Low levels of HDL-cholesterol and peripheral artery disease: Protocol for systematic review and meta-analysis

Purpose of review: HDL-C is believed to retard the formation of atherosclerotic lesions by removing excess cholesterol from cells and preventing endothelial dysfunction. However, there are no systematic analyses or well-conducted meta-analyses to evaluate the relationship between very low HDL-Cand stroke. Patients with peripheral artery disease (PAD) have been found to suffer from diabetes, obesity, lipid abnormalities, including elevated levels of total and LDL-cholesterol as well as triglyceride levels. The aim of this study is to examine this association of very low HDL-Cwith stroke in different ages and sex. Recent findings: The update systematic review and meta-analysis will be conducted using published studies that will be identified from electronic databases (ie, PubMed, EMBASE, Web of Science, and Google Scholar. Studies that (1) examined the association between very low HDL-Cand stroke, (2) had a longitudinal or prospective cohort design, (3) will conducted among in adults aged 34 to 70 years., (4) provided sufficient data for calculating odds ratios (ORs) or relative risk with a 95% CI, (5) were published as original articles written in English or other languages, and (6) have been published until January 2019 will be included. Study selection, data collection, quality assessment and statistical syntheses will be conducted based on discussions among investigators. Summary: Ethics approval was not required for this study because it was based on published studies. The results and findings of this study will be submitted and published in a scientific peer-reviewed journal. Strengths and limitations of this study: This systematic review and meta-analysis will offer better understanding regarding the association between metabolic syndrome and peripheral artery disease (PAD). The findings from this study will be useful for assessing of very low HDL-Cand the risk factors in PAD, and determining approaches for prevention of PAD the future. An improved understanding of this relationship may help to inform public health PAD prevention strategies. Included studies may have substantially different methodologies, which could limit our ability to draw reliable conclusions from the existing evidence base. Depending on the results, confounding factors that were not adjusted for the selected studies and low generalizability situations can be limitations. To minimize these limitations, we will evaluate the heterogeneity between the studies, perform sensitivity analysis and meta-regression. *Correspondence to: Leonardo Roever, Federal University of Uberlândia, Department of Clinical Research, Uberlândia, Brazil, E-mail: leonardoroever@hotmail.com

provide information on low and very low HDL levels (<20; 20-30; 30-40 vs. >40 as ref. according to sex) in cardioembolic and a control group (healthy controls or other PAD subtypes).
Data will be extracted using a standardized template. We will use the PICOS (Population, Intervention, Comparator, Outcomes and Study design) framework, originally devised to formulate a research question, as a basis to develop data extraction criteria. As this is an aetiological study, 'exposure' will replace 'intervention' and 'study characteristics' will replace 'study design' . Data items on the following five domains will be extracted:

Outcomes
Primary outcomes: All patients with PAD (relative risk (RR), and odds ratio (OR). Studies will be included in the review if the primary outcome was any PAD, clinically diagnosed or self-reported, and was the patient's first or subsequent PAD.

Secondary outcomes:
The association of EAT with PAD, by EAT measurement method. The secondary outcome measures include changes in pain free walking distance (PFWD) and quality of life (QOL).
For studies meeting the inclusion criteria, we will additionally assess the following outcomes: TIA (a transient episode of neurological dysfunction caused by focal brain, spinal cord or retinal ischemia without acute infarction) and subtypes of stroke (ischemic vs haemorrhagic). Most strokes (approximately 85%) are ischemic (an episode of neurological dysfunction caused by focal, cerebral, spinal or retinal infarction), compared with haemorrhagic (neurological dysfunction caused by a focal collection of blood within or on the surface of the brain). Eligibility criteria may be further developed, in an iterative process, after preliminary searches.

Study design
This is a systematic review and meta-analysis protocol of prospective cohort studies, following the PRISMA-P (Preferred Reporting Items for Systematic Reviews and Meta-Analysis protocols) guideline [14]. The systematic review and meta-analysis will be reported according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guideline [15]. The whole process of study selection is summarized in the PRISMA flow diagram (Figure 1).

Search strategy
A systematic review of the literature will be conducted. A language restriction shall not be applied to the search. If there are relevant non-English abstracts, attempts shall be made to translate them wherever Background HDL-C is believed to retard the formation of atherosclerotic lesions by removing excess cholesterol from cells and preventing endothelial dysfunction and a very low HDL-C increased risk of cardiovascular events [1]. It is in large part the results of unbalanced diet, low socioeconomic and cultural levels, stress and sedentary lifestyle. Although the literature on the very low HDL-C and the risk factors for PAD has been increasing, to our knowledge, a systematic review of the association between very low of HDL-C and risk of PAD has not yet been conducted [1][2][3][4][5][6][7][8][9][10][11]. This study aims to systematically access the association between a very low HDL-C and the PAD in adults aged 34 to 70 years; and to provide a framework to further understand these factors in order to better target prevention strategies.

Method
This systematic review of the literature will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations. The databases PubMed, Embase, Web of Science, Google Scholar, and Cochrane will be searched for articles [12]. Our search will focus on cohort, case-control and cross-sectional studies examining the association between very low HDL-Cand PAD. The primary outcome is PAD. Two reviewers will independently screen articles, extract relevant data and assess the quality of the studies.
The aim of this analysis is to investigate whether there is an association between HDL-C levels and PAD in the adult population with cardiovascular outcomes. We plan to look at the prevalence of very low HDL-C levels in PAD individuals and to analyse whether low and very low HDL levels (<20; 20-30; 30-40 vs. >40 mg/dL as ref. according to sex) in PAD might to be additional risk factor and predictor of CVD events, and mortality (CV-mortality, mortality and all-cause mortality). [1][2]

Systematic review registration
The study is registered with PROSPERO (CRD42018083417). This protocol conforms to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines. [13][14]

Objectives
The primary objective is to identify and summarize the association with of very low of HDL-C levels and with PAD risk in adults (34-70 years) in different ages and sexes.

Eligibility criteria
The PICOS strategy (population, intervention (changed to exposure for the purposes of this review of observational studies), comparator, outcome, study characteristics) was used to define the eligibility criteria for this study. Inclusion criteria: studies will be considered if they include: ischemic PAD or transient ischemic attack patients in the diagnosis of the low and very low HDL levels (<20; 20-30; 30-40 vs. >40 mg/dL as ref. according to sex) is made during hospitalization. To evaluate the association between of the low and very low HDL levels (<20; 20-30; 30-40 vs. >40 mg/dL as ref. according to sex) and etiology PAD;defined according to a validated classification. Exclusion: Reviews or abstracts from congresses/conferences, letters, editorials, case reports, interventional studies or clinical trials. We excluded studies that did not possible. The following bibliographic databases (Embase, PubMed-MEDLINE, Web of Science, Cochrane Library, and Google Scholar) will be searched for articles published until January 2019.
Our search focuses on studies examining the association between very low HDL-C diagnosis and PAD risk in adults (34-70 years)11. At each step of the selection process, reasons for inclusion/exclusion will be recorded in the PRISMA Flowchart [13].

Data collection
A record will be kept of all searches and search decisions to ensure reproducibility. Search results will be exported to a citation management program (EndNote ver. 7.0). Duplicates will be removed and retained separately. The resulting references will be exported separately to the two reviewers for independent review using MS Excel.

Selection of studies
Two authors (LR, FCV) will independently screen all titles and abstracts identified through the literature searches and will exclude all records clearly not meeting inclusion criteria. Disagreements will be resolved by consensus. The selection process will be pilot tested to ensure a high degree of agreement between reviewers. Full text of the remaining studies will then be retrieved. The same two authors (LR, FCV) will independently assess the papers for fulfilment of inclusion criteria. In case of differences of opinion regarding study inclusion, a third review author (GBZ) will serve as arbiter. To avoid double counting, if multiple publications based on the same cohort of participants are retrieved, only the study reporting the largest sample size will be used. The reasons for excluding papers for which the full text was retrieved will be documented.

Data extraction and management
A data extraction form will be used to collect details from the included studies. The form includes information on study design, patient population, and presence of PAD. Two review authors (LR and FCV) will independently extract the data. The data extraction form will be pilot tested on several papers to ensure consistency and that all relevant information is being captured. If necessary, a statistician will review the extraction of data to further ensure quality and reliability. Authors will be contacted for missing data.
In terms of the study results, unadjusted and fully adjusted effect estimates for the association between very low HDL-C and PAD will be recorded. Details of the confounders measured and adjusted for will also be noted. Results of any additional stratified analyses will also be recorded. Where possible, results from additional subgroup analyses with evidence regarding our non-primary objectives will also be recorded, for example, the association between very low HDL-C and the secondary outcomes (PAD).

Assessment of methodological quality
Two investigators (LR and FCV) will independently assess each selected study for study quality using the Newcastle-Ottawa Quality Assessment Scale (NOS). The NOS evaluates cohort studies based on eight items categorized into the following three groups: (1) selection of the study cases, (2) comparability of the population, and (3) ascertainment of whether the exposure or outcome includes any risk of bias (i.e., selection bias or bias from lost to follow-up). The NOS is scored ranging from 0 to 9, and studies with scores ≥ 7 are considered as high quality [16]. Discrepancy of quality assessment among the investigators will be solved by discussion and consensus among all authors.

Data synthesis and statistical analysis
We anticipate that there may be significant heterogeneity in the prevalence of very low HDL-C features of PAD. There are several factors that could contribute to such heterogeneity. The relative risk (RR), and odds ratio (OR) are the way the result will be expressed statistically.
These factors include the following: differences in demographic and clinical features (e.g., age, hypertension, renal disease, smoking, duration and severity of diabetes) among study cohorts; differences in definitions of HDL-C. An I2 statistic will be calculated for the studies to be included in each proposed meta-analysis (i.e. for each neuroradiology correlate of interest) with values of 25, 50, and 75% suggesting low, moderate, or high degrees of heterogeneity, respectively, which report a dichotomized (i.e., present or absent) or categorical (i.e., absent, mild, moderate, severe) shall be harmonized for meta-analysis if deemed appropriate by our statistician. Other types of rating scales shall not be included in a meta-analysis and the data based on any such data scale would be presented in narrative form.
If significant heterogeneity between studies, as determined by consultation with our statistician, prevents meaningful pooling of the data, we will limit ourselves to providing a narrative description of observed trends. Given the heterogeneity of the populations studied, assumption of a fixed effect size across populations would not be justified, thus analyses would be performed using a random effects model. Given the dichotomized (presence or absence) or categorical (severity measure) nature of our data of, meta-analysis will be performed a random effects analysis. We will also add funnel graphs, publication bias analysis and a meta-regression analysis.
If there are sufficient data to allow such analyses (in principle from as few as a single high-quality study, but if possible, by pooling data from multiple studies), we will perform subgroup analyses for participants with renal disease and participants with hypertension. In addition, if enough data are available, we shall perform subgroup analyses by age and diabetes duration. Funding sources and conflict of interest will be extracted from included studies. Statistical analysis will be performed using RevMan software.

Strategy for data synthesis
The data of interest presented as continuous (mean value and SD) will be used to perform meta-analysis to obtain the standardized mean difference (SMD) and 95% confidence interval (CI). Cocharn's Q-statistic and I-squared test will be used to test for heterogeneity between the included studies. If a I-squared value will be greater than 50% or a p value of the Q-test will be less than 0.05, indicating maximal heterogeneity among the included studies, a random-effect model will be put into use. A sensitivity analysis based on multivariate metaanalysis and multivariate meta-regression will be performed if data allow.

Analysis of subgroups or subsets
The subgroup meta-analyses will be conduct according to the prespecified study-level characteristics using a fixed-effects meta-analysis and if there is substantial heterogeneity, we will use the random effects model. The sources included location, sex, age, method of HDL-C assessment, the definition of PAD. We also will conduct sensitivity analyses to evaluate the potential sources of heterogeneity in the analyses.

Summary of evidence
We will produce a narrative synthesis of the main results extracted from articles in full text. A summary of the included studies will provide information on the authors, study design, participants, number and age of the subjects, theoretical structure (if relevant), alcohol consumption (as primary outcome of interest), main findings, Study information. Special emphasis will be placed on the identification of very low of HDL-C and the risk of PAD. In the presentation of the results, we will try to separate the factors for which the evidence of causality is strong (from longitudinal studies) and factors for which the causal nature of the relationship is less secure (cross-sectional data). A graphical summary of all the data they represent will be provided and consider the number of studies that provide evidence of a factor and the relative strength of the association presented based on study design and quality assessment. The membership level will be evaluated based on adjusted data

Discussion
This systematic review will synthesize research evidence to establish whether the risk of developing PAD is relatively high in adults with very low HDL-C. Strengths and limitations will be highlighted in the identified evidence. Strength of observational data may include large sample size, high rate of follow-up and frequency of PAD more likely to be representative of the population at risk. Limitations may include the quality of data extracted which may not allow studies to be combined in a meta-analysis. This may be overcome by presenting the findings in a descriptive manner. This review will be conducted in collaboration with an experienced librarian who helped appraise the search criteria, refine the keywords and MeSH terms and identify appropriate database(s). To the best of our knowledge, no reviews have been published exploring the study question; however, if a review addressing a similar question is published, it will be incorporated in this review and added in a meta-analysis if feasible.

Study design
If it is necessary, we will update this protocol in the future. We will submit the original protocol, final protocol and summary of changes as a supplement

Implications of results
This systematic review will provide an updated and quantifiable estimate of the risk of PAD in adults with low HDL-C. Furthermore, the systematic search will identify where future research is required. For instance, this review may inform a prognostic study which may be useful in understanding the course and factors associated with PAD development.

Amendments
If it is necessary, we will update this protocol in the future. We will submit the original protocol, final protocol and summary of changes as a supplement.

Ethics and dissemination Ethical issues
No ethical approval is required because this study includes no confidential personal data or interventions with the patients.

Publication plan
The procedures of this systematic review and NAM will be conducted in accordance with the PRISMA-compliant guideline. The results of this systematic review will be submitted to a peer-reviewed journal for publication. study idea and devised the study methodology. LR, ASRB, ACPC and ESR participated in the design and coordination of the study. LR was primarily responsible for protocol writing and developed the search strategy. LR and FCV will screen identified literature, conduct data extraction and analyses the review findings. All authors read the drafts, provided comments and agreed on the final version of the manuscript.