Background To study the effect of retinal/ vitreoretinal surgeries on corneal elevations.
Background
To study the effect of retinal/ vitreoretinal surgeries on corneal elevations.Methods Patients who underwent retinal/ vitreoretinal surgeries were divided into 3 groups. Scleral buckling was performed in 11 eyes (Group 1). In 8 (25%) eyes, vitreoretinal surgery was performed along with scleral buckling (Group 2). In 12 eyes, pars plana vitrectomy was performed for vitreous hemorrhage (Group 3). An encircling element was used in all the eyes. The parameters evaluated were best-corrected visual acuity (BCVA), change in axial length, and corneal topographic changes on Orbscan topography system II, preoperative and at 12 weeks following surgery.
Methods
Patients who underwent retinal/ vitreoretinal surgeries were divided into 3 groups. Scleral buckling was performed in 11 eyes (Group 1). In 8 (25%) eyes, vitreoretinal surgery was performed along with scleral buckling (Group 2). In 12 eyes, pars plana vitrectomy was performed for vitreous hemorrhage (Group 3). An encircling element was used in all the eyes. The parameters evaluated were best-corrected visual acuity (BCVA), change in axial length, and corneal topographic changes on Orbscan topography system II, preoperative and at 12 weeks following surgery.Results There was a statistically significant increase in anterior corneal elevation in all the three groups after surgery (p = 0.003, p = 0.008 & p = 0.003 respectively). The increase in posterior corneal elevation was highly significant in all the three groups after surgery (p = 0.0000, p = 0.0001 & p = 0.0001 respectively). The increase in the posterior corneal elevation was more than the increase in the anterior elevation and was significant statistically in all the three groups (group I: p = 0.02; group II: p = 0.01; group III: p = 0.008).
Results
There was a statistically significant increase in anterior corneal elevation in all the three groups after surgery (p = 0.003, p = 0.008 & p = 0.003 respectively). The increase in posterior corneal elevation was highly significant in all the three groups after surgery (p = 0.0000, p = 0.0001 & p = 0.0001 respectively). The increase in the posterior corneal elevation was more than the increase in the anterior elevation and was significant statistically in all the three groups (group I: p = 0.02; group II: p = 0.01; group III: p = 0.008).Conclusions Retinal/ vitreoretinal surgeries cause a significant increase in the corneal elevations and have a greater effect on the posterior corneal surface.
Conclusions
Retinal/ vitreoretinal surgeries cause a significant increase in the corneal elevations and have a greater effect on the posterior corneal surface.

Background Changes in corneal curvature and axial length have been reported following scleral buckling procedure using keratometer [ 1 - 7 ]. Videokeratography has also been used to assess the corneal surface changes after buckling procedure[ 8 ]. All these studies have reported a change in the corneal curvature in its anterior surface. However, no study has been performed to evaluate the posterior corneal topographic changes with scanning slit topography system following retinal surgeries. Hence, we made an attempt to study the effect of various retinal surgeries on posterior corneal surface using Orbscan II topography system.
Background
Changes in corneal curvature and axial length have been reported following scleral buckling procedure using keratometer [ 1 - 7 ]. Videokeratography has also been used to assess the corneal surface changes after buckling procedure[ 8 ].All these studies have reported a change in the corneal curvature in its anterior surface. However, no study has been performed to evaluate the posterior corneal topographic changes with scanning slit topography system following retinal surgeries. Hence, we made an attempt to study the effect of various retinal surgeries on posterior corneal surface using Orbscan II topography system.Methods A prospective study was performed by enrolling patients admitted in the Retina Services of Rajendra Prasad Centre for Ophthalmic Sciences, New Delhi for retinal/ vitreoretinal surgeries. Thirty one eyes of 31 patients who underwent retinal/ vitreoretinal surgery and came for regular follow-up as per schedule during the period between December 2001 and October 2002 were included in the study. Only those patients were included who had not undergone any previous ocular surgery & did not have any corneal pathology. An informed consent was obtained from all the patients. The patients were evaluated preoperatively on parameters of best-corrected visual acuity (BCVA), axial length (AL) measured by Ultrasound A-scan instrument (Sonomed, Inc., NY) and detailed corneal examination on slit-lamp biomicroscope and on Orbscan topography system II (Bausch and Lomb, Salt Lake City, Utah). The parameters that were evaluated by Orbscan topography system were anterior elevation, posterior elevation and simulated keratometry. All surgeries were performed by a single surgeon (LV) under local anesthesia by peribulbar injection of 6 ml of 2% xylocaine and 2 ml of 0.5% bupivacaine. Eyes with fresh retinal detachment with clear media and absence of advanced proliferative vitreoretinopathy underwent scleral buckling procedure (Group I, n = 11). In all these eyes, the break/ s were localized, cryotherapy was performed and subretinal fluid was drained. Only circumferential buckle of silicone of style 276 (Labtician Ophthalmics, Inc., Oakville, Canada) was used and radial buckle or sponge was not used in any eye. The size of the buckle was 90° to 360° depending upon the requirement in individual cases. Encircling element of style 240 (Labtician Ophthalmics, Inc., Oakville, Canada) was used in all the eyes undergoing scleral buckling. In eyes with associated vitreous hemorrhage or advanced proliferative vitreoretinopathy changes along with retinal detachment, vitreoretinal surgery was performed along with scleral buckling (Group II, n = 8). Pars plana vitrectomy was performed and vitreoretinal membranes were removed either by peeling or by segmentation or delamination. Air fluid exchange was performed followed by Air-Silicone oil exchange. In eyes with only vitreous hemorrhage without the presence of retinal detachment, only pars plana vitrectomy was performed (Group III, n = 12). An encircling element of style 240 (Labtician Ophthalmics, Inc., Oakville, Canada) was used in all these eyes to counter the anterior traction that could not be fully released by vitrectomy in order to avoid lens damage. The intraoperative details including the nature of surgery, size of the buckle, encircling element, drainage of subretinal fluid, vitrectomy & use of silicone oil or gas, were noted. Postoperative treatment included topical ciprofloxacin 0.3% QID, topical dexamethasone 0.1% QID and topical Homatropine 2% QID. The patients were evaluated at 12 weeks following surgery on similar (preoperative) parameters. Statistical analysis The data of all the patients were managed on an excel spreadsheet. All the entries were checked for any possible keyboard error. Preoperative and postoperative measurements in the three retinal surgery groups were summarized by mean and standard deviation. Changes following surgery within each group were assessed using paired 't' test. Preoperative and postoperative values in the three groups were compared using one way analysis of variance (ANOVA), followed by bon ferroni correction for multiple comparison. For the three retinal surgery groups, median was computed for increase in various parameters due to surgery. Kruskal Wallis one way analysis of variance was used to compare median increase in the three groups. STATA 7.0 statistical software was used for data analysis. In this study, p-value smaller than 0.05 was considered as statistically significant.
Methods
A prospective study was performed by enrolling patients admitted in the Retina Services of Rajendra Prasad Centre for Ophthalmic Sciences, New Delhi for retinal/ vitreoretinal surgeries. Thirty one eyes of 31 patients who underwent retinal/ vitreoretinal surgery and came for regular follow-up as per schedule during the period between December 2001 and October 2002 were included in the study. Only those patients were included who had not undergone any previous ocular surgery & did not have any corneal pathology. An informed consent was obtained from all the patients.The patients were evaluated preoperatively on parameters of best-corrected visual acuity (BCVA), axial length (AL) measured by Ultrasound A-scan instrument (Sonomed, Inc., NY) and detailed corneal examination on slit-lamp biomicroscope and on Orbscan topography system II (Bausch and Lomb, Salt Lake City, Utah). The parameters that were evaluated by Orbscan topography system were anterior elevation, posterior elevation and simulated keratometry.All surgeries were performed by a single surgeon (LV) under local anesthesia by peribulbar injection of 6 ml of 2% xylocaine and 2 ml of 0.5% bupivacaine. Eyes with fresh retinal detachment with clear media and absence of advanced proliferative vitreoretinopathy underwent scleral buckling procedure (Group I, n = 11). In all these eyes, the break/ s were localized, cryotherapy was performed and subretinal fluid was drained. Only circumferential buckle of silicone of style 276 (Labtician Ophthalmics, Inc., Oakville, Canada) was used and radial buckle or sponge was not used in any eye. The size of the buckle was 90° to 360° depending upon the requirement in individual cases. Encircling element of style 240 (Labtician Ophthalmics, Inc., Oakville, Canada) was used in all the eyes undergoing scleral buckling.In eyes with associated vitreous hemorrhage or advanced proliferative vitreoretinopathy changes along with retinal detachment, vitreoretinal surgery was performed along with scleral buckling (Group II, n = 8). Pars plana vitrectomy was performed and vitreoretinal membranes were removed either by peeling or by segmentation or delamination. Air fluid exchange was performed followed by Air-Silicone oil exchange. In eyes with only vitreous hemorrhage without the presence of retinal detachment, only pars plana vitrectomy was performed (Group III, n = 12). An encircling element of style 240 (Labtician Ophthalmics, Inc., Oakville, Canada) was used in all these eyes to counter the anterior traction that could not be fully released by vitrectomy in order to avoid lens damage.The intraoperative details including the nature of surgery, size of the buckle, encircling element, drainage of subretinal fluid, vitrectomy & use of silicone oil or gas, were noted. Postoperative treatment included topical ciprofloxacin 0.3% QID, topical dexamethasone 0.1% QID and topical Homatropine 2% QID.The patients were evaluated at 12 weeks following surgery on similar (preoperative) parameters.Statistical analysis The data of all the patients were managed on an excel spreadsheet. All the entries were checked for any possible keyboard error. Preoperative and postoperative measurements in the three retinal surgery groups were summarized by mean and standard deviation. Changes following surgery within each group were assessed using paired 't' test. Preoperative and postoperative values in the three groups were compared using one way analysis of variance (ANOVA), followed by bon ferroni correction for multiple comparison. For the three retinal surgery groups, median was computed for increase in various parameters due to surgery. Kruskal Wallis one way analysis of variance was used to compare median increase in the three groups. STATA 7.0 statistical software was used for data analysis. In this study, p-value smaller than 0.05 was considered as statistically significant.
Statistical analysis
The data of all the patients were managed on an excel spreadsheet. All the entries were checked for any possible keyboard error. Preoperative and postoperative measurements in the three retinal surgery groups were summarized by mean and standard deviation. Changes following surgery within each group were assessed using paired 't' test. Preoperative and postoperative values in the three groups were compared using one way analysis of variance (ANOVA), followed by bon ferroni correction for multiple comparison. For the three retinal surgery groups, median was computed for increase in various parameters due to surgery. Kruskal Wallis one way analysis of variance was used to compare median increase in the three groups. STATA 7.0 statistical software was used for data analysis. In this study, p-value smaller than 0.05 was considered as statistically significant.Results The mean age of the patients was 45.96 ± 15.17 (range: 18–78) years and majority (83.87%) of the patients (N = 31) were males. Of these, right eye was operated in 19 (61.29%) patients. The mean preoperative best-corrected visual acuity (BCVA) was hand motion close to face in 26 eyes; counting finger near to face in 4 eyes and 1/60 on snellen's acuity chart in 1 eye. The mean decimal postoperative BCVA was 0.20 ± 0.12 at 12 weeks follow-up after surgery. The mean preoperative anterior corneal elevation as recorded by Orbscan II topography system in group I was 0.006 ± 0.007 mm, which increased to 0.024 ± 0.013 mm at 12 weeks after surgery (p= 0.003). In group II, it increased from 0.009 ± 0.006 mm preoperatively to 0.021 ± 0.010 mm at 12 weeks (p= 0.008) and in group III, it increased from 0.003 ± 0.004 mm preoperatively to 0.012 ± 0.007 mm at 12 weeks follow-up (p = 0.003). On comparative evaluation between the groups, the change in anterior corneal elevation was significant between group I and III (p = 0.04). The mean posterior elevation in group I increased from a preoperative value of 0.016 ± 0.010 mm to 0.043 ± 0.007 mm at 12 weeks after surgery (p= 0.0000). In group II, it increased from 0.014 ± 0.006 mm preoperatively to 0.043 ± 0.007 mm at 12 weeks (p= 0.0001) and in group III it increased from a preoperative value of 0.012 ± 0.005 mm to 0.029 ± 0.006 mm at 12 weeks after surgery (p = 0.0001). A comparative analysis between the groups indicated that the increase in posterior corneal elevation between groups I & III and groups II & III were found to be highly significant (I vs III: p= 0.001; II vs III: p= 0.001). Again, the increase in the posterior corneal elevation was greater than the increase in the anterior elevation in all the 3 groups and on comparative evaluation, the difference in the increase in posterior and anterior elevation was significant statistically in each group (group I: p = 0.02; group II: p = 0.01; group III: p = 0.008). The mean corneal astigmatism in group I increased from 0.89 ± 0.54D preoperatively to 2.50 ± 1.39D at 12 weeks follow-up (p= 0.004). In group II, the average corneal astigmatism increased from 0.87 ± 0.30D to 3.38 ± 2.15D at 12 weeks (p= 0.01) and in group III, the mean preoperative and postoperative corneal astigmatism was 0.85 ± 0.55D and 1.37 ± 0.87D respectively (p= 0.02). A comparative analysis of the change in corneal astigmatism following surgery between groups II & III was significant statistically (p= 0.02). The mean preoperative axial length in group I was 23.27 ± 0.79 mm which increased to 23.98 ± 0.76 mm at 12 weeks after surgery (p= 0.009). The mean preoperative and postoperative (12 weeks follow-up) axial length in group II were 23.92 ± 1.32 mm and 25.94 ± 2.96 mm respectively (p= 0.03). The mean preoperative and postoperative axial length in group III were 22.69 ± 0.87 mm and 22.71 ± 0.83 mm respectively (p= 0.79). Comparative analysis of increase in axial length following surgery between groups I & III and groups II & III were found to be significant statistically (I & III: p = 0.003; II & III: p = 0.003).
Results
The mean age of the patients was 45.96 ± 15.17 (range: 18–78) years and majority (83.87%) of the patients (N = 31) were males. Of these, right eye was operated in 19 (61.29%) patients.The mean preoperative best-corrected visual acuity (BCVA) was hand motion close to face in 26 eyes; counting finger near to face in 4 eyes and 1/60 on snellen's acuity chart in 1 eye. The mean decimal postoperative BCVA was 0.20 ± 0.12 at 12 weeks follow-up after surgery.The mean preoperative anterior corneal elevation as recorded by Orbscan II topography system in group I was 0.006 ± 0.007 mm, which increased to 0.024 ± 0.013 mm at 12 weeks after surgery (p= 0.003). In group II, it increased from 0.009 ± 0.006 mm preoperatively to 0.021 ± 0.010 mm at 12 weeks (p= 0.008) and in group III, it increased from 0.003 ± 0.004 mm preoperatively to 0.012 ± 0.007 mm at 12 weeks follow-up (p = 0.003). On comparative evaluation between the groups, the change in anterior corneal elevation was significant between group I and III (p = 0.04).The mean posterior elevation in group I increased from a preoperative value of 0.016 ± 0.010 mm to 0.043 ± 0.007 mm at 12 weeks after surgery (p= 0.0000). In group II, it increased from 0.014 ± 0.006 mm preoperatively to 0.043 ± 0.007 mm at 12 weeks (p= 0.0001) and in group III it increased from a preoperative value of 0.012 ± 0.005 mm to 0.029 ± 0.006 mm at 12 weeks after surgery (p = 0.0001). A comparative analysis between the groups indicated that the increase in posterior corneal elevation between groups I & III and groups II & III were found to be highly significant (I vs III: p= 0.001; II vs III: p= 0.001).Again, the increase in the posterior corneal elevation was greater than the increase in the anterior elevation in all the 3 groups and on comparative evaluation, the difference in the increase in posterior and anterior elevation was significant statistically in each group (group I: p = 0.02; group II: p = 0.01; group III: p = 0.008).The mean corneal astigmatism in group I increased from 0.89 ± 0.54D preoperatively to 2.50 ± 1.39D at 12 weeks follow-up (p= 0.004). In group II, the average corneal astigmatism increased from 0.87 ± 0.30D to 3.38 ± 2.15D at 12 weeks (p= 0.01) and in group III, the mean preoperative and postoperative corneal astigmatism was 0.85 ± 0.55D and 1.37 ± 0.87D respectively (p= 0.02). A comparative analysis of the change in corneal astigmatism following surgery between groups II & III was significant statistically (p= 0.02).The mean preoperative axial length in group I was 23.27 ± 0.79 mm which increased to 23.98 ± 0.76 mm at 12 weeks after surgery (p= 0.009). The mean preoperative and postoperative (12 weeks follow-up) axial length in group II were 23.92 ± 1.32 mm and 25.94 ± 2.96 mm respectively (p= 0.03). The mean preoperative and postoperative axial length in group III were 22.69 ± 0.87 mm and 22.71 ± 0.83 mm respectively (p= 0.79). Comparative analysis of increase in axial length following surgery between groups I & III and groups II & III were found to be significant statistically (I & III: p = 0.003; II & III: p = 0.003).Discussion Retinal surgery with or without the use of encircling and buckling elements for external tamponade can alter the shape of the globe. This may cause changes in the refractive status of the eye. Scleral buckling is known to cause a change in the shape of the sclera and can cause induced refractive changes, including astigmatic and nonastigmatic changes [ 5 - 10 ]. We have used Orbscan slit scanning system II to evaluate the corneal topographic changes following retinal/ vitreoretinal surgeries. The data accumulated by Orbscan may be limited by factors such as the accuracy of the system which is ± 20 μm, the measurement noise which leads to both positive and negative difference in the height of the posterior corneal surface and the necessity of aligning the posterior surface before and after surgery which may be a source of error[ 11 , 12 ]. However, this is the best tool available to study the posterior corneal elevation. In the present study, there was a significant increase in both anterior and posterior corneal elevation as detected by scanning slit topography (Orbscan II topography system) following surgery. This increase in the anterior and posterior corneal elevation is probably due to the use of encircling element and/ or buckle in retinal surgeries resulting in corneal steepening. We noted that the change in the posterior elevation was more significant than anterior elevation. A comparative analysis between the three groups indicated that there was no significant difference in the anterior elevation; however the posterior elevation was significantly more in eyes with buckle. It is possible that the buckle and the encircling element have a greater effect on the posterior corneal surface. The increase in the anterior and posterior corneal elevation might be one of the contributing factors for the non improvement of visual acuity in an eye that has undergone retinal/ vitreoretinal surgery. This change might escape detection by routine videokeratography. The anterior protrusion of the corneal back surface induces an increase in the negative power of the corneal surface. Assessing the corneal surface by keratometry or placido disc videokeratography may provide inadequate information regarding refractive change caused by corneal surface alteration that results in retinal/ vitreoretinal surgery. There was an increase in mean corneal astigmatism following surgery in all the groups. This is due to the effect of encircling element or buckle on the corneal surface. Studies have reported that induced astigmatism has been associated with radial scleral buckles[ 5 , 13 ], circumferential buckles[ 14 ], medial rectus disinsertion[ 15 ], anterior location of the scleral buckle[ 13 ], and use of sponge material rather than hard silicone[ 1 ]. In our study, a comparative analysis between the groups indicated that buckle causes more astigmatic changes than encircling element. In retinal/ vitreoretinal surgeries, the encircling band creates a circular indentation of the eye, thereby increasing its anteroposterior axial length; the myopic shift may be upto 3 diopters (D)[ 9 , 10 , 13 , 16 ]. An increase in axial length by 0.54 mm[ 9 ] and 1.7 mm[ 14 ] has been reported following scleral buckling in two studies. In the present study, there was an increase in the mean axial length of the eyes in all the three groups (Table 1). This increase in axial length may be attributed to an anteroposterior elongation of the eyeball secondary to the transverse compression by the buckle and/ or an encircling element. The postoperative increase in axial length was found to be more pronounced in eyes with buckle. Buckle being wider and thicker results in greater indentation and thereby a greater increase in axial length than encircling element.
Discussion
Retinal surgery with or without the use of encircling and buckling elements for external tamponade can alter the shape of the globe. This may cause changes in the refractive status of the eye. Scleral buckling is known to cause a change in the shape of the sclera and can cause induced refractive changes, including astigmatic and nonastigmatic changes [ 5 - 10 ].We have used Orbscan slit scanning system II to evaluate the corneal topographic changes following retinal/ vitreoretinal surgeries. The data accumulated by Orbscan may be limited by factors such as the accuracy of the system which is ± 20 μm, the measurement noise which leads to both positive and negative difference in the height of the posterior corneal surface and the necessity of aligning the posterior surface before and after surgery which may be a source of error[ 11 , 12 ]. However, this is the best tool available to study the posterior corneal elevation.In the present study, there was a significant increase in both anterior and posterior corneal elevation as detected by scanning slit topography (Orbscan II topography system) following surgery. This increase in the anterior and posterior corneal elevation is probably due to the use of encircling element and/ or buckle in retinal surgeries resulting in corneal steepening. We noted that the change in the posterior elevation was more significant than anterior elevation. A comparative analysis between the three groups indicated that there was no significant difference in the anterior elevation; however the posterior elevation was significantly more in eyes with buckle. It is possible that the buckle and the encircling element have a greater effect on the posterior corneal surface. The increase in the anterior and posterior corneal elevation might be one of the contributing factors for the non improvement of visual acuity in an eye that has undergone retinal/ vitreoretinal surgery. This change might escape detection by routine videokeratography. The anterior protrusion of the corneal back surface induces an increase in the negative power of the corneal surface. Assessing the corneal surface by keratometry or placido disc videokeratography may provide inadequate information regarding refractive change caused by corneal surface alteration that results in retinal/ vitreoretinal surgery.There was an increase in mean corneal astigmatism following surgery in all the groups. This is due to the effect of encircling element or buckle on the corneal surface. Studies have reported that induced astigmatism has been associated with radial scleral buckles[ 5 , 13 ], circumferential buckles[ 14 ], medial rectus disinsertion[ 15 ], anterior location of the scleral buckle[ 13 ], and use of sponge material rather than hard silicone[ 1 ]. In our study, a comparative analysis between the groups indicated that buckle causes more astigmatic changes than encircling element.In retinal/ vitreoretinal surgeries, the encircling band creates a circular indentation of the eye, thereby increasing its anteroposterior axial length; the myopic shift may be upto 3 diopters (D)[ 9 , 10 , 13 , 16 ]. An increase in axial length by 0.54 mm[ 9 ] and 1.7 mm[ 14 ] has been reported following scleral buckling in two studies. In the present study, there was an increase in the mean axial length of the eyes in all the three groups (Table 1). This increase in axial length may be attributed to an anteroposterior elongation of the eyeball secondary to the transverse compression by the buckle and/ or an encircling element. The postoperative increase in axial length was found to be more pronounced in eyes with buckle. Buckle being wider and thicker results in greater indentation and thereby a greater increase in axial length than encircling element.Conclusions Retinal/ vitreoretinal surgeries result in an increase in the elevation of the corneal surfaces. These changes are more pronounced on posterior corneal surface.
Conclusions
Retinal/ vitreoretinal surgeries result in an increase in the elevation of the corneal surfaces. These changes are more pronounced on posterior corneal surface.Declaration of competing interest None declared.
Declaration of competing interest
None declared.Individual contribution of authors RS designed the study and performed the data collection. NS wrote the manuscript. LKV performed the retinal surgeries. RMP performed the statistical analysis and RBV followed up the patients. All authors read and approved the final manuscript.
Individual contribution of authors
RS designed the study and performed the data collection. NS wrote the manuscript. LKV performed the retinal surgeries. RMP performed the statistical analysis and RBV followed up the patients.All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background The non-thrombotic effects of thrombin in cardiovascular tissues, as mediated via the protease activated receptors (PARs), and particularly PAR-1, have been the focus of much recent research. The aims of this study were to evaluate the effects of thrombin, a specific PAR-1 activating peptide (PAR1-AP), and a PAR-1 antagonist on human umbilical artery tone in vitro.
Background
The non-thrombotic effects of thrombin in cardiovascular tissues, as mediated via the protease activated receptors (PARs), and particularly PAR-1, have been the focus of much recent research. The aims of this study were to evaluate the effects of thrombin, a specific PAR-1 activating peptide (PAR1-AP), and a PAR-1 antagonist on human umbilical artery tone in vitro.Methods Human umbilical artery samples were obtained from 17 women at term. Arterial rings were suspended under physiologic conditions for isometric recording. The in vitro effects of thrombin (0.5 units/mL to 3 units/mL), PAR1-AP TFLLR-NH2 [10(-9) to 10(-6) M], and PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH2) [10(-9) M to 10(-5) M] on umbilical artery tone were measured.
Methods
Human umbilical artery samples were obtained from 17 women at term. Arterial rings were suspended under physiologic conditions for isometric recording. The in vitro effects of thrombin (0.5 units/mL to 3 units/mL), PAR1-AP TFLLR-NH2 [10(-9) to 10(-6) M], and PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH2) [10(-9) M to 10(-5) M] on umbilical artery tone were measured.Results Both thrombin and TFLLR-NH2 exerted a potent cumulative vasodilatory effect on human umbilical artery resistance (P < 0.001). The mean net maximal inhibition (MMI) for thrombin was 53.05% (n = 6; SEM = 1.43) at tissue bath concentration of 3 units/mL. The MMI with TFLLR-NH2 was 61.50 % (n = 6; SEM = 1.43) at bath concentration of 10(-6) M. In comparison to vehicle control, the PAR-1 antagonist did not show a significant relaxant or contractile effect (P > 0.05).
Results
Both thrombin and TFLLR-NH2 exerted a potent cumulative vasodilatory effect on human umbilical artery resistance (P < 0.001). The mean net maximal inhibition (MMI) for thrombin was 53.05% (n = 6; SEM = 1.43) at tissue bath concentration of 3 units/mL. The MMI with TFLLR-NH2 was 61.50 % (n = 6; SEM = 1.43) at bath concentration of 10(-6) M. In comparison to vehicle control, the PAR-1 antagonist did not show a significant relaxant or contractile effect (P > 0.05).Conclusion These findings highlight a potential role for thrombin and PAR-1 receptors in vascular regulation of feto-placental blood flow in normal pregnancy, and in association with the vascular lesions associated with IUGR and pre-eclampsia.
Conclusion
These findings highlight a potential role for thrombin and PAR-1 receptors in vascular regulation of feto-placental blood flow in normal pregnancy, and in association with the vascular lesions associated with IUGR and pre-eclampsia.

Background In disorders resulting in poor fetal growth, and in pre-eclampsia, thrombotic lesions are frequently observed in the maternal and fetal vascular components of the placenta, [ 1 - 3 ] and hence have been implicated in the pathophysiology of these conditions. In addition, it has been reported that in vivo generation of thrombin, in maternal plasma, is higher in patients with small for gestation age fetuses and with pre-eclampsia, than in normal pregnancy [ 4 ]. It is well established, for many years, that thrombin plays a role in blood coagulation, but its effects in many other cell and tissue types (smooth muscle cells, endothelial cells, lymphocytes) [ 5 , 6 ] have been the subject of more recent attention. It is now apparent that thrombin can regulate target cells by cleaving and activating a family of G-protein-coupled protease-activated receptors (PARs)[ 5 - 7 ]. This proteolytic cleavage of PARs is mediated by a family of enzymes that require serine within the active site i.e. serine proteases [ 5 ]. There are 4 major PAR subtypes (PAR1-4) with diverse reported functions in various tissues [ 6 ]. PAR activation has been closely linked to inflammation [ 6 , 8 ], contraction of vascular [ 5 ] and non-vascular [ 9 , 10 ] smooth muscle, and platelet activation [ 6 ]. Although PAR-1, PAR-3 and PAR-4 [ 11 ] are all known to be thrombin receptors, the mechanism of activation by thrombin at these different PARs varies [ 12 , 13 ]. There is growing evidence, from vascular tissue studies in several animal models, that non-thrombotic thrombin-mediated signalling events are central to the response to the disease process typical of vascular lesion formation in atherosclerosis [ 14 ]. The direct effects of thrombin on vascular cells, via the PAR receptors, and particularly PAR-1, have been the main focus of investigation for this hypothesis. PARs 1, 3 and 4 are activated by thrombin [ 11 ], but PAR-1 is activated at low thrombin concentrations and most of what is known about thrombin signalling downstream of the receptors, has been derived from studies of PAR-1 [ 14 ]. While other PAR subtypes are present in human arterial vessels, it appears that PAR-1 is primarily involved in endothelium-dependent relaxation to thrombin and trypsin [ 15 ]. To our knowledge, there are no data outlining the potential effects of thrombin, or specific PAR-1 receptor modulation, on the feto-placental circulation, despite the critical role of thrombin in disorders of this vasculature. The aims of this study were to evaluate the direct effects of thrombin, the specific PAR-1 activating peptide (PAR1-AP), TFLLR-NH 2  (Thr-Phe-Leu-Leu-Arg-NH 2 ), and the PAR-1 specific antagonist (N-trans cinnamoyl -p-fluoroPhe-p-quanidinoPhe-Leu-Arg-Orn-NH 2 ) on human umbilical artery tone in vitro.
Background
In disorders resulting in poor fetal growth, and in pre-eclampsia, thrombotic lesions are frequently observed in the maternal and fetal vascular components of the placenta, [ 1 - 3 ] and hence have been implicated in the pathophysiology of these conditions. In addition, it has been reported that in vivo generation of thrombin, in maternal plasma, is higher in patients with small for gestation age fetuses and with pre-eclampsia, than in normal pregnancy [ 4 ]. It is well established, for many years, that thrombin plays a role in blood coagulation, but its effects in many other cell and tissue types (smooth muscle cells, endothelial cells, lymphocytes) [ 5 , 6 ] have been the subject of more recent attention. It is now apparent that thrombin can regulate target cells by cleaving and activating a family of G-protein-coupled protease-activated receptors (PARs)[ 5 - 7 ]. This proteolytic cleavage of PARs is mediated by a family of enzymes that require serine within the active site i.e. serine proteases [ 5 ]. There are 4 major PAR subtypes (PAR1-4) with diverse reported functions in various tissues [ 6 ]. PAR activation has been closely linked to inflammation [ 6 , 8 ], contraction of vascular [ 5 ] and non-vascular [ 9 , 10 ] smooth muscle, and platelet activation [ 6 ]. Although PAR-1, PAR-3 and PAR-4 [ 11 ] are all known to be thrombin receptors, the mechanism of activation by thrombin at these different PARs varies [ 12 , 13 ].There is growing evidence, from vascular tissue studies in several animal models, that non-thrombotic thrombin-mediated signalling events are central to the response to the disease process typical of vascular lesion formation in atherosclerosis [ 14 ]. The direct effects of thrombin on vascular cells, via the PAR receptors, and particularly PAR-1, have been the main focus of investigation for this hypothesis. PARs 1, 3 and 4 are activated by thrombin [ 11 ], but PAR-1 is activated at low thrombin concentrations and most of what is known about thrombin signalling downstream of the receptors, has been derived from studies of PAR-1 [ 14 ]. While other PAR subtypes are present in human arterial vessels, it appears that PAR-1 is primarily involved in endothelium-dependent relaxation to thrombin and trypsin [ 15 ]. To our knowledge, there are no data outlining the potential effects of thrombin, or specific PAR-1 receptor modulation, on the feto-placental circulation, despite the critical role of thrombin in disorders of this vasculature. The aims of this study were to evaluate the direct effects of thrombin, the specific PAR-1 activating peptide (PAR1-AP), TFLLR-NH 2  (Thr-Phe-Leu-Leu-Arg-NH 2 ), and the PAR-1 specific antagonist (N-trans cinnamoyl -p-fluoroPhe-p-quanidinoPhe-Leu-Arg-Orn-NH 2 ) on human umbilical artery tone in vitro.Methods The study was carried out in the Department of Obstetrics and Gynaecology, University College Hospital Galway, Ireland between May 2002 and April 2003. Sections of human umbilical cord approximately 10 cm in length were excised from the proximal segment of the cord (i.e. closest to the placental attachment) immediately after elective cesarean delivery. Samples were obtained from 17 women after elective cesarean section at term and from one patient after normal vaginal delivery. All pregnancies were uncomplicated and there was no evidence of hypertensive disease or intrauterine fetal growth restriction. The mean maternal age was 32.47 years (range 25–40 years). The median period of gestation was 38 weeks (range 37–42 weeks). The reasons for cesarean section included previous cesarean section (n = 9), breech presentation (n = 6), previous myomectomy (n = 1) and unstable fetal lie (n = 1). At the time of recruitment 4 women were nulliparous and 13 women were parous. Samples were immediately placed in cold buffered Krebs Henseleit physiological salt solution (pH 7.4) of the following composition: potassium chloride 4.7 mmol/L, sodium chloride 118 mmol/L, magnesium sulphate 1.2 mmol/L, calcium chloride 1.2 mmol/L, potassium phosphate 1.2 mmol/L, sodium bicarbonate 25 mmol/L and glucose 11 mmol/L. Maternal written informed consent was obtained prior to tissue collection, and the tissue collection procedure was approved by the Research Ethics Committee at University College Hospital Galway. Umbilical arteries were carefully dissected free of Wharton's Jelly and cut in rings 4–5 mm in axial length. Rings were suspended individually on stainless steel hooks inserted into their lumens and mounted under 2 g (30 mN) of isometric tension, in glass-jacketed tissue baths, as previously described [ 16 , 17 ]. Each bath-contained 10 mL of oxygenated (95% O 2  / 5% CO 2 ) Krebs Henseleit physiological salt solution (PSS) at 37°C and pH 7.4. Rings were allowed to equilibrate for 90 minutes with regular washouts of PSS. During this interval, spontaneous tone developed. After the equilibration period, the vessel rings were challenged with 60 mM potassium chloride (KCl). Three washouts with PSS were carried out once the maximum response had reached a plateau, and a 20 minute recovery period was allowed in order that baseline be attained again. The KCl challenge was performed three times. After the last KCl challenge, 40 minutes recovery was allowed, and contraction was then stimulated by bath exposure of the vessel rings to 5-hydroxytriptamine (5-HT) (10 -7  M). Once maximum contractile response to 5-HT was attained, the rings were allowed to remain at plateau for 20 minutes. Concentration- effect experiments were performed by cumulative additions of thrombin, the PAR1-AP, or the PAR-1 antagonist to the tissue bath. Thrombin was added to the tissue bath at an initial concentration of 0.5 units/mL, and this was increased at 20-minute intervals to 1 unit/mL, 2 units/mL and 3 units/mL respectively. The bath concentrations ranges investigated for PAR1-AP were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L and 1 micromol/L (i.e. 10 -9  - 10 -6 M), and for PAR-1 antagonist were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L, 1 micromol/L and 10 micromol/L (i.e. 10 -9 -10 -5 M), all at 20-minute intervals. The effects of thrombin, PAR1-AP and PAR-1 antagonist were demonstrated by expressing the mean amplitude calculated during the 20-minute period following addition of each drug concentration, as a percentage of the mean amplitude obtained in the 20 minutes prior to any drug addition. This measurement represents percentage contractility or tone, and subtracted from 100%, provides the percentage relaxation value for each bath concentration of vehicle and study compounds. The net relaxant effect of each compound was calculated by subtracting the percentage contractility value calculated for its respective vehicle (for thrombin, PAR1-AP or PAR-1 antagonist), control experiment, at each similar bath concentration. All of the umbilical artery samples used for experimentation for each compound were obtained from different women (i.e. n = 6 for PAR1-AP), for example, was achieved by using umbilical artery samples from 6 different women). The allocation of umbilical artery samples for the different experiments was entirely random. Fresh Krebs Henseleit physiological salt solution was made and buffered daily. KCl solutions were prepared on the day of experimentation. A stock solution of 5-HT (Sigma-Aldrich, Dublin, Ireland) was made up in de-ionised water and diluted with Krebs solution. Thrombin was purchased from Sigma-Aldrich (Dublin, Ireland) and a stock solution of 1100 U/ml prepared in de-ionised water and stored at -20°C. PAR1-AP was purchased from Tocris Cookson Ltd (Bristol, UK) and a stock solution of 1 millimol/L was prepared using deionized water, with subsequent dilutions in PSS. The PAR-1 antagonist was prepared and assayed by methods previously described [ 18 ]. The IC 50  value for antagonist inhibition of platelet aggregation stimulated with 1 micromol/L SFLLRN agonist was determined to be 0.1 micromol/L. It was prepared as a 10 millimol/L stock solution in dimethysulphoxide (DMSO) and stored in room temperature protected from direct light. Final bath concentrations of DMSO, at the highest concentration of PAR-1 antagonist, did not exceed 1%, for both study and vehicle control strips. Serial dilutions of thrombin, PAR1-AP and the PAR-1 antagonist were made using Krebs Henseleit physiological salt solutions. Comparisons of measurements of amplitude for each bath concentration of thrombin, PAR1-AP and the PAR-1 antagonist, or respective control vehicle, were made using a one-way ANOVA. Post-hoc comparisons were made using the Tukey HSD test. A P value <0.05 was accepted as statistically significant. The statistical package SPSS version 10 was used for statistical calculations.
Methods
The study was carried out in the Department of Obstetrics and Gynaecology, University College Hospital Galway, Ireland between May 2002 and April 2003. Sections of human umbilical cord approximately 10 cm in length were excised from the proximal segment of the cord (i.e. closest to the placental attachment) immediately after elective cesarean delivery. Samples were obtained from 17 women after elective cesarean section at term and from one patient after normal vaginal delivery. All pregnancies were uncomplicated and there was no evidence of hypertensive disease or intrauterine fetal growth restriction. The mean maternal age was 32.47 years (range 25–40 years). The median period of gestation was 38 weeks (range 37–42 weeks). The reasons for cesarean section included previous cesarean section (n = 9), breech presentation (n = 6), previous myomectomy (n = 1) and unstable fetal lie (n = 1). At the time of recruitment 4 women were nulliparous and 13 women were parous.Samples were immediately placed in cold buffered Krebs Henseleit physiological salt solution (pH 7.4) of the following composition: potassium chloride 4.7 mmol/L, sodium chloride 118 mmol/L, magnesium sulphate 1.2 mmol/L, calcium chloride 1.2 mmol/L, potassium phosphate 1.2 mmol/L, sodium bicarbonate 25 mmol/L and glucose 11 mmol/L. Maternal written informed consent was obtained prior to tissue collection, and the tissue collection procedure was approved by the Research Ethics Committee at University College Hospital Galway.Umbilical arteries were carefully dissected free of Wharton's Jelly and cut in rings 4–5 mm in axial length. Rings were suspended individually on stainless steel hooks inserted into their lumens and mounted under 2 g (30 mN) of isometric tension, in glass-jacketed tissue baths, as previously described [ 16 , 17 ]. Each bath-contained 10 mL of oxygenated (95% O 2  / 5% CO 2 ) Krebs Henseleit physiological salt solution (PSS) at 37°C and pH 7.4. Rings were allowed to equilibrate for 90 minutes with regular washouts of PSS. During this interval, spontaneous tone developed. After the equilibration period, the vessel rings were challenged with 60 mM potassium chloride (KCl). Three washouts with PSS were carried out once the maximum response had reached a plateau, and a 20 minute recovery period was allowed in order that baseline be attained again. The KCl challenge was performed three times. After the last KCl challenge, 40 minutes recovery was allowed, and contraction was then stimulated by bath exposure of the vessel rings to 5-hydroxytriptamine (5-HT) (10 -7  M). Once maximum contractile response to 5-HT was attained, the rings were allowed to remain at plateau for 20 minutes. Concentration- effect experiments were performed by cumulative additions of thrombin, the PAR1-AP, or the PAR-1 antagonist to the tissue bath. Thrombin was added to the tissue bath at an initial concentration of 0.5 units/mL, and this was increased at 20-minute intervals to 1 unit/mL, 2 units/mL and 3 units/mL respectively. The bath concentrations ranges investigated for PAR1-AP were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L and 1 micromol/L (i.e. 10 -9  - 10 -6 M), and for PAR-1 antagonist were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L, 1 micromol/L and 10 micromol/L (i.e. 10 -9 -10 -5 M), all at 20-minute intervals.The effects of thrombin, PAR1-AP and PAR-1 antagonist were demonstrated by expressing the mean amplitude calculated during the 20-minute period following addition of each drug concentration, as a percentage of the mean amplitude obtained in the 20 minutes prior to any drug addition. This measurement represents percentage contractility or tone, and subtracted from 100%, provides the percentage relaxation value for each bath concentration of vehicle and study compounds. The net relaxant effect of each compound was calculated by subtracting the percentage contractility value calculated for its respective vehicle (for thrombin, PAR1-AP or PAR-1 antagonist), control experiment, at each similar bath concentration. All of the umbilical artery samples used for experimentation for each compound were obtained from different women (i.e. n = 6 for PAR1-AP), for example, was achieved by using umbilical artery samples from 6 different women). The allocation of umbilical artery samples for the different experiments was entirely random.Fresh Krebs Henseleit physiological salt solution was made and buffered daily. KCl solutions were prepared on the day of experimentation. A stock solution of 5-HT (Sigma-Aldrich, Dublin, Ireland) was made up in de-ionised water and diluted with Krebs solution. Thrombin was purchased from Sigma-Aldrich (Dublin, Ireland) and a stock solution of 1100 U/ml prepared in de-ionised water and stored at -20°C. PAR1-AP was purchased from Tocris Cookson Ltd (Bristol, UK) and a stock solution of 1 millimol/L was prepared using deionized water, with subsequent dilutions in PSS. The PAR-1 antagonist was prepared and assayed by methods previously described [ 18 ]. The IC 50  value for antagonist inhibition of platelet aggregation stimulated with 1 micromol/L SFLLRN agonist was determined to be 0.1 micromol/L. It was prepared as a 10 millimol/L stock solution in dimethysulphoxide (DMSO) and stored in room temperature protected from direct light. Final bath concentrations of DMSO, at the highest concentration of PAR-1 antagonist, did not exceed 1%, for both study and vehicle control strips. Serial dilutions of thrombin, PAR1-AP and the PAR-1 antagonist were made using Krebs Henseleit physiological salt solutions.Comparisons of measurements of amplitude for each bath concentration of thrombin, PAR1-AP and the PAR-1 antagonist, or respective control vehicle, were made using a one-way ANOVA. Post-hoc comparisons were made using the Tukey HSD test. A P value <0.05 was accepted as statistically significant. The statistical package SPSS version 10 was used for statistical calculations.Results A recording from control experiments (i.e. without addition of vehicle, thrombin, PAR1-AP or PAR-1 antagonist) demonstrating umbilical artery tone due to bath exposure of the ring to serotonin, for the entire duration of an experiment, is shown in Figure  1A . The mean net spontaneous relaxation of tone observed was 14.39% (SEM = 2.76). Thrombin exerted a potent and cumulative vasodilatory effect on umbilical artery tone in comparison to simultaneous vehicle (PSS) only experiments. A representative recording of the effects of thrombin is shown in Figure  1B . At bath concentrations at or greater than 0.5 units/mL thrombin exerted a significant vasodilatory effect. The net vasodilatory effects of thrombin are provided in Table  1 . The mean net inhibition of tone observed, at maximum thrombin concentration investigated (i.e. 3 units/mL) was 53.5% (SEM = 4.62; n = 6; P < 0.001). Figure 1 Representative recording of  A)  Serotonin induced contraction of umbilical artery,  B)  Serotonin induced contraction followed by cumulative additions of Thrombin and  C)  Serotonin induced contraction followed by cumulative additions of PAR1-AP. Table 1 Net inhibitory effect of thrombin, PAR-1 and PAR1-AP on human umbilical artery tone. Thrombin (n = 6) (% ± SEM) PAR1-AP (n = 6) (% ± SEM) PAR-1 Antagonist (n = 6) (% ± SEM) (P Value) 0.5 U/ml #28.90 ± 2.60 10 -9 M *23.91 ± 5.64 10 -9 M 2.83 ± 3.05 (NS) (0.630) 1 U/ml *35.39 ± 3.91 10 -8 M *37.32 ± 2.29 10 -8 M 3.80 ± 2.33 (NS) (0.471) 2 U/ml *44.72 ± 2.31 10 -7 M *52.39 ± 1.28 10 -7 M 5.60 ± 3.74 (NS) (0.228) 3 U/ml *53.51 ± 4.62 10 -6 M *61.50 ± 1.43 10 -6 M 7.03 ± 5.74 (NS) (0.148) 10 -5 M 6.87 ± 4.48 (NS) (0.815) Values presented represent the mean inhibitory effects on umbilical artery tone i.e. after adjusting for control / vehicle experiments. The values provided represent % inhibition ± standard error of the mean (SEM). The PAR1-AP used was Threonine-Phenylalanine-Leucine-leucine-Arginine-NH 2 . The PAR-1 antagonist used was Ser- pFPhe-pGPe-Leu-Arg-Orn-NH 2 . The values were compared with amplitude measurements observed prior to drug addition (NS not significant, # P < 0.01, * P < 0.001). TFLLR-NH 2 , the PAR1-AP, similarly exerted a cumulative vasodilatory effect on umbilical artery tone. A representative recording of the cumulative effects of PAR1-AP are shown in Figure  1C , with the mean difference in amplitude measurements (i.e. in comparison to simultaneous control experiments) provided in Table  1 . The mean net inhibition of tone observed, at maximum PAR1-AP concentration investigated (i.e. 10 -6 M / 1 micromol/L) was 61.5% (SEM = 1.43; n= 6; P < 0.001). For the PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH 2 , no relaxation of umbilical artery tone in vitro was observed, in comparison to vehicle only control experiments. Figure  2A  demonstrates a representative recording of umbilical artery tone, after exposure to vehicle only (i.e. DMSO added cumulatively). The mean net relaxation of tone observed with vehicle was 70.28% (SEM = 2.98). In Figure  2B , a representative recording of the effects of cumulatively increasing bath exposure of arterial rings to the PAR-1 antagonist is shown. The mean net maximal inhibition exerted was 6.87% (SEM = 1.57; n = 6; P = 0.280). Finally, there was no observed difference in the vasodilatory effects of thrombin, PAR1-AP or the PAR-1 antagonist in relation to parity. Figure 2 Representative recordings of  A)  umbilical artery tone after serotonin induced contraction with exposure to vehicle only (i.e. DMSO added cumulatively), and  B)  the effects of cumulatively increasing bath exposure of arterial rings to Ser- pFPhe-pGPe-Leu-Arg-Orn-NH 2  after serotonin induced contraction.
Results
A recording from control experiments (i.e. without addition of vehicle, thrombin, PAR1-AP or PAR-1 antagonist) demonstrating umbilical artery tone due to bath exposure of the ring to serotonin, for the entire duration of an experiment, is shown in Figure  1A . The mean net spontaneous relaxation of tone observed was 14.39% (SEM = 2.76). Thrombin exerted a potent and cumulative vasodilatory effect on umbilical artery tone in comparison to simultaneous vehicle (PSS) only experiments. A representative recording of the effects of thrombin is shown in Figure  1B . At bath concentrations at or greater than 0.5 units/mL thrombin exerted a significant vasodilatory effect. The net vasodilatory effects of thrombin are provided in Table  1 . The mean net inhibition of tone observed, at maximum thrombin concentration investigated (i.e. 3 units/mL) was 53.5% (SEM = 4.62; n = 6; P < 0.001).Net inhibitory effect of thrombin, PAR-1 and PAR1-AP on human umbilical artery tone.Thrombin (n = 6)(% ± SEM)PAR1-AP (n = 6)(% ± SEM)PAR-1 Antagonist (n = 6)(% ± SEM)(P Value)0.5 U/ml0.5 U/ml10 -9 M10 -9 M10 -9 M10 -9 M1 U/ml1 U/ml10 -8 M10 -8 M10 -8 M10 -8 M2 U/ml2 U/ml10 -7 M10 -7 M10 -7 M10 -7 M3 U/ml3 U/ml10 -6 M10 -6 M10 -6 M10 -6 M10 -5 M10 -5 MValues presented represent the mean inhibitory effects on umbilical artery tone i.e. after adjusting for control / vehicle experiments. The values provided represent % inhibition ± standard error of the mean (SEM). The PAR1-AP used was Threonine-Phenylalanine-Leucine-leucine-Arginine-NH 2 . The PAR-1 antagonist used was Ser- pFPhe-pGPe-Leu-Arg-Orn-NH 2 . The values were compared with amplitude measurements observed prior to drug addition (NS not significant, # P < 0.01, * P < 0.001).TFLLR-NH 2 , the PAR1-AP, similarly exerted a cumulative vasodilatory effect on umbilical artery tone. A representative recording of the cumulative effects of PAR1-AP are shown in Figure  1C , with the mean difference in amplitude measurements (i.e. in comparison to simultaneous control experiments) provided in Table  1 . The mean net inhibition of tone observed, at maximum PAR1-AP concentration investigated (i.e. 10 -6 M / 1 micromol/L) was 61.5% (SEM = 1.43; n= 6; P < 0.001).For the PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH 2 , no relaxation of umbilical artery tone in vitro was observed, in comparison to vehicle only control experiments. Figure  2A  demonstrates a representative recording of umbilical artery tone, after exposure to vehicle only (i.e. DMSO added cumulatively). The mean net relaxation of tone observed with vehicle was 70.28% (SEM = 2.98). In Figure  2B , a representative recording of the effects of cumulatively increasing bath exposure of arterial rings to the PAR-1 antagonist is shown. The mean net maximal inhibition exerted was 6.87% (SEM = 1.57; n = 6; P = 0.280). Finally, there was no observed difference in the vasodilatory effects of thrombin, PAR1-AP or the PAR-1 antagonist in relation to parity.Discussion This study demonstrates that thrombin exerts a potent vasodilatory effect on human umbilical artery vasculature in vitro. While it is known that thrombin has an inhibitory effect on vessel tone in other animal and human vascular tissue types [ 19 - 21 ] this is the first report, to our knowledge, of the direct effects of thrombin on human umbilical artery vasculature. Receptors for thrombin, the PAR family of receptors, are present on vascular smooth muscle cells, and on endothelial cells [ 14 , 22 ], with the effects of thrombin mediated mainly via the PAR 1, 3 and 4 receptor subtypes [ 11 ]. The potential physiological function of thrombin in mediating vascular tone in the umbilical circulation in normal pregnancy is unknown. Our findings also raise questions in relation to the role of the non-thrombotic effects of thrombin in the feto-placental vasculature in disorders of pregnancy such as pre-eclampsia and intrauterine growth restriction, which are classically associated with thrombotic lesions or a relative excess of thrombin [ 1 - 3 ]. The possibility that the vasodilatory effect of thrombin may serve to counteract the diminished perfusion associated with the pathophysiology of these conditions, at least in the early stages of disease, is one hypothesis. A further theory is that the much enhanced uterine contractility elicited by thrombin and PAR1-AP [ 13 ] may be concomitantly associated with a feto-placental vasodilatory effect to maintain good utero-placental blood flow. We have not elucidated the exact mechanism, or mechanisms, of the thrombin mediated alteration in vascular tone in human umbilical artery. We have however demonstrated that a similar effect is elicited by bath exposure of the arterial rings to the specific PAR1-AP, TFLLR-NH 2 . PAR1-APs are recently designed small synthetic peptide ligands which mimic the effects of proteases by binding directly to the activation site of the PAR-1 receptor, bypassing the need for proteolytic cleavage of the receptor. Soluble peptide ligands, as PAR-APs are, can vary greatly in potency as agonists in comparison with proteases [ 10 ]. TFLLR-NH 2  is reportedly one of the more selective PAR1-APs and is deemed most preferable for purposes of studying the physiologic role of PAR-1 [ 10 ]. In view of the fact that PAR-1 function is central to a thrombin-mediated effect in other tissues [ 11 , 14 , 15 ] and that a PAR-1 mediated effect in human umbilical artery vasculature is similar to that elicited by thrombin, the findings from this study are suggestive of a major role for PAR-1 in mediating the vasodilatory effect of thrombin in umbilical artery vasculature. Other possible mechanisms, and the potential role of PARs 3 and 4, have not been evaluated in this study. The PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH 2 , did not alter umbilical artery tone. This peptide is a competitive antagonist i.e., it competes for the agonist binding site and does not appear to have any other activity [ 23 ]. The direct effects of PAR-antagonism had not previously been evaluated on umbilical artery preparations in vitro, and hence their inclusion in this study. We have also previously demonstrated that this specific PAR-1 antagonist exerted a relaxant effect on human myometrium in vitro, the mechanism of which is unknown [ 24 ]. The results observed here serve to confirm that N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH 2  is inactive towards the PAR-1 receptor. The true role of PAR antagonists, in such experiments, is for the purpose of investigation of the potential effects of an agonist at a PAR subtype. Pre-incubation of the arterial rings with PAR-1 antagonist, for example, should alter the response elicited by PAR1-AP. Such experiments, with selective blockade of the PAR subtypes, in normal and diseased pregnancies, are a subject of further studies. There are some limitations to this study. All umbilical cord artery samples were obtained at elective cesarean section. This was included in the design of the study in order to maintain uniformity in terms of mode of delivery, and, to avoid using cord samples that may have undergone excessive traction in the third stage of labor at vaginal delivery. It is unknown whether this latter point is valid, or not, in studies using in vitro umbilical preparations. We are currently performing comparative studies from samples obtained at vaginal delivery to further evaluate this matter. Secondly, the effects of thrombin and PAR-1 modulation, on smaller vessels in the feto-placenta circulation (i.e. placental arteries), and in association with disorders of pregnancy, would compliment our knowledge of the importance of this pathway. Finally, there are limitations in extrapolating from in vitro experiments to the in vivo situation, but the experiments conducted here represent a reliable and valid in vitro model for these vascular preparations.
Discussion
This study demonstrates that thrombin exerts a potent vasodilatory effect on human umbilical artery vasculature in vitro. While it is known that thrombin has an inhibitory effect on vessel tone in other animal and human vascular tissue types [ 19 - 21 ] this is the first report, to our knowledge, of the direct effects of thrombin on human umbilical artery vasculature. Receptors for thrombin, the PAR family of receptors, are present on vascular smooth muscle cells, and on endothelial cells [ 14 , 22 ], with the effects of thrombin mediated mainly via the PAR 1, 3 and 4 receptor subtypes [ 11 ]. The potential physiological function of thrombin in mediating vascular tone in the umbilical circulation in normal pregnancy is unknown. Our findings also raise questions in relation to the role of the non-thrombotic effects of thrombin in the feto-placental vasculature in disorders of pregnancy such as pre-eclampsia and intrauterine growth restriction, which are classically associated with thrombotic lesions or a relative excess of thrombin [ 1 - 3 ]. The possibility that the vasodilatory effect of thrombin may serve to counteract the diminished perfusion associated with the pathophysiology of these conditions, at least in the early stages of disease, is one hypothesis. A further theory is that the much enhanced uterine contractility elicited by thrombin and PAR1-AP [ 13 ] may be concomitantly associated with a feto-placental vasodilatory effect to maintain good utero-placental blood flow.We have not elucidated the exact mechanism, or mechanisms, of the thrombin mediated alteration in vascular tone in human umbilical artery. We have however demonstrated that a similar effect is elicited by bath exposure of the arterial rings to the specific PAR1-AP, TFLLR-NH 2 . PAR1-APs are recently designed small synthetic peptide ligands which mimic the effects of proteases by binding directly to the activation site of the PAR-1 receptor, bypassing the need for proteolytic cleavage of the receptor. Soluble peptide ligands, as PAR-APs are, can vary greatly in potency as agonists in comparison with proteases [ 10 ]. TFLLR-NH 2  is reportedly one of the more selective PAR1-APs and is deemed most preferable for purposes of studying the physiologic role of PAR-1 [ 10 ]. In view of the fact that PAR-1 function is central to a thrombin-mediated effect in other tissues [ 11 , 14 , 15 ] and that a PAR-1 mediated effect in human umbilical artery vasculature is similar to that elicited by thrombin, the findings from this study are suggestive of a major role for PAR-1 in mediating the vasodilatory effect of thrombin in umbilical artery vasculature. Other possible mechanisms, and the potential role of PARs 3 and 4, have not been evaluated in this study.The PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH 2 , did not alter umbilical artery tone. This peptide is a competitive antagonist i.e., it competes for the agonist binding site and does not appear to have any other activity [ 23 ]. The direct effects of PAR-antagonism had not previously been evaluated on umbilical artery preparations in vitro, and hence their inclusion in this study. We have also previously demonstrated that this specific PAR-1 antagonist exerted a relaxant effect on human myometrium in vitro, the mechanism of which is unknown [ 24 ]. The results observed here serve to confirm that N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH 2  is inactive towards the PAR-1 receptor. The true role of PAR antagonists, in such experiments, is for the purpose of investigation of the potential effects of an agonist at a PAR subtype. Pre-incubation of the arterial rings with PAR-1 antagonist, for example, should alter the response elicited by PAR1-AP. Such experiments, with selective blockade of the PAR subtypes, in normal and diseased pregnancies, are a subject of further studies.There are some limitations to this study. All umbilical cord artery samples were obtained at elective cesarean section. This was included in the design of the study in order to maintain uniformity in terms of mode of delivery, and, to avoid using cord samples that may have undergone excessive traction in the third stage of labor at vaginal delivery. It is unknown whether this latter point is valid, or not, in studies using in vitro umbilical preparations. We are currently performing comparative studies from samples obtained at vaginal delivery to further evaluate this matter. Secondly, the effects of thrombin and PAR-1 modulation, on smaller vessels in the feto-placenta circulation (i.e. placental arteries), and in association with disorders of pregnancy, would compliment our knowledge of the importance of this pathway. Finally, there are limitations in extrapolating from in vitro experiments to the in vivo situation, but the experiments conducted here represent a reliable and valid in vitro model for these vascular preparations.Conclusion In conclusion, thrombin exerts a potent vasodilatory effect on umbilical artery preparations in vitro. A similar effect is also observed using a specific PAR1-AP. The potential non-thrombotic role of thrombin, and PAR subtype modulation, in regulation of the feto-placental circulation in normal pregnancy, and in pregnancies complicated by hypertensive disease or intrauterine growth restriction, is highlighted by these findings.
Conclusion
In conclusion, thrombin exerts a potent vasodilatory effect on umbilical artery preparations in vitro. A similar effect is also observed using a specific PAR1-AP. The potential non-thrombotic role of thrombin, and PAR subtype modulation, in regulation of the feto-placental circulation in normal pregnancy, and in pregnancies complicated by hypertensive disease or intrauterine growth restriction, is highlighted by these findings.Authors' contributions AJO'L performed the experiments and wrote the manuscript. CJO'S and NR performed the experiments. AMF analysed the data and wrote the manuscript. JTE provided the PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH 2 ) and wrote the manuscript. JJM designed, supervised the study and wrote the manuscript. All authors read and approved the final manuscript.
Authors' contributions
AJO'L performed the experiments and wrote the manuscript. CJO'S and NR performed the experiments. AMF analysed the data and wrote the manuscript. JTE provided the PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH 2 ) and wrote the manuscript. JJM designed, supervised the study and wrote the manuscript. All authors read and approved the final manuscript.