RESUMO
Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age-related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post-mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age-related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length-independent telomere damage in cardiomyocytes activates the classical senescence-inducing pathways, p21CIP and p16INK4a, and results in a non-canonical senescence-associated secretory phenotype, which is pro-fibrotic and pro-hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age-related myocardial dysfunction and in the wider setting to ageing in post-mitotic tissues.
Assuntos
Cardiomegalia/patologia , Senescência Celular , Dano ao DNA , Fibrose/patologia , Mitose , Miócitos Cardíacos/patologia , Encurtamento do Telômero , Envelhecimento , Animais , Cardiomegalia/etiologia , Feminino , Fibrose/etiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Monoaminoxidase/fisiologia , Miócitos Cardíacos/metabolismo , Fenótipo , RNA/fisiologia , Ratos Sprague-Dawley , Telomerase/fisiologiaRESUMO
The purpose of this study was to characterize changes in antioxidant and age-related gene expression in aorta and aortic valve with aging, and test the hypothesis that increased mitochondrial oxidative stress accelerates age-related endothelial and aortic valve dysfunction. Wild-type (MnSOD(+/+)) and manganese SOD heterozygous haploinsufficient (MnSOD(+/-)) mice were studied at 3 and 18 mo of age. In aorta from wild-type mice, antioxidant expression was preserved, although there were age-associated increases in Nox2 expression. Haploinsufficiency of MnSOD did not alter antioxidant expression in aorta, but increased expression of Nox2. When compared with that of aorta, age-associated reductions in antioxidant expression were larger in aortic valves from wild-type and MnSOD haploinsufficient mice, although Nox2 expression was unchanged. Similarly, sirtuin expression was relatively well-preserved in aorta from both genotypes, whereas expression of SIRT1, SIRT2, SIRT3, SIRT4, and SIRT6 were significantly reduced in the aortic valve. Expression of p16(ink4a), a marker of cellular senescence, was profoundly increased in both aorta and aortic valve from MnSOD(+/+) and MnSOD(+/-) mice. Functionally, we observed comparable age-associated reductions in endothelial function in aorta from both MnSOD(+/+) and MnSOD(+/-) mice. Interestingly, inhibition of NAD(P)H oxidase with apocynin or gp91ds-tat improved endothelial function in MnSOD(+/+) mice but significantly impaired endothelial function in MnSOD(+/-) mice at both ages. Aortic valve function was not impaired by aging or MnSOD haploinsufficiency. Changes in antioxidant and sirtuin gene expression with aging differ dramatically between aorta and aortic valve. Furthermore, although MnSOD does not result in overt cardiovascular dysfunction with aging, compensatory transcriptional responses to MnSOD deficiency appear to be tissue specific.
Assuntos
Envelhecimento/metabolismo , Aorta Torácica/enzimologia , Valva Aórtica/enzimologia , Mitocôndrias/enzimologia , Estresse Oxidativo , Superóxido Dismutase/deficiência , Transcrição Gênica , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Animais , Antioxidantes/farmacologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Aorta Torácica/fisiopatologia , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Cálcio/metabolismo , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Genótipo , Haploinsuficiência , Heterozigoto , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Osteogênese , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fenótipo , Sirtuínas/genética , Sirtuínas/metabolismo , Superóxido Dismutase/genética , Vasodilatação , Vasodilatadores/farmacologiaRESUMO
BACKGROUND AND AIMS: The overall goal of this study was to determine the effects of MnSOD-deficiency on vascular structure and function in hypercholesterolemic mice. Previous work suggested that increases in mitochondrial-derived reactive oxygen species (ROS) can exacerbate vascular dysfunction and atherosclerosis. It remains unknown, however, how MnSOD-deficiency and local compensatory mechanisms impact atherosclerotic plaque composition. METHODS AND RESULTS: We used a hypercholesterolemic mouse model (ldlr-/-/ApoB100/100; LA), either wild-type for MnSOD (LA-MnSOD+/+) or MnSOD-haploinsufficient (LA-MnSOD+/-), that was fed a western diet for either 3 or 6 months. Consistent with previous reports, reductions of MnSOD did not significantly worsen hypercholesterolemia-induced endothelial dysfunction in the aorta. Critically, dramatic impairment of vascular function with Nox2 inhibition or catalase pretreatment suggested the presence of a significant NO-independent vasodilatory mechanism in LA-MnSOD+/- mice (e.g. H2O2). Despite remarkably well-preserved overall vascular relaxation, loss of mitochondrial antioxidant capacity in LA-MnSOD+/- mice significantly increased osteogenic signalling and vascular calcification compared to the LA-MnSOD+/+ littermates. CONCLUSIONS: Collectively, these data are the first to suggest that loss of mitochondrial antioxidant capacity in hypercholesterolemic mice results in dramatic upregulation of NADPH oxidase-derived H2O2. While this appears to be adaptive in the context of preserving overall endothelium-dependent relaxation and vascular function, these increases in ROS appear to be remarkably maladaptive and deleterious in the context of vascular calcification.
Assuntos
Peróxido de Hidrogênio , Calcificação Vascular , Animais , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias , Estresse Oxidativo , Espécies Reativas de Oxigênio , Superóxido Dismutase , Calcificação Vascular/genética , Calcificação Vascular/prevenção & controle , VasodilataçãoRESUMO
Background: While a small number of studies suggest that oxidative stress has an influential role in fibrocalcific aortic valve disease (FCAVD), the roles of specific antioxidant enzymes in progression of this disease remain poorly understood. Here, we focused on selectively altering mitochondrial-derived oxidative stress-which has been shown to alter progression of a myriad of age-associated diseases-on the progression of molecular and phenotypic drivers of FCAVD. Methods: We generated low-density lipoprotein receptor-deficient, Apolipoprotein B100-only mice (LA) that were either haploinsufficient for MnSOD (LA-MnSOD +/-) or genetically overexpressing MnSOD (LA-MnSOD Tg/0). After 6 months of Western diet feeding, mice underwent echocardiography to assess valvular and cardiac function and tissues were harvested. Quantitative-RT PCR, immunohistochemistry, and histopathology were used to measure changes in molecular pathways related to oxidative stress, calcification, and fibrosis. Results: While reductions in MnSOD increased oxidative stress, there was not an overt phenotypic effect of MnSOD deficiency on valvular and cardiac function in LA-MnSOD +/- mice. While markers of canonical bone morphogenetic protein signaling tended to increase in valve tissue from LA-MnSOD +/- (e.g., p-SMAD1/5/8 and osterix), we did not observe statistically significant increases in osteogenic signaling. We did, however, observe highly significant reductions in expression of osteopontin, which were associated with significant increases in calcium burden in LA-MnSOD +/- mice. Reciprocally, genetically increasing MnSOD did not preserve valve function in LA-MnSOD Tg/0, but we did observe slight reductions in p-SMAD1/5/8 levels compared to their non-transgenic littermates. Interestingly, overexpression of MnSOD dramatically increased expression of osteopontin in valve tissue from LA-MnSOD Tg/0 mice, but was not sufficient to attenuate calcium burden when compared to their LA-MnSOD 0/0 littermates. Conclusions: Collectively, this study demonstrates that maintenance of mitochondrial antioxidant capacity is important in preventing accelerated disease progression in a mouse model of FCAVD, but that effectively altering mitochondrial antioxidant capacity as a monotherapeutic approach to slow key histopathological and molecular drivers of FCAVD remains biologically and therapeutically challenging.
RESUMO
Objective: Sirtuin deacetylases are major regulators of organismal aging, and while depletion of sirtuin 6 (SIRT6) in mice results in a profound progeroid phenotype, the role of SIRT6 in the regulation of vasomotor function is unknown. Thus, our objective was to test the hypothesis that reductions in SIRT6 elicit endothelial dysfunction in young, genetically altered mice. Results and Approach: We used young (3 month old), littermate-matched, SIRT6 wild-type (WT), and SIRT6 heterozygous (HET) mice. SIRT6 expression (qRT-PCR) was reduced by 50% in HET mice. Carotid vessel responses to acetylcholine, sodium nitroprusside, U46619, and serotonin were examined in isolated organ chamber baths. Relaxation in response to acetylcholine (ACH) was impaired in HET mice compared to littermate-matched WT controls (67 ± 3% versus 76 ± 3%, respectively; p < 0.05), while responses to sodium nitroprusside were unchanged. Short-term incubation of carotid rings with the NAD(P)H oxidase inhibitor, apocynin, significantly improved in vessels from HET mice but not their WT littermates. Peak tension generated in response to either U46619 or serotonin was significantly blunted in HET mice compared to their WT littermates. Conclusion: These data suggest that SIRT6 is a key regulator of vasomotor function in conduit vessels. More specifically, we propose that SIRT6 serves as a tonic suppressor of NAD(P)H oxidase expression and activation, as inhibition of NAD(P)H oxidase improved endothelial function in SIRT6 haploinsufficient mice. Collectively, SIRT6 activation and/or histone acetyltransferase inhibition may be useful therapeutic approaches to reduce endothelial dysfunction and combat age-associated cardiovascular disease.
RESUMO
Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.
Assuntos
Procedimentos Cirúrgicos Robóticos , Animais , Rim/diagnóstico por imagem , Imageamento por Ressonância Magnética , Camundongos , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , UltrassonografiaRESUMO
Angiotensin II (Ang II) is an oligopeptide of the renin-angiotensin system, and Ang II-induced vascular smooth muscle cell (VSMC) proliferation is an important pathophysiological process involved in atherosclerosis; however, the underlying mechanism remains unclear. Orai1 and Stim1 are the main components of store-operated Ca2+ entry (SOCE), which has an important effect on VSMC proliferation. In the present study, we showed that Ang II-induced human coronary smooth muscle cell (HCSMC) proliferation was associated with increased calcium entry. The expression of Orai1, but not that of Stim1, was significantly upregulated in Ang II-treated HCSMCs. However, knockdown of Orai1 or Stim1 decreased HCSMC proliferation and SOCE activity in Ang II-treated HCSMCs. Orai1 was significantly downregulated in HCSMCs transfected with short interfering RNA (siRNA) against NOX2 or NF-κB. Transfection with siRNA against NOX2 or p65 also decreased Ang II-induced HCSMCs SOCE activation and proliferation. These findings suggested that Ang II upregulated Orai1 via the NF-κB and NOX2 pathways, leading to increased SOCE and HCSMC proliferation. The molecular factors mediating Ang II-induced SOCE upregulation are potential therapeutic targets for the prevention of Ang II-sensitive or Ang II-dependent HCSMC proliferation.
Assuntos
Angiotensina II/farmacologia , Cálcio/metabolismo , Músculo Liso Vascular/citologia , Proteína ORAI1/metabolismo , Angiotensina II/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/citologia , Humanos , NADPH Oxidase 2/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Store depletion has been shown to induce Ca2+ entry by Na+/Ca+ exchange (NCX) 1 reversal in proliferative vascular smooth muscle cells (VSMCs). The study objective was to investigate the role of transient receptor potential canonical (TRPC) channels in store depletion and NCX1 reversal in proliferative VSMCs. In cultured VSMCs, expressing TRPC1, TRPC4, and TRPC6, the removal of extracellular Na+ was followed by a significant increase of cytosolic Ca2+ concentration that was inhibited by KBR, a selective NCX1 inhibitor. TRPC1 knockdown significantly suppressed store-operated, channel-mediated Ca2+ entry, but TRPC4 knockdown and TRPC6 knockdown had no effect. Separate knockdown of TRPC1, TRPC4, or TRPC6 did not have a significant effect on thapsigargin-initiated Na+ increase in the peripheral regions with KBR treatment, but knockdown of both TRPC4 and TRPC6 did. Stromal interaction molecule (STIM)1 knockdown significantly reduced TRPC4 and TRPC6 binding. The results demonstrated that TRPC4-TRPC6 heteromultimerization linked Ca2+ store depletion and STIM1 accumulation with NCX reversal in proliferative VSMCs.
Assuntos
Miócitos de Músculo Liso/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Trocador de Sódio e Cálcio/antagonistas & inibidores , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/deficiênciaRESUMO
BACKGROUND: Na+/Ca2+ exchange (NCX) reversal-mediated Ca2+ entry is a critical pathway for stimulating proliferation in many cell lines. However, the role of reverse-mode NCX1 in neointima formation and atherosclerosis remains unclear. The aims of the present study were to investigate the functional role of NCX1 in the pathogenesis of atherosclerosis and vascular smooth muscle cell (VSMC) proliferation, and to determine the interaction between NCX1 and store depletion in VSMCs. METHODS: A rat balloon injury model was established to examine the effect of the knockdown of NCX1 on neointima formation after injury. VSMCs were cultured to verify that NCX1 knockdown suppressed serum-induced VSMC proliferation. RESULTS: The results showed that balloon injury induced neointima formation and upregulated NCX1 expression at 7 and 14 days after injury in rat carotid arteries (1.18- and 1.45-fold, respectively). A lentivirus vector expressing short hairpin (sh)RNA against rat NCX1 dramatically downregulated NCX1, proliferating cell nuclear antigen (PCNA) and Ki-67 expression, and suppressed neointima formation in vivo (62% at 7 days and 70% at 14 days). KB-R7943 (an inhibitor of reverse-mode NCX1) and NCX1 knockdown significantly inhibited serum-induced VSMC proliferation (65% at 72 hours and 41% at 72 hours, respectively), determined according to PCNA and Ki-67 expression and cell counting in vitro, and markedly suppressed store depletion-mediated Ca2+ entry and peripheral cytosolic Na+ transients in VSMCs. CONCLUSIONS: Reverse-mode NCX1 is activated by store depletion and is required for proliferative VSMC proliferation and neointima formation after arterial injury.
Assuntos
Aterosclerose/metabolismo , Artérias Carótidas , Neointima/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Lesões do Sistema Vascular/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Proliferação de Células , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Silenciamento de Genes , Antígeno Ki-67/metabolismo , Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Tioureia/análogos & derivados , Tioureia/farmacologiaRESUMO
While reports suggest a single dose of senolytics may improve vasomotor function, the structural and functional impact of long-term senolytic treatment is unknown. To determine whether long-term senolytic treatment improves vasomotor function, vascular stiffness, and intimal plaque size and composition in aged or hypercholesterolemic mice with established disease. Senolytic treatment (intermittent treatment with Dasatinib + Quercetin via oral gavage) resulted in significant reductions in senescent cell markers (TAF(+) cells) in the medial layer of aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques. While senolytic treatment significantly improved vasomotor function (isolated organ chamber baths) in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice. Genetic clearance of senescent cells in aged normocholesterolemic INK-ATTAC mice phenocopied changes elicited by D+Q. Senolytics tended to reduce aortic calcification (alizarin red) and osteogenic signaling (qRT-PCR, immunohistochemistry) in aged mice, but both were significantly reduced by senolytic treatment in hypercholesterolemic mice. Intimal plaque fibrosis (picrosirius red) was not changed appreciably by chronic senolytic treatment. This is the first study to demonstrate that chronic clearance of senescent cells improves established vascular phenotypes associated with aging and chronic hypercholesterolemia, and may be a viable therapeutic intervention to reduce morbidity and mortality from cardiovascular diseases.
Assuntos
Envelhecimento/patologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Senescência Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Quercetina/farmacologia , Sistema Vasomotor/fisiopatologia , Animais , Dano ao DNA , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Hipercolesterolemia/complicações , Hipercolesterolemia/patologia , Camundongos , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1(-/Δ) mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1(-/∆) mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.
Assuntos
Envelhecimento/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Osteoporose/prevenção & controle , Quercetina/farmacologia , Transcriptoma , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/patologia , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Senescência Celular/genética , Classe I de Fosfatidilinositol 3-Quinases , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Combinação de Medicamentos , Endonucleases/genética , Endonucleases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Efrinas/genética , Efrinas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Coração/efeitos dos fármacos , Coração/fisiopatologia , Disco Intervertebral/química , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Knockout , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidor 2 de Ativador de Plasminogênio/genética , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
AIMS: Myxomatous mitral valve disease (MMVD) is associated with leaflet thickening, fibrosis, matrix remodelling, and leaflet prolapse. Molecular mechanisms contributing to MMVD, however, remain poorly understood. We tested the hypothesis that increased transforming growth factor-ß (TGF-ß) signalling and reactive oxygen species (ROS) are major contributors to pro-fibrotic gene expression in human and mouse mitral valves. METHODS AND RESULTS: Using qRT-PCR, we found that increased expression of TGF-ß1 in mitral valves from humans with MMVD (n = 24) was associated with increased expression of connective tissue growth factor (CTGF) and matrix metalloproteinase 2 (MMP2). Increased levels of phospho-SMAD2/3 (western blotting) and expression of SMAD-specific E3 ubiquitin-protein ligases (SMURF) 1 and 2 (qRT-PCR) suggested that TGF-ß1 signalling occurred through canonical signalling cascades. Oxidative stress (dihydroethidium staining) was increased in human MMVD tissue and associated with increases in NAD(P)H oxidase catalytic subunits (Nox) 2 and 4, occurring despite increases in superoxide dismutase 1 (SOD1). In mitral valves from SOD1-deficient mice, expression of CTGF, MMP2, Nox2, and Nox4 was significantly increased, suggesting that ROS can independently activate pro-fibrotic and matrix remodelling gene expression patterns. Furthermore, treatment of mouse mitral valve interstitial cells with cell permeable antioxidants attenuated TGF-ß1-induced pro-fibrotic and matrix remodelling gene expression in vitro. CONCLUSION: Activation of canonical TGF-ß signalling is a major contributor to fibrosis and matrix remodelling in MMVD, and is amplified by increases in oxidative stress. Treatments aimed at reducing TGF-ß activation and oxidative stress in early MMVD may slow progression of MMVD.