Dysregulation of micro-RNA 143-3p as a Biomarker of Carotid Atherosclerosis and the Associated Immune Reactions During Disease Progression

Atherosclerosis commonly remains undiagnosed until disease manifestations occur. The disease is associated with dysregulated micro(mi)RNAs, but how this is linked to atherosclerosis-related immune reactions is largely unknown. A mouse model of carotid atherosclerosis, human APOB100-transgenic Ldlr−/− (HuBL), was used to study the spatiotemporal dysregulation of a set of miRNAs. Middle-aged HuBL mice with established atherosclerosis had decreased levels of miR-143-3p in their carotid arteries. In young HuBL mice, early atherosclerosis was observed in the carotid bifurcation, which had lower levels of miR-15a-5p, miR-143-3p, and miR-199a-3p, and higher levels of miR-155-5p. The dysregulation of these miRNAs was reflected by specific immune responses during atheroprogression. Finally, levels of miR-143-3p were 70.6% lower in extracellular vesicles isolated from the plasma of patients with carotid stenosis compared to healthy controls. Since miR-143-3p levels progressively decrease when transitioning between early and late experimental carotid atherosclerosis, we propose it as a biomarker for atherosclerosis. Graphical Abstract Low levels of miR-143-3p in plasma extracellular vesicles can serve as a biomarker for atherosclerosis, and dysregulation of microRNAs is linked to the immune reactions associated with disease progression Supplementary Information The online version contains supplementary material available at 10.1007/s12265-024-10482-1.


Mouse experiments
The human APOB100-transgenic Ldlr tm1Her (HuBL, European mutant mouse archive 09689) strain carries the full-length human APOB100 gene, in which codon 2153 has been converted from glutamine to leucine to prevent the formation of apolipoprotein B48, thus generating only apolipoprotein B100 [1,2].From the same in-house breeding colony, Ldlr tm1Her mice (Jax strain 002207) lacking the human APOB100-transgene, were used for comparing HuBL mice to a model of milder hypercholesterolemia.
The mice were euthanized by a CO 2 overdose and blood was collected by cardiac puncture in ethylenediaminetetraacetic acid-coated tubes.The vasculature was perfused with ice-cold sodium chloride (9 mg/ml) before the organs were collected.The aortic arch was fixed in buffered 4% formaldehyde solution for later pinning and staining with Sudan IV (Sigma-Aldrich), as previously described [3].Microdissected carotid arteries were stored in RNAlater reagent (Thermo Fisher) for RNA isolation.The spleen and iliac lymph nodes were snap-frozen for RNA isolation.The heart was preserved in optimal cutting temperature compound and the proximal part of the aortic root was sectioned using a CryoStar NX50 cryostat (Thermo Fisher).Five µm thick sections were fixed in buffered 4% formaldehyde solution and stained with 0.5% Oil Red O (Sigma-Aldrich).Hematoxylin was used to counterstain the nuclei of the cells.Then, the tissues were mounted with an aqueous mounting medium, and micrographs were acquired using a light microscope (Leica DM LB2).The mediastinal and renal lymph nodes were collected in phosphate-buffered saline with 0.1% bovine serum albumin and 1 mM ethylenediaminetetraacetic acid for flow cytometry.

Cell and plasma analyses
Single-cell suspensions were prepared from spleens and CD3 + T cells isolated by negative selection with antibodies to CD11b, CD16/32, CD45R, and Ter-119 (Dynabeads untouched mouse T cells kit, Invitrogen).Flow cytometry was performed using a fixable near-infrared dead cell stain kit (Thermo Fisher).After Fc-block (anti-CD16/32, BD Biosciences), fluorophore-labeled primary antibodies were used for extracellular staining (Table S2).Samples were acquired on a Cytek Northern Lights 3000 spectral flow cytometer and data were analyzed using FlowJo software (Tree Star).Whole blood, splenocyte, and lymph node single-cell suspensions were analyzed on a Vet animal blood counter (Scil).
Plasma cholesterol and triglycerides were analyzed using enzymatic colorimetric kits (Randox).

RNA isolation, reverse transcription, and quantitative polymerase chain reaction
The small non-coding miRNAs and the coding messenger RNAs were isolated from plasma extracellular vesicles, mouse carotids, lymph nodes, and cells using the mirVana miRNA isolation kit with phenol (Thermo Fisher), following the manufacturer's protocol and adapting the volumes of the lysis binding buffer for each sample: 100 μL for the carotids, lymph nodes, and splenic CD3 + cells, and 300 μL for splenic CD3 -cells.Total RNA quality was analyzed on a BioAnalyzer instrument (Agilent Technologies) and quantified by 260 nm absorbance using a Nanodrop 1000 spectrophotometer (Thermo Fisher).
Reverse transcription was performed with a high-capacity cDNA reverse transcription kit for the mRNA and using the TaqMan advanced miRNA cDNA synthesis kit for the miRNA.The amplification of the cDNA was performed by real-time PCR using TaqMan universal master mix and pre-manufactured primers and probes (assay-on-demand) for the genes of interest and Hprt mRNA, miR-191-5p, and miR-16-5p as internal controls, in a QuantStudio 7 Pro real-time PCR System using the QuantStudio Design & Analysis Software 2.6.0 (all from Applied Biosystems).Data were analyzed using the relative abundance of mRNA or miRNA targets, normalized with the endogenous gene and relative to the control, calculated as follows: Relative quantification (RQ) = 2 −ΔΔCt ; ΔCt (cycle threshold) = Ct (miRNA target) − Ct (endogenous control); ΔΔCt = [ΔCt (for sample) − ΔCt (for the control group)].Amplification of internal controls was performed simultaneously with all samples.

Fig. S1
Fig. S1 Detection of miR-143-3p in extracellular vesicles stratified according to sex and diabetes.(a) miRNAs in extracellular vesicles were isolated from the plasma of patients with advanced atherosclerosis.Levels of miR-143-3p were quantified by real-time PCR and stratified according to sex (Mann-Whitney test, males n=21, females n=7) (b) Levels of miR-143-3p stratified according to type 2 diabetes mellitus (Mann-Whitney test, non-diabetic n=18, diabetic n=10).

Fig. S3
Fig. S3 Flow cytometric phenotyping of aorta-associated lymph nodes.(a) Schematic overview of the experimental setup.(b) Cell counts in the pooled mediastinal and renal lymph nodes.(c) Relative germinal center B cell frequencies.(d-e) Relative fraction and absolute number of plasma cells.(f) Gating strategy for flow cytometry analysis of B cells.(g-h) Number of CD8 + and CD4 + T cells.(i) Relative frequency of CD44 + CD62L -T EM cells among CD4 + T-helper cells.(j) Number of CD4 + CD44 + CD62L -T EM cells.(k-l) Frequency of T EM cells with intermediate and high PD1 expression, respectively.(m) Gating strategy for flow cytometry analysis of T cells.(n) Linear regression between miR-199-3p in splenic CD3 -cells and germinal center B cell frequency, PD1 high T EM cell frequency, and PD1 high effector T EM cell number.Female human APOB100-transgenic Ldlr -/-(HuBL) mice, 11 weeks n=6, 46 weeks n=4, GC=germinal center, EM=effector/memory, PD1=programmed cell death 1, int=intermediate.

Table S1 .
Basic characteristics of patients with advanced carotid atherosclerosis.

Table S3 .
List of assay-on-demand used for real-time PCR.

Table S4 .
Basic characteristics of female HuBL mice.

Table S5 .
Basic characteristics of 52-week-old male mice.