Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy

Abstract

Mutations in LMNA, the gene that encodes lamin A and C, causes LMNA-related dilated cardiomyopathy (DCM) or cardiolaminopathy. LMNA is expressed in endothelial cells (ECs); however, little is known about the EC-specific phenotype of LMNA-related DCM. Here, we studied a family affected by DCM due to a frameshift variant in LMNA. Human induced pluripotent stem cell (iPSC)–derived ECs were generated from patients with LMNA-related DCM and phenotypically characterized. Patients with LMNA-related DCM exhibited clinical endothelial dysfunction, and their iPSC-ECs showed decreased functionality as seen by impaired angiogenesis and nitric oxide (NO) production. Moreover, genome-edited isogenic iPSC lines recapitulated the EC disease phenotype in which LMNA-corrected iPSC-ECs showed restoration of EC function. Simultaneous profiling of chromatin accessibility and gene expression dynamics by combining assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) as well as loss-of-function studies identified Krüppel-like factor 2 (KLF2) as a potential transcription factor responsible for the EC dysfunction. Gain-of-function studies showed that treatment of LMNA iPSC-ECs with KLF2 agonists, including lovastatin, rescued the EC dysfunction. Patients with LMNA-related DCM treated with lovastatin showed improvements in clinical endothelial dysfunction as indicated by increased reactive hyperemia index. Furthermore, iPSC-derived cardiomyocytes (iPSC-CMs) from patients exhibiting the DCM phenotype showed improvement in CM function when cocultured with iPSC-ECs and lovastatin. These results suggest that impaired cross-talk between ECs and CMs can contribute to the pathogenesis of LMNA-related DCM, and statin may be an effective therapy for vascular dysfunction in patients with cardiolaminopathy.

A protocol for transdifferentiation of human cardiac fibroblasts into endothelial cells via activation of innate immunity

“…This step sorts the iECs from the transdifferentiation protocol. CD144, also known as VE-Cadherin has been widely used for the isolation of ECs derived from embryonic stem cells (ESCs) and iPSCs ( Liu et al., 2020 ; Sayed et al., 2020 ; Sayed et al., 2015 ). Aspirate the spent medium and dissociate the cells using 1 mL TrypLE express per well for 10 mins at 37°C.…”

“…We recommend dedicating 2-wells for each downstream phenotypic characterization. The most widely used assays to assess the function of ECs are listed here: a) In vitro angiogenesis assay to assess the capacity to form network of tubular structures; b) Nitric oxide assay to assess the capacity to produce NO; c) Acetylated LDL-uptake assay to assess the capacity to uptake Ac-LDL; d) In vitro scratch assay to assess the migration capacity; e) In vivo angiogenesis assay to assess the capacity to from capillaries ( Huang et al., 2021 ; Liu et al., 2018 ; Paik et al., 2018 ; Sayed et al., 2020 ; Zhang et al., 2019b ).…”

Building Multi-Dimensional Induced Pluripotent Stem Cells-Based Model Platforms to Assess Cardiotoxicity in Cancer Therapies

“…Crosstalk between myocytes and non-myocytes are essential for maintaining a physiological balance and cardiovascular tone. Endothelial cells support cardiac metabolism, contractility and survival ( Brutsaert, 2003 ; Sayed et al, 2020 ); whereas fibroblast play a key role in metabolic, structural, electrical and mechanical maturation ( Giacomelli et al, 2017 ; Giacomelli et al, 2020 ). Therefore, it is intuitive that these major non-myocyte cell populations may play a significant role in disease and drug-related toxicities.…”

Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity

“…To further enrich ECs, the differentiated cells can be sorted for the CD31 positive population ( Liu et al, 2018a ). In a recent study from our lab, we demonstrated that iPSC-ECs exhibit a wide range of properties characteristic of primary ECs ( Sayed et al, 2020 ). Specifically, iPSC-ECs showed morphological similarity to primary ECs with a cobblestone cell shape.…”