Milk Fat Globule Epidermal Growth Factor VIII Fragment Medin in Age-Associated Arterial Adverse Remodeling and Arterial Disease.

Medin, a small 50-amino acid peptide, is an internal cleaved product from the second discoidin domain of milk fat globule epidermal growth factor VIII (MFG-E8) protein. Medin has been reported as the most common amylogenic protein in the upper part of the arterial system, including aortic, temporal, and cerebral arterial walls in the elderly. Medin has a high affinity to elastic fibers and is closely associated with arterial degenerative inflammation, elastic fiber fragmentation, calcification, and amyloidosis. In vitro, treating with the medin peptide promotes the inflammatory phenotypic shift of both endothelial cells and vascular smooth muscle cells. In vitro, ex vivo, and in vivo studies demonstrate that medin enhances the abundance of reactive oxygen species and reactive nitrogen species produced by both endothelial cells and vascular smooth muscle cells and promotes vascular endothelial dysfunction and arterial stiffening. Immunostaining and immunoblotting analyses of human samples indicate that the levels of medin are increased in the pathogenesis of aortic aneurysm/dissection, temporal arteritis, and cerebrovascular dementia. Thus, medin peptide could be targeted as a biomarker diagnostic tool or as a potential molecular approach to curbing the arterial degenerative inflammatory remodeling that accompanies aging and disease.

Inflammatory Role of Milk Fat Globule-Epidermal Growth Factor VIII in Age-Associated Arterial Remodeling.

Background Age-associated aortic remodeling includes a marked increase in intimal medial thickness (IMT), associated with signs of inflammation. Although aortic wall milk fat globule-epidermal growth factor VIII (MFG-E8) increases with age, and is associated with aortic inflammation, it is not known whether MFG-E8 is required for the age-associated increase in aortic IMT. Here, we tested whether MFG-E8 is required for the age-associated increase in aortic IMT. Methods and Results To determine the role of MFG-E8 in the age-associated increase of IMT, we compared aortic remodeling in adult (20-week) and aged (96-week) MFG-E8 (-/-) knockout and age matched wild-type (WT) littermate mice. The average aortic IMT increased with age in the WT from 50±10 to 70±20 µm (P<0.0001) but did not significantly increase with age in MFG-E8 knockout mice. Because angiotensin II signaling is implicated as a driver of age-associated increase in IMT, we infused 30-week-old MFG-E8 knockout and age-matched littermate WT mice with angiotensin II or saline via osmotic mini-pumps to determine whether MFG-E8 is required for angiotensin II-induced aortic remodeling. (1) In WT mice, angiotensin II infusion substantially increased IMT, elastic lamina degradation, collagen deposition, and the proliferation of vascular smooth muscle cells; in contrast, these effects were significantly reduced in MFG-E8 KO mice; (2) On a molecular level, angiotensin II treatment significantly increased the activation and expression of matrix metalloproteinase type 2, transforming growth factor beta 1, and its downstream signaling molecule phosphorylated mother against decapentaplegic homolog 2, and collagen type I production in WT mice; however, in the MFG-E8 knockout mice, these molecular effects were significantly reduced; and (3) in WT mice, angiotensin II increased levels of aortic inflammatory markers phosphorylated nuclear factor-kappa beta p65, monocyte chemoattractant protein 1, tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 molecular expression, while in contrast, these inflammatory markers did not change in knockout mice. Conclusions Thus, MFG-E8 is required for both age-associated proinflammatory aortic remodeling and also for the angiotensin II-dependent induction in younger mice of an aortic inflammatory phenotype observed in advanced age. Targeting MFG-E8 would be a novel molecular approach to curb adverse arterial remodeling.

Age-associated proinflammatory elastic fiber remodeling in large arteries.

Elastic fibers are the main components of the extracellular matrix of the large arterial wall. Elastic fiber remodeling is an intricate process of synthesis and degradation of the core elastin protein and microfibrils accompanied by the assembly and disassembly of accessory proteins. Age-related morphological, structural, and functional proinflammatory remodeling within the elastic fiber has a profound effect upon the integrity, elasticity, calcification, amyloidosis, and stiffness of the large arterial wall. An age-associated increase in arterial stiffness is a major risk factor for the pathogenesis of diseases of the large arteries such as hypertensive and atherosclerotic vasculopathy. This mini review is an update on the key molecular, cellular, functional, and structural mechanisms of elastic fiber proinflammatory remodeling in large arteries with aging. Targeting structural and functional integrity of the elastic fiber may be an effective approach to impede proinflammatory arterial remodeling with advancing age.

Proinflammatory Arterial Stiffness Syndrome: A Signature of Large Arterial Aging.

Age-associated structural and functional remodeling of the arterial wall produces a productive environment for the initiation and progression of hypertension and atherosclerosis. Chronic aging stress induces low-grade proinflammatory signaling and causes cellular proinflammation in arterial walls, which triggers the structural phenotypic shifts characterized by endothelial dysfunction, diffuse intimal-medial thickening, and arterial stiffening. Microscopically, aged arteries exhibit an increase in arterial cell senescence, proliferation, invasion, matrix deposition, elastin fragmentation, calcification, and amyloidosis. These characteristic cellular and matrix alterations not only develop with aging but can also be induced in young animals under experimental proinflammatory stimulation. Interestingly, these changes can also be attenuated in old animals by reducing low-grade inflammatory signaling. Thus, mitigating age-associated proinflammation and arterial phenotype shifts is a potential approach to retard arterial aging and prevent the epidemic of hypertension and atherosclerosis in the elderly.

Proinflammation: the key to arterial aging.

Arterial aging is the major contributing factor to increases in the incidence and prevalence of cardiovascular disease, due mainly to the presence of chronic, low-grade, 'sterile' arterial inflammation. Inflammatory signaling driven by the angiotensin II cascade perpetrates adverse age-associated arterial structural and functional remodeling. The aged artery is characterized by endothelial disruption, enhanced vascular smooth muscle cell (VMSC) migration and proliferation, extracellular matrix (ECM) deposition, elastin fracture, and matrix calcification/amyloidosis/glycation. Importantly, the molecular mechanisms of arterial aging are also relevant to the pathogenesis of hypertension and atherosclerosis. Age-associated arterial proinflammation is to some extent mutable, and interventions to suppress or delay it may have the potential to ameliorate or retard age-associated arterial diseases.

Calpain-1 regulation of matrix metalloproteinase 2 activity in vascular smooth muscle cells facilitates age-associated aortic wall calcification and fibrosis.

Age-associated central arterial wall stiffness is linked to extracellular matrix remodeling, including fibrosis and vascular calcification. Angiotensin II induces both matrix metalloproteinase 2 (MMP2) and calpain-1 expression and activity in the arterial wall. However, the role of calpain-1 in MMP2 activation and extracellular matrix remodeling remains unknown. Dual histo-immunolabeling demonstrates colocalization of calpain-1 and MMP2 within old rat vascular smooth muscle cells. Overexpression of calpain-1 induces MMP2 transcripts, protein levels, and activity, in part, by increasing the ratio of membrane type 1 MMPs to tissue inhibitor of metalloproteinases 2. These effects of calpain-1 overexpression-induced MMP2 activation are linked to increased collagen I and III production and vascular calcification. In addition, overexpression of calpain-1 also induces transforming growth factor-ß1/Smad signaling, elastin degradation, alkaline phosphatase activation, and total calcium content but reduces the expression of calcification inhibitors, osteopontin, and osteonectin, in cultured vascular smooth muscle cells in vitro and in carotid artery rings ex vivo. Furthermore, both calpain-1 and collagen II increase with aging within human aortic intima. Interestingly, in aged human aortic wall, both calpain-1 and collagen II are highly expressed in artherosclerotic plaque areas compared with grossly normal areas. Cross-talk of 2 proteases, calpain-1 and MMP2, leads to secretion of active MMP2, which modulates extracellular matrix remodeling via enhancing collagen production and facilitating vascular calcification. These results establish calpain-1 as a novel molecular candidate to retard age-associated extracellular matrix remodeling and its attendant risk for hypertension and atherosclerosis.

Chronic matrix metalloproteinase inhibition retards age-associated arterial proinflammation and increase in blood pressure.

Age-associated arterial remodeling involves arterial wall collagen deposition and elastin fragmentation, as well as an increase in arterial pressure. This arterial remodeling is linked to proinflammatory signaling, including transforming growth factor-ß1, monocyte chemoattractant protein 1, and proendothelin 1, activated by extracellular matrix metalloproteinases (MMPs) and orchestrated, in part, by the transcriptional factor ets-1. We tested the hypothesis that inhibition of MMP activation can decelerate the age-associated arterial proinflammation and its attendant increase in arterial pressure. Indeed, chronic administration of a broad-spectrum MMP inhibitor, PD166739, via a daily gavage, to 16-month-old rats for 8 months markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by the following: (1) inhibition of the age-associated increases in aortic gelatinase and interstitial collagenase activity in situ; (2) preservation of the elastic fiber network integrity; (3) a reduction of collagen deposition; (4) a reduction of monocyte chemoattractant protein 1 and transforming growth factor-ß1 activation; (5) a diminution in the activity of the profibrogenic signaling molecule SMAD-2/3 phosphorylation; (6) inhibition of proendothelin 1 activation; and (7) downregulation of expression of ets-1. Acute exposure of cultured vascular smooth muscle cells in vitro to proendothelin 1 increased both the transcription and translation of ets-1, and these effects were markedly reduced by MMP inhibition. Furthermore, infection of vascular smooth muscle cells with an adenovirus harboring a full-length ets-1 cDNA increased activities of both transforming growth factor-ß1 and monocyte chemoattractant protein 1. Collectively, our results indicate that MMP inhibition retards age-associated arterial proinflammatory signaling, and this is accompanied by preservation of intact elastin fibers, a reduction in collagen, and blunting of an age-associated increase in blood pressure.

Increased aortic calpain-1 activity mediates age-associated angiotensin II signaling of vascular smooth muscle cells.

BACKGROUND: Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, has been linked to an age-associated increase in migration capacity of vascular smooth muscle cells (VSMC), and to other proinflammatory features of arterial aging. Calpain-1 activation is required for MMP2 expression in fibroblasts and is induced in cardiomyocytes by Ang II. The consequences of engagement of calpain-1 with its substrates, however, in governing the age-associated proinflammatory status within the arterial wall, remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: The present findings demonstrate that transcription, translation, and activity of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC from old (30-mo) rats compared to young (8-mo). Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II increases within the aged arterial wall. To further explore the relationship of calpain-1 to Ang II, we chronically infused Ang II into young rats, and treated cultured aortic rings or VSMC with Ang II. We also constructed adenoviruses harboring calpain-1 (CANP1) or its endogenous inhibitor calpastatin (CAST) and infected these into VSMC. Ang II induces calpain-1 expression in the aortic walls in vivo and ex vivo and VSMC in vitro. The Ang II mediated, age-associated increased MMP2 activity and migration in VSMC are both blocked by calpain inhibitor 1 or CAST. Over-expression of calpain-1 in young VSMC results in cleavage of intact vimentin, and an increased migratory capacity mimicking that of old VSMC, which is blocked by the MMP inhibitor, GM6001. CONCLUSIONS/SIGNIFICANCE: Calpain-1 activation is a pivotal molecular event in the age-associated arterial Ang II/MMP2 signaling cascade that is linked to cytoskeleton protein restructuring, and VSMC migration. Therefore, targeting calpain-1 has the potential to delay or reverse the arterial remodeling that underlies age-associated diseases i.e. atherosclerosis.