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 as well as children. 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 video microscopy and cultured cells under flow conditions), genetically modified mouse models of human disease, human tissue samples from lung and blood and induced pluripotent stem cells (iPSCs) differentiated to vascular cells to learn how we can activate molecular programs to regenerate lost microvessels and to reverse the obliterative changes in the pulmonary circulation. Over the past decade our research has led to two novel compounds in clinical trial: A Phase 1 trial with the elastase inhibitor human recombinant elafin was completed, and planning is underway for a Phase 2 study in PAH patients; and low dose FK-506 treatment is approved for a Phase 2 Clinical Trial. Our studies encompass other monogenic disorders where obstruction occurs in pulmonary arteries (Alagille, Williams syndromes) and has implications for systemic vascular diseases as well.
Elastase, Elafin, Chronic Inflammation and Altered Immunity: We build on previous studies in the Rabinovitch Laboratory that 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, and 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. We are currently investigating whether a reduction in BMPR2 increases a long non-coding (lnc) RNA on the X chromosome, Xist, inactivating the X chromosome. Xist and HERV-K share a similar SPEN binding site, and we speculate that increased Xist levels would result in reduced availability of SPEN to bind and repress HERV-K, leading to a pro-inflammatory monocyte.
We found that neutrophils from PAH patients when compared to those from healthy donors as controls, show profound alterations in adhesion, migration, trans-endothelial migration, 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 an interferon response attributed to heightened expression of the HERV-K envelope, and the consequence of HERV-K mediated sequelae were explored in an animal model of pulmonary hypertension. We are now developing an assay to determine the efficacy of a recombinant endogenous neutrophil elastase (NE) inhibitor, elafin, including the determination of NE activity in the plasma and neutrophils of PAH patients, and if this activity can serve as a plasma biomarker for elafin. Further studies explore the mechanism of elastase mediated NET formation and the proteins with which elafin interacts to prevent NET formation. We also investigate 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. Single cell mass cytometry (CYTOF) analyses (with the Nolan/Fantl labs) are used to study the abnormal activation of immune cells in blood from PAH patients, and multiplex ion based imaging (MIBI) analyses (with the Angelo Lab) is used to localized these cells to the occluded pulmonary arteries in the lung tissue from PAH patients.
Genetics, Epigenetics and Metabolism in Pulmonary Arterial Hypertension: We continue to use induced pluripotent stem cells (iPSCs) -derived endothelial and smooth muscle cells and now inflammatory cells from patients with pulmonary hypertension and other monogenic vascular disorders combined with a variety of ‘omics’ technologies as surrogates for native pulmonary arterial and other vascular cells and inflammatory cells, to elucidate modifying factors that impact penetrance of a mutation in causing PAH. In SMC, EC and iPSCs, 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 underlie a host of mutations, 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. (see below). We have embarked on the characterization of extracellular vesicles from genetically-edited autologous cells that can restore normal vascular function. Studies are underway investigating mutations in TBX4, the second most common genetic cause of pediatric PAH; the role of aldehyde dehydrogenase ALDH3A1 and SOX17 as flow-responsive factors that regulate gene accessibility and regulation; and the role of FOXF1, a transcription factor associated with DNA repair and angiogenesis in the pathogenesis of PHA.
The role of biomechanical forces in the normal modulation of endothelial function and plasticity is investigated using flow systems to study the impact of laminar flow, disturbed flow and high shear on endothelial cell chromatin structure, transcription factor accessibility and gene regulation. ATAC-Seq and RNA-Seq analyses show pronounced changes in chromatin accessibility and gene expression when PAEC are exposed to shear stress, and HiChIP and the ABC method (with the Engreitz lab) reveal important enhancer promoter interactions that impact gene expression. We study the pathological effects of high shear stress levels, such as observed in children predisposed to PAH associated with congenital heart disease or in small vessels that are occluded in idiopathic PAH. 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. Companion studies use a mouse model of HSS induced by an aortocaval shunt to evaluate agents that can treat or reverse disease. 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. In collaboration with the Skylar-Scott Lab, we are 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 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 a perfusion system, different shear stress and strain of vessels can be tested by controlling flow rate and internal pressure, respectively. By using this platform, we can study gene expression changes and extracellular matrix remodeling in vessels according to the diverse physiological conditions.
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. 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 (SMC) and fibroblasts. Abnormalities in elastic fiber assembly underlie both pulmonary arterial hypertension as well as systemic vascular diseases including aneurysm formation. We are investigating the role of BMPR2 in SMC of the vessel wall, and have shown that mice with SMC deletion of Bmpr2 have a lethal congenital heart defect. When we delete Bmpr2 only in the SMC of pulmonary arteries, we find the SMC are hyper-proliferative, resistant to apoptosis, and less contractile. We related these changes to a pAkt-mediated increase in β-Catenin and cMyc. The same observation was made in complementary studies of SMC isolated from lungs of PAH patients with a BMPR2 mutation. We are applying novel genetic technologies such as in vivo perturb seq to understand at a single cell level, how mutations linked to PAH converge of similar pathways of gene dysregulation. Genetic studies are also investigating families with congenital heart defects and pulmonary vascular malformations or severe pulmonary hypertension. For example, we use induced pluripotent stem cells (iPSCs) to uncover the mechanism of arterial and venous dysfunction in hypoplastic left heart Syndrome (HLHS), 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.
Our research program is funded by the NIH and private foundations; please see our funded projects under https://med.stanford.edu/rabinovitchbland/research/funding-sources.html
Research Contribution from the Bland Lab at Stanford
The Bland lab focused 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 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.