Erratum: Induced Endothelial Cells Enhance Osteogenesis and Vascularization of Mesenchymal Stem Cells

Adequate vascularization remains one of the major challenges in bone tissue engineering. Since the microvascular endothelium is of benefit to osteogenesis and vascularization when in direct contact with bone marrow mesenchymal stem cells (BM-MSCs), we investigated whether endothelial cells induced from BM-MSCs have the same effect on BM-MSCs in vitro and in vivo. BM-MSCs were isolated, characterized and induced into endothelial-like cells (induced endothelial cells, IECs) in endothelial cell growth medium 2. BM-MSCs and IECs were co-cultured with direct contact. In vitro, IECs were evaluated in terms of their characteristics of endothelial cells and their effects on the osteogenic potential of BM-MSCs by cell morphology, immunofluorescent staining, alkaline phosphatase activity and osteocalcin synthesis. In vivo, scaffolds consisting of β-tricalcium phosphate co-seeded with IECs and BM-MSCs were transplanted into mouse dorsal pockets, and a histological analysis was performed to determine the extent of new bone and blood vessel formation. Isolated BM-MSCs were positive for the markers CD105 and CD29 and negative for hematopoietic markers CD34, CD45 and CD14. They were able to differentiate into adipocytes, osteocytes and chondrocytes in respective media. Immunofluorescent analysis with von Willebrand factor and CD31 staining showed that BM-MSCs could differentiate into endothelial cells. The alkaline phosphatase activity and the osteocalcin content of the co-culture group were obviously higher than those of any other group (p < 0.05). Histologically, newly formed bone and vessels were more evident in the culture group (p < 0.05). Our findings suggest that IECs could efficiently stimulate the in vitro differentiation of osteoblast-like cells and promote osteogenesis in vivo by direct contact with BM-MSCs

Supplementary Material for: Evidence of an Association between Age-Related Functional Modifications and Pathophysiological Changes in Zebrafish Heart

<b><i>Background:</i></b> Zebrafish have become a valuable model for the study of developmental biology and human disease, such as cardiovascular disease. It is difficult to discriminate between disease-related and age-related alterations. <b><i>Objective:</i></b> This study was aimed to investigate the effects and potential mechanisms of age-related cardiac modifications in an older zebrafish population. <b><i>Methods:</i></b> In this study, we calculated the survival rate and measured the spinal curvature through the aging process. A swimming challenge test was performed and showed that swimming capacity and endurance dramatically dropped in older fish groups. <b><i>Results:</i></b> To find out the effect of stress on zebrafish during the aging process, we recorded electrocardiograms on zebrafish and showed that during stress, aging not only led to a significant reduction in heart rate, but also caused other age-related impairments, such as arrhythmias and ST-T depression. Echocardiography showed a marked increase in end-diastolic ventricular dimensions and in isovolumic relaxation time and a notably slower mean and peak velocity of the bulboventricular valve in older zebrafish, but stroke volume and cardiac output were not different in young and old zebrafish. Both <i>nppa</i> and <i>nppb</i> (cardiac fetal genes for natriuretic factor) expression detected by real-time polymerase chain reaction analysis increased in older fish compared to the younger group. Histological staining revealed fibrosis within cardiomyocytes and an increase in ventricular myocardial density and a decrease in epicardial vessel dimensions in older fish hearts that may correlate with a deterioration of cardiac function and exercise capacity. <b><i>Conclusion:</i></b> These data suggest that cardiac functional modifications in zebrafish are comparable to those in humans and may partly be due to changes in the cardiovascular system including cardiac fetal gene reprogramming, myocardial density, and epicardial vessel dimensions

Supplementary Material for: Loss of Pten in renal tubular cells leads to water retention by upregulating AQP2

Introduction: Phosphatase and tensin (PTEN) is a multifunctional gene associated with the normal development and physiological function of various tissues including the kidney. However, its role in renal tubular reabsorption function has not been well elucidated. Methods: We generated a renal tubule-specific Pten knockout mouse model by crossing Ptenfl/fl mice with Ksp-Cre transgenic mice, evaluated the effect of Pten loss on renal tubular function, and investigated the underlying mechanisms. Results: Pten loss resulted in abnormal renal structure and function and water retention in multiple organs. Our results also demonstrated that aquaporin-2 AQP2, an important water channel protein, was upregulated and concentrated on the apical plasma membrane of collecting duct cells, which could be responsible for the impaired water balance in Pten loss mice. The regulation of Pten loss on AQP2 was mediated by Protein kinase B (AKT) activation. Discussion/Conclusions: Our results reveal a connection between PTEN gene inactivation and water retention, suggesting the importance of PTEN in normal kidney development and function

Supplementary Material for: Complement Factor I Polymorphism Is Not Associated with Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy in a Chinese Population

<b><i>Purpose:</i></b> To identify the associations of the two complement factor I (CFI) polymorphisms <i>rs10033900</i> and <i>rs2285714</i> with risk of neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in a Chinese case-control study. <b><i>Methods:</i></b> A total of 900 subjects - 300 controls, 300 cases with nAMD and 300 cases with PCV - were included in the present study. Genomic DNA was extracted from venous blood leukocytes. The allelic variants of <i>rs10033900</i> and <i>rs2285714</i> were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The differences in allele distribution between the cases and controls were tested by a χ² test with age and gender adjusted for by logistic regression analysis. We also performed a meta-analysis of the case-control studies of <i>rs10033900</i> and <i>rs2285714</i> based on the currently available evidence from the literature. The meta-analysis was conducted via an inverse-variance, fixed-effects model, as previously described. <b><i>Results:</i></b> No statistically significant association was observed between the two polymorphisms of CFI and AMD risk, including nAMD, PCV and combined AMD (p > 0.05 for all comparisons). By meta-analysis, we detected significant associations between both of the SNPs and late AMD, which is consistent with previous results (odds ratio, OR, <i>rs10033900</i> = 0.814, p <i>rs10033900</i> < 0.001; OR <i>rs2285714</i> = 1.221, p <i>rs2285714</i> < 0.001). For <i>rs2285714</i>, the results of the meta-analysis were less reliable due to its heterogeneity. <b><i>Conclusions:</i></b> In our case-control study, neither of the two SNPs most studied (<i>rs10033900</i> or <i>rs2285714</i>) in the <i>CFI</i> gene was a risk factor for developing nAMD or PCV in a Chinese population. Additional large, comprehensive and well-designed association studies are needed to better understand the role of ethnicity and other gene interactions in the association between the <i>CFI</i> gene and AMD

Supplementary Material for: Palmitic Acid Curcumin Ester Facilitates Protection of Neuroblastoma against Oligomeric Aβ₄₀ Insult

<b><i>Background/Aims:</i></b> The generation of reactive oxygen species (ROS) caused by amyloid-β (Aβ) is considered to be one of mechanisms underlying the development of Alzheimer’s disease. Curcumin can attenuate Aβ-induced neurotoxicity through ROS scavenging, but the protective effect of intracellular curcumin on neurocyte membranes against extracellular Aβ may be compromised. To address this issue, we synthesized a palmitic acid curcumin ester (P-curcumin) which can be cultivated on the cell membrane and investigated the neuroprotective effect of P-curcumin and its interaction with Aβ. <b><i>Methods:</i></b> P-curcumin was prepared through chemical synthesis. Its structure was determined via nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). An MTT assay was used to assess Aβ cytotoxicity and the protective effect of P-curcumin on SH-SY5Y cells. The effect of P-curcumin on Aβ-induced ROS production <i>in vitro</i> and <i>in vivo</i> were assessed based on changes in dichlorofluorescein (DCF) fluorescence. A spectrophotometric method was employed to detect lipid peroxidation. To mimic the interaction of P-curcumin on cell membranes with Aβ, liposomes were prepared by thin film method. Finally, the interactions between free P-curcumin and P-curcumin cultivated on liposomes and Aβ were determined via spectrophotometry. <b><i>Results:</i></b> A novel derivative, palmitic acid curcumin ester was prepared and characterized. This curcumin, cultivated on the membranes of neurocytes, may prevent Aβ-mediated ROS production and may inhibit the direct interaction between Aβ and the cellular membrane. Furthermore, P-curcumin could scavenge Aβ-mediated ROS as curcumin <i>in vitro</i> and <i>in vivo</i>, and had the potential to prevent lipid peroxidation. Morphological analyses showed that P-curcumin was better than curcumin at protecting cell shape. To examine P-curcumin’s ability to attenuate direct interaction between Aβ and cell membranes, the binding affinity of Aβ to curcumin and P-curcumin was determined. The association constants for free P-curcumin and curcumin were 7.66 × 10⁴ M^-1 and 7.61 × 10⁵ M^-1, respectively. In the liposome-trapped state, the association constants were 3.71 × 10⁵ M^-1 for P-curcumin and 1.44× 10⁶ M^-1 for curcumin. With this data, the thermodynamic constants of P-curcumin association with soluble Aβ <i>(ΔH, ΔS</i>, and Δ<i>G</i>) were also determined. <b><i>Conclusion:</i></b> Cultivated curcumin weakened the direct interaction between Aβ and cell membranes and showed greater neuroprotective effects against Aβ insult than free curcumin

Supplementary Material for: Identification of Subpathway Signatures For Ovarian Cancer Prognosis by Integrated Analyses of High-Throughput miRNA and mRNA Expression

<b><i>Background/Aims:</i></b> Ovarian cancer (OC) causes more death and serious conditions than any other female reproductive cancers, and many expression signatures have been identified for OC prognoses. However, no significant overlap is found among signatures from different studies, indicating the necessity of signature identifications at the functional level. <b><i>Methods:</i></b> We performed an integrated analyses of miRNA and gene expressions to identify OC prognostic subpathways (pathway regions). Using The Cancer Genome Atlas data set, we identified core prognostic subpathways, and calculated subpathway risk scores using both miRNA and gene components. Finally, we performed global risk impact analyses to optimize core subpathways using the random walk algorithm. <b><i>Results:</i></b> Subpathway-level analyses displayed more robust results than the gene- and miRNA-level analyses. Moreover, we verified the advantage of core subpathways over the entire pathway-based results and their prognostic performance in two independent validation data sets. Based on the global impact score, 13 subpathway signatures were selected and a combined subpathway-based risk score was further calculated for OC patient prognoses. <b><i>Conclusions:</i></b> Overall, it was possible to systematically perform integrated analyses of the expression levels of miRNAs and genes to identify prognostic subpathways and infer subpathway risk scores for use in OC clinical applications

Erratum: Endoplasmic Reticulum Stress Predicts Clinical Response to Cyclosporine Treatment in Primary Membranous Nephropathy

<b><i>Background:</i></b> Little is known about the endoplasmic reticulum stress (ERS) marker glucose regulated protein 78 (GRP78) and calcineurin in the kidney in primary membranous nephropathy (PMN) and if they could predict post-cyclosporine treatment outcome. <b><i>Methods:</i></b> This is a retrospective study using a dataset of biopsy-confirmed PMN from Peking Union Medical College Hospital from 1996 to 2014. Seventy-six adult patients treated with cyclosporine as primary immunosuppression for at least 6 months were studied. Immunohistochemistry was used to detect GRP78 and calcineurin in the kidney. Serum calcineurin was assayed by ELISA. Patients were grouped into no-remission (NR, n = 17), partial remission (PR, n = 39), or complete remission (CR, n = 20) at the end of 6 months of treatment. <b><i>Results:</i></b> There was no difference of initial dose of cyclosporine among NR, PR, and CR groups. Kidney calcineurin expression in PMN was significantly increased compared to that in controls (p < 0.0083). The glomerular GRP78 in NR PMN was higher than that in control, CR and PR patients (p < 0.0083). Kidney calcineurin expression and GRP78 expression was positively correlated. However, there were no differences in either serum calcineurin levels or kidney calcineurin expressions among NR, PR or CR groups. There was a negative correlation between serum calcineurin activity and whole kidney calcineurin expression (p = 0.034) or glomerular calcineurin expression (p = 0.007). Neither kidney calcineurin nor GRP78 expression was correlated with proteinuria. <b><i>Conclusions:</i></b> ERS marker GRP78 in the glomeruli but not serum or kidney calcineurin expression could be a useful marker in PMN to negatively predict the response to cyclosporine treatment at the sixth month

Supplementary Material for: Early Blood Pressure Reduction in Acute Ischemic Stroke with Various Severities: A Subgroup Analysis of the CATIS Trial

<b><i>Background:</i></b> Clinical trials have generally showed a neutral effect of blood pressure (BP) reduction on clinical outcomes among acute ischemic stroke patients. We conducted a prespecified subgroup analysis to assess whether disease severity modifies the effect of early antihypertensive treatment on death and disability among patients with acute ischemic stroke. <b><i>Methods:</i></b> In the China Antihypertensive Trial in Acute Ischemic Stroke, 4,071 patients with acute ischemic stroke and elevated BP were randomly assigned to receive antihypertensive treatment or to discontinue all hypertension medications within 48 h of symptom onset. The primary outcome was a combination of death and major disability at 14 days or hospital discharge. In this subgroup analysis, participants were categorized into 3 groups according to their baseline NIH Stroke Scale (NIHSS) scores (0-4, 5-15, or ≥16). <b><i>Results:</i></b> At 24 h after randomization, mean systolic BP differences (95% CIs) were -8.5 (-10.0 to -7.1), -9.8 (-11.4 to -8.3), and -9.1 (-14.4 to -3.8) mm Hg between the treatment and control groups (all p values <0.001) for patients with a baseline NIHSS score of 0-4, 5-15, and ≥16, respectively. At day 7 after randomization, the corresponding mean systolic BP differences were -9.3 (-10.5 to -8.2), -9.1 (-10.3 to -7.8), and -10.1 (-15.1 to -5.1) mm Hg between the treatment and control groups (all p values <0.001). The primary outcome was not significantly different between the treatment and control groups at day 14 or hospital discharge among all NIHSS subgroups (p value for homogeneity = 0.66). ORs (95% CI) associated with treatment were 1.14 (0.87-1.49, p = 0.33), 1.04 (0.86-1.25, p = 0.70), and 0.67 (0.18-2.44, p = 0.54) for patients with a baseline NIHSS score of 0-4, 5-15, and ≥16, respectively. The composite outcome of death and major disability at 3-month follow-up did not differ between the 2 comparison groups for all NIHSS subgroups. In addition, vascular events and recurrent stroke were not significantly different between the 2 comparison groups at the 3-month follow-up visit among all NIHSS subgroups except that there was a suggestive risk reduction for recurrent stroke among those with an NIHSS score of 5-15 (OR 0.45, 95% CI 0.20-0.99, p = 0.05). <b><i>Conclusion:</i></b> Early BP reduction with antihypertensive medications did not reduce or increase the risk of death, major disabilities, recurrent instances of stroke, and vascular events in acute ischemic stroke patients with a variety of disease severities

Supplementary Material for: ABCA1 rs1883025 Polymorphism Shows No Association with Neovascular Age-Related Macular Degeneration or Polypoidal Choroidal Vasculopathy in a Northern Chinese Population

<b><i>Purpose:</i></b> To analyze the association between ABCA1 rs1883025 variants with neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in a northern Chinese population. <b><i>Methods:</i></b> The study enrolled 900 subjects, including 300 controls, 300 cases with nAMD and 300 cases with PCV. Genomic DNA was extracted from venous blood leukocytes. Single-nucleotide polymorphisms in the ABCA1 (rs1883025) gene were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. <b><i>Results:</i></b> The ABCA1 rs1883025 polymorphism was not significantly associated with nAMD (22.5%; p > 0.05) or PCV (20.8%; p > 0.05) in a northern Chinese population. The association remained insignificant after adjustment for age and gender differences (p > 0.05). <b><i>Conclusions:</i></b> This study suggests that ABCA1 rs1883025 variants are not associated with nAMD or PCV in a Chinese population, which is likely due to an ethnic difference

Supplementary Material for: rs4711751 and rs1999930 Are Not Associated with Neovascular Age-Related Macular Degeneration or Polypoidal Choroidal Vasculopathy in the Chinese Population

<b><i>Purpose:</i></b> rs1999930 and rs4711751 have recently been identified as novel variants associated with advanced age-related macular degeneration (AMD) in populations of European ancestry. We aimed to investigate whether these two single nucleotide polymorphisms (SNPs) were associated with neovascular AMD (nAMD) or with polypoidal choroidal vasculopathy (PCV), a variant of AMD in Asians, using a Chinese case-control study. <b><i>Methods:</i></b> A total of 900 subjects, including 300 controls, 300 cases with nAMD and 300 cases with PCV, were included in the present study. Genomic DNA was extracted from venous blood leukocytes. The allelic variants of rs1999930 and rs4711751 were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The differences in allele distribution between cases and controls were tested by a χ² test, with additional adjustments for age and gender using logistic regression. The statistical power was also calculated. Values of p < 0.05 were considered statistically significant. <b><i>Results:</i></b> No statistically significant association was observed between the two polymorphisms of nAMD or PCV phenotype (p > 0.05 for all comparisons). The difference remained insignificant after correction for age and gender (p > 0.05 for all comparisons). The statistical powers to detect the association between these two SNPs and nAMD or PCV range from 0.05 to 0.36, assuming conventional levels of statistical significance. <b><i>Conclusions:</i></b> In the present study, we could not replicate the reported association of these two SNPs and either nAMD or PCV in a Chinese population, suggesting that they are unlikely to be a major AMD and PCV susceptibility gene locus in the Chinese population. Considering the low power value, a large sample size is required to draw more reliable conclusions