Abstract: Objectives. We sought to assess the effects of a high loading dose of rosuvastatin (40 mg) on acute inflammatory response after coronary stenting. Methods. Patients with stable coronary disease without statin use (≥7 days) and undergoing elective percutaneous coronary intervention (PCI) in a native coronary artery were randomized to receive a loading dose of rosuvastatin (n = 64) or not (n = 61). Blood samples were obtained before statin intake (time point A), 3 hours after medication (time point B), and 3 hours after PCI (time point C). The primary goal was the comparison in the variation of the serum inflammatory markers and their gene expression at the different time points between the two groups. Results. Baseline clinical, angiographic, and procedural characteristics did not significantly differ between the groups, except for the more frequent use of postdilation in the control group (73.4% vs 90.2%; P=.02). Patients pretreated with statin showed a reduction in the serum levels of interleukin (IL)-1β (Δ = -0.491 pg/mL; Pinteraction<.001), IL-6 (Δ = -0.209 pg/mL; Pinteraction<.001), and plasminogen activator inhibitor 1 (Δ = -1.573 pg/mL; Pinteraction<.001) as well as in their genetic expression, which was not observed in the control group. Regarding high-sensitivity c-reactive protein, there was no significant variation in its value from time point A to C in patients pretreated with statin (P=.58) while it significantly increased in the control group (P=.04). Conclusion. Among patients with stable coronary artery disease undergoing PCI with stents, pretreatment with high dose of rosuvastatin resulted in significant reduction in the serum levels of important inflammatory markers and their genetic expression.
J INVASIVE CARDIOL 2020 June 22 (Epub Ahead of Print).
Key words: statin, inflammation, neointimal hyperplasia, vessel remodeling
Restenosis is the arterial wall’s exacerbated healing response to mechanical injury after percutaneous coronary intervention (PCI) and comprises two main processes — neointimal hyperplasia (ie, smooth muscle migration/proliferation, extracellular matrix deposition) and vessel remodeling. Since the advent of stents, vessel negative remodeling has virtually disappeared due to mechanical properties of these metallic devices.1,2 Conversely, inflammation has taken the lead as the main mechanism responsible for excessive neointimal tissue formation after local barotrauma secondary to stent deployment.
Three phases are described in the restenosis process: (1) inflammation; (2) cellular proliferation and granulation; and (3) vessel remodeling, secondary to the synthesis of extracellular matrix.3,4 Platelet aggregation, inflammatory cell infiltration, release of growth factors, medial smooth muscle cell (SMC) modulation and proliferation, proteoglycan deposition, and extracellular matrix remodeling were identified as the major milestones in the temporal sequence of this response. Based on animal studies, Forrester et al3 proposed a paradigm for neointimal hyperplasia as a general “wound-healing” response. The magnitude of the acute inflammatory healing response might correlate with the occurrence of early and late negative events, including restenosis and stent thrombosis after PCI.3
The production of cytokines (interleukin [IL]-1, IL-6, IL-8, tumor necrosis factor [TNF]-α, transforming growth factor [TGF]-β, monocyte chemoattractant protein [MCP]-1/C-C motif chemokine ligand [CCL]-2, plasminogen activator inhibitor [PAI]-1) stimulates the migration of leukocytes to the vessel wall, through the thin layer of platelets and fibrin, formed after vessel trauma caused by stent implantation.5-10 Other cytokines and growth factors are released to the blood, activating N(ε)-(carboxymethyl)lysine, which will induce the production of the extracellular matrix in the subintimal layer.4
Systemic medications able to minimize this inflammatory response might lead to a reduction in restenosis and other negative events after PCI. We sought to investigate the role of preloading with high-dose rosuvastatin in the reduction of serum levels of inflammatory markers and their genetic expression after PCI with metallic stents.
Study design and population. This is a single-center, prospective, randomized trial aimed to determine the acute effects of preloading with a high dose of statin (40 mg rosuvastatin) in the serum levels of inflammatory markers (IL-1, IL-6, IL-8, TNF-α, TGF-β, MCP-1/CCL-2, PAI-1, and high-sensitivity C-reactive protein (hsCRP) of patients with coronary artery disease (CAD) submitted to PCI with bare-metal stent (BMS) or drug-eluting stent (DES) implantation.
Patients undergoing PCI were eligible for the study if they had stable CAD and at least one lesion amenable to stent implantation. Those who were enrolled should not have taken statins for at least 7 days prior to PCI. Those in chronic use of the medication had to go through a 7-day “wash-out” period after signing the informed consent. Statin (80 mg atorvastatin) was reinitiated 24 hours after the procedure.
We excluded patients with acute coronary syndrome and those in recent use (within 30 days) of corticosteroids and/or non-steroidal anti-inflammatory drugs. Women were excluded if they were pregnant, breastfeeding, or under age 45 years and did not use contraceptive methods. We also excluded patients with restenotic lesions or lesions located at venous/arterial grafts.
Patients were randomized in a 1:1 ratio to receive either a loading dose of 40 mg rosuvastatin (statin group) or not (control group). The allocation list was generated in random permuted blocks of variable size, while randomization was implemented through a web-based automated randomization system. Written informed consent was obtained from all patients and the local ethics committees approved this study.
Study procedures. The study flow chart is shown in Supplemental Figure S1 (supplemental materials available at www.invasivecardiology.com). Data were obtained at baseline, on the day of PCI, at hospital discharge, and at 1 month, 6 months, and 12 months post procedure. Blood samples for dosage of serum inflammatory markers and gene expression were obtained 3 hours before and 3 hours after the oral administration of rosuvastatin and then repeated 3 hours post PCI. CK-MB mass was obtained before the procedure (<24 hours) and repeated 12-18 hours after PCI.
Cytokine concentrations were assessed with Luminex 100 multiplex assays (Luminex Corporation) and their gene expression with the PAXgene Blood RNA kit, version 2 (Qiagen GmbH). This analysis was conducted by an independent molecular biology and genetics lab, blinded to patient and procedure characteristics. After the procedures, all patients were prescribed with statin according to current guidelines.
Stenting procedure. All interventions were performed according to the current standard guidelines. The strategy to pre- and/or postdilate was left to the operator’s discretion. Multiple stenting procedures were allowed. The choice of stent type was at the operator’s discretion and varied according to the enrollment period. In the first months of enrollment, only BMS options were available at our public institution. In the remaining period, zotarolimus-eluting stents were available.
During the procedure, intravenous heparin (70-100 IU/kg) was administered after sheath insertion to maintain an activated clotting time >250 seconds. A 12-lead electrocardiogram (ECG) was obtained before the procedure, immediately afterward, and 24 hours post PCI.
All patients enrolled in the study were pretreated with a loading dose of 300 mg clopidogrel and 200 mg aspirin, followed by 75 mg clopidogrel daily for a minimum of 1 month after BMS and 6 months after DES implantation, and 100 mg aspirin daily, indefinitely.
Study objectives and definitions. The primary objective of the present analysis was to compare the variation (Δ) in the inflammatory serum marker levels (IL-1, IL-6, IL-8, TNF-α, TGF-β, MCP-1/CCL-2, PAI-1, and hsCRP) and their gene expression, at different time points, between patients receiving or not receiving a loading dose of rosuvastatin. Blood samples for dosage of serum markers and genetic expression were obtained 3 hours before and after statin administration, as well as 3 hours post PCI. Our secondary objective was to report the 1-year major adverse cardiac event (MACE) and stent thrombosis rates.
Deaths were defined as cardiac in origin unless a non-cardiac origin could be clearly established by clinical and/or pathological study. Myocardial infarction (MI) diagnosis was based on either the development of new pathological Q-waves in ≥2 contiguous ECG leads, and/or elevation of CK-MB mass >5x the upper normal limit post procedure during the index hospitalization (periprocedure or type 4a MI), or cardiac enzyme elevation >2x the upper normal limit thereafter (spontaneous MI). Target-lesion revascularization (TLR) was based on the presence of symptoms and/or signs of ischemia.
Stent thrombosis was classified as definite, probable, or possible according to the definitions proposed by the Academic Research Consortium,11 and was stratified as acute (<24 hours), subacute (24 hours to 30 days), or late (1 to 12 months) and very late (>12 months).
Clinical follow-up, by office appointment or phone call, was scheduled at 1 month, 6 months, and 12 months after stent implantation, and then annually based on information entered on case report forms at the time of the office visit/telephone contact. All phone follow-up data were collected by the same person especially trained for this task and blinded to the procedure results. Individual patient data were coded to prevent the identification of study participants. Adverse events were adjudicated by an independent committee of three cardiologists not involved in the procedures.
Sample size. Sample size was based on an anticipated 25% reduction in the serum levels of IL-6 in the treatment group. This assumption was based on a previous study with patients in chronic use of statin, who presented a 50% reduction in the inflammatory markers as compared to those who did not use this medication.5 Among all the inflammatory markers analyzed in the present trial, IL-6 was the one that needed a larger sample size to determine the potential anti-inflammatory benefit of the statin. Therefore, the sample size assumption was based on its expected variation. A total of 55 patients in each cohort would result in a 90% statistical power and a 2-tailed α of 5%.
Statistical analysis. Data are presented as mean ± 1 standard deviation or frequencies. Categorical variables were compared with the Chi-square test. When the expected frequency was <5, Fisher’s exact test was used. For continuous variables comparison, t-test was used. In case of non-normal distribution, Mann-Whitney test was the alternative. Variation in the inflammatory markers and their genetic expression were compared by analysis of variance (ANOVA) with repeated measures, taking into account two factors: (1) treatment (active vs control); and (2) moment of evaluation (before and after PCI).
Finally, to determine the independent predictors of MACE, a multivariable logistic regression model was built using a stepwise (forward/backward) procedure, with independent variables entered into the model at the .20 significance level and removed at the .10 level. Variables were eligible for inclusion in the multivariable logistic regression model-building process if the variable was present for 90% of the subjects in the analysis, they had a P-value <.20 from the univariable analysis, and if highly correlated with another variable (r>0.50 and P<.05), had the higher level of significance. All statistical analyses were performed with SPSS, version 20 (SPSS).
Study participants and PCI procedures. Between March and December 2015, a total of 130 patients were enrolled and randomly allocated into two groups: (1) the treatment group (n = 65), pretreated with loading dose of 40 mg rosuvastatin; and (2) the control group (n = 65), with patients who did not receive the study medication prior to PCI. One patient from the treatment cohort and 4 patients from the control group were excluded due to problems with the blood samples, which precluded the assessment of inflammatory markers and gene expression. Therefore, the final study population comprised 125 patients (64 in the statin group and 61 in the control group).
Table 1 displays the main baseline clinical characteristics. No significant differences were observed between the two groups regarding age, gender, or the presence of CAD risk factors, such as diabetes, hypertension, smoking, and dyslipidemia.
Almost all patients had been taking statins in both cohorts, most for >1 month. A low-potency statin (simvastatin) was the most frequently used (>80% in both cohorts). All patients interrupted the medication for a minimum of 7 days prior to randomization (during the wash-out period).
Angiographic and procedure characteristics are displayed in Table 2. Most patients had single-vessel disease and there was a predominance of lesions located in the proximal/mid segments of the treated vessels. Thrombolysis in Myocardial Infarction (TIMI) 3 flow was achieved in >98% of the cases in both cohorts. Most PCI procedures were performed by radial access (65.6% in the statin group vs 68.8% in the control group; P=.01) with single-stent implantation (68.7% in the statin group vs 69.6% in the control group; P=.84). DES options were deployed in about 70% of all cases (73.4% in the statin group vs 67.2% in the control group; P=.56). Postdilation was less frequently performed in the statin group (73.4% vs 90.2% in the control group; P=.02).
Inflammatory markers and gene expression. Table 3 displays the mean values of serum inflammatory markers 3 hours prior to randomization and 3 hours post PCI, as well as their relative variations (treatment/control). Table 4 displays the gene expression for the two groups at the same time points.
In the treatment cohort, there was a significant reduction in the levels of IL-1β (Δ = -0.491 pg/mL; Pinteraction<.001), IL-6 (Δ = -0.209; Pinteraction<.001), PAI-1 (Δ = -1.573 pg/mL; Pinteraction<.001) as well as in their genetic expression. In the control group, a contrary variation was observed, with a marked increase in the serum levels and gene expression of these markers. IL-8 serum levels and gene expression did not significantly vary in both groups.
TGF-β serum levels were marked differently at baseline between the two cohorts. In the statin group, there was a trend to reduction in the serum levels of this marker (Δ = -0.583 pg/mL; Pinteraction=.10) not accompanied by its gene expression (0.859 vs 9.849; P=.61).
Serum levels of TNF-α increased in the statin group (Δ = +1.177 pg/mL; Pinteraction<.01), which was not followed by a significant increase in its genetic expression when compared with the control group (0.310 vs 0.235, respectively;P=.82).
MCP-1 serum levels increased in both cohorts, but were more accentuated in the treatment population (Δ = +1.347 pg/mL; Pinteraction<.01). The gene expression was higher in the treatment cohort as well, but did not achieve statistical significance (0.709 vs 0.512; P=.29).
Regarding hsCRP, there was no significant variation in its value from time point A to time point C among patients pretreated with statin (from 0.86 ± 1.88 mg/L to 0.88 ± 1.75 mg/L; P=.58), while there was a significant increase in the control group (from 0.76 ± 0.74 mg/L to 0.94 ± 0.73 mg/L; P=.04).
Clinical outcomes. Periprocedure MI rate was higher in the control group (23% in the control group vs 4.7% in the statin group; P<.01) and correlated only with the increase in the serum level of IL-6 (Figure 1). No other MACE was observed during this period in both cohorts. Patients pretreated with statin had a shorter hospitalization length (1.4 ± 0.8 days in the control group vs 1.0 ± 0.2 days in the statin group; P<.01).
Complete 1-year clinical follow-up was obtained in all cases at 12.4 ± 3.0 months. Figure 2 shows the survival free of MACE curves for the two groups. Despite the reduction in the absolute number of MACE in the statin group, it did not achieve statistical significance (hazard ratio, 0.67; 95% confidence interval, 0.24-1.7; P=.14). Furthermore, there was a trend toward less restenosis among patients pretreated with rosuvastatin (6.6% in the control group vs 1.6% in the statin group; P=.09).
To the best of our knowledge, this is the first study to demonstrate that in stable CAD patients who are not in chronic use of statins, preloading with a high dose of rosuvastatin reduces the serum levels of inflammatory markers (including IL-1β, IL-6, and PAI-1), accompanied by the reduction in their gene expression. This finding might reduce periprocedure myocardial injury and impact the occurrence of long-term MACE in patients treated with metallic stents.
Despite the success of cholesterol-lowering therapies, researchers have long known that atherosclerosis is not solely a disease of cholesterol deposition and that inflammation plays a key role in its pathogenesis. Previous studies have shown that chronic use of statins has a beneficial effect in terms of inflammation reduction. The CARE study was a randomized, placebo-controlled trial of pravastatin 40 mg daily among 4159 individuals with a history of MI and elevated levels of cholesterol (low-density lipoprotein [LDL], 115-175 mg/dL).12 In a cohort of 472 randomly selected individuals from the trial, those treated with pravastatin experienced a mean decrease in hsCRP of 21.6% over 5 years of follow-up compared with placebo.12 Likewise, among 1702 patients with no history of cardiovascular disease in the PRINCE study, those randomized to pravastatin 40 mg daily experienced a 16.9% reduction in hsCRP at 24 weeks compared with no reduction in the placebo group.13 These effects were independent of LDL cholesterol reduction.
More potent statins have a more marked effect on hsCRP. In the JUPITER study, a total of 17,802 men and women free of cardiovascular disease with low levels of LDL cholesterol (<130 mg/dL) but elevated levels of hsCRP (≥2.0 mg/L) were randomized to either rosuvastatin 20 mg daily or placebo.14 Overall, rosuvastatin led to a 37% median hsCRP reduction (P<.001) vs placebo.15 Similarly, PROVE-IT TIMI 22 randomized 3745 individuals with an acute coronary syndrome to either atorvastatin 80 mg or pravastatin 40 mg daily, with a primary outcome of recurrent MI or coronary-related death.16 Following 30 days of therapy, 57.5% of individuals treated with atorvastatin achieved an hsCRP <2 mg/L vs 44.9% of those treated with pravastatin.17 In the present study, while the baseline values for hsCRP were not markedly elevated (reflecting the low risk of the enrolled population), we showed that statin pretreatment prevented hsCRP elevation after PCI with stent implantation. In our analysis, the effects were observed a few hours after statin intake, suggesting an early (acute) effect as well.
Inflammatory signals involved in atherosclerosis, including cholesterol crystals, hypoxia, and turbulent flow, activate the NLRP3 inflammasome, which is a multi-protein assembly that integrates these signals and specifically activates the IL-1β isoform.18 Furthermore, IL-1 triggers increased vascular SMC proliferation.19 IL-1 also upregulates IL-6, another proinflammatory cytokine that induces hepatocytes to synthesize and release different acute-phase reactants, including CRP, fibrinogen, and plasminogen-activator inhibitor.18 Recently, several new drugs have been developed to target IL-1 signaling. Among them, canakinumab was particularly attractive since known atherosclerosis risk factors upregulate IL-1β via the NLRP3 inflammasome. Moreover, canakinumab may be less likely to impair host immune function, since signaling via IL-1α remains intact. In a pilot study, this drug led to a significant decrease in hsCRP, fibrinogen, and IL-6 with no impact on LDL cholesterol or other lipid measures among 556 diabetic individuals at high risk for cerebrovascular disease.20 Later, in a larger, randomized trial (CANTOS),21 this new drug was shown to significantly reduce hsCRP and IL-1β serum levels and resulted in a 15% reduction in MACE rates (P<.01). In the present analysis, a high loading dose of rosuvastatin was associated with a reduction in serum levels and gene expression of both IL-1β and IL-6.
Study limitations. This study has some potential limitations. The sample size is insufficient to determine the clinical impact of the laboratory findings. We did not investigate the role of the loading dose of rosuvastatin in patients in chronic use of statin, who represent the vast majority of the population currently referred to PCI. Since all patients were prescribed statin post PCI, it is difficult to determine the impact of the preloading dose at 1-year follow-up.
Among patients with stable CAD undergoing PCI with stents, pretreatment with a high dose of rosuvastatin resulted in a signficant reduction in the serum levels of inflammatory markers (IL-1β, IL-6, PAI-1, and hsCRP) and their genetic expression. Of note, the increase in IL-6 levels correlated with the occurrence of periprocedural MI.
From the 1Instituto Dante Pazzanese de Cardiologia, São Paulo, Brazil; 2São Paulo University (USP), São Paulo, Brazil; 3Federal University of São Paulo (UNIFESP)São Paulo, Brazil; and 4Federal University of Mato Grosso, Cuiabá, Brazil.
Funding: This study was partially funded by Fundação de Amparo a Pesquisa de São Paulo (FAPESP 2012/ 22989-1), a government health agency.
Disclosure: The authors have completed and returned the ICMJE Form for Disclosure of Potential Conflicts of Interest. The authors report no conflicts of interest regarding the content herein.
Manuscript submitted February 9, 2020, provisional acceptance given February 18, 2020, final version accepted March 4, 2020.
Address for correspondence: Juliano R. Slhessarenko, MD, Av. Dr. Dante Pazzanese, 500-Vila Mariana, São Paulo, SP, Brasil, CEP 04012-180. Email: firstname.lastname@example.org
- Labinaz M, Pels K, Hoffert C, Aggarwal S, O’Brien ER. Time course and importance of neoadventitial formation in arterial remodeling following balloon angioplasty of porcine coronary arteries. Cardiovasc Res. 1999;41:255-266.
- Costa MA, de Wit LE, de Valk V, et al. Indirect evidence for a role of a subpopulation of activated neutrophils in the remodelling process after percutaneous coronary intervention. Eur Heart J. 2001;22:580-586.
- Forrester JS, Fishbein M, Helfant R, Fagin J. A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies. J Am Coll Cardiol. 1991;17:758-769.
- Libby P, Schwartz D, Brogi E, Tanaka H, Clinton SK. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation. 1992;86:III47-III52.
- Sardella G, Mariani P, D’Alessander M, et al. Early elevation of interleukin-1beta and interleukin-6 levels after bare or drug-eluting stent implantation in patients with stable angina. Thromb Res. 2006;117:659-664.
- Schillinger M, Exner M, Mlekusch W, et al. Balloon angioplasty and stent implantation induce a vascular inflammatory reaction. J Endovasc Ther. 2002;9:59-66.
- Tsakiris DA, Tschöpl M, Jäger K, Haefeli WE, Wolf F, Marbet GA. Circulating cell adhesion molecules and endothelial markers before and after transluminal angioplasty in peripheral arterial occlusive disease. Atherosclerosis. 1999;142:193-200.
- McDonald RA, Hata A, MacLean MR, Morrell NW, Baker AH. MicroRNA and vascular remodelling in acute vascular injury and pulmonary vascular remodeling. Cardiovasc Res. 2012;93:594-604.
- Costa MA, Simon DI. Molecular basis of restenosis and drug eluting stents. Circulation. 2005;111:2257-2273.
- Von der Thüsen JH, Kuiper J, van Berkel TJ, Biessen EA. Interleukins in atherosclerosis: molecular pathways and therapeutic potential. Pharmacol Rev. 2003;55:133-166.
- Cutlip DE, Windecker S, Mehran R, et al; Academic Research Consortium. Clinical end points in coronary stent trials: a case for standardized definitions. Circulation. 2007;115:2344-2351.
- Sacks FM, Pfeffer MA, Moye LA, et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med. 1996;335:1001-1009.
- Albert MA, Danielson E, Rifai N, Ridker PM. Effect of statin therapy on C-reactive protein levels: the pravastatin inflammation/CRP evaluation (PRINCE): a randomized trial and cohort study. JAMA. 2001:286:64-70.
- Ridker PM, Danielson E, Fonseca FAH, et al. Rosuvastatin to prevent vascular events in men and women with elevated c-reactive protein. N Engl J Med. 2008:359:2195-2207.
- Ridker PM, Danielson E, Fonseca FA, et al; JUPITER Trial Study Group.Reduction in C-reactive protein and LDL cholesterol and cardiovascular event rates after initiation of rosuvastatin: a prospective study of the JUPITER trial. Lancet. 2009;373:1175-1182.
- Cannon CP, Braunwald E, McCabe CH, et al; the Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction 22 Investigators. Intensive versus moderate lipid lowering with statins after acute coronary syndromes. N Engl J Med. 2004:350:1495-1504.
- Ridker PM, Morrow DA, Rose LM, Rifai N, Cannon CP, Braunwald E. Relative efficacy of atorvastatin 80 mg and pravastatin 40 mg in achieving the dual goals of low-density lipoprotein cholesterol <70 mg/dL and C-reactive protein <2 mg/L: an analysis of the PROVE-IT TIMI-22 trial. J Am Coll Cardiol. 2005;45:1644-1648.
- Libby P. Interleukin-1 β as a target for atherosclerosis therapy. J Am Coll Cardiol. 2017:70:2278-2289.
- Libby P, Warner SJ, Friedman GB. Interleukin 1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest. 1988;81:487-498.
- Ridker PM, Howard CP, Walter V, et al. Effects of interleukin-1β inhibition with canakinumab on hemoglobin A1c, lipids, C-reactive protein, interleukin-6, and fibrinogen: a phase IIb randomized, placebo-controlled trial. Circulation. 2012;126:2739-2748.
- Ridker PM, Everett BM, Thuren T, et al; CANTOS Trial Group. Anti-inflammatory therapy with canakinumab for atherosclerotic disease. N Engl J Med. 2017;377:1119-1131.