The Lipopeptide MALP-2 Promotes Collateral Growth

Abstract

Beyond their role in pathogen recognition and the initiation of immune defense, Toll-like receptors (TLRs) are known to be involved in various vascular processes in health and disease. We investigated the potential of the lipopeptide and TLR2/6 ligand macrophage activating protein of 2-kDA (MALP-2) to promote blood flow recovery in mice. Hypercholesterolemic apolipoprotein E (Apoe)-deficient mice were subjected to microsurgical ligation of the femoral artery. MALP-2 significantly improved blood flow recovery at early time points (three and seven days), as assessed by repeated laser speckle imaging, and increased the growth of pre-existing collateral arteries in the upper hind limb, along with intimal endothelial cell proliferation in the collateral wall and pericollateral macrophage accumulation. In addition, MALP-2 increased capillary density in the lower hind limb. MALP-2 enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) release from endothelial cells and improved the experimental vasorelaxation of mesenteric arteries ex vivo. In vitro, MALP-2 led to the up-regulated expression of major endothelial adhesion molecules as well as their leukocyte integrin receptors and consequently enhanced the endothelial adhesion of leukocytes. Using the experimental approach of femoral artery ligation (FAL), we achieved promising results with MALP-2 to promote peripheral blood flow recovery by collateral artery growth.

Keywords: TLR2/6; blood flow recovery; collateral growth; femoral artery ligation.

Figure 1

MALP-2 improved the perfusion recovery and collateral growth in the hind limb following femoral artery ligation (FAL) in hypercholesterolemic Apoe-deficient mice. (a) Following the FAL, the perfusion recovery was determined by laser Speckle perfusion imaging for C57BL/6, BALB/c and hypercholesterolemic Apoe-KO mice (12 weeks on a high fat diet (HFD)) treated with MALP-2 or PBS (control) pre/post the FAL, after three and seven days and, in Apoe-KO mice, after 10 days. Data are expressed as the ratio of the ligated and the non-ligated hind limb. ** P < 0.01, N = 4–7. (b) Representative laser speckle perfusion images indicate the effect of MALP-2 compared to the control (PBS) on perfusion recovery in the ligated hind limbs of Apoe-KO mice pre/post the FAL and after 3, 7 and 10 days. (c) Representative haematoxilin-eosin staining of cross sections of collateral arteries in the adductor muscle of the ligated and the non-ligated hind limbs of hypercholesterolemic Apoe-KO mice treated with MALP-2 or PBS (control) 10 days after the FAL and the corresponding morphometric analysis of the collateral diameter and wall area. Scale bar = 10 µm. * P < 0.05 vs. control, N = 6–14 collaterals.

Figure 2

MALP-2 increased pericollateral macrophage accumulation, endothelial cell proliferation and downstream angiogenesis following FAL. This shows the representative immunostaining of cross sections of collateral arteries in the adductor muscle and the calf muscle of the ligated hind limb in hypercholesterolemic Apoe-KO mice treated with MALP-2 and PBS (control) 3, 7 and 10 days after the FAL and the corresponding quantitative analysis. (a) CD68 staining to assess the accumulation of macrophages around the collateral (α-SMA indicates the media of the collateral wall). Scale bar = 25 µm. (b) Ki67 staining to determine the portion of proliferating CD31-positive collateral endothelial cells (white arrow heads). Scale bar = 25 µm. (c) CD31 indicates capillary density in the calf muscle. Scale bar = 50 µm. * P <0.05, ** P <0.01 vs. control, N = up to 20 collaterals, n.d. = not detected.

Figure 3

MALP-2 up-regulated inflammatory genes in the upper hind limb muscle. Tissue pieces of the adductor muscles of C57BL/6 mice were isolated and stimulated ex vivo with MALP-2 (1 µg/mL); Ccl2, Gm-csf, Il-1β and Tnf-α mRNA levels were analyzed after the indicated times by (a) real-time PCR and (b) the corresponding protein in the supernatant after 6 h by ELISA. CXCL12 mRNA levels were analyzed (c) in tissue pieces of the adductor muscle of C57BL/6 mice ex vivo and in (d) MyEND cells following MALP-2 stimulation (1 µg/mL) after the indicated times by real-time PCR. * P < 0.05, ** P < 0.01 vs. control, N = 4–6.

Figure 4

MALP-2 improved NO-dependent vascular relaxation in the mesenteric arteries of C57BL/6 mice. (a) The relaxation response to acetylcholine (ACh 0.001–10 µM) during phenylephrine-induced (PE, 10 µM) contraction in mesenteric arteries incubated with MALP-2 or PBS (control), N = 6. (b) The relaxation response to ACh (0.01–10 µM) during K⁺-induced (60 mM) contraction in mesenteric arteries incubated with indomethacin (10 µM, COX-inhibitor) and MALP-2 or PBS, N = 3. (c) The relaxation response to ACh (0.001–10 µM) in the presence of L-NAME (100 µM, NOS inhibitor) and indomethacin (10 µM). A.U.C. = area under the curve, * P < 0.05 vs. control, N = 3.

Figure 5

MALP-2 enhanced the endothelial cell-derived NO release. MyEnd cells were stimulated with MALP-2 (1 µg/mL); (a) the AKT phosphorylation (p-AKT) as well as (b) the eNOS phosphorylation (p-eNOS) were analyzed after the indicated times by Western blot and (c) the NO release was analyzed with the Griess reagent. The numbers between panels indicate fold-change vs. unstimulated after normalization to total AKT or eNOS, respectively. β-Actin was used as the loading control. * P < 0.05 vs. control, N = 4–5.

Figure 6

MALP-2 up-regulated endothelial adhesion molecules and enhanced the endothelial adhesion of monocytic cells. (a) The MyEnd cells were stimulated with MALP-2 (1 µg/mL) and the VCAM-1, ICAM-1, E-selectin and P-selectin mRNA levels were analyzed after the indicated times by real-time PCR. * P < 0.05, ** P < 0.01 vs. control, N = 6–8. (b) The MyEnd cells were stimulated with MALP-2 (1 µg/mL) and the VCAM-1 protein expression was analyzed after the indicated times by Western blot. β-Actin was used as the loading control. The numbers between panels indicate fold-change vs. unstimulated after normalization to β-Actin. * P < 0.05 vs. control, N = 4–5. (c) Fluorescence images depicting calcein-AM-labeled J774A.1 cells on a MyEnd monolayer with or without pretreatment with MALP-2 (1 µg/mL) for 6 h with an additional adhesion time of 1 h and the corresponding quantitative analysis. Pictures before and after washing are shown. Scale bar = 100 µm, ** P < 0.01 vs. control, N = 3.