A simplified aortic ring assay: A useful ex vivo method to assess biochemical and functional parameters of angiogenesis

Abstract

We present a simplified method for conducting aortic ring assays which yields robust sprouting and high reproducibility targeted towards matrix biologists studying angiogenesis and extracellular matrix signaling. Main adjustments from previously established protocols include embedding aortic rings between two layers of 3D type I collagen matrix and supplementing with vascular endothelial media. We also introduce a concise and effective staining protocol for obtaining high-resolution images of intracellular and extracellular matrix proteins along with a more accurate protocol to quantify angiogenesis. Importantly, we present a novel method to perform biochemical analyses of vessel sprouting without contamination from the aortic ring itself. Overall, our refined method enables detection of low abundance and phosphorylated proteins and provides a straightforward ex vivo angiogenic assay that can be easily reproduced by those in the matrix biology field.

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering

“…The various experimental methods for stimulating and quantifying angiogenesis have been reviewed by Tahergorabi and Khazaei, 59 Hasan et al 60 and Norrby; 61 however, the CAM has not superseded these methods in some areas of research, as the ex vivo aortic ring assay 62 and hind limb ischaemia model 63 are still used, depending on the question being asked and the ultimate research goal. The hindlimb ischaemia model is focused on finding therapies for peripheral vascular disease using methods such as growth factors and stem cells, and would not be a suitable method for scaffold assessment as the model does not imitate the clinical scenario.…”

Endorepellin evokes an angiostatic stress signaling cascade in endothelial cells

“…Thoracic aortae from 6-7-week-old wildtype C57/BL6 mice (Jackson Laboratories) were surgically isolated, cleaned off the fibro-adipose tissue, and sectioned serially into 0.5-1 mm rings. Rings were sandwiched between two 3-D collagen type I gel layers (1 mg/ml) in 48-well plate and incubated in EGM-2 media (Lonza) at 37°C, as described elsewhere (48,79). Following the initial sprouting, i.e., three days after embedding, the rings were given either acute or chronic treatment.…”

“…Sprouting of the rings was measured as the radial distance using the low-magnification (5X) phasecontrast images (79). We subtracted the background and highlighted the sprouts using the threshold function.…”

“…During the immunofluorescence analyses the rings were labeled with IB4, CD31, P-PERK and GADD45 and Z-stack images of the rings were captured with Zeiss LSM780 NLO confocal/multiphoton microscope and the images were analyzed using ImageJ software. Two methods of analyses were used: fluorescence of the traced-out sprouts (79) and intensity profile of the sprouts, both using ImageJ. Briefly, fluorescence intensity was uniformly normalized across all conditions using the threshold function, and regions of interest outlining the sprouts were selected using the polygon drawing tool.…”

Collagen hydrogels loaded with fibroblast growth factor-2 as a bridge to repair brain vessels in organotypic brain slices

“…Therefore, it is beneficial to come up with alternative in vitro extracellular matrices with a precisely defined composition to clearly identify the effects of experimental conditions without interference of matrix variability. Type I collagen was used as an extracellular matrix in ex vivo models of aortic ring organ cultures for evaluating endothelial cell sprouting and angiogenesis (Kapoor et al 2020 ). Collagen is a natural extracellular matrix protein with remarkable biocompatibility and no toxicity (for a review see: Ucar and Humpel 2018 ).…”