Research Models for Studying Vascular Calcification

Calcification of the vessel wall contributes to high cardiovascular morbidity and mortality. Vascular calcification (VC) is a systemic disease with multifaceted contributing and inhibiting factors in an actively regulated process. The exact underlying mechanisms are not fully elucidated and reliable treatment options are lacking. Due to the complex pathophysiology, various research models exist evaluating different aspects of VC. This review aims to give an overview of the cell and animal models used so far to study the molecular processes of VC. Here, in vitro cell culture models of different origins, ex vivo settings using aortic tissue and various in vivo disease-induced animal models are summarized. They reflect different aspects and depict the (patho)physiologic mechanisms within the VC process

Pathophysiologie der vaskulären Kalzifizierung: Mechanismen und Therapieoptionen

Vessel calcifications are a main risk factor for the development of cardiovascular diseases, a leading cause of morbidity and mortality in western societies. Even though vascular calcifications have been researched for centuries, patients still suffer from a huge therapeutic gap, because neither an effective prophylaxis, nor an effective treatment is available. Currently, consent exists that vascular calcification is an active cellular process that resembles bone formation. Vascular Smooth Muscle Cells (VSMCs) and their transdifferentiation into osteoblast like cells are considered decisive in the formation of vascular calcifications. Several stimuli can induce osteoblastic transdifferentiation, including cellular aging processes, oxidative stress and inflammation. These factors can be linked into a concept termed inflammaging via the formation of damage associated molecular patterns, persistent DNA damage and induction of a senescence associated secretory phenotype. The objective of this dissertation was to analyse the effects of the cellular stressors Azathioprine and Doxorubicin and the pro-inflammatory cytokine interleukin 1ß on the induction of osteoblastic transdifferentiation of VSMCs and vascular calcification in different experimental settings. The cellular stressors induce the formation of reactive oxygen species, cellular senescence, secretion of pro-inflammatory cytokines such as interleukin 1ß and interleukin 6, upregulation of NLRP3 inflammasome components as well as osteoblastic transdifferentiation and calcification of VSMCs. Stimulation with interleukin 1ß induced a pro-inflammatory auto-loop, osteoblastic transdifferentiation and calcification of VSMCs, but did not induce markers of senescence in VSMCs apart from components of the cellular senescence associated secretory phenotype. For both, cellular stressors and interleukin 1ß, the NLRP3 inflammasome is decisively involved in the calcification process, as inhibition of NLRP3 significantly reduces the calcification induced by cellular stressors and interleukin 1ß. This dissertation is in line with other recent research that emphasizes the involvement of inflammaging in the pathology of vascular calcification. Several therapeutic targets arise from the insight that oxidative stress, inflammation and senescence are crucially involved in the pathophysiology of VSMC calcification. These new therapeutic approaches can help to meet the high unmet clinical need of patients suffering from vascular calcifications

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

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-1 beta 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

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

Calcification and chronic inflammation of the vascular wall is a high-risk factor for cardiovascular mortality, especially in patients with chronic uremia. For the reduction or prevention of rapid disease progression, no specific treatment options are currently available. This study aimed to evaluate an adenine-based uremic mouse model for studying medial vessel calcification and senescence-associated secretory phenotype (SASP) changes of aortic tissue to unravel molecular pathogenesis and provide a model for therapy testing. The dietary adenine administration induced a stable and similar degree of chronic uremia in DBA2/N mice with an increase of uremia blood markers such as blood urea nitrogen, calcium, creatinine, alkaline phosphatase, and parathyroid hormone. Also, renal fibrosis and crystal deposits were detected upon adenine feeding. The uremic condition is related to a moderate to severe medial vessel calcification and subsequent elastin disorganization. In addition, expression of osteogenic markers as Bmp-2 and its transcription factor Sox-9 as well as p21 as senescence marker were increased in uremic mice compared to controls. Pro-inflammatory uremic proteins such as serum amyloid A, interleukin (I1)-1 beta, and I1-6 increased. This novel model of chronic uremia provides a simple method for investigation of signaling pathways in vascular inflammation and calcification and therefore offers an experimental basis for the development of potential therapeutic intervention studies

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

Calcification and chronic inflammation of the vascular wall is a high-risk factor for cardiovascular mortality, especially in patients with chronic uremia. For the reduction or prevention of rapid disease progression, no specific treatment options are currently available. This study aimed to evaluate an adenine-based uremic mouse model for studying medial vessel calcification and senescence-associated secretory phenotype (SASP) changes of aortic tissue to unravel molecular pathogenesis and provide a model for therapy testing. The dietary adenine administration induced a stable and similar degree of chronic uremia in DBA2/N mice with an increase of uremia blood markers such as blood urea nitrogen, calcium, creatinine, alkaline phosphatase, and parathyroid hormone. Also, renal fibrosis and crystal deposits were detected upon adenine feeding. The uremic condition is related to a moderate to severe medial vessel calcification and subsequent elastin disorganization. In addition, expression of osteogenic markers as Bmp-2 and its transcription factor Sox-9 as well as p21 as senescence marker were increased in uremic mice compared to controls. Pro-inflammatory uremic proteins such as serum amyloid A, interleukin (Il)-1β, and Il-6 increased. This novel model of chronic uremia provides a simple method for investigation of signaling pathways in vascular inflammation and calcification and therefore offers an experimental basis for the development of potential therapeutic intervention studies. Graphical abstractOpen Access funding enabled and organized by Projekt DEAL.Ernst und Berta Grimmke Stiftung http://dx.doi.org/10.13039/501100008436Berlin-Institute-of-HealthSonnenfeld Stiftung http://dx.doi.org/10.13039/100010121DynAge Focus AreaNanchong school science and technology strategic cooperation projectCharité - Universitätsmedizin Berlin (3093

Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists

To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall-degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7-38% are orphan genes, including genes that encode secreted effector-like proteins. Convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a \u27symbiosis toolkit\u27, with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes

A Novel Protocol for Detection of Senescence and Calcification Markers by Fluorescence Microscopy

Vascular calcification and stiffening of the arterial wall is a systemic phenomenon that is associated with aging and it can be increased by several risk factors. The underlying mechanisms, especially the pathways of cellular senescence, are under current investigation. Easily manageable in vitro settings help to study the signaling pathways. The experimental setting presented here is based on an in vitro model using rat vascular smooth muscle cells and the detection of senescence and osteoblastic markers via immunofluorescence and RNAscope™. Co-staining of the senescence marker p21, the osteoblastic marker osteopontin, detection of senescence-associated heterochromatin foci, and senescence-associated β-galactosidase is possible within one test approach requiring fewer cells. The protocol is a fast and reliable evaluation method for multiplexing of calcifying and senescence markers with fluorescence microscopy detection. The experimental setting enables analysis on single cell basis and allows detection of intra-individual variances of cultured cells

Research Models for Studying Vascular Calcification

Calcification of the vessel wall contributes to high cardiovascular morbidity and mortality. Vascular calcification (VC) is a systemic disease with multifaceted contributing and inhibiting factors in an actively regulated process. The exact underlying mechanisms are not fully elucidated and reliable treatment options are lacking. Due to the complex pathophysiology, various research models exist evaluating different aspects of VC. This review aims to give an overview of the cell and animal models used so far to study the molecular processes of VC. Here, in vitro cell culture models of different origins, ex vivo settings using aortic tissue and various in vivo disease-induced animal models are summarized. They reflect different aspects and depict the (patho)physiologic mechanisms within the VC process.</jats:p

Long-Term Treatment of Azathioprine in Rats Induces Vessel Mineralization

Medial vascular calcification (mVC) is closely related to cardiovascular disease, especially in patients suffering from chronic kidney disease (CKD). Even after successful kidney transplantation, cardiovascular mortality remains increased. There is evidence that immunosuppressive drugs might influence pathophysiological mechanisms in the vessel wall. Previously, we have shown in vitro that mVC is induced in vascular smooth muscle cells (VSMCs) upon treatment with azathioprine (AZA). This effect was confirmed in the current study in an in vivo rat model treated with AZA for 24 weeks. The calcium content increased in the aortic tissue upon AZA treatment. The pathophysiologic mechanisms involve AZA catabolism to 6-thiouracil via xanthine oxidase (XO) with subsequent induction of oxidative stress. Proinflammatory cytokines, such as interleukin (IL)-1ß and IL-6, increase upon AZA treatment, both systemically and in the aortic tissue. Further, VSMCs show an increased expression of core-binding factor α-1, alkaline phosphatase and osteopontin. As the AZA effect could be decreased in NLRP3−/− aortic rings in an ex vivo experiment, the signaling pathway might be, at least in part, dependent on the NLRP3 inflammasome. Although human studies are necessary to confirm the harmful effects of AZA on vascular stiffening, these results provide further evidence of induction of VSMC calcification under AZA treatment and its effects on vessel structure.</jats:p