- Email: [email protected]
Urinary trypsin inhibitor reduced inflammatory response after stent injury in minipig
Pathology – Research and Practice 208 (2012) 344–349
Contents lists available at SciVerse ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.de/prp
Original article
Urinary trypsin inhibitor reduced inflammatory response after stent injury in minipig J.Y. Kong ∗ , T.Q. Wang, G.H. Jiang, L. Li, F.P. Wang Department of Emergency, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Hei Long Jiang, China
a r t i c l e
i n f o
Article history: Received 25 January 2012 Received in revised form 1 March 2012 Accepted 6 March 2012 Keywords: Urinary trypsin inhibitor Stent Restenosis Inflammation
a b s t r a c t This study investigated whether urinary trypsin inhibitor (UTI) inhibits neointimal formation by reducing inflammatory response after stent injury. Twenty minipigs having undergone oversized bare material stent implantation in the left anterior descending artery were randomly subdivided into two groups: a UTI group (n = 10) and a control group (n = 10). Two systemic markers of inflammation (serum macrophage chemoattractant protein-1 and interleukin-6 levels measured by ELISA) were increased after stent implantation, and two days after stem implantation, their levels were positively correlated with the maximal percentage of area stenosis on day 28 (r2 = 0.889 and 0.743, respectively). This effect was abolished by UTI administration. Twenty-eight days after implantation, morphometric analysis of the stented arteries revealed significantly reduced luminal stenosis (38 ± 6% vs. 64 ± 12%, P < 0.05), a neointimal area (3.22 ± 0.57 mm2 vs. 5.21 ± 1.04 mm2 , P < 0.05), neointimal thickness (0.31 ± 0.13 mm vs. 0.46 ± 0.16 mm, P < 0.05), and an inflammatory score of 1.02 ± 0.05 vs. 1.30 ± 0.08 in UTI-treated animals as compared with controls. Twenty-eight days after stenting, arterial nuclear factor-B expression was 36.93 ± 7.16% in all of the cells in controls and 23.32 ± 4.54% in UTI-treated minipigs. UTI could reduce neointimal formation after stenting by inhibiting the local and the systemic inflammatory response. Percutaneous coronary intervention could benefit from precocious anti-inflammatory treatment. © 2012 Elsevier GmbH. All rights reserved.
Introduction In-stent restenosis (ISR) remains a challenge for interventional cardiologists [16,17]. Systemic inflammation characterizes the response to vascular injury after percutaneous coronary intervention (PCI) [9,16,33]. Stent implantation, in particular, precipitates arterial intimal cellular proliferation and extracellular matrix synthesis largely mediated by inflammatory processes [13,35]. Inflammation is a major force in the pathophysiology of atherothrombosis. In addition to its presumed role in induction, progression and destabilization of the atherosclerotic lesion, the inflammatory response that follows vascular injury is of major importance to the restenotic process. Measurement of cytokine and acute phase proteins after stenting, such as C-reactive protein, may be important for the identification of high-risk subjects and for the development of specific treatment tailored to individual patients [14,16]. Urinary trypsin inhibitor (UTI) can inhibit the production and cascade of inflammatory cytokines and improve the inflammatory
symptom. It has been widely used for the treatment of non-specific inflammatory response diseases clinically [1–3,8,18,21,28,32]. Some previous studies have demonstrated the effect of UTI on inflammatory response after coronary artery bypass grafting with cardiopulmonary bypass; however, only few data on UTI and inflammatory response after stent injury are available [11,36]. The present study investigated whether UTI was able to reduce neointimal formation by inhibiting the inflammatory response in minipigs subjected to stent injury. Material and methods All animal care and procedures conducted in the present study conformed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Harbin Medical University (2009-X023). Vascular injury model
∗ Corresponding author. Tel.: +86 45186651587; fax: +86 45186651587. E-mail address: [email protected] (J.Y. Kong). 0344-0338/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2012.03.008
Twenty minipigs (20–25 kg) were intubated after sedation with ketamine [20 mg/kg, intramuscular (i.m.)] and diazepam
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
(0.4 mg/kg, i.m.) followed by 3% sodium pentobarbital through the marginal ear vein [25 mg/kg, (intravenous)]. Coronary angiography was carried out using 6-F guiding catheters after intracoronary administration of nitroglycerin (200 g). One bare-metal stent [(BMS), 18 mm in length and 3.0–3.5 mm in diameter; Lepu Medical Company, Beijing, China] was placed in the left anterior descending artery of each pig at high pressure (12–14 atm.) for >30 s. The stent-to-artery ratio was maintained at 1.2:1. All animals were pretreated with clopidogrel (150 mg loading dose followed by 75 mg/day) and aspirin (300 mg loading dose followed by 100 mg/day) for three days before the procedure. The animals were randomly assigned to either the control or the UTI group (Techpool Biochemical Pharmaceutical Co., Ltd., 10,000 U/kg/d, continuous administration 28 days) after injury. Arterial blood was collected in citrate-containing tubes 24 h prior to the procedure and at 2, 7, 14, and 28 days after injury. The serum was separated and stored at −70 ◦ C for further analysis. After thawing, the macrophage chemoattractant protein (MCP-1) and interleukin-6 (IL-6) levels were measured in triplicate with a commercial ELISA kit (Austria, Bender). Quantitative coronary analyses (QCA) Angiograms were performed during the initial procedure and on day 28. A computerized coronary angiography analysis system (GE Company, Germany) was used for QCA by two experienced cardiologists who were not aware of the treatment protocol. Discrepancies were resolved by mutual consensus. The late lumen loss (minimum LD immediately after interventional procedure minus the minimum LD at follow-up) was calculated.
345
Immunohistochemistry Immunohistochemical staining was performed on the paraffinembedded sections. After deparaffinization and hydration of specimens, endogenous peroxidase activity was blocked, and the specimens were fixed by immersion in 0.3% H2 O2 in methanol for 20 min. Immunohistochemical staining was performed with a mouse monoclonal antibody against pig nuclear factor-B (NF-B; Santa Cruz Biotechnology, Santa Cruz, CA) using the labeled streptavidin biotin complex method (Simple-stain MAX-PO kit, Nichirei, Tokyo, Japan). After blocking with 10% rabbit or goat serum, the slides were incubated overnight with a primary antibody at 4 ◦ C in a moisture chamber. The slides were washed with Tris-buffered saline (TBS) and incubated with a biotinylated secondary antibody at room temperature for 30 min. After washing with TBS, the slides were incubated with streptavidin at room temperature for 30 min and visualized with 3,3 -diaminobenzidine. Statistics The data was expressed as the mean ± SD. Statistical significance was evaluated using independent-samples t-test for comparisons between two groups. Significance was established at a value of P < 0.05. All statistics were calculated with SPSS v18.0 software. Results Twenty minipigs underwent successful implantation of 20 BMSs in the left anterior descending artery. All minipigs survived until they were killed.
Tissue harvest and processing for histology
QCA
The minipigs were killed after 28 days by intravenous injection of potassium chloride. The hearts were excised and pressure-perfused with 0.9% heparinized saline, followed by pressure-perfusion fixation in 10% neutral buffered formalin at 80–100 mm Hg via the aortic stump. On day 28, after implantation, vessels with stents from every group (n = 10 for each group) were cut into five 3 mm long pieces, fixed in 10% buffered formalin and embedded in glycol methacrylate or in paraffin. Cross sections from proximal, distal, and medial pieces were stained with hematoxylin and eosin and Verhoeff van Gieseon for morphometric analysis. Sections from the other pieces were embedded in paraffin and used for immunohistochemical analysis. The neointima from histology slides was morphometrically analyzed by an independent observer, and the neointimal thickness and the area of external elastic lamina, internal elastic lamina and lumen of each section were measured using digital morphometry. Neointimal percent area stenosis (AS%) was calculated as 100 × (1 − [stenotic lumen area/internal elastic lamina area]). The histological evaluation included the degree of injury score and inflammatory score. The injury score was determined according to the previously published method [29]: 0 = no injury; 1 = break in the internal elastic membrane; 2 = perforation of the media; and 3 = perforation of the external elastic membrane to the adventitia. The average injury score for each segment was calculated by dividing the sum of injury scores by the total number of struts at the section examined. Assessment of the pathological score for strut-associated inflammation was graded as follows [6]: 0, none; 1, scattered inflammatory cells; 2, inflammatory cells encompassing 50% of a strut in at least 25–50% of the circumference of the artery; and 3, inflammatory cells surrounding a strut in at least 25–50% of the circumference of the artery.
Table 1 summarizes the results of QCA. There were no significant differences in the reference vessel diameter between the two groups. During follow-up, the late lumen loss in the UTI group was significantly less than that in the control group (Fig. 1A and B). Effect of UTI on stent-induced intimal hyperplasia Intimal hyperplasia in the arteries of the control group implanted with stents was relatively significant as expected, with an intimal area of 5.21 ± 1.04 mm2 , intimal thickness of 0.46 ± 0.16 mm, and AS of 64% ± 12%. UTI treatment significantly decreased the neointimal area by 38% (to 3.22 ± 0.57 mm2 ), the intimal thickness by 33% (to 0.31 ± 0.13 mm), and the AS% by 41% (to 38% ± 6%) (Fig. 2A and B, P < 0.05 for intimal area, intimal thickness, and AS%). The histological analysis is illustrated in Fig. 3A–D. On day 28, the control group had the highest inflammatory score compared to the UTI group (1.30 ± 0.08 vs. 1.02 ± 0.05, P < 0.001). There was no significant difference in the injury score (P > 0.05) between both groups.
Table 1 QCA results. Parameter
Control group (n = 10)
Reference diameter (mm) Immediate MLD (mm) Follow-up MLD (mm) Late lumen loss (mm)
2.73 2.93 1.77 1.16
± ± ± ±
0.15 0.13 0.27 0.33
UTI group (n = 10) 2.74 3.05 2.26 0.79
± ± ± ±
0.16 0.33 0.30 0.34
P 0.89 0.31 <0.001 0.03
Data are mean ± SD. P < 0.05 indicates a significant difference. UTI = urinary trypsin inhibitor; MLD = minimal lumen diameter. Late lumen loss = immediate MLD − follow-up MLD.
346
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
Fig. 1. Left anterior oblique angiograms 4 weeks after stent placement. Comparison between control group (A) and UTI group (B). (A) Revealed significant in-stent restenosis.
Fig. 2. Intimal hyperplasia 28 days after stent injury (H&E staining, ×20). Control group (A) was thicker than UTI group (B).
Serum MCP-1 and IL-6 The results are presented in Table 2. The increase in MCP-1 and IL-6 levels in the serum was highest two days after stenting. Then they began to decline, but on day 28 they were also higher than the baseline levels in the control group. UTI could decrease the levels of MCP-1 and IL-6 after stenting, and on day 28 their levels were near to the baseline levels.
In the control group, two days after injury, the highest increase in MCP-1 and IL-6 was positively correlated with the maximal AS% on day 28 (r2 = 0.873 and 0.769, respectively, Fig. 4). Immunohistochemical staining for NF-ÄB Fig. 5A–C shows the immunohistochemical staining of NF-B of the two groups. The quantification of NF-B staining in the intima
Table 2 Serum MCP-1 and IL-6 levels (pg/mL). Time
MCP-1
IL-6
Control group Before stenting After stenting 2 days After stenting 7 days After stenting 14 days After stenting 28 days
77.74 270.75 147.93 143.03 128.31
± ± ± ± ±
12.78 28.07 30.65 13.04 12.93
UTI group 70.32 188.09 101.56 106.66 80.72
± ± ± ± ±
14.84 29.79 17.75 19.48 12.90
P
Control group
0.246 <0.001 <0.001 <0.001 <0.001
115.80 327.44 247.25 170.27 154.10
± ± ± ± ±
16.44 45.09 50.40 39.15 42.95
UTI group 120.90 239.70 163.90 135.60 122.10
± ± ± ± ±
P 19.32 35.96 24.87 18.88 17.86
Data are mean ± SD. P < 0.05 indicates a significant difference. MCP-1 = macrophage chemoattractant protein; IL-6 = interleukin-6; UTI = urinary trypsin inhibitor.
0.53 <0.001 <0.001 0.02 0.04
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
347
Fig. 3. Twenty-eight-day high-power photomicrographs (H&E staining, ×200) of the pathology of control group (A) and UTI group (B) after stent implantation. Severe inflammatory reaction to control group with mononuclear cells surrounding the stent struts (#) and fibrin deposition (*). (C) Inflammation scores for two groups, *P < 0.001. (D) Injury scores for two groups, P > 0.05.
of the stented arteries at follow-up showed that in the control group, NF-B-positive cells represented 36.93% ± 7.16% of the total number of vascular smooth muscle cells (VSMCs) as compared with only 23.32% ± 4.54% of the UTI group.
Discussion In recent years, research has increasingly concentrated on the role of inflammation in the pathophysiology of ISR, and a
Fig. 4. The serum levels of MCP-1 (A) and IL-6 (B) on day 2 after injury were positively correlated with the maximal AS% on day 28 (r2 = 0.889 and 0.743, respectively).
348
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
Fig. 5. Immunohistochemical staining for NF-B of VSMCs of each group. (A) Control and (B) UTI group, NF-b-positive VSMCs were indicated by their nuclear brown staining (×400). (C) The percentage of NF-b-positive VSMCs was lower in the UTI group compared to the control group.
few prospective observational studies suggested that inflammation contributed to ISR [12,20]. Coronary stenting was a strong inflammatory stimulus, and the acute systemic response to local inflammation produced by coronary stenting was a feature of PCI [15]. An experimental study showed that leukocyte recruitment could be detected within 15 min after stent deployment at the level of the coronary segment injured by the stent [33]. Local inflammation caused by stent deployment also elicited a systemic inflammatory response initially mediated by inflammatory leukins, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-␣. These molecules caused the liver to produce acute-phase reactants (such as CRP) that rapidly increase in the blood and might directly amplify the inflammatory stimulus [33]. NF-B could mediate the expression of many inflammatory cytokines [10], which played an important role in smooth muscle cell proliferation and differentiation. Antisense oligonucleotide to the P65 subunit of NF-B was found to inhibit human VSMC adherence and proliferation and to prevent neointima formation in rat carotid arteries [4]. MCP-1 and IL-6 were the inflammatory factors regulated by the NF-B signaling pathway. Oshima’s clinical trials concluded that MCP-1 production at stented coronary arterial sites was associated with an increased risk of restenosis six months post-stent implantation [22]. Cipollone et al. also confirmed that the MCP-1 plasma level measured 15 days after PCI was the only statistically significant independent predictor of restenosis [7]. IL-6 was the predominant determinant for production of acute phase proteins (for example, C reactive protein), and showed proinflammatory properties in addition to cellular effects (for example, smooth muscle hyperplasia) which were associated with restenosis. IL-6 concentrations which increased in unstable angina were also elevated after angioplasty, suggesting that IL-6 might therefore be a sensitive marker for the postprocedure initial inflammatory response, and also a possible predictor of later restenosis [5,19,31]. In the present animal study, the early increase
in serum MCP-1 and IL-6 and their correlation with further intimal growth might indicate that systemic secretion was associated with the late growth effect, whereas increased NF-B expression and inflammatory score supported a local effect. Few data on the effect of UTI in the cardiovascular area are available. Some studies reported that administration of ulinastatin before cardiopulmonary bypass could decrease the inflammatory response and the complication during coronary artery bypass grafting [11,36]. UTI could also prevent the development of coronary artery lesions by reversing the imbalance in favor of matrix metalloproteinase activation in patients with Kawasaki disease [27,30]. From the results of this study, we found that UTI not only inhibited the local NF-B expression and inflammatory reaction surrounding the stent struts, but also reduced the serum levels of MCP-1 and IL6. All these observations might be the major mechanisms of UTI in the inhibition of neointimal hyperplasia after stent injury. Previous studies have demonstrated that statins were suitable for restenosis prevention via reduction of smooth muscle cell proliferation, anti-inflammatory effects, and improvement of endothelial function [26,34]. However, they could not inhibit intimal hyperplasia and restenosis but promote plaque regression in normocholesterolemic patients undergoing coronary stenting [25]. Valsartan could also prevent restenosis after stenting of type B2/C lesion, however, the restenosis rate was relatively high with lowdose valsartan [23,24]. It was necessary to find an ideal agent as an adjuvant could limit ISR after stenting procedures for all patients carrying a high risk of restenosis. The present study suggests the potential future clinical applications of UTI for the treatment of coronary artery disease. It also suggests that inhibition of inflammatory response pathway activation might be one of major targets for prevention of ISR, and PCI could benefit from precocious antiinflammatory treatment. This study was limited to observations in the lesions produced in healthy vessels whose relevance to human clinical condition was
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
uncertain. Also, the observation period was only 28 days, and it remains to be determined how long the beneficial effect would be maintained in animals. Therefore, a prospective clinical study should be performed in order to evaluate the effect of UTI on human coronary stenosis. Conclusions UTI could inhibit neointimal hyperplasia after stent implantation. The underlying mechanism was probably related to the inhibition of local and systemic inflammatory response. Funding This work was supported by the Heilongjiang Provincial Office of Public Health. Conflict of interest None. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10.1016/j.prp.2012.03.008. References [1] K. Akashi, T. Ishimaru, T. Shibuya, M. Harada, Y. Niho, Human urinary proteinase inhibitor in the treatment of P carinii pneumonia, Chest 99 (1991) 1055–1056. [2] S. Ambiru, M. Miyazaki, K. Sasada, H. Ito, F. Kimura, K. Nakagawa, H. Shimizu, K. Ando, N. Nakajima, Effects of perioperative protease inhibitor on inflammatory cytokines and acute-phase proteins in patients with hepatic resection, Dig. Surg. 17 (2000) 337–343. [3] S. Aosasa, S. Ono, H. Mochizuki, H. Tsujimoto, C. Ueno, A. Matsumoto, Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor alpha production of monocytes, Shock 15 (2001) 101–105. [4] M.V. Autieri, T.L. Yue, G.Z. Ferstein, E. Ohlstein, Antisense oligonucleotide to the P65 subunit of NF-B inhibit human vascular smooth muscle cell adherence and proliferation and prevent neointima formation in rat carotid arteries, Biochem. Biophys. Res. Commun. 213 (1995) 827–836. [5] L.M. Biasucci, A. Vitelli, G. Liuzzo, S. Altamura, G. Caligiuri, C. Monaco, A.G. Rebuzzi, G. Ciliberto, A. Maseri, Elevated levels of interleukin-6 in unstable angina, Circulation 94 (1996) 874–877. [6] M. Cilingiroglu, J. Elliott, D. Patel, F. Tio, H. Matthews, M. McCasland, B. Trauthen, J. Elicker, S.R. Bailey, Long-term effects of novel biolimus eluting DEVAXAXXESS plus nitinol self-expanding stent in a porcine coronary model, Catheter. Cardiovasc. Interv. 68 (2006) 271–279. [7] F. Cipollone, M. Marini, M. Fazia, B. Pini, A. Iezzi, M. Reale, L. Paloscia, G. Materazzo, E. D’Annunzio, P. Conti, F. Chiarelli, F. Cuccurullo, A. Mezzetti, Elevated circulating levels of monocyte chemoattractant protein-1 in patients with restenosis after coronary angioplasty, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 327–334. [8] P. Damas, J.L. Canivet, D. de Groote, Y. Vrindts, A. Albert, P. Franchimont, M. Lamy, Sepsis and serum cytokine concentrations, Crit. Care Med. 25 (1997) 405–412. [9] A. Gaspardone, F. Versaci, Coronary stenting and inflammation, Am. J. Cardiol. 96 (2005) 65L–70L. [10] P.B. Gogo Jr., D.J. Schneider, M.W. Watkins, E.F. Terrien, B.E. Sobel, H.L. Dauerman, Systemic inflammation after drug-eluting stent placement, J. Thromb. Thrombolysis 19 (2005) 87–92. [11] Y.F. Jiang, W.W. Wang, W.L. Ye, Y.F. Ni, J. Li, X.L. Chen, S.W. Jin, Q.Q. Lian, Effects of alprostadil and ulinastatin on inflammatory response and lung injury after cardiopulmonary bypass in pediatric patients with congenital heart diseases, Zhonghua Yi Xue Za Zhi. 88 (2008) 2893–2897. [12] G. Karthikeyan, B. Bhargava, Prevention of restenosis after coronary angioplasty, Curr. Opin. Cardiol. 19 (2004) 500–509. [13] R. Kornowski, M.K. Hong, F.O. Tio, O. Bramwell, H. Wu, M.B. Leon, In-stent restenosis: contributions of inflammatory responses and arterial Injury to neointimal hyperplasia, J. Am. Coll. Cardiol. 31 (1998) 224–230.
349
[14] M. Kozinski, A. Krzewina-Kowalska, J. Kubica, M. Zbikowska-Gotz, G. Dymek, R. Piasecki, A. Sukiennik, G. Grzesk, M. Bogdan, M. Chojnicki, A. Dziedziczko, G. Sypniewska, Percutaneous coronary intervention triggers a systemic inflammatory response in patients treated for in-stent restenosis – comparison with stable and unstable angina, Inflamm. Res. 54 (2005) 187–193. [15] J.J. Li, Inflammatory response, drug-eluting stent and restenosis, Chin. Med. J. (Engl.) 121 (2008) 566–572. [16] J.J. Li, S.P. Nie, C.Y. Zhang, Z. Gao, X. Zheng, Y.L. Guo, Is inflammation a contributor for coronary stent restenosis? Med. Hypotheses 68 (2007) 945–951. [17] J.J. Li, B. Xu, Y.J. Yang, W.H. Ma, J.L. Chen, S.B. Qiao, X.W. Qin, M. Yao, H.B. Liu, Y.J. Wu, J.Q. Yuan, J. Chen, S.J. You, J. Dai, R. Xia, R.L. Gao, A comparison of angiographic and clinical outcomes after sirolimus-eluting versus paclitaxel-eluting stents for the treatment of in-stent restenosis, Chin. Med. J. (Engl.) 119 (2006) 1059–1064. [18] S.D. Lin, R. Endo, A. Sato, Y. Takikawa, K. Shirakawa, K. Suzuki, Plasma and urine levels of urinary trypsin inhibitor in patients with acute and fulminant hepatitis, J. Gastroenterol. Hepatol. 17 (2002) 140–147. [19] G. Liuzzo, A. Buffon, L.M. Biasucci, J.R. Gallimore, G. Caligiuri, A. Vitelli, S. Altamura, G. Ciliberto, A.G. Rebuzzi, F. Crea, M.B. Pepys, A. Maseri, Enhanced inflammatory response to coronary angioplasty in patients with severe unstable angina, Circulation 98 (1998) 2370–2376. [20] H.C. Lowe, S.N. Oesterle, L.M. Khachigian, Coronary in-stent restenosis: current status and future strategies, J. Am. Coll. Cardiol. 39 (2002) 183–193. [21] K. Nakatani, S. Takeshita, H. Tsujimoto, Y. Kawamura, I. Sekine, Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury, J. Leukoc. Biol. 69 (2001) 241–247. [22] S. Oshima, H. Ogawa, S. Hokimoto, S. Nakamura, K. Noda, T. Saito, H. Soejima, K. Takazoe, F. Ishibashi, H. Yasue, Plasma monocyte chemoattractant protein-1 antigen levels and the risk of restenosis after coronary stent implantation, Jpn. Circ. J. 65 (2001) 261–264. [23] S. Peters, Comparison of efficacy of low- (80 mg/day) and high(160–320 mg/day) dose valsartan in the prevention of in-stent restenosis after implantation of bare-metal stents in type B2/C coronary artery lesions, Am. J. Cardiovasc. Drugs 8 (2008) 83–87. [24] S. Peters, B. Götting, M. Trümmel, H. Rust, A. Brattström, Valsartan for prevention of restenosis after stenting of type B2/C lesion: the VAL-PREST trial, J. Invasive Cardiol. 13 (2001) 93–97. [25] A.S. Petronio, G. Amoroso, U. Limbruno, B. Papini, M. De Carlo, A. Micheli, N. Ciabatti, M. Mariani, Simvastatin does not inhibit intimal hyperplasia and restenosis but promotes plaque regression in normocholesterolemic patients undergoing coronary stenting: a randomized study with intravascular ultrasound, Am. Heart J. 149 (2005) 520–526. [26] F. Prati, F. Imola, P. Corvo, C. Cernigliaro, T. Fouadi, V. Ramazzotti, A. Manzoli, A. Pappalardo, M. Cera, The RESTART project: high-dose atorvastatin helps control RESTenosis AfteR sTenting procedures at high risk for restenosis. Results from a prospective study and a matched control group comparison, EuroIntervention 3 (2007) 89–94. [27] T. Kanai, T. Ishiwata, T. Kobayashi, H. Sato, M. Takizawa, Y. Kawamura, H. Tsujimoto, K. Nakatani, N. Ishibashi, M. Nishiyama, Y. Hatai, Y. Asano, T. Kobayashi, S. Takeshita, S. Nonoyama, With Kawasaki disease: a retrospective study ulinastatin, a urinary trypsin inhibitor, for the initial treatment of patients, Circulation 124 (2011) 2822–2828. [28] N. Sato, K. Murakami, K. Ishida, K. Ikeda, K. Saito, Pulmonary hypertension and polymorphonuclear leukocyte elastase after esophageal cancer operations, Ann. Thorac. Surg. 51 (1991) 754–758. [29] R.S. Schwartz, K.C. Huber, J.G. Murphy, W.D. Edwards, A.R. Camrud, R.E. Vlietstra, D.R. Holmes, Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model, J. Am. Coll. Cardiol. 19 (1992) 267–274. [30] H. Senzaki, S. Masutani, J. Kobayashi, T. Kobayashi, H. Nakano, H. Nagasaka, N. Sasaki, H. Asano, S. Kyo, Y. Yokote, Circulating matrix metalloproteinases and their inhibitors in patients with Kawasaki disease, Circulation 104 (2001) 860–863. [31] T. Suzuki, S. Ishiwata, K. Hasegawa, K. Yamamoto, T. Yamazaki, Raised interleukin 6 concentrations as a predictor of postangioplasty restenosis, Heart 83 (2000) 578. [32] T. Tani, H. Abe, Y. Endo, K. Hanasawa, M. Kodama, Effects of a urinary trypsin inhibitor on acute circulatory insufficiency after surgical operation, Am. J. Surg. 175 (1998) 142–145. [33] F. Versaci, A. Gaspardone, Prevention of restenosis after stenting: the emerging role of inflammation, Coron. Artery Dis. 15 (2004) 307–311. [34] D.H. Walter, V. Schachinger, M. Elsner, S. Mach, W. Auch-Schwelk, A.M. Zeiher, Effect of statin therapy on restenosis after coronary stent implantation, Am. J. Cardiol. 85 (2000) 962–968. [35] F.G. Welt, C. Rogers, Inflammation and restenosis in the stent era, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 1769–1776. [36] Q. Zhou, G. Wang, C. Gao, T. Chen, Effect of ulinastatin on perioperative inflammatory response to coronary artery bypass grafting with cardiopulmonary bypass, Zhong Nan Da Xue Xue Bao Yi Xue Ban. 35 (2010) 107–110.
Contents lists available at SciVerse ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.de/prp
Original article
Urinary trypsin inhibitor reduced inflammatory response after stent injury in minipig J.Y. Kong ∗ , T.Q. Wang, G.H. Jiang, L. Li, F.P. Wang Department of Emergency, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Hei Long Jiang, China
a r t i c l e
i n f o
Article history: Received 25 January 2012 Received in revised form 1 March 2012 Accepted 6 March 2012 Keywords: Urinary trypsin inhibitor Stent Restenosis Inflammation
a b s t r a c t This study investigated whether urinary trypsin inhibitor (UTI) inhibits neointimal formation by reducing inflammatory response after stent injury. Twenty minipigs having undergone oversized bare material stent implantation in the left anterior descending artery were randomly subdivided into two groups: a UTI group (n = 10) and a control group (n = 10). Two systemic markers of inflammation (serum macrophage chemoattractant protein-1 and interleukin-6 levels measured by ELISA) were increased after stent implantation, and two days after stem implantation, their levels were positively correlated with the maximal percentage of area stenosis on day 28 (r2 = 0.889 and 0.743, respectively). This effect was abolished by UTI administration. Twenty-eight days after implantation, morphometric analysis of the stented arteries revealed significantly reduced luminal stenosis (38 ± 6% vs. 64 ± 12%, P < 0.05), a neointimal area (3.22 ± 0.57 mm2 vs. 5.21 ± 1.04 mm2 , P < 0.05), neointimal thickness (0.31 ± 0.13 mm vs. 0.46 ± 0.16 mm, P < 0.05), and an inflammatory score of 1.02 ± 0.05 vs. 1.30 ± 0.08 in UTI-treated animals as compared with controls. Twenty-eight days after stenting, arterial nuclear factor-B expression was 36.93 ± 7.16% in all of the cells in controls and 23.32 ± 4.54% in UTI-treated minipigs. UTI could reduce neointimal formation after stenting by inhibiting the local and the systemic inflammatory response. Percutaneous coronary intervention could benefit from precocious anti-inflammatory treatment. © 2012 Elsevier GmbH. All rights reserved.
Introduction In-stent restenosis (ISR) remains a challenge for interventional cardiologists [16,17]. Systemic inflammation characterizes the response to vascular injury after percutaneous coronary intervention (PCI) [9,16,33]. Stent implantation, in particular, precipitates arterial intimal cellular proliferation and extracellular matrix synthesis largely mediated by inflammatory processes [13,35]. Inflammation is a major force in the pathophysiology of atherothrombosis. In addition to its presumed role in induction, progression and destabilization of the atherosclerotic lesion, the inflammatory response that follows vascular injury is of major importance to the restenotic process. Measurement of cytokine and acute phase proteins after stenting, such as C-reactive protein, may be important for the identification of high-risk subjects and for the development of specific treatment tailored to individual patients [14,16]. Urinary trypsin inhibitor (UTI) can inhibit the production and cascade of inflammatory cytokines and improve the inflammatory
symptom. It has been widely used for the treatment of non-specific inflammatory response diseases clinically [1–3,8,18,21,28,32]. Some previous studies have demonstrated the effect of UTI on inflammatory response after coronary artery bypass grafting with cardiopulmonary bypass; however, only few data on UTI and inflammatory response after stent injury are available [11,36]. The present study investigated whether UTI was able to reduce neointimal formation by inhibiting the inflammatory response in minipigs subjected to stent injury. Material and methods All animal care and procedures conducted in the present study conformed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Harbin Medical University (2009-X023). Vascular injury model
∗ Corresponding author. Tel.: +86 45186651587; fax: +86 45186651587. E-mail address: [email protected] (J.Y. Kong). 0344-0338/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2012.03.008
Twenty minipigs (20–25 kg) were intubated after sedation with ketamine [20 mg/kg, intramuscular (i.m.)] and diazepam
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
(0.4 mg/kg, i.m.) followed by 3% sodium pentobarbital through the marginal ear vein [25 mg/kg, (intravenous)]. Coronary angiography was carried out using 6-F guiding catheters after intracoronary administration of nitroglycerin (200 g). One bare-metal stent [(BMS), 18 mm in length and 3.0–3.5 mm in diameter; Lepu Medical Company, Beijing, China] was placed in the left anterior descending artery of each pig at high pressure (12–14 atm.) for >30 s. The stent-to-artery ratio was maintained at 1.2:1. All animals were pretreated with clopidogrel (150 mg loading dose followed by 75 mg/day) and aspirin (300 mg loading dose followed by 100 mg/day) for three days before the procedure. The animals were randomly assigned to either the control or the UTI group (Techpool Biochemical Pharmaceutical Co., Ltd., 10,000 U/kg/d, continuous administration 28 days) after injury. Arterial blood was collected in citrate-containing tubes 24 h prior to the procedure and at 2, 7, 14, and 28 days after injury. The serum was separated and stored at −70 ◦ C for further analysis. After thawing, the macrophage chemoattractant protein (MCP-1) and interleukin-6 (IL-6) levels were measured in triplicate with a commercial ELISA kit (Austria, Bender). Quantitative coronary analyses (QCA) Angiograms were performed during the initial procedure and on day 28. A computerized coronary angiography analysis system (GE Company, Germany) was used for QCA by two experienced cardiologists who were not aware of the treatment protocol. Discrepancies were resolved by mutual consensus. The late lumen loss (minimum LD immediately after interventional procedure minus the minimum LD at follow-up) was calculated.
345
Immunohistochemistry Immunohistochemical staining was performed on the paraffinembedded sections. After deparaffinization and hydration of specimens, endogenous peroxidase activity was blocked, and the specimens were fixed by immersion in 0.3% H2 O2 in methanol for 20 min. Immunohistochemical staining was performed with a mouse monoclonal antibody against pig nuclear factor-B (NF-B; Santa Cruz Biotechnology, Santa Cruz, CA) using the labeled streptavidin biotin complex method (Simple-stain MAX-PO kit, Nichirei, Tokyo, Japan). After blocking with 10% rabbit or goat serum, the slides were incubated overnight with a primary antibody at 4 ◦ C in a moisture chamber. The slides were washed with Tris-buffered saline (TBS) and incubated with a biotinylated secondary antibody at room temperature for 30 min. After washing with TBS, the slides were incubated with streptavidin at room temperature for 30 min and visualized with 3,3 -diaminobenzidine. Statistics The data was expressed as the mean ± SD. Statistical significance was evaluated using independent-samples t-test for comparisons between two groups. Significance was established at a value of P < 0.05. All statistics were calculated with SPSS v18.0 software. Results Twenty minipigs underwent successful implantation of 20 BMSs in the left anterior descending artery. All minipigs survived until they were killed.
Tissue harvest and processing for histology
QCA
The minipigs were killed after 28 days by intravenous injection of potassium chloride. The hearts were excised and pressure-perfused with 0.9% heparinized saline, followed by pressure-perfusion fixation in 10% neutral buffered formalin at 80–100 mm Hg via the aortic stump. On day 28, after implantation, vessels with stents from every group (n = 10 for each group) were cut into five 3 mm long pieces, fixed in 10% buffered formalin and embedded in glycol methacrylate or in paraffin. Cross sections from proximal, distal, and medial pieces were stained with hematoxylin and eosin and Verhoeff van Gieseon for morphometric analysis. Sections from the other pieces were embedded in paraffin and used for immunohistochemical analysis. The neointima from histology slides was morphometrically analyzed by an independent observer, and the neointimal thickness and the area of external elastic lamina, internal elastic lamina and lumen of each section were measured using digital morphometry. Neointimal percent area stenosis (AS%) was calculated as 100 × (1 − [stenotic lumen area/internal elastic lamina area]). The histological evaluation included the degree of injury score and inflammatory score. The injury score was determined according to the previously published method [29]: 0 = no injury; 1 = break in the internal elastic membrane; 2 = perforation of the media; and 3 = perforation of the external elastic membrane to the adventitia. The average injury score for each segment was calculated by dividing the sum of injury scores by the total number of struts at the section examined. Assessment of the pathological score for strut-associated inflammation was graded as follows [6]: 0, none; 1, scattered inflammatory cells; 2, inflammatory cells encompassing 50% of a strut in at least 25–50% of the circumference of the artery; and 3, inflammatory cells surrounding a strut in at least 25–50% of the circumference of the artery.
Table 1 summarizes the results of QCA. There were no significant differences in the reference vessel diameter between the two groups. During follow-up, the late lumen loss in the UTI group was significantly less than that in the control group (Fig. 1A and B). Effect of UTI on stent-induced intimal hyperplasia Intimal hyperplasia in the arteries of the control group implanted with stents was relatively significant as expected, with an intimal area of 5.21 ± 1.04 mm2 , intimal thickness of 0.46 ± 0.16 mm, and AS of 64% ± 12%. UTI treatment significantly decreased the neointimal area by 38% (to 3.22 ± 0.57 mm2 ), the intimal thickness by 33% (to 0.31 ± 0.13 mm), and the AS% by 41% (to 38% ± 6%) (Fig. 2A and B, P < 0.05 for intimal area, intimal thickness, and AS%). The histological analysis is illustrated in Fig. 3A–D. On day 28, the control group had the highest inflammatory score compared to the UTI group (1.30 ± 0.08 vs. 1.02 ± 0.05, P < 0.001). There was no significant difference in the injury score (P > 0.05) between both groups.
Table 1 QCA results. Parameter
Control group (n = 10)
Reference diameter (mm) Immediate MLD (mm) Follow-up MLD (mm) Late lumen loss (mm)
2.73 2.93 1.77 1.16
± ± ± ±
0.15 0.13 0.27 0.33
UTI group (n = 10) 2.74 3.05 2.26 0.79
± ± ± ±
0.16 0.33 0.30 0.34
P 0.89 0.31 <0.001 0.03
Data are mean ± SD. P < 0.05 indicates a significant difference. UTI = urinary trypsin inhibitor; MLD = minimal lumen diameter. Late lumen loss = immediate MLD − follow-up MLD.
346
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
Fig. 1. Left anterior oblique angiograms 4 weeks after stent placement. Comparison between control group (A) and UTI group (B). (A) Revealed significant in-stent restenosis.
Fig. 2. Intimal hyperplasia 28 days after stent injury (H&E staining, ×20). Control group (A) was thicker than UTI group (B).
Serum MCP-1 and IL-6 The results are presented in Table 2. The increase in MCP-1 and IL-6 levels in the serum was highest two days after stenting. Then they began to decline, but on day 28 they were also higher than the baseline levels in the control group. UTI could decrease the levels of MCP-1 and IL-6 after stenting, and on day 28 their levels were near to the baseline levels.
In the control group, two days after injury, the highest increase in MCP-1 and IL-6 was positively correlated with the maximal AS% on day 28 (r2 = 0.873 and 0.769, respectively, Fig. 4). Immunohistochemical staining for NF-ÄB Fig. 5A–C shows the immunohistochemical staining of NF-B of the two groups. The quantification of NF-B staining in the intima
Table 2 Serum MCP-1 and IL-6 levels (pg/mL). Time
MCP-1
IL-6
Control group Before stenting After stenting 2 days After stenting 7 days After stenting 14 days After stenting 28 days
77.74 270.75 147.93 143.03 128.31
± ± ± ± ±
12.78 28.07 30.65 13.04 12.93
UTI group 70.32 188.09 101.56 106.66 80.72
± ± ± ± ±
14.84 29.79 17.75 19.48 12.90
P
Control group
0.246 <0.001 <0.001 <0.001 <0.001
115.80 327.44 247.25 170.27 154.10
± ± ± ± ±
16.44 45.09 50.40 39.15 42.95
UTI group 120.90 239.70 163.90 135.60 122.10
± ± ± ± ±
P 19.32 35.96 24.87 18.88 17.86
Data are mean ± SD. P < 0.05 indicates a significant difference. MCP-1 = macrophage chemoattractant protein; IL-6 = interleukin-6; UTI = urinary trypsin inhibitor.
0.53 <0.001 <0.001 0.02 0.04
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
347
Fig. 3. Twenty-eight-day high-power photomicrographs (H&E staining, ×200) of the pathology of control group (A) and UTI group (B) after stent implantation. Severe inflammatory reaction to control group with mononuclear cells surrounding the stent struts (#) and fibrin deposition (*). (C) Inflammation scores for two groups, *P < 0.001. (D) Injury scores for two groups, P > 0.05.
of the stented arteries at follow-up showed that in the control group, NF-B-positive cells represented 36.93% ± 7.16% of the total number of vascular smooth muscle cells (VSMCs) as compared with only 23.32% ± 4.54% of the UTI group.
Discussion In recent years, research has increasingly concentrated on the role of inflammation in the pathophysiology of ISR, and a
Fig. 4. The serum levels of MCP-1 (A) and IL-6 (B) on day 2 after injury were positively correlated with the maximal AS% on day 28 (r2 = 0.889 and 0.743, respectively).
348
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
Fig. 5. Immunohistochemical staining for NF-B of VSMCs of each group. (A) Control and (B) UTI group, NF-b-positive VSMCs were indicated by their nuclear brown staining (×400). (C) The percentage of NF-b-positive VSMCs was lower in the UTI group compared to the control group.
few prospective observational studies suggested that inflammation contributed to ISR [12,20]. Coronary stenting was a strong inflammatory stimulus, and the acute systemic response to local inflammation produced by coronary stenting was a feature of PCI [15]. An experimental study showed that leukocyte recruitment could be detected within 15 min after stent deployment at the level of the coronary segment injured by the stent [33]. Local inflammation caused by stent deployment also elicited a systemic inflammatory response initially mediated by inflammatory leukins, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-␣. These molecules caused the liver to produce acute-phase reactants (such as CRP) that rapidly increase in the blood and might directly amplify the inflammatory stimulus [33]. NF-B could mediate the expression of many inflammatory cytokines [10], which played an important role in smooth muscle cell proliferation and differentiation. Antisense oligonucleotide to the P65 subunit of NF-B was found to inhibit human VSMC adherence and proliferation and to prevent neointima formation in rat carotid arteries [4]. MCP-1 and IL-6 were the inflammatory factors regulated by the NF-B signaling pathway. Oshima’s clinical trials concluded that MCP-1 production at stented coronary arterial sites was associated with an increased risk of restenosis six months post-stent implantation [22]. Cipollone et al. also confirmed that the MCP-1 plasma level measured 15 days after PCI was the only statistically significant independent predictor of restenosis [7]. IL-6 was the predominant determinant for production of acute phase proteins (for example, C reactive protein), and showed proinflammatory properties in addition to cellular effects (for example, smooth muscle hyperplasia) which were associated with restenosis. IL-6 concentrations which increased in unstable angina were also elevated after angioplasty, suggesting that IL-6 might therefore be a sensitive marker for the postprocedure initial inflammatory response, and also a possible predictor of later restenosis [5,19,31]. In the present animal study, the early increase
in serum MCP-1 and IL-6 and their correlation with further intimal growth might indicate that systemic secretion was associated with the late growth effect, whereas increased NF-B expression and inflammatory score supported a local effect. Few data on the effect of UTI in the cardiovascular area are available. Some studies reported that administration of ulinastatin before cardiopulmonary bypass could decrease the inflammatory response and the complication during coronary artery bypass grafting [11,36]. UTI could also prevent the development of coronary artery lesions by reversing the imbalance in favor of matrix metalloproteinase activation in patients with Kawasaki disease [27,30]. From the results of this study, we found that UTI not only inhibited the local NF-B expression and inflammatory reaction surrounding the stent struts, but also reduced the serum levels of MCP-1 and IL6. All these observations might be the major mechanisms of UTI in the inhibition of neointimal hyperplasia after stent injury. Previous studies have demonstrated that statins were suitable for restenosis prevention via reduction of smooth muscle cell proliferation, anti-inflammatory effects, and improvement of endothelial function [26,34]. However, they could not inhibit intimal hyperplasia and restenosis but promote plaque regression in normocholesterolemic patients undergoing coronary stenting [25]. Valsartan could also prevent restenosis after stenting of type B2/C lesion, however, the restenosis rate was relatively high with lowdose valsartan [23,24]. It was necessary to find an ideal agent as an adjuvant could limit ISR after stenting procedures for all patients carrying a high risk of restenosis. The present study suggests the potential future clinical applications of UTI for the treatment of coronary artery disease. It also suggests that inhibition of inflammatory response pathway activation might be one of major targets for prevention of ISR, and PCI could benefit from precocious antiinflammatory treatment. This study was limited to observations in the lesions produced in healthy vessels whose relevance to human clinical condition was
J.Y. Kong et al. / Pathology – Research and Practice 208 (2012) 344–349
uncertain. Also, the observation period was only 28 days, and it remains to be determined how long the beneficial effect would be maintained in animals. Therefore, a prospective clinical study should be performed in order to evaluate the effect of UTI on human coronary stenosis. Conclusions UTI could inhibit neointimal hyperplasia after stent implantation. The underlying mechanism was probably related to the inhibition of local and systemic inflammatory response. Funding This work was supported by the Heilongjiang Provincial Office of Public Health. Conflict of interest None. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10.1016/j.prp.2012.03.008. References [1] K. Akashi, T. Ishimaru, T. Shibuya, M. Harada, Y. Niho, Human urinary proteinase inhibitor in the treatment of P carinii pneumonia, Chest 99 (1991) 1055–1056. [2] S. Ambiru, M. Miyazaki, K. Sasada, H. Ito, F. Kimura, K. Nakagawa, H. Shimizu, K. Ando, N. Nakajima, Effects of perioperative protease inhibitor on inflammatory cytokines and acute-phase proteins in patients with hepatic resection, Dig. Surg. 17 (2000) 337–343. [3] S. Aosasa, S. Ono, H. Mochizuki, H. Tsujimoto, C. Ueno, A. Matsumoto, Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor alpha production of monocytes, Shock 15 (2001) 101–105. [4] M.V. Autieri, T.L. Yue, G.Z. Ferstein, E. Ohlstein, Antisense oligonucleotide to the P65 subunit of NF-B inhibit human vascular smooth muscle cell adherence and proliferation and prevent neointima formation in rat carotid arteries, Biochem. Biophys. Res. Commun. 213 (1995) 827–836. [5] L.M. Biasucci, A. Vitelli, G. Liuzzo, S. Altamura, G. Caligiuri, C. Monaco, A.G. Rebuzzi, G. Ciliberto, A. Maseri, Elevated levels of interleukin-6 in unstable angina, Circulation 94 (1996) 874–877. [6] M. Cilingiroglu, J. Elliott, D. Patel, F. Tio, H. Matthews, M. McCasland, B. Trauthen, J. Elicker, S.R. Bailey, Long-term effects of novel biolimus eluting DEVAXAXXESS plus nitinol self-expanding stent in a porcine coronary model, Catheter. Cardiovasc. Interv. 68 (2006) 271–279. [7] F. Cipollone, M. Marini, M. Fazia, B. Pini, A. Iezzi, M. Reale, L. Paloscia, G. Materazzo, E. D’Annunzio, P. Conti, F. Chiarelli, F. Cuccurullo, A. Mezzetti, Elevated circulating levels of monocyte chemoattractant protein-1 in patients with restenosis after coronary angioplasty, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 327–334. [8] P. Damas, J.L. Canivet, D. de Groote, Y. Vrindts, A. Albert, P. Franchimont, M. Lamy, Sepsis and serum cytokine concentrations, Crit. Care Med. 25 (1997) 405–412. [9] A. Gaspardone, F. Versaci, Coronary stenting and inflammation, Am. J. Cardiol. 96 (2005) 65L–70L. [10] P.B. Gogo Jr., D.J. Schneider, M.W. Watkins, E.F. Terrien, B.E. Sobel, H.L. Dauerman, Systemic inflammation after drug-eluting stent placement, J. Thromb. Thrombolysis 19 (2005) 87–92. [11] Y.F. Jiang, W.W. Wang, W.L. Ye, Y.F. Ni, J. Li, X.L. Chen, S.W. Jin, Q.Q. Lian, Effects of alprostadil and ulinastatin on inflammatory response and lung injury after cardiopulmonary bypass in pediatric patients with congenital heart diseases, Zhonghua Yi Xue Za Zhi. 88 (2008) 2893–2897. [12] G. Karthikeyan, B. Bhargava, Prevention of restenosis after coronary angioplasty, Curr. Opin. Cardiol. 19 (2004) 500–509. [13] R. Kornowski, M.K. Hong, F.O. Tio, O. Bramwell, H. Wu, M.B. Leon, In-stent restenosis: contributions of inflammatory responses and arterial Injury to neointimal hyperplasia, J. Am. Coll. Cardiol. 31 (1998) 224–230.
349
[14] M. Kozinski, A. Krzewina-Kowalska, J. Kubica, M. Zbikowska-Gotz, G. Dymek, R. Piasecki, A. Sukiennik, G. Grzesk, M. Bogdan, M. Chojnicki, A. Dziedziczko, G. Sypniewska, Percutaneous coronary intervention triggers a systemic inflammatory response in patients treated for in-stent restenosis – comparison with stable and unstable angina, Inflamm. Res. 54 (2005) 187–193. [15] J.J. Li, Inflammatory response, drug-eluting stent and restenosis, Chin. Med. J. (Engl.) 121 (2008) 566–572. [16] J.J. Li, S.P. Nie, C.Y. Zhang, Z. Gao, X. Zheng, Y.L. Guo, Is inflammation a contributor for coronary stent restenosis? Med. Hypotheses 68 (2007) 945–951. [17] J.J. Li, B. Xu, Y.J. Yang, W.H. Ma, J.L. Chen, S.B. Qiao, X.W. Qin, M. Yao, H.B. Liu, Y.J. Wu, J.Q. Yuan, J. Chen, S.J. You, J. Dai, R. Xia, R.L. Gao, A comparison of angiographic and clinical outcomes after sirolimus-eluting versus paclitaxel-eluting stents for the treatment of in-stent restenosis, Chin. Med. J. (Engl.) 119 (2006) 1059–1064. [18] S.D. Lin, R. Endo, A. Sato, Y. Takikawa, K. Shirakawa, K. Suzuki, Plasma and urine levels of urinary trypsin inhibitor in patients with acute and fulminant hepatitis, J. Gastroenterol. Hepatol. 17 (2002) 140–147. [19] G. Liuzzo, A. Buffon, L.M. Biasucci, J.R. Gallimore, G. Caligiuri, A. Vitelli, S. Altamura, G. Ciliberto, A.G. Rebuzzi, F. Crea, M.B. Pepys, A. Maseri, Enhanced inflammatory response to coronary angioplasty in patients with severe unstable angina, Circulation 98 (1998) 2370–2376. [20] H.C. Lowe, S.N. Oesterle, L.M. Khachigian, Coronary in-stent restenosis: current status and future strategies, J. Am. Coll. Cardiol. 39 (2002) 183–193. [21] K. Nakatani, S. Takeshita, H. Tsujimoto, Y. Kawamura, I. Sekine, Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury, J. Leukoc. Biol. 69 (2001) 241–247. [22] S. Oshima, H. Ogawa, S. Hokimoto, S. Nakamura, K. Noda, T. Saito, H. Soejima, K. Takazoe, F. Ishibashi, H. Yasue, Plasma monocyte chemoattractant protein-1 antigen levels and the risk of restenosis after coronary stent implantation, Jpn. Circ. J. 65 (2001) 261–264. [23] S. Peters, Comparison of efficacy of low- (80 mg/day) and high(160–320 mg/day) dose valsartan in the prevention of in-stent restenosis after implantation of bare-metal stents in type B2/C coronary artery lesions, Am. J. Cardiovasc. Drugs 8 (2008) 83–87. [24] S. Peters, B. Götting, M. Trümmel, H. Rust, A. Brattström, Valsartan for prevention of restenosis after stenting of type B2/C lesion: the VAL-PREST trial, J. Invasive Cardiol. 13 (2001) 93–97. [25] A.S. Petronio, G. Amoroso, U. Limbruno, B. Papini, M. De Carlo, A. Micheli, N. Ciabatti, M. Mariani, Simvastatin does not inhibit intimal hyperplasia and restenosis but promotes plaque regression in normocholesterolemic patients undergoing coronary stenting: a randomized study with intravascular ultrasound, Am. Heart J. 149 (2005) 520–526. [26] F. Prati, F. Imola, P. Corvo, C. Cernigliaro, T. Fouadi, V. Ramazzotti, A. Manzoli, A. Pappalardo, M. Cera, The RESTART project: high-dose atorvastatin helps control RESTenosis AfteR sTenting procedures at high risk for restenosis. Results from a prospective study and a matched control group comparison, EuroIntervention 3 (2007) 89–94. [27] T. Kanai, T. Ishiwata, T. Kobayashi, H. Sato, M. Takizawa, Y. Kawamura, H. Tsujimoto, K. Nakatani, N. Ishibashi, M. Nishiyama, Y. Hatai, Y. Asano, T. Kobayashi, S. Takeshita, S. Nonoyama, With Kawasaki disease: a retrospective study ulinastatin, a urinary trypsin inhibitor, for the initial treatment of patients, Circulation 124 (2011) 2822–2828. [28] N. Sato, K. Murakami, K. Ishida, K. Ikeda, K. Saito, Pulmonary hypertension and polymorphonuclear leukocyte elastase after esophageal cancer operations, Ann. Thorac. Surg. 51 (1991) 754–758. [29] R.S. Schwartz, K.C. Huber, J.G. Murphy, W.D. Edwards, A.R. Camrud, R.E. Vlietstra, D.R. Holmes, Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model, J. Am. Coll. Cardiol. 19 (1992) 267–274. [30] H. Senzaki, S. Masutani, J. Kobayashi, T. Kobayashi, H. Nakano, H. Nagasaka, N. Sasaki, H. Asano, S. Kyo, Y. Yokote, Circulating matrix metalloproteinases and their inhibitors in patients with Kawasaki disease, Circulation 104 (2001) 860–863. [31] T. Suzuki, S. Ishiwata, K. Hasegawa, K. Yamamoto, T. Yamazaki, Raised interleukin 6 concentrations as a predictor of postangioplasty restenosis, Heart 83 (2000) 578. [32] T. Tani, H. Abe, Y. Endo, K. Hanasawa, M. Kodama, Effects of a urinary trypsin inhibitor on acute circulatory insufficiency after surgical operation, Am. J. Surg. 175 (1998) 142–145. [33] F. Versaci, A. Gaspardone, Prevention of restenosis after stenting: the emerging role of inflammation, Coron. Artery Dis. 15 (2004) 307–311. [34] D.H. Walter, V. Schachinger, M. Elsner, S. Mach, W. Auch-Schwelk, A.M. Zeiher, Effect of statin therapy on restenosis after coronary stent implantation, Am. J. Cardiol. 85 (2000) 962–968. [35] F.G. Welt, C. Rogers, Inflammation and restenosis in the stent era, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 1769–1776. [36] Q. Zhou, G. Wang, C. Gao, T. Chen, Effect of ulinastatin on perioperative inflammatory response to coronary artery bypass grafting with cardiopulmonary bypass, Zhong Nan Da Xue Xue Bao Yi Xue Ban. 35 (2010) 107–110.