3D ex vivo analysis and culture of PDR fibrovascular tissues reveal novel patterns in proliferative diabetic retinopathy

Session Details

Session Title: Free Paper Session 24: Vascular Diseases & Diabetic Retinopathy VII

Session Date/Time: Sunday 10/09/2017 | 10:00-11:30

Paper Time: 10:12

Venue: Room 117

First Author: : S.Loukovaara FINLAND

Co Author(s): :    E. Gucciardo   A. Korhonen   B. Martins   H. Vihinen   E. Jokitalo   K. Lehti              

Abstract Details

Purpose:

Proliferative diabetic retinopathy (PDR) is a major microvascular complication and a vision-threatening cause of blindness in diabetic individuals. To date, vitreous samples and histological sections have provided information of the involved pathogenic factors. Processes related to inflammation, stem cell recruitment and abnormal vascular differentiation bear a promise for improved therapy, but remain mechanistically unclear due to lack of diabetic mouse models with similar proliferative disease.

Setting:

We developed an ex vivo model for the human disease by using surgically excised, patient-derived PDR tissue biopsies.

Methods:

The diabetic eyes underwent transconjunctival microincision vitreoretinal surgery by 23- or 20-gauge three-port pars plana vitrectomy using the CONSTELLATION Vision System (Alcon Laboratories, Inc., Fort Worth, TX, USA). For ex vivo culture, neovascular tufts were obtained from 39 vitrectomized eyes using segmentation and delamination techniques and intraocular end-gripping microforceps (MaxGrip Alcon® Laboratories, Inc., Fort Worth, TX, USA). Immunohistochemistry: Rabbit polyclonal antibodies against NG2 (Millipore), Ki67 (Leica Microsystems), GFAP (Dako), Prox-1 (ReliaTech), rabbit monoclonal antibodies against Cleaved Caspase-3 (5A1E, Cell Signalling), ERG (EP111, Dako and CM421C, Biocare Medicals for IHC-P), and mouse monoclonal antibodies against α-SMA (1A4, Sigma-Aldrich), CD31 (JC70A, Dako), CD34 (QBEND10, Dako), CD117 (K45, Thermo Scientific), CD45 (2B11 + PD7/26, Dako) and CD68 (ImmunoWay), as well as goat polyclonal antibodies against Prox-1 (R&D Systems) were used. Vitreous analysis: For vascular endothelial growth factor-A (VEGFA), fibroblast growth factor-2 (FGF2, bFGF), vascular endothelial growth factor-C (VEGFC) and transforming growth factor-β (TGFβ) measurements, commercially available Quantikine ELISA kits (R&D Systems) were used according to manufacturer’s instructions. Ex vivo culture, 3D spheroid assay, whole-mount immunofluorescence and electron microscopy were performed.

Results:

Intra and inter-patient variability in the irregular neovasculature ranged from dense tortuous endothelial structures to plain pericyte-deficient endothelial capillaries with gaps in their thin walls. Moreover, spatially-confined apoptosis and proliferation were coupled with previously unappreciated three-dimensional patterns of sphere-like vascular islets as well as vascular and perivascular progenitor cells with varying stem cell markers. Neovascular explants retained these unique features and the heterogenous multicellularity upon ex vivo culture. These tissue properties and outgrowth into surrounding matrix were sensitive to the known angiogenic and fibrotic factors VEGFA, bFGF, and TGFβ as well as to VEGFC also found at variable levels in corresponding vitreous. Interestingly, besides CD31+ endothelial cells and NG2+ pericytes, populations of Prox-1+ cells contributed to capillary-like sprouting.

Conclusions:

Our results warrant future investigation of mechanisms and treatment responses in the multicellular PDR microenvironment, like in the described model, whereby the interrelated processes of pathological angiogenesis, fibrosis, and inflammation are reproduced ex vivo. 

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