The Pathogenic Mechanisms of and Novel Therapies for Lamin A/C-Related Dilated Cardiomyopathy Based on Patient-Specific Pluripotent Stem Cell Platforms and Animal Models.

Variants (pathogenic) of the LMNA gene are a common cause of familial dilated cardiomyopathy (DCM), which is characterised by early-onset atrioventricular (AV) block, atrial fibrillation and ventricular tachyarrhythmias (VTs), and progressive heart failure. The unstable internal nuclear lamina observed in LMNA-related DCM is a consequence of the disassembly of lamins A and C. This suggests that LMNA variants produce truncated or alternative forms of protein that alter the nuclear structure and the signalling pathway related to cardiac muscle diseases. To date, the pathogenic mechanisms and phenotypes of LMNA-related DCM have been studied using different platforms, such as patient-specific induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and transgenic mice. In this review, point variants in the LMNA gene that cause autosomal dominantly inherited forms of LMNA-related DCM are summarised. In addition, potential therapeutic targets based on preclinical studies of LMNA variants using transgenic mice and human iPSC-CMs are discussed. They include mitochondria deficiency, variants in nuclear deformation, chromatin remodelling, altered platelet-derived growth factor and ERK1/2-related pathways, and abnormal calcium handling.

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.

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.

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.

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.

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.

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 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.

The epidemiology of COVID-19 cases and the successful containment strategy in Hong Kong-January to May 2020.

BACKGROUND: Hong Kong, a Special Administrative Region of China, recorded its first confirmed coronavirus disease 2019 (COVID-19) case on 23 January 2020. We reviewed the case epidemiology and the various public health measures implemented from January to May 2020. METHODS: The epidemiological and clinical characteristics of the cases recorded in different phases of the epidemic were described and compared, and the effectiveness of the public health measures implemented were reviewed using the changes in the daily number of confirmed cases and the interval from symptom onset to hospital admission. RESULTS: Between January and May 2020, 1084 confirmed COVID-19 cases were reported, about 70% of which had a history of travel during the incubation period. The case fatality ratio was 0.4%. The local epidemic progressed through four phases: (1) preparedness and imported infection from mainland China, (2) local transmission, (3) imported infection from overseas countries associated with local transmission, and (4) controlled imported infection with limited local transmission, with an eventual reduction of the daily case number and minimization of the onset-to-admission interval. Various public health measures, including enhanced surveillance, border control, and social distancing, were introduced in phases in response to the prevailing local and global situations. DISCUSSION: The overall containment strategy in Hong Kong led to a stabilization of the number of cases and the absence of a community-wide outbreak during the 4.5 m after the first case was reported. This strategy of containment might serve as an example for future planning of preparedness and response against novel infectious agents.

Varicella vaccine dose depended effectiveness and waning among preschool children in Hong Kong.

In Hong Kong, universal varicella vaccination was introduced in July 2014 with a two-dose schedule but the vaccines had been available in the private market since 1996. With data from varicella notification and surveys on immunization coverage, we used the screening method to estimate dose-specific varicella vaccine effectiveness (VE) among preschool children in Hong Kong before universal vaccination. We estimated the VE of one- and two-dose varicella vaccination against all notified varicella as 69.4% (95% confidence interval (95% CI) 69.5-71.2) and 93.4% (95% CI 91.7-94.7), respectively. We found that VE did not decrease with time since receipt. Varicella vaccine was more effective against complications (85.4% [95% CI 48.8-95.8] for one dose and 100% [95% CI -Inf to 100] for two doses) and against hospital admission (75.2% [95% CI 53.4-86.8] for one dose and 93.1% [95% CI 47.1-99.1] for two doses). Lower protection of one-dose varicella vaccine resulted in breakthrough varicella. Under universal vaccination, second-dose varicella vaccine (given as combined measles, mumps, rubella and varicella vaccine) was first scheduled for children when they reach primary one (about 6 years of age) and was recently advanced to 18 months of age. Shortening the interval between the first dose and second dose of varicella vaccination should reduce breakthrough varicella and outbreaks in preschool.

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.

Catheter-Based Splanchnic Denervation for Treatment of Hypertensive Cardiomyopathy.

Emerging preclinical data suggest that splanchnic sympathetic nerve activation may play an important role in the pathophysiology of hypertension. We sought to determine the potential therapeutic application of catheter-based splanchnic denervation in a clinically relevant large animal model of hypertensive cardiomyopathy (hCMP). Sustained elevated blood pressure was induced in adult pigs using a combination of intravenous infusion of Ang II (angiotensin II) and subcutaneous implantation of deoxycorticosterone acetate pellets to establish a large animal model of hCMP. Serial changes in cardiac echocardiographic and invasive hemodynamic parameters and neurohumoral biomarkers were investigated in animals with hypertension alone (n=9) and hypertension with catheter-based splanchnic denervation (n=6). Another 6 pigs without hypertension induction served as controls. At 10 weeks, hypertensive animals developed sustained elevated blood pressure and phenotype of hCMP with significant systolic and diastolic dysfunction, and left ventricular remodeling and hypertrophy as determined by invasive hemodynamic and echocardiogram assessments, respectively, and increased venoarterial norepinephrine gradient over the myocardium, kidneys, and splanchnic organs compared with baseline. Catheter-based splanchnic denervation decreased the venoarterial norepinephrine gradient over the splanchnic organs associated with the reduced splenic sympathetic nerve innervation; attenuated the elevated blood pressure, left ventricular remodeling, and hypertrophy; and preserved left ventricular systolic and diastolic function at 20 weeks in pigs with hCMP. Our results provide novel mechanistic insight into the role of splenic sympathetic nerve innervation in hypertension and important proof-of-principle data for the therapeutic application of catheter-based splanchnic denervation in a large animal model of hCMP.

Seasonal influenza vaccine effectiveness at primary care level, Hong Kong SAR, 2017/2018 winter.

The 2017/18 winter influenza season in Hong Kong started in early January 2018, predominated by influenza B/Yamagata. We collaborated with private medical practitioners of our sentinel surveillance system to collect respiratory specimens and clinical information from patients with influenza-like illness for estimation of the influenza vaccine effectiveness (VE) using the test-negative case-control design. We found that the overall VE was 59.1% (95%CI 41.1 to 71.8%) against all influenza and 53.5% (95%CI 35.4 to 74.6%) against influenza B. Seasonal influenza vaccine provided moderate to good protection against laboratory-confirmed influenza infection at primary care level in Hong Kong in the 2017/18 winter influenza season.

Genetic Regulation of Pigment Epithelium-Derived Factor (PEDF): An Exome-Chip Association Analysis in Chinese Subjects With Type 2 Diabetes.

Elevated circulating levels of pigment epithelium-derived factor (PEDF) have been reported in patients with type 2 diabetes (T2D) and its associated microvascular complications. This study aimed to 1) identify the genetic determinants influencing circulating PEDF levels in a clinical setting of T2D, 2) examine the relationship between circulating PEDF and diabetes complications, and 3) explore the causal relationship between PEDF and diabetes complications. An exome-chip association study on circulating PEDF levels was conducted in 5,385 Chinese subjects with T2D. A meta-analysis of the association results of the discovery stage (n = 2,936) and replication stage (n = 2,449) was performed. The strongest association was detected at SERPINF1 (p.Met72Thr; Pcombined = 2.06 × 10-57; ß [SE] -0.33 [0.02]). Two missense variants of SMYD4 (p.Arg131Ile; Pcombined = 7.56 × 10-25; ß [SE] 0.21 [0.02]) and SERPINF2 (p.Arg33Trp; Pcombined = 8.22 × 10-10; ß [SE] -0.15 [0.02]) showed novel associations at genome-wide significance. Elevated circulating PEDF levels were associated with increased risks of diabetic nephropathy and sight-threatening diabetic retinopathy. Mendelian randomization analysis showed suggestive evidence of a protective role of PEDF on sight-threatening diabetic retinopathy (P = 0.085). Our study provided new insights into the genetic regulation of PEDF and further support for its potential application as a biomarker for diabetic nephropathy and sight-threatening diabetic retinopathy. Further studies to explore the causal relationship of PEDF with diabetes complications are warranted.

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.

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

An Exome-Chip Association Analysis in Chinese Subjects Reveals a Functional Missense Variant of GCKR That Regulates FGF21 Levels.

Fibroblast growth factor 21 (FGF21) is increasingly recognized as an important metabolic regulator of glucose homeostasis. Here, we conducted an exome-chip association analysis by genotyping 5,169 Chinese individuals from a community-based cohort and two clinic-based cohorts. A custom Asian exome-chip was used to detect genetic determinants influencing circulating FGF21 levels. Single-variant association analysis interrogating 70,444 single nucleotide polymorphisms identified a novel locus, GCKR, significantly associated with circulating FGF21 levels at genome-wide significance. In the combined analysis, the common missense variant of GCKR, rs1260326 (p.Pro446Leu), showed an association with FGF21 levels after adjustment for age and sex (P = 1.61 × 10-12; ß [SE] = 0.14 [0.02]), which remained significant on further adjustment for BMI (P = 3.01 × 10-14; ß [SE] = 0.15 [0.02]). GCKR Leu446 may influence FGF21 expression via its ability to increase glucokinase (GCK) activity. This can lead to enhanced FGF21 expression via elevated fatty acid synthesis, consequent to the inhibition of carnitine/palmitoyl-transferase by malonyl-CoA, and via increased glucose-6-phosphate-mediated activation of the carbohydrate response element binding protein, known to regulate FGF21 gene expression. Our findings shed new light on the genetic regulation of FGF21 levels. Further investigations to dissect the relationship between GCKR and FGF21, with respect to the risk of metabolic diseases, are warranted.

Generation of Human Liver Chimeric Mice with Hepatocytes from Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

Familial hypercholesterolemia (FH) causes elevation of low-density lipoprotein cholesterol (LDL-C) in blood and carries an increased risk of early-onset cardiovascular disease. A caveat for exploration of new therapies for FH is the lack of adequate experimental models. We have created a comprehensive FH stem cell model with differentiated hepatocytes (iHeps) from human induced pluripotent stem cells (iPSCs), including genetically engineered iPSCs, for testing therapies for FH. We used FH iHeps to assess the effect of simvastatin and proprotein convertase subtilisin/kexin type 9 (PCSK9) antibodies on LDL-C uptake and cholesterol lowering in vitro. In addition, we engrafted FH iHeps into the liver of Ldlr-/-/Rag2-/-/Il2rg-/- mice, and assessed the effect of these same medications on LDL-C clearance and endothelium-dependent vasodilation in vivo. Our iHep models recapitulate clinical observations of higher potency of PCSK9 antibodies compared with statins for reversing the consequences of FH, demonstrating the utility for preclinical testing of new therapies for FH patients.