Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC B3tLFkD43jZ8fDYkYQtMQdk S0066-782X2018001600562 S0066-782X2018005013102 10.5935/abc.20180163 Original Article Effects of Rosuvastatin on Apolipoprotein J in Balloon-Injured Carotid Artery in Rats Yang Ning 1 Dong Bo 1 Yang Jinyu 1 Li Yang 1 Kou Lu 1 Liu Yue 1 Qin Qin 1 Tianjin Chest Hospital Department of Cardiovascular Tianjin China Department of Cardiovascular, Tianjin Chest Hospital, Tianjin - China Mailing Address: Qin Qin, Nº. 261 Taierzhuang South Road, Jinnan District, Tianjin. 300222, Tianjin - China E-mail: qinqintj@163.com

Author contributions

Conception and design of the research and Writing of the manuscript: Yang N, Qin Q; Acquisition of data: Yang N, Dong B, Yang J, Li Y, Kou L, Liu Y; Analysis and interpretation of the data: Yang N, Dong B, Yang J, Li Y, Kou L; Statistical analysis: Yang N, Dong B; Critical revision of the manuscript for intellectual content: Yang N, Dong B, Yang J, Li Y, Qin Q.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 21 09 2018 10 2018 111 4 562 568 07 12 2017 07 05 2018 09 05 2018 This 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 cited Abstract Background:

Restenosis after percutaneous coronary intervention in coronary heart disease remains an unsolved problem. Clusterin (CLU) (or Apolipoprotein [Apo] J) levels have been reported to be elevated during the progression of postangioplasty restenosis and atherosclerosis. However, its role in neointimal hyperplasia is still controversial.

 Objective:

To elucidate the role Apo J in neointimal hyperplasia in a rat carotid artery model in vivo with or without rosuvastatin administration.

 Methods:

Male Wistar rats were randomly divided into three groups: the control group (n = 20), the model group (n = 20) and the statin intervention group (n = 32). The rats in the intervention group were given 10mg /kg dose of rosuvastatin. A 2F Fogarty catheter was introduced to induce vascular injury. Neointima formation was analyzed 1, 2, 3 and 4 weeks after balloon injury. The level of Apo J was measured by real-time PCR, immunohistochemistry and western blotting.

 Results:

Intimal/medial area ratio (intimal/medial, I/M) was increased after balloon-injury and reached the maximum value at 4weeks in the model group; I/M was slightly increased at 2 weeks and stopped increasing after rosuvastatin administration. The mRNA and protein levels of Apo J in carotid arteries were significantly upregulated after rosuvastatin administration as compared with the model group, and reached maximum values at 2 weeks, which was earlier than in the model group (3 weeks).

 Conclusion:

Apo J served as an acute phase reactant after balloon injury in rat carotid arteries. Rosuvastatin may reduce the neointima formation through up-regulation of Apo J. Our results suggest that Apo J exerts a protective role in the restenosis after balloon-injury in rats.

 Keywords: Coronary Artery Dsease Percutaneous Coronary Intervention Rosuvastatin Calcium Apolipoprotein J Coronary Reestenosis Rats Health and Family Planning Commission, Science and Technology of Tianjin 2015KR07 Tianjin Health Industry 13KG131 This work was funded by the Health and Family Planning Commission, Science and Technology of Tianjin, (Award Nº. 2015KR07), and the Tianjin Health Industry (Award Nº. 13KG131). Introduction

Coronary heart disease (CHD) is one of the most common cardiovascular diseases with high morbidity and mortality. Major effective techniques for myocardial revascularization are percutaneous coronary intervention (PCI) and coronary bypass surgery. Percutaneous transluminal coronary angioplasty (PTCA) is an effective treatment for CHD, but its effect in long-term is influenced by a high restenosis rate. Although drug eluting stents (DES) combined with dual antiplatelet therapy greatly reduce the occurrence of restenosis, the incidence rate still exceeds 10%.1,2 The mechanism underlying restenosis after PCI has been widely studied worldwide, but effective cellular or molecular targets for the treatment of restenosis after PCI urgently needs to be identified.

Clusterin (CLU), or Apolipoprotein (Apo) J, is a heterodimeric glycoprotein, which is composed of α and β subunits linked by disulfide bond.3,4 The coding gene of Apo J is located on chromosome 8p21-p12, mainly encoding two isoforms - secretory CLU (sCLU) and nuclear CLU (nCLU).5 Apo J has been reported to be induced during the progression of postangioplasty restenosis and atherosclerosis.6-9 However, the role of Apo J in neointimal hyperplasia is still controversial. It has been reported that Apo J could stimulate the proliferation and migration of vascular smooth muscle cell (VSMC) in CLU-knockout mice by inhibiting the expression of p53 and p21, and promote restenosis.10,11 On the contrary, Kim et al.12 revealed that the overexpression of sCLU can inhibit the migration and proliferation of VSMC and inhibit the apoptosis of cells. In view of existing paradoxical findings, we aimed to elucidate the role Apo J in neointimal hyperplasia in a rat carotid artery model in vivo with or without rosuvastatin intervention.

 Methods Animals

Male Wistar rats weighing 350-400 g were randomly divided into three groups: control group (n = 20), model group (n = 20) and intervention (statin) group (n = 32). All animals were then randomly divided into 4 groups - to be evaluated at 1, 2, 3 or 4 weeks after balloon injury. The study was approved by the Ethics Committee of Tianjin Chest Hospital.

 Balloon injury

The rats were weighed on the day of operation, and randomly divided into three groups. The rats in the intervention group were given 10 mg/kg dose of rosuvastatin. A 2F Fogarty catheter was introduced to induce vascular injury as previously reported.13 Briefly, the rats were anesthetized after intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 mL/100 g body weight. A 2F balloon catheter was inserted into aortic outlet of carotid artery. The balloon was then inflated and pulled back 3 times to denude the endothelium.

At 1, 2, 3 and 4 weeks after surgery, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 ml/100 g body weight. Then, the animals were sacrificed by intravenous administration of 2-3 mL of potassium chloride solution via subclavian vein; 0.3 cm of the right carotid artery was fixed in 10% neutral formalin for pathological examination, and the other part was frozen immediately in liquid nitrogen and stored at -80ºC for further use.

 Hematoxylin-eosin (HE) staining

Vascular specimens were fixed in 10% formaldehyde solution for 3-4h. Routine dehydration and paraffin embedding were performed. The sections were cut evenly and the thickness was 4 µm. The injury of blood vessels was observed under light microscope.

 Immunohistochemistry (IHC) assay

The level of Apo J was assessed by IHC in rat carotid artery. The primary antibody (polyclonal rabbit anti-human Apo J IgG) was purchased from Santa Cruz, Inc. (Cat No. sc-8354). The secondary antibody (labeled goat anti-rat/rabbit IgG polymer) was purchased from Maixin BioTech (Fuzhou, China). All photos were captured and saved using the ISCapture system, and data collection and analysis are performed using the Image Pro Plus 6 image processing software.

 Enzyme-linked immunosorbent assay (ELISA)

Venous blood was collected and centrifugated at 3000r/min for 10 min. The supernatant was collected using a micropipette and stored in the refrigerator at -20ºC for use. The samples were then thawed at room temperature for ELISA. ELISA was performed using a commercial kit (Rat Competitive ELISA for Apolipoprotein J A 252 SC), following the manufacturers’ instructions.

 Real-time polymerase chain reaction (PCR)

The mRNA level of Apo J was detected by real-time PCR in rat carotid artery. RNA was extracted by Trizol one-step extraction method, and reverse transcription was performed. Primers used for amplification for Apo J were as follows: Forward, TAA GGA GAT TCA GAA CGC CG; reverse, ATC CCT GGT GTC ATC TAG AG. Primers for the control GAPDH were as follows: Forward, GTG ATG CTG GTG CCG AGT AG; reverse, GGT GGC AGT GAT GGC GTG C. Real-time PCR reactions were prepared following the instructions of SYBR® Premix Ex Taq™ system (Perfect Real Time). The mRNA levels in each sample were calculated by 2-ΔΔCt.

 Western blotting

Proteins were extracted from 30 mg of rat carotid artery. Briefly, proteins were separated using SDS-PAGE with 10% separation gel and 5% concentrated gel. Then the separated proteins were transferred into polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and incubated with antibodies. Relative levels of Apo J were analyzed using Image Lab analysis software. β-actin was used as inner control. Bands were quantified using QUANTITY ONE software (Bio-Rad, Hercules, CA, USA).

 Statistical analysis

Statistical analysis was performed using SPSS 20.0. Quantitative data were expressed by mean ± standard deviation (SD). The difference between two groups was compared using independent-samples t test; comparisons between three groups were analyzed using one-way ANOVA (analysis of variance). P < 0.05 was considered statistically significant.

 Results Survival and success rates of rat carotid artery model

Among the 52 rats of the model group and the intervention group, 2 died during operation by suffocation, and 2 died for arterial hemorrhage 12h after operation. Therefore, 47 rats survived with approximately 90% survival rate. The pathological examination showed intimal hyperplasia and thickening in the experimental group, suggesting that the model was successfully constructed. The mean operation time was 34.19 ± 6.09 min. The feasibility and success rate of this model can be highly reproducible if surgical procedures are properly performed.

 Level of serum Apo J

There was no significant difference in serum Apo J level before and after operation in the intervention group (Table 1). There was no significant difference in the level of serum Apo J at 1, 2, 3 and 4 weeks before (F= 1.002, p = 0.408) of after (F= 0.189, p = 0.903) operation.

Serum levels of apolipoprotein J (Apo J) before and after operation in the statin intervention group
Time points Pre-operation Post-operation t* p#
n Apo J n Apo J
1 7 13.498 ± 3.015 7 10.317 ± 3.567 1.802 0.097
2 7 14.062 ± 4.538 7 11.516 ± 1.762 1.383 0.192
3 8 11.234 ± 2.740 8 11.117 ± 3.104 0.08 0.937
4 8 14.143 ± 4.609 8 11.205 ± 3.579 1.424 0.176
F$ 1.002 0.189
P# 0.408 0.903

t test used to compare the differences between the two groups;

F one-way ANOVA (analysis of variance) to compare the difference between all four groups.

p value < 0.05 was considered statistically significant.

 Statin intervention inhibited intimal hyperplasia

Results of pathological examination showed that no intimal hyperplasia was observed in the control group (Figure 1). In the model group and intervention group, the intima was slightly thickened 1 week after operation, and further thickened 2 weeks after operation. During 3 weeks after operation, the degree of intimal hyperplasia was further aggravated in the model group; however, this change was not as marked as at 2 weeks after operation, and the cells gradually became paralleled. During 4 weeks after operation, the degree of intimal hyperplasia was further aggravated in the model group, but no significant changes were observed regarding the degree of intimal hyperplasia as compared with week 3 (Figure 2 and 3).

Hematoxylin-eosin (HE) staining in the control group. Upper, magnification 40×; lower, magnification 400×.

Hematoxylin-eosin (HE) staining in the model group and in intervention group 1 week (w), 2 weeks, 3 weeks and 4 weeks after balloon injury of rat carotid arteries; magnification 40×.

Hematoxylin-eosin (HE) staining in the model group and in intervention group 1 week (w), 2 weeks, 3 weeks and 4 weeks after balloon injury of rat carotid arteries; magnification 400×.

Intimal and medial membrane areas were measured using Image Pro Plus 6, and intimal/medial area ratio (intimal/medial, I/M) was used to indicate the degree of intimal hyperplasia. As shown in Table 2, the I/M was close to 0 in the control group and was significantly different from that in the model group and the intervention group at all time points (1, 2, 3 and 4 weeks). There were significant differences of I/M between different time points in the model group, and I/M reached the maximum at the fourth week. No significant difference of I/M was observed between 2, 3 and 4 weeks post-surgery in the intervention group, and I/M in the intervention group was significantly lower than that in the model group (Table 2). Taken together, our results suggest that rosuvastatin could significantly inhibit intimal hyperplasia in rats.

Intimal/medial (I/M) area ratio in the study groups
Time points Control group Model group Statin intervention group t* p#
n I/M n I/M n I/M
1 5 0.04 ± 0.07 5 0.63 ± 0.40γ 5 0.42 ± 0.04γ 10.066 < 0.001
2 5 0.01 ± 0.02 4 1.08 ± 0.29▲ 4 1.29 ± 0.31∆▲ 39.639 < 0.001
3 5 0.03 ± 0.03 4 1.81 ± 0.11aβ 4 1.47 ± 0.54∆β 37.142 < 0.001
4 5 0.05 ± 0.04 4 2.61 ± 1.12abθ 4 1.50 ± 0.26∆θc 20.287 < 0.001
F$ 0.741 9.432 21.393
P# 0.543 < 0.001 < 0.001

t test used to compare the differences between the two groups.

F one-way ANOVA (analysis of variance) to compare the difference between all four groups.

p value < 0.05 was considered statistically significant

 Level of Apo J in carotid arteries

The mRNA levels of Apo J were measured by real-time PCR. Our results showed that the Apo J mRNA level was strikingly increased 2 weeks after operation, reached to a peak at the 3rd week, and decreased at the 4th week post-surgery in the model group. In intervention group, the Apo J mRNA level was strikingly increased and reached to a peak at the 2nd week, and decreased at the 3rd and 4th week post-surgery in the intervention group. In addition, the mRNA level of Apo J was higher in the intervention group than in the model group at the 1st week after operation. At the 2nd week post-surgery, the mRNA level of Apo J was strikingly increased in both groups and was significantly higher in the stain-intervention group than the model group (Table 3). Similar results have been observed in the protein levels of Apo J as shown in Figure 4. Our results showed that rosuvastatin could significantly increase the expression level of Apo J in balloon-injured rat carotid arteries.

Relative (2-ΔΔCt) levels of apolipoprotein J mRNA
Time points Control group Model group Statin intervention group t* p#
n 2-ΔΔCt n 2-ΔΔCt n 2-ΔΔCt
1 5 0.958 ± 0.251 5 0.641 ± 0.296 6 1.275 ± 0.468a 4.212 0.039
2 5 0.948 ± 0.090 4 7.804 ± 1.328a▲ 6 10.040 ± 2.086∆b 52.279 < 0.001
3 5 1.004 ± 0.196 4 8.011 ± 2.306aβ 6 7.327 ± 2.869∆*β 15.31 < 0.001
4 5 1.048 ± 0.349 4 3.429 ± 1.119abcθ 6 2.413 ± 0.492* #θ 14.212 0.001
F$ 0.182 29.266 31.336
P# 0.907 < 0.001 < 0.001

t value was calculated using independent-samples t test to compare the difference between two groups.

F value was calculated using one-way ANOVA (analysis of variance) to compare the difference among the four groups.

p value (probability value) < 0.05 is considered to be statistically significant.

Western blotting of apolipoprotein J (Apo J) protein levels 1 week (w), 2 weeks, 3 weeks and 4 weeks after balloon injury of rat carotid arteries.

Discussion

In the present study, we found that I/M increased after balloon-injury and reached the maximum at 4w in the model group; also, I/M was slightly increased at 2w and stopped increasing after rosuvastatin administration. Our results suggest that rosuvastatin could significantly reduce the degree of intimal hyperplasia in balloon-injured carotid arteries in rats. The levels of Apo J mRNA and protein in carotid arteries were significantly upregulated after rosuvastatin administration as compared with the model group, and reached to maximum at 2 weeks, which was earlier than the in the model group. Our results suggest that rosuvastatin may inhibit intimal hyperplasia through upregulation of Apo J after balloon-injury in rats.

Apo J has been reported to be closely related to cardiovascular diseases, such as atherosclerosis and restenosis after angioplasty.14,15 Ishikawa et al.7 revealed the distribution of Apo J in the extracellular matrix of endarterium in human atherosclerotic aorta, and its potential protective role against human atherosclerosis by cholesterol transport from the aortic wall to the liver. It has been reported that ApoJ is increased in tissue injury and cell stress, and plays vital role in protection against oxidative stress, cell lysis and apoptotic cell death.16-21 Additionally, ApoJ could be observed in active tissue remodeling. These findings indicate that ApoJ may act as an acute phase reactant. In the present study, we observed a marked neointimal thickening 2 weeks post-surgery, with proliferation and migration VSMCs observed by HE staining in the model group. The proliferation and migration of VSMC were the most active at week 3, and decrease at week 4. In the meantime, the mRNA and protein levels of Apo J were significantly increased at week 2, reached a peak at 3 weeks after operation, and then decreased at 4 weeks. The results showed high expression of Apo J in the phase of active proliferation and migration of VSMCs. Consistent with other studies, our results suggest that Apo J may be an acute phase reactant after balloon-injury in rat carotid arteries.

In-stent restenosis after interventional procedures has become one of the most urgent problems to be solved worldwide. Rosuvastatin, a potent hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been reported to reduce neointimal thickening after vascular endothelial injury in rats13 In the present study, the rats in the intervention group received intragastric administration of rosuvastatin (10 mg/kg/d). In accordance with other studies,22-24 we found that rosuvastatin significantly reduced the neointima formation.

It has been reported that secreted isoform of Apo J (sCLU) could inhibit the proliferation and migration of VSMCs.12,25 Kim et al.12 also found that Apo J could significantly inhibit neointimal hyperplasia using adenovirus-mediated overexpression of Apo J in rats. In the present study, we found that the mRNA and protein levels of ApoJ in carotid arteries were significantly upregulated after rosuvastatin administration as compared with the model group. Moreover, Apo J reached a maximum at week 2 after rosuvastatin administration, and that was earlier than the model group which reached peak expression at the third week. These results suggest that rosuvastatin may increase the level of Apo J in the balloon-injured carotid arteries, which indirectly indicates a protective role of Apo J against restenosis after balloon-injury in rats.

 Conclusion

Our results showed that Apo J served as an acute phase reactant after balloon-injury in rat carotid arteries. Rosuvastatin may reduce the neointima formation through further up-regulation of Apo J. Our findings suggest that Apo J exerts a protective role against restenosis after balloon-injury in rats.

Sources of Funding

This work was funded by the Health and Family Planning Commission, Science and Technology of Tianjin, (Award Nº. 2015KR07), and the Tianjin Health Industry (Award Nº. 13KG131).

Study Association

This study is not associated with any thesis or dissertation work.

Ethics approval and consent to participate

This study was approved by the Ethics Committee on Animal Experiments of the Tianjin Chest Hospital under the protocol number 2015-006-05

 Acknowledgements

We greatly appreciate the help of Tianjin Cardiovascular Disease Research Institute on the animal experiment.

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Contribuição dos autores

Concepção e desenho da pesquisa e Redação do manuscrito: Yang N, Qin Q; Obtenção de dados: Yang N, Dong B, Yang J, Li Y, Kou L, Liu Y; Análise e interpretação dos dados: Yang N, Dong B, Yang J, Li Y, Kou L; Análise estatística: Yang N, Dong B; Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Yang N, Dong B, Yang J, Li Y, Qin Q

Potencial conflito de interesses

Declaro não haver conflito de interesses pertinentes.

 Resumo Fundamento:

A reestenose após intervenção coronária percutânea (ICP) após doença coronariana continua um problema não solucionado. Estudos relataram que os níveis de clusterina (CLU), também chamada de apolipoproteína (Apo) J, encontram-se elevados na progressão da reestenose pós-angioplastia e na aterosclerose. Contudo, seu papel na hihperplasia neointimal ainda é controverso.

 Objetivo:

Elucidar o papel da Apo J na hiperplasia neointimal na artéria carótida utilizando um modelo experimental com ratos in vivo, com e sem intervenção com rosuvastatina.

 Métodos:

ratos Wistar machos foram divididos aleatoriamente em três grupos - grupo controle (n = 20), grupo modelo (n = 20), e grupo intervenção com estatina (n = 32). Os ratos no grupo intervenção receberam 10 mg/kg de rosuvastatina. Um cateter Fogarty 2 F foi introduzido para induzir lesão vascular. A formação de neoíntima foi analisada 1, 2, 3 e 4 semanas após lesão com balão. Concentrações de Apo J foram medidas por PCR em tempo real, imuno-histoquímica e western blotting.

 Resultados:

A razão área íntima/média (I/M) aumentou após a lesão com balão e atingiu o valor máximo 4 semanas pós-lesão no grupo modelo; observou-se um pequeno aumento na I/M na semana 2, que cessou após a administração de rosuvastatina. Os níveis de mRNA e proteína da Apo J nas artérias carótidas aumentaram significativamente após administração de rosuvastatina em comparação ao grupo modelo, atingindo o máximo na semana 2, mais cedo em comparação ao grupo modelo (semana 3).

 Conclusão:

A Apo J atuou como reagente de fase aguda após lesão com balão nas artérias carótidas de ratos. A rosuvastatina pode reduzir a formação de neoíntoma por aumento de Apo J. Nossos resultados sugerem que a Apo J exerce um papel protetor na reestenose após lesão com balão em ratos.

 Palavras-chave: Doença da Artéria Coronária Intervenção Coronária Percutânea Rosuvastatina Cálcica Apolipoproteína J Reestenose Coronária Ratos Introdução

A doença coronária é uma das doenças cardiovasculares mais comuns, com alta morbidade e mortalidade. As principais técnicas consideradas eficazes para revascularização do miocárdio são a intervenção coronária percutânea (ICP) e a cirurgia de bypass coronário. A angioplastia coronária transluminal percutânea (ACTP) é um método eficaz para o tratamento da doença coronária, mas seu efeito em longo prazo é influenciado por uma alta taxa de reestenose. Apesar de stents farmacológicos, combinados à terapia antiplaquetária dupla, reduzirem a ocorrência de reestenose, a taxa de incidência ainda excede 10%.1,2 O mecanismo da reestenose após a IPC tem sido amplamente estudado em todo o mundo, porém, alvos moleculares ou celulares eficazes para o tratamento da reestenose após ICP precisam ser identificados urgentemente.

A clusterina (CLU), ou apolipoproteína (Apo) J, é uma glicoproteína heterodimérica composta por subunidades α e β, ligadas por uma ponte dissulfeto.3,4 O gene que codifica a Apo J está localizado no cromossomo 8p21-p12, e codifica duas principais isoformas - CLU secretória (CLUs) e CLU nuclear (CLUn).5 Estudos prévios relataram que a Apo J é induzida durante a progressão da reestenose pós-angioplastia e da aterosclerose.6-9 No entanto, o papel da Apo J na hiperplasia neointimal é ainda controverso. Há estudos relatando que a Apo J pode estimular a proliferação e a migração de células do músculo liso vascular (CMLV) em ratos knockouts para CLU inibindo a expressão de p53 e p21, e promover reestenose.10,11 Por outro lado, Kim et al.12 revelaram que a superexpressão de CLUs pode inibir a migração e a proliferação de CMLV e inibir a apoptose celular. Em vista dos resultados controversos, nosso objetivo foi elucidar o papel da Apo J na hiperplasia neointimal, usando a artéria carótida de ratos in vivo, com ou sem administração de rosuvastatina.

 Métodos Animais

Ratos Wistar machos pesando 350-400g foram divididos aleatoriamente em três grupos - grupo controle (n = 20), grupo modelo (n = 20) e grupo intervenção (estatina) (n = 32). Os ratos de todos os grupos foram então divididos aleatoriamente em 4 grupos conforme momento de avaliação após lesão induzida com balão -1, 2 3 ou 4 semanas. O estudo foi aprovado pelo comitê de ética do hospital de tórax Tianjin.

 Lesão com balão

Os ratos foram pesados no dia da cirurgia e divididos aleatoriamente em três grupos. No grupo intervenção, os ratos receberam 10mg/kg de rosuvastatina. Um cateter de Fogarty 2F foi introduzido para induzir lesão vascular como descrito anteriormente.13 Em resumo, os ratos foram anestesiados após injeção intraperitoneal de hidrato de cloral 10% na dose 0,3 mL/100 g de peso corporal. Um cateter balão 2F foi inserido na artéria carótida, saída para a aorta. O balão foi então inflado, desinflado, e puxado para trás três vezes para expor o endotélio.

Após 1, 2, 3 ou 4 semanas, os ratos foram anestesiados por injeção intraperitoneal de hidrato de cloral 10% na dose 0,3 mL/100 g de peso corporal e sacrificados administrando-se 2-3mL de solução de cloreto de potássio via veia subclávia. A artéria carótida direita foi removida; 0,3 cm foi fixada em formalina neutra a 10% para análise patológica, e a outra parte imediatamente congelada em nitrogênio líquido e armazenado a -80ºC para análise futura.

 Coloração com hematoxilina e eosina (HE)

As amostras do vaso foram fixadas em solução de formaldeído por 3-4 horas. Foram realizadas hidratação e inclusão em parafina de rotina. As seções foram cortadas uniformemente em uma espessura de 4 µm. A lesão dos vasos sanguíneos foi analisada em microscópio.

 Teste de imuno-histoquímica (IHQ)

Os níveis de Apo J foram avaliadas por IHQ na artéria carótida do rato. O anticorpo primário (IgG de coelho anti-Apo J humano) foi comprado da empresa Santa Cruz, Inc. (número no catálogo sc-8354). O anticorpo secundário (anticorpo produzido em cabra anti IgG de rato/coelho) foi adquirido da empresa Maixin BioTech (Fuzhou, China). Todas as fotos foram capturadas e salvas usando o sistema ISCapture, e a coleta e análise de dados realizadas pelo programa de processamento de imagens Image Pro Plus 6.

 Ensaio de imunoabsorção enzimática (ELISA)

Amostras de sangue venoso foram coletadas e centrifugadas a 3000r/min. O sobrenadante foi coletado usando uma micropipeta e armazenado em geladeira a -20ºC. As amostras foram então descongeladas à temperatura ambiente para serem submetidas ao ELISA, o qual foi realizado usando um kit comercial (Rat Competitive ELISA for Apolipoprotein J A 252 SC) seguindo instruções do fabricante.

 Reação em cadeia da polimerase (PCR) em tempo real (Real-time PCR)

Os níveis de mRNA da Apo J foram detectadas por PCR em tempo real na artéria carótida do rato. O RNA foi extraído pelo método de extração em fase única com reagente Trizol, e realizada transcrição reversa. Os primers usados para amplificação da Apo J foram: forward, TAA GGA GAT TCA GAA CGC CG; reverse, ATC CCT GGT GTC ATC TAG AG. Os primers para o controle GAPDH foram: forward, GTG ATG CTG GTG CCG AGT AG; reverse, GGT GGC AGT GAT GGC GTG C. As reações de PCR em tempo real foram preparadas seguindo as instruções do sistema SYBR® Premix Ex Taq™ (Perfect Real Time). Os níveis de mRNA em cada amostra foram calculados como 2-ΔΔCt.

 Western blotting

As proteínas foram extraídas de 30mg da artéria carótida de ratos. Em resumo, as proteínas foram separadas por SDS-PAGE com gel de separação a 10% e gel concentrado a 5%. Em seguida, as proteínas separadas foram transferidas para membranas PVDF (fluoreto de polivinilideno). As membranas foram bloqueadas e incubadas com anticorpos. Níveis relativos de Apo J foram analisados pelo programa de análise Image Lab. Beta-actina foi usada como controle. As bandas foram quantificadas pelo programa QUANTITY ONE (Bio-Rad, Hercules, CA, EUA).

 Análise estatística

A análise estatística foi realizada usando o programa SPSS 20.0. Dados quantitativos foram expressados por média ± desvio padrão (DP). Diferenças entre os dois grupos foram comparadas pelo teste t para amostras independentes. Comparações entre três grupos foram analisadas pela análise de variância simples (one-way ANOVA). Um p < 0,05 foi considerado estatisticamente significativo.

 Resultados Taxas de sobrevida e de sucesso do modelo

Entre os 52 ratos do grupo modelo e do grupo intervenção, 2 ratos morreram durante a operação por asfixia, e 2 morreram por hemorragia arterial 12 horas após operação. Portanto, 47 ratos sobreviveram, com uma taxa de sobrevida aproximada de 90%. O exame patológico mostrou hiperplasia e espessamento da camada íntima no grupo experimental, sugerindo que o modelo foi construído com sucesso. O tempo médio de operação foi de 34,19 ± 6,09 min. A taxa de viabilidade e de sucesso do modelo deste modelo pode ter alta reprodutibilidade se os procedimentos cirúrgicos forem realizados adequadamente.

 Níveis de Apo J sérico

Não houve diferença significativa nos níveis séricos de Apo J antes e após a operação no grupo intervenção (Tabela 1). Não houve diferença significativa nos níveis séricos de Apo J na 1ª, 2ª, 3ª ou 4ª semana antes (F= 1,002, p = 0,408) ou após (F= 0,189, p = 0,903) o procedimento.

Níveis séricos de apolipoproteína J (Apo J) antes e após a operação no grupo intervenção
Time points Pré-operação Pós-operação t* p#
n Apo J n Apo J
1 7 13,498±3,015 7 10,317±3,567 1,802 0,097
2 7 14,062±4,538 7 11,516±1,762 1,383 0,192
3 8 11,234±2,740 8 11,117±3,104 0,08 0,937
4 8 14,143±4,609 8 11,205±3,579 1,424 0,176
F$ 1,002 0,189
P# 0,408 0,903

teste t para comparar diferença entre os dois grupos.

valor F calculado usando análise de variância simples (one-way ANOVA) para comparar a diferença entre os quatro grupos.

p < 0.05 considerado estatisticamente significativo.

 Intervenção com estatina inibiu hiperplasia intimal

Os resultados da avaliação patológica mostraram que não foi houve hiperplasia intimal no grupo controle (Figura 1). No grupo modelo e no grupo intervenção, a camada íntima estava ligeiramente espessada 1 semana após a operação, e ainda mais espessada 2 semanas após a operação. Durante 3 semanas após o procedimento, o grau de hiperplasia intimal aumentou no grupo modelo; no entanto, essa mudança não foi tão marcante como a observada na semana 2 após a operação, e as células tornaram-se paralelas gradualmente. Durante 4 semanas após a operação, o grau de hiperplasia intimal agravou-se no grupo modelo, mas não foram observadas mudanças significativas quanto ao grau de hiperplasia intimal em comparação à semana 3 (Figura 2 e 3).

Coloração de hematoxilina-eosina (HE) no grupo controle. Aumento de 40x (acima) e de 400x (abaixo).

Coloração de hematoxilina-eosina (HE) no grupo modelo e no grupo intervenção controle em 1 semana (sem), 2 semanas, 3 semanas e 4 semanas após lesão com balão de artérias carótidas de ratos; aumento de 40x.

Coloração de hematoxilina-eosina (HE) no grupo modelo e no grupo intervenção controle em 1 semana (sem), 2 semanas, 3 semanas e 4 semanas após lesão com balão de artérias carótidas de ratos; aumento de 400 x.

As áreas da membrana íntima e média foram medidas usando o Image Pro Plus 6, e a razão área íntima/média (íntima/média, I/M) foi usada para indicar o grau de hiperplasia intimal. Como mostrado na Tabela 2, a I/M foi próxima a 0 no grupo controle e foi significativamente diferente da I/M no grupo modelo e no grupo intervenção em todos os tempos (1, 2, 3 e 4 semanas). Não houve diferenças significativas da I/M entre os diferentes tempos no grupo modelo, e a I/M alcançou o valor máximo na quarta semana. Não foi observada diferença de I/M no grupo intervenção entre as semanas 2, 3 e 4 pós-cirurgia no grupo intervenção, e I/M no grupo intervenção foi significativamente mais baixa que aquela no grupo modelo (Tabela 2). Nossos resultados, como um todo, sugerem que a intervenção com rosuvastatina poderia inibir de maneira significativa a hiperplasia intimal em ratos.

Razão área íntima/média (I/M) nos grupos do estudo
Tempos Grupo controle Grupo modelo Grupo intervenção t* p#
n I/M n I/M n I/M
1 5 0,04 ± 0,07 5 0,63 ± 0,40γ 5 0,42 ± 0,04γ 10,066 < 0,001
2 5 0,01 ± 0,02 4 1,08 ± 0,29▲ 4 1,29 ± 0,31∆▲ 39,639 < 0,001
3 5 0,03 ± 0,03 4 1,81 ± 0,11aβ 4 1,47 ± 0,54∆β 37,142 < 0,001
4 5 0,05 ± 0,04 4 2,61 ± 1,12abθ 4 1,50 ± 0,26∆θc 20,287 < 0,001
F$ 0,741 9,432 21,393
P# 0,543 < 0,001 < 0,001

teste t para comparar diferença entre os dois grupos.

valor F calculado usando análise de variância simples (one-way ANOVA) para comparar a diferença entre os quatro grupos.

p < 0.05 considerado estatisticamente significativo.

 Níveis de Apo J nas artérias carótidas

Os níveis de mRNA de Apo J foram medidos por PCR em tempo real. Os resultados mostraram que o nível de mRNA de Apo J sofreu um grande aumentou 2 semanas após a cirurgia, atingiu o pico na terceira semana, e diminuiu na quarta semana pós-cirurgia no grupo modelo. No grupo intervenção, o nível de mRNA de Apo J sofreu um grande aumentou e atingiu o pico na semana 2, diminuindo nas semanas 3 e 4 pós-cirurgia no grupo intervenção. Além disso, o nível de mRNA de Apo J foi maior no grupo intervenção que no grupo modelo na primeira semana pós-operação. Na segunda semana pós-cirurgia, o nível de mRNA de Apo J aumentou consideravelmente em ambos os grupos, e foi significativamente mais alto no grupo intervenção que no grupo modelo (Tabela 3). Resultados similares foram observados nos níveis de proteína de Apo J como mostrado na Figura 4. Nossos resultados mostraram que a rosuvastatina pode aumentar de maneira significativa a expressão de Apo J nas artérias carótidas de ratos lesionadas com balão.

Níveis relativos (2-ΔΔCt) de mRNA da apolipoproteína J
Tempos Grupo controle Grupo modelo Grupo intervenção t* p#
n 2-ΔΔCt n 2-ΔΔCt n 2-ΔΔCt
1 5 0,958 ± 0,251 5 0,641 ± 0,296 6 1,275 ± 0,468a 4,212 0,039
2 5 0,948 ± 0,090 4 7,804 ± 1,328a▲ 6 10,040 ± 2,086∆b 52,279 < 0,001
3 5 1,004 ± 0,196 4 8,011 ± 2,306aβ 6 7,327 ± 2,869∆*β 15,31 < 0,001
4 5 1,048 ± 0,349 4 3,429 ± 1,119abcθ 6 2,413 ± 0,492* #θ 14,212 0,001
F$ 0,182 29,266 31,336
P# 0,907 < 0,001 < 0,001

teste t para comparar diferença entre os dois grupos.

valor F calculado usando análise de variância simples (one-way ANOVA) para comparar a diferença entre os quatro grupos.

p < 0.05 considerado estatisticamente significativo.

Western blotting dos níveis de proteína da apolipoproteína J (Apo J) 1 semana (sem), 2 semanas, 3 semanas e 4 semanas após lesão com balão de artérias carótidas de ratos.

Discussão

No presente estudo, encontramos que a I/M aumentou após a lesão com balão e atingiu o valor máximo na quarta semana no grupo modelo; além disso, I/M estava aumentada na semana 2 e tal aumento cessou após administração de rosuvastatina. Nossos resultados sugerem que a rosuvastatina pode reduzir significativamente o grau de hiperplasia intimal nas artérias carótida lesionadas com balão em ratos. Os níveis de mRNA e de proteína de Apo J nas artérias carótidas foram aumentados pela rosuvastatina quando comparados ao grupo modelo, atingindo o máximo na segunda semana, mais cedo que no grupo modelo. Nossos resultados sugerem que a rosuvastatina pode inibir a hiperplasia intimal pelo aumento de Apo J após lesão com balão em ratos.

Estudos relataram que a Apo J está intimamente relacionada a doenças cardiovasculares, tais como aterosclerose e reestenose após angioplastia.14,15 Ishikawa et al.7 apresentaram a distribuição de Apo J na matriz extracelular da camada íntima da aorta aterosclerótica de humanos, e seu potencial papel protetor na aterosclerose por meio do transporte de colesterol da parede aórtica para o fígado. A Apo J está aumentada na lesão tissular e estresse celular e tem uma função vital na proteção contra estresse oxidativo, lise celular e morte celular apoptótica.16-21 Além disso, observou-se Apo J na remodelagem ativa do tecido. Esses achados indicam que a Apo J pode atuar como um reagente de fase aguda. No presente estudo, observamos um espessamento marcante da camada neoíntima duas semanas após a cirurgia, com proliferação e migração de CMLV identificadas por coloração de HE no grupo modelo. A proliferação e a migração de CMLV foram mais ativas na terceira semana após cirurgia, e diminuíram na quarta semana. Ao mesmo tempo, os níveis de mRNA e de proteína da Apo J aumentaram significativamente na semana 2, alcançou um pico na terceira semana após operação, e diminuiu na quarta semana. Os resultados mostraram alta expressão de Apo J na fase de proliferação ativa e migração de CMLV. Consistente com outros estudos, nossos resultados sugerem que a Apo J pode ser um reagente de fase aguda após lesão com balão nas artérias carótidas de ratos.

Reestenose de stent após procedimentos intervencionistas tornou-se um dos problemas mais urgentes a serem resolvidos em todo o mundo. A rosuvastatina, um potente inibidor da enzima HMG-CoA (hidroximetilglutaril-CoA) redutase, parece reduzir o espessamento neointimal após lesão endotelial vascular em ratos. No presente estudo, os ratos do grupo intervenção receberam administração intragástrica de rosuvastatina (10 mg/kg/d). De acordo com outros estudos,22-24 encontramos que a rosuvastatina reduziu significativamente a formação de neoíntima.

Estudos prévios relataram que a isoforma secretada da Apo J (CLUs) pode inibir a proliferação e a migração de CMLV.12,25 Kim et al.12 também relataram que a Apo J poderia inibir significativamente a hiperplasia neointimal por meio da superexpressão de Apo J mediada por adenovírus em ratos. Em nosso estudo, observamos que níveis de mRNA e proteína da Apo J nas artérias carótidas aumentaram após a administração de rosuvastatina em comparação ao grupo controle. Ainda, a Apo J atingiu o máximo na segunda semana após a administração de rosuvastatina, e tal fato ocorreu mais cedo em comparação ao grupo modelo (terceira semana). Esses resultados sugerem que a rosuvastatina pode aumentar os níveis de Apo J nas artérias carótidas com lesões induzidas por balão, o que indiretamente indica que a Apo J exerce função protetora contra reestenose após lesão por balão em ratos.

 Conclusão

Nossos resultados mostraram que a Apo J serviu como um reagente de fase aguda após lesão nas artérias carótidas de ratos induzida por balão. A rosuvastatina pode reduzir a formação de neoíntima pelo aumento nos níveis de Apo J. Nossos resultados sugerem que a Apo J exerce papel protetor contra reestenose após lesão por balão em ratos.

Fontes de financiamento

O presente estudo foi financiado pela Comissão de Saúde e Planejamento Familiar Ciência e Tecnologia (Health and Family Planning Commission, Science and Technology) de Tianjin (concessão/prêmio número 2015KR07), e Indústria da Saúde de Tianjin (Tianjin Health Industry) (concessão/ prêmio número 13KG131).

Vinculação acadêmica

Não há vinculação deste estudo a programas de pós-graduação.

Aprovação ética e consentimento informado

Este estudo foi aprovado pela Comissão de Ética em Experimentação Animal do Tianjin Chest Hospital sob o número de protocolo 2015-006-05.

 Agradecimentos

Agradecemos muito o Instituto de Pesquisa sobre Doença Cardiovascular de (Cardiovascular Disease Research Institute) de Tianjin pela ajuda no experimento com os animais.