Endothelin receptors and signaling mechanisms science business media

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.

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.

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.

4. Role of the Actin Cytoskeleton in the Endothelium

6. 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.

9. 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.

Conn, M. P. (2012). Laboratory Methods in Cell Biology: Biochemistry and Cell Culture. Academic Press.

Flamme, S. E., & Kowalczyk, A. P. (2008). Cell Junctions: Adhesion, Development and Disease. John Wiley & Sons.

Michael, C. (2012). Imaging and Spectroscopic Analysis of Living Cells: Optical and Spectroscopic Techniques. Academic Press.

Pollock, D. M., & Highsmith, R. F. (2013). Endothelin Receptors and Signaling Mechanisms. Springer Science & Business Media.