Mechanisms For Microcalcification Of Heart Valves

Calcified aortic valve disease(CAVD)is a widespread degenerative disease,and it is also one of the most common indications for adult valve surgery.At present,the effects of drug therapy on valve calcification at the middle and late stages are not good.Therefore,early intervention in the early stage of valve calcification has become of great interest for research.CAVD has been previously considered as a passive degenerative process.Recent studies reveal that valve calcification is an active biological process,similar to bone formation,and multiple osteogenic related factors get involved.The analysis of bone tissue showed that calcium and phosphorus ions first entered into the stromal vesicles and formed minerals in their membranes.After that,the ruptured vesicles released hydroxyapatite into extracellular matrix as a new nucleation site and continued to crystallize.The release of matrix vesicles with HA crystals has been found during the calcification of valves,however,the underlying mechanisms for formation of calcified nodules through release of matrix vesicles remain unknown.Magnesium ions(Mg2++)as one of the major components of human bone has been widely used to promote the growth and regeneration of bone.On the other hand,Mg2+ion can inhibit the formation of HA crystals from amorphous calcium phosphate.However,the effects of Mg2+ ions on the calcification of heart valves is still unclear.This paper mainly analyzes the calcification process in the early stage of aortic valve calcification,and discusses the specific role of magnesium ion in the calcification stage of valve.The specific content is divided into the following aspects:(1)On the basis of enzymolysis,the primary interstitial cells were extracted from the 68-month-old pig valves.By controlling the digestion time of type Ⅱ and type Ⅲ collagenase,the primary interstitial cells were obtained after repeated digestion and centrifugation,and cultured in vitro;(2)The 3-8 generations of valve interstitial cells were induced by high calcium(2.7 mM Ca2+)high phosphates(2.5 mM PO43-)medium,and a 2D cell calcification model was established in vitro.The results of alizarin red staining,scanning electron microscopy and real-time quantitative PCR showed that the interstitial cells could be induced successfully.On the second day,a small amount of calcium nodules composed of calcified particles grew on the surface and inside of the cells,ranging in size from tens of nanometers to several microns.After 4 days,the calcified granules increased significantly,forming calcium nodules of micron size;(3)With the increase of magnesium ion concentration,the calcification was improved gradually,which indicated that magnesium ion inhibited the calcification of valve stromal cells;(4)It was found that there were apatite like particles in the vesicles of the high calcium and high phosphates group,but no similar particles in the high magnesium group.The difraction results showed that the components in the vesicles were amorphous.To sum up,the calcification process similar to osteogenesis can occur in the interstitial cells of aortic valve.The calcification may form amorphous in MVs,and then crystallize and nucleate outside.Magnesium ion can inhibit the calcification of valve interstitial cells successfully,which proves that magnesium ion can inhibit the calcification of valve.The possible mechanism is that magnesium ion can directly inhibit the transformation and differentiation of VICs into osteoblast like cells,and at the same time,it can not form apatite in MVs,which provides a new idea for the follow-up study of anti calcification.