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

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 α1-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.

Dexamethasone Preconditioning in Cardiac Procedures Reduces Decreased Antithrombin Activity and Is Associated to Beneficial Outcomes: Role of Endothelium

“…To confirm the pro-angiogenic activity of AT in ECs from large vessels such as aorta, another assay was performed using rat aortic rings cultured in Matrigel ® ( Vicente et al, 2016 ) and the results obtained were similar to those previously shown with hAECs. Incubation with AT potentiates the angiogenic growth by increasing the length of the capillary-like network formed.…”

“…Male Wistar rats (270–300 g) bred in our animal facility were anesthetized with isoflurane and killed by decapitation. Segments of rat thoracic aorta of were aseptically removed and cleaned from adipose tissue as previously described ( Vicente et al, 2016 ). Aortic rings (1 mm) were sectioned in a Petri dish on ice and rinsed with five consecutive washes of Krebs solution (NaCl 118 mM, KCl 4.7 mM, CaCl 2 1.8 mM, KH 2 PO 4 1.2 mM, NaHCO 3 25.0 mM, glucose 11.0 mM).…”

“…The cultures were photographed daily from day 3 to day 6 using an inverted microscope Leica DM IL LED coupled to a Leica Digital Camera, at appropriate magnification (25×). The length of the longest vessel sprouting from the rat aortic rings was measured as the distance in the x- and y -axis, (taking the outer surface of the ring as the starting point) using Leica Microsystems LAS Software V3.7.0 (Heerbrugg, Switzerland) as has been described elsewhere ( Vicente et al, 2016 ).…”

Sex-Dependent End-of-Life Mental and Vascular Scenarios for Compensatory Mechanisms in Mice with Normal and AD-Neurodegenerative Aging

“…Segments of MCA and the descending thoracic aorta were dissected in ice-cold physiological salt solution (PSS; composition in mM: NaCl 112.0; KCl 4.7; CaCl 2 2.5; KH 2 PO 4 1.1; MgSO 4 1.2; NaHCO 3 25.0 and glucose 11.1) supplemented with amphotericin B (15 mg/l) (Biowhittaker ® , Lonza, Basel, Switzer-land) and gentamicine (30mg/l) (Genta-gobens ® , Laboratorios Normon SA, Tres Cantos, Madrid, Spain) and gassed with 95% O 2 and 5% CO 2 . Afterwards, vessels were immersed into Matrigel ® (50 µL; BD Bioscience, San Jose, CA, USA) following the protocol previously described [34,35]. Angiogenic growth was measured using an inverted microscope equipped with a camera (10× objective; TE2000 Nikon Eclipse-S, Nikon España, Madrid, Spain) at day 4, 5, 6, and 6 after seeding.…”

“…Angiogenic growth was measured using an inverted microscope equipped with a camera (10× objective; TE2000 Nikon Eclipse-S, Nikon España, Madrid, Spain) at day 4, 5, 6, and 6 after seeding. The longest vessel sprouting from the artery's outer surfacy (starting point) determined the angiogenic growth [34,35], and at least three fields per arterial segment were measured.…”