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Arterial cyclic stretch regulates Lamtor1 and promotes neointimal hyperplasia via circSlc8a1/miR-20a-5p axis in vein grafts.

ABSTRACT: Rationale: Neointimal hyperplasia caused by dedifferentiation and proliferation of venous smooth muscle cells (SMCs) is the major challenge for restenosis after coronary artery bypass graft. Herein, we investigated the role of Lamtor1 in neointimal formation and the regulatory mechanism of non-coding RNA underlying this process. Methods: Using a "cuff" model, veins were grafted into arterial system and Lamtor1 expression which was correlated with the activation of mTORC1 signaling and dedifferentiation of SMCs, were measured by Western blot. Whole transcriptome deep sequencing (RNA-seq) of the grafted veins combined with bioinformatic analysis identified highly conserved circSlc8a1 and its interaction with miR-20a-5p, which may target Lamtor1. CircSlc8a1 was biochemically characterized by Sanger sequencing and resistant to RNase R digestion. The cytoplasmic location of circSlc8a1 was shown by fluorescence in situ hybridization (FISH). RNA pull-down, luciferase assays and RNA immunoprecipitation (RIP) with Ago2 assays were used to identify the interaction circSlc8a1 with miR-20a-5p. Furthermore, arterial mechanical stretch (10% elongation) was applied in vitro. Results: In vivo, Lamtor1 was significantly enhanced in grafted vein and activated mTORC1 signaling to promote dedifferentiation of SMCs. Arterial mechanical stretch (10% elongation) induced circSlc8a1 expression and positively regulated Lamtor1, activated mTORC1 and promoted SMC dedifferentiation and proliferation. Local injection of circSlc8a1 siRNA or SMC-specific Lamtor1 knockout mice prevented neointimal hyperplasia in vein grafts in vivo. Conclusions: Our study reveals a novel mechanobiological mechanism underlying the dedifferentiation and proliferation of venous SMCs in neointimal hyperplasia. CircSlc81/miR-20a-5p/Lamtor1 axis induced by arterial cyclic stretch may be a potential clinical target that attenuates neointimal hyperplasia in grafted vessels.

SUBMITTER: Liu JT

PROVIDER: S-EPMC9274756 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Models of arterial injury in rodents have been invaluable to our current understanding of vessel restenosis and play a continuing role in the development of endovascular interventions for cardiovascular disease. Mechanical distention of the vessel wall and denudation of the vessel endothelium are the two major modes of vessel injury observed in most clinical pathologies and are critical to the reproducible modelling of progressive neointimal hyperplasia. The current models which have dominated this research area are the mouse wire carotid or femoral injury and the rat carotid balloon injury. While these elicit simultaneous distension of the vessel wall and denudation of the luminal endothelium, each model carries limitations that need to be addressed using a complementary injury model. Wire injuries in mice are highly technical and procedurally challenging due to small vessel diameters, while rat balloon injuries require permanent blood vessel ligation and disruption of native blood flow. Complementary models of vascular injury with reproducibility, convenience, and increased physiological relevance to the pathophysiology of endovascular injury would allow for improved studies of neointimal hyperplasia in both basic and translational research. In this study, we developed a new surgical model that elicits vessel distention and endothelial denudation injury using sequential steps using microforceps and a standard needle catheter inserted via arteriotomy into a rat common carotid artery, without requiring permanent ligation of branching arteries. After 2 weeks post-injury this model elicits highly reproducible neointimal hyperplasia and rates of re-endothelialisation similar to current wire and balloon injury models. Furthermore, evaluation of the smooth muscle cell phenotype profile, inflammatory response and extracellular matrix within the developing neointima, showed that our model replicated the vessel remodelling outcomes critical to restenosis and those becoming increasingly focused upon in the development of new anti-restenosis therapies.

Project description:BackgroundThe microbiome has a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia development has been linked to inflammation, a direct role of the microbiota in neointimal hyperplasia has not yet been established. Germ-free (GF) mice are an invaluable model for studying causative links between commensal organisms and the host. We hypothesized that GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally raised (CONV-R) mice.MethodsTwenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers at five days. Systemic inflammation was assessed by multiplex immunoassays of serum. CONV-R mice treated in the same manner served as the control cohort. GF and CONV-R mice were compared using standard statistical methods.ResultsAll GF mice remained sterile during the entire study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia development compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media) area. The collagen content of the neointimal lesions appeared qualitatively similar on Masson's trichrome staining. There was significantly reduced Ki67 immunoreactivity in the media and adventitia of GF carotid arteries 5 days after ligation. GF mice also had increased arterial infiltration of anti-inflammatory M2 macrophages compared to CONV-R mouse arteries and a reduced proportion of mature neutrophils. GF mice had significantly reduced serum IFN-?-inducible protein (IP)-10 and MIP-2 5 days after carotid ligation, suggesting a reduced systemic inflammatory response.ConclusionsGF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which is likely related to altered kinetics of wound healing and acute inflammation. Recognizing the role of commensals in the regulation of arterial remodeling will provide a deeper understanding of the pathophysiology of restenosis and support strategies to treat or reduce restenosis risk by manipulating microbiota.

Project description:Increasing evidence has suggested a critical role for endothelial-to-mesenchymal transition (EndoMT) in a variety of pathological conditions. MicroRNA-200c-3p (miR-200c-3p) has been implicated in epithelial-to-mesenchymal transition. However, the functional role of miR-200c-3p in EndoMT and neointimal hyperplasia in artery bypass grafts remains largely unknown. Here we demonstrated a critical role for miR-200c-3p in EndoMT. Proteomics and luciferase activity assays revealed that fermitin family member 2 (FERM2) is the functional target of miR-200c-3p during EndoMT. FERMT2 gene inactivation recapitulates the effect of miR-200c-3p overexpression on EndoMT, and the inhibitory effect of miR-200c-3p inhibition on EndoMT was reversed by FERMT2 knockdown. Further mechanistic studies revealed that FERM2 suppresses smooth muscle gene expression by preventing serum response factor nuclear translocation and preventing endothelial mRNA decay by interacting with Y-box binding protein 1. In a model of aortic grafting using endothelial lineage tracing, we observed that miR-200c-3p expression was dramatically up-regulated, and that EndoMT contributed to neointimal hyperplasia in grafted arteries. MiR-200c-3p inhibition in grafted arteries significantly up-regulated FERM2 gene expression, thereby preventing EndoMT and reducing neointimal formation. Importantly, we found a high level of EndoMT in human femoral arteries with atherosclerotic lesions, and that miR-200c-3p expression was significantly increased, while FERMT2 expression levels were dramatically decreased in diseased human arteries. Collectively, we have documented an unexpected role for miR-200c-3p in EndoMT and neointimal hyperplasia in grafted arteries. Our findings offer a novel therapeutic opportunity for treating vascular diseases by specifically targeting the miR-200c-3p/FERM2 regulatory axis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Project description:ObjectiveInflammation is a key driver of excessive neointimal hyperplasia within vein grafts. Recent work demonstrates that specialized proresolving lipid mediators biosynthesized from omega-3 polyunsaturated fatty acids, such as resolvin D1 (RvD1), actively orchestrate the process of inflammation resolution. We investigated the effects of local perivascular delivery of RvD1 in a rabbit vein graft model.MethodsIpsilateral jugular veins were implanted as carotid interposition grafts through an anastomotic cuff technique in New Zealand white rabbits (3-4 kg; N = 80). RvD1 (1 μg) was delivered to the vein bypass grafts in a perivascular fashion, using either 25% Pluronic F127 gel (Sigma-Aldrich, St. Louis, Mo) or a thin bilayered poly(lactic-co-glycolic acid) (PLGA) film. No treatment (bypass only) and vehicle-loaded Pluronic gels or PLGA films served as controls. Delivery of RvD1 to venous tissue was evaluated 3 days later by liquid chromatography-tandem mass spectrometry. Total leukocyte infiltration, macrophage infiltration, and cell proliferation were evaluated by immunohistochemistry. Elastin and trichrome staining was performed on grafts harvested at 28 days after bypass to evaluate neointimal hyperplasia and vein graft remodeling.ResultsPerivascular treatments did not influence rates of graft thrombosis (23%), major wound complications (4%), or death (3%). Leukocyte (CD45) and macrophage (RAM11) infiltration was significantly reduced in the RvD1 treatment groups vs controls at 3 days (60%-72% reduction; P < .01). Cellular proliferation (Ki67 index) was also significantly lower in RvD1-treated vs control grafts at 3 days (40%-50% reduction; P < .01). Treatment of vein grafts with RvD1-loaded gels reduced neointimal thickness at 28 days by 61% vs bypass only (P < .001) and by 63% vs vehicle gel (P < .001). RvD1-loaded PLGA films reduced neointimal formation at 28 days by 50% vs bypass only (P < .001). RvD1 treatment was also associated with reduced collagen deposition in vein grafts at 28 days.ConclusionsLocal perivascular delivery of RvD1 attenuates vein graft hyperplasia without associated toxicity in a rabbit carotid bypass model. This effect appears to be mediated by both reduced leukocyte recruitment and decreased cell proliferation within the graft. Perivascular PLGA films may also impart protection through biomechanical scaffolding in this venous arterialization model. Our studies provide further support for the potential therapeutic role of specialized proresolving lipid mediators such as D-series resolvins in modulating vascular injury and repair.

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Arterial cyclic stretch regulates Lamtor1 and promotes neointimal hyperplasia via circSlc8a1/miR-20a-5p axis in vein grafts.

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