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ELN expression was decreased in elastin insufficiency (EI) induced pluripotent stem cells (iPSC)-smooth muscle cells (SMCs). A, ELN mRNA expression by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was 20%-46% lower in patient iPSCSMCs compared with all control SMCs. B, Parallel reaction monitoring mass spectrometry of the sum of peak area of 4 normalized elastin peptides showed lower elastin formation in all patient iPS-SMCs compared to controls (not statistically significant for ELN2). C, Quantification of 3 elastin peptides upstream of the elastin variants and one elastin peptide downstream of the elastin variants showed lower abundance of both upstream and downstream peptides in all patient cells (n=3 independent experiments). *P<0.05, patient vs control SMCs; †P<0.05, patient vs control SMCs for fourth peptide only. WS indicates Williams syndrome.
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Objective
Elastin gene deletion or mutation leads to arterial stenoses due to vascular smooth muscle cell (SMC) proliferation. Human induced pluripotent stem cells–derived SMCs can model the elastin insufficiency phenotype in vitro but show only partial rescue with rapamycin. Our objective was to identify drug candidates with superior efficacy in r...
Contexts in source publication
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... measured ELN mRNA expression in all iPSC-SMCs using RT-qPCR. Compared with control SMCs, elastin mRNA levels were significantly lower in all patient SMCs ( Figure 3A). Elastin protein expression was then quantified by parallel reaction monitoring mass spectrometry using the sum of 4 different peptides. ...
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... was significantly lower in all EI patient SMCs compared to controls except for ELN2-SMCs which showed a nonsignificantly lower ELN expression. WS3 had the lowest levels compared to controls ( Figure 3B). To determine if the difference in ELN mRNA expression between ELN1 and ELN2-SMCs could be related to failure of nonsense-mediated decay of truncated elastin in ELN2, we quantified the amount of elastin peptides separately. ...
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... peptides upstream of ELN1 (p.Tyr595X) and ELN2 (p.Ala288fs) variants and one peptide downstream of the variants, that is, VPGALAAAK (644, 652) were quantified. Because the downstream peptide was present in both ELN mutation patients, it indicates that mutated mRNA was being eliminated and that the baseline phenotype was likely not related to translation of structurally abnormal protein in ELN mutant SMCs ( Figure 3C). In summary, we successfully differentiated patient iPSCs into SMCs and recapitulated the abnormal EI phenotype. ...
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... measured ELN mRNA expression in all iPSC-SMCs using RT-qPCR. Compared with control SMCs, elastin mRNA levels were significantly lower in all patient SMCs ( Figure 3A). Elastin protein expression was then quantified by parallel reaction monitoring mass spectrometry using the sum of 4 different peptides. ...
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... was significantly lower in all EI patient SMCs compared to controls except for ELN2-SMCs which showed a nonsignificantly lower ELN expression. WS3 had the lowest levels compared to controls ( Figure 3B). To determine if the difference in ELN mRNA expression between ELN1 and ELN2-SMCs could be related to failure of nonsense-mediated decay of truncated elastin in ELN2, we quantified the amount of elastin peptides separately. ...
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... peptides upstream of ELN1 (p.Tyr595X) and ELN2 (p.Ala288fs) variants and one peptide downstream of the variants, that is, VPGALAAAK (644, 652) were quantified. Because the downstream peptide was present in both ELN mutation patients, it indicates that mutated mRNA was being eliminated and that the baseline phenotype was likely not related to translation of structurally abnormal protein in ELN mutant SMCs ( Figure 3C). In summary, we successfully differentiated patient iPSCs into SMCs and recapitulated the abnormal EI phenotype. ...
Citations
... For example, the mTOR inhibitor everolimus improves in vitro smooth muscle cell differentiation of stem cells derived from individuals with elastin insufficiency because of ELN mutations. 66 Our results suggest that targeting DD with everolimus is a compelling topic of future research. ...
... One potential therapy would be everolimus, a mammalian target of rapamycin (mTOR) antagonist that was shown to rescue defective elastin phenotypes in stem cell-derived vascular smooth muscle cells in individuals with ELN gene mutations that caused elastin insufficiency. 66 It should be noted that the direction of causality in the association of diverticulitis and decreased elastin is not definitively established in our analyses: it is possible chronic inflammation in individuals with diverticulitis leads to elastin degradation. 71 However, none of the individuals in our analysis who had emergency diverticulitis surgery had prior episodes of diverticulitis or gastrointestinal inflammatory conditions, leading us to believe it is more likely that decreased elastin density led to diverticulitis rather than the reverse. ...
Diverticular disease is a common and morbid complex phenotype influenced by both innate and environmental risk factors. We performed the largest genome-wide association study meta-analysis for diverticular disease, identifying 126 novel loci. Employing multiple downstream analytic strategies, including tissue and pathway enrichment, statistical fine-mapping, allele-specific expression, protein quantitative trait loci and drug-target investigations, and linkage disequilibrium score regression, we prioritized causal genes and produced several lines of evidence linking diverticular disease to connective tissue biology and colonic motility. We substantiated these findings by integrating single-cell RNA sequencing data, showing that prioritized diverticular disease-associated genes are enriched for expression in colonic smooth muscle, fibroblasts, and interstitial cells of Cajal. In quantitative analysis of surgical specimens, we found a substantial reduction in the density of elastin present in the sigmoid colon in severe diverticulitis.
... 69,71 To date, iPSC-SMCs have been used to study pathological mechanisms in several vascular diseases, such as hypertension, 72 atherosclerosis, 73 and supravalvular aortic stenosis among others. 74,75 Pericytes Pericytes together with SMCs stabilize newly formed vasculature through physical and molecular interactions with the endothelium. In the heart, the absence or depletion of pericytes can result in vascular leakage and hemorrhaging in the microvasculature. ...
An ensemble of in vitro cardiac tissue models has been developed over the past several decades to aid our understanding of complex cardiovascular disorders using a reductionist approach. These approaches often rely on recapitulating single or multiple clinically relevant end points in a dish indicative of the cardiac pathophysiology. The possibility to generate disease-relevant and patient-specific human induced pluripotent stem cells has further leveraged the utility of the cardiac models as screening tools at a large scale. To elucidate biological mechanisms in the cardiac models, it is critical to integrate physiological cues in form of biochemical, biophysical, and electromechanical stimuli to achieve desired tissue-like maturity for a robust phenotyping. Here, we review the latest advances in the directed stem cell differentiation approaches to derive a wide gamut of cardiovascular cell types, to allow customization in cardiac model systems, and to study diseased states in multiple cell types. We also highlight the recent progress in the development of several cardiovascular models, such as cardiac organoids, microtissues, engineered heart tissues, and microphysiological systems. We further expand our discussion on defining the context of use for the selection of currently available cardiac tissue models. Last, we discuss the limitations and challenges with the current state-of-the-art cardiac models and highlight future directions.
... Human undifferentiated iPSCs reprogrammed from skin fibroblasts of a WBS patient [WS1-iPSC line C from (65,66)], an ELN mutant non-syndromic SVAS patient [ELN1 from (66)] and a control human [CT2 from (66)] were used to generate iPSC-SMC progenitor cell lines via an embryoid body stage (65,66). The age and sex of iPSC donors are provided in Supplemental Table 2. ...
... Human undifferentiated iPSCs reprogrammed from skin fibroblasts of a WBS patient [WS1-iPSC line C from (65,66)], an ELN mutant non-syndromic SVAS patient [ELN1 from (66)] and a control human [CT2 from (66)] were used to generate iPSC-SMC progenitor cell lines via an embryoid body stage (65,66). The age and sex of iPSC donors are provided in Supplemental Table 2. ...
... Human undifferentiated iPSCs reprogrammed from skin fibroblasts of a WBS patient [WS1-iPSC line C from (65,66)], an ELN mutant non-syndromic SVAS patient [ELN1 from (66)] and a control human [CT2 from (66)] were used to generate iPSC-SMC progenitor cell lines via an embryoid body stage (65,66). The age and sex of iPSC donors are provided in Supplemental Table 2. ...
Obstructive arterial diseases including supravalvular aortic stenosis (SVAS), atherosclerosis and restenosis share two important features: an abnormal or disrupted elastic lamellae structure and excessive smooth muscle cells (SMCs). However, the relationship between these pathological features is poorly delineated. SVAS is caused by heterozygous loss-of-function, hypomorphic or deletion mutations in the elastin gene ELN, and SVAS patients and elastin mutant mice display increased arterial wall cellularity and luminal obstructions. Pharmacological treatments for SVAS are lacking as underlying pathobiology is inadequately defined. Herein, using human aortic vascular cells, mouse models as well as aortic samples and SMCs derived from induced pluripotent stem cells of ELN-deficient patients, we demonstrated that elastin insufficiency induced epigenetic changes, upregulating the Notch pathway in SMCs. Specifically, reduced elastin increased levels of γ-secretase, activated NOTCH3 intracellular domain and downstream genes. Notch3 deletion or pharmacological inhibition of γ-secretase attenuated aortic hypermuscularization and stenosis in Eln(-/-) mutants. Eln(-/-) mice expressed higher levels of Notch ligand JAGGED1 (JAG1) in aortic SMCs and endothelial cells (ECs). Finally, Jag1 deletion in SMCs, but not ECs, mitigated the hypermuscular and stenotic phenotype in the aorta of Eln(-/-) mice. Our findings reveal that NOTCH3 pathway upregulation induced pathological aortic SMC accumulation during elastin insufficiency and provide potential therapeutic targets for SVAS.
... In particular, hiPSCderived models for bicuspid valvular and aortic calcification identified mutations in the notch homolog 1 (NOTCH1) gene (Garg et al., 2005;Theodoris et al., 2015). Additionally, the generation of hiPSC-derived SMCs allowed to recapitulate the multiple features of supravalvular aortic stenosis (SVAS) including mutations in the elastin (ELN) gene (Ge et al., 2012;Kinnear et al., 2020). Thus, hiPSC-derived models provide a great tool to investigate cellular function as well as the molecular mechanisms behind such diseases, ultimately leading to new therapeutic approaches (Ge et al., 2012;Chailangkarn and Muotri, 2017;Kinnear et al., 2020). ...
... Additionally, the generation of hiPSC-derived SMCs allowed to recapitulate the multiple features of supravalvular aortic stenosis (SVAS) including mutations in the elastin (ELN) gene (Ge et al., 2012;Kinnear et al., 2020). Thus, hiPSC-derived models provide a great tool to investigate cellular function as well as the molecular mechanisms behind such diseases, ultimately leading to new therapeutic approaches (Ge et al., 2012;Chailangkarn and Muotri, 2017;Kinnear et al., 2020). ...
Induced pluripotent stem cells (iPSCs) originate from the reprogramming of adult somatic cells using four Yamanaka transcription factors. Since their discovery, the stem cell (SC) field achieved significant milestones and opened several gateways in the area of disease modeling, drug discovery, and regenerative medicine. In parallel, the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) revolutionized the field of genome engineering, allowing the generation of genetically modified cell lines and achieving a precise genome recombination or random insertions/deletions, usefully translated for wider applications. Cardiovascular diseases represent a constantly increasing societal concern, with limited understanding of the underlying cellular and molecular mechanisms. The ability of iPSCs to differentiate into multiple cell types combined with CRISPR-Cas9 technology could enable the systematic investigation of pathophysiological mechanisms or drug screening for potential therapeutics. Furthermore, these technologies can provide a cellular platform for cardiovascular tissue engineering (TE) approaches by modulating the expression or inhibition of targeted proteins, thereby creating the possibility to engineer new cell lines and/or fine-tune biomimetic scaffolds. This review will focus on the application of iPSCs, CRISPR-Cas9, and a combination thereof to the field of cardiovascular TE. In particular, the clinical translatability of such technologies will be discussed ranging from disease modeling to drug screening and TE applications.
... Additional studies suggest that hypertension arises in Eln +/mice as part of a developmental adaptation to normalize vessel wall stress, capitalizing on the increased pressures to prop open the narrow, stiff elastin-insufficient vessels 103 , although more recent studies indicate that reactive oxygen species (ROS) production may also have a role 74 . Several additional molecular and cellular mechanisms impact the pathogenesis of elastin arteriopathy, including mechanistic target of rapamycin (mTOR) perturbation of smooth muscle mechanosensing [104][105][106] , and the adaptive immune system 29 . ...
... Additionally, patient-derived iPSCs may soon offer platforms through which to test novel therapeutics for impact on unique WS transcriptional pathways and phenotypes. Initial investigations incorporating this technology have been used to screen for medications that impact the increased smooth muscle cell proliferation seen in WS 105 . Rather than a goal of preventing stenosis, future studies that focus on resolving or reducing existing stenoses may be more relevant to treatment and lead to improved clinical outcomes, especially in young children who often come to medical attention with stenosis already present. ...
Williams syndrome (WS) is a relatively rare microdeletion disorder that occurs in as many as 1:7,500 individuals. WS arises due to the mispairing of low-copy DNA repetitive elements at meiosis. The deletion size is similar across most individuals with WS and leads to the loss of one copy of 25–27 genes on chromosome 7q11.23. The resulting unique disorder affects multiple systems, with cardinal features including but not limited to cardiovascular disease (characteristically stenosis of the great arteries and most notably supravalvar aortic stenosis), a distinctive craniofacial appearance, and a specific cognitive and behavioural profile that includes intellectual disability and hypersociability. Genotype–phenotype evidence is strongest for ELN, the gene encoding elastin, which is responsible for the vascular and connective tissue features of WS, and for the transcription factor genes GTF2I and GTF2IRD1, which are known to affect intellectual ability, social functioning and anxiety. Mounting evidence also ascribes phenotypic consequences to the deletion of BAZ1B, LIMK1, STX1A and MLXIPL, but more work is needed to understand the mechanism by which these deletions contribute to clinical outcomes. The age of diagnosis has fallen in regions of the world where technological advances, such as chromosomal microarray, enable clinicians to make the diagnosis of WS without formally suspecting it, allowing earlier intervention by medical and developmental specialists. Phenotypic variability is considerable for all cardinal features of WS but the specific sources of this variability remain unknown. Further investigation to identify the factors responsible for these differences may lead to mechanism-based rather than symptom-based therapies and should therefore be a high research priority.
... Protocol-directed hiPSC-SMC differentiation is quite different in 3D (Xie et al., 2007;Ge et al., 2012;Kinnear et al., 2013;Wang et al., 2014;Kinnear et al., 2020) and 2D (Huang et al., 2006;Patsch et al., 2015;Yang et al., 2016) culture systems. Cells derived from the outgrowth of human EBs cultured in SMC differentiation condition, which was only composed of Dulbecco modified eagle medium (DMEM) + 5% fetal bovine serum (FBS) and a gelatin-coated surface, produced SMCs expressing smooth muscle MHC (SMMHC) and α-smooth muscle actin (α-SMA) (Xie et al., 2007). ...
... This produced highly homogenous SMC-like cells (around 96%) (Ge et al., 2012). This protocol has been used to differentiate elastin mutant hiPSC into SMCs to model elastin insufficiency phenotype in SMCs (Kinnear et al., 2020) and Williams-Beuren syndrome in vitro (Kinnear et al., 2013). Furthermore, the same protocol has efficiently induced SMCs used for manufacturing of macroporous and nanofibrous poly(L-lactic acid) scaffold (Wang et al., 2014). ...
Human pluripotent stem cells (hPSCs) are derived from human embryos (human embryonic stem cells) or reprogrammed from human somatic cells (human induced pluripotent stem cells). They can differentiate into cardiovascular cells, which have great potential as exogenous cell resources for restoring cardiac structure and function in patients with heart disease or heart failure. A variety of protocols have been developed to generate and expand cardiovascular cells derived from hPSCs in vitro. Precisely and spatiotemporally activating or inhibiting various pathways in hPSCs is required to obtain cardiovascular lineages with high differentiation efficiency. In this concise review, we summarize the protocols of differentiating hPSCs into cardiovascular cells, highlight their therapeutic application for treatment of cardiac diseases in large animal models, and discuss the challenges and limitations in the use of cardiac cells generated from hPSCs for a better clinical application of hPSC-based cardiac cell therapy.
... Given the phenotypic and genetic heterogeneity of CHD, international collaborations in clinical and biological data sharing across pediatric and adult centers will be needed(28). Finally, innovations in fetal cardiac interventions, in endogenous regeneration strategies, and in gene editing should be explored with the ultimate goal of developing biological therapies that can change the natural history of CHD and improve lifelong outcomes(29,30).PHENOME. An aging patient with CHD carries a unique combination of cardiostructural, electroanatomic, syndromic, and extracardiac alterations.Along with physical examination, electrocardiography, and laboratory assessment, cardiovascular imaging with or without invasive hemodynamic assessment has been the mainstay of phenotypic characterization and therapeutic decision making. ...
More than 90% of patients with congenital heart disease (CHD) are nowadays surviving to adulthood and adults account for over two-thirds of the contemporary CHD population in Western countries. Although outcomes are improved, surgery does not cure CHD. Decades of longitudinal observational data are currently motivating a paradigm shift toward a lifespan perspective and proactive approach to CHD care. The aim of this review is to operationalize these emerging concepts by presenting new constructs in CHD research. These concepts include long-term trajectories and a life course epidemiology framework. Focusing on a precision health, we propose to integrate our current knowledge on the genome, phenome, and environome across the CHD lifespan. We also summarize the potential of technology, especially machine learning, to facilitate longitudinal research by embracing big data and multicenter lifelong data collection.
... As most therapeutic discoveries obtained from animal models are ineffective in human clinical trials, 129 patient-specific iPSCs which carry both the causal genetic defects and permissive genetic background have been increasingly used as a powerful tool for disease modeling, drug discovery, and regenerative medicine. [130][131][132] To date, patient-specific iPSC-VSMCs (induced pluripotent stem cell VSMCs) have been used to model a handful of vascular diseases such as Hutchinson-Gilford progeria syndrome, [133][134][135][136][137][138][139] supravalvular aortic stenosis, [140][141][142][143] hypertension, 144 aortic aneurysm, 126,145,146 and atherosclerosis. 147 In general, these studies demonstrated that patient-derived iPSC-VSMCs could recapitulate several pathological features of vascular diseases. ...
The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.
... A model of BAV has also demonstrated that NC-VSMCs, but not PM-VSMCs, from patients with BAV and TAA have defects in differentiation and contractile function . Currently published iPSC models for SVAS, however, did not use lineage-specific protocols in their investigation (Ge et al., 2012;Kinnear et al., 2013Kinnear et al., , 2020. In a 3D model of SVAS, a lineage-specific protocol also was not used, although the investigators inferred lineage based on responsiveness to cytokines . ...
... This work established robust high throughput assays for pharmacogenomics studies, paving the way for future studies which may incorporate the use of isogenic controls. A recent report of a model for SVAS has used an iPSC model to test the effect of different classes and combinations of drugs, finding that mTOR inhibitor everolimus was the most effective at rescuing the disease phenotype (Kinnear et al., 2020). Interestingly, they found that combination therapy using everolimus and additional classes of drugs was not beneficial. ...
Thoracic aortic diseases, whether sporadic or due to a genetic disorder such as Marfan syndrome, lack effective medical therapies, with limited translation of treatments that are highly successful in mouse models into the clinic. Patient-derived induced pluripotent stem cells (iPSCs) offer the opportunity to establish new human models of aortic diseases. Here we review the power and potential of these systems to identify cellular and molecular mechanisms underlying disease and discuss recent advances, such as gene editing, and smooth muscle cell embryonic lineage. In particular, we discuss the practical aspects of vascular smooth muscle cell derivation and characterization, and provide our personal insights into the challenges and limitations of this approach. Future applications, such as genotype-phenotype association, drug screening, and precision medicine are discussed. We propose that iPSC-derived aortic disease models could guide future clinical trials via “clinical-trials-in-a-dish”, thus paving the way for new and improved therapies for patients.
Abdominal aortic aneurysm (AAA) is a highly lethal cardiovascular disease that currently lacks effective pharmacological treatment given the complex pathophysiology of the disease. Here, single‐cell RNA‐sequencing data from patients with AAA and a mouse model are analyzed, which reveals pivotal pathological changes, including the M1‐like polarization of macrophages and the loss of contractile function in smooth muscle cells (SMCs). Both cell types express the integrin αvβ3, allowing for their dual targeting with a single rationally designed molecule. To this end, a biocompatible nanodrug, which is termed EVMS@R‐HNC, that consists of the multifunctional drug everolimus (EVMS) encapsulated by the hepatitis B virus core protein modifies to contain the RGD sequence to specifically bind to integrin αvβ3 is designed. Both in vitro and in vivo results show that EVMS@R‐HNC can target macrophages as well as SMCs. Upon binding of the nanodrug, the EVMS is released intracellularly where it exhibits multiple functions, including inhibiting M1 macrophage polarization, thereby suppressing the self‐propagating inflammatory cascade and immune microenvironment imbalance, while preserving the normal contractile function of SMCs. Collectively, these results suggest that EVMS@R‐HNC presents a highly promising therapeutic approach for the management of AAA.
