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

Jiang L, Wang M, Zhang J, Monticone RE, Telljohann R, Spinetti G, Pintus G, Lakatta EG - PLoS ONE (2008)

Bottom Line: 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.The consequences of engagement of calpain-1 with its substrates, however, in governing the age-associated proinflammatory status within the arterial wall, remains unknown.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America.

ABSTRACT

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.

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Calpain-1 transcriptome, protein abundance, and activity increase in the aged aortic wall.A. Average data of calpain-1 transcriptome. B. Western blots for calpain-1 protein of rat intact aortae (upper panel); average data (lower panel). C. Dual en face fluorescence staining for α-SMA (green), calpain-1 (red) in the medial aortic sections from young (upper panels) and old rats (lower panels). Nuclei were counterstained with DAPI (blue). Merged image is depicted in right panel (yellow-blue). Magnification: ×400. D. Western blots for calpain substrate α-II spectrin from aortic lysates (left panels). Average cleaved spectrin fraction (right panel). *p<0.05, old vs. young.
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pone-0002231-g001: Calpain-1 transcriptome, protein abundance, and activity increase in the aged aortic wall.A. Average data of calpain-1 transcriptome. B. Western blots for calpain-1 protein of rat intact aortae (upper panel); average data (lower panel). C. Dual en face fluorescence staining for α-SMA (green), calpain-1 (red) in the medial aortic sections from young (upper panels) and old rats (lower panels). Nuclei were counterstained with DAPI (blue). Merged image is depicted in right panel (yellow-blue). Magnification: ×400. D. Western blots for calpain substrate α-II spectrin from aortic lysates (left panels). Average cleaved spectrin fraction (right panel). *p<0.05, old vs. young.

Mentions: The quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the transcript level of calpain-1 increased 1.8 fold within the aortic wall in old (30 mo) compared to young (8 mo) rats (Fig. 1A); Western blot analysis indicated that calpain-1 protein abundance increased 1.3-fold (p<0.05) with age (Fig. 1B). In addition, dual immunolabeling of en face medial aortic sections demonstrated that increased calpain-1 protein within old rats was co-localized with α-smooth muscle actin (α-SMA), a marker of VSMC, stained cells (Fig. 1C, lower right-most panel). The levels of cleaved spectrin fragments predict calpain activity. In aortic lysates of old rats Fig. 1D (left panel) three conspicuous spectrin bands, designated as intact (250 kDa) and cleaved spectrin fragments (145, and 150 kDa) respectively, are observed. In contrast, only intact spectrin was detected in young rat aortic lysates. Thus, on the basis of cleaved spectrin fragments old rats have a 3.3 fold increase in aortic calpain activity compared to their young counterparts (Fig. 1D, right panel).


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

Jiang L, Wang M, Zhang J, Monticone RE, Telljohann R, Spinetti G, Pintus G, Lakatta EG - PLoS ONE (2008)

Calpain-1 transcriptome, protein abundance, and activity increase in the aged aortic wall.A. Average data of calpain-1 transcriptome. B. Western blots for calpain-1 protein of rat intact aortae (upper panel); average data (lower panel). C. Dual en face fluorescence staining for α-SMA (green), calpain-1 (red) in the medial aortic sections from young (upper panels) and old rats (lower panels). Nuclei were counterstained with DAPI (blue). Merged image is depicted in right panel (yellow-blue). Magnification: ×400. D. Western blots for calpain substrate α-II spectrin from aortic lysates (left panels). Average cleaved spectrin fraction (right panel). *p<0.05, old vs. young.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2373882&req=5

pone-0002231-g001: Calpain-1 transcriptome, protein abundance, and activity increase in the aged aortic wall.A. Average data of calpain-1 transcriptome. B. Western blots for calpain-1 protein of rat intact aortae (upper panel); average data (lower panel). C. Dual en face fluorescence staining for α-SMA (green), calpain-1 (red) in the medial aortic sections from young (upper panels) and old rats (lower panels). Nuclei were counterstained with DAPI (blue). Merged image is depicted in right panel (yellow-blue). Magnification: ×400. D. Western blots for calpain substrate α-II spectrin from aortic lysates (left panels). Average cleaved spectrin fraction (right panel). *p<0.05, old vs. young.
Mentions: The quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the transcript level of calpain-1 increased 1.8 fold within the aortic wall in old (30 mo) compared to young (8 mo) rats (Fig. 1A); Western blot analysis indicated that calpain-1 protein abundance increased 1.3-fold (p<0.05) with age (Fig. 1B). In addition, dual immunolabeling of en face medial aortic sections demonstrated that increased calpain-1 protein within old rats was co-localized with α-smooth muscle actin (α-SMA), a marker of VSMC, stained cells (Fig. 1C, lower right-most panel). The levels of cleaved spectrin fragments predict calpain activity. In aortic lysates of old rats Fig. 1D (left panel) three conspicuous spectrin bands, designated as intact (250 kDa) and cleaved spectrin fragments (145, and 150 kDa) respectively, are observed. In contrast, only intact spectrin was detected in young rat aortic lysates. Thus, on the basis of cleaved spectrin fragments old rats have a 3.3 fold increase in aortic calpain activity compared to their young counterparts (Fig. 1D, right panel).

Bottom Line: 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.The consequences of engagement of calpain-1 with its substrates, however, in governing the age-associated proinflammatory status within the arterial wall, remains unknown.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus