Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy.

Heiner Kremer,Elena Beltramo,Martin C Harmsen,Harald Klueter,Stefanie Uhlig,Agnese Fiori,Hans-Peter Hammes,Carsten Sticht,Julian Gebauer,Lothar Steeb,Karen Bieback,Susanne Elvers-Hornung

doi:10.3389/fcell.2020.00387

Abstract

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features.

Highlights

Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus with a risk of causing blindness (Stitt et al, 2016; Hammes, 2018)
Others and we have shown that Adipose-derived stromal cells (ASC) resist hyperglycemic stress and restore the angiogenic properties of endothelial cells (EC) which were suppressed by hyperglycemia (Rajashekhar et al, 2014; Hajmousa et al, 2018; Fiori et al, 2020)
To compare the basic characteristics of ASC and human retinal microvascular pericytes (HRMVPC), cell morphology, growth curves, and expression of typical mesenchymal stromal cells (MSC) markers were assessed. Both cell types had a typical fibroblastoid morphology, but both immortalized Bmi-HRMVPC, used first for comparison, and HRMVPC, used later to account for potential donor differences, appeared more elongated and slender with an apparently higher light diffraction at the cell borders (Figure 1A). Both HRMVPC detached within seconds after adding trypsin, in contrast to ASC that needed approximately 5 min to detach

Summary

Introduction

Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus with a risk of causing blindness (Stitt et al, 2016; Hammes, 2018). The early stage of the disease, known as non-proliferative DR, is diagnosed by microvascular abnormalities. These are the consequence of a sequela of detrimental events, which involve the whole neurovascular retina and include pericyte dropout, basal lamina thickening, and endothelial, neuronal, and glial dysfunction (Stitt et al, 2016; Fiori et al, 2018; Hammes, 2018). To avoid the associated side effects, cell-based therapies have been suggested (Stitt et al, 2011)

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