Stressor-Induced “Inflammaging” of Vascular Smooth Muscle Cells via Nlrp3-Mediated Pro-inflammatory Auto-Loop

Abstract

Calcification of the vessel wall as one structural pathology of aged vessels is associated with high cardiovascular mortality of elderly patients. Aging is linked to chronic sterile inflammation and high burden of reactive oxygen species (ROS), leading to activation of pattern recognition receptors (PRRs) such as Nlrp3 in vascular cells. The current study investigates the role of PRR activation in the calcification of vascular smooth muscle cells (VSMCs). Therefore, in vitro cell culture of primary rat VSMCs and ex vivo aortic stimulations were used to analyze osteogenic, senescence and inflammatory markers via real-time PCR, in situ RNA hybridization, Western Blot, photometric assays and histological staining. Induction of ROS and DNA-damage by doxorubicin induces a shift of VSMC phenotype toward the expression of osteogenic, senescence and inflammatory proteins. Induction of calcification is dependent on Nlrp3 activity. Il-1β as a downstream target of Nlrp3 induces the synthetic, pro-calcifying VSMC phenotype. Inhibition of PRR with subsequent reduction of chronic inflammation might be an interesting target for reduction of calcification of VSMCs, with subsequent reduction of cardiovascular mortality of patients suffering from vessel stiffness.

Molecular Imaging and Quantification of Smooth Muscle Cell and Aortic Tissue Calcification In Vitro and Ex Vivo with a Fluorescent Hydroxyapatite-Specific Probe

“…Multiplexing on a single cell level not only allows reduction of primary cells in the thought of the ThreeR (3R), referring to the guiding principles “Replace, Reduce, Refine” for more ethical use of animals for scientific research formulated by Russel and Burch [ 13 ], but also permits a direct comparison of the markers of interest in the signaling cascade in one cell [ 14 ]. As already known from our own and others’ previous studies, doxorubicin is a potent inducer of DNA damage and calcification in VSMC and was therefore used as a positive control [ 12 , 14 , 15 ], in addition to the stimulation of cells with COM and CAM as before.…”

“…In addition, co-staining of several markers, e.g., DNA damage via γH2A.X, allows single-cell-based visualization and direct comparison of, e.g., a senescence or osteogenic marker in a single cell with HAP crystals surrounding the cell [ 14 ]. For induction of DNA damage, the known inducer doxorubicin was used [ 12 , 14 , 15 ]. The combination of OsteoSense™ detection with other fluorescence-labeled senescence or osteogenic markers is also possible and therefore offers advantages concerning a reduced primary cell consumption according to the 3R principles [ 13 ] and additional experimental read out information.…”

Uremic mouse model to study vascular calcification and “inflamm-aging”

“…In addition, studies have shown that the pathways of Il-1β and Il-6 are interconnected [ 36 ]. Recently, we could show that Il-6 is induced during calcification progression, but not directly induces mineralization, while Il-1β induces an osteogenic driven auto-loop in smooth muscle cells [ 37 ]. Il-1β and Il-6 are known middle-sized uremic toxins influencing osteogenic differentiation [ 9 ].…”

Proteomics Revealed That Mitochondrial Function Contributed to the Protective Effect of Herba Siegesbeckiae Against Cardiac Ischemia/Reperfusion Injury

“…The activated caspase-1 then promotes the maturation and release of interleukin (IL)-1β and IL-18, and induces the synthesis of more amount of inflammatory factors, such as IL-6 and tumor necrosis factor (TNF)-α, thus leading to an inflammatory cascade response ( 15 , 16 ). Moreover, activated caspase-1 can shear and activate Gasdermin D (GSDMD) protein, causing pyroptosis and aggravating mitochondrial damage ( 17 ). Recent studies in heart failure with preserved ejection fraction mice model and Sirt3-KO mice demonstrated that mitochondrial overacetylation promotes ASC aggregation into perinuclear mitochondria and NLRP3 inflammasome formation, implying that NLRP3 inflammasome activation is sensitive to mitochondrial function ( 18 ).…”