Choroid Sprouting Assay: An Ex Vivo Model of Microvascular Angiogenesis

Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth in vivo. Vascular endothelial cells cultured in vitro are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible ex vivo angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research

CRISPR-based genome editing in primary human pancreatic islet cells

Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the KATP channel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.We gratefully acknowledge organ donors and their families, Canadian organ procurement organizations, particularly the Human Organ Procurement and Exchange (HOPE) program and the Trillium Gift of Life Network, and islet procurement through the Alberta Diabetes Institute Islet Core, Integrated Islet Distribution Program (U.S. NIH UC4 DK098085). R.J.B. was supported by a postdoctoral fellowship from JDRF (3-PDF-2018-584-A-N) and is on leave from the Animal Biotechnology Laboratory, Facultad de Agronomía, Universidad de Buenos Aires/INPA CONICET, CABA, Argentina. Work in the CRG and ICL was funded by the Wellcome Trust (WT101033), Medical Research Council (MR/L02036X/1), European Research Council Advanced Grant (789055), Ministerio de Ciencia e Innovación (RTI2018-095666-B-I00) and Marató TV3 #201611. Work in the University of Alberta was supported by a Foundation Grant from the Canadian Institutes of Health Research (CIHR: 148451, MacDonald). Work in the Kim lab was supported by NIH awards (R01 DK107507; R01 DK108817; U01 DK123743 to S.K.K.), and JDRF Northern California Center of Excellence (to S.K.K. and M. Hebrok). Work here was also supported by NIH grant P30 DK116074 (S.K.K.

Choroid Sprouting Assay: An <i>Ex Vivo</i> Model of Microvascular Angiogenesis

<div><p>Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth <i>in vivo</i>. Vascular endothelial cells cultured <i>in vitro</i> are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible <i>ex vivo</i> angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research.</p></div

Suggested Conditions for Choroid Microvascular Sprouting Assay

<p>Suggested Conditions for Choroid Microvascular Sprouting Assay</p

Choroid Sprouting Standardization and Optimization

<p>Choroid Sprouting Standardization and Optimization</p

Mouse and rat central and peripheral choroid sprouting: intra- and inter-animal variability.

<p>(a) Five replicates from 5 animals were compared for the intra-animal (from the same eye of the same animal) variability and inter-animal variability of choroid sprouting. Scale bar: 500 µm. (b) The sprouting from the peripheral mouse choroid is more consistent than central mouse choroid (n = 5) (c) Flow cytometry analysis of choroid sprouting cell populations. About 60% of the cell population from the choroid sprouts stained positive for both CD-31 and isolectin, indicating ECs/macrophages. 36% of the cell population is isolectin-positive but did not stain for CD-31. (d) The extending growth cone resembles vascular tube formation <i>in vivo</i> and stains positively with isolectin GS (arrow head) surrounded by chondroitin sulfate proteoglycan neuron-glial antigen 2 (NG2) positive pericytes (arrow). Scale bar: 10 µm. (e) Real time-PCR analysis of choroid sprouts and aortic ring sprouts indicates that the expression of endothelial marker VE-cadherin is not significantly different between the two assays when normalized to CD31 expression (n = 12) p = 0.97; unpaired T-test. (f) The expression of NG2 by real time PCR is higher in choroid sprouts compared to aortic ring sprouts (n = 8) *** p<0.0001; unpaired t-test. (g) The rate of choroid sprouting is not correlated to the size of the choroid tissue embedded within the rage of 0.2 mm² to 0.8 mm².</p

RPE cells promote choroidal endothelial sprouting in endothelial-selective media.

<p>Three different cell culture media (CSC, EGM-2 and DMEM) were compared for standardizing choroidal tissue response. (A) Complete CSC medium with growth factor Boost promotes rapid sprouting. An intact RPE layer on choroid further accelerates sprouting (n = 5–12) p<0.0001 compared to choroid without RPE. (B) RPE cells also potentiate choroid sprouting in EGM-2 medium (n = 6 for each time point) p<0.0001, (C) However, in DMEM there is no difference in sprouting rate (n = 6–18, p = 0.1) regardless of the presence of RPE. The sprouting in DMEM medium contained RPE-like contamination (arrows) and the sprouts did not form growth cones as shown in CSC or EGM-2 medium. All comparisons are 2-way ANOVA with Bonferroni correction. Scale bar: 500 µm.</p

The age and strain of the animal affects the rate of choroidal sprouting in explant culture.

<p>(A&B) Choroid with or without RPE from P8 animals sprouts significantly faster than that from P240 aging animals (n = 6–18, p<0.0001 with RPE; n = 6–12, p = 0.0002 without RPE). (C) The choroid explants from 129S6/SvEvTac mice (n = 10) grow significantly faster than choroid explants from C57BL/6J mice (n = 12) at day 7 p = 0.0017. All comparisons are 2-way ANOVA with Bonferroni corrections.</p