Abstract 658: Unraveling the Controversy of Bisphosphonates as Vascular Calcification Therapy Using a Nanoanalytical Approach

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jessica L Ruiz ◽  
Joshua D Hutcheson ◽  
Elena Aikawa
Keyword(s):  
Vascular Calcification ◽  
Plaque Rupture ◽  
Three Dimensional ◽  
Building Blocks ◽  
Underlying Mechanism ◽  
Pharmacological Intervention ◽  
Fibrous Cap ◽  
Collagen Hydrogel ◽  
Increased Risk

Vascular calcification significantly predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates, a proposed therapy for vascular calcification, paradoxically indicated increased risk in patients with prior acute events. We recently demonstrated that calcifying extracellular vesicles (EVs) released by cells within the plaque aggregate and nucleate calcific mineral, but the underlying mechanism and the potential for pharmacological intervention remain poorly understood. We hypothesize that bisphosphonates block EV aggregation and arrest existing mineral growth, freezing calcifications in a high-risk morphology that hastens plaque rupture. This study visualized for the first time EV aggregation and calcification at single-EV resolution, via scanning electron microscopy. Three-dimensional (3-D) collagen hydrogels incubated with calcifying EVs modeled fibrous cap calcification, serving as an in vitro platform to image mineral nucleation and test candidate drugs for the potential to inhibit or reverse vascular calcification. EVs aggregated along and between collagen fibrils. Energy-dispersive x-ray spectroscopy (EDS) confirmed that EV aggregates contained calcium and phosphorous, the building blocks of calcific mineral (vs. internal collagen control, p<0.001). The addition of the bisphosphonate ibandronate decreased the EDS-detected amount of calcium (4.32% by weight (wt%) vs. 2.36 wt%, p<0.001) and phosphorous (4.26 wt% vs. 1.94 wt%, p<0.001) comprising EV aggregates. Further, ibandronate reduced the size (21.5 μm 2 vs. 14.2 μm 2 , p=0.012) and changed the morphology of calcific EV aggregates (Figure). These findings agree with our hypothesis that bisphosphonates alter EV-driven calcification, and confirm that our 3-D collagen hydrogel system is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

scholarly journals Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model

2021 ◽  
Vol 118 (14) ◽  
pp. e1811725118
Author(s):  
Jessica L. Ruiz ◽  
Joshua D. Hutcheson ◽  
Luis Cardoso ◽  
Amirala Bakhshian Nik ◽  
Alexandra Condado de Abreu ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Cardiovascular Events ◽  
Plaque Rupture ◽  
Three Dimensional ◽  
Element Analysis ◽  
Fibrous Cap ◽  
3D Collagen ◽  
Atherosclerotic Plaque Rupture

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE−/− mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

scholarly journals Three-Dimensional Biofabrication Models of Endometriosis and the Endometriotic Microenvironment

2020 ◽  
Author(s):  
Jillian R. H. Wendel ◽  
Xiyin Wang ◽  
Lester J. Smith ◽  
Shannon M. Hawkins
Keyword(s):  
Cell Line ◽  
Three Dimensional ◽  
Uterine Cavity ◽  
Building Blocks ◽  
Cell Types ◽  
In Vitro Models ◽  
Inflammatory Factors ◽  
Biologically Relevant ◽  
Increased Risk

Endometriosis occurs when endometrial-like tissue grows outside the uterine cavity, leading to pelvic pain, infertility, and increased risk of ovarian cancer. The present study describes the optimization and characterization of cellular spheroids as building blocks for Kenzan scaffold-free method biofabrication and proof-of-concept models of endometriosis and the endometriotic microenvironment. The spheroid building blocks must be a specific diameter (~500 m), compact, round, and smooth to withstand Kenzan biofabrication. Under optimized spheroid conditions for biofabrication, the endometriotic epithelial-like cell line, 12Z, expressed high levels of estrogen-related genes and secreted high amounts of endometriotic inflammatory factors that were independent of TNF stimulation. Heterotypic spheroids, composed of 12Z and T-HESC, an immortalized endometrial stromal cell line, self-assembled into a biologically relevant pattern, consisting of epithelial cells on the outside of the spheroids and stromal cells in the core. 12Z spheroids were biofabricated into large three-dimensional constructs alone, with HEYA8 spheroids, or as heterotypic spheroids with T-HESC. These three-dimensional biofabricated constructs containing multiple monotypic or heterotypic spheroids represent the first scaffold-free biofabricated in vitro models of endometriosis and the endometriotic microenvironment. These efficient and innovative models will allow us to study the complex interactions of multiple cell types within a biologically relevant microenvironment.

scholarly journals Three-Dimensional Biofabrication Models of Endometriosis and the Endometriotic Microenvironment

Biomedicines ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 525
Author(s):  
Jillian R. H. Wendel ◽  
Xiyin Wang ◽  
Lester J. Smith ◽  
Shannon M. Hawkins
Keyword(s):  
Cell Line ◽  
Three Dimensional ◽  
Uterine Cavity ◽  
Building Blocks ◽  
Cell Types ◽  
In Vitro Models ◽  
Inflammatory Factors ◽  
Biologically Relevant ◽  
Increased Risk

Endometriosis occurs when endometrial-like tissue grows outside the uterine cavity, leading to pelvic pain, infertility, and increased risk of ovarian cancer. The present study describes the optimization and characterization of cellular spheroids as building blocks for Kenzan scaffold-free method biofabrication and proof-of-concept models of endometriosis and the endometriotic microenvironment. The spheroid building blocks must be of a specific diameter (~500 μm), compact, round, and smooth to withstand Kenzan biofabrication. Under optimized spheroid conditions for biofabrication, the endometriotic epithelial-like cell line, 12Z, expressed high levels of estrogen-related genes and secreted high amounts of endometriotic inflammatory factors that were independent of TNFα stimulation. Heterotypic spheroids, composed of 12Z and T-HESC, an immortalized endometrial stromal cell line, self-assembled into a biologically relevant pattern, consisting of epithelial cells on the outside of the spheroids and stromal cells in the core. 12Z spheroids were biofabricated into large three-dimensional constructs alone, with HEYA8 spheroids, or as heterotypic spheroids with T-HESC. These three-dimensional biofabricated constructs containing multiple monotypic or heterotypic spheroids represent the first scaffold-free biofabricated in vitro models of endometriosis and the endometriotic microenvironment. These efficient and innovative models will allow us to study the complex interactions of multiple cell types within a biologically relevant microenvironment.

scholarly journals The CAD risk locus 9p21 increases the risk of vascular calcification in an iPSC-derived VSMC model

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anja Trillhaase ◽  
Beatrice Schmidt ◽  
Marlon Märtens ◽  
Undine Haferkamp ◽  
Jeanette Erdmann ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Vascular Calcification ◽  
Alizarin Red ◽  
Vitro Assay ◽  
Human Ipscs ◽  
Increased Risk ◽  
Risk Locus ◽  
9P21 Locus ◽  
Cad Risk

Abstract Background Coronary artery disease (CAD) is the leading cause of death worldwide. Chromosome locus 9p21 was the first to be associated with increased risk of CAD and coronary artery calcification (CAC). Vascular calcification increases the risk for CAD. Vascular smooth muscle cells (VSMCs) are one of the major cell types involved in the development of vascular calcification. Methods So far, mainly animal models or primary SMCs have been used to model human vascular calcification. In this study, a human in vitro assay using iPSC-derived VSMCs was developed to examine vascular calcification. Human iPSCs were derived from a healthy non-risk (NR) and risk (R) donor carrying SNPs in the 9p21 locus. Additionally, 9p21 locus knockouts of each donor iPSC line (NR and R) were used. Following differentiation, the iPSC-derived VSMCs were characterized based on cell type, proliferation, and migration rate, along with calcium phosphate (CaP) deposits. CaP deposits were confirmed using Calcein and Alizarin Red S staining and then quantified. Results The data demonstrated significantly more proliferation, migration, and CaP deposition in VSMCs derived from the R and both KO iPSC lines than in those derived from the NR line. Molecular analyses confirmed upregulation of calcification markers. These results are consistent with recent data demonstrating increased calcification when the 9p21 murine ortholog is knocked-out. Conclusion Therefore, in conclusion, genetic variation or deletion of the CAD risk locus leads to an increased risk of vascular calcification. This in vitro human iPSC model of calcification could be used to develop new drug screening strategies to combat CAC.

Vitamin K deficiency: an emerging player in the pathogenesis of vascular calcification and an iatrogenic consequence of therapies in advanced renal disease

2020 ◽  
Vol 319 (4) ◽  
pp. F618-F623
Author(s):  
David S. Levy ◽  
Rickinder Grewal ◽  
Thu H. Le
Keyword(s):  
Bone Metabolism ◽  
Vascular Calcification ◽  
Vitamin K ◽  
Hospital Admissions ◽  
Fibroblast Growth Factor 23 ◽  
Vitamin K Deficiency ◽  
Increased Risk ◽  
K Deficiency ◽  
Human Aortic Valve

Vascular calcification is a known complication of chronic kidney disease (CKD). The prevalence of vascular calcification in patients with non-dialysis-dependent CKD stages 3–5 has been shown to be as high as 79% ( 20 ). Vascular calcification has been associated with increased risk for mortality, hospital admissions, and cardiovascular disease ( 6 , 20 , 50 , 55 ). Alterations in mineral and bone metabolism play a pivotal role in the pathogenesis of vascular calcification in CKD. As CKD progresses, levels of fibroblast growth factor-23, parathyroid hormone, and serum phosphorus increase and levels of 1,25-(OH)2 vitamin D decrease. These imbalances have been linked to the development of vascular calcification. More recently, additional factors have been found to play a role in vascular calcification. Matrix G1a protein (MGP) in its carboxylated form (cMGP) is a potent inhibitor of vascular calcification. Importantly, carboxylation of MGP is dependent on the cofactor vitamin K. In patients with CKD, vitamin K deficiency is prevalent and is exacerbated by warfarin, which is frequently used for anticoagulation. Insufficient bioavailability of vitamin K reduces the amount of cMGP available, and, therefore, it may lead to increased risk of vascular calcification. In vitro studies have shown that in the setting of a high-phosphate environment and vitamin K antagonism, human aortic valve interstitial cells become calcified. In this article, we discuss the pathophysiological consequence of vitamin K deficiency in the setting of altered mineral and bone metabolism, its prevalence, and clinical implications in patients with CKD.

scholarly journals The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues

2021 ◽  
Author(s):  
Marina Spoerrer ◽  
Delf Kah ◽  
Richard C Gerum ◽  
Barbara Reischl ◽  
Danyil Huraskin ◽  
...  
Keyword(s):  
Striated Muscle ◽  
Contractile Function ◽  
Three Dimensional ◽  
Mechanical Damage ◽  
Underlying Mechanism ◽  
Intermediate Filament Protein ◽  
Wild Type ◽  
Age Related ◽  
Contractile Forces

Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronized contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared to tissues grown from wild-type cells. Within one week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than five seconds, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fiber breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

scholarly journals The emerging role of iron in heart failure and vascular calcification in CKD

2020 ◽  
Author(s):  
Paola Ciceri ◽  
Mario Cozzolino
Keyword(s):  
Heart Failure ◽  
Iron Deficiency ◽  
Vascular Calcification ◽  
Strong Association ◽  
In Vivo Models ◽  
Risk Of Death ◽  
Increased Risk ◽  
Cv Disease

Abstract Iron deficiency is a frequent comorbidity of cardiovascular (CV) diseases and nearly 50% of patients with heart failure (HF) with or without anaemia have low levels of available iron. There is a strong association between anaemia and the increase in mortality and hospitalizations in patients with CV disease and HF. Moreover, anaemia and chronic kidney disease (CKD) often coexist in patients with HF, with anaemia increasing the risk of death in these subjects and with a further increased risk in CKD population. The evidence that the treatment of iron deficiency and the increase in haemoglobin are associated with a better prognosis in HF patients has elicited new interest in the utilization of iron in HF and CKD patients. One of the central players in CV disease is vascular calcification (VC), which has been recognized as a major independent risk factor for incident CV disease and overall mortality in chronic disease patients. In this review, we summarize the evidences generated by clinical trials aimed to study the effect of iron deficiency correction, the effect of iron-based phosphate binder in in vivo models of kidney failure and the effect of iron in in vitro models of VC, trying to give an overview of the present knowledge on iron effect and its mechanisms of action.

scholarly journals Host metabolism dysregulation and cell tropism identification in human airway and alveolar organoids upon SARS-CoV-2 infection

2020 ◽  
Author(s):  
Rongjuan Pei ◽  
Jianqi Feng ◽  
Yecheng Zhang ◽  
Hao Sun ◽  
Lian Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Neutralizing Antibody ◽  
Underlying Mechanism ◽  
Alveolar Type ◽  
Physiological Conditions ◽  
Therapeutic Drugs ◽  
Distal Airway

AbstractThe coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.

scholarly journals Partial Hepatectomy-Induced Upregulation of miR-1907 Accelerates Liver Regeneration by Activation Autophagy

2018 ◽  
Vol 2018 ◽  
pp. 1-9
Author(s):  
Tianfei Lu ◽  
Jun Hao ◽  
Chuan Shen ◽  
Guangxiang Gu ◽  
Jianjun Zhang ◽  
...  
Keyword(s):  
Body Weight ◽  
Liver Regeneration ◽  
Partial Hepatectomy ◽  
Weight Ratio ◽  
Underlying Mechanism ◽  
Hepatocyte Proliferation ◽  
Pharmacological Intervention ◽  
Body Weight Ratio

Liver regeneration after partial hepatectomy (PH) is a highly orchestrated biological process in which synchronized hepatocyte proliferation is induced after massive liver mass loss. Hepatocyte proliferation could be regulated by multiple signals, such as miRNAs and autophagy, but underlying mechanism remains unclear. Here a functional miRNA during liver regeneration was identified and its underlying mechanism was delineated in vitro and in vivo. We found that miR-1907 was highly upregulated during liver regeneration after 2/3 PH at various timepoints. The level of miR-1907 was also increased in normal liver cell line treated with HGF at different concentrations. Functionally, miR-1907 enhanced hepatocyte proliferation in vitro and in vivo, and the liver/body weight ratio in miR-1907-overexpressed mice was significantly higher in comparison to the control mice after 2/3 PH. Forced expression of miR-1907 promoted autophagy activation of hepatocyte. Importantly, autophagy inhibition significantly attenuated miR-1907-induced hepatocyte proliferation and the liver/body weight ratio. Finally, GSK3β, a suppressor of autophagy signaling, was identified as the direct target gene of miR-1907. Taken together, miR-1907 accelerates hepatocyte proliferation during liver regeneration by activating autophagy; thus pharmacological intervention regulating miR-1907/autophagy axis may be therapeutically beneficial in liver transplantation and liver failure by inducing liver regeneration.

Immunomodulatory derivatives induce PU.1 down-regulation, myeloid maturation arrest, and neutropenia

Blood ◽  
2010 ◽  
Vol 115 (3) ◽  
pp. 605-614 ◽  
Author(s):  
Rekha Pal ◽  
Sara A. Monaghan ◽  
Andrea Cortese Hassett ◽  
Markus Y. Mapara ◽  
Peter Schafer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
Venous Thromboembolism ◽  
Platelet Aggregation ◽  
Underlying Mechanism ◽  
Cathepsin G ◽  
Immunomodulatory Drugs ◽  
Maturation Arrest ◽  
Increased Risk

AbstractThe immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide yield high response rates in patients with multiple myeloma, but the use of IMiDs in multiple myeloma is associated with neutropenia and increased risk for venous thromboembolism (VTE) by mechanisms that are unknown. We show that IMiDs down-regulate PU.1, a key transcription factor involved in granulocyte differentiation in vitro and in patients treated with lenalidomide. Loss of PU.1 results in transient maturation arrest with medullary accumulation of immature myeloid precursors and subsequent neutropenia. Accumulation of promyelocytes leads to high levels of the platelet aggregation agonist, cathepsin G stored in the azurophilic granules of promyelocytes. High levels of cathepsin G subsequently may increase the risk of VTE. To our knowledge, this is the first report investigating the underlying mechanism of IMiD-induced neutropenia and increased risk of VTE in multiple myeloma.

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