α-Gal Nanoparticles Mediated Homing of Endogenous Stem Cells for Repair and Regeneration of External and Internal Injuries by Localized Complement Activation and Macrophage Recruitment

Abstract

This review discusses a novel experimental approach for the regeneration of original tissue structure by recruitment of endogenous stem-cells to injured sites following administration of α-gal nanoparticles, which harness the natural anti-Gal antibody. Anti-Gal is produced in large amounts in all humans, and it binds the multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) presented on α-gal nanoparticles. In situ binding of anti-Gal to α-gal nanoparticles activates the complement system and generates complement cleavage chemotactic-peptides that rapidly recruit macrophages. Macrophages reaching anti-Gal coated α-gal nanoparticles bind them via Fc/Fc receptor interaction and polarize into M2 pro-reparative macrophages. These macrophages secrete various cytokines that orchestrate regeneration of the injured tissue, including VEGF inducing neo-vascularization and cytokines directing homing of stem-cells to injury sites. Homing of stem-cells is also directed by interaction of complement cleavage peptides with their corresponding receptors on the stem-cells. Application of α-gal nanoparticles to skin wounds of anti-Gal producing mice results in decrease in healing time by half. Furthermore, α-gal nanoparticles treated wounds restore the normal structure of the injured skin without fibrosis or scar formation. Similarly, in a mouse model of occlusion/reperfusion myocardial-infarction, near complete regeneration after intramyocardial injection of α-gal nanoparticles was demonstrated, whereas hearts injected with saline display ~20% fibrosis and scar formation of the left ventricular wall. It is suggested that recruitment of stem-cells following anti-Gal/α-gal nanoparticles interaction in injured tissues may result in induction of localized regeneration facilitated by conducive microenvironments generated by pro-reparative macrophage secretions and "cues" provided by the extracellular matrix in the injury site.

Keywords: alpha-gal nanoparticles; macrophage recruitment; myocardium regeneration; natural anti-Gal antibody; stem-cell homing; wound healing.

Figure 1

Illustration of the α-gal nanoparticle as a spheric lipid bilayer studded with α-gal glycolipids (α-gal epitopes illustrated as rectangles on glycolipid molecules) (A) and of the hypothesized outcomes of anti-Gal binding to α-gal epitopes on the nanoparticles (B). The various steps, following anti-Gal/α-gal nanoparticles interaction, are detailed below. Macrophages are colored green and stem cells-red. Modified from ref. [57].

Figure 2

Demonstration of macrophage recruitment by α-gal nanoparticles, in various tissues of anti-Gal producing α1,3galactosyltransferase knockout (GT-KO) mice. (A) Macrophage recruitment 24 h after intradermal injection of 10 mg α-gal nanoparticles. The empty oval area is the space formed by the injection of α-gal nanoparticles. The nanoparticles were dissolved by alcohol during the processing for hematoxylin & eosin staining (H&E × 100). (B) Macrophages identified at the injection site, 4 days post injection, by specific staining with the anti-F4/80 antibody coupled to peroxidase (HRP) (×200). (C) The intradermal injection site after 7 days is full of many large macrophages containing vacuoles, suggesting activation of these cells (H&E × 400). (D) Individual macrophages, similar to those in (C) migrating into polyvinyl alcohol (PVA) sponge disc containing 10 mg α-gal nanoparticles and implanted subcutaneously into GT-KO mouse for 7 days. The many vacuoles in the cytoplasm of the macrophages are of the anti-Gal coated α-gal nanoparticles internalized by the macrophages (Wright staining, ×1000). (E). Mouse healthy heart, 4 days following two injections of α-gal nanoparticles (each 10 μl of 10 mg/mL nanoparticles). Recruited macrophages are identified by arrows (H&E × 20). (F). Infiltration of macrophages into an area near a sciatic nerve branch (oval structure), 4 days post injection of α-gal nanoparticles (H&E × 200). Modified from ref. [57].

Figure 3

Flow cytometry of cells recruited into PVA sponge discs containing α-gal nanoparticles. (A). The large majority of the cells is macrophages as indicated by the staining of most cells with antibodies to two biomarkers of macrophages, CD11b and CD14. No staining was observed for T or B lymphocytes (not shown). (B). Analysis of the recruited macrophages state of polarization. The large size macrophages (CD11b^pos/F4/80^pos) were positive also for IL-10 and Arginase-1 but were negative for IL-12, implying that the majority of the recruited cells were M2 macrophages. Modified from ref. [57].

Figure 4

Scanning electron microscopy (SEM) of macrophages. (A). An adherent macrophage lacking α-gal epitopes and incubated with α-gal nanoparticles coated with the anti-Gal antibody, then washed and processed for SEM. Note the multiple α-gal nanoparticles binding to the macrophage via Fc/Fc receptor interactions and covering the surface of the macrophage. (B). A macrophage incubated with α-gal nanoparticles in the absence of anti-Gal. No nanoparticles bind to the macrophage cell membrane in the absence of the antibody. Adapted with permission from ref. [57].

Figure 5

Representative examples of colonies formed within 5 days of culturing cells obtained from PVA sponge discs containing 10 mg/mL α-gal nanoparticles that were implanted subcutaneously for 7 days in GT-KO mice producing anti-Gal. The frequency of colony forming cells was approximately one in 5 × 10⁴ and 1 × 10⁵ recruited macrophages. The number of cells per colony was ~300 (A) and ~1000 (B) (×100). Reproduced with permission from ref. [57].

Figure 6

Formation of fibrocartilage in PVA sponge discs containing α-gal nanoparticles (10 mg/mL) and microscopic porcine meniscus cartilage homogenate as ECM (50 mg/mL), implanted subcutaneously for 5 weeks in anti-Gal producing GT-KO mice. (A). PVA sponge disc section for demonstration of fibrocartilage growth (stained red) in areas marked with rectangles (H&E ×10). (B). Magnification of the inset in (A) demonstrating the fibrocartilage growth (red). The PVA sponge material is stained purple-red. (C). Mason-trichrome staining of the collagen fibers blue in an area similar to that in (B) (×100). (D). The inset in (C) (×200) demonstrates fibrocartilage formation consisting of multiple collagen fibers. The nuclei of the few fibrochondroblasts are stained purple. (E). Control sponge disc containing only meniscus cartilage ECM homogenate, displaying adipocytes and no fibrocartilage formation (Mason-trichrome × 100). (F). Structure of porcine meniscus fibrocartilage displaying parallel organization of fibrochondrocytes and the collagen fibers they produce (H&E, ×200). Representative sections are from 5 GT-KO mice per group. Reprinted with permission from ref. [57].

Figure 7

Effect of topical application of α-gal nanoparticles on healing of wounds, determined as proportion of regenerating epidermis covering the wound. (Aa) Representative morphology on Day 6 of saline treated oval 6 × 9 mm full thickness excisional wounds. No healing and regeneration are observed. (Ab). Wound as in (Aa), treated with α-gal nanoparticles. Note the complete healing of the wound which is covered by regenerating epidermis. (Ac) Histology of the wound in (Aa) (H&E × 100). (Ad) Histology of the wound in (Ab) (H&E × 100). (B) Healing of wounds was evaluated with α-gal nanoparticles (10 mg/mL) (closed columns), α-gal liposomes, which are large α-gal nanoparticles (1–10 μm, hatched columns), 10 mg GT-KO pig liposomes lacking α-gal epitopes (gray columns), or saline (open columns). Mean + SD from ≥5 mice/group. On Day 6, 20 mice were evaluated per each group. Modified from ref. [57].

Figure 8

Histology of GT-KO mouse wounds treated with α-gal nanoparticles or with saline and inspected 28 days post treatment. Wounds were stained with H&E or with Masson trichrome for detection of de novo produced collagen. Whereas the saline treated wounds display distinct fibrosis and scar formation (dense connective tissue, hyperplastic epidermis, absence of skin appendages), the α-gal nanoparticles treated wounds display restoration of normal skin structure including growth of hair, loose connective tissue, adipose tissue and smooth muscle cells (×100). Reprinted from ref. [57] with permission.

Figure 9

GT-KO mouse hearts undergoing MI by occlusion/reperfusion, treated post-MI with two intramyocardial injections of 10 μl saline or α-gal nanoparticles (10 mg/mL) and inspected 28 days post treatment. (A). The hearts were sectioned and stained with Masson trichrome for identification of scar tissue. Representative saline treated hearts displayed scar formation and thinning of the ventricular wall whereas α-gal nanoparticles treated hearts displayed near complete regeneration of the ventricular wall. (B). Planimetry studies of fibrosis and scar formation in hearts from 10 saline treated and 20 α-gal nanoparticles treated hearts. Mean ± S.E., **** p < 0.0001. Adapted from ref. [19].

Figure 10

Regeneration (A–D) or fibrosis (E,F) of GT-KO mouse hearts undergoing MI by occlusion/reperfusion. (A). Heart treated with α-gal nanoparticles, 4 days post treatment. Arrows indicated macrophage infiltration in the two injection sites (H&E × 10). (B). Heart treated with α-gal nanoparticles, 7 days post treatment. Arrows indicated the peak infiltration of macrophages (H&E × 10). (C). Magnification of infiltration site in heart as in (B). Red circles mark mitotic figures at various stages of cell cycle (H&E × 200). (D). Near complete regeneration of a treated heart after 14 days (Masson trichrome × 10). (E). Saline treated heart after 4 days displays peak infiltration of macrophages, marked by arrows (H&E × 10). (F). Saline treated heart after 14 days displaying thinning of the anterior ventricular wall which also displays fibrosis, indicated by the blue-grey stained collagen (Masson trichrome × 10). Modified from ref. [19].