Posttranscriptional Regulation of 14q32 microRNAs by RNA Binding Proteins CIRBP and HADHB during Vascular Regeneration after Ischemia:Posttranscriptional regulation of 14q32 microRNAs

After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the microRNA genes’ order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32, and we aimed to identify these RBPs. To identify proteins responsible for this posttranscriptional regulation, we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of cold-inducible RBP (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) to the precursors of late-upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hindlimb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329 but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB−/− 3T3 cells, which display reduced expression of miR-329 and miR-495 but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs. Keywords: microRNA, 14q32, microRNA cluster, miR-329, miR-495, HADHB, CIRBP, RNA binding proteins, ischemia, hindlimb ischemia mode

CCR7-CCL19/CCL21 Axis is Essential for Effective Arteriogenesis in a Murine Model of Hindlimb Ischemia

Craipeau Maria. Le conformiste Metello Les clowns Les faucons Five Easy Pieces Performance. In: Raison présente, n°19, Juillet – Août – Septembre 1971. Télévision, pouvoir et liberté. pp. 115-116

Protease-activated receptor (PAR)2, but not PAR1, is involved in collateral formation and anti-inflammatory monocyte polarization in a mouse hind limb ischemia model

In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model. PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered. PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patient

Revascularization upon ischemia is PAR2-dependent.

<p>(A) SMA staining was performed to detect collaterals in the ischemic adductor muscles and subsequently quantified. Mean collateral number in WT mice was compared to PAR1-/- mice (B) and PAR2-/- mice. (C) Capillary density was quantified after CD31 staining of ischemic calf muscles. Mean capillary number of WT was compared to PAR1-/- mice (D) and PAR2-/- mice. (E) Images represent the CD31 and SMA stainings of ischemic hind limbs of WT, PAR1-/- and PAR2-/- mice. Arrowheads indicate vessels. Bar, 100 µm. * p<0.05.</p

Increased accumulation of repair-associated macrophages surrounding collaterals in ischemic hind limbs is PAR2-dependent.

<p>(A) Stainings of CD206-positive macrophages (green) and SMA-positive vessels (red) in non-ischemic (control) and ischemic (ligated) hind limbs of WT, PAR1-/- and PAR2-/- mice are shown. Nuclei were visualized with DAPI (blue). Arrows indicate single macrophages in the non-ischemic adductor. Quantification of the average number of repair-associated macrophages per vessel is indicated on the right. (B) Correlation between the number of CD206-positive macrophages in the ischemic tissues and the expression of CD11b and (C) CD115 on monocytes. ** p<0.01, *** p<0.001.</p

PAR2 mediates blood flow restoration in a hind limb ischemia model.

<p>The average blood perfusion ratio between ischemic and non-ischemic hind limbs was measured with laser Doppler before, directly after ligation, at day 3 and every week in a period of four weeks. Representative images of laser Doppler measurements of the ligated hind limbs are shown. (A) The ratio measured in PAR1-/- mice (n = 10) compared to the ratio measured in WT mice (n = 9). (B) The blood perfusion in PAR2-/- mice (n = 7) was compared to the ratio measured in WT mice (n = 10). * p<0.05, ** p<0.01, *** p<0.001.</p

Representative images of the pial circulation in PAR2^-/- (A) and WT mice (B).

<p>To illustrate, several, but not all collateral arterioles have been indicated with white asterisks. Following exsanguination and maximal dilation of the dorsal cerebral circulation, Microfil™ was used as a casting agent, after which the whole brain was fixated in 4% PFA and subsequently stained in Evans Blue for contrast. (C) Pial collateral density was calculated in PAR2^-/- and WT mice by dividing the sum of collaterals between anterior, middle and posterior cerebral arteries by the surface area of the cerebral hemispheres. Areas were excluded when they were damaged, had poor filling with Microfil™, or were otherwise uncountable. All values are presented as the mean ± SEM.</p