Methodological Approach to Use Fresh and Cryopreserved Vessels as Tools to Analyze Pharmacological Modulation of the Angiogenic Growth

Autores de IIS La Fe

• Diana Vicente Miralles

  Autor

• Vanessa Segura Caballer

  Autor

• Vicente Mirabet Lis

  Autor

• Pilar D'ocon Navaza

  Autor

Participantes ajenos a IIS La Fe

• Hernández B
• Pascual D
• Fornaciari G
• Monto F
• Montesinos MC

Grupos

• Investigación Clínica y Traslacional en Cáncer

  

  Leader

  José Gómez Codina

• Regeneración y Trasplante Cardíaco

  

  Leader

  Pilar Sepúlveda Sanchis

Abstract

The sprouting of new vessels is greatly influenced by the procedure chosen. We sought to optimize the experimental conditions of the angiogenic growth of fresh and cryopreserved vessels cultured in Matrigel with the aim to use this system to analyze the pharmacological modulation of the process. Segments of second-order branches of rat mesenteric resistance arteries, thoracic aorta of rat or mouse, and cryopreserved rat aorta and human femoral arteries were cultured in Matrigel for 7-21 days in different mediums, as well as in the absence of endothelial or adventitia layer. Quantification of the angiogenic growth was performed by either direct measurement of the mean length of the neovessels or by calcein AM staining and determination of fluorescence intensity and area. Fresh and cryopreserved arterial rings incubated in Matrigel exhibited a spontaneous angiogenic response that was strongly accelerated by fetal calf serum. Addition of vascular endothelial growth factor, fibroblast growth factor, endothelial growth factor, or recombinant insulin-like growth factor failed to increase aortic sprouting, unless all were added together. Removal of adventitia, but not the endothelial layer, abrogated the angiogenic response of aortic rings. Determination of the mean neovessel length is an easy and accurate method to quantify the angiogenic growth devoid of confounding factors, such as inclusion of other cellular types surrounding the neovessels. Activity of a alpha(1)-adrenoceptor agonist ( phenylephrine) and its inhibition by a selective antagonist (prazosin) were analyzed to prove the usefulness of the Matrigel system to evaluate the pharmacological modulation of the angiogenic growth.

Keywords

• angiogenesis; cryopreservation; arterial ring assay; human vessels; matrigel