The role of macrophage phenotype in vascularization of tissue engineering scaffolds

Abstract

Angiogenesis is crucial for the success of most tissue engineering strategies. The natural inflammatory response is a major regulator of vascularization, through the activity of different types of macrophages and the cytokines they secrete. Macrophages exist on a spectrum of diverse phenotypes, from "classically activated" M1 to "alternatively activated" M2 macrophages. M2 macrophages, including the subsets M2a and M2c, are typically considered to promote angiogenesis and tissue regeneration, while M1 macrophages are considered to be anti-angiogenic, although these classifications are controversial. Here we show that in contrast to this traditional paradigm, primary human M1 macrophages secrete the highest levels of potent angiogenic stimulators including VEGF; M2a macrophages secrete the highest levels of PDGF-BB, a chemoattractant for stabilizing pericytes, and also promote anastomosis of sprouting endothelial cells in vitro; and M2c macrophages secrete the highest levels of MMP9, an important protease involved in vascular remodeling. In a murine subcutaneous implantation model, porous collagen scaffolds were surrounded by a fibrous capsule, coincident with high expression of M2 macrophage markers, while scaffolds coated with the bacterial lipopolysaccharide were degraded by inflammatory macrophages, and glutaraldehyde-crosslinked scaffolds were infiltrated by substantial numbers of blood vessels, accompanied by high levels of M1 and M2 macrophages. These results suggest that coordinated efforts by both M1 and M2 macrophages are required for angiogenesis and scaffold vascularization, which may explain some of the controversy over which phenotype is the angiogenic phenotype.

Keywords: Angiogenesis; Collagen; Foreign body response; Inflammation; Macrophage.

Figure 1. Derivation and characteristics of macrophages

(a) Peripheral blood monocytes were differentiated to macrophages (M0) and polarized to 3 different phenotypes (M1, M2a, M2c). (b) Gene expression of some known markers of macrophage phenotype (RT-PCR using monocytes/macrophages from n = 9 human donors). Data are shown as Mean ± SEM. ^*p<0.05, ^**p<0.01, ^***p<0.001.

Figure 2. Flow cytometric analysis of macrophage phenotype markers

(a) The percentage of cells staining positively for the surface marker, and (b) mean fluorescent intensity per cell (n = 3–5 human donors). Data are shown as Mean ± SEM. ^*p<0.05, ^**p<0.01, ^***p<0.001.

Figure 3. Gene expression and protein secretion levels of different macrophage phenotypes

(a) Gene expression of proteins typically associated with the initiation of angiogenesis (VEGF, FGF, IL8, RANTES), the maturation of growing blood vessels (PDGFBB, HBEGF), and remodeling of the vascular network (MMP9, TIMP3) (RT-PCR using monocytes/macrophages from n = 9 human donors). (b) ELISA of macrophage-conditioned media for levels of protein secretion (n = 4–6 human donors). (c) Enzymatic activity for MMP-9 was confirmed by gel zymography (representative gel shown, n = 5 human donors). Data are presented as Mean ± SEM. ^*p<0.05, ^**p<0.01, ^***p<0.001.

Figure 4. Functionality of macrophage-secreted factors in angiogenesis in vitro

An in vitro sprouting assay was used to assess HUVEC organization on Matrigel® in macrophage-conditioned media. Networks were analyzed using the Angiogenesis Analyzer macro in ImageJ following background subtraction in MATLAB. Data are shown as Mean ± SEM (3–5). ⁰Non-significant differences compared to control group (Basal media only); ^#Significantly different from control group. ^*p<0.05, ^**p<0.01, ^***p<0.001.

Figure 5. Organization of HUVECs on fibrin gel when cultured in macrophage-conditioned media for 4 days

The behavior of endothelial cells in media conditioned by a single macrophage phenotype was compared to that of endothelial cells cultured in media that was switched after 24hrs from one macrophage phenotype to another. Cell nuclei were stained with DAPI (blue) and actin filaments were stained with fluorescent phalloidin (green). Experiments were repeated three times. Scale bars are 500μm.

Figure 6. Relationships between macrophage phenotype and scaffold vascularization in vivo after 10 days in a subcutaneous implantation model in mice

(a) Gross view of scaffolds upon explantation. Scale bar is 2mm. (b) H&E staining. Scale bars are 100μm. (c) Immunohistochemical staining for the endothelial cell marker CD31. Scale bars are 100μm. Representative images are shown from n= 4–6 replicates.

Figure 7. Immunohistochemical analysis of macrophage phenotype markers

Sections of explanted scaffolds with surrounding tissue were stained for multiple markers of M1 and M2 macrophage phenotypes in combination with the pan-macrophage marker F480. M1 markers are TNFa (a), iNOS (b), and CCR7 (c). M2 markers are CD206 (a), Arg1 (b), and CD163 (d). Scale bars are 100μm. Representative images are shown from n= 4–6 replicates. (e) Delete primary controls. Glutaraldehyde-crosslinked exhibited substantial autofluorescence.

Figure 8. Proposed model of macrophage-mediated angiogenesis

M1 macrophages promote sprouting of blood vessels via secretion of VEGF, bFGF, IL8, RANTES, and TNFa. M2a macrophages promote fusion of blood vessels through as-yet unidentified secreted factors. M2a macrophages may also regulate the actions of M1 macrophages via production of TIMP3, and may recruit pericytes via secretion of PDGF-BB, although this was not directly assessed in this study. M2c macrophages may function in vascular remodeling, given their high levels of production of MMP9.