Int J Stem Cells.  2022 Nov;15(4):395-404. 10.15283/ijsc21149.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Repair SU5416-Injured Emphysema by Inhibiting Apoptosis via Rescuing VEGF-VEGFR2-AKT Pathway in Rats

• Chen Q
• Lv L
• Zheng C
• Pan H
• Xu J
• Lin J
• Deng Z
• Qian W

• KMID: 2536209
• DOI: http://doi.org/10.15283/ijsc21149

Abstract

Background and Objectives
Chronic obstructive pulmonary disease (COPD) is a common, frequently-occurring disease and poses a major health concern. Unfortunately, there is current no effective treatment for COPD, particularly emphysema. Recently, experimental treatment of COPD using mesenchymal stem cells (MSCs) mainly focused on bone marrow-derived MSCs (BM-MSCs). Human umbilical cord-derived MSCs (hUC-MSCs) have more advantages compared to BM-MSCs. However, studies on the role of hUC-MSCs in management of COPD are limited. This study sought to explore the role of hUC-MSCs and its action mechanisms in a rat model of VEGF receptor blocker SU5416-injured emphysema.
Methods and Results
hUC-MSCs were characterized by immunophenotype and differentiation analysis. Rats were div-ided into four groups: Control, Control＋MSC, SU5416 and SU5416＋MSC. Rats in model group were administered with SU5416 for three weeks. At the end of the second week after SU5416 administration, model group were infused with 3×10⁶ hUC-MSCs through tail vein. After 14 days from hUC-MSCs transplantation, rats were euthanized and data were analyzed. HE staining and mean linear intercepts showed that SU5416-treated rats exhibited typical emphysema while emphysematous changes in model rats after hUC-MSCs transplantation disappeared completely and were restored to normal phenotype. Furthermore, hUC-MSCs inhibited apoptosis as shown by TUNEL and Western blotting. ELISA and Western blotting showed hUC-MSCs rescued VEGF-VEGFR2-AKT pathway in emphysematous lungs.
Conclusions
The findings show that hUC-MSCs effectively repair the emphysema injury. This study provides the first evidence that hUC-MSCs inhibit apoptosis via rescuing VEGF- VEGFR2-AKT pathway in a rat model of emphysema.

Keyword

COPD; Emphysema; SU5416; hUC-MSCs; VEGF-VEGFR2-AKT pathway; Apoptosis

Figure

• 

  Fig. 1 Characterization of hUC-MSCs. (A) Flow cytometry analysis of surface antigens of hUC-MSCs. hUC-MSCs were negative for CD11b, CD19 and CD34, but positive for CD29, CD73 and CD90. (B) Differentiation potential of hUC-MSCs. (a) Osteogenic differentiation results using Alizarin Red staining, numerous cells became Alizarin Red positive. (b) Adipogenic differentiation results using Oil Red O staining, some of the cells contained numerous Oil Red O-positive lipid droplets. (c) Non-sti-mulated control cells grown in regular medium. (a∼c) Magnification, ×100; scale bar, 200 μm.

• 

  Fig. 2 hUC-MSCs repaired SU5416-induced emphysema. (A) Representative results of HE staining in lung sections chosen from each group. There was no significant difference between the Control group (CON) and the Control＋MSC group (CON＋MSC). Emphysematous changes were observed in the group treated with SU5416 (SU). hUC-MSCs transplantation repaired SU5416-induced emphysema (SU＋MSC). Magnification, ×100; scale bar, 200 μm. (B) Morphometric analysis of the mean linear intercept (MLI). Values of MLI are presented as mean±SEM. *p<0.05 compared with the Control group. #p<0.05 compared with the SU5416 (SU) group. n=9.

• 

  Fig. 3 hUC-MSCs inhibited apoptosis of SU5416-treated lungs. (A) Re-presentative results of TUNEL assay using lung sections chosen from each group. There was no significant difference between the Control (CON) group and the Control＋MSC (CON＋MSC) group. Increased TUNEL-positive cells were observed in the group treated with SU5416 (SU). hUC-MSCs reduced TUNEL-positive cells (SU＋MSC). Magnification, ×400; scale bar, 50 μm. (B) Quantitative results of TUNEL assay showing apoptosis index of the four groups. Data are presented as mean±SEM. ****p<0.0001 compared with the Control (CON) group, ####p<0.0001 compared with the SU5416 (SU) group. n=3. (C) Representative results of active caspase-3 and PCNA as determined by western blotting. hUC-MSCs inhibited increased levels of active caspase-3 but not affect proliferation in the SU5416-treated models.

• 

  Fig. 4 hUC-MSCs rescued VEGF expression in SU5416-induced emphysema. (A) VEGF protein level in BALF was determined by ELISA. Data are presented as mean±SEM. **p<0.01 compared with the Control (CON) group, ##p<0.01 compared with the SU5416 (SU) group. n=9. (B) VEGF protein level in lungs as determined by western blotting.

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  Fig. 5 hUC-MSCs rescued the expression of p-VEGFR2 and p-AKT in emphysema induced by SU5416. The protein expressions of p-VEGFR2, VEGFR2, p-AKT and AKT in the lungs were determined by western blotting. Compared with the control group (CON), p-VEGFR2 and p-AKT in the model group (SU) were significantly reduced, but both of them in the hUC-MSCs transplantation group (SU＋MSC) returned to the level of the Control group. There was no significant change in the expression of VEGFR2 and AKT in each group.

• 

  Fig. 6 hUC-MSCs did not alter the expression of pro-inflammatory cytokines in SU5416-induced emphysema. (A∼C) RQ-PCR detect IL-1β, IL-6 and TNFα mRNA expression in lungs, respectively. (D∼F) ELISA detect IL-1β, IL-6 and TNFα protein expression in BALF, respectively. hUC-MSCs had no significant effect on inflammatory response in normal or damaged lung tissues.

Reference

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