Serum levels of soluble receptor activator for nuclear factor kB ligand play a crucial role in the association of osteoprotegerin with coronary artery disease.

Osteoprotegerin (OPG) is a soluble decoy receptor for receptor activator of nuclear factor kB ligand (RANKL), and is implicated in the pathogenesis of atherosclerosis. The aim of the present study was to examine the hypothesis that serum OPG concentrations are increased in patients with stable coronary artery disease (CAD) at different serum levels of soluble RANKL (sRANKL). The study used a case-control design in which consecutively hospitalized individuals were recruited. Fasting blood samples were taken upon admission for serum testing. Participants with previously diagnosed CAD that was asymptomatic or had controlled symptoms constituted the stable CAD group, whereas patients with negative coronary computed tomography angiography results constituted the control non-CAD group. Exclusion criteria included recent acute coronary syndrome, severe heart failure, CAD-complicating autoimmune, blood or thyroid diseases, cancer, elevated temperature with or without infection, severe liver or kidney dysfunction, abnormal calcium metabolism, recent surgery and trauma history. A total of 118 individuals were included in the study. Smoothed plots generated using the recursive method and multivariate models showed that the incidence of stable CAD increased with serum OPG level up to the turning point of 18 pg/ml. This trend was observed at both high [odds ratio (OR), 1.61; 95% confidence interval (CI), 1.04-2.50; P=0.032) and low sRANKL concentrations (OR, 1.52; 95% CI, 1.06-2.17; P=0.022) after adjustment for cardiovascular risk factors. In conclusion, serum OPG levels ≤18 pg/ml are positively associated with stable CAD, regardless of sRANKL levels. In addition, at the same serum OPG level, higher sRANKL levels are associated with a greater incidence of stable CAD compared with lower sRANKL levels. This study identified the relationship between OPG, sRANKL, and stable CAD, and established the reference range for future clinical use.

Recombinant interleukin-2 stimulates lymphocyte recovery in patients with severe COVID-19.

A recently identified type of pneumonia, referred to as coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2, has rapidly spread worldwide. Lymphopenia and a proinflammatory cytokine storm frequently occur in patients with severe COVID-19. However, to the best of our knowledge, no specific immunomodulatory therapy for COVID-19 has been reported to date. In the present retrospective case-control study, the potential therapeutic effect of recombinant human interleukin-2 (rIL-2) in patients with severe COVID-19 was demonstrated. A total of 59 patients with severe COVID-19 were admitted to the Union Hospital of Tongji Medical College (Wuhan, China) between 29th January 2020 and 29th February 2020 and were included in the present study. In total, 20 patients received subcutaneous injection of rIL-2 (1 million IU per day) for 7-10 days in addition to regular treatment and were classified as the rIL-2 group. Furthermore, 20 of the 39 patients receiving regular treatment, without the intervention of rIL-2, were matched as the control group. Patients in these two groups were subjected to propensity score matching in terms of clinical characteristics such as age, sex, symptoms, signs, laboratory data and comorbidities. Changes in the lymphocyte count, as well as IL-6 and C-reactive protein (CRP) levels, were analyzed at the time of admission and discharge and any differences between the rIL-2 and non-rIL-2 groups were determined. The results demonstrated an increase in the lymphocyte count and a decrease in CRP levels in the rIL-2 group compared with that in the non-rIL-2 group. The difference in the change of the lymphocyte count between the rIL-2 group and non-rIL-2 group was statistically significant (P<0.01). Although CRP levels were decreased to a greater extent in the rIL-2 group, the difference between the two groups was not statistically significant (P>0.05). Collectively, the present results suggested that administration of rIL-2 may be a prospective adjuvant therapy for patients with severe COVID-19 and its effects may be mediated by increasing lymphocyte numbers.

N­acetylcysteine inhibits atherosclerosis by correcting glutathione­dependent methylglyoxal elimination and dicarbonyl/oxidative stress in the aorta of diabetic mice.

In diabetic animal models, high plasma/tissue levels of methylglyoxal (MG) are implicated in atherosclerosis. N­acetylcysteine (NAC) is a cysteine prodrug that replenishes intracellular glutathione (GSH) levels, which can increase the elimination of MG in diabetes mellitus (DM). The present study investigated the anti­atherosclerotic role of NAC in DM and aimed to determine whether the mechanism involved GSH­dependent MG elimination in the aorta. Apolipoprotein­E knockdown (ApoE­/­) mice injected with streptozotocin for 5 days exhibited enhanced atherosclerotic plaque size in the aortic root; notably, a high­lipid diet aggravated this alteration. NAC treatment in the drinking water for 12 weeks decreased the size of the atherosclerotic lesion, which was associated with a reduction in MG­dicarbonyl stress and oxidative stress, as indicated by decreased serum malondialdehyde levels, and increased superoxide dismutase­1 and glutathione peroxidase­1 levels in the diabetic aorta. Endothelial damage was also corrected by NAC, as indicated by an increase in the expression levels of phosphorylated (p­)Akt and p­endothelial nitric oxide synthase (eNOS) in the aorta, as well as nitric oxide (NO) in the serum. In addition, MG­treated human umbilical vein endothelial cells (HUVECs) exhibited increased reactive oxygen species and decreased antioxidant enzyme expression levels. NAC treatment corrected the alteration in HUVECs induced by MG, whereas the protective role of NAC was blocked via inhibition of GSH. These findings indicated that the diabetic aorta was more susceptible to atherosclerotic lesions compared with non­diabetic ApoE­/­ mice. Furthermore, NAC may offer protection against atherosclerotic development in DM by altering aortic and systemic responses via correcting GSH­dependent MG elimination, leading to decreased oxidative stress and restoration of the p­Akt/p­eNOS pathway in the aorta.

Role of Wnt/ß-Catenin Pathway in the Arterial Medial Calcification and Its Effect on the OPG/RANKL System.

In this study, the hypothesis that Wnt/ß-catenin pathway is involved in the arterial calcification by regulating the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) system was tested. The ß-catenin expression was measured in the warfarin-induced calcified arteries and the osteoblast-like cells differentiating from smooth muscle cells (SMCs) by immunohistochemistry and Western blotting. The Wnt/ß-catenin pathway was activated or inhibited by lithium chloride (LiCl) or dickkopf 1 (DKK1) in vitro and in vivo. Then the calcification level was determined by von Kossa staining, Ca2+ content assay, and alkaline phosphatase (ALP) activity assay. The expression levels of osteocalcin, OPG and RANKL were detected by Western blotting or real-time PCR. The results showed that in calcified arteries and OBL cells, the activation of Wnt/ß-catenin pathway significantly enhanced the calcification as evidenced by increased von Kossa stains, Ca2+ contents, ALP activities, and osteocalcin expression levels (P<0.05), and it promoted the RANKL expression (P<0.05), but slightly affected the OPG expression. These results indicated that the activation of Wnt/ß-catenin pathway worsens the arterial calcification, probably by promoting the RANKL expression.

Correlation between Histone Deacetylase 9 and Regulatory T Cell in Patients with Chronic Heart Failure.

Heart failure (HF) is the end stage of various kinds of cardiovascular diseases and leads to a high mortality worldwide. Numerous studies have demonstrated that frequencies of CD4+CD25+Foxp3+ regulatory T cells (Tregs) are reduced in HF patients and properly expanding Tregs attenuates HF progression. Histone deacetylase (HDAC) 9 has been revealed to contribute to several cardiovascular and cerebrovascular diseases. Plenty of studies showed that HDAC9 negatively regulated the number and function of Tregs. Thus, we aim to investigate the expression of HDAC 9 in patients with chronic heart failure (CHF) and the relationship among HDAC9, Tregs and CHF. Our research showed a reduced number of Tregs and an increased expression of HDAC9 mRNA in CHF patients. Patients with CHF were divided into two groups by heart function grade of New York Heart Association (NYHA), we found that the HDAC9 mRNA expression level in NYHA grade II-III group were lower than that in NYHA grade IV group. More importantly, the correlation study suggested that the expression of HDAC9 mRNA was negatively correlated to Tregs frequency and left ventricular ejection fraction (LVEF), whereas positively correlated to larger left ventricular end-diastolic dimension (LVEDD) and B-type natriuretic peptide (BNP) in patients with CHF. The correlation studies also showed a positive correlation between HDAC9 and the severity of CHF. Our research suggests that HDAC9 may be a new indicator for assessing CHF and it may offer a new direction for research of CHF.

Indirect co­culture of vascular smooth muscle cells with bone marrow mesenchymal stem cells inhibits vascular calcification and downregulates the Wnt signaling pathways.

Vascular calcification (VC) is widely considered to be a crucial clinical indicator of cardiovascular disease. Recently, certain properties of mesenchymal stem cells (MSCs) have been hypothesized to have potential in treating cardiovascular diseases. However, their effect on the initiation and progression of VC remains controversial. The present study aimed to investigate whether MSCs indirectly mediate VC and their impact on the Wnt signaling pathways. A Transwell system was selected to establish the indirect co­culture environment, and hence, vascular smooth muscle cells (VSMCs) were indirectly co­cultured in the presence or absence of MSCs at a ratio of 1:1. Osteogenic medium (OS) was added to imitate a calcifying environment. Fourteen days later, VSMCs in the lower layers of the Transwell plates were harvested. Alkaline phosphatase activity and calcium nodules were markedly increased in calcific VSMCs induced by OS. However, these parameters were significantly decreased in VSMCs by indirectly co­culturing with MSCs in the same medium. Furthermore, the messenger RNA expression levels of osteopontin and osteoprotegerin were notably increased in VSMCs cultured in OS, but reduced by indirect interaction with MSCs. In addition, the activities of canonical and noncanonical Wnt ligands, wingless­type MMTV integration site family, number 5A (Wnt5a), receptor tyrosine kinase­like orphan receptor 2 (Ror2) and ß­catenin, which are important in the process of VC, were downregulated by indirect contact with MSCs in OS. Thus, indirect co­culture with MSCs inhibits VC and downregulates the Wnt signaling pathways.

The function and meaning of receptor activator of NF-κB ligand in arterial calcification.

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.

Osteoprotegerin inhibits calcification of vascular smooth muscle cell via down regulation of the Notch1-RBP-Jκ/Msx2 signaling pathway.

OBJECTIVE: Vascular calcification is a common pathobiological process which occurs among the elder population and in patients with diabetes and chronic kidney disease. Osteoprotegerin, a secreted glycoprotein that regulates bone mass, has recently emerged as an important regulator of the development of vascular calcification. However, the mechanism is not fully understood. The purpose of this study is to explore novel signaling mechanisms of osteoprotegerin in the osteoblastic differentiation in rat aortic vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VSMCs were isolated from thoracic aorta of Sprague Dawley rats. Osteoblastic differentiation of VSMCs was induced by an osteogenic medium. We confirmed by Von Kossa staining and direct cellular calcium measurement that mineralization was significantly increased in VSMCs cultured in osteogenic medium; consistent with an enhanced alkaline phosphatase activity. This osteoblastic differentiation in VSMCs was significantly reduced by the addition of osteoprotegerin in a dose responsive manner. Moreover, we identified, by real-time qPCR and western blotting, that expression of Notch1 and RBP-Jκ were significantly up-regulated in VSMCs cultured in osteogenic medium at both the mRNA and protein levels, these effects were dose-dependently abolished by the treatment of osteoprotegerin. Furthermore, we identified that Msx2, a downstream target of the Notch1/RBP-Jκ signaling, was markedly down-regulated by the treatment of osteoprotegerin. CONCLUSION: Osteoprotegerin inhibits vascular calcification through the down regulation of the Notch1-RBP-Jκ signaling pathway.

Effects of alendronate on the Notch1­RBP­Jκ signaling pathway in the osteogenic differentiation and mineralization of vascular smooth muscle cells.

Cardiovascular disease and osteoporosis are major causes of mortality in the elderly. Alendronate, a bisphosphonate, is widely used in the treatment of osteoporosis and may be used to inhibit vascular calcification. However, its mechanisms are not completely understood. The present study aimed to explore novel signaling mechanisms behind the action of alendronate in the osteoblastic differentiation of rat aortic vascular smooth muscle cells (VSMCs). The osteoblastic differentiation of VSMCs was induced by an osteogenic medium. Using von Kossa staining and direct cellular calcium content determination, mineralization was found to be significantly increased in VSMCs induced with osteogenic medium, consistent with an enhanced alkaline phosphatase activity. Osteoblastic differentiation in VSMCs was significantly reduced by the action of alendronate in a dose­dependent manner. In addition, the expression of Notch1 and RBP­Jκ was significantly upregulated in VSMCs cultured with osteogenic medium at the mRNA and protein levels. The effects of Notch1­RBP­Jκ were inhibited by treatment with alendronate in a dose­dependent manner. In summary, results of the current study indicate that alendronate inhibits vascular calcification through downregulation of the Notch1­RBP­Jκ signaling pathway.

The Wnt5a/Ror2 pathway is associated with determination of the differentiation fate of bone marrow mesenchymal stem cells in vascular calcification.

Accumulating evidence have demonstrated that mesenchymal stem cells (MSCs) are involved in the initiation and progression of various vascular diseases. Canonical Wnt signaling controls the fate of MSCs, and plays an important role in vascular calcification. However, vascular calcification can be inhibited by the non-canonical Wnt signaling pathway Wnt5a/Ror2. This study aimed to investigate whether the Wnt5a/Ror2 pathway is associated with determination of the differentiation fate of MSCs in vascular calcification. Direct co-cultures were established by seeding smooth muscle cells (SMCs) or calcified SMCs and MSCs together at ratios of SMCs or calcified SMCs 15x104; SMCs or calcified SMCs 5x104: MSCs 10x104, SMCs or calcified SMCs 10x104: MSCs 5x104. Osteosynthesis-inducing medium (OS) was added to the culture medium in the groups of MSCs with non-calcified SMCs. Cells were cultured for nine days. Osteoblastic differentiation was evaluated by cell morphology and the activity of alkaline phosphatase (ALP) in cell lysates and ALP staining. Furthermore, we investigated the inhibition of Wnt signaling, and observed that the members of the non-canonical signaling pathway Wnt5a/Ror2 were expressed in each group. Additionally, MSCs cultured in culture media with OS did not differentiate into an osteoblast phenotype when in direct contact with non-calcified SMCs, irrespective of the number of MSCs. However, an osteoblast phenotype was observed when MSCs were cultured in media without OS differentiation towards direct contact with calcified SMCs, and the levels of osteoblastic markers had a direct correlation with the number of MSCs. Of note, the Wnt5a protein was associated with the levels of calcification, thus, although rarely detected in non-calcification, Ror2 mRNA in the non-calcified groups was significantly higher compared to that in the calcified groups. Therefore, the Wnt5a/Ror2 pathway is associated with determination of the differentiation fate of bone marrow mesenchymal stem cells in vascular calcification.

Implication of receptor activator of NF-κB ligand in Wnt/ß-catenin pathway promoting osteoblast-like cell differentiation.

Recent studies showed that activation of Wnt/ß-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/ß-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/ß-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/ß-catenin pathway was activated by LiCl, the expression of RANKL was significantly increased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/ß-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/ß-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.