TET2 Expression in Pediatric Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare severe pulmonary arteriopathy
with poor survival due to development of right-sided heart failure. PAH can
occur in children (pediatric PAH) and adults. When PAH is diagnosed, pulmonary
vasodilators that are used as a therapy, improve survival, but the disease
cannot be cured, leaving heart and lung transplantation as the ultimate
treatment option. We thus need additional therapies for PAH.
PAH is an inflammatory vasculopathy. A master-regulator of inflammation is
interleukin (IL)-1β. The secretion of IL-1β, and also IL-18, is controlled by
inflammasomes. While serum IL-1β and IL-18 are elevated in adult PAH patients,
suggestive of inflammasome activation, it is unclear whether the inflammasome
is activated in pediatric PAH. Our findings in a rat model of pediatric PAH
have shown that specifically the NLRP3 inflammasome is activated during PAH and
that its inhibition ameliorates pediatric PAH. We now ask whether the NLRP3
inflammasome is activated in pediatric PAH and may be a therapeutic target; and
at the same time we aim to identify biomarkers for inflammasome activation in
pediatric PAH. Recent studies in adult PAH have suggested that low expression
of ten eleven translocation 2 (TET2) in peripheral blood mononuclear cells
(PBMCs) may be a biomarker for inflammation-associated PAH. We now aim to
define whether this is also the case in pediatric PAH.

We aim to identify if pediatric PAH patients have increased plasma IL-1β and
IL-18 compared to control participants, as well as increased caspase-1 cleavage
and NLRP3, IL-1β and IL-18 mRNA expression in PBMCs, reflective of inflammasome
activation. We also aim to compare TET2 mRNA expression in PBMCs between the
pediatric PAH group and the control group in order to interrogate its potential
as biomarker for inflammation-associated pediatric PAH.
We hypothesize that PAH patients show increased serum concentrations of IL-1β,
IL-18, as well as increased caspase-1 cleavage and NLRP3, IL-1β, and IL-18 mRNA
expression and decreased mRNA expression of TET2 in PBMCs.

The current study is a single-center, cross-sectional, case-control
observational study. The study population consists of PAH patients and control
individuals who will be children seen at the outpatient clinic of the Beatrix
Children*s Hospital. We will obtain blood from PAH patients and control
patients during an already planned venipuncture at an outpatient visit or as
part of a pre-operative blood work-up for hospital admission. From each
participant, we will collect 15 ml of blood for monocyte and serum isolation.
10 ml will be withdrawn for monocyte isolation, and another 5 ml will be
collected for serum analysis during a venipuncture already performed as part of
their routine clinical care. Processing and storage of the samples will occur
in the Laboratory of Pediatrics in the UMCG.

The control group consists of patients seen at the outpatient clinic of the
Beatrix Children*s Hospital. These patients already undergo venipuncture during
an outpatient visit or during pre-operative blood work-up as part of standard
care. Control participants are recruited from the departments of general
pediatrics, endocrinology, nephrology, and gastroenterology. Inclusion criteria
for control participants involve the absence of a diagnosis related to
pulmonary vascular disease, heart failure, an infection, inflammation or an
immunological comorbidity. All participants with an acute infection or
inflammatory condition with fever (determined through anamnesis) during the
last week before the blood collection, or other systemic symptoms on the day of
blood collection, will be excluded. After blood collection, participants with a
serum of CRP >10 mg/L or monocytes >10% of white blood cell count will be
excluded.

This study has important potential benefits for the pediatric PAH population.
The identification of the IL-1β and IL-18, being the main products of
inflammasome activation, in the serum of the patients has not been reported in
the pediatric population. Validating our preclinical findings in a pediatric
patient population would reinforce our understanding of the role of
inflammation in the pathophysiology of this disease. Understanding the
mechanisms through which PAH progresses allows for identification of potential
therapeutic targets in the future.
Furthermore, describing the role of inflammation in PAH can also result in
better clinical monitoring of the disease. As mentioned before, current
monitoring tools have major limitations in the pediatric population and only
reflect mechanical strain. A biomarker reflecting pathophysiological disease
progression directly, obtained in a minimally invasive way could allow us to
monitor the disease by processes connected to the vascular remodeling of the
pulmonary vasculature compared to the current biomarkers reflecting the right
sided heart failure. Decreased TET2 expression is upstream of NLRP3 activation,
and as such could be used to track inflammation and disease progression.

Regarding the risks, venipuncture has low physical risk, including redness,
bleeding and infection. Venipuncture may cause discomfort and even traumatize
pediatric patients who have a history of extensive hospital admissions. To
minimize the burden, we collect blood samples during an already planned
venipuncture as part of standard care. The amount of blood that will be
withdrawn is negligible (15 ml), even in children. The venipuncture is
performed by specialized personnel. We thus believe that our study adds minimal
burden to the subjects.

Our pediatric PAH cohort also consists of children that have conditions that
impair their decision-making (at least 15% of our cohort) and these children
are therefore not legally competent to make their own decisions. Considering
that these patients already undergo venipuncture for standard care, we consider
the additional burden of participation of these individuals minimal.