Role of PECAM-1 in arteriogenesis and specification of preexisting collaterals

Abstract

Rationale: Hemodynamic forces caused by the altered blood flow in response to an occlusion lead to the induction of collateral remodeling and arteriogenesis. Previous work showed that platelet endothelial cell adhesion molecule (PECAM)-1 is a component of a mechanosensory complex that mediates endothelial cell responses to shear stress.

Objective: We hypothesized that PECAM-1 plays an important role in arteriogenesis and collateral remodeling.

Methods and results: PECAM-1 knockout (KO) and wild-type littermates underwent femoral artery ligation. Surprisingly, tissue perfusion and collateral-dependent blood flow were significantly increased in the KO mice immediately after surgery. Histology confirmed larger caliber of preexisting collaterals in the KO mice. Additionally, KO mice showed blunted recovery of perfusion from hindlimb ischemia and reduced collateral remodeling, because of deficits in shear stress-induced signaling, including activation of the nuclear factor κB pathway and inflammatory cell accumulation. Partial recovery was associated with normal responses to circumferential wall tension in the absence of PECAM-1, as evidenced by the upregulation of ephrin B2 and monocyte chemoattractant protein-1, which are 2 stretch-induced regulators of arteriogenesis, both in vitro and in vivo.

Conclusions: Our findings suggest a novel role for PECAM-1 in arteriogenesis and collateral remodeling. Furthermore, we identify PECAM-1 as the first molecule that determines preexisting collateral diameter.

Figure 1. Plantar perfusion after hindlimb ischemia

(A) Doppler images of plantar perfusion before (Pre), immediately after (Acute), 7 days (7d) and 3 weeks after hindlimb ischemia. Pseudo-color scale: black=0, white=1000, in arbitrary units. (B) Ratio of plantar perfusion (ligated vs. sham control side) quantified from the Doppler images. Values are means ± SE. * p<0.05, compared with the respective time point of PECAM-1^+/+; # p<0.05, compared with acute perfusion.

Figure 2. Lumen diameter and endothelial cell morphometry in collateral vessels

(A) Collateral lumen diameter in sham and ligated side 3 weeks after hindlimb ischemic surgery. Values are means ± SE. (B) Silver staining of isolated collaterals from PECAM-1^+/+ and PECAM-1^−/− mice. A single EC is highlighted in each vessel. Gray arrow indicates flow direction. Scale bar: 50μm. The diagram arrows indicate the width and length of the highlighted ECs. (C) Width and length of collateral ECs. Values are means ± SE. The n number in panel C are cells measured in the collateral endothelium of PECAM-1^+/+ and PECAM-1^−/− animals.

Figure 3. Cell proliferation during arteriogenesis

(A) PCNA-positive cells (red) in preexisting collaterals 3 days (3D) after surgery. Slides were co-stained for smooth muscle cell α-actin (green) and DAPI (blue). Bar, 20μm; (B) Percentage of PCNA-positive ECs; (C) Total number of ECs per collateral intima; and (D) Total number of smooth muscle cells per collateral media. Values are means ± SE.

Figure 4. Leukocyte accumulation in collaterals during arteriogenesis

(A) CD45-positive cells (red) in preexisting collaterals 3 days (3D) after surgery. Slides were co-stained for smooth muscle cell α-actin (green) and DAPI (blue). Bar, 20μm; (B) CD45-positive cell count per collateral area. Values are means +/− SE

Figure 5. Inflammatory signaling during arteriogenesis

(A, B, C) Expression of NF-κB p65 subunit (red, A), ICAM-1 (red, B), or VCAM-1 (red, C) in preexisting collaterals 24 hours (24H) after surgery. Slides were co-stained for smooth muscle cell α-actin (green) and DAPI (blue). Bar, 20μm.

Figure 6. Effects of cyclic stretch on endothelial cell alignment in vitro

(A) PECAM-1 reconstituted (PE-RC) or knockout (PE-KO) endothelial cells were exposed to cyclic stretch (15% strain, 1Hz, 24 hours) and stained with TRITC-phalloidin, or β-catenin (red) and DAPI (blue, inset images). Arrows show the direction of stretch. Bar: 50μm. (B) Quantification of orientation of F-actin stress fibers. Values are mean ± S.E.

Figure 7. Expression of MCP-1 and ephrin B2 during arteriogenesis and in response to cyclic stretch or laminar shear stress in vitro

(A, B) Expression of MCP-1 (A, red) or ephrin B2 (B, red) in preexisting collaterals 3 days (3D) after hindlimb ischemic surgery. Slides were co-stained for smooth muscle cell α-actin (green) and DAPI (blue). Bar, 20μm. (C) Relative mRNA expression levels of MCP-1 and ephrin B2 in ECs subjected to cyclic stretch or laminar shear stress. Static ECs in plates were controls. Values are expressed as mean ± S.E. (D) Western blots and quantification of ephrin B2 in cyclic stretch- or laminar shear stress-stimulated ECs. The protein blots were quantified and normalized to α-tubulin. Values are mean ± S.E.