Systemic Sclerosis Center of Research Translation

Systemic Sclerosis Associated PAH: The Role of the Oxidant State

Maria Trojanowska, PhD Project 2 Principal Investigator

Elena Goncharova, PhD Co- Investigator

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive increase in pulmonary vascular resistance that can eventually lead to right ventricular failure and death.  In systemic sclerosis (SSc), PAH is a leading cause of morbidity and mortality. Moreover, patients with SSc-PAH have a poorer prognosis than patients with idiopathic PAH. To improve treatment options for SSc-PAH, a better understanding of SSc-PAH pathogenesis is needed. Although the exact pathophysiology of PAH is unknown, abnormal cellular metabolism caused by altered mitochondrial dynamics, is now considered an important contributor to pathological alterations in PAH. During the prior grant period, we demonstrated increased endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in circulating immune cells (PBMCs) from SSc patients with limited disease (lcSSc), with even higher levels in lcSSc-PAH patients. We also demonstrated highly fragmented mitochondria in lcSSc PBMCs; extending these studies to lungs from SSc-PAH patients undergoing transplantation, we observed highly increased UPR markers in endothelial cells (EC) and macrophages and evidence of increased oxidation. Given the complex nature of PAH, a therapeutic agent capable of modulating several key pathways would be an attractive addition to the treatment regimen of PAH. As a possible candidate, we have focused on dimethyl fumarate (DMF), an agent that augments the intrinsic cellular antioxidant response and was recently approved (Tecfidera®) for treatment of multiple sclerosis. Using the rodent-hypoxia model of PH, we found DMF prevented and reversed hemodynamic changes, muscularization of pulmonary vessels, and right ventricular hypertrophy. DMF also attenuated lung damage caused by oxidative stress and reduced immune cell infiltration into lungs of treated mice. To translate these results to human SSc-PAH, we propose:

1. Determine the role of altered mitochondrial dynamics on activation of immune cells in lcSSc
2. Determine the molecular characteristics of freshly isolated PAEC from lcSSc patients with and without PAH undergoing right heart catheterization
3. Determine the effect of DMF on the function of distal resistance pulmonary arteries from explanted lungs of patients with SSc-PAH undergoing transplantation.

These complementary studies will develop better understanding of the role of the redox state in the pathogenesis of SSc-PAH, validate anti6oxidants, such as DMF, as potential treatment for this entity, and develop collaborative disease models between Boston University and the University of Pittsburgh Medical Centers, two expert centers with long-standing interest in SSc and SScPAH.

Targeting Pro-Fibrotic E3 Ligases in Systemic Sclerosis

Daniel J. Kass, MD  Project 3- Principle-Investigator

Beibei Bill Chen, PhD Project 3- Co-Investigator

Systemic sclerosis (SSc) is a progressive multi-organ system fibrotic disorder associated with autoimmunity. The leading causes of SSc-related death are pulmonary hypertension (PAH) and interstitial lung disease (ILD). PAH affects 10-15% of SSc patients. The prevalence of ILD varies from 40 to 65% in cohort studies. SSc-ILD is the subject of CORT Project #3. Common to many of the SSc disease phenotypes, include the skin and the lung, is the observation that fibroblasts, the principal effector cells of fibrosis, assume the contractile myofibroblast phenotype, synthesize matrix, and exhibit a pathologically increased lifespan. A traditional approach to discover novel genes involved in the pathogenesis of pulmonary fibrosis has been to perform genome-wide screening to uncover dysregulated RNA species that may be involved in the pathogenesis of disease. However, this approach may miss the critical role of proteins that are NOT regulated at the level of transcription but rather through protein degradation, particularly through the ubiquitin-proteasome system (UPS) mediated by ubiquitin E3 enzymes. Our recent studies using a large number of idiopathic pulmonary fibrosis (IPF) and SSc lung samples have uncovered an array of several, previously unsuspected, molecular targets. In particular, we have identified two highly novel profibrotic signaling pathways in SSc fibroblasts: First is the loss of a new collagen 1 gene repressor, E2 transcriptional factor 8 (E2F8) by a ubiquitin E3 ligase, Fbxo16. This ultimately leads to increased collagen synthesis in SSc fibroblasts. The second is a new protein isoform, termed FIEL1 (Fibrosis-Inducing E3 Ligase 1), which potently stimulates the central pro-fibrotic cytokine, TGFβ, signaling pathway through the site-specific ubiquitination and degradation of the TGFβ inhibitor PIAS4. Further, we have developed a first-in-class small molecule inhibitor towards FIEL1 that is highly effective in ameliorating fibrosis in mice. Thus, we hypothesize that dysregulation of members of E3 ubiquitin ligase system drives the fibrotic phenotype in SSc. In this project, we propose to screen SSc-ILD lungs and lung fibroblasts derived from SSc-ILD lungs for dysregulated expression of members of the SCF- and HECT-domain ubiquitin E3 ligases and to assay their function in mediating the pathologic myofibroblast phenotype. We will then design small molecule inhibitors for these E3 ligases and test them for effectiveness in animal models of lung fibrosis as well as our unique ex vivo diseased human lung perfusion and culture systems here at the University of Pittsburgh. These proposed studies will help us discover a new set of potentially druggable targets underlying the pathobiology of fibrotic pathways in SSc. These studies will provide a fundamental platform for a unique and potentially transformative initiative in SSc-ILD. Our project will interact very closely with the other CORT projects and cores to discover the overlapping biology of dysregulated ubiquitin E3 ligases in SSc-PAH and SSc-skin disease.

Mauricio Rojas, MD
Lung Tissue Core – Lead

The Specific Aims for the Core are:

1. To enrich our current biorepository of lung, skin and bone marrow stem cells procured from normal individuals and individuals with SSc.
2. To assist program investigators in the design and implementation of experiments using biological specimens from the biobank.

Research Plan: The bio-bank will provide tissue samples of lung, skin and bone marrow stem cells procured from normal individuals and individuals with SSc. We will include the collection of lung and skin fibroblasts, bone marrow derived stem cells, endothelial cells, and smooth muscle cells, as well as tissue samples for RNA, DNA, protein and histopathology analyses. The ex-vivo lung perfusion sub-core will provide a preclinical translational model for the test of therapeutic candidates. The ex-vivo 3-D human lung tissue model sub-core will provide a collection of pulmonary arteries for biomechanical analyses, and agarose embedded tissue for ex-vivo 3D lung organ culture.

Significance and synergy: Comparative studies between human and mice of responses to systemic inflammation demonstrate marked differences and poor correlations between the two species, confirming the difficulties in the use of animal models to mimic human diseases. There is a need of more preclinical studies using human specimens to answer fundamental questions in complex diseases such as systemic sclerosis. All Research Projects will use resources of the lung tissue core to:

1. test mechanistic hypotheses of the pathogenesis of SSc
2. test the efficacy of new therapeutics in established ex vivo lung perfusion model and 3-D human lung tissue model
3. evaluate molecular mechanisms in human biological samples.

Robyn Domsic, MD, MPH Clinical Core – Lead

Robert Simms, MD

The essential function of the Clinical Core is to provide prospectively collected, longitudinal clinical data and associated biosamples on a well-characterized cohort of systemic sclerosis (SSc) patients. This clinical data will be linked by date to the biologic specimens essential to the completion of the projects. The Clinical Core will build on two existing, dedicated Scleroderma Center longitudinal SSc patient cohorts at the University of Pittsburgh and Boston University Medical Center in order to accomplish this goal. These observational cohorts will enroll consecutive SSc patients presenting to each institution. Cohort databases will be combined in the UPMC Rheumatic Disease Data Management System (RDMS). Blood samples for serum, plasma and PBMC RNA will be obtained at clinical visits, and linked to clinical data to support all projects. Skin biopsies will be collected from patients with diffuse cutaneous SSc for single cell RNA-seq and development of a prognostic skin biomarker (Project #1). Catheter tips from patients undergoing right heart catheterization and associated clinical information, including right heart hemodynamics, will be collected to characterize endothelial cells from SSc patients with pulmonary arterial hypertension (Project #2). In specific aim 1 the Clinical Core will continue collecting clinical data and harmonize data collection in a two-center observational, clinical data repository of SSc patients, supporting Projects #1, #2 and #3. This clinical data will be collected prospectively at each SSc Center clinic visit. Clinical data will be available corresponding to the time of SSc patient tissue or blood sample ascertainment. Clinical data will include medical history, SSc-related symptoms, physical examination, objective testing and patient reported outcomes (PROs). In specific aim 2 the core will provide blood, skin and pulmonary vascular biospecimens, accompanied by full SSc-associated autoantibody gold standard testing for Projects #1, #2 and #3. The Clinical Core will facilitate linking the clinical data in either cross-sectional (such as first SSc clinic visit) or longitudinal fashion, and thus examine whether project mechanistic findings (protein or mRNA levels) correlate with longitudinal disease assessments (pharmacodynamic biomarkers), or with the change in disease from the initial biological assessment (prognostic biomarkers). The UPMC Scleroderma Center is the only dedicated SSc Center that has historically been able to complete all gold standard SSc associated autoantibody testing. Thus, the Clinical Core will help project investigators to assess the relationship between the patients’ autoantibody status, and clinical and biological outcomes. In specific aim 3 the Clinical Core will provide project investigators in Projects #1, #2 and #3 with the statistical support necessary to investigate associations between the clinical data with the proposed mechanistic and prognostic analyses of all three Projects.

Dr. Michael Whitfield

Application of high-throughput gene expression technology to systemic sclerosis (SSc) skin biopsies, isolated SSc cell lines and peripheral blood cell (PBC) samples has shown that it will be an important tool for understanding the diversity in rheumatic diseases, as well as changes to the underlying gene expression pathways. The Translational Genomics and Data Integration (TGDI) core will use novel bioinformatic and genomic methods that have been developed and already successfully implemented in the core PI’s laboratory to analyze SSc samples and healthy controls. High quality RNA will be prepared and analyzed by RNA-sequencing using protocols established in the PIs laboratory. All data are processed using standard and novel methods that use a combination of algorithms that test for differential gene expression, enriched pathways analysis and put the changes into the context of the all publicly available SSc highthroughput data. The TGDI core provides network analyses using a Scleroderma Specific Network (SSN) to analyze data from cells lines, mouse models, clinical trials, and single cell RNA-seq (scRNA-seq), thus providing a measure of how well a therapy eliminates the aberrant gene expression we observe in SSc. The goals of this core are to 1) Provide high quality RNA-seq analyses for individual projects and process the resulting data in a rigorously controlled analysis pipeline to provide differential gene expression and patient subset assignments, 2) provide a systems biology and network analysis of gene expression data in SSc using our novel SSN, and 3) perform metaanalyses
of SSc clinical trials using both existing data as well as new data generated as part of the CORT research projects.

High-throughput gene expression analysis has allowed the definition of subsets of SSc and identified deregulated pathways that can be targeted therapeutically. Recent studies have shown that a patient’s subset or activated pathways at baseline can be predictive of clinical response. This core, provides high-throughput studies, data analyses for CORT investigators, bioinformatics and systems biology analyses of SSc samples.