Experimental research on the relationship between the stiffness and the expressions of fibronectin proteins and adaptor proteins of rat trabecular meshwork cells

Authors state that they adhered to the tenets of the Declaration of Helsinki or the NIH statement for the use of Animals in Research. This research was approved according to relevant laws and institutional regulations. The Rat TM cells were supported by YanYu trade co., LTD. Shanghai, China. The supplier’s catalog number for the cells is FS02436. The primary cultures were established by those cells migrating from the excised Rat trabecular tissue [32]. Spindle cells cultures generated confirmation to progress through morphological and biochemical changes characteristic of the cell type (see Fig. 1.). Further, rat TM cells were identified and assayed by WB analysis with rabbit anti rat FN and NSE antibody (see Fig. 1.). In our experiment, the third generation of TM cells was selected as the experimental object.

Cell samples were homogenously inoculated in Petri dishes. After 12 h for cell adherent, Petri dishes were randomly divide into seven groups, each group two dishes. We marked them as D1, D2 and D3 for DEX (dexamethasone; Solarbio; Beijing; China) concentration at 10⁻⁶ M, 10⁻⁵ M and 10⁻⁴ M and M1, M2 and M3 for MIF (mifepristone; abcam; Cambridge; UK) concentration at 10⁻⁶ M, 10⁻⁵ M and 10⁻⁴ M. The other two dishes were marked as C for blank control group. After 48 h of cell cultivation, seven cells were randomly selected from each Petri dish for AFM cell mechanics test. We obtained cells force curve. The other group cells were used only for WB analysis. The expression of FN and LNK protein was recorded.

The total protein was extracted with the RIPA (Applygen, Beijing, China) from TM cells subjected to different experimental conditions. The concentration of total proteins was monitored with BCA Protein Assay Kit (Dinao; Beijing; China). The proteins were diluted with RIPA buffer to the desired concentration and then mixed with an appropriate amount of loading buffer (Thermo, Pittsburgh, USA) with the proportion of 5:1. The protein mixture was taken to boil for 5 min at the temperature of 9 °C. The protein mixture (20 μg each lane) were separated using 10% SDS-PAGE gel electrophoresis (Beyotime Biotechnology, China) at a constant voltage of 80 V for 2.5 h at 4 °C and transferred to PVDF membranes (Millipore, USA) at a constant current of 250 mA for 1 h at 4 °C. And incubated at 4 °C overnight with the following primary antibodies: Anti-Fibronectin antibody, Anti-LNK antibody, Anti-NSE antibody (Abcam, Cambridge, UK). Membranes were incubated with 1:2000 dilution of peroxidase-conjugated goat anti-mouse IgG secondary antibody (Applygen, Beijing, China) for 1 h at room temperature. Finally, the membranes were incubated in ECL reagent (Pierce, Thermo, USA) for HRP detection and then exposed to autoradiography film (Bio-Rad, USA) for band visualization.

In our tests β-actin was used as an internal control. In fact, studies had tested the effects of 0.1 mM DEX for 10 days and found no significant change in β-actin protein expression in TM cells [33, 34]. Since both DEX and MIF are counteractive pattern with glucocorticoid receptors and the action time is short. We speculate that β-actin protein expression will not change together. We did three separated WB analysis for statistical analysis.

By measuring indentation at a given force, the local stiffness of the sample in terms of Young’s modulus can be extracted from the recorded force curve, where a model for the tip–sample contact mechanics [35‐38] and the Hertz model [39] were applied. The force curve consists of an approach–retract cycle between the tip and the sample during which the cantilever deflection is measured as a function of the relative motion [40]. Elasticity information on a biological sample can be obtained by exploiting the loading force curve [41].

Based on literature, where 1–3 cells [42], 6 cells [43] were applied to measure the stiffness by AFM, in this study, 7 single cells were randomly selected from each Petri dish for the atomic force microscope (AFM, NTEGRA, NT-MDT, Russia) cell mechanics test. We adopted spherical probe, probe models is MLCT - O10 - A, Bruker, sphere radius r = 2500 nm, cantilever spring constant k = 0.05 N/m, loading speed is 500 nm/s. When the spring constants of the cantilever and the sample are comparable, once the tip is in contact with the sample, upon further approaching the sample will undergo indentation, resulting in a minor cantilever deflection. Then the indentation force can be obtained. Indentation depth should select about 1/10 the thickness of the sample because a very thin sample may cause the tip to feel the underlying stiff substrate. In order to guarantee the consistency and certainty of the test, the axis along which the force is administered by the AFM tip is perpendicular to the nuclei surface to avoid lateral forces (see Fig. 2).

We can choose cells under the inverted microscope. For each cell the loading force curve and unloading force curve were obtained. The loading force curve for each cell was used to determine the elastic modules. The typical force curves were shown in Fig. 2, where the horizontal axis shows the indentation depth. The vertical axis shows the force to load. Since the contact points of the AFM tip and the cells were different, we did the translation process and all contact point at 8000 nm, and this process did not alter the stiffness value.

For cells, basically meet the assumption of Hertz theory, applies to the model of spherical tip [44]. Hertz model is most widely used for the characteristic of the elastic modulus of cells [45].

We used the Hertz model for loading force curve fitting and obtained young’s modulus E for each cell.

The formula F represents the force (nN) applied to the sample, and v is poison ratio, typically take in biological samples v choose 0.5. E (GPa) represents elastic modules.δ(nm) represents indentation depth. In our curve fitting, sphere radius R = 2500 nm, poison ratio is 0.5, a and b represent the start point for curve fitting. We applied the Hertz equation for non-linear curve fitting to obtain the E value. This process and results were shown in Fig. 3.

FN protein is a hot spot of research for TM cells. FN protein plays a major role in cell morphology and function. It is discovered abundantly in TM beams, with slightly higher levels in the basement membranes of TM beam cells [49, 50]. Stiffness of TM cells after treated with DEX has different degree increase (2-fold for human patients TM [51], 3.5-fold for rabbit TM [52], and more than 2-fold for rat TM in this study). It develops a fibril with considerable elasticity [52, 53]. The increase of FN means elastic fibers increased in the ECM, and thus enhanced the elastic modulus of the cell. Besides, our elastic modulus result was derived from AFM probe direct perception of cells, cell stiffness is closely linked to the measured elastic modulus. DEX (MIF) treated cell’s FN expression increase (decrease), the corresponding elastic modulus also increase (decrease) accordingly. The stiffness alternation may correlate with the expression of FN protein.

LNK is an adaptor protein. It is an important regulator of TM cell kinetics, including the ability to cell growth, mobilization, and recruitment for cell regeneration [54]. Its function involves endothelial cell activation, regulation and control of the cytoskeleton. It also involves adjusting its structural characteristics. Signal transduction mechanism and biological function. It can be compatible with the formation of macromolecular compounds, to participate in the integration and intracellular signal transmission. It can be speculated that the increased amount of LNK expression makes the above functions enhanced; as a result, increase macromolecular compounds may have a close relationship with cell stiffness increase.

It has reported that aqueous humor outflow resistance may increase by DEX treatment [55]. Study reported that after topical administration of 0.1% DEX in vivo for 3 weeks, the elastic modulus of TM in D-treated eyes was 3.89 ± 2.55 kPa, which was significantly larger than that in control eyes. Therefore, by DEX treatment both TM cell stiffness and aqueous humor outflow resistance increase. Opposite to DEX [56], MIF is a kind of cortical antagonist, may occur the opposite effect. We found that MIF can dramatically decrease cell stiffness. If by MIF treatment aqueous humor outflow resistance decrease, we can assert that cell stiffness and aqueous humor outflow resistance are positively correlative. In fact, MIF treatment can lower IOP [57, 58]. On the other hand, our results indicate that the expressions of FN and LNK decrease after MIF treatment. It follows that ECM deposition in the TM reduces. This may decrease the aqueous humor resistance through the trabecular meshwork. During glaucoma, high IOP may be correlated with rat TM cell’s oxidative stress. Oxidation may lead to TM endothelial cell decay, tissue malfunction and subclinical inflammation, these changes reduced TM outflow facility and resulted increased IOP [48]. The mechanisms responsible for the aqueous humor that increases during glaucoma are different affecting the trabecular cells and inducing an IOP increase. According to the discussion above, we infer that there is a positive correlation relationship between the change in cell stiffness and aqueous outflow resistance. This assertion needs to be further studied by combining with the animal in vivo experiments.