Bone morphogenetic protein signaling is required for RAD51-mediated maintenance of genome integrity in vascular endothelial cells.

The integrity of blood vessels is fundamental to vascular homeostasis. Inactivating mutations in the bone morphogenetic protein (BMP) receptor type II (BMPR2) gene cause hereditary vascular disorders, including pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia, suggesting that BMPR2 and its downstream signaling pathway are pivotal to the maintenance of vascular integrity through an unknown molecular mechanism. Here we report that inactivation of BMPR2 in pulmonary vascular endothelial cells results in a deficit of RAD51, an enzyme essential for DNA repair and replication. Loss of RAD51, which causes DNA damage and cell death, is also detected in animal models and human patients with pulmonary arterial hypertension. Restoration of BMPR2 or activation of the BMP signaling pathway rescues RAD51 and prevents DNA damage. This is an unexpected role of BMP signaling in preventing the accumulation of DNA damage and the concomitant loss of endothelial integrity and vascular remodeling associated with vascular disorders

Accurate genetic diagnosis of Finnish pulmonary arterial hypertension patients using oligonucleotide-selective sequencing

The genetic basis of pulmonary arterial hypertension (PAH) among Finnish PAH patients is poorly understood. We adopted a novel-targeted next-generation sequencing (NGS) approach called Oligonucleotide-Selective Sequencing (OS-Seq) and developed a custom data analysis and interpretation pipeline to identify pathogenic base substitutions, insertions, and deletions in seven genes associated with PAH (BMPR2, BMPR1B, ACVRL1, ENG, SMAD9, CAV1, and KCNK3) from Finnish PAH patients. This study represents the first clinical study with OS-Seq technology on patients suffering from a rare genetic disorder. We analyzed DNA samples from 21 Finnish PAH patients, whose BMPR2 and ACVRL1 mutation status had been previously studied using Sanger sequencing. Our sequencing panel covered 100% of the targeted base pairs with >15× sequencing depth. Pathogenic base substitutions were identified in the BMPR2 gene in 29% of the Finnish PAH cases. Two of the pathogenic variant-positive patients had been previously tested negative using Sanger sequencing. No clinically significant variants were identified in the six other PAH genes. Our study validates the use of targeted OS-Seq for genetic diagnostics of PAH and revealed pathogenic variants that had been previously missed using Sanger sequencing.Peer reviewe

FOXL2, GATA4, and SMAD3 Co-Operatively Modulate Gene Expression, Cell Viability and Apoptosis in Ovarian Granulosa Cell Tumor Cells

Peer reviewe

Molecular Genetic Mechanisms of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is one of the most severe vascular diseases. In addition to increased vasoconstriction there are significant histopathological changes in small pulmonary arteries of PAH patients. These histopathological changes include endothelial cell (EC) injury, vascular inflammation, medial hypertrophy, intimal hyperplasia, and plexogenic arteriopathy. Changes in EC phenotype are suggested to play an important role in the development of occlusive vascular lesions. This phenotypic switch involves apoptosis-resistance, hyperproliferative capacity, and changes in energy metabolism. Somatic genomic abnormalities have also been identified in vascular lesions from patients with PAH. Mutations in bone morphogenetic protein receptor 2 (BMPR2) have been tightly linked to the pathogenesis of PAH. The genetic landscape of PAH-associated mutations in Finnish population is poorly understood. In this study, we utilized a novel targeted next-generation sequencing approach called Oligonucleotide-Selective sequencing (OS-Seq) to evaluate the role of known PAH genes, confirming a relatively high incidence of BMPR2 mutations among analyzed Finnish PAH-patients. Pulmonary ECs from the patients with PAH are identified with severe somatic chromosomal abnormalities. The role of genomic instability during pathogenesis of PAH and the mechanisms behind these changes in ECs are yet unknown. The genomic instability in IPAH patient ECs and the occlusive vasculopathy resemble a neoplastic process. In this study we revealed that loss of BMPR2 in pulmonary ECs leads to increased susceptibility to DNA damage and establish a link between Breast cancer 1 and BMPR2. BMPR2 deficiency-associated loss of DNA damage control could play a role in PAH -associated EC dysfunction, genomic instability and altered cellular phenotypes. One of the critical transcription factors downstream of BMPR2 is peroxisome proliferator-activated receptor gamma (PPARγ). In mice the TIE2 promoter mediated EC-specific loss of functional PPARγ (TIE2CrePPARγflox/flox) leads to dysregulation of EC genes such as apelin and development of PAH. The role of PPARγ in vascular regeneration or angiogenesis has remained poorly understood. We show that dysfunctional PPARγ in ECs leads to attenuation of angiogenic capacity and migration defect. Purinergic signaling is one potential modulator of pulmonary vascular homeostasis. ATP is known to regulate a variety of cellular processes, including vascular remodeling. In response to cell damage, ATP is released into extracellular space and through several physiological mechanisms cells can regulate their basal ATP niche contributing to the growth and microenvironment of the cells. The role of ATP in vascular modulation is well-known yet its association with pathophysiology of PAH is still undefined. In ECs the CD39 enzyme is the most prominent ATP hydrolyzing enzyme. We showed that PAH patients manifest suppression of CD39 leading to excess ATP niche in vascular wall. Our results suggest that attenuated CD39 activity is tightly linked to vascular remodelling and phenotypic switch of ECs.Keuhkovaltimoverenpainetauti on harvinainen, mutta vakava ja nopeasti etenevä tauti. Tällä hetkellä tautiin ei ole tarjolla parantavaa lääkitystä, mutta nykyisillä lääkehoidoilla voidaan hidastaa taudin etenemistä ja lievittää potilaan oireita. Monen potilaan kohdalla keuhkonsiirto on lopulta ainoa hengen pelastava toimenpide. Keuhkovaltimoverenpainetaudin patobiologia kohdistuu pienten keuhkovaltimoiden uudelleen muovautumiseen. Tämän seurauksena nämä verisuonet ahtautuvat ja keuhkoverenkierron vastus nousee pysyvästi. Taudin edetessä tästä seuraa sydämen oikean puolen vaikea toimintahäiriö. BMPR2-signaloinnin toimintahäiriöt ovat tunnusomaisia taudin kehityksessä. Suomalaisten keuhkovaltimoverenpainetautia sairastavien potilaiden geneettinen tausta on vielä heikosti tunnettu. Hyödyntäen uuden sukupolven sekvensointimenetelmää, nimeltään Oligonucleotide-Selective sequencing, osoitamme suhteellisen korkean BMPR2 mutaatioiden esiintyvyyden tutkituilla potilailla. BMPR2-signaloinnin häiriintyminen herkistää endoteelisolun ajautumisen apoptoosiin ja solujen uudistumiskyvyn heikentymiseen. Tautia sairastavien potilaiden ahtautuneista verisuonista on löytynyt somaattisia mutaatioita geeneistä, jotka säätelevät solun geneettistä tasapainoa. BMPR2 toimintahäiriön vaikutus geneettisen epätasapainon syntymiseen näissä soluissa on vielä epäselvää. Näytämme, että keuhkovaltimoverenpainetautia sairastavilta potilailta peräisin olevat endoteelisolut ovat huomattavan herkkiä DNA-vaurioille ja BMPR2:sen ilmentyminen on tiukasti sidottu DNA-vaurioiden valvontaan endoteelisoluissa. Tärkeitä BMPR2-signaloinnin vasteita välittää PPARγ, joka säätelee solullista erilaistumista, kehitystä ja aineenvaihduntaa. PPARγ:n tarkka rooli verisuonien uudismuodostuksessa on edelleenkin epäselvä. Näytämme, että PPARγ on merkittävä tekijä verisuonten endoteelisolujen henkiinjäämis- ja lisääntymisvasteissa ja hiirillä endoteelispesifinen PPARγ toimintahäiriö johtavaa keuhkovaltimoverenpainetaudin ja luunkovettumistaudin kehittymiseen ja endoteelisolujen vaurioitumiseen, joka puolestaan johtaa verisuonten uudelleenmuodostuksen toimintahäiriöön. Vasteena soluvauriolle, ATP vapautuu solujen ulkopuoliseen tilaan ja useiden fysiologisten mekanismien kautta solut voivat säädellä ATP:n perustilaa soluissa ja siten vaikuttaa solujen kasvuun ja niiden mikroympäristöön. Osoitamme, että keuhkovaltimoverenpainetautia sairastavilla potilailla on merkittävästi alentunut CD39 ilmentyminen ja solun ulkopuolinen ATP-pitoisuus on huomattavasti kohonnut. Tämä johtaa fenotyypin muutoksiin endoteeli- ja lihassolupatologiassa

Extracellular ATP protects endothelial cells against DNA damage

Cell damage can lead to rapid release of ATP to extracellular space resulting in dramatic change in local ATP concentration. Evolutionary, this has been considered as a danger signal leading to adaptive responses in adjacent cells. Our aim was to demonstrate that elevated extracellular ATP or inhibition of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39) activity could be used to increase tolerance against DNA-damaging conditions. Human endothelial cells, with increased extracellular ATP concentration in cell proximity, were more resistant to irradiation or chemically induced DNA damage evaluated with the DNA damage markers gamma H2AX and phosphorylated p53. In our rat models of DNA damage, inhibiting CD39-driven ATP hydrolysis with POM-1 protected the heart and lung tissues against chemically induced DNA damage. Interestingly, the phenomenon could not be replicated in cancer cells. Our results show that transient increase in extracellular ATP can promote resistance to DNA damage.Peer reviewe

Genetics and genotype-phenotype correlations in Finnish patients with dilated cardiomyopathy

Aims Genetic analysis among patients with dilated cardiomyopathy (DCM) is becoming an important part of clinical assessment, as it is in hypertrophic cardiomyopathy (HCM). The genetics of DCM is complex and therefore next-generation sequencing strategies are essential when providing genetic diagnostics. To achieve maximum yield, the diagnostic approach should include comprehensive clinical phenotyping combined with high-quality, high-coverage deep sequencing of DCM-associated genes and clinical variant classification as a basis for defining true yield in genetic testing. Our study has combined a novel sequencing strategy and clinical interpretation to analyse the yield and genotype-phenotype correlations among well-phenotyped Finnish DCM patients.Despite our increased understanding of the genetic basis of dilated cardiomyopathy (DCM), the clinical utility and yield of clinically meaningful findings of comprehensive next-generation sequencing (NGS)-based genetic diagnostics in DCM has been poorly described. We utilized a high-quality oligonucleotide-selective sequencing (OS-Seq)-based targeted sequencing panel to investigate the genetic landscape of DCM in Finnish population and to evaluate the utility of OS-Seq technology as a novel comprehensive diagnostic tool. Methods and results Using OS-Seq, we targeted and sequenced the coding regions and splice junctions of 101 genes associated with cardiomyopathies in 145 unrelated Finnish patients with DCM. We developed effective bioinformatic variant filtering strategy and implemented strict variant classification scheme to reveal diagnostic yield and genotype-phenotype correlations. Implemented OS-Seq technology provided high coverage of the target region (median coverage 410x and 99.42% of the nucleotides were sequenced at least 15x read depth). Diagnostic yield was 35.2% (familial 47.6% and sporadic 25.6%, P = 0.004) when both pathogenic and likely pathogenic variants are considered as disease causing. Of these, 20 (53%) were titin (TTN) truncations (non-sense and frameshift) affecting all TTN transcripts. TTN truncations accounted for 20.6% and 14.6% of the familial and sporadic DCM cases, respectively. Conclusion Panel-based, high-quality NGS enables high diagnostic yield especially in the familial form of DCM, and bioinformatic variant filtering is a reliable step in the process of interpretation of genomic data in a clinical setting.Peer reviewe

Bone morphogenetic protein signaling is required for RAD51-mediated maintenance of genome integrity in vascular endothelial cells.

The integrity of blood vessels is fundamental to vascular homeostasis. Inactivating mutations in the bone morphogenetic protein (BMP) receptor type II (BMPR2) gene cause hereditary vascular disorders, including pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia, suggesting that BMPR2 and its downstream signaling pathway are pivotal to the maintenance of vascular integrity through an unknown molecular mechanism. Here we report that inactivation of BMPR2 in pulmonary vascular endothelial cells results in a deficit of RAD51, an enzyme essential for DNA repair and replication. Loss of RAD51, which causes DNA damage and cell death, is also detected in animal models and human patients with pulmonary arterial hypertension. Restoration of BMPR2 or activation of the BMP signaling pathway rescues RAD51 and prevents DNA damage. This is an unexpected role of BMP signaling in preventing the accumulation of DNA damage and the concomitant loss of endothelial integrity and vascular remodeling associated with vascular disorders