3D ARTIFICIAL VESSELS ARRAY-ON-A-CHIP FOR HIGH-THROUGHPUT SCREENING

In a collaborative project with Dr. Mark Skylar-Scott Lab, Dr. Song is developing an in-vitro micro-physiological system mimicking the 3D multi-layer structure of endothelial and smooth muscle cells to study the disease mechanism and treatment of pulmonary arterial hypertension (PAH). Free-standing vessels can be fabricated on a chip in situ based on 3D printing and tissue engineering technologies using a circular fibrin scaffold with smooth muscle cells embedded and lined by endothelial cells. By connecting the free-standing 3D tubular vessels array with perfusion system, different shear stress and strain of vessels can be tested by controlling flow rate and internal pressure, respectively. By using this platform, Dr. Song is studying gene expression changes and extracellular matrix remodeling in vessels according to the diverse physiological conditions.

ALDEHYDE DEHYDROGENASE ALDH3A1 AND SOX17 ARE FLOW RESPONSIVE FACTORS THAT REGULATE GENE ACCESSIBILITY AND GENE REGULATION

Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression, and determine cell function in health and disease.  The Rabinovitch Laboratory has revealed that aldehyde dehydrogenase ALDH1A3 is increased in smooth muscle cells (SMC) from PAH patients and this enzyme coordinates energy metabolism with chromatin remodeling and gene expression important in aberrantly proliferating cells. The goal of this study is to determine whether an isoform ALDH3A1 has a similar role in coordinating energy metabolism with chromatin remodeling and gene regulation to maintain homeostasis in pulmonary arterial endothelial cells. The role of the transcription factor SOX17, in which a mutation is linked to PAH in patients with a congenital heart defect and high shear stress, is also investigated as a flow sensitive gene. The regulation of SOX17 in response to flow and the role of SOX17 targets in endothelial homeostasis are studied

DNA DAMAGE IN PULMONARY ARTERIAL HYPERTENSION (PAH)

The role of DNA damage in causing persistent PAH is investigated in relation to loss of BMPR2 the most common genetic cause of PAH. While there is some evidence of DNA damage in pulmonary arterial endothelial cells (EC) with reduced BMPR2, oxidant stress (reoxygenation after hypoxia) brings out further DNA damage judged by multiple assays. A similar phenotype is seen when there is loss of the DNA damage sensor ATM. There is unrepaired DNA damage in response to oxidant stress and mice with loss of ATM in EC have persistent PAH. RNA Seq analyses revealed many common genes, but a reduction in FOXF1, a transcription factor associated with DNA repair and angiogenesis appeared responsible for the pulmonary hypertension. Targeted pulmonary endothelial delivery of Foxf1 to mice with loss of Bmpr2 in EC prevented persistent DNA damage and pulmonary hypertension.

Immunofluorescence reveals DNA damage in EC-Bmpr2-/- mice.  We previously showed EC-Bmpr2-/- mice have persistent PH after reoxygenation, a PH mouse model where mice are exposed to 10% hypoxia for 3 weeks followed by recovery in room-air for 4 weeks. (Diebold et al. Cell metabolism 2015). Confocal microscopy images show foci of DNA damage, indicated by gH2AX (stained green), in pulmonary artery EC (labeled by tdTomato, red) in lung tissue sections from EC-Bmpr2-/- mice (Figure1). Ataxia-telangiectasia mutated (ATM) plays an important role as a sensor in the DNA damage response. Dr. Isobe showed that loss of Atm induced DNA damage in EC, and that EC-Atm deficient mice have persistent PH following reoxygenation

Foxf1 is a transcription factor that is reduced by DNA damage in the reoxygenation PH model.  RNA seq of pulmonary endothelial cells of EC-Bmpr2-/- and EC-Atm-/- mice revealed that the angiogenesis genes were commonly decreased compared with control mice. Motif analysis showed that Foxf1 was enriched in genes that were decreased in these cells.

FOXF1 gene therapy improves persistent PH.  FOXF1 is important for the development of vessels, and FOXF1 mutation is known to cause alveolar capillary dysplasia and pulmonary arterial hypertension. It has been reported that Foxf1 expresses in EC of arteries and capillaries in lung, is a target of p53, and is active in DNA damage repair.  Immunohistochemistry showed FOXF1 was decreased in EC of PAH patients (Figure 2). Dr. Isobe is investigating whether Foxf1 gene therapy improves DNA damage repair, and restores EC health, and reverses persistent PH in the reoxygenation PH model.

the role of exosome-ExtraceLLULAR VESICLE signaling in the maintenance of vascular homeostasis

The role of exosome (extracellular vesicle, EV) signaling in the maintenance of vascular homeostasis is investigated. These studies led to the identification of BMPR2 as a critical mediator of EV biogenesis during hypoxia, and the observation that aberrant EV signaling contributes to the pathogenesis of PAH. Endothelial EVs can be engineered to correct a BMPR2 deficiency, and BMPR2 dependent microRNA cargo plays an instrumental role in repressing aberrant smooth muscle cell (SMC) proliferation during hypoxia. These studies show that EVs are an emerging class of pharmacological agents to treat PAH. Future studies will involve developing strategies to optimize the therapeutic index of EVs with improved targeting and reduced immunogenicity.

Mapping, programming, and correcting gene regulatory sequences for Alagille Syndrome
(Joint project with the lab of Jesse Engreitz)
Mauro Lago do Campo, PhD, Aiqin Cao, PhD and Lingli Wang, MD (Rabinovitch Lab) with Michael Montgomery, PhD and Tri Nguyen, PhD (Engreitz Lab)

Alagille Syndrome is caused by heterozygous loss-of-function mutations in JAG1 (95% of cases) or its receptor NOTCH2 (2% of cases). The resulting reduction in JAG1-NOTCH2 signaling, particularly in vascular cells, leads to life-threatening complications including biliary atresia, severe hypoplastic central and peripheral pulmonary arteries, extensive proximal and distal pulmonary arterial stenoses, and right-sided congenital heart defects such as Tetralogy of Fallot. Despite repeated corrective surgical procedures in infancy and early childhood, many patients with Alagille Syndrome experience refractory pulmonary artery hypoplasia and stenoses that ultimately lead to right heart failure.  This project explores the potential of genome therapy for Alagille Syndrome, by engineering regulatory DNA sequences to turn up the production of the functional, unaffected allele of JAG1. This is an attractive approach as it would be applicable to the 95% of Alagille Syndrome patients that have loss-of-function variants in JAG1 and would be the first proof of concept strategy for other diseases linked to haploinsufficiency. JAG1 is a dose-sensitive gene, where reduction to 50% normal expression levels is sufficient to affect cellular function in vitro and lead to Alagille-like phenotypes in mouse models. Although, it has never been tested whether restoration of JAG1 function in human patient cells would fully restore healthy gene expression programs and cellular phenotypes, this is a promising strategy even if  Alagille patients carry additional disease-causing variants that cooperate with JAG1 to affect the penetrance and expressivity of Alagille Syndrome phenotypes.  Our preliminary work demonstrated that exposure of endothelial cells to physiological laminar shear stress induces JAG1 and elastin, and that loss of JAG1-NOTCH2 signaling decreases ELN under this condition (Figure 1 below).  Elastin is critical to blood vessel health, and loss of elastin could underlie vascular dysfunction in Alagille Syndrome.

We will characterize the molecular and cellular phenotypes of Alagille versus healthy iPSC-derived endothelial and smooth muscle cells.  With expertise developed by Dr. Engreitz and his group, the study team will combine single-cell 10X Multiome with the BPNet and Activity-by-Contact computational models to build a genome-wide nucleotide-resolution map of transcription in these cells, that will be cultured under conditions of static, physiologic, and pathological flow, to identify key regulators of JAG1.  We will systematically mutate regulatory DNA sequences in the JAG1 promoter and identify sequences that can increase JAG1 gene expression by ~2-fold.  As a test for the potential therapy, we will use CRISPR prime editing to directly correct coding point mutations in iPSCs from Alagille patients and examine the effects of the correction on JAG1 signaling and downstream cellular phenotypes. We will conduct similar experiments introducing regulatory edits to the JAG1 promoter to up-regulate JAG1 expression from the unaffected allele (Figure 2 below).