Mouse Model Of Marfan Syndrome Research Articles

Allopurinol blocks aortic aneurysm in a mouse model of Marfan syndrome via reducing aortic oxidative stress.

Increasing evidence indicates that redox stress participates in MFS aortopathy, though its mechanistic contribution is little known. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mouse aortae. Here we address the contribution of xanthine oxidoreductase (XOR), which catabolizes purines into uric acid and ROS in MFS aortopathy. In aortic samples from MFS patients, XOR protein expression, revealed by immunohistochemistry, increased in both the tunicae intima and media of the dilated zone. In MFS mice (Fbn1C1041G/+), aortic XOR mRNA transcripts and enzymatic activity of the oxidase form (XO) were augmented in the aorta of 3-month-old mice but not in older animals. The administration of the XOR inhibitor allopurinol (ALO) halted the progression of aortic root aneurysm in MFS mice. ALO administrated before the onset of the aneurysm prevented its subsequent development. ALO also inhibited MFS-associated endothelial dysfunction as well as elastic fiber fragmentation, nuclear translocation of pNRF2 and increased 3'-nitrotyrosine levels, and collagen maturation remodeling, all occurring in the tunica media. ALO reduced the MFS-associated large aortic production of H2O2, and NOX4 and MMP2 transcriptional overexpression. Allopurinol interferes in aortic aneurysm progression acting as a potent antioxidant. This study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and warrants the evaluation of ALO therapy in MFS patients.

No prominent role for complement C1-esterase inhibitor in Marfan syndrome mice.

Marfan syndrome (MFS) is a connective tissue disorder causing aortic aneurysm formation. Currently, only prophylactic aortic surgery and blood pressure lowering drugs are available to reduce the risk for aortic rupture. Upon whole genome sequencing (WGS) of a Marfan family, we identified a complement gene C1R variant (p.Ser152Leu), which associated with the severe aortic patients. Therefore, we assessed the role of complement activation in MFS aortic tissue. Expression of various complement genes and proteins was detected in human and murine MFS aneurysm tissue, which prompted us to study complement inhibition in MFS mice. Treatment of the Fbn1C1041G/+ MFS mice with human plasma-derived C1-esterase inhibitor Cetor® resulted in reduced complement deposition, decreased macrophage influx in the aorta and lower circulating TNFα levels. However, in line with previous anti-inflammatory treatments, complement inhibition did not change aortic dilatation rate in this MFS mouse model. Thus, while complement factors/C3 activation were detected in human/murine MFS aorta, Cetor® had no effect on aortic dilatation in MFS mice, indicating that complement inhibition is not a suitable treatment strategy in MFS.

Abstract P1112: Inhibition Of Wnt Signaling Protects Against Myxomatous Valve Disease In A Mouse Model Of Marfan Syndrome

Introduction: Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome (MFS) includes valve leaflet thickening and increased immune cell infiltration. However, the underlying mechanisms by which immune cells are recruited in MFS valves remain undefined. Hypothesis: Inhibition of Wnt signaling prevents immune cell recruitment and MVD progression in MFS valves. Methods: Mice with the mutation of Fibrillin 1 ( Fbn1 C1039G/+ ) recapitulate histopathological features of MFS and Wnt signaling activity was detected in TCF/Lef-lacZ reporter mice. Inhibition of Wnt signaling was achieved by conditional induction of the soluble Wnt inhibitor Dkk1 in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ; Fbn1 C1039G/+ mice. Dietary doxycycline was administered for one month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for immune cells were performed. Results: Wnt signaling as detected by TCF/Lef-reporters is activated early in mitral valve disease progression, prior to immune cell infiltration in Fbn1 C1039G/+ mice. Inhibition of Wnt signaling by Dkk1 induction at 1-month of age reduced valve leaflet thickness with decreased CD45+ leukocytes in Fbn1 C1039G/+ mice. However, later Wnt inhibition starting at 2 months did not alter valve leaflet thickness. Conclusions: Inhibition of early Wnt signaling ameliorates myxomatous valve degeneration and immune cell infiltration previously demonstrated to be required for progression of MVD.

Effect of the General Inhibitor of Nitric Oxide Synthase (L‐NAME) On Cardiac and Aortic Function and Structure in A Mouse Model of Marfan Syndrome‐Associated Aortic Aneurysm

Marfan Syndrome (MFS) is an autosomal‐dominant connective tissue disorder caused by missense mutations in the gene that encodes for Fibrillin‐1 (FBN1). FBN1 is an extracellular matrix glycoprotein that functions as a scaffolding protein during the formation and maturation of elastic fibers. The mechanism underlying MFS pathogenesis stems from a crosstalk between the Angiotensin‐II pathway and over activation of transforming growth factor‐beta signaling. There are several clinical manifestations seen in MFS, but the most serious and life‐threatening is the aortic aneurysm due to degradation and fragmentation of aortic wall elastin fibers. If left untreated, aortic aneurysm progression may lead to dissection and rupture.Previous studies in the MFS mouse model have shown that the progression of aortic aneurysm is associated with a significant decline in endothelial function and nitric oxide (NO) production in the aortic wall, and that overexpression of endothelial nitric oxide synthase (eNOS) could delay the progression of aortic root aneurysm in MFS mice. A recent study in our lab has also shown that mild aerobic exercise can block aortic aneurysm in MFS mice. It is well established that exercise can improve endothelial function mainly through an increase in eNOS‐mediated NO production. Therefore, we tested the hypothesis that mild aerobic exercise prevents aortic aneurysm progression in the mouse model of MFS mainly through increasing the production of NO within the aortic wall.To test this hypothesis, C57BL/6 WT and MFS mice were subjected to daily treadmill exercise (at 55% of VO2max, starting at 4 weeks of age) in the absence or presence of the general inhibitor of NO production, N(G)‐Nitro‐L‐arginine methyl ester (L‐NAME) at 0.05 g per 100 mL of drinking water. At 6‐months of age, in vivo imaging of the aortic root diameter and pulse wave velocity (PWV) as an index of wall stiffness, was performed using a high‐resolution Vevo2100 ultrasound system. The aortic root diameter at the sinus of Valsalva, and pulse wave velocity were both increased in MFS mice compared to WT, however MFS mice subjected to treadmill exercise had significantly decreased aortic root diameter and pulse wave velocity compared to sedentary MFS mice. In situ analysis of aortic wall integrity was analyzed at 6 months by Van Geison staining. Our results revealed that MFS mice had an increase in elastin fragment count and a decrease in elastin fiber length compared to WT. Interestingly, MFS mice subjected to exercise had fewer elastin fragment count and an increase in elastin fiber length compared to sedentary MFS mice. Interestingly, L‐NAME treatment reversed the protective effects of exercise on aortic root diameter and pulse wave velocity in MFS mice to a level that was not different than MFS sedentary groups. This study provides preliminary evidence that mild aerobic exercise blocks the progression of aortic root dilation in MFS mice through increasing the production of NO within the aortic wall, and further emphasizes the value of increased physical activity in aneurysm population.

Characterization of cerebral arteries function and structure in a mouse model of Marfan syndrome: effects of exercise and Losartan treatment

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin‐1 (FBN1) gene. This mutation manifests in a variety of phenotypic changes in the musculoskeletal, cardiovascular, and pulmonary systems, with a notable vascular effect leading to aortic aneurysm, dissection, and rupture. This results in a significant increase in morbidity and mortality in these patients. Studies have shown that there is a connection between MFS and intracranial aneurysms (IA). The prevalence of IA in patients with aortic disease is quadrupled compared to that in the general population. There is a modestly increased prevalence of ischemic stroke in hospitalized patients with MFS when compared with healthy controls. The reduced cerebral blood flow triggered by cardiac and peripheral vascular dysfunction could further make the brain more vulnerable to vascular dementia and Alzheimer’s pathology. Studies have found that 50% school‐age children with MFS had one or more neuropsychologic deficits. Despite the reported neurological complications in MFS patients, our understanding of cerebrovascular function and structure in MFS is very limited. Losartan, an AT1R blocker has shown positive effects on slowing the progression of aortic root aneurysm in the well‐established mouse model of MFS (Fbn1 +/‐ p. Cys1041Gly). Alternatively, the cardiovascular benefits of moderate exercise training have been well documented in the literature. In addition, studies have shown that aerobic exercise can improve cognitive function, decrease neuropsychiatric and neurodegenerative symptoms. In this study we aim to investigate the impact of MFS pathogenesis and Losartan treatment and mild aerobic exercise on other vessels such as the posterior cerebral artery, coronary and pulmonary arteries.At 4 weeks of age, male and female mice were divided into experimental groups: control (Ctrl), MFS, MFS + Losartan, MFS + exercise. MFS mice received 0.6 g/L of Losartan in drinking water or subjected to an exercise regimen of 8m/min, 30min/day, 5days/week. At 6 months of age, in vivo ultrasound imaging was performed to measure aortic pulse wave velocity (PWV), and peak blood flow of the coronary, pulmonary and posterior cerebral arteries. Blood pressure (BP) measurements were also taken using the tail‐cuff method.Our results showed that PWV, an index of aortic stiffness is increased in MFS mice compared to Ctrl, while Losartan and exercise reduced the PWV in MFS mice bringing it back to the values of healthy Ctrl. The posterior cerebral artery peak blood flow was significantly reduced in MFS and MFS + Losartan groups compared to Ctrl. No significant difference was observed in the MFS + exercise group compared to Ctrl. The coronary and pulmonary peak blood flow showed no significant change between MFS and control groups and MFS and intervention groups. There was also no statistical significance in the systolic and diastolic BP between the groups.This study provides an early insight into the disease progression of MFS as well as investigates the preliminary potential effects of Losartan and exercise on peak blood flow in coronary, pulmonary and cerebral arteries in the well‐established MFS mouse model.

Increased Neurovascular Vulnerability to Mild Traumatic Brain Injury in a Mouse Model of Marfan Syndrome

Over 69 million traumatic brain injuries (TBIs) are reported annually worldwide, where the geriatric population are at greatest risk for prolonged morbidity and mortality. Cerebrovascular aging contributes to this increased vulnerability through extracellular matrix (ECM) impairment, vascular wall weakening and stiffening, blood‐brain barrier (BBB) permeability, and exacerbated cytokine production, yet the mechanisms behind these contributions are minimally investigated. In order to study these targets efficiently and with minimal variability, an animal model presenting these cerebrovascular alterations is required. Fibrillin‐1 (Fbn1) protein plays a role in elasticity and inflammation, and its mutation is known to lead to ECM impairment and vessel wall weakening induced by increased availability in transforming growth factor‐b (TGF‐b) and downstream production of matrix metalloproteinases (MMPs)‐2/‐9. In mice, this mutation induces vascular dysfunction by 6M of age and is a well‐established model of Marfan Syndrome.In this study, Fbn1+/‐ were utilized to test the hypothesis that Fbn1 mutation accelerates cerebrovascular rigidity, BBB permeability, and neurological alterations associated with aging, leaving the brain more vulnerable to diffuse TBI. In 6M‐ and 12M‐old Fbn1+/‐ and C57BL/6 wildtype (WT) male and female mice, posterior cerebral artery (PCA) blood flow (n=8‐10), PCA rupture point (N=3‐5/group), BBB permeability (N=2‐3/group), injury viability (N=5‐8/group), neurobehavioral severity scale (NSS) outcomes (N=10‐11/group), and microglial perturbation (N=1/group) were assessed.Our data shows that 6M‐old Fbn1+/‐ mice have decreased PCA blood flow (p<0.05), compromised PCA wall strength (p<0.05), increased BBB permeability (p<0.05), and elevated NSS scores (p<0.05) as compared to 6M‐old WT, and similar to 12M‐old WT mice. Preliminary investigation of iba‐1 staining for microglia, a supported assessment of neuroinflammation, suggests increased microglial activation in 6M‐old Fbn1+/‐ mice compared to age‐matched WT mice. To investigate vulnerability to mild TBI (mTBI), varying pulse pressures (1.11‐1.32atm) were applied to the brains of 6M‐old WT (1.25‐1.32atm) and Fbn1+/‐ (1.17‐1.21atm) mice using midline fluid percussion injury, where Fbn1+/‐ mice required lowered pressure to induce mTBI righting reflex times (5‐10 minutes).These data support that cerebrovascular aging contributes to outcome after TBI. Furthermore, this study demonstrates that Fbn1 mutation plays a critical role in the vulnerability of the aged brain to mTBI. Using this novel approach of a Marfan Syndrome mouse model that isolates cerebrovascular aging allows for the mechanistic study of potential Fbn1 downstream pathways that could serve as a therapeutic target for prevention and management of post‐concussive symptoms.

Evaluation of the effects of mild aerobic exercise and Losartan treatment on aortic endothelial function in a mouse model of Marfan syndrome

Marfan Syndrome (MFS) is an autosomal dominant disorder in a family of connective tissue disorders. It is well established that vascular complications in MFS is associated with endothelial dysfunction, leading to a whole host of phenotypic abnormalities. A predominant clinical concern is the development of aortic aneurysm with possible subsequent dissection and rupture.In the present study, we have utilized the ex‐vivo isometric myograph technique to examine the reactivity of isolated aortic segments from control and MFS mice in the presence or absence of different in‐vivo treatments and pharmacological agents. More specifically, we assessed the impact of mild aerobic exercise on endothelial function, aortic root growth, and aortic wall elasticity in a well‐established mouse model of MFS‐associated aortic aneurysm with special focus on nitric oxide production. Nitric oxide, as frequently demonstrated in the literature, plays a crucial role in maintaining normal vascular structure and function. In the current study, we examine the potential effects of mild aerobic exercise in the absence or presence of a potent general inhibitor of nitric oxide synthase (NOS), N5‐[imino(nitroamino)methyl]‐L‐ornithine, methyl ester, monohydrochloride (L‐NAME) on aortic contraction and endothelium‐mediated vasorelaxation in 6‐month‐old MFS mice carrying Fbn1 mutation and age‐ and sex‐matched C57BL/6J wildtype (WT) controls (male and females).At 4‐6 weeks of age, MFS mice were subjected to daily treadmill exercise (at 55% of VO2max, 8m/min, 30min/day, 5days/week) and/or drug treatment for a duration of 5 months. At 6 months of age, 2mm descending aortic rings were collected and mounted onto a small chamber myograph in order to measure the vascular reactivity in response to different vasoconstricting and vasodilating agents.Our study shows marked reduction in the phenylephrine (PE)‐induced contraction [50 µmol] and aortic wall rupture point in MFS mice aorta as compared to healthy controls. Treatment with either aerobic exercise or Losartan, an angiotensin II type 1 receptor blocker (ARB), rescued rupture point closer to WT control, but led to further decrease in PE contraction compared to MFS sedentary mice, mainly due to further increase in NO production within the aortic wall. In addition, direct inhibition of NO production via L‐Name, a nitric oxide synthase (NOS) inhibitor, effectively reduced PE‐induced contraction in MFS mice aorta that were subjected to exercise or Losartan treatment, further confirming that both exercise and Losartan treatment cause an increase in NO production.This study highlights the importance of endothelial function in improving the function and structure of aortic wall during the progression of aortic aneurysm in MFS mice and establish the notion that the protective effects of aerobic exercise and Losartan treatments are potentially through improving endothelium‐mediated nitric oxide production.

Latent TGFβ-binding proteins 1 and 3 protect the larval zebrafish outflow tract from aneurysmal dilatation.

ABSTRACTAortic root aneurysm is a common cause of morbidity and mortality in Loeys-Dietz and Marfan syndromes, where perturbations in transforming growth factor beta (TGFβ) signaling play a causal or contributory role, respectively. Despite the advantages of cross-species disease modeling, animal models of aortic root aneurysm are largely restricted to genetically engineered mice. Here, we report that zebrafish devoid of the genes encoding latent-transforming growth factor beta-binding protein 1 and 3 (ltbp1 and ltbp3, respectively) develop rapid and severe aneurysm of the outflow tract (OFT), the aortic root equivalent. Similar to syndromic aneurysm tissue, the distended OFTs display evidence for paradoxical hyperactivated TGFβ signaling. RNA-sequencing revealed significant overlap between the molecular signatures of disease tissue from mutant zebrafish and a mouse model of Marfan syndrome. Moreover, chemical inhibition of TGFβ signaling in wild-type animals phenocopied mutants but chemical activation did not, demonstrating that TGFβ signaling is protective against aneurysm. Human relevance is supported by recent studies implicating genetic lesions in LTBP3 and, potentially, LTBP1 as heritable causes of aortic root aneurysm. Ultimately, our data demonstrate that zebrafish can now be leveraged to interrogate thoracic aneurysmal disease and identify novel lead compounds through small-molecule suppressor screens.This article has an associated First Person interview with the first author of the paper.

Impact of Notch3 Activation on Aortic Aneurysm Development in Marfan Syndrome.

Background The leading cause of mortality in patients with Marfan syndrome (MFS) is thoracic aortic aneurysm and dissection. Notch signaling is essential for vessel morphogenesis and function. However, the role of Notch signaling in aortic pathology and aortic smooth muscle cell (SMC) differentiation in Marfan syndrome (MFS) is not completely understood. Methods RNA-sequencing on ascending aortic tissue from a mouse model of MFS, Fbn1mgR/mgR, and wild-type controls was performed. Notch 3 expression and activation in aortic tissue were confirmed with real-time RT-PCR, immunohistochemistry, and Western blot. Fbn1mgR/mgR and wild-type mice were treated with a γ-secretase inhibitor, DAPT, to block Notch activation. Aortic aneurysms and rupture were evaluated with connective tissue staining, ultrasound, and life table analysis. Results The murine RNA-sequencing data were validated with mouse and human MFS aortic tissue, demonstrating elevated Notch3 activation in MFS. Data further revealed that upregulation and activation of Notch3 were concomitant with increased expression of SMC contractile markers. Inhibiting Notch3 activation with DAPT attenuated aortic enlargement and improved survival of Fbn1mgR/mgR mice. DAPT treatment reduced elastin fiber fragmentation in the aorta and reversed the differentiation of SMCs. Conclusions Our data demonstrated that matrix abnormalities in the aorta of MFS are associated with increased Notch3 activation. Enhanced Notch3 activation in MFS contributed to aortic aneurysm formation in MFS. This might be mediated by inducing a contractile phenotypic change of SMC. Our results suggest that inhibiting Notch3 activation may provide a strategy to prevent and treat aortic aneurysms in MFS.

Hyperkyphosis is not dependent on bone mass and quality in the mouse model of Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant disease affecting cardiovascular, ocular and skeletal systems. It is caused by mutations in the fibrillin-1 (FBN1) gene, leading to structural defects of connective tissue and increased activation of TGF-β. Angiotensin II (ang-II) is involved in TGF-β activity and in bone mass regulation. Inhibition of TGF-β signaling by blockage of the ang-II receptor 1 (AT1R) via losartan administration leads to improvement of cardiovascular and pulmonary phenotypes, but has no effect on skeletal phenotype in the haploinsufficient mouse model of MFS mgR, suggesting a distinct mechanism of pathogenesis in the skeletal system. Here we characterized the skeletal phenotypes of the dominant-negative model for MFS mgΔlpn and tested the effect of inhibition of ang-II signaling in improving those phenotypes. As previously shown, heterozygous mice present hyperkyphosis, however we now show that only males also present osteopenia. Inhibition of ang-II production by ramipril minimized the kyphotic deformity, but had no effect on bone microstructure in male mutant animals. Histological analysis revealed increased thickness of the anterior longitudinal ligament (ALL) of the spine in mutant animals (25.8±6.3 vs. 29.7±7.7μm), coupled with a reduction in type I (164.1±8.7 vs. 139.0±4.4) and increase in type III (86.5±10.2 vs. 140.4±5.6) collagen in the extracellular matrix of this ligament. In addition, we identified in the MFS mice alterations in the erector spinae muscles which presented thinner muscle fibers (1035.0±420.6 vs. 655.6±239.5μm2) surrounded by increased area of connective tissue (58.17±6.52 vs. 105.0±44.54μm2). Interestingly, these phenotypes were ameliorated by ramipril treatment. Our results reveal a sex-dependency of bone phenotype in MFS, where females do not present alterations in bone microstructure. More importantly, they indicate that hyperkyphosis is not a result of osteopenia in the MFS mouse model, and suggest that incompetent spine ligaments and muscles are responsible for the development of that phenotype.

Age and sex dependency of thoracic aortopathy in a mouse model of Marfan syndrome.

Thoracic aortic aneurysm is one of the manifestations of Marfan syndrome (MFS) that is known to affect men more severely than women. However, the incidence of MFS is similar between men and women. The aim of this study is to show that during pathological aortic dilation, sex-dependent severity of thoracic aortopathy in a mouse model of MFS translates into sex-dependent alterations in cells and matrix of the ascending aorta, consequently affecting aortic biomechanics. Fibrillin-1 C1041G/+ (Het) mice were used as a mouse model of MFS. Ultrasound measurements from 3 to 12 mo showed increased aortic diameter in Het aorta, with larger percentage increase in diameter for males compared with females. Immunohistochemistry showed decreased contractile smooth muscle cells in Het aortic wall compared with healthy aorta, which was accompanied by decreased contractility measured by wire myography. Elastin autofluorescence, second-harmonic generation microscopy of collagen fibers, and passive biomechanical assessments using myography showed more severe damage to elastin fibers, increased medial fibrosis, and increased stiffness of the aortic wall in MFS males but not females. Male and female Het mice showed increased expression of Sca-1-positive adventitial progenitor cells versus controls at young ages. In agreement with clinical data, Het mice demonstrate sex-dependent severity of thoracic aortopathy. It was also shown that aging exacerbates the disease state especially for males. Our findings suggest that female mice are protected from progression of aortic dilation at early ages, leading to a lag in aneurysm growth.NEW & NOTEWORTHY Male Fbn1C1041G/+ mice show more severe thoracic aortic changes compared with females, especially at 12 mo of age. Up to 6 mo of age, Sca-1+ smooth muscle progenitor cells are more abundant in the adventitia of both male and female Fbn1 Het mice compared with wild types (WTs). Male and female Het mice show similar patterns of expression of Sca-1+ cells at early ages.

Abstract 122: Pharmacological Inhibition Of Macrophage Infiltration Prevents Myxomatous Valve Degeneration In Marfan Syndrome

Introduction: Myxomatous valve degeneration (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. Currently, there is no medical therapy for the treatment of MVD. MVD in a mouse model of Marfan syndrome (MFS) is characterized leaflet thickening and increased macrophage infiltration, which are reduced with loss of C-C chemokine receptor type 2 (CCR2). However, the specific contributions of macrophages to pathological extracellular matrix (ECM) remodeling and underlying mechanisms are unknown. Hypothesis: Inhibition of macrophage infiltration by a CCR2 inhibitor blocks ECM abnormalities and MVD progression in mitral valves of MFS mice by suppressing the response to cytokine/chemokines. Methods: Mice with the mutation of Fibrillin 1 (Fbn1 C1039G/+ ) recapitulate histopathological features of MFS. Here, we tested the efficacy of a selective CCR2 antagonist RS504393 in the valves of MFS mice in the initiation (1-month-old) and the progression (2-month-old) of MVD, respectively. MFS mice were intraperitoneally injected with RS504393 at 2 mg/kg/d for 30 days. Histological evaluation and immunofluorescence for macrophages and ECM were performed. RNAseq was performed in mitral valves from 2-month old Fbn1 C1039G/+ mice with CCR2 knockout (CCR2 RFP/RFP ). Results: MFS valves revealed ECM abnormalities characterized by collagen fragmentation and proteoglycan accumulation. RS504393 treatment reduced infiltrating macrophages (MHCII+, CCR2+) in myxomatous valves. Remarkably, RS504393 was protective against both the initiation and the progression of MVD, detected by decreased mitral valve thickness and prevention of pathological ECM remodeling in MFS mice. RNAseq data confirmed increased leukocyte activation involved in immune response and abnormal extracellular matrix remodeling in MFS valves. CCR2 deficiency blocked macrophage infiltration and inhibited the response to cytokines in Fbn1 C1039G/+ valves. Conclusions: Our results show that macrophage infiltration is critical for progressive MVD. Moreover, CCR2 inhibition ameliorates MVD progression by preventing immune response. Thus, the CCR2 inhibitor RS504393 is a potential pharmacological candidate to treat MVD in MFS.

Nitro-oleic acid reduces thoracic aortic aneurysm progression in a mouse model of Marfan syndrome.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the Fibrillin-1 gene. It is associated with formation of thoracic aortic aneurysms that can potentially be a life-threatening condition due to aortic rupture or dissection. Excessive non-canonical transforming growth factor beta signalling, mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2), as well as inducible nitric oxide synthase (NOS2)-dependent nitric oxide production, have been identified to drive aortic pathology in MFS through induction of elastin fragmentation and smooth muscle cell apoptosis. Despite promising results in animal studies, specific pharmacological interventions approved for clinical use in patients with MFS-related aortic disease are rare. Nitro-oleic acid (NO2-OA) is an endogenously generated signalling modulator, which is available as an oral compound and has been shown to inhibit ERK1/2 activation and NOS2 expression in different disease models, thereby exerting promising therapeutic effects. In this study, we investigated whether NO2-OA decreases aortic dilation in MFS. Eight-week-old MFS (Fbn1C1041G/+) mice were treated with NO2-OA or vehicle for 4 weeks via subcutaneously implanted osmotic minipumps. Echocardiography indicated progressive ascending aortic dilation and wall stiffening in MFS mice, which was significantly attenuated by NO2-OA treatment. This protective effect was mediated by inhibition of aortic ERK1/2, Smad2 as well as nuclear factor kappa B overactivation and consequent attenuation of elastin fragmentation by matrix metalloproteinase 2, apoptosis, and collagen deposition. Critically, the therapeutic efficacy of NO2-OA in MFS was further emphasized by demonstrating its capability to reduce lethal aortic complications in Fbn1C1041G/+ mice challenged with Angiotensin II. NO2-OA distinctly attenuates progression of aortic dilation in MFS via modulation of well-established disease-mediating pathways, thereby meriting further investigation into its application as a therapeutic agent for the treatment of this condition.

Anti-TGFβ (Transforming Growth Factor β) Therapy With Betaglycan-Derived P144 Peptide Gene Delivery Prevents the Formation of Aortic Aneurysm in a Mouse Model of Marfan Syndrome.

We investigated the effect of a potent TGFβ (transforming growth factor β) inhibitor peptide (P144) from the betaglycan/TGFβ receptor III on aortic aneurysm development in a Marfan syndrome mouse model. We used a chimeric gene encoding the P144 peptide linked to apolipoprotein A-I via a flexible linker expressed by a hepatotropic adeno-associated vector. Two experimental approaches were performed: (1) a preventive treatment where the vector was injected before the onset of the aortic aneurysm (aged 4 weeks) and followed-up for 4 and 20 weeks and (2) a palliative treatment where the vector was injected once the aneurysm was formed (8 weeks old) and followed-up for 16 weeks. We evaluated the aortic root diameter by echocardiography, the aortic wall architecture and TGFβ signaling downstream effector expression of pSMAD2 and pERK1/2 by immunohistomorphometry, and Tgfβ1 and Tgfβ2 mRNA expression levels by real-time polymerase chain reaction. Marfan syndrome mice subjected to the preventive approach showed no aortic dilation in contrast to untreated Marfan syndrome mice, which at the same end point age already presented the aneurysm. In contrast, the palliative treatment with P144 did not halt aneurysm progression. In all cases, P144 improved elastic fiber morphology and normalized pERK1/2-mediated TGFβ signaling. Unlike the palliative treatment, the preventive treatment reduced Tgfβ1 and Tgfβ2 mRNA levels. P144 prevents the onset of aortic aneurysm but not its progression. Results indicate the importance of reducing the excess of active TGFβ signaling during the early stages of aortic disease progression.

Abnormal mitochondria is associated with aortic aneurysm in a mouse model of Marfan Syndrome

Aortic aneurysms, or the pathogenic remodeling and stiffening of the aorta extracellular matrix (ECM), are usually undetectable until excessive expansion, dissection, or rupture occurs. The understanding of the molecular mechanisms of aneurysm and dissection remains incomplete, but heritable syndromes such as Marfan Syndrome (MFS), where genetic mutations result in aneurysm phenotypes, offer models to discover functional pathways. Recent work in our lab and others have identified a potential association between reduced mitochondrial regulators, such as PGC1alpha, mitochondrial content, and aneurysm. These associations have not been fully described to date in a model of classical MFS driven by Fibrillin-1 (FBN1) mutation. We hypothesized that mitochondrial content and integrity would be disrupted in the MFS aorta and isolated MFS smooth muscle cell models. Western blot for several key mitochondrial proteins associated with oxidative phosphorylation (NDUFB8, SDHB, UQCRC2, MTCO1, ATP5A) demonstrated significant reduction in the aortic root of MFS mice compared to wild type (WT) mice. Consistent with these in vivo findings, we observed a reduced total quantity of mitochondria relative to total cell volume in isolated primary MFS relative to WT ascending aortic smooth muscle cells. Furthermore, analysis of mitochondrial morphology revealed that MFS primary lines have more fragmented mitochondrial networks than WT lines, indicating abnormal structural regulation of mitochondrial networks in MFS. Through these initial studies we have found indicators of potential dysfunction in the networks of MFS mitochondria. Future work will elucidate the functional consequences of these abnormalities and decipher their potential role in the pathogenesis of aneurysm progression in MFS.

Pharmacological Inhibition of Macrophage Infiltration Prevents Myxomatous Valve Degeneration in Marfan Syndrome

Background Myxomatous valve degeneration (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome (MFS) is characterized leaflet thickening and increased macrophage infiltration which are reduced with loss of C-C chemokine receptor type 2 (CCR2). However, the specific contributions of macrophages to pathological extracellular matrix (ECM) remodeling and the underlying mechanisms are unknown. Methods Mice with the mutation of Fibrillin 1 (Fbn1C1039G/+) recapitulate histopathological features of MFS. ECM remodeling in mitral valves was assessed by detecting collagen and proteoglycan in Fbn1C1039G/+; CCR2RFP/RFP mice. Here, we tested the efficacy of a selective CCR2 antagonist RS504393 in the valves of MFS model in the initiation (1-month-old) and the progression (2-month-old) of MVD, respectively. MFS mice were intraperitoneally injected with RS504393 at 2 mg/kg/d for 1 month. Histological evaluation and immunofluorescence for macrophages and ECM were performed. Results MFS valves revealed ECM abnormalities characterized by collagen fragmentation and proteoglycan accumulation. Deficiency of CCR2 inhibited ECM abnormalities in MFS mitral valves. RS504393 treatment reduced infiltrating macrophages (MHCII+, CCR2+) in myxomatous valves. Remarkably, RS504393 was protective against both the initiation and the progression of MVD, detected by decreased mitral valve thickness and prevented pathological ECM remodeling in MFS mice. Conclusion Our results suggest the deficiency of CCR2 inhibits macrophage infiltration and abnormal ECM remodeling. In addition, the CCR2 inhbitor RS504393 is a potential pharmacological candidate to treat MVD in MFS.

Evaluation of the effects of Marfan pathogenesis and losartan treatment on coronary and cerebral arteries in the well‐established Marfan Syndrome mouse model

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin-1 (FBN1) gene. This mutation manifests in a variety of phenotypic changes with a notable cardiovascular effect leading to aortic aneurysm, dissection, and rupture. This results in a significant increase in morbidity and mortality in these patients. The angiotensin II type I receptor (AT1R) signalling pathway has previously been shown to contribute to the cardiovascular effects in the progression of this disease. Prior studies have demonstrated the protective effects of Losartan, an AT1R blocker, on slowing the progression of aortic root aneurysm in the well-established mouse model of MFS (Fbn1 +/- p. Cys1041Gly). In this study, we are further investigating the impact of MFS pathogenesis and Losartan treatment on other vessels such as the posterior cerebral artery and coronary artery. Mice (male + female) were divided into experimental groups: Control, MFS, MFS + 0.6g/L losartan. Drug therapy consisted of 0.6g/L of losartan in drinking water. Water intakes were recorded to ensure the mice were receiving the treatment. At 7 months of age, in vivo ultrasound imaging was performed to measure aortic diameters, aortic pulse wave velocity (PWV), and peak blood flow of the coronary and posterior cerebral arteries. In order to assess elastin fragmentation in the aortic wall, 5μm aortic cross-sections were subjected to Van Geison elastin staining. Blood pressure (BP) measurements were also obtained, using the tail-cuff method. Our results demonstrate that Losartan reduces aortic root diameters at the sinus of Valsalva and reduced the PWV, an index of aortic stiffness, in MFS mice at 7 months of age. Losartan also reduces elastin fragmentation in the aortic wall of MFS mice. The systolic coronary peak flow demonstrates a statistically significant decrease in MFS when compared to CTRL. Losartan treatment has no effect on the systolic coronary peak flow. There is no statistical significance in diastolic coronary peak flow between CTRL vs. MFS and MFS vs. MFS + Losartan. There is no statistical significance in comparison of the posterior cerebral artery peak flow between CTRL to MFS and MFS to MFS + Losartan. There is also no statistical significance in the systolic and diastolic BP between the groups. Our results show that Losartan has beneficial effects in delaying the progression of aortic aneurysm, reduces vessel wall stiffness, and reduces elastin fragmentation in the aortic root. This study also provides an early insight into the disease progression of MFS in the less investigated coronary and cerebral arteries, as well as investigates the preliminary potential effects of Losartan on peak flow in coronary and cerebral vessels in the well-established MFS mouse model.

Extracellular Tuning of Mitochondrial Respiration Leads to Aortic Aneurysm.

Marfan syndrome (MFS) is an autosomal dominant disorder of the connective tissue caused by mutations in the FBN1 (fibrillin-1) gene encoding a large glycoprotein in the extracellular matrix called fibrillin-1. The major complication of this connective disorder is the risk to develop thoracic aortic aneurysm. To date, no effective pharmacologic therapies have been identified for the management of thoracic aortic disease and the only options capable of preventing aneurysm rupture are endovascular repair or open surgery. Here, we have studied the role of mitochondrial dysfunction in the progression of thoracic aortic aneurysm and mitochondrial boosting strategies as a potential treatment to managing aortic aneurysms. Combining transcriptomics and metabolic analysis of aortas from an MFS mouse model (Fbn1c1039g/+) and MFS patients, we have identified mitochondrial dysfunction alongside with mtDNA depletion as a new hallmark of aortic aneurysm disease in MFS. To demonstrate the importance of mitochondrial decline in the development of aneurysms, we generated a conditional mouse model with mitochondrial dysfunction specifically in vascular smooth muscle cells (VSMC) by conditional depleting Tfam (mitochondrial transcription factor A; Myh11-CreERT2Tfamflox/flox mice). We used a mouse model of MFS to test for drugs that can revert aortic disease by enhancing Tfam levels and mitochondrial respiration. The main canonical pathways highlighted in the transcriptomic analysis in aortas from Fbn1c1039g/+ mice were those related to metabolic function, such as mitochondrial dysfunction. Mitochondrial complexes, whose transcription depends on Tfam and mitochondrial DNA content, were reduced in aortas from young Fbn1c1039g/+ mice. In vitro experiments in Fbn1-silenced VSMCs presented increased lactate production and decreased oxygen consumption. Similar results were found in MFS patients. VSMCs seeded in matrices produced by Fbn1-deficient VSMCs undergo mitochondrial dysfunction. Conditional Tfam-deficient VSMC mice lose their contractile capacity, showed aortic aneurysms, and died prematurely. Restoring mitochondrial metabolism with the NAD precursor nicotinamide riboside rapidly reverses aortic aneurysm in Fbn1c1039g/+ mice. Mitochondrial function of VSMCs is controlled by the extracellular matrix and drives the development of aortic aneurysm in Marfan syndrome. Targeting vascular metabolism is a new available therapeutic strategy for managing aortic aneurysms associated with genetic disorders.

AAV-mediated AP-1 decoy oligonucleotide expression inhibits aortic elastolysis in a mouse model of Marfan syndrome.

Marfan syndrome is one of the most common inherited disorders of connective tissue caused by fibrillin-1 mutations, characterized by enhanced transcription factor AP-1 DNA binding activity and subsequently abnormally increased expression and activity of matrix-metalloproteinases (MMPs). We aimed to establish a novel adeno-associated virus (AAV)-based strategy for long-term expression of an AP-1 neutralizing RNA hairpin (hp) decoy oligonucleotide (dON) in the aorta to prevent aortic elastolysis in a murine model of Marfan syndrome. Using fibrillin-1 hypomorphic mice (mgR/mgR), aortic grafts from young (9 weeks old) donor mgR/mgR mice were transduced ex vivo with AAV vectors and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Grafts were explanted after 30 days. For in vitro studies, isolated primary aortic smooth muscle cells (SMCs) from mgR/mgR mice were used. Elastica-van-Giesson staining visualized elastolysis, reactive oxygen species (ROS) production was assessed using dihydroethidine staining. RNA F.I.S.H. verified AP-1 hp dON generation in the ex vivo transduced aortic tissue. MMP expression and activity were assessed by western blotting and immunoprecipitation combined with zymography.Transduction resulted in stable therapeutic dON expression in endothelial and SMCs. MMP expression and activity, ROS formation as well as expression of monocyte chemoattractant protein-1 were significantly reduced. Monocyte graft infiltration declined and the integrity of the elastin architecture was maintained. RNAseq analysis confirmed the beneficial effect of AP-1 neutralization on the pro-inflammatory environment in SMCs. This novel approach protects from deterioration of aortic stability by sustained delivery of nucleic acids-based therapeutics and further elucidated how to interfere with the mechanism of elastolysis.

The Molecular Genetics of Marfan Syndrome.

Marfan syndrome (MFS) is a complex connective tissue disease that is primarily characterized by cardiovascular, ocular and skeletal systems disorders. Despite its rarity, MFS severely impacts the quality of life of the patients. It has been shown that molecular genetic factors serve critical roles in the pathogenesis of MFS. FBN1 is associated with MFS and the other genes such as FBN2, transforming growth factor beta (TGF-β) receptors (TGFBR1 and TGFBR2), latent TGF-β-binding protein 2 (LTBP2) and SKI, amongst others also have their associated syndromes, however high overlap may exist between these syndromes and MFS. Abnormalities in the TGF-β signaling pathway also contribute to the development of aneurysms in patients with MFS, although the detailed molecular mechanism remains unclear. Mutant FBN1 protein may cause unstableness in elastic structures, thereby perturbing the TGF-β signaling pathway, which regulates several processes in cells. Additionally, DNA methylation of FBN1 and histone acetylation in an MFS mouse model demonstrated that epigenetic factors play a regulatory role in MFS. The purpose of the present review is to provide an up-to-date understanding of MFS-related genes and relevant assessment technologies, with the aim of laying a foundation for the early diagnosis, consultation and treatment of MFS.