In Situ Blood Vessel Regeneration Using SP (Substance P) and SDF (Stromal Cell-Derived Factor)-1α Peptide Eluting Vascular Grafts

Abstract

Objective: The objective of this study was to develop small-diameter vascular grafts capable of eluting SDF (stromal cell-derived factor)-1α-derived peptide and SP (substance P) for in situ vascular regeneration.

Approach and results: Polycaprolactone (PCL)/collagen grafts containing SP or SDF-1α-derived peptide were fabricated by electrospinning. SP and SDF-1α peptide-loaded grafts recruited significantly higher numbers of mesenchymal stem cells than that of the control group. The in vivo potential of PCL/collagen, SDF-1, and SP grafts was assessed by implanting them in a rat abdominal aorta for up to 4 weeks. All grafts remained patent as observed using color Doppler and stereomicroscope. Host cells infiltrated into the graft wall and the neointima was formed in peptides-eluting grafts. The lumen of the SP grafts was covered by the endothelial cells with cobblestone-like morphology, which were elongated in the direction of the blood flow, as discerned using scanning electron microscopy. Moreover, SDF-1α and SP grafts led to the formation of a confluent endothelium as evaluated using immunofluorescence staining with von Willebrand factor antibody. SP and SDF-1α grafts also promoted smooth muscle cell regeneration, endogenous stem cell recruitment, and blood vessel formation, which was the most prominent in the SP grafts. Evaluation of inflammatory response showed that 3 groups did not significantly differ in terms of the numbers of proinflammatory macrophages, whereas SP grafts showed significantly higher numbers of proremodeling macrophages than that of the control and SDF-1α grafts.

Conclusions: SDF-1α and SP grafts can be potential candidates for in situ vascular regeneration and are worthy for future investigations.

Keywords: endothelial cells; regeneration; stem cells; substance P; tissue engineering.

Figure 1.

Scanning electron microscope (SEM) micrographs of vascular grafts. Control (A, B), SDF (stromal cell–derived factor)-1α (C, D), and SP (substance P; E, F). Scale bar, 30 µm (A, C, E) and 15 µm (B, D, F). The fiber size was measured by using at least 100 fibers per groups. The average diameter of microfibers was found to be 7.069±0.6159 µm, 8.1607±0.5871 µm, and 8.9246±1.031 µm in control, SDF-1, and SP grafts, respectively (G). H, Cell proliferation in vitro. Data are shown as mean±SD (n=5 per group) and evaluated by 1-way ANOVA followed by Tukey post hoc analysis. I, J, Pictures of vascular grafts before implantation. K, Picture of an implanted graft. Col indicates collagen; and PCL, polycaprolactone.

Figure 2.

In vitro cell migration assay. Negative control (A), PCL/Col (B), SDF-1 (C), and SP (D). Only a few cells were recruited toward negative and positive control groups (migrated cell density per hpf, negative control, 4508.86±576.54, n=5 and positive control, 28966.78±2999.39 µm²/mm², n=5; E). On the contrary, SDF-1 and SP groups recruited higher numbers of MSCs (migrated cell density per hpf, µm² per mm², SDF-1, 157889.74±21188.30 µm²/mm², n=5 and SP, 135415.88±14519.59 µm²/mm², n=5). Data are shown as mean±SEM and evaluated by Kruskal-Wallis test followed by Dunn multiple comparisons test. *P=0.0065, **P=0.0283, #P=0.0292, and ##P=0.0103. Scale bar, 500 µm. COL indicates collagen; hpf, high power field; MSC, mesenchymal stem cell; PCL, polycaprolactone; SDF, stromal cell–derived factor; and SP, substance P.

Figure 3.

Patency of vascular grafts at 4 wk. A, Color Doppler images of vascular grafts were captured before the explanation of grafts (n=5 per group). B, Stereomicrographs of grafts retrieved 4 wk after implantation. The luminal surface of the explanted grafts was smooth and free of platelet aggregates or thrombi. Data are shown as mean±SEM (n=5 per group) and evaluated by 1-way ANOVA followed by Tukey post hoc analysis (C). SDF indicates stromal cell–derived factor; and SP, substance P.

Figure 4.

Histological analysis of vascular grafts (n=5 per group) explanted 4 wk after implantation. A–I, H&E staining, (J–L) Masson trichrome staining, and (M–O), DAPI staining. Grafts were well-integrated with the surrounding tissues as demonstrated using H&E staining. SP and SDF-1 grafts showed neointimal formation on the luminal side (E, F, H, I). MT staining showed the regeneration of collagen in the neointima and the graft wall. Cross-sections were also stained with DAPI to discern cellularization. Control (M), SDF-1 (N), and SP (O). Cell infiltration was qualitatively and quantitatively analyzed based on the DAPI-stained sections. For quantitative analysis of cell density, 10 high power field (hpf) images were used per sample and 5 samples were used per group. Control and SDF-1 grafts did not significantly differ in terms of the cell density per hpf (control, 1473.83±49.69; SDF-1, 1350.22±63.36), whereas SP grafts showed significantly higher cell density per hpf than that of the control and SDF-1 grafts (1857.86±50.18; P). Data are presented as mean±SEM and evaluated by 1-way ANOVA followed by Tukey post hoc analysis. *P=0.00001081 and **P=0.00086931. SDF indicates stromal cell–derived factor; and SP, substance P.

Figure 5.

Endothelialization of vascular grafts explanted after 4 wk (n=5 per group). Explanted grafts were observed with scanning electron microscope (SEM) and also stained with vWF (von Willebrand factor). A, Scanning electron micrographs of explanted grafts and (B) immunofluorescence staining of vascular grafts with vWF antibody. SP grafts revealed the formation of endothelial cells (ECs) with cobblestone-like morphology on the luminal side. Scale bars have been shown on images. SP and SDF-1 grafts also showed a confluent layer of vWF-positive ECs on the luminal side. Scale bar, 200 µm. SDF indicates stromal cell–derived factor; and SP, substance P.

Figure 6.

Smooth muscle cells (SMCs) regeneration in vascular grafts retrieved at 4 wk (n=5 per group). Explanted grafts were stained with α-smooth muscle actin (α-SMA) antibody. The α-SMA⁺ cells have been indicated by arrows (A–I). The numbers of α-SMA⁺ cells were counted and found to be 8.6±1.47, 19.67±0.88, and 24.17±1.93 per hpf in control, SDF-1, and SP grafts, respectively (J). SDF-1 and SP grafts showed significantly higher numbers of α-SMA⁺ cells than that of the control grafts (SDF-1 vs control, P=0.00172 and SP vs control, P=0.00517; E, F, H, I). Scale bar, 100 µm. Data are shown as mean±SEM and evaluated by 1-way ANOVA followed by Tukey post hoc analysis. *P=0.00172 and **P=0.00517. COL indicates collagen; hpf, high power field; PCL, polycaprolactone; SDF, stromal cell–derived factor; and SP, substance P.

Figure 7.

Stem cell recruitment in vascular grafts explanted at 4 wk (n=5 per group). Explanted grafts were stained with anti-Sca-1 antibody. Control grafts show only a few numbers of Sca-1⁺ cells (1.5±0.5 per hpf; A–C). On the contrary, SDF-1 and SP grafts showed higher numbers of Sca-1⁺ cells (D–I). The numbers of recruited cells were also quantified by using image J and found to be 7.0±1.53 and 14.0±1.52 per hpf in SDF-1 and SP grafts, respectively (J). Data are shown as mean±SEM and evaluated by Kruskal-Wallis test followed by Dunn multiple comparisons test. *P=0.0338. COL indicates collagen; hpf, high power field; PCL, polycaprolactone; Sca, stem cell antigen; SDF, stromal cell–derived factor; and SP, substance P.

Figure 8.

Angiogenesis in the explanted grafts at 4 wk (n=5 per group). Explanted grafts were stained with laminin antibody (A–I). The laminin⁺ area was calculated by using image J and found to be 1300.5±181.5 µm², 5587.62±369.99 µm², and 6985.0±580.08 µm² per hpf in control, SDF-1, and SP grafts, respectively (J). Scale bar, 50 µm. Data are shown as mean±SEM and evaluated by 1-way ANOVA followed by Tukey post hoc analysis. *P=0.00205 and #P=0.000167031. COL indicates collagen; hpf, high power field; PCL, polycaprolactone; Sca, stem cell antigen; SDF, stromal cell–derived factor; and SP, substance P.

Figure 9.

Inflammatory response to explanted grafts at 4 wk (n=5 per group). Explanted grafts were stained with CD68 and CD206 antibodies (A–F). The numbers of CD68⁺ macrophages were counted and found to be 4.86±0.83, 5.64±0.54, and 4.17±0.56 per hpf in control, SDF-1, and SP grafts, respectively (G). The numbers of CD206⁺ (M2) macrophages were found to be 8.45±1.11, 5.12±0.45, and 22.21±1.18 per hpf in control, SDF-1, and SP grafts, respectively (H). Scale bar, 50 µm. Data are shown as mean±SEM and evaluated by Kruskal-Wallis test followed by Dunn multiple comparisons test. *P=0.0020 and **P≤0.0001. COL indicates collagen; hpf, high power field; PCL, polycaprolactone; Sca, stem cell antigen; SDF, stromal cell–derived factor; and SP, substance P.