Elastase, Elafin, Chronic Inflammation, & Altered Immunity
Mir S Adil, PharmD, PhD and Shalina Taylor, PhD
NEUTROPHIL DYSFUNCTION IN PULMONARY ARTERIAL HYPERTENSION (PAH)
Dr. Taylor, a former postdoctoral Fellow, found that neutrophils from PAH patients when compared to those from healthy donors as controls, show profound alterations in adhesion, migration, trans-endothelial migration, degranulation, and release of chromatin as extracellular traps (NETosis). Heightened production and release of elastase explained the propensity to NETosis that cause tissue damage. Proteomic analyses identified increased vinculin, explaining increased adhesion and impaired migration. PAH plasma levels of endogenous retroviral protein HERV-K dUTPase are elevated and HERV-K dUTPase can stimulate an increase in vinculin in a neutrophil cell line. Transcriptomic analyses of PAH vs. control neutrophils revealed a type I interferon response attributed to heightened expression of the endogenous retrovirus HERV-K envelope; in fact, the phenotype of the neutrophil related to heightened elastase and interferon can also be attributed to HERV-K. Dr. Taylor also tested the consequence of HERV-K mediated sequelae in an animal model of pulmonary hypertension.
Dr. Mir Adil is extending the studies and developing an assay to determine the efficacy of a recombinant endogenous neutrophil elastase (NE) inhibitor, elafin, developed as a therapy for patients suffering from PAH. Dr. Adil will test NE activity in the plasma and in neutrophils isolated from blood of PAH patients, and if this activity can serve as a plasma biomarker for the effect of elafin. He will also investigate the mechanism of elastase mediated neutrophil extracellular trap (NET) formation and the proteins with which elafin interacts to prevent NET formation. These studies will play a very important role in an upcoming Phase 2 Clinical Trial using human recombinant Elafin to treat PAH.
Mir S Adil, PharmD, PhD
HUMAN ENDOGENOUS RETROVIRUS K (HERV-K) AND ENDOTHELIAL CELL BIOLOGY IN PULMONARY ARTERIAL HYPERTENSION (PAH)
Studies in the Rabinovitch Laboratory revealed that loss of BMPR2 resulting from a PAH associated mutation plays a critical role in the pathogenesis of PAH. For example, loss of BMPR2 in endothelial cells increases GM-CSF resulting in enhanced monocyte recruitment. BMPR2 deficient monocytes have elevated STAT1 signifying a pro-inflammatory phenotype which can be attributed to an increase in expression of human endogenous retrovirus dUTPase. Dr. Adil's research investigates whether a reduction in BMPR2 increases a long non-coding (lnc) RNA on the X chromosome, responsible for inactivation of the X chromosome. An increase in this lnc RNA Xist, could result in its enhanced binding to the deacetylase SPEN. Since Xist and HERV-K share the a similar SPEN binding site, we speculate that this would result in reduced availability of SPEN to bind and repress HERV-K, leading to a pro-inflammatory monocyte.
Xin Zhou, PhD and Toshie Saito, MD
DENDRITIC CELLS AND THE PATHOGENESIS OF PULMONARY ARTERIAL HYPERTENSION (PAH)
Dr. Toshie Saito, a former postdoctoral fellow and research associate discovered a novel link between viral infection, immunity and inflammation in PAH. SAMHD1, a protein that is known to be protective against HIV infection, was present as an antigen highly expressed in immune complexes in PAH lungs. This led to the insight that human endogenous retrovirus K (HERV-K) is linked to the pathology of PAH.
Single cell mass cytometry (CYTOF) analyses revealed abnormal activation of dendritic cells in PAH patients. In collaboration with a current postdoctoral fellow Dr. Xin Zhou, single cell RNA-seq confirmed abnormal activation of cDC1 dendritic cells and a highly inflammatory cDC2 dendritic cell subpopulation in PAH. Multiplex ion based imaging (MIBI) analyses, described under Dr. Selena Ferrian, localized these cells to the occluded pulmonary arteries in the lung tissue from PAH patients.
UMAP of Conventional dendritic cell population and composition in cells from three PAH patients and four healthy donors. The human conventional dendritic cell selected from PBMC of seven donors by negative sorting. The left cluster represents cDC2 by high expression of CD1C, and the right cluster represents cDC1 by high expression of CLEC9A. Bar graph on right panel shows the composition of cells identified from the UMAP. PAH shows increased percentage of S100A9 proinflammatory DC, and a decreased cDC1 in total.
Selena Ferrian, PhD, with Toshie Saito, MD and Aiqin Cao, PhD
HIGH-DIMENSIONAL SINGLE-CELL IMAGING MAPS DISTINCT INFLAMMATORY CELL SUBSETS TO PULMONARY ARTERIAL HYPERTENSION (PAH) VASCULOPATHY
Recent studies from our group and others support the role of chronic inflammation and altered immunity in the pathogenesis of PAH. Developing a successful immunotherapy to reverse PAH pathology requires a comprehensive knowledge of the immune cell landscape, its microenvironmental complexities, and how communication with the vascular cells leads to the initiation and progression of disease. We therefore embarked on a collaboration with the laboratory of Michael Angelo at Stanford, led by former post-doctoral fellow Selena Ferrian with former lab member Toshie Saito, to map the immune landscape in pulmonary arteries (PAs) in lung tissue and establish spatial relationships that regulate vascular pathology. Dr. Ferrian used multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to interrogate the immune landscape in PAs from idiopathic (IPAH) and hereditary (HPAH) PAH patients. Massive immune infiltration in I/HPAH was observed with intramural infiltration linked to PA occlusive changes. The spatial context of CD141+ dendritic cells expressing SAMHD1, TIM-3 and IDO-1 and MPO+ neutrophils within an immune microenvironment was associated with greater SMC mass in HPAH. Monocyte-derived dendritic cells (Mo-DC) were linked to inflamed endothelial cells and proliferating SMCs. Experimental data with Aiqin Cao in cultured cells reinforced causal relationships between neutrophil extracellular vesicles and mo-DC in mediating SMC proliferation. These studies set the stage for further analyses of the pathways by which mo-DCs induce SMC proliferation and by which the myeloid cells in particular play a key pathogenetic role in PAH.
Interrogation of pulmonary arteries in pulmonary arterial hypertension patients and donor controls. On the left, Representative MIBI image overlay from a PA section selected based on paired hematoxylin and eosin (H&E), along with the corresponding MIBI image scanned (carbon) and the MIBI-TOF workflow for cell segmentation and localization of immune cells with respect to the vascular architecture (vessel mask), scale bars = 100μm. Vim, Vimentin.
Middle and right: Dot plots showing the neutrophil count and frequencies of cDC1 co-expressing IDO-1, TIM-3 and SAMHD1 in hereditary PAH (HPAH) and idiopathic PAH (IPAH) subgroups.
Rebecca Harper, PhD, Xin Zhou, PhD, Mir Adil, PharmD, PhD, Aiqin Cao, PhD
MONOCYTES AND MACROPHAGES IN THE DEVELOPMENT AND PERSISTENCE OF PULMONARY ARTERIAL HYPERTENSION
Monocytes and macrophages contribute to the development and persistence of chronic inflammation seen in pulmonary arterial hypertension (PAH). Single cell RNAseq revealed a consistent upregulation of STAT1 in classical, intermediate and non-classical monocytes, consistent with an interferon response resulting from elevated levels of the endogenous retrovirus, HERV-K. Quite remarkable was a reduced expression of CD14 in classical monocytes, impaired LPS mediated stimulation and a propensity to apoptosis that is related to loss of BMPR2. These relationships are being investigated using induced pluripotent stem cells differentiated into monocytes, monocyte derived dendritic cells and macrophages.
Xin Zhou, PhD
THE MICROBIOME, IMMUNE SYSTEM AND THE PULMONARY ARTERIAL HYPERTENSION (PAH)
Multi-omics studies aim to identify the relationship between the host microbiome and immune system. Diseases such as prediabetes and viral/bacterial infections are normally accompanied with inflammation. However, the nature of the inflammation is highly personalized. For example, the Integrated Human Microbiome Project (https://hmpdacc.org/ihmp/) allowed Dr. Zhou and his colleagues to identify an interaction pattern between human gut microbiome and IL-17 producing cells, which indicated a beneficial role of such cells in maintaining the gut homeostasis. PAH is also characterized as a disease associated with a personalized inflammatory signature. Microbiome dysbiosis is therefore being investigated in the blood with the view that this will inform the nature of the inflammation as it relates to progression of disease and to therapeutic targets.
Hypothesized model. In PAH, the detailed interactions between cytokine production and the microbiome and the mechanistic role of the interaction in the development of PAH is yet to be understood.
Genetics, Epigenetics and Metabolism in Pulmonary Arterial Hypertension
Aiqin Cao, PhD
USING STEM CELLS TO UNCOVER THE MECHANISM OF ARTERIAL AND VENOUS DYSFUNCTION IN HYPOPLASTIC LEFT HEART SYNDROME
Hypoplastic left heart syndrome (HLHS) is a severe form of single ventricle congenital heart disease (CHD) characterized by the underdevelopment of the left ventricle, mitral valve, aortic valve, and ascending aorta. Abnormalities in HLHS endothelial cells may contribute to pulmonary hypertension that is a major impediment to successful surgical interventions in HLHS patients. Understanding the molecular and cellular origins of HLHS related pulmonary hypertension could contribute to the development of EC-targeted therapeutic interventions. Human induced pluripotent stem cells (iPSCs) obtained from both healthy individuals and HLHS patients, were differentiated into arterial and venous endothelial cells (ECs). RNA-seq was performed to understand the molecular and functional defects in HLHS patient vs. control iPSC-derived arterial and venous ECs. We have found more differentially expressed genes in pulmonary venous endothelial cells that reflect this left sided lesion.
Heatmap of gene expression in artery and vein endothelial cells from three Donor controls vs three patients with hypoplastic left heart syndrome (HLHS).
Mauro Lago-Docampo, PhD
UNCOVERING THE BASIS OF TBX4-MEDIATED PULMONARY ARTERIAL HYPERTENSION (PAH)
TBX4 is the second most common genetic cause of pediatric PAH, and while we see both gain-of-function (GOF) or loss-of-function (LOF) mutations in TBX4 in PAH patients, the molecular mechanism leading to disease development is unknown. The hypothesis being tested is that GOF and LOF TBX4 mutations lead to dysregulation of vascular smooth muscle cell (SMC) gene expression and function, and that TBX4 dysregulation in SMC causes endothelial cell (EC) dysfunction, resulting in PAH. In SMC with endogenous TBX4 deleted using CRISPR/Cas9, wild type TBX4 or GOF or LOF tagged constructs will be introduced and proximal proteomics applied to assess changes in the interaction with co-activators and chromatin remodelers. ChIP-Seq and ATAC-Seq will be used to identify alterations in DNA binding related to differential gene expression, determined by RNA-Seq. CRISPR-editing of TBX4 mutations in SMC will determine how gene expression is impacted by the SMC genetic background. To find modulators of penetrance, induced pluripotent stem cells (iPSCs) differentiated to SMC from TBX4 mutation patients and unaffected carriers will be used in -omic and functional SMC characterization. Vessel-like fibrin tubes that incorporate SMC and are lined by EC will be used to address non-cell autonomous changes in EC gene expression under physiologic flow.
Jan-Renier Moonen, MD, PhD
SHEAR STRESS REGULATION OF THE ENDOTHELIAL CHROMATIN LANDSCAPE: DEFINING VULNERABILITY TO DYSFUNCTION AND DISEASE
This project addresses the role of biomechanical forces in the normal modulation of endothelial function and plasticity, Findings from this study reveal that normal laminar shear stress is an important determinant of the endothelial chromatin landscape that directs KLF2/4 mediated gene expression. ATAC-Seq and RNA-Seq analyses show pronounced changes in chromatin accessibility and gene expression when PAEC are exposed to shear stress. Studies reveal important enhancer promoter interactions that impact gene expression using HiChIP and the ABC method. In some cases, enhancers skip genes to regulate others more distally located. KLF2/4 interact with the SWI/SNF chromatin remodeling complex in executing changes of chromatin accessibility as shown by proximity ligation analyses.
KLF4 interacts with the SWI-SNF nucleosome remodeling complex to increase chromatin accessibility. Image of KLF4-BRG1 Proximity Ligation Assay (PLA) in healthy rat lung tissue (350 M thick sections). Interaction of KLF4 with BRG1(magenta) is apparent at straight sites of the vasculature that are exposed to laminar shear stress (LSS; upper left insert), while sites at bifurcations which are exposed to disturbed shear stress (DSS) do not show the KLF4-BRG1 interaction (bottom right insert). Grey, vWF (pseudo-color); green, aSMA; blue, nuclei stained with DAPI. Quantification on the right, showing the number of foci per nucleus in regions of DSS or LSS at 6 random bifurcations. Scale bar, 20 mm, and 10 mm in the higher magnifications. Data shown as the mean±sem. ****P<0.0001 by Student’s two-tailed t-test.
Tsutomu Shinohara, MD, PhD with Jan Renier Moonen, MD PhD
PULMONARY VASCULAR REMODELING BY VESSEL WALL SHEAR STRESS
The pathological effects of high shear stress levels (HSS, up to 100 dyn/cm2), such as observed in children predisposed to PAH associated with congenital heart disease or as in small vessels that are occluded in idiopathic PAH are studied. The values used for pathological high shear stress were modeled by a collaboration with the Marsden Lab. A process of endothelial to mesenchymal transition has been identified in response to HSS, with loss of endothelial (EC) genes and gain of smooth muscle (SMC) genes, and a mechanism related to loss of ERG was identified. A mouse model of HSS induced by an aortocaval shunt shows progressive PAH that can be reversed by targeting the ERG gene to the pulmonary arterial vasculature.
New observations show that Jagged1-Notch2 interaction regulates elastin under laminar shear stress, but that this is perturbed with mutations seen in Jagged1 or Notch 2 in patients with Alagille syndrome, or when there is reduced ERG1 under high shear stress.
Endothelial mesenchymal transition in PAEC in response to HSS. PAEC isolated form lungs of healthy donors exposed to high shear stress (HSS) show decreased levels of endothelial cell markers (PECAM1 and CHD5, stained green) and increased levels of smooth muscle cell markers ACTA2 and FSP-1 (stained red) compared to PAEC exposed to laminar shear stress (LSS). Nuclei are stained with DAPI (blue). On the right, a quantification of the protein levels, relative to the number of nuclei.
Jason Szafron, PhD
Mechanobiological mechanisms and compositional differences driving differential remodeling in the PA tree during development of PAH
In a collaboration with the Marsden lab, computational modeling tools are used to simulate the evolution of the geometry and composition of blood vessels as PAH develops. These Studies investigate whether different orders of the PA tree experience varying deviations from their normal mechanobiological state in response to a perturbation in mechanical loading, such as that occurring in patients with congenital heart disease. The objective is to recapitulate the time course of vascular growth and remodeling (G&R) in each order of the PA tree in the murine aortocaval model of PAH and to distinguish maladaptive and adaptive changes in the vessels’ geometry and microstructure. Growth and remodeling simulations along with computational fluid dynamics link these structural and hemodynamic changes over time. Jason is supported by a Parker B Francis fellowship pulmonary and critical care research. This project is also and this project is supported in part by an MCHRI Pilot Grant (Co-PIs: Alison Marsden and Marlene Rabinovitch) "Elucidating the role of structure and morphometry on mechanobiological drivers of pulmonary arterial hypertension".
Pre-clinical imaging and computational fluid dynamics reveal differential loading in the PA tree during PAH development. (A) Increased blood flow from an aorto-caval shunt (ACS) induces pulmonary vascular remodeling in a murine model of PAH with geometric changes observable from pre-clinical MRI. (B) Geometries are reconstructed to perform computational fluid dynamics simulations that determine mechanobiological stimuli that modulate cell phenotype, such as wall shear stress (WSS). (C) These metrics are extrapolated down the PA tree to the pre-capillary arteries to identify differences in loading that drive differential remodeling, such as large increases in WSS for smaller PAs while the larger vessels maintain normal levels of WSS.
A coupled framework for hemodynamic evolution with computational growth and remodeling. Strahler diameter-defined morphometric trees are used to generate the bifurcating vessel tree (black lines, decreasing thickness for decreasing order). Inlet flow and outlet pressure are prescribed as boundary conditions. For each vessel order, mean pressure and mean flow are passed to the G&R framework to determine the evolving average diameter. Examples of this exchange are shown for the orders N, N-2, and 1. Mean values are passed for each order, shown here with cylinders representing vessels of decreasing size. Each vessel order can have its own composition, represented here by different relative amounts of smooth muscle (red), collagen (blue), and elastin (black) constituents. An example of mean hemodynamic calculation is shown for order N-2, marked in orange, where values from all segments of that order are averaged to pass to the G&R framework. Intramural stress and wall shear stress are calculated in the G&R framework for each order. Configurations of vessel order N are shown over time to demonstrate the multiplicative combination of deformations that determines stretch of each constituent.
Seo Woo Song, PhD
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.
Chongyang Zhang, PhD
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
Top: Compared to healthy distal and medial precapillary arteries (left), the increased shear stress of blood flow in PAH is associated with vascular remodeling (right), which impacts functions of endothelial cells.
Bottom: Our hypothesis (on the left) is that SOX17 as an important transcription factor regulates expression of target genes that are important for endothelial homeostasis in response to flow. On the right we show that the mRNA level of SOX17 was induced in primary human pulmonary artery endothelial cells subjected to physiological laminar shear stress (LSS).
Sarasa Isobe, MD, PhD
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.
Lingli Wang, MD
MICE WITH LOSS OF BMPR2 IN SMOOTH MUSCLE CELLS HAVE PERSISTENT PULMONARY HYPERTENSION DURING RECOVERY FROM HYPOXIA
These studies investigate the role of BMPR2 in the smooth muscle cells (SMC) of the vessel wall. While mice with SMC deletion of Bmpr2 can have a lethal congenital heart defect (see top image), the limited penetrance of this defect results in survivors used in this study. Subsequently, a mouse with inducible deletion of Bmpr2 in smooth muscle cells was used. Smooth muscle cells from pulmonary arteries (PASMC) of these mice are hyper-proliferative and apoptosis-resistant, and show reduced contractility. The same observation was made in complementary studies of human PASMC obtained from lungs of PAH patients with a BMPR2 mutation (see bottom image). The impaired contractility is related to an increase in β-Arrestin2 causing a reduction in RhoA and Rac1. The hyper-proliferation and apoptosis-resistance of PASMC with reduced BMPR2 is related to a pAkt-mediated increase in β-Catenin and cMyc. This study was recently published, Wang L et al., “Dysregulated Smooth Muscle Cell BMPR2 - ARRB2 Axis Causes Pulmonary Hypertension”. Circulation Research 2023;132:545–564 (PMCID: PMC10008520).
Double Outlet Right Ventricle (DORV) and Ventricular Septal Defect (VSD) were found in newborn mice with BMPR2 deleted in SMCs. Serial sections through the heart of newborn mice, stained with H&E. Littermate controls on the top show normal structure of the heart. Newborn mice with BMPR2 deleted in smooth muscle cells (KO; SM22a-Cre/Bmpr2 homo-floxed mice) show congenital heart defects, such as Double Outlet Right Ventricle (DORV, bottom left) and Ventricular Septal Defect (VSD, bottom right).
PV = pulmonary valve; RV = right ventricle; LV = left ventricle; PA = pulmonary artery; PT = pulmonary trunk.
b-Arrestin2 and Active b-Catenin (ABC) Expression in Human SPA from a Donor (top row) and a PAH Patient with BMPR2 Mutation. Representative images of immunofluorescence staining for smooth muscle marker SMA (Green), nuclei (DAPI, blue) and either b-Arrestin2 or b-Catenin (Red). PASMCs with b-Arrestin2 or b-Catenin positive staining are limited in the Donor lung. In contrast, widely distributed b-Arrestin2 staining primarily co-localized with SMA to the cytoplasm, and b-Catenin to the nuclei, of PASMCs in the PAH Patient with a BMPR2 Mutation.
David Marciano, PhD
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.
PET/CT imaging of Gallium-68 radiolabeled exosome biodistribution in vivo. The isolated, labeled exosomes were injected into a mouse, and live imaging experiments performed in collaboration with Guillem Pratx laboratory at Stanford.
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).
Figure 1: Physiological laminar shear stress (LSS) induces Jagged 1 (JAG1) and Elastin (ELN) in endothelial cells; Loss of JAG1-NOTCH2 signaling decreases ELN under LSS. (a) 4D Flow MRI shows how a pulmonary stenosis results in progressive pathological high shear stress (HSS) (from Pewowaruk, Ann Biomed Eng. 2019). (b) Histopathology of a normal pulmonary artery (left) and the region associated with pulmonary stenosis (right). Note the well organized and thick elastic laminae (left) versus disorganized and fragmented appearance of multiple thin elastic laminae (right). (c) Microscopic images show endothelial cell alignment in response to static (ST), laminar shear stress (LSS) and high shear stress (HSS) conditions. (d) LSS induces JAG1 and ELN but not NOTCH2 mRNA in pulmonary artery endothelial cells. (e,f) Reducing JAG1 with small interfering RNA (siJAG1) under LSS decreases ELN mRNA by qPCR and protein by Western blot. (g) JAG1 is a ligand of Notch signaling. Once JAG1 binds the NOTCH2 receptor, γ-secretase induces NOTCH2 intracellular domain (N2ICD) which binds the DNA-binding transcription factor RBPJ. (h) Suppression of NOTCH signaling by siNOTCH2, γ-secretase inhibitor DAPT or siRBPJ, all decrease ELN mRNA under LSS by qPCR. N=5 biological replicates, *P<0.05, **P<0.01.
Figure 2: Schematic representation of the project.
(a) Alagille syndrome (ALGS) is caused by heterozygous JAG1 loss-of-function mutations. (b) Work flow: iPSCs from healthy/ALGS individuals will be differentiated into vascular endothelial cells (iPSC-EC) and smooth muscle cells (iPSC-SMC), and exposed to the three fluid flow conditions (ST, LSS, HSS). We will build a nucleotide-resolution map of gene regulation in iPSC-EC and SMC (1). Pooled CRISPR prime editing screens will be used to dissect the regulatory logic of the JAG1 promoter (2). We will apply genome editing to restore proper JAG1 expression (3).