Effect of Adrenocorticotropic Hormone on Vascular Endothelial Growth Factor Release in Children Study

Problem: Chronic steroid use causes a wide range of side effects, of which bone disease and
adrenal suppression cause significant morbidity. Bone disease, which includes osteopenia,
fractures, and osteonecrosis, is very common. In patients on chronic steroids, fractures can
occur in up to 30-50%, low bone mineral density (BMD) can occur in up to 50%, and up to 40%
have some degree of osteonecrosis. Another common side effect of steroid use is suppression
of the HPA axis. This can cause a patient's endogenous cortisol and ACTH production to be
reduced, which can take up to several months to return to baseline after discontinuing
steroids. Diagnosing adrenal suppression can be difficult. Literature exists showing that
ACTH can stimulate release of VEGF (in vitro and in animal in vivo studies), which can both
be protective against bone disease and be used as a marker of exogenous cosyntropin
administration. The primary goal of this study is to show that ACTH can increase VEGF levels
in healthy humans.

Bone disease: As stated above, patients taking chronic steroids are at high risk for
significant bone effects. Glucocorticoids cause osteoblast apoptosis and decreased function
while simultaneously decreasing apoptosis of osteoclasts, overall resulting in decreased bone
formation and higher resorption. This leads to low BMD and fractures. Osteonecrosis can also
be due to glucocorticoid causing decreased angiogenesis in high risk areas of bone (i.e. the
femoral head). In rabbits,one study demonstrated that ACTH use protects against osteonecrosis
by stimulating osteoblasts to release VEGF, which maintains good blood flow to these high
risk areas of bone. Another study demonstrated that Cushing Syndrome patients with
ACTH-producing pituitary tumors had less BMD loss than those with adrenal cortisol-producing
tumors. This outcome points toward ACTH being protective against osteopenia (even in a high
steroid state). The mechanism for this protective effect is unclear but could be through ACTH
stimulation of VEGF. It is unknown whether ACTH increases VEGF in humans and if it does the
dose needed and timeframe of the response need to be determined.

Low-dose ACTH stimulation test (LDAST): There are several methods to evaluate adrenal
insufficiency (AI), however the LDAST is best for diagnosing central AI and steroid-induced
adrenal suppression (SIAS). The Metyrapone test is very specific, but it carries the risk of
causing acute AI and requires a hospitalization to administer. The insulin tolerance test is
the gold standard for diagnosing AI, but also carries the risk of causing hypoglycemia. The
"standard" or high-dose (250mcg) ACTH stimulation test is a good test as well for diagnosing
primary AI, but can result in false negatives that miss patients with central AI or SIAS,
which can have significant morbidity (sensitivity for central AI is only 73%). Primary AI can
also be diagnosed with an elevated ACTH level, but central AI and SIAS usually have a low to
normal ACTH. Therefore, the LDAST test was created to help increase the rate of patients with
central AI being diagnosed, with a sensitivity for central AI at 93%. However, there are
several limitations to the LDAST. Cosyntropin is dispensed in 250 mcg vials, which is used
for the high-dose test. and must be diluted to 1 mcg for the LDAST. The medication also runs
the risk of sticking to IV tubing. Therefore, it is occasionally not truly given to the
patient, which can cause a false positive result (the lack of cortisol response to ACTH is
not due to AI, but due to never receiving cosyntropin). Due to these limitations, the
specificity for diagnosing central AI is 90%. This can cause the interpreting physician to
diagnose AI, and prescribe a hydrocortisone, when the patient did not truly have AI.

If the LDAST had a positive control to show that the cosyntropin appropriately reached the
patient, it would help to allow the endocrinologist to recognize a false positive result. As
stated above, VEGF is stimulated by ACTH in animals. If VEGF levels were measured with
cortisol levels, and they rose above a set threshold, the interpreting physician could feel
more comfortable knowing that the test was administered appropriately. For VEGF to be a good
control value, it would need to have a significant rise in response to cosyntropin, and would
need to rise quickly and after one dose (the LDAST lasts one hour) and be independent of the
cortisol response. In the in vitro study above, steroid-treated cells had significant rise in
VEGF within one hour of ACTH treatment, and VEGF stayed elevated for up to four hours. In a
study looking at whether VEGF could be a diagnostic biomarker to differentiate acute stroke
in adults versus stroke mimics, there was a significant elevation of VEGF at the time of
stroke presentation compared to the average normal value (peak median 1700 pg/mL with
interquartile range of 1500-1900; baseline median 466 with interquartile range of 392-649).
The mechanism for rise in VEGF was postulated to be due to a hypoxia stimulus in this case.
However, it seems that VEGF can be acutely stimulated (potentially within one hour of a
stimulus) and has the ability to rise to several standard deviations above the normal median
baseline value in humans.

VEGF: Vascular Endothelial Growth Factor is a cytokine glycoprotein that is responsible for
angiogenesis, or the formation of new blood vessels. It can also maintain blood vessel
density, thickness, and permeability, and it is vital for endothelial cell survival. VEGF is
a family of cytokines, with VEGF-A being the prototype and most common. It is found in the
lungs, kidneys, heart, adrenals, bone, brain, and several other organs. Within the adrenal,
VEGF has been shown to be stimulated by ACTH and can be protective against atrophy in steroid
use. Important in regards to this study, an animal study showed that VEGF can be stimulated
by ACTH outside the adrenal glands, namely in osteoblasts in bone. Osteoblasts have an MC2R
receptor that is stimulated by ACTH, leading to a rise in VEGF levels.

Inclusion Criteria:

- Subjects will be between the ages of 9-18 years old on the day of testing

Exclusion Criteria:

- Currently taking any medication other than over-the-counter medications
(over-the-counter medications will be stopped on the day of the study)

- Steroid use within the prior six months (including IV, oral, inhaled, and intranasal
steroids)

- Oral Contraceptive Pill use within the prior six months

- Any chronic medical conditions

- Pregnancy