Diederik van der Feen, MD
Kazuya Miyagawa, MD, PhD
ENDOTHELIAL-SMOOTH MUSCLE INTERACTION AND ENDOTHELIAL METABOLISM PROMOTING VESSEL STABILITY
Postdoctoral fellow Kazuya Miyagawa, MD, PhD, is investigating an interaction between endothelial cells and smooth muscle cells and the role of this interaction in endothelial metabolism and vessel stability in the pathogenesis of pulmonary arterial hypertension. Specifically, he is addressing a BMPR2-Notch1 dependent change in endothelial metabolism that mediates epigenetic modification of endothelial cells and vessel stability.
Immunofluorescence image of Notch1 intracellular domain in pulmonary artery (PA) endothelial cells (PAEC) with or without contact co-culture with PA smooth muscle cells (PASMC). In contact with PASMC, PAEC Notch1 is activated but when both cell types has loss of BMPR2 (BMPR2 mutation), Notch1 is not activated (red marked).
Jan-Renier (JR) Moonen, MD, PhD
SHEAR STRESS REGULATION OF THE ENDOTHELIAL CHROMATIN LANDSCAPE: DEFINING VULNERABILITY TO DYSFUNCTION AND DISEASE.
Jan-Renier 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 or genetic mutations such as those of BMPR2. He investigates the regulation of chromatin accessibility changes in PAEC exposed to different flow conditions using an in vitro perfusion system. His main goal is to identify the chromatin remodeling complexes that modify chromatin landscapes that either protect against, or are permissive for endothelial vulnerability to dysfunction and endothelial-mesenchymal transition. Preliminary data obtained by ATAC-Seq and RNA-Seq show global changes in chromatin accessibility of PAEC when exposed to LSS that coincide with altered gene expression profiles. By identifying the transcription factors that regulate 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 particularly vulnerable to disease. As a first step, Jan-Renier is exploring such approaches using targeted adenoviral delivery to modulate the identified regulatory factors in endothelial cells in experimental models for PAH.
Pin-I Chen, PhD
GPCRs, AMPHETAMINE AND PULMONARY HYPERTENSION
Pin-I Chen, PhD, postdoctoral fellow focuses on a sub-category of pulmonary hypertension associated with abuse of amphetamine derivatives. She has elucidated the signaling mechanism whereby these compounds subvert cell survival, and amplify inflammatory responses and DNA damage. She is also establishing an animal model with chronic infusion of amphetamine.
Top Figure: A schematic model showing amphetamine-induced GPCR signaling cross-talk with endothelial cell survival pathways and inflammatory responses via recruitment of β-arrestin platforms.
Bottom Figure: Red: 53BP1, the protein recruited to a focus of DNA damage. Green: phospho gammaH2AX an indicator of DNA damage. Yellow: overlap of 53BP1 and phospho gammaH2AX. The micrographs demonstrate that DNA damage in endothelial cells induced by Doxorubicin (Dox) is amplified by amphetamine (AMPH).
Lingli Wang, MD
GENETICALLY MODIFIED MURINE MODELS OF DISEASE
Lingli Wang, MD, 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 of the vessel wall and found that these cells are hypocontractile and hyperproliferative owing to impaired RhoA and heightened beta catenin.
Dan Li, PhD
PASMCs INVOLVING SEVERE OBLITERATION OF PA IN PAH
Post-doctoral fellow Dan Li Ph.D, is focused on the pathological features of hyperproliferative pulmonary arterial smooth muscle cells (PASMCs) involving severe obliteration of pulmonary arteries (PA) in pulmonary arterial hypertension (PAH). In the preliminary RNA-Seq analyses, she found aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) is upregulated in the PAH PASMCs among 87 significantly changed genes compared with control PASMCs. ALDH1A3 is a key enzyme in acetaldehyde metabolism that can contribute to the source of acetyl-CoA that catalyzes H3K27ac and H3K9ac resulting in chromatin remodeling necessary for the expression of genes linked to the hyperproliferative phenotype. In this study, the histone marks, H3K27ac and H3K9ac were also increased in PAH PASMCs. Her overarching goal is to integrate metabolic changes with chromatin remodeling and gene regulation to better understand the pathobiology of the hyperproliferative PASMC phenotype.
Diederik van der Feen, MD
A REVERSIBLE AND IRREVERSIBLE RESPONSE TO HAEMODYNAMIC UNLOADING IN FLOW-INDUCED PULMONARY ARTERIAL HYPERTENSION – VASCULAR PROFILES ASSOCIATED WITH THE REVERSIBILITY OF PULMONARY VASCULAR DISEASE.
Diederik developed a model for flow-induced pulmonary arterial hypertension that shows a reversible and an irreversible response to haemodynamic unloading. He has shown that this phenotypical reversibility switch coincided with a notable change in vascular cellular profile. Diederik’s work involves integrative transcriptomics complemented by cellular deconvolution algorithms developed at Stanford University. Using this strategy, he aims to uncover cell-specific signalling pathways that may underlie the transitions that cause irreversible PAH. His model further enables to test compounds that interfere upon these pathways in vivo in order to reverse “irreversible” PAH.
Roger Thompson, MRCP, PhD
DOUBLE STRANDED RNA (dsRNA) IN PULMONARY VASCULAR REMODELLING in PAH
Vascular remodelling is the key pathological feature of PAH but the triggers and regulatory pathways involved remain poorly understood. Recent data have identified Toll-like receptor 3 (TLR3) as a potential regulator of vascular remodelling through its sensing of endogenous damage signals. I have investigated the role of TLR3 and its ligand, double stranded RNA (dsRNA) in pulmonary vascular remodelling. My unpublished data show that TLR3 deficiency markedly exacerbates PAH in the SUGEN/hypoxia (SuHx) mouse model. In human cell culture, I have also shown that dsRNA suppresses PDGF-induced proliferation of pulmonary artery smooth muscle cells (PASMCs). I therefore hypothesise that pathways activated by endogenous dsRNA protect against aberrant pulmonary vascular remodelling in PAH and targeting these pathways will generate new opportunities to slow or reverse disease progression. The goal of this project is to determine how pathways associated with dsRNA sensing regulate vascular remodelling in PAH. Furthermore, the project aims to characterise endogenous dsRNA signals in the context of PAH and to establish whether dsRNA expression and stability is altered in disease.
Shalina Taylor, PhD
NEUTROPHIL FUNCTIONS DURING PULMONARY ARTERIAL HYPERTENSION (PAH)
Shalina’s research focuses on determining whether neutrophil functions are abnormal during Pulmonary Arterial Hypertension (PAH) i.e. migration, trans-endothelial migration, ROS generation, and NETosis. Furthermore, she plans to address whether these functions are exaggerated by the interaction with Pulmonary Arterial Endothelial Cells (PA EC’s) and whether this is Elafin inhibitable. She will also apply transcriptomics and proteomics data to better characterize the phenotype of neutrophils in PAH.
Nancy Ferriera Tojais, PhD
CELL EXTRACELLULAR MATRIX INTERACTIONS AND REGULATION OF ELASTIN ASSEMBLY IN VASCULAR DEVELOPMENT AND PATHOLOGY
Nancy Ferreira Tojais, PhD, postdoctoral fellow investigated the factors that cause susceptibility of elastin to degradation and has discovered the role of BMPR2 signaling in regulating appropriate scaffolding of the elastin fiber via fibrillin1 and the role of the proteoglycan decorin in restricting elastin assembly in development and impairing elastin repair in disease. Figure shows that BMP stimulates fluorescent elastin fiber formation in lung fibroblasts.
Aiqin Cao, PhD
Aiqin’s current project is studying the role of BMPR2 in pulmonary artery smooth muscle cells and adventitial fibroblasts specifically addressing the necessary role of BMPR2 is in elastin protein production by TGFβ1. She is also working on the post-translational modifications (PTMs) of PPARγ in pulmonary arterial endothelial cells (PAEC) from patients with pulmonary arterial hypertension. She will determine the extent to which the PTMs derange PPARγ function causing PAEC vulnerability to apoptosis and genomic instability, and to evaluate these modifications as future targets for therapeutic intervention.
Toshie Saito, MD and Shoichiro Otsuki, MD, PhD
IMMUNITY, INFLAMMATION AND PULMONARY HYPERTENSION
Toshie Saito, MD, research associate, 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 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. She is currently investigating the cellular mechanisms that are involved in this pathobiology. In a related project, Shoichiro Otsuki, MD, PhD, is studying pulmonary endothelial cells in culture and the effect of HERV-K on endothelial cell biology.
Toshie’s second project is a comprehensive profiling of immune cells in peripheral blood from PAH patients and controls (healthy volunteers) using single cell mass cytometry (CyTOF), in collaboration with the labs of Dr. Garry Nolan and Dr. Wendy Fantl. The ultimate goal is to eventually discover innovative personalized therapies for suffering patients.
Top Figure: Confocal microscopy images of representative lung sections from a PAH patient and a control: Cells immunolabeled for HERV-K envelope protein or HERV-K dUTPase (green), macrophages (CD68+, red) and nuclei (DAPI, blue). Dashed line indicates vessel boundary. Elastin auto-fluorescence appears pink. (Taken from Saito et al, Circulation 2017, in press)
Bottom Figure: Proposed model: The endogenous retrovirus HERV-K is expanded, possibly as a result of an environmental or genotoxic stress. The product, HERV-K dUTPase, and the subsequent activation of vascular, inflammatory and immune cells lead to adverse vascular remodeling and PAH. (Taken from Saito et al, Circulation 2017, in press)
Mingxia Gu, MD, PhD
MECHANISM UNDERLYING THE REDUCED PENETRANCE OF BMPR2 MUTATION IN FPAH USING PATIENT-SPECIFIC IPSC DERIVED ENDOTHELIAL CELLS (IPSC-ECS)
Post-doctoral fellow Mingxia Gu, MD, PhD, aims to uncover the mechanism underlying the reduced penetrance of BMPR2 mutation in FPAH using patient-specific iPSC derived endothelial cells (iPSC-ECs). She is working on a cohort of nine individuals from three FPAH families carrying three different BMPR2 mutations (Family 1: c.354T>G p.C118W; Family 2: c.2504delC p.T835fs; Family 3: c.G350A p.C117Y) and three gender matched controls. Each family included one to three FPAH patients with a BMPR2 mutation, and one unaffected mutation carrier with identical BMPR2 mutation (Mut. Carrier). She has identified compensatory p-p38 signaling pathway which leads to preserved endothelial functions in iPSC-ECs from Mut. Carriers. She will computationally integrate RNA-Seq and ChIP-Seq analyses to further define how the landscape of histone marks is related to gene expression networks involved in preserved EC functions in Mut. Carriers. Since reduced function and expression of BMPR2 is also seen in patients with idiopathic and associated forms of PAH, these studies reveal compensatory mechanisms that might be targeted in developing novel therapies for all forms of PAH.
Silin Sa, PhD
INDUCED PLURIPOTENT STEM CELLS AND PULMONARY HYPERTENSION
Research Associate Silin Sa, PhD, compared pulmonary artery endothelial cells (PAEC) to endothelial cells derived from induced pluripotent stem cells (iPSC-EC) from the same individuals and determines whether abnormalities in PAEC of PAH patients compared with those of healthy individuals (controls) are recapitulated in the iPSC-EC from PAH patients vs. iPSC-EC from controls (Sa et al., Am J Respir Crit Care Med. 2017 Apr 1;195(7):930-941), to establish whether PAH patient-specific iPSC-EC could serve as surrogates for native PAEC in drug testing, potentially tailored to a specific patient.
Native PAEC and iPSC-EC are Morphologically Similar
Native pulmonary artery endothelial cells (PAEC) isolated and cultured from explanted lungs, and endothelial cells derived from induced pluripotent stem cells differentiated from skin fibroblasts (iPSC-EC) exhibit similar cobblestone morphology characteristic of endothelial cells, incorporate acetylated low-density lipoprotein (Ac-LDL, green) and express the endothelial marker CD144 (green). Blue: Nuclei, DAPI
Ajay Bhatia, MD
He joined the lab in the summer of 2016 and is investigating how the altered hemodynamics seen in patients with pulmonary arterial hypertension associated with congenital heart disease (APAH-CHD) contributes to disease pathophysiology. Our lab has leveraged microfluidic technologies and high-throughput sequencing methods to attempt to understand why certain patients with CHD develop disease while others do not. Specifically, we have been studying how primary pulmonary endothelial cells (PAEC) derived from patients with APAH-CHD are affected by disturbed flow conditions, including oscillatory shear stress and high laminar shear stress.
Alignment of pulmonary arterial endothelial cells under laminar shear stress (LSS) conditions at 15dyn/cm2 compared to oscillatory shear stress (OSS) conditions at +/- 3 dyn/cm2. Quantitative analysis in the two bottom panels demonstrate that the PAEC are well aligned with the direction of flow under LSS, but do not align under OSS.
Shuai Mao, MD
Shuai Mao is studying the functional abnormalities and altered gene expression patterns of smooth muscle cells differentiated from induced pluripotent stem cells. His goal is to investigate whether smooth muscle cells differentiated from pluripotent cells could serve as surrogates to test emerging therapies. He will compare smooth muscle cells differentiated from induced pluripotent stem cells with native pulmonary arterial smooth muscle cells from the same PAH patient. A second aim is to compare cells from patients with idiopathic or heritable pulmonary arterial hypertension with cells from healthy subjects (unused donor lungs) to determine whether the idiopathic and hereditary PAH cells share functional abnormalities and altered gene expression vs. controls.