Restenosis following vascular injury remains a pressing clinical problem. Mesenchymal stem cells (MSCs) promise as a main actor of cell-based therapeutic strategies. The possible therapeutic role of MSCs in vascular stenosis in vivo has been poorly investigated so far. We tested the effectiveness of allogenic bone marrow-derived MSCs in reduction of stenosis in a model of rat carotid arteriotomy. MSCs were expanded in vitro retaining their proliferative and differentiation potentiality. MSCs were able to differentiate into adipocyte and osteocyte mesenchymal lineage cells, retained specific antigens CD73, CD90, and CD105, expressed smooth muscle alpha-actin, were mainly in proliferative phase of cell cycle and showed limited senescence. WKY rats were submitted to carotid arteriotomy and to venous administration with 5 x 10(6) MSCs. MSCs in vivo homed in injured carotids since 3 days after arteriotomy but not in contralateral uninjured carotids. Lumen area in MSC-treated carotids was 36% greater than in control arteries (P = 0.016) and inward remodeling was limited in MSC-treated carotids (P = 0.030) 30 days after arteriotomy. MSC treatment affected the expression level of inflammation-related genes, inducing a decrease of IL-1beta and Mcp-1 and an increase of TGF-beta in injured carotids at 3 and 7 days after arteriotomy (P < 0.05). Taken together, these results indicate that allogenic MSC administration limits stenosis in injured rat carotids and plays a local immunomodulatory action.

The reciprocal parent of origin-specific expression of H19 and IGF2 is controlled by the H19/IGF2:IG-DMR (IC1), whose maternal allele is unmethylated and acts as a CTCF-dependent insulator. In humans, internal IC1 deletions are associated with Beckwith–Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), depending on their parental origin. These genetic mutations result in aberrant DNA methylation, deregulation of IGF2/H19 and disease with incomplete penetrance. However, the mechanism linking the microdeletions to altered molecular and clinical phenotypes remains unclear. To address this issue, we have previously generated and characterized two knock-in mouse lines with the human wild-type (hIC1wt) or mutant (hIC1∆2.2) IC1 allele replacing the endogenous mouse IC1 (mIC1). Here, we report an additional knock-in line carrying a mutant hIC1 allele with an internal 1.8 kb deletion (hIC1∆1.8). The phenotype of these mice is different from that of the hIC1∆2.2-carrying mice, partially resembling hIC1wt animals. Indeed, proper H19 and Igf2 imprinting and normal growth phenotype were evident in the mice with maternal transmission of hIC1Δ1.8, while low DNA methylation and non-viable phenotype characterize its paternal transmission. In contrast to hIC1wt, E15.5 embryos that paternally inherit hIC1Δ1.8 displayed variegated hIC1 methylation. In addition, increased Igf2 expression, correlating with increased body weight, was found in one third of these mice. Chromatin immunoprecipitation experiments in mouse embryonic stem cells carrying the three different hIC1 alleles demonstrate that the number of CTCF target sites influences its binding to hIC1, indicating that in the mouse, CTCF binding is key to determining hIC1 methylation and Igf2 expression.

Obesity is a pathophysiological disorder associated with adiposity accumulation, oxidative stress, and chronic inflammation state that is progressively increasing in younger population worldwide, negatively affecting male reproductive skills. An emerging topic in the field of male reproduction is circRNAs, covalently closed RNA molecules produced by backsplicing, actively involved in a successful spermatogenesis and in establishing high-quality sperm parameters. However, a direct correlation between obesity and impaired circRNA cargo in spermatozoa (SPZ) remains unclear. In the current work, using C57BL6/J male mice fed with a high-fat diet (HFD, 60% fat) as experimental model of oxidative stress, we investigated the impact of HFD on sperm morphology and motility as well as on spermatic circRNAs. We performed a complete dataset of spermatic circRNA content by a microarray strategy, and differentially expressed (DE)-circRNAs were identified. Using a circRNA/miRNA/target network (ceRNET) analysis, we identified circRNAs potentially involved in oxidative stress and sperm motility pathways. Interestingly, we demonstrated an enhanced skill of HFD sperm in backsplicing activity together with an inefficient epididymal circRNA biogenesis. Fused protein in sarcoma (FUS) and its ability to recruit quaking (QKI) could be involved in orchestrating such mechanism.

Background Peripheral artery disease (PAD) is highly prevalent in people with type 2 diabetes and associates with chronic limb ischemia and poor prognosis. Understanding the mechanisms of impaired blood vessel growth in diabetic patients is of paramount importance to develop new angiogenic therapies in this setting. Dysregulation of epigenetic mechanisms of gene transcription in vascular cells contributes to cardiovascular disease development but is currently not targeted by therapies. Apabetalone (RVX-208) – an FDA approved small molecule inhibitor of the epigenetic readers bromodomain and extra-terminal (BET) proteins – has recently shown to modulate transcriptional programs implicated in vascular inflammation and atherosclerosis. Purpose To investigate RVX-208 effects in modulating angiogenic response and post-ischemic vascularization in diabetes. Methods Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) for 48 hours in presence of RVX-208 (20μM) or vehicle (DMSO). Scratch and tube formation assays were performed to investigate the impact of RVX-208 on angiogenic properties of HAECs. T1D mice (streptozotocin-induced diabetes) and T2D mice (Lepdb/db) were orally treated with apabetalone or vehicle for 5 days. Hindlimb ischemia was induced in T1D mice & blood flow recovery analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Sprouting and matrigel plug assays were performed in Lepdb/db mice. Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings. Results HG impaired HAECs migration and tube formation as compared to NG, whereas treatment with RVX-208 rescued HG-induced impairment of angiogenic properties. Real time PCR arrays in HG-treated HAECs showed that RVX-208 treatment prevents the dysregulation of genes implicated in endothelial migration, sprouting and inflammation, namely the anti-angiogenic molecule thrombospondin (THBS1), VEGF-A, IL-1β, IL-6, VCAM-1, and CXCL1. Of interest, both gene silencing of BET protein (BRD4) or its pharmacological inhibition by RVX-208 reduced THBS1 expression while restoring VEGFA levels in HG-treated HAECs. ChIP assays showed the enrichment of both BRD4 and the active chromatin mark H3K27Ac on THBS1 promoter. Mechanistic experiments uncovered the inhibitory role of THBS1 on VEGFA signalling, as also confirmed by STRING analysis. Treatment of T1D mice with RVX-208 improved blood flow reperfusion and vascular density at 14 days as compared to vehicle-treated animals. Moreover, RVX-208 restored endothelial sprouting in T2D-Lepdb/db mice. Of clinical relevance, THBS1 was upregulated while VEGFA expression was reduced in gastrocnemius muscle specimens from T2D patients with PAD as compared to non-diabetic controls. Conclusion In vivo targeting of BET-proteins by RVX-208 may represents a novel therapeutic approach to boost post-ischemic neovascularization in diabetes. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich