Hypoxia-induced miR-210 modulates inflammatory response and fibrosis upon acute peripheral ischemia
Authors: Zaccagnini G , Greco S , Longo M , Maimone B , Voellenkle C , Fuschi P , Carrara M , Creo P , Maselli D , Spinetti G , Tirone M , Mazzone M , Martelli F 
Published: November 2020

Abstract

Introduction
We previously demonstrated in mouse models of peripheral and heart ischemia that miR-210 is necessary and sufficient to stimulate blood perfusion recovery, as well as arteriolar and capillary density increase following ischemic damage.

Aim
To clarify the role of inflammatory cells in miR-210-induced angiogenesis.

Methods and results
In a mouse model of acute limb ischemia, miR-210 loss-of-function was obtained by administration of complementary anti-miR-210 LNA oligonucleotides (anti-210), while doxycycline-inducible miR-210 transgenic mice (Tg210) were used for gain-of-function experiments. Transcriptomic and gene ontology analysis in ischemic gastrocnemius muscles upon miR-210 blocking displayed an enrichment of categories related not only to angiogenesis, but also to inflammation, suggesting that miR-210 decrease prompted a pro-inflammatory and anti-regenerative response. Accordingly, immunofluorescence staining of ischemic muscles of anti-210 treated mice, showed an increased presence of granulocytes (Scr = 28±7, anti-210 = 108±17 cells/mm2, p&lt;0.001), T and B lymphocytes (Scr = 32±8 SE, anti-210 = 112±19 cells/mm2, p&lt;0.003; Scr = 45±10, anti-210 = 103±14 cells/mm2, p&lt;0.006, respectively) and macrophages (Scr = 17±1, anti-210 = 27±4 cells/mm2; p&lt;0.03), with a higher M1/M2 macrophage ratio (Scr = 0.6±0.1, anti-210 = 1.7±0.3; p&lt;0.02). Conversely macrophages (WT = 17±2, Tg210 = 5±1 cells/mm2, p&lt;0.003) and M1/M2 ratio (WT = 1.0±0.1, Tg210 = 0.3±0.1, p&lt;0.02) were decreased in ischemic TG-210 mice. To clarify the role of inflammatory cells in miR-210-induced angiogenesis, bone-marrow (BM) transplantation experiments were performed. Tg210 mice transplanted with WT BM cells (BM-wt/R-Tg210), compared with WT mice transplanted with Tg210 BM (BM-Tg210/R-wt) showed increased blood perfusion (vascularity ratio: BM-wt/R-Tg210 = 0.8±0.1, BM-Tg210/R-wt = 0.6±0.1; p&lt;0.01) and capillary density after ischemia (BM-wt/R-Tg210 = 497±41, BM-Tg210/R-wt = 212±32 cap./mm2; P&lt;0.00001). Thus, miR-210 expression in the muscle and not in the BM-derived cells was crucial for miR-210-stimulated angiogenesis. Interestingly, BM-wt/R-Tg210 mice also showed increased fibrotic areas (sirius red staining: BM-wt/R-Tg210 = 12±2, BM-Tg210/R-wt = 22±3 A.U.; p&lt;0.01), characterized by α-SMA+, vimentin+ and collagen V+ stainings. Fibrotic regions were enriched in cells double-positive for both CD206/α-SMA and CD45/α-SMA, as well as in phospho-Smad3+ cells, suggesting the activation of the TGFβ pathway. In vitro experiments showed higher expression of α-SMA and collagens in TG-210 BM-derived macrophages compared to WT, both in the presence and in the absence of TGFβ (α-SMA: w/o TGFβ 2.3±1.4 fold increase p&lt;0.004; TGFβ+ 11.3±2 fold increase p&lt;0.003).

Conclusions
Collectively, these data show that a miR-210 enriched milieu was sufficient to improve angiogenesis after ischemia. Moreover, a context-dependent regulation by miR-210 of the inflammatory response and of fibrosis were identified.

Funding Acknowledgement
Type of funding source: Public hospital(s). Main funding source(s): Italian Ministry of health: Ricerca Corrente and 5X1000