Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells

Abstract

Aims/hypothesis: The immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis.

Methods: We investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs).

Results: ASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-κB dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1.

Conclusions/interpretation: The ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy.

Keywords: Adipose tissue-derived stromal cells; Diabetic retinopathy; High glucose; Oxidative stress.

Fig. 1

Ultrastructure of ASC-induced vascular network. HUVECs were seeded on confluent monolayers of ASCs. After 5 days, 0.5 μm sections were stained with toluidine blue and analysed by light microscopy, and 60 nm sections of glutaraldehyde-fixed and plastic-embedded co-cultures were analysed by transmission electron microscopy. (a–c) Representative light micrographs: (a) Planar, parallel section (i.e. the top view of the culture) showing the formation of a vascular network (arrows, endothelial cells) demarcated by the dotted lines. Lumens (L) have formed, which are aligned by ASCs (black arrowheads) in close contact. (b) Cross section of the lumen-containing 3D vascular structures in between (interrupted) layers of ASCs. (c) Enlargement of a vascular structure with several aligned ASCs (arrows, endothelial cells; black arrowheads, ASCs). (d–i) Transmission electron micrographs of the vascular structures: (d) a vascular structure consisting of endothelial cells (arrows) and lumen is depicted by the dotted lines with surrounding ASCs (black arrowheads). (e) Specific cell–cell connections with tight junctions between endothelial cells (white arrowheads), with lumen formation on top of the ASCs. (f) ASCs deposit extracellular matrix (black asterisks), which forms a basement membrane-like structure between the endothelial cells and the ASCs. (f, g) Peg-and-socket connections are shown by the lightning symbols, and the inset in (g) shows intracellular filaments (white asterisks), indicative of contractility, similar to smooth muscle cells, i.e. hinting at the maturation of ASCs to pericytes. (h, i) Detailed views of the endothelial cell–cell connections and basal membrane formation around the endothelial cells, i.e. the vascular structure with connected ASCs. Scale bar, 5 μm. Lumen (L); endothelial cells (arrows); ASCs (black arrowheads); endothelial cell–cell connections (white arrowheads); extracellular matrix formation (black asterisks); intracellular filaments representative for smooth muscle cell phenotype (white asterisks); peg-and-socket connections of ASCs with endothelial cells (lightning symbols)

Fig. 2

ASCs enhance hypoxia-driven angiogenesis in the ROP mouse model. (a) Scheme of the ROP model. Mouse pups were exposed to hyperoxia (75% O₂) from P7 to P12 and subsequently transferred to room air (21% O₂). This causes hypoxia at room air that results in extensive retinal neovascularisation at P17. ASCs were injected into the vitreous at P12 to evaluate their influence on hypoxia-driven neovascularisation. (b, c) Representative micrographs of CM-DiI-labelled ASCs (red, white arrows) co-localising with the endothelial layer (lectin, green); magnification × 20. (d–f) Micrographs of EGFP-tagged ASCs (green) co-localising with the endothelial layer (lectin, red) in the pericytic position. The scale bars in (d–f) are 100 μm, 25 μm and 15 μm, respectively. (g, h) Histological analysis of the effects of ASC injection on hypoxia-induced retinal neovascularisation; magnification × 20. Neovascularisation was assessed histologically by counting the endothelial cell nuclei anterior to the inner limiting membrane. (g) Histological features of retinal neovascularisation in the control group. (h) Histological features of retinal neovascularisation in ASC-injected mice. Intravitreal injection of ASCs in the ROP mouse model increased the number of neovascular tufts extending into the vitreous (black arrows). (i) Hypoxia-driven neovascularisation in the retinas was enhanced by 54% in animals injected with ASCs (PBS vs ASC, 8.3 ± 0.62 vs 12.8 ± 0.96). The graph shows the mean number of neovascularisation nuclei per section per animal, ***p < 0.001. GCL, ganglion cell layer; ILM, inner limiting membrane; INL, inner nuclear layer; neovascul., neovascularised; ONL, outer nuclear layer

Fig. 3

ASCs modulate the ROP micro-environment. Gene expression analyses normalised to Gapdh of (a) dissected ROP retinas at P13 (grey bars) compared with the control retinas at P13 (white bars). The expression levels of Vegfa, Fgf2, and Col4a1 were increased. In addition, an inflammatory response was induced, as measured by increased expression of Il1b and Ccl2. (b) To assess the ASC-guided changes to the ROP micro-environment at P19, gene expression in ROP retinas of eyes with ASC injection (grey bars) were compared with controls (white bars). We observed increased expression of Angpt1 and Fgf2, and decreased expression of Angpt2 in ROP retinas of eyes with ASC injection. The expression of Vegfa, Pdgfb and Col4a1 was similar to controls. The inflammatory response was modulated by normalised Il1b expression, while the expression of Tnf, Cxcl15 and Ccl2 was increased. *p < 0.05, **p < 0.01 vs control retinas. Graphs show the means ± SEM from retinas of five animals in each group; experiments were performed in triplicate

Fig. 4

Anti-inflammatory and anti-apoptotic effects of ASCs depend on chronic HG preconditioning. (a, b) Expression of 36 genes, normalised to ACTB, in ASCs after acute exposure to HG (7 days) or chronic HG (more than 21 days maintenance in HG) compared with NG-exposed controls. Grey bars, acute HG exposure; white bars, chronic HG exposure. Graphs represent data ± SEM from n = 4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs NG-exposed control. (c, d) The inflammatory response in HG conditions was induced in ASCs and measured by ELISA to detect PGE2 and CCL2 in ASC-Cme. LPS and TNF-α were used as positive-stimulated controls. Celecoxib (10 mmol/l) was used as an inhibitor of COX2 in acute-HG-treated ASCs. Graphs show mean ± SEM from n = 5 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs NG control; ††p < 0.01, †††p < 0.001 vs chronic HG. Genes encode the following proteins: TNF, tumour necrosis factor; IL1B, IL-1β; IL1A, IL-1α; IL6, IL-6; CXCL8, C-X-C motif chemokine ligand 8 (also known as IL-8); CCL2, chemokine (C-C motif) ligand 2; PTGS2, prostaglandin-endoperoxide synthase 2; CXCL12, C-X-C motif chemokine 12; NFKB1, NF-κB subunit 1; MMP1, matrix metallopeptidase 1; ICAM1, intercellular adhesion molecule 1; IDO1, indoleamine 2,3-dioxygenase 1; IL1R1, IL-1 receptor type 1; IL1RN, IL-1 receptor antagonist; IL10RA, IL-10 receptor subunit α; IL10RB, IL-10 receptor subunit β; SLC2A1, solute carrier family 2 member 1 (also known as GLUT1); SLC2A4, solute carrier family 2 member 4 (also known as GLUT4); VEGFA, vascular endothelial growth factor A; KDR, kinase insert domain receptor/VEGF receptor 2; TGFB1, TGF-β1; FGF2, fibroblast growth factor 2; IGF, IGF; ANGPT1, angiopoietin 1; ANGPT2, angiopoietin 2; TIE1, tyrosine kinase with immunoglobulin like and EGF like domains 1; TEK, TEK receptor tyrosine kinase (also known as Tie2); PDGFRB, platelet derived growth factor receptor β; RGS5, regulator of G-protein signalling 5; TAGLN, transgelin; ACTA1, actin α1; PECAM1, platelet/endothelial cell adhesion molecule 1 (skeletal muscle); CNN1, calponin 1; MCAM, melanoma cell adhesion molecule (also known as CD146); DES, desmin; THY1, Thy-1 cell surface antigen (also known as CD90)

Fig. 5

The antioxidant role of ASC-Cme, combined with declining NF-κB activation, promotes cell viability of HG-challenged BRECs. (a) ASC-Cme promotes BREC viability following HG-induced apoptosis (viability ∼88% vs 94.6%, HG vs NG; 88% vs 93.9%, HG vs Cme). (b) HG-induced apoptosis was normalised by ASC-Cme (annexin V-positive cells ∼11.2% vs 4.6%, HG vs NG; 11.2% vs 4.8%, HG vs Cme). (c) Necrosis (ethidium [ET] homodimer lll-positive cells ∼0.73% vs 0.45%, HG vs NG; 0.73% vs 0.25% HG vs Cme). (a–c) LPS was used as a positive control. (d) The histogram shows the representative increase of fluorescence intensity (by DCF) after exposure to HG or H₂O₂ control compared with NG or ASC-Cme and NAC treatment. (e) HG induces ROS in BRECs. Total cellular ROS production was measured by DCF. ASC-Cme suppressed ROS production in the presence of HG, compared with HG alone. The ROS inhibitor NAC and H₂O₂ were used as negative and positive controls, respectively. (f) NF-κB activation by conditioned BREC medium in THP1-XBlue-MD2-CD14 cells, and mediated by ASC-Cme. NF-κB activation was significantly higher in cells treated with BREC conditioned medium under HG conditions (BREC under HG), compared with unstimulated control. This response was almost absent when cells were treated with ASC-Cme alongside BREC conditioned medium under HG conditions (BREC under Cme). LPS was used as a positive control for THP1-XBlue-MD2-CD14 cells and induced NF-κB activation. RPMI-1640 medium, NG-DMEM and HG-DMEM were used as controls, which had no effect on activation of THP1-XBlue-MD2-CD14 cells. Absorbance values were plotted to express NF-κB activation with arbitrary units. *p < 0.05, **p < 0.01, ***p < 0.001 vs NG control; †p < 0.05, ††p < 0.01, †††p < 0.001 vs HG. Values are mean ± SEM (n = 4 in a–c, f and n = 5 in e). Cme, ASC-Cme

Fig. 6

ASC-Cme downmodulates the main inflammatory genes in HG-challenged BRECs, compared with NG-treated controls. (a–r) HG upregulated the expression of the main genes related to inflammation (excluding KLF4, n). The upregulation of TNF, IL1B, IL1A, IL6, CXCL8, VCAM1, SELE, ICAM1, VEGFA, VEGFB, PDGFB, CCL2, PTGS2, and NOS3 was significantly modulated (reduced) by ASC-Cme. ASC-Cme upregulated the gene expression of ANGPT1, KLF4 and NOS2. Values are mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs NG control; †p < 0.05, ††p < 0.01 vs HG

Fig. 7

(a) THP-1 cell adhesion to BRECs exposed to HG, with and without ASC-Cme. The intensity of fluorescence of adherent THP-1 cells (mean ± SEM, n = 3, fold change of NG-treated control) in resting or activated BRECs was measured. A significant decrease in adhered THP-1 cells occurred after treatment of HG-challenged BRECs with ASC-Cme, compared with HG treatment alone. (b) Scratch wound healing assay to study interaction in the BREC monolayer under NG, HG and ASC-Cme conditions. The percentage of covered area between the wound edges was analysed. The percentage signifies the remaining gap size 30 h after making the scratches, compared with the initial gap size. The gap width decreased in a similar pattern in all three groups (NG, closed circles; HG, open circles; ASC-Cme, open diamond) and was not significantly delayed in HG conditions. Values are mean ± SEM. **p < 0.01, ***p < 0.001 vs NG control; †p < 0.05 vs HG

Fig. 8

Therapeutic actions of ASCs in the normalisation of HG-challenged BRECs: a roadmap to the treatment of diabetic retinopathy. (a) BRECS under HG conditions and (b) with the addition of ASC-Cme. This model was corroborated in HG-activated BRECs by the suppression of ROS production by ASC-Cme, together with a reduction in monocyte adhesion and improved cell survival. ASCs preconditioned in HG can rescue dysfunctional retinal endothelium through suppression of the inflammatory and proangiogenic genes induced by glucose-induced oxidative stress, and can stabilise their vascular networks via pericytic function. EC, endothelial cell; ICAM1, intercellular adhesion molecule 1; PS, phosphatidylserine; SELE, selectin E; VCAM1, vascular cell adhesion molecule