Efficacy of Statin Therapy in Pulmonary Arterial Hypertension: A Systematic Review and Meta-Analysis

Since the evidence regarding statin therapy in PAH has not been conclusive, we assessed the impact of statin therapy in PAH through a systematic review and meta-analysis of available studies. We searched selected databases up to August 1, 2015 to identify the studies investigating the effect of statin administration on PAH. Meta-analysis was performed using either a fixed-effects or random-effect model according to I2 statistic. Meta-analysis of 8 studies with 665 patients did not suggest any significant improvement in 6-min walking distance (6MWD) by statin therapy (weighed mean difference [WMD]: −6.08 m, 95% confidence interval [CI]: −25.66, 13.50, p = 0.543; Q = 8.41, I2 = 28.64%). Likewise, none of the other indices including pulmonary arterial pressure (WMD: −0.97 mmHg, 95%CI: −4.39, 2.44, p = 0.577; Q = 14.64, I2 = 79.51%), right atrial pressure (WMD: 1.01 mmHg, 95%CI: −0.93, 2.96, p = 0.307; Q = 44.88, I2 = 95.54%), cardiac index (WMD: 0.05 L/min/m2, 95%CI: −0.05, 0.15, p = 0.323; Q = 3.82, I2 = 21.42%), and pulmonary vascular resistance (WMD: −1.42 dyn*s/cm5, 95%CI: −72.11, 69.27, p = 0.969; Q = 0.69, I2 = 0%) was significantly altered by statin therapy. In conclusion, the results of the meta-analysis did not show a statistically significant effect of statin therapy in the improvement of 6MWD, pulmonary arterial pressure, right atrial pressure, cardiac index and pulmonary vascular resistance.

hypertension due to left heart disease, pulmonary hypertension due to lung diseases and/or hypoxia, chronic thromboembolic pulmonary hypertension and other pulmonary artery obstructions, and pulmonary hypertension with unclear and/or multifactorial mechanisms 1 . Several prognostic factors such as right ventricular failure, higher World Health Organization functional classification (WHO FC), shorter 6-min walk distance (6MWD) and hemodynamic factors such as right atrial pressure, and brain natriuretic peptide (BNP) levels are useful for PAH prognosis 2,3 . Furthermore, the American College of Chest Physicians' (CHEST) Guideline suggests that the diagnosis of PAH should be made in a systematic and consistent manner with the use of a combination of WHO FC, exercise capacity, echocardiographic, laboratory and hemodynamic variables, and care should be provided by experts in the management of PAH. (Grade CB) 4 . However, the prognosis for patients with PAH remains poor, especially without appropriate treatment 5 .
The pleiotropic effects of statins through induction of endothelial nitric oxide (NO) expression as well as anti-inflammatory and antiproliferative mechanisms possibly confer various clinical advantages beyond the reduction of serum cholesterol levels 8,9 . Since statins have been suggested as potential drugs for PAH treatment 10 , we assessed the impact of statin therapy on multiple parameters in PAH in this systematic review and meta-analysis.

Methods
This study was designed according to the 2009 preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines 11 . Due to the study design (meta-analysis) no Institutional Review Board (IRB) approval, as well as no patients' informed consents were obtained.
Search Strategy. PubMed-Medline, SCOPUS, Web of Science and Google Scholar databases were searched using the following search terms in titles and abstracts (also in combination with MESH terms): (atorvastatin OR simvastatin OR rosuvastatin OR fluvastatin OR pravastatin OR pitavastatin OR lovastatin OR cerivastatin OR "statin therapy" OR statins OR statin) AND ("pulmonary arterial hypertension" OR "pulmonary hypertension" OR "pulmonary artery hypertension" OR "pulmonary vascular disease" OR "pulmonary heart disease" OR "pulmonary cardiac disease" OR PAH). The wild-card term "* " was used to increase the sensitivity of the search strategy. The literature search was limited to articles published in English. The search was limited to studies in humans. The literature was searched from inception to August 1, 2015. Two reviewers (MRG and AGB) evaluated each article independently. Disagreements were resolved by discussion with a third party (MB).

Study Selection.
Original studies were included if they met the following criteria: (i) being a clinical study with either observational or interventional design, (ii) recruiting patients with a clinical diagnosis of PAH according to echocardiography regardless of the etiology (congenital heart disease, connective tissue disease, chronic thrombembolism or iatrogenic), and, (iii) investigating the impact of statin therapy on a valid disease activity index including 6MWD, pulmonary arterial pressure, right atrial pressure, cardiac index and pulmonary vascular resistance.
Exclusion criteria were: (i) non-clinical studies, (ii) lack of a statin-free control group in the study design, and, (iii) lack of sufficient information on baseline or follow-up indices of PAH.
Data extraction. Eligible studies were reviewed and the following data were abstracted: 1) first author's name 2) year of publication 3) study location 4) study design 5) number of participants in the statin and groups 5) age, gender and body mass index (BMI) of study participants 6) type and duration of statin therapy and 7) baseline and follow-up values of PAH-related indices.
Data extraction was performed independently by 2 reviewers; disagreements were resolved by a third reviewer.
Quality assessment. Methodological quality of the included studies was assessed using the Newcastle-Ottawa Scale (NOS) 12 . In this context, topic items of each eligible study are assessed: that is, the selection of the studied patients, the comparability of the studied populations and the ascertainment of the exposure. A study can be awarded a maximum of one point for each item. Risk-of-bias assessment was performed independently by 2 reviewers; disagreements were resolved by a third reviewer.
Quantitative Data Synthesis. Meta-analysis was conducted using Comprehensive Meta-Analysis (CMA) V2 software (Biostat, NJ) 13 . Net changes in measurements (change scores) were calculated as follows: measure at end of follow-up − measure at baseline. For single-arm cross-over trials, net change in each efficacy measure was calculated by subtracting the value after control intervention from that reported after treatment. Standard deviations (SDs) of the mean difference were calculated using the following formula: SD = square root [(SD pre-treatment ) 2 + (SD post-treatment ) 2 − (2R × SD pre-treatment × SD post-treatment ), assuming a correlation coefficient (R) = 0.5. If the outcome measures were reported in median and range or 95% CI, mean and standard SD values were estimated using the method described by Wan et al. 14 . Where standard error of the mean (SEM) was only reported, SD was estimated using the following formula: SD = SEM × sqrt (n), where n is the number of subjects.
A random-effects model (using DerSimonian-Laird method) and the generic inverse variance method were used to compensate for the heterogeneity of studies in terms of demographic characteristics of populations being studied and also differences in study design and type of statin being studied 15 . Heterogeneity was quantitatively Scientific RepoRts | 6:30060 | DOI: 10.1038/srep30060 assessed using I 2 index. Effect sizes were expressed as weighted mean difference (WMD) and 95% confidence interval (CI). In order to evaluate the influence of each study on the overall effect size, sensitivity analysis was conducted using leave-one-out method, i.e. removing one study each time and repeating the analysis.
Publication bias. Potential publication bias was explored using visual inspection of Begg's funnel plot asymmetry, Begg's rank correlation, and Egger's weighted regression. Duval and Tweedie "trim and fill" was used to adjust the analysis for the effects of publication bias 16 .

Flow and characteristics of included studies. On the basis of a database search 859 published studies
were found, 310 of which were non-clinical or review articles and thus they were not included in further analysis. We screened 549 articles but 533 of them were excluded because they did not meet inclusion criteria. Out of 16 eligible papers, 6 were excluded since they were not conducted in subjects with PAH, one of them did not assess any of the pre-specified efficacy measures, and one was not a clinical study. Finally, we included 8 articles in this meta-analysis [17][18][19][20][21][22][23][24] (Fig. 1).
Most of the analyzed studies (7/8) were randomized, double-or triple-blind and placebo-controlled. This meta-analysis comprised 665 patients with PAH receiving either statins (rosuvastatin, simvastatin, pravastatin or atorvastatin) (311 patients) or placebo (354 patients) for 6 months. In analyzed patients, PAH was idiopathic, heritable, associated with congenital heart disease, atrial-septal defects or connective tissue disease, and many of them suffered from chronic obstructive pulmonary disease. The PAH-related indices 6MWD, pulmonary arterial pressure, right atrial pressure, cardiac index and pulmonary vascular resistance were measured in 6, 6, 2, 4 and 2 analyzed studies, respectively ( Table 1).
Risk of bias assessment. The quality of the included studies assessed by the Newcastle-Ottawa Scale (NOS) 12 is shown in Table 2.
Effect of statin therapy on PAH-related indices. Meta-analysis of included studies did not suggest any significant improvement in 6MWD by statin therapy (WMD: − 6.08 m, 95% CI: − 25.66, 13.50, p = 0.543; Q = 8.41, I 2 = 28.64%) ( Fig. 2A). This effect was robust in sensitivity analysis and the statistical significance was not influenced by any single study included in the meta-analysis (Fig. 2B). Likewise, none of the other indices including pulmonary arterial pressure (WMD: − 0.97 mmHg, 95% CI: Publication bias. The funnel plot of the study standard error by effect size (mean difference) for the meta-analysis of statin effects on 6MWD was asymmetric suggesting a potential publication bias (Fig. 4). Using "trim and fill" correction, two potentially missing studies were imputed on the left side of funnel plot, yielding an effect size of − 11.72 m (95% CI:  Barreto et al. 17 Kawut et al. 18 Lee et al. 19 Liu et al. 20 Moosavi et al. 21 Reed et al. 22 Wilkins et al. 23 Zeng et al. 24 Year  17 Kawut et al. 18 Lee et al. 19 Liu et al. 20 Moosavi et al. 21 Reed et al. 22 Wilkins et al. 23 Zeng et al. 24 Follow   Table 1. Main characteristics of the included studies. Data are presented as mean ± SD and range in brackets. ABBREVIATIONS: 6-MWD -6 minutes walk distance; COPD -chronic obstructive pulmonary disease; FEV1 -forced expiratory volume in one second % of vital capacity; NYHA -New York Heart Association; PAH -pulmonary arterial hypertension; PAP -pulmonary arterial pressure; PFT -pulmonary function test; WHO -world health organization; *mean change; * * change in patients with idiopathic pulmonary arterial hypertension and pulmonary arterial hypertension associated with connective tissue disorder; * * * change in patients with pulmonary arterial hypertension associated with congenital heart disease.

Discussion
To our knowledge, the current systematic review and meta-analysis is the first to evaluate the effects of statins on PAH. Contrary to the findings from some studies 18,25 , this meta-analysis of 8 studies did not suggest any significant improvement in 6MWD, pulmonary arterial pressure, right atrial pressure, cardiac index, and pulmonary vascular resistance by statin therapy. The lack of benefit was robust in sensitivity analysis and the statistical significance was not influenced by any single study included in the meta-analysis. These findings are of clinical interest, since the use of statins in PAH treatment is thought to be beneficial due to their antiproliferative, anti-inflammatory, and pro-apoptotic pleiotropic effects as well as the ability to restore endothelial vasoactive mediator production 26 . Various studies linked statin effects with the pathogenesis of pulmonary hypertension and various biomarkers 27 , through analyzing the relation between statins, endothelin-1 and PAH 28,29 , statins, asymmetric dimethylarginine and PAH [30][31][32] , statins, D-dimers and PAH 33,34 , statins, von Willebrand factor antigen and PAH 35,36 or fibrinogen and PAH 37,38 . Until now, the results obtained using statins on PAH parameters in different animal models have been contradictory. Some experimental studies suggested that statins might attenuate the development or even regress established experimental PAH by decreasing proliferation and increasing apoptosis of pathological smooth muscle cells in the medial walls and neointima of  . Forest plot detailing weighted mean difference and 95% confidence intervals for the impact of statin therapy on 6-min walking distance ( Fig. 2A). Lower plot (Fig. 2B) shows leave-one-out sensitivity analysis.
pulmonary arteries and reducing right ventricular hypertrophy 39,40 . Other experimental studies on murine models reported an inhibition of progression of emphysema and pulmonary hypertension related to tobacco exposure after statin therapy 41,42 . One experimental study showed that low doses of fluvastatin have beneficial effects on adventitial fibroblasts from chronic hypoxic animals through actions on the Rac1-p38 MAP kinase-signaling pathway 43 . Another experimental study showed that rosuvastatin improved ischemia-reperfusion injury by decreasing macrophage infiltration and up-regulating endothelial nitric oxide synthase 44 . In contrast, atorvastatin neither improved survival nor reduced PAH, vascular remodeling and right ventricular hypertrophy in an experimental study on a rat monocrotaline PAH model 45 . However, some experimental studies reported a decrease of right ventricular (RV) hypertrophy associated with statin therapy in PAH models 46 , but no study showed a reduction in pulmonary arterial pressure 47,48 . An experimental study on guinea pigs exposed to cigarette smoking during 6 months showed no effects of simvastatin on small airway remodeling 49 .  (Fig. 3A), right atrial pressure (RAP) (Fig. 3B), cardiac index (Fig. 3C), and pulmonary vascular resistance (PVR) (Fig. 3D).
Scientific RepoRts | 6:30060 | DOI: 10.1038/srep30060 The data on humans from short-term randomized trials are few and contradictory. In an open-label observational trial, simvastatin was successfully used as adjunctive therapy in patients with PAH associated with various pathologies 25 . In another study, atorvastatin reduced pulmonary artery pressure and raised the migration and adhesion of endothelial progenitor cells (EPCs) in patients with chronic pulmonary heart disease 20 . Other studies described a decreased frequency of exacerbations and intubations 50 , a reduced decline of pulmonary function 51 and decreased pulmonary pressures associated with improved exercise capacity after statin treatment 19 in patients with chronic obstructive pulmonary diseases. A multicenter propensity score study on 2,363 patients showed for the first time that statins improve survival in PAH patients by decreasing one-year all-cause mortality 52 .
Similar to our results obtained in this meta-analysis, and in contrast to previous arguments, another study did not find any effect of statins on 6MWD 23 , but the short duration of this trial and background therapy of PAH patients may be potential causes for the lack of effects. Another trial on 220 PAH patients showed no benefit of atorvastatin on cardio-pulmonary hemodynamics, 6 MWD and survival at 6 months of treatment 24 , suggesting that atorvastatin should not be prescribed as specific treatment in PAH.
Limitations. This meta-analysis has several limitations. First, the studies were relatively small and heterogeneous concerning study design, patients characteristics, major outcomes, PAH etiology and severity. Second, the patients included were classified in various functional classification groups. Third, the positive effects of statins in one study might have been overlapped by lack of effects of statins in the rest of studies since the effects of four statins (rosuvastatin, simvastatin, pravastatin and atorvastatin) were analyzed. Fourth, the patients received supportive drugs such as diuretics, warfarin, and digoxin, which could have influenced the results. The follow-up of the study was relatively short (6 months), so the long-term effects cannot be concluded and it is possible that longer therapy with statin might have been effective. Taking into account the given small sample size and short duration of the included studies, the meta-analysis could not also look at CVD outcomes that would be of the greatest interest. Finally, in the most of included studies PAH was diagnosed mainly based on echocardiography, which is also an important limitation.
In conclusion, the results of this meta-analysis of available studies did not show a statistically significant effect of statin therapy in the improvement of 6 MWD, pulmonary arterial pressure, right atrial pressure, cardiac index and pulmonary vascular resistance. Additional large, long term and well-designed trials are necessary to investigate the impact of statins for the treatment of PAH including their use as pulmonary vascular antiproliferative agents.