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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 and vimentin interactions within VSMC.A. Photomicrographs of dual labeling for calpain-1 (green) and vimentin (red) within VSMC from young (left panels) and old rats (right panels); merged image (yellow bottom panels). B. Casein zymogram of VSMC from young and old VSMC infected with a CANP1 or CAST virus (upper panel). Western blots of vimentin from young and old VSMC infected with CANP1 or CAST virus (lower panel). Proteolytic fragments are indicated by arrows and designated I-V. C. Fluorescence labeled calpain-1 protein of VSMC isolated from young rat aortae. VSMC were isolated from the aorta of 8-month-old rat, allowed to adhere on a fibrogen matrix, fixed with methanol and acetone, and stained with calpain-1 antibody (red). The nuclei were counterstained with DAPI (blue). Magnification × 400.
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pone-0002231-g006: Calpain-1 and vimentin interactions within VSMC.A. Photomicrographs of dual labeling for calpain-1 (green) and vimentin (red) within VSMC from young (left panels) and old rats (right panels); merged image (yellow bottom panels). B. Casein zymogram of VSMC from young and old VSMC infected with a CANP1 or CAST virus (upper panel). Western blots of vimentin from young and old VSMC infected with CANP1 or CAST virus (lower panel). Proteolytic fragments are indicated by arrows and designated I-V. C. Fluorescence labeled calpain-1 protein of VSMC isolated from young rat aortae. VSMC were isolated from the aorta of 8-month-old rat, allowed to adhere on a fibrogen matrix, fixed with methanol and acetone, and stained with calpain-1 antibody (red). The nuclei were counterstained with DAPI (blue). Magnification × 400.

Mentions: With aging, alterations of VSMC cytoskeletal proteins such as α-SMA and vimentin occur concomitantly with changes in VSMC migration rates [35]. Fig. 6A demonstrates that within the old VSMC increased calpain-1 colocalizes with vimentin (Fig. 6A, lower right panel, yellow color) and α-SMA (data not shown). Casein zymography (Fig. 6B, upper panel) confirms that calpain-1 but not calpain-2 activity in VSMC increase with age. Over-expression of calpastatin did not affect calpain-1 activity in young VSMC, although it decreased calpain-1 activity in old cells. Moreover, calpain-2 activity was not affected by over-expression of calpastatin in either young or old VSMC. These inconsistent effects may be due to different calcium requirements for calpastatin binding to calpain-1 or calpain-2 [36]. Cytosolic calcium concentrations ([Ca 2+]) are known to increase with aging [37]. A single vimentin cDNA encodes a 57-kDa intact protein [27]. In addition, we observed intact vimentin protein only in early passage young VSMC, but not in old cells, which showed only small vimentin fragments (Fig. 6B). Further, several small fragments are disproportionately distributed in young and old cells: while both young and old cells share fragments II and III, fragment I is more abundant in young cells (Fig. 6B, 8 mo control), while fragment IV is unique to old cells (Fig. 6B, 30 mo controls).


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 and vimentin interactions within VSMC.A. Photomicrographs of dual labeling for calpain-1 (green) and vimentin (red) within VSMC from young (left panels) and old rats (right panels); merged image (yellow bottom panels). B. Casein zymogram of VSMC from young and old VSMC infected with a CANP1 or CAST virus (upper panel). Western blots of vimentin from young and old VSMC infected with CANP1 or CAST virus (lower panel). Proteolytic fragments are indicated by arrows and designated I-V. C. Fluorescence labeled calpain-1 protein of VSMC isolated from young rat aortae. VSMC were isolated from the aorta of 8-month-old rat, allowed to adhere on a fibrogen matrix, fixed with methanol and acetone, and stained with calpain-1 antibody (red). The nuclei were counterstained with DAPI (blue). Magnification × 400.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2373882&req=5

pone-0002231-g006: Calpain-1 and vimentin interactions within VSMC.A. Photomicrographs of dual labeling for calpain-1 (green) and vimentin (red) within VSMC from young (left panels) and old rats (right panels); merged image (yellow bottom panels). B. Casein zymogram of VSMC from young and old VSMC infected with a CANP1 or CAST virus (upper panel). Western blots of vimentin from young and old VSMC infected with CANP1 or CAST virus (lower panel). Proteolytic fragments are indicated by arrows and designated I-V. C. Fluorescence labeled calpain-1 protein of VSMC isolated from young rat aortae. VSMC were isolated from the aorta of 8-month-old rat, allowed to adhere on a fibrogen matrix, fixed with methanol and acetone, and stained with calpain-1 antibody (red). The nuclei were counterstained with DAPI (blue). Magnification × 400.
Mentions: With aging, alterations of VSMC cytoskeletal proteins such as α-SMA and vimentin occur concomitantly with changes in VSMC migration rates [35]. Fig. 6A demonstrates that within the old VSMC increased calpain-1 colocalizes with vimentin (Fig. 6A, lower right panel, yellow color) and α-SMA (data not shown). Casein zymography (Fig. 6B, upper panel) confirms that calpain-1 but not calpain-2 activity in VSMC increase with age. Over-expression of calpastatin did not affect calpain-1 activity in young VSMC, although it decreased calpain-1 activity in old cells. Moreover, calpain-2 activity was not affected by over-expression of calpastatin in either young or old VSMC. These inconsistent effects may be due to different calcium requirements for calpastatin binding to calpain-1 or calpain-2 [36]. Cytosolic calcium concentrations ([Ca 2+]) are known to increase with aging [37]. A single vimentin cDNA encodes a 57-kDa intact protein [27]. In addition, we observed intact vimentin protein only in early passage young VSMC, but not in old cells, which showed only small vimentin fragments (Fig. 6B). Further, several small fragments are disproportionately distributed in young and old cells: while both young and old cells share fragments II and III, fragment I is more abundant in young cells (Fig. 6B, 8 mo control), while fragment IV is unique to old cells (Fig. 6B, 30 mo controls).

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