normal and there was no bleeding. We checked rats’ vital status and incisions every day. We maintained the environment cool, changed their bedding regularly, and gave them sufficient fodder and water. Three days after the operation, 400,000 IU penicillin were delivered via intramuscular injection to every rat on a daily basis.
Sham group: we merely simulated the operation process. Jugular veins were dissociated and collateral vessels were ligatured, without dividing or transplanting; Control group: jugular arteriovenous graft on the same side; α-CA group: jugular arteriovenous graft and application of α-CA glue to grafted veins; RPM group: jugular arteriovenous graft and application of RPM to grafted veins; α-CA-RPM group: jugular arteriovenous graft on the same side and application of α-CA-RPM to grafted veins.
Collection of samples
Blood samples were taken preoperatively at 0 h and postoperatively at 12 h, 36 h, and 4 weeks after operation. Serum was collected by centrifugation and stored at -80°C until cytokine analysis. Four weeks later, we collected each group’s vein sample. Fully anaesthetized rats were fixed on the operating table, heparinized as previously described and operated in the same way through the same path. We observed grafted veins’ shapes and circulation and ligatured and isolated vessels at both ends of cannulas; we then removed intact and fresh veins and washed lumens fully with normal saline. Samples with HE staining and immunohistochemistry were placed in microtubes full of paraformaldehyde. Samples with RT-PCR were placed in microtubes full of RNA-EZ regents and then kept in the fridge at -80°C. Rats were euthanized by cervical dislocation method and handled properly.
Enzyme-linked immunosorbent assay for ET-1
ET-1 was determined by ELISA Kits (R&D, USA) using 50 µl of serum for the assay. Three measurements were performed for each blood sample. The ELISA plate was read at 450 nm in a plate reader.
Histological examination of graft tissue
Immersed in formalin, grafted veins were cut into 4 mm sections. Hematoxylin-eosin (HE) staining was subsequently performed using a hematoxylin and eosin staining kit (Beyotime Biotechnology, Shang Hai, China). Olympus microscope image acquisition system was used to collect section images (×100 objective lens) and measure intima thickness. Two independent researchers performed the measurements and data analysis. Sections were selected randomly from grafted and non-grafted veins; we then measured 16 points’ thickness and calculated the mean. Three sections were selected and measured from every rat. We then calculated intima thickness.
Determination of proliferation index
Tissue sections were incubated with the immunohistochemistry analysis kit for proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight. After washing with phosphate-buffered saline (PBS) (DAKO, Glostrup, Denmark) and incubating with the secondary antibody, color was developed using the DAB system. The tissue sections were dehydrated and installed on slides. All images (×200 objective lens) were captured by Olympus microscope image acquisition system and SPOT Digital Camera (Diagnostic Instruments, Sterling Heights, MI). PCNA-positive cells were counted in the intima. A total of 10 observation views were used to calculate the average percentage of PCNA-positive cells for each rat.
RT-PCR
Total RNA of the vessel tissues was isolated by the TRIzol Kit (Life Technology, USA). The RNA was reverse-transcripted to cDNA using the RNA reverse transcription kit (Promega, USA). 2 µg total RNA and 1 µl of random primer were denatured at 70°C for 10 min and annealed at 4°C for 10 min, and then 2 µl of 10× buffer, 2 µl of MgCl2 (20.8 mol/l) and 1 µl of reverse transcriptase were added to the reaction system. Double distilled water (ddH20) was added to bring the volume to 20 µl. The condition for cDNA synthesis was 37°C for 1 h and 4°C for 10 min. The PCR also contained 10 µl 2× SYBR Mixture (Takara, Japan), 7 µl ddH20 and 1 µl forward and 1 µl reverse primers. The PCR conditions were 95°C for 5 min, 95°C for 15 s, 60°C for 60 s, and 40 cycles. The sequences of the primers used for RT-PCR were as follows: Forward, 5'-CATCTCCGTGGTCCTGAAGT-3' and reverse, 5'-GGCAAGGGAAACGTCTAGTG-3' for von Willebrand factor; forward, 5'-CAGAGTCCAGCACAATACCAG-3' and reverse, 5'-GACCCAGATTATGTTTGAGACC for α-Smooth Muscle Actin ; and forward, 5'-ACATGAATGACCTCGTCTCTGA-3' and reverse, 5'-CCTCTTCTTCTGCCTCCTCTCC-3' for GAPDH. The instrument for quantitative real-time PCR was purchased from ABI (USA).
Statistical analysis
All data were analyzed using statistical analysis software SPSS 17.0. Data are presented as mean ± standard deviation. Because data showed a normal distribution, comparison among multiple groups was analyzed by single-factor analysis of variance (ANOVA) and comparison between two groups was conducted by Fisher's least significant difference (LSD) test. A value of p < 0.05 was considered statistically significant.
ResultsRats survived well 4 weeks after operation
Operating procedure as previously described was used to build models of grafted rat jugular vein on carotid artery on one side. Post-operation, the transplanted veins were well filled and the blood vessels beat well; the glue was spread evenly over the surface of the veins in the α-CA and α-CA-RPM group. Rats’ vital status and incisions were checked every day. Subsequently, we found that one rat in the RPM group and one rat in the α-CA group had died of low temperature 2 weeks after operation and the other rats survived and recovered well with strong pulse in grafted veins. The rats were euthanized 4 weeks after surgery; notably, there were only 2 rats who presented venous occlusion, one in the α-CA group and one the RPM group. Correspondingly, blood flow in other grafted veins was patent. Veins in the sham group slightly expanded. What is more, veins in the control group had new granulation tissue, thickened tubes, edema, and light stiffness; however, veins in the α-CA, RPM, and α-CA-RPM groups had few fresh tissues which were easily separated, with no obvious expansion and clear boundary from the surrounding, and the glue was not fully degraded (Figure 1).
Rats survived well 4 weeks after operation. Operating procedure as previously described was used to build models of grafted rat jugular vein on carotid artery on one side. Post-operation, the transplanted veins are well filled and the blood vessels beat well, and the glue was spread evenly over the surface of the veins in the α-CA and α-CA-RPM groups (arrow). Four weeks after operation, veins in the sham group slightly expanded; the control group had new granulation tissue, thickening tubes, edema and light stiffness; the α-CA group had few fresh tissues which were easily separated, with no obvious expansion and clear boundary from the surrounding, and the glue was not fully degraded (arrow); the RPM group had clear boundaries from the surrounding tissue, and they were fresh and no obvious expansion. The general form of α-CA-RPM group was similar to α-CA group.
α-CA-RPM reduced intimal thickening of the vein graft
In order to observe what impacts each group’s intervention had on intimal hyperplasia, grafted veins were stained with HE 4 weeks after surgery. Afterwards, we used computer image analysis system to analyze intimal hyperplasia. This showed that the intima of the control group was strikingly thicker than that of α-CA group, RPM group, and α-CA-RPM group; the difference was statistically significant (91.3 ± 3.9, 133.6 ± 8.0, 50.6 ± 5.4 vs. 233.6 ± 29.1 µm, p < 0.01; Figure 2B, C, D, E and F); the intima of the RPM group was thicker than that of α-CA group; the difference was statistically significant (133.6 ± 8.0 vs. 91.3 ± 3.9 µm, p < 0.05; Figure 2C, D and F); the intima of α-CA group and RPM groups was thicker than that of α-CA-RPM group; the difference was statistically significant (50.6 ± 5.4 vs. 91.3 ± 3.9 µm, 133.6 ± 8.0 µm, p < 0.05; Figure 2 C, D, E and F). What is more, as shown in Figure 3, our results from immunohistochemical staining of PCNA demonstrate that the control, α-CA, RPM, and α-CA-RPM groups had a significantly higher proliferating index than the sham group (p < 0.01; Figure 3A, B, C, D, E and F), and the percentage of PCNA-positive cells in the α-CA, RPM, and α-CA-RPM groups was significantly less than in the control group (p < 0.01; Figure 3B, C, D, E and F). Moreover, it is worth noting that the proliferating index in the α-CA-RPM group was markedly less than in the α-CA or RPM group (p < 0.01; Figure 3C, D, E and F). Taken together, our results strongly demonstrate that α-CA, RPM, and α-CA-RPM inhibit intimal hyperplasia in vein grafts, and the effect of α-CA-RPM is stronger than that of α-CA or RPM.
α-CA-RPM reduced intimal thickening of the vein graft. The vessel tissue was harvested 4 weeks after the operation, fixed in formalin, sliced to 4 <italic>µ</italic>m tissue sections and stained with HE. Images (×100 objective lens) were collected and analyzed by Olympus micro-imaging system. The rats were divided into 5 group: Sham group (A), control group (B), α-cyanoacrylate group (C), Rapamycin group (D) and α-CA-RPM group (E). (F) Represented the statistical graph of each group’s intima thickness. The intima of control group was dramatically thicker than that of α-CA, RPM and α-CA-RPM groups; the difference was statistically significant (91.3 ± 3.9, 133.6 ± 8.0, 50.6 ± 5.4 vs. 233.6 ± 29.1 <italic>µ</italic>m, p < 0.01); the intima of the RPM group was thicker than that of α-CA group (133.6 ± 8.0 vs. 91.3 ± 3.9 <italic>µ</italic>m, p < 0.05); the intima of α-CA group and RPM group was thicker than that of α-CA-RPM group (50.6 ± 5.4 vs. 91.3 ± 3.9 <italic>µ</italic>m, 133.6 ± 8.0 <italic>µ</italic>m, p < 0.05). * The control group had obvious difference with other groups, p < 0.05. <sup>#</sup> The α-cyanoacrylate group had obvious difference with other groups, p < 0.05. <sup>△</sup> The rapamycin group had obvious difference with other groups, p < 0.05. <sup>☆</sup> The α-CA-RPM group had obvious difference with other groups, p < 0.05.
α-CA-RPM decreased the proliferating index of vein graft. The vessel tissue was harvested 4 weeks after the operation, fixed with formalin, sliced to 4 <italic>µ</italic>m tissue sections and stained with the primary antibody anti-PCNA. Images (×200 objective lens) were collected and analyzed by Olympus micro-imaging system. Likewise, the rats were also divided into 5 group: Sham group (A), control group (B), α-cyanoacrylate group (C), Rapamycin group (D) and α-CA-RPM group (E). (F) Represented the statistical graph of each group’s PCNA proliferation index. * The control group had obvious difference with other groups, p < 0.01. <sup>#</sup> The α-cyanoacrylate group had obvious difference with other groups, p < 0.01. <sup>△</sup> The rapamycin group had obvious difference with other groups, p < 0.01. <sup>☆</sup> The α-CA-RPM group had obvious difference with other groups, p < 0.01.
α-CA-RPM diminished intimal hyperplasia and inflammatory responses
In order to further study the mechanism through which the three intervening methods prevent intimal hyperplasia, we examined the value of αSMA and vWF in grafted veins 4 weeks after surgery. The αSMA values in the α-CA, RPM, and α-CA-RPM groups were much lower than in the control group, as detected by RT-PCR (p < 0.01; Figure 4A). The αSMA values in the α-CA-RPM group were lower than in the α-CA and RPM groups (p < 0.01; Figure 4A). Similar results were found in the value of vWF in α-CA-RPM group (p < 0.01; Figure 4B). This result verified that α-CA, RPM, and α-CA-RPM inhibition might reduce intimal hyperplasia by blocking αSMA and vWF over-expression.
α-CA-RPM diminished the expression of αSMA and vWF and inflammatory responses. Four weeks after the surgery, RT-PCR was used to detect αSMA and vWF in grafted veins. (A) Value of αSMA in α-CA, RPM, and α-CA-RPM groups was much lower than in the control group, as detected by RT-PCR. Value of αSMA in α-CA-RPM group was lower than that of α-CA group and RPM group (p < 0.01). (B) Similar results were found in the value of vWF in α-CA-RPM group (p < 0.01). (C) The serum levels of ET-1 are shown for each group at different times. The level of ET-1 in the control group was significantly higher than that in the α-CA, RPM and α-CA-RPM groups 36 hours and 4 weeks after operation (96.1 ± 7.9 ng/l vs. 84.0 ± 10.9 ng/l, 79.5 ± 5.7 ng/l and 72.7 ± 9.9 ng/l; 99.7 ± 7.7 ng/l vs. 87.1 ± 13.3 ng/l, 65.4 ± 23.4 ng/l and 43.7 ± 20.1 ng/l; p < 0.05, respectively). Additionally, at 4 weeks after surgery, the level of ET-1 of the α-CA-RPM group was significantly lower than that of the α-CA, RPM, and control groups, (43.7 ± 20.1 ng/l vs. 87.1 ± 13.3 ng/l, 65.4 ± 23.4 ng/l and 99.5 ± 7.7 ng/l; p < 0.05, respectively). * The control group had obvious difference with other groups, p < 0.01. <sup>#</sup> The α-cyanoacrylate group had obvious difference with sham group, control group and α-CA-RPM group, p < 0.01. <sup>△</sup> The rapamycin group had obvious difference with sham group, control group and α-CA-RPM groups, p < 0.01. <sup>☆</sup> The α-CA-RPM group had obvious difference with other groups, p < 0.01.
In order to investigate the effect of α-CA-RPM on inflammatory responses, we performed ELISA assays to examine serum levels of ET-1. We found the ET-1 level of the control, α-CA, RPM, and α-CA-RPM groups gradually increased 36 hours after operation; those of the control, α-CA, and RPM groups was still high 4 weeks after operation and that of the α-CA-RPM group had basically returned to normal. The ET-1 level in the control group was significantly higher than that of the α-CA, RPM, and α-CA-RPM groups 36 hours and 4 weeks after operation (96.1 ± 7.9 ng/l vs. 84.0 ± 10.9 ng/l, 79.5 ± 5.7 ng/l, and 72.7 ± 9.9 ng/l; 99.7 ± 7.7 ng/l vs. 87.1 ± 13.3 ng/l, 65.4 ± 23.4 ng/l, and 43.7 ± 20.1 ng/l; p < 0.05, respectively). Additionally, at 4 weeks after surgery, the ET-1 level of the α-CA-RPM group was significantly lower than in the α-CA, RPM, and control groups (43.7 ± 20.1 ng/l vs. 87.1 ± 13.3 ng/l, 65.4 ± 23.4 ng/l, and 99.5 ± 7.7 ng/l; p < 0.05, respectively) (Figure 4C). These findings indicate that α-CA, RPM, and α-CA-RPM seem to reduce inflammatory responses and that α-CA-RPM is more effective.
Discussion
The main finding of our study is that the application of α-CA, RPM, or α-CA-RPM can improve the patency of the vein graft in rat models by inhibiting intimal hyperplasia.8 More importantly, the use of RPM combined with α-CA is more effective than either α-CA or RPM alone.
The complicated remodeling process of vessels leads to restenosis of vein grafts, but the exact mechanism is not explicit. Studies have shown that restenosis is related to dysfunction of intima endothelial cells, proliferation, immigration of vascular smooth muscle cells, adventitial fibroblasts, inflammatory reaction, shear force, and hemodynamic changes.9,10 The pathological process of restenosis of bridge vessel may include early thrombosis, intimal hyperplasia, and atherosclerosis; intimal hyperplasia is the most important reason. When separating, ligaturing, dividing, transplanting, and revascularizing bridge vessels, factors such as aggregation of platelets and neutrophils, release of cytokine and chemokine, activation of transduction pathway, and enzymatic reaction may prompt hyperplasia of vascular smooth muscle cells and accumulation of endothelial cells. All these factors are expected to result in intimal hyperplasia, and restenosis of grafted vessels follows.11,12
In 1963, Parsonnet and colleagues first pointed out that extravascular supporters could enhance the patency rate of grafted veins.5 Several foundational and clinical tests have proved that extravascular supporters could inhibit intimal hyperplasia and enhance patency rate. Four extravascular supporters have been widely used in foundational and clinical tests, i.e., nitinol extravascular stent, polymeric extravascular stent, fibrin glue extravascular supporter, and α-CA. It is acknowledged that α-CA not only can prevent post-transplantation vessels from expansion, but also can prompt vascular smooth muscle cells’ migration to vascular outer membrane.13,14 Stimulated by α-CA, many neutrophils and monocytes aggregated to adventitia, especially mononuclear phagocytes which can release amounts of chemotactic factors to attract vascular smooth muscle cells and fibroblast immigration and colonization.15 A series of changes mentioned above will activate a range of antiatherosclerotic factors: NO, PGI2, cAMP and cGMP. They can also decrease intimal cholesterol and inhibit pro-atherosclerotic factors.16 Outcomes in our experiment revealed that veins in the α-CA group had few fresh tissues and were easy to separate and had clear boundary from the surrounding tissues 4 weeks after the operation. The glue was not fully degraded and the intima of α-CA group was thinner than in the control group. Additionally, the percentage of PCNA-positive cells was significantly less than in the control group. Most importantly, α-CA as the extravascular supporter was able to inhibit intimal hyperplasia and enhance the patency rate.
The proliferation, immigration, and secretion of vascular smooth muscle cells are key to intimal hyperplasia, which contribute to restenosis of vein grafts. Although certain drugs are effective for inhibiting intimal hyperplasia by inhibiting cytokinin and regulating cell cycle, severe toxic reactions and side effects limit their extensive use, as a consequence of which local application becomes particularly significant. RPM, colchicine, and other drugs are used locally on grafted veins. After anastomosis, these drugs are smeared evenly on grafted veins. RPM can accelerate vascular smooth muscle cells’ apoptosis by inhibiting the transformation of cells from G1 to S phase, thus suppressing vascular smooth muscle cells’ proliferation and immigration. Additionally, RPM protects endothelial cell function and reduces the release of vasoactive peptide when endothelial cells get injured.17-19 Furthermore, RPM can also inhibit the differentiation, proliferation, and immigration of endothelial progenitor cells (EPC) and reduce NOS-mRNA expression in EPC.20,21 Our results verified that veins in the RPM group had clear boundaries from the surrounding tissue; they were also fresh and clearly not expanded. Moreover, the intima of the RPM group was thinner than the control group’s and the percentage of PCNA-positive cells was remarkably lower than in the control group. In summary, RPM may inhibit intimal hyperplasia and enhance patency rate.
This study aimed to experiment the combination of an extravascular supporter and a local drug application. We chose α-CA as the extravascular supporter, RPM as the local application, and α-CA-RPM as the combination. α-CA-RPM was used in rat models of autogenous vein graft to stimulate grafted veins’ pathophysiological process after CABG. Interestingly, we found the percentage of PCNA-positive cells in the α-CA-RPM group was markedly less than in the control, α-CA, and RPM groups, which indicated that α-CA-RPM was more effective in inhibiting intimal hyperplasia than either α-CA or RPM separately. We concluded that α-CA-RPM can combine the effectiveness of extravascular supporters and local drugs and thus better inhibit intimal hyperplasia. Meanwhile, α-CA is an ideal carrier for the formulation of long-term control drug release which surrounds the vein graft tightly so that RPM will be released slowly and no RPM will be wasted.
The endothelin-1 (ET-1) has been implicated in the pathogenesis of restenosis and vascular hypertrophy via enhancing aggregation of platelets and neutrophils, release of cytokine and chemokine, accumulation of endothelial cells, and promotiong of vascular smooth muscle cell migration towards the intimal layer.22 Our results indicate that α-CA, RPM, and α-CA-RPM can stabilize endothelial cell function and diminish the release of ET-1 to inhibit intimal hyperplasia. An endothelin A/B receptor antagonist contributed to reduction of intimal hyperplasia in an organ culture of human saphenous veins and prevented neointimal development of coronary angioplasty in pigs, which is in accordance with our experiment.23,24 αSMA is the specific protein of vascular smooth muscle cells and the expression of αSMA can reflect the hyperplasia of vascular smooth muscle cells. In our experiment, we examined the values of αSMA in grafted veins with RT-PCR and found that the values in the α-CA, RPM, and α-CA-RPM groups was lower than in the control group. Notably, the value of the α-CA-RPM group was lower than that of the α-CA and RPM groups. A study in which the αSMA component of vascular progenitor cells correlated with the coronary artery Gensini score also made the same point.25 An experiment in a swine model of arteriovenous bypass grafting also provided tangible evidence to support this point of view.26
The vWF is a glycoprotein encoded by the short arm of chromosome 12 and can be combined with collagen fibers and platelets; it is closely related to a range of cardiovascular diseases such as atherosclerosis, acute coronary syndrome, and atrial fibrillation.27 vWF directly stimulates vascular smooth muscle cell proliferation, resulting in a direct dose-response effect. It also accelerates intimal hyperplasia in intact endothelium without platelet activation or platelet-derived growth factor release.28 Likewise, we found the vWF values of the α-CA, RPM, and α-CA-RPM groups was lower than that of the control group, and the α-CA-RPM group was lower than the α-CA or RPM groups. Our results in rats have been supported by experiments in other animals, such as an efficacy study in dogs and intimal hyperplasia of rabbit carotid arteries.29,30 These results demonstrate that α-CA, RPM, and α-CA-RPM might reduce intimal hyperplasia by blocking ET-1, αSMA, and vWF overexpression.
Our results show that rapamycin combined with α-cyanoacrylate contributes to inhibiting intimal hyperplasia and is more effective for vascular patency than individual use of either α-CA or RPM in rat models 4 weeks after operation. The long-term effects of α-CA-RPM on vein graft remodeling are still unclear. Our team will conduct further research on intimal hyperplasia pathophysiological processes in pigs after CABG and the impacts of related interventions on grafted veins.
Conclusion
Our results confirmed that α-CA-RPM contributes to inhibiting intimal hyperplasia and is more effective for vascular patency than individual use either α-CA or RPM in rat models of artery bypass grafting. The positive effects appear to be associated with decreased intimal thickening, reduced cell proliferation in the vein graft, and decreased inflammatory responses. Although the shor-term effects of α-CA-RPM seem promising, the long-term effects and clinical significance of α-CA-RPM in CABG need to be studied in the future.
Sources of Funding
This study was funded by National Natural Science Foundation of China.
Study Association
This article is part of the thesis of master submitted by Tianshu-Chu, from Anhui Medical University.
Ethics approval and consent to participate
This study was approved by the Ethics Committee on Animal Experiments of the Anhui Animal Ehtics Committee under the protocol number ah56743.
Acknowledgements
The authors would like to thank XR Huang and DK Huang for their expert technique.
References1 Blaas I, Heinz K, Wurtinger P, Turkcan A, Tepekoylu C, Grimm M, et al. Vein graft thrombi, a niche for smooth muscle cell colonization - a hypothesis to explain the asymmetry of intimal hyperplasia. J Thromb Haemost. 2016;14(5):1095-104.BlaasIHeinzKWurtingerPTurkcanATepekoyluCGrimmMVein graft thrombi, a niche for smooth muscle cell colonization - a hypothesis to explain the asymmetry of intimal hyperplasiaJ Thromb Haemost2016145109511042 Hameau DR, Veas PN, Mendez LM, Martinez RG. Focal arterialization and neoatherosclerosis of a saphenous vein graft. Improving our understanding of late graft failures. Arq Bras Cardiol. 2016;107(5):495-6.HameauDRVeasPNMendezLMMartinezRGFocal arterialization and neoatherosclerosis of a saphenous vein graft. Improving our understanding of late graft failuresArq Bras Cardiol201610754954963 Sur S, Sugimoto JT, Agrawal DK. Coronary artery bypass graft: why is the saphenous vein prone to intimal hyperplasia? Can J Physiol Pharmacol. 2014;92(7):531-45.SurSSugimotoJTAgrawalDKCoronary artery bypass graft: why is the saphenous vein prone to intimal hyperplasia?Can J Physiol Pharmacol20149275315454 Taggart DP, Ben Gal Y, Lees B, Patel N, Webb C, Rehman SM, et al. A randomized trial of external stenting for saphenous vein grafts in coronary artery bypass grafting. Ann Thorac Surg. 2015;99(6):2039-45.TaggartDPBen GalYLeesBPatelNWebbCRehmanSMA randomized trial of external stenting for saphenous vein grafts in coronary artery bypass graftingAnn Thorac Surg2015996203920455 Parsonnet V, Lari AA, Shah IH. New stent for support of veins in arterial grafts. Arch Surg. 1963 Oct;87:696-702.ParsonnetVLariAAShahIHNew stent for support of veins in arterial graftsArch Surg196310876967026 Yao J, Zhang Y, Hu Q, Zeng D, Hua F, Meng W, et al. Optimization of paeonol-loaded poly(butyl-2-cyanoacrylate) nanocapsules by central composite design with response surface methodology together with the antibacterial properties. Eur J Pharm Sci. 2017 Apr 1;101:189-99.YaoJZhangYHuQZengDHuaFMengWOptimization of paeonol-loaded poly(butyl-2-cyanoacrylate) nanocapsules by central composite design with response surface methodology together with the antibacterial propertiesEur J Pharm Sci20170411011891997 Dai L, Gao M, Gu C, Zhang F, Yu Y. Perivenous application of cyanoacrylate tissue sealants reduces intimal and medial thickening of the vein graft and inflammatory responses in a rabbit model of carotid artery bypass grafting. Eur J Cardiothorac Surg. 2016;49(2):675-81.DaiLGaoMGuCZhangFYuYPerivenous application of cyanoacrylate tissue sealants reduces intimal and medial thickening of the vein graft and inflammatory responses in a rabbit model of carotid artery bypass graftingEur J Cardiothorac Surg20164926756818 Zou Y, Dietrich H, Hu Y, Metzler B, Wick G, Xu Q. Mouse model of venous bypass graft arteriosclerosis. Am J Pathol. 1998;153(4):1301-10.ZouYDietrichHHuYMetzlerBWickGXuQMouse model of venous bypass graft arteriosclerosisAm J Pathol19981534130113109 Jeremy JY, Dashwood MR, Mehta D, Izzat MB, Shukla N, Angelini GD. Nitric oxide, prostacyclin and cyclic nucleotide formation in externally stented porcine vein grafts. Atherosclerosis. 1998;141(2):297-305.JeremyJYDashwoodMRMehtaDIzzatMBShuklaNAngeliniGDNitric oxide, prostacyclin and cyclic nucleotide formation in externally stented porcine vein graftsAtherosclerosis1998141229730510 Shi Y, O'Brien JE Jr, Mannion JD, Morrison RC, Chung W, Fard A, et al. Remodeling of autologous saphenous vein grafts. The role of perivascular myofibroblasts. Circulation. 1997;95(12):2684-93.ShiYO'BrienJEJrMannionJDMorrisonRCChungWFardARemodeling of autologous saphenous vein grafts. The role of perivascular myofibroblastsCirculation199795122684269311 Owens CD, Rybicki FJ, Wake N, Schanzer A, Mitsouras D, Gerhard-Herman MD, et al. Early remodeling of lower extremity vein grafts: inflammation influences biomechanical adaptation. J Vasc Surg. 2008;47(6):1235-42.OwensCDRybickiFJWakeNSchanzerAMitsourasDGerhard-HermanMDEarly remodeling of lower extremity vein grafts: inflammation influences biomechanical adaptationJ Vasc Surg20084761235124212 Masuda H, Kawamura K, Nanjo H, Sho E, Komatsu M, Sugiyama T, et al. Ultrastructure of endothelial cells under flow alteration. Microsc Res Tech. 2003;60(1):2-12.MasudaHKawamuraKNanjoHShoEKomatsuMSugiyamaTUltrastructure of endothelial cells under flow alterationMicrosc Res Tech200360121213 Vijayan V, Shukla N, Johnson JL, Gadsdon P, Angelini GD, Smith FC, et al. Long-term reduction of medial and intimal thickening in porcine saphenous vein grafts with a polyglactin biodegradable external sheath. J Vasc Surg. 2004;40(5):1011-9.VijayanVShuklaNJohnsonJLGadsdonPAngeliniGDSmithFCLong-term reduction of medial and intimal thickening in porcine saphenous vein grafts with a polyglactin biodegradable external sheathJ Vasc Surg20044051011101914 George SJ, Izzat MB, Gadsdon P, Johnson JL, Yim AP, Wan S, et al. Macro-porosity is necessary for the reduction of neointimal and medial thickening by external stenting of porcine saphenous vein bypass grafts. Atherosclerosis. 2001;155(2):329-36.GeorgeSJIzzatMBGadsdonPJohnsonJLYimAPWanSMacro-porosity is necessary for the reduction of neointimal and medial thickening by external stenting of porcine saphenous vein bypass graftsAtherosclerosis2001155232933615 Owens CD. Adaptive changes in autogenous vein grafts for arterial reconstruction: clinical implications. J Vasc Surg. 2010;51(3):736-46.OwensCDAdaptive changes in autogenous vein grafts for arterial reconstruction clinical implicationsJ Vasc Surg201051373674616 Angelini GD, Lloyd C, Bush R, Johnson J, Newby AC. An external, oversized, porous polyester stent reduces vein graft neointima formation, cholesterol concentration, and vascular cell adhesion molecule 1 expression in cholesterol-fed pigs. J Thorac Cardiovasc Surg. 2002;124(5):950-6.AngeliniGDLloydCBushRJohnsonJNewbyACAn external, oversized, porous polyester stent reduces vein graft neointima formation, cholesterol concentration, and vascular cell adhesion molecule 1 expression in cholesterol-fed pigsJ Thorac Cardiovasc Surg2002124595095617 Kong J, Deng Y, Dong Q, Liu W, Lu Y. Colchicine reduces restenosis after balloon angioplasty treatment for in-stent restenosis. Arch Med Res. 2015;46(2):101-6.KongJDengYDongQLiuWLuYColchicine reduces restenosis after balloon angioplasty treatment for in-stent restenosisArch Med Res201546210110618 Miao LF, Yin YP, Cui YL, Chen LF, Zeng Y, Huang CL, et al. Efficacy and mechanism of local delivery of rapamycin and rapamycin-loaded poly(lactic-co-glycolic) acid nanoparticles on coronary restenosis of injury-stenosis model of minipigs. Zhonghua Yi Xue Za Zhi. 2016;96(1):36-42.MiaoLFYinYPCuiYLChenLFZengYHuangCLEfficacy and mechanism of local delivery of rapamycin and rapamycin-loaded poly(lactic-co-glycolic) acid nanoparticles on coronary restenosis of injury-stenosis model of minipigsZhonghua Yi Xue Za Zhi2016961364219 Guo M, Zhang Y, Tian D, Wu X, Chen Z, Ma L, et al. Inhibitory effect of topical application of 5-fluorouracil on intimal hyperplasia of vein graft. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009;23(8):940-6.GuoMZhangYTianDWuXChenZMaLInhibitory effect of topical application of 5-fluorouracil on intimal hyperplasia of vein graftZhongguo Xiu Fu Chong Jian Wai Ke Za Zhi200923894094620 Wang XT, Venkatraman S, Boey F, Loo SC, Tan LP. Effects of controlled-released sirolimus from polymer matrices on human coronary artery smooth muscle cells. J Biomater Sci Polym Ed. 2007;18(11):1401-14.WangXTVenkatramanSBoeyFLooSCTanLPEffects of controlled-released sirolimus from polymer matrices on human coronary artery smooth muscle cellsJ Biomater Sci Polym Ed200718111401141421 Taniwaki M, Raber L, Magro M, Kalesan B, Onuma Y, Stefanini GG, et al. Long-term comparison of everolimus-eluting stents with sirolimus- and paclitaxel-eluting stents for percutaneous coronary intervention of saphenous vein grafts. EuroIntervention. 2014;9(12):1432-40.TaniwakiMRaberLMagroMKalesanBOnumaYStefaniniGGLong-term comparison of everolimus-eluting stents with sirolimus- and paclitaxel-eluting stents for percutaneous coronary intervention of saphenous vein graftsEuroIntervention20149121432144022 Tsujino M, Hirata Y, Eguchi S, Watanabe T, Chatani F, Marumo F. Nonselective ETA/ETB receptor antagonist blocks proliferation of rat vascular smooth muscle cells after balloon angioplasty. Life Sci. 1995;56(25):PL449-54.TsujinoMHirataYEguchiSWatanabeTChataniFMarumoFNonselective ETA/ETB receptor antagonist blocks proliferation of rat vascular smooth muscle cells after balloon angioplastyLife Sci19955625PL449PL45423 Sanmartin M, Fernandez-Ortiz A, Fantidis P, Aragoncillo P, Fernandez-Durango R, Rollin R, et al. Effects of bosentan on neointimal response following coronary angioplasty. Eur J Clin Invest. 2003;33(9):762-8.SanmartinMFernandez-OrtizAFantidisPAragoncilloPFernandez-DurangoRRollinREffects of bosentan on neointimal response following coronary angioplastyEur J Clin Invest200333976276824 Aziz O, Rahman MS, Hadjianastassiou VG, Kokotsakis J, Vitali M, Cherian A, et al. Novel applications of Dermabond (2-octyl -cyanoacrylate) in cardiothoracic surgery. Surg Technol Int. 2007 Apr 5;16:46-51.AzizORahmanMSHadjianastassiouVGKokotsakisJVitaliMCherianANovel applications of Dermabond (2-octyl -cyanoacrylate) in cardiothoracic surgerySurg Technol Int200704516465125 Wang CH, Hsieh IC, Chen SJ, Wang JS, Cherng WJ, Chen CC, et al. VE-Cadherin(low)alpha-smooth muscle actin+ component of vascular progenitor cells correlates with the coronary artery Gensini score. Circ J. 2012;76(2):477-84.WangCHHsiehICChenSJWangJSCherngWJChenCCVE-Cadherin(low)alpha-smooth muscle actin+ component of vascular progenitor cells correlates with the coronary artery Gensini scoreCirc J201276247748426 Goldstone RN, McCormack MC, Khan SI, Salinas HM, Meppelink A, Randolph MA, et al. Photochemical tissue passivation reduces vein graft intimal hyperplasia in a swine model of arteriovenous bypass grafting. J Am Heart Assoc. 2016;5(8):pii:e003856.GoldstoneRNMcCormackMCKhanSISalinasHMMeppelinkARandolphMAPhotochemical tissue passivation reduces vein graft intimal hyperplasia in a swine model of arteriovenous bypass graftingJ Am Heart Assoc201658pii:e00385627 Lenting PJ, Christophe OD, Denis CV. von Willebrand factor biosynthesis, secretion, and clearance: connecting the far ends. Blood. 2015;125(13):2019-28.LentingPJChristopheODDenisCVvon Willebrand factor biosynthesis, secretion, and clearance: connecting the far endsBlood2015125132019202828 Qin F, Impeduglia T, Schaffer P, Dardik H. Overexpression of von Willebrand factor is an independent risk factor for pathogenesis of intimal hyperplasia: preliminary studies. J Vasc Surg. 2003;37(2):433-9.QinFImpedugliaTSchafferPDardikHOverexpression of von Willebrand factor is an independent risk factor for pathogenesis of intimal hyperplasia: preliminary studiesJ Vasc Surg200337243343929 Azuma H, Niimi Y, Terada T, Hamasaki H. Accelerated endothelial regeneration and intimal hyperplasia following a repeated denudation of rabbit carotid arteries: morphological and immunohistochemical studies. Clin Exp Pharmacol Physiol. 1995;22(10):748-54.AzumaHNiimiYTeradaTHamasakiHAccelerated endothelial regeneration and intimal hyperplasia following a repeated denudation of rabbit carotid arteries: morphological and immunohistochemical studiesClin Exp Pharmacol Physiol1995221074875430 Wang X, Zhao Y, Fu Z, He Y, Xiang D, Zhang L. Prelining autogenic endothelial cells in allogeneic vessels inhibits thrombosis and intimal hyperplasia: an efficacy study in dogs. J Surg Res. 2011;169(1):148-55.WangXZhaoYFuZHeYXiangDZhangLPrelining autogenic endothelial cells in allogeneic vessels inhibits thrombosis and intimal hyperplasia an efficacy study in dogsJ Surg Res20111691148155Artigo OriginalRapamicina em Combinação com α-Cianoacrilato Contribui à Inibição de Hiperplasia Intimal em Modelos em RatosTianshu-ChuCongrong-GaoZhiwei-zhaoFei-LingAyu-SunYuanbiao-ZhengJing-Cao0000-0002-9424-6049GeJianjunAnhui Medical University, Hefei - ChinaCorrespondência: Jianjun Ge, 17 # Lujiang Road, Hefei 230001, P.R.China. E-mail: SLGejianjun@outlook.com
Contribuição dos autores
Concepção e desenho da pesquisa: Tianshu-Chu, Congrong-Gao, Zhiwei-zhao; obtenção de dados: Tianshu-Chu, Fei-Ling, Ayu-Sun; análise e interpretação dos dados: Tianshu-Chu, Jing-Cao, Yuanbiao-Zheng, Jianjun Ge; análise estatística: Tianshu-Chu, Congrong-Gao, Zhiwei-zhao; redação do manuscrito: Tianshu-Chu, Congrong-Gao, Fei-Ling, Ayu-Sun; revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Tianshu-Chu, Congrong-Gao, Zhiwei-zhao, Fei-Ling, Ayu-Sun, Jianjun Ge.
Potencial conflito de interesses
Declaro não haver conflito de interesses pertinentes.
ResumoFundamento:
Reestenose de enxertos venosos tem um impacto adverso na circulação de pontagens e no prognóstico de pacientes após a cirurgia de revascularização miocárdica.
Objetivos:
Nós utilizamos α-cianoacrilato (α-CA) como suporte extravascular, rapamicina/sirolimus (RPM) como aplicação local e a combinação dos dois (α-CA-RPM) em modelos de enxerto venoso autógeno em ratos para estimular mudança no enxerto venoso. O objetivo do nosso estudo foi observar o efeito de α-CA, RPM e α-CA-RPM na hiperplasia venosa.
Métodos:
Cinquenta ratos Sprague Dawley (SD) saudáveis foram randomizados nos 5 grupos seguintes: sham, controle, α-CA, RPM e α-CA-RPM. O procedimento operacional descrito subsequentemente foi utilizado para construir modelos de enxertos da veia jugular na artéria carótida em ratos, em um lado. O nível de endotelina-1 (ET-1) foi determinado por ensaio de imunoabsorção enzimática (ELISA). As veias enxertadas foram observadas a olho nu 4 semanas após; as veias frescas foram observadas via microscópio e software de processamento de imagem com coloração hematoxilina-eosina (HE) e imuno-histoquímica depois de serem fixadas e armazenadas; α-actina do músculo liso (αSMA) e o fator de von Willebrand (vWF) foram medidos com reação em cadeia da polimerase-transcriptase reversa (RT-PCR). Realizaram-se as comparações com análise de variância de fator único (ANOVA) e o teste de diferença mínima significativa (LSD) de Fisher, com p < 0,05 sendo considerado estatisticamente significante.
Resultados:
Nós achamos que a espessura intimal nos grupos α-CA, RPM e α-CA-RPM era menor que no grupo controle (p < 0,01) e a espessura no grupo α-CA-RPM era notavelmente menor que nos grupos α-CA e RPM (p < 0,05).
Conclusão:
A combinação de RPM e α-CA contribui à inibição de hiperplasia em modelos em ratos e é mais efetivo para patência vascular que uso individual de α-CA ou RPM.
Palavras-chave:Revascularização Miocárdica/cirurgiaCianoacrilatosSirolimusHiperplasiaOclusão de Enxerto VascularGrau de Desobstrução VascularRatosIntrodução
A cirurgia de revascularização miocárdica (CRM) é uma das principais terapias para doença coronariana. Porém, 40% dos vasos de pontagem estão totalmente obstruídos e 30% do fluxo sanguíneo vascular é reduzido após a CRM, o que seriamente impacta a sobrevida e o prognóstico do paciente.1,2 Mecanismos de reestenose incluem trombose, hiperplasia intimal e aterosclerose. A imigração de células endoteliais e células musculares lisas vasculares é vital para a hiperplasia intimal, que é a principal causa de reestenose.3
Embora os fármacos que inibem a citocinina e regulam o ciclo celular possam contribuir à inibição da hiperplasia intimal, os efeitos colaterais sistêmicos são prejudiciais para os pacientes. Portanto, a aplicação local é muito importante. A rapamicina (sirolimus) é amplamente utilizada para a anti-rejeição após operações de transplante, e os stents farmacológicos são amplamente utilizados em artérias coronárias. Pesquisadores verificaram que a aplicação de rapamicina em veias enxertadas é eficaz na inibição da hiperplasia intimal, pois inibe a proliferação e promove a apoptose de células musculares lisas.4
Em 1963, Parsonnet e seus colegas observaram que suporte perivenoso era eficaz para a patência vascular.5 Subsequentemente, pesquisadores clínicos descobriram que o suporte perivenoso pode aumentar as taxas de patência pela redução da hiperplasia intimal nas veias enxertadas. O α-CA, que é líquido à temperatura ambiente, é inofensivo para o corpo humano. O tempo de degradação é de 1 a 3 meses, dependendo da dosagem. O α-CA é usado em cirurgia para tratar sangramentos e suturar feridas.6
Usualmente, o α-CA e a RPM são utilizados como suporte perivenoso e aplicação local, respetivamente. Investigamos de forma inovadora o processo fisiopatológico da hiperplasia da neoíntima em veias enxertadas após CRM usando modelos de enxerto venoso autógeno em ratos, com o intuito de encontrar novos métodos para inibir a hiperplasia intimal.
MétodosReagente e método
O α-CA (99% n-octil-α-cianoacrilato + n-butil-α-cianoacrilato) foi adquirido da Beijing Fuaile Science and Technology Development Co. (Beijing, China). A RPM foi adquirida da Selleck Company. Dissolvemos 8 mg de RPM em 1 ml de α-CA (obtidos com pipeta) em um tubo Eppendorf estéril. Após isso, utilizou-se um agitador magnético para misturá-los, obtendo o α-CA-RPM de 8mg/ml, que foi armazenado sob refrigeração de 2 a 8°C. RPM era hidrosolvente e foi preparado pelo mesmo método.7
Modelos e grupos
Cinquenta ratos SD (fornecidos pelo Anhui Lab Animal Research Center e identificados pelo comitê de ética médica da Anhui Medical University), machos e fêmeas, entre 10 e 12 semanas de idade, com peso entre 220-280 g, foram randomizados (desenho completamente randomizado) em 5 grupos, cada grupo contendo 10 ratos, e alimentados durante 4 semanas após a operação. O procedimento operacional e o tamanho da amostra foram determinados de acordo com experimentos pilotos e estudos anteriores, conforme descrito abaixo.
Procedimento operacional: utilizou-se uma injeção intraperitoneal de 10% chloralic hydras para anestesiar os ratos. A heparinização foi induzida por uma injeção de heparina (700 UI/Kg) na veia caudal. Uma incisão vertical de aproximadamente 1 cm foi feita no meio do pescoço (desviada para o lado da operação) e as veias foram dissociadas em um lado. Epitheca de 1-2 mm foram obtidas com uma agulha de punção arterial 20G vermelha (BD Company), usada como cânula. A artéria carótida foi isolada até os ramos. Em seguida, duas linhas de sutura e hemoclips foram colocados em ambas as extremidades da artéria para bloquear o fluxo sanguíneo. O meio da artéria foi isolado e cuidadosamente virado para 1-1,2 mm acima da cânula. Utilizou-se uma sutura de seda 6/0 para ligar e fixar a veia para isolá-la das artérias; desta maneira, foi possível abrir os grampos vasculares. Verificamos que o pulso das veias enxertadas era normal e sem sangramento e em seguida suturamos a incisão. Diariamente, verificamos o estado vital e as incisões de todos os ratos. Mantivemos o ambiente fresco, trocamos os ninhos regularmente e demos forragem e água suficiente. Três dias após a operação, cada rato começou a receber uma injeção intramuscular de 400.000 IU de penicilina, diariamente.
Grupo sham: apenas simulou-se o processo de operação. As veias jugulares foram dissociadas e os vasos colaterais foram ligados, sem divisão ou transplante; Grupo controle: enxerto arteriovenoso jugular no mesmo lado; Grupo α-CA: enxerto arteriovenoso jugular e aplicação de cola α-CA nas veias enxertadas; Grupo RPM: enxerto arteriovenoso jugular e aplicação de RPM nas veias enxertadas; Grupo α-CA-RPM: enxerto arteriovenoso jugular no mesmo lado e aplicação de α-CA-RPM nas veias enxertadas.
Coleta de amostras
Amostras de sangue foram coletadas no pré-operatório às 0 h e no pós-operatório às 12 h, 36 h e 4 semanas após a operação. O soro foi obtido por centrifugação e foi armazenado a -80°C até a análise de citocinas. Quatro semanas depois, coletamos uma amostra de veia de cada grupo. Os ratos foram completamente anestesiados, fixados na mesa de operação, heparinizados como descrito anteriormente e operados da mesma maneira pelo mesmo caminho. Observamos as formas e a circulação das veias enxertadas, dos vasos com ligadura e dos vasos isolados nas duas extremidades das cânulas; em seguida, removemos as veias intactas e frescas e lavamos os lúmenes completamente com solução salina normal. As amostras com coloração HE e imuno-histoquímica foram colocadas em microtubos cheios de paraformaldeído e as amostras com RT-PCR foram colocadas em microtubos cheios de regentes de RNA-EZ e mantidas na geladeira a -80°C. Os ratos foram sacrificados pelo método de deslocamento cervical e adequadamente manipulados.
Ensaio de imunoabsorção enzimática para ET-1
ET-1 foi determinado com kits ELISA (R&D, USA) utilizando 50 µl de soro para o ensaio. Foram realizadas 3 medidas para cada amostra de sangue e a placa de ELISA foi lida a 450 nm em um leitor de placas.
Exame histológico do tecido enxertado
As veias enxertadas foram imersas em formalina e cortadas em seções de 4 mm. Subsequentemente, realizou-se coloração hematoxilina-eosina (HE) utilizando um kit de coloração hematoxilina e eosina (Beyotime Biotechnology, Shang Hai, China). O sistema de aquisição de imagem do microscópio Olympus foi utilizado para obter imagens de seção (lente objetiva 100 ×) e medir a espessura da íntima. As medidas e a análise de dados foram realizadas por 2 pesquisadores independentes. As seções foram selecionadas aleatoriamente de veias enxertadas e não-enxertadas; em seguida medimos 16 pontos de espessura e calculamos a média. Três seções foram selecionadas e medidas de cada rato. Em seguida, calculamos a espessura da íntima.
Determinação do índice de proliferação
As seções de tecido foram incubadas com o kit de análise imuno-histoquímica para o antígeno nuclear de células em proliferação (PCNA) (Santa Cruz Biotechnology, Dallas, TX, EUA) a 4°C durante uma noite. Após a lavagem com solução salina tamponada com fosfato (PBS) (DAKO, Glostrup, Dinamarca) e incubação com o anticorpo secundário, realizou-se a coloração com DAB. As secções de tecido foram desidratadas e instaladas em lâminas. Todas as imagens (lente objetiva 200 ×) foram captadas pelo sistema de aquisição de imagem do microscópio Olympus e pela SPOT Digital Camera (Diagnostic Instruments, Sterling Heights, MI, EUA). Células positivas para PCNA foram contadas na íntima. Utilizou-se um total de 10 vistas de observação para calcular a percentagem média de células positivas para PCNA para cada rato.
RT-PCR
O RNA total dos tecidos dos vasos foi isolado com o Kit TRIzol (Life Technology, USA). A transcrição reversa foi utilizada para obter cDNA a partir do RNA por meio do Kit de Transcrição Reversa de RNA (Promega, EUA). Um total de 2 µg de RNA e 1 µl de primer randômico foram desnaturados a 70°C durante 10 min e anelados a 4 °C durante 10 min. Em seguida, 2 µL de tampão 10 ×, 2 µl de MgCl2 (20,8 mol/l) e 1 µL de transcriptase reversa foram adicionados ao sistema de reação. Acrescentou-se água duplamente destilada (ddH2O) para levar o volume a 20 µl. As condições para síntese do cDNA eram de 37°C durante 1 h e 4°C durante 10 min. O PCR também continha 10 µl de 2 × SYBR Mixture (Takara, Japão), 7 µl de ddH2O, 1 µl de forward primer e 1 µl de reverse primer. As condições de PCR eram de 95°C durante 5 min, 95°C durante 15 s, 60°C durante 60 s e 40 ciclos. As sequências dos primers utilizados para RT-PCR eram as seguintes: Forward, 5'-CATCTCCGTGGTCCTGAAGT-3' e reverse, 5'-GGCAAGGGAAACGTCTAGTG-3' para fator de von Willebrand; forward, 5'-CAGAGTCCAGCACAATACCAG-3' e reverse, 5'-GACCCAGATTATGTTTGAGACC para α-actina do músculo liso; e forward, 5'-ACATGAATGACCTCGTCTCTGA-3' e reverse, 5'-CCTCTTCTTCTGCCTCCTCTCC-3' para GAPDH. O instrumento para PCR quantitativo em tempo real foi adquirido de ABI (EUA).
Análise estatística
Todos os dados foram analisados com o software de análise estatística SPSS 17.0. Os dados são expressos como média ± desvio padrão. Visto que os dados mostraram uma distribuição normal, a comparação entre os vários grupos foi analisada por análise de variância de fator único (ANOVA) e a comparação entre dois grupos foi realizada pelo teste de diferença mínima significativa (LSD) de Fisher. Um valor de p < 0,05 foi considerado estatisticamente significante.
ResultadosOs ratos sobreviveram bem 4 semanas após a operação
O procedimento de operação descrito anteriormente foi utilizado para construir modelos de enxerto da veia jugular na artéria carótida em ratos, em um lado. No pós-operatório, as veias transplantadas estavam bem preenchidas e os vasos sanguíneos batiam bem; a cola estava uniformemente espalhada na superfície das veias dos grupos α-CA e α-CA-RPM. O estado vital e as incisões dos ratos foram verificados todos os dias. Subsequentemente, descobrimos que um rato no grupo RPM e um no grupo α-CA tinham morrido de temperatura baixa 2 semanas depois da operação; os outros ratos sobreviveram e recuperaram-se bem, com pulso forte nas veias enxertadas. Os ratos foram sacrificados 4 semanas após a cirurgia; notavelmente, apenas 2 ratos apresentaram oclusão venosa, um no grupo α-CA e um no grupo RPM. Correspondentemente, o fluxo sanguíneo nas outras veias enxertadas era patente. As veias no grupo sham tinham se expandido levemente. Além disso, as veias do grupo controle tinham novo tecido de granulação, tubos mais espessos, edema e rigidez leve; porém, as veias dos grupos α-CA, RPM e α-CA-RPM tinham poucos tecidos novos que eram facilmente separados, sem expansão óbvia e claramente divididos dos tecidos ao redor e a cola não estava completamente degradada (Figura 1).
Os ratos sobreviveram bem 4 semanas após a operação. O procedimento de operação descrito anteriormente foi utilizado para construir modelos de enxertos da veia jugular na artéria carótida em ratos, em um lado. Após a operação, as veias transplantadas estavam bem preenchidas e os vasos sanguíneos batiam bem; a cola estava uniformemente espalhada na superfície das veias dos grupos α-CA e α-CA-RPM (flecha). Quatro semanas após a operação, as veias no grupo sham tinham se expandido levemente; as veias no grupo controle tinham novo tecido de granulação, tubos mais espessos, edema e rigidez leve; no grupo α-CA havia poucos tecidos novos que eram facilmente separados, sem expansão óbvia e claramente divididos dos tecidos ao redor e a cola não estava completamente degradada (flecha); no grupo RPM os tecidos estavam claramente divididos dos tecidos ao redor e eram frescos e sem expansão óbvia. A forma geral do grupo α-CA-RPM era similar à do grupo α-CA.
α-CA-RPM reduziu espessamento intimal dos enxertos venosos
Com a finalidade de observarmos os impactos que a intervenção de cada grupo teve na hiperplasia intimal, realizamos coloração das veias enxertadas 4 semanas após a operação. Após isso, utilizamos um sistema de análise de imagens de computador para analisar hiperplasia intimal. Isto revelou que a íntima do grupo controle era notavelmente mais espessa que a dos grupos α-CA, RPM e α-CA-RPM; a diferença era estatisticamente significativa (91,3 ± 3,9, 133,6 ± 8,0, 50,6 ± 5,4 vs. 233,6 ± 29,1 µm; p < 0,01; Figura 2B, C, D, E e F); a íntima do grupo RPM era mais espessa do que a do grupo α-CA; a diferença era estatisticamente significativa (133,6 ± 8,0 vs. 91,3 ± 3,9 µm; p < 0,05; Figura 2C, D e F); a íntima dos grupos α-CA e RPM era mais espessa do que a do grupo α-CA-RPM; a diferença era estatisticamente significativa (50,6 ± 5,4 vs. 91,3 ± 3,9 µm, 133,6 ± 8,0 µm; p < 0,05; Figura 2 C, D, E e F). Além disso, conforme descrito na Figura 3, os nossos resultados da coloração imuno-histoquímica para PCNA demonstram que os grupos controle, α-CA, RPM e α-CA-RPM tinham um índice de proliferação significativamente mais alto que o grupo sham (p < 0,01; Figura 3A, B, C, D, E e F) e a percentagem de células positivas para PCNA nos grupos α-CA, RPM e α-CA-RPM era significativamente menor que no grupo controle (p < 0,01; Figura 3B, C, D, E e F). Ademais, é notável que o índice de proliferação no grupo α-CA-RPM era marcadamente menor que nos grupos α-CA e RPM (p < 0,01; Figura 3C, D, E e F). Em conjunto, os nossos resultados demonstram que α-CA, RPM e α-CA-RPM inibem hiperplasia intimal em enxertos venosos; o efeito de α-CA-RPM é mais forte do de α-CA ou RPM.
α-CA-RPM reduziu espessamento intimal dos enxertos venosos. Quatro semanas após a operação, os tecidos foram coletados, fixados em formalina, cortados em seções de 4 <italic>µ</italic>m e coloridos com HE. Imagens (lente objetiva 100 ×) foram obtidas e analisadas com o sistema micro-imagem de Olympus. Os ratos foram divididos em 5 grupos: grupo sham (A), grupo controle (B), grupo α-CA (C), grupo RPM (D) e grupo α-CA-RPM (E). (F) representa o gráfico estatístico a espessura intimal de cada grupo. A íntima do grupo controle era notavelmente mais espessa que a dos grupos α-CA, RPM e α-CA-RPM; a diferença era estatisticamente significativa (91,3 ± 3,9, 133,6 ± 8,0, 50,6 ± 5,4 vs., 233,6 ± 29,1 <italic>µ</italic>m; p < 0,01); a íntima do grupo RPM era mais espessa da do grupo α-CA; a diferença era estatisticamente significativa (133,6 ± 8,0 vs. 91,3 ± 3,9 <italic>µ</italic>m; p < 0,05); a íntima dos grupos α-CA e RPM era mais espessa da do grupo α-CA-RPM; a diferença era estatisticamente significativa (50,6 ± 5,4 vs. 91,3 ± 3,9 <italic>µ</italic>m, 133,6 ± 8,0 <italic>µ</italic>m; p < 0,05). * Havia diferenças óbvias entre o grupo controle e os demais grupos, p < 0,05. <sup>#</sup> Havia diferenças óbvias entre o grupo α-CA e os demais grupos, p < 0,05. <sup>△</sup> Havia diferenças óbvias entre o grupo RPM e os demais grupos, p < 0,05. <sup>☆</sup> Havia diferenças óbvias entre o grupo α-CA-RPM e os demais grupos, p < 0,05.
α-CA-RPM diminuiu o índice de proliferação do enxerto venoso. Quatro semanas após a operação, os tecidos foram coletados, fixados em formalina, cortados em seções de 4 <italic>µ</italic>m e coloridos com o anticorpo primário anti-PCNA. Imagens (lente objetiva 200 ×) foram obtidas e analisadas com o sistema micro-imagem de Olympus. Os ratos foram divididos em 5 grupos: grupo sham (A), grupo controle (B), grupo α-CA (C), grupo RPM (D) e grupo α-CA-RPM (E). (F) representa o gráfico estatístico do índice de proliferação de PCNA de cada grupo. * Havia diferenças óbvias entre o grupo controle e os demais grupos, p < 0,01. <sup>#</sup> Havia diferenças óbvias entre o grupo α-CA e os demais grupos, p < 0,01. <sup>△</sup> Havia diferenças óbvias entre o grupo RPM e os demais grupos, p < 0,01. <sup>☆</sup> Havia diferenças óbvias entre o grupo α-CA-RPM e os demais grupos, p < 0,01.
α-CA-RPM diminuiu hiperplasia intimal e respostas inflamatórias
Com a finalidade de estudar o mecanismo pelo qual os três métodos de intervenção inibem hiperplasia intimal mais profundamente, nós examinamos os valores de αSMA e vWF nas veias enxertadas 4 semanas após a cirurgia. Os valores de αSMA nos grupos α-CA, RPM e α-CA-RPM eram muito menores que no grupo controle, conforme detectado por RT-PCR (p < 0,01; Figura 4A). Os valores de αSMA no grupo α-CA-RPM eram menores que nos grupos α-CA e RPM (p < 0,01; Figura 4A). Encontraram-se resultados similares nos valores de vWF no grupo α-CA-RPM (p < 0,01; Figura 4B). Este resultado verificou que a inibição por α-CA, RPM e α-CA-RPM pode reduzir hiperplasia intimal por bloquearem a sobre-expressão de αSMA e vWF. Notamos que os níveis de ET-1 aumentaram gradualmente nos grupos controle, α-CA, RPM e α-CA-RPM 36 horas após a operação; os dos grupos controle, α-CA e RPM ainda estavam altos 4 semanas após a operação e os do grupo e α-CA-RPM tinham basicamente voltado ao normal. Os níveis de ET-1 no grupo controle eram significativamente mais altos que os dos grupos α-CA, RPM e α-CA-RPM 36 horas e 4 semanas após a operação (96,1 ± 7,9 ng/l vs. 84,0 ± 10,9 ng/l, 79,5 ± 5,7 ng/l e 72,7 ± 9,9 ng/l; 99,7 ± 7,7 ng/l vs. 87,1 ± 13,3 ng/l, 65,4 ± 23,4 ng/l e 43,7 ± 20,1 ng/l; p < 0,05, respectivamente). Adicionalmente, 4 semanas após a operação, os níveis de ET-1 no grupo α-CA-RPM eram significativamente menores que nos grupos α-CA, RPM e controle (43,7 ± 20,1 ng/l vs. 87,1 ± 13,3 ng/l, 65,4 ± 23,4 ng/l e 99,5 ± 7,7 ng/l; p < 0,05, respectivamente) (Figura 4C). Estes resultados indicam que α-CA, RPM e α-CA-RPM parecem reduzir respostas inflamatórias e que α-CA-RPM é mais efetivo.
α-CA-RPM diminuiu a expressão de αSMA e vWF e respostas inflamatórias. Quatro semanas após a operação, realizou-se RT-PCR para detectar αSMA e vWF nas veias enxertadas. (A) Os valores de αSMA nos grupos α-CA, RPM e α-CA-RPM eram muito menores que no grupo controle, conforme detectado por RT-PCR. Os valores de αSMA no grupo α-CA-RPM eram menores que nos grupos α-CA e RPM (p < 0,01). (B) Encontraram-se resultados similares nos valores de vWF no grupo α-CA-RPM (p < 0,01). (C) Mostram-se os níveis séricos de ET-1 para cada grupo em diversos momentos. Os níveis de ET-1 o grupo controle eram significativamente mais altos que os dos grupos α-CA, RPM e α-CA-RPM 36 horas e 4 semanas após a operação (96,1 ± 7,9 ng/l vs. 84,0 ± 10,9 ng/l, 79,5 ± 5,7 ng/l e 72,7 ± 9,9 ng/l; 99,7 ± 7,7 ng/l vs. 87,1 ± 13,3 ng/l, 65,4 ± 23,4 ng/l e 43,7 ± 20,1 ng/l; p < 0,05, respectivamente). Adicionalmente, 4 semanas após a operação, os níveis de ET-1 no grupo α-CA-RPM eram significativamente menores que nos grupos α-CA, RPM e controle (43,7 ± 20,1 ng/l vs. 87,1 ± 13,3 ng/l, 65,4 ± 23,4 ng/l e 99,5 ± 7,7 ng/l; p < 0,05, respectivamente). * Havia diferenças óbvias entre o grupo α-CA e os demais grupos, p < 0,01. <sup>#</sup> Havia diferenças óbvias entre o grupo α-CA e os grupos sham, controle e α-CA-RPM, p < 0,01. <sup>△</sup> Havia diferenças óbvias entre o grupo RPM e os grupos sham, controle e α-CA-RPM, p < 0,01.<sup>☆</sup> Havia diferenças óbvias entre o grupo α-CA-RPM e os demais grupos, p < 0,01.
Discussão
O achado principal do nosso estudo é que a aplicação de α-CA, RPM ou α-CA-RPM pode melhorar a patência de modelos de enxerto venoso em ratos por meio da inibição de hiperplasia intimal.8 Mais importante, o uso de RPM em combinação com α-CA é mais efetivo que o uso isolado de α-CA ou RPM.
O processo complicado de remodelação de vasos conduz à reestenose de enxertos venosos, mas o mecanismo exato não é explícito. Estudos têm mostrado que a reestenose está relacionada à disfunção de células endoteliais da íntima, à proliferação, à migração de células musculares lisas vasculares, fibroblastos adventícios, reações inflamatórias, força de cisalhamento e mudanças hemodinâmicas.9,10 O processo patológico de reestenose de um pontagem pode incluir trombose precoce, hiperplasia intimal e aterosclerose; a hiperplasia intimal é a causa mais importante. Quando se separa, liga, divide, transplanta e revasculariza pontagens, fatores como agregação de plaquetas e neutrófilos, liberação de citocinas e quimiocinas, ativação da via de transdução e reação enzimática podem desencadear hiperplasia de células musculares lisas vasculares e acumulação de células endoteliais. Todos esses fatores podem resultar em hiperplasia intimal e a reestenose dos vasos enxertados decorre em sequência.11,12
Em 1963, Parsonnet e seus colegas indicaram, pela primeira vez, que suportes extravasculares podiam melhorar as taxas de patência das veias enxertadas.5 Numerosos ensaios básicos e clínicos têm comprovado que suportes extravasculares podem inibir hiperplasia intimal e melhorar as taxas de patência. Quatro suportes extravasculares têm sido amplamente utilizados em ensaios básicos e clínicos, i.e. stent extravascular de nitinol, stent extravascular polimérico, cola de fibrina e α-CA. Reconhece-se que α-CA pode não apenas inibir a expansão dos vasos após o transplante mas também estimular a migração das células musculares lisas vasculares para a membrana externa vascular.13,14 Estimulados por α-CA, muitos neutrófilos e monócitos agregam à adventícia, especialmente fagócitos mononucleares que podem liberar quantidades de fatores quimiotáticos para atrair a imigração e colonização de células musculares lisas vasculares e fibroblastos.15 Uma série de mudanças mencionadas acima ativará vários fatores anti-ateroscleróticos: NO, PGI2, cAMP e cGMP. Eles também podem diminuir colesterol intimal e inibir fatores pro-ateroscleróticos.16 Os resultados do nosso experimento revelaram que as veias do grupo α-CA tinham poucos tecidos frescos que eram facilmente separados, sem expansão óbvia e claramente divididos dos tecidos ao redor 4 semanas após a operação. A cola não estava completamente degradada e a íntima do grupo α-CA era menos espessa que a do grupo controle. Além disso, a percentagem de células positivas para PCNA era significativamente menor que no grupo controle. Sobretudo, como suporte extravasuclar α-CA foi capaz de inibir hiperplasia intimal e melhorar a taxa de patência.
A proliferação, imigração e secreção de células musculares lisas vasculares são fundamentais à hiperplasia intimal, que contribui à reestenose de enxertos venosos. Embora certos fármacos possam ser efetivos em inibir a hiperplasia intimal por meio da inibição da citocinina e a regulação do ciclo celular, reações tóxicas severas e efeitos colaterais limitam o seu uso extensivo; consequentemente, a aplicação local torna-se particularmente significativa. A RPM, colchicina e outros fármacos são utilizados localmente em veias enxertadas. Após a anastomose, o fármaco é aplicado uniformemente nas veias enxertadas. A RPM pode acelerar a apoptose de células musculares lisas vasculares por meio da inibição da transformação das células da fase G1 à fase S, deste modo suprimindo a proliferação e imigração das células musculares lisas vasculares. Adicionalmente, RPM protege a função das células endoteliais e reduz a liberação de peptídeo vasoativo quando as células endoteliais são lesadas.17-19 Além disso, a RPM também pode inibir a diferenciação, proliferação e imigração de células progenitoras endoteliais (CPE) e reduz a expressão de NOS-mRNA em CPE.20,21 Os nossos resultados verificaram que as veias no grupo RPM claramente divididos dos tecidos do entorno eram frescas e não tinham se expandido. Ademais, a íntima do grupo RPM era menos espessa do que a do grupo controle e a percentagem de células positivas para PCNA era marcadamente menor que no grupo controle. Em resumo, RPM pode inibir a hiperplasia intimal e melhorar a taxa de patência.
O presente estudo visou experimentar a combinação de um suporte extravascular e uma aplicação local. Nós escolhemos α-CA como o suporte extravascular, RPM como a aplicação local e α-CA-RPM como a combinação. Utilizou-se α-CA-RPM em modelos de enxerto venoso autógeno em ratos para estimular o processo patofisiológico após CRM. É de interesse que a percentagem de células positivas para PCNA no grupo α-CA-RPM era marcadamente menor que nos grupos controle, α-CA e RPM, o que indica que α-CA-RPM era mais efetivo em inibir a hiperplasia intimal que α-CA ou RPM, isoladamente. Nós concluímos que α-CA-RPM pode combinar a efetividade de suportes extravasculares e aplicações locais e que inibe a hiperplasia intimal melhor. Entretanto, α-CA é um veículo ideal para a formulação de um fármaco com liberação controlada a longo prazo que circunda o enxerto venoso estavelmente de maneira que a RPM seja liberada lentamente e sem desperdício.
A endotelina-1 (ET-1) tem sido implicada na patogênese da reestenose e a hipertrofia vascular por meio do aumento da agregação de plaquetas e neutrófilos, a liberação de citocinas e quimiocinas, o acúmulo de células endoteliais e a promoção da migração de células musculares lisas vasculares em direção à camada intimal.22 Os nossos resultados indicam que α-CA, RPM e α-CA-RPM podem estabilizar a função celular endotelial e diminuir a liberação de ET-1 para inibir a hiperplasia intimal. Uma antagonista dos receptores de endotelina A/B contribuiu para a redução da hiperplasia intimal em uma cultura de órgãos de veias safenas humanas e impediu o desenvolvimento neointimal da angioplastia coronariana em porcos, o qual está de acordo com o nosso experimento.23,24 A αSMA é a proteína específica das células musculares lisas vasculares e a sua expressão pode refletir a hiperplasia das células musculares lisas vasculares. Examinamos o valor de αSMA nas veias enxertadas com RT-PCR e verificamos que os valores nos grupos α-CA, RPM e α-CA-RPM eram menores que no grupo controle. Notavelmente, o valor do grupo α-CA-RPM era menor que nos grupos α-CA e RPM. Um estudo no qual o componente de αSMA das células progenitoras vasculares foi correlacionado com o escore de Gensini da artéria coronária também fez a mesma observação.25 Um experimento em um modelo suíno de desvio arteriovenoso também forneceu evidências tangíveis que apóiam este ponto de vista.26
O vWF é uma glicoproteína codificada pelo braço curto do cromossomo 12 que pode ser combinada com fibras de colágeno e plaquetas; está intimamente relacionada com uma série de doenças cardiovasculares, tais como aterosclerose, síndrome coronariana aguda e fibrilação atrial.27 O vWF estimula a proliferação das células musculares lisas vasculares diretamente, o que resulta em efeito de dose-resposta direto. Também acelera a hiperplasia intimal no endotélio intacto sem ativação plaquetária ou liberação de fator de crescimento derivado de plaquetas.28 Similarmente, nós achamos que os valores de vWF nos grupos α-CA, RPM e α-CA-RPM eram menores que no grupo controle e o do grupo α-CA-RPM era menor que nos grupos α-CA e RPM. Os nossos resultados em ratos são corroborados por experimentos em outros animais, por exemplo, um estudo de eficácia em cachorros e um estudo sobre hiperplasia intimal de artérias carótidas de coelhos.2930 Estes resultados demonstram que α-CA, RPM e α-CA-RPM podem reduzir a hiperplasia intimal por bloquearem a sobre-expressão de αSMA e vWF.
Os nossos resultados mostram que rapamicina em combinação com α-cianoacrilato contribui à inibição de hiperplasia intimal e é mais efetivo para patência vascular que o uso individual de α-CA ou RPM em modelos em ratos 4 semanas após operação. Os efeitos a longo prazo de α-CA-RPM em remodelação de enxertos venosos ainda não estão claros. A nossa equipe conduzirá pesquisa futura em porcos após CRM e os impactos de intervenções relacionadas às veias enxertadas.
Conclusão
Os nossos resultados confirmaram que α-CA-RPM contribui à inibição de hiperplasia intimal e é mais efetivo para patência vascular que o uso individual de α-CA ou RPM em modelos de cirurgia de revascularização miocárdica em ratos. Os efeitos positivos parecem ser associados à diminuição de espessamento intimal, à redução de proliferação celular no enxerto venoso e à diminuição de respostas inflamatórias. Embora os efeitos a curto prazo de α-CA-RPM pareçam promissores, os efeitos a longo prazo e o significado clínico de α-CA-RPM em CRM precisam ser estudados futuramente.
Fontes de financiamento
O presente estudo foi financiado pelo National Natural Science Foundation of China.
Vinculação acadêmica
Este artigo é parte de dissertação de Mestrado de Tianshu-Chu pela Anhui Medical University.
Aprovação ética e consentimento informado
Este estudo foi aprovado pela Comissão de Ética em Experimentação Animal da Anhui Animal Ehtics Committee sob o número de protocolo ah56743.
Agradecimentos
Os autores gostariam de agradecer XR Huang e DK Huang pela sua técnica especializada.