Bone Marrow-Derived Mesenchymal Stem Cells Repair Necrotic Pancreatic Tissue and Promote Angiogenesis by Secreting Cellular Growth Factors Involved in the SDF-1 α /CXCR4 Axis in Rats

Abstract

Acute pancreatitis (AP), a common acute abdominal disease, 10%-20% of which can evolve into severe acute pancreatitis (SAP), is of significant morbidity and mortality. Bone marrow-derived mesenchymal stem cells (BMSCs) have been reported to have a potential therapeutic role on SAP, but the specific mechanism is unclear. Therefore, we conducted this experiment to shed light on the probable mechanism. We validated that SDF-1α significantly stimulated the expressions of VEGF, ANG-1, HGF, TGF-β, and CXCR4 in BMSCs, which were inhibited by its receptor agonist, AMD3100. The capacities of proliferation, migration, and repair of human umbilical vein endothelial cells were enhanced by BMSCs supernatant. Meanwhile, BMSCs supernatant could also promote angiogenesis, especially after the stimulation with SDF-1α. In vivo, the migration of BMSCs was regulated by SDF-1α/CXCR4 axis. Moreover, transplanted BMSCs could significantly alleviate SAP, reduce the systematic inflammation (TNF-α↓, IL-1β↓, IL-6↓, IL-4↑, IL-10↑, and TGF-β↑), and promote tissue repair and angiogenesis (VEGF↑, ANG-1↑, HGF↑, TGF-β↑, and CD31↑), compared with the SAP and anti-CXCR4 groups. Taken together, the results showed that BMSCs ameliorated SAP and the SDF-1α/CXCR4 axis was involved in the repair and regeneration process.

Figure 1

The SDF-1α/CXCR4 axis could promote the expressions of cellular growth factors in BMSCs and induce angiogenesis in vitro. ((a), (d), and (e)) The CXCR4 expression of BMSCs was significantly unregulated after being stimulated by combined SDF-1α protein, which could be inhibited by the CXCR4 receptor agonist, AMD3100. ((b)–(d), (f)-(g)) Recombined SDF-1α protein could significantly promote BMSCS expression of VEGF, ANG-1, HGF, and TGF-β and the effect could also blocked by AMD3100. Moreover, there was a dose-effect relationship between combined SDF-1α protein and CXCR4, VEGF, ANG-1, HGF, and TGF-β expressions. Data are expressed as mean ± SD (^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001 for SDF-1α (10 ng/mL or 100 ng/mL) versus NC, ^† P < 0.05, ^†† P < 0.01, and ^††† P < 0.001 for SDF-1α (10 ng/mL or 100 ng/mL) versus SDF-1α (10 ng/mL or 100 ng/mL) + AMD3100 10 μg/mL). (h) BMSCs supernatant could significantly promoted angiogenesis, especially after being pretreated with SDF-1α and this increase was partly offset by AMD3100. Both VEGF siRNA and Ang-1 siRNA significantly weakened proangiogenic capacity of BMSCs. Data are expressed as mean ± SD (^## P < 0.01 for NC versus PBS, ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001 for SDF-1α (10 ng/mL or 100 ng/mL), Ang-1 siRNA, or VEGF siRNA versus NC) (NC, normal control, SDF-1α, stromal cell derived factor-1a, CXCR4, CXC chemokine receptor 4, VEGF, vascular endothelial growth factor, and ANG-1, angiopoietin-1).

Figure 2

The BMSCs supernatant could promote the proliferation, migration, and repair of human umbilical-vein endothelial cell line (EA.hy926 cells). (a) The methylene blue staining (magnification, ×200) is indicating that the BMSCs supernatant could significantly promote the migration of EA.hy926 cells. Data are expressed as mean ± SD (^* P < 0.05 for DMEM-LG versus DMEM-LG + BMSCs supernatant). (b) The significant increase of the growth of EA.hy926 cells was investigated in MTT test after being given BMSCs supernatant. (c) Impaired EA.hy926 cells could be repaired by BMSCs supernatant. Data are expressed as mean ± SD (^# P > 0.05 for sham versus normal, ^* P < 0.05 and ^** P < 0.01 for DMEM-LG versus DMEM-LG + BMSCs supernatant at each corresponding time point). Data analysis was performed by a one-way ANOVA. (BMSCs, bone marrow-derived mesenchymal stem cells, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,S-diphenyltetrazolium bromide).

Figure 3

There is similarity in the proliferation and CXCR4 expression between unlabeled and SPION-labeled BMSCs ([Fe³⁺] = 25 g/mL). (a) The Prussian blue staining shows that BMSCs were labeled by SPION successfully. (b) The MTT tests are showing that the growth activity of SPION-labeled BMSCs ([Fe³⁺] = 25 g/mL) slowed slightly compared with that of the unlabeled BMSCs, which was also significantly stronger than that of other SPION-labeled BMSCs ([Fe³⁺] = 50, 75, or 100 g/mL). Data are expressed as mean ± SD (^&& P < 0.01 and ^&&& P < 0.001 for normal control versus SPION-labeled BMSCS ([Fe³⁺] = 100 g/mL), ^$$ P < 0.01 and ^$$$ P < 0.001 for normal control versus SPION-labeled BMSCS ([Fe³⁺] = 75 g/mL), and ^† P < 0.05 and ^††† P < 0.001 for normal control versus SPION-labeled BMSCS ([Fe³⁺] = 50 g/mL) at each corresponding time point). ((c)-(d)) CXCR4 expression was detected by immunofluorescence and western blotting and similar between unlabeled and SPION-labeled BMSCs (P > 0.05). (SPION, superparamagnetic iron oxide nanoparticles, BMSCs, bone marrow-derived mesenchymal stem cells).

Figure 4

Prussian blue staining was performed for detecting the SPION-labeled BMSCs in pancreatic and lung tissues. (a) The Prussian blue staining of pancreatic tissue indicates that the cells were stained blue (as indicated by black arrows), gradually increased, and peaked on postoperative days 5–7, when the formation of tubular complexes was also maximal (as indicated by red arrows). However, the migration was partly inhibited and the trend was not obviously investigated in anti-CXCR4 group. (b) The lung tissues were stained by Prussian blue and the blue particles (as indicated by black arrows) were gradually decreasing in both BMSCs and anti-CXCR4 groups. (c) The blue particles in lung, pancreas, liver, spleen, and small intestine were counted and analyzed between BMSCs and anti-CXCR4 groups. The result showed that the number of blue particles of pancreas in BMSCs group was significantly more than in anti-CXCR4 group at postoperative days 1, 3, 5, and 7. Data are expressed as mean ± SD (^** P < 0.01 and ^*** P < 0.001 for BMSCs versus anti-CXCR4 at each corresponding time point).

Figure 5

The transplanted SPION-labeled BMSCs were tracked by MRI in vivo. (a) [Fe³⁺] was detected by MRI and the result showed that the SPION-labeled BMSCs ([Fe³⁺] = 25 g/mL) appeared high signals (white spots) on T1WI and low signals (dark spots) on T2WI. (b) The SPION-BMSCs ([Fe³⁺] = 25 g/mL) was tracked by MRI in vivo and the photos show that the white spots (inside of red circle) were increasing gradually and peaked on postoperative days 5–7. Instead, the white spots decreased on postoperative day 5 in anti-CXCR4 group compared with BMSCs group. (c) The SPION-BMSCs ([Fe³⁺] = 25 g/mL) were detected in SAP rats at postoperative day 7 by T1WI and T2WI and the result was showing that the high signals (white spots), which appeared in T1WI, became low signals (dark spots) in T2WI (SPION, superparamagnetic iron oxide nanoparticles, T1WI, T1-weighted imaging, and T2WI, T2-weighted imaging).

Figure 6

Transplanted BMSCs could reduce severe acute pancreatitis (SAP), promoting the formation of tubular complexes and inhibiting systematic inflammatory response, being involved in the SDF-1α/CXCR4 axis. ((a), (b)) The H&E staining of pancreatic tissues (magnification, ×100) is showing that the edema, infiltration, and necrosis were significantly reduced in the SAP+BMSCs group compared with SAP and SAP+anti-CXCR4 BMSCs groups at postoperative days 1, 4, and 7, respectively. A large number of tubular complexes were also investigated in SAP+BMSCs group (as indicated by red arrow in a 400x magnified picture). Serum amylase activity was also significantly reduced in SAP+BMSCs group than in normal, SAP, and anti-CXCR4 BMSCs groups at postoperative days 1, 4, and 7, respectively. (c) The levels of serum proinflammatory cytokines are significantly lower in SAP+BMSCs than in SAP and SAP+anti-CXCR4 BMSCs groups. In contrast, the levels of serum anti-inflammatory cytokines are significantly higher in SAP+BMSCs than in SAP and SAP+anti-CXCR4 BMSCs groups. Data are expressed as mean ± SD (^# P > 0.05 for sham versus normal, ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001 for SAP versus Normal, ^† P < 0.05, ^†† P < 0.01, and ^††† P < 0.001 for SAP versus SAP+BMSCs, ^$ P < 0.05, ^$$ P < 0.01, and ^$$$ P < 0.001 for SAP+BMSCs versus SAP+anti-CXCR4 BMSCs). Data analysis was performed by Student's test (BMSCs, bone marrow-derived mesenchymal stem cells, H&E, hematoxylin-eosin).

Figure 7

qRT-PCR or western-blot assays are showing that the expressions of ANG-1, VEGF, HGF, TGF-β, and CD31 in pancreatic tissue were significantly higher in SAP+BMSCs group. ((a), (c), and (d)) The expression of ANG-1 was investigated higher in SAP+BMSCs group than in normal, SAP, and SAP+anti-CXCR4 BMSCs groups. ((b), (c), and (e)) There was a significant higher expression of VEGF in SAP+BMSCs group than in normal, SAP, and SAP+anti-CXCR4 BMSCs groups at posttransplant days 1 and 4, with a subsequent decrease at day 7. ((f)–(h)) The mRNA level of HGF, TGF-β, and CD31 was significantly higher in SAP+BMSCs group than in normal, SAP, and SAP+anti-CXCR4 BMSCs groups. Data are expressed as mean ± SD (^# P > 0.05 for sham versus NC, ^* P < 0.05 and ^** P < 0.01 for SAP+BMSCs versus NC, ^† P < 0.05, ^†† P < 0.01, and ^††† P < 0.001 for SAP+BMSCs versus SAP, ^$ P < 0.05, ^$$ P < 0.01, and ^$$$ P < 0.001 for SAP+BMSCs versus SAP+anti-CXCR4 BMSCs at each corresponding time point) (NC, normal control, ANG-1, angiopoietin-1, VEGF, vascular endothelial growth factor, SAP, severe acute pancreatitis, and BMSCs, bone marrow-derived mesenchymal stem cells).

Figure 8

Transplantation BMSCs could promote angiogenesis in damaged pancreatic tissue, which was inhibited by anti-CXCR4 treatment. ((a), (d)) VEGF expression was assayed in pancreatic tissue by immunohistochemistry and the result showed that VEGF expression was higher in SAP+BMSCs group than in normal, SAP, and anti-CXCR4 groups at postoperative days 1 and 4. On the contrary, it was lower in SAP+BMSCs group than in SAP group at postoperative day 7. ((b)–(d)) vWf and CD31 expressions in pancreatic tissue were showed to be significantly higher in SAP+BMSCs group than in normal, SAP, and SAP+anti-CXCR4 BMSCs groups. Data are expressed as mean ± SD (^# P > 0.05 for sham versus NC, ^* P < 0.05 and ^** P < 0.01 for SAP+BMSCs versus NC, ^† P < 0.05, ^†† P < 0.01, and ^††† P < 0.001 for SAP+BMSCs versus SAP, ^$ P < 0.05, ^$$ P < 0.01, and ^$$$ P < 0.001 for SAP+BMSCs versus SAP+anti-CXCR4 BMSCs at each corresponding time point) (NC, normal control, VEGF, vascular endothelial growth factor, SAP, severe acute pancreatitis, BMSCs, bone marrow-derived mesenchymal stem cells, vWF, von Willebrand factor, and CD31, platelet endothelial cell adhesion molecule-1).