Christopher W. Moehle

Smooth muscle phenotypic modulation is an early event in aortic aneurysms.

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.

Genetic inactivation of IL-1 signaling enhances atherosclerotic plaque instability and reduces outward vessel remodeling in advanced atherosclerosis in mice

Clinical complications of atherosclerosis arise primarily as a result of luminal obstruction due to atherosclerotic plaque growth, with inadequate outward vessel remodeling and plaque destabilization leading to rupture. IL-1 is a proinflammatory cytokine that promotes atherogenesis in animal models, but its role in plaque destabilization and outward vessel remodeling is unclear. The studies presented herein show that advanced atherosclerotic plaques in mice lacking both IL-1 receptor type I and apolipoprotein E (Il1r1⁻/⁻Apoe⁻/⁻ mice) unexpectedly exhibited multiple features of plaque instability as compared with those of Il1r1⁺/⁺Apoe⁻/⁻ mice. These features included reduced plaque SMC content and coverage, reduced plaque collagen content, and increased intraplaque hemorrhage. In addition, the brachiocephalic arteries of Il1r1⁻/⁻Apoe⁻/⁻ mice exhibited no difference in plaque size, but reduced vessel area and lumen size relative to controls, demonstrating a reduction in outward vessel remodeling. Interestingly, expression of MMP3 was dramatically reduced within the plaque and vessel wall of Il1r1⁻/⁻Apoe⁻/⁻ mice, and Mmp3⁻/⁻Apoe⁻/⁻ mice showed defective outward vessel remodeling compared with controls. In addition, MMP3 was required for IL-1-induced SMC invasion of Matrigel in vitro. Taken together, these results show that IL-1 signaling plays a surprising dual protective role in advanced atherosclerosis by promoting outward vessel remodeling and enhancing features of plaque stability, at least in part through MMP3-dependent mechanisms.

PDGF-DD, a novel mediator of smooth muscle cell phenotypic modulation, is upregulated in endothelial cells exposed to atherosclerosis-prone flow patterns

Platelet-derived growth factor (PDGF)-BB is a well-known smooth muscle (SM) cell (SMC) phenotypic modulator that signals by binding to PDGF alphaalpha-, alphabeta-, and betabeta-membrane receptors. PDGF-DD is a recently identified PDGF family member, and its role in SMC phenotypic modulation is unknown. Here we demonstrate that PDGF-DD inhibited expression of multiple SMC genes, including SM alpha-actin and SM myosin heavy chain, and upregulated expression of the potent SMC differentiation repressor gene Kruppel-like factor-4 at the mRNA and protein levels. On the basis of the results of promoter-reporter assays, changes in SMC gene expression were mediated, at least in part, at the level of transcription. Attenuation of the SMC phenotypic modulatory activity of PDGF-DD by pharmacological inhibitors of ERK phosphorylation and by a small interfering RNA to Kruppel-like factor-4 highlight the role of these two pathways in this process. PDGF-DD failed to repress SM alpha-actin and SM myosin heavy chain in mouse SMCs lacking a functional PDGF beta-receptor. Importantly, PDGF-DD expression was increased in neointimal lesions in the aortic arch region of apolipoprotein C-deficient (ApoE(-/-)) mice. Furthermore, human endothelial cells exposed to an atherosclerosis-prone flow pattern, as in vascular regions susceptible to the development of atherosclerosis, exhibited a significant increase in PDGF-DD expression. These findings demonstrate a novel activity for PDGF-DD in SMC biology and highlight the potential contribution of this molecule to SMC phenotypic modulation in the setting of disturbed blood flow.

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.

Adenosine 2A receptor modulates inflammation and phenotype in experimental abdominal aortic aneurysms

Activation of the adenosine 2A receptor (A2AR) reduces inflammation in models of acute injury but contribution in development of chronic abdominal aortic aneurysms (AAAs) is unknown. Elastase perfusion to induce AAA formation in A2AR‐knockout (A2ARKO) and C57BL6/J wild‐type (WT) mice resulted in nearly 100% larger aneurysms in A2ARKO compared to WT at d 14 (P < 0.05), with evidence of greater elastin fragmentation, more immune cell infiltration, and increased matrix metallatoproteinase (MMP) 9 expression (P<0.05). Separately, exogenous A2AR antagonism in elastase‐perfused WT mice also resulted in larger aneurysms (P<0.05), while A2AR agonism limited aortic dilatation (P<0.05). Activated Thy‐1.2+ T lymphocytes from WT mice treated in vitro with A2AR antagonist increased cytokine production, and treatment with A2AR agonist decreased cytokine production (P<0.05 for all). Primary activated CD4+ T lymphocytes from A2ARKO mice exhibited greater chemotaxis (P<0.05). A2AR antagonist increased chemotaxis of activated CD4+ cells from WT mice in vitro, and A2AR agonist reduced this effect (P<0.05). A2AR activation attenuates AAA formation partly by inhibiting immune cell recruitment and reducing elastin fragmentation. These findings support augmenting A2AR signaling as a putative target for limiting aneurysm formation.—Bhamidipati, C. M., Mehta, G. S., Moehle, C. W., Meher, A. K., Su, G., Vigneshwar, N. G., Barbery, C., Sharma, A. K., Kron, I. L., Laubach, V. E., Owens, G. K., Upchurch Jr., G. R., Ailawadi, G. Adenosine 2A receptor modulates inflammation and phenotype in experimental abdominal aortic aneurysms. FASEB J. 27, 2122–2131 (2013). www.fasebj.org

Krüppel-like Factors KLF2, KLF4, and KLF5: Central Regulators of Smooth Muscle Function

The vascular smooth muscle cell (SMC) plays a vital role in mammalian physiology through its regulation of blood pressure via contraction and relaxation. In response to vascular injury, it is capable of rapidly and reversibly modulating its pheno-type to a cell type capable of performing a number of functions key to wound healing and vascular inflammation including migration, proliferation, matrix synthesis, chem-okine production, and protein synthesis. Recent work has identified three Kruppel-like factors—KLF2, KLF4, KLF5—as intricately involved in all of these processes. This review provides a brief overview of the role these factors play in regulating these and other key SMC functions.