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Background: Aortic dilation, stiffening, and dissection are common and potentially lethal complications of Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), which involve abnormal transforming growth factor beta (TGF-β) signalling. The relation of aortic dimensions, stiffness, and biomarker levels is unknown. The objective of this study was to measure aortic dimensions, stiffness, TGF-β and matrix metalloproteinase (MMP) levels, and endothelial function in patients with MFS, and to compare TGF-β levels in patients with MFS receiving different therapeutic regimens. Methods: This was a cohort study of 40 MFS and 4 LDS patients and 87 control participants. Aortic dimension and stiffness indexes, including pulse wave velocity (PWV), were measured using echocardiography and Doppler. Total and free TGF-β and MMP blood levels were measured using Quantikine (R&D Systems, Inc, Minneapolis, MN) and Quanterix (Billerica, MA) kits. Endothelial function was measured using brachial artery flow-mediated dilation. Results: PWV was increased in patients with MFS. There were increased MMP-2 levels in those with MFS but no increase in free or total TGF-β or MMP-9 levels compared with control participants. There was no difference in TGF-β levels between MFS patients receiving no medications, angiotensin receptor blockers, and β-blockers. PWV correlated most strongly with age. Endothelial function showed premature gradual decline in patients with MFS. Conclusions: Despite the increased PWV, monitoring aortic stiffness or TGF-β levels would not be helpful in patients with MFS. TGF-β levels were not increased and the increased MMP-2 levels suggest consideration of a different therapeutic target. Résumé: Contexte: La dilatation, la rigidification et la dissection de l’aorte sont des complications fréquentes et parfois mortelles du syndrome de Marfan (SM) et du syndrome de Loeys-Dietz (SLD), qui sont tous deux dûs à une anomalie de la voie de signalisation du facteur de croissance transformant bêta (TGF-β). On ne connaît pas la relation entre les dimensions et la rigidité de l’aorte et la présence de biomarqueurs. Notre étude visait à mesurer les dimensions et la rigidité de l’aorte, les taux de TGF-β et de métalloprotéases matricielles (MMP) et la fonction endothéliale chez des patients atteints du SM, et à les comparer aux taux de TGF-β observés chez des patients également atteints de SM, mais recevant un autre traitement. Méthodologie: Il s’agissait d’une étude de cohorte menée auprès de 40 patients atteints du SM et de quatre patients atteints du SLD, ainsi que de 87 témoins. Les indices des dimensions et de la rigidité aortiques, y compris la vitesse d’onde de pouls (VOP), ont été mesurés par échocardiographie et par échographie Doppler. Les taux sanguins de TGF-β et de MMP totaux et libres ont été mesurés à l’aide de trousses Quantikine (R&D Systems, Inc, Minneapolis, MN) et Quanterix (Billerica, MA). La fonction endothéliale a été mesurée par dilatation liée au flux dans l’artère brachiale. Résultats: La VOP était plus élevée chez les patients atteints du SM. On a aussi observé une hausse des taux de MMP-2 chez les patients atteints de SM, mais aucune augmentation des taux de TGF-β ou de MMP-9 libres ou totaux comparativement aux témoins. Il n’y avait pas de différence entre les taux de TGF-β chez les patients atteints de SM ne recevant aucun traitement, ceux qui prenaient un antagoniste des récepteurs de l’angiotensine et ceux qui prenaient un bêtabloquant. La VOP été plus fortement corrélée avec l’âge. La fonction endothéliale a affiché un déclin progressif prématuré chez les patients atteints du SM. Conclusions: Malgré l’augmentation de la VOP, il ne semble pas utile de surveiller la rigidité aortique ni les taux de TGF-β en cas de SM. Les taux de TGF-β n’étaient pas plus élevés chez les patients atteints du SM, et la hausse des taux de MMP-2 indique qu’il conviendrait de choisir une autre cible thérapeutique.

Abstract Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the FBN1 gene that produces wide disease phenotypic variability. The lack of ample genotype–phenotype correlation hinders translational study development aimed at improving disease prognosis. In response to this need, an induced pluripotent stem cell (iPSC) disease model has been used to test patient-specific cells by a proteomic approach. This model has the potential to risk stratify patients to make clinical decisions, including timing for surgical treatment. The regional propensity for aneurysm formation in MFS may be related to distinct smooth muscle cell (SMC) embryologic lineages. Thus, peripheral blood mononuclear cell (PBMC)-derived induced pluripotent stem cells (iPSC) were differentiated into lateral mesoderm (LM, aortic root) and neural crest (NC, ascending aorta/transverse arch) SMC lineages to model MFS aortic pathology. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) proteomic analysis by tandem mass spectrometry was applied to profile LM and NC iPSC SMCs from four MFS patients and two healthy controls. Analysis revealed 45 proteins with lineage-dependent expression in MFS patients, many of which were specific to diseased samples. Single protein-level data from both iPSC SMCs and primary MFS aortic root aneurysm tissue confirmed elevated integrin αV and reduced MRC2 in clinical disease specimens, validating the iPSC iTRAQ findings. Functionally, iPSC SMCs exhibited defective adhesion to a variety of extracellular matrix proteins, especially laminin-1 and fibronectin, suggesting altered cytoskeleton dynamics. This study defines the aortic embryologic origin-specific proteome in a validated iPSC SMC model to identify novel protein markers associated with MFS aneurysm phenotype. Translating iPSC findings into clinical aortic aneurysm tissue samples highlights the potential for iPSC-based methods to model MFS disease for mechanistic studies and therapeutic discovery in vitro.

Background Male patients with Marfan syndrome have a higher risk of aortic events and root dilatation compared with females. The role androgens play during Marfan syndrome aneurysm development in males remains unknown. We hypothesized that androgens potentiate transforming growth factor beta induced Erk (extracellular‐signal‐regulated kinase)/Smad activation, contributing to aneurysm progression in males. Methods and Results Aortic diameters in Fbn1C1039G/+ and littermate wild‐type controls were measured at ages 6, 8, 12, and 16 weeks. Fbn1C1039G/+ males were treated with (1) flutamide (androgen receptor blocker) or (2) vehicle control from age 6 to 16 weeks and then euthanized. p‐Erk1/2, p‐Smad2, and matrix metalloproteinase (MMP) activity were measured in ascending/aortic root and descending aorta specimens. Fbn1C1039G/+ male and female ascending/aortic root‐derived smooth muscle cells were utilized in vitro to measure Erk/Smad activation and MMP‐2 activity following dihydrotestosterone, flutamide or transforming growth factor beta 1 treatment. Fbn1C1039G/+ males have increased aneurysm growth. p‐Erk1/2 and p‐Smad2 were elevated in ascending/aortic root specimens at age 16 weeks. Corresponding with enhanced Erk/Smad signaling, MMP‐2 activity was higher in Fbn1C1039G/+ males. In vitro smooth muscle cell studies revealed that dihydrotestosterone potentiates transforming growth factor beta‐induced Erk/Smad activation and MMP‐2 activity, which is reversed by flutamide treatment. Finally, in vivo flutamide treatment reduced aneurysm growth via p‐Erk1/2 and p‐Smad2 reduction in Fbn1C1039G/+ males. Conclusions Fbn1C1039G/+ males have enhanced aneurysm growth compared with females associated with enhanced p‐Erk1/2 and p‐Smad2 activation. Mechanistically, in vitro smooth muscle cell studies suggested that dihydrotestosterone potentiates transforming growth factor beta induced Erk/Smad activation. As biological proof of concept, flutamide treatment attenuated aneurysm growth and p‐Erk1/2 and p‐Smad2 signaling in Fbn1C1039G/+ males.

Marfan syndrome (MFS) is an autosomal-dominant disorder of connective tissue caused by mutations in the fibrillin-1 (FBN1) gene. Mortality is often due to aortic dissection and rupture. We investigated the structural and functional properties of the heart and aorta in a [Fbn1C1039G/+] MFS mouse using high-resolution ultrasound (echo) and optical coherence tomography (OCT). Echo was performed on 6- and 12-month old wild type (WT) and MFS mice (n = 8). In vivo pulse wave velocity (PWV), aortic root diameter, ejection fraction, stroke volume, left ventricular (LV) wall thickness, LV mass and mitral valve early and atrial velocities (E/A) ratio were measured by high resolution echocardiography. OCT was performed on 12-month old WT and MFS fixed mouse hearts to measure ventricular volume and mass. The PWV was significantly increased in 6-mo MFS vs. WT (366.6 ± 19.9 vs. 205.2 ± 18.1 cm/s; p = 0.003) and 12-mo MFS vs. WT (459.5 ± 42.3 vs. 205.3 ± 30.3 cm/s; p< 0.0001). PWV increased with age in MFS mice only. We also found a significantly enlarged aortic root and decreased E/A ratio in MFS mice compared with WT for both age groups. The [Fbn1C1039G/+] mouse model of MFS replicates many of the anomalies of Marfan patients including significant aortic dilation, central aortic stiffness, LV systolic and diastolic dysfunction. This is the first demonstration of the direct measurement in vivo of pulse wave velocity non-invasively in the aortic arch of MFS mice, a robust measure of aortic stiffness and a critical clinical parameter for the assessment of pathology in the Marfan syndrome.