Senescence Of Vascular Smooth Muscle Cells Research Articles

EP09.05-01 Ionizing Radiation Induces Vascular Smooth Muscle Cell Senescence through Activating NF-κB/CTCF/p16 Pathway

EP09.05-01 Ionizing Radiation Induces Vascular Smooth Muscle Cell Senescence through Activating NF-κB/CTCF/p16 Pathway

PPARγ activation by fisetin mitigates vascular smooth muscle cell senescence via the mTORC2-FoxO3a-autophagy signaling pathway

Cellular senescence is caused by diverse stimuli and contributes to cardiovascular diseases. Several studies have indicated that PPARγ acts as a key mediator of lipid metabolism and shown that it has a protective effect on vascular biology. Nevertheless, the mechanism responsible for the anti-aging effects of PPARγ has not been fully elucidated in vascular smooth muscle cell (VSMC). Furthermore, although mTOR complex 2 (mTORC2) is known to be involved in cellular senescence and autophagy, relatively few studies have investigated its effects as compared with mTOR complex 1 (mTORC1). Therefore, we focused on mTORC2 function and investigated the relationship between PPARγ and mTORC2, and the anti-aging mechanism in VSMC. We found PPARγ activation dose-dependently mitigated the hydrogen peroxide (H2O2)-induced senescence. Treatment of fisetin induced the translocation of PPARγ from cytosol to nuclear and inhibited VSMC senescence. Moreover, activated PPARγ increased PTEN transcription, leading to inhibition of the mTORC2 signaling pathway. We determined mTORC2 activation contributed to senescence by suppressing the FoxO3a-autophagy signaling pathway, and dual knockdown of mTORC1 and mTORC2 decreased cellular senescence and increased autophagy activation more than respective single knockdown. Finally, fisetin acted as a PPARγ activator and inhibited VSMC senescence through the mTORC2-FoxO3a-autophagy signaling pathway. These results demonstrate PPARγ is associated with cellular senescence and that fisetin has an anti-aging effect via PPARγ activation and mTORC2 inhibition in VSMC. These results demonstrate that the mTORC2 signaling pathway regulates autophagy and cellular senescence, which suggests mTORC2 should be considered a significant target for preventing cellular senescence and age-related diseases.

Senolytics prevent caveolar CaV 3.2-RyR axis malfunction in old vascular smooth muscle.

Aging is a major risk factor for cardiovascular diseases. Our previous studies demonstrate that aging impairs the caveolar T-type CaV 3.2-RyR axis for extracellular Ca2+ influx to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). We hypothesize that the administration of senolytics, which can selectively clear senescent cells, could preserve the caveolar CaV 3.2-RyR axis in aging VSMCs. In this study, 10-month-old mice were administered the senolytics cocktail consisting of dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi-weekly for 4 months. Using VSMCs from mouse mesenteric arteries, we found that Ca2+ sparks were diminished after caveolae disruption by methyl-β-cyclodextrin (10 mM) in cells from D + Q treated but not vehicle-treated 14-month-old mice. D + Q treatment promoted the expression of CaV 3.2 in 14-month-old mesenteric arteries. Structural analysis using electron tomography and immunofluorescence staining revealed the remodeling of caveolae and co-localization of CaV 3.2-Cav-1 in D + Q treatment aged mesenteric arteries. In keeping with theoretical observations, Cav 3.2 channel inhibition by Ni2+ (50 μM) suppressed Ca2+ in VSMCs from the D + Q group, with no effect observed in vehicle-treated arteries. Our study provides evidence that age-related caveolar CaV 3.2-RyR axis malfunction can be alleviated by pharmaceutical intervention targeting cellular senescence. Our findings support the potential of senolytics for ameliorating age-associated cardiovascular disease.

65P Ionizing radiation induces vascular smooth muscle cell senescence through activating NF-κB-CTCF-p16 pathway

65P Ionizing radiation induces vascular smooth muscle cell senescence through activating NF-κB-CTCF-p16 pathway

Lack of activity of the mitochondrial large-conductance calcium-regulated potassium channels in senescent vascular smooth muscle cells.

A limited number of studies have shown functional changes in mitochondrial ion channels in aging and senescent cells. We have identified, for the first time, mitochondrial large-conductance calcium-regulated potassium channels in human smooth muscle mitochondria. This channel, with a conductance of 273 pS, was regulated by calcium ions and membrane potential. Additionally, it was activated by the potassium channel opener NS11021 and blocked by paxilline. Importantly, we have shown that senescence of these cells induced by hydrogen peroxide treatment leads to the disappearance of potassium channel protein levels and channel activity measured by the single channel patch-clamp technique. Our data suggest that disturbances in the expression of mitochondrial large conductance calcium-regulated potassium channels may be hallmarks of cellular senescence and contribute to the misregulation of mitochondrial function in senescent cells.

Antibody-mediated PCSK9 neutralization worsens outcome after bare-metal stent implantation in mice

AimsDespite advances in pharmacotherapy and device innovation, in-stent restenosis (ISR) and stent thrombosis (ST) remain serious complications following percutaneous coronary intervention (PCI) procedure with stent implantation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme involved in plasma cholesterol homeostasis and recently emerged as a therapeutic target for hypercholesterolemia. Antibody-based PCSK9 inhibition is increasingly used in different subsets of patients, including those undergoing PCI. However, whether PCSK9 inhibition affects outcome after stent implantation remains unknown. Methods and results12 to 14 weeks old C57Bl/6 mice underwent carotid artery bare-metal stent implantation. Compared to sham intervention, stent implantation was associated with increased expression of several inflammatory mediators, including PCSK9. The increase in PCSK9 protein expression was confirmed in the stented vascular tissue, but not in plasma. To inhibit PCSK9, alirocumab was administered weekly to mice before stent implantation. After 6 weeks, histological examination revealed increased intimal hyperplasia in the stented segment of alirocumab-treated animals compared to controls. In vitro, alirocumab promoted migration and inhibited the onset of senescence in primary human vascular smooth muscle cells (VSMC). Conversely, it blunted the migration and increased the senescence of endothelial cells (EC). ConclusionAntibody-based PCSK9 inhibition promotes in-stent intimal hyperplasia and blunts vascular healing by increasing VSMC migration, while reducing that of EC. This effect is likely mediated, at least in part, by a differential effect on VSMC and EC senescence. The herein-reported data warrant additional investigations concerning the use of PCSK9 inhibitors in patients undergoing PCI with stent implantation.

Abstract P3044: MicroRNA-127-3p Regulates Inflammatory Response In Injury-induced Vascular Remodeling And Cellular Senescence In Vascular Smooth Muscle Cells

The accumulation of senescent cells in the vasculature over the lifespan contributes to vascular remodeling due to the severely impaired functional properties of the cells. MicroRNAs (miRs) are promising therapeutical targets as they exhibit diverse and cell-specific functions and can be effectively targeted in vivo . Here, we propose that targeting miR-127-3p might be an attractive approach to enhance vascular healing and regeneration in a cell-specific manner, thereby limiting angioplasty-related complications. The initial screenings revealed miR-127-3p as a highly regulated miR in the process of restenosis 7 and 21 days (p<0.001) after wire induced-injury and in aged (20 months) C57BL/6 mice (p<0.05) in vivo . Additionally, replicative senescent vascular smooth muscle cells (SMC) and human coronary artery endothelial cells (EC) showed an enhanced expression of miR-127-3p (p<0.05) compared to non-senescent cells. Functional effects of miR-127-3p on SMCs and ECs were assessed via transfection with either pre-miR or antagomir. Overexpression of miR-127-3p in vitro reduced SMC proliferation (p<0.05) and altered migrational capacity. Downregulation of miR-127-3p resulted in a reverse effect. In contrast, cellular functions were not altered in ECs. We further identified potential targets with effects on inflammation and cellular aging by in silico analysis and verified them on mRNA and protein levels by qRT-PCR and western blot analysis. As a direct target of miR-127-3p, the senescence-associated gene CD44 was identified by TargetScan. Expression of CD44 was altered on mRNA and protein levels after overexpression respectively downregulation of miR-127-3p in SMCs. Further, p14arf and laminB1 were downregulated after overexpression of miR-127-3p in SMCs, contrary to the effect after downregulation of miR-127-3p with increased mRNA levels of p14arf and laminB1. In this study, we identified miR-127-3p as a contributing factor in SMC function during the development of cellular senescence and show that it might therewith also be a modulating factor in the process of restenosis. However, further studies need to be carried out to validate the potential of miR-127-3p as a therapeutic target in vascular aging and remodeling in vitro and in vivo .

The role of miR-127-3p, miR-367-3p, and miR-148a-3p for vascular smooth muscle cell senescence in the context of vascular remodeling

The role of miR-127-3p, miR-367-3p, and miR-148a-3p for vascular smooth muscle cell senescence in the context of vascular remodeling

Smooth Muscle Heterogeneity and Plasticity in Health and Aortic Aneurysmal Disease.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aorta, which plays a critical role in the maintenance of aortic wall integrity. VSMCs have been suggested to have contractile and synthetic phenotypes and undergo phenotypic switching to contribute to the deteriorating aortic wall structure. Recently, the unprecedented heterogeneity and diversity of VSMCs and their complex relationship to aortic aneurysms (AAs) have been revealed by high-resolution research methods, such as lineage tracing and single-cell RNA sequencing. The aortic wall consists of VSMCs from different embryonic origins that respond unevenly to genetic defects that directly or indirectly regulate VSMC contractile phenotype. This difference predisposes to hereditary AAs in the aortic root and ascending aorta. Several VSMC phenotypes with different functions, for example, secreting VSMCs, proliferative VSMCs, mesenchymal stem cell-like VSMCs, immune-related VSMCs, proinflammatory VSMCs, senescent VSMCs, and stressed VSMCs are identified in non-hereditary AAs. The transformation of VSMCs into different phenotypes is an adaptive response to deleterious stimuli but can also trigger pathological remodeling that exacerbates the pathogenesis and development of AAs. This review is intended to contribute to the understanding of VSMC diversity in health and aneurysmal diseases. Papers that give an update on VSMC phenotype diversity in health and aneurysmal disease are summarized and recent insights on the role of VSMCs in AAs are discussed.

DPP4 inhibition impairs senohemostasis to improve plaque stability in atherosclerotic mice.

Senescent vascular smooth muscle cells (VSMCs) accumulate in the vasculature with age and tissue damage, and secrete factors that promote atherosclerotic plaque vulnerability and disease. Here, we report increased levels and activity of dipeptidyl peptidase 4 (DPP4), a serine protease, in senescent VSMCs. Analysis of the conditioned media from senescent VSMCs revealed a unique senescence-associated secretory phenotype (SASP) signature comprising many complement and coagulation factors; silencing or inhibiting DPP4 reduced these factors and increased cell death. Serum samples from persons with high risk for cardiovascular disease contained high levels of DPP4-regulated complement and coagulation factors. Importantly, DPP4 inhibition reduced senescent cell burden and coagulation and improved plaque stability, while single-cell resolution of senescent VSMCs reflected the senomorphic and senolytic effects of DPP4 inhibition in murine atherosclerosis. We propose that DPP4-regulated factors could be exploited therapeutically to reduce senescent cell function, reverse senohemostasis, and improve vascular disease.

FISETIN ENHANCES PPAR GAMMA EXPRESSION TO PREVENT VASCULAR SMOOTH MUSCLE CELL SENESCENCE VIA MTORC2-AKT-FOXO3A SIGNALING PATHWAY

Objective: Cellular senescence is caused by diverse stimuli and is contributed to cardiovascular diseases such as atherosclerosis, coronary heart disease, and hypertension. Several studies have indicated that PPAR gamma is mainly a key mediator in lipid metabolism and adipocyte differentiation. In addition, PPAR gamma has a protective effect in vascular biology and regulates the inflammation and reactive oxygen species (ROS). Nevertheless, the anti-aging mechanism of PPAR gamma has not been elucidated in vascular smooth muscle cell (VSMC). Although mTORC2 is involved in the cellular senescence and autophagy, there are significantly fewer studies than mTORC1. Therefore, we focused on mTORC2 function and then aimed to investigate the correlation between PPAR gamma and mTORC2, and to identify the anti-aging mechanism in VSMC. Design and method: To generate the oxidative stress-induced VSMC senescence, we treated with hydrogen peroxide (H2O2). The anti-aging effect and underlying mechanism of fisetin were evaluated by Western blot analysis, senescence-associated beta-galactosidase (SA-beta-gal) staining, flow cytometry, immunofluorescence analysis, and immunoprecipitation analysis. Results: We found hydrogen peroxide (H2O2) induced not only PPAR gamma translocation from nuclear to cytosol but also mTORC2 activation with VSMC senescence. We determined mTORC2 activation contributed to the senescence by suppressing FoxO3a-autophagy signaling pathway. Furthermore, the double knockdown of mTORC1 and mTORC2 decreased the cellular senescence and increased the autophagy activation more than single knockdown. However, fisetin acted as a PPAR gamma activator, leading to the inhibition of VSMC senescence through mTORC2-Akt (Ser473)-FoxO3a signaling pathway which is related to autophagy. Conclusions: These results demonstrate PPAR gamma is associated with cellular senescence and fisetin has anti-aging effect via PPAR gamma activation and mTORC2 inhibition. Moreover, mTORC2 signaling pathway could regulate autophagy, which suggests mTORC2 is considered a significant target for preventing cellular senescence and aging-related disease.

MicroRNA-19b-3p dysfunction of mesenchymal stem cell-derived exosomes from patients with abdominal aortic aneurysm impairs therapeutic efficacy

Senescence of vascular smooth muscle cells (VSMCs) contributes to the formation of abdominal aortic aneurysm (AAA). Although mesenchymal stem cell exosomes (MSC-EXO) have been confirmed to restrict the development of AAA, their biological activity depends largely on the physiological state of the MSCs. This study aimed to compare the effects of adipose-derived MSC-EXO from healthy donors (HMEXO) and AAA patients (AMEXO) on senescence of VSMCs in AAA and explore the underlying mechanisms. An ApoE-/- mouse model of AAA was used to investigate the therapeutic effects of HMEXO, AMEXO or miR-19b-3p-AMEXO on AAA development. This in vitro model of AAA was established by treating VSMCs with Ang II (Angiotensin II). The senescence of VSMCs was determined by senescence-associated β-galactosidase (SA-β-gal) staining. The morphology of mitochondria in VSMCs was examined by MitoTracker staining. HMEXO exhibited superior capacity compared with AMEXO to inhibit VSMC senescence and attenuate AAA formation in Ang II–treated ApoE-/- mice. In vitro, both AMEXO and HMEXO inhibited Ang II-induced VSMC senescence via downregulation of mitochondrial fission. Notably, compared with HMEXO, the ability of AMEXO to inhibit VSMC senescence was significantly decreased. miRNA sequencing and the expression of miR-19b-3p was significantly decreased in AMEXO compared with HMEXO. Luciferase assay suggested that MST4 (Mammalian sterile-20-like kinase 4) is a potential target of miR-19b-3p. Mechanistically, miR-19b-3p in HMEXO ameliorated VSMC senescence by inhibiting mitochondrial fission via regulation of the MST4/ERK/Drp1 signaling pathway. Overexpression of miR-19b-3p in AMEXO improved their beneficial effect on AAA formation. Our study reveals that MSC-exosomal miR-19b-3p exerts protective effects against Ang II-induced AAA and VSMC senescence via regulation of the MST4/ERK/Drp1 pathway. The pathological state of AAA patients alters the miRNA components of AMEXO and impairs their therapeutic benefits.

Targeting Vascular Smooth Muscle Cell Senescence: A Novel Strategy for Vascular Diseases.

Vascular diseases are a major threat to human health, characterized by high rates of morbidity, mortality, and disability. VSMC senescence contributes to dramatic changes in vascular morphology, structure, and function. A growing number of studies suggest that VSMC senescence is an important pathophysiological mechanism for the development of vascular diseases, including pulmonary hypertension, atherosclerosis, aneurysm, and hypertension. This review summarizes the important role of VSMC senescence and senescence-associated secretory phenotype (SASP) secreted by senescent VSMCs in the pathophysiological process of vascular diseases. Meanwhile, it concludes the progress of antisenescence therapy targeting VSMC senescence or SASP, which provides new strategies for the prevention and treatment of vascular diseases.

Mitochondrial Homeostasis in VSMCs as a Central Hub in Vascular Remodeling.

Vascular remodeling is a common pathological hallmark of many cardiovascular diseases. Vascular smooth muscle cells (VSMCs) are the predominant cell type lining the tunica media and play a crucial role in maintaining aortic morphology, integrity, contraction and elasticity. Their abnormal proliferation, migration, apoptosis and other activities are tightly associated with a spectrum of structural and functional alterations in blood vessels. Emerging evidence suggests that mitochondria, the energy center of VSMCs, participate in vascular remodeling through multiple mechanisms. For example, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)-mediated mitochondrial biogenesis prevents VSMCs from proliferation and senescence. The imbalance between mitochondrial fusion and fission controls the abnormal proliferation, migration and phenotypic transformation of VSMCs. Guanosine triphosphate-hydrolyzing enzymes, including mitofusin 1 (MFN1), mitofusin 2 (MFN2), optic atrophy protein 1 (OPA1) and dynamin-related protein 1 (DRP1), are crucial for mitochondrial fusion and fission. In addition, abnormal mitophagy accelerates the senescence and apoptosis of VSMCs. PINK/Parkin and NIX/BINP3 pathways alleviate vascular remodeling by awakening mitophagy in VSMCs. Mitochondrial DNA (mtDNA) damage destroys the respiratory chain of VSMCs, resulting in excessive ROS production and decreased ATP levels, which are related to the proliferation, migration and apoptosis of VSMCs. Thus, maintaining mitochondrial homeostasis in VSMCs is a possible way to relieve pathologic vascular remodeling. This review aims to provide an overview of the role of mitochondria homeostasis in VSMCs during vascular remodeling and potential mitochondria-targeted therapies.

Fisetin alleviates cellular senescence through PTEN mediated inhibition of PKCδ-NOX1 pathway in vascular smooth muscle cells

Reactive oxygen species (ROS) are a key risk factor of cellular senescence and age-related diseases, and protein kinase C (PKC) has been shown to activate NADPH oxidases (NOXs), which generate ROS. Although PKC activation induces oxidative stress, leading to the cellular dysfunction in various cell types, the correlation between PKC and senescence has not been reported in vascular smooth muscle cell (VSMC). Several studies have indicated cellular senescence is accompanied by phosphatase and tensin homolog (PTEN) loss and that an interaction exists between PTEN and PKC. Therefore, we aimed to determine whether PTEN and PKC are associated with VSMC senescence and to investigate the mechanism involved. We found hydrogen peroxide (H2O2) decreased PTEN expression and increased PKCδ phosphorylation. Moreover, H2O2 upregulated the NOX1 subunits, p22phox and p47phox, and induced VSMC senescence via p53-p21 signaling pathway. We identified PKCδ activation contributed to VSMC senescence through activation of NOX1 and ROS production. However, fisetin inhibited cellular senescence induced by the PTEN-PKCδ-NOX1-ROS signaling pathway, and this anti-aging effect was attributed to reduced ROS production caused by suppressing NOX1 activation. These results suggest that the PTEN-PCKδ signaling pathway is directly related to senescence via NOX1 activation and that the downregulation of PKCδ by flavonoids provides a potential means of treating age-associated diseases.

BMF-AS1/BMF Promotes Diabetic Vascular Calcification and Aging both In Vitro and In Vivo.

Vascular calcification and aging often increase morbidity and mortality in patients with diabetes mellitus (DM); however, the underlying mechanisms are still unknown. In the present study, we found that Bcl-2 modifying factor (BMF) and BMF antisense RNA 1 (BMF-AS1) were significantly increased in high glucose-induced calcified and senescent vascular smooth muscle cells (VSMCs) as well as artery tissues from diabetic mice. Inhibition of BMF-AS1 and BMF reduced the calcification and senescence of VSMCs, whereas overexpression of BMF-AS1 and BMF generates the opposite results. Mechanistic analysis showed that BMF-AS1 interacted with BMF directly and up-regulated BMF at both mRNA and protein levels, but BMF did not affect the expression of BMF-AS1. Moreover, knocking down BMF-AS1 and BMF suppressed the calcification and senescence of VSMCs, and BMF knockout (BMF-/-) diabetic mice presented less vascular calcification and aging compared with wild type diabetic mice. In addition, higher coronary artery calcification scores (CACs) and increased plasma BMF concentration were found in patients with DM, and there was a positive correlation between CACs and plasma BMF concentration. Thus, BMF-AS1/BMF plays a key role in promoting high glucose-induced vascular calcification and aging both in vitro and in vivo. BMF-AS1 and BMF represent potential therapeutic targets in diabetic vascular calcification and aging.

Clearance of Stress-Induced Premature Senescent Cells Alleviates the Formation of Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a multifactorial disease characterized by various pathophysiological processes, including chronic inflammation, oxidative stress, and proteolytic activity in the aortic wall. Stress-induced premature senescence (SIPS) has been implicated in regulating these pathophysiological processes, but whether SIPS contributes to AAA formation remains unknown. Here, we detected SIPS in AAA from patients and young mice. The senolytic agent ABT263 prevented AAA development by inhibiting SIPS. Additionally, SIPS promoted the transformation of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a synthetic phenotype, whereas inhibition of SIPS by the senolytic drug ABT263 suppressed VSMC phenotypic switching. RNA sequencing and single-cell RNA sequencing analysis revealed that fibroblast growth factor 9 (FGF9), secreted by stress-induced premature senescent VSMCs, was a key regulator of VSMC phenotypic switching and that FGF9 knockdown abolished this effect. We further showed that the FGF9 level was critical for the activation of PDGFRβ/ERK1/2 signaling, facilitating VSMC phenotypic change. Taken together, our findings demonstrated that SIPS is critical for VSMC phenotypic switching through the activation of FGF9/PDGFRβ/ERK1/2 signaling, promoting AAA development and progression. Thus, targeting SIPS with the senolytic agent ABT263 may be a valuable therapeutic strategy for the prevention or treatment of AAA.

Structural dysregulation of the pulmonary autograft was associated with a greater density of p16INK4A-vascular smooth muscle cells

The present study tested the hypothesis that a senescent phenotype of vascular smooth muscle cells (VSMCs) may represent the seminal event linked to maladaptive pulmonary autograft remodeling of a small number of patients that underwent the Ross procedure. The diameter of the pulmonary autograft (47±4 mm) of three male patients was significantly greater compared to the pulmonary artery (26±1 mm) excised from bicuspid aortic valve (BAV) patients. The pulmonary autograft was associated with a neointimal region and the adjacent medial region was significantly thinner compared to the pulmonary artery of BAV patients. Structural dysregulation was evident as elastin content of the medial region was significantly reduced in the pulmonary autograft compared to the pulmonary artery of BAV patients. By contrast, collagen content of the medial region of the pulmonary autograft and the pulmonary artery of BAV patients was not significantly different. Reduced medial elastin content of the pulmonary autograft was associated with increased protein levels of matrix metalloproteinase-9. The latter phenotype was not attributed to a robust inflammatory response as the percentage of Mac-2(+)-infiltrating monocytes/macrophages was similar between groups. A senescent phenotype was identified as protein levels of the cell cycle inhibitor p27kip1 were upregulated and the density of p16INK4A/non-muscle myosin IIB(+)-VSMCs was significantly greater in the pulmonary autograft compared to the pulmonary artery of BAV patients. Thus, senescent VSMCs may represent the predominant cellular source of increased matrix metalloproteinase-9 protein expression translating to maladaptive pulmonary autograft remodeling characterized by elastin degradation, medial thinning and neointimal formation.

Lin28b delays vasculature aging by reducing platelet-derived growth factor-beta resistance in senescent vascular smooth muscle cells

Background and aimsPlatelet-derived growth factor-β (PDGFB) is an important mediator of vascular smooth muscle cell (VSMC) proliferation, and PDGFB resistance is observed in senescent VSMCs. Lin28b is a stemness regulator in the embryo; however, its role in vasculature aging and VSMC senescence is unknown. We aimed to investigate whether Lin28b could restore the VSMC response to PDGFB and delay vasculature aging. MethodsApoE−/− mice were fed a high-fat diet for different weeks to establish an aging model. PDGFB resistance was observed using EdU staining in vessel culture in vitro. Quantitative polymerase chain reaction and in situ hybridization were used to detect let-7 expression. Senescence was identified by Western blotting, senescence-associated beta-galactosidase activity or Sudan Black B staining, and VSMC function was determined using CCK-8, migration, and enzyme-linked immunosorbent assays. ResultsVessels from aged mice showed poor responses to PDGFB stimulation compared with those from young mice; similar results were found in senescent VSMCs. The expression levels of Lin28b and PDGF receptor-β were downregulated in aging vasculature and senescent VSMCs, whereas let-7 family levels increased with aging and VSMC passage growth. Transfection of VSMCs with let-7c induced PDGFB resistance and accelerated VSMC senescence, whereas blocking let-7c restored PDGFB reactions in VSMCs. Overexpression of Lin28b protein by lentivirus resulted in the restoration of PDGFB reactions and delayed VSMC senescence, which was blocked by a let-7c mimic. ConclusionsThis study reveals the role of Lin28b in delaying vasculature aging by decreasing senescent VSMC PDGFB resistance mediated by let-7.

Vascular smooth muscle cell senescence accelerates medin aggregation via small extracellular vesicle secretion and extracellular matrix reorganization.

Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid peptide. Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid accumulation in the extracellular matrix (ECM). To determine the mechanisms of AMA formation with age, we explored the impact of vascular smooth muscle cell (VSMC) senescence, EV secretion, and ECM remodeling on medin accumulation. Medin was detected in EVs secreted from primary VSMCs. Small, round medin aggregates colocalized with EV markers in decellularized ECM in vitro and medin was shown on the surface of EVs deposited in the ECM. Decreasing EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age and VSMC senescence increased EV secretion, increased EV medin loading and triggered deposition of fibril-like medin. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition, which led to enhanced EV-ECM binding and accelerated medin aggregation. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalized with EVs and medin. Isolated EVs selectively bound to HSPG2 in the ECM and its knock-down decreased formation of fibril-like medin structures. These data identify VSMC-derived EVs and HSPG2 in the ECM as key mediators of medin accumulation, contributing to age-associated AMA development.