Contraction generated upon application o f RGD-containing synthetic decapeptide.

In order to test the importance o f RGD-mediated integrin-matrix ligand cell binding a decapeptide, RGDSPASSKP was added as a synthetic competitive blocking agent. It functions by mimicking the cell binding sequence present on a Fn molecule, and hence blocks integrin-ligand interactions.

Apphcation o f Img/ml RGDS eliminated contraction completely over the first 6 hours (Figure 41a). Rate o f contraction registered during 0-4 hours was negative indicating a net feiU in force (-4.25 dynes/hour/million cells) compared to untreated controls (8.14 dynes/hour/milhon cells) (Figure 41b). However, following a recovery o f contraction fi*om 6 hours post-seeding, rates o f contraction for RGDS treated cells rapidly increased (4-8 hours), contracting 3.6 times faster (15.7 dynes/hour/million cells) than untreated controls (4.37 dynes/hour/milhon cells). Interestingly, this sudden increase in contraction rate was also observed on apphcation o f low dose anti-a5 pi-antibody and anti-Fn- antibody (refer to section 3.3.1.2 and 3.3.1.3). Cells treated with 1 mg/ml RGDS eventuaUy regained force generation (8 hours onwards) to reach levels o f net force seen in controls with contraction rates to match. Reduction o f the inhibitor concentration to 0.5 mg/ml induced a 1 hour inhibition of contraction, which was foUowed by a gradual, partial recovery. Maximal peak contraction using this particular concentration was attained by 10.5 hours (27 dynes) remaining constant thereafter. Recovery levels o f contraction achieved using 0.5 mg/ml RGDS were 40% o f those generated by untreated controls, which contracted to a maximum o f 67 dynes. Rates o f contraction were reduced throughout, except during the 8-12 hour period when rates for RGDS treated cells were comparable to untreated controls (1.6 and 1.56 dynes/hour/mihion ceUs, respectively).

Further reduction o f the inhibitor concentration to 0.25 mg/ml produced levels o f contraction similar to those seen in untreated controls. The characteristic rapid initial generation o f force seen using low concentrations o f anti-a5 pi-antibody and anti-Fn- antibody (refer to section 3.3.1.2 and 3.3.1.3) was again noted using this inhibitor. Rates acquired for the first 4 hours o f contraction show that inhibitor treated cells were contracting approximately 1.5 times faster than untreated control cells, ie.: 11.82

dynes/hour/ million cells compared to 8.14 dynes/hour/million cells. Rates throughout the remainder of the experiment decreased and were comparable to untreated controls. Interestingly, rates of contraction seen using all low dose treatments (ie,: anti-a5pl- antibody, anti-Fn-antibody and RGD peptide) generated comparable increases in initial contraction rates (0-4 hours). All low dose inhibitors caused the cells to contract 1.4 to

1.6 times faster than untreated control cells.

Data representing the rapid recovery seen at 6 hours using a concentration of Img/ml appears to suggest that initial rates were dose dependent, providing a linear relationship between dose and inhibition of contraction. However, final contraction force attained generated a bell shaped curve relationship between dose and inhibition of contraction, with 0.5mg/ml inducing maximum inhibition.

a 6 0 -20 -4 0 20 12 14 16 18 0 2 4 6 8 10 T i m e ( h o u r s )

a Untreated bmg/ml -A- 0 Smg/ml O 0.25nig/ml

b

- 1 0

0 -4 12-16 16-20

T im e (h o u rs)

B untreated □ Img/ml 0.5m g/nil □ 0 . 2 5 m g /m l

Figure 41. Figure 41a represents the contraetile data generated upon the apphcation of a Fn specific RGD-type synthetic decapeptide (RGDSPASSKP). Although 1 mg/ml caused the greatest initial inhibition, 0.5mg/ml appeared to induce maximum inhibition. Figure 41b shows this more clearly with 1 mg/ml recovering rapidly, and 0.5mg/ml partially but consistently inhibiting contraction throughout the 20 hours.

3.3.1.5 Contraction generated upon application of anti-aS-antibody

Recovery of contraction (post 6-8 hours) was a feature of all the treatments, with the exception of certain concentrations of the anti-fibronectin reeeptor-antibody (a5pl). To test whether this difference was caused by the broad specificity of the p 1 subunit present in the fibronectin receptor antibody (ie,: inhibition of pi containing integrins may have blocked other cell binding activity), a specific antibody to the a5 subunit was added.

Application of 0.1 mg/ml anti-a5-antibody induced a limited inhibition of cellular contraction which lasted approximately 5 hours, compared to the complete elimination of contraction observed with the anti-a5pi-antibody (Figure 42a). Initial contraction rates displayed were low compared to untreated controls (-1.64 and 8.14 dynes/hour/million cells, respectively) (Figure 42b). However, as recovery of contraction began, rates increased with the treated cells contracting faster (6.56 dynes/hour/million cells) than untreated control cells (4.37 dynes/hour/million cells) between 4-8 hours. Although the

treated cells contracted only marginally faster than the untreated control cells, it is a characteristic that was constantly evident throughout the remaining experimental period. Levels o f contraction attained at the end o f the 20 hour period were comparable to controls (59.73 and 66.98 dynes, respectively). A ten-fold reduction o f antibody concentration gave a weak and transient inhibition o f contraction that lasted for 1 hour. Recovery was rapid with elevated contraction rates over the initial 8 hours. Speed of force generation during the first half o f this period was similar to that exhibited by untreated controls (7.8 and 8.14 dynes/ hour/million cells, respectively), however during the latter half o f the 8 hours contraction period, rates o f treated cells remained high (8.75 dynes/hour/million cells), whilst untreated controls fell (4.37 dynes/hour/million cells), hence propelling treated cells towards control levels o f contraction. Upon attaining control levels o f contraction, treated cells dramatically slowed down their rate of contraction and displayed a fall in tension. Again, the increase in force seen between 4-8 hours using 0.01 mg/ml anti-a5-antibody, was similar to the effect of low dose concentrations o f anti-a5 pi-antibody, anti-Fn-antibody and the synthetic RODS peptide (refer to section 3.3.1.2 to 3.3.1.4).

The trend observed that low doses o f inhibitor generate a rapid initial rate o f contraction, may be explained by cell-substrate adhesiveness and consequential cell migratory speeds (DiMilla et al. 1991). A possible explanation is that low doses o f inhibitor slightly reduce the adherence o f cells to the substrate. This would in effect increase the migratory speed o f the cells (ie,: no strong attachments to ECM and therefore more motile) and in doing so enhance traction. Thus, explaining the elevated contraction rates. However, as cells V)C^irt bo generate forces in excess o f controls (ie,: exceed control levels o f contraction) an internal tensional homeostasis mechanism is activated and prevents them fi*om doing so. Hence, increased rates o f contraction tend to be followed by a fall in tension.

a 3 20 -20 -40 tim e (h o u rs) ■ untreated 0 kng/ml A- O.Okng/ml I control tim e (h o u rs) 12-16 I 0 kng/m l I 0 0 kng/ml 16-20

Figure 42. Figure 42a indicates contractile profiles for cells treated with anti-a5-antibody (part of the FnR complex). Limited inhibition of cellular contraction was seen using 0.1 mg/ml anti-a5-antibody, compared to the complete elimination of contraction observed with the anti-a5 pi-antibody. Figure 42b shows the rate of contraction compared to untreated cell seeded controls. Cells treated with anti-a5-antibody distinctly contract faster than controls to reach tensional homeostasis.