Isogenic Human-Induced Pluripotent Stem-Cell-Derived Cardiomyocytes Reveal Activation of Wnt Signaling Pathways Underlying Intrinsic Cardiac Abnormalities in Rett Syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by MeCP2 mutations. Nonetheless, the pathophysiological roles of MeCP2 mutations in the etiology of intrinsic cardiac abnormality and sudden death remain unclear. In this study, we performed a detailed functional studies (calcium and electrophysiological analysis) and RNA-sequencing-based transcriptome analysis of a pair of isogenic RTT female patient-specific induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) that expressed either MeCP2wildtype or MeCP2mutant allele and iPSC-CMs from a non-affected female control. The observations were further confirmed by additional experiments, including Wnt signaling inhibitor treatment, siRNA-based gene silencing, and ion channel blockade. Compared with MeCP2wildtype and control iPSC-CMs, MeCP2mutant iPSC-CMs exhibited prolonged action potential and increased frequency of spontaneous early after polarization. RNA sequencing analysis revealed up-regulation of various Wnt family genes in MeCP2mutant iPSC-CMs. Treatment of MeCP2mutant iPSC-CMs with a Wnt inhibitor XAV939 significantly decreased the ß-catenin protein level and CACN1AC expression and ameliorated their abnormal electrophysiological properties. In summary, our data provide novel insight into the contribution of activation of the Wnt/ß-catenin signaling cascade to the cardiac abnormalities associated with MeCP2 mutations in RTT.

Mendelian Randomization Focused Analysis of Vitamin D on the Secondary Prevention of Ischemic Stroke.

BACKGROUND AND PURPOSE: Experimental studies showed vitamin D (Vit-D) could promote vascular regeneration and repair. Prior randomized studies had focused mainly on primary prevention. Whether Vit-D protects against ischemic stroke and myocardial infarction recurrence among subjects with prior ischemic insults was unknown. Here, we dissected through Mendelian randomization any effect of Vit-D on the secondary prevention of recurrent ischemic stroke and myocardial infarction. METHODS: Based on a genetic risk score for Vit-D constructed from a derivation cohort sample (n=5331, 45% Vit-D deficient, 89% genotyped) via high-throughput exome-chip screening of 12 prior genome-wide association study-identified genetic variants of Vit-D mechanistic pathways (rs2060793, rs4588, and rs7041; F statistic, 73; P<0.001), we performed a focused analysis on prospective recurrence of myocardial infarction (MI) and ischemic stroke in an independent subsample with established ischemic disease (n=441, all with prior first ischemic event; follow-up duration, 41.6±14.3 years) under a 2-sample, individual-data, prospective Mendelian randomization approach. RESULTS: In the ischemic disease subsample, 11.1% (n=49/441) had developed recurrent ischemic stroke or MI and 13.3% (n=58/441) had developed recurrent or de novo ischemic stroke/MI. Kaplan-Meier analyses showed that genetic risk score predicted improved event-free survival from recurrent ischemic stroke or MI (log-rank, 13.0; P=0.001). Cox regression revealed that genetic risk score independently predicted reduced risk of recurrent ischemic stroke or MI combined (hazards ratio, 0.62 [95% CI, 0.48-0.81]; P<0.001), after adjusted for potential confounders. Mendelian randomization supported that Vit-D is causally protective against the primary end points of recurrent ischemic stroke or MI (Wald estimate: odds ratio, 0.55 [95% CI, 0.35-0.81]) and any recurrent or de novo ischemic stroke/MI (odds ratio, 0.64 [95% CI, 0.42-0.91]) and recurrent MI alone (odds ratio, 0.52 [95% CI, 0.30-0.81]). CONCLUSIONS: Genetically predicted lowering in Vit-D level is causal for the recurrence of ischemic vascular events in persons with prior ischemic stroke or MI.

High-sensitivity troponin I and B-type natriuretic peptide biomarkers for prediction of cardiovascular events in patients with coronary artery disease with and without diabetes mellitus.

BACKGROUND: High-sensitivity troponin I (hs-Tnl) and B-type natriuretic peptide (BNP) are promising prognostic markers for coronary artery disease (CAD). This prospective cohort study investigated whether a combination of these cardiac biomarkers with conventional risk factors would add incremental value for the prediction of secondary major adverse cardiovascular events (MACEs) in patients with CAD, with and without type 2 diabetes mellitus (T2DM). METHODS: Baseline plasma level of hs-Tnl and BNP was measured in 2275 Chinese patients with stable CAD. Patients were monitored for new-onset of MACE over a median of 51 months. Cox proportional hazard model and area under the receiver operating characteristic curve (AUC) were used to assess the association of cardiac biomarkers with MACE and their predictive values in relationship with or without T2DM. RESULTS: During the follow up period 402 (18%) patients experienced a new-onset MACE with hs-Tnl and BNP level significantly higher than in those without MACE. In multivariable analyses, patients with elevated hs-Tnl (hazard ratio, 1.75 [95% CI 1.41-2.17]; P < 0.001) and BNP (hazard ratio, 1.42 [95% CI 1.15-1.75]; P = 0.001) were significantly associated with an increased risk of MACE after adjustment for variables of a risk factor model of age, sex, T2DM and hypertension. The risk factor model had an AUC of 0.64 for MACE prediction. The AUC significantly increased to 0.68 by the addition of hs-Tnl to the risk factor model. Subgroup analyses showed that hs-Tnl and BNP remained significant predictors of MACE in both patients with and without T2DM in multivariable models with higher risk of MACE evident in those without T2DM. Among patients without T2DM, addition of each biomarker yielded greater predictive accuracy than in T2DM patients, with AUC further increased to 0.75 when a combination of hs-Tnl and BNP was added to the risk factor model (age, sex and hypertension). CONCLUSIONS: Elevated hs-Tnl and BNP level are independent predictors of new-onset MACE in CAD patients, irrespective of diabetes status. Among CAD patients without T2DM, a combination of cardiac biomarkers hs-Tnl and BNP yield the greatest predictive value beyond conventional risk factors.

Age-Biomarkers-Clinical Risk Factors for Prediction of Cardiovascular Events in Patients With Coronary Artery Disease.

Objective- In patients with stable coronary artery disease, conventional risk factors provide limited incremental predictive value for cardiovascular events. We sought to investigate whether a panel of cardiometabolic biomarkers alone or combined with conventional risk factors would exhibit incremental value in the prediction of cardiovascular events. Approach and Results- In the discovery cohort, we measured serum adiponectin, A-FABP (adipocyte fatty acid-binding protein), lipocalin-2, FGF (fibroblast growth factor)-19 and 21, plasminogen activator inhibitor-1, and retinol-binding protein-4 in 1166 Chinese coronary artery disease patients. After a median follow-up of 35 months, 170 patients developed new-onset major adverse cardiovascular events (MACE). In the model with age ≥65 years and conventional risk factors, area under the curve for predicting MACE was 0.68. Addition of lipocalin-2 to the age-clinical risk factor model improved predictive accuracy (area under the curve=0.73). Area under the curve further increased to 0.75 when a combination of lipocalin-2, A-FABP, and FGF-19 was added to yield age-biomarkers-clinical risk factor model. The adjusted hazard ratio on MACEs for lipocalin-2, A-FABP, and FGF-19 levels above optimal cutoffs were 2.23 (95% CI, 1.62-3.08), 1.99 (95% CI, 1.43-2.76), and 1.65 (95% CI, 1.15-2.35), respectively. In the validation cohort of 1262 coronary artery disease patients with type 2 diabetes mellitus, the age-biomarkers-clinical risk factor model was confirmed to provide good discrimination and calibration over the conventional risk factor alone for prediction of MACE. Conclusions- A combination of the 3 biomarkers, lipocalin-2, A-FABP, and FGF-19, with clinical risk factors to yield the age-biomarkers-clinical risk factor model provides an optimal and validated prediction of new-onset MACE in patients with stable coronary artery disease.

Exome-chip association analysis reveals an Asian-specific missense variant in PAX4 associated with type 2 diabetes in Chinese individuals.

AIMS/HYPOTHESIS: Genome-wide association studies (GWASs) have identified many common type 2 diabetes-associated variants, mostly at the intronic or intergenic regions. Recent advancements of exome-array genotyping platforms have opened up a novel means for detecting the associations of low-frequency or rare coding variants with type 2 diabetes. We conducted an exomechip association analysis to identify additional type 2 diabetes susceptibility variants in the Chinese population. METHODS: An exome-chip association study was conducted by genotyping 5640 Chinese individuals from Hong Kong, using a custom designed exome array, the Asian Exomechip. Single variant association analysis was conducted on 77,468 single nucleotide polymorphisms (SNPs). Fifteen SNPs were subsequently genotyped for replication analysis in an independent Chinese cohort comprising 12,362 individuals from Guangzhou. A combined analysis involving 7189 cases and 10,813 controls was performed. RESULTS: In the discovery stage, an Asian-specific coding variant rs2233580 (p.Arg192His) in PAX4, and two variants at the known loci, CDKN2B-AS1 and KCNQ1, were significantly associated with type 2 diabetes with exome-wide significance (p discovery < 6.45 × 10-7). The risk allele (T) of PAX4 rs2233580 was associated with a younger age at diabetes diagnosis. This variant was replicated in an independent cohort and demonstrated a stronger association that reached genome-wide significance (p meta-analysis [p meta] = 3.74 × 10-15) in the combined analysis. CONCLUSIONS/INTERPRETATION: We identified the association of a PAX4 Asian-specific missense variant rs2233580 with type 2 diabetes in an exome-chip association analysis, supporting the involvement of PAX4 in the pathogenesis of type 2 diabetes. Our findings suggest PAX4 is a possible effector gene of the 7q32 locus, previously identified from GWAS in Asians.

Amelioration of X-Linked Related Autophagy Failure in Danon Disease With DNA Methylation Inhibitor.

BACKGROUND: Danon disease is an X-linked disorder that leads to fatal cardiomyopathy caused by a deficiency in lysosome-associated membrane protein-2 (LAMP2). In female patients, a later onset and less severe clinical phenotype have been attributed to the random inactivation of the X chromosome carrying the mutant diseased allele. We generated a patient-specific induced pluripotent stem cell (iPSCs)-based model of Danon disease to evaluate the therapeutic potential of Xi-chromosome reactivation using a DNA methylation inhibitor. METHODS: Using whole-exome sequencing, we identified a nonsense mutation (c.520C>T, exon 4) of the LAMP2 gene in a family with Danon disease. We generated iPSC lines from somatic cells derived from the affected mother and her 2 sons, and we then differentiated them into cardiomyocytes (iPSC-CMs) for modeling the histological and functional signatures, including autophagy failure of Danon disease. RESULTS: Our iPSC-CM platform provides evidence that random inactivation of the wild-type and mutant LAMP2 alleles on the X chromosome is responsible for the unusual phenotype in female patients with Danon disease. In vitro, iPSC-CMs from these patients reproduced the histological features and autophagy failure of Danon disease. Administration of the DNA demethylating agent 5-aza-2'-deoxycytidine reactivated the silent LAMP2 allele in iPSCs and iPSC-CMs in female patients with Danon disease and ameliorated their autophagy failure, supporting the application of a patient-specific iPSC platform for disease modeling and drug screening. CONCLUSIONS: Our iPSC-CM platform provides novel mechanistic and therapeutic insights into the contribution of random X chromosome inactivation to disease phenotype in X-linked Danon disease.

Generation of Induced Cardiospheres via Reprogramming of Skin Fibroblasts for Myocardial Regeneration.

Recent pre-clinical and clinical studies have suggested that endogenous cardiospheres (eCS) are potentially safe and effective for cardiac regeneration following myocardial infarction (MI). Nevertheless the preparation of autologous eCS requires invasive myocardial biopsy with limited yield. We describe a novel approach to generate induced cardiospheres (iCS) from adult skin fibroblasts via somatic reprogramming. After infection with Sox2, Klf4, and Oct4, iCS were generated from mouse adult skin fibroblasts treated with Gsk3ß inhibitor-(2'Z,3'E)- 6-Bromoindirubin-3'-oxime and Oncostatin M. They resembled eCS, but contained a higher percentage of cells expressing Mesp1, Isl1, and Nkx2.5. They were differentiated into functional cardiomyocytes in vitro with similar electrophysiological properties, calcium transient and contractile function to eCS and mouse embryonic stem cell-derived cardiomyocytes. Transplantation of iCS (1 × 106 cells) into mouse myocardium following MI had similar effects to transplantation of eCS but significantly better than saline or fibroblast in improving left ventricular ejection fraction, increasing anterior/septal ventricular wall thickness and capillary density in the infarcted region 4 weeks after transplantation. No tumor formation was observed. iCS generated from adult skin fibroblasts by somatic reprogramming and a cocktail of Gsk3ß inhibitor-6-Bromoindirubin-3'-oxime and Oncostatin M may represent a novel source for cell therapy in MI. Stem Cells 2016;34:2693-2706.

Patient-specific induced-pluripotent stem cells-derived cardiomyocytes recapitulate the pathogenic phenotypes of dilated cardiomyopathy due to a novel DES mutation identified by whole exome sequencing.

In this paper, we report a novel heterozygous mutation of A285V codon conversion on exon 4 of the desmin (DES), using whole exome sequencing (WES) in an isolated proband with documented dilated cardiomyopathy (DCM). This mutation is predicted to cause three-dimensional structure changes of DES. Immunohistological and electron microscopy studies demonstrated diffuse abnormal DES aggregations in DCM-induced-pluripotent stem cell (iPSC)-derived cardiomyocytes, and control-iPSC-derived cardiomyocytes transduced with A285V-DES. DCM-iPSC-derived cardiomyocytes also exhibited functional abnormalities in vitro. This is the first demonstration that patient-specific iPSC-derived cardiomyocytes can be used to provide histological and functional confirmation of a suspected genetic basis for DCM identified by WES.

Remodelling of cardiac sympathetic re-innervation with thoracic spinal cord stimulation improves left ventricular function in a porcine model of heart failure.

AIMS: Thoracic spinal cord stimulation (SCS) has been shown to improve left ventricular ejection fraction (LVEF) in heart failure (HF). Nevertheless, the optimal duration (intermittent vs. continuous) of stimulation and the mechanisms of action remain unclear. METHODS AND RESULTS: We performed chronic thoracic SCS at the level of T1-T3 (50 Hz, pulse width 0.2 ms) in 30 adult pigs with HF induced by myocardial infarction and rapid ventricular pacing for 4 weeks. All the animals were treated with daily oral metoprolol succinate (25 mg) plus ramipril (2.5 mg), and randomized to a control group (n = 10), intermittent SCS (4 h ×3, n = 10) or continuous SCS (24 h, n = 10) for 10 weeks. Serial measurements of LVEF and +dP/dt and serum levels of norepinephrine and B-type natriuretic peptide (BNP) were measured. After sacrifice, immunohistological studies of myocardial sympathetic and parasympathetic nerve sprouting and innervation were performed. Echocardiogram revealed a significant increase in LVEF and +dP/dt at 10 weeks in both the intermittent and continuous SCS group compared with controls (P < 0.05). In both SCS groups, there was diffuse sympathetic nerve sprouting over the infarct, peri-infarct, and normal regions compared with only the peri-infarct and infarct regions in the control group. In addition, sympathetic innervation at the peri-infarct and infarct regions was increased following SCS, but decreased in the control group. Myocardium norepinephrine spillover and serum BNP at 10 weeks was significantly decreased only in the continuous SCS group (P < 0.05). CONCLUSIONS: In a porcine model of HF, SCS induces significant remodelling of cardiac sympathetic innervation over the peri-infarct and infarct regions and is associated with improved LV function and reduced myocardial norepinephrine spillover.

COVID-19 outbreaks in residential care homes in Hong Kong and effectiveness of vaccine against severe outcomes.

Background: COVID-19 outbreaks in residential care homes for the elderly (RCHEs) and for persons with disability (RCHDs) have caused significant morbidity and mortality during 5th epidemic in Hong Kong. This article reviewed COVID-19 outbreaks situation and estimated the effectiveness of receiving at least two-dose of COVID-19 vaccine in preventing severe outcomes. Methods: To estimate attack rates and vaccination coverage, documentation on COVID-19 infection and their vaccination records of residential care homes (RCH) residents reported between December 31, 2021 and May 31, 2022 were reviewed, and infected cases were follow-up for 4 weeks for severe outcomes or death. Correlation between vaccination coverage against attack rate by types of homes was examined. Infected RCH residents with available information were included in the analysis of vaccine effectiveness against severe outcomes and death. Results: COVID-19 vaccination coverage was low in RCHDs (median 0.46, IQR: 0.24-0.76) and very low in RCHEs (median 0.08, IQR: 0.00-0.19). Higher attack rates were recorded among RCHE residents (median 0.84, IQR: 0.64-0.93) and higher case fatality rate (CFR: 28.1%) than in RCHDs (median 0.58, IQR: 0.31-0.84; CFR: 3.9%). The attack rate decreased when vaccination coverage increased for both RCHEs (ρ = -0.131, p < 0.001) and RCHDs (ρ = -0.333, p < 0.001). Comparing with infected residents who were unvaccinated/vaccinated with one-dose, receiving at least two-dose was estimated to be effective in reducing severe outcomes in 31% and 36% of infected RCHE and RCHD residents respectively; with greater reduction in mortality among RCHD than RCHE residents (54% and 38%, respectively). Vaccine effectiveness of two-dose of BNT162b2 against severe outcomes and death are higher than that of CoronaVac. Conclusions: Increasing COVID-19 vaccination could have significant impact on reducing the risk of COVID-19 outbreaks in RCHs. At least two-dose of COVID-19 vaccine is still effective in reducing severe outcomes and death among infected residents in RCHs during Omicron epidemic.

The city-wide full-scale interactive application of sewage surveillance programme for assisting real-time COVID-19 pandemic control - A case study in Hong Kong.

The paper discusses the implementation of Hong Kong's tailor-made sewage surveillance programme led by the Government, which has demonstrated how an efficient and well-organized sewage surveillance system can complement conventional epidemiological surveillance to facilitate the planning of intervention strategies and actions for combating COVID-19 pandemic in real-time. This included the setting up of a comprehensive sewerage network-based SARS-CoV-2 virus surveillance programme with 154 stationary sites covering 6 million people (or 80 % of the total population), and employing an intensive monitoring programme to take samples from each stationary site every 2 days. From 1 January to 22 May 2022, the daily confirmed case count started with 17 cases per day on 1 January to a maximum of 76,991 cases on 3 March and dropped to 237 cases on 22 May. During this period, a total of 270 "Restriction-Testing Declaration" (RTD) operations at high-risk residential areas were conducted based on the sewage virus testing results, where over 26,500 confirmed cases were detected with a majority being asymptomatic. In addition, Compulsory Testing Notices (CTN) were issued to residents, and the distribution of Rapid Antigen Test kits was adopted as alternatives to RTD operations in areas of moderate risk. These measures formulated a tiered and cost-effective approach to combat the disease in the local setting. Some ongoing and future enhancement efforts to improve efficacy are discussed from the perspective of wastewater-based epidemiology. Forecast models on case counts based on sewage virus testing results were also developed with R2 of 0.9669-0.9775, which estimated that up to 22 May 2022, around 2,000,000 people (~67 % higher than the total number of 1,200,000 reported to the health authority, due to various constraints or limitations) had potentially contracted the disease, which is believed to be reflecting the real situation occurring in a highly urbanized metropolis like Hong Kong.

Interferons induce the expression of IFITM1 and IFITM3 and suppress the proliferation of rat neonatal cardiomyocytes.

Cardiovascular diseases have been one of the leading killers among the human population worldwide. During the heart development, cardiomyocytes undergo a transition from hyperplastic to hypertrophic growth with an unclear underlying mechanism. In this study, we aim to investigate how interferons differentially stimulate the interferon-inducible transmembrane (IFITM) family proteins and further be involved in the process of heart development. The expression levels of three IFITM family members, IFITM1, IFITM2, and IFITM3 were investigated during Sprague-Dawley rat myocardial development and differentiation of H9C2 cardiomyocytes. The effects of interferon-α, -ß, and -γ on DNA synthesis in H9C2 cells were also characterized. Up-regulation of IFITM1 and IFITM3 were observed during the heart development of Sprague-Dawley rat and the differentiation of H9C2 cells. Moreover, interferon-α and -ß induce the expression of IFITM3 while interferon-γ up-regulates IFITM1. Finally, interferon-α and -ß were demonstrated to inhibit DNA synthesis during H9C2 cell differentiation. Our results indicated interferons are potentially involved in the differentiation and cell proliferation during heart development.

Thoracic spinal cord stimulation improves cardiac contractile function and myocardial oxygen consumption in a porcine model of ischemic heart failure.

BACKGROUND: Prior experimental studies show that thoracic spinal cord stimulation (SCS) improves left ventricular (LV) ejection fraction (LVEF). The mechanism of this improvement in the LV contractile function after SCS and its effects on the myocardial oxygen consumption remains unknown. METHODS AND RESULTS: We performed thoracic SCS (T1-T2 level) followed by 4 weeks of rapid ventricular pacing in 9 adult pigs with ischemic heart failure (HF) induced by myocardial infarction (MI). At 24 hours off-pacing, detailed echocardiogram and invasive hemodynamic assessment were performed to determine LV contractile function and myocardial oxygen consumption. Serum norepinephrine level was measured before and after SCS. SCS was performed on 2 occasions for 15 minutes, 30 minutes apart (recovery) with 50 Hz frequency (pulse width 0.2 millisecond, 90% of motor threshold at 2 Hz output). Echocardiogram revealed significant decrease in LVEF (33.8 ± 1.8% vs 66.5 ± 1.7%, P < 0.01) after induction of MI and HF. Compared with MI and HF, acute SCS significantly increased LVEF and +dP/dt (all P < 0.05). Withdrawal of SCS during recovery decreased +dP/dt, but not LVEF that increased again with repeated SCS. Myocardial oxygen consumption also significantly decreased during SCS compared with MI and HF (P = 0.006) without any change in serum norepinephrine level (P = 0.9). Speckle tracking imaging showed significant improvement in global and regional circumferential strains over the infarcted mid and apical regions, decreased in time to peak circumferential strain over the lateral and posterior wall after SCS, and the degree of intraventricular dyssynchrony during SCS compared with MI and HF (P < 0.05). CONCLUSIONS: In a porcine model of ischemic HF, acute SCS improved global and regional LV contractile function and intraventricular dyssynchrony, and decreased myocardial oxygen consumption without elevation of norepinephrine level.

Reversal of endothelial progenitor cell dysfunction in patients with type 2 diabetes using a conditioned medium of human embryonic stem cell-derived endothelial cells.

BACKGROUND: The potential clinical application of bone marrow or peripheral blood-derived progenitor cells for cardiovascular regeneration in patients with diabetes mellitus (DM) is limited by their functional impairment. We sought to determine the mechanisms of impaired therapeutic efficacy of peripheral blood-derived progenitor cells in type 2 DM patients and evaluated the use of cell-free conditioned medium obtained from human embryonic stem cell-derived endothelial-like cells (ESC-ECs) to reverse their functional impairment. METHODS: The angiogenic potential of late outgrowth endothelial cells (OECs) and cytokine profile of the conditional medium of proangiogenic cells (PACs) derived from peripheral blood-mononuclear cells of healthy control and DM patients and ESC-ECs was compared by in vitro tube formation assay and a multiplex bead-based immunoassay kit, respectively. The in vivo angiogenic potential of ESC-ECs derived conditioned medium in rescuing the functional impairment of PB-PACs in DM patients was investigated using a hindlimb ischemia model. RESULTS: Human ESC-ECs had similar functional and phenotypic characteristics as OECs in healthy controls. Cytokine profiling showed that vascular endothelial growth factor, stromal cell-derived factor 1 and placental growth factor were down-regulated in PACs from DM patients. Tube formation assay that revealed functional impairment of OECs from DM patients could be rescued by ESC-ECs conditioned medium. Administration of ESC-ECs conditioned medium restored the therapeutic efficacy of PB-PACs from DM patients in a mouse model of hindlimb ischemia. CONCLUSIONS: Our results showed that peripheral blood-derived progenitor cells from DM patients have impaired function because of defective secretion of angiogenic cytokines, which could be restored by supplementation of ESC-ECs conditioned medium.

Liposome-encapsulated curcumin attenuates HMGB1-mediated hepatic inflammation and fibrosis in a murine model of Wilson's disease.

BACKGROUND AND AIMS: Wilson's disease (WD) is an inherited disorder of copper metabolism with predominant hepatic manifestations. Left untreated, it can be fatal. Current therapies focus on treating copper overload rather than targeting the pathophysiology of copper-induced liver injuries. We sought to investigate whether liposome-encapsulated curcumin (LEC) could attenuate the underlying pathophysiology of WD in a mouse model of WD. APPROACH AND RESULTS: Subcutaneous administration in a WD mouse model with ATP7B knockout (Atp7b-/-) resulted in robust delivery of LEC to the liver as determined by in-vitro and in-vivo imaging. Treatment with LEC attenuated hepatic injuries, restored lipid metabolism and decreased hepatic inflammation and fibrosis, and thus hepatosplenomegaly in Atp7b-/- mice. Mechanistically, LEC decreased hepatic immune cell and macrophage infiltration and attenuated the hepatic up-regulation of p65 by preventing cellular translocation of high-mobility group box-1 (HMGB-1). Moreover, decreased translocation of HMGB1 was associated with reduced splenic CD11b+/CD43+/Ly6CHi inflammatory monocyte expansion and circulating level of proinflammatory cytokines. Nevertheless there was no change in expression of oxidative stress-related genes or significant copper chelation effect of LEC in Atp7b-/- mice. CONCLUSION: Our results indicate that treatment with subcutaneous LEC can attenuate copper-induced liver injury in an animal model of WD via suppression of HMGB1-mediated hepatic and systemic inflammation. These findings provide important proof-of-principle data to develop LEC as a novel therapy for WD as well as other inflammatory liver diseases.

A review of epidemiology and public health control measures of COVID-19 variants in Hong Kong, December 2020 to June 2021.

Background: The emergence of coronavirus disease 2019 (COVID-19) variants posed considerable threats to the global public health. We reviewed the epidemiology of variant cases and control measures implemented in Hong Kong. Methods: The epidemiological characteristics and the temporal trend of the COVID-19 variant cases and local clusters in Hong Kong, and the corresponding public health control measures were reviewed. Results: Between December 2020 and June 2021, 393 variant cases were reported, including 153, 59 and 79 cases of Alpha, Beta and Delta variants with no Gamma variant. The vast majority (378, 96.2%) were imported cases. Since early June 2021, Delta variant had taken over Alpha as the dominant strain. Public health control measures, including risk-stratified quarantine and testing requirements for inbound travellers, banning of flights from extremely high-risk areas, enhanced contact tracing and quarantine, were implemented. Among the 3 clusters involving local transmissions, 2 were linked to imported cases while the source of the remaining one was unknown. Discussion: Amid the global surges of COVID-19 variants, Hong Kong had continued to limit and prevent the occurrence of community-wide outbreak. Ongoing control strategies should be constantly reviewed and adjusted in response to the global and local COVID-19 situation.

CRISPR-targeted genome editing of human induced pluripotent stem cell-derived hepatocytes for the treatment of Wilson's disease.

BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by loss-of-function mutations in ATP7B, which encodes a copper-transporting protein. It is characterized by excessive copper deposition in tissues, predominantly in the liver and brain. We sought to investigate whether gene-corrected patient-specific induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) could serve as an autologous cell source for cellular transplantation therapy in WD. METHODS: We first compared the in vitro phenotype and cellular function of ATP7B before and after gene correction using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) in iHeps (derived from patients with WD) which were homozygous for the ATP7B R778L mutation (ATP7BR778L/R778L). Next, we evaluated the in vivo therapeutic potential of cellular transplantation of WD gene-corrected iHeps in an immunodeficient WD mouse model (Atp7b -/- / Rag2 -/- / Il2rg -/- ; ARG). RESULTS: We successfully created iPSCs with heterozygous gene correction carrying 1 allele of the wild-type ATP7B gene (ATP7BWT/-) using CRISPR/Cas9 and ssODNs. Compared with ATP7BR778L/R778L iHeps, gene-corrected ATP7BWT/- iHeps restored i n vitro ATP7B subcellular localization, its subcellular trafficking in response to copper overload and its copper exportation function. Moreover, in vivo cellular transplantation of ATP7BWT/- iHeps into ARG mice via intra-splenic injection significantly attenuated the hepatic manifestations of WD. Liver function improved and liver fibrosis decreased due to reductions in hepatic copper accumulation and consequently copper-induced hepatocyte toxicity. CONCLUSIONS: Our findings demonstrate that gene-corrected patient-specific iPSC-derived iHeps can rescue the in vitro and in vivo disease phenotypes of WD. These proof-of-principle data suggest that iHeps derived from gene-corrected WD iPSCs have potential use as an autologous ex vivo cell source for in vivo therapy of WD as well as other inherited liver disorders. LAY SUMMARY: Gene correction restored ATP7B function in hepatocytes derived from induced pluripotent stem cells that originated from a patient with Wilson's disease. These gene-corrected hepatocytes are potential cell sources for autologous cell therapy in patients with Wilson's disease.

Mendelian randomization analysis of vitamin D in the secondary prevention of hypertensive-diabetic subjects: role of facilitating blood pressure control.

BACKGROUND: Vitamin D (Vit-D) promotes vascular repair and its deficiency is closely linked to the development of type 2 diabetes mellitus (T2DM) and hypertension. Whether genetially predicted vitamin D status (serological 25-hydroxyvitamin D [25(OH)D]) confers secondary protection against cardiovascular diseases (CVD) among high-risk hypertensive-diabetic subjects was unknown. METHODS: This is a prospective, individual-data, two-sample Mendelian randomization study. We interrogated 12 prior GWAS-detected SNPs of comprehensive Vit-D mechanistic pathways using high-throughput exome chip analyses in a derivation subcohort (n = 1460) and constructed a genetic risk score (GRS) (rs2060793, rs4588, rs7041; F-statistic = 32, P < 0.001) for causal inference of comprehensive CVD hard clinical endpoints in an independent sample of hypertensive subjects (n = 3746) with prevailing co-morbid T2DM (79%) and serological 25(OH)D deficiency [< 20 ng/mL] 45%. RESULTS: After 55.6 ± 28.9 months, 561 (15%) combined CVD events including myocardial infarction, unstable angina, ischemic stroke, congestive heart failure, peripheral vascular disease, and cardiovascular death had occurred. Kaplan-Meier analysis showed that genetically predicted reduced vitamin D status was associated with reduced event-free survival from combined CVD events (log-rank = 13.5, P = 0.001). Multivariate-adjusted per-allele increase in GRS predicted reduced combined CVD events (HR = 0.90 [0.84 to 0.96], P = 0.002). Mendelian randomization indicates that increased Vit-D exposure, leveraged through each 1 ng/mL genetically instrumented rise of serum Vit-D, protects against combined CVD events (Wald's estimate: OR = 0.86 [95%CI 0.75 to 0.95]), and myocardial infarction (OR = 0.76 [95%CI 0.60 to 0.90]). Furthermore, genetically predicted increase in Vit-D status ameliorates risk of deviation from achieving guideline-directed hypertension control (JNC-8: systolic target < 150 mmHg) (OR = 0.89 [95%CI 0.80 to 0.96]). CONCLUSIONS: Genetically predicted increase in Vit-D status [25(OH)D] may confer secondary protection against incident combined CVD events and myocardial infarction in a hypertensive-diabetic population where serological 25(OH)D deficiency is common, through facilitating blood pressure control.

Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice.

BACKGROUND: Aging and aging-related disorders impair the survival and differentiation potential of bone marrow mesenchymal stem cells (MSCs) and limit their therapeutic efficacy. Induced pluripotent stem cells (iPSCs) may provide an alternative source of functional MSCs for tissue repair. This study aimed to generate and characterize human iPSC-derived MSCs and to investigate their biological function for the treatment of limb ischemia. METHODS AND RESULTS: Human iPSCs were induced to MSC differentiation with a clinically compliant protocol. Three monoclonal, karyotypically stable, and functional MSC-like cultures were successfully isolated using a combination of CD24(-) and CD105(+) sorting. They did not express pluripotent-associated markers but displayed MSC surface antigens and differentiated into adipocytes, osteocytes, and chondrocytes. Transplanting iPSC-MSCs into mice significantly attenuated severe hind-limb ischemia and promoted vascular and muscle regeneration. The benefits of iPSC-MSCs on limb ischemia were superior to those of adult bone marrow MSCs. The greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment after transplantation to induce vascular and muscle regeneration via direct de novo differentiation and paracrine mechanisms. CONCLUSIONS: Functional MSCs can be clonally generated, beginning at a single-cell level, from human iPSCs. Patient-specific iPSC-MSCs can be prepared as an "off-the-shelf" format for the treatment of tissue ischemia.

Epigenetic regulation of neonatal cardiomyocytes differentiation.

The relationship between DNA methylation, histone modifications and terminal differentiation in cardiomyocytes was investigated in this study. The upregulation of methylation-related proteins, including DNA methyltransferase (DNMT) 1, methyl-CpG binding domain proteins 1, 2 and 3, and the increase in global methylation during rat neonatal heart development were observed. Moreover, an increase in DNA synthesis and a delay in differentiation were found in 5-azacytidine (5-azaC)-treated cardiomyocytes. Increase in acetylation of H3-K9, H4-K5, H4-K8 and methylation of H3-K4 suggested a more accessible chromatin structure in 5-azaC-treated cells. Furthermore, methyl-CpG-binding protein 2 was found to be upregulated in differentiated cardiomyocytes. Overexpression of this protein resulted in an increase of global methylation levels. Therefore, we suggest that a hypermethylated genome and a more compact chromatin structure are formed during terminal differentiation of cardiomyocytes.