Nano-hydroxyapatite Accelerates Vascular Calcification Via Lysosome Impairment And Autophagy Dysfunction In Smooth Muscle Cells

Background:Vascular calcification（VC）is a common characteristic of aging,diabetes,chronic renal failure,and atherosclerosis.Vascular calcification（VC）is a physiological process that predominantly involves the apoptosis of vascular smooth muscle cells（VSMCs）and the phenotypic transformation of VSMCs into osteoblast-like cells.During calcification,cytoplasmic Ca²⁺and Pi incorporate with alkaline phosphatase（ALP）into matrix vesicles,which bud off the plasma membrane and associate with extracellular proteins,such as collagen.Crystals initially form octacalcium phosphate,which reorganizes and stimulates the epitaxial growth of highly insoluble hydroxyapatite（HAp）;HAp then repeats nucleation and crystallization in the same approach and expands the deposition area.Studies have showed that nanosized hydroxyapatite（n HAp or nano-HAp）can induce VC,however the underlying mechanisms remain unknown.Recently,nanoparticles were identified to affect autophagy.And autophagy plays a role in vascular calcification according to previous studies.However,there lacks evidence that nano-HAp could induce autophagy changes in VSMCs.Therefore,we hypothesized that nano-HAp might promote extracellular calcification deposition of VSMCs via modulating autophagy pathway.Objective:To clarify the specific role of n HAp in the occurrence and development of vascular calcification,and provide a new idea for the prevention and control of vascular calcification.To verify whether n HAp can cause vascular calcification in vivo and in vitro models,and to study whether n HAp can cause extracellular calcification of smooth muscle cells by affecting changes in autophagy function in vitro,and to further explore the mechanism.Methods:Human calcified aorta specimens were collected and the characteristics of calcified aorta were identified by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）and immunofluorescence techniques.n HAp was synthesized and extracted from human calcified plaques respectively,and identified and compared by SEM,XRD and FT-IR.Mouse VSMCs were isolated and amplified.n HAp was added to culture medium in vitro.The Alizarin Red staining,calcium content,the expressions of osteogenic genes Runx2 and OPN,and alkaline phosphatase（ALP）activity were detected to demonstrate the role of n HAp in SMCs.n HAp was implanted subcutaneously and smeared on the surface of abdominal aorta respectively in C57BL/6J mice.Micro-CT scanning and pathological section staining were performed to determine the effect of n HAp on smooth muscle cell calcification in vivo.In vitro,TEM,immunofluorescence staining and Western blot analysis were used to determine the mechanism of n HAp-induced smooth muscle cell calcification.Results:We found nanoscale HAp in human calcification specimens,n HAp adhered to the surface of vascular cells,and n HAp was found in intracellular lysosomes.The expression of Runx2,a calcification marker in VSMCs near calcification deposition,is obvious.The above research results laid a foundation for the follow-up research.Synthetic n HAp and n HAp extracted from human specimens were similar in morphology,size and chemical composition,which provided support for the subsequent experimental study to replace human n HAp with synthetic n HAp.In vitro,after n HAp treatment of SMCs,the expressions of calcification markers Runx2,OPN and ALP activity were significantly increased compared with the control group,and n HAp resulted in a large amount of calcium deposition extracellularly,which was significantly higher than that in the normal calcification medium group.These studies suggested that n HAp can induce osteoblastic transformation of SMCs and accelerate calcification.In vivo,n HAp induced calcium deposition in subcutaneous grafts and abdominal aorta and increased Runx2 expression in SMCs.In order to elucidate the mechanism of n HAp-induced calcification,TEM showed that n HAp could be endocytosis into lysosomes of SMCs treated with both human origin and synthetic n HAp,and the number of autophagosomes increased significantly compared with the blank control group.After n HAp treatment,LC3-II and p62 in cells were increased.LC3 dual fluorescent adenovirus confirmed that n HAp induced the blockage of autophagy flux in SMCs.Immunofluorescence co-staining of LC3 and lysosomal markers（Rab7,LAMP1）showed that n HAp did not affect the fusion of autophagosomes and lysosomes.Therefore,the next step is to explore the function of lysosomes.After n HAp treatment,acidizing capacity of lysosomes decreased,the mature phase of CTSD decreased,and the immature phase increased.EGFR degradation experiments showed that n HAp affected the degradation of EGFR in lysosome,suggesting that the lysosome dysfunction affected autophagy degradation.The co-localization rate of lysosomal pump V-ATPase and LAMP1 decreased,suggesting that the damage of proton pump may be the reason for the decreased capacity of lysosomal acidification.Exosomes from supernatant of VSMCs were extracted,and n HAp promoted the release of exosomes,and calcium content in exosomes was significantly increased.Moreover,LC3 and LAMP1 in exosomes were significantly increased in n HAp treatment group.Exosome release inhibitor GW4869 can down-regulate exosome release induced by n HAp and inhibit calcium deposition.Finally,the mechanism of osteoblastic transformation of SMCs was discussed.n HAp treatment of VSMCs can activate c-Jun amino-terminal kinase/c-Jun（JNK/c-Jun）pathway phosphorylation,JNK phosphorylation inhibitor SP600125 can down-regulate the expression of osteogenesis markers Runx2 and OPN as well as ALP activity,n HAp-induced calcium deposition is also decreased.Conclusions:On the one hand,n HAp promotes the osteogenic transformation of smooth muscle cells by activating the JNK/c-Jun signaling pathway;On the other hand,n HAp impairs the function of lysosome by destroying the lysosome pump,affecting autophagy degradation,leading to intracellular accumulation of autophagosomes and autophagosomes,promoting the release of exosomes,and accelerating vascular calcification deposition.