Mitochondria are dynamic organelles regulating metabolism, cell death, and energy production. Therefore, maintaining mitochondrial health is critical for cellular homeostasis. Mitophagy and mitochondrial reorganization via fission and fusion are established mechanisms for ensuring mitochondrial quality. In recent years, mitochondrial-derived vesicles (MDVs) have emerged as a novel cellular response. MDVs are shed from the mitochondrial surface and can be directed to lysosomes or peroxisomes for intracellular degradation. MDVs may contribute to cardiovascular disease (CVD) which is characterized by mitochondrial dysfunction. In addition, evidence suggests that mitochondrial content is present in extracellular vesicles (EVs). Herein, we provide an overview of the current knowledge on MDV formation and trafficking. Moreover, we review recent findings linking MDV and EV biogenesis and discuss their role in CVD. Finally, we discuss the role of vesicle-mediated mitochondrial transfer and its potential cardioprotective effects.
IntroductionVascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using in vitro mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways.Materials and methods and resultsHuman coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs–a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease–a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and in silico drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). In vitro validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP.ConclusionOur direct comparison results of two in vitro calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification in vitro. We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in in vitro studies using different calcification protocols.
Introduction: Vascular calcification (VC) is a significant risk factor for cardiovascular morbidity and mortality. Based on the mineral deposition site in the arterial wall, VC classifies into intimal and medial calcification. We hypothesize that distinct in vitro mineralization methods promote specific intracellular signalling pathways in vascular smooth muscle cells (SMC), reflecting both VC types. Methods and Results: Human coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium-phosphate medium (CaP) to induce calcification. OM resembles SMCs differentiation in intimal calcification - a key process in atherosclerotic plaque remodeling. CaP is associated with chronic kidney disease - a risk factor for medial calcification. Transcriptomics revealed a distinct gene expression profile of OM and CaP-calcifying SMCs, that share 6.9% and 11.3% of their genes, respectively. The 109 shared dysregulated genes between OM and CaP-calcifying SMCs highlighted enriched pathways related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated a different metabolic profile in OM and CaP-calcifying SMCs. We observed decreased mitochondrial respiration and glycolysis (-57,3% p=0.029) by CaP and increased mitochondrial respiration without altered glycolysis by OM. Subsequent kinome and in silico drug repurposing analysis (Connectivity Map) revealed a distinct role of protein kinase C (PKC). Validation using prostratin, a specific PKC activator, demonstrated differential effects on matrix mineralization that was decreased by OM (-69,3%, p<0.001) and increased by CaP (+69,6%, p=0.044). Conclusions: In conclusion, OM and CaP-induced SMC calcification underlies differential mechanisms in vitro. Therefore, research should distinguish between the two aspects of VC and increased knowledge about the underlying pathophysiological mechanisms may open the possibility for preventing intimal and medial calcification.
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