Expression of Pro- and Anti-inflammatory Cytokines During Anti-PCSK9 in Familial Hypercholesterolemia

Study Design Non-randomized, non-controlled, before-after comparison, quasiexperimental, single-center study.

Study Population Patients older than 18 years, with diagnosis statin-resistant FH (SR-FH), who were attended at the Cardiology Department, Centro Médico Nacional "20 de Noviembre ISSSTE", Mexico City. SR-FH was defined as symptomatic cardiovascular disease accompanied by LDL-C concentration higher than 160 mg/dL despite maximally tolerated statin dose (Ito MK, McGowan MP, Moriarty PM. Management of familial hypercholesterolemias in adult patients: recommendations from the National Lipid Association Expert Panel on Familial Hypercholesterolemia. J Clin Lipidol. 2011;5(3 Suppl):S38-S45. doi: 10.1016/j.jacl.2011.04.001.). We excluded patients with infections, neoplasia or under oncological therapy. The study was approved by the Research, Biosafety and Ethics Committee of the Centro Médico Nacional 20 de Noviembre ID 061.2019.

Clinical-Demographic characteristics Clinical-demographic characteristics like age, gender, past diseases and medication use were collected from a direct interview and verified through the medical record. Body mass index (BMI) was calculated as weight/height2 (kg/m2). Blood pressure was obtained while the patient was in a seated position and was considered to be the mean of three readings taken 5 min apart using a Welch Allyn 767 mobile aneroid sphygmomanometer (Welch Allyn Inc.; Skaneateles Falls, NY, USA). Diabetes Mellitus was defined according to the guidelines from the American Diabetes Association with one of the following conditions (repeated for confirmation at a separate date): 1) Hb A1C ≥ 6.5%; 2) fasting glucose ≥ 126 mg/dl; or 3) 2-h plasma glucose ≥ 200 mg/dl during an oral glucose tolerance test.

Complete Blood Count and Lipid Profile Following a 12-h fast, venous blood samples (4 ml) were collected into BD Vacutainer® tubes (Becton, Dickinson \& Co., Franklin Lakes, NJ, USA) containing 1.8 mg ethylenediaminetetra-acetic acid/ml of blood. Standard blood cell count was performed immediately. For clinical chemistry blood simple was centrifuged at 1 200 g for 5 min, at 4°C, separated into fractions and plasma was stored at -80°C prior to analyses. Blood cell count and plasma triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined using routine clinical laboratory equipment and a standard auto-analyser (Synchron CX®9 PRO Clinical System; Beckman Coulter, Brea, CA, USA). Atherogenic index was calculated as log triglyceride / HDL-C.

Flow Cytometry Blood sample was processed following standardized protocol; then, centrifuged to obtain leucocyte population. After three washes, cells were fixed in 4% paraformaldehyde and stored for 12 hours. Flow cytometry analysis (MACSQuant Analyzer 10 - Miltenyi Biotec) was performed in a total of 1,000,000 events collected for each analysis, and corresponding isotype controls were used to set appropriate regions. Cells subpopulations were identified by their specific marker combination (CD14+, CD163+ for M1-macrophages; CD14+, CD206+ for M2-macrophages; CD16+, CXCR4 receptor and CD16+, CXCR2 for monocytes.

Plasma Soluble Biomarkers An aliquot of blood sample was immediately centrifuged at 5000rpm/5 min and plasma was collected. Analytes (fraktaline, IL-1, IL-4, IL-6, IL-8, IL-10, MCP-1 and TNF-alpha), using commercially available multiplexing assay (HCYTOMAG-60-08K, MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay Merc, Millipore Massachusetts, USA), and following manufacturer instructions; and read in Multiplex reader (MAGPIX System, Millipore, Austin, Texas).

Follow up and Study Endpoints Follow-up was conducted after 6 months of therapy, through programmed medical evaluations and blood analyses. Primary endpoint consisted of modification in: 1) serum lipids; while secondary end points included: 1) pro-inflammatory mediators (neutrophils, lymphocytes, NtLR, soluble pro-inflammatory cytokines); and 2) atherogenic index.

Statistical analysis

Quatitative data were resumed as mean ± SD; while categorical data as n(%).One-way T-test was applied. Statistical significance was considered if p <0.05.