The effect of TNF-α inhibitor treatment on microRNAs and endothelial function in collagen induced arthritis

Abstract

Chronic inflammation causes dysregulated expression of microRNAs. Aberrant microRNA expression is associated with endothelial dysfunction. In this study we determined whether TNF-α inhibition impacted the expression of miRNA-146a-5p and miRNA-155-5p, and whether changes in the expression of these miRNAs were related to inflammation-induced changes in endothelial function in collagen-induced arthritis (CIA). Sixty-four Sprague-Dawley rats were divided into control (n = 24), CIA (n = 24) and CIA+etanercept (n = 16) groups. CIA and CIA+etanercept groups were immunized with bovine type-II collagen, emulsified in incomplete Freund's adjuvant. Upon signs of arthritis, the CIA+etanercept group received 10mg/kg of etanercept intraperitoneally, every three days. After six weeks of treatment, mesenteric artery vascular reactivity was assessed using wire-myography. Serum concentrations of TNF-α, C-reactive protein, interleukin-6, vascular adhesion molecule-1 (VCAM-1) and pentraxin-3 (PTX-3) were measured by ELISA. Relative expression of circulating miRNA-146a-5p and miRNA-155-5p were determined using RT-qPCR. Compared to controls, circulating miRNA-155-5p, VCAM-1 and PTX-3 concentrations were increased, and vessel relaxation was impaired in the CIA (all p<0.05), but not in the CIA+etanercept (all p<0.05) groups. The CIA group had greater miRNA-146a-5p expression compared to the CIA+etanercept group (p = 0.005). Independent of blood pressure, miRNA-146a-5p expression was associated with increased PTX-3 concentrations (p = 0.03), while miRNA-155-5p expression was associated with impaired vessel relaxation (p = 0.01). In conclusion, blocking circulating TNF-α impacted systemic inflammation-induced increased expression of miRNA-146a-5p and miRNA-155-5p, which were associated with endothelial inflammation and impaired endothelial dependent vasorelaxation, respectively.

Fig 1. Circulating concentrations of biomarkers of endothelial function in control, CIA and CIA+etanercept groups.

Circulating concentrations of miR146a-5p (A), miR 155-5p (B), VCAM-1 (C) and PTX-3 (D) in control, CIA, and CIA+etanercept groups. Data presented as mean ± SEM or median (IQR). *p<0.05 versus control, **p<0.001 versus control. ^†p<0.05 versus CIA, ^††p<0.001 versus CIA, using a Kruskal-Wallis test or a two-way ANOVA followed by Tukey post-hoc tests. CIA: collagen-induced arthritis; VCAM-1: Vascular adhesion molecule-1; PTX-3: Pentraxin-3.

Fig 2. The effects of inflammation and TNF-α inhibitor treatment on vessel responses to vasoactive substances.

The cumulative dose-response curves to (A) potassium chloride, (B) phenylephrine (C) acetylcholine and (D) sodium nitroprusside in the second branch of mesenteric arteries, in control, CIA and CIA+etanercept groups. Contractile responses are expressed as a percentage of the KCl maximal response. Relaxation responses are expressed as a percentage of the Phe (10 μM)-induced contraction. Data expressed as means ± SEM. *p<0.01 control and CIA+etanercept versus CIA, ^†p<0.05 CIA+etanercept versus control; using repeated-measures ANOVA followed by Tukey post-hoc tests.

Fig 3. The effects of TNF-α inhibitor treatment on the sensitivity (LogEC₅₀) and maximum responses (E_max) to vasodilatory substances in mesenteric arteries.

The sensitivity (LogEC₅₀) to acetylcholine (A) and sodium nitroprusside (B) and the maximal relaxation response (E_max) to acetylcholine (C) and sodium nitroprusside (D) in the second branch of mesenteric arteries, in control, CIA and CIA+etanercept groups. Data expressed as means ± SEM. **p<0.001 versus control; ^†† p<0.001 versus CIA; using two-way ANOVA and Tukey post-hoc tests.