Mendelian Randomization Analysis
This study employed the TSMR approach, considering COPD as the exposure factor and both stroke and myocardial infarction as outcome variables to establish causal links. Data related to COPD, ischemic stroke (IS) [27], and acute myocardial infarction (AMI) were respectively sourced from FinnGen Biobank, MEGASTROKE, and the MRC-IEU organizations (Table 1).
Table 1
Characteristics of data sources and strength of IVs used in the Mendelian randomization study
Exposures/Outcomes | GWAS ID | Consortium | Ethnicity | Sample Sizes | Number of SNPs | Year |
COPD | finn-b-J10_COPD | FinnGen Biobank | European | 193,638 | 16,380,382 | 2021 |
Ischemic stroke | ebi-a-GCST006907 | MEGASTROKE | European | 150,765 | 8,418,349 | 2018 |
Acute myocardial infarction | ukb-b−3469 | MRC-IEU | European | 463,010 | 9,851,867 | 2018 |
GWAS:Genome-Wide Association Studies,COPD:Chronic obstructive pulmonary disease,SNPs: single nucleotide polymorphisms, NA: Not Available. |
To conduct the analysis, we compiled single nucleotide polymorphisms (SNPs) associated with COPD at a significant level of P < 5 × 10− 6. In order to ensure the independence of these SNPs, we established a linkage disequilibrium threshold (r2) of 0.01 and a genetic distance of 5000 kb for the selection process. We identified the phenotypes associated with the remaining SNPs using the Human Genotype-Phenotype Association Database (www.phenoscanner.medschl.cam.ac.uk), [28] with a special emphasis on excluding SNPs whose corresponding phenotypes were significantly associated with the outcomes. The datasets for both the exposure and outcome variables were combined, while palindromic SNPs were removed to ensure the integrity of the analysis. In the meanwhile, to mitigate interference from reverse causality, reverse MR analysis was employed using SNPs related to IS and AMI.
Mice
8-week-old male SPF grade ApoE knockout mice (ApoE−/−) were purchased from the Experimental Animal Center of Hangzhou Medical College in Zhejiang, China (Licence Number: SYXK(Zhe)2019-0011) and housed in a specific pathogen-free (SPF) facility in a controlled environment. All experimental protocols were approved by the Zhejiang Provincial Experimental Animal Center Animal Welfare Ethics Committee (Ethics Number: ZJCLA-IACUC-20030066). All methods were carried out in accordance with relevant guidelines and regulations. All animal experiments were taken place in SPF Animal Laboratory at Zhejiang Chinese Medical University. The mice were randomly divided into the following groups:
Control group: Mice were given a normal diet, and equivalent doses of saline were instilled during the PPE and LPS induction for the COPD model.
COPD group: Mice were instilled with 1.2 IU of PPE via the airway once a week for a total of 4 times. Two weeks after the final PPE instillation, 200 µg of LPS was instilled once a week, for a total of 2 times.
HFD group: To establish the AS model, mice were fed a HFD (comprising 3% cholesterol, 0.5% sodium cholate, 0.2% propylthiouracil, 5% sugar, 10% lard, and 81.3% base diet) for 7 weeks.
HFD + COPD group: Alongside initiating HFD, COPD interventions were given as well. Mice received 1.2 IU of PPE instilled via the airway once a week for a total of 4 times. One week after the last PPE instillation, 200 µg of LPS was instilled once a week for a total of 2 times.
HFD + COPD + BAY11-7082 group: While establishing the combined COPD and AS model, mice were given intraperitoneal injections of the NF-κB inhibitor BAY11-7082 (HY-13453, MedChemExpress) (5 mg/kg) on the same day as the PPE instillation. Subsequently, injections were given every other day until the end of the experiment.
HFD + COPD + NS-398 group: While establishing the combined COPD and AS model, mice were given intraperitoneal injections of the COX-2 inhibitor NS-398 (HY-13913, MedChemExpress) (5 mg/kg) on the same day as the PPE instillation. Subsequently, injections were given every other day until the end of the experiment.
Cells
Primary mouse aorta endothelial cells were used for this study. The aortic endothelial cells were then purified using anti-CD31 coupled magnetic beads. [29] (Fig. 5A-B) Simultaneously, serum was extracted from both the COPD group of mice and the control group of mice. The cells were randomly divided into the following groups:
Control group: cells received no special treatment.
NS group: cells received serum from the control group mice.
CS group: cells received serum from COPD mice.
CS + BAY11-7082 group: cells received BAY11-7082 while receiving serum from COPD mice.
CS + BAY11-7082 + oe-NC group: cells received serum from COPD mice, BAY11-7082, and transfected empty plasmid simultaneously.
CS + BAY11-7082 + oe-COX-2 group: cells received serum from COPD mice, BAY11-7082, and transfected COX-2 expressing plasmid (purchased from Santa Cruz Biotechnology) simultaneously.
Pulmonary Function Test
Mice were anaesthetized by intraperitoneal injection using a 3% pentobarbital sodium solution. After the tracheal cannula was connected to the airway of the plethysmograph (PFT, DSI), the measurements of functional residual capacity (FRC), resistance index (RI), dynamic lung compliance (Cdyn), and minute ventilation (MV) were taken. At the end of these experiments, all mice were euthanized by CO2 asphyxiation.
Mice Lipid Profile
The following mouse kits were used: Total Cholesterol (TC), Triglyceride (TG), High-Density Lipoprotein Cholesterol (HDL-C), and Low-Density Lipoprotein Cholesterol (LDL-C) (A111-1-1, A110-1-1, A112-1-1 and A113-1-1, Nanjing Jiancheng Bioengineering Institute). These serum samples then underwent testing on a fully-automated biochemistry analyser (COBAS INTEGRA 800, Roche, Switzerland).
Mice Tissue
The lung tissue and aortic roots of the different groups of mice were evaluated using Hematoxylin and Eosin (H&E) (C0105S, Beyotime) staining to assess the pathological changes in lung tissues and the size of arteriosclerotic plaques. Additionally, Oil Red O (G1015-100ML, Servicebio) staining was employed to evaluate lipid accumulation in the aorta.
Cells Functions
Cells were seeded in a 96-well plate, treated with CCK-8 (CA1210, Solarbio) solution, and absorbance at 450 nm was measured to determine viability. Apoptosis was evaluated using flow cytometry (Attune™ NxT, Thermo Fisher Scientific), where cells were stained with Annexin V-FITC (C1062S, Beyotime) and Propidium Iodide (PI), and apoptotic cells were detected based on fluorescence microscope (M205 FCA, Leica). Cells were seeded in Matrix-Gel™ chambers, cultured, and stained with crystal violet for migration assessment. Tubule formation potential was investigated by starving cells, seeding them in Matrigel-coated wells, and observing the formation of tube networks.
RT-qPCR
Total RNA was extracted from the cells using TRIzol reagent. cDNA was then synthesized using PrimeScript reverse transcriptase following the manufacturer's protocol. The amplified products were COX-2 forward primer: 5'-AGGACTCTGCTCACGAAGGA-3', COX-2 reverse primer: 5'- TGACATGATTGGAACAGCA-3', GAPDH forward primer: 5'-ACCCTTAAGAGGATGCTGC-3', and GAPDH reverse primer: 5'- CCCAATACGGCCAAATCCGT-3'. The gene expression levels were quantified using the 2^-ΔΔCQ method and normalized to the internal reference gene GAPDH.
ELISA
The relevant inflammatory factors in the mice aortic tissues, serum, and cells from each group were detected using enzyme-linked immunosorbent assay (ELISA) kits for IL-6, IL-1β, TNF-α, PGE2, MCP-1, VCAM-1, and ICAM-1(SEKM-0007, SEKM-0002, SEKM-0034, SEKM-0173, SEKM-0108, SEKM-0037, SEKM-0132). Analyses were performed according to the kit instructions.
Western Blot
Proteins were extracted from the aortic tissues of various mouse groups and from the cells of each group using RIPA lysis buffer (D3910201, Sigma) supplemented with 1% Phenylmethanesulfonyl fluoride (PMSF). Proteins were then separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), with an equal amount of protein (30µg) loaded per lane, and subsequently transferred onto PVDF membranes (IPVH00010, Millipore). These membranes were incubated overnight at 4°C with antibodies diluted at 1:3000 for p-p65, p65, COX-2, caspase 3, Bcl-2, Bax, and GAPDH (3033, 8242, 4842, 9662, 3498, 2772 and 5174, CST). This was followed by a 1-hour incubation with Horseradish Peroxidase (HRP) conjugated secondary antibodies. Densitometric analysis was conducted using ImageJ software.
4. Statistical Analysis
We utilized the software packages "TwoSampleMR" and "MRPRESSO" with R version 4.3.1 to conduct MR analysis. We utilized several methods including inverse variance weighting (IVW), [30, 31] MR-Egger, [32, 33] Simple mode, weighted mode and weighted median method. [34] The IVW method was used as the primary analysis. We assessed heterogeneity by employing Cochran’s Q method, considering heterogeneity to be significant at p < 0.05.[35] We utilized the MR-Egger intercept test [33] and the MR-PRESSO global test to identify and remove any potential outliers with horizontal pleiotropic effects that might have significantly influenced the estimation results. [36]
This study utilized SPSS 22.0 software for the in vivo and in vitro experiments statistical analysis. Quantitative data were assessed for normality and presented as the mean ± standard deviation (x̅ ± SD). The T test was employed to analyse differences between groups, and statistical significance was determined when P < 0.05. Graphs were generated using GraphPad Prism 8.0 (GraphPad, USA).