Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Martin-Luther-Universität Halle-Wittenberg Background and Purpose The accumulation of senescent cells in the vasculature contributes to impaired vascular function over the lifetime. Micro RNAs (miRs) are promising therapeutic targets due to their cell-specific functions. Targeting miR-127-3p emerges as an attractive approach to enhance vascular healing and limit angioplasty-related complications. Here, we focus on elucidating the role of miR-127-3p in vascular smooth muscle cells (VSMC) to characterize their role in cellular function and vascular senescence. Methods Utilizing reverse transcription and qRT-PCR we checked expression levels of miR-127-3p in human and murine aortic VSMCs. After overexpression and downregulation via transfection of pre-miR and antagomiR, we evaluated the influence of miR-127-3p on cellular functions such as proliferation and migration of human (non-) replicative senescent VSMCs. Potential targets were selected via target prediction analysis and verified on mRNA and protein levels. Morphological changes were visualized via high-resolution fluorescence microscopy. Senescence marker expression was checked after transfection by qRT-PCR. Results MiR-127-3p is highly upregulated in femoral artery tissue on day 10 after wire-induced injury in C57Bl6 mice, serving as restenosis model (p<0.001) and aged C57BL/6 mice (3 months vs. 22 months, p<0.0001). Replicative senescent human VSMC exhibit increased miR-127-3p expression (p<0.05). In vitro, miR-127-3p overexpression reduces proliferation in senescent (p<0.001) and non-senescent (p<0.01) cells. It hinders migration in non-senescent cells (p<0.0001) but has a reverse effect in senescent cells (p<0.01). Potential targets affecting cellular functions, inflammatory pathways, and cellular aging were identified as mTOR, AXL, and TEAD3 (p<0.05). Transfection of pre-miR leads to increase in cell size and the acquisition of a flattened morphology of human VSMC. P14arf and laminB1, as senescence-associated genes, showed downregulation after overexpression of miR-127-3p on mRNA level (p<0.05). When VSMCs were stimulated with anti-miR-127-3p, increased mRNA levels of p14arf and laminB1 were detected (p<0.05). Conclusion This study identifies miR-127-3p as a contributor to cellular functions including cellular proliferation and migration and seems to have opposing effects in non- and replicative senescent VSMC. Further research is needed to validate miR-127-3p as a therapeutic target for vascular aging and remodeling in vitro and in vivo.

Abstract Funding Acknowledgements None. Smooth muscle cell (SMC) apoptosis is a contributory factor to plaque rupture in atherosclerosis, thereby promoting plaque destabilization through various mechanisms such as thrombogenic activation and plaque microcalcification. Immunohistochemical analysis has revealed elevated expression of proteins associated with intrinsic apoptosis, such as BCL2L11 (Bim), in unstable atherosclerotic plaques. Our hypothesis posits that Bim is a direct target of miR-92b-3p, an understudied microRNA, prompting an investigation into its role in regulating apoptosis in SMCs. Our objective is to discern a novel therapeutic axis for preventing plaque rupture by employing miRNA-based targeted interventions. Within a model of primary human vascular cells, we employed various in vitro techniques, including PCR, Western Blot, Live cell imaging, and ELISA. Lipotransfection facilitated transient modulation of miR-92b-3p, using both knockdown and elevation, while an siRNA transfection model was also utilized. MiR-92b-3p exhibits robust expression in late atherosclerotic lesions in vivo (p<0.001) and demonstrates heightened levels in smooth muscle cells (SMCs) compared to endothelial cells (ECs) in vitro (p<0.01) and in vivo in respective tissues (p<0.01). Bim, a direct target of miR-92b-3p, experiences upregulation post-miR-92b-3p knockdown and downregulation following pre-miR-92b-3p introduction into SMCs, both at mRNA and protein levels (p<0.01, p<0.01). Apoptosis increases in growth-medium cultured SMCs after miR-92b-3p knockdown (p<0.05), but not in senescent SMCs, ECs, or basal-medium treated SMCs. Live-cell imaging reveals a reduction in SMC count after anti-miR-92b-3p transfection (p<0.01), accompanied by cell shrinkage indicative of apoptosis 72 hours post-transfection (p<0.05). Growth factor stimulation results in miR-92b-3p upregulation (p>0.05) and Bim downregulation in SMCs. SiRNA knockdown of BIM in SMCs abolishes apoptosis induction through miR-92b-3p knockdown (p<0.05). Conversely, in apoptotic SMCs, miR-92b-3p upregulation protects cells from apoptosis (p<0.05). MiR-92b-3p effectively modulates mitochondrial apoptosis in SMCs by targeting BIM. This regulatory impact is exclusive to proliferative SMCs, known for their deleterious involvement in atherosclerotic plaques. Cumulatively, the elevation of miR-92b-3p via targeted miRNA therapeutics or miRNA-drug interactions in the advanced stages of atherosclerosis presents a potential therapeutic avenue for averting plaque rupture.

Abstract Funding Acknowledgements None. Background and purpose MicroRNAs (miRs) have emerged as potential therapeutic targets in cardiovascular disease, given their ability to modulate multiple signaling pathways and cellular processes. Neointima formation as an angioplasty-related complication remains a major issue calling for innovative therapeutic options and is predominantly mediated by phenotypic and functional changes of vascular smooth muscle cells. Here, we propose that targeting miR-31-5p might be an effective approach to selectively modulate smooth muscle cell functions to enhance vascular healing and regeneration. Methods C57J/BlL6 mice (n=6, 3 months, male) were subjected to wire-induced femoral artery injury. At days 10 and 21 after injury, femoral arteries were harvested for miRNA analysis. Expression levels of miR-31-5p were confirmed by qRT-PCR in human coronary artery endothelial cells (HCAEC) and -smooth muscle cells (HCASMC). Further, the effect of miR-31-5p on cellular functions was investigated on HCAECs and HCASMCs in vitro, including migration (scratch assay), proliferation (BrdU assay), and apoptosis. Possible targets of miR-31-5p were identified in silico by structured literature research and target binding prediction and confirmed on mRNA and protein levels. High-resolution microscopy analysis was used to investigate morphological changes. Results Expression analysis revealed a significant upregulation of miR-31-5p (p<0.0001) in murine femoral artery neointimal tissue at 7 and 21 days. In vitro, miR-31-5p was significantly upregulated in HCASMC (p<0,05) but not HCAEC following serum stimulation. On a functional level, miR-31-5p revealed dissenting effects on HCAEC and HCASMC. Whereas migration and proliferation are not altered in HCAECs, knockdown of miR-31-5p significantly reduced migration (p<0,01), proliferation (p<0,05), and increased apoptosis (p<0,05) in HCASMCs. Additionally, the knockdown of miR-31-5p influenced the phenotypical switch of HCASMC towards a contractile phenotype. Under these conditions, the release of cytokines like TNF-alpha and IL1-beta was also significantly reduced (p<0,05) in HCASMCs. Systematic in silico target screens suggested DKK1 and PIAS3 as potential direct targets. Indeed, knockdown of miR-31-5p resulted in a significant upregulation of DKK1 in HCASMC (p<0,05) on mRNA level and DKK1 (p<0,05) and PIAS3 (p<0,05) on mRNA and protein level. An overexpression resulted in a significant downregulation of DKK1 in HCASMC (p<0,05) on mRNA level. Conclusion In conclusion, we report that miR-31-5p is robustly upregulated during neointima formation and seems to exert a functional neointima formation-promoting role in HCASMC rather than HCAEC. Mechanistically, DKK1 and PIAS3 as direct targets of miR-31-5p can mediate the observed effects. Thus, miR-31-5p might represent an attractive target to selectively modulate HCASMC dysfunction following vascular intervention, limiting neointima formation in treated vessels.

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): University Hospital Halle, Martin-Luther-University Halle-Wittenberg The dysfunctionality of endothelial cells (EC) and smooth muscle cells (SMC) contributes to the progression of vascular remodeling and subsequent cardiovascular pathologies. Several microRNAs (miRNA) have been revealed as key regulators of EC and SMC function, therefore suggesting them as potential targets in the modulation of vascular remodeling. Here, we investigate the potential role of miR-32-5p in vascular cell function and its potential as a therapeutic target in vascular remodeling. Initial screenings by qRT-PCR analysis for miR-32-5p expression were performed in murine femoral artery tissue on day 10 and 21 after wire-induced injury in C57Bl6 mice, serving as restenosis model. MiR-32-5p expression was assessed using qRT-PCR in human vascular endothelial (HVECs) and human vascular smooth muscle cells (HVSMC). The role of miR-32-5p on cellular functions, as migration and proliferation, was determined by performing scratch wound and BrdU assays. Potential miR-32-5p downstream targets were identified from literature and their expression levels upon interference with miR-32-5p evaluated on mRNA and protein levels. MiR-32-5p expression was decreased over time in the wire-induced injury model. In vitro, miR-32-5p expression was significantly decreased in replicative senescent HVSMC and HVEC (p<0,05) and relatively higher in HVSMC than in HVEC for all conditions. Under growth conditions an elevated expression of miR-32-5p in HVEC and significantly increased expression in HVSMC was detected (p<0,01). Proliferation in HVSMC increased significantly (p<0,05) by pre-miR-32-5p transfection, but remained unaffected in HVEC. Migration of HVECs and HVSMCs was improved by miR-32-5p and decelerated after antagomiR interference. Systemic literature target research revealed several potential targets in both cell types, of which the following were significantly downregulated in HVSMC after pre-miR transfection including PIK3r3 (p<0,05), KLF4 (p<0,05), CD69 (p<0,01), MAP2K4 (p<0,05) and BCL211 (p<0,01). Additionally, CD69 and KLF4 expression increased significantly either in HVECs (p<0,05). Our results suggest that miR-32-5p may be a substantial regulator in HVEC and HVSMC senescence and function and subsequently in vascular remodeling. To fully evaluate its role in the pathogenesis of atherosclerosis and therefore, its therapeutic potential in cardiovascular diseases, further investigations are needed. Additional experiments with the purpose of investigating miR-32-5p-target interactions and apoptotic effects will be performed shortly.

Background and Aims: The accumulation of senescent vascular smooth muscle cells (VSMC) in the vasculature over the lifespan contributes significantly to vascular remodeling. Severely impaired functional properties characterize senescent cells. MicroRNAs are key regulators in various pathological processes. Therefore, identifying microRNAs that contribute to vascular remodeling processes during the development of cellular senescence can result in new therapeutic approaches. Methods: Screenings of primary vascular cells in vitro and in vivo were performed to reveal regulated microRNAs. Utilizing qRT-PCR expression levels of microRNAs were determined in human and murine VSMC. Functional effects of the microRNAs on human VSMC were assessed via transfection of pre-miR and antagomir. We verified potential targets with established effects on cellular aging on the mRNA level and the protein level. Results: The initial screenings revealed miR-127-3p, miR-367-3p, and miR-148a-3p as highly regulated microRNAs in the setting of replicative senescence in vitro and in aged (20 months) C57BL6 mice in vivo. Senescent cells showed compared to non-senescent human and murine VSMCs alterations in expression levels of the microRNAs. Overexpression of the microRNAs in vitro led to an increased VSMC proliferation and total cell count. A reverse effect was shown after the knockdown. Data revealed respective alterations in target mRNA levels and protein levels of the identified targets after overexpression and downregulation of the microRNAs. Conclusions: MiR-127-3p, miR-367-3p, and miR-148a-3p are contributing factors in VSMC function during the development of cellular senescence. Consequently, future studies should elicit the potential of the microRNAs as targets in vascular aging and remodeling in vivo.

Background and Aims: To improve the healing after myocardial infarction (MI), a well-coordinated angiogenic response is necessary. MicroRNAs (miRNAs) were identified as regulators of angiogenesis, however, strategies to improve angiogenesis by targeting miRNAs are challenging. Here, we evaluate novel miRNA candidates concerning their therapeutic potential toward neovascularization after MI. Methods: The respective microRNAs (miRNA 127-3p, 148a-3p, miRNA 367-3p) were characterized regarding gene/protein expression for their contribution to EC function, i.e. proliferation, migration, and metabolism after either pre- or anti-miR transfection. In consecutive experiments, tube and sprouting formation ability in vitro and the angiogenic capacity of ECs in vivo were determined. Results: Under hypoxic conditions, as observed in the MI zones, all three miRNAs were upregulated in ECs. For miRNA-127-3p and 148a-3p, this upregulation under hypoxia is independent of senescence, whereas miRNA 367-3p is upregulated especially in senescent ECs under hypoxia. After transfection of pre-miR 367-3p, an improvement in migration capacity, as assessed per scratch wound assay, was observed. Pre-miR 367-3p and pre-miR-148a-3p promoted migration whereas anti-miRs of the same microRNAs reduced the migration. Pre and anti-miR 127-3p showed no effect on migration. Overexpression of pre-miRs of all miRNAS increased the metabolic activity of ECs. Conclusions: Summarizing different expression levels of miRNA 127-3p, 148a-3p and miRNA 367-3p exerted distinct effects on EC function, especially under hypoxic conditions. Our experimental data regarding cellular function show that these miRNAs are involved in the angiogenetic response and hold the potential to serve as therapeutic targets. In future experiments, we will evaluate the microRNA potential on MI healing in vivo.

Background and Aims: MicroRNAs (miRs) play a crucial role in vascular regeneration, a key factor in CVD-development. Although few miRs are characterized in the cardiovascular context, consequently investigating new miRs could lead to new therapeutic strategies. Here, we evaluate miR-31-5p and its influence on cellular functions in vascular cells such as endothelial and smooth muscle cells, especially in the context of cellular aging.

Background and Aims: Smooth muscle cells (SMC) are of central importance to vascular homeostasis and the development of vascular remodelling. They influence pathological processes induced by vascular aging and senescence like atherosclerosis. Here, we identified miR-92b-3p as a robustly regulated miR in senescent SMC in vitro and in vivo. Methods: We used vascular smooth muscle cells (VSMC) and human umbilical vein endothelial cells (HUVECS) to assess the effect of miR-92b-3p on vasculature in vitro. Methods included expression analysis of miR-92b-3, multiple functional assays to assess proliferation, apoptosis and migration and identification of possible targets on mRNA and protein level. Results: We demonstrate that contrasting the expression in HUVECS miR-92b-3p expression levels in SMCs were downregulated during replicative senescence. Similar changes of miRNA 92b-3p expression were observed in murine ECs and VSMCs. Elevating miR-92b-3p levels in senescent and non-senescent VSMCs, using miR-92b-3p mimics enhanced the migrational capacity in these cells. Degradation of the reduced expression levels of miR-92b-3p in VSMCs via specific antimiRs resulted in reduction of the proliferative and migrational capacities of senescent and non-senescent SMCs. Apoptosis was increased following knock down of miR-92b-3p. The determined target genes were confirmed to be altered in their expression following over expression or knock down respectively. Conclusions: MiR-92b-3p differs in its expression in aging and in different vascular cell types. It is indicated that miR-92b-3p regulates senescence progression in SMC, since reconstitution of miR-92b-3p levels in SMC partially reduces senescence-induced functional impairments. The miR-92b-3p might be a target for future therapeutic options influencing vascular aging and subsequent pathologies.