26 Isolation and Culture of Endothelial Cells from Large Vessels

The blocking and antibody solutions are pipetted directly on the tissue section and covered with a small coverslip made by cutting a section of Parafilm. The coverslips are removed from the slide by immersing the slide in PBST in a vertical staining jar. Slides are washed in a Wheaton staining dish on a rocking platform agitator such as a VariMix platform (Thermolyne). 28. The new solution pushes the previous solution down out of the capillary gap.

Acknowledgments

We are grateful to Christian Young for excellent technical assistance and suggestions. The work of the authors was supported by Canadian Institutes of Health Research grants 82790 and 102606, a Canada Research Chair on Hypertension and Vascular Research from Canadian Institutes of Health Research/Government of Canada, and the Canada Fund for Innovation (all to E.L.S.). K.K.M is supported by CIHR grant 115000 and is a Junior 2 Chercheur Boursier of the FRQ-S.

References

1. Schiffrin EL (2008) The flame that lights the fire: oxidative stress, inflammation, and renal damage in angiotensin II-induced hypertension.

Hypertension 52:205–206 2. Schiffrin EL (2010) T lymphocytes: a role in hypertension? Curr Opin Nephrol Hypertens 19:181–186 3. Schiffrin EL (2013) The immune system: role in hypertension. Can J Cardiol 29(5):543–548 4. Barhoumi T, Kasal DA, Li MW, Shbat L,

Laurant P, Neves MF, Paradis P, Schiffrin

EL (2011) T Regulatory lymphocytes prevent angiotensin II-induced hypertension and vascular injury. Hypertension 57:469–476 5. De Ciuceis C, Amiri F, Brassard P, Endemann

DH, Touyz RM, Schiffrin EL (2005) Reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin II-infused macrophage colonystimulating factor-deficient mice: evidence for a role in inflammation in angiotensininduced vascular injury. Arterioscler Thromb

Vasc Biol 25:2106–2113 6. Guzik TJ, Hoch NE, Brown KA, McCann LA,

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Zdanovich E, Rehman A, Neves MF, Laurant

P, Paradis P, Schiffrin EL (2012) T regulatory lymphocytes prevent aldosterone-induced vascular injury. Hypertension 59:324–330 8. Ko EA, Amiri F, Pandey NR, Javeshghani D,

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EL (2009) Aldosterone induces arterial stiffness in absence of oxidative stress and endothelial dysfunction. J Hypertens 27:2192–2200 11. Matrougui K, Zakaria AE, Kassan M, Choi S,

Nair D, Gonzalez-Villalobos RA, Chentoufi

AA, Kadowitz P, Belmadani S, Partyka M (2011) Natural regulatory T cells control coronary arteriolar endothelial dysfunction in hypertensive mice. Am J Pathol 178:434–441 12. Peters CE, Woodside SM, Eaves AC (2005)

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Chapter 26

Isolation and Culture of Endothelial Cells from Large Vessels

Augusto C. Montezano, Karla B. Neves, Rheure A.M. Lopes, and Francisco Rios

Abstract

The endothelium, which is at the interface between circulating blood and the vascular wall, comprises a simple squamous layer of cells that lines the inner surface of all blood vessels. Endothelial cells are highly metabolically active and play an important role in many physiological functions, including control of vasomotor tone, blood cell trafficking, vascular permeability, and maintenance of vascular integrity (Mensah, Vascul Pharmacol 46(5):310–314, 2007; Yetik-Anacak and Catravas, Vascul Pharmacol 45(5):268–276, 2006). Endothelial cells are characteristically ‘quiescent’ in that they do not actively proliferate, with the average lifespan of an endothelial cell being >1 year. The endothelium is very sensitive to mechanical stimuli (stretch, shear stress, pressure), humoral agents (angiotensin II (Ang II), endothelin-1 (ET-1), aldosterone, bradykinin, thromoxane) and chemical factors (glucose, reactive oxygen species (ROS)) and responds by releasing endothelial-derived mediators, such as nitric oxide (NO), prostacyclin (PGI2), platelet-activating factor (PAF), C-type atrial natriuretic peptide (ANP), and ET-1 to regulate vascular tone, prevent thrombosis and inflammation, and maintain structural integrity. Primary culture of endothelial cells is an important tool in dissecting the role of the endothelium in many physiological or pathological responses. This chapter describes the explant method for culture of endothelial cells from large vessels. Cells derived by the protocol described here can be used for cell biology and molecular biology studies in hypertension and other cardiovascular diseases where endothelial function may be impaired.

Key words Endothelial cells, Primary culture, Large vessels

1 Introduction

Endothelial cells (ECs) are the inner layer of cells of all blood vessels becoming the unique interface between the blood and the vessel wall. ECs form a continuous monolayer which plays a crucial role in the immune response, coagulation, growth regulation, production of extracellular matrix components, and it is an essential modulator of blood vessel tone [1–5].

The endothelium plays a key role in vascular homeostasis [6]. ECs produce and release several vasoactive molecules that modulate vascular smooth muscle cells tone. Furchgott and Zawadzki

Rhian M. Touyz and Ernesto L. Schiffrin (eds.), Hypertension: Methods and Protocols, Methods in Molecular Biology, vol. 1527, DOI 10.1007/978-1-4939-6625-7_26, © Springer Science+Business Media LLC 2017 345

were the first to demonstrate an endothelium-derived relaxing factor, nitric oxide (NO) [7], a molecule converted from l-arginine by of endothelial NO synthase (eNOS) [8]. NO leads to cGMPmediated vasodilatation when diffuses to the vascular smooth muscle cells and activates guanylate cyclase. The endothelium also can sustain vasodilator tone mediating hyperpolarization of vascular smooth muscle cells in a NO-independent manner, which promotes increase in potassium conductance and consequent propagation of depolarization of vascular smooth muscle cells [9]. Alterations in endothelial function strongly regulate pathological processes such as atherosclerosis, thrombosis, inflammation, or vascular wall remodeling [4, 6]. Over recent years, the understanding of the endothelial cell biology has increased with the possibility to grow endothelial cells in vitro [10]. In this chapter we describe methods to isolate endothelial cells by the explant technique. Cells isolated by this protocol can not only be used for molecular biology techniques but also for electrophysiology studies [11].

2 Materials

1. Ham’s F12 nutrient mix (pH 7.2) containing: penicillin/ streptomycin (1×), HEPES (15 mM), l-glutamine (1 mM), sodium bicarbonate (14 mM). 2. Endothelial Cell Medium: Dulbecco’s Modified Eagle Medium (DMEM) (pH 7.2) containing: low d-glucose (5.5 mM), penicillin/streptomycin (1×), HEPES (25 mM), l-glutamine (4 mM), sodium bicarbonate (44 mM), sodium pyruvate (1 mM), fetal bovine serum (10 %). Add to 1 L of DMEM: 10 mL of nonessential amino acids solution (MEM— 10 mM—100×), 1 mL of heparin (10,000 U.S.P. units/mL), 30 mg of endothelial cell growth supplement from bovine neural tissue (ECGS). 3. Matrigel. 4. Dispase (use 0.2 mL/10 cm2—10 U) (see Note 1). 5. 6-well plate. 6. Surgical material: toothed forceps, straight fine forceps, angled fine forceps, small sharp scissors, microdissecting scissors.

3 Methods

1. Prior to the vessel collection, prepare the Matrigel in a 6-well plate. Dilute the Matrigel, following manufacturer’s instructions, in endothelial cell medium and make a layer of Matrigel in each well spreading it evenly.

4 Notes

2. Carefully dissect the vessel from anesthetized and heparinized mice and place it in complete Ham’s F12 nutrient mix. 3. In a petri dish, carefully clean the vessels of periadventitial fat and connective tissue (see Note 2). 4. Open the vessel longitudinally and cut it into squares of 2–3 mm length each (see Note 3). 5. Place each piece of vessel with the intima side facing the

Matrigel layer in each well. 6. Place the 6-well plate with the vessel pieces in a 37 °C incubator for 24 h (see Note 4). 7. After the 24 h, add carefully (through the side of the well), 300–400 μL of endothelial cell medium, avoiding that the

Matrigel dries up. 8. Change medium every second day. Be careful to not remove the vessel pieces from the Matrigel. 9. After 6–8 days, remove the vessel pieces with a pipette. 10. Allow cells to grow on the Matrigel until the 12th day. 11. Remove cells from the Matrigel with dispase (10 U). Remove excess of endothelial cells medium and add dispase. Incubate for 60 min at 37 °C. With a P1000, pipette up and down to remove cells and disperse them from the Matrigel (see Note 5). 12. Add more endothelial cell medium and centrifuge the cell suspension at 300 × g for 5 min. 13. Aspirate the supernatant and remove as much Matrigel as possible. 14. Resuspend the cell pellet in endothelial cell medium (growth medium) and grow the cells as needed (see Notes 6 and 7).

1. Concentration of dispase may differ accordingly to different manufacturers and vessel of choice (It may vary from 100 to 10 U). Trypsin will not work since the endothelial cell medium contains FBS. 2. Do not use pre-digestion protocols to facilitate the cleaning of adventitial and connective tissue. It may harm endothelial cells. 3. Smaller pieces are not stable in the Matrigel. Bigger pieces generate poor extraction of endothelial cells. 4. 24 h is the necessary time for the stabilization of the vessel pieces in the Matrigel layer. 5. This is a crucial step. If endothelial cells are not viable after this step, one should decrease the dispase concentration or incubation