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.

Development of a novel murine model of aortic aneurysms using peri-adventitial elastase.

BACKGROUND: Our aim was to establish a novel model of abdominal aortic aneurysms (AAA) in mice using application of peri-adventitial elastase. METHODS: C57BL/6J male mice underwent infrarenal peri-adventitial application of either (1) sodium chloride (control; n = 7), (2) porcine pancreatic elastase (PPE; n = 14), or (3) PPE and doxycycline (PPE + doxycycline 200 mg/kg; n = 11) for 14 days. Aortas were analyzed by video micrometry, immunohistochemistry, qualitative polymerase chain reaction, and zymography. Groups underwent Mann-Whitney U comparisons. RESULTS: At day 14 compared with baseline, control animals had minimal aortic dilation, whereas fusiform aneurysms were seen in PPE (control, 20 ± 3%; PPE, 82 ± 15%; P ≤ .003). Doxycycline abrogated aneurysm formation (PPE, 82 ± 15%; PPE + doxycycline, 37 ± 10%; P ≤ .03). Compared with control and PPE + doxycycline, immunohistochemistry demonstrated greater elastin fiber degradation, macrophage infiltration, and matrix metalloproteinase-9 expression in PPE. Ki-67 and cleaved caspase-3 were lower in control versus PPE. The loss of smooth muscle marker expression seen with PPE was preserved in PPE + doxycycline. Zymography confirmed that both MMP-2 and -9 were more active in PPE than PPE + doxycycline. CONCLUSION: Peri-adventitial application of elastase is a simple, reproducible in vivo model of aneurysm formation leading to consistent infrarenal aortic aneurysm development by day 14, with inflammatory cell infiltration and MMP upregulation. Doxycycline inhibits AAA progression in this model via limiting matrix degradation and preserving differentiated smooth muscle cells.

Interleukin-1ß 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-1ß represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1ß modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1ß- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1ß distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1ß exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1ß-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-1ß-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-1ß- 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.

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.

Bone marrow-derived MCP1 required for experimental aortic aneurysm formation and smooth muscle phenotypic modulation.

OBJECTIVES: This study tested the hypothesis that monocyte chemotactic protein 1 (MCP1) is required for abdominal aortic aneurysm (AAA) and smooth muscle phenotypic modulation in a mouse elastase perfusion model. METHODS: Infrarenal aortas of C57BL/6 (wild type [WT]) and MCP1 knockout (KO) mice were analyzed at 14 days after perfusion. Key cellular sources of MCP1 were identified using bone marrow transplantation. Cultured aortic smooth muscle cells (SMCs) were treated with MCP1 to assess its potential to directly regulate SMC contractile protein expression and matrix metalloproteinases (MMPs). RESULTS: Elastase perfused WT aortas had a mean dilation of 102% (n = 9) versus 53.7% for MCP1KO aortas (n = 9, P < .0001) and 56.3% for WT saline-perfused controls (n = 8). Cells positive for MMP9 and Mac-2 were nearly absent in the KO aortas. Complimentarily, the media of the KO vessels had abundant differentiated smooth muscle and intact elastic fibers and markedly less MMP2. Experiments in cultured SMCs showed MCP1 can directly repress smooth muscle markers and induce MMP2 and MMP9. Bone marrow transplantation studies showed that KO of MCP1 in bone marrow-derived cells protects from AAA formation. Moreover, KO in the bone was significantly more protective than global KO, suggesting an unexpected benefit to selectively depleting MCP1 in bone marrow-derived cells. CONCLUSIONS: These results have shown that MCP1 derived from bone marrow cells is required for experimental AAA formation and that retention of nonbone marrow MCP1 limits AAA compared with global depletion. This protein contributes to macrophage infiltration into the AAA and can act directly on SMCs to reduce contractile proteins and induce MMPs.

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.

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.