Are elevated prolactin levels harmful for the retinal and sublingual microvascular bed?

Besides being a classical pituitary hormone, recent observations indicate that
prolactin could have several other functions as well. In illustration,
prolactin may have a pro-coagulant effect. Additionally prolactin has been
shown to modulate the immune function. Furthermore it may play a role in new
vessel formation through its effect on the vascular endothelium. Full-length
prolactin stimulates angiogenesis in vivo and in vitro, whereas 16 kDa
N-terminal prolactin fragments suppress this process.
Imaging of the retina and sublingual tissue provides insight in the status of
the vascular endothelium and can highlight the different phases of
angiogenesis. There are several techniques to easily show the vasculature in
the retina and the sublingual vasculature.
Of interest, elevated persistent prolactin levels are permitted in older
patients with a prolactinoma. In case this study shows that prolactin
stimulates angiogenesis in the retina and in the sublingual tissues, it is
likely that angiogenic processes elsewhere in the body are also stimulated by
prolactin. This would imply that for patients who have comorbidities in which
angiogenesis plays a role, a high prolactin level is extra unfavourable.
When this study will indicate that prolactin stimulates vessel formation, we
plea for a study in which the effect of prolactin lowering on angiogenesis in
patients with a prolactinoma will be investigated. This would in particular be
interesting for older patients who have co-morbidity in which angiogenesis
plays a role in the pathology, whose prolactin levels are currently not
agressively lowered.
When this study shows that prolactin stimulates new vessel formation, it could
also be interesting to study whether patients without prolactinoma and
co-morbidity in which angiogenesis plays a role, would profit from prolactin
lowering therapy. In healthy patients without a prolactinoma there is such a
variation in physiological prolactin levels, that it can be worthwile to lower
prolactin.

Primary objective: To evaluate whether patients with a prolactinoma have signs
of altered vascular function and/or morphology in the retina or sublingual
tissues in comparison to healthy controls.

Secondary Objectives:
1. To evaluate whether there is a difference in plasma in inflammatory markers,
in coagulation factors, in markers of insulin resistance and in lipids between
patients with a prolactinoma and healthy controls.
2. To evaluate whether patients with a prolactinoma have more albuminuria in
comparison to healthy controls.
3. To evaluate the extent of tube formation after a patient*s plasma is
incubated in the in vitro angiogenesis assay, compared to the extent of tube
formation upon incubation with plasma from healthy controls.
4. To evaluate whether levels of 16 kDa prolactin fragments in patients with a
prolactinoma are different from levels in healthy controls.

Patients visiting the outpatient clinic from the department of Endocrinology or
Internal Medicine with an active prolactinoma will be included in this
case-control study. They should enter and finish the study before start of
prolactin lowering medication. Patients will be asked to bring a person who
will serve as control.
The research physician will perform an anamnesis and physical examination.
Blood will be withdrawn and SDF is done. Furtehrmore, patients and controls are
asked to collect their urine for 24 hours in order to assess the extent of
micro-albuminuria.
A second visit to the AMC is planned for fluoresceine angiography and OCT.

Blood withdrawal
At the first visit blood was withdrawn (40 ml). Factors that play a role in
angiogenesis, inflammation, coagulation and insulin resistance and colesterol
will be studied.

In vitro angiogenesis assay
Endothelial cells will be resuspended in growth medium with patient*s or
control*s plasma, with or without prolactin receptor antagonist. After 16
hours, the extent of endothelial tube formation will be quantified.

Sidestream Dark Field (SDF)
Investigators will use SDF imaging to analyze functional changes in the
sublingual microcirculation. Five consecutive videoclips will be made of
different sites of the sublingual microcirculation. Video clips will be
analyzed blindly and randomly by a resident ophthalmology, using different
approaches, namely a validated semi-quantitative score of bloodflow, and a
quantitative score of vascular capillary density and vessel diameter.

Fluorescein angiography (FAG)
Fluorescein angiography will be conducted in conjunction with colour fundus
photography at baseline. Investigators will use digital fluorescein angiograms
to determine presence or absence of retinal vascular abnormalities. The
following angiographic and photographic procedures are to be followed. After
pupil dilation with tropicamide 1% and prior to fluorescein dye injection, 35
degree red-free and colour photographs will be taken of the maculae of one eye.
Next, 5 ml of fluorescein 10% dye will be injected IV into the antecubital
vein. Photographs of the study eye will be taken during the early transit phase
from 15 to 45 seconds, at 60 to 90 seconds, and at 5 and 10 minutes after
completion of the fluorescein dye injection. Photographs that are taken of the
macula of the study eye during the fluorescein transit will typically include 1
stereoscopic pair at 3-5 minutes. Photographs of the macula of the fellow eye
will be taken at 2 and 10 minutes after fluorescein dye injection. Photographs
will be analyzed afterwards by two blinded ophthalmologists.

Optical coherence tomography (OCT)
An OCT scan will be made of the macular region of the retina of the research
eye. It will give clues about retinal thickness and vascular leakage of retinal
capillaries. In conduction with FAG, the OCT scan will be made. This will take
5 minutes. The images will be analyzed afterwards.

Collection of urine
Patients will be asked to collect urine for 24 hours, in order to assess the
extent of microalbuminuria. Normally not much protein is filtererd by the
kidney. However, in case of renal damage proteins will be filtered more easily.

Participation of healthy controles.
Patients will be asked to bring someone they know iwho s willing to to serve as
a control. In case that patients will not be able to find someone who can serve
as control, we will find those patients through advertisement (please check
appendix for advertisement).

No additional harm is expected due to sublingual and retinal evaluation (SDF,
OCT and fluorescein angiography). These methods are routinely performed and
part of several prior study protocols in the Academic Medical Centre, Amsterdam
(department of Ophthalmology). Patients should stay fasted for the first study
visit to the AMC until blood is withdrawn, and should not take any drinks
containing caffein until SDF is done.
As mentioned in the Introduction section, additional knowledge is needed in
order to evaluate the biological action of elevated prolactin levels on
angiogenesis-related pathology, in order to assess the long term effects of
persistent increased prolactin levels that are currently permitted after middle
age (in both male and female patients with prolactinoma).