Mouse Model Of Marfan Syndrome Research Articles

Increased upper airway collapsibility in a mouse model of Marfan syndrome

Marfan syndrome (MFS) is a genetic disorder caused by mutations in the FBN1 gene that codifies for fibrilin-1. MFS affects elastic fiber formation and the resulting connective tissue shows abnormal tissue laxity and organization. Although an increased prevalence of obstructive sleep apnea among patients with MFS has been described, the potential effects of this genetic disease on the collapsible properties of the upper airway are unknown. The aim of this study was to assess the collapsible properties of the upper airway in a mouse model of MFS Fbn1(C1039G/+) that is representative of most of the clinical manifestations observed in human patients. The upper airway in wild-type and Marfan mice was cannulated and its critical pressure (Pcrit) was measured in vivo by increasing the negative pressure through a controlled pressure source. Pcrit values from MFS mice were higher (less negative) compared to wild-type mice (−3.1±0.9cmH2O vs. −7.8±2.0cm H2O) suggesting that MFS increases the upper airway collapsibility, which could in turn explain the higher prevalence of OSA in MFS patients.

Morphological phenotyping of mouse hearts using optical coherence tomography.

Transgenic mouse models have been instrumental in the elucidation of the molecular mechanisms behind many genetically based cardiovascular diseases such as Marfan syndrome (MFS). However, the characterization of their cardiac morphology has been hampered by the small size of the mouse heart. In this report, we adapted optical coherence tomography (OCT) for imaging fixed adult mouse hearts, and applied tools from computational anatomy to perform morphometric analyses. The hearts were first optically cleared and imaged from multiple perspectives. The acquired volumes were then corrected for refractive distortions, and registered and stitched together to form a single, high-resolution OCT volume of the whole heart. From this volume, various structures such as the valves and myofibril bundles were visualized. The volumetric nature of our dataset also allowed parameters such as wall thickness, ventricular wall masses, and luminal volumes to be extracted. Finally, we applied the entire acquisition and processing pipeline in a preliminary study comparing the cardiac morphology of wild-type mice and a transgenic mouse model of MFS.

IN VIVO MEASUREMENTS OF BIOPHYSICAL PROPERTIES OF A HEART AND AORTA IN A MOUSE MODEL OF MARFAN SYNDROME

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue caused by mutations in the fibrillin-1 (FBN1) gene. Cardiovascular complications are the most life-threatening manifestations in MFS patients, and death is often sudden due to aortic dissection and rupture in young patients. In the present study, we aim to investigate the structural and functional properties of the heart and aorta in a well-established MFS mouse model by using a high resolution (50-MHz) ultrasound system. Six- and twelve-month old WT (Fbn1 +/+) and MFS [Fbn1 (C1039G/+)] mice (n = 8) were scanned using the Vevo 2100 ultrasound system (VisualSonics®). We measured several key variables including pulse wave velocity (PWV), aortic root diameter, ejection fraction, stroke volume, left ventricular (LV) wall thickness, LV mass, mitral valve early velocity/atrial velocity (E/A) ratio and LV strain analysis. Our results indicate that PWV was significantly increased in 6-mo MFS vs. WT (366.6 ± 19.9 vs. 205.2 ± 18.1 cm/s; p < 0.001) and 12-mo MFS vs. WT (459.5 ± 42.3 vs. 205.3 ± 30.3 m/s; p = 0.001) and the increased PWV directly proportional to age in MFS mice (R-squared =0.356; p = 0.02). Regarding the LV structure, interventricular septal thickness (N) (2.52 ± 0.14 vs. 2.08 ± 0.10 μm/g; p = 0.03) and LV mass (N) (3.06 ± 0.16 vs. 2.46 ± 0.09 μm/g; p = 0.007) were significantly increased in 6-month MFS mice compared with WT, as well the LV anterior wall (N) (2.72 ± 0.26 vs. 2.07 ± 0.11 μm/g; p = 0.045) and posterior wall (N) (2.35 ± 0.16 vs. 1.91 ± 0.06 μm/g; p = 0.03) thickness were significantly increased in 12-month MFS mice. We also found a significantly enlarged aortic root, decreased E/A ratio, prolonged isovolumic relaxation time (IVRT) and increased myocardial performance index (MPI) in MFS mice compared with WT for both two age groups. (N, data normalized with mice body weight.) This study shows significant aortic dilation and central aortic stiffness in the MFS mouse, which are associated with LV hypertrophy and diastolic dysfunction. Moreover, the symptoms progressed with increasing age from six months to twelve months. It is not clear whether the increased LV muscle mass and abnormal diastolic function are due to the abnormal after-load caused either by increased aortic stiffness or by abnormal myocardial connective tissue.

Regional changes in elastic fiber organization and transforming growth factor β signaling in aortas from a mouse model of marfan syndrome.

In Marfan Syndrome (MFS), development of thoracic aortic aneurysms (TAAs) is characterized by degeneration of the medial layer of the aorta, including fragmentation and loss of elastic fibers, phenotypic changes in the smooth muscle cells, and an increase in the active form of transforming growth factor-β (TGFβ), which is thought to play a major role in development and progression of the aneurysm. We hypothesized that regional difference in elastic fiber fragmentation contributes to TGFβ activation and hence the localization of aneurysm formation. The fibrillin-1-deficient mgR/mgR mouse model of MFS was used to investigate regional changes in elastin fiber fragmentation, TGFβ activation and changes in gene expression as compared to wild-type littermates. Knockdown of Smad 2 and Smad 3 with shRNA was used to determine the role of the specific transcription factors in gene regulation in aortic smooth muscle cells. We show increased elastin fiber fragmentation in the regions associated with aneurysm formation and altered TGFβ signaling in these regions. Differential effects of Smad 2 and Smad 3 were observed in cultured smooth muscle cells by shRNA-mediated knockdown of expression of these transcription factors. Differential signaling through Smad 2 and Smad 3 in regions of active vascular remodeling likely contribute to aneurysm formation in the mgR/mgR model of MFS. Increased elastin fiber fragmentation in these regions is associated with these changes as compared to other regions of the thoracic aorta and may contribute to the changes in TGFβ signaling in these regions.

Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy.

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.

Medical Treatment of Aortic Aneurysms in Marfan Syndrome and other Heritable Conditions

Thoracic aortic aneurysms can be triggered by genetic disorders such as Marfan syndrome (MFS) and related aortic diseases as well as by inflammatory disorders such as giant cell arteritis or atherosclerosis. In all these conditions, cardiovascular risk factors, such as systemic arterial hypertension, may contribute to faster rate of aneurysm progression. Optimal medical management to prevent progressive aortic dilatation and aortic dissection is unknown. β-blockers have been the mainstay of medical treatment for many years despite limited evidence of beneficial effects. Recently, losartan, an angiotensin II type I receptor antagonist (ARB), has shown promising results in a mouse model of MFS and subsequently in humans with MFS and hence is increasingly used. Several ongoing trials comparing losartan to β-blockers and/or placebo will better define the role of ARBs in the near future. In addition, other medications, such as statins and tetracyclines have demonstrated potential benefit in experimental aortic aneurysm studies. Given the advances in our understanding of molecular mechanisms triggering aortic dilatation and dissection, individualized management tailored to the underlying genetic defect may be on the horizon of individualized medicine. We anticipate that ongoing research will address the question whether such genotype/pathogenesis-driven treatments can replace current phenotype/syndrome-driven strategies and whether other forms of aortopathies should be treated similarly. In this work, we review currently used and promising medical treatment options for patients with heritable aortic aneurysmal disorders.

Abstract 487: Resveratrol as an Inhibitor of Aortic Root Dilatation in Marfan Mice

Rationale: Marfan syndrome (MFS) is an autosomal connective tissue disorder caused by mutations in the Fibrillin-1 (FBN1) gene. Marfan patients are at risk for aortic aneurysm development and dissection, with often lethal consequences. Treatment consists of aortic root replacement surgery and beta-blocker medication. Recently, losartan was proven to be effective in inhibition of aortic root dilatation in MFS patients. The beneficial effect of losartan suggests the involvement of angiotensin II receptor 1 (AT1R) signaling as a cause for aortic pathology. Angiotensin II (Ang-II) can induce cell senescence, in contrast to sirtuin-1 (SIRT-1), which is an NAD-dependent deacetylase and prevents senescence. Among other functions, resveratrol can activate SIRT-1, which prompted us to investigate the effectiveness of resveratrol treatment in MFS mice. Methodology: In the current study we investigate whether senescence plays a role in aortic dilatation and if resveratrol treatment inhibits aortic root dilatation in the Fbn1 C1039G/+ MFS mouse model. Eight week old mice were treated for two months with resveratrol. Aortic root diameter and senescence were measured. In addition, aortic root pathology was analyzed by different immunohistochemical stainings. Results: Senescence correlates positively with aorta dilatation. Resveratrol inhibits aortic root dilatation, even more efficiently than losartan. The number of inflammatory cells (CD45) and the signaling pathways involving phosphorylated (p-)SMAD2 or p-ERK1/2 are decreased in aortic root sections of losartan treated MFS mice. In contrast, these were not affected in the resveratrol treated MFS mice. Resveratrol did result in a diminished number of elastic lamina breaks and an increased number of nuclear SIRT-1 expressing cells. Conclusions: Senescence is involved in aortic dilatation in MFS mice. Resveratrol efficiently inhibits aortic root dilatation, yet via a different mechanism of action than is considered for losartan, which opens new possibilities for pharmacological treatment of MFS patients.

Analysis of disease progression-associated gene expression profile in fibrillin-1 mutant mice: new insight into molecular pathogenesis of marfan syndrome.

Marfan syndrome (MFS) is a dominantly inherited connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1) and is characterized by aortic dilatation and dissection, which is the primary cause of death in untreated MFS patients. However, disease progression-associated changes in gene expression in the aortic lesions of MFS patients remained unknown. Using a mouse model of MFS, FBN1 hypomorphic mouse (mgR/mgR), we characterized the aortic gene expression profiles during the progression of the MFS. Homozygous mgR mice exhibited MFS-like phenotypic features, such as fragmentation of elastic fibers throughout the vessel wall and were graded into mgR1–4 based on the pathological severity in aortic walls. Comparative gene expression profiling of WT and four mgR mice using microarrays revealed that the changes in the transcriptome were a direct reflection of the severity of aortic pathological features. Gene ontology analysis showed that genes related to oxidation/reduction, myofibril assembly, cytoskeleton organization, and cell adhesion were differentially expressed in the mgR mice. Further analysis of differentially expressed genes identified several candidate genes whose known roles were suggestive of their involvement in the progressive destruction of aorta during MFS. This study is the first genome-wide analysis of the aortic gene expression profiles associated with the progression of MFS. Our findings provide valuable information regarding the molecular pathogenesis during MFS progression and contribute to the development of new biomarkers as well as improved therapeutic strategies.

Abstract 522: TGF-beta Neutralization Augments Development of Angiotensin II-induced Aneurysms in Both Ascending and Abdominal Aortic Regions

Introduction and Objectives Angiotensin II (AngII) infusion induces ascending and abdominal aortic aneurysms (AAs) in mice. In a mouse model of Marfan Syndrome expressing Fbn1 C1039G/+ , ascending AAs were reduced by administration of a transforming growth factor-beta (TGF-beta) neutralizing antibody. In contrast, administration of TGF-beta neutralizing antibodies to AngII-infused mice increased aortic rupture. The purpose of this study was to compare the effects of TGF-beta neutralization on formation and progression of AngII-induced ascending and abdominal AAs. Methods and Results Male C57BL/6 mice were fed a normal diet and infused subcutaneously with AngII (1,000 ng/kg/min). Five days prior to initiating infusion, mice were injected i.p. with either a mouse monoclonal TGF-beta antibody (1D11) or an isotype matched IgG at a dose of either 0.3 or 5 mg/kg x 3/per week. 1D11 administration significantly decreased serum TGF-beta concentrations. TGF-beta neutralization at 5 mg/kg greatly increased the incidence of aortic rupture, which was attributed to rupture in both the ascending and abdominal regions. For mice that remained viable after 28 days of infusion, there were equivalent increases in aortic dilation in both the ascending and abdominal regions. Prior to rupture, aortic diameters determined by ultrasound demonstrated no significant effect on AngII-induced dilation of the ascending or abdominal aorta. We also studied the effects of TGF-beta neutralization in mice with established AngII-induced AAs following AngII-infusion for 28 days. C57BL/6 mice were injected with the mouse TGF-beta neutralizing antibody or IgG control (5 mg/kg x 3/per week, n=10 per group), while AngII infusion was continued for a further 28 days. Although TGF-beta antibody administration significantly decreased serum TGF-beta concentrations in mice with established AAs, there was no effect on aortic rupture or dilation of either the ascending or abdominal aortic region. Conclusion TGF-beta inhibition augmented AngII-induced aortic rupture in both the ascending and abdominal regions but had no effect on dilation. Furthermore, TGF-beta neutralization had no effect on either aortic rupture or expansion in established AAs.

Effect of Angiotensin II Receptor Blocker on Experimental Periodontitis in a Mouse Model of Marfan Syndrome

Marfan syndrome is an autosomal dominant disease characterized by aneurysm and dilatation of the aortic root, tall stature, and ectopia lentis. These manifestations reflect excessive signaling of transforming growth factor beta (TGF-β). Moreover, cases are frequently associated with severe periodontitis, which is a chronic inflammation of the gingiva, periodontal ligament, and alveolar bone. Recently, angiotensin II receptor blockers (ARBs) were discovered to be an effective drug class that can prevent aortic aneurysm and dilation in Marfan syndrome by inhibiting TGF-β signaling. To investigate the effect of ARB on the progression of periodontitis, the application of a potent ARB, telmisartan, was examined in a mouse model of Marfan syndrome (MgΔ). Six-week-old male heterozygous MgΔ and wild-type mice were challenged with Porphyromonas gingivalis, which causes chronic periodontitis, with and without telmisartan application. After infection, alveolar bone resorption was measured by micro-computed tomography (μCT), and inflammatory cytokine levels were examined. Infection of Porphyromonas gingivalis induced alveolar bone resorption in both MgΔ and wild-type mice. The amount of resorption was significantly larger in the former than the latter. Immunoarray and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α) levels were significantly higher in infected MgΔ mice than infected wild-type mice. Telmisartan treatment significantly suppressed the alveolar bone resorption of infected MgΔ mice. Telmisartan also significantly decreased levels of TGF-β, IL-17, and TNF-α in infected MgΔ mice to levels seen in infected wild-type mice. This study suggests that ARB can prevent the severe periodontitis frequently seen in Marfan syndrome.

Modulation of Transforming Growth Factor-β Signaling and Extracellular Matrix Production in Myxomatous Mitral Valves by Angiotensin II Receptor Blockers

Little is known about the pathophysiology of myxomatous degeneration of the mitral valve, the pathological hallmark of mitral valve prolapse, associated with symptomatic mitral regurgitation, heart failure, and death. Excess transforming growth factor (TGF)-β signaling is known to cause mitral valve degeneration and regurgitation in a mouse model of Marfan syndrome. We examined if TGF-β signaling is dysregulated in clinical specimens of sporadic mitral valve prolapse compared with explanted nondiseased mitral valves and we tested the effects of angiotensin II receptor blockers on TGF-β signaling in cultured human mitral valve cells. Operative specimens, cultured valve tissues, and cultured valvular interstitial cells were obtained from patients with mitral valve prolapse undergoing mitral valve repair or from organ donors without mitral valve disease. Increased extracellular matrix in diseased valve tissue correlated with an upregulation of TGF-β expression and signaling as evidenced by SMAD2/3 phosphorylation. Both TGF-β ligand and signaling mediators colocalized primarily to valvular interstitial cells suggesting autocrine/paracrine activation. In cultured valve tissue, exogenous TGF-β increased basal extracellular matrix production, whereas serological neutralization of TGF-β inhibited disease-driven extracellular matrix overproduction. TGF-β-induced extracellular matrix production in cultured valvular interstitial cells was dependent on SMAD2/3 and p38 signaling and was inhibited by angiotensin II receptor blockers. TGF-β has a profibrotic role in the pathogenesis of sporadic mitral valve prolapse. Attenuation of TGF-β signaling by angiotensin II receptor blockers may represent a mechanistically based strategy to modulate the pathological progression of mitral valve prolapse in patients.

Targeting collagen strands by photo-triggered triple-helix hybridization

Collagen remodeling is an integral part of tissue development, maintenance, and regeneration, but excessive remodeling is associated with various pathologic conditions. The ability to target collagens undergoing remodeling could lead to new diagnostics and therapeutics as well as applications in regenerative medicine; however, such collagens are often degraded and denatured, making them difficult to target with conventional approaches. Here, we present caged collagen mimetic peptides (CMPs) that can be photo-triggered to fold into triple helix and bind to collagens denatured by heat or by matrix metalloproteinase (MMP) digestion. Peptide-binding assays indicate that the binding is primarily driven by stereo-selective triple-helical hybridization between monomeric CMPs of high triple-helical propensity and denatured collagen strands. Photo-triggered hybridization allows specific staining of collagen chains in protein gels as well as photo-patterning of collagen and gelatin substrates. In vivo experiments demonstrate that systemically delivered CMPs can bind to collagens in bones, as well as prominently in articular cartilages and tumors characterized by high MMP activity. We further show that CMP-based probes can detect abnormal bone growth activity in a mouse model of Marfan syndrome. This is an entirely new way to target the microenvironment of abnormal tissues and could lead to new opportunities for management of numerous pathologic conditions associated with collagen remodeling and high MMP activity.

Marfan syndrome: Progress report

Marfan syndrome is a multi-system connective tissue disorder, with primary involvement of the cardiovascular, ocular and skeletal systems. This autosomal heritable disease is mainly attributable to a defect in the FBN1 gene. Until 2010, the clinical diagnosis of Marfan syndrome was based on the Ghent criteria of 1996. Recently, the Ghent criteria have been revised. The revised guidelines of 2010 place more emphasis on aortic root dilatation, ectopia lentis and FBN1 mutation testing in the diagnostic assessment of Marfan syndrome. Although the revised Ghent criteria of 2010 are easier to apply, they do raise some issues that need to be addressed.In addition to adjustments in the diagnosis of Marfan syndrome, there is progress in the understanding of the pathophysiology in Marfan syndrome, leading to new treatment strategies. Losartan, an angiotensin II receptor type 1 blocker, has been shown to inhibit transforming growth factor beta signal transduction and thereby prevent aortic root aneurysms in a mouse model of Marfan syndrome. This article will provide a critical appraisal of the revised Ghent nosology in 2010 and will highlight future perspectives regarding the treatment of Marfan syndrome.

MMP-2 Regulates Erk1/2 Phosphorylation and Aortic Dilatation in Marfan Syndrome

Aneurysm and dissection of the ascending thoracic aorta are the main cardiovascular complications of Marfan syndrome (MFS) resulting in premature death. Studies using mouse models of MFS have shown that activation of transforming growth factor-beta (TGF-β) and the concomitant upregulation of matrix metalloproteinases (MMPs) contribute to aneurysm development. Our previous study showed that doxycycline delayed aneurysm rupture in a mouse model of MFS, Fbn1(mgR/mgR). Losartan has been shown to prevent aneurysms in another mouse model of MFS, Fbn1(C1039G/+), through inhibition of the Erk1/2 pathway. However, the role of MMP-2 in MFS and effect of losartan on the lifespan of MFS mice remain unknown. We investigated the role of MMP-2 in MFS and compared the effects of losartan and doxycycline on aortic dilatation and survival in Fbn1(mgR/mgR) mice. By life table analysis, we found that losartan and doxycycline improved the survival of Fbn1(mgR/mgR) mice. Gelatin zymography and Western blot data showed that only doxycycline inhibited MMP-2 expression, whereas both drugs decreased Erk1/2 phosphorylation. When combined, only one of nine mice died within the 30-week study; aortic histology and diameter were normalized and the effects on Smad2 phosphorylation was additive. To further explore the role of MMP-2 in MFS, we created MMP-2-deficient Fbn1(mgR/mgR) mice. MMP-2 deletion inhibited activation of TGF-β and phosphorylation of Erk1/2 and Smad2 and prolonged the lifespan of the mice. These studies demonstrated that inhibition of MMP-2 by doxycycline delayed the manifestations of MFS, in part, through its ability to decrease active TGF-β and the noncanonical signaling cascade downstream of TGF-β. This study further suggested that targeting TGF-β signaling at different points might be a more effective strategy for inhibiting disease progression.

Oxidative stress and unfolded protein response (UPR) in advanced aortic disease from Marfan Syndrome (MFS) transgenic mice

We investigated the occurrence and mechanisms of oxidative stress in aortae from MFS mouse model (mgÄFBN1). Aortic dilation, elastic fiber rupture and collagen deposition markedly increased from 3–6 months(mo). ROS production (by 3 methods) increased in aortae from 6‐mo, but not 1 or 3‐mo‐old MFS mice, mainly at crossa. Nox4 mRNA increased after 1mo, while Nox2 only at 6mo. To assess oxidative stress mechanisms, we investigated UPR markers in MFS aortae. Expression of KDEL chaperones increased at 6mo, but not 1 or 3mo. Protein disulfide isomerase(PDI), which associates with and regulates Nox(es), equally increased. Also, PDI expression topography correlated with ROS production sites (DMPO adduct antibody), mainly at adventitia in crossa and media in abdominal aorta. Adventitial PDI switch in crossa correlated with diffuse collagen accumulation. Moreover, yeast‐two‐hybrid screening disclosed PDI interaction with fibrillin‐1(FBN1); in cultured fibroblasts, such association was mainly intra, but not extracellular. FBN1 immunodetection decreased by~80% and co‐localized with PDI in MFS adventitia. PDI/UPR marker changes were not systemic and absent in kidney and liver. Losartan prevented aortic remodeling, but ROS production remained increased in aorta at 6mo. Thus, ROS production increases with MFS aortic disease progression, in close temporal and topographic correlation with PDI expression and UPR.

Novel pharmacological strategies to prevent aortic complications in Marfan syndrome

The Marfan syndrome (MFS) is a systemic connective tissue disorder caused by mutations in the FBN1 gene. Recent molecular studies, most performed in mouse models, revealed that the MFS is more a developmental abnormality with broad and complex effects on the morphogenesis and function of multiple organ systems. FBN1 haploinsufficiency and dysregulated transforming growth factor-beta (TGF-β) signaling seem to be critical for clinical manifestations in MFS including aortic root dilatation. Aortic root aneurysm and aortic dissection represent the main causes of morbidity and mortality in MFS. Most importantly, TGF-β antagonism through angiotensin II type 1 receptor blockers (ARBs), for example losartan, has been shown to prevent and possibly reverse aortic root dilatation in a mouse model of MFS. A first human study on a small pediatric cohort confirmed those promising results in reducing the aortic root growth over a follow-up period of 12 to 47 months. So, a large multicenter trial has been set up and results should be available soon. Other therapeutic strategies which might be combined with losartan include traditional β-blockade, doxycyclin and statins. Such management could offer the first potential for primary prevention of clinical manifestations in MFS.

Pravastatin Reduces Marfan Aortic Dilation

Statins attenuate aortic root dilation in a mouse model of Marfan syndrome.

Altered tissue behavior of a non-aneurysmal descending thoracic aorta in the mouse model of Marfan syndrome.

Aortic aneurysm is predominantly found in the ascending aorta in patients with Marfan syndrome (MFS). However, descending aortic disease has emerged as a problem since people are living longer because of improved medical and surgical management of the ascending aorta. Diagnostic procedures before disease onset and the mechanisms involved in the transition of normal aortic tissue to aneurysm remain unclear. We determined signs of descending aortic disease before disease onset in mice with a mutation in the fibrillin 1 gene (Fbn1(+/C1039G)), a validated mouse model of disease susceptibility and progression of aortic aneurysm of MFS. We analyzed a tubular unfixed non-aneurysmal descending thoracic aorta from 8-month-old wild-type and Fbn1(+/C1039G) mice by a tubular biaxial tester that works in conjunction with a two-photon nonlinear microscope. Fbn1(+/C1039G) mouse aorta was more compliant in the circumferential direction. Two-photon imaging showed defective organization of adventitial collagen fibers in the pressurized aortas of Fbn1(+/C1039G) mice. Moreover, disruption in the elastic lamina was noted in the absence of aneurysms in pressurized aortas but not unpressurized aortas of Fbn1(+/C1039G) mice. At the molecular level, this altered tissue behavior in non-aneurysmal descending aortas of Fbn1(+/C1039G) mice was accompanied by an increasing trend of canonical but not noncanonical, transforming growth factor-β (TGFβ) signaling. Finally, assays of in vitro collagen lattice formation in mouse wild-type and TGFβ1-deficient embryonic fibroblasts indicate that TGFβ1 can regulate collagen organization. The ability to reveal the presence of altered biomechanics and microstructure coupled with subtle changes in TGFβ signaling provides a novel surrogate measure of tissue susceptibility to aneurysm before disease onset.

ADAMTSL6β Protein Rescues Fibrillin-1 Microfibril Disorder in a Marfan Syndrome Mouse Model through the Promotion of Fibrillin-1 Assembly

Marfan syndrome (MFS) is a systemic disorder of the connective tissues caused by insufficient fibrillin-1 microfibril formation and can cause cardiac complications, emphysema, ocular lens dislocation, and severe periodontal disease. ADAMTSL6β (A disintegrin-like metalloprotease domain with thrombospondin type I motifs-like 6β) is a microfibril-associated extracellular matrix protein expressed in various connective tissues that has been implicated in fibrillin-1 microfibril assembly. We here report that ADAMTSL6β plays an essential role in the development and regeneration of connective tissues. ADAMTSL6β expression rescues microfibril disorder after periodontal ligament injury in an MFS mouse model through the promotion of fibrillin-1 microfibril assembly. In addition, improved fibrillin-1 assembly in MFS mice following the administration of ADAMTSL6β attenuates the overactivation of TGF-β signals associated with the increased release of active TGF-β from disrupted fibrillin-1 microfibrils within periodontal ligaments. Our current data thus demonstrate the essential contribution of ADAMTSL6β to fibrillin-1 microfibril formation. These findings also suggest a new therapeutic strategy for the treatment of MFS through ADAMTSL6β-mediated fibrillin-1 microfibril assembly.

Pravastatin Reduces Marfan Aortic Dilation

The sequelae of aortic root dilation are the lethal consequences of Marfan syndrome. The root dilation is attributable to an imbalance between deposition of matrix elements and metalloproteinases in the aortic medial layer as a result of excessive transforming growth factor-beta signaling. This study examined the efficacy and mechanism of statins in attenuating aortic root dilation in Marfan syndrome and compared effects to the other main proposed preventative agent, losartan. Marfan mice heterozygous for a mutant allele encoding a cysteine substitution in fibrillin-1 (C1039G) were treated daily from 6 weeks old with pravastatin 0.5 g/L or losartan 0.6 g/L. The end points of aortic root diameter (n=25), aortic thickness, and architecture (n=10), elastin volume (n=5), dp/dtmax (maximal rate of change of pressure) (cardiac catheter; n=20), and ultrastructural analysis with stereology (electron microscopy; n=5) were examined. The aortic root diameters of untreated Marfan mice were significantly increased in comparison to normal mice (0.161 ± 0.001 cm vs 0.252 ± 0.004 cm; P<0.01). Pravastatin (0.22 ± 0.003 cm; P<0.01) and losartan (0.221 ± 0.004 cm; P<0.01) produced a significant reduction in aortic root dilation. Both drugs also preserved elastin volume within the medial layer (pravastatin 0.23 ± 0.02 and losartan 0.29 ± 0.03 vs untreated Marfan 0.19 ± 0.02; P=0.01; normal mice 0.27 ± 0.02). Ultrastructural analysis showed a reduction of rough endoplasmic reticulum in smooth muscle cells with pravastatin (0.022 ± 0.004) and losartan (0.013 ± 0.001) compared to untreated Marfan mice (0.035 ± 0.004; P<0.01). Statins are similar to losartan in attenuating aortic root dilation in a mouse model of Marfan syndrome. They appear to act through reducing the excessive protein manufacture by vascular smooth muscle cells, which occurs in the Marfan aorta. As a drug that is relatively well-tolerated for long-term use, it may be useful clinically.