What is the clinical evidence from this study?

Study design: Other. Population: Aortic Valve Calcification. Intervention: Lipopolysaccharide and inorganic phosphate vs. Media alone. Primary outcome: Calcium accumulation (percentage area of positive Alizarin Red staining).

October 1, 2021Open Access

Organ Culture Model of Aortic Valve Calcification

Key Result

Incubation of intact porcine aortic valve leaflets with lipopolysaccharide and inorganic phosphate induced calcified nodules, which were inhibited by adenosine in a concentration-dependent manner.

Structured PICO

Does adenosine prevent calcification induced by lipopolysaccharide and inorganic phosphate in an ex-vivo porcine aortic valve leaflet model?

Population

Ex vivo organ culture model using intact porcine aortic valve leaflets to study calcification mechanisms.

Intervention

Lipopolysaccharide (100 ng/mL) and inorganic phosphate (3 mM) to induce calcification, with or without adenosine (10^-8 to 10^-5 M) to assess pharmacological inhibition, cultured for 14 days

Comparator

Media alone (negative control) or lipopolysaccharide and inorganic phosphate without adenosine (positive control)

Outcome

Calcification quantified by the percentage area of positive Alizarin Red staining viewed under polarized lightsurrogate

An ex-vivo organ culture model using intact porcine aortic valve leaflets successfully demonstrates calcification induced by lipopolysaccharide and phosphate, which can be pharmacologically inhibited by adenosine.

Limitations

The effect of LPS and phosphate takes at least 2 weeks to develop.
The time-course experiment was based on data from only 2 animals.
Relies on the use of porcine tissue, which may not respond in an identical manner to human tissue.

Abstract

A significant amount of knowledge has been gained with the use of cell-based assays to elucidate the mechanisms that mediate heart valve calcification. However, cells used in these studies lack their association with the extra-cellular matrix or the influence of other cellular components of valve leaflets. We have developed a model of calcification using intact porcine valve leaflets, that relies upon a biological stimulus to drive the formation of calcified nodules within the valve leaflets. Alizarin Red positive regions were formed in response to lipopolysaccharide and inorganic phosphate, which could be quantified when viewed under polarized light. Point analysis and elemental mapping analysis of electron microscope images confirmed the presence of nodules containing calcium and phosphorus. Immunohistochemical staining showed that the development of these calcified regions corresponded with the expression of RUNX2, osteocalcin, NF-kB and the apoptosis marker caspase 3. The formation of calcified nodules and the expression of bone markers were both inhibited by adenosine in a concentration-dependent manner, illustrating that the model is amenable to pharmacological manipulation. This organ culture model offers an increased level of tissue complexity in which to study the mechanisms that are involved in heart valve calcification.

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