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.

Modeling Treatment Response for Lamin A/C Related Dilated Cardiomyopathy in Human Induced Pluripotent Stem Cells.

BACKGROUND: Precision medicine is an emerging approach to disease treatment and prevention that takes into account individual variability in the environment, lifestyle, and genetic makeup of patients. Patient-specific human induced pluripotent stem cells hold promise to transform precision medicine into real-life clinical practice. Lamin A/C (LMNA)-related cardiomyopathy is the most common inherited cardiomyopathy in which a substantial proportion of mutations in the LMNA gene are of nonsense mutation. PTC124 induces translational read-through over the premature stop codon and restores production of the full-length proteins from the affected genes. In this study we generated human induced pluripotent stem cells-derived cardiomyocytes from patients who harbored different LMNA mutations (nonsense and frameshift) to evaluate the potential therapeutic effects of PTC124 in LMNA-related cardiomyopathy. METHODS AND RESULTS: We generated human induced pluripotent stem cells lines from 3 patients who carried distinctive mutations (R225X, Q354X, and T518fs) in the LMNA gene. The cardiomyocytes derived from these human induced pluripotent stem cells lines reproduced the pathophysiological hallmarks of LMNA-related cardiomyopathy. Interestingly, PTC124 treatment increased the production of full-length LMNA proteins in only the R225X mutant, not in other mutations. Functional evaluation experiments on the R225X mutant further demonstrated that PTC124 treatment not only reduced nuclear blebbing and electrical stress-induced apoptosis but also improved the excitation-contraction coupling of the affected cardiomyocytes. CONCLUSIONS: Using cardiomyocytes derived from human induced pluripotent stem cells carrying different LMNA mutations, we demonstrated that the effect of PTC124 is codon selective. A premature stop codon UGA appeared to be most responsive to PTC124 treatment.

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.

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.

Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons.

Patients with Danon disease may suffer from severe cardiomyopathy, skeletal muscle dysfunction as well as varying degrees of mental retardation, in which the primary deficiency of lysosomal membrane-associated protein-2 (LAMP2) is considerably associated. Owing to the scarcity of human neurons, the pathological role of LAMP2 deficiency in neural injury of humans remains largely elusive. However, the application of induced pluripotent stem cells (iPSCs) may shed light on overcoming such scarcity. In this study, we obtained iPSCs derived from a patient carrying a mutated LAMP2 gene that is associated with Danon disease. By differentiating such LAMP2-deficient iPSCs into cerebral cortical neurons and with the aid of various biochemical assays, we demonstrated that the LAMP2-deficient neurons are more susceptible to mild oxidative stress-induced injury. The data from MTT assay and apoptotic analysis demonstrated that there was no notable difference in cellular viability between the normal and LAMP2-deficient neurons under non-stressed condition. When exposed to mild oxidative stress (10 µM H2O2), the LAMP2-deficient neurons exhibited a significant increase in apoptosis. Surprisingly, we did not observe any aberrant accumulation of autophagic materials in the LAMP2-deficient neurons under such stress condition. Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.

Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese.

Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10(-7)), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci-PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser-also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD.

Attenuation of hind-limb ischemia in mice with endothelial-like cells derived from different sources of human stem cells.

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (P>0.05). While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05), the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05). Compared with medium, transplanting BM-EC (n = 6), HUVEC (n = 6), hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as "off-the-shelf" format for the treatment of tissue ischemia.

Randomized controlled trial of vitamin D supplement on endothelial function in patients with type 2 diabetes.

BACKGROUND: Suboptimal vitamin D status is associated with endothelial dysfunction and an increased risk of cardiovascular diseases but it is unclear whether vitamin D supplementation is beneficial. The aim was to investigate the effect of vitamin D supplementation on endothelial function in patients with type 2 diabetes mellitus (DM). METHODS: In a double-blind, placebo-controlled trial, we randomized 100 type 2 DM patients to vitamin D supplement (5000 IU/day, n = 50) or placebo (controls, n = 50) for 12 weeks. Assessment of vascular function with brachial artery flow-mediated dilatation (FMD), circulating levels of endothelial progenitor cells (EPCs) and brachial-ankle pulse wave velocity, and metabolic parameter, high-sensitivity C-reactive protein (hsCRP) and oxidative stress markers were performed before and after the supplementation. RESULTS: After 12 weeks, vitamin D treated patients had significant increases in serum 25-hydroxyvitamin D [25(OH)D] concentration (treatment effect 34.7 ng/mL, 95% CI 26.4-42.9, P < 0.001) and serum ionized calcium (treatment effect 0.037 mmol/L, 95% CI 0.007-0.067, P = 0.018); decreased serum parathyroid hormone concentration (treatment effect -0.55 pmol/L, 95% CI -1.08 to -0.02, P = 0.042) compared to patients who received placebo. Nevertheless, vitamin D supplementation did not improve vascular function as determined by FMD, circulating EPC count or baPWV (all P > 0.05). Furthermore, hsCRP, oxidative stress markers, low- and high-density lipoprotein and glycated hemoglobin were also similar between two groups (all P > 0.05). CONCLUSION: In patients with type 2 DM, 12 weeks oral supplementation of vitamin D did not significantly affect vascular function or serum biomarkers of inflammation and oxidative stress. CLINICAL TRIAL NUMBER: HKCTR-867, www.hkclinicaltrials.com.

Modeling of lamin A/C mutation premature cardiac aging using patient­specific induced pluripotent stem cells.

AIMS: We identified an autosomal dominant non­sense mutation (R225X) in exon 4 of the lamin A/C (LMNA) gene in a Chinese family spanning 3 generations with familial dilated cardiomyopathy (DCM). In present study, we aim to generate induced pluripotent stem cells derived cardiomyocytes (iPSC­CMs) from an affected patient with R225X and another patient bearing LMNA frame­shift mutation for drug screening. METHODS AND RESULTS: Higher prevalence of nuclear bleb formation and micronucleation was present in LMNA(R225X/WT) and LMNA(Framshift/WT) iPSC­CMs. Under field electrical stimulation, percentage of LMNA­mutated iPSC­CMs exhibiting nuclear senescence and cellular apoptosis markedly increased. shRNA knockdown of LMNA replicated those phenotypes of the mutated LMNA field electrical stress. Pharmacological blockade of ERK1/2 pathway with MEK1/2 inhibitors, U0126 and selumetinib (AZD6244) significantly attenuated the pro­apoptotic effects of field electric stimulation on the mutated LMNA iPSC­CMs. CONCLUSION: LMNA­related DCM was modeled in­vitro using patient­specific iPSC­CMs. Our results demonstrated that haploinsufficiency due to R225X LMNA non­sense mutation was associated with accelerated nuclear senescence and apoptosis of iPSC­ CMs under electrical stimulation, which can be significantly attenuated by therapeutic blockade of stress­related ERK1/2 pathway.

Vitamin D deficiency is associated with depletion of circulating endothelial progenitor cells and endothelial dysfunction in patients with type 2 diabetes.

CONTEXT: Vitamin D (Vit-D) deficiency is associated with type 2 diabetes mellitus (DM) and endothelial dysfunction. The relationship of Vit-D deficiency with circulating endothelial progenitor cells and endothelial dysfunction in type 2 DM patients nonetheless remains unclear. OBJECTIVE: We aimed to investigate the cross-sectional association of Vit-D status with brachial flow-mediated dilation (FMD) and circulating endothelial progenitor cell (EPC) numbers in type 2 DM patients. DESIGN, SETTING, AND PARTICIPANTS: We conducted a cross-sectional study of 280 patients (59% male, aged 68 ± 10 yr) with type 2 DM recruited in outpatient clinics during the winter period. MAIN OUTCOME MEASURE: We measured serum 25-hydroxyvitamin D [25(OH)D] by an ELISA kit, circulating CD34+/kinase insert domain-containing receptor (KDR)+ and CD133+/KDR+ EPCs by flow cytometry and brachial artery FMD by vascular ultrasound, respectively. RESULTS: The mean serum 25(OH)D concentration was 25.00 ± 9.17 ng/ml, and 34.3% of patients had Vit-D deficiency [25(OH)D < 20 ng/ml]. Serum 25(OH)D concentration had a significant correlation with hemoglobin A1c level [B = -0.018, 95% confidence interval (CI) -0.035 to -0.002, P = 0.032]. Patients with Vit-D deficiency status had significantly lower brachial FMD (mean difference -1.43%, 95% CI -2.31 to -0.55, P = 0.001) and CD133+/KDR+EPC counts (mean difference -0.12%, 95% CI -0.21 to -0.019, P = 0.022) than those with sufficient Vit-D status after adjustment for age, sex, and cardiovascular risk factors, including hemoglobin A1c levels. CONCLUSIONS: Our results demonstrate that serum 25(OH)D status was significantly associated with brachial artery FMD and circulating CD133+/KDR+EPCs. This suggests that Vit-D deficiency might contribute to depletion of EPCs and endothelial dysfunction in patients with type 2 DM.

Triiodothyronine promotes cardiac differentiation and maturation of embryonic stem cells via the classical genomic pathway.

Embryonic stem cells (ESCs) can differentiate into functional cardiomyocytes and thus represent a promising cell source for cardiac regenerative therapy. Nevertheless, the therapeutic application of ESC-derived cardiomyocytes is limited by the low efficacy of the current protocol for cardiac differentiation and their immature phenotypes. Although thyroid hormone is essential for normal cardiac development and function, its role in the cardiac differentiation of ESCs, as well as the maturation of ESC-derived cardiomyocytes, remains unclear. In this study, we examined the cardiac differentiation of murine ESCs in the presence of T(3) for 7 d using flow cytometry, RT-PCR, cellular electrophysiology study, and confocal calcium imaging. Compared with control conditions, T(3) supplementation increased the number of ESC-derived cardiomyocytes and was accompanied by up-regulation of a panel of cardiac markers, including Nkx2.5, myosin light chain-2V, as well as alpha- and beta-myosin heavy chain. More importantly, electrophysiological study revealed that ESC-derived cardiomyocytes exhibited more adult-like phenotypes after T(3) supplementation based on action potential characteristics. They also exhibited more adult-like calcium homeostasis properties. These phenotypic changes were associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase-2a and ryanodine receptor-2 expression. In addition, the classical (genomic) pathway was shown to be involved in T(3)-induced cardiac differentiation of ESCs. Our results show that T(3) supplementation promotes cardiac differentiation of ESCs and enhances maturation of electrophysiological, as well as calcium homeostasis, properties of ESC-derived cardiomyocytes.

Role of circulating endothelial progenitor cells in patients with rheumatoid arthritis with coronary calcification.

OBJECTIVE: Patients with rheumatoid arthritis (RA) are prone to premature atherosclerosis. We hypothesize that depletion of circulating endothelial progenitor cells (EPC) related to RA can contribute to the development of atherosclerosis. METHODS: We studied coronary calcifications by multidetector computed tomography and their relationship with different subtypes of circulating EPC in 70 patients with RA and 35 age- and sex-matched controls (mean age 54.1 +/- 10.2 yrs, 87% were women). The presence of coronary atherosclerosis was defined as an Agatston score > or = 10. Four subpopulations of EPC were determined by flow cytometry on the basis of surface expression of CD34, CD133, and KDR antigen: CD34+, CD34/KDR+, CD133+, and CD133/KDR+ EPC, respectively. RESULTS: Among those with RA, 15 patients (21%) had coronary atherosclerosis. The mean Agatston score was higher (61.8 +/- 201.7 vs 0.14 +/- 0.69; p = 0.01) and coronary atherosclerosis was more prevalent (21.4% vs 0%; p < 0.01) in patients with RA compared to controls. RA patients with coronary atherosclerosis were older (66.2 +/- 6.9 vs 51.5 +/- 16.2 yrs; p < 0.01), had higher prevalence of hypertension (46.7% vs 14.5%; p = 0.01), and had lower CD133/KDR+ (0.45% +/- 0.28% vs 0.89% +/- 0.81%; p < 0.01) and CD133+ EPC levels (0.74% +/- 0.39% vs 1.22% +/- 0.83%; p < 0.01), but similar CD34/KDR+ and CD34+ EPC levels (all p > 0.05) compared to those without. Multiple logistic regression revealed that older age (OR 1.25, 95% CI 1.10-1.41, p < 0.01) and lower CD133/KDR+ EPC (OR 0.07, 95% CI 0.00-0.97, p < 0.01) were independent predictors for coronary atherosclerosis in patients with RA. CONCLUSION: Our results demonstrated that RA patients with coronary atherosclerosis have significantly lower levels of CD133/KDR+ and CD133+ EPC than those without. In addition to older age, lower levels of circulating CD133/KDR+ EPC also predicted occurrence of coronary atherosclerosis in RA patients.

Early postnatal sound exposure induces lasting neuronal changes in the inferior colliculus of senescence accelerated mice (SAMP8): a morphometric study on GABAergic neurons and NMDA expression.

Senescence-acceleration-prone mice (SAMP8) provide a model to study the influence of early postnatal sound exposure upon the aging auditory midbrain. SAMP8 were exposed to a 9-kHz monotone of either 53- or 65-dB sound pressure level during the first 30 postnatal days, the neurons in the auditory midbrain responding selectively to 9 kHz were localized by c-fos immunohistochemistry and the following parameters were compared to control SAMP8 not exposed to sound: mortality after sound exposure, dendritic spine density, and quantitative neurochemical alterations in this 9-kHz isofrequency lamina. For morphometric analysis, animals were examined at 1, 4, and 8 months of age. Serial sections of the inferior colliculus were Golgi impregnated or stained immunohistochemically for the expression of epsilon1 subunit of NMDA receptor or GABA. Mortality after exposure to 53 dB was the same as in controls, but was markedly increased from 7 months of age onward after postnatal exposure to 65 dB. No gross morphological alterations were observed in the auditory midbrain after sound exposure. However, sound exposure to 53 or 65 dB significantly reduced dendritic spine density by 11% at 4 months or by 11-17% both at 1 and 4 months of age, respectively. The effect of sound exposure upon neurons expressing the NMDAepsilon1 subunit was dose-dependent. Increasing with age until 4 months in control mice and remaining essentially stable thereafter, the percentage of NMDAepsilon1-immunoreactive neurons was significantly elevated by 40-66% in 1- and 8-month-old SAMP8 exposed to 53 dB, whereas no significant effect of 65 dB was apparent. The proportion of GABAergic cells declined with age in controls. It was significantly decreased at 1 month after 53 and 65 dB sound exposure. In contrast, it was elevated at later stages, being significantly increased at 4 months after exposure to 53 dB and at 8 months after exposure to 65 dB. The total cell number in the 9-kHz isofrequency lamina of SAMP8 decreased with age, but was not affected by exposure to either 53 or 65 dB. The present results indicate that early postnatal exposure to a monotone of mild intensity has long-term effects upon the aging auditory brain stem. Some of the changes induced by sound exposure, e.g., decline in spine density, are interpreted as accelerations of the normal aging process, whereas other effects, e.g., increased NMDAepsilon1 expression after 53 dB and elevated GABA expression after both 53 and 65 dB, are not merely explicable by accelerated aging.