Osteopontin in Pulmonary Hypertension

Abstract

Pulmonary hypertension (PH) is a pathological condition with multifactorial etiology, which is characterized by elevated pulmonary arterial pressure and pulmonary vascular remodeling. The underlying pathogenetic mechanisms remain poorly understood. Accumulating clinical evidence suggests that circulating osteopontin may serve as a biomarker of PH progression, severity, and prognosis, as well as an indicator of maladaptive right ventricular remodeling and dysfunction. Moreover, preclinical studies in rodent models have implicated osteopontin in PH pathogenesis. Osteopontin modulates a plethora of cellular processes within the pulmonary vasculature, including cell proliferation, migration, apoptosis, extracellular matrix synthesis, and inflammation via binding to various receptors such as integrins and CD44. In this article, we provide a comprehensive overview of the current understanding of osteopontin regulation and its impact on pulmonary vascular remodeling, as well as consider research issues required for the development of therapeutics targeting osteopontin as a potential strategy for the management of PH.

Full text

Citation: Mamazhakypov, A.;
Maripov, A.; Sarybaev, A.S.;
Schermuly, R.T.; Sydykov, A.
Osteopontin in Pulmonary
Hypertension. Biomedicines 2023,11,
1385. https://doi.org/10.3390/
biomedicines11051385
Academic Editor: Giuseppe
Cappellano
Received: 28 March 2023
Revised: 1 May 2023
Accepted: 5 May 2023
Published: 7 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biomedicines
Review
Osteopontin in Pulmonary Hypertension
Argen Mamazhakypov 1, Abdirashit Maripov 2, Akpay S. Sarybaev 2, Ralph Theo Schermuly 1
and Akylbek Sydykov 1, *
1Department of Internal Medicine, Excellence Cluster Cardio-Pulmonary Institute (CPI), Member of the
German Center for Lung Research (DZL), Justus Liebig University of Giessen, 35392 Giessen, Germany
2Department of Mountain and Sleep Medicine and Pulmonary Hypertension, National Center of Cardiology
and Internal Medicine, Bishkek 720040, Kyrgyzstan
*Correspondence: akylbek.sydykov@innere.med.uni-giessen.de
Abstract:
Pulmonary hypertension (PH) is a pathological condition with multifactorial etiology,
which is characterized by elevated pulmonary arterial pressure and pulmonary vascular remodeling.
The underlying pathogenetic mechanisms remain poorly understood. Accumulating clinical evidence
suggests that circulating osteopontin may serve as a biomarker of PH progression, severity, and
prognosis, as well as an indicator of maladaptive right ventricular remodeling and dysfunction.
Moreover, preclinical studies in rodent models have implicated osteopontin in PH pathogenesis.
Osteopontin modulates a plethora of cellular processes within the pulmonary vasculature, including
cell proliferation, migration, apoptosis, extracellular matrix synthesis, and inflammation via binding
to various receptors such as integrins and CD44. In this article, we provide a comprehensive overview
of the current understanding of osteopontin regulation and its impact on pulmonary vascular
remodeling, as well as consider research issues required for the development of therapeutics targeting
osteopontin as a potential strategy for the management of PH.
Keywords: osteopontin; pulmonary hypertension; biomarkers; right heart failure
1. Introduction
Pulmonary hypertension (PH) is a condition affecting pulmonary vasculature [
1
]
and is hemodynamically defined as a mean pulmonary artery pressure (mPAP) greater
than 20 mmHg at rest as assessed by right heart catheterization [
2
]. PH is classified into
five main clinical groups: pulmonary arterial hypertension (PAH), PH resulting from left
heart disease, PH resulting from chronic lung disease or hypoxia, chronic thromboembolic
PH (CTEPH), and PH with unclear or multifactorial mechanisms [
2
]. PH represents
a chronic and ultimately fatal pulmonary vascular disorder of multifactorial origin [
3
].
The primary pathological characteristics of PH include persistent pulmonary vascular
constriction and excessive obstructive pulmonary vascular remodeling [4]. At the cellular
level, initial pathological mechanisms of pulmonary vascular remodeling are characterized
by dysfunction and apoptosis of pulmonary artery endothelial cells (PAECs). At later
stages, hyperproliferation and apoptosis resistance of pulmonary artery smooth muscle
cells (PASMCs) lead to structural changes in the pulmonary vasculature, elevation in
pulmonary arterial pressure (PAP), and pulmonary vascular resistance, which ultimately
culminate in right ventricular (RV) failure [5].
Despite the multitude of studies conducted in the field of PH research, the underlying
mechanisms of this condition remain an unresolved issue. A plethora of pathological
processes, such as an inherent genetic predisposition [
6
], aberrant metabolic processes [
7
,
8
],
perturbations in apelin signaling pathways [
9
], aberrations in calcium signaling path-
ways [
10
], DNA damage [
11
], mitochondrial dysfunction [
12
], and dysregulation of micro
RNAs (miRNAs) [
13
], has been implicated in the pathogenesis of PH. Various matricellular
Biomedicines 2023,11, 1385. https://doi.org/10.3390/biomedicines11051385 https://www.mdpi.com/journal/biomedicines

Biomedicines 2023,11, 1385 2 of 19
proteins, including osteopontin, have been identified as critical mediators in the patho-
genesis of pulmonary vascular remodeling. Matricellular proteins, as a class of inducible,
multifunctional, secretory, and non-structural proteins, can act as cytokines on a plethora of
cellular mechanisms and function as extracellular matrix proteins located between cells [
14
].
In this article, we provide a comprehensive overview of the recent advancements in our
understanding of the role of osteopontin in the pathogenesis of pulmonary vascular remod-
eling, gleaned from various experimental
in vitro
studies and animal models, and clinical
studies. We will also delve into the challenges faced in the investigation of osteopontin in
PH and explore its potential as a viable therapeutic target.
2. Osteopontin Signaling
The name “osteopontin”, which is composed of two words, was proposed by Oldberg
and colleagues [
15
]. The prefix osteo- is derived from “osteon”, the Greek word for bone,
and reflects the fact that osteopontin was first isolated from the mineralized bone matrix
of bovines as a bone sialoprotein I [
16
]. The suffix -pontin is derived from “pons”, the
Latin word for bridge, and reflects osteopontin’s role as a linking protein between cells and
hydroxyapatite in the matrix [
15
]. Osteopontin is also known as secreted phosphoprotein 1,
uropontin, and early T-lymphocyte activation-1.
As soon as osteopontin was isolated, it was revealed that the matricellular protein
osteopontin is a cytokine that is synthesized and expressed by a wide array of cells and
tissues in the body. The tissues and organs expressing osteopontin include kidney, inner ear,
brain, heart, lung, vessels, skin, and bone marrow [
17
], as well as luminal epithelial surfaces
of the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung,
breast, salivary glands, and sweat glands [
18
]. Osteopontin is also found in biological fluids,
such as blood, milk, urine, and seminal fluid [17]. Osteopontin expression is considerably
upregulated in response to a variety of pathological processes, including inflammation,
mechanical stress, and tissue injury and repair. In various cardiovascular diseases, synthesis
of osteopontin is induced in smooth muscle cells and cardiomyocytes [17].
Osteopontin plays a multifarious role in biological processes such as inflammation, im-
munological response, wound healing, cellular adhesion, migration, survival, and biomin-
eralization. Binding of osteopontin to integrins activates signaling pathways that regulate
diverse cellular functions including cell proliferation, adhesion, invasion, migration, and
fibrosis [19].
Osteopontin also interacts with CD44, a ubiquitously expressed cell-surface receptor,
which exists as a number of isoforms [
20
]. It is encoded by 20 exons, 10 of which are
constant and form the invariant extracellular domain of the smallest standard isoform [
21
].
The variant isoforms are generated by alternative splicing and may contain in addition
to 10 constant exons a single variant exon or a combination of variant exons [
21
]. CD44
has been shown to be involved in cell–matrix and cell–cell interactions [
22
]. Although
hyaluronic acid is considered as a principal ligand for CD44, other extracellular-matrix
proteins including serglycin, collagen, fibronectin, chondroitin sulfate, laminin, and osteo-
pontin also may serve as ligands for CD44 [
22
]. Osteopontin does not interact with the
standard isoform of CD44, but rather binds to its variant isoforms [
23
], such as CD44v6
and CD44v7 [24,25].
Variant CD44 isoforms play critical roles in the development of various cancers
through their interaction with osteopontin [
21
]. The exact role of the osteopontin-mediated
activation of CD44 variants in cardiovascular diseases remains poorly explored and the
literature is scarce. It was demonstrated that the osteopontin–CD44v6 interaction mediates
calcium deposition in valve interstitial cells from patients with noncalcified aortic valve
sclerosis [26].
Histological studies revealed CD44 expression in lung plexiform lesions from patients
with idiopathic PAH (IPAH) [
27
]. Another study showed that adventitial fibroblasts isolated
from chronically hypoxic hypertensive calves displayed increased expression of CD44 along
with
α
V
β
3 and osteopontin [
28
]. However, these studies did not investigate which CD44

Biomedicines 2023,11, 1385 3 of 19
isoforms were upregulated, and did not demonstrate an interaction between CD44 and
osteopontin. Recently, CD44v7–10, CD44v8–10, CD44v9–10, and CD44v10 transcripts
were detected in lungs of mice subjected to chronic hypoxia [
29
]. In PAH patients, the
CD44v8–10 variant was expressed by endothelial-to-mesenchymal transition-like PAECs in
pulmonary vessels with neointimal hyperplasia or occluded vessels, including plexiform
lesions [
29
]. It remains, however, to be elucidated whether osteopontin interacts with these
CD44 variants and which signaling pathways are activated in the setting of PH.
Recent studies showed that osteopontin serves as a ligand for CD275, which regulates
immune responses by activated T cells [
30
]. Alternative splicing of osteopontin generates
three isoforms: osteopontin-a (the full-length isoform), osteopontin-b (lacking exon 5),
and osteopontin-c (lacking exon 4) [
31
]. Alternative translation of osteopontin produces
two isoforms: a cell-secreted full-length and a shortened intracellular protein lacking the
N-terminal signal sequence [
31
]. In addition, osteopontin undergoes numerous posttransla-
tional modifications, such as serine/threonine phosphorylation, sulfation, O-glycosylation,
glutamination, and proteolytic processing, which can subsequently determine the func-
tional variability of osteopontin [
32
]. To date, thrombin, matrix metalloproteinases (MMPs),
caspase-8/3, plasmin, cathepsin D, and enterokinase have been identified as proteases
that cleave osteopontin at different sites, resulting in the formation of several fragments
of different sizes and with variable functions [
33
]. Taken together, the ultimate roles and
functions of osteopontin are impacted by multiple factors, spanning from gene transcription
to protein translation, posttranslational modification, and proteasomal processing of the
final protein product.
Osteopontin-mediated cellular signaling pathways are well explored in cancer dis-
eases. Thus, interaction of osteopontin with cell surface receptors, integrins, and/or CD44
activates JNK, Ras/Raf/MEK/ERK, PI3K/AKT, JAK/STAT, NF-
κ
B, and TIAM1/Rac1
signaling pathways, leading to enhancement of various malignant properties of cancer
cells [
34
]. Unfortunately, the role of signaling pathways activated by osteopontin in pul-
monary hypertension has been poorly studied.
3. Osteopontin as a Biomarker of Pulmonary Hypertension and Right
Ventricular Failure
Circulating biomarkers are of significant importance in clinical medicine as they can
be utilized for diagnostic, prognostic, or therapeutic response monitoring purposes across a
broad range of conditions, including PH [
35
,
36
]. Osteopontin, a secreted circulating protein,
serves as a valuable biomarker due to its accessibility through non-invasive methods such
as peripheral blood sampling, which allows frequent and repeated measurements during
the course of disease or treatment implementation. Circulating osteopontin has been
established as a valuable biomarker of disease severity and adverse outcomes in patients
with various cardiovascular conditions including heart failure [37–39].
PH patients with different etiologies, including IPAH [
40
,
41
], PAH associated with
congenital heart diseases (CHD-PAH) [
42
], PAH associated with connective tissue dis-
eases (CTD-PAH) [
43
] and PH associated with COPD [
44
], display increased osteopontin
expression in the pulmonary vasculature and lung tissue. Osteopontin was found to be
one of the top five overregulated genes in the explanted lung tissues of patients with PH
of various etiologies, and its expression was directly correlated with the severity of the
hemodynamic conditions [
41
]. Another study demonstrated that osteopontin was one of
the nine hub genes in the genome transcriptome datasets evaluated in the lung tissues
of PAH patients [
45
]. In contrast, a recent study demonstrated decreased osteopontin
expression in the lung tissue of PAH patients [46].
Elevated circulating osteopontin levels were reported in patients with various forms
of PH, including IPAH [
47
], CHD-PAH [
42
], CTD-PAH [
43
], and CTEPH [
48
]. Circulating
osteopontin levels were associated with PH development in CTD patients [
43
] and CHD
patients [
42
]. Increased osteopontin levels were also reported in other cardiac conditions
complicated with PH and RV failure, such as dilated cardiomyopathy (DCM) [
49
]. In a

Biomedicines 2023,11, 1385 4 of 19
parallel comparison study, it was found that the levels of circulating osteopontin in PH
patients increased to a similar extent as they did in patients with DCM and left ventricular
hypertrophy compared to healthy controls [
47
]. Several studies demonstrated a correla-
tion of circulating osteopontin levels with a number of hemodynamic parameters such as
mPAP [
42
], pulmonary artery dispensability index [
50
], right atrial pressure [
51
], cardiac
index [
42
], and total pulmonary vascular resistance [
42
] in PH patients. Similarly, corre-
lation of osteopontin levels with pulmonary hemodynamics and several RV remodeling
parameters was demonstrated in patients with DCM [49].
An increase in circulating osteopontin was associated with development of Eisen-
menger syndrome in CHD-PAH patients [
42
]. In PAH patients, baseline circulating os-
teopontin levels predicted survival [
51
–
53
] and increased with worsening of the disease
condition as assessed by six-minute walking distance (6MWD) [
50
,
51
] and New York Heart
Association Functional Classification (NYHA-FC) [51,52].
Patients with CTEPH had higher osteopontin plasma concentrations compared to
patients with pulmonary embolism (PE) [
48
]. Findings of a higher risk for CTEPH develop-
ment during follow-up in patients with lower osteopontin levels at diagnosis of acute PE
suggest that osteopontin might play an important role in thrombus resolution and thus
in the development of CTEPH after PE [
48
]. Interestingly, pulmonary endarterectomy in
CTEPH patients was associated with a further elevation of circulating osteopontin [48].
Studies have demonstrated that circulating osteopontin levels predict RV dysfunction
and remodeling in PAH patients [
50
,
54
]. PH patients with maladaptive RV remodeling
displayed higher levels of circulating osteopontin compared to those with adapted RV and
left heart failure patients with DCM and left ventricular hypertrophy [
47
]. Interestingly,
circulating osteopontin levels were significantly higher in CTEPH patients with maladap-
tive RV remodeling compared to those with adapted RV and to IPAH patients with both
adapted and maladapted RV [47].
Taken together, the accumulated evidence suggests that osteopontin might serve
as a valuable biomarker for detection of perturbations in pulmonary hemodynamics,
exacerbation of RV dysfunction, and prediction of adverse outcomes in patients with
various forms of PH (Table 1and Figure 1).
Table 1.
Summary of clinical studies evaluating circulating osteopontin levels in patients with
various forms of pulmonary hypertension. Abbreviations: PA, pulmonary artery; PH, pulmonary
hypertension; IPAH, idiopathic pulmonary arterial hypertension; RV, right ventricle; NYHA-FC, New
York Heart Association functional classification; WHO-FC, World Health Organization functional
class; RVEDD, RV end-diastolic diameter; TAPSE, tricuspid annulus plane systolic excursion; RV-S
´
,
tricuspid annulus systolic velocity; RVD1, RV basal diameter; RVD2, RV mid-diameter; NT-proBNP,
N-terminal pro-b-type natriuretic peptide; 6MWD, six-minute walking distance; PAP, pulmonary
artery pressure; mPAP, mean PAP; sPAP, systolic PAP; RVD, right ventricular dysfunction; PA,
pulmonary artery; CAD, coronary artery disease; DCM, dilated cardiomyopathy; CTD, connective
tissue disease; CHD, congenital heart disease, COPD, chronic obstructive pulmonary disease; MRI,
magnetic resonance imaging; CI, cardiac index; TPVR, total pulmonary vascular resistance; CTEPH,
chronic thromboembolic pulmonary hypertension; LVH, left ventricular hypertrophy.
Subjects Main Findings Studies
IPAH (n = 35)
Circulating osteopontin levels correlated with WHO-FC and 6MWD. A cut-off of
osteopontin (53.4 ng/mL) predicted significant differences in survival at 4.0 ±
2.2-year follow-up.
[53]
IPAH (n = 95) (retrospective
cohort (n = 70), prospective
cohort (n = 25), control (n = 40)
In both retrospective and prospective cohorts, circulating osteopontin levels correlated
with mPAP and NT-BNP. In the retrospective cohort, osteopontin levels also correlated
with age, 6MWD, and NYHA class. Multivariate Cox analysis demonstrated that
baseline osteopontin levels were independent predictors of mortality.
[51]

Biomedicines 2023,11, 1385 5 of 19
Table 1. Cont.
Subjects Main Findings Studies
PH (n = 71), control (n = 40)
Patients with advanced right heart failure revealed higher levels of circulating
osteopontin compared to less symptomatic ones (NYHA III-IV vs. NYHA I-II).
Osteopontin was a strong independent predictor of all-cause mortality within 24
months of follow-up.
[52]
PH (PAH + CTEPH) (n = 71)
Circulating osteopontin levels correlated with RVEDD, TAPSE, and RV-S
´
. Patients
with RV dysfunction had higher levels of osteopontin compared to those without RV
dysfunction (956 ng/mL vs. 628 ng/mL). ROC analysis revealed that an osteopontin
concentration of 694.2 ng⁄mL detects RV dilatation.
[54]
PH (PAH + CTEPH) (n = 62),
control (n = 12)
Circulating osteopontin levels in PH patients were elevated compared to those in
healthy control subjects. Circulating osteopontin levels predicted decreased 6MWD.
Osteopontin levels were associated with NT-proBNP, RVEDD (echo), RVD (MRI),
and PA distensibility index.
[50]
CAD-COPD (n = 131)
Circulating osteopontin levels correlated with mPAP and 6MWD. Osteopontin
levels > 43 ng/mL were a statistically significant predictor of PH in patients
with CAD-COPD.
[44]
CHD (n = 22), CHD-PAH
(n = 25), control (n = 24)
Circulating osteopontin levels increase with the development of PAH and
Eisenmenger syndrome. Circulating osteopontin levels correlated with mPAP, CI,
and TPVR.
[42]
DCM (n = 70) (DCM without
RVD (n = 15) and with RVD
(n = 55))
Circulating osteopontin levels in DCM patients correlated with RVD1, RVD2, and sPAP.
[49]
PH (n = 62), DCM (n = 34),
LVH (LVH; n = 47), control
(n = 38)
Circulating osteopontin levels were higher in PH, DCM, and LVH patients
compared to those in the controls. Osteopontin concentrations in PH patients with
maladaptive RV were significantly higher than in those with adaptive RV of CTEPH
origin. In PH patients, osteopontin levels were correlated with TAPSE/sPAP,
RVEDD, mPAP, PVR, NT-pro-BNP, NYHA-FC.
[47]
CTD (n = 113)
CTD-PAH patients showed significantly higher circulating osteopontin levels than
patients with CTD alone. Osteopontin levels were independently associated with
PAH diagnosis.
[43]
Although the level of osteopontin in the circulation is elevated in PH patients, the
specific origin of this increase is still unknown. Several lines of evidence point to the pul-
monary vasculature and heart as possible sources of circulating osteopontin. Osteopontin
release from the heart into the coronary circulation in proportion to the left ventricular (LV)
systolic function and volumes was revealed in patients with a previous anterior wall my-
ocardial infarction [
55
]. Plasma osteopontin levels decreased significantly in heart failure
patients following heart transplantation [
38
]. However, it is hypothesized that the elevated
osteopontin may come from both the RV myocardium and the pulmonary vasculature since
both of these compartments are impacted in PH [
52
]. Based on the studies of LV failure, in
which the myocardium is considered the primary source of circulating osteopontin, it can
be expected that both the RV and pulmonary vasculature may contribute to the rise in the
circulating osteopontin in PH. Indeed, lung contribution to circulating osteopontin was
clearly demonstrated previously by measuring the transpulmonary osteopontin gradient
in heart failure patients [
56
] and there is clinical and experimental evidence of increased
osteopontin expression in the lungs [
41
,
57
] as well as in the right ventricle [
57
]. The pos-
sibility that other organs contribute to elevated circulating osteopontin levels, at least in
advanced disease stages with multiple organ dysfunction [
56
], needs further evaluation.
However, the relative contribution of each of these compartments at various stages of the
disease needs to be established. Studies that specifically examine the osteopontin levels in
the circulation, RV and LV myocardium, and pulmonary vasculature, as well as the relation-
ship between osteopontin and disease severity, will be important in shedding light on the
origin of osteopontin in this condition. Identification of the source of elevated osteopontin

Biomedicines 2023,11, 1385 6 of 19
in PH and understanding the underlying mechanisms may help in development of new
diagnostic and therapeutic strategies for patients with this debilitating condition.
Biomedicines 2023, 11, x FOR PEER REVIEW 5 of 20
circulating osteopontin levels, at least in advanced disease stages with multiple organ
dysfunction [56], needs further evaluation. However, the relative contribution of each of
these compartments at various stages of the disease needs to be established. Studies that
specifically examine the osteopontin levels in the circulation, RV and LV myocardium,
and pulmonary vasculature, as well as the relationship between osteopontin and disease
severity, will be important in shedding light on the origin of osteopontin in this condi-
tion. Identification of the source of elevated osteopontin in PH and understanding the
underlying mechanisms may help in development of new diagnostic and therapeutic
strategies for patients with this debilitating condition.
Figure 1. Clinical role of osteopontin. In patients with pulmonary hypertension (PH) of various
etiologies (1), changes to the pulmonary vessels and the right ventricle (RV) (2) cause enhanced
osteopontin release into the bloodstream. Circulating osteopontin can be measured in plasma or
serum samples (3). Numerous studies (4) have found that osteopontin levels are increased in PH
patients, and these elevated levels are linked to invasive hemodynamic alterations, changes in the
functional and structural parameters of the RV, and adverse outcomes. CHD-PAH, pulmonary ar-
tery hypertension associated with congenital heart disease; IPAH, idiopathic pulmonary artery
hypertension; COPD, chronic obstructive pulmonary disease; DCM, dilated cardiomyopathy; CTD,
connective tissue disease; CTEPH, chronic thromboembolic pulmonary hypertension; RA, right
atrium.
Table 1. Summary of clinical studies evaluating circulating osteopontin levels in patients with
various forms of pulmonary hypertension. Abbreviations: PA, pulmonary artery; PH, pulmonary
hypertension; IPAH, idiopathic pulmonary arterial hypertension; RV, right ventricle; NYHA-FC,
New York Heart Association functional classification; WHO-FC, World Health Organization func-
tional class; RVEDD, RV end-diastolic diameter; TAPSE, tricuspid annulus plane systolic excur-
sion; RV-S´, tricuspid annulus systolic velocity; RVD1, RV basal diameter; RVD2, RV mid-diameter;
NT-proBNP, N-terminal pro-b-type natriuretic peptide; 6MWD, six-minute walking distance; PAP,
pulmonary artery pressure; mPAP, mean PAP; sPAP, systolic PAP; RVD, right ventricular dys-
function; PA, pulmonary artery; CAD, coronary artery disease; DCM, dilated cardiomyopathy;
CTD, connective tissue disease; CHD, congenital heart disease, COPD, chronic obstructive pul-
monary disease; MRI, magnetic resonance imaging; CI, cardiac index; TPVR, total pulmonary
vascular resistance; CTEPH, chronic thromboembolic pulmonary hypertension; LVH, left ventric-
ular hypertrophy.
Figure 1.
Clinical role of osteopontin. In patients with pulmonary hypertension (PH) of various etiologies
(
1
), changes to the pulmonary vessels and the right ventricle (RV) (
2
) cause enhanced osteopontin
release into the bloodstream. Circulating osteopontin can be measured in plasma or serum samples
(
3
). Numerous studies (
4
) have found that osteopontin levels are increased in PH patients, and these
elevated levels are linked to invasive hemodynamic alterations, changes in the functional and structural
parameters of the RV, and adverse outcomes. CHD-PAH, pulmonary artery hypertension associated with
congenital heart disease; IPAH, idiopathic pulmonary artery hypertension; COPD, chronic obstructive
pulmonary disease; DCM, dilated cardiomyopathy; CTD, connective tissue disease; CTEPH, chronic
thromboembolic pulmonary hypertension; RA, right atrium.
4. Osteopontin in Pulmonary Vascular Cells
4.1. Osteopontin in Pulmonary Artery Endothelial Cells
Osteopontin plays a crucial role in both the physiology and pathophysiology of en-
dothelial cells in the systemic vasculature. It serves as a vital mediator in the intricate
physiological and pathological processes that govern endothelial cells in the pathogenesis
of several cardiovascular diseases. Various factors, including aldosterone [
58
], vascular
endothelial cell growth factor (VEGF) [
59
], and hypoxia [
60
], regulate osteopontin expres-
sion in endothelial cells, and vice versa, it regulates various functions of endothelial cells.
Osteopontin stimulates endothelial cell differentiation and angiogenesis via its SVVYGLR
fragment [
61
]. It promotes angiogenesis by mediating endothelial cell attachment to the
ECM [
62
] and synthesis of angiogenic factors by endothelial cells [
63
]. Osteopontin en-
hances VEGF expression through AKT and ERK signaling pathways to induce endothelial
cell motility, proliferation, and tube formation via the
α
v
β
3-integrin receptor [
64
]. In turn,
VEGF augments expression of osteopontin and
α
v
β
3-integrin in endothelial cells and stimu-
lates integrin-dependent endothelial cell migration [
59
]. A conserved sequence comprising
nine amino acids (RSKSKKFRR) located at the thrombin cleavage site of osteopontin pro-
motes endothelial cell migration, proliferation, and tube formation
in vitro
[
65
]. The crucial
function of osteopontin in neovascularization is further corroborated by the findings of the
compromised angiogenic capacity of endothelial cells derived from osteopontin knockout

Biomedicines 2023,11, 1385 7 of 19
mice, which is partially restored by exogenous administration of osteopontin [
66
]. Besides
angiogenesis, osteopontin influences several other endothelial cell functions. It increases
vascular permeability by downregulating expression of the tight junction proteins ZO-1
and claudin-5 in endothelial cells [
67
], induces endothelial-to-mesenchymal transition via
CD44 receptor in response to shear stress [
68
] and regulates endothelial cell apoptosis [
69
].
The precise role of osteopontin in the functional regulation of PAECs and its interplay
in the pathogenesis of pulmonary vascular remodeling still remains largely unknown. It
can be hypothesized that elevated circulating osteopontin levels in PH patients may exert
effects on PAECs similar to those on endothelial cells in systemic vasculature, potentially
leading to alterations in endothelial cell physiology leading to subsequent pathological
pulmonary vascular remodeling. This is due to the established fact that endothelial cell
dysfunction plays a crucial role in the pathogenesis of PH, particularly in the early stages
of the disease [
70
]. However, the precise mechanisms by which osteopontin affects PAEC
physiology and its potential contribution to the development of PH remains a subject of
future research.
4.2. Osteopontin in Pulmonary Artery Smooth Muscle Cells
Osteopontin critically influences physiology of systemic vascular smooth muscle cells
(SMCs) through modulation of a myriad of cellular processes, including cell prolifera-
tion [
71
], apoptosis [
72
], migration [
73
,
74
], and neointima formation [
75
]. Various factors,
including hypoxia [
76
], platelet-derived growth factor (PDGF) [
77
], hyperglycemia [
78
],
mechanical stress [
79
], aldosterone [
80
], and reactive oxygen species (ROS) [
81
], regulate
osteopontin expression in SMCs.
In vitro
studies revealed that the cell proliferation rate of
SMCs is directly related to osteopontin expression levels in these cells [
82
], suggesting a di-
rect role of osteopontin in mediating cell proliferation. Autocrine expression of osteopontin
contributed to PDGF-induced SMC migration [
83
] and regulated adhesion of SMCs to the
ECM [
84
] and production of MMPs [
85
]. Osteopontin increased ECM synthesis in SMCs
via activating the p38 MAPK signaling pathway [
86
]. Production of osteopontin in SMCs
was inhibited by a cyclic guanosine monophosphate (cGMP)-dependent protein kinase, an
important mediator of nitric oxide (NO) and cGMP signaling [
87
]. Pharmacological agents
enhancing NO-cGMP signaling might thus potentially attenuate osteopontin expression.
Available data suggest that osteopontin regulates various cellular functions of PASMCs,
similar to the systemic vascular SMCs. Osteopontin was found to be as one of the most
highly overregulated matricellular proteins in senescent PASMCs [
40
]. Its expression in
these cells was associated with an augmented cell migration and proliferation [40], which
were markedly reduced in the presence of neutralizing anti-osteopontin antibodies [40].
Multiple factors with known pathological roles in the pulmonary vascular remodeling,
including acidic fibroblast growth factor [
88
], angiotensin-II [
88
], transforming growth
factor-
β
[
89
], PDGF-BB [
90
], hypoxia [
91
,
92
], sphingosine-1-phosphate [
93
], and mechanical
stretch [
42
], induce osteopontin expression in PASMCs through various signaling pathways.
Acidic fibroblast growth factor induced osteopontin expression in PASMCs via activat-
ing Ras/JNK and Ras/MEK1/2 signaling pathways [
88
], while hypoxia did so via ERK
and p38MAPK signaling pathways [
92
] and mechanical stretch via AKT-ERK signaling
pathways [
42
]. In turn, osteopontin modulates various functions of PASMCs. In partic-
ular, it promoted PASMC proliferation and migration in a dose-dependent manner via
αVβ3-integrin
mediated AKT and ERK1/2 signaling pathways [
42
]. Furthermore, activa-
tion of the calcineurin/NFATc3 signaling pathway by sphingosine-1-phosphate upregulated
osteopontin expression and stimulated PASMCs proliferation, which was suppressed by
the activation of the proliferator-activated receptor gamma (PPAR-γ) [93].
4.3. Osteopontin in Pulmonary Artery Adventitial Fibroblasts
Recent research demonstrated the significance of vascular adventitia and adventitial
fibroblasts in maintaining vascular homeostasis of both systemic [
94
] and pulmonary
vasculature [
95
]. The adventitial fibroblasts are involved in the regulation of various

Biomedicines 2023,11, 1385 8 of 19
cellular processes, including ECM remodeling, inflammation, and angiogenesis, which are
critical for maintaining the structural and functional integrity of the vasculature [
94
,
95
]. In
the systemic vasculature, osteopontin plays a significant role in SMC-mediated regulation
of adventitial fibroblast functions [
96
]. Integrin
β
3 was found to be the responsible receptor
for promoting osteopontin-mediated adventitial fibroblast migration [
97
]. The role of
osteopontin in the physiology of pulmonary vascular adventitial fibroblasts was studied
primarily in the bovine model of PH. Adventitial fibroblasts isolated from calves with
severe hypoxia-induced PH exhibited high proliferative, migratory, and pro-invasive
capabilities. These functional alterations of adventitial fibroblasts were mediated via
activated ERK1/2 and AKT signaling pathways that correlated with high osteopontin
expression [
28
]. Inhibition of osteopontin expression with a specific small interfering RNA
or neutralizing antibodies led to an attenuation of proliferative, migratory, and invasive
capabilities of adventitial fibroblasts [28].
4.4. Osteopontin in Pulmonary Vascular Macrophages
The literature pertaining to the specific functions of osteopontin in the functional
dynamics of macrophages and their interactions in the pathogenesis of PH is scarce. In
hypoxia, osteopontin can act as an inflammatory cytokine, thereby promoting the chemo-
taxis of various inflammatory cells and modulating the inflammatory milieu in the affected
tissue. Although circulating monocytes do not express osteopontin, it is among the most
highly expressed genes in activated macrophages [
98
]. Additionally, it serves as a pow-
erful chemotactic stimulus for these immune cells [
99
]. Osteopontin regulates key func-
tions of macrophages, including migration, survival, phagocytosis, and pro-inflammatory
cytokine production [
100
,
101
]. These effects are mainly mediated via interaction with
α4/α9-integrins by its SLAYGLR domain [102].
The potent pro-inflammatory properties of osteopontin are further augmented fol-
lowing its cleavage by thrombin, which leads to the generation of an N-terminal fragment
containing two integrin-binding domains, namely the RGD and SVVYGLR motifs [
103
].
Osteopontin can contribute to pulmonary vascular remodeling through facilitating attrac-
tion and retention of macrophages and T lymphocytes in areas of inflammation within
pulmonary vessels [
104
]. A single-cell RNA sequencing demonstrated that enhanced os-
teopontin expression in lung tissues from patients with systemic sclerosis was enriched in
macrophages [
98
], suggesting that osteopontin signaling in these cells might contribute to
the pulmonary vascular remodeling in PAH associated with systemic sclerosis.
Thus, available data suggest that osteopontin might contribute to the chemotaxis of
leukocytes and other inflammatory cells to the remodeled pulmonary vasculature and
thereby be involved in the pathogenesis of the disease. However, the intricacies of osteopon-
tin function within pulmonary vascular macrophages and other immunocompetent cells
and their contribution to pulmonary vascular remodeling remain enigmatic, warranting
future investigation.
4.5. Osteopontin in Intercellular Communications of Vascular Cells in Pulmonary
Vascular Remodeling
Elevated circulating osteopontin levels in PH can be attributed to its enhanced syn-
thesis by pulmonary vascular cells. The intricate regulation of osteopontin expression by
various factors including hypoxia, growth factors, mechanical stress, and inflammation
in these cells highlights its complexity. Direct effects of osteopontin on pulmonary vas-
cular cells through both paracrine and autocrine mechanisms suggest its potential role
in regulating cellular proliferation, migration, and other functions in these cells, thereby
potentially serving as a key mediator in intercellular communication within the pulmonary
vasculature (Figure 2). These effects of osteopontin on pulmonary vascular cells adversely
affect the disease and may determine the impact of circulating osteopontin on the status
and outcome of PAH patients. However, the intricacy of these interactions, particularly
concerning the extent of their contribution and hierarchical involvement in the pathological

Biomedicines 2023,11, 1385 9 of 19
processes of pulmonary vascular remodeling, presents a significant challenge in precisely
defining the cell-specific role of osteopontin in pulmonary vascular cells.
Biomedicines 2023, 11, x FOR PEER REVIEW 9 of 20
cells and their contribution to pulmonary vascular remodeling remain enigmatic, war-
ranting future investigation.
4.5. Osteopontin in Intercellular Communications of Vascular Cells in Pulmonary Vascular
Remodeling
Elevated circulating osteopontin levels in PH can be attributed to its enhanced syn-
thesis by pulmonary vascular cells. The intricate regulation of osteopontin expression by
various factors including hypoxia, growth factors, mechanical stress, and inflammation
in these cells highlights its complexity. Direct effects of osteopontin on pulmonary vas-
cular cells through both paracrine and autocrine mechanisms suggest its potential role in
regulating cellular proliferation, migration, and other functions in these cells, thereby
potentially serving as a key mediator in intercellular communication within the pulmo-
nary vasculature (Figure 2). These effects of osteopontin on pulmonary vascular cells
adversely affect the disease and may determine the impact of circulating osteopontin on
the status and outcome of PAH patients. However, the intricacy of these interactions,
particularly concerning the extent of their contribution and hierarchical involvement in
the pathological processes of pulmonary vascular remodeling, presents a significant
challenge in precisely defining the cell-specific role of osteopontin in pulmonary vascular
cells.
Figure 2. Regulation of osteopontin expression and effects of osteopontin in pulmonary vascular
cells. Several factors regulate osteopontin expression in pulmonary vascular cells. In adventitial
fibroblasts, hypoxia induces osteopontin expression, which has implications in cell proliferation
and migration. In pulmonary artery smooth muscle cells, many factors regulate osteopontin ex-
pression, including hypoxia, PDGF (platelet-derived growth factor), TGF-beta (transforming
growth factor beta), FGF1 (fibroblast growth factor 1), Ang-II (angiotensin II), S1P (sphingosine
1-phosphate), and mechanical stress, all of which also regulate cell proliferation and migration.
However, the exact factors regulating osteopontin in pulmonary vascular endothelial cells and
Figure 2.
Regulation of osteopontin expression and effects of osteopontin in pulmonary vascular
cells. Several factors regulate osteopontin expression in pulmonary vascular cells. In adventitial
fibroblasts, hypoxia induces osteopontin expression, which has implications in cell proliferation and
migration. In pulmonary artery smooth muscle cells, many factors regulate osteopontin expression,
including hypoxia, PDGF (platelet-derived growth factor), TGF-beta (transforming growth factor
beta), FGF1 (fibroblast growth factor 1), Ang-II (angiotensin II), S1P (sphingosine 1-phosphate), and
mechanical stress, all of which also regulate cell proliferation and migration. However, the exact
factors regulating osteopontin in pulmonary vascular endothelial cells and macrophages have not
been identified. While osteopontin regulates macrophage chemotaxis, its role in endothelial cells
remains unknown.
5. Osteopontin in Animal Models of Pulmonary Hypertension
Utilization of experimental models for the study of PH has been a crucial methodology
for understanding the underlying mechanisms and causes of this condition, as well as
for the preclinical advancement of the currently approved therapeutic approaches. The
established models of PH continue to play a vital role in facilitating further research and
exploration of potential innovative treatment options for the management of PH [
105
,
106
].
Increased lung expression of osteopontin was demonstrated in several rodent PH mod-
els, including hypoxia exposure in mice [
107
,
108
] and rats [
46
,
88
,
104
] and monocrotaline
injection in rats [
109
,
110
]. These studies suggested that osteopontin release from remod-
eled pulmonary vessels could account for elevated circulating osteopontin levels [
110
].
Examination of lung tissues revealed generation of osteopontin in the remodeled vessels by
pulmonary fibroblasts [
28
,
104
] and PASMCs [
110
,
111
]. In addition, osteopontin levels may

Biomedicines 2023,11, 1385 10 of 19
reflect the extent of pulmonary vascular remodeling. For example, atrial natriuretic peptide
null mice exhibit augmented lung osteopontin expression, which is associated with an inten-
sive pulmonary vascular remodeling [
107
], while mice with mutated transforming growth
factor-beta receptor type 2 display attenuated pulmonary vascular remodeling and blunted
osteopontin expression in response to hypoxia exposure [
108
]. In addition, attenuation
of lung osteopontin expression may indicate beneficial effects of a particular drug tested
in animal models of PH. For example, pioglitazone, an activator of PPAR-
γ
, improved
pulmonary vascular and RV remodeling in monocrotaline rats along with attenuating
osteopontin expression in the lung [
109
]. Similarly, the beneficial effects of calcium-sensing
receptor antagonist NPS2390 [
111
] and serotonin transporter inhibitor fluoxetine [
110
] in
animal PH models was associated with decreased lung tissue osteopontin expression along
with improvement in pulmonary hemodynamics. Collectively, the pulmonary osteopontin
expression level corresponds to the extent of pulmonary vascular remodeling in multiple
animal PH models. These studies demonstrate that osteopontin can be utilized as a means
of monitoring the impact of potential pharmacological agents in rodent PH models.
Several experimental models, including aging-associated and high-flow-induced PH,
have nicely demonstrated the pivotal role of osteopontin in the pathogenesis of pulmonary
vascular remodeling. Mice lacking osteopontin were protected from age-induced pul-
monary vascular remodeling and displayed attenuated pulmonary vascular remodeling
in response to hypoxia exposure [
40
]. In a rat model of shunt-induced PH, pulmonary
expression of osteopontin and its two main receptors
α
V
β
3-integrin and CD44 increased
along with the disease worsening [
42
]. Interestingly, application of an
α
v
β
3-integrin antag-
onist in surgically corrected shunt rats accelerated recovery from the pulmonary vascular
remodeling and RV failure [42].
There is still a lack of studies investigating the specific actions of osteopontin within
individual pulmonary vascular cells using genetically modified mice lacking or overexpress-
ing osteopontin in the experimental PH models (Figure 3). Using cell-specific conditional
knockout animals in addition to global osteopontin knockout animals may add more value
to exploring cell-specific roles of osteopontin in PH. In addition, conditional gene knockouts
allow overcoming some of the limitations of conventional knockouts, such as early embryonic
lethality, compensation by another gene product during development, and alterations of other
organ systems. Another key advantage of conditional gene knockouts is that by knocking out
the gene of interest in specific tissues or cell types, it is possible to assign a phenotype to a
particular cell type. Further, gene inactivation at a specific time point allows determining the
role of the protein in the initiation vs. progression of the disease.
Effects of osteopontin in severe PH models, such as SuHx or monocrotaline injection,
are yet to be investigated. Moreover, there are no experimental studies investigating the
effects of the administration of recombinant osteopontin to increase its levels or the use
of osteopontin-neutralizing antibodies to decrease its levels in circulation in PH models
(Figure 3). Cumulatively, the biological significance of osteopontin in animal PH models
requires further investigation in order to fully comprehend its role in the development and
progression of pulmonary vascular remodeling. This information will provide a conclusive
answer regarding the potential benefits of manipulating osteopontin levels for managing
PH patients.

Biomedicines 2023,11, 1385 11 of 19
Biomedicines 2023, 11, x FOR PEER REVIEW 11 of 20
Effects of osteopontin in severe PH models, such as SuHx or monocrotaline injec-
tion, are yet to be investigated. Moreover, there are no experimental studies investigating
the effects of the administration of recombinant osteopontin to increase its levels or the
use of osteopontin-neutralizing antibodies to decrease its levels in circulation in PH
models (Figure 3). Cumulatively, the biological significance of osteopontin in animal PH
models requires further investigation in order to fully comprehend its role in the devel-
opment and progression of pulmonary vascular remodeling. This information will pro-
vide a conclusive answer regarding the potential benefits of manipulating osteopontin
levels for managing PH patients.
Figure 3. Possible implications of experimental pulmonary hypertension models in future studies.
(A) Among many available mouse and rat pulmonary hypertension models, listed here (hypoxia,
shunt, monocrotaline, sugen plus hypoxia (SuHx), pulmonary artery banding (PAB)), osteopon-
tin-targeted studies were performed only in hypoxia-exposed global osteopontin knockout mice
and in a rat model of shunt-induced pulmonary hypertension using osteopontin receptor (αVβ3)
antagonist. Osteopontin-oriented in vivo studies in other pulmonary hypertension models are still
missing. There have been no studies with cell-specific osteopontin deletion or overexpression in
rodent pulmonary hypertension models. —indicates that animal models with the corresponding
osteopontin manipulations are available; NA—studies with the corresponding animal models and
osteopontin manipulations are not available. (B) No experimental studies have evaluated effects of
Figure 3.
Possible implications of experimental pulmonary hypertension models in future studies.
(
A
) Among many available mouse and rat pulmonary hypertension models, listed here (hypoxia,
shunt, monocrotaline, sugen plus hypoxia (SuHx), pulmonary artery banding (PAB)), osteopontin-
targeted studies were performed only in hypoxia-exposed global osteopontin knockout mice and
in a rat model of shunt-induced pulmonary hypertension using osteopontin receptor (
α
V
β
3) an-
tagonist. Osteopontin-oriented
in vivo
studies in other pulmonary hypertension models are still
missing. There have been no studies with cell-specific osteopontin deletion or overexpression in
rodent pulmonary hypertension models.
Biomedicines 2023, 11, x FOR PEER REVIEW 11 of 20
Effects of osteopontin in severe PH models, such as SuHx or monocrotaline injec-
tion, are yet to be investigated. Moreover, there are no experimental studies investigating
the effects of the administration of recombinant osteopontin to increase its levels or the
use of osteopontin-neutralizing antibodies to decrease its levels in circulation in PH
models (Figure 3). Cumulatively, the biological significance of osteopontin in animal PH
models requires further investigation in order to fully comprehend its role in the devel-
opment and progression of pulmonary vascular remodeling. This information will pro-
vide a conclusive answer regarding the potential benefits of manipulating osteopontin
levels for managing PH patients.
Figure 3. Possible implications of experimental pulmonary hypertension models in future studies.
(A) Among many available mouse and rat pulmonary hypertension models, listed here (hypoxia,
shunt, monocrotaline, sugen plus hypoxia (SuHx), pulmonary artery banding (PAB)), osteopon-
tin-targeted studies were performed only in hypoxia-exposed global osteopontin knockout mice
and in a rat model of shunt-induced pulmonary hypertension using osteopontin receptor (αVβ3)
antagonist. Osteopontin-oriented in vivo studies in other pulmonary hypertension models are still
missing. There have been no studies with cell-specific osteopontin deletion or overexpression in
rodent pulmonary hypertension models. —indicates that animal models with the corresponding
osteopontin manipulations are available; NA—studies with the corresponding animal models and
osteopontin manipulations are not available. (B) No experimental studies have evaluated effects of
—indicates that animal models with the corresponding
osteopontin manipulations are available; NA—studies with the corresponding animal models and
osteopontin manipulations are not available. (
B
) No experimental studies have evaluated effects of
recombinant osteopontin application or osteopontin-neutralizing antibodies. In order to elucidate the
cell specific roles of osteopontin, the Cre/LoxP system can be utilized using cell-specific promoter
systems. Further, to better characterize the role of osteopontin in such models it is recommended to
use invasive catheterization (
C
) to measure right atrial (RA) pressure, right ventricular (RV) systolic
pressure (RVSP), RV diastolic pressure (RVDP), aortic pressure, left ventricular (LV) systolic pressure
(LVSP) and LV diastolic pressure (LVDP). Echocardiographic imaging (
D
) of the heart is also war-
ranted to inform additional characteristics of the RV including both systolic and diastolic functions,
including the following parameters: the ratio of pulmonary artery acceleration time to pulmonary
artery ejection time (PAAT/PAET), tricuspid annulus systolic excursion (TAPSE), RV annulus systolic

Biomedicines 2023,11, 1385 12 of 19
velocity (RV-S
´
), RV internal diameter (RVID), RV wall thickness (RVWT), stroke volume (SV), cardiac
output (CO), LV eccentricity index (LVEI), tricuspid valve inflow velocities (TV E/A), and tricuspid
annulus lateral velocities (TV E
´
/A
´
). Following the terminal catheterization and echocardiography
assessments, lung and heart tissues (
E
) can be evaluated ex vivo for pulmonary artery (PA) wall
thickness, muscularization and inflammation, as well as lung capillary density. RV tissue can be
assessed for RV fibrosis, cardiomyocyte hypertrophy, inflammation, and angiogenesis. Furthermore,
lung and RV tissues can be studied for the expression of genes and proteins involved in various
pathological processes including inflammation, extracellular matrix (ECM) synthesis and endothelial-
to-mesenchymal transition (EndMT). Finally, the exact cellular roles of osteopontin can be studied
in vitro
(
F
) using cell culture techniques under both osteopontin loss- and gain-of-function conditions
to assess cell proliferation, migration, and apoptosis. Employing such strategies in rodent pulmonary
hypertension models, and ex vivo tissue and
in vitro
cell culture experiments may be necessary to
fully characterize the role of osteopontin in pulmonary hypertension.
6. Osteopontin in Right Ventricular Remodeling
Numerous investigations utilizing both
in vivo
animal models and human clinical
studies have demonstrated a crucial role for osteopontin in the pathogenesis of LV failure of
various etiologies [
112
]. However, there remains a paucity of data pertaining to the specific
role of osteopontin in RV dysfunction and failure. Accumulating evidence suggests that os-
teopontin may play a significant role in RV pathologies. Similar to the pulmonary vascular
remodeling, remodeled RV may represent an important source of circulating osteopontin.
A correlation between the plasma concentrations of osteopontin and the expression levels
within the hypertrophied RV myocardium was demonstrated in monocrotaline and SuHx
rat PH models [
113
,
114
]. Recent transcriptomic analysis of RV tissues from monocrotaline
and SuHx rats revealed that osteopontin was one of the top nine overregulated genes [
115
],
suggesting that pressure overload induces osteopontin expression in the RV.
The proposition that osteopontin may negatively impact the RV is partially predicated
upon the findings of various studies that reported a reduction in osteopontin expression
following treatment with pharmacological agents that improve pulmonary hemodynamics
and RV function, such as PPAR-
γ
activator pioglitazone [
109
] and estrogen receptor-
β
agonist 17
β
-estradiol [
113
]. While it is possible that this decrease in osteopontin expression
is simply a reflection of the reduced afterload, which subsequently mitigates the stress
exerted upon the RV wall, it cannot be entirely ruled out that these agents may exert a direct
impact on the RV and thereby attenuate osteopontin expression. Therefore, it is crucial to
perform further investigations utilizing the afterload-independent experimental models of
RV failure, in which the concentrations of osteopontin are either enhanced via exogenous
administration or decreased through the utilization of genetically modified knockout
organisms or neutralizing antibodies, in order to either confirm or refute such hypotheses.
7. Osteopontin as a Treatment Target in Pulmonary Hypertension
Despite the wealth of data implicating osteopontin in the pathogenesis of PH, stud-
ies employing specific therapeutic approaches directly targeting osteopontin, such as
osteopontin-neutralizing antibodies or osteopontin aptamers in preclinical PH models, are
still lacking. Studies based on the cancer pathologies demonstrated a therapeutic potential
of various approaches to suppress osteopontin using aptamers, antibodies, and small molec-
ular as well as the miRNA-based medications [
116
]. Among these many options, aptamers
have emerged as one of the promising strategies to target osteopontin more specifically and
precisely. Osteopontin aptamers effectively block osteopontin function
in vitro
[
117
] and
in vivo
[
118
] and thereby provide therapeutic benefits. Similarly, osteopontin-neutralizing
antibodies suppressed osteopontin in various heart failure models [
119
,
120
]. Blocking
osteopontin receptors represents another interesting approach to inhibit its effects. Appli-
cation of an
α
V
β
3-integrin antagonist accelerated recovery from the pulmonary vascular
remodeling and RV failure in surgically corrected shunt rats [
42
]. Taken together, despite
the accumulating evidence implicating osteopontin in the pathogenesis of PH, the field

Biomedicines 2023,11, 1385 13 of 19
has yet to fully explore the potential therapeutic utility of osteopontin inhibition through
various modalities, including but not limited to: RNA interference utilizing small inter-
fering RNAs, short hairpin RNAs, aptamers, monoclonal antibodies, and small molecular
inhibitors. Further investigations in animal models are imperative in order to evaluate the
potential benefits of osteopontin inhibition in the management of PH.
8. Future Experimental Perspectives
Available data suggest that osteopontin plays a pivotal role in pulmonary vascular
remodeling by regulating various functions of pulmonary vascular cells. However, they
do not provide a definitive clue on the precise roles of osteopontin in PH. Consequently,
several issues need to be addressed in future studies. First, although enhanced osteopontin
expression in lung and heart tissues was demonstrated in several animal PH models, the
specific roles of osteopontin were studied in only a few of them. These models include
hypoxia-exposed global osteopontin knockout mice [
40
] and a rat model of shunt-induced
PH that used an osteopontin receptor (
α
V
β
3) antagonist [
42
]. In both models, blocking
osteopontin was associated with improved pulmonary hemodynamics and pulmonary
vascular remodeling. Nevertheless, there is still a lack of experimental studies examin-
ing the
in vivo
role of osteopontin in other PH models such as monocrotaline injection,
SuHx, and pulmonary artery banding models. Moreover, no studies examined effects of
cell-specific osteopontin deletion or overexpression, recombinant osteopontin application,
or osteopontin-neutralizing antibodies in rodent PH models. Utilization of the Cre/LoxP
system with cell-specific promoters could provide information on the cell-specific roles of
osteopontin
in vivo
, ultimately determining the main cell type in the pulmonary vascu-
lature involved in the development of PH. Such precise cell-specific strategies may have
implications for the development of successful osteopontin-specific therapeutics.
It is crucial to employ in future studies state-of-the-art techniques, including magnetic
resonance imaging, echocardiography, and catheterization, to better characterize the effects
of osteopontin loss- and gain-of-function strategies on pulmonary hemodynamics and
cardiac function/structure. Then, lung and RV tissues can be subjected to multi-omics
analysis to give insights into the upstream and downstream signaling pathways underlying
osteopontin-mediated pathological processes in pulmonary vascular remodeling. PASMCs
are the most frequently used pulmonary vascular cell type to study the cellular roles of
osteopontin in PH. The exact regulators of osteopontin and its functional effects in other
cell types in the pulmonary vasculature remain unclear. Thus, the exact cellular roles
of osteopontin require further study using
in vitro
cell culture techniques under both
osteopontin loss- and gain-of-function conditions (Figure 3).
Osteopontin may be deemed a viable therapeutic target for the treatment of PH. How-
ever, prior to the advancement of osteopontin-directed therapeutics in PH, the precise
functions of full-length osteopontin and its various fragments and their implications in the
pathogenesis of PH must be carefully elucidated. This will allow identification of those
osteopontin fragments that should be specifically targeted. Such a precise, accurate ma-
nipulation of the osteopontin system will contribute to the amelioration of the pulmonary
vascular and RV remodeling and affect positively the natural trajectory of PH, ultimately
resulting in improved outcomes for PH.
9. Summary
The purpose of this review was to illuminate the crucial role of osteopontin and re-
lated signaling pathways in the pathogenesis of pulmonary vascular remodeling. Strong
evidence has accumulated for the critical role of osteopontin in pulmonary vascular re-
modeling. Osteopontin is a key component in the regulation of complex signaling events
that drive aberrant cellular functions in the pulmonary vasculature. Its direct actions on
pulmonary vascular cells, such as augmented cellular proliferation, migration, and ECM
synthesis, are considered salient mechanisms underlying osteopontin-mediated pathologi-
cal pulmonary vascular remodeling. Its pro-inflammatory actions promote infiltration of

Biomedicines 2023,11, 1385 14 of 19
inflammatory cells into the pulmonary vascular wall, thereby further negatively affecting
the pulmonary vascular remodeling. These pivotal cellular contributions of osteopontin in
PH are further corroborated by studies utilizing rodent PH models, in which osteopontin
deficiency resulted in the amelioration of PH phenotype in mice exposed to hypoxia or
promoted pulmonary vascular reverse remodeling in a shunt model of PH. In PAH patients,
osteopontin has become an established marker of altered pulmonary hemodynamics and
adverse outcomes. A strong relationship was demonstrated between its circulating levels
and the extent of alterations in pulmonary hemodynamics and RV structure and function,
and functional capacity in PH patients. Owing to its role in the pathogenesis of pulmonary
vascular remodeling, osteopontin may represent a viable target for the treatment of PAH.
While specific strategies such as silencing osteopontin expression or inhibiting its activity
have yet to be utilized in rodent PH models, such approaches may provide a valuable in-
sight into the potential utility of osteopontin suppression in the treatment of this condition.
Further research is necessary to fully understand the mechanisms by which osteopontin
regulates adverse pulmonary vascular remodeling and to address numerous unanswered
questions in this field.
Author Contributions:
Conceptualization, A.M. (Argen Mamazhakypov) and A.S.S.; writing—original
draft preparation, A.M. (Argen Mamazhakypov) and A.S.S.; writing—review and editing, A.M. (Argen
Mamazhakypov), A.M. (Abdirashit Maripov), A.S., R.T.S., and A.S.S.; visualization, A.M. (Argen
Mamazhakypov) and A.S.S.; supervision, A.S.S.; funding acquisition, R.T.S. and A.S. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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