Hypothesis and Rationale for this Study
The microvascular is of great significance in the body by acting as a barrier and hence selectively allowing permeability of solutes and fluids. Permeability of the endothelium is actively regulated by the junctions located between the endothelial cells with the junctions being adhesive in nature. Numerous studies indicate that in order to maintain stability of the junctions, a cortical actin belt plays a significant role. Contrary to this statement however, generation of centripetal tension within the junctions is attributable to actin stress fibers. It is this tension that is directly linked to weakening of the junctions. The larger part of this theory however bases on such studies where there is treatment of endothelial cells with known mediators of inflammation all in attempt to upsurge endothelial permeability. Thereafter, the cells are fixed and F-actin labeled for microscopic observation.
Studies of the various mechanisms that are the determinants of endothelial barrier integrity can be alternatively conducted using live-cell imaging which altogether facilitates incorporation of actin cytoskeleton which is dynamic in nature. This method is however advantageous as it gives room for assessment of the implications on actin structures found in endothelial cells with such implications arising from various inflammatory stimuli. Assessment is to be conducted on before and after treatment and on the same set of living cells. This study was therefore conducted to ascertain the above and an experiment was carried out for the same purpose.
- Key Steps of the Procedure Used in this Experiment Behind These Steps.
When conducting the experiment, the procedure adopted involved several steps which are; transfecting the Human Umbilical Vein Endothelial Cells (HUVEC) using GFP-actin, setting up of the stage heater and the live-cell imaging chamber. Each of the steps mentioned have been discussed in details henceforth.
2.1 Transfection of HUVEC using GFP-actin
There is a wide variety of methods that adoptable in the transfection of HUVEC. In this experiment however, we adopted the Nucleofector system (Gallo & Lanier, 2010). This system however calls for speedy working as an intervention for improving viability of the cells and transfection efficiency. The requirement for each transfection is 5 x 105 HUVEC that is to be seeded on two coverslips with a corning number, 1, 22 x 50 mm. The Nucleofector will then effect combination of chemical reagents and electroporation so as to transfect plasmid DNA. Typically, the efficiency of expression achieved will be >50%.
Sterilization of glass coverslips is done in a biological safety hood by use of ethanol with a concentration of 70%. They are then picked using sterile forceps to avoid contamination and then air-dried. Once they are dry, each coverslip is placed in individual 10 cm cover plate. A 300 μL bead of warm gelatin solution is pipetted at the center of the cover slip and allowed to stand for approximately five minutes before being aspirated. One 1.5 ml microfuge tube per transfection is prepared and placed in an incubator containing 5% CO2 and a temperature of 37°C. HUVEC is then detached using 0.25% trypsin EDTA and collected into 15ml conical tube. The volume of the cell suspension is however adjusted so as to obtain a pellet containing 5 x 105 cells multiplied by the total number of transfections that is to be performed. After centrifuging, the supernatant is aspirated to eject as much media as possible.
The pellet is then re-suspended using the basic Nucleofector solution that could be from either the Primary Endothelial Cell Nucleofector cell or the HUVEC. This step however requires quick action as the mentioned solutions are highly toxic. GFP-β-actin vector plasmid is then added to the samples. Per transfection, 0.2-2 μg of GFP-actin plasmid DNA per 5 x 105 cells is added. 100 μL of the cell suspension is added into a Nucleofector Cuvette. It is then covered and severally as a way of ensuring that the suspension has gotten to the bottom of the Cuvette. The cuvette is placed in a slot designed for it in the Nucleofector II device. Program A-034 is then run for HUVEC.
2.2 Setting up of Stage Heater and Live-Cell Imaging Chamber
Used in this experiment is an RC22 (Warner Instruments open diamond bath) (Goldman & Spector, 2005) that is placed in a PH-1 stage heater (Conn, 2012). The feeding system is of gravity flow. Enough medium is aliquoted to be used throughout the experiment. The most appropriate amount for this experiment is 50ml of albumin physiological salt solution that is to be added for every hour that the experiment lasts. APSS is added to the gravity flow system that is built using an intravenous that is connected to the Warner instruments. The flow rate applied is approximately 40ml per hour. To the diamond bath, grease is applied on the outer edge of the underside using an applicator that is tipped with cotton.
From the incubator, one cell covered coverslip is removed and the coverslip lifted gently using forceps. The backside is gently touched using a Kim wipe so that any excess medium can be soaked up while at the same time keeping the side that is cell covered wet. The diamond bath is then placed over the coverslip with the cells face up to form a chamber. The chamber is quickly placed into the stage heater and the clamps hand-tightened over the chamber. It is however of much importance to conduct this move quickly. When done, pipette 1ml of the medium when done in a bid to avoid drying out of the cells.
- Why Endothelial cells for this study?
The whole study was aimed at treating endothelial cells with known inflammatory mediators to increase endothelial permeability all in attempt to understand the impact of the mentioned mediators on endothelial barrier function. The cells were however chosen as they are the most susceptible to various inflammatory mediators (Jaffe, 2012). They were therefore the most appropriate and no other cells could have substituted them.
- Role of the Actin Cytoskeleton in the Endothelium
Maintenance of Endothelial integrity carried out by intercellular junctions located in between endothelial cells (Shirao & Sekino, 2017). These junctions maintain solute movements from the bloodstream into the tissues and vice-versa. They also facilitate the flow of white blood cells into the tissue and more so into locations where inflammation or infection has occurred. During the initial formation of the junctions, there is perpendicular organisation of actin filaments to the membrane by accumulation of actin nucleating complexes locally. Into the later stages of junction maturation, actin fibers develop parallel to the endothelium membrane leading to formation of peripheral actin rim (Flamme & Kowalczyk, 2008).
- Advantages and disadvantages of using GFP-ß-actin
The major advantage is that green fluorescent proteins are not toxic to cells (Chalfie & Kain, 2005). GFP Beta conjugated actin therefore bears the same advantage. Additionally, their detection requires no permeabilization or fixation of cells. GFP protein have a barrel- like structure and are tight and thus ensures that its fluorescence capacity is not affected by any kind of attachment.
Negatively noting, it is impossible to control the amplification of the GFP signal (Sullivan, 2007). This results to possible prevention of detection of low levels of expression. Folding of GFP into fluorescent and active form again tends to be slow.
- kymograph analysis
A kymograph analysis involves representation of moving structures’ dynamics in two dimensional figures (Michael, 2012). They give room for reading out directly the direction, intensity and speed of the structures being analyzed. It was therefore used in this study to aid in analysis of movement of actin fiber over time. It is the only way that detailed analysis of actin cytoskeleton responding to stimuli of an inflammatory mediator could be established.
- Function of Thrombin
Research have proven that PAR1, PAR2 and PAR3 receptors are expressed in Human Umbilical Vein Endothelial Cells (Pollock & Highsmith, 2013). These receptors are in a capacity of being activated by trypsin and thrombin. Thrombin was therefore used in this experiment to activate the various receptors found in HUVEC to effect the effectiveness of the inflammatory stimuli. Before treatment with thrombin, formation and regression of local lamellipodia was observed along the endothelial cells edges. After treatment with thrombin, there was interruption of formation of lamellipodia and turnover.
- Role of actin Stress Fibers
Located in non-muscle cells are actin bundles which are contractile in nature known as stress fibers (Jeanteur, 2012). They play an important role in formation prior to maintenance of adhesion which could be either cell-ECM (Extracellular Matrix) or cell-cell (Tanishita & Yamamoto, 2016). They are therefore actively involved in formation of tight junctions, focal adhesions and adherens junctions. There are also three types of actin stress fibers that are involved during migration of cell following certain chemical or mechanical stimuli. They are, transverse arcs, dorsal and ventral stress fibers. Actin stress fibers also play avital role in Mechanotransduction and Morphogenesis.
- Most Important Finding of this Study
The most significant finding was establishment of the origin of stress fibers in HUVEC. Through imaging of live cells, it was established that periphery of the cell is the origin of most stress fibers which resembled transverse arc fibers commonly found in migrating cell.
It goes without saying that this approach is of great significance to other researchers worldwide. It is through this method that contribution of actin cytoskeletal dynamics to a wide range of endothelial cell activities have been assessed in details. Such activities include, maintenance of barrier function, junctional maturation, intercellular junctions’ formation, migration and mitosis. This method is also able to bring into the limelight the behavior of endothelial actin cytoskeleton prior to treatment with thrombin and after the same. In conclusion, understanding of structural mechanisms and signals that play a vital role in barrier integrity can be deeply understood through live cell imaging of endothelial cells.
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