Clinical Heterogeneity of Pulmonary Arterial Hypertension Associated With Variants in TBX4

Background: The knowledge of hereditary predisposition has changed our understanding of Pulmonary Arterial Hypertension. Genetic testing has been widely extended and the application of Pulmonary Arterial Hypertension specific gene panels has allowed its inclusion in the diagnostic workup and increase the diagnostic ratio compared to the traditional sequencing techniques. This is particularly important in the differential diagnosis between Pulmonary Arterial Hypertension and Pulmonary Venoocclusive Disease. Methods: Since November 2011, genetic testing is offered to all patients with idiopathic, hereditable and associated forms of Pulmonary Arterial Hypertension or Pulmonary Venoocclusive Disease included in the Spanish Registry of Pulmonary Arterial Hypertension. Herein, we present the clinical phenotype and prognosis of all Pulmonary Arterial Hypertension patients with disease-associated variants in TBX4. Results: Out of 579 adults and 45 children, we found in eight patients from seven families, disease-causing associated variants in TBX4. All adult patients had a moderate-severe reduction in diffusion capacity. However, we observed a wide spectrum of clinical presentations, including Pulmonary Venoocclusive Disease suspicion, interstitial lung disease, pulmonary vascular abnormalities and congenital heart disease. Conclusions: Genetic testing is now essential for a correct diagnosis work-up in Pulmonary Arterial Hypertension. TBX4-associated Pulmonary Arterial Hypertension has marked clinical heterogeneity. In this regard, a genetic study is extremely useful to obtain an accurate diagnosis and provide appropriate management.This project was founded by Project "Bases Gene´tico Moleculares de la Medicina de Precisio´n en la Hipertensio´n Arterial Pulmonar". Funder: Instituto Carlos III. Ministerio de Economı´a y Competitividad. https://www.isciii.es/Paginas/Inicio.aspx Award number: PI 18/01233 Grant Recipient: P E-

Análisis funcionales y nuevos marcadores para la Hipertensión Arterial Pulmonar

Pulmonary Arterial Hypertension (PAH) is a rare disease characterized by progressive obliteration of the pulmonary precapillary arteries, leading to an increase in pulmonary vascular resistance that causes right heart failure, and subsequently, death. In the last decade, mass sequencing has increased the number of genes involved in PAH, doubling the number of known genes. This quantitative increase in the available data has shown low efficiency in classifying variants, resulting in high levels of variants of uncertain significance, which show as a problem for both diagnosis and genetic counseling. To improve the classification of variants into PAH-related genes, we will perform functional studies in new genes (ABCC8) and others with broad known involvement but no functional analysis (TBX4). The ABCC8 gene encodes for the regulatory subunit of an ATP-dependent potassium channel known as SUR1, which needs binding to the pore-forming protein Kir6, the involvement of this channel in diseases such as diabetes is known, but as it fits exactly into the pathogenesis of PAH it is for now diffuse. ABCC8 is a gene of complex structure, consisting of 39 small exons with broad intronic regions. Due to these characteristics, most of the mutations found in the Spanish HAP registry (REHAP), are found in the vicinity of regions bordering exons and introns. To determine whether these seemingly change-of-direction mutations can induce exon jump events, we will make minigenes for 11 variants detected in REHAP. On the other hand, TBX4 is one of the genes with the greatest involvement in pediatric PAH, although in recent years the proportion of variants in adult forms has increased considerably. The function of TBX4 is well known in embryonic development, but its role in adults is not. For this reason, we collected 51 variants in TBX4 for their functional study, by analyzing their transcriptional activity and cell location, as well as minigenes for the cut and splice variants. At present, the diagnosis of PAH is produced using invasive and expensive means that require the hospitalization of the patient, no marker allows the diagnosis quickly and easily. In the last decade the use of circulating microRNAs (miRNAs) as biomarkers has begun to be proposed, miRNAs are small non-coding RNA molecules of 16-22 base pairs that regulate posttranscriptional activity by binding to the 3 'UTR region of messenger RNA. stable in the blood and their levels in this fluid are affected in pathological processes. Which makes them potential biomarkers for countless diseases, such as PAH, which due to the processes of remodeling, proliferation and inhibition of apoptosis that occurs in endothelial cells and smooth muscle of the pulmonary precapillary arteries, makes it a strong candidate for the development of a model for its diagnosis based on circulating miRNAs. To perform this model, microRNA-Seq will be used, from total RNA from patient plasma and controls. Initially a differential expression analysis will be done, and then the results will be validated using qPCR using specific probes. Once we have a set of miRNAs with differentiated expression between the two groups, a variable number of miRNAs will be proposed for prevalence in a cohort belonging to REHAP, to finally be validated in 100 PAH patients and 100 controls using digital droplet PCR. Machine learning methods will be used to assess different combinations of circulating miRNAs. Besides, the effect of more deregulated miRNAs will be assessed in vitro using arterial endothelial cells.A Hipertensión Arterial Pulmonar (HAP) é unha enfermidade rara caracterizada pola obliteración progresiva das arterias precapilares pulmonares, conducindo a un aumento da resistencia vascular pulmonar que provoca insuficiencia cardíaca dereita, e posteriormente, a morte. Na última década a secuenciación masiva permitiu aumentar o número de xenes implicados na HAP, duplicando o número de xenes coñecidos. Este aumento cuantitativo dos datos dispoñibles evidenciou baixa eficiencia para clasificar variantes, obtendo niveis altos de variantes de significado incerto, que amosan coma un problema para tanto á hora de diagnosticar coma dar consello xenético. Co obxectivo de mellorar a clasificación de variantes en xenes relacionados coa HAP, faremos estudos funcionais en xenes novos (ABCC8) e outros con ampla implicación coñecida pero sen análises funcionais (TBX4). O xene ABCC8 codifica para a subunidade reguladora dunha canle de potasio dependente de ATP coñecida como SUR1, que precisa da unión coa proteína formadora de poro Kir6, a implicación desta canle en enfermidades como a diabetes é coñecida, pero como encaixa exactamente na patoxénese da HAP é por agora difuso. ABCC8 é un xene de estrutura complexa, formado por 39 exóns de pequeno tamaño con rexións intrónicas amplas. Debido a estas características, a maior parte das mutacións atopadas no rexistro español de HAP (REHAP), atópanse en proximidade de rexións limítrofes entre exóns e intróns. Co obxectivo de determinar se estas mutacións aparentemente de cambio de sentido poden chegar a inducir eventos de salto de exóns, faremos minixenes para 11 variantes detectadas no REHAP. Por outra banda, TBX4 é un dos xenes con maior implicación en HAP pediátrica, aínda que nos últimos anos a proporción de variantes nas formas adultas aumentou considerablemente. A función de TBX4 é ben coñecida no desenvolvemento embrionario, pero o seu papel nos adultos non. Por este motivo, recompilamos 51 variantes en TBX4 para o seu estudo funcional, mediante a análise da súa actividade transcricional e localización celular, ademais de minixenes para as variantes de corte e empalme. Na actualidade, o diagnóstico da HAP prodúcese utilizando medios invasivos e custosos que esixen o internamento do paciente, non existe ningún marcador que permita o diagnóstico dunha forma rápida e sinxela. Nesta última década comezouse a propor o uso de microARNs (miRNAs) circulantes como biomarcadores, os miRNAs son pequenas moléculas de ARN non codificante de 16-22 pares de bases que regulan a actividade postranscricional uníndose á rexión 3’ UTR do ARN mensaxeiro, son moi estables no sangue e seus niveis neste líquido vense afectados nos procesos patolóxicos. O que os converte en biomarcadores potenciais para infinidade de enfermidades, coma a HAP, que debido aos procesos de remodelación, proliferación e inhibición da apoptose que ocorre nas células endoteliais e do músculo liso das arterias precapilares pulmonares, vólvea unha firme candidata para o desenvolvemento dun modelo para a súa diagnose baseado nos miRNAs circulantes. Para realizar este modelo utilizarase a microRNA-Seq, a partir de ARN total provinte de plasma de pacientes e controles. Nun primeiro momento farase unha análise de expresión diferencial, e despois, os resultados validaranse mediante qPCR utilizando sondas específicas. Unha vez teñamos un conxunto de miRNAs con expresión diferenciada entre ambos grupos, propoñerase un número variable de miRNAs para a súa prevalidación nunha cohorte pertencente ao REHAP, para poder finalmente facer unha validación en 100 doentes de HAP e 100 controis utilizando droplet digital PCR. Para valorar distintas combinacións de miRNAs circulantes utilizaranse métodos de machine learning. Ademáis, o efecto dos miRNAs máis desregulados sen función coñecida avaliarase in vitro utilizando células de endotelio arterial.La hipertensión arterial pulmonar (HAP) es una enfermedad rara caracterizada por la obliteración progresiva de las arterias precapilares pulmonares, lo que lleva a un aumento de la resistencia vascular pulmonar que provoca insuficiencia cardíaca derecha y, posteriormente, la muerte. En la última década, la secuenciación masiva ha aumentado el número de genes implicados en la HAP, duplicando el número de genes conocidos. Este incremento cuantitativo de los datos disponibles ha mostrado una baja eficiencia en la clasificación de variantes, resultando en altos niveles de variantes de significado incierto, que se muestran como un problema tanto para el diagnóstico como para el consejo genético. Con el fin de mejorar la clasificación de variantes en genes relacionados con la HAP, realizaremos estudios funcionales en genes nuevos (ABCC8) y otros con implicación conocida, pero sin análisis funcionales (TBX4). El gen ABCC8 codifica para la subunidad reguladora de un canal de potasio dependiente de ATP conocido como SUR1, que necesita unirse a la proteína formadora de poros Kir6, la participación de este canal en enfermedades como la diabetes es conocida, pero cómo encaja exactamente en la patogénesis de la HAP es por ahora un enigma. ABCC8 es un gen de estructura compleja, que consta de 39 exones pequeños con amplias regiones intrónicas. Por estas características, la mayoría de las mutaciones encontradas en el registro español de HAP (REHAP), se encuentran en las proximidades de regiones limítrofes entre exones e intrones. Con el fin de determinar si estas mutaciones aparentemente de cambio de sentido pueden inducir eventos de salto de exón, crearemos minigenes para las 11 variantes detectadas en la REHAP. Por otro lado, TBX4 es uno de los genes con mayor implicación HAP pediátrica, aunque en los últimos años la proporción de variantes en formas adultas ha aumentado considerablemente. La función de TBX4 está bien caracterizada durante el desarrollo embrionario, pero su papel en adultos no está claro. Por esta razón, recopilamos 51 variantes en TBX4 para su estudio funcional, analizando su actividad transcripcional y ubicación celular, así como minigenes para las variantes de corte y empalme. En la actualidad el diagnóstico de HAP se produce mediante medios invasivos y costosos que requieren la hospitalización del paciente, no existe ningún marcador que permita el diagnóstico de una manera rápida y sencilla. En la última década se ha comenzado a proponer el uso de microARN circulantes (miARN) como biomarcadores, los miARN son pequeñas moléculas de ARN no codificantes de 16-22 pares de bases que regulan la actividad postranscripcional uniéndose a la región 3 'UTR del ARN mensajero, son muy estables en sangre y sus niveles en este líquido se ven afectados en procesos patológicos. Lo que los convierte en potenciales biomarcadores de innumerables enfermedades, como la HAP, que debido a los procesos de remodelación, proliferación e inhibición de la apoptosis que se producen en las células endoteliales y el músculo liso de las arterias precapilares pulmonares, los convierte en buenos candidatos para el desarrollo de un modelo para su diagnóstico basado en miARN circulantes. Para realizar este modelo, se utilizará microRNA-Seq a partir de ARN total de plasma de paciente y controles. Inicialmente se realizará un análisis de expresión diferencial y luego se validarán los resultados mediante qPCR utilizando sondas específicas. Una vez que tengamos un conjunto de miRNAs con expresión diferencial entre ambos grupos, se propondrá un número variable de miRNAs para hacer una prevalidación en una cohorte perteneciente al REHAP, para finalmente hacer una validación en 100 pacientes con HAP y 100 controles mediante droplet digital PCR. Se utilizarán métodos de machine learning para evaluar diferentes combinaciones de miARN circulantes. Además, el efecto de miARN más desregulados sin función conocida se evaluará in vitro utilizando células endoteliales.Xunta de Galicia | Ref. ED481A-2018/304Instituto de Salud Carlos III | Ref. PI18/01233Xunta de Galicia | Ref. ED431C 2018/5

Common Variation in EDN1 Regulatory Regions Highlights the Role of PPARγ as a Key Regulator of Endothelin in vitro

14 pages, 7 figures, 1 table.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 (EDN1) is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the most common targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of patients with PAH, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors (TFs) predicted for these regions [peroxisome proliferator-activated receptor γ (PPARγ), Krüppel-Like Factor 4 (KLF4), and vitamin D receptor (VDR)] to assess EDN1 expression in cell culture and validate the binding sites. First, we detected a single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR; rs397751713) and another in the 3'regulatory region (rs2859338) that altered luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPARγ could bind to the rs397751713 and VDR to rs2859338. By using siRNAs and luciferase assays, we determined that PPARγ binds differentially to rs397751713. PPARγ and VDR Knock-Down (KD) increased the EDN1 mRNA levels and EDN1 production in porcine aortic endothelial cells (PAECs), while PPARγ and KLF4 KD increased the EDN1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter EDN1 levels. We were able to validate that PPARγ binds in rs397751713 and is a key regulator of EDN1. In addition, KLF4 and VDR regulate EDN1 production in a cell-dependent manner, but VDR does not bind directly to the regions we studiedThis work was funded by the Cardiovascular Research Network of Instituto de Salud Carlos III de Madrid (RD06/0003/0012) Spanish Ministry of Science and Innovation PI18/01233 and Janssen Pharmaceuticals. CINBIO has financial support from Xunta de Galicia and the European Union (European Regional Development Fund-ERDF) (PO FEDER ED431G/02). ML-D and LM-M are supported by a Xunta de Galicia predoctoral fellowship (ED481A-2018/304; IN606A-2020/006). CS is supported by a Ministerio de Universidades FPU predoctoral fellowship (FPU19/00175)Peer reviewe

ALMS1 regulates TGF-β signaling and morphology of primary cilia

In this study, we aimed to evaluate the role of ALMS1 in the morphology of primary cilia and regulation of cellular signaling using a knockdown model of the hTERT-RPE1 cell line. ALMS1 depletion resulted in the formation of longer cilia, which often displayed altered morphology as evidenced by extensive twisting and bending of the axoneme. Transforming growth factor beta/bone morphogenetic protein (TGF-β/BMP) signaling, which is regulated by primary cilia, was similarly affected by ALMS1 depletion as judged by reduced levels of TGFβ-1-mediated activation of SMAD2/3. These results provide novel information on the role of ALMS1 in the function of primary cilia and processing of cellular signaling, which when aberrantly regulated may underlie Alström syndrome.Ministerio de Educación, Cultura y Deporte | Ref. FPU12/01442Ministerio de Educación, Cultura y Deporte | Ref. FPU13/01835Ministerio de Educación, Cultura y Deporte | Ref. FPU17/01567Ministerio de Educación, Cultura y Deporte | Ref. FPU19/00175Ministerio de Economía y Competitividad | Ref. PI15/00049Ministerio de Ciencia, Innovación y Universidades | Ref. PI19/00332Xunta de Galicia | Ref. ED481A-2018/304Xunta de Galicia | Ref. ED431G2019-0

Novel genetic and molecular pathways in pulmonary arterial hypertension associated with connective tissue disease

Pulmonary Arterial Hypertension (PAH) is a severe complication of Connective Tissue Disease (CTD), with remarkable morbidity and mortality. However, the molecular and genetic basis of CTD-PAH remains incompletely understood. This study aimed to screen for genetic defects in a cohort of patients with CTD-PAH, using a PAH-specific panel of 35 genes. During recruitment, 79 patients were studied, including 59 Systemic Sclerosis patients (SSc) and 69 females. Disease-associated variants were observed in nine patients: 4 pathogenic/likely pathogenic variants in 4 different genes (TBX4, ABCC8, KCNA5 and GDF2/BMP9) and 5 Variants of Unknown Significance (VUS) in 4 genes (ABCC8, NOTCH3, TOPBP1 and CTCFL). One patient with mixed CTD had a frameshift pathogenic variant in TBX4. Two patients with SSc-PAH carried variants in ABCC8. A patient diagnosed with Systemic Lupus Erythematous (SLE) presented a pathogenic nonsense variant in GDF2/BMP9. Another patient with SSc-PAH presented a pathogenic variant in KCNA5. Four patients with SSc-PAH carried a VUS in NOTCH1, CTCFL, CTCFL and TOPBP1, respectively. These findings suggest that genetic factors may contribute to Pulmonary Vascular Disease (PVD) in CTD patients.Instituto de Salud Carlos III | Ref. PI 18/0123

Characterization of rare ABCC8 variants identified in Spanish pulmonary arterial hypertension patients

Pulmonary Arterial Hypertension (PAH) is a rare and fatal disease where knowledge about its genetic basis continues to increase. In this study, we used targeted panel sequencing in a cohort of 624 adult and pediatric patients from the Spanish PAH registry. We identified 11 rare variants in the ATP-binding Cassette subfamily C member 8 (ABCC8) gene, most of them with splicing alteration predictions. One patient also carried another variant in SMAD1 gene (c.27delinsGTAAAG). We performed an ABCC8 in vitro biochemical analyses using hybrid minigenes to confirm the correct mRNA processing of 3 missense variants (c.211C > T p.His71Tyr, c.298G > A p.Glu100Lys and c.1429G > A p.Val477Met) and the skipping of exon 27 in the novel splicing variant c.3394G > A. Finally, we used structural protein information to further assess the pathogenicity of the variants. The results showed 11 novel changes in ABCC8 and 1 in SMAD1 present in PAH patients. After in silico and in vitro biochemical analyses, we classified 2 as pathogenic (c.3288_3289del and c.3394G > A), 6 as likely pathogenic (c.211C > T, c.1429G > A, c.1643C > T, c.2422C > A, c.2694 + 1G > A, c.3976G > A and SMAD1 c.27delinsGTAAAG) and 3 as Variants of Uncertain Significance (c.298G > A, c.2176G > A and c.3238G > A). In all, we show that coupling in silico tools with in vitro biochemical studies can improve the classification of genetic variants.Instituto de Salud Carlos III | Ref. RD06/0003/0012Instituto de Salud Carlos III | Ref. PI18/01233Xunta de Galicia | Ref. ED431G-2019/06Xunta de Galicia | Ref. ED431C 2018/54Xunta de Galicia | Ref. ED481A-2018/304Ministerio de Economía y Competitividad (España) | Ref. RYC-2015-1824

Clinical heterogeneity of pulmonary arterial hypertension associated with variants in TBX4

Background The knowledge of hereditary predisposition has changed our understanding of Pulmonary Arterial Hypertension. Genetic testing has been widely extended and the application of Pulmonary Arterial Hypertension specific gene panels has allowed its inclusion in the diagnostic workup and increase the diagnostic ratio compared to the traditional sequencing techniques. This is particularly important in the differential diagnosis between Pulmonary Arterial Hypertension and Pulmonary Venoocclusive Disease. Methods Since November 2011, genetic testing is offered to all patients with idiopathic, hereditable and associated forms of Pulmonary Arterial Hypertension or Pulmonary Venoocclusive Disease included in the Spanish Registry of Pulmonary Arterial Hypertension. Herein, we present the clinical phenotype and prognosis of all Pulmonary Arterial Hypertension patients with disease-associated variants in TBX4. Results Out of 579 adults and 45 children, we found in eight patients from seven families, disease-causing associated variants in TBX4. All adult patients had a moderate-severe reduction in diffusion capacity. However, we observed a wide spectrum of clinical presentations, including Pulmonary Venoocclusive Disease suspicion, interstitial lung disease, pulmonary vascular abnormalities and congenital heart disease. Conclusions Genetic testing is now essential for a correct diagnosis work-up in Pulmonary Arterial Hypertension. TBX4-associated Pulmonary Arterial Hypertension has marked clinical heterogeneity. In this regard, a genetic study is extremely useful to obtain an accurate diagnosis and provide appropriate managementInstituto de Salud Carlos III | Ref. PI 18/0123

Running title: Novel loss of function KCNA5 variants in PAH

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in PAH patients, but their functional consequences and potential impact on the disease are largely unknown. Herein, we aimed to characterize the functional consequences of 7 KCNA5 variants found in a cohort of PAH patients. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with WT or mutant Kv1.5 cDNA. Flow cytometry, western blot and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells (hPASMC). KCNA5 variants found in PAH patients (namely, p.Arg184Pro and p.Gly384Arg) resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modelling analyses. The p.Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with p.Arg184Pro or p.Gly384Arg variants decreased apoptosis of hPASMCs compared with the WT, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1 %. Our data indicate that some KCNA5 variants found PAH patients have critical consequences for channel function supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.This work was supported by Fundación Contra la Hipertensión Pulmonar (FCHP); Ministerio de Ciencia e Innovación [PID2020-117939RB-I00 to AC, PID2019-104366RB-C21 to TG, PID2019-107363RB-I00 to FPV]; Comunidad de Madrid [B2017/BMD-3727 to AC] and Instituto de Salud Carlos III [PI18/01233, PI21/01593] with funds from the European Union (Fondo Europeo de Desarrollo Regional FEDER); and by an annual grant by the FEDER foundation (Federación Española de Enfermedades Raras).Peer reviewe

Customized massive parallel sequencing panel for diagnosis of pulmonary arterial hypertension

Pulmonary arterial hypertension is a very infrequent disease, with a variable etiology and clinical expressivity, making sometimes the clinical diagnosis a challenge. Current classification based on clinical features does not reflect the underlying molecular profiling of these groups. The advance in massive parallel sequencing in PAH has allowed for the describing of several new causative and susceptibility genes related to PAH, improving overall patient diagnosis. In order to address the molecular diagnosis of patients with PAH we designed, validated, and routinely applied a custom panel including 21 genes. Three hundred patients from the National Spanish PAH Registry (REHAP) were included in the analysis. A custom script was developed to annotate and filter the variants. Variant classification was performed according to the ACMG guidelines. Pathogenic and likely pathogenic variants have been found in 15% of the patients with 12% of variants of unknown significance (VUS). We have found variants in patients with connective tissue disease (CTD) and congenital heart disease (CHD). In addition, in a small proportion of patients (1.75%), we observed a possible digenic mode of inheritance. These results stand out the importance of the genetic testing of patients with associated forms of PAH (i.e., CHD and CTD) additionally to the classical IPAH and HPAH forms. Molecular confirmation of the clinical presumptive diagnosis is required in cases with a high clinical overlapping to carry out proper management and follow up of the individuals with the disease.Instituto de Salud Carlos III (España) | Ref. FISPI18/01233Xunta de Galicia | Ref. ED481A-2018/30

Reduced FOXF1 links unrepaired DNA damage to pulmonary arterial hypertension

Abstract Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common genetic cause of PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxia (reoxygenation), as do mice with EC-specific deletion of Bmpr2 (EC-Bmpr2 -/-) and persistent pulmonary hypertension. Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm -/-). Gene expression analysis of EC-Atm -/- and EC-Bmpr2 -/- lung EC reveals reduced Foxf1, a transcription factor with selectivity for lung EC. Reducing FOXF1 in control PAEC induces DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repairs DNA damage and restores angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2 -/- mice repairs DNA damage, induces angiogenesis and reverses pulmonary hypertension