The overarching goal of this grant is to investigate the mechanisms underlying pathologic pulmonary arterial smooth muscle cell (PASMC) proliferation and migration in pulmonary arterial hypertension (PAH). Specifically, we are investigating a novel signaling pathway underlying PASMC dysfunction and pulmonary vascular remodeling in PAH, a disease which continues to cause significant morbidity and mortality owing to the lack of therapies designed to reverse vascular remodeling. Our data suggests that, in PAH, increased calpain activity in PASMCs leads to activation of Ca2+/calmodulin dependent protein kinase II (CaMKII), which then induces increased activity of the Na+/H+ exchanger (NHE), resulting in pathologically elevated PASMC proliferation and migration and pulmonary vascular remodeling. We plan to investigate this pathway using PASMCs isolated from PAH patients as well as the Sugen/hypoxia rodent model of PAH. Through this work, we hope to uncover novel mechanisms underlying pathologic PASMC function in PAH which could hopefully be translated into new therapeutic targets.

​​​​​​​Team: John Huetsch, Larissa Shimoda
Funding: NHLBI K08HL133475 (Huetsch)
Links: https://projectreporter.nih.gov/project_info_description.cfm?aid=9538254&icde=42474874&ddparam=&ddvalue=&ddsub=&cr=1&csb=default&cs=ASC&pball=

Pulmonary arterial hypertension (PAH) in children or adults is a progressive and fatal disease characterized by sustained elevations of pulmonary artery pressure of unknown etiology. We lack simple, minimally invasive more lung/vascular specific, objective, repeatable, generalizable and less expensive measures of PAH to improve outcomes. We now have pilot data that the pulmonary angiogenic protein, hepatoma derived growth factor (HDGF), is a significant circulating predictor of PAH severity and survival. The overall goal of this proposal is to elucidate the in vitro and in vivo mechanistic role of HDGF in PAH and potential as a circulating new measure of PAH therapeutic response and survival. The significance of the proposed studies is that by linking HDGF to pulmonary endothelial/smooth muscle cellular proliferation, patient survival, and response to treatment, a critical step in the function of HDGF is revealed and provides the basis for new therapeutic, diagnostic and prognostic strategies in PAH. HDGF could fill an important gap in the current clinical care and treatment of PAH patients for improved risk stratification, assessment of response to therapy, clinical worsening exceeding current clinical care measures.

Team: Allen Everett, Rachel Damico
Funding: NHLBI R01HL135114 (Everett)
Links: https://projectreporter.nih.gov/project_info_description.cfm?aid=9395937&icde=39401413&ddparam=&ddvalue=&ddsub=&cr=2&csb=default&cs=ASC&pball=

This study aims to assess the impact of dimethyl fumarate, an immunomodulatory therapy, in patients with pulmonary arterial hypertension related to scleroderma. Dimethyl fumarate is an FDA-approved medication that is used to treat psoriasis and multiple sclerosis. The medication has multiple effects on the immune system that could be particularly relevant in scleroderma.

Clinical Trials Number: NCT02981082

This study aims to assess the impact of initial combination therapy with macitentan, tadalafil, and selexipag to the combination of macitentan and tadalafil alone in patients with newly-diagnosed pulmonary arterial hypertension who have not been treated with other medications. All of the medications in this study are FDA-approved to treat pulmonary arterial hypertension.

Clinical Trial Number: NCT02558231

It is well established that physiologic, imaging, and circulating biomarkers of right ventricular (RV) dysfunction predict adverse outcomes in pulmonary arterial hypertension (PAH), yet our understanding of the genetic, molecular, and cellular determinants of RV failure is nascent. In this context, our global objectives are to identify and characterize novel molecular determinants and clinical biomarkers of RV function and failure and define both their contribution to disease phenotype and their potential for exploitation as therapeutic targets. We have demonstrated that endostatin (ES), a potent inhibitor of angiogenesis (i.e., angiostatic factor), is elevated in PAH, associated with adverse hemodynamic and functional status, and, importantly, with increased mortality in PAH. Further, we have identified multiple genetic variants in the gene encoding ES (Col18a1) that are associated with potential altered protein function, protein expression, cardiac function, and outcomes in PAH. The objectives of the grant are to further define the relationship between ES and cardiopulmonary structure and function in disease, to further define and characterize the molecular and cellular effects of variants in the gene encoding ES (Col18a1), and to provide necessary preclinical data on the impact of ES antagonism as a potential therapeutic target in PAH.

Team: Rachel Damico, Paul Hassoun, Todd Kolb
Funding: NHLBI R01HL132153 (Damico, Hassoun)
Links: https://projectreporter.nih.gov/project_info_description.cfm?aid=9398149&icde=39401184&ddparam=&ddvalue=&ddsub=&cr=1&csb=default&cs=ASC&pball=

Pulmonary hypertension due to any cause portends a poor prognosis. While recent advances in therapies have improved functional capacity and reduced hospitalizations and disease progression, PH remains incurable and imparts a significant burden on patients with this disease. Assessing the patient’s experience with this disease using measures of health-related quality of life (HRQOL) is an important yet understudied area in pulmonary hypertension. In particular, none of the commonly employed HRQOL tools have been fully validated in PH. In study, we will determine the validity of three separate measures of HRQOL in patients with various forms of PH and assess the responsiveness of these measures to medical therapy.

Team: Rubina Khair, Stephen Mathai, Paul Hassoun
Funding: NHLBI U01 HL125175 (supplement) (Khair, Mathai, Hassoun)

Started in 2003, the JHPHP Patient Registry is one of the largest patient registries for patients with PH in the country, with over 3000 participants. This registry includes patients with all forms of pulmonary hypertension and captures demographic, physiologic, hemodynamic, and imaging data. Importantly, measures of patient important outcomes, such as health-related quality of life, depression, and anxiety are also collected as part of the registry.

Team: Stephen Mathai, Rachel Damico, Todd Kolb, Paul Hassoun
Funding: Johns Hopkins Pulmonary Hypertension Program

The overall objective of this R01 proposal is to investigate mechanisms that direct the immune system to resolve severe acute lung inflammation. Despite thorough investigation into the injury and inflammation that drive ARDS, no targeted therapies promote its resolution. Regulatory T cells (Tregs) — a subset of CD4+ lymphocytes that suppress exuberant immune system activation—resolve inflammation in mouse models of lung injury. However, the mechanisms that promote Treg function following lung injury remain unknown. Preliminary data identified in Tregs the Lgp2 locus, which encodes the immune regulatory protein LGP2, as a novel site that augments Treg responses to inflammation. DNA methylation at this site is dynamic and contributes to repression of the Dhx58 locus following lung injury. Thus, the hypothesis of Treg DNA hypomethylation at the Lgp2 locus will increase LGP2 protein levels, enhance Treg pro-repair function, and promote resolution of acute lung inflammation. To specifically test our hypothesis we will employ transgenic mice including strains with Dhx58 deficiency as well as Treg-specific Uhrf1 deficiency. Accomplishment of this proposal will uncover mechanisms controlling Treg function during resolution of acute lung injury that could be translated for therapeutic benefit in ARDS.

Team: Franco D’Alessio, Rachel Damico
Funding: NHLBI R01HL131812 (D’Alessio)
Links: https://projectreporter.nih.gov/project_info_description.cfm?aid=9309225&icde=39401594&ddparam=&ddvalue=&ddsub=&cr=1&csb=default&cs=ASC&pball=

Resistance to apoptosis, or programmed cell death, in both the endothelial and smooth muscle cells (EC and SMC, respectively) is a primary feature of the remodeling process in pulmonary hypertension. Although studies in pulmonary vascular cells from patients and animals with pulmonary hypertension describe significant abnormalities that would normally induce apoptosis, these cells are resistant to both endogenous and exogenous apoptotic stimuli. Based on new preliminary data, our work tests the hypothesis that SMC and EC survival during PH is promoted by increased Na+/H+ exchange isoform 1 (NHE1) activity, which limits expression of the endoplasmic reticulum stress signal, CHOP, and prevents activation of caspases, the final executioners of apoptosis. In this project, we are testing whether inhibition of NHE1 can re-sensitize ECs and PASMCs to endogenous apoptotic signals to specifically target abnormal cells and de-remodel the pulmonary vasculature in pulmonary hypertension.

Team: Larissa Shimoda, Mahendra Damarla, John Huetsch
Funding: NHLBI R01HL073859 (Shimoda)
Links: https://projectreporter.nih.gov/project_info_description.cfm?aid=9503750&icde=42587761&ddparam=&ddvalue=&ddsub=&cr=1&csb=default&cs=ASC&MMOpt=

This study aims to assess the impact of anastrozole, an oral medication that is FDA-approved to treat breast cancer, on symptoms and walking ability in patients with pulmonary arterial hypertension. Anastrozole inhibits the conversion of other hormones to estrogen; high estrogen levels have been implicated in the development of pulmonary arterial hypertension and may explain the higher incidence of this disease in women.

Clinical Trial Number: NCT03229499

This multi-center registry is supported by the Pulmonary Hypertension Association and aims to collect demographic, physiologic, hemodynamic, and quality of life data on patients with newly diagnosed pulmonary arterial hypertension or chronic thromboembolic pulmonary arterial hypertension.

Team: Stephen Mathai, Rachel Damico, Todd Kolb, Paul Hassoun
Funding: none

This multi-center registry is supported by the Pulmonary Hypertension Association and aims to collect demographic, physiologic, hemodynamic, and quality of life data on patients with newly diagnosed pulmonary arterial hypertension or chronic thromboembolic pulmonary arterial hypertension.

Team: Stephen Mathai, Rachel Damico, Todd Kolb, Paul Hassoun
Funding: none

Various forms of heart and lung disease lead to pulmonary vascular disease that ultimately progresses from pulmonary vascular remodeling to cause pulmonary hypertension and right ventricular dysfunction. The genetic, molecular, and cellular processes driving these phenomena are not well understood. Using high throughput -omic methodology, bioinformatics, and network biology, this project will broadly search for homologies and differences across the spectrum of diseases associated with pulmonary hypertension with the goal of defining new meaningful subclassifications of pulmonary vascular disease.

Team: Paul Hassoun, Stephen Mathai, Todd Kolb, Rachel Damico
Funding: NHLBI U01 HL125175 (Hassoun, Mathai)
Links: NCT02980887

This study aims to assess the impact of rituximab, an immunomodulatory therapy, on pulmonary arterial hypertension in patients with scleroderma. Scleroderma is an autoimmune disease characterized by overproduction of collagen that leads to fibrosis of many tissues in the body. This disease can affect the heart and lungs and lead to pulmonary arterial hypertension. Rituximab, an FDA-approved drug that is used to treat many types of cancers, may reduce collagen production through disruption of B cells and is thus being studied in patients with scleroderma.

Clinical Trials Number: NCT01086540

Morphometric studies revealed that the development of pulmonary hypertension is associated with structural remodeling of the small pulmonary arteries, characterized by thickening of the smooth muscle cell layer and extension of new muscle around previously non-muscular precapillary arterioles. While the former is thought to be due to pulmonary arterial smooth muscle cell (PASMC) hypertrophy, hyperplasia and resistance to apoptosis, the latter is believed to result from PASMC migration. Our laboratory identified a new candidate as a regulator of PASMC migration, proliferation and survival: aquaporins (AQPs). AQPs are a family of proteins that form transmembrane channels that facilitate the transport of water into and out of cells. We have evidence that aquaporin 1 (AQP1), the first family member identified, is expressed in PASMCs, induced by hypoxia and required for PASMC migration and proliferation. Moreover, we have generated exciting data indicating a critical role for the AQP1 C-terminal tail, but not water transport, in controlling these cellular processes. Our preliminary data also indicate that increased AQP1 protein is associated with elevated b-catenin expression, a protein that regulates migratory, proliferative and survival responses in PASMCs. How AQP1 regulates b-catenin levels is unknown, but we show that the AQP1 cytoplasmic tail is required. This project involves identifying the mechanisms by which AQP1 is upregulated in vascular cells during pulmonary hypertension and how AQP1 modulates b-catenin expression to control vascular remodeling.

Team: Larissa Shimoda, Mahendra Damarla, Karthik Suresh
Funding: NHLBI R01HL126514 (Shimoda)
Links: https://projectreporter.nih.gov/project_info_description.cfm?aid=9392925&icde=42587761&ddparam=&ddvalue=&ddsub=&cr=3&csb=default&cs=ASC&pball=

The objectives of this grant are to investigate mechanisms underlying abnormal endothelial migration/proliferation in pulmonary arterial hypertension (PAH). PAH is a lethal disease
characterized by abnormal migration and proliferation of endothelial cells (ECs) in the distal blood vessels of the lung. There are currently no therapies that target the underlying endothelial dysfunction in PAH. Reactive oxygen species (ROS) and intracellular calcium (Ca2+) are important mediators of migration and proliferation in ECs and both are known to be elevated in PAH, but the mechanisms that link ROS and Ca2+ influx to the transformation of normal ECs to the abnormal phenotype seen in PAH is unknown. Our prior published work and preliminary data in ECs isolated from humans with PAH (hPAH-ECs) and rats undergoing Sugen/Hypoxia (SuHx), an experimental form of PAH (rPAH-ECs), suggest that in ECs: 1) elevations in ROS increase [Ca2+]i by activating the calcium channel TRPV4; 2) regulation of TRPV4 phosphorylation by the Src kinase Fyn tethered to the cell membrane by its anchor, CD36, is critical for activation of TRPV4; 3) baseline ROS levels, cytosolic Ca2+, migration and proliferation are elevated in rPAH- and hPAH-ECs, and attenuated by quenching of ROS or inhibition of TRPV4; 4) rPAH-ECs exhibit evidence of mitochondrial dysfunction that may represent the source of ROS elevations in PAH; and 5) loss of CD36 or Fyn attenuates development of PAH in a murine SuHx model. Thus, we hypothesize that phosphorylation of TRPV4 by CD36-tethered Fyn is required for activation of this channel by elevated cytosolic ROS that occur due to mitochondrial dysfunction, promoting EC migration and proliferation. Using rat and human PAH-ECs (and normoxic controls) we will study the role of mtROS-induced TRPV4 activation in promoting MVEC dysfunction in vitro and PAH in vivo.

Team: Karthik Suresh and Larissa Shimoda
Funding: NHLBI K08HL132055 (Suresh)
Links: https://projectreporter.nih.gov/project_info_description.cfm?aid=9526537&icde=42563610&ddparam=&ddvalue=&ddsub=&cr=1&csb=default&cs=ASC&pball=

This study aims to assess the impact of inhaled treprostinil, an FDA-approved therapy for pulmonary arterial hypertension, in patients with pulmonary hypertension related to interstitial lung disease.

Clinical Trial Number: NCT02630316