Kylie M. Drake

Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension

Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH.

Altered MicroRNA processing in heritable pulmonary arterial hypertension: an important role for Smad-8.

RATIONALE Heritable pulmonary arterial hypertension (HPAH) is primarily caused by mutations of the bone morphogenetic protein (BMP) type-II receptor (BMPR2). Recent identification of mutations in the downstream mediator Smad-8 (gene, SMAD9) was surprising, because loss of Smad-8 function in canonical BMP signaling is largely compensated by Smad-1 and -5. We therefore hypothesized that noncanonical pathways may play an important role in PAH. OBJECTIVES To determine whether HPAH mutations disrupt noncanonical Smad-mediated microRNA (miR) processing. METHODS Expression of miR-21, miR-27a, and miR-100 was studied in pulmonary artery endothelial (PAEC) and pulmonary artery smooth muscle cells (PASMC) from explant lungs of patients with PAH. MEASUREMENTS AND MAIN RESULTS SMAD9 mutation completely abrogated miR induction, whereas canonical signaling was only reduced by one-third. miR-21 levels actually decreased, suggesting that residual canonical signaling uses up or degrades existing miR-21. BMPR2 mutations also led to loss of miR induction in two of three cases. HPAH cells proliferated faster than other PAH or controls. miR-21 and miR-27a each showed antiproliferative effects in PAEC and PASMC, and PAEC growth rate after BMP treatment correlated strongly with miR-21 fold-change. Overexpression of SMAD9 corrected miR processing and reversed the hyperproliferative phenotype. CONCLUSIONS HPAH-associated mutations engender a primary defect in noncanonical miR processing, whereas canonical BMP signaling is partially maintained. Smad-8 is essential for this miR pathway and its loss was not complemented by Smad-1 and -5; this may represent the first nonredundant role for Smad-8. Induction of miR-21 and miR-27a may be a critical component of BMP-induced growth suppression, loss of which likely contributes to vascular cell proliferation in HPAH.

The lysosomal inhibitor, chloroquine, increases cell surface BMPR-II levels and restores BMP9 signalling in endothelial cells harbouring BMPR-II mutations

Pulmonary arterial hypertension (PAH) is characterized by dysregulated pulmonary artery endothelial cell (PAEC) proliferation, apoptosis and permeability. Loss-of-function mutations in the bone morphogenetic protein receptor type-II (BMPR-II) are the most common cause of heritable PAH, usually resulting in haploinsufficiency. We previously showed that BMPR-II expression is regulated via a lysosomal degradative pathway. Here, we show that the antimalarial drug, chloroquine, markedly increased cell surface expression of BMPR-II protein independent of transcription in PAECs. Inhibition of protein synthesis experiments revealed a rapid turnover of cell surface BMPR-II, which was inhibited by chloroquine treatment. Chloroquine enhanced PAEC expression of BMPR-II following siRNA knockdown of the BMPR-II transcript. Using blood outgrowth endothelial cells (BOECs), we confirmed that signalling in response to the endothelial BMPR-II ligand, BMP9, is compromised in BOECs from patients harbouring BMPR-II mutations, and in BMPR-II mutant PAECs. Chloroquine significantly increased gene expression of BMP9-BMPR-II signalling targets Id1, miR21 and miR27a in both mutant BMPR-II PAECs and BOECs. These findings provide support for the restoration of cell surface BMPR-II with agents such as chloroquine as a potential therapeutic approach for heritable PAH.

Correction of Nonsense BMPR2 and SMAD9 Mutations by Ataluren in Pulmonary Arterial Hypertension

Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.

Increased Mutagen Sensitivity and DNA Damage in Pulmonary Arterial Hypertension

RATIONALE Pulmonary arterial hypertension (PAH) is a serious lung condition characterized by vascular remodeling in the precapillary pulmonary arterioles. We and others have demonstrated chromosomal abnormalities and increased DNA damage in PAH lung vascular cells, but their timing and role in disease pathogenesis is unknown. OBJECTIVES We hypothesized that if DNA damage predates PAH, it might be an intrinsic cell property that is present outside the diseased lung. METHODS We measured DNA damage, mutagen sensitivity, and reactive oxygen species (ROS) in lung and blood cells from patients with Group 1 PAH, their relatives, and unrelated control subjects. MEASUREMENTS AND MAIN RESULTS Baseline DNA damage was significantly elevated in PAH, both in pulmonary artery endothelial cells (P < 0.05) and peripheral blood mononuclear cells (PBMC) (P < 0.001). Remarkably, PBMC from unaffected relatives showed similar increases, indicating this is not related to PAH treatments. ROS levels were also higher (P < 0.01). DNA damage correlated with ROS production and was suppressed by antioxidants (P < 0.001). PBMC from patients and relatives also showed markedly increased sensitivity to two chemotherapeutic drugs, bleomycin and etoposide (P < 0.001). Results were consistent across idiopathic, heritable, and associated PAH groups. CONCLUSIONS Levels of baseline and mutagen-induced DNA damage are intrinsically higher in PAH cells. Similar results in PBMC from unaffected relatives suggest this may be a genetically determined trait that predates disease onset and may act as a risk factor contributing to lung vascular remodeling following endothelial cell injury. Further studies are required to fully characterize mutagen sensitivity, which could have important implications for clinical management.

Endothelial Chromosome 13 Deletion in Congenital Heart Disease–associated Pulmonary Arterial Hypertension Dysregulates SMAD9 Signaling

Supported in part by the National Heart, Lung and Blood Institute of the National Institutes of Health under award numbers R01HL098199, R03HL110831, and RC37HL60917. Author Contributions: K.M.D. and M.A.A. designed the study; acquired, analyzed, and interpreted data; and wrote the manuscript. S.A.C. and S.C.E. acquired samples, established cells, and reviewed the clinical information. R.M.T. reviewed and interpreted the pathology. All authors contributed to writing the manuscript. This letter has an online supplement, which is accessible from this issues table of contents at www.atsjournals.org Author disclosures are available with the text of this letter at www.atsjournals.org.

Feasibility of mesenchymal stem cell culture expansion for a phase I clinical trial in multiple sclerosis

Background Multiple sclerosis is an inflammatory, neurodegenerative disease of the central nervous system for which therapeutic mesenchymal stem cell transplantation is under study. Published experience of culture-expanding multiple sclerosis patients’ mesenchymal stem cells for clinical trials is limited. Objective To determine the feasibility of culture-expanding multiple sclerosis patients’ mesenchymal stem cells for clinical use. Methods In a phase I trial, autologous, bone marrow-derived mesenchymal stem cells were isolated from 25 trial participants with multiple sclerosis and eight matched controls, and culture-expanded to a target single dose of 1–2 × 106 cells/kg. Viability, cell product identity and sterility were assessed prior to infusion. Cytogenetic stability was assessed by single nucleotide polymorphism analysis of mesenchymal stem cells from 18 multiple sclerosis patients and five controls. Results One patient failed screening. Mesenchymal stem cell culture expansion was successful for 24 of 25 multiple sclerosis patients and six of eight controls. The target dose was achieved in 16–62 days, requiring two to three cell passages. Growth rate and culture success did not correlate with demographic or multiple sclerosis disease characteristics. Cytogenetic studies identified changes on one chromosome of one control (4.3%) after extended time in culture. Conclusion Culture expansion of mesenchymal stem cells from multiple sclerosis patients as donors is feasible. However, culture time should be minimized for cell products designated for therapeutic administration.

Genomic stability of pulmonary artery endothelial colony-forming cells in culture

Pulmonary vascular remodeling, including proliferation and migration of pulmonary artery endothelial cells (PAEC), is a pathologic hallmark of pulmonary arterial hypertension (PAH). Multiple studies have shown evidence of increased levels of DNA damage and lineage-specific genetic changes in PAH lung vascular cells, suggesting increased genomic instability. Highly proliferative endothelial colony-forming cell (ECFC) clones can be isolated from PAEC. Here we utilized ECFC to track chromosomal copy number of 20 PAH and eight control clones across serial passages using genome-wide microarrays. All PAH clones were genomically stable for at least 20–22 population doublings. At very late passages, ECFC developed a highly aneuploid karyotype, but this was generally associated with senescence and was common to both PAH and controls. We also utilized ECFC to isolate the chromosomally abnormal cells from a mixed population of PAH PAEC. Analysis of PAEC harboring two different changes affecting chromosomes 1 and X demonstrated that both abnormalities were present in the same clone, indicating they originated in a common ancestral cell. In a second case, with a partial duplication of chromosome 17, clones carrying the duplication were more frequent at later passages than chromosomally normal clones from the same PAEC culture, suggesting the rearrangement may confer a proliferative advantage. Overall, this small study suggests that endothelial cells from PAH lungs are stable in culture, but that when chromosome abnormalities do occur, they may confer a selective advantage that allows expansion of the abnormal cell population and could contribute to lung vascular remodeling in vivo.