Novel approaches to pulmonary arterial hypertension drug discovery.
Expert opinion on drug discovery
2016; 11 (4): 407-414
Loss of PPAR? in endothelial cells leads to impaired angiogenesis.
Journal of cell science
2016; 129 (4): 693-705
Introduction Pulmonary arterial hypertension (PAH) is a rare disorder associated with abnormally elevated pulmonary pressures that, if untreated, leads to right heart failure and premature death. The goal of drug development for PAH is to develop effective therapies that halt, or ideally, reverse the obliterative vasculopathy that results in vessel loss and obstruction of blood flow to the lungs. Areas Covered This review summarizes the current approach to candidate discovery in PAH and discusses the currently available drug discovery methods that should be implemented to prioritize targets and obtain a comprehensive pharmacological profile of promising compounds with well-defined mechanisms. Expert opinion To improve the successful identification of leading drug candidates, it is necessary that traditional pre-clinical studies are combined with drug screening strategies that maximize the characterization of biological activity and identify relevant off-target effects that could hinder the clinical efficacy of the compound when tested in human subjects. A successful drug discovery strategy in PAH will require collaboration of clinician scientists with medicinal chemists and pharmacologists who can identify compounds with an adequate safety profile and biological activity against relevant disease mechanisms.
View details for DOI 10.1517/17460441.2016.1153625
View details for PubMedID 26901465
Cyclosporine Does Not Prevent Microvascular Loss in Transplantation but Can Synergize With a Neutrophil Elastase Inhibitor, Elafin, to Maintain Graft Perfusion During Acute Rejection
AMERICAN JOURNAL OF TRANSPLANTATION
2015; 15 (7): 1768-1781
Tie2 promoter-mediated loss of peroxisome proliferator-activated receptor gamma (PPARγ) in mice leads to osteopetrosis and pulmonary arterial hypertension. Vascular disease is associated with loss of PPARγ in pulmonary microvascular endothelial cells (PMVEC), we evaluated the role of PPARγ in PMVEC functions, such as angiogenesis and migration. The role of PPARγ in angiogenesis was evaluated in Tie2CrePPARγ(flox/flox) and wild type (WT) mice, and in mouse and human PMVECs. RNA-sequencing and bioinformatic approaches were utilized to reveal angiogenesis-associated targets for PPARγ. Tie2CrePPARγ(flox/flox) mice showed an impaired angiogenic capacity. Analysis of endothelial progenitor-like cells using bone marrow transplantation combined with evaluation of isolated PMVECs revealed that loss of PPARγ attenuates the migration and angiogenic capacity of mature PMVECs. PPARγ-deficient human PMVECs showed a similar migration defect in culture. Bioinformatic and experimental analyses revealed E2F1 as a novel target of PPARγ in the regulation of PMVEC migration. Disruption of the PPARγ-E2F1 axis was associated with a dysregulated Wnt pathway related to the GSK3β interaction protein. In conclusion, PPARγ plays an important role in sustaining angiogenic potential in mature PMVECs through E2F1-mediated gene regulation.
View details for DOI 10.1242/jcs.169011
View details for PubMedID 26743080
Perlecan heparan sulfate deficiency impairs pulmonary vascular development and attenuates hypoxic pulmonary hypertension.
2015; 107 (1): 20-31
The loss of a functional microvascular bed in rejecting solid organ transplants is correlated with fibrotic remodeling and chronic rejection; in lung allografts, this pathology is predicted by bronchoalveolar fluid neutrophilia which suggests a role for polymorphonuclear cells in microcirculatory injury. In a mouse orthotopic tracheal transplant model, cyclosporine, which primarily inhibits T cells, failed as a monotherapy for preventing microvessel rejection and graft ischemia. To target neutrophil action that may be contributing to vascular injury, we examined the effect of a neutrophil elastase inhibitor, elafin, on the microvascular health of transplant tissue. We showed that elafin monotherapy prolonged microvascular perfusion and enhanced tissue oxygenation while diminishing the infiltration of neutrophils and macrophages and decreasing tissue deposition of complement C3 and the membrane attack complex, C5b-9. Elafin was also found to promote angiogenesis through activation of the extracellular signal-regulated kinase (ERK) signaling pathway but was insufficient as a single agent to completely prevent tissue ischemia during acute rejection episodes. However, when combined with cyclosporine, elafin effectively preserved airway microvascular perfusion and oxygenation. The therapeutic strategy of targeting neutrophil elastase activity alongside standard immunosuppression during acute rejection episodes may be an effective approach for preventing the development of irreversible fibrotic remodeling.
View details for DOI 10.1111/ajt.13189
View details for Web of Science ID 000356494300013
View details for PubMedID 25727073
Activation of the Wnt/Planar Cell Polarity Pathway Is Required for Pericyte Recruitment during Pulmonary Angiogenesis.
American journal of pathology
2015; 185 (1): 69-84
Excessive vascular cell proliferation is an important component of pulmonary hypertension (PH). Perlecan is the major heparan sulfate (HS) proteoglycan in the vascular extracellular matrix. It binds growth factors, including FGF2, and either restricts or promotes cell proliferation. In this study, we have explored the effects of perlecan HS deficiency on pulmonary vascular development and in hypoxia-induced PH.In normoxia, Hspg2(Δ3/Δ3) mice, deficient in perlecan HS, had reduced pericytes and muscularization of intra-acinar vessels. Pulmonary angiography revealed a peripheral perfusion defect. Despite these abnormalities, right ventricular systolic pressure (RVSP) and myocardial mass remained normal. After 4 weeks of hypoxia, increases in the proportion of muscularized vessels, RVSP, and right ventricular hypertrophy were significantly less in Hspg2(Δ3/Δ3) compared with wild type. The early phase of hypoxia induced a significantly lower increase in fibroblast growth factor receptor-1 (FGFR1) protein level and receptor phosphorylation, and reduced pulmonary artery smooth muscle cell (PASMC) proliferation in Hspg2(Δ3/Δ3). At 4 weeks, FGF2 mRNA and protein were also significantly reduced in Hspg2(Δ3/Δ3) lungs. Ligand and carbohydrate engagement assay showed that perlecan HS is required for HS-FGF2-FGFR1 ternary complex formation. In vitro, proliferation assays showed that PASMC proliferation is reduced by selective FGFR1 inhibition. PASMC adhesion to fibronectin was higher in Hspg2(Δ3/Δ3) compared with wild type.Perlecan HS chains are important for normal vascular arborization and recruitment of pericytes to pulmonary vessels. Perlecan HS deficiency also attenuates hypoxia-induced PH, where the underlying mechanisms involve impaired FGF2/FGFR1 interaction, inhibition of PASMC growth, and altered cell-matrix interactions.
View details for DOI 10.1093/cvr/cvv143
View details for PubMedID 25952902
Optical Coherence Tomography of Pulmonary Arterial Walls in Humans and Pigs (Sus scrofa domesticus).
2015; 65 (3): 217-224
Pericytes are perivascular cells localized to capillaries that promote vessel maturation, and their absence can contribute to vessel loss. Whether impaired endothelial-pericyte interaction contributes to small vessel loss in pulmonary arterial hypertension (PAH) is unclear. Using 3G5-specific, immunoglobulin G-coated magnetic beads, we isolated pericytes from the lungs of healthy subjects and PAH patients, followed by lineage validation. PAH pericytes seeded with healthy pulmonary microvascular endothelial cells failed to associate with endothelial tubes, resulting in smaller vascular networks compared to those with healthy pericytes. After the demonstration of abnormal polarization toward endothelium via live-imaging and wound-healing studies, we screened PAH pericytes for abnormalities in the Wnt/planar cell polarity (PCP) pathway, which has been shown to regulate cell motility and polarity in the pulmonary vasculature. PAH pericytes had reduced expression of frizzled 7 (Fzd7) and cdc42, genes crucial for Wnt/PCP activation. With simultaneous knockdown of Fzd7 and cdc42 in healthy pericytes in vitro and in a murine model of angiogenesis, motility and polarization toward pulmonary microvascular endothelial cells were reduced, whereas with restoration of both genes in PAH pericytes, endothelial-pericyte association was improved, with larger vascular networks. These studies suggest that the motility and polarity of pericytes during pulmonary angiogenesis are regulated by Wnt/PCP activation, which can be targeted to prevent vessel loss in PAH.
View details for DOI 10.1016/j.ajpath.2014.09.013
View details for PubMedID 25447046
Oxido-reductive regulation of vascular remodeling by receptor tyrosine kinase ROS1
JOURNAL OF CLINICAL INVESTIGATION
2014; 124 (12): 5159-5174
Pulmonary arterial hypertension (PAH) is a devastating disorder characterized by progressive elevation of the pulmonary pressures that, in the absence of therapy, results in chronic right-heart failure and premature death. The vascular pathology of PAH is characterized by progressive loss of small (diameter, less than 50 μm) peripheral pulmonary arteries along with abnormal medial thickening, neointimal formation, and intraluminal narrowing of the remaining pulmonary arteries. Vascular pathology correlates with disease severity, given that hemodynamic effects and disease outcomes are worse in patients with advanced compared with lower-grade lesions. Novel imaging tools are urgently needed that demonstrate the extent of vascular remodeling in PAH patients during diagnosis and treatment monitoring. Optical coherence tomography (OCT) is a catheter-based intravascular imaging technique used to obtain high-resolution 2D and 3D cross-sectional images of coronary arteries, thus revealing the extent of vascular wall pathology due to diseases such as atherosclerosis and in-stent restenosis; its utility as a diagnostic tool in the assessment of the pulmonary circulation is unknown. Here we show that OCT provides high-definition images that capture the morphology of pulmonary arterial walls in explanted human lungs and during pulmonary arterial catheterization of an adult pig. We conclude that OCT may facilitate the evaluation of patients with PAH by disclosing the degree of wall remodeling present in pulmonary vessels. Future studies are warranted to determine whether this information complements the hemodynamic and functional assessments routinely performed in PAH patients, facilitates treatment selection, and improves estimates of prognosis and outcome.
View details for PubMedID 26141446
View details for PubMedCentralID PMC4485630
Targeting the Wnt signaling pathways in pulmonary arterial hypertension
DRUG DISCOVERY TODAY
2014; 19 (8): 1270-1276
Targeting the Wnt signaling pathways in pulmonary arterial hypertension.
Drug discovery today
2014; 19 (8): 1270-1276
Angioplasty and stenting is the primary treatment for flow-limiting atherosclerosis; however, this strategy is limited by pathological vascular remodeling. Using a systems approach, we identified a role for the network hub gene glutathione peroxidase-1 (GPX1) in pathological remodeling following human blood vessel stenting. Constitutive deletion of Gpx1 in atherosclerotic mice recapitulated this phenotype of increased vascular smooth muscle cell (VSMC) proliferation and plaque formation. In an independent patient cohort, gene variant pair analysis identified an interaction of GPX1 with the orphan protooncogene receptor tyrosine kinase ROS1. A meta-analysis of the only genome-wide association studies of human neointima-induced in-stent stenosis confirmed the association of the ROS1 variant with pathological remodeling. Decreased GPX1 expression in atherosclerotic mice led to reductive stress via a time-dependent increase in glutathione, corresponding to phosphorylation of the ROS1 kinase activation site Y2274. Loss of GPX1 function was associated with both oxidative and reductive stress, the latter driving ROS1 activity via s-glutathiolation of critical residues of the ROS1 tyrosine phosphatase SHP-2. ROS1 inhibition with crizotinib and deglutathiolation of SHP-2 abolished GPX1-mediated increases in VSMC proliferation while leaving endothelialization intact. Our results indicate that GPX1-dependent alterations in oxido-reductive stress promote ROS1 activation and mediate vascular remodeling.
View details for DOI 10.1172/JCI77484
View details for Web of Science ID 000345677200011
View details for PubMedID 25401476
Loss of Bone Morphogenetic Protein Receptor 2 Is Associated with Abnormal DNA Repair in Pulmonary Arterial Hypertension
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY
2014; 50 (6): 1118-1128
Whole-Exome Sequencing Reveals TopBP1 as a Novel Gene in Idiopathic Pulmonary Arterial Hypertension
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
2014; 189 (10): 1260-1272
Pulmonary arterial hypertension (PAH) is a life-threatening disorder that is associated with elevated pulmonary pressures and right heart failure resulting from progressive loss and thickening of small pulmonary arteries. Despite their ability to improve symptoms, current therapies fail to prevent disease progression, leaving lung transplantation as the only therapy in end-stage PAH. To overcome the limitations of current therapies, there is an active search for disease-modifying agents capable of altering the natural history of, and improving clinical outcomes in, PAH. The Wnt signaling pathways have emerged as attractive treatment targets in PAH given their role in the preservation of pulmonary vascular homeostasis and the recent development of Wnt-specific compounds and biological therapies capable of modulating pathway activity. In this review, we summarize the literature describing the role of Wnt signaling in the pulmonary circulation and discuss promising advances in the field of Wnt therapeutics that could lead to novel clinical therapies capable of preventing and/or reversing pulmonary vascular pathology in patients with this devastating disease.
View details for DOI 10.1016/j.drudis.2014.06.014
View details for PubMedID 24955837
Leukotrienes in pulmonary arterial hypertension.
2014; 58 (2-3): 387-393
Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disorder characterized by progressive loss of pulmonary microvessels. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable and ∼15% of patients with IPAH, their low penetrance (∼20%) suggests that other unidentified genetic modifiers are required for manifestation of the disease phenotype. Use of whole-exome sequencing (WES) has recently led to the discovery of novel susceptibility genes in heritable PAH, but whether WES can also accelerate gene discovery in IPAH remains unknown.To determine whether WES can help identify novel gene modifiers in patients with IPAH.Exome capture and sequencing was performed on genomic DNA isolated from 12 unrelated patients with IPAH lacking BMPR2 mutations. Observed genetic variants were prioritized according to their pathogenic potential using ANNOVAR.A total of nine genes were identified as high-priority candidates. Our top hit was topoisomerase DNA binding II binding protein 1 (TopBP1), a gene involved in the response to DNA damage and replication stress. We found that TopBP1 expression was reduced in vascular lesions and pulmonary endothelial cells isolated from patients with IPAH. Although TopBP1 deficiency made endothelial cells susceptible to DNA damage and apoptosis in response to hydroxyurea, its restoration resulted in less DNA damage and improved cell survival.WES led to the discovery of TopBP1, a gene whose deficiency may increase susceptibility to small vessel loss in IPAH. We predict that use of WES will help identify gene modifiers that influence an individual's risk of developing IPAH.
View details for DOI 10.1164/rccm.201310-17490C
View details for Web of Science ID 000336017200018
View details for PubMedID 24702692
View details for PubMedCentralID PMC4225850
Perioperative pharmacological management of pulmonary hypertensive crisis during congenital heart surgery.
2014; 4 (1): 10-24
Leukotrienes (LTs) are lipid mediators derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism and are markers and mediators of pulmonary inflammation. Research over the past two decades has established that LTs modulate inflammation in pulmonary arterial hypertension (PAH). The purpose of this review was to summarize the current knowledge of LTs in the pathophysiology of PAH and to highlight a recent study that advances our understanding of how leukotriene B4 (LTB4) specifically contributes to pulmonary vascular remodeling. The results of these studies suggest that pharmacological inhibition of LT pathways, especially LTB4, has high potential for the treatment of PAH.
View details for DOI 10.1007/s12026-014-8492-5
View details for PubMedID 24570092
Tie2-dependent VHL knockdown promotes airway microvascular regeneration and attenuates invasive growth of Aspergillus fumigatus
JOURNAL OF MOLECULAR MEDICINE-JMM
2013; 91 (9): 1081-1093
Pulmonary hypertensive crisis is an important cause of morbidity and mortality in patients with pulmonary arterial hypertension secondary to congenital heart disease (PAH-CHD) who require cardiac surgery. At present, prevention and management of perioperative pulmonary hypertensive crisis is aimed at optimizing cardiopulmonary interactions by targeting prostacyclin, endothelin, and nitric oxide signaling pathways within the pulmonary circulation with various pharmacological agents. This review is aimed at familiarizing the practitioner with the current pharmacological treatment for dealing with perioperative pulmonary hypertensive crisis in PAH-CHD patients. Given the life-threatening complications associated with pulmonary hypertensive crisis, proper perioperative planning can help anticipate cardiopulmonary complications and optimize surgical outcomes in this patient population.
View details for DOI 10.1086/674885
View details for PubMedID 25006417
MiR-133a Modulates Osteogenic Differentiation of Vascular Smooth Muscle Cells
2013; 154 (9): 3344-3352
MicroRNAs: promising therapeutic targets for the treatment of pulmonary arterial hypertension.
Expert opinion on therapeutic targets
2013; 17 (5): 557-564
Microvascular ischemia and infections are associated with the development of chronic rejection following lung transplantation. The von Hippel-Lindau protein (VHL) controls protein levels of hypoxia-inducible factors (HIFs), regulates vascular repair, and improves tissue perfusion. Here, we studied the role of VHL in microvascular repair by orthotopically transplanting tracheas into mice with VHL haplodeficiency in Tie2 lineage cells. We showed that VHL haplodeficiency prolonged airway microvascular perfusion and promoted tissue blood flow through the production of the angiogenic factors, SDF-1 and angiopoietin 1. VHL-haplodeficient pulmonary endothelial cells exhibited increased angiogenic activity, resistance to serum deprivation-induced cell death, and enhanced microvascular repair. By contrast, in recipient mice with HIF-1α deficiency in Tie2 lineage cells, microvascular repair was significantly diminished and suggested that recipient-derived HIF-1α normally participates in the repair of alloimmune-mediated microvascular damage. To evaluate the translational impact of our findings, we compared VHL-haplodeficient mice with wild-type controls using a model of Aspergillus airway infection. In 83 % of the VHL-haplodeficient recipients, Aspergillus fumigatus was noninvasive in contrast to 75 % of wild-type mice in which the mold was deeply invasive. Our study demonstrated that stabilization of HIF-1α in angiogenic cells, through Tie2 cell VHL haplodeficiency, promoted airway microvascular regeneration and vascular normalization and thereby minimized tissue ischemia and hypoxia. By also mitigating the virulence of A. fumigatus, a common pathogen and itself a risk factor for the development of lung transplant rejection, the selective enhancement of HIF-1α expression has the prospect of offering several novel therapeutic effects to transplant recipients.Microvascular loss and prolonged ischemia occurs with acute rejection. Von Hippel-Lindau (VHL) protein controls hypoxia inducible factors (HIFs). In tracheal allografts, VHL haplodeficient Tie2 cells promote neovascularization. Reduced transplant ischemia limits Aspergillus invasion.
View details for DOI 10.1007/s00109-013-1063-8
View details for Web of Science ID 000324068100007
Loss of adenomatous poliposis coli-a3 integrin interaction promotes endothelial apoptosis in mice and humans.
2012; 111 (12): 1551-1564
MicroRNAs (miRNAs) are small noncoding RNAs that not only regulate gene expression during normal development but can also be active players in several diseases. To date, several studies have demonstrated a possible role for specific miRNAs in the regulation of pulmonary vascular homeostasis suggesting that novel therapeutic agents which target these modulators of gene expression could serve to treat pulmonary arterial hypertension (PAH). AREAS COVERED: The characterization of miRNA-mediated gene modulation in the pulmonary circulation is expanding very rapidly. This review summarizes current relevant findings on the role of miRNAs in the pathogenesis of PAH and expands on the potential use of agents that target these molecules as future disease-modifying therapies. EXPERT OPINION: Further understanding of miRNA biology and function in the pulmonary circulation will serve to further enhance our understanding of their contribution to the pathogenesis of PAH. The implementation of a systems biology approach will help accelerate the discovery of miRNAs that influence angiogenesis and cellular responses to vascular injury. Experimental characterization of these miRNAs using in vitro and in vivo methods will be required to validate the biological roles of these miRNAs prior to the consideration of their use as therapeutic targets in future clinical trials.
View details for DOI 10.1517/14728222.2013.765863
View details for PubMedID 23379818
Loss of Adenomatous Poliposis Coli-alpha 3 Integrin Interaction Promotes Endothelial Apoptosis in Mice and Humans
2012; 111 (12): 1551-?
Role of miR-148a in Hepatitis B Associated Hepatocellular Carcinoma
2012; 7 (4)
Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins promote pulmonary angiogenesis by recruiting the Wnt/β-catenin pathway, we proposed that β-catenin activation could reduce loss and induce regeneration of small pulmonary arteries (PAs) and attenuate PH.This study aims to establish the role of β-catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis after injury.To assess the impact of β-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (Apc(Min/+)) mouse, where reduced APC causes constitutive β-catenin elevation. Surprisingly, hypoxic Apc(Min/+) mice displayed greater PH and small PA loss compared with control C57Bl6J littermates. PA endothelial cells isolated from Apc(Min/+) demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase-1 and phospho Akt. We found that PA endothelial cells from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin, and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC.We show that APC is integral to PA endothelial cells adhesion and survival and is reduced in PA endothelial cells from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.
View details for DOI 10.1161/CIRCRESAHA.112.267849
View details for PubMedID 23011394
View details for PubMedCentralID PMC3821702
Hepatitis B virus encoded X antigen (HBx) is a trans-regulatory protein that alters the activity of selected transcription factors and cytoplasmic signal transduction pathways. HBx transcriptionally up-regulates the expression of a unique gene, URG11, which in turn transcriptionally up-regulates β-catenin, thereby contributing importantly to hepatocarcinogenesis. HBx and URG11 also alter the expression of multiple microRNAs, and by miRNA array analysis, both were shown to promote the expression of miR-148a. Elevated miR-148a was also seen in HBx positive liver samples from infected patients. To study the function of miR-148a, anti-148a was introduced into HepG2 and Hep3B cells stably expressing HBx or stably over-expressing URG11. Anti-miR-148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Introduction of anti-miR-148a increased PTEN protein and mRNA expression, suggesting that PTEN was targeted by miR-148a. Anti-miR-148a failed to suppress PTEN expression when co-transfected with reporter gene mutants in the 3'UTR of PTEN mRNA. Introduction of anti-miR-148a also resulted in depressed Akt signaling by HBx and URG11, resulting in decreased expression of β-catenin. Thus, miR-148a may play a central role in HBx/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type.
View details for DOI 10.1371/journal.pone.0035331
View details for Web of Science ID 000305014500055
View details for PubMedID 22496917