Werkbezoek Philadelphia USA

  • Aanvrager: De heer P. Prabhala

Laminin a4 and a5 as regulators of airway inflammation and remodelling in allergic asthma. 7.1.16.134CO (3.2.12.079) Dr. P. Prabhala (Pavan) University of Groningen, Department of Clinical Pharmacy and Pharmacology & Department of Molecular Pharmacology, The Netherlands Airway remodelling, including changes in the extracellular matrix (ECM) and increased airway smooth muscle (ASM) mass and contractility contributes to the pathology of asthma. Laminins form one of the main ECM components of the basement membranes, including those of the endothelium and ASM.

Introduction
Airway remodelling, including changes in the extracellular matrix (ECM) and increased airway smooth muscle (ASM) mass and contractility contributes to the pathology of asthma. Laminins form one of the main ECM components of the basement membranes, including those of the endothelium and ASM. Importantly, one of the main aims of the project (3.2.12.079) was to investigate the role of laminin a4 and a5 in human ASM cell proliferation and in the expression of contractile marker proteins in vitro (aim #4). Previously, we have shown that laminin a4 promotes both a contractile and pro-fibrotic ASM phenotype as evidenced by a reduced sm-a-actin and fibronectin mRNA expression in stable laminin a4-deficient human telomerase reverse transcriptase (hTERT) ASM cell lines1. mRNA expression of sm-MHC and calponin was unchanged. Conversely, laminin a5 deficiency did not significantly affect fibronectin and sm-a-actin mRNA, but increased calponin and sm-MHC mRNA abundance. In addition, laminin a4, but not a5 deficient cells, attenuated TGF-ß1-induced upregulation of fibronectin mRNA and protein expression. Moreover, expression of laminin a5 appears to be reduced in biopsies of asthmatic patients and correlates with lung function (FEV1). Hence, to investigate whether changes in expression of contractile marker proteins are also associated with changes in contractile function of single airway smooth muscle cells, we would like to visit the lab of Dr. Pera to perform wrinkling assay experiments with our laminin deficient cell lines.
Methods
Wrinkling assay
To identify contractile cells, stably transduced hTERT cells (scrambled, laminin a4 shRNA and laminin a5 shRNA cells) will be seeded on deformable silicone substrates (stiffness 5 kPa; Excellness Biotech, Lausanne, Switzerland). Cells will be seeded at a density of 30.000 cells/well onto substrates coated with 10 µg/mL fibronectin isolated from human plasma. Twenty-four hours after seeding, 10 random fields will be photographed using phase contrast microscopy and the percentage of contractile cells inducing wrinkles will be determined using ImageJ software. Protocol according to that described in (1)
Results
Initially the assay did not work and the cells showed no sign of wrinkling. The cells showed no signs of contractile phenotype development, as none of the cells developed wrinkles (deformations in the silicone substrate). No wrinkles were identified by Dr. Pera or myself in three repetitions of the experiment. This result was at odds with what we predicted based on the results from previous publications. We subsequently began the process of trouble shooting the protocol, Dr. Pera and myself discussed the various issues that could affect the assay and we decided to repeat the experiment by changing one variable at a time. We started by reducing the number of cells so that individual cells could be more easily viewed, and the appearance of wrinkles would be more prominent. This improved the assay and we were able to better view individual cells. However, this did not increase the appearance of wrinkles. The next variable we aimed to test was the extracellular matrix (ECM) protein on which the cells were seeded. From our experiments and the previous experiments of Dr. Pera, we decided to opt for fibronectin as our ECM protein on which to seed our cells (as part of the protocol). During our trouble shooting we decided to replace the fibronectin with collagen 1. This unfortunately made no change to the way the cells were seeded and it also made no impact the ability of the cells to generate wrinkles. This was then followed by treating the substrates with UV light for a short time to increase their sensitivity (2). This also appeared to be ineffective in generating wrinkles within the substrate.
At this point we had exhausted the main variables within the protocol and we had to then question whether or not we had the appropriate substrates upon which to seed the cells in order to generate and view the wrinkles. We then decided to manually stimulate the substrate alone, in the absence of cells to see if it was possible to generate wrinkles. Hence, with a 200µL pipette tip a scratch was manually made across the silicone substrate to see if the manual scratch force would cause the substrate to develop wrinkles. To our surprise this deliberate effort to generate wrinkles was also unsuccessful as the substrate just split evenly along the length of the scratch leaving no wrinkles behind. As a result of this negative result we proceeded to contact the manufacturer, ExCellness Biotech to determine whether or not we had received the correct substrate in accordance with the previous publication. The sales representative then alerted us to the fact that the substrates that we ordered were the same as the previous publication except that they had ordered the plates through the company but the plates were generated at the Univeristy of Toronto under the guidance of Professor Boris Hinz. Although we had informed the sales representative at this company about our intentions on how we wanted to use the substrates, he made no mention of this issue when we ordered the plates.
Subsequently, we collaborated directly with Professor Hinz and we attained the correct wrinkling substrates. Just to be certain we ordered the substrates at different stiffnesses as we wanted to be extra careful in proceeding with this experiment. Upon receiving the new substrates we applied our modified protocol from the trouble shooting above and the results are indicated below Figure 1a and b. We used normal untransfected hTERT airway smooth muscle cells. The boxes in Figures 1a and 1b represent the appearance of endogenous wrinkles in the absence of any stimulation. This result is in line with the previous publications (1, 2). Figure 1a is a randomly photographed representative section of the well from a substrate stiffness of 5kPa at 4x magnification. Figure 1b is also a randomly photographed representative section of a well with a substrate stiffness of 5kPa at 10x magnification. We also have images from different stiffnesses (2kPa, 10kPa, 15kPa and 20kPa). The 2kPa well was too soft and the substrate disintegrated from the gentle pressure of pipetting solution upon the well. The rest of the higher stiffnesses did not produce wrinkles as the cells were unable to overcome the higher stiffness to produce pronounced wrinkles as seen at a stiffness of 5kPa.
Discussion
The results were largely negative, but we were able to adequately resolve the problem and there is now a bright future to progress with the study. One of the main aims of the project (3.2.12.079) was to investigate the role of laminin a4 and a5 in human ASM cell proliferation and in the expression of contractile marker proteins in vitro (aim #4). In our previous studies we showed that the roles of laminin a4 and laminin a5 were different, where laminin a4 promotes both a contractile and pro-fibrotic ASM phenotype as evidenced by a reduced sm-a-actin and fibronectin mRNA expression in stable laminin a4-deficient human telomerase reverse transcriptase (hTERT) ASM cell lines. Conversely, laminin a5 deficiency did not significantly affect fibronectin and sm-a-actin mRNA, but increased calponin and sm-MHC mRNA abundance. Hence, we wanted to see if contractile protein expression was associated with a change in contractile function in individual stably transduced human telomerase reverse transcriptase airway smooth muscle cells.
We unfortunately did not see any wrinkles (deformations induced in the silicone substrate) as a result of the increase in stiffness of the ASM cells due to the increase in contractile protein expression. This phenomenon of wrinkling should have been present at a basal level. Hence, there should have been a small population of cells producing wrinkles irrespective of their stimulation or transduction with shRNA for the scrambled control, laminin a4 and laminin a5. This is due to the endogenous phenotype plasticity of ASM cells. Hence in a given population of cells, some should endogenously produce wrinkles. Since we did not see this endogenous level of wrinkling, we concluded that the problem was not with the cells and that the problem lay within the protocol. We decided to go for 10 000 cells per well since this allowed us to view individual cells better and subsequently it would allow us to view the wrinkles produced by individual cells to be viewed better. We then looked at the ECM protein that was used as the coating agnet. Collagen was chosen as an alternative to fibronectin to make sure that the fibronectin was not an issue in the protocol, and collagen has been shown previously to be a medium on which ASM cells can be grown. We then decided to add UV light to the protocol as a publication showed that the substrates need to be treated with UV light in order to become more sensitive and become more receptive to wrinkles (2).
Currently the cells have been safely stored in liquid nitrogen in Philadelphia in the lab of Dr. Pera and he will assist us in completing the experiments now that I am back in the Netherlands.