U.S. patent application number 15/305879 was filed with the patent office on 2017-03-30 for method of testing for pulmonary hypertension.
This patent application is currently assigned to TOHOKU UNIVERSITY. The applicant listed for this patent is TOHOKU UNIVERSITY. Invention is credited to Nobuhiro KIKUCHI, Kimio SATOH, Hiroaki SHIMOKAWA.
Application Number | 20170089924 15/305879 |
Document ID | / |
Family ID | 54332266 |
Filed Date | 2017-03-30 |
United States Patent
Application |
20170089924 |
Kind Code |
A1 |
SHIMOKAWA; Hiroaki ; et
al. |
March 30, 2017 |
METHOD OF TESTING FOR PULMONARY HYPERTENSION
Abstract
A primary object of the present invention is to provide a method
for conveniently and accurately testing for pulmonary hypertension.
To achieve this object, the present invention provides a method for
testing for pulmonary hypertension using as an indicator the
concentration of selenoprotein P protein in a sample derived from a
subject.
Inventors: |
SHIMOKAWA; Hiroaki; (Miyagi,
JP) ; SATOH; Kimio; (Miyagi, JP) ; KIKUCHI;
Nobuhiro; (Miyagi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TOHOKU UNIVERSITY |
Miyagi |
|
JP |
|
|
Assignee: |
TOHOKU UNIVERSITY
Miyagi
JP
|
Family ID: |
54332266 |
Appl. No.: |
15/305879 |
Filed: |
March 31, 2015 |
PCT Filed: |
March 31, 2015 |
PCT NO: |
PCT/JP2015/060198 |
371 Date: |
December 14, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/68 20130101;
G01N 33/6893 20130101; G01N 2333/47 20130101; G01N 2800/12
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2014 |
JP |
2014-088531 |
Claims
1. A method of testing for pulmonary hypertension comprising
performing a test for pulmonary hypertension using as an indicator
the concentration of selenoprotein P protein in a sample derived
from a subject.
2. The method according to claim 1 comprising: [1] measuring the
concentration of selenoprotein P protein in a sample derived from a
subject; and [2] assessing the presence or absence of pulmonary
hypertension and/or the risk of pulmonary hypertension, based on
the results obtained in [1].
3. The method according to claim 2, wherein when the concentration
of selenoprotein P protein is higher than a predetermined cutoff
value, the subject is assessed as having pulmonary hypertension
and/or as being at risk for developing pulmonary hypertension.
4. The method according to claim 1, wherein the sample derived from
a subject is plasma.
5. A biomarker for detecting pulmonary hypertension, comprising
selenoprotein P protein.
6. A test kit for pulmonary hypertension, comprising a means for
measuring the concentration of selenoprotein P protein in a sample
derived from a subject.
7. A method for treating pulmonary hypertension comprising the
steps of: (i) testing for pulmonary hypertension using as an
indicator the concentration of selenoprotein P protein in a sample
derived from a subject; and (ii) subjecting a subject in which
pulmonary hypertension is detected to a procedure for treating
pulmonary hypertension and/or preventing progression of pulmonary
hypertension.
8. The method according to claim 2, wherein the sample derived from
a subject is plasma.
9. The method according to claim 3, wherein the sample derived from
a subject is plasma.
Description
TECHNICAL FIELD
[0001] The present invention mainly relates to a method of testing
for pulmonary hypertension. The present invention further relates
to a biomarker for detecting pulmonary hypertension, and a test kit
for pulmonary hypertension.
BACKGROUND ART
[0002] Pulmonary hypertension is a disease in which pulmonary
artery blood pressure is increased due to a variety of causes.
According to WHO classification or the like, pulmonary hypertension
is classified into pulmonary arterial hypertension (PAH), pulmonary
hypertension due to left heart disease, pulmonary hypertension due
to lung disease and/or hypoxia, chronic thromboembolic pulmonary
hypertension (CTEPH)), and pulmonary hypertension of unknown cause
due to a combination of factors (Non-patent Literature (NPL) 1 to
5). In Japan, pulmonary hypertension includes many diseases
designated as intractable diseases (specified diseases). In the
case of patients at the terminal stage, lung transplantation may be
necessary.
[0003] For example, in pulmonary arterial hypertension, pulmonary
artery blood pressure abnormally increases due to stenosis of
distal pulmonary arteries. Abnormal growth and denaturation of
endothelial cells and vascular smooth muscle cells are considered
to cause stenosis of distal pulmonary arteries. Pulmonary arterial
hypertension is a lethal disease with a five-year survival rate of
20% in untreated patients. Early diagnosis is difficult, even for
cardiologists. When diagnosed, patients are often already at the
terminal stage. Although there are still no means other than lung
transplantation for saving critically ill patients, the number of
donors is limited, and there are many cases in which patients die
while awaiting transplantation. The disease advances quickly in
many younger-age cases, and death due to right heart failure
frequently occurs, even when multidrug therapy is used. In recent
years, there has been an increase in the number of pulmonary
hypertension patients in Japan. However, the causes of this disease
have yet to be fully clarified.
[0004] With the progression of therapeutic methods in recent years,
if pulmonary hypertension can be accurately diagnosed, sufficient
therapeutic effects can be expected. In particular, if the disease
is found early, therapeutic effects as well as prognostic
improvement can be expected. For example, if pulmonary arterial
hypertension is found at an early stage and an appropriate
procedure is performed, the five-year survival rate is about
50%.
[0005] However, subjective symptoms in the early stage of pulmonary
hypertension patients are limited to nonspecific symptoms, such as
shortness of breath during exertion, and fatigue. Therefore,
doctors must determine, mainly through medical interviews, the
probability that the patient has pulmonary hypertension.
Furthermore, to accurately test for pulmonary hypertension, right
heart catheterization, lung ventilation-blood flow scintigraphy,
chest imaging CT, and like tests must be performed. Specifically,
hospitalization is necessary to perform pulmonary hypertension
tests, thus imposing a great burden on both patients and health
care providers.
[0006] Under such circumstances, there has been desired a method
for easy and accurate testing for pulmonary hypertension, the
method being performed on an outpatient basis by blood sampling or
the like mainly for the purpose of screening.
[0007] Selenoprotein P protein is one of the Se-containing proteins
(a member of the selenoprotein family), and is a protein containing
multiple selenocysteine residues. It has also been found that
selenoprotein P protein is one of the causes of insulin resistance
in type 2 diabetes, and that extracellular stimulation of
selenoprotein P protein suppresses intracellular AMPK activity in
hepatocytes (NPL 6). However, selenoprotein P protein, including
its signaling pathway, remains largely unknown.
CITATION LIST
NPL
[0008] NPL 1: The Japanese Circulation Society: Pulmonary
Hypertension Treatment Guidelines (revised 2012) [0009] NPL 2: J Am
Col Cardiol, 2009; 54 (suppl): S78-84 [0010] NPL 3: Circulation,
2009; 119: 2250-2294 [0011] NPL 4: Eur Heart J, 2009; 30: 2493-2537
[0012] NPL 5: Euro Respir J, 2009; 34: 1219-1263 [0013] NPL 6: Cell
Metabolism, 2010; 12: 483-495 [0014] NPL 7: J Am Col Cardiol, 2013;
62 (25 suppl): D34-41
SUMMARY OF INVENTION
Technical Problem
[0015] A main object of the present invention is to provide a
method of testing for pulmonary hypertension, and, in particular, a
method capable of easily and accurately testing for pulmonary
hypertension. The present invention further relates to a biomarker
for detecting pulmonary hypertension, and a test kit for pulmonary
hypertension.
Solution to Problem
[0016] The present inventors conducted extensive research to
achieve the above. As a result, the inventor found that pulmonary
hypertension can be tested by using as an indicator the
concentration of selenoprotein P protein in a sample derived from a
subject. The present invention has been accomplished through
further research based on this finding.
[0017] Specifically, the present invention includes the following.
[0018] Item 1: A method of testing for pulmonary hypertension
comprising performing a test for pulmonary hypertension using as an
indicator the concentration of selenoprotein P protein in a sample
derived from a subject. [0019] Item 2: The method according to
claim 1 comprising: [0020] [1] measuring the concentration of
selenoprotein P protein in a sample derived from a subject; and
[0021] [2] assessing the presence or absence of pulmonary
hypertension and/or the risk of pulmonary hypertension, based on
the results obtained in [1]. [0022] Item 3: The method according to
claim 2, wherein when the concentration of selenoprotein P protein
is higher than a predetermined cutoff value, the subject is
assessed as having pulmonary hypertension and/or as being at risk
for developing pulmonary hypertension. [0023] Item 4: The method
according to any one of claims 1 to 3, wherein the sample derived
from a subject is plasma. [0024] Item 5: A biomarker for detecting
pulmonary hypertension, comprising selenoprotein P protein. [0025]
Item 6: A test kit for pulmonary hypertension, comprising a means
for measuring the concentration of selenoprotein P protein in a
sample derived from a subject. [0026] Item 7: A method for treating
pulmonary hypertension comprising the steps of: [0027] (i) testing
for pulmonary hypertension using as an indicator the concentration
of selenoprotein P protein in a sample derived from a subject; and
[0028] (ii) subjecting a subject in which pulmonary hypertension is
detected to a procedure for treating pulmonary hypertension and/or
preventing progression of pulmonary hypertension.
Advantageous Effects of Invention
[0029] The present invention provides a method for easily and
accurately testing for pulmonary hypertension. Because, for
example, the testing method of the present invention can be
performed by testing patients' blood, and can be performed on an
outpatient basis, little burden is imposed on both patients and
health care providers.
[0030] The testing method of the present invention is expected to
allow pulmonary hypertension patients to be proactively tested, and
thus receive effective therapy early.
BRIEF DESCRIPTION OF DRAWINGS
[0031] FIG. 1 shows the results of measuring the plasma
concentration of selenoprotein P protein in pulmonary hypertension
patients (PH, N=146) and control patients (Control, N=19), which
are shown in a box plot. The plasma concentration (ng/ml) of
selenoprotein P protein in pulmonary hypertension patients
increased. P<0.001
[0032] FIG. 2 shows the results of detecting selenoprotein P
protein in pulmonary blood vessel smooth muscle cells derived from
idiopathic pulmonary arterial hypertension patients. FIG. 2 shows
the results of four independent trials under each of the following
conditions: at normoxia; at hypoxia; and at hypoxia in the presence
of fasudil (hypoxia+fasudil). In FIG. 2, the upper column shows the
detection of selenoprotein P protein in whole cell lysates by
western blot analysis, whereas the lower column shows the detection
of selenoprotein P protein in conditioned medium by western blot
analysis.
[0033] FIG. 3 shows the results of measuring the plasma
concentration of selenoprotein P protein in pulmonary hypertension
patients (PH, n=203) and control patients (Control, n=20), which
are shown in a box plot.
[0034] FIG. 4 shows the results of measuring the plasma
concentration of selenoprotein P protein in patients with each type
of pulmonary hypertension, which are shown in a box plot. The
asterisks and circles in FIG. 4 represent the outliers.
DESCRIPTION OF EMBODIMENTS
1. Method of Testing for Pulmonary Hypertension Pulmonary
Hypertension
[0035] The testing method of the present invention is to test for
pulmonary hypertension.
[0036] Pulmonary hypertension includes diseases in which increased
pulmonary arterial pressure (mPAP, for example, 25 mmHg or more) is
observed as a clinical observation. Pulmonary hypertension include
pulmonary arterial hypertension (PAH), pulmonary hypertension due
to left heart disease, pulmonary hypertension due to lung disease
and/or hypoxia, chronic thromboembolic pulmonary hypertension
(CTEPH), other pulmonary hypertension of unknown cause due to a
combination of factors, and the like.
[0037] An example of a pathological condition of pulmonary
hypertension is pulmonary arterial hypertension. Examples of
significant clinical observations of pulmonary arterial
hypertension include increased pulmonary artery pressure (mPAP, for
example, 25 mmHg or more) and normal pulmonary wedge pressure
(right atrial pressure) (PCWP, for example, 15 mmHg or less) (these
can be measured, for example, by right heart catheterization);
pulmonary blood flow distribution anomalies are not observed (these
can be measured by, for example, lung ventilation-blood flow
scintigraphy).
[0038] Pulmonary arterial hypertension is a lethal disease in which
blood vessel endothelial function deterioration, vascular smooth
muscle cell growth, inflammatory cell infiltration, etc., have a
complex interplay in the progression of wall-thickening or
narrowing of pulmonary microvessels (pulmonary vascular
remodeling). Its early diagnosis is difficult, even for
cardiologists. When introduced to an institution for lung
transplantation, patients are often already at the terminal stage.
Although there are still no means other than lung transplantation
for saving critically ill patients, the number of donors is
limited, and there are many cases in which patients die while
awaiting transplantation. Among these, the disease advances quickly
in many idiopathic pulmonary arterial hypertension patients
(Idiopathic PAH, IPAH), and shows advanced pulmonary artery
remodeling characterized by abnormal growth of pulmonary artery
vascular smooth muscle cells. Therefore, control of right heart
failure is difficult, even with multidrug therapy, and there are
still many cases in which patients die at a young age. Accordingly,
early diagnosis and treatment are necessary before the clinical
stage advances.
[0039] Pulmonary arterial hypertension includes pathological
conditions such as idiopathic pulmonary arterial hypertension
(IPAH)), heritable pulmonary arterial hypertension,
drug/poison-induced pulmonary arterial hypertension, pulmonary
arterial hypertension associated with other pathological
conditions, persistent pulmonary hypertension of newborns,
connective tissue disease, portopulmonary hypertension (portoPH),
congenital heart diseases (shunt PAH), and the like.
[0040] Pathological conditions of pulmonary hypertension other than
pulmonary arterial hypertension are also known. Specifically,
reference can be made to guidelines in Japan (NPL 1), guidelines in
Europe and America (NPL 2 to 5), etc.
[0041] The 2013 Nice Conference redefined pulmonary hypertension,
and reclassified clinical classification (in this specification,
this is referred to as "Nice Classification") (NPL 1, NPL 7).
Testing Method
[0042] The testing method of the present invention uses as an
indicator the concentration of selenoprotein P protein in a sample
derived from a subject.
[0043] "Testing" as used herein includes testing for the presence
or absence of pulmonary hypertension and testing for the risk of
pulmonary hypertension. Preferably, "testing" refers to testing for
the presence or absence of pulmonary hypertension. "Testing for the
risk" includes testing and determination of the presence or absence
of a probability of developing pulmonary hypertension in the
future. "Testing" can be paraphrased as "determination" or
"diagnosis."
Sample and Subject
[0044] In the testing method of the present invention, the sample
is obtained from a subject.
[0045] The sample is derived from a subject to be tested by the
testing method. The subject is not particularly limited, and
examples thereof include mammals including humans. Examples of
non-human mammals include mice, rats, dogs, cats, cattle, sheep,
horses, and the like. The subject of the testing method of the
present invention is preferably a human. When the subject is a
human, examples of subjects include patients having any subjective
symptom that is characteristic of pulmonary hypertension, such as
shortness of breath during exertion, or chest discomfort, and
preferably patients suspected to have pulmonary hypertension.
[0046] When the subject is a human, the sex, age, and race of the
subject are not particularly limited. In a preferable embodiment of
the present invention, the subject is an Asian person (e.g.,
Japanese, Chinese, or South Korean) and is particularly preferably
Japanese. In one preferable embodiment of the present invention,
the subject is female in view of a statistically larger number of
patients.
[0047] The sample is preferably a blood sample derived from the
subject. Specific examples of the blood sample include blood (whole
blood), blood-derived serum and plasma, and the like. The blood
sample is preferably plasma. Plasma is a part of blood obtained by
removing corpuscular components from blood. For example, it can be
obtained as a supernatant when subjected to centrifugal separation
under conditions in which blood is not solidified (for example, in
the presence of sodium citrate).
[0048] The blood vessel from which the blood sample is derived is
not limited. The blood can be collected from a systemic circulation
blood vessel (arteries (peripheral arteries), veins (peripheral
veins), and capillaries) or a pulmonary circulation blood vessel
(pulmonary arteries, pulmonary veins, and pulmonary capillaries).
From the viewpoint of collecting blood easily, it is preferable
that the blood is collected from a systemic circulation blood
vessel, in particular, from a vein (peripheral vein). Blood can be
collected from a pulmonary circulation blood vessel upon right
heart catheterization.
[0049] The testing method of the present invention preferably
comprises the steps of: [0050] [1] measuring the concentration of
selenoprotein P protein in a sample derived from a subject; and
[0051] [2] assessing the presence and/or absence of pulmonary
hypertension or the risk of pulmonary hypertension, based on the
results obtained in [1] above.
Concentration of Selenoprotein P Protein in Sample
[0052] In the testing method of the present invention, the
concentration of selenoprotein P (SeP) protein in a sample is used
as an indicator.
[0053] Selenoprotein P protein is a known protein. Human
selenoprotein P protein is one of the Se-containing proteins (a
member of selenoprotein family) encoded on the selenoprotein P,
plasma, 1 (SEPP1) gene locus, and is a protein containing multiple
selenocysteine residues. Selenoprotein P protein is known to mainly
secrete from the liver extracellularly. The "P" in the term refers
to "plasma."
[0054] Selenoprotein P protein is known to be a cause of insulin
resistance in type 2 diabetes mellitus. It has also been found that
extracellular stimulation of selenoprotein P protein suppresses
intracellular AMPK activity in hepatocytes (NPL 6). However,
selenoprotein P protein, including its signaling pathway, remains
largely unknown. In particular, selenoprotein P protein's
involvement in pulmonary hypertension is unknown.
[0055] For example, the amino acid sequences of human (Homo
sapiens) selenoprotein P protein and mouse (Mus musculus)
selenoprotein P protein, as well as the amino acid sequences of
mRNA encoding these proteins, have been registered at GenBank
provided by the National Center for Biotechnology Information
(NCBI), under the following accession numbers (it should be
understood that when multiple revisions have been registered, each
number refers to the latest revision): [0056] Human selenoprotein P
Proteins: NP_005401, NP_001078955, NP_001087195; [0057] Human
selenoprotein P mRNA: NM_005410, NM_001085486, NM_001093726; [0058]
Mouse selenoprotein P Proteins: NP_033181, NP_001036078,
NP_001036079; and [0059] Mouse selenoprotein P mRNA: NM_009155,
NM_001042613, NM_001042614.
[0060] The means for measuring the blood concentration of
selenoprotein P protein can be suitably selected by persons skilled
in the art. Examples of preferable means include immunoassay using
an antibody specifically detecting selenoprotein P protein (i.e.,
an antibody specifically binding to selenoprotein P protein)
(including full length of antibody (immunoglobulin protein)
molecules and fragments such as F(ab) and F(ab').sub.2). Examples
of immunoassay include ELISA, EIA, western blot, and the like.
Among these, techniques that can perform a quantitative test, such
as ELISA, are preferable. The antibody that can be used in
immunoassay is not particularly limited as long as it can
specifically detect selenoprotein P protein in a sample.
[0061] The concentration of selenoprotein P protein in a sample can
also be measured by using a commercially available reagent. For
example, a commercially available product can be used as a reagent
for determining the concentration of selenoprotein P protein in a
sample. Examples of commercially available products include, but
are not limited to, a Human Selenoprotein P, SEPP1 ELISA Kit
(produced by Cusabio, Model No.: CSB-EL021018HU). Alternatively, as
antibodies for detecting selenoprotein P protein, commercially
available antibodies specifically binding to anti-selenoprotein P,
such as an anti-selenoprotein P antibody (produced by Santa Cruz
Biotechnology, Model No.: sc-30162), can also be used.
[0062] The determined concentration of selenoprotein P protein in a
sample may be an absolute value and a relative value. Preferably,
an absolute value of the selenoprotein P protein concentration in a
sample is determined.
Assessment Criteria
[0063] In the testing method of the present invention, the presence
or absence of and/or the risk of pulmonary hypertension can be
assessed, based on the measured concentration of selenoprotein P
protein in samples. The assessment criteria for the testing method
can be suitably selected by persons skilled in the art.
[0064] A predetermined cutoff value can be used as assessment
criteria. For example, when the concentration of selenoprotein P
protein is used as an indicator, if the concentration of
selenoprotein P protein in a sample is higher than a predetermined
cutoff value, the patient can be determined to have pulmonary
hypertension, or have a probability of developing pulmonary
hypertension.
[0065] The cutoff value can be determined by various statistical
analysis techniques. Examples of the cutoff value include a median
or mean value in pulmonary hypertension, a value determined based
on ROC curve analysis (e.g., Youden's index), and the like.
Multiple cutoff values can also be set.
[0066] When the concentration of selenoprotein P protein is used as
an indicator, the cutoff value can be set to, for example, about 10
.mu.g/ml. The concentration of selenoprotein P protein in healthy
subjects is usually in the range of about 3 to 8 .mu.g/ml.
Other Indicators
[0067] The testing method of the present invention may be combined
with another known or future testing method for pulmonary
hypertension.
[0068] In one embodiment of the present invention, the testing
method may comprise a step of measuring a tricuspid regurgitation
pressure gradient (TRPG) in a subject. The TRPG can be measured by
cardiac ultrasonography. The TRPG refers to a difference between a
pressure of blood squeezed out of the heart and a pressure of blood
having passed through the valve, which can be calculated by
echocardiography. In general, when the TRPG is about 50 mmHg or
more, the subject can be determined to have a high probability of
having pulmonary hypertension. In the testing method according to
this embodiment of the present invention, the subject can be
determined to have a high probability of having pulmonary
hypertension not only when the TRPG is about 50 mmHg or more, but
also when the TRPG is about 50 mmHg or less and the measured
concentration of selenoprotein P protein in a sample is equal to or
higher than a predetermined cutoff value.
[0069] Pulmonary hypertension in a subject is thus tested.
[0070] The subject determined to have pulmonary hypertension or
have a risk of developing pulmonary hypertension by the testing
method of the present invention is preferably subjected to a
thorough examination of the presence or absence of pulmonary
hypertension and/or identification of a lesion by right cardiac
catheterization, lung ventilation-blood flow scintigraphy, chest
computed tomography (CT), lung arteriography, optical coherence
tomography (OCT), or the like.
[0071] In particular, the patient determined to have a high
probability of having pulmonary hypertension by a thorough
examination is preferably subjected to an appropriate procedure for
treating and/or preventing progression of pulmonary hypertension.
For example, when pulmonary hypertension is pulmonary arterial
hypertension, examples of appropriate procedures include
administration of endothelin receptor antagonists (e.g., bosentan,
ambrisentan), administration of prostaglandin I.sub.2
(prostacyclin) preparations, administration of phosphodiesterase-5
(PDE-5) inhibitors (e.g., sildenafil and tadalafil), and the like.
These treatments can be used singly, or in a combination of two or
more. From the viewpoint of high therapeutic effects, combination
therapy, in particular, triple-combination therapy, is
preferable.
[0072] Although it is unnecessary for a subject who was not
assessed as having pulmonary hypertension or as being at risk of
developing pulmonary hypertension to receive immediate care,
receiving sufficient follow-up is preferable.
2. Kit
[0073] The present invention further provides a kit for testing for
pulmonary hypertension.
[0074] The kit of the present invention includes a means for
measuring the concentration of selenoprotein P protein in a sample
derived from a subject.
[0075] Examples of means for measuring the concentration of
selenoprotein P protein include the above-mentioned means for
performing immunoassay etc. using an antibody that specifically
detects selenoprotein P protein. Specific examples include
antibodies that specifically detect selenoprotein P protein and/or
other reagents for performing immunoassay, such as ELISA, EIA, or
western blot.
[0076] The kit of the present invention may further include other
components, if necessary. Examples of such other components
include, but are not limited to, instruments for collecting a
sample (e.g., syringes), positive control samples (e.g., samples
derived from patients confirmed to have pulmonary hypertension),
negative control samples (e.g., samples derived from patients
confirmed not to have pulmonary hypertension (healthy subjects)),
and the like. The kit may also include a procedure manual for
performing the test method.
[0077] The kit of the present invention can be produced by
appropriately preparing the above-mentioned components according to
a usual method.
[0078] Although the usage of the kit is not particularly limited,
the kit is preferably used in the testing method described above.
When the kit is used in the testing method described above, the
testing for pulmonary hypertension can easily be conducted.
3. Method for Treatment
[0079] The present invention further provides a method for treating
pulmonary hypertension. The "treatment" or "treating" as used
herein includes treatments for pulmonary hypertension and
maintenance therapies for symptomatic relief and recurrence
prevention.
[0080] The method for treatment of the present invention comprises
the steps of: [0081] (i) testing for pulmonary hypertension using
as an indicator the concentration of selenoprotein P protein in a
sample derived from a subject; and [0082] (ii) subjecting the
subject to a treatment for treating pulmonary hypertension and/or
preventing progression of pulmonary hypertension.
[0083] In step (i), the testing method described in the
above-mentioned section "1. Method of Testing for Pulmonary
Hypertension" is performed.
[0084] Subsequently, the subject in whom pulmonary hypertension was
detected is subjected to a treatment for treating pulmonary
hypertension and/or preventing progression of pulmonary
hypertension (step (ii)).
[0085] Between steps (i) and (ii), there may be conducted a
thorough examination of the presence or absence of a cardiovascular
disease and/or identification of a lesion by a thorough
examination, such as cardiac catheterization or angiography
(including X-ray angiography, computed tomographic (CT)
angiography, and nuclear magnetic resonance (MR) angiography).
[0086] As a procedure for treating pulmonary hypertension and/or
preventing progression of a cardiovascular disease, a known
appropriate procedure is performed. For example, when the pulmonary
hypertension is pulmonary arterial hypertension, procedures
include, for example, administration of endothelin receptor
antagonists (e.g., bosentan, ambrisentan); administration of
prostaglandin I.sub.2 (prostacyclin) preparations; administration
of phosphodiesterase-5 (PDE-5) inhibitors (e.g., sildenafil and
tadalafil); and the like. These treatments can be used singly, or
in a combination of two or more. From the viewpoint of high
therapeutic effects, combination therapy, in particular,
triple-combination therapy, is preferable.
[0087] Thus, pulmonary hypertension is treated.
EXAMPLES
[0088] The present invention is described in more detail below with
reference to Examples etc. However, the present invention is not
limited to these examples.
[0089] The statistical analysis and production of box plots were
performed using IBM SPSS statistics.
Example 1
Test for Pulmonary Hypertension
[0090] The plasma concentration of selenoprotein P protein in
pulmonary hypertension patients and non-pulmonary hypertension
patients was measured. The pulmonary hypertension patients as
subjects were patients diagnosed as having pulmonary arterial
hypertension.
Patient Group
[0091] The blood sampled from the following patient groups was
subjected to testing. [0092] (i) Non-pulmonary hypertension patient
(non-PH) group (control group): 19 persons (a group of healthy
subjects who were suspected of having a heart disease and underwent
a thorough examination, including cardiac catheterization, and in
whom no lesion was observed); and [0093] (ii) a group of pulmonary
hypertension (PH) patients: 146 persons.
[0094] This research was approved beforehand by the Medical Ethics
Committee of Tohoku University, and performed with the informed
consent and approval of every patient.
Method
[0095] Plasma was separated from peripheral vein-derived blood
samples and pulmonary artery-derived blood samples obtained from
the above-mentioned patient groups at right heart catheterization.
The plasma concentration of selenoprotein P protein was measured by
ELISA using a Human Selenoprotein P, SEPP1 ELISA kit (produced by
Cusabio Inc.; Model No. CSB-EL021018HU).
Results and Discussion
[0096] FIG. 1 shows the results. As shown in FIG. 1, it became
clear that pulmonary hypertension patients have a plasma
concentration of selenoprotein P protein significantly higher than
that of non-pulmonary hypertension patients.
Example 2
Pulmonary Hypertension Test
[0097] The plasma concentration of selenoprotein P protein was
measured in pulmonary hypertension patients and in non-pulmonary
hypertension patients in the same manner as in Example 1.
Patient Group
[0098] The blood sampled from the following patient groups was
tested. [0099] (i) Non-pulmonary hypertension patient (non-PH)
group (control group): 20 persons; and [0100] (ii) pulmonary
hypertension patient (PH) group: 183 persons.
[0101] The detailed breakdown of the pulmonary hypertension
patients is as follows: [0102] connective tissue disease [CPAH;
Group 1, 4)-1, Nice Classification]: 25 persons; [0103] chronic
thromboembolic pulmonary hypertension [CTEPH; Group 4, Nice
Classification]: 51 persons; [0104] idiopathic pulmonary arterial
hypertension [IPAH; Group 1, 1, Nice Classification]: 58 persons;
[0105] pulmonary hypertension associated with lung diseases and/or
hypoxemia [Lung-PAH; Group 3, Nice Classification]: 10 persons;
[0106] pulmonary hypertension associated with unknown multi-factor
mechanisms [non-categorized; Group 5, Nice Classification]: 13
persons; [0107] portopulmonary hypertension [portoPH; Group 1,
4)-3, Nice Classification]: 6 persons; and [0108] congenital heart
disease [shunt PAH; Group 1, 4)-4, Nice Classification]: 20
persons.
[0109] This research was approved beforehand by the Medical Ethics
Committee of Tohoku University, and performed with the informed
consent and approval of every patient.
Method
[0110] Plasma was separated from the blood samples obtained from
the above patient groups in the same manner as in Example 1. The
selenoprotein P protein concentration in plasma was measured.
Results and Discussion
[0111] FIGS. 3 and 4 show the results. FIG. 3 shows the measurement
results of all of the pulmonary hypertension patients. FIG. 4 shows
the results of each type of pulmonary hypertension. The results
revealed that irrespective of the type of pulmonary hypertension,
all types of pulmonary hypertension patients had a significantly
higher plasma concentration of selenoprotein P protein than
non-pulmonary hypertension patients.
Reference Example 1
[0112] The response of pulmonary artery smooth muscle cells (PASMC)
in idiopathic pulmonary arterial hypertension (IPAH) patients
(hereinafter also referred to as IPAH-PASMC) to hypoxia stimulation
was observed.
Material
[0113] Primary cell lines were established from pulmonary vascular
smooth muscle in idiopathic pulmonary arterial hypertension
patients, in accordance with the following document: Ogawa A,
Nakamura K, Matsubara H, Fujio H, Ikeda T, Kobayashi K, Miyazaki I,
Asanuma M, Miyaji K, Miura D, Kusano K F, Date H, Ohe T,
Circulation, 2005; 112: 1806-1812. The pulmonary vascular smooth
muscle in idiopathic pulmonary arterial hypertension patients was
isolated from pulmonary arteries having an outer diameter of 1.5 mm
or less.
Culture Conditions
[0114] IPAH-PASMC was cultured in D-MEM medium supplemented by 10%
FBS (fetal bovine serum) at 37.degree. C. under constant
temperature and constant humidity conditions at 95% air and 5%
CO.sub.2 in accordance with a usual method. In this test, passage 4
to 7 cells at a confluence of 70 to 80% were used.
Test Method
[0115] The cells to be used for the test were cultured for 24 hours
under one of the following conditions: at normoxia (oxygen
concentration: 21%); at hypoxia (oxygen concentration: 2%); and at
hypoxia in the presence of fasudil (hydroxyfasudil concentration:
10 .mu.M).
[0116] After culturing, whole cell lysates and a conditioned medium
were prepared for each sample, and selenoprotein P protein was
detected by western blot analysis.
Results and Discussion
[0117] FIG. 2 shows the results. It became clear that the pulmonary
blood vessel smooth muscle cells derived from idiopathic pulmonary
arterial hypertension patients promote extracellular secretion of
selenoprotein P protein (into the medium) at hypoxia than at
normoxia. In the presence of fasudil, which is an inhibitor of Rho
kinase (ROCK), enhanced secretion of selenoprotein P protein was
not observed, even at hypoxia.
[0118] The above experimental results suggest that in the pulmonary
artery vascular smooth muscle cells derived from pulmonary arterial
hypertension patients, selenoprotein P protein is secreted Rho
kinase-dependently by hypoxia stimulation.
* * * * *