Type I Diabetes Delays Perfusion and Engraftment of 3D Constructs by Impinging on Angiogenesis; Which can be Rescued by Hepatocyte Growth Factor Supplementation
The biggest bottleneck for cell-based regenerative therapy is the lack of a functional vasculature to support the grafts. This problem is exacerbated in diabetic patients, where vessel growth is inhibited. To address this issue, we aim to identify the causes of poor vascularization in 3D engineered tissues in diabetes and to reverse its negative effects.
We used 3D vascularized constructs composed of microvessel fragments containing all cells present in the microcirculation, embedded in collagen type I hydrogels. Constructs were either cultured in vitro or implanted subcutaneously in non-diabetic or in a type I diabetic (streptozotocin-injected) mouse model. We used qPCR, ELISA, immunostaining, FACs and co-culture assays to characterize the effect of diabetes in engineered constructs.
We demonstrated in 3D vascularized constructs that perivascular cells secrete hepatocyte growth factor (HGF), driving microvessel sprouting. Blockage of HGF or HGF receptor signaling in 3D constructs prevented vessel sprouting. Moreover, HGF expression in 3D constructs in vivo is downregulated in diabetes; while no differences were found in HGF receptor, VEGF or VEGF receptor expression. Low HGF expression in diabetes delayed the inosculation of graft and host vessels, decreasing blood perfusion and preventing tissue engraftment. Supplementation of HGF in 3D constructs, restored vessel sprouting in a diabetic milieu.
We show for the first time that diabetes affects HGF secretion in microvessels, which in turn prevents the engraftment of engineered tissues. Exogenous supplementation of HGF, restores angiogenic growth in 3D constructs showing promise for application in cell-based regenerative therapies.
KeywordsEndothelial cell Tissue engineering Microvessel Regenerative medicine Revascularization Angiogenesis Hepatocyte growth factor Blood perfusion Diabetes Anastomosis Inosculation
This work was supported by grants from the Canadian Institutes of Health Research (CIHR), Institute of Circulatory and Respiratory Health (137352 and PJT153160) and the Heart and Stroke Foundation of Canada (G-14-0006265) to S.S.N; NIH Grant (EB007556) to J.B.H. A Discovery grant from the Natural Sciences and Engineering Research Council (RGPIN 06621-2017) and an Early Researcher Award from the Ministry of Research, Innovation and Science (ER17-13-149) to S.S.N. supported R.H.
SSN designed the experiments, coordinated the project and contributed to the writing of the manuscript. JBH supported HGF blockage assays. WA designed and performed experiment and contributed to manuscript writing. RH contributed to performing experiments and writing manuscript and YA contributed to performing experiments.
Conflict of interest
J.B.H. is an inventor on a patent regarding the use of adipose-derived microvessels and has equity interest with Advanced Solutions Life sciences, which is commercializing isolated microvessel technology. A version of this technology was used as an experimental model in this manuscript. This equity was obtained after the work for this manuscript was completed. W.A., R.H., Y.A. and S.S.N. declare no conflict of interest.
All animal studies were carried out in accordance with Institutional guidelines and approved by the Animal Care Committee at the University Health Network (ID 2420 and 2427). No human subjects were used in this study.
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