A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition &lt;em&gt;In Vitro&lt;/em&gt;

Jaminon, Armand M. G.; Rapp, Nikolas; Akbulut, Asim Cengiz; Dzhanaev, Robert; Reutelingsperger, Chris; Jahnen‐Dechent, Willi; Schurgers, Leon J.

doi:10.3791/64029

Cited by 2 publications

(2 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ectopic calcification poses a significant clinical challenge that currently lacks effective diagnostic and treatment options 31 . Previously we demonstrated the use of fluorescence labeled fetuin-A, a major circulating inhibitor of calcification, for the detection of microcalcifications in mouse tissues 13 and in cell culture 32 . Here, we further enhanced fluorescent fetuin-A staining with genetically labeled derivates.…”

Section: Discussionmentioning

confidence: 99%

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions

Dzhanaev¹,

Hasberg²,

Gorgels³

et al. 2023

Theranostics

Self Cite

View full text Add to dashboard Cite

Rationale: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel diagnostic tools capable of detecting nascent calcium phosphate deposits are highly desirable. Methods: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate, mEmerald or mRuby3, respectively. The proteins were expressed in mammalian cell lines. Sequence optimization resolved folding issues and increased sensitivity of mineral binding. Chimeric proteins were tested for their ability to detect calcifications in cell cultures and tissue sections retrieved from calcification-prone mice. Results: We employed novel genetically labeled fetuin-A-based fluorescent proteins for the detection of ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity. Conclusion: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fluorescent probes for imaging microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. They also surpass in continuous live cell imaging the chemically fluorescence labeled fetuin-A, which we established previously.

show abstract

Section: Discussionmentioning

confidence: 99%

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions

Dzhanaev¹,

Hasberg²,

Gorgels³

et al. 2023

Theranostics

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, evaluation of calcium density through CACS with non-contrast computed tomography was reviewed by focusing on 3 main areas: (1) CAC scan acquisition parameters; (2) pathophysiology of calcified plaques; and (3) epidemiologic evidence relating calcium density to ASCVD outcomes [ 35 ]. Future and present development in this area include automated cardiovascular risk categorization through artificial intelligence-driven coronary calcium quantification in cardiac PET acquired attenuation correction CCT [ 36 ] or machine learning approach for cardiovascular risk and CACS [ 37 ] or even new in vitro tools [ 38 ].…”

Section: Calcium Score and Atherosclerosismentioning

confidence: 99%

Subclinical Hypertension-Mediated Organ Damage (HMOD) in Hypertension: Atherosclerotic Cardiovascular Disease (ASCVD) and Calcium Score

Rizzoni

Agabiti-Rosei

Ciuceis

et al. 2022

High Blood Press Cardiovasc Prev

View full text Add to dashboard Cite

Calcium controls numerous events within the vessel wall. Permeability of the endothelium is calcium dependent, as are platelet activation and adhesion, vascular smooth muscle proliferation and migration, and synthesis of fibrous connective tissue. Double-helix computerized tomography is a noninvasive technique that can detect, measure, and compare coronary calcification in the coronary arteries. Despite some convincing evidence about the prognostic value and usefulness of coronary artery calcium score (CACS) in the stratification of cardiovascular risk in the high risk general population and also in hypertensive patients, current guidelines for the management of hypertension, do not include such evaluation among the recommended procedures to be performed in the majority of patients even with the intent to detect hypertension-mediated organ damage (HMOD) in an early phase. On the contrary, the European Society of Cardiology guidelines for the diagnosis and management of chronic coronary syndromes, the 2019 ACC/AHA Guideline on the Primary Prevention of Cardiovascular Disease, and the 2018 Cholesterol Clinical Practice Guidelines indicate that the evaluation of CACS may be of some usefulness in specific subpopulations, although this view is not accepted in the US Preventive Services Task Force document. Very recently, the European Society of Cardiology Guidelines on cardiovascular disease prevention in clinical practice stated that CACS estimation may be considered to improve risk classification around treatment decision thresholds. In conclusion, the use of CACS as a diagnostic tool is still controversial. While some evidence exists about is ability to improve stratification of cardiovascular risk in primary prevention, in particular in selected patients who are at intermediate or borderline risk of atherosclerotic cardiovascular disease, there is insufficient evidence to use it as a standard means to assess HMOD.

show abstract

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition <em>In Vitro</em>

Cited by 2 publications

References 6 publications

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions

Subclinical Hypertension-Mediated Organ Damage (HMOD) in Hypertension: Atherosclerotic Cardiovascular Disease (ASCVD) and Calcium Score

Contact Info

Product

Resources

About