Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells

Abstract

Background: Promoting survival of transplanted cells or endogenous precursors is an important goal. We hypothesized that a novel approach to promote vascularization would be to create injectable microenvironments within the myocardium that recruit endothelial cells and promote their survival and organization.

Methods and results: In this study we demonstrate that self-assembling peptides can be injected and that the resulting nanofiber microenvironments are readily detectable within the myocardium. Furthermore, the self-assembling peptide nanofiber microenvironments recruit progenitor cells that express endothelial markers, as determined by staining with isolectin and for the endothelial-specific protein platelet-endothelial cell adhesion molecule-1. Vascular smooth muscle cells are recruited to the microenvironment and appear to form functional vascular structures. After the endothelial cell population, cells that express alpha-sarcomeric actin and the transcription factor Nkx2.5 infiltrate the peptide microenvironment. When exogenous donor green fluorescent protein-positive neonatal cardiomyocytes were injected with the self-assembling peptides, transplanted cardiomyocytes in the peptide microenvironment survived and also augmented endogenous cell recruitment.

Conclusions: These experiments demonstrate that self-assembling peptides can create nanofiber microenvironments in the myocardium and that these microenvironments promote vascular cell recruitment. Because these peptide nanofibers may be modified in a variety of ways, this approach may enable injectable tissue regeneration strategies.

Figure 1

Cells spontaneously populate cell-free peptide microenvironment after 7 days. a, H&E stain of LV wall of adult male mouse 3 hours after injection of microenvironment. Few nuclei were evident within the microenvironment border, denoted by arrows, although there were some red blood cells. b, H&E of LV wall of adult male mouse 7 days after microenvironment injection shows many nuclei within the microenvironment. c, To confirm microenvironment regions and nuclei content, a biotinylated microenvironment was injected, and tissue was stained with streptavidin—Texas red and DAPI (blue). At 3 hours after injection, there were relatively few nuclei within the microenvironment; borders are denoted by dotted lines as determined by Texas red staining and light microscopy. d, Similarly to b, there were many cells within the microenvironment after 7 days, and the border is defined by streptavidin—Texas red staining. Bars represent 20 μm.

Figure 2

Endothelial cells spontaneously populate cell-free peptide microenvironment after 7 days and organize at later time points. a, Many cells (nuclei=blue, DAPI) within the peptide microenvironment (dotted lines as determined by light microscopy) stained positively with the endothelial cell marker isolectin-FITC (green) 7 days after injection. b, Endothelial cells were still present within the microenvironment 14 days after injection and appeared to be elongated in shape. c, Twenty-one days after injection, the endothelial cells appeared to be clustered within sections of the peptide microenvironment. d, Distinct capillary-like structures (arrows) were seen within the microenvironment 28 days after injection. e, Endothelial cell phenotype was confirmed by immunohistochemical staining for PECAM-1 (CD31, brown staining) within the peptide microenvironment at 28 days. Note the vessels within the gel highlighted by arrows that contain red blood cells. Bars represent 20 μm.

Figure 3

Smooth muscle cells populate the peptide microenvironment and form mature, vessel-like structures. a, At 14 days after injection, the cells (blue=DAPI) in the microenvironment stained positively with an α-smooth muscle actin antibody (red) and showed features of organization. b, At 28 days after injection, several arteriole-like structures were seen within the microenvironment in all samples. Bars represent 10 μm.

Figure 4

Putative myocyte precursors spontaneously populate the peptide microenvironment with a later time course than endothelial cell presence. a, There were few cells (blue=DAPI) within the peptide microenvironment that stain positively for α-sarcomeric actin (red) 7 days after injection. b, However, there was an increase in myocyte staining after 14 days, which continued to 21 days (c) and 28 days (d) after injection. Note that many of the myocytes were small cells and remained so at all time points. Bars represent 20 μm. e, Differences in endothelial and myocyte density time courses. ***P<0.001 vs 14-, 21-, and 28-day endothelial cells (EC); **P<0.01, 14 days vs 21 days; 21 days vs 7 and 28 days; 28 days vs 7, 14, and 21 days.

Figure 5

Endothelial cells do not populate Matrigel to the same degree as the self-assembling peptide microenvironment. Isolectin (green) and α-sarcomeric actin (red) staining of Matrigel sections 7 days (a) and 28 days (b) after injection. Note that much of the gel area is unpopulated. Blue=DAPI; bars represent 20 μm.

Figure 6

Implantation of GFP myocytes results in increased endogenous myocyte density within the peptide microenvironment. a, α-Sarcomeric actin (red) staining of the peptide microenvironment region 7 days after injection. There were many endogenous putative myocyte precursors within the microenvironment. b, Merged image from 7 days after injection showing that relatively few α-sarcomeric actin—positive cells are GFP positive, as denoted by arrows (green=GFP, merged=yellow). c, Larger, doublenucleated endogenous myocytes (red, arrows) were visible within the microenvironment 28 days after injection, while there were still small endogenous myocytes. d, Difference in myocyte recruitment in GFP myocyte—injected samples and microenvironment alone. **P<0.01 vs microenvironment alone at same time point.

Figure 7

Potential cardiomyocyte phenotype. a, High-magnification image of cells within the GFP myocyte—embedded microenvironment 7 days after injection, demonstrating positive staining for Nkx2.5 (red; arrows) within the nucleus (DAPI; blue). b, Different peptide microenvironment section of same mouse stained with DAPI (blue) and secondary antibody alone. Note the absence of background staining within the peptide microenvironment. Bars represent 10 μm.

Figure 8

Spontaneous embryonic stem cell differentiation in the injectable microenvironment. Image of a mouse heart section injected with myosin heavy chain—eGFP embryonic stem cells embedded within the peptide microenvironment after 14 days. Green=eGFP, blue=DAPI. Bars represent 20 μm. The bright green cells show that some embryonic stem cells differentiate into cardiac myocytes and survive in the microenvironment.