Anatomically specific reactive oxygen species production participates in Marfan syndrome aneurysm formation

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1^C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin‐enhanced chemiluminescence (LGCL), Verhoeff's elastin‐Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1^C1039G/+ AS‐ or DES‐derived smooth muscle cells (SMC) were treated with anti‐TGF‐β antibody, angiotensin II (AngII), anti‐TGF‐β antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1^C1039G/+ AS aorta, but absent in normal‐sized DES aorta. Fbn1^C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1^C1039G/+‐derived AS SMC had increased NADPH activity compared to DES‐derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF‐β dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF‐β dependent.     

Aortic and Cardiac Structure and Function Using High-Resolution Echocardiography and Optical Coherence Tomography 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. Mortality is often due to aortic dissection and rupture. We investigated the structural and functional properties of the heart and aorta in a [Fbn1^C1039G/+] 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 [Fbn1^C1039G/+] 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.     

Alpha 2(I) collagen deficient oim mice have altered biomechanical integrity, collagen content, and collagen crosslinking of their thoracic aorta.

Collagen and elastin are the primary determinants of vascular integrity, with elastin hypothesized to be the major contributor to aortic compliance and type I collagen the major contributor to aortic strength and stiffness. Type I collagen is normally heterotrimeric composed of two alpha1(I) and one alpha2(I) collagen chains, alpha1(I)(2)alpha2(I). Recent investigations have reported that patients with recessively inherited forms of Ehlers Danlos syndrome that fail to synthesize proalpha2(I) chains have increased risks of cardiovascular complications. To assess the role of alpha2(I) collagen in aortic integrity, we used the osteogenesis imperfecta model (oim) mouse. Oim mice, homozygous for a COL1A2 mutation, synthesize only homotrimeric type I collagen, alpha1(I)3. We evaluated thoracic aortas from 3-month-old oim, heterozygote, and wildtype mice biomechanically for circumferential breaking strength (Fmax) and stiffness (IEM), histologically for morphological differences, and biochemically for collagen content and crosslinking. Circumferential biomechanics of oim and heterozygote descending thoracic aortas demonstrated the anticipated reduced Fmax and IEM relative to wildtype mice. Histological analyses of oim descending aortas demonstrated reduced collagen staining relative to wildtype aortas suggesting decreased collagen content, which hydroxyproline analyses of ascending and descending oim aortas confirmed. These findings suggest the reduced oim thoracic aortic integrity correlates with the absence of the alpha2(I)collagen chains and in part with reduced collagen content. However, oim ascending aortas also demonstrated a significant increase in pyridinoline crosslinks/collagen molecule as compared to wildtype ascending aortas. The role of increased collagen crosslinks is uncertain; increased crosslinking may represent a compensatory mechanism for the decreased integrity.     

A new mouse model for marfan syndrome presents phenotypic variability associated with the genetic background and overall levels of Fbn1 expression

Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mgΔloxPneo, carrying the same internal deletion of exons 19-24 as the mgΔ mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression.     

Generation of Fbn1 conditional null mice implicates the extracellular microfibrils in osteoprogenitor recruitment

Loss-of-function experiments in mice have yielded invaluable mechanistic insights into the pathogenesis of Marfan syndrome (MFS) and implicitly, into the multiple roles fibrillin-1 microfibrils play in the developing and adult organism. Unfortunately, neonatal death from aortic complications of mice lacking fibrillin-1 (Fbn1(-/-) mice) has limited the scope of these studies. Here, we report the creation of a conditional mutant allele (Fbn1(fneo) ) that contains loxP sites bordering exon1 of Fbn1 and an frt-flanked neo expression cassette downstream of it. Fbn1(fneo/+) mice were crossed with FLPeR mice and the resulting Fbn1(Lox/+) progeny were crossed with Fbn1(+/-) ;CMV-Cre mice to generate Fbn1(CMV-/-) mice, which were found to phenocopy the vascular abnormalities of Fbn1(-/-) mice. Furthermore, mating Fbn1(Lox/+) mice with Prx1-Cre or Osx-Cre mice revealed an unappreciated role of fibrillin-1 microfibrils in restricting osteoprogenitor cell recruitment. Fbn1(Lox/+) mice are, therefore, an informative genetic resource to further dissect MFS pathogenesis and the role of extracellular fibrillin-1 assemblies in organ development and homeostasis.     

Early Impairment of Lung Mechanics in a Murine Model of Marfan Syndrome

Early morbidity and mortality in patients with Marfan syndrome (MFS) -a connective tissue disease caused by mutations in fibrillin-1 gene- are mainly caused by aorta aneurysm and rupture. However, the increase in the life expectancy of MFS patients recently achieved by reparatory surgery promotes clinical manifestations in other organs. Although some studies have reported respiratory alterations in MFS, our knowledge of how this connective tissue disease modifies lung mechanics is scarce. Hence, we assessed whether the stiffness of the whole lung and of its extracellular matrix (ECM) is affected in a well-characterized MFS mouse model (FBN1^C1039G/+). The stiffness of the whole lung and of its ECM were measured by conventional mechanical ventilation and atomic force microscopy, respectively. We studied 5-week and 9-month old mice, whose ages are representative of early and late stages of the disease. At both ages, the lungs of MFS mice were significantly more compliant than in wild type (WT) mice. By contrast, no significant differences were found in local lung ECM stiffness. Moreover, histopathological lung evaluation showed a clear emphysematous-like pattern in MFS mice since alveolar space enlargement was significantly increased compared with WT mice. These data suggest that the mechanism explaining the increased lung compliance in MFS is not a direct consequence of reduced ECM stiffness, but an emphysema-like alteration in the 3D structural organization of the lung. Since lung alterations in MFS are almost fully manifested at an early age, it is suggested that respiratory monitoring could provide early biomarkers for diagnosis and/or follow-up of patients with the Marfan syndrome.     

基质金属蛋白酶3、9在升主动脉瘤中的表达

目的探讨基质金属蛋白酶3、9(MMP-3、MMP-9)在升主动脉瘤发病机制中的作用.方法将40只幼年Wistar大鼠随机分为对照组和实验组,制备升主动脉缩窄鼠模型.于术后3-5个月取升主动脉,采用HE染色和免疫组化技术,观察升主动脉形态学变化及MMP-3、MMP-9的蛋白表达.结果升主动脉瘤中MMP-3、MMP-9表达强阳性.结论 MMP-3、MMP-9在升主动脉瘤成因中有可能有重要作用.     

Single exposure to radiation produces early anti-angiogenic effects in mouse aorta

Radiation exposure can increase the risk for many non-malignant physiological complications, including cardiovascular disease. We have previously demonstrated that ionizing radiation can induce endothelial dysfunction, which contributes to increased vascular stiffness. In this study, we demonstrate that gamma radiation exposure reduced endothelial cell viability or proliferative capacity using an in vitro aortic angiogenesis assay. Segments of mouse aorta were embedded in a Matrigel-media matrix 1 day after mice received whole-body gamma irradiation between 0 and 20 Gy. Using three-dimensional phase contrast microscopy, we quantified cellular outgrowth from the aorta. Through fluorescent imaging of embedded aortas from Tie2GFP transgenic mice, we determined that the cellular outgrowth is primarily of endothelial cell origin. Significantly less endothelial cell outgrowth was observed in aortas of mice receiving radiation of 5, 10, and 20 Gy radiation, suggesting radiation-induced endothelial injury. Following 0.5 and 1 Gy doses of whole-body irradiation, reduced outgrowth was still detected. Furthermore, outgrowth was not affected by the location of the aortic segments excised along the descending aorta. In conclusion, a single exposure to gamma radiation significantly reduces endothelial cell outgrowth in a dose-dependent manner. Consequently, radiation exposure may inhibit re-endothelialization or angiogenesis after a vascular injury, which would impede vascular recovery.     

No Beneficial Effect of General and Specific Anti-Inflammatory Therapies on Aortic Dilatation in Marfan Mice

Aims

Patients with Marfan syndrome have an increased risk of life-threatening aortic complications, mostly preceded by aortic dilatation. In the FBN1 ^C1039G/+ Marfan mouse model, losartan decreases aortic root dilatation. We recently confirmed this beneficial effect of losartan in adult patients with Marfan syndrome. The straightforward translation of this mouse model to man is reassuring to test novel treatment strategies. A number of studies have shown signs of inflammation in aortic tissue of Marfan patients. This study examined the efficacy of anti-inflammatory therapies in attenuating aortic root dilation in Marfan syndrome and compared effects to the main preventative agent, losartan.

Methods and Results

To inhibit inflammation in FBN1 ^C1039G/+ Marfan mice, we treated the mice with losartan (angiotensin II receptor type 1 inhibitor), methylprednisolone (corticosteroid) or abatacept (T-cell-specific inhibitor). Treatment was initiated in adult Marfan mice with already existing aortic root dilatation, and applied for eight weeks. Methylprednisolone- or abatacept-treated mice did not reveal a reduction in aortic root dilatation. In this short time frame, losartan was the only treatment that significantly reduced aorta inflammation, transforming growth factor-beta (TGF-β) signaling and aortic root dilatation rate in these adult Marfan mice. Moreover, the methylprednisolone-treated mice had significantly more aortic alcian blue staining as a marker for aortic damage.

Conclusion

Anti-inflammatory agents do not reduce the aortic dilatation rate in Marfan mice, but possibly increase aortic damage. Currently, the most promising therapeutic drug in Marfan syndrome is losartan, by blocking the angiotensin II receptor type 1 and thereby inhibiting pSmad2 signaling.     

Calpain-2 Compensation Promotes Angiotensin II-Induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice

Background and Objective

Recently, we demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development.

Methodology/Results

To investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr −/− mice that were either calpain-1 +/+ or −/− were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and −/− mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an actin binding cytoskeletal protein in aorta.

Conclusion

Calpain-2 compensates for loss of calpain-1, and both calpain isoforms are involved in AngII-induced aortic aneurysm formation in mice.     

Comparative study of flow in right-sided and left-sided aortas: numerical simulations in patient-based models

A right-sided aorta is a rare malformation which may be associated with other various types of congenital heart disease. We utilised haemodynamic, echocardiographic measurements, computerised tomography and image reconstruction software packages that were integrated in a computational fluid dynamics model to determine blood flow patterns in patient-based aortas. In the left-sided aorta, a systolic clockwise rotational component was present, while helical flow was depicted in the aortic arch that was converted in the descending aorta as counter-rotating vortices with accompanying retrograde flow. The right-sided configuration has not altered the orientation of the three-dimensional vortex, but intensification of polymorphic flow patterns, alterations in wall shear stress distribution and development of a lateral pressure gradient at the area of an aneurysmal anomaly was observed. Moreover, increments of Reynolds, Womersley and Dean numbers were evident. These phenomena along with the formation of the aneurysm might influence cardiovascular risk in patients with right-sided aortas.     

MCP-1, ICAM-1 and VCAM-1 are present in early aneurysmal dilatation in experimental rats

Recent studies have suggested that inflammation actively participates in ascending aortic aneurysm formation. The aim of the present study was to evaluate the expression changes of adhesion molecules and MMPs in an experimental model of ascending aortic aneurysm induced by ascending aorta banding in Wistar rats. Twelve rats developed aortic dilation after ascending aorta banding treatment, while nine normal animals underwent surgery without banding were used as controls. Light microscope and scanning electron microscope showed that the wall of the ascending aorta became disorganized as well as infiltration by inflammatory cells in aneurysmal rats. By using immunohistochemical techniques, a significant increase in the immunostaining of MCP-1 was observed in the aneurysmal wall as compared to the normal aortic wall. Under similar experimental conditions, we also found that the immunostaining of ICAM-1 and VCAM-1 was markedly increased in the aneurysmal wall. In addition, gelatin zymographic analysis showed that the expression and activities of MMP-2 and MMP-9 were remarkably enhanced in the ascending aorta of ascending aortic aneurysmal rats as compared to normal rats. These results demonstrate that MCP-1, ICAM-1 and VCAM-1 are involved in the pathogenesis of ascending aortic aneurysm and an increase in the immunostaining and activity of MMP-2 and MMP-9 may promote the progression of ascending aortic aneurysm.     

Earlier accumulation of calcium, phosphorus, and magnesium in the coronary artery in comparison with the ascending aorta, aortic valve, and mitral valve

To explore reasons for a high accumulation of Ca and P occurring in the coronary artery of Thai with aging, the authors investigated age-related changes of elements in the coronary artery, ascending aorta near the heart, and cardiac valves in single individuals, and the relationships in the elements between the coronary artery and either the ascending aorta or cardiac valves. After an ordinary dissection by medical students at Chiang Mai University was finished, the anterior descending arteries of the left coronary artery, ascending aortas, mitral valves, and aortic valves were resected from the subjects. The subjects consisted of 17 men and 9 women, ranging in age from 46 to 76 yr. The element content was analyzed by inductively coupled plasma-atomic emission spectrometry. The average content of Ca and P was the highest in the coronary artery and decreased in the order aortic valve, ascending aorta, and mitral valve. The Ca, P, and Mg content increased in the coronary artery in the fifties and in the ascending aorta, aortic valve, and mitral valve in the sixties. It should be noted that the accumulation of Ca, P, and Mg occurred earlier in the coronary artery than in the ascending aorta, aortic valve, and mitral valve. It was found that with respect to the Ca, P, Mg, and Na contents, the coronary artery correlated well with both the aortic valve and ascending aorta, especially with the aortic valve, but it did not correlate with the mitral valves. This finding suggests that the accumulation of Ca, P, Mg, and Na occurs in the coronary artery together with the aortic valve and ascending aorta, but not together with the mitral valve. Because regarding the accumulation of Ca, P, and Mg, the ascending aorta and aortic valve are preceded by the coronary artery, it is unlikely that the accumulation of Ca, P, and Mg spreads from the ascending aorta or aortic valve to the coronary artery.     

The effects of angiotensin II on the coupled microstructural and biomechanical response of C57BL/6 mouse aorta

RationaleAbdominal aortic aneurysm (AAA) is a complex disease that leads to a localized dilation of the infrarenal aorta, the rupture of which is associated with significant morbidity and mortality. Animal models of AAA can be used to study how changes in the microstructural and biomechanical behavior of aortic tissues develop as disease progresses in these animals. We chose here to investigate the effect of angiotensin II (AngII) in C57BL/6 mice as a first step towards understanding how such changes occur in the established ApoE^?/? AngII infused mouse model of AAA.ObjectiveThe objective of this study was to utilize a recently developed device in our laboratory to determine how the microstructural and biomechanical properties of AngII-infused C57BL/6 wildtype mouse aorta change following 14 days of AngII infusion.MethodsC57BL/6 wildtype mice were infused with either saline or AngII for 14 day. Aortas were excised and tested using a device capable of simultaneously characterizing the biaxial mechanical response and load-dependent (unfixed, unfrozen) extracellular matrix organization of mouse aorta (using multiphoton microscopy). Peak strains and stiffness values were compared across experimental groups, and both datasets were fit to a Fung-type constitutive model. The mean mode and full width at half maximum (FWHM) of fiber histograms from two photon microscopy were quantified in order to assess the preferred fiber distribution and degree of fiber splay, respectively.ResultsThe axial stiffness of all mouse aorta was found to be an order of magnitude larger than the circumferential stiffness. The aortic diameter was found to be significantly increased for the AngII infused mice as compared to saline infused control (p=0.026). Aneurysm, defined as a percent increase in maximum diameter of 30% (defined with respect to saline control), was found in 3 of the 6 AngII infused mice. These three mice displayed adventitial collagen that lacked characteristic fiber crimp. The biomechanical response in the AngII infused mice showed significantly reduced circumferential compliance. We also noticed that the ability of the adventitial collagen fibers in AngII infused mice to disperse in reaction to circumferential loading was suppressed.ConclusionsCollagen remodeling is present following 14 days of AngII infusion in C57BL/6 mice. Aneurysmal development occurred in 50% of our AngII infused mice, and these dilatations were accompanied with adventitial collagen remodeling and decreased circumferential compliance.     

Transgenic expression of an altered angiotensin type I AT1 receptor resulting in marked modulation of vascular type I collagen

The angiotensin II (AngII) type I receptor (AT1) was modified by replacing its third intracellular loop and C-terminal tail with the corresponding regions from the bradykinin B2 receptor. Transgenic mice were produced that overexpress this mutated receptor (AB3T). Considerably less collagen content in the intact aorta and in primary aortic smooth muscle cells (aSMCs) cultures was observed in the transgenic mice. On the other hand, elastin content remained unchanged as measured by Western blot, and insoluble amino acid quantitation. The contraction of isolated aortas also remained unaltered. The aSMCs derived from the transgenic mice showed a reduction in AngII responsive type I collagen production. In aSMCs from transgenic mice, the cascade of Akt to the mammalian target rapamycin (mTOR) to p70 S6 kinase (p70S6K) was not AngII activated, while in the aSMCs from wild-type (WT) mice the cascade was AngII activated. Angiotensin activation of Smad2 and Stat3 was also reduced in the AB3T aSMCs. However, no change in the effect of transforming growth factor β (TGFβ) on type I collagen production was observed. Also, the activation of ERK and JNK and G-protein linked signaling remained unaltered in response to AngII. Akt and PI3K activation inhibitors blocked AngII-stimulated type I collagen expression in WT aSMCs, whereas ERK inhibitor had no such effect. Our results point to an Akt/mTOR/p70S6K regulation of collagen production by AngII with participation of Smad2 and Stat3 cascades in this process.