An adequate education

blood pressure

VEGF is known to have a vasodilatory and hypotensive effect (Henry TD et al., 2003). We were concerned that uterine artery administration of Ad.VEGF-A165might lead to a sudden and detrimental drop in maternal blood pressure. In order to assess the safety of adenoviral mediated VEGF gene transfer, maternal haemodynamic parameters were monitored telemetrically continuously 3 days

before and 4 to 7 days after Ad.VEGF-A165vector injection in 4 animals. For these experiments, a PhysioTel^®Implantable Transmitter with a blood-pressure sensitive catheter (PA-D70 device, Data Sciences International Tilburg,

Netherlands) was inserted into the maternal carotid artery just before placement of flow probes around the uterine arteries. Blood pressure and heart rate were

measured for 3 days before vector injection in order to derive baseline values (to compare the post-injection values with) and to examine the diurnal variation.

Measurements were then taken continuously round the clock after vector injection over the period that would coincide with the time of maximum gene expression, which is normally from 12 hours post-infection to 1 week.

To study the long-term effects of Ad.VEGF-A165vector injection, I also recorded maternal haemodynamic parameters daily until the end of gestation.

Measurements were taken for 1 hour at the same time of the day in each animal to avoid diurnal variation. It was not possible to continuously monitor all 4 animals until the end of pregnancy because only one animal could be monitored at one time.

There was no significant change in maternal mean arterial pressure (MAP) from pre-injection baseline values in the time period immediately following the vector injection. Five days after vector injection, the MAP had fallen from 92.78+6.34 mmHg to 90.41+8.05 mmHg (n=4, paired t-test p=0.28). In normal ovine pregnancy, there is a small non-significant fall in MAP during the third trimester (105-136 days) (Kitanaka T et al., 1989). Maternal HR was not significantly different and changed from 106+9.4 beats per minute (bpm) to 108+8.6 bpm (p=0.46) in the five days following Ad.VEGF-A165administration.

The MAP and heart rate at term were not significantly different than the values at vector injection. The following figures (3.4-3.7) summarize the changes in MAP and heart rate short term and long term after the administration of the vector.

Figure 3.4 – Diurnal variation in maternal blood pressure before and after administration of Ad.VEGF-A165and Ad.LacZ to the uterine arteries of

pregnant sheep

A blood pressure-sensitive haemodynamic catheter was implanted in the carotid artery of mid-gestation pregnant sheep. The uterine arteries were injected with Ad.VEGF-A₁₆₅and Ad.LacZ contra-laterally one week later. The maternal blood pressure was monitored continuously day and night for 2-5 days before, and 3-7 days after the administration of the vector. There was no significant acute change in maternal blood pressure in the period following the vector injection.

Purple = Systolic blood pressure,Green = Mean arterial pressure;Red = Diastolic pressure

Figure 3.5 – Daily variation in maternal blood pressure before and after administration of Ad.VEGF-A165and Ad.LacZ to the uterine arteries of

pregnant sheep

A blood pressure-sensitive haemodynamic catheter was implanted in the carotid artery of mid-gestation pregnant sheep. The uterine arteries were injected with Ad.VEGF-A₁₆₅and Ad.LacZ contra-laterally one week later. The maternal blood pressure was monitored for one hour each morning at the same time of the day, before and after the administration of the vector, until term.

There was no significant change in maternal blood pressure following the vector injection.Purple

= Systolic blood pressure,Green = Mean arterial pressure;Red = Diastolic pressure

Figure 3.6 – Diurnal variation in maternal heart rate before and after administration of Ad.VEGF-A165and Ad.LacZ to the uterine arteries of

pregnant sheep

A blood pressure-sensitive haemodynamic catheter was implanted in the carotid artery of mid-gestation pregnant sheep. The uterine arteries were injected with Ad.VEGF-A165and Ad.LacZ contra-laterally one week later. The maternal heart rate was monitored continuously day and night for 2-5 days before, and 3-7 days after the administration of the vector. There was no significant acute change in maternal heart rate in the period following the vector injection.

Figure 3.7 – Daily variation in maternal heart rate before and after administration of Ad.VEGF-A165and Ad.LacZ to the uterine arteries of

pregnant sheep

A blood pressure-sensitive haemodynamic catheter was implanted in the carotid artery of mid-gestation pregnant sheep. The uterine arteries were injected with Ad.VEGF-A₁₆₅and Ad.LacZ contra-laterally one week later. The maternal heart rate was monitored for one hour each morning at the same time of the day, before and after the administration of the vector, until term. There was no significant change in maternal heart rate following the vector injection.

Overall this data shows that there was no statistically significant effect seen on systemic maternal haemodynamics upon vector administration either short-term or long-term, suggesting there is unlikely to be any acute or chronic clinically important manifestation. A clinically important effect may be one wherein the magnitude of difference may be 20 mm Hg or greater, as in the case of severe haemorrhage. However, our experimental animals showed no

detrimental signs of health. They resumed normal feeding a couple of hours after surgery, were active and displayed no evident signs of distress.

3.5 Local administration of Ad.VEGF-A

to the uterine arteries does not lead to significant changes in fetal blood pressure

In order to further assess the safety of Ad.VEGF-A165administration to the uterine arteries of pregnant sheep, fetal haemodynamic parameters were

monitored telemetrically (n=4). For these experiments, a PhysioTel^®Implantable Transmitter with two blood-pressure sensitive catheters (D70-PCTP device, Data Sciences International Tilburg, Netherlands) was used, with one catheter being chronically implanted in the fetal carotid while the other one was placed in the amniotic cavity. This device can transmit in a range slightly exceeding one meter and hence, we arranged our two receivers in a way to maximize free animal movement while maintaining a good signal. The monitoring room set-up is graphically illustrated in Figure 2.1. Fetal blood pressure and heart rate data were transmitted telemetrically to the receivers which were in turn connected by cable to a computer in the adjacent room. Uploaded traces were viewed using the Dataquest software (Data Sciences International, Tilburg, Netherlands) then, selected traces were copied onto a database (Microsoft Office Excel 2003 software, Microsoft Corporation, USA) for further analysis. The difference between the fetal arterial and amniotic fluid pressure was used to calculate the true fetal BP. Data were collected continuously for 3 days before and 7 days after vector injection and then for 1 hour a day until the end of gestation (n=2 ewes).

During the monitoring schedule, short intermittent periods of signal loss were initially noticed when the ewe strayed away from the receivers. After repositioning of the receivers, this was kept to a minimum. Using this set-up, the signal could be detected by one of the two receivers at any one time, providing data for hourly monitoring in 100% of the diurnal and daily monitoring periods.

3.5.1 Fetal blood pressure and heart rate can be measured telemetrically long term

In the first procedure we performed, the catheter was placed into the common carotid artery. Fetal BP and HR data were collected and appeared to be in the normal range for the first 11 days after catheter placement. Adenovirus vector injection was performed uneventfully 7 days after catheter placement. On the 12^thday after catheter placement, there was a sudden drop in BP and HR followed by fetal death. Post-mortem examination performed 48 hours later showed no fetal abnormality, haemorrhage or structural damage, and culture of tissues from the uterus and fetus revealed no evidence of microbial invasion. The tip of the catheter was found lying in the bicarotid trunk where it may have caused a partial obstruction of blood flow to both common carotid arteries. As there is some evidence that cannulation of the fetal sheep carotid arteries can result in ischemic brain damage especially when bilateral obstruction has occurred (Dodic M et al., 1998), we attempted to cannulate the second fetus through the femoral artery as an alternative route. On examining the fetal femoral artery at

hysterotomy, this proved impossible to achieve at this gestational age due to the discrepancy between the small diameter of the femoral artery when compared to the catheter tip. The second fetus was thus cannulated through the common carotid artery as before. Fetal death again occurred suddenly, on the 8^thday after catheter placement, and one day after vector injection. The catheter tip was observed at the bicarotid trunk in a similar way to the preceding fetus. There were no abnormal findings at post mortem examination.

In the subsequent two procedures we adjusted our technique to thread the catheter tip further upstream the carotid artery in an attempt to bypass the

bicarotid trunk and reach the descending aorta (Figure 3.8). We used the distance between the point of insertion and the middle third of the sternum as an estimate of the threading distance which was approximately 7cm in both fetuses. The cannulation of the common carotid artery in these two fetuses was uncomplicated, and both survived until the end of gestation. At scheduled post mortem

examination in one fetus (138 days of gestation), the catheter tip was found to be

lying in the right common carotid artery just below the point of insertion. This migration of the catheter tip might have been a result of fetal growth and movement. We were not able to ascertain the position of the catheter tip in the other fetus, which was unexpectedly born overnight on the day of the scheduled post mortem at term (139 days of gestation). During birth the catheter was pulled out from the carotid artery without obvious haemorrhage or structural damage to the fetal neck. This lamb survived until planned post mortem examination at four months of age.

Figure 3.8 – Vascular anatomy of the neck arteries of fetal sheep

1 – Position of blood pressure sensitive catheter tip in first two experiments (placement in the right common carotid artery) which resulted in fetal death due to downward migration of catheter tip and obstruction of the bicarotid trunk. 2 – Position of catheter tip in subsequent two experiments (placement in the descending aorta) which allowed long-term monitoring of fetal haemodynamics.

Picture adapted and modified from

http://www.wikidoc.org/index.php/Image:Aortic_variations_truebovine.jpg

3.5.2 Short-term and long-term changes in fetal blood pressure

There was no significant change in fetal MAP from baseline values in the time period immediately following the vector injection. Three days after vector injection, the fetal MAP had fallen from 38.29+3.17 mmHg at baseline to 37.97+3.72 mmHg (n=3, paired t-test, p=0.28). Fetal heart rate was not

significantly different and changed from 178+12.4 bpm to 171+10.9 bpm in the three days following Ad.VEGF-A165administration. In one fetal sheep, the change in MAP and heart rate was investigated during vector injection and subsequent occlusion. The fetal MAP and heart rate increased by a maximum of 8mm Hg and 14 bpm respectively 3 minutes after occlusion and remained elevated until 30 minutes after release of occlusion. Figure 3.9 summarize the changes in MAP and heart rate short-term and long-term after vector injection.

Figure 3.9 – Representative figures showing changes in fetal blood pressure and heart rate after administration of Ad.VEGF-A165and Ad.LacZ to the

uterine arteries of one pregnant sheep (UA29)

A blood pressure-sensitive haemodynamic catheter was implanted in the carotid artery of fetal sheep between 100-110 days gestational age (term=145 days). The maternal uterine arteries were

injected with Ad.VEGF-A₁₆₅and Ad.LacZ contra-laterally one week later. The fetal blood pressure and heart rate were monitored continuously round the clock for 3 days before and 5 days after the administration of the vector (A and C respectively). Fetal blood pressure and heart rate were also monitored for one hour each morning at the same time of the day, before and after the administration of the vector, until term (B and D respectively). There were no significant or detrimental changes in fetal haemodynamics following vector administration.Purple = Systolic blood pressure,Green = Mean arterial pressure;Red = Diastolic pressure;Blue = Heart Rate

Overall this data shows that there is no statistically significant effect seen on systemic maternal haemodynamics upon vector administration either short-term or long-short-term, suggesting there is unlikely to be any acute or chronic clinically important manifestation.

3.6 Ad.VEGF-A

transduction of uterine arteries in the pregnant sheep results in long term changes in vascular reactivity.

I investigated the vascular responses of uterine arteries transduced with Ad.VEGF-A165and Ad.LacZ long-term, approximately 30-45 days after local administration of the adenovirus vector. At 4 – 7 days after adenovirus-mediated gene transfer of VEGF to the uterine arteries, a significant reduction in the contractile response and a significant enhancement of the relaxation response were seen (David AL et al., 2008). We therefore studied both types of response in these long term transduced vessels.

3.6.1 Local over-expression of VEGF-A₁₆₅in the uterine arteries of pregnant sheep results in a diminished contractile response in twin and singleton pregnancies.

Organ bath experiments were conducted on the 2^ndand 3^rdbranches of the main uterine artery. I initially studied all the four branches (main, 1^st, 2^ndand 3^rd branches). The magnitude of the contractile responses generated in the main uterine artery and its 1^stbranch however were very large resulting in deformation of the L-shaped pins of the organ bath. Hence, I studied only the 2^ndand 3^rd branches for our pharmacology experiments. This is in keeping with published