Heparin-binding epidermal growth factor-like growth factor and mesenchymal stem cells act synergistically to prevent experimental necrotizing enterocolitis

Abstract

Background: We have shown that administration of heparin-binding EGF (epidermal growth factor)-like growth factor (HB-EGF) protects the intestines from experimental necrotizing enterocolitis (NEC). We have also demonstrated that systemically administered mesenchymal stem cells (MSC) can engraft into injured intestines. This study investigated the effects of HB-EGF on MSC in vitro, and whether MSC and HB-EGF can act synergistically to prevent NEC in vivo.

Study design: In vitro, the effect of HB-EGF on MSC proliferation, migration, and apoptosis was determined. In vivo, rat pups received MSC either intraperitoneally (IP) or intravenously (IV). Pups were assigned to 1 of 7 groups: Group 1, breast-fed; Group 2, experimental NEC; Group 3, NEC+HB-EGF; Group 4, NEC+MSC IP; Group 5, NEC+HB-EGF+MSC IP; Group 6, NEC+MSC IV; or Group 7, NEC+HB-EGF+MSC IV. Mesechymal stem cell engraftment, histologic injury, intestinal permeability, and mortality were determined.

Results: Heparin-binding EGF-like growth factor promoted MSC proliferation and migration, and decreased MSC apoptosis in vitro. In vivo, MSC administered IV had increased engraftment into NEC-injured intestine compared with MSC administered IP (p < 0.05). Heparin binding EGF-like growth factor increased engraftment of IP-administered MSC (p < 0.01) and IV-administered MSC (p < 0.05). Pups in Groups 3 to 7 had a decreased incidence of NEC compared with nontreated pups (Group 2), with the lowest incidence in pups treated with HB-EGF+MSC IV (p < 0.01). Pups in Group 7 had a significantly decreased incidence of intestinal dilation and perforation, and had the lowest intestinal permeability, compared with other treatment groups (p < 0.01). Pups in all experimental groups had significantly improved survival compared with pups exposed to NEC, with the best survival in Group 7 (p < 0.05).

Conclusions: Heparin-binding EGF-like growth factor and MSC act synergistically to reduce injury and improve survival in experimental NEC.

Figure 1

Effect of HB-EGF on mesenchymal stem cell (MSC) proliferation, migration and apoptosis. (A) MSC proliferation assay. Statistical symbols for Student’s t test are: ^{† ‡}p<0.05 vs NA; ^§ p<0.01 vs NA; ^§ p<0.05 vs ^†. (B) MSC migration assay. Statistical symbols for Student’s t test are: ^{† ‡ §}p<0.01 vs NA; ^§ p<0.05 vs ^†. (C) MSC apoptosis assay. Statistical symbols for Student’s t test are: ^†p<0.01 vs NA; ^∥p<0.05 vs ^{‡ §}; ^∥p<0.01 vs ^†. Values represent mean ± SD. Each experiment was performed 4 times with each sample tested in duplicate. YFP, yellow fluorescent protein; MSC, mesenchymal stem cell; HB-EGF, heparin binding EGF-like growth factor; NA, no addition.

Figure 2

Effect of HB-EGF on mesenchymal stem cell (MSC) engraftment into intestines subjected to experimental necrotizing enterocolitis. Shown are representative images from pups subjected to: (A) NEC+MSC IP; (B) NEC+HB-EGF+MSC IP; (C) NEC+MSC IV; or (D) NEC+HB-EGF+MSC IV. Cell nuclei are indicated by blue fluorescent (DAPI) staining and engrafted YFP-positive mesenchymal stem cells are indicated by green fluorescent staining (white arrows). Magnification x100. (E) Quantification of MSC engraftment. Values represent mean ± SD, n=12-16 animals in each group. Statistical symbols for 1-way ANOVA with Turkey test are: ^†p<0.01 vs *; ^§ p<0.05 vs ^‡; ^‡p<0.05 vs *. HB-EGF, heparin binding EGF-like growth factor; NEC, necrotizing enterocolitis; YFP, yellow fluorescent protein; MSC, mesenchymal stem cells; IP, intraperitoneal; IV, intravenous.

Figure 3

Necrotizing enterocolitis (NEC) evaluation. (A) Gross view of the small intestine subjected to experimental NEC. The proximal small intestine is to the left and the cecum is to the right. pneumatosis; intestinal narrowing; perforation; intraluminal bleeding; intestinal dilation. (B) Histological scoring of intestines from neonatal rats. Magnification x100) (a) Grade 0: uninjured intestines; (b) Grade 1: epithelial cell lifting or separation; (c) Grade 2: epithelial cell sloughing to mid villus level; (d) Grade 3: necrosis of entire villus; (e) Grade 4: transmural necrosis. Grade 2 or higher injury is defined as being consistent with experimental NEC; Grade 3 or 4 injury is defined as being consistent with severe NEC. (C) Incidence of NEC. Statistical symbols for Fisher’s exact test are: ^†p<0.01 vs BF; ^{§ ∥ #} p<0.05 vs ^†; ^{‡ ¶}p<0.01 vs ^†. (D) Incidence of severe NEC. Statistical symbols for Fisher’s exact test are assigned as follows: ^†p<0.01 vs BF; ^{‡ § ∥ ¶} p<0.05 vs ^†; ^#p<0.01 vs ^{† ‡}. In (C) and (D), the numbers of animals in each group are as follows: BF (n=10), NEC (n=38), NEC+HB-EGF (n=43), NEC+MSC IP (n=25), NEC+HB-EGF+MSC IP (n= 26), NEC+MSC IV (n=27), NEC+HB-EGF+MSC IV (n=28). NEC, necrotizing enterocolitis; BF, breast fed; HB-EGF, heparin binding EGF-like growth factor; MSC, mesenchymal stem cells; IP, intraperitoneal; IV, intravenous.

Figure 4

Effect of HB-EGF and mesenchymal stem cells (MSC) on gut barrier function. The concentration of serum FD70 represents intestinal permeability. Higher permeability represents worsened gut barrier function. Values represent mean ± SD; n=9 for each group. Statistical symbols for 1-way ANOVA with Turkey test are: ^†p<0.01 vs BF; ^{‡ § ∥ ¶ #}p<0.01 vs ^†; ^#p<0.05 vs ^{§ ∥ ¶}; ^#p<0.01 vs ^‡. BF, breast fed; HB-EGF, heparin binding EGF-like growth factor; MSC, mesenchymal stem cells; FD, fluorescein isothiocyanate (FITC)-labeled dextran.

Figure 5

Rat pup 96-hour survival. Statistical symbols for Fisher’s exact test are: †p<0.01 vs BF; ^{‡ § ∥ ¶} p<0.05 vs ^†; ^#p<0.01 vs ^†; ^#p<0.05 vs ^‡ (Fisher’s exact test). The numbers of animals in each group are as follows: BF (n=10), NEC (n=38), NEC+HB-EGF (n= 43), NEC+MSC IP (n=25), NEC+HB-EGF+MSC IP (n=26), NEC+MSC IV (n=27), and NEC+HB-EGF+MSC IV (n=28). BF, breast fed; NEC, necrotizing enterocolitis; HB-EGF, heparin binding EGF-like growth factor; MSC, mesenchymal stem cells; IP, intraperitoneal; IV, intravenous.