Vascular outgrowth of normal and atherosclerotic aortic grafts in modified fibrin gels : a clinically translatable model

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Collins, Scott Forrest

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The success of regenerative cardiac therapy requires reestablishing a capable blood supply via vasculature. The objective of this study was to develop an optimal scaffold formulation for de novo collateral vessel growth of aortic grafts using modified fibrin clots. This ex vivo vascular outgrowth model can be used to interrogate the complex cell or tissue interactions on the angiogenic front as vessels are formed. Based on formulation constraints, the methods used here may provide a clinically applicable option for guided collateral formation. Once understood, the methods and procedures can be tested and modified as necessary for in vivo, in situ regenerative therapy. Aortic segments from wild-type (C57BL/6J) and apolipoprotein-E deficient (ApoE) atherosclerosis-prone mice were cultured in a 3D environment created by various formulations of PEGylated fibrin. Aortic outgrowth was assessed and the optimal formulation was chosen to test the formation of de novo vascular circuits -- the first step necessary for collateral artery formation. The cultures were examined by conventional and confocal microscopy as well as by optical coherence tomography. Experiments testing the relationship between fibrin PEGylation and aortic vascular outgrowth showed that PEGylating fibrinogen prior to clot formation increased outgrowth over non-PEG control (n=6, p<.05) at lower fibrin concentrations. Lowering fibrin concentration to 10, 5, or 2.5mg/ml resulted in significantly higher outgrowth that was 1.92, 2.04, or 2.20 times that of 20mg/ml PEGylated fibrin gels. When multiple aortic segments are cultured in proximity, microvascular outgrowths visually anastamose suggesting that aorta-aorta conduits can be formed in fibrin based hydrogels. Anastomosing circuits appeared between wild-type aortic segments as well as between wild-type and atherosclerotic prone ApoE knockout segments. Fibrin gels, with or without PEGylation, form scaffolds suitable for regenerative vascular outgrowth ex vivo in normal and atherogenic environments. PEGylating fibrin prior to thrombin-initiated polymerization will allow the incorporation of growth factors or other bioactive components, making this a customizable therapy for guided collateral formation. Additionally, the incorporation of PEG itself does not limit and may actually increase the outgrowth from aortic segments in lower density gels. Finally, PEGylated fibrin gels offer an environment that will promote vascular extensions that visually anastamose, making this a viable model for ex vivo collateral formation.



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