Smooth muscle phenotypic modulation is an early event in aortic aneurysms

Abstract

Objectives: Vascular smooth muscle cells can undergo profound changes in phenotype, defined by coordinated repression of smooth muscle cell marker genes and production of matrix metalloproteinases in response to injury. However, little is known of the role of smooth muscle cells in aortic aneurysms. We hypothesized that smooth muscle cells undergo phenotypic modulation early in the development of aortic aneurysms.

Methods: Abdominal aortas from C57B6 mice (n = 79) were perfused with elastase or saline (control) and harvested at 1, 3, 7, or 14 days. Aortas were analyzed by means of quantitative polymerase chain reaction and immunohistochemistry for smooth muscle cell marker genes, including SM22A, smooth muscle alpha-actin, and matrix metalloproteinases 2 and 9. In complimentary experiments human aneurysms (n = 10) and control aorta (n = 10) were harvested at the time of surgical intervention and analyzed.

Results: By 14 days, aortic diameter was larger after elastase perfusion compared with control diameter (100% +/- 9.6% vs 59.5% +/- 18.9%, P = .0002). At 7 days, elastase-perfused mice had a 78% and 85% reduction in SM22 alpha and smooth muscle alpha-actin expression, respectively, compared with that seen in control animals well before aneurysms were present, and these values remained repressed at 14 days. Immunohistochemistry confirmed less SM22 alpha and smooth muscle alpha-actin in experimental aneurysms at 14 days in concert with increased matrix metalloproteinase 2 and 9 expression at 7 and 14 days. Similarly, human aneurysms had less SM22 alpha and smooth muscle alpha-actin and increased matrix metalloproteinase 2 and 9 staining, compared with control values, as determined by means of quantitative polymerase chain reaction.

Conclusions: Aneurysms demonstrate smooth muscle cell phenotypic modulation characterized by downregulation of smooth muscle cell marker genes and upregulation of matrix metalloproteinases. These events in experimental models occur before aneurysm formation. Targeting smooth muscle cells to a reparative phenotype might provide a novel therapy in the treatment of aortic aneurysms.

FIGURE 1

A, Elastase perfusion of the infrarenal abdominal aorta. B, An explanted abdominal aortic aneurysm 14 days after elastase perfusion. C, Graph demonstrating aortic aneurysm formation at 14 days after elastase (and not saline) perfusion. Importantly, at 7 days, no differences in aortic diameter are evident between elastase- and saline-treated animals.

FIGURE 2

Quantitative polymerase chain reaction analysis of smooth muscle marker genes at 7 and 14 days after elastase/saline perfusion. SM22α and SM α-actin expression is downregulated in elastase-treated aortas at 7 and 14 days after perfusion. Standard error bars are shown.

FIGURE 3

Histochemistry and immunohistochemistry in mice 7 days after elastase or saline perfusion. Little differences in elastin are evident by means of Movat staining, with intact elastin in both treatment groups. Some early degradation might be present in regions of the aorta (blue arrowheads) after elastase perfusion compared with that seen after saline perfusion. Expression of matrix metalloproteinase (MMP) 2 and 9 appears to be upregulated at 7 days after elastase compared with that seen after saline. Minimal activated (cleaved) caspase-3 staining is present in either group (brown positive stain, methyl green nuclear counterstain). Images were obtained at 25× magnification, except for caspase-3, which was obtained at 10× magnification.

FIGURE 4

Histochemistry and immunohistochemistry in mice 14 days after elastase or saline perfusion. Movat staining demonstrated destruction of the medial elastin layer in elastase-treated animals. SM α-actin and SM22α (arrowheads) are downregulated compared with that seen in saline controls. Expression of matrix metalloproteinase (MMP) 2 and 9 is markedly increased in elastase-treated animals. There is little activated (cleaved) caspase-3 staining in either elastase- or saline-treated animals (brown staining positive, methyl green nuclear counterstain). Images were obtained at 25× magnification, except for caspase-3, which was obtained at 10× magnification.

FIGURE 5

Quantitative polymerase chain reaction in human ascending aortic aneurysms and control ascending aortic tissue. SM α-actin expression is significantly decreased in ascending aortic aneurysms, whereas matrix metalloproteinase (MMP) 2 expression is increased compared with that seen in control aortas. Standard error bars are shown.

FIGURE 6

Immunohistochemistry in human ascending and abdominal aortic aneurysms (AA) and control ascending aortas. SM α-actin and SM22α are downregulated in aortic aneurysms compared with that seen in control aortas. Expression of matrix metalloproteinase (MMP) 2 and 9 is increased in aneurysmal tissue. Apoptosis (caspase-3) is evident in ascending and abdominal aortic aneurysms and, to a lesser extent, in control aortas. All images were obtained at 10× magnification.