Intravenous delivery of mesenchymal stromal cells reverses Müller cell endoplasmic reticulum stress in diabetic retinopathy

Abstract

Background: Mesenchymal stromal cells (MSCs) have emerged as a promising disease-modifying therapy for the complications of diabetes mellitus (DM), including diabetic retinopathy (DR). However, the optimal treatment regimen remains unclear, and challenges persist regarding the timing, route of delivery and the mechanisms underlying the therapeutic effects. This study focused on human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs), to elucidate their retinal protective effects, and investigate the underlying mechanisms by which a single intravenous injection might ameliorate the pathological alterations of DR.

Methods: Two time points after the development of DM were chosen for the in vivo experiments to study the effects of the intervention after different times of exposure to hyperglycemia. hUC-MSCs were injected via the tail vein at 8 and 16 weeks after STZ injection. Retinal samples were collected 2 weeks post-treatment to analyze the therapeutic effect of MSCs on DR. In vitro experiments were conducted using a Müller cell line and a retinal microvascular endothelial cell line cultured under high-glucose conditions, with treatment by hUC-MSCs conditioned media (MSC-CM), to explore the underlying mechanisms.

Results: After a single intravenous injection of hUC-MSCs at week 16 and not 8 weeks post-STZ injection, retinal tissue showed improved thickness of the inner nuclear layer. There was also an increase in the number of acellular capillaries observed in retinal flat mounts of diabetic animals which was improved in the DM and MSC treatment group. MSC treatment reduced high glucose induced activation markers (GFAP and Vimentin) of Müller cells and alleviated endoplasmic reticulum (ER) stress. VEGF expression was also reduced in the retina. MSC-conditioned media also reversed high glucose-induced expression of VEGF in Müller cells. Finally, in a retinal microvascular endothelial cell line, high glucose concentrations, demonstrated increased ER stress which was reduced by MSC conditioned media.

Conclusions: Single Intravenous injection of hUC-MSC to DM animals could alleviate DR via reducing Müller cell and endothelial cell activation and ER stress, and thus might represent a promising therapy for DR.

Keywords: Diabetic retinopathy; Endoplasmic reticulum stress; Human umbilical cord mesenchymal stromal cells; Müller cells.

Fig. 1

Schematic of in vivo experimental design summarizing the design of two experiments to investigate the effects of hUC-MSCs on retinal disease in streptozotocin (STZ) -induced diabetic C57BL/6 mice model at two time points after induction of DM

Fig. 2

Individual layer thickness of the retina in three groups at timepoint 1 (A, B) and timepoint 2 (C). Error bars represent SD. D Retinal layer thickness statistics at two timepoints. E Number of cells at two timepoints. F-G Effect of MSC treatment on the number of acellular capillaries in the retina of mice at two timepoints. Error bars represent SD. N = 6; *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 500 μm in A; 100 μm in B and C; 50 μm in F. INL, inner nuclear layer; ONL, outer nuclear layer

Fig. 3

Quantification of GFAP and Vimentin intensity expressed as mean integrated intensity at timepoint 1 (A, C) and timepoint 2 (B, D) measured by ImageJ. (Data represent combined mean ± SD from N = 4 animals/group, Scale bars: 20 μm in A and B, ***p < 0.001, ****p < 0.0001)

Fig. 4

The expression of CHOP, GRP78 and VEGF in retinal tissue at two timepoints after induction of diabetes mellitus. A Immunohistochemical staining of CHOP, GRP78 and VEGF in the retina tissues in three groups at two timepoints. B Quantitative immunohistochemical staining of retinal sections at timepoint 1. C Quantitative immunohistochemical staining of retinal sections at timepoint 2. Data represent as mean ± SD, N = 6/group, Scale bars: 100 μm in A, ***p < 0.001;****p < 0.0001

Fig. 5

The three animal groups at time point 2 were examined using electron microscopy to observe the morphology of ER and mitochondria in Müller cells located in the inner nuclear layer (A and B). mRNA expression of GFAP, TGF-β and FGF in three groups at Timepoint 1 (C) and Timepoint 2 (D). mRNA expression of ATF4, ATF6, CHOP and GRP78 in three groups at Timepoint 1 (E) and Timepoint 2 (F). The red arrow indicates enlarged, swollen ER, while the yellow arrow indicates swollen mitochondria with disrupted internal structures. N = 6, Scale bars: 2 μm in A; 10 μm in B. Error bars represent SD. *p < 0.05,**p < 0.01;****p < 0.0001

Fig. 6

CCK-8 results of Müller cells treated with different time and glucose concentration gradients (A). Müller cell status after treatment with different stem cell conditioned media (B). Scale bars: 100 μm

Fig. 7

A mRNA expression of XBP1, ATF4, ATF6, CHOP and GRP78 in five groups in Müller cells. B mRNA expression of XBP1, ATF4, ATF6, CHOP and GRP78 in five groups in Mouse retinal microvascular endothelial cells. C-J Western blots of ER stress related proteins in Müller cells. Representative bands (left panels) and graphical representations of the densitometric quantification of ER stress-related proteins (IRE1, ATF6, XBP1, CHOP) and housekeeping proteins (β-actin, Tubulin) from Western blots of Müller cells from groups of control, mannitol, high glucose, high glucose + MSC medium and high glucose + MSC medium, N = 3. Western blots of VEGF proteins in Müller cells, N = 4