Rabinovitch Research Overview
Our research program seeks to identify the cellular and molecular programs that are dysregulated in vascular disease with a focus on pulmonary arterial hypertension (PAH). This condition can be a fatal complication in children with heart defects, but can also arise as a potentially fatal disease of unknown etiology, primarily in young women. PAH is characterized by progressive loss and occlusion of the distal pulmonary microcirculation, and is associated with endothelial dysfunction, exuberant proliferation of de-differentiated smooth muscle-like cells, and chronic perivascular inflammation, all contributing to increased resistance to flow and culminating in heart failure. There are treatments for PAH that improve survival and quality of life. However these therapies primarily dilate blood vessels and do not cure PAH because they do not alter the underlying pathological mechanisms that cause the obliteration of the lung blood vessels. Our studies use high throughput genomic technologies, a variety of cell biology platforms including confocal and videomicroscopy, genetically modified mouse models of human disease, human tissue samples from lung and blood and induced pluripotent stem cells differentiated to vascular cells to learn how we can activate molecular programs to regenerate lost microvessels and to reverse the obliterative changes. Over the past decade our research has led to two novel compounds in clinical trial, the elastase inhibitor human recombinant elafin is approved for a Phase I and low dose FK-506 for a Phase II.
Our research projects have explored the link between the genetic mutation causing loss of function of bone morphogenetic protein receptor (BMPR)2 and perturbation of Wingless (Wnt) signaling, PPARg-mediated gene regulation, RNA translation, cross-talk with other cell surface receptors (PDGF, RAGE), all of which lead to endothelial and smooth muscle cell dysfunction and chronic inflammation, and propensity to unresolved DNA damage. We also address how expansion of endogenous retroviral elements in myeloid cells can lead to the activation and production of neutrophil elastase and interferon in the vessel wall. We use single cell RNA Seq to establish lung vascular and inflammatory cells that are transformed or abnormally expanded. Flow systems are introduced to study the impact of laminar flow, disturbed flow and high shear on endothelial cell chromatin structure, transcription factor accessibility and gene regulation.
We continue to use induced pluripotent stem cells derived endothelial and smooth muscle cells from patients with pulmonary hypertension combined with a variety of ‘omics’ technologies as surrogates for native pulmonary arterial cells in drug screening platforms, to develop biomarkers of disease and to elucidate modifying factors that impact penetrance of a mutation in causing PAH. We are applying innovative bioinformatics methods of analysis to integrate very large publicly available data sets with novel data sets derived from state-of-the-art transcriptomic and metabolomic technologies, to generate a powerful systems biology approach to characterize PAH and to find common aberrant pathways in the cells that could be targeted therapeutically. In so doing we have uncovered novel mechanisms underlying the propensity to unrepaired DNA damage in endothelial cells, proliferation in smooth muscle cells and homing of inflammatory cells to the adventitia. The impact of these abnormalities in causing vascular disease is tested in genetically modified mice and rats. We have embarked on the characterization of exosomes from genetically-edited autologous cells that can restore normal vascular function.
We are embarking on a clinical trial in which daily subcutaneous injection of human recombinant elafin will be investigated first in healthy controls but later as a treatment for patients with PAH, because this agent both inhibits elevated elastase activity and inflammation and activates the critical BMPR2 pathway that promotes normal blood vessel function. In parallel, studies, patient-specific pluripotent cell-derived endothelial cells are expected to identify individuals that might best respond to elafin therapy. Mass spectrometry at the single cell level in circulating immune/inflammatory cells (CyTOF) and in the tissues (MIBI) are being developed to produce biomarkers of therapeutic efficacy and new insights into PAH pathobiology.
NIH funded research uses induced pluripotent stem cells in collaboration with the laboratory of Dr. Joseph Wu and next generation sequencing in collaboration with the laboratory of Dr. Michael Snyder. Our team compares endothelial cells (ECs) derived from induced pluripotent stem cells (iPSC) with native ECs, to improve our understanding of pulmonary hypertension. Towards this goal, we are comparing gene variants (by Exome/whole genome sequencing), epigenetic changes (DNA methylation, by Methyl-Seq) and RNA expression (by RNA-Seq) in iPSC-ECs derived from skin fibroblasts or blood cells, iPSCs derived from pulmonary arterial EC, with native PAECs from the same PAH patients or controls. Our second goal is to use iPSC-ECs derived from blood or skin of PAH patients to correct gene variants and to screen novel therapies to determine whether the EC functions of these cells normalize.
This collaborative grant was the gateway into our use of integrative Omics as a discovery tool for pulmonary hypertension. We are developing and applying innovative bioinformatics methods of analysis to integrate very large publicly available data sets with novel data sets derived from state-of-the-art transcriptomic and metabolomic technologies, to generate a powerful systems biology approach to characterize PAH: Big-data analysis of publicly available PAH data sets will lead to the development of a common PAH module. We will probe the transcriptomes and metabolomes of vascular endothelial and smooth muscle cells, fibroblasts, and inflammatory cells (T cells, B cells and macrophages) isolated from explanted human PAH and healthy lungs. The data sets generated will be used to find common aberrant pathways in the cells that could be targeted therapeutically in PAH. Companion studies in rodents focus on the relationship of the pathways identified to the evolution of PAH. Combining metabolomics and transcriptomic data will extend our analysis to interactions between metabolites and genes, as well as between pathways, and emergent dominant processes will be prioritized for hypothesis testing. Animal models approximating these PAH pathways will be developed to test relevance to human PAH that could be used to explore therapies, beginning with those that repress critical pathways. We have a companion NIH funded Career Development Program that funds post-doctoral research training for PhD and physician-scientists.
An NIH Translational Program Project with the laboratories of Drs. Richard Bland and Mark Nicolls addressed the vulnerable microcirculation in pulmonary hypertension, chronic lung disease of prematurity and lung transplant rejection and to work toward translation of novel therapies including the elastase inhibitor elafin. Together with the Nolan, Robinson, Utz and Kodadek Groups on an NIH Proteomics Initiative we embarked on studies of autoimmunity and its relationship to the development of pulmonary hypertension. We use high throughput immunophenotyping and mass element flow cytometry (CyTOF) to identify abnormalities in the immune system and in the response to viral infection. A second cycle of this grant is under final funding consideration by the NIH, and will explore Elafin as a therapeutic agent for pulmonary arterial hypertension, through Phase I and Phase II clinical trials.
Other investigator-initiated research focuses on the pivotal role of PPARgamma as a transcription factor activated by BMPR2, and its novel role in DNA damage and repair. These studies led to investigations linking p53, with PPARgamma and the DNA repair machinery .We have also uncovered a role for amphetamine-stimulated G-protein Coupled Receptor (GPCR) signaling in amplifying DNA damage.
We are engaged in creating genetically modified mice with conditional cell-specific deletion of genes, fate mapped reporter genes to trace lineage and heterozygosity to understand vascular and cardiac phenotypes. We are then positioned to use these mice to reverse disease. We address many features of altered signaling related to mutated BMPR2. Recently we have shown that alterations in normal functioning of BMPR2 impairs vascular elastic fiber assembly and increases susceptibility to degradation by elastase. Well-assembled elastin maintains the integrity and distensibility of vessels and prevents abnormal proliferation of underlying smooth muscle cells and fibroblasts. Abnormalities in elastic fiber assembly underlie both pulmonary arterial hypertension as well as systemic vascular diseases including aneurysm formation. Genetic studies are also investigating families with congenital heart defects and pulmonary vascular malformations or severe pulmonary hypertension.
Building on our investigations on the role of immunity and BMPR2 dysfunction in PAH, we are collaborating with the lab of Dr. Mark Nicolls to address the hypothesis that injured pulmonary artery EC with dysfunctional BMPR2 signaling recruit and activate macrophages (MØs) that amplify leukotrienes (LTB4) and endogenous retroviral elements of the HERV-K family, and that these immune factors work in concert to sustain inflammation and promote severe PAH by apoptosis and endothelial to mesenchymal transition (EndMT). We will evaluates how LTB4 may be autonomously produced by injured PAECs, which in turn activate MØs to stimulate further LTB4 biosynthesis to cause PAEC apoptosis and EndMT when BMPR2 signaling is impaired. We then plan to evaluate whether PAECs from PAH patients including those with reduced BMPR2 function, secrete factors in response to oxidant or inflammatory injury, that amplify HERV-K expression in monocytes. In rat and mouse models, we will investigate the vulnerability of BMPR2-deficient animals to develop severe PH and EndMT following a disease-inducing stimulus including LTB4 and HERV-K dUTPase.
Our program is a member of the Pulmonary Hypertension Breakthrough Initiative (PHBI), funded by the NIH and the Cardiovascular Medical Research and Education Fund (CMREF). The network accrues specimens of PAH patients and healthy controls (unused donor lungs), which are highly integrated with pathologic, genetic, and genomic subphenotypes pertaining to lung and blood specimens. The explanted lungs are used for the isolation of pulmonary cells and the banking of fixed and frozen tissue sections. Since its establishment in 2006, the PHBI successfully developed a novel and unique infrastructure, whose success relied on the active participation of a highly integrated network of university-based sites with extensive expertise in each of the spheres of competency: excellence in clinical care of PAH (including patient accruals), lung transplantation, pathology, genetics, genomics, and cell isolation. The Stanford Transplant Preparation Center, led by Dr. Rabinovitch and co-directed by Dr. Zamanian, is charged with the harvest and preparation of the explanted lungs for processing in accordance with the Network’s protocols, in such a manner that it will be useful to investigators for the broadest spectrum of studies.
Bland Research Overview
The Bland lab research program focuses on lung growth and development, and the adverse impact of prolonged mechanical ventilation on the incompletely formed lung, which in very premature infants often leads to a life-threatening condition that was first described as bronchopulmonary dysplasia (Northway WH Jr et al, New Engl J Med 276: 357-368, 1967). This form of neonatal chronic lung disease is the leading cause of long-term hospitalization and recurrent respiratory disorders seen in tiny infants who have been born at less than 28 weeks of gestation. Failed alveolar formation and disordered lung elastin are prominent histological features of this disease, which in some ways resembles adult emphysema. We study the effects of mechanical ventilation, with either air or 40% oxygen, on genes and proteins that regulate lung growth and development in newborn mice, whose alveoli and pulmonary capillaries form mainly after birth at term gestation.
As elastin plays a crucial role in lung growth and development (elastin-null mice die soon after birth from cardiorespiratory failure related to defective alveolar and lung vascular formation), our group has a special interest in studying the effects of prolonged mechanical ventilation (cyclic lung stretch) with oxygen-rich gas (which is often needed to sustain life of extremely premature infants) on genes that regulate elastin synthesis and assembly, which in turn can affect lung septation and angiogenesis. As mechanical ventilation of developing lungs can trigger release of proteolytic enzymes that break down elastin, we recently completed studies showing that intrapulmonary treatment of newborn mice with elafin, a potent inhibitor of serine elastase activity, prevented the adverse effects of mechanical ventilation on lung growth. In addition, we recently discovered that intrapulmonary treatment of newborn mice with a neutralizing antibody to TGFß preserved VEGF signaling, prevented apoptosis and promoted lung growth during mechanical ventilation. These studies provide strong rationale for developing novel strategies to treat or prevent neonatal chronic lung disease, and perhaps other respiratory disorders that exhibit similar pathological features in older children and adults.