Fig 2: IL6 but not IL8 levels correlate with miR34a expression in healthy subjects. The expression levels of miR-34a were evaluated by qRT-PCR and IL6 or IL8 by ELISA in the serum of 20-90 years-old healthy subjects. Scatter plots showing correlation between age and miR-34a (A), age and IL6 (B), miR34a and IL6 (C), age and IL8 (D), miR-34a and IL8 (E). Variables showed a skewness distribution and were log-transformed. r = Pearson’s coefficient; n = 127–128.

Fig 3: Cytokine production in splenocyte co-cultures.Spleen cells were stimulated with LPS in the presence of LET cells and/or retinol. Culture components are indicated below each bar. Cytokines that were reproducibly elevated in the presence of LET cells and retinol are shown, including GM-CSF (Panel A), MCP-1 (Panel B), and IL-6 (Panel C). IFN? levels were reduced in the presence of LET cells and retinol (Panel D). Panel E: IgA levels are shown following splenocyte/LET co-cultures in the presence of LPS and retinol, with and without neutralization using anti-IL-6 and/or anti-GMC-SF antibodies. ‘++’ indicates that antibodies were added to cultures at levels exceeding manufacturers’ recommendations, either 5× (for anti-IL-6 antibodies) or 2× (for anti-GM-CSF antibodies). Panel F: Levels of GM-CSF, MCP-1 and IL-6 are shown in LET cell cultures (without splenocytes) in the presence or absence of LPS.

Fig 4: IL6 levels increase and correlate with miR-34a expression during vascular aging and human aortic smooth muscle cells (HASMCs) senescence. (A) IL6 expression in aortas of 2.5-month-old (young) and 21-month-old (old) mice analyzed by qRT-PCR and normalized to HPRT levels. Values represent the means ± SD. *, p <0.05; Mann Whitney test; n = 5 young, 3 old mice. (B) Correlation analysis between miR-34a and IL6 levels in mice aortas. r = Pearson’s coefficient; n = 5 young (red), 3 old (black) mice. (C,D) The miR-34a and IL6 mRNA expression in replicative young human aortic smooth muscle cells (HASMCs), isolated from different donors of indicated age (year-old; yo), was evaluated by qRT-PCR and normalized to corresponding U6 and GAPDH levels, respectively. Correlation analysis between (C) age (Age; years) and miR-34a or IL6 mRNA levels and (D) miR-34a and IL6 mRNA levels. r = Pearson’s coefficient; n = 3 donors. (E,F) miR-34a and IL6 mRNA expression in HASMCs isolated from donors of indicated age (year-old; yo) at young replicative (P5) and senescent (P15) passages evaluated by qRT-PCR and normalized to corresponding U6 and GAPDH levels, respectively. *, p <0.05; paired t test; n = 3 donors.

Fig 5: IL6 expression in Mir34a+/+ and Mir34a-/- mice early after vitamin D treatment. Twelve-week-old Mir34a+/+ and Mir34a-/- mice were treated subcutaneously with either vitamin D (vit D) or a mock solution (Ctrl) for three consecutive days and sacrificed 3 days after the first injection (Day 3). (A) IL6 expression was analyzed by qRT-PCR and normalized to HPRT levels in the kidney and abdominal aorta. Values are mean ± SD. ** p < 0.01, *** p < 0.001; 1-way ANOVA with a Bonferroni post hoc test; n = 6, 5, 6–7 and 4–5. (B) Representative images of thoracic aorta sections stained for IL6 expression with a specific antibody. Bar = 20 µm. (C) Bars show quantification of the percentage of IL6 positive area to the total thoracic aortic area relative to (B). Values are mean ± SD; ***, p < 0.001; 1-way ANOVA followed by Bonferroni’s multiple comparison test; n = 4, 4, 4 and 3.