Proteomic Analysis in Aortic Media of Patients With Marfan Syndrome Reveals Increased Activity of Calpain 2 in Aortic Aneurysms

Abstract

Background-Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation. Methods and Results-Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibrilassociated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls.Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin. Conclusions-Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta. Key Words: aneurysm Ⅲ aorta Ⅲ calpain Ⅲ Marfan syndrome Ⅲ proteomics M arfan syndrome (MFS) is a disorder of connective tissue caused by mutations in the fibrillin-1 gene (FBN-1) that may lead to abnormal regulation of transforming growth factor-␤ (TGF-␤) signaling. 1 Clinical manifestation implies principally the cardiovascular, skeletal, and ocular systems, and other systems such as skin, lung, and dura may also be affected. Cardiovascular defects involve dilatation of the ascending aortic root leading to aortic aneurysm and dissection, which are the main cause of morbidity and mortality in MFS, and mitral valve prolapse with or without regurgitation. 2 Clinical Perspective on p 991Molecular mechanisms of aneurysm formation and its treatment are the subject of intensive research. Three key proteins seem to directly contribute to aneurysm formation. First, defective fibrillin-1 is thought to alter targeting and sequestration of TGF-␤, leading to increased TGF-␤ activity. 1 Blocking TGF-␤ activity with antagonists such as TGF-␤-neutralizing antibodies prevents aneurysm formation in a mouse model of MFS. 3 Second, angiotensin II (Ang II) is upregulated in MFS. 4 Treatment of patients with Ang II type 1 (AT 1 ) receptor (AT 1 R) blocker or angiotensin-converting enzyme (ACE) inhibitors reduces progression of aortic d...

Vascular Smooth Muscle Cell Phenotypic Changes in Patients With Marfan Syndrome

“…61 Changes in the VSMC phenotype are also reported in other aortic aneurysms 21,22 in a murine model of MFS. 62 Our results are largely coincident with those obtained after the proteomic analysis of the tunica media of human Marfan aneurysms, 63 which report strong upregulation of calponin1, filamin A, and vinculin among other contractile proteins. However, this coincidence does not extend to transgelin (SM22α), which in the study by Pilop et al 63 was downregulated, but that in our Marfan aortic aneurysms showed variability and even reduced at transcriptional level in cultured Marfan cells.…”

“…62 Our results are largely coincident with those obtained after the proteomic analysis of the tunica media of human Marfan aneurysms, 63 which report strong upregulation of calponin1, filamin A, and vinculin among other contractile proteins. However, this coincidence does not extend to transgelin (SM22α), which in the study by Pilop et al 63 was downregulated, but that in our Marfan aortic aneurysms showed variability and even reduced at transcriptional level in cultured Marfan cells. Moreover, VSMC explanted from heterozygous TGFβR2 mutations decreased global expression of contractile proteins and failed to increase their expression after exposure to the cytokine, which was attributed to the disruption of TGF-β signaling.…”

Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation

“…Marfan syndrome is caused by mutations in fibrillin-1 gene, and its most serious manifestation is aortic aneurysm and dissection of the ascending aorta (45). Importantly, MFAP4 protein is up-regulated in ascending aortic samples from patients suffering from Marfan syndrome (46), suggesting that MFAP4 might play an important role in Marfan syndrome pathology and aneurysm formation.…”

Increased expression of calpain and elevated activity of calcineurin in the myocardium of patients with congestive heart failure

“…A previous study demonstrated that calpains were involved in the development of Ang IIinduced vascular remodelling (4). Additionally, the activation of calpains has been identified in several types of cardiovascular diseases, including aortic aneurysm and diabetic cardiomyopathy (17,18). Furthermore, inhibition of calpains by treatment with a pharmacological inhibitor or overexpression of calpastatin may protect the heart from ischemiareperfusion injury (19)(20)(21).…”