Abstract 13025: Methotrexate Carried in Lipid Core Nanoparticles Prevented the Dilation and Dissection of the Aortic Arch in Mice With Marfan Syndrome by Increasing the Bioavailability of Intracellular Adenosine

Introduction: Mutations in the fibrillin-1 gene result in aneurysms and rupture of the aorta, the main cause of mortality in patients with Marfan syndrome (MFS). Inflammation is associated with dissection and rupture of the aorta. We showed that methotrexate (MTX) carried in lipid core nanoparticles (LDE) had powerful anti-inflammatory effects on atherosclerotic lesions and myocardial infarction. Hypothesis: To investigate whether treatment with LDE-MTX can prevent the development of aortic arch lesions in MFS. Methods: mgΔloxPneo MFS murine model and wild-type mice were allocated in 3 groups of treatment: LDE only; MTX in commercial formulation; LDE-MTX. The treatment occurred weekly at a dose of 1mg/kg ip, between the 3rd and 6th month of life, period in which both dilation and dissection of the aortic arch are observed. After 12 weeks, animals were submitted to analysis of the aortic arch. Results: Compared to LDE and MTX groups, LDE-MTX treatment showed smaller lumen area of ascending and descending aorta and aortic arch segments in MFS mice. LDE-MTX also decreased collagen volume fraction and the amount of aortic dissections, but did not affect the thickness of the vessel wall and the number of elastic fiber ruptures. Protein expression of the inflammatory markers CD68 (macrophages), CD3 (lymphocytes) and tumor necrosis factor alpha, of the apoptotic marker caspase 3 and of type 1 collagen were lower in LDE-MTX group when compared to LDE and MTX. Moreover, treatment with LDE-MTX reduced TGF-β, ERK and SMAD3 protein expression. Of note, CD68 and CD3 protein expression were positively correlated with the lumen area of the aortic arch (r2=0.36; p<0.001), indicating the importance of inflammation for the aortic dilation. The increase in bioavailability of intracellular adenosine was suggested by higher expression of A2a adenosine receptor and lower levels of adenosine deaminase in the aortic arch. Conclusions: Possibly by increasing the bioavailability of intracellular adenosine, LDE-MTX treatment had the ability to reduce the processes of inflammation, apoptosis and fibrosis that are consequent to fibrillin-1 mutation. By these means, LDE-MTX may conceivably prevent the development of the dilation and dissection in the aortic arch, the hallmarks of MFS.

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Methotrexate associated with a lipid core nanoparticle prevented the dilation and dissection of the aortic arch in mice with Marfan syndrome

European Heart Journal ◽

10.1093/ehjci/ehaa946.3777 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

M.C Guido ◽

N.M Lopes ◽

C.I Albuquerque ◽

E.R Tavares ◽

L Jensen ◽

...

Keyword(s):

Protein Expression ◽

Aortic Arch ◽

Marfan Syndrome ◽

Murine Model ◽

Volume Fraction ◽

Elastic Fiber ◽

Funding Source ◽

Lumen Area ◽

Lipid Core ◽

Fibrillin 1

Abstract Introduction Mutations in the fibrillin-1 gene result in dilation, dissection and rupture of the aorta, the main cause of mortality in patients with Marfan syndrome (MFS). Inflammation is associated with greater susceptibility to dissection and rupture of the aorta. Previously, we showed that methotrexate (MTX) carried in lipid core nanoparticles (LDE) had powerful anti-inflammatory and anti-proliferative effects on rheumatoid arthritis and atherosclerotic lesions induced in rabbits and improved myocardial infarction morphological and functional aspects in rats with ligation of left coronary artery. When MTX is associated to LDE, the uptake of the drug by the cells is increased several-fold, which conceivably endows MTX with enhanced action mechanisms and the drug toxicity is diminished. Purpose To investigate whether treatment with LDE-MTX can prevent the development of aortic arch lesions in a murine model of MFS. Methods mgΔloxPneo MFS murine model and wild-type mice were allocated in 3 groups of treatment: LDE only; MTX in commercial formulation; LDE-MTX. The treatment occurred weekly at a dose of 1mg/kg ip, between the 3rd and 6th month of life, period in which both dilation and dissection of the aortic arch are observed. After 12 weeks, the animals were submitted to echocardiography, morphometry and protein expression of the aortic arch. Results Compared to LDE and MTX groups, LDE-MTX treatment showed smaller lumen area of ascending and descending aorta and aortic arch segments in MFS mice. LDE-MTX also decreased the collagen volume fraction and the amount of aortic dissections, but did not affect the thickness of the vessel wall and the number of elastic fiber ruptures. The protein expression of the inflammatory markers CD68 (macrophages), CD3 (lymphocytes) and tumor necrosis factor alpha, of the apoptotic marker caspase 3 and of type 1 collagen were lower in MFS LDE-MTX group when compared to MFS mice treated with LDE and MTX. Moreover, treatment with LDE-MTX reduced TGF-β, ERK and the SMAD3 protein expression. Of note, CD68 and CD3 protein expression was positively correlated with the lumen area of the aortic arch (r2=0.36; p<0.001), indicating the importance of inflammation for the aortic dilation. The increase in bioavailability of intracellular adenosine in MFS animals treated with LDE-MTX was suggested by the higher expression of A2a adenosine receptor and the lower expression of adenosine deaminase in the aortic arch. Conclusion Possibly by increasing the bioavailability of intracellular adenosine, LDE-MTX treatment had the ability to reduce the processes of inflammation, apoptosis and fibrosis that are consequent to fibrillin-1 mutation. By these means, LDE-MTX may conceivably prevent the development of the dilation and dissection in the aortic arch, the hallmarks of MFS, in this animal model. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)

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Daunorubicin associated to lipid core nanoparticles reduces atherosclerotic lesions, inflammation and cardiotoxicity in atherosclerosis rabbit model

European Heart Journal ◽

10.1093/ehjci/ehaa946.3792 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

C.I Albuquerque ◽

E.R Tavares ◽

M.C Guido ◽

N.M Lopes ◽

R.V Baroni ◽

...

Keyword(s):

Body Weight ◽

Protein Expression ◽

Post Treatment ◽

Heart Weight ◽

Funding Source ◽

Lipid Core ◽

Atherosclerotic Lesions ◽

Apoptotic Factors ◽

Aortic Lesions ◽

Core Nanoparticles

Abstract Introduction Antiproliferative agents used in cancer chemotherapy have low toxicity and increased pharmacological action when carried into lipid core nanoparticles (LDE). LDE has similar structure to LDL and binds to LDL receptors through apo E it acquires in contact with plasma. Previously, we showed that treatment of rabbits with atherosclerosis with drugs such as taxanes and etoposide associate to LDE had pronounced reduction of the atherosclerotic lesions. In this study, we hypothesized whether daunorubicin (DNR) that belongs to another class of anti-cancer drugs, with a distinct mechanism of action and that has strong cardiotoxicity could also have effects against atherosclerosis. Methods Sixteen New Zealand male rabbits were fed with a 1% cholesterol diet for 8 weeks to induce atherosclerosis. After 4 weeks, the animals were treated weekly with LDE-DNR (6mg/kg, iv, n=9) or with LDE only (n=7). In addition, 3 animals without any intervention were used as a control group. Chow consumption, lipid and hematological profiles, body weight, and echocardiography were evaluated at baseline, pre-treatment and post-treatment. Morphometry and protein expression were performed to analyze the aortas. Results There was no difference in food intake and body weight for control, LDE and LDE-DNR groups. Total cholesterol, HDL-C, non-HDL-C and triglycerides increased in LDE and LDE-DNR groups when comparing baseline and post-treatment. Red blood cells decreased in LDE group at post-treatment in comparison to baseline, while LDE-DNR presented no such difference. Regarding aortic lesions of LDE-DNR group was 50% lower than LDE group. The protein expression of the inflammatory markers CD68, TNF-α and IL-6 in LDE-DNR was lower than LDE. Pro-apoptotic factors caspase 3, caspase 9 and BAX were also lower in LDE-DNR compared to LDE. Protein expression of vascular endothelial growth factor (VEGF) and vascular cell adhesion molecule (VCAM) was lower in the LDE-DNR group when compared to the LDE group. To test the cardiotoxicity of LDE-DNR, echocardiography was performed and observed that under LDE-DNR both systolic and diastolic function were preserved with no difference in cardiac mass in LDE-DNR when compared to control and LDE groups. However, LDE showed a significant increase in relative heart weight compared to control while LDE-DNR had no such difference. Treatment with LDE-DNR did not induce observable hematological toxicity. Conclusion The treatment with LDE-DNR reduced aortic lesions, diminished inflammatory and pro-apoptotic factors, diminished VCAM expression in the aorta and promoted cardiac preservation in a rabbit atherosclerosis model. In conclusion, LDE-DNR can be eligible for future developments in the quest for novel effective and safe treatments for atherosclerosis. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): FAPESP (Fundaçao de Amparo a Pesquisa do Estado de Sao Paulo)

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Ocular Phenotype of Mice with Impaired Fibrillin-1 Function on Hypercholesterolemic Apolipoprotein E-Deficient Background

Current Trends in Ophthalmology ◽

10.18314/ctoy.v1i1.1265 ◽

2018 ◽

Vol 1 (1) ◽

pp. 75-79

Author(s):

Emmi Kokki ◽

Tommi Karttunen ◽

Sanna Kettunen ◽

Kati Kinnunen ◽

Guido R.Y. De Meyer ◽

...

Keyword(s):

Apolipoprotein E ◽

Marfan Syndrome ◽

High Fat Diet ◽

Elastic Fiber ◽

Retinal Vessel ◽

High Fat ◽

Vessel Occlusion ◽

Collagen Formation ◽

Fibrillin 1 ◽

Retinal Vessel Occlusion

Aim: Transgenic mice with an elastic fiber mutation (C1039G+/-) in the fibrillin-1 gene and apolipoprotein E deficiency express vulnerable atherosclerotic plaque formation in the aorta and coronary and carotid arteries. The fibrillin-1 gene mutation alone leads to impaired fibrillin-1 function common to Marfan syndrome. The aim was to study for the first time the potential effects of atherosclerosis and vulnerable plaques in mouse eye and spontaneous retinal vessel occlusions in these mice.Methods: Apolipoprotein E-deficient and fibrillin-1 mutated mice (ApoE-/-/Fbn1C1039G+/-) were used for the study. ApoE-/- littermates served as controls. Optical coherence tomography and fluorescein angiography were used to study the retina and retinal vessels before and after the 12-week high-fat diet. Series of staining were performed to study morphology, apoptosis, glial fibrillary acidic protein activation, collagen formation, inflammatory cell infiltration and drusen formation.Results: No pathological changes were found in hypercholesterolemic ApoE-/-/Fbn1C1039G+/- mice in imaging studies nor in the histological stainings. There was neither abnormality in the retinal morphology nor any detectable biomarkers.Conclusion: ApoE-/-/Fbn1C1039G+/- mice did not present the ocular signs of Marfan syndrome.12-week high-fat diet is not sufficient to induce retinal vessel occlusion on 20 to 23 weeks old mice. This indicates that the mechanistic background of retinal vessel occlusion is more complex.

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444Plasma levels of microfibrillar associated protein type 4 (MFAP4) are predictive for aortic dissection in patients with Marfan Syndrome

European Heart Journal ◽

10.1093/eurheartj/ehz747.0110 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

M M Van Andel ◽

S Wanga ◽

X Yin ◽

P Skroblin ◽

D R Koolbergen ◽

...

Keyword(s):

Aortic Dissection ◽

Marfan Syndrome ◽

Elastic Fiber ◽

Aortic Distensibility ◽

High Plasma ◽

Type B ◽

Tgf Beta ◽

Fibrillin 1 ◽

Aortic Dissections

Abstract Aim Marfan syndrome is a disorder with mutations in the fibrillin-1 gene, leading to elastic fiber degradation and increased TGF-beta signaling. The life-threatening feature of Marfan is aneurysm formation with a risk of fatal aortic dissections. In a proteomics screen, we identified MFAP4, a protein involved in fibrillin-1 and elastic fiber formation, to be increased in the Marfan aorta. We aim to study the role of MFAP4 in Marfan aortic disease. Methods and results MFAP4 co-localizes in the aorta with elastin and collagen fibers. In vitro experiments show that MFAP4 expression is upregulated by TGF-beta, which could explain the increased MFAP4 protein levels in the Marfan aorta. In a substudy of 96 Marfan patients from the COMPARE trial, MFAP4 levels correlate with aortic root diameter (r=0.30, p=0.01). Patients previously enrolled in the COMPARE trial were retrospectively analyzed. Cardiovascular events, including aortic dissection, were assessed. Plasma samples were prospectively collected at time of inclusion in the study and analyzed retrospectively on MFAP4. In the 7 years of follow up, 5 Type B dissections occurred, all of them in patients in the upper tertile of plasma MFAP4. High plasma MFAP4 associates with poor dissection-free survival (Figure 1). Moreover, the aortic distensibility as measure for aortic stiffness and damage, was calculated throughout the aorta from available MRI images of these patients. Interestingly, in the descending thoracic aorta where type B dissections occur, the aortic distensibility is significantly lower (indicating decreased aortic elasticity) in Marfan patients with high plasma MFAP4, thus associating with aortic damage. Figure 1 Conclusion MFAP4, a protein involved in extracellular matrix assembly, is elevated in the Marfan aorta. High plasma MFAP4 seems to reflect aortic damage and predicted type B aortic dissections in up to 7 years follow up.

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P2577Losartan diminishes the expression of TGF-beta and improves cardiomyopathy in mice with Marfan syndrome

European Heart Journal ◽

10.1093/eurheartj/ehz748.0904 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

M C Guido ◽

V Debbas ◽

N M Lopes ◽

J C A Ferreira-Filho ◽

V M C Salemi ◽

...

Keyword(s):

Growth Factor ◽

Protein Expression ◽

Marfan Syndrome ◽

Coronary Arteries ◽

Murine Model ◽

Elastic Fibers ◽

Control Group ◽

Losartan Group ◽

Fibrillin 1 ◽

Losartan Treatment

Abstract Introduction Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene, leading to abnormal signaling of the transforming growth factor beta (TGF-β). This results in aortic root dilation, dissection and rupture, which are the main causes of morbidity and mortality of MFS patients. Current treatment with losartan, an angiotensin-II receptor-1 blocker, has shown beneficial effect on aortic disease in MFS murine models. However, the mechanisms whereby the treatment with losartan improves cardiac remodeling and function of the left ventricle (LV) in MFS are still unknown. Purpose To investigate the effects of losartan on mechanisms of the cardiomyopathy in mice with MFS. Methods mgΔloxPneo MFS murine model from C129/sv background was utilized in this study. To evaluate the effect of the treatment with losartan on the LV of MFS and wild-type mice, animals were allocated in 2 groups of treatments: Losartan group: mice were kept with water supplemented with losartan (0.6g/L); Untreated control group: mice were kept with water only. The animals received treatment from 1 month of age until completing 6 months. After five months of treatment, LV echocardiography was performed, and fragments of LV tissue were analyzed by morphometry and protein expression analysis by Western blot. Results Losartan MFS mice showed decrease in interventricular septum and posterior wall thickness and LV mass. Furthermore, losartan prevented aortic and mitral regurgitation, arrhythmia, bradycardia, septal hypokinesia and pulmonary hypertension when compared with control MFS mice. Systolic and diastolic LV function were not different between groups. Collagen volume fraction and the disorganization and disruptions of the elastic fibers in coronary arteries were lower in losartan treatment than in controls. The protein expression of pro-apoptotic factors (BAX/Bcl-2 and caspase 3 and 9), proliferating cell nuclear antigen and hypoxia-inducible factor 1 and 2α were lower in losartan treatment, whereas the expression of vascular endothelial growth factor was increased in losartan group when compared with control MFS mice. Moreover, the treatment with losartan strongly reduced the TGF-β, ERK and p38MAPK protein expression compared to controls. Conclusion In this murine model of MFS, losartan treatment has the ability to change several pathophysiological mechanisms related with the fibrillin-1 mutation, by decreasing apoptosis, cell proliferation and increasing angiogenesis. Overall, the treatment resulted in improved structural rearrangement and attenuation of the rupture of elastic fibers in the coronary arteries, of the cardiac hypertrophy and myocardial fibrosis. These effects were possibly related with the decreased TGF-β expression by ERK and p38MAPK signaling pathways by losartan. Our findings shed a new light on the mechanisms of action of losartan on MFS cardiomyopathy. Acknowledgement/Funding FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)

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Faculty Opinions recommendation of A nonsense mutation in the fibrillin-1 gene of a Marfan syndrome patient induces NMD and disrupts an exonic splicing enhancer.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008392.105207 ◽

2002 ◽

Author(s):

Lynne Maquat

Keyword(s):

Marfan Syndrome ◽

Nonsense Mutation ◽

Syndrome Patient ◽

Exonic Splicing Enhancer ◽

Fibrillin 1 ◽

Splicing Enhancer

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MIP-T3 Expression Associated with Defects of Ciliogenesis in Airway of COPD Patients

Canadian Respiratory Journal ◽

10.1155/2020/1350872 ◽

2020 ◽

Vol 2020 ◽

pp. 1-6

Author(s):

Wen-Jun Wang ◽

Shi-Fang Yang ◽

Zhi-Rui Gao ◽

Ze-Ru Luo ◽

Yuan-Ling Liu ◽

...

Keyword(s):

Protein Expression ◽

Bronchial Epithelium ◽

Smoking History ◽

Chronic Obstructive ◽

Healthy Smoker ◽

Necrosis Factor Alpha ◽

Copd Patients ◽

Structural Abnormalities ◽

Abnormal Cilia ◽

Nonsmoker Group

Introduction. Some studies have found that cilia were shorter in COPD smokers than in nonsmokers or healthy smokers. However, the structural abnormalities of cilia and the cause of such abnormalities in COPD patients still remain unknown. Tumor necrosis factor alpha receptor 3 interacting protein 1 (MIP-T3) may play an important role in the progress of ciliary protein transporting. Objectives. This study aimed at exploring the dominated structural abnormalities of cilia and the involvement of MIP-T3 in the pathogenesis of cilia of COPD patients. Methods. Patients who accepted pulmonary lobectomy were divided into 3 groups: the chronic obstructive pulmonary disease (COPD) smoker group, the healthy smoker group, and the nonsmoker group, according to smoking history and pulmonary function. The ultrastructure of cilia and the percentage of abnormal cilia were analyzed using a transmission electron microscope. Real-time PCR, immunohistochemical staining, and western blotting in bronchial epithelium were used to determine MIP-T3 mRNA and protein expression. The relationship between the percentage of abnormal cilia and lung function and MIP-T3 protein expression was analyzed. Results. Patients in the COPD smoker group had increased percentage of abnormal cilia comparing to both the healthy smoker group and the nonsmoker group (both P values <0.05). MIP-T3 expression was significantly declined in the COPD smoker group (P value <0.05). Moreover, the percentage of abnormal cilia was negatively correlated with FEV1, FEV1/FVC ratio, and FEV1%pred (all P values <0.05). Moreover, the MIP-T3 protein expression was positively correlated with the percentage of abnormal cilia (P value <0.05). Conclusions. Our results suggested that the abnormal ciliary ultrastructure, which was common in COPD patients, might be due to MIP-T3 downregulation.

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