Mingxia Gu, MD, PhD
Yifei (Simon) Miao, PhD
Integrating single-cell RNA-seq of iPSC-ECs and of human heart tissue from patients with congenital heart disease (CHD) reveals abnormal endothelial and endocardial cell (EC) functions contributing to impaired development of the valves and cardiac chambers. More
By using single cell RNA-sequencing, transcriptomic information from CHD patient-specific induced pluripotent stem cell (iPSC)-derived endocardial and endothelial cells (upper left) and patient’s heart tissue (upper right) are used to pinpoint abnormalities that impair heart development. Upper left: tSNE-plot indicated different cell subpopulation distribution between patient and control iPSC-EC; upper right: various cell populations in heart tissue from CHD patient illustrated by UMAP.
High-Throughput drug screening for improved EC and SMC function in PAH. More
iPSC-ECs generated from six PAH patients were exposed to 4,500 FDA approved compounds and assayed for improved cell survival following serum withdrawal using the caspase 3/7 assay, which gave a reproducible luminescent signal that can be detected by the plate reader in a high- throughput manner. Only drugs showing (i) more then 50% improvement, (ii) a positive dose response, (iii) no toxicity, and (iv) no interference with the assay, were chosen for further investigation. Top candidate drugs were then tested for their efficacy in reversing SMC and EC dysfunction such as aberrant cell proliferation and impaired angiogenesis. The LINCS database was used to identify a pathway targeted by the agents that improved function of PAH cells. Those compounds most promising in cell and organ culture studies are also tested in a rat model of severe pulmonary hypertension.
Dr. Mingxia Gu, Instructor in Pediatric Cardiology, investigates the role of the endocardium and endothelium in congenital heart disease, the genetic and epigenetic basis of the variable penetrance of the cardiac defect, and the response to therapy.
MODELING CONGENITAL HEART DISEASE WITH INDUCED PLURIPOTENT STEM CELLS (iPSC)
Dr. Gu is uncovering the role of the endocardium and endothelium is causing Hypoplastic Left Heart Syndrome (HLHS), a severe form of congenital heart disease (CHD). Together with research associate Dr. Yifei Miao, single cell RNA sequencing is being applied to patient-specific induced pluripotent stem cells (iPSC) and to heart tissues obtained from HLHS patients. Novel observations pointing to the abnormal differentiation of the endocardium have been made and current studies address how this might affect formation of heart valves and heart muscle. In collaboration with Dr. Jinjing Li, abnormalities in the transcriptome are being related to gene variants analyzed by whole exome sequencing through the Pediatric Cardiac Genetics Consortium (PCGC) database.
REPURPOSING DRUGS FOR PAH USING IPSC AS A SCREENING TOOL
Dr. Gu has also used patient-specific iPSC derived vascular cells combined with a bioinformatic approach.to identify drugs that could be repurposed to reverse the vascular remodeling in pulmonary arterial hypertension (PAH). She has developed a high-throughput phenotypic screen to identify compounds that reverse PAH endothelial cell (EC) and smooth muscle cell (SMC) dysfunction in a patient-specific manner. Induced pluripotent stem cell derived ECs (i-EC) generated from six PAH patients were exposed to 4,500 compounds and assayed for improved cell survival following serum withdrawal using the caspase assay. Three novel compounds appear to reverse the abnormal function of iPSC-derived vascular cells from six patients thus far tested. Efficacy of these agents has been confirmed in an animal model. Another 13 compounds were identified as related to patient-specific drug response. Dr. Gu has initiated a collaboration with Calibr in San Diego to use their library of 14,000 compounds to develop automated screening of complex endothelial functions such as angiogenesis and cell migration.
Marcy Martin, PhD
THREE-DIMENSIONAL ORGANOID GENESIS TO MODEL PULMONARY ARTERIAL HYPERTENSION
Dr. Marcy Martin is a postdoctoral fellow evaluating 3D organoid genesis as a method to not only provide a model of disease for in vitro characterization, but also to create a physiological platform that will be useful in investigating microenvironments such as hypoxia and inflammation or drugs and toxins associated with pulmonary arterial hypertension (PAH), and to test the efficacy of novel therapies. The organoids establish an arterial, capillary and venous system in continuity with the circulation of the host animal. This system will allow assessment of the contribution of genetic disorders such as BMPR2 mutations to pathology in arteries, as well as capillaries and veins. The vascular organoids will be assessed under conditions of blood flow for morphologic perturbations that can be related to altered transcriptional regulation at the single cell level. Furthermore, ingrafting the vascular organoids into mice will provide key information to the hemodynamic regulation between control donors and BMPR2 mutants. Vascular organoids from PAH patients would be expected to have an exacerbated response to hypoxia and pro-inflammatory cytokines.
Equal concentrations of induced pluripotent stem cells, primary pulmonary arterial endothelial cells and smooth muscle cells were aggregated in suspension for 10 days. Aggregates were then implanted into Matrigel for 10 more days to produce a vascular organoid. Endothelial cells were immunostained with VE-Cadherin (green); smooth muscle cells were stained with SM22a (cyan), and extracellular matrix was stained with collagen type IV (red).
Shalina Taylor, PhD
NETosis in Human Neutrophils Isolated from PAH and Healthy Donors. More
NETs were induced by treatment with PMA and monitored over a period of 60min minutes. Top: immunofluorescent images showing SYTOX Green staining of externalized decondensed neutrophil chromatin, with DAPI (blue) staining for the nuclei. Bottom: NETs release was quantified by the spread area of the NETs, normalized to healthy donor neutrophils. NETosis is heightened in PAH neutrophils. The increased NETosis in PAH neutrophils was abrogated in response to treatment by the elastase inhibitor Elafin. Data are presented as mean±SEM, n=30-60 cells. **P<0.001 compared to Donor neutrophils, by one-way ANOVA with Bonferonni’s.
NEUTROPHIL FUNCTIONS DURING PULMONARY ARTERIAL HYPERTENSION (PAH)
Dr. Shalina Taylor, a postdoctoral fellow, investigates neutrophil function in patients with PAH. Comparing neutrophils of PAH patients with those of healthy donor, she identified profound alterations in adhesion, migration, trans-endothelial migration, degranulation, and NETosis. Heightened production and release of elastase explained the propensity to NETosis. Proteomic analyses identified vinculin as a critical factor in explaining increased adhesion and impaired migration, and transcriptomic analyses revealed a type I interferon response attributed to heightened expression of the endogenous retrovirus HERV-K; in fact, the phenotype of the neutrophil related to heightened elastase and vinculin expression can also be attributed to HERV-K. Current studies are aimed at testing the consequence of HERV-K mediated sequelae in an animal model of pulmonary hypertension.
Toshie Saito, MD
Peripheral blood mononuclear cells (PBMCs) were analyzed by CyTOF (single cell mass cytometry). Unbiased clustering system algorithm “Spade” was used to cluster subpopulations of PBMCs. SAMHD1 level is shown as a color code (low: blue to high: red).
DENDRITIC CELLS AND THE PATHOGENESIS OF PULMONARY ARTERIAL HYPERTENSION
Dr. Toshie Saito discovered a novel target of immune complexes in lungs from PAH patients, that links viral infection, immunity and inflammation with pulmonary hypertension. In her studies, Toshie identified SAMHD1, a well-known host protective factor of HIV infection, as an antigen of immune complexes in lungs from patients. This led to the insight that human endogenous retrovirus K (HERV-K) is linked to the pathology of pulmonary arterial hypertension. This work was published in 2017 (Saito et al, Circulation. 2017; 136:1920-1935).
CyTOF analyses have focused her attention on abnormal activation of dendritic cells in PAH patients compared to controls (healthy volunteers). Single cell RNAseq is currently being carried out to further investigate the nature of the aberrant dendritic cell in PAH, and MIBI is being used to localize these cells to the occluded pulmonary arteries in the lung tissue from PAH patients.
Shoichiro Otsuki, MD, PhD
Exposing PAECs to HERV-K dUTPase induces changes consistent with endothelial to mesenchymal transition (EndMT). More
Top: PAEC exposed to 10 µg/mL HERV-K dUTPase for 20 days undergo morphological changes from the cobblestone appearance typical of endothelial cells, to a smooth-cell like elongated shape, in a disordered monolayer. Scale bar, 100 µm.
Bottom: After 10 days with HERV-K dUTPase, PAEC in culture exhibit fragmented VE Cadherin (green), changed from the continuous VE cadherin staining typical of endothelial cells. This is accompanied by an increase in the smooth muscle cell marker alpha-SMA (red).
HUMAN ENDOGENOUS RETROVIRUS K (HERV-K) AND ENDOTHELIAL CELL BIOLOGY IN PULMONARY ARTERIAL HYPERTENSION
Following the finding (T. Saito et al. Circulation 2017), that human endogenous retrovirus K (HERV-K) is linked to the pathology of pulmonary arterial hypertension, Dr. Shoichiro Otsuki, a postdocoral fellow, aims to elucidate the mechanisms by which HERV-K induces a PAH-related phenotype in pulmonary artery endothelial cells (PAECs). Shoichiro discovered that an HERV-K protein, HERV-K dUTPase, induces changes in PAECs that are consistent with endothelial to mesenchymal transition (EndMT). He is investigating the molecular mechanism by which this transformation is induced, and its relevance to the pathological changes in PAECs that are observed in PAECs of PAH patients. He has already established the role of NFkappa B and TLR4 activation downstream of HERV-K dUTPase. Dr. Otsuki has also established the impact of HERV-K upregulation in macrophages in inducing endothelial to mesenchymal transition.
Rebecca Harper, PhD
Graph based clustering of single cell RNA sequencing of human peripheral blood mononuclear cells. Each dot represents a single cell, and each color represents a cluster. Clusters represent a group of cells that are genetically different from other clusters.
THE ROLE OF MONOCYTES AND MACROPHAGES IN THE DEVELOPMENT AND PERSISTENCE OF PULMONARY ARTERIAL HYPERTENSION
Dr. Rebecca Harper, a postdoctoral fellow, is examining the role of monocytes and macrophages and how they contribute to the development and persistence of the chronic inflammation seen in pulmonary arterial hypertension (PAH). She hypothesizes that in the context of PAH, monocytes recruited from the bone marrow have an altered gene and protein expression profile which produce invasive, pro-inflammatory macrophages that contribute to vasculature remodeling in the lungs. Using cutting-edge technologies such as single cell RNAseq, she has recently discovered a dysregulation of CD14 in two monocyte populations, with a depletion of one sub-population. Furthermore, she has shown that when stimulated, control, but not CD14+, monocytes from PAH patients up-regulate transcription of BMPR2. Future studies aim to examine the consequence of this dysregulated CD14 pathway and how this may impact other immune cells such as NK and T cells.
Jan-Renier Moonen, MD, PhD
Schematic representation of the proposed mechanism for the regulation of gene expression by Laminar or Disturbed flow
Three-dimensional image of a rat pulmonary artery.
300µm sections of agarose embedded rat pulmonary tissue were stained for the endothelial cell marker, vWF (red) and the smooth muscle cell marker, aSMA (green). Nuclei are depicted in blue (DAPI stain). The image is a 3D reconstruction of a 100µm Z-stack.
SHEAR STRESS REGULATION OF THE ENDOTHELIAL CHROMATIN LANDSCAPE: DEFINING VULNERABILITY TO DYSFUNCTION AND DISEASE
Postdoctoral fellow Dr. Jan-Renier Moonen studies the role of biomechanical forces in modulating endothelial function and plasticity, and how this contributes to the onset and progression of pulmonary arterial hypertension and other occlusive vascular diseases. His central hypothesis is that shear stress is an important determinant of the endothelial chromatin landscape by directing gene expression profiles that determine the vulnerability of endothelial cells to environmental risk factors such as elevated shear stress or genetic mutations such as those of BMPR2. He investigates the regulation of chromatin accessibility changes in pulmonary artery endothelial cells (PAEC) exposed to different flow conditions using an in vitro perfusion system. His main goal is to identify the chromatin remodeling complexes that that either protect against, or cause endothelial dysfunction. ATAC-Seq and RNA-Seq analyses show pronounced changes in chromatin accessibility of PAEC when exposed to shear stress, which relate to altered gene expression profiles. By identifying the transcription factors that regulate the chromatin accessibility changes and uncovering the remodeling enzymes with which they interact, he ultimately aims to identify therapeutic approaches that protect vascular regions that are vulnerable to disease. Together with Tsutomu Shinohara, Jan-Renier also studies the pathological effects of very high shear stress levels (up to 170dyne/cm2) such as observed in children predisposed to pulmonary arterial hypertension associated with congenital heart disease.
Tsutomu Shinohara, MD, PhD
PAEC exposed to very high shear stress show enhanced alignment with the direction of the flow compared to PAEC exposed to laminar shear stress; under static conditions, the cells are not aligned. More
PAEC were isolated form lungs of healthy donors. Nuclei are stained with DAPI (Blue) and actin in stress fibers is stained green. The graph shows the percent of the cells at each angle, relative to the direction of the flow.
PULMONARY VASCULAR REMODELING BY VESSEL WALL SHEAR STRESS
Postdoctoral fellow Dr. Tsutomu Shinohara asks what leads to the disordered development of the pulmonary artery and pulmonary arterial hypertension (PAH) observed in congenital heart disease (CHD) patients with L-R shunt. Based on that clinical question, his current research focuses on the mechanisms leading to dysfunction of primary pulmonary artery endothelial cells (PAEC) exposed to very high shear stress, modeled by a collaboration with the Marsden lab and based upon MRI data from patients with PAH associated with congenital heart disease. Dr. Shinohara uses an apparatus where software-controlled flow rates and shear stress can create conditions of laminar shear stress (LSS, 15 dyn/cm2) or very high shear stress (VHSS, 170dyn/cm2), using the same culture conditions. This model is revealing heightened production of reactive oxygen species that result from uncoupling of eNOS.
Dan Li, PhD
Nuclear ALDH1A3 is increased in smooth muscle cells in PAH.
Confocal microscopy was applied to PAH vs. control lung tissues sections stained for ALDH1A3, (red) and SMC (SM22alpha, green). Nuclei were stained with DAPI (blue). We see a striking increase in nuclear ALDH1A3 expression in PAH vs. control PA SMC.
PULMONARY ARTERY (PA) SMOOTH MUSCLE CELLS INVOLVEMENT IN THE SEVERE OBLITERATION OF PA IN PULMONARY ARTERIAL HYPERTENSION
Postdoctoral fellow Dr. Dan Li focuses on the pathological features of hyperproliferative pulmonary arterial smooth muscle cells (PASMCs), that contribute to the severe obliteration of pulmonary arteries (PA) in pulmonary arterial hypertension (PAH). In RNA-Seq analyses, she found aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) is upregulated in PASMCs from lungs of PAH patients compared with PASMCs of healthy controls. ALDH1A3 is a key enzyme in acetaldehyde metabolism, and is a source of acetyl-CoA. Acetyl-CoA catalyzes the acetylation of histones, resulting in chromatin remodeling that can enhance the expression of genes linked to the hyperproliferative phenotype. Dan found that the histone mark H3K27ac is increased in PAH PASMCs, and decreased with loss of the expression of ALDH1A3. From transcription factor binding motif analysis, she found that nuclear transcription factor Y targets 66 down-regulated genes, including PKM2, DLD and IDH1, that are responsible for accumulation of acetyl-coA and production of energy, and CCNA2 and CDC20, that regulate cell cycle.
Using an in vivo mice model, where ALDH1A3 is reduced in smooth muscle cells, Dan showed decreased development of pulmonary hypertension following exposure to chronic hypoxia. This study reveals a novel mechanism for understanding the biological process, w linking the enzymatic activity and gene expression via chromatin remodeling, and may lead to new targets for PAH therapy.
Sarasa Isobe, MD, PhD
Immunofluorescence reveals DNA damage in pulmonary arterial endothelial cells of PAH patients. More
Confocal microscopy images show foci of DNA damage, indicated by gammaH2AX (stained green), in PAEC (labeled by von-Willebrand factor, vWF, stained red) in lung tissue sections from PAH patients and controls. Arrowheads indicate cells in insets. (Figure 5A from Li et al., 2019, Cell Reports 26, 1333–1343).
DNA DAMAGE IN PULMONARY HYPERTENSION
Unrepaired DNA damage induces genomic instability and causes carcinogenesis and aging. Dr. Sarasa Isobe investigates the role of DNA damage in causing persistent inflammation and how it contributes to the development of pulmonary arterial hypertension. A study recently published by CG Li, a postdoctoral fellow in our lab, uncovered a DNA damage response function for PPARγ through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase, UBR5 (Li et al., 2019, Cell Reports 26, 1333–1343). Dr. Isobe’s study builds upon the observation that pulmonary arterial endothelial cells (PAEC) from PAH patients show disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. She is investigating how DNA damage contributes to the cellular dysfunction associated with pulmonary arterial hypertension using PAEC from PAH patients and BMPR2-/- mice.
Lingli Wang, MD
Double Outlet Right Ventricle (DORV) and Ventricular Septal Defect (VSD) were found in newborn mice with BMPR2 deleted in SMCs. More
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; SM22alpha-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.
β-Arrestin2 and Active β-Catenin (ABC) Expression in Human SPA from a Donor (top row) and a PAH Patient with BMPR2 Mutation (bottom) More
Representative images of immunofluorescence staining for smooth muscle marker SMA (Green), nuclei (DAPI, blue) and either β-Arrestin2 or β-Catenin (Red). PASMCs with β-Arrestin2 or β-Catenin positive staining are limited in the Donor lung. In contrast, widely distributed β-Arrestin2 staining primarily co-localized with SMA to the cytoplasm, and β-Catenin to the nuclei, of PASMCs in the PAH Patient with a BMPR2 Mutation.
GENETICALLY MODIFIED MURINE MODELS OF DISEASE
Dr. Lingli Wang, Senior Life Science Research Associate (Lab Manager) is creating genetically modified mice to conditionally delete and fate map cells, to be used in investigating pulmonary hypertension pathophysiology. She has investigated 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. When exposed to hypoxia, these SMC-Bmpr2 deleted mice developed persistent pulmonary hypertension, and increased muscularity of distal vessels that did not regress during return to room-air. Smooth muscle cells from pulmonary arteries (PASMCs) of these mice are hyper-proliferation and apoptosis-resistance, and show reduced contractility. The same observation was made in complementary studies of human PASMCs obtained from lungs of PAH patients with a BMPR2 mutation (see bottom image). Dr. Wang found that 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.
Aiqin Cao, PhD
Impaired resolution of DNA damage foci (γH2AX) following Dox, when there is a reduction in the DNA repair target genes of PPARγ-P53 mediated transcription (e.g. GADD45B). More
Immunoblots with densitometry for gammaH2AX, representing DNA damage foci, in cultured human PAEC. PAEC were transfected with non-targeting (Con) siRNA or siRNA targeting PPARγ, p53 or GADD45B, DNA damage was induced with Dox for 16 hours, the allowed to recover for 24 hours. Bars represent mean±s.e.m of n=3. *p<0.05 vs. vehicle with Con siRNA, #p<0.05 vs. Dox with Con siRNA, by 1-way ANOVA with FDR post-hoc analysis.
DNA DAMAGE REPAIR IN PULMONARY ARTERY ENDOTHELIAL CELLS, AND ITS DYSREGULATION IN PULMONARY ARTERIAL HYPERTENSION
Endothelial dysfunction is associated with impaired BMPR2 signaling, and PPARγ and tumor suppressor p53 are transcription factors that mediate gene regulation downstream of BMPR2 in PAEC. Genomic instability and DNA damage are associated with PAH pathogenesis. PPARγ, and p53 both play critical roles in DNA damage repair, and restoration of PPARγ or p53 signaling in the pulmonary vasculature reverses experimental PH. Research Associate Dr. Aiqin Cao builds upon investigations carried out by Dr. Jan Hennigs, a former postdoctoral fellow in the lab, on the interactions between PPARγ and p53 in PAEC. Dr. Cao’s studies show how these interactions are induced by exposing the PAECs to genotoxic conditions such as doxorubicin (Dox), or by increasing p53 using Nutlin-3 in PAEC. She shows how altered PPARγ-P53 interactions cause PAEC vulnerability to apoptosis and genomic instability. The project assesses whether reversal of DNA damage in PAH PAEC restores gene regulation and thus improves PAEC function. These studies will lead us to assess ways in which we can reverse DNA damage and pulmonary hypertension by restoring normal function of the BMPR2-PPARγ axis.