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Objective: To delineate temporal and spatial dynamics of vascular smooth muscle cell (SMC) transcriptomic changes during aortic aneurysm development in Marfan syndrome (MFS). Approach and Results: We performed single-cell RNA sequencing to study aortic root/ascending aneurysm tissue from Fbn1 C1041G/ + (MFS) mice and healthy controls, identifying all aortic cell types. A distinct cluster of transcriptomically modulated SMCs (modSMCs) was identified in adult Fbn1 C1041G/ + mouse aortic aneurysm tissue only. Comparison with atherosclerotic aortic data (ApoE −/− mice) revealed similar patterns of SMC modulation but identified an MFS-specific gene signature, including plasminogen activator inhibitor-1 ( Serpine1 ) and Kruppel-like factor 4 ( Klf4 ). We identified 481 differentially expressed genes between modSMC and SMC subsets; functional annotation highlighted extracellular matrix modulation, collagen synthesis, adhesion, and proliferation. Pseudotime trajectory analysis of Fbn1 C1041G/ + SMC/modSMC transcriptomes identified genes activated differentially throughout the course of phenotype modulation. While modSMCs were not present in young Fbn1 C1041G/ + mouse aortas despite small aortic aneurysm, multiple early modSMCs marker genes were enriched, suggesting activation of phenotype modulation. modSMCs were not found in nondilated adult Fbn1 C1041G/ + descending thoracic aortas. Single-cell RNA sequencing from human MFS aortic root aneurysm tissue confirmed analogous SMC modulation in clinical disease. Enhanced expression of TGF-β (transforming growth factor beta)-responsive genes correlated with SMC modulation in mouse and human data sets. Conclusions: Dynamic SMC phenotype modulation promotes extracellular matrix substrate modulation and aortic aneurysm progression in MFS. We characterize the disease-specific signature of modSMCs and provide temporal, transcriptomic context to the current understanding of the role TGF-β plays in MFS aortopathy. Collectively, single-cell RNA sequencing implicates TGF-β signaling and Klf4 overexpression as potential upstream drivers of SMC modulation.

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF‐β driven via Smad (canonical) and ERK (non‐canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF‐β‐driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF‐β‐dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF‐β‐dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a ‘mixed’ contractile‐synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.

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 Fbn1 C1039G/+ and littermate wild‐type controls were measured at ages 6, 8, 12, and 16 weeks. Fbn1 C1039G/+ 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. Fbn1 C1039G/+ 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. Fbn1 C1039G/+ 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 Fbn1 C1039G/+ 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 Fbn1 C1039G/+ males. Conclusions Fbn1 C1039G/+ 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 Fbn1 C1039G/+ males.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root widening and aneurysm if unmanaged. We have previously reported doxycycline, a nonselective matrix metalloproteinases (MMPs) inhibitor, to attenuate aortic root widening and improve aortic contractility and elasticity in MFS mice. We were also first to use multiphoton microscopy, a non-invasive and label-free imaging technique, to quantify and link the aortic ultrastructure to possible changes in the skin dermis. Here, we aimed to assess the effects of long-term doxycycline treatment on the aortic ultrastructure and skin dermis of MFS mice through immunohistochemical evaluation and quantification of elastic and collagen content and morphology using multiphoton microscopy. Our results demonstrate a rescue of aortic elastic fiber fragmentation and disorganization accompanied by a decrease in MMP-2 and MMP-9 expression within the aortic wall in doxycycline-treated MFS mice. At 12 months of age, reduced skin dermal thickness was observed in both MFS and control mice, but only dermal thinning in MFS mice was rescued by doxycycline treatment. MMP-2 and MMP-9 expression was reduced in the skin of doxycycline-treated MFS mice. A decrease in dermal thickness was found to be positively associated with increased aortic root elastin disorganization and wall thickness. Our findings confirm the beneficial effects of doxycycline on ultrastructural properties of aortic root as well as on skin elasticity and structural integrity in MFS mice.

Aortic aneurysm is the most life-threatening complication in Marfan syndrome (MFS) patients. Doxycycline, a nonselective matrix metalloproteinases inhibitor, was reported to improve the contractile function and elastic fiber structure and organization in a Marfan mouse aorta using ex vivo small chamber myography. In this study, we assessed the hypothesis that a long-term treatment with doxycycline would reduce aortic root growth, improve aortic wall elasticity as measured by pulse wave velocity, and improve the ultrastructure of elastic fiber in the mouse model of MFS. In our study, longitudinal measurements of aortic root diameters using high-resolution ultrasound imaging display significantly decreased aortic root diameters and lower pulse wave velocity in doxycycline-treated Marfan mice starting at 6 months as compared to their non-treated MFS counterparts. In addition, at the ultrastructural level, our data show that long-term doxycycline treatment corrects the irregularities of elastic fibers within the aortic wall of Marfan mice to the levels similar to those observed in control subjects. Our findings underscore the key role of matrix metalloproteinases during the progression of aortic aneurysm, and provide new insights into the potential therapeutic value of doxycycline in blocking MFS-associated aortic aneurysm.

Regular low-impact physical activity is generally allowed in patients with Marfan syndrome, a connective tissue disorder caused by heterozygous mutations in the fibrillin-1 gene. However, being above average in height encourages young adults with this syndrome to engage in high-intensity contact sports, which unfortunately increases the risk for aortic aneurysm and rupture, the leading cause of death in Marfan syndrome. In this study, we investigated the effects of voluntary (cage-wheel) or forced (treadmill) aerobic exercise at different intensities on aortic function and structure in a mouse model of Marfan syndrome. Four-week-old Marfan and wild-type mice were subjected to voluntary and forced exercise regimens or sedentary lifestyle for 5 mo. Thoracic aortic tissue was isolated and subjected to structural and functional studies. Our data showed that exercise improved aortic wall structure and function in Marfan mice and that the beneficial effect was biphasic, with an optimum at low intensity exercise (55–65% V̇o2max) and tapering off at a higher intensity of exercise (85% V̇o2max). The mechanism underlying the reduced elastin fragmentation in Marfan mice involved reduction of the expression of matrix metalloproteinases 2 and 9 within the aortic wall. These findings present the first evidence of potential beneficial effects of mild exercise on the structural integrity of the aortic wall in Marfan syndrome associated aneurysm. Our finding that moderate, but not strenuous, exercise protects aortic structure and function in a mouse model of Marfan syndrome could have important implications for the medical care of young Marfan patients. NEW & NOTEWORTHY The present study provides conclusive scientific evidence that daily exercise can improve aortic health in a mouse model of Marfan syndrome associated aortic aneurysm, and it establishes the threshold for the exercise intensity beyond which exercise may not be as protective. These findings establish a platform for a new focus on promoting regular exercise in Marfan patients at an optimum intensity and create a paradigm shift in clinical care of Marfan patients suffering from aortic aneurysm complications.

Several behavioral studies report that adolescent rats display a preference for nicotine compared with adults. However, age-related pharmacokinetic differences may confound the interpretation of these findings. Thus, differences in pharmacokinetic analyses of nicotine were investigated. Nicotine was administered via acute s.c. (1.0 mg base/kg) or i.v. (0.2 mg base/kg) injection to early adolescent (EA; postnatal day 25) and adult (AD; postnatal day 71) male Wistar rats. Nicotine and its primary metabolite, cotinine, and additional metabolites nornicotine, nicotine-1′-N-oxide, trans-3′-hydroxycotinine, and norcotinine were sampled from 10 minutes to 8 hours (plasma) and 2 to 8 hours (brain) post nicotine and analyzed by liquid chromatography–tandem mass spectrometry. Following s.c. nicotine, the EA cohort had lower levels of plasma nicotine, cotinine, and nicotine-1′-N-oxide at multiple time points, resulting in a lower area under the plasma concentration-time curve (AUC) for nicotine (P < 0.001), cotinine (P < 0.01), and nicotine-1′-N-oxide (P < 0.001). Brain levels were also lower for these compounds. In contrast, the EA cohort had higher plasma and brain AUCs (P < 0.001) for the minor metabolite nornicotine. Brain-to-plasma ratios varied for nicotine and its metabolites, and by age. Following i.v. nicotine administration, similar age-related differences were observed, and this route allowed detection of a 1.6-fold-larger volume of distribution and 2-fold higher plasma clearance in the EA cohort compared with the AD cohort. Thus, unlike in humans, there are substantial age differences in nicotine pharmacokinetics such that for a given nicotine dose, adolescent rats will have lower plasma and brain nicotine compared with adults, suggesting that this should be considered when interpreting animal model data.

Introduction: Aortic aneurysm is the most life-threatening complication in Marfan syndrome (MFS), a connective tissue disorder caused by mutations in fibrillin-1 gene. We have previously reported that doxycycline, an antibiotic and a general inhibitor of matrix metalloproteinases (MMPs), could improve aortic contractility and elastin fibers structure and organization in a mouse model of MFS. However, the in vivo effects of MMPs inhibition on the progression of aneurysm and pulse wave velocity (PWV) have not been investigated. In this study, we assessed the hypothesis that a long-term treatment with a sub-antibiotic dose of doxycycline would delay the progression of aortic aneurysm and improve the PWV in the Marfan mouse. Methods: In this study, we have used a high-resolution, high-contrast ultrasound imaging technique to conduct a longitudinal study in order to measure aortic diameter (dilatation) and PWV (indication of aortic elasticity/stiffness) in the animals. Marfan (n=12) and control (n=13) mice were given a sub-antibiotic dose of doxycycline in their drinking water at a concentration of 0.24 g/L/day beginning at 6 weeks of age, whereas another group of Marfan (n=12) and control (n=12) mice were given plain drinking water for comparison. At 3, and 6 months of age, aortic diameter and PWV of the experimental animals were measured by ultrasound imaging. Results: At 3 months of age, measurement of aortic root diameter showed significant dilatation in both treated and non-treated Marfan mice as compared to control groups (p < 0.01). However, at 6 months of age, the aortic root diameter in treated Marfan mice was not different from those in controls. The same observation was made for the Sinus of Valsalva region. Especially in this region, treated Marfan aorta showed significantly reduced diameter compared to non-treated Marfan groups (p < 0.05). With respect to the measurement of PWV, a significant increase was observed in 3- and 6-month Marfan mice aorta vs. control (p < 0.01). Interestingly, doxycycline treatment normalized the PWV in 6-month old Marfan mice compared to non-treated Marfan aorta. Conclusions: Our findings show that long-term doxycycline treatment has protective effects in terms of reduction of aortic dilatation and stiffness in Marfan mice.

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder presented with multiple cafe-au-lait spots, Lisch nodules,multiple nerve sheath tumors, and cardiovascular complications such as ischemia...

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder, characterized by multiple cafe-au-lait spots, iris harmartomas, multiple nerve sheath tumors. Patients also present with heart and cereb...

In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.

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.