Human umbilical cord mesenchymal stem cells improve the pregnancy outcomes of preeclampsia rats via inducing placental angiogenesis

Abstract

Background: Preeclampsia is considered to be a serious complication unique to pregnancy caused by placental dysplasia. Although interventions such as antihypertensive drugs and magnesium sulfate can partially mitigate maternal risk, the ultimate remedy remains delivery. In recent years, mesenchymal stem cells (MSCs) have become promising therapeutic approach in the treatment of ischemic diseases. Therefore, this study aimed to investigate the effect of human umbilical cord mesenchymal stem cells (hucMSCs) on pregnancy outcomes and placental angiogenesis in N-nitro-L-arginine methyl ester (L-NAME) induced preeclampsia rats.

Methods: The expression of mesenchymal markers in hucMSCs was analyzed by flow cytometry. Multipotent differentiation of hucMSCs was identified, respectively. HucMSCs were injected into the preeclampsia rat by the tail vein. SBP was measured at the rat tail artery using an automatic noninvasive blood pressure monitor. The proteinuria levels were measured using the BCA method. RT-qPCR and ELISA were used to assess the mRNA expression and plasma concentrations of soluble FMS-like tyrosine kinase 1 (sFlt-1), placental growth factor (PLGF), and vascular endothelial growth factor (VEGF). Placental tissues were collected for immunohistochemistry and pathological analysis.

Results: HucMSCs were positive for CD90, CD73, and CD105, and could differentiate into osteoblasts, adipocytes and chondrocytes. PE rats treated with hucMSCs showed a lowering of SBP and proteinuria and a higher fetal and placental mass. The microvascular density (MVD), mRNA expression, and plasma concentrations of VEGF and PLGF were increased in the hucMSCs-treated group, while the sFlt-1 levels were decreased.

Conclusion: HucMSCs may promote placental angiogenesis and improve the pregnancy outcomes of preeclampsia rats by regulating the balance of pro-angiogenic and antiangiogenic factors.

Keywords: angiogenesis; human umbilical cord mesenchymal stem cells; placental vasculature; preeclampsia; pregnancy outcomes.

FIGURE 1

Identification of human umbilical cord mesenchymal stem cells. (a) The analysis of mesenchymal marker expression in hucMSCs by flow cytometry. (b) The morphology of the hucMSCs (× 40, bar = 200 μm). (c) Image of osteogenic differentiation by alizarin red S staining (× 100, bar = 100 μm). (d) Image of adipogenic differentiation by oil red O staining (× 200, bar = 50 μm). (e) Image of chondrogenic differentiation by alcian blue staining (× 100, bar = 100 μm). hucMSCs, human umbilical cord mesenchymal stem cells. CD, cluster of differentiation; PE, phycoerythrin; APC, allophycocyanin; HLA, human leukocyte antigen; FITC, fluorescein isothiocyanate.

FIGURE 2

Schematic illustrations of the animal study. (a) The vaginal plug or sperm was observed on GD1. The arrows indicate vaginal plug and sperm. (b) Animal experiment timeline was described by figdraw.

FIGURE 3

HucMSCs lower the SBP and 24-h urine protein concentrations in PE rats. (a-e) The SBP of different group rats was measured on GD7, GD10, GD14, GD17, and GD19. (f) The ΔSBP between GD17 and GD14 in different group rats. (g) The ΔSBP between GD19 and GD14 in different group rats. (h, i) The 24-h urinary protein concentrations of different group rats were analyzed with a BCA kit on GD7 and GD19. n = 9. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 4

HucMSCs improve the pregnancy outcomes of PE rats. (a) The image of fetuses in different groups. (b) The number of fetuses in different groups. (c) The weight of fetuses in different groups. (d) The weight of placentas in different groups. n = 9. ^∗p < 0.05; ^∗∗p < 0.01.

FIGURE 5

HucMSCs improve the pathological changes of the placental tissue in PE rats. H&E staining was used to analyze the pathological changes of placental tissue in different groups (×200, bar = 50 μm). (a) NP: In the normal group, the infiltration of placental trophoblast cells was regular, more blood vessels were recast, and the blood vessels in the labyrinth layer were abundant. (b) PE + vehicle: The trophoblast giant cells proliferated, trophoblast cells vacuolated seriously, recast blood vessels were reduced, and blood vessels in the labyrinth layer were reduced. (c) PE + hucMSCs: The vacuolation of trophoblast cells and rare recast blood vessels were improved, and the blood vessels in the labyrinthic layer were increased. n = 9.

FIGURE 6

HucMSCs induce placenta angiogenesis in PE rats. (a-c) Immunohistochemistry was used to analyze the expression of CD31 in each group (×400). The scale bar represents 20 μm. (d) Quantification of MVD in different groups. (e-g) RT-qPCR was used to analyze the mRNA expression of sFlt-1, PLGF, and VEGF in placental tissues of different group rats. (h-j) The concentrations of sFlt-1, PLGF, and VEGF in rat plasma was measured by ELISA. n = 9, P < 0.05 was considered statistically significant. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.