Exploring the use of venous blood samples for diagnosis and monitoring of patients with pulmonary hypertension, and determining the genetic base of disease.

Pulmonary hypertension (PH) is a fatally progressive disease leading to right
heart failure and death. Lung ECs from PH patients exhibit unbalanced
production of vaso-constrictive, proliferation inducing vascular mediators such
as endotheline-1 and thromboxane, in excess of the countering vasodilators
prostaglandin and nitric oxide, ultimately leading to vasoconstriction,
proliferation and a progressive increase in pulmonary vascular pressure and
resistance.

Diagnosis of PH and assessment of hemodynamic status in response to treatment
is done by invasive right heart catheterisation. An elevation of mean pulmonary
arterial pressure exceeding 25 mmHg confirms the presence of PH. This procedure
is a recurrent burden for the patient, as the effect of treatment is under
constant evaluation and many catheterisations are needed. Direct assessments of
the primary disease process in PH, such as proliferation of lung microvascular
endothelial cells (MVECs), is only rarely possible as it requires biopsy of
lung tissue. Therefore, an alternative method to assess the vasculature of
patients with PH is warranted. These methods would not only aid in the
management of patients, but also in research on the pathophysiology of PH,
which now relies on tissue samples obtained after lung transplantation or
autopsy.

In this study, we will explore the use of venous blood sampling as an
alternative for invasive diagnostic tests. Platelets will be isolated from
blood to study expression patterns of DNA and RNA and quantify processes of
proliferation and inflammation. Endothelial colony forming cels (ECFCs) will be
isolated from venous blood to cell to determine whether these cells are valid
alternatives for lung MVECs.


Third part of the study is completely optional; subjects choose wether they
want to participate in this study. It is possible to participate in the rest of
the study, without undergoing genetical research. Using this research, we aim
to identify mutations explaining and predicting the onset of pulmonary
hypertension. Hereby we'll be able to identify patient groups at risk in the
future. We perform this research in collaboration with the university of
Cambridge. All subjects will be offered a consult with the clinical genetics,
who will explain the possible consequences of genetic screening. Patients
choose wether they want to be informed about possible relevant mutations
explaining the onset of pulmonary hypertension. Accidental findings will never
be shared with the patient.

The main objective of this study is to obtain valuable biomarkers that are
useful in the prediction of the presence of (subtypes of PH) and/or treatment
responses. Nucleic acid expression patterns will be determined in plasma and
peripheral blood. ECFCs from patients with PH are compared to ECFCs from
control subjects regarding key phenotypic abnormalities and their reaction to
un-physiologically high flow. In addition we'll use genetic sreening to
identify the genetic basis of pulmonary hypertension. This part of the study is
' optional' and will be performed in collaboration with the University of
Cambridge.

The VU University Medical Center (VUmc) is the national referral center for
suspected PH patients, who are all potential study candidates. Although the
routine diagnostic work-up at the time of diagnosis and during work-up is
extensive (including invasive hemodynamic assessment), patients are highly
motivated to undergo these investigations, as they have a strong desire to know
the exact nature of their disease and response to therapy. In this setting and
infrastructure, patients undergo diagnostic right heart catheterization to
confirm the diagnosis of PH or the establish the current hemodynamic status
under treatment. During this essential clinical procedure, a 80 mL blood draw
can be considered as a minimal burden.

As a control group, age matched healthy subjects will be included. Blood will
be drawn by venous punction, which is a very low risk, routine procedure done
by experienced doctors from the VUmc. All mentioned techniques are operational
at our institute. We aim to include 50 samples from healthy control subjects.
Control subjects will be recruited from our department, from healthy volunteers
and out of the direct environment of the patient (relatives, friends), from
patients undergoing a right heart catheterization for diagnostic purposes, but
with a normal hemodynamic profile and healthy volunteers. Experiments will be
performed on samples matched for age, gender and smoking status.

The procedure of extracting nucleic acids from plasma/platelets and culturing
ECFCs from blood samples is an operational procedure in our research lab. ECFCs
from patients with PH have successfully been grown. For the purpose of our
research we will examine prospectively collected ECFCs from PAH patients and
control subjects, as well as MVECs cultured from explanted PAH lung tissue and
lungs derived from major lung resections for cancer treatment. We will
prospectively collect ECFCs and MVECs from single patients (blood draw prior to
lung transplantation or lung surgery), but our ECFC-MVEC comparisons will also
be done on unrelated sample pairs. In all cell types, proliferation rates,
apoptosis resistance and protein expression will be determined. In addition,
ECFC reaction to flow will be quantified and compared to MVEC. The underlying
signalling cascades and genome expression leading to abnormal EC behaviour will
be investigated. Furthermore, for those patients who have consented, genetic
screening will be performed on PBMCs (whole genome and exome sequencing). This
part of the study will be done in collaboration with the University of
Cambridge.

Overall benefit
By the development of blood derived biomarkers we can decrease the invasiveness
of patient diagnosis of PH and follow up. By these methods we expect to be able
to diagnose earlier, provide better follow up and identify groups at risk as
well as predict treatment response in individual patients.

Risk assessment
Venous punction will be done by highly qualified medical doctors of the
Department of Pulmonology VUMc. Occasionally punction can cause a hematoma. The
total amount of blood withdrawn will be 80 ml per patient for the complete
study. The total time taken for the procedure will not exceed 10 min.