Mitochondrial dysfunction is implicated in a wide range of disorders, including cancer, neurodegeneration, and cardiovascular diseases. Conventional assays typically assess mitochondrial function by measuring bulk respiration rates across thousands of cells. While informative, these approaches cannot resolve the behavior of individual mitochondria, may overlook rare mitochondrial events, and often lack sensitivity to subtle changes. Fluorescence microscopy provides single-organelle resolution but is usually low throughput; for example, imaging 1000 cells can require from 20 minutes to an entire day depending on imaging mode. Additionally, analysis of fluorescence images frequently relies on manually selected thresholds, introducing potential bias and variability due to differences in signal-to-background ratios across cells. Here, we present a High-throughput Mitochondrial Imaging Platform (H-MIP) combined with a deep learning segmentation model, enabling rapid imaging and automated characterization of mitochondrial morphology in millions of mitochondria from tens of thousands of cells. We demonstrate the utility of H-MIP by assessing mitochondrial morphology following treatment with Mdivi-1, an inhibitor of mitochondrial division. Furthermore, we employ an in vitro disease model (vascular calcification) to show that mitochondria become elongated in vascular smooth muscle cells undergoing pathological calcification. Overall, H-MIP provides a scalable and robust tool for investigating mitochondrial structure, with the potential to accelerate therapeutic discovery targeting mitochondrial health. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/J004316/1, BBS/E/D/20221657, BBS/E/RL/230001C, BB/Y513982/1 British Heart Foundation, RE/18/5/34216 Wellcome Trust, grant 210752 European Research Council, 866411 & 101113551 & 101213822 German Research Foundation, TRR-359

Chronic kidney disease-mineral and bone disorder (CKD-MBD) presents with extra-skeletal calcification and renal osteodystrophy (ROD). However, the pathophysiology of ROD remains unclear. Here we examine the hypothesis that stalled mitophagy within osteocytes of CKD-MBD mouse models contributes to bone loss. RNA-seq analysis revealed an altered expression of genes associated with mitophagy and mitochondrial function in tibia of CKD-MBD mice. The expression of mitophagy regulators, p62/SQSTM1, ATG7 and LC3 was inconsistent with functional mitophagy, and in mito-QC reporter mice with ROD, there was a two to three-fold increase in osteocyte mitolysosomes. To determine if uremic toxins were potentially responsible for these observations, treatment of cultured osteoblasts with uremic toxins revealed increased mitolysosome number and mitochondria with distorted morphology. Membrane potential and oxidative phosphorylation were also decreased, and oxygen-free radical production increased. The altered p62 /SQSTM1 and LC3-II expression was consistent with impaired mitophagy machinery, and the effects of uremic toxins were reversible by rapamycin. A causal link between uremic toxins and the development of mitochondrial abnormalities and ROD was established by showing that a mitochondria-targeted antioxidant (MitoQ) and the charcoal adsorbent AST-120 were able to mitigate the uremic toxin-induced mitochondrial changes and improve bone health. Overall, our study shows that impaired clearance of damaged mitochondria may contribute to the ROD phenotype. Targeting uremic toxins, oxygen-free radical production and the mitophagy process may offer novel routes for intervention to preserve bone health in patients with CKD-MBD. This would be timely as our current armamentarium of anti-fracture medications for patients with severe CKD-MBD is limited.

Autophagy is a highly conserved cellular recycling process essential for homeostasis in all eukaryotic cells. Lipid accumulation and its regulation by autophagy are key areas of research for understanding metabolic disorders in human and model mammals. However, the role of autophagy in lipid regulation remains poorly characterized in non-model fish species of importance to food production, which could be important for managing health and welfare in aquaculture. Addressing this knowledge gap, we investigate the role of autophagy in lipid regulation using a macrophage-like cell line (SHK-1) from Atlantic salmon (Salmo salar L.), the world's most commercially valuable farmed finfish. Multiple lines of experimental evidence reveal that the autophagic pathway responsible for lipid droplet breakdown is conserved in Atlantic salmon cells. We employed global lipidomics and proteomics analyses on SHK-1 cells subjected to lipid overload, followed by treatment with rapamycin to induce autophagy. This revealed that activating autophagy via rapamycin enhances storage of unsaturated triacylglycerols and suppresses key lipogenic proteins, including fatty acid elongase 6, fatty acid binding protein 2 and acid sphingomyelinase. Moreover, fatty acid elongase 6 and fatty acid binding protein 2 were identified as possible cargo for autophagosomes, suggesting a critical role for autophagy in lipid metabolism in fish. Together, this study establishes a novel model of lipotoxicity and advances understanding of lipid autophagy in fish cells, with significant implications for addressing fish health issues in aquaculture.

Myxomatous mitral valve degeneration (MMVD) is one of the most important age-dependent degenerative heart valve disorders in both humans and dogs. It is characterized by the aberrant remodeling of extracellular matrix (ECM), regulated by senescent myofibroblasts (aVICs) transitioning from quiescent valve interstitial cells (qVICs), primarily under TGFB1/TGF-beta 1 control. In the present study, we found senescent aVICs exhibited impaired macroautophagy/autophagy as evidenced by compromised autophagy flux and immature autophagosomes. MTOR-dependent autophagy induced by rapamycin and torin-1 attenuated cell senescence and decreased the expression of cyclin-dependent kinase inhibitors (CDKIs) CDKN2A/p16INK4A and CDKN1A/p21CIP1. Furthermore, induction of autophagy in aVICs by ATG (autophagy related) gene overexpression restored autophagy flux, with a concomitant reduction in CDKN1A and CDKN2A expression and senescence-associated secretory phenotype (SASP). Conversely, autophagy deficiency induced CDKN1A and CDKN2A accumulation and SASP, whereas ATG re-expression alleviated senescent phenotypic transformation. Notably, CDKN1A and CDKN2A localized to autophagosomes and lysosomes following MTOR antagonism or MG132 treatment. SQSTM1/p62 was identified as the autophagy receptor to selectively sequester CDKN1A and CDKN2A cargoes for autophagic degradation. Our findings are the first demonstration that CDKN1A and CDKN2A are degraded through SQSTM1-mediated selective autophagy, independent of the ubiquitin-proteasome pathway. These data will inform development of therapeutic strategies for the treatment of canine and human MMVD, and for the treatment of Alzheimer disease, Parkinson disease and other age-related degenerative disorders.Abbreviations: ACTA2/alpha-SMA: actin alpha 2, smooth muscle; AKT: AKT serine/threonine kinase; aVICs: activated valve interstitial cells; ATG: autophagy related; baf-A1: bafilomycin A1; BrdU, bromodeoxyuridine; BSA: bovine serum albumin; CDKIs, cyclin-dependent kinase inhibitors; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; co-IP: co-immunoprecipitation; DMSO: dimethylsulfoxide; ECM, extracellular matrix; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; eGFP: green fluorescent protein; ELISA: enzyme-linked immunosorbent assay; HEK-293T, human embryonic kidney 293T; HRP: horseradish peroxidase; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LIR: MAP1LC3/LC3-interacting region; MFS: Marfan syndrome; MKI67/Ki-67: marker of proliferation Ki-67; MMVD: myxomatous mitral valve degeneration; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; OE: overexpression; PBST, phosphate-buffered saline with 0.1% Tween-20; PCNA: proliferating cell nuclear antigen; PIK3CA/PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PLA: proximity ligation assays; PSMA1: proteasome 20S subunit alpha 1; PSMB5: proteasome 20S subunit beta 5; qVICs: quiescent valve interstitial cells; qRT-PCR: quantitative real-time PCR; SA-GLB1/beta-gal: SA-senescence-associated GLB1/beta-galactosidase; ROS: reactive oxygen species; SASP: senescence-associated secretory phenotype; RPS6KB1/p70 S6K: ribosomal protein S6 kinase B1; SMAD: SMAD family member; SQSTM1/p62: sequestosome 1; STEM: scanning transmission electron microscopy; TGFB: transforming growth factor beta; TGFBR: transforming growth factor beta receptor; TP53/p53: tumor protein p53; UPS: ubiquitin-proteasome system; WT, wild-type.

Aims Calcific aortic valve disease (CAVD) is a common heart valve disease with significant clinical consequences. The mechanisms that drive the pathogenesis of CAVD remain to be fully elucidated. N6-methyladenosine (m6A), the most prevalent RNA epigenetic regulator, has recently been implicated in cardiovascular disease, but its role in CAVD has yet to be investigated. In this study, we investigated the potential function of m6A modification in CAVD.Methods and results Using clinical samples from CAVD patients in combination with human valve interstitial cell (hVIC) calcification model, we screened the expression of m6A modulators and discovered that alkB homolog 5, RNA demethylase (ALKBH5), a key m6A demethylase, was significantly down-regulated in calcified hVICs and human aortic valves. Consistently, increased m6A levels were seen in calcified hVICs, and treatment with 3-deazaadenosine (DAA), an inhibitor of m6A modification, significantly reduced hVIC osteogenic differentiation and calcification. In addition, we showed that silencing of ALKBH5 expression increased global m6A levels and accelerated hVIC osteogenic differentiation and calcification, whereas overexpression of ALKBH5 resulted in the opposite effect. We demonstrated that ALKBH5 directly modulate m6A levels of TGFBR2 and its mRNA stability, leading to altered TGFBR2 expression and SMAD2 signalling in hVICs. We further showed that inhibition of TGFBR2 or knockdown of SMAD2 attenuated ALKBH5 knockdown-induced hVIC osteogenic differentiation and calcification. The expression of the m6A reader protein YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) was up-regulated during the process of hVIC calcification. Intriguingly, we revealed that the ALKBH5 silencing-induced increased hVIC osteogenic differentiation and calcification were abolished after knockdown of YTHDF1. These data suggest a potential role YTHDF1 in aortic valve calcification.Conclusion This study showed that ALKBH5 attenuated aortic valve calcification through the TGFBR2/SMAD2 signalling pathway via direct m6A modification of TGFBR2.

Individuals diagnosed with Parkinson’s disease (PD) often exhibit heightened susceptibility to cardiac dysfunction, reflecting a complex interaction between these conditions. The involvement of mitochondrial dysfunction in the development and progression of cardiac dysfunction and PD suggests a plausible commonality in some aspects of their molecular pathogenesis, potentially contributing to the prevalence of cardiac issues in PD. Mitochondria, crucial organelles responsible for energy production and cellular regulation, play important roles in tissues with high energetic demands, such as neurons and cardiac cells. Mitochondrial dysfunction can occur in different and non-mutually exclusive ways; however, some mechanisms include alterations in mitochondrial dynamics, compromised bioenergetics, biogenesis deficits, oxidative stress, impaired mitophagy, and disrupted calcium balance. It is plausible that these factors contribute to the increased prevalence of cardiac dysfunction in PD, suggesting mitochondrial health as a potential target for therapeutic intervention. This review provides an overview of the physiological mechanisms underlying mitochondrial quality control systems. It summarises the diverse roles of mitochondria in brain and heart function, highlighting shared pathways potentially exhibiting dysfunction and driving cardiac comorbidities in PD. By highlighting strategies to mitigate dysfunction associated with mitochondrial impairment in cardiac and neural tissues, our review aims to provide new perspectives on therapeutic approaches.

Alterations in ABCC6 underpin pseudoxanthoma elasticum (PXE), a rare heritable disorder characterized by the degeneration of elastic fibers in the skin, eyes, and cardiovascular system. Given that ABCC6 is predominantly expressed in hepatocytes, research over the past 2 decades has traditionally focused on a liver-centric hypothesis underpinning PXE. However, an alternate cellular hypothesis in which resident cells in affected tissues play a prominent role has recently gained traction. Using a multipronged approach through histological, molecular, and pathological assessment, the study by Brampton et al (2024) highlights for the first time a direct contribution of bone marrow–derived ABCC6, revealing a new axis through which immune cells dramatically influence the development of the calcification phenotype in PXE.

Vascular calcification is frequently seen in patients with chronic kidney disease (CKD), and significantly increases cardiovascular mortality and morbidity. Sirt7, a NAD+-dependent histone deacetylases, plays a crucial role in cardiovascular disease. However, the role of Sirt7 in vascular calcification remains largely unknown. Using in vitro and in vivo models of vascular calcification, this study showed that Sirt7 expression was significantly reduced in calcified arteries from mice administered with high dose of vitamin D3 (vD3). We found that knockdown or inhibition of Sirt7 promoted vascular smooth muscle cell (VSMC), aortic ring and vascular calcification in mice, whereas overexpression of Sirt7 had opposite effects. Intriguingly, this protective effect of Sirt7 on vascular calcification is dependent on its deacetylase activity. Unexpectedly, Sirt7 did not alter the osteogenic transition of VSMCs. However, our RNA-seq and subsequent studies demonstrated that knockdown of Sirt7 in VSMCs resulted in increased intracellular reactive oxygen species (ROS) accumulation, and induced an Nrf-2 mediated oxidative stress response. Treatment with the ROS inhibitor N-acetylcysteine (NAC) significantly attenuated the inhibitory effect of Sirt7 on VSMC calcification. Furthermore, we found that knockdown of Sirt7 delayed cell cycle progression and accelerated cellular senescence of VSMCs. Taken together, our results indicate that Sirt7 regulates vascular calcification at least in part through modulation of ROS and cellular senescence of VSMCs. Sirt7 may be a potential therapeutic target for vascular calcification.

PI3K/AKT/mTOR signalling contributes to several cardiovascular disorders. The aim of this study was to examine the PI3K/AKT/mTOR pathway in myxomatous mitral valve disease (MMVD). Double-immunofluorescence examined expression of PI3K and TGF-β1 in canine valves. Valve interstitial cells (VICs) from healthy or MMVD dogs were isolated and characterized. Healthy quiescent VICs (qVICs) were treated with TGF-β1 and SC-79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve-derived aVICs were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K) was modulated using siRNA and gene overexpression. SA-β-gal and TUNEL staining were used to identify cell senescence and apoptosis, and qPCR and ELISA to examine for senescence-associated secretory phenotype. Protein immunoblotting was used to examine expression of phosphorylated and total proteins. TGF-β1 and PI3K are highly expressed in mitral valve tissues. Activation of PI3K/AKT/mTOR and increased expression of TGF-β are found in aVICs. TGF-β transitions qVICs to aVICs by upregulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. TGF-β-induced PI3K/AKT/mTOR signalling contributes to MMVD pathogenesis and plays crucial roles in the regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.

Recent advancements in the bone biology field have identified a novel bone-metabolism axis. In this review, we highlight several novel studies that further our knowledge of new endocrine functions of bone; explore remaining unanswered questions; and discuss translational challenges in this complex era of bone biology research.

Arterial calcification is an important characteristic of cardiovascular disease. It has key parallels with skeletal mineralization; however, the underlying cellular mechanisms responsible are not fully understood. Mitochondrial dynamics regulate both bone and vascular function. In this study, we therefore examined mitochondrial function in vascular smooth muscle cell (VSMC) calcification. Phosphate (Pi)-induced VSMC calcification was associated with elongated mitochondria (1.6-fold increase, p < 0.001), increased mitochondrial reactive oxygen species (ROS) production (1.83-fold increase, p < 0.001) and reduced mitophagy (9.6-fold decrease, p < 0.01). An increase in protein expression of optic atrophy protein 1 (OPA1; 2.1-fold increase, p < 0.05) and a converse decrease in expression of dynamin-related protein 1 (DRP1; 1.5-fold decrease, p < 0.05), two crucial proteins required for the mitochondrial fusion and fission process, respectively, were noted. Furthermore, the phosphorylation of DRP1 Ser637 was increased in the cytoplasm of calcified VSMCs (5.50-fold increase), suppressing mitochondrial translocation of DRP1. Additionally, calcified VSMCs showed enhanced expression of p53 (2.5-fold increase, p < 0.05) and β-galactosidase activity (1.8-fold increase, p < 0.001), the cellular senescence markers. siRNA-mediated p53 knockdown reduced calcium deposition (8.1-fold decrease, p < 0.01), mitochondrial length (3.0-fold decrease, p < 0.001) and β-galactosidase activity (2.6-fold decrease, p < 0.001), with concomitant mitophagy induction (3.1-fold increase, p < 0.05). Reduced OPA1 (4.1-fold decrease, p < 0.05) and increased DRP1 protein expression (2.6-fold increase, p < 0.05) with decreased phosphorylation of DRP1 Ser637 (3.20-fold decrease, p < 0.001) was also observed upon p53 knockdown in calcifying VSMCs. In summary, we demonstrate that VSMC calcification promotes notable mitochondrial elongation and cellular senescence via DRP1 phosphorylation. Furthermore, our work indicates that p53-induced mitochondrial fusion underpins cellular senescence by reducing mitochondrial function.

The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.

A link between increased glycolysis and vascular calcification has recently been reported, but it remains unclear how increased glycolysis contributes to vascular calcification. We therefore investigated the role of PFKFB3, a critical enzyme of glycolysis, in vascular calcification. We found that PFKFB3 expression was upregulated in calcified mouse VSMCs and arteries. We showed that expression of miR-26a-5p and miR-26b-5p in calcified mouse arteries was significantly decreased, and a negative correlation between Pfkfb3 mRNA expression and miR-26a-5p or miR-26b-5p was seen in these samples. Overexpression of miR-26a/b-5p significantly inhibited PFKFB3 expression in VSMCs. Intriguingly, pharmacological inhibition of PFKFB3 using PFK15 or knockdown of PFKFB3 ameliorated vascular calcification in vD3 -overloaded mice in vivo or attenuated high phosphate (Pi)-induced VSMC calcification in vitro. Consistently, knockdown of PFKFB3 significantly reduced glycolysis and osteogenic transdifferentiation of VSMCs, whereas overexpression of PFKFB3 in VSMCs induced the opposite effects. RNA-seq analysis and subsequent experiments revealed that silencing of PFKFB3 inhibited FoxO3 expression in VSMCs. Silencing of FoxO3 phenocopied the effects of PFKFB3 depletion on Ocn and Opg expression but not Alpl in VSMCs. Pyruvate or lactate supplementation, the product of glycolysis, reversed the PFKFB3 depletion-mediated effects on ALP activity and OPG protein expression in VSMCs. Our results reveal that blockade of PFKFB3-mediated glycolysis inhibits vascular calcification in vitro and in vivo. Mechanistically, we show that FoxO3 and lactate production are involved in PFKFB3-driven osteogenic transdifferentiation of VSMCs. PFKFB3 may be a promising therapeutic target for the treatment of vascular calcification.

Calcific aortic valve disease (CAVD) is the most common heart disease of the developed world. It has previously been established that metformin administration reduces arterial calcification via autophagy; however, whether metformin directly regulates CAVD has yet to be elucidated. In the present study we investigated whether metformin alleviates valvular calcification through the autophagy-mediated recycling of Runx2. Calcification was reduced in rat valve interstitial cells (RVICs) by metformin treatment (0.5–1.5 mM) ( P < 0.01), with a marked decrease in Runx2 protein expression compared to control cells ( P < 0.05). Additionally, upregulated expression of Atg3 and Atg7 (key proteins required for autophagosome formation), was observed following metformin treatment (1 mM). Blocking autophagic flux using Bafilomycin-A1 revealed colocalisation of Runx2 with LC3 puncta in metformin treated RVICs ( P < 0.001). Comparable Runx2 accumulation was seen in LC3 positive autolysosomes present within cells that had been treated with both metformin and hydroxychloroquine in combination ( P < 0.001). Mechanistic studies employing three-way co-immunoprecipitation with Runx2, p62 and LC3 suggested that Runx2 binds to LC3-II upon metformin treatment in VICs. Together these studies suggest that the utilisation of metformin may represent a novel strategy for the treatment of CAVD.

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1 −/− mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1 −/− mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1 −/− mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1 −/− mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1 −/−  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.

BackgroundTGFβ signaling appears to contribute to the pathogenesis of myxomatous mitral valve disease (MMVD) in both dogs and humans. However, little is known about the extent of the downstream signaling changes that will then affect cell phenotype and function in both species.ObjectiveIdentify changes in downstream signals in the TGFβ pathway in canine MMVD and examine the effects of antagonism of one significant signal (SMAD2 was selected).Materials and methodsCanine cultures of normal quiescent valve interstitial cells (qVICs) and disease-derived activated myofibroblasts (aVICs) (n = 6) were examined for TGFβ signaling protein expression using a commercial antibody array. Significant changes were confirmed, and additional proteins of interest downstream in the TGFβ signaling pathway and markers of cell phenotype were examined (PRAS40, S6K, elF4E IRS-1, αSMA, and VIM), using protein immunoblotting. RT-PCR examined expression of gene markers of VIC activation (ACTA2, TAGLN, and MYH10; encoding the proteins αSMA, SM22, and Smemb, respectively). Attenuation of pSMAD2 in aVICs was examined using a combination of RNA interference technology (siRNA) and the SMAD7 (antagonizes SMAD2) agonist asiaticoside.ResultsThe antibody array identified significant changes (P < 0.05) in 19 proteins, of which six were phosphorylated (p). There was increased expression of pSMAD2 and pRAC1 and decreased expression of pmTOR, pERK1/2, and pAKT1. Expression of pPRAS40 and pIRS-1 was increased, as was the mTOR downstream transcription factor pS6K, with increased expression of peIF4E in aVICs, indicating negative feedback control of the PI3K/AKT/mTOR pathway. SMAD2 antagonism by siRNA and the SMAD7 agonist asiaticoside decreased detection of pSMAD by at least 50%, significantly decreased expression of the aVIC gene markers ACTA2, TAGLN, and MYH10, and pαSMA, pAKT2, and pERK1, but had no effect on pS6K, pERK2, or pVIM expression in aVICs. SMAD2 antagonism transitioned diseased aVICs to normal qVICs, while maintaining a mesenchymal phenotype (VIM+) while concurrently affecting non-canonical TGFβ signaling.ConclusionMMVD is associated with changes in both the canonical and non-canonical TGFβ signaling pathway. Antagonism of SMAD2 transitions diseased-activated myofibroblasts back to a normal phenotype, providing data that will inform studies on developing novel therapeutics to treat MMVD in dogs and humans.