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Vascular calcification (VC) is a common characteristic of aging, diabetes, chronic renal failure, and atherosclerosis. The basic component of VC is hydroxyapatite (HAp). Nano-sized HAp (nHAp) has been identified to play an essential role in the development of pathological calcification of vasculature. However, whether nHAp can induce calcification in vivo and the mechanism of nHAp in the progression of VC remains unclear. We discovered that nHAp existed both in vascular smooth muscle cells (VSMCs) and their extracellular matrix (ECM) in the calcified arteries from patients. Synthetic nHAp had similar morphological and chemical properties as natural nHAp recovered from calcified artery. nHAp stimulated osteogenic differentiation and accelerated mineralization of VSMCs in vitro. Synthetic nHAp could also directly induce VC in vivo. Mechanistically, nHAp was internalized into lysosome, which impaired lysosome vacuolar H+-ATPase for its acidification, therefore blocked autophagic flux in VSMCs. Lysosomal re-acidification by cyclic-3′,5′-adenosine monophosphate (cAMP) significantly enhanced autophagic degradation and attenuated nHAp-induced calcification. The accumulated autophagosomes and autolysosomes were converted into calcium-containing exosomes which were secreted into ECM and accelerated vascular calcium deposit. Inhibition of exosome release in VSMCs decreased calcium deposition. Altogether, our results demonstrated a repressive effect of nHAp on lysosomal acidification, which inhibited autophagic degradation and promoted a conversion of the accumulated autophagic vacuoles into exosomes that were loaded with undissolved nHAp, Ca2+, Pi and ALP. These exosomes bud off the plasma membrane, deposit within ECM, and form calcium nodules. Vascular calcification was thus accelerated by nHAP through blockage of autophagic flux in VSMCs., Graphical abstract The basic component of vascular calcification is hydroxyapatite (HAp). Nano-sized HAp (nHAp, nano-HAp) has been identified to play a role in development of vascular calcification. Nano-HAp was found in the calcified vasculature. Here we demonstrate the possible roles of nano-HAp in vascular calcification. When nHAp was endocytosed by VSMCs and located in early endosomes, osteogenic trans-differentiation was enhanced with more Runx2 and ALP expression. Lysosomal vacuolar H+-ATPase was damaged by nHAp when endosomes fused with lysosomes, therefore lysosomal acidification was impaired. However, nHAp did not affect autophagosome-lysosome fusion. Such formed autolysosome could not be degraded. The blockage of autophagic flux promoted a conversion of the accumulated autophagic vacuoles into exosomes that were loaded with undissolved nHAp, Ca2+, Pi and ALP. These exosomes buded off the plasma membrane, deposited within ECM, and formed calcium nodules. Vascular calcification was thus accelerated by nHAp through blockage of autophagic flux in VSMCs.Image 1, Highlights • We first demonstrated that nHAp was internalized into the vascular cell in human calcified aorta. • Nano-HAp impairs lysosomal acidification and degradation, and causesblockage of autophagy flux in VSMCs. • The accumulated autophagosomes and autolysosomes induced by nHAp in VSMCs are converted into exosomes which promote calcification development.

BackgroundThe Dietary precursor has been identified as a contributor in the development of cardiovascular disease. However, it is inconsistent if dietary precursors could affect the process of cardiovascular disease.MethodsHere we performed Mendelian randomization (MR) analysis of the data from genome-wide association study of European ancestry to evaluate the independent effects of three dietary precursors on cardiovascular disease (CVD), myocardial infarction (MI), heart failure (HF), atrial fibrillation (AF), and valvular disease (VHD). Inverse variance weighting method was used for the MR estimation. Sensitivity was determined by MR-PRESSO analysis, weighted median analysis, MR-Egger analysis, and Leave-one-out analysis.ResultsWe found that elevated choline level had a causal relationship with VHD [odds ratio (OR) = 1.087, 95% confidence interval (CI), 1.003–1.178, P = 0.041] and MI (OR = 1.250, 95% CI, 1.041–1.501, P = 0.017) by single-variable MR analysis. Furthermore, elevated carnitine level was associated with MI (OR = 5.007, 95% CI, 1.693–14.808, P = 0.004) and HF (OR = 2.176, 95% CI, 1.252–3.780, P = 0.006) risk. In addition, elevated phosphatidylcholine level can increase the risk of MI (OR = 1.197, 95% CI, 1.026–1.397, P = 0.022).ConclusionOur data show that choline increases VHD or MI risk, carnitine increases the risk of MI or HF, and phosphatidylcholine increases HF risk. These findings suggest the possibility that decrease in choline level in circulation may be able to reduce overall VHD or MI risk, reduce in carnitine level could be decrease MI and HF risks as well as decrease in phosphatidylcholine could reduce MI risk.

Age-related diseases such as cardiovascular diseases portend disability, increase health expenditures, and cause late-life mortality. Synthetic agonists of growth hormone-releasing hormone (GHRH) exhibit several favorable effects on heart function and remodeling. Here we assessed whether GHRH agonist MR409 can modulate heart function and systemic parameters in old mice. Starting at the age of 15 months, mice were injected subcutaneously with MR409 (10 µg/day, n = 8) or vehicle (n = 7) daily for 6 months. Mice treated with MR409 showed improvements in exercise activity, cardiac function, survival rate, immune function, and hair growth in comparison with the controls. More stem cell colonies were grown out of the bone marrow recovered from the MR409-treated mice. Mitochondrial functions of cardiomyocytes (CMs) from the MR409-treated mice were also significantly improved with more mitochondrial fusion. Fewer β-gal positive cells were observed in endothelial cells after 10 passages with MR409. In Doxorubicin-treated H9C2 cardiomyocytes, cell senescence marker p21 and reactive oxygen species were significantly reduced after cultured with MR409. MR409 also improved cellular ATP production and oxygen consumption rate in Doxorubicin-treated H9C2 cells. Mitochondrial protein OPA1 long isoform was significantly increased after treatment with MR409. The effects of MR409 were mediated by GHRH receptor and protein kinase A (PKA). In short, GHRH agonist MR409 reversed the aging-associated changes with respect of heart function, mobility, hair growth, cellular energy production, and senescence biomarkers. The improvement of heart function may be related to a better mitochondrial functions through GHRH receptor/cAMP/PKA/OPA1 signaling pathway and relieved cardiac inflammation.

Background: fear of COVID-19 is widespread among the population, especially among college students because of their increased exposure to the media information overload of the COVID-19 outbreak. The Fear of COVID-19 scale (FCV-19 S) is a relatively short instrument used to evaluate fears surrounding the COVID-19 pandemic. However, the validity and reliability of the Fear of COVID-19 Scale have not been fully investigated in Chinese university student groups. Objectives: this study assessed the cross-cultural adaptability and reliability of the FCV-19S for Chinese university students. Methods: a Chinese version of Fear of COVID-19 Scale (C-FCV-19S) was generated using the translation-backward translation method. Psychometric properties of the C-FCV-19S, including internal consistency, split-half reliability, construct reliability, convergent validity, and diagnostic accuracy, were evaluated. The Patient Health Questionnaire (PHQ) and Generalized Anxiety Disorder Questionnaire (GAD-7) scales were also used to evaluate participants for depression and anxiety. Results: the C-FCV-19S has acceptable internal consistency (Cronbach’s alpha: 0.872) and satisfactory split-half reliability (correlation coefficient: 0.799). Using the exploratory factor analysis (EFA), we examined the construct reliability (KMO = 0.920). The confirmatory factor analysis (CFA) confirmed that the bifactor model of scale (including general factor, factor1: the awareness of COVID-19 and physiological arousal, factor 2: fear-related thinking) had a good fit index (χ2/df =6.18, RMSEA= 0.067, SRMR = 0.028, GFI = 0.986, TLI = 0.970 and CFI= 0.988). Using depression-positive and anxiety-positive scores as reference criteria, we found that the areas under the curve were 0.70 and 0.68, respectively, and that the optimal cutoff scores of the C-FCV-19S was 17.5 (sensitivity: 66.3% and 58.7%, respectively). Conclusions: the validity and reliability of C-FCV-19S are satisfactory, and the optimal cutoff point was 17.5. The C-FCV-19S can be applied adopted in Chinese university students.

Qi Liu,1,* Pingping Xiang,1,* Mingyao Chen,1 Yi Luo,1 Yun Zhao,1,2 Jinyun Zhu,1 Wangwei Jing,1 Hong Yu1 1Department of Cardiology, Cardiovascular Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, 310009, People’s Republic of China; 2The Affiliated Cardiovascular Hospital of Qingdao University, Qingdao, Shandong Province, 266071, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hong YuDepartment of Cardiology, Cardiovascular Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 JieFang Road, Hangzhou, Zhejiang Province, 310009, People’s Republic of ChinaTel +86-571-87783992Fax +86-571-88002709Email yuvascular@zju.edu.cnPurpose: The deposition of hydroxyapatite (HAp) crystals plays an important role in the development of vascular calcification (VC). This study aimed to demonstrate the effects of nanosized HAp (nHAp) on vascular smooth muscle cells (VSMCs) and VC progression.Methods: Transmission electron microscopy (TEM) was used to examine cellular uptake of nHAp. Cell viability was determined using CCK-8 assay kit. Mitochondrial impairment and reactive oxygen species were detected by TEM and fluorescence dye staining, respectively. Cell apoptosis was detected by Western blot analysis and Annexin V staining. Mouse model of VC was built via applying nHAp on the surface of abdominal aorta. Calcification was visualized by Alizarin red and von Kossa staining.Results: We found that nHAp could promote osteogenic transformation of VSMCs by elevating expression of runt-related factor 2 (Runx2), osteopontin (OPN) and alkaline phosphatase (ALP), impairing function and morphology of mitochondria and inducing apoptosis of VSMCs. More phosphorylation of c-Jun N-terminal protein kinase/c-JUN (JNK/c-JUN) in VSMCs was detected after mixing nHAp with VSMCs. HAp-induced osteogenic transformation of VSMCs was blocked by JNK inhibitor SP600125, resulted in decreased ALP activity, less Runx2 and OPN expressions. SP600125 also inhibited apoptosis of VSMCs. Application of nHAp to outside of aorta induced osteogenic transformation and apoptosis of VSMCs, and significant deposition of calcium on the vessel walls of mice, which can be effectively attenuated by SP600125.Conclusion: JNK/c-JUN signaling pathway is critical for nHAp-induced calcification, which could be a potential therapeutic target for controlling the progression of VC.Keywords: vascular calcification, hydroxyapatite, apoptosis, mitochondria, osteogenic transformation, JNK

Background: Vascular calcification (VC) is a common characteristic of aging, diabetes, chronic renal failure, and atherosclerosis. The basic component of VC is hydroxyapatite (HAp). Nano-sized HAp (nHAp) has been identified as the initiator of pathological calcification of vasculature. However, whether nHAp can induce calcification in vivo and the mechanism of nHAp in the progression of VC remains unclear.Results: We discovered that nHAp existed both in vascular smooth muscle cells (VSMCs) and their extracellular matrix (ECM) in the calcified arteries from patients. Synthetic nHAp had similar morphological and chemical properties as natural nHAp recovered from calcified artery. nHAp induced rapid progression of VC by stimulating osteogenic differentiation and accelerating mineralization of VSMCs in vitro. Synthetic nHAp could also directly induce VC in vivo. Mechanistically, nHAp was internalized into lysosome, which impaired lysosome vacuolar H+-ATPase for its acidification, therefore blocked autophagic flux in VSMCs. The accumulated autophagosomes and autolysosomes were converted into calcium-containing exosomes which were secreted into ECM and accelerated vascular calcium deposit. Inhibition of exosome release in VSMCs decreased calcium deposition. Conclusions: Our results illustrated a novel mechanism of nHAp-induced vascular calcification. Understanding the role of nHAp in autophagy-lysosome-exosome pathway in SMCs could have great clinical significance in preventing the progression of VC.