Cardiovascular disease (CVD) correlates with inflammation and a reduction in circulating endothelial progenitor cells (cEPCs). Recently, CVD was shown to be the main cause of mortality in individuals with type 1 diabetes (T1DM). In animals, miR-342 was shown to exert an anti-inflammatory effect in CVD. Hypothesis: miR-342-3p/-5p are downregulated in subclinical CVD (T1DM), whereas inflammatory cytokines are upregulated. We studied miR -342 -3p/5p in plasma/peripheral blood mononuclear cells (PBMCs) in 29 T1DM and 20 controls (HC). Vascular health was measured by fibronectin adhesion assay (FAA), cEPCs (CD45dimCD34+133+ cells) and by assessing inflammation and tissue inhibition of metalloproteases (TIMP-1). In T1DM IL-7, IL-8, TNFα and VEGF-C were increased in plasma. MiR-342-3p/-5p were downregulated in PBMCs in T1DM, but not in plasma. PANX2, chemokine receptors CXCR1/2 mRNAs, were increased in PBMCs in T1DM. MiR-342-3p was negatively correlated with TIMP-1, IL-6, IL-8, TNF-α, HbA1c and CXCR2, whilst miR-342-5p was negatively correlated with TIMP-1, IL-6, IL-8 and HbA1c. There was a positive correlation among miR-342-3p, FAA and cEPCs, and between miR-342-5p and cEPCs. ROC curve analyses showed significant downregulation of miR-342-3p/-5p at HbA1c > 46.45 mmol/mol, indicating their potential as biomarkers for subclinical CVD. Our findings validated animal studies and confirmed the proangiogenic properties of miR-342-3p/-5p. MiR-342-3p/-5p-based intervention or monitoring may prove to be beneficial in managing CVD.

Background Type 1 diabetes (T1DM) is associated with premature cardiovascular disease (CVD) and a pro-inflammatory state whilst the proangiogenic miR-126-3p/-5p may play a role in CVD. Animal studies established miR-126 to be pro-angiogenic. We hypothesised miR-126-3p/-5p are reduced in T1DM whilst pro-inflammatory cytokines are increased. Methods 29 well controlled, T1DM patients without CVD and 20 healthy controls (HCs) were studied. MiR-126-3p/-5p were assayed in plasma and peripheral blood mononuclear cells (PBMCs) whilst Chemokine C-X-C Receptor 1/2 (CXCR1/2) mRNA in PBMCs by real-time quantitative PCR. Cytokines were assayed by the Mesoscale Discovery. Ingenuity Pathway Analysis (IPA) was used to predict target genes, cellular functions and pathological states regulated by miR-126-3p/-5p. IPA generated both direct and indirect causations between different targets and analysed whether these effects would be inhibitory or stimulatory based on the published evidence. Results T1DM patients had a relatively good diabetic control (HbA1c = 7.4 ± 0.7% or 57.3 ± 7.6 mmol/mol). Homeostatic cytokine IL-7, pro-inflammatory cytokines IL-8 and TNF-α, and vascular endothelial growth factor-C (VEGF-C) were increased in T1DM, versus HCs; p = 0.008, p = 0.003, p = 0.041 and p = 0.013 respectively. MiR-126-5p was significantly upregulated in PBMCs in T1DM versus HCs; p = 0.01, but not in plasma. MiR-126-3p was unchanged. CXCR1/2 were elevated in T1DM versus HCs; p = 0.009 and p < 0.001 respectively. MiR-126-5p was positively correlated with CXCR1/2, and with HbA1c whilst negatively correlated with circulating endothelial progenitor cells (CD34+CD133+CD45dim) and fibronectin adhesion assay in a combined group of T1DM patients and HCs; p = 0.028 p = 0.049 p = 0.035 p = 0.047 and p = 0.004 respectively. IPA predicted miR-126-5p to be anti-inflammatory through the inhibition of chemokine C–C motif ligand 27, chymotrypsin-like elastase 2A and IL-7, whilst miR-126-3p had no direct anti-inflammatory effect. Simultaneously IPA predicted IL-7 as the most upstream cytokine target. Conclusions T1DM without apparent CVD or diabetic complications is an inflammatory state characterised not only by raised pro-inflammatory cytokines but also by increased receptor CXCR1/2 and miR-126-5p. MiR-126-5p upregulation may represent a compensatory response. Pro-miR-126-5p therapies or anti-IL-7 therapies may be a new option to reduce both inflammation and CVD risk in T1DM. Further research is required in a large prospective study in patients with T1DM.

Background In spite of clinical progress, cardiovascular disease (CVD) remains the predominant cause of mortality worldwide. Overexpression studies in animals have proven miR-424-5p to have anti-angiogenic properties. As type 1 diabetes mellitus (T1DM) without CVD displays endothelial dysfunction and reduced circulating endothelial progenitor cells (cEPCs), it offers a model of subclinical CVD. Therefore, we explored miR-424-5p, cytokines and vascular health in T1DM. Methods Twenty-nine well-controlled T1DM patients with no CVD and 20-matched controls were studied. Cytokines IL8, TNF-α, IL7, VEGF-C, cEPCs/CD45dimCD34+CD133+ cells and ex-vivo proangiogenic cells (PACs)/fibronectin adhesion assay (FAA) were measured. MiR-424-5p in plasma and peripheral blood mononuclear cells (PBMC) along with mRNAs in PBMC was evaluated. Results We found an elevation of IL7 (p = 0.008), IL8 (p = 0.003), TNF-α (p = 0.041), VEGF-C (p = 0.013), upregulation of mRNA CXCR1 (p = 0.009), CXCR2 (p < 0.001) and reduction of cEPCs (p < 0.001), PACs (p < 0.001) and FAA (p = 0.017) in T1DM. MiR-424-5p was upregulated in T1DM in PBMC (p < 0.001). MiR-424-5p was negatively correlated with cEPCs (p = 0.006), PACs (p = 0.005) and FAA (p < 0.001) and positively with HbA1c (p < 0.001), IL7 (p = 0.008), IL8 (p = 0.017), VEGF-C (p = 0.007), CXCR1 (p = 0.02) and CXCR2 (p = 0.001). ROC curve analyses showed (1) miR-424-5p to be a biomarker for T1DM (p < 0.001) and (2) significant upregulation of miR-424-5p, defining subclinical CVD, occurred at HbA1c of 46.5 mmol/mol (p = 0.002). Conclusion We validated animal research on anti-angiogenic properties of miR-424-5p in T1DM. MiR-424-5p may be a biomarker for onset of subclinical CVD at HbA1c of 46.5 mmol/mol (pre-diabetes). Thus, miR-424-5p has potential use for CVD monitoring whilst anti-miR-424-5p-based therapies may be used to reduce CVD morbidity/mortality in T1DM.

Abstract BACKGROUND Cardiovascular disease (CVD), the leading causes of death worldwide, correlates with inflammation and shortens life span in type 1 diabetes (T1DM). In animals, miR-342 were shown to be anti-inflammatory in CVD. We hypothesise: proangiogenic miR-342-3p/-5p are downregulated in T1DM whereas inflammatory cytokines are upregulated.METHODS We studied plasma/peripheral blood mononuclear cells (PBMCs) in 29 T1DM and 20 controls (HC). Vascular health was measured by Fibronectin Adhesion Assay (FAA), CD45 dim CD34 + 133 + cells (flow cytometry) and Tissue inhibitor of metalloproteases (TIMP-1). Inflammatory markers IL-7, IL-8, TNFα and VEGF-C were assayed by Mesoscale assay, mRNA for chemokine receptor CXCR1/2, PANX2 and miR 342-3p/5p by qRT-PCR. Analysis: Unpaired Student t test, Pearson correlation and Ingenuity Pathway Analysis (IPA) to predict miR-342-3p/-5p targets.RESULTS In T1DM, pro-inflammatory cytokines IL-8 and TNF-α, IL-7 and growth factor VEGF-C were increased versus HCs; p=0.008, p=0.003, p=0.041 and p=0.013 respectively. MiR-342-3p/-5p were downregulated in PBMCs in T1DM versus HC; p=0.01, but not in plasma. PANX2, CXCR1 and CXCR2 mRNA were increased in PBMCs in T1DM versus HCs; p=0.004, p=0.017 and p<0.001 respectively. MiR-342-3p correlated negatively with TIMP-1, IL-6, IL-8, TNF-α, HbA 1c ; p=0.006, p=0.031, p=0.029, p=0.038, p=0.001, p=0.031 whilst miR-342-5p with TIMP-1, IL-6, IL-8, HbA 1c , p=0.005, p=0.034, p=0.029, p=0.001, p=0.042 respectively. There was positive correlation between miR-342-3p and FAA, CXCR1, CXCR2; p=0.006, p=0.019, p=0.001 and between miR-342 - 5p and FAA, CXCR2; p=0.038, p=0.036 respectively. IPA predicted miR-342-3p/-5p to be anti-inflammatory through the indirect regulation of mitogen-activated protein 3 kinase 1, inhibitor of nuclear factor kappa B kinase regulatory subunit Gamma, Surfactant protein A1 and PANX2 as well as predicting IL-8, TNF-α, IL-7 and glucose to activate the inflammatory response. ROC curve analyses showed: miR-342-3p/-5p to be a biomarker for T1DM: p=0.01, p =0.006 respectively (2) significant downregulation of miR-342-3p/-5p has occured at HbA 1c > 46.45 mmol/mol, p=0.006, p =0.005 respectively.CONCLUSION Our findings validated animal studies. Downregulation of miR-342-3p/-5p by hyperglycaemia and increase in cytokines confirmed inflammation in T1DM. MiR-342-3p/5p correlated with indices of vascular health and defined early CVD at HbA1c of 46.5 mmol/mol cut off point. MiR-342-3p/-5p based either intervention or monitoring may prove to be beneficial in CVD in T1DM.