Inhibiting Integrin α5 Cytoplasmic Domain Signaling Reduces Atherosclerosis and Promotes Arteriogenesis

Abstract

Background: Fibronectin in endothelial basement membranes promotes endothelial inflammatory activation and atherosclerosis but also promotes plaque stability and vascular remodeling. The fibronectin receptor α5 subunit is proinflammatory through binding to and activating phosphodiesterase 4D5, which inhibits anti-inflammatory cyclic adenosine monophosphate and protein kinase A. Replacing the α5 cytoplasmic domain with that of α2 resulted in smaller atherosclerotic plaques. Here, we further assessed plaque phenotype and compensatory vascular remodeling in this model.

Methods and results: α5/2 mice in the hyperlipidemic apolipoprotein E null background had smaller plaques in the aortic root, with reduced endothelial NF-κB activation and inflammatory gene expression, reduced leukocyte content, and much lower metalloproteinase expression. However, smooth muscle cell content, fibrous cap thickness, and fibrillar collagen were unchanged, indicating no shift toward vulnerability. In vivo knockdown of phosphodiesterase 4D5 also decreased endothelial inflammatory activation and atherosclerotic plaque size. α5/2 mice showed improved recovery from hindlimb ischemia after femoral artery ligation.

Conclusions: Blocking the fibronectin-Integrin α5 pathway reduces atherosclerotic plaque size, maintains plaque stability, and improves compensatory remodeling. This pathway is therefore a potential therapeutic target for treatment of atherosclerosis.

Keywords: arteriogenesis; atherosclerosis; fibronectin; inflammation; matrix metalloprotease; phosphodiesterase 4D5; plaque vulnerability.

Figure 1

Inflammatory signaling in early atherosclerosis. Sections through the aortic root from ApoE^−/− and α5/2;ApoE^−/− mice on a high‐fat Western diet for 4 weeks were stained for (A) fibronectin (ApoE^−/− mice, n=5; α5/2;ApoE^−/− mice, n=5), (B) VCAM‐1 (ApoE^−/− mice, n=4; α5/2;ApoE^−/− mice, n=4), (C) CD45 (ApoE^−/− mice, n=4; α5/2;ApoE^−/− mice, n=3), or (D) CD68 (ApoE^−/− mice, n=5; α5/2;ApoE^−/− mice, n=4). Nuclei were counterstained with DAPI (blue). Right, Quantification of staining. Values are means±SD (*P<0.05 compared with ApoE^−/−; unpaired 2‐tailed Student's t test). APOE indicates ApoE; FN, fibronectin; WT, wild type.

Figure 2

Advanced atherosclerosis. Aortic roots from ApoE^−/− and α5/2;ApoE^−/− mice on a high‐fat diet for 16 weeks. A, hematoxylin and eosin–stained aortic root sections with quantification of total plaque area (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=9); and necrotic core area (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=9). Necrotic areas are highlighted with dotted blue lines. B, Oil Red staining of aortic roots and quantification of the stained area (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=9). Values are means±SD (*P<0.05, **P<0.01 compared with ApoE^−/−; unpaired 2‐tailed Student's t test). APOE indicates ApoE; WT, wild type.

Figure 3

Plaque inflammatory markers. Aortic roots from ApoE^−/− and α5/2;ApoE^−/− mice on a high‐fat diet for 16 weeks were stained for (A) phospho‐S536 NF‐κB p65 (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=9), (B) VCAM‐1; (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=9), (C) ICAM‐1 (ApoE^−/− mice, n=7; α5/2;ApoE^−/− mice, n=9), (D) CD45 (ApoE^−/− mice, n=8; α5/2;ApoE^−/− mice, n=9), (E) CD68 (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=9), and (F) F480 (ApoE^−/− mice, n=7; α5/2;ApoE^−/− mice, n=7). For quantification, values are means±SD (*P<0.05, **P<0.01 compared with ApoE^−/−; unpaired 2‐tailed Student's t test). Nuclei are counterstained with DAPI (blue).

Figure 4

Matrix and matrix remodeling. Aortic root sections from ApoE^−/− and α5/2;ApoE^−/− mice on a high‐fat diet for 16 weeks were stained as indicated, with nuclei counterstained with DAPI (blue). Adjacent graphs show quantification as means±SD. A, Picrosirius red (ApoE^−/− mice, n=6; α5/2;ApoE^−/− mice, n=6). B, Anti‐fibronectin (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=9). C, Anti‐MMP9 (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=8). D, Anti‐MMP2 (ApoE^−/− mice, n=9; α5/2;ApoE^−/− mice, n=9). NS, not significant; ****P<0.0001; **P<0.005 compared with ApoE^−/−; unpaired 2‐tailed Student's t test). MMP indicates matrix metalloproteinase.

Figure 5

Fibrous caps. Aortic root sections from WT;ApoE^−/− and α5/2;ApoE^−/− mice on a high‐fat diet for 16 weeks were stained for smooth muscle actin (SMA). Quantification shows the percentage of the plaque area stained positive for SMA (ApoE^−/− mice, n=7; α5/2;ApoE^−/− mice, n=9) and fibrous cap thickness (ApoE^−/− mice, n=8; α5/2;ApoE^−/− mice, n=9). Values are means±SD (NS, Not significant compared with ApoE^−/−; unpaired 2‐tailed Student's t test). APOE indicates ApoE; WT, wild type.

Figure 6

Endothelial in vivo PDE4D5 knockdown. PDE4D siRNA or luciferase siRNA were packaged into endothelial‐specific nanoparticles and administered intravenously into WT (C57BL6) mice. A, Intimal RNA was isolated and assayed for PDE4D and proinflammatory gene expression by quantitative real‐time polymerase chain reaction. B, Outline of PDE4D siRNA protocol for atherosclerosis. ApoE^−/− mice on a high‐fat diet were serially injected with nanoparticle‐packaged PDE4D or luciferase siRNA and examined at 8 weeks. C, Plasma lipid levels. D, Aortic root sections were stained for Oil Red O, and the positively stained area was quantified (siLUC; n=5 mice; siPDE4D, n=5 mice). *P<0.05 compared with ApoE^−/− by unpaired 2‐tailed Student's t test. FN indicates fibronectin; HDL, high‐density lipoprotein; HFD, high‐fat diet; LDL, low‐density lipoprotein; LUC, luciferase; MMP, matrix metalloproteinase; PDE, phosphodiesterase; si, small interfering [RNA]; WT, wild type.

Figure 7

In vivo PDE4D (4D5) knockdown and plaque inflammatory markers. ApoE^−/− mice on a high‐fat diet were serially injected with nanoparticle‐packaged PDE4D or luciferase siRNA, and examined at 8 weeks. Aortic root sections were stained for (A) VCAM‐1 (siLUC, n=4 mice; siPDE4D, n=5 mice); (B) ICAM‐1 (siLUC, n=4 mice; siPDE4D, n=4 mice); (C) CD45 (siLUC, n=4 mice; siPDE4D, n=5 mice); and (D) CD68 (siLUC, n=5 mice; siPDE4, n=4 mice), as indicated. For quantification, values are means±SD *P<0.05 compared with ApoE^−/− by unpaired 2‐tailed Student's t test. LUC indicates luciferase; PDE, phosphodiesterase; si, small interfering [RNA].

Figure 8

Blood flow recovery after hindlimb ischemia. A, Representative laser Doppler images of flow in the hind limbs before surgery and at the indicated times after femoral artery ligation. B, Quantification from multiple mice (WT mice, n=9; α5/2 mice, n=9). Values are means±SD. *P<0.05 by unpaired 2‐tailed Student's t test. WT indicates wild type.

Figure 9

Arterial vasculature after hindlimb ischemia. Analysis of vasculature in WT and mutant mice 21 days after surgery. A, Sections through the calf region were stained with PECAM antibody (CD31) to mark blood vessels in the lower leg. Capillary density was then quantified (WT mice, n=4; α5/2 mice, n=4). B, Representative microCT images of vasculature. The number of vessels within each size range was quantified as described in Methods (WT mice, n=5; α5/2 mice, n=5; *P<0.001 by 2‐way ANOVA, multiple comparisons). C, Sections through the upper leg were stained for smooth muscle actin to label arteries and vessel diameter measured (WT mice, n=4; α5/2 mice, n=4). For all panels, values are means±SD. *P<0.05, **P<0.005 compared with WT mice by unpaired 2‐tailed Student's t test. CT indicates computed tomography; Lig, ligated; PECAM, platelet/endothelial cell adhesion molecule; WT, wild type.