Modulation of human uterine smooth muscle cell collagen contractility by thrombin, Y-27632, TNF alpha and indomethacin

Thrombin, hirudin, TNFα, indomethacin and rat tail collagen type I were purchased from Sigma (Dublin, Ireland). Y-27632 was from Calbiochem (San Diego, CA, USA). Stock solutions of thrombin, hirudin, TNFα and Y-27632 were dissolved in deionised water. A stock solution of 100 μM indomethacin was made in ethanol. Dulbecco's Modified Eagle Medium (DMEM), F-12 media, and foetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA, USA).

Human primary uterine smooth muscle cells, hUtSMCs (Cambrex, Wokingham, Berkshire, UK) were expanded in DMEM high glucose with 10% FBS (Invitrogen, USA). Myometrial human telomerase reverse transcriptase (hTERT-HM) cells kindly provided by Dr. Jennifer C. Condon [6] were cultured in DMEM-F-12/10% FBS (Invitrogen, USA). HEK293 cells were maintained in DMEM medium (Invitrogen, USA).

Collagen gels were prepared from rat tail Type 1 collagen (Sigma, Ireland), to a final concentration of 1.5 mg/ml, and seeded in 24 well culture dishes, with 150,000 hUtSMC (passages 5–8), hTERT-HM or HEK293 cells per well, based on the technique described by Dallot et al. (4). Cells in collagen gels were allowed to equilibrate overnight in serum free DMEM medium. Gels were released from the culture dishes and various test agents of interest were added to the serum free media, inhibitors were added 30 min prior to treatment and release. The vehicle controls for thrombin, hirudin, TNFα, and Y-27632 were serum free media and for the indomethacin experiments it was 5% indomethacin, in serum free medium. Gel images were captured over time, using a FluorchemTM 8900 imager (Alpha Innotech Corporation, San Leandro, CA, USA) and the area (cm²) of the gels measured using Image J software http://​rsb.​info.​nih.​gov/​ij/​. For each condition, collagen contraction was determined in quadruplicate, at a minimum. Each experiment was performed at least 3 times. Results were expressed as mean gel area (cm2) ± the standard error of the mean (SEM). 10% (vol/vol) FBS was used as a positive control for contraction in all experiments, and un-stimulated cells the negative control. The original area of the collagen gels was the area (2 cm²) of a well of a 24 well plate, before release from the sides of the wells. A decrease in gel area correlates with an increase in contractility and an increase in gel area correlates with relaxation or inhibition of contractility. Percentage increase or decrease in contractility was compared to that of the unstimulated or basal contraction. One way ANOVA with Tukey's post-hoc analysis, were used to statistically analyse the data (GraphPad Prism software 5, GraphPad Software, Inc., La Jolla, CA, USA), P values < 0.05 were considered to be statistically significant.

Fluorescence microscopy was performed on hUtSMC embedded collagen gels (equilibrated overnight and 30–105 min post release/treatment). Gels were washed in 1× phosphate buffered saline (PBS) and fixed in 1% paraformaldehyde and permeabilised with 1% TritonX-100/1 × PBS. The blocking solution was 1% bovine serum albumin (BSA)/PBS. The primary antibody was a 1/100 dilution of a fluorescein isothiocyanate (FITC) labelled monoclonal mouse anti-human SMα-Actin antibody (Sigma, Ireland) in 1%BSA (wt/vol)/1 × PBS in 500 μl at 4°C overnight. The gels were washed and then incubated in 1 × PBS 1/400 dilution of secondary anti-mouse antibody in 1%BSA (wt/vol)-1 × PBS. Fluorescent images were obtained using the DP70 fluorescence microscope (Olympus, Tokyo, Japan).

Samples (collagen cell lattices, 1–5 hrs post release/treatment, with at least 8 hrs pre-equilibration) were washed in 0.1 M phosphate buffer and then fixed in 2.5% (vol/vol) glutaraldehyde. The gels were then washed in phosphate buffer and dehydrated in 50–100% ethanol. Hexamethyldisilazane was added and samples were air-dried and mounted on scanning electron microscopy stubs using a carbon pad, gold coat. Samples were scanned on an S-4700 scanning electron microscope (Hitachi, Japan).

hUtSMC embedded collagen gels were digested with diethyl pyrocarbonate (DEPC) treated collagenase I (Sigma, Ireland) at 37°C for 30 mins and centrifuged at 4°C and subsequently washed and centrifuged at 4°C with 1 × PBS (DEPC treated) and sterile RNase free water. Total RNA was isolated from the cell pellet using the RNeasy mini RNA isolation kit (Qiagen, Crawley, West Sussex, UK) and DNase 1-treated (Invitrogen, USA).

RNA (1 μg) was reverse transcribed into complementary DNA (cDNA) with SuperScript III (Invitrogen, USA). Control RNA samples, in which no reverse transcriptase was added, were included to confirm that no genomic DNA contamination was present.

1 μl of the 20 μl RT reaction was used as template for PCR, performed with 1.25 U Taq DNA polymerase (Bioline Ltd., Taunton, MA, USA), 0.2 mM dNTPs and 0.2 μM of each primer. cDNA amplification was performed by an initial denaturation step of 5 minutes at 95°C followed by 28–40 cycles of denaturation at 94°C for 1 min, annealing at 55–60°C for 1 min and elongation at 72°C for 30 s–1 min, and a final extension step at 72°C for 10 minutes. The sequences of the oligonucleotide primers (MWG, Ebersberg, Germany) were:

5'-CAACTCCATCATGAAGTGTGA-3'

5'-GCCATGCCAATCTCATC-3' (Accession M10277)

5'-GTGCAACACTTGAGTGGCTAT-3'

5'-AGCAATTTGCCTGGTGAATGAT-3' [19]

5'-ACAAGGGAAGTATGGCTATGGA-3'

5'-GGTCTTTTCGTATCCCACCTTTC-3' [19]

5'-TTGAAGGCAAAGACATGGCAG-3'

5'-CCATCTGAAGGCCAATGACAT-3' [19]

5'-CGAAGACGAAAGGAAACAAGGT-3'

5'-GCTTGGGGTCGTAGAGGTG-3' [19]

5'-CAAAACCTGACACCTCCAGTT-3'

5'-GCCACATGGTAAGGCATAATGA-3' [19]

5'-ACAACAGCATCATGAAGTGT-3'

5'-CCAGTAGCCTATTTCAGATT-3' (Refseq NM_001613)

Real time fluorescence PCR was performed using the Applied Biosystems StepOne Plus™ Real Time PCR System Relative Standard Curve method (ABI, Foster City, CA, USA). Standard curves were created for both a housekeeping gene (ACTB) and the gene of interest, using DNA of known concentration as template, with ABI Fast Sybr Green (2×) and Fast Optical 96 well reaction plates (ABI, USA). The concentration of each primer was 0.4 μM and template cDNA was 1/10–1/25 dilutions of cDNA from RNA isolated from collagen embedded and treated hUtSMCs. cDNA amplification was performed by an initial step of 95°C for 20 seconds, followed by 40 cycles of denaturation at 95°C for 3 seconds, annealing at 60°C for 30 seconds. The sequences of the oligonucleotide primers for ACTA2, TAGLN and PTGS2 were as above and the sequences for the additional oligonucleotide primers for real time PCR were:

5'-CAAATGCCACCTTAGATCCCC-3'

5'-CTTCTGAGATGAATGCAGGAAGT-3' [19]

5'-GGGCATGGGTCAGAAGGATT-3'

5'-AGTTGGTGACGATGCCGTG-3' (Accession M10277)

Fluorescence data was acquired at the end of each PCR cycle. Melting curve analysis was performed by an initial denaturation step of 95°C for 15 seconds and 60°C for 1 minute and 95°C for 15 seconds. Fluorescence was measured continually during the melting curve cycle. Each reaction was performed in triplicate. With the relative standard curve method, the StepOne Plus™ Real Time computer software (ABI, USA) measured amplification of F2R and the endogenous control, the housekeeping gene ACTB in samples, reference sample (unstimulated collagen embedded cells) and in the standard dilution series, all on the same reaction plate. Measurements were normalised using the endogenous control, ACTB, where the F2R quantity mean sample value is divided by the corresponding ACTB value. Data from the standard dilution series were used to generate the standard curve. Using the standard curve, the software interpolated target quantity in the samples and the reference sample (un-stimulated collagen embedded cells). The software determined the relative quantity of target in each sample by comparing target quantity in each sample to target quantity in the reference sample. Mean ACTB normalised starting RNA relative quantities ± SEM from treated collagen embedded hUtSMCs were compared to control or unstimulated cells in collagen gels ± SEM and fold changes calculated. Statistical analyses, student t-tests or one way ANOVA with Tukeys post hoc tests were performed using Graphpad Prism 5 software (GraphPad Software, Inc., USA). P values < 0.05 were considered to be statistically significant.

The collagen cultured primary hUtSMCs (to passage 8) expressed mRNA for the smooth muscle differentiation markers TAGLN, CNN1 and ACTA2, plus ESR1 (Figure 1A(i–iv)). Cultured hUtSMCs also expressed PTGS2 (Figure 1A(v)) and OXTR (oxytocin receptor) mRNA (data not shown). The hTERT-HM cells expressed TAGLN, PGR, ACTA2, CNN1 and PTGS2 (Figure 1B(i–v)). The hUtSMC and hTERT-HM cells caused basal contraction of collagen gels whereas the non-contractile HEK293 cells did not (Figure 1C).

TNFα (10 ng/ml) increased collagen contractility, with a decrease in gel area of 9.5% (P < 0.05) to a peak of 47.3% (P < 0.001), 6 to 26 hrs respectively, after treatment, in comparison to un-stimulated collagen embedded hUtSMCs (Figure 2A). Indomethacin (5 μM), a non-selective COX inhibitor, added 30 minutes prior to TNFα treatment, inhibited the TNFα-induced contraction, resulting in an increase in collagen area of 28% (P < 0.001), at 2 hours 30 minutes. Indomethacin (5 μM) alone, inhibited basal contraction; a 23% increase (P < 0.001) in gel area was observed in comparison to control hUtSMC embedded collagen gels, 2.5 hours after its addition (Figure 2B).

Thrombin (1 U/ml) induced a 15.5% increase (P < 0.001) in contractility after 30 minutes which increased to 17.3% at 1 hour (P < 0.01) (Figure 3A). A bar chart representation from a separate experiment illustrated a 29.3% (P < 0.05) decrease in collagen gel area after thrombin application alone, at 1 hr 30 mins. Hirudin (10 U/ml), a thrombin inhibitor, inhibited the thrombin induced contraction, while alone it did not affect basal levels of contractility (Figure 3B).

Total RNA was isolated from collagen embedded hUtSMCs after a contractility experiment in which cells cultured overnight in collagen in serum free medium were stimulated with 1 U/ml thrombin for 2 hours, resulting in a 35% increase (P < 0.001) in contractility (Figure 3D). RNA was isolated immediately after the area measurement for the 2 hr time-point was acquired.

Relative quantitative expression analysis was performed by real-time fluorescence RT-PCR. In order to minimise any undue experimental error from sources such as pipetting inaccuracies, analysis of each gene was performed in triplicate. The collagen embedded hUtSMCs demonstrated expression of the thrombin receptor F2R and the housekeeping gene, ACTB mRNA. RT-PCR product specificity was confirmed using melting curve analysis. Amplification curve crossing points were determined for each gene generated within the initial phase of exponential amplification.

The mean quantities per starting mRNA, for the treated and control cells for F2R and the housekeeping gene (ACTB) were interpolated from the corresponding CT values from the standard curves, using the relative standard curve method StepOne software (ABI, USA). These ACTB normalised values, were averaged and values determined for both treated (n = 3) and non-treated (n = 3). The mean ACTB normalised RNA quantities for treated and control ± SEM, for F2R were 0.215 ± 0.025, 0.09083 ± 0.01282, this is graphically represented in Figure 3C), with a resultant relative fold increase of 2.36 in the thrombin treated versus the untreated collagen gels.

Thrombin (1 U/ml) caused a significant decrease in gel area (Figure 4A and 4B), from 36.3% at 15 mins (P < 0.001) to 17.1% at 35 mins, in comparison to basal contraction of hTERT-HM cells in the collagen gels (Figure 4B). Hirudin (10 U/ml) blocked the thrombin-induced increase, with a return to basal levels of contractility at all time-points analysed (Figure 4A and 4B). Hirudin alone had no significant effect on contractility (Figure 4B). The application of Y-27632 (2 μM) to the hTERT-HM collagen lattices resulted in an inhibition or decrease of basal contraction, with a 24.7% (P < 0.001) at 20 mins, to 73% (P < 0.001) at 4 hr 20 min increase in gel area, in comparison to the untreated collagen hTERT-HM gels. The contractile effect of thrombin was reduced by Y-27632, with a decrease in contractility in the Thr + Y-27632 treated cells compared to those treated with thrombin alone, ranging from 25% at 30 min (P < 0.001) to 49% at 1 hr (P < 0.01) and 1 hr 50 min (P < 0.001), back to 25% at 4 hr 30 min (P < 0.01) (Figure 4A).

The next aim was to analyse the effect of a longer incubation time and a greater concentration of Y-27632 on thrombin induced and basal contractility. The application of Y-27632 (10 μM) resulted in a decrease in contractility (increase in gel area) over basal levels, of 22.2% (P < 0.001) at 15 mins to 75.4% (P < 0.001) at 24 hrs. The contractile effect of thrombin was antagonised by the application of 10 μM Y-27632 with a decrease in contractility in the Thr+Y-27632 treated gels versus those with thrombin treatment alone, ranging from 14% at 25 min (P < 0.001) to 31.8% at 2 hr 40 min (P < 0.001), 56.3% (P < 0.001) to 99% at 24 hrs. On addition of the thrombin inhibitor hirudin, to Thr+Y-27632, contractility levels returned to those of Y-27632 alone (Figure 4B).

Fluorescence microscopy revealed SMα-Actin FITC immunolabelling of hUtSMCs within the collagen gels in Figure 5(A) and 5(B). It was apparent that the cells demonstrated typical smooth muscle characteristics with an elongated shape and were forming networks.

Scanning electron microscopic analysis confirmed the incorporation of the hUtSMCs within the 3D structure of collagen fibres. The micrographs demonstrated the cells entrapped within the collagen meshwork, as indicated (Figure 6(A–D). In Figure 6A multiple cells are demonstrated. An individual cell was visible with collagen fibrils entwined about it (Figure 6D).