See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/298420271

Hyaluronic Acid in Inflammation and Tissue Regeneration

Article · March 2016

CITATIONS READS
38 669

5 authors, including:

Małgorzata Litwiniuk Alicja Krejner-Bienias

Medical University of Warsaw Medical University of Warsaw
22 PUBLICATIONS   197 CITATIONS    19 PUBLICATIONS   87 CITATIONS   

SEE PROFILE SEE PROFILE

Anibal Raul Gauto Tomasz Grzela

Eisenhower Medical Center Medical University of Warsaw
6 PUBLICATIONS   88 CITATIONS    100 PUBLICATIONS   1,168 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Proteasome inhibitors in tumor biology View project

Limb Salvage View project

All content following this page was uploaded by Alicja Krejner-Bienias on 03 January 2019.

The user has requested enhancement of the downloaded file.

 REVIEW

Hyaluronic Acid in Inflammation and

Tissue Regeneration

Malgorzata Litwiniuk, MD; Alicja Krejner; and Tomasz

Grzela, PhD

E
Wounds 2016;28(3):78-88 Abstract: Hyaluronic acid (HA), the main component of extracellular
matrix, is considered one of the key players in the tissue regenera-

From the Department of Histology
and Embryology, Medical University
of Warsaw, Warsaw, Poland
AT
tion process. It has been proven to modulate via specific HA recep-
tors, inflammation, cellular migration, and angiogenesis, which are
the main phases of wound healing. Studies have revealed that most
HA properties depend on its molecular size. High molecular weight

Address correspondence to:
Malgorzata Litwiniuk, MD
Department of Histology and
Embryology
Medical University of Warsaw
Warsaw, Poland
malgosia.litwiniuk@gmail.com
Disclosure: The authors disclose
D
IC
HA displays anti-inflammatory and immunosuppressive properties,
whereas low molecular weight HA is a potent proinflammatory mol-
ecule. In this review, the authors summarize the role of HA polymers
of different molecular weight in tissue regeneration and provide a
PL
short overview of main cellular receptors involved in HA signaling.
In addition, the role of HA in 2 major steps of wound healing is
examined: inflammation and the angiogenesis process. Finally, the
antioxidative properties of HA are discussed and its possible clinical
U
implication presented.
Key words: hyaluronic acid, extracellular matrix, wound healing, in-

no financial or other conflicts of

interest. flammation
T

H
O

yaluronic acid (HA) is a member of a large family of glycosami-

noglycans (GAGs), which are the main components of the extra-
cellular matrix (ECM). Unique features that distinguish HA from
N

other GAGs are its simple structure and large molecular size. The HA mol-
ecule is composed of D-glucuronic acid and N-acetyl-D-glucosamine bound
with β-glycosidic linkages. This simple molecular unit repeated thousands
O

of times forms a structure of a very long linear polymer, with molecular

weight reaching 5 x 106 kDa.1,2 Hyaluronic acid is synthesized by HA syn-
thases on the inner surface of the cellular membrane and translocated into
D

extracellular space along with the elongation of the polymeric chain. This
is a unique method of synthesis, different from other GAGs that are synthe-
sized at the intracellular space. Hyaluronic acid is also the only GAG not
linked to a core protein, and it does not undergo any postsynthetic modifi-
cations. Long hyaluronan polymers have the ability to bind large amounts
of water. Hygroscopic and viscoelastic properties of HA make it a perfect
component of vitreous fluid, joint fluid, and derma. Hyaluronan in its native
form of a very long polymer is known as high molecular weight (HMW) hy-
aluronan. However, in certain conditions, it can be decomposed into small
fragments referred to as low molecular weight HA (LMWHA).3 Hyaluronan
turnover is a rapid process, as the half-life of HA molecules in the blood-
78 WOUNDS® www.woundsresearch.com

stream is only about 2-5 minutes.4 Fragmentation of hyal-
uronan is controlled by enzymes called hyaluronidases.
Hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2)
are responsible for HA degradation in somatic tissues. At
first Hyal-2, which is a cell-membrane linked enzyme, de-
grades hyaluronan to fragments with molecular weight
reaching 20 kDa. These HA molecules are subsequently
endocytosed and delivered to lysosomes, where further
digestion is performed by Hyal-1.5 In injured tissue, free
radicals are also able to decompose HA polymers into
smaller fragments. 6
It has been shown that HA fragments of different
molecular sizes can display different, sometimes op-
posing properties. For example, it has been well docu-
mented that HMWHA displays anti-inflammatory and
immunosuppressive properties, whereas LMWHA is a
potent proinflammatory molecule. A proposed hypoth-
esis suggests LMWHA may have a different biological
function than HMWHA, such as to inform cells about
stress conditions.7
PL
Wound healing is a complex biological process, com-
prised of a series of sequential events aimed at repairing
U
injured tissue. Extracellular matrix components play an
important role in regulation of all phases of tissue repair,
D
T
O
N
O
D
Figure 1. Main hyaluronic acid receptors and their localization in the cell. LYVE-1: lymphatic vessel endothelial receptor 1; CD44:
antigen, a type of transmembrane glycoprotein; RHAMM: receptor for hyaluronan-mediated motility; TLR-4: toll-like receptor-4
Litwiniuk et al
including cellular migration, inflammation, angiogenesis,
remodeling, and scar formation. Studies have revealed
that not only do they provide a wound microenviron-
ment, but are also involved in various signaling pathways
that are activated in the wound bed during the healing
process. Hyaluronic acid is a basic ECM component with
some unique properties that make it a key player in tis-
sue regeneration.
E
Hyaluronic Acid Signaling
CD44. The CD44 antigen, a type 1 transmembrane
AT
glycoprotein, is the main receptor for hyaluronan. It is
present on cell membranes of almost all human cells,8
and consists of 10 constant and 10 variant exons insert-
ed in various combinations at a single extramembrane
IC
site (Figure 1). That gives a great number of different
splicing variants, which may vary in function and prop-
erties.9 A specific function of CD44 is the capability to
bind and internalize HA; however, it can interact with
other ligands, such as fibronectin, collagens, osteopon-
tin, and matrix metalloproteinases (MMPs). It has proven
to work as a docking agent for MMP9 and as a growth
factor reservoir.10 In a wound environment, CD44 is re-
sponsible for cellular internalization of HA degradation
Vol. 28, No. 3 March 2016 79
 Litwiniuk et al

products. This CD44 function has been demonstrated in
lung inflammation, where blocking CD44 on the surface
of macrophages resulted in impaired clearance and de-
lay in the healing process.11 Therefore, CD44 is involved
in the control of exaggerated inflammatory response.
The other role of CD44 signaling in the wound environ-
ment is to induce fibroblast migration from surrounding
tissue into the wounded area. It is noteworthy that nei-
ther CD44 nor HA alone can induce cell migration and
Smaller hyaluronan fragments can also interact with
CD44, but the effects they induce in target cells are dif-
ferent than those caused by HMWHA. The current hy-
pothesis that could explain this phenomenon is that
HMWHA is able to cluster the receptors on the cell
membrane surface to modulate the receptor activity.14
It has also been proven that HA binding to the CD44 re-
ceptor provides the effect of coating the cell membrane.
This protective layer of hyaluronan on a cell surface is

E
promote wound healing; an interaction between HA and able to mask cell death receptors and consequently pre-
CD44 is essential to activate this process.12 The ability vent the cell from reaching apoptosis.15 Small HA frag-

of hyaluronan particles to bind with the CD44 receptor
depends on its molecular size. It has been proven that
avidity of binding of hyaluronan oligomers to CD44 in-
creases with an oligomer size of up to 38 sugars.13
AT
ments do not display these kinds of properties.
Stimulation of CD44 triggers a signaling cascade as-
sociated with 2 tyrosine kinases: p185 human epidermal
growth factor receptor 2 (HER2)16 and c-Src kinase.17

IC
PL
U
D
T
O
N
O
D

Figure 2. Main signaling pathways activated in the cells by hyaluronic acid. TLR: toll-like receptor; HARE: hyaluranon receptor
for endocytosis; RHAMM: receptor for hyaluranon-mediated motility; CD44: antigen, a type 1 transmembrane receptor; LYVE-1:
lymphatic vessel endothelial receptor 1; MyD88: myeloid differentiation primary response gene 88; NFkB: nuclear factor kappa-
light-chain enhancer of activated B cells; Ras: protein; Src: kinase; p185HER2: tyrosine kinase p185 human epidermal growth
factor receptor 2; IL: interleukin; TGF-β: transforming growth factor beta

80 WOUNDS® www.woundsresearch.com

Activation of p185HER2 leads to increased cell growth,
whereas c-Src kinase activity is responsible for phos-
phorylation of cytoskeleton proteins and induction of
cell motility. In addition to phosphoinositide-3 kinase/
phosphoinositide-dependent kinase 1/protein kinase
B pathway, Ras protein signaling pathways are also in-
volved in CD44 cytoplasmic signaling18,19 (Figure 2).
Receptor for hyaluronan-mediated motility. Recep-
tor for hyaluronan-mediated motility (RHAMM), also
known as CD168, is present in several cell types includ-
ing endothelial cells and various tumor cell lines.20 Simi-
lar to CD44, RHAMM also exists in several isoforms, pro-
duced by alternative splicing, that can be distributed on
the cell surface, or intracellularly, within the cytoplasm
or in the nucleus21 (Figure 1). Several kinases, such as
Src kinase, focal adhesion kinase, extracellular-signal-
regulated kinases (ERK) 1/2 and protein kinase C, are
involved in RHAMM signaling. This receptor is also asso-
ciated with the Ras protein and the Ras signaling path-
way22,23 (Figure 2). Intracellular RHAMM interacts with
PL
cytoskeletal proteins, such as actin filaments and micro-
tubules, and activates the previously mentioned protein
U
kinases, which result in cell movement stimulation.24
These properties of RHAMM-HA complex are impor-
D
tant in tissue repair and the inflammation process. It has
been demonstrated that the blocking of hyaluronan-me-
diated RHAMM signaling in smooth muscle cell cultures
T
inhibited cell ability of migration.25 The receptor for
hyaluronan-mediated motility is also highly expressed in
O
fibroblasts, and it has been proven that activation of this
receptor stimulates fibroblast proliferation in vitro.26
Tolg and colleagues26 have shown in a murine model
N
that RHAMM-deficient fibroblasts display reduced mi-
gration capacity and healing potential. At the same time,
activation of RHAMM induces an inflammatory response
O
in the wound environment which can have a negative
influence on the wound healing process. Zaman and
coauthors24 have shown RHAMM signaling was critical
D
for the activation of macrophages during incubation
with HA. These results are consistent with the findings
of Tolg and coauthors,27 which have proven that block-
age of HA signaling with a RHAMM-mimetic peptide was
able to reduce inflammation and fibrogenesis in an exci-
sional skin wound model.27 The implication of RHAMM
and RHAMM signaling in the wound healing process re-
quires further investigation.
Recent studies have shown that cell surface RHAMM
can interact with CD44 while regulating signal transduc-
tion, thus may influence cell motility and wound heal-
Litwiniuk et al
Keypoints
• A specific function of CD44 is the capability to
bind and internalize HA; however, it can interact
with other ligands, such as fibronectin, collagens,
osteopontin, and matrix metalloproteinases.
• 
Intracellular receptors for hyaluronan-mediated
motility interacts with cytoskeletal proteins, such
as actin filaments and microtubules, and activates
the previously mentioned protein kinases, which
E
result in cell movement stimulation.24
• 
Recent studies have shown that cell surface
AT
RHAMM can interact with CD44 while regulating
signal transduction, thus may influence cell motil-
ity and wound healing;22 however, all of the coop-
erative mechanisms between RHAMM and CD44
IC
are not clearly understood and require further in-
vestigation.29
ing.22 The receptor for hyaluronan-mediated motility
expression has been shown to play a key role in the
translocation of CD44 to the cell surface, formation of
CD44-ERK1/2 complexes, and subsequent ERK1/2 ac-
tivation. Hamilton et al28 observed elevated levels of
ERK1/2 activation in invasive breast cancer cells upon
growth factor/motogenic stimulation when cell surface
CD44 and RHAMM were coexpressed and coassociated
with each other. It is postulated that while CD44 and
RHAMM can independently regulate cell behavior via
HA signaling, in some cases they cooperate or at least
have overlapping functions. However, all of the coop-
erative mechanisms between RHAMM and CD44 are not
clearly understood and require further investigation.29
Toll-like receptors. Toll-like receptors (TLRs) are a
group of highly conserved molecules that allow the im-
mune system to sense common bacteria and viruses and
to coordinate an early host defense against these patho-
gens. The human TLR family consists of 10 receptors
comprised of different pathogen-associate molecular
patterns (PAMPs) that are characteristic of various mi-
crobial classes. For example, TLR4 recognizes lipopoly-
saccharide (LPS), which is an integral component of the
outer membranes of gram-negative bacteria, whereas
TLR2 recognizes the peptidoglycan and lipopeptide,
which are cell membrane components of gram-positive
bacteria. Recent studies30 suggest TLRs can be activated
not only by PAMPs but also by some endogenous mol-
ecules, called damage associated molecular patterns
(DAMPs).30 They include heat shock proteins, members
of the S100 protein family, high mobility group box-1
Vol. 28, No. 3 March 2016 81
 Litwiniuk et al
Keypoints
• In acute wounds, small hyaluronan fragments ac-
cumulating at the site of injury activate the im-
mune system to manage rupture in tissue integrity;
however, in chronic wounds an excessive inflam-
matory response proves to be a negative phenom-
enon that prevents that wound from healing.
• Unlike small hyaluronic acid (HA) fragments,
hyaluronan in its native form of a large polymer
displays anti-inflammatory and immunosupresive
properties.
• The CD44 signaling pathway plays an important
role in the regulation of inflammation by high mo-
lecular weight hyaluronic acid (HMWHA).
• The exact mechanism in which HMWHA interacts
with TLR receptors, leading to inhibition of inflam-
matory cascade, is not known.
(HMGB1) and ECM breakdown products such as low-
molecular weight hyaluronan fragments (Figure 2). To
PL
date, it has been confirmed that LMWHA is recognized
by TLR2 and TLR4.30
U
Hyaluronan receptor for endocytosis and lymphatic
vessel endothelial receptor 1. Hyaluronan receptor for
D
endocytosis (HARE), also known as stabilin 2, is respon-
sible for clearance of HA, as well as other GAGs, from sys-
temic circulation.31,32 Hyaluronan receptor for endocyto-
T
sis is present on the inner surface of endothelial cells in
vascular and lymphatic vessels (Figure 1). Interaction of
O
HA with HARE leads to nuclear factor kappa-light-chain-
enhancer of activated B cells (NF-κB), signaling pathway
activation33 (Figure 2).
N
Lymphatic vessel endothelial receptor 1 (LYVE-1)
typically occurs in the lymphatic system and has been
detected in lymphatic endothelial cells, liver sinusoi-
O
dal endothelial cells, and in reticular cells of the lymph
nodes.34,35 The biological function of LYVE-1 is the ab-
sorption of HA from tissue to the lymph, that helps to
D
regulate tissue hydration level.36
The involvement of HARE and LYVE-1 in tissue regen-
eration has not yet been well documented and requires
further study.
Hyaluronic Acid and Inflammation
It has been proven that HMWHA displays anti-inflam-
matory activity, whereas low molecular weight degrada-
tion products of HA can induce inflammation.36 It has
been reported that LMWHA may elicit various proinflam-
matory responses such as activation of murine alveolar
82 WOUNDS® www.woundsresearch.com
macrophages,37 or induction of irreversible phenotypic
and functional maturation of human dendritic cells.38,39
Further studies have shown that small hyaluronan frag-
ments increase the expression and protein production of
several cytokines such as MMP-12, plasmogen activator
inhibitor-1,40,41 macrophage inflammatory protein (MIP)-
1α, MIP-1β, monocyte chemoattractrant-1, keratinocyte
chemoattractant, interleukin (IL)-8, and IL-12 by macro-
phages.42-44 The obvious candidate to activate this series
E
of events in immune system cells is CD44, the main re-
ceptor for hyaluronan. Nevertheless, macrophages from
AT
CD44 null mice still respond to small HA fragments treat-
ment, which is consistent with the fact that other recep-
tors, such as TLRs, are also involved in HA signaling.45
Several studies have concentrated on TLRs and TLR
IC
signaling as a key player responsible for proinflamma-
tory properties of LMWHA. Several authors have con-
firmed, using in vitro studies, that LMWHA is able to
bind to TLR receptors and consequently initiate the
signaling cascade, leading to the production of pro-
inflammatory cytokines and chemokines in various
types of cells.46 In immune cells from injured tissues,
TLR2 and TLR4 activation by LMWHA leads to initia-
tion of MyD88-dependent NFκB signaling cascade and
pro-inflammatory cytokine gene expression.14,47 Iwata
and coauthors48 have shown that LMWHA stimulates
B lymphocytes via TLR4 receptor to IL-6 and TGF beta
production. Induction by LMWHA TLR-related myeloid
differentiation primary response gene 88 (MyD88)/
NFκB signaling was also confirmed in breast tumor
cells. Bourguignon et al49 have shown that small HA
fragments stimulate CD44 association with TLR2, TLR4,
and MyD88, leading to NF-κB-specific transcriptional
activation and the expression of proinflammatory cyto-
kines IL-1β and IL-8 in the human breast cell line.Taken
together, these reports suggest that LMWHA induces
inflammation via activation of TLR receptors and initia-
tion of MyD88/NFκB signaling which leads to produc-
tion of proinflammatory cytokines and chemokines.
In physiological conditions, the activation of immune
system cells is crucial for proper wound healing. In
acute wounds, small hyaluronan fragments accumulat-
ing at the site of injury activate the immune system to
manage rupture in tissue integrity; however, in chronic
wounds a constant excessive inflammatory response
proves to be a negative phenomenon that actually pre-
vents that wound from healing.
Unlike small HA fragments, hyaluronan in its native
form of a large polymer displays anti-inflammatory and

immunosupresive properties.The anti-inflammatory po-
tential of HMWHA has been well documented in osteo-
arthritis. Since HA is a basic component of normal sy-
novial fluid and the concentration of HA is decreased in
joints affected with osteoarthritis, intra-articular injec-
tion of HMWHA has been used to treat this condition.
Therapeutic effects of such supplementation have been
well documented in a number of clinical studies. Sever-
al authors investigated the influence of HMWHA on the
expression of proinflammatory and anti-inflammatory
cytokines by synoviocytes. High molecular weight HA
was able to inhibit IL-1β expression in synoviocytes in
a rabbit model of osteoarthritis.50 Also the IL-1 depen-
dent expression of MMP-1 and MMP-3 was reduced in
human synoviocytes by HMWHA treatment. In a large
study, Wang and coauthors51 analyzed the influence of
HMWHA on gene expression of various inflammatory
cytokines by human fibroblast-like synoviocytes (FLS)
in patients with early-stage of osteoarthritis. They re-
ported the downregulation of IL-8 and iNOS gene ex-
PL
pression in unstimulated FLS and aggrecanase-2, and
tumor necrosis factor alpha (TNFα) gene expression in
U
IL-1-stimulated FLS. Blocking the CD44 receptor with
anti-CD44 antibody inhibited the down-regulatory ef-
D
fects of HMWHA on gene expression.49 This may sug-
gest the important role of CD44 signaling pathway in
the regulation of inflammation by HMWHA. Campo et
T
al52 studied the involvement of TLR receptors in this
process. They have reported that HMWHA was able to
O
significantly diminish TLR4, TLR2, MyD88 and NF-kB
expression and protein synthesis in synoviocytes in
murine model of osteoarthritis. The have also observed
N
reduced mRNA expression and protein production for
TNFα, IL-1β, IL-17, MMP-13 and inducible nitrous oxide
synthase gene in arthritic mice treated with HMWHA.52
O
Interestingly, all of the effects mentioned above were
present only when HMWHA was administered in an
early inflammatory phase of osteoarthritis. This may
D
suggest that along with disease progression, signaling
pathways apart from the TLR-dependent ones could be
involved in the chronic inflammation state. The exact
mechanism in which HMWHA interacts with TLR recep-
tors, leading to inhibition of inflammatory cascade is
not known. Campo et al52 have proposed that HA poly-
mers of high molecular mass may mask TLR2 and TLR4
via its polymerized structure and subsequently prevent
the stimulation of these receptors.
Antioxidant properties of hyaluronic acid. Oxidative
stress is caused by the imbalance between the amount
Litwiniuk et al
Keypoints
• Findings suggest high molecular weight hyaluronic
acid (HMWHA) should be proposed to patients
treated with certain ocular medications to reduce
the risk of ocular surface impairment.54,55
• Recently, antioxidant properties of HA have also
been taken into consideration in various patholo-
gies, including osteoarthritis.59
• Numerous studies have shown that HA signaling
E
plays an important role in angiogenesis regulation,
mainly by influencing endothelial cell behavior.
AT
of reactive oxygen species (ROS) and antioxidant capac-
ity. When present in excess, ROS has noxious effects on
cellular proteins, lipids, and DNA. It is postulated that es-
IC
pecially high molecular weight forms of HA can protect
from the effects of ROS.
Different antioxidant effects of HMWHA include the
decrease in ultraviolet B-induced apoptosis and ethyl-
enediaminetetraacetic acid-induced oxidative damage
of DNA.15,53 Moreover, HMWHA diminishes apopto-
sis and oxidative stress triggered with benzalkonium
chloride and sodium lauryl sulfate detergents, which
are widely used in ophthalmic preparations. It is sug-
gested that HMWHA should be proposed to patients
treated with ocular medications with the ingredients
aforementioned to reduce the risk of ocular surface im-
pairment.54,55 What is more, it has been demonstrated
that treatment with eye drops containing HA reduces
oxidative stress in the conjunctiva of patients with dry
eye disease.56 Despite numerous studies, the HMWHA
mechanisms of oxidative stress reduction are still not
completely understood. High molecular weight HA
possesses hydroxyl functional groups, which can pre-
sumably absorb ROS. Furthermore, HMWHA interacts
with the CD44 receptor. It is suggested that due to this
interaction, HMWHA may activate pathways involved
in the regulation of cellular redox status and intracel-
lular ROS generation.57 Moreover, HMWHA forms a cy-
toprotective coat on a cell membrane and, as a result,
protects the cell from apoptosis. The antioxidant func-
tion of HMWHA contributes to the attenuation of DNA
damage in human leukocytes during oxidative burst.
It is postulated that HA binds to the CD44 receptor
on the surface of monocytes and granulocytes and is
endocytosed. Subsequently, HA neutralizes intracellu-
lar ROS and attenuates the DNA damage. Polyanionic
HA molecules chelate Fe2+ and Cu2+ ions, which are
required in Fenton’s reaction; in the absence of these
Vol. 28, No. 3 March 2016 83
 Litwiniuk et al
Keypoints
• Hyaluronic acid (HA) is present through all steps
of the wound healing process not only as a com-
ponent of the wound environment, but as a factor
that actively modulates tissue regeneration.
• A rich natural source of HA are fetal membranes,
especially the jelly substance from the umbilical
cord and the amnion.
• This component of the amnion is one of the factors
responsible for its beneficial actions observed in
chronic wound therapy.74
ions, hydroxyl radical, which is highly reactive with
DNA, cannot be generated. Furthermore, HA may also
neutralize ROS outside the leukocytes and protect
neighboring cells.58
Using an intra-articular injection of HA in the thera-
py of osteoarthritis is not a novel idea. Hyaluronic acid
determines elastic properties and viscosity of synovial
fluid. Recently, antioxidant properties of HA have also
PL
been taken into consideration in various pathologies, in-
cluding osteoarthritis.59 According to Yu et al,60 HA intra-
U
articular injection reduces ROS levels in synovial fluid
of patients suffering from osteoarthritis. In addition, HA
D
suppresses hydrogen peroxide-induced cell death in
human chondrocytes through an intracellular signaling
pathway. The proteomic analysis of human osteoarthri-
T
tis chondrocytes cultured in stress conditions have re-
vealed the up-regulated expression of proteins involved
O
in stress response and apoptotic pathways: transaldol-
ase, annexin A1, and elongation factor 2. This effect was
suppressed by adding HA to the culture medium.60
N
As previously mentioned, the majority of HA biologi-
cal actions are strongly associated with its molecular
size. Therefore, when it comes to its antioxidative po-
O
tential, the integrity of the polymeric structure of HA
should always be considered. Most studies focus on
the underlying antioxidant properties of HMWHA or
D
just HA.54-57 However, Ke and colleagues61 demonstrat-
ed strong antioxidant functions of LMWHA, which has
shown protective effects against ROS both in vitro and
in vivo, and inhibits lipid peroxidation and scavenges
free radicals. Moreover, LMWHA raises total antioxidant
capacity in immunosuppressed mice.62 The mechanisms
of the LMWHA antioxidant effect remain unknown and
need further study.
The effects of the antioxidant properties of HA are
also of interest in different research fields. Najafi et al63
have proposed to add HA to semen extenders used in
84 WOUNDS® www.woundsresearch.com
cryopreservation of animal sperm. Major medical ap-
plications include the treatment of dry eye disease and
osteoarthritis.56,59
Hyaluronan in angiogenesis. Angiogenesis is an
essential part of the proliferation phase of the wound
healing process. During this phase, endothelial cells
(EC) migrate from established vessels into surrounding
tissues where they proliferate and create new cell-to-cell
attachments as well as tubule structures of new capillar-
E
ies.7 Interactions between EC and ECM components are
crucial in the regulation of new vessel formation.
AT
Numerous studies have shown that HA signaling plays
an important role in angiogenesis regulation, mainly by
influencing EC behavior. Both HMWHA and LMWHA are
potent regulators of angiogenesis, however they display
IC
opposite effects on EC proliferation and motility. It has
been proven that LMWHA stimulates vascular EC pro-
liferation, migration, and tubule formation in vitro, as
well as in various in vivo models of angiogenesis.63 At
the same time HMWHA displays antiangiogenic proper-
ties by inhibiting EC proliferation, motility, and sprout
formation.63
The exact molecular mechanisms determining the
proangiogenic or antiangiogenic effects of different HA
forms have not been fully elucidated. CD44 as well as
RHAMM, 2 main receptors for HA, are present on the
surface of the endothelial cells. In vitro experiments
demonstrated both anti-RHAMM and anti-CD44 antibod-
ies blocked the EC ability to form tubule-like structures
in matrigel.64 Blocking the CD44 signaling by anti-CD44
antibody inhibited EC proliferation in vitro.65 Cao and
coauthors66 have reported angiogenesis was signifi-
cantly affected in CD44 knockout mice. The earliest
studies concentrated on the HA-mediated mitogenesis
via the MAPK/ERK signaling pathway activation. It has
been shown that CD44 and RHAMM stimulated by HA
oligomers create a complex with ERK 1/2, that leads to
constant ERK 1/2 activation and increases cell motility
of invasive breast cancer cell lines.67 Similar ERK 1/2
pathway activation by LMWHA has also been reported
in other tumor cell lines, but also in various types of
ECs, such as human umbilical vein ECs (HUVECs), hu-
man micro vessels endothelial cells, or human pulmo-
nary artery ECs.68
Interestingly, recent reports suggest that the promo-
tion of EC proliferation caused by LMWHA may also be
associated with expression of other signaling molecules,
such as ezrin. This protein, also known as cytovillin or
villin-2 belongs to the ERM protein family,which also

includes moesin, radixin, and merlin. Ezrin is a linkage
protein between the plasma membrane and actin skel-
eton, thus it plays a key role in cell surface adhesion and
migration. Ezrin can interact with cellular C-terminus
of CD44 receptor. Studies have shown that ezrin pro-
motes cell adhesion and migration, whereas merlin in-
hibits cell proliferation. Mo and coauthors68 have shown
that LMWHA promoted the proliferation of HUVECs. At
the same time, expression of ezrin mRNA and protein
was significantly increased in HUVECs pretreated with
LMWHA. In contrast, no difference in ezrin and merlin
expressions have been observed in HUVECs incubated
with HMWHA.69
The proangiogenic effects of LMWHA and antiangio-
genic properties of HMWHA have been reported in nu-
merous studies. Surprisingly, in several types of tumor
xenografts, injection of hyaluronan oligomers inhibited
rather than stimulated tumor growth. This may suggest
a context-dependent response to different forms of HA
and a possible role of the microenvironment in this pro-
PL
cess. Fuchs et al70 have investigated the influence of HM-
WHA and LMWHA on chemokine (C-X-C motif) ligand
U
12/chemokine receptor type 4 (CXCR4) signaling. Che-
mokine (C-X-C motif) ligand 12 (CXCL12) can stimulate
D
angiogenesis by activating CXCR4 present on the EC
surface.70 In wound closure assays, adding the CXCL12
to the culture medium significantly increased cell migra-
T
tion and induced faster wound closure. This effect was
statistically augmented when cells were preincubated
O
with HMWHA. In vitro studies have shown that CXCR4
activation by CXCL12 was significantly increased in HU-
VECs pretreated with HMWHA, whereas preincubation
N
with LMWHA blocked CXCL12 signaling in these cells.
A new, interesting way of CD44 signaling regulation
has been reported by Yang and coauthors.71 Using sev-
O
eral renal and breast cancer cell lines, the researchers
demonstrated that HMWHA was able to stimulate clus-
tering of CD44 into large conglomerates, whereas LM-
D
WHA disrupted these structures.72 It is postulated that
clusters may influence CD44 signaling by stabilizing
complexes; this receptor forms with various signaling
molecules. Nevertheless, the possible impact of CD44
clustering on its biological function requires further in-
vestigation.
Conclusion
Hyaluronic acid is present through all steps of the
wound healing process not only as a component of
the wound environment, but as a factor that actively
Litwiniuk et al
modulates tissue regeneration. Along with studies that
revealed the unique properties of HA, some attempts
have been made to apply it in clinical practice, especial-
ly in chronic wound treatment. Extensive literature can
be found concerning exogenous applications of HA to
wounds. Various HA sources, formulations, and delivery
systems have been used in clinical trials.73 A rich natural
source of HA are fetal membranes, especially the jelly
substance from the umbilical cord and the amnion. The
E
amniotic membrane has been used in traditional medi-
cine, and also in the form of a commercially available
AT
dressing in wound treatment.72 Studies have revealed
the amniotic membrane, even after various processing
and preservation procedures, contains high amounts of
HMWHA. This component of the amnion is one of the
IC
factors responsible for its beneficial actions observed in
chronic wound therapy.74 An effective wound healing
therapy remains one of the greatest challenges of mod-
ern clinical medicine. Hyaluronic acid as a biologically
active molecule that regulates tissue repair process on
multiple levels should be considered as a safe and effec-
tive option to be used in skin repair.
References
1. Agren UM, Tammi RH, Tammi MI. Reactive oxygen spe-
cies contribute to epidermal hyaluronan catabolism
in human skin organ culture. Free Radic Biol Med.
1997;23(7):996-1001.
2. Vigetti D, Karousou E, Viola M, Deleonibus S, De Luca G,
Passi A. Hyaluronan: biosynthesis and signaling. Biochim
Biophys Acta. 2014;1840(8):2452-2459.
3. Aya KL, Stern R. Hyaluronan in wound healing: re-
discovering a major player. Wound Repair Regen.
2014;22(5):579-593.
4. Reed RK, Laurent UB, Fraser JR, Laurent TC. Removal rate
of [3H] hyaluronan injected subcutaneously in rabbits.
Am J Physiol. 1990;259(2 Pt 2):532-535.
5. Stern R, Jedrzejas M. Hyaluronidases: their genom-
ics, structures, and mechanisms of action. Chem Rev.
2006;106(3):818-839.
6. Longaker MT, Chiu ES, Adzick NS, Stern M, Harrison MR,
Stern R. Studies in fetal wound healing. V. A prolonged
presence of hyaluronic acid characterizes fetal wound
fluid. Ann Surg. 1991;213(4):292-296.
7. Turley EA, Noble PW, Bourguignon LY. Signaling
properties of hyaluronan receptors. J Biol Chem.
2002;277(7):4589-4592.
8. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B.
CD44 is the principal cell surface receptor for hyaluro-
Vol. 28, No. 3 March 2016 85
 Litwiniuk et al
nate. Cell. 1990;61(7):1303-1313.
9. Lesley J, Hyman R, Kincade PW. CD44 and its interaction
with extracellular matrix. Adv Immunol. 1993;54:271–
335.
10. Sherman L, Sleeman J, Herrlich P, Ponta H. Hyaluronate
receptors: key players in growth, differentiation, migra-
tion and tumor progression. Curr Opin Cell Biol. 1994;
6(5):726-733.
11. Liang J, Jiang D, Griffith J, et al. CD44 is a negative regula-
tor of acute pulmonary inflammation and lipopolysac-
charide-TLR signaling in mouse macrophages. J Immu-
nol. 2007;178(4):2469-2475.
12. Svee K, White J, Vaillant P, et al. Acute lung injury fibro-
blast migration and invasion of a fibrin matrix is medi-
ated by CD44. J Clin Invest. 1996;98(8):1713-1727.
13. Lesley J, Hascall VC, Tammi M, Hyman R. Hyaluro-
nan binding by cell surface CD44. J Biol Chem.
2000;275(35):26967-26975.
PL
14. Jiang D, Liang J, Fan J, et al. Regulation of lung injury and
repair by Toll-like receptors and hyaluronan. Nat Med.
2005;11(11):1173-1179.
15. Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung
U
MC. Interaction between the adhesion receptor, CD44,
and the oncogene product, p185HER2, promotes
D
human ovarian tumor cell activation. J Biol Chem.
1997;272(44):27913-27918.
16. Bourguignon LYW, Zhu H, Shao L, Chen YW. CD44 in-
T
teraction with c-Src kinase promotes cortactin-me-
diated cytoskeleton function and hyaluronic acid-
O
dependent ovarian tumor cell migration. J Biol Chem.
2001;276(10):7327-7336.
17. Vigetti D, Viola M, Karousou E, et al. Hyaluronan–CD44–
N
ERK1/2 regulate human aortic smooth muscle cell mo-
tility during aging. J Biol Chem. 2008;283(7):4448-4458.
18. Bourguignon LY, Gilad E, Brightman A, Diedrich F, Sin-
O
gleton P. Hyaluronan–CD44 interaction with leukemia-
associated RhoGEF and epidermal growth factor recep-
tor promotes Rho/Ras co-activation, phospholipase C
D
epsilon-Ca2+ signaling, and cytoskeleton modification
in head and neck squamous cell carcinoma cells. J Biol
Chem. 2006;281(20):14026-14040.
19. Hardwick C, Hoare K, Owens R, et al. Molecular cloning
of a novel hyaluronan receptor that mediates tumor cell
motility. J Cell Biol. 1992;117(6):1343-1350 .
20. Entwistle J, Hall CL, Turley EA. HA receptors: regula-
tors of signalling to the cytoskeleton. J Cell Biochem.
1996;61(4):569-577.
21. Nikitovic D, Kouvidi K, Karamanos NK, Tzanakakis GN.
The roles of hyaluronan/RHAMM/CD44 and their re-
86 WOUNDS® www.woundsresearch.com
spective interactions along the insidious pathways of
fibrosarcoma progression. Biomed Res Int. 2013;2013.
DOI:10.1155/2013/929531.
22. Kouvidi K, Berdiaki A, Nikitovic D, et al. Role of receptor
for hyaluronic acid-mediate motility (RHAMM) in low
molecular weight hyaluronan (LMWHA)-mediated fibro-
sarcoma cell adhesion. J Biol Chem. 2011;286(44):38509-
38520.
23. Hall CL, Collis LA, Bo AJ, et al. Fibroblasts require protein
E
kinase C activation to respond to hyaluronan with in-
creased locomotion. Matrix Biol. 2001;20(3):183-192.
AT
24. Zaman A, Cui Z, Foley JP, et al. Expression and role of the
hyaluronan receptor RHAMM in inflammation after bleo-
mycin injury. Am J Respir Cell Mol Biol. 2005;33(5):447-
454.
IC
25. Tolg C, Poon R, Fodde R, Turley EA, Alman BA. Genetic
deletion of receptor for hyaluronan-mediated motility
(Rhamm) attenuates the formation of aggressive fibro-
matosis (desmoid tumor). Oncogene. 2003;22(44):6873-
6882.
26. Tolg C, Hamilton SR, Nakrieko KA, et al. Rhamm-/- fibro-
blasts are defective in CD44-mediated ERK1,2 moto-
genic signaling, leading to defective skin wound repair. J
Cell Biol. 2006;175(6):1017-1028.
27. Tolg C, Hamilton SR, Zalinska E, et al. A RHAMM mimetic
peptide blocks hyaluronan signaling and reduces inflam-
mation and fibrogenesis in excisional skin wounds. Am J
Pathol. 2012;181(4):1250-1270.
28. Hamilton SR, Fard SF, Paiwand FF, et al. The hyaluronan
receptors CD44 and RHAMM (CD168) form complexes
with ERK 1,2 that sustain high basal motility in breast
cancer cells. J Biol Chem. 2007;282(22):16667-16680.
29. Misra S, Hascall V, Markwald R, Ghatak S. Interactions
between hyaluronan and its receptors (CD44, RHAMM)
regulate the activities of inflammation and cancer. Front
Immunol. 2015;6:201. DOI: 0.3389/fimmu.2015.00201.
30. Scheibner K, Lutz MA, Boodoo S, Fenton MJ, Powell JD,
Horton MR. Hyaluronan fragments act as an endogenous
danger signal by engaging TLR2. J Immunol. 2006;
177(2):1272-1281.
31. Harris EN, Kyosseva SV, Weigel JA, Weigel PH. Expres-
sion, processing, and glycosaminoglycan binding ac-
tivity of the recombinant human 315-kDa hyaluronic
acid receptor for endocytosis (HARE). J Biol Chem.
2007;282(5):2785-2797.
32. Zhou B, Weigel JA, Fauss L, Weigel PH. Identification of
the hyaluronan receptor for endocytosis (HARE). J Biol
Chem. 2000; 275(48):37733-37741.
33. Pandey MS, Baggenstoss BA, Washburn J, Harris EN, Wei-

gel PH.The hyaluronan receptor for endocytosis (HARE)
activates NF-κB-mediated gene expression in response
to 40-400-kDa, but not smaller or larger, hyaluronans. J
Biol Chem. 2013;288(20):14068-14079.
34. Mouta Carreira C, Nasser SM, di Tomaso E, et al. LYVE-1 is
not restricted to the lymph vessels: expression in normal
liver blood sinusoids and down-regulation in human liv-
er cancer and cirrhosis. Cancer Res. 2001;61:8079-8084.
35. Wróbel T, Dziegiel P, Mazur G, Zabel M, Kuliczkowski K,
Szuba A. LYVE-1 expression on high endothelial venules
(HEVs) of lymph nodes. Lymphology. 2005; 38(3):107-
110.
36. Prevo R, Banerji S, Ferguson DJ, Clasper S, Jackson
DG. Mouse LYVE-1 is an endocytic receptor for hyal-
uronan in lymphatic endothelium. J Biol Chem. 2001;
276(22):19420-19430.
37. Nobel PW, McKee CM, Cowman M, Shin HS. Hyaluronan
fragments activate an NF-kappa B/I-kappa B alpha au-
toregulatory loop in murine macrophages. J Exp Med.
1996; 183(5):2373-2378.
PL
38. Tremeer C, Benedix F, Sleeman J, et al. Oligosaccharides
of hyaluronan activate dendritic cells via toll-like recep-
U
tor 4. J Exp Med. 2002;195(1):99-111.
39. Termeer CC, Hennies J, Voith U, et al. Oligosaccharides
D
of hyaluronan are potent activators of dendritic cells. J
Immunol. 2000;165(4):1863-1870.
40. Horton MR, Olman MA, Bao C, et al. Regulation of plas-
T
minogen activator inhibitor-1 and urokinase by hyaluro-
nan fragments in mouse macrophages. Am J Physiol
O
Lung Cell Mol Physiol. 2000;279(4):L707-L715.
41. Horton MR, Shapiro S, Bao C, Lowenstein CJ, Noble PW.
Induction and regulation of macrophage metalloelastase
N
by hyaluronan fragments in mouse macrophages. J Im-
munol. 1999;162(7):4171-4176.
42. Horton MR, Burdick MD, Strieter RM, Bao C, Noble PW.
O
Regulation of hyaluronan-induced chemokine gene
expression by IL-10 and IFN-gamma in mouse macro-
phages. J Immunol. 1998;160(6):3023-3030.
D
43. Hodge-Dufour J, Noble PW, et al. Induction of IL-12 and
chemokines by hyaluronan requires adhesion-depen-
dent priming of resident but not elicited macrophages. J
Immunol. 1997;159(5):2492-2500.
44. McKee CM, Penno MB, Cowman M, et al. Hyaluronan
(HA) fragments induce chemokine gene expression in
alveolar macrophages. The role of HA size and CD44. J
Clin Invest. 1996;98(10):2403-2413.
45. Teder P,Vandivier RW, Jiang D, et al. Resolution of lung in-
flammation by CD44. Science. 2002; 296(5565):155-158.
46. Litwiniuk M, Grzela T, Samaha R. Chronic inflammation
Litwiniuk et al
in venous leg ulcer–problems and perspectives. Cent
Eur J Immunol. 2009;34(4):247-51.
47. Suga H, Sugaya M, Fujita H, et al. TLR4, rather than TLR2,
regulates wound healing through TGF-b and CCL5 ex-
pression. J Dermatol Sci. 2014; 73(2):117-124.
48. Iwata Y, Yoshizaki A, Komura K, et al. CD19, a response
regulator of B lymphocytes, regulates wound heal-
ing through hyaluronan-induced TLR4 signaling. Am J
Pathol. 2009;175(2):649-660.
E
49. Bourguignon LY, Wong G, Earle CA, Xia W. Interaction of
low molecular weight hyaluronan with CD44 and toll-
AT
like receptors promotes the actin filament-associated
protein 110-actin binding and MyD88-NFκB signaling
leading to proinflammatory cytokine/chemokine pro-
duction and breast tumor invasion. Cytoskeleton (Hobo-
IC
ken). 2011;68(12):671-693.
50. Takahashi, R.S. Goomer, F. Harwood, T. Kubo, Y. Hirasawa,
D. Amiel. The effects of hyaluronan on matrix metallo-
proteinase-3 (MMP-3), interleukin-1beta(IL-1beta), and
tissue inhibitor of metalloproteinase-1 (TIMP-1) gene
expression during the development of osteoarthritis.
Osteoarthritis Cartilage. 1999;7(2):182-190.
51. Wang CT, Lin YT, Chiang BL, Lin YH, Hou SM. High mo-
lecular weight hyaluronic acid down-regulates the gene
expression of osteoarthritis-associated cytokines and
enzymes in fibroblast-like synoviocytes from patients
with early osteoarthritis. Osteoarthritis Cartilage.
2006;14(12):1237-1247.
52. Campo GM, Avenoso A, Nastasi G, et al. Hyaluronan re-
duces inflammation in experimental arthritis by modu-
lating TLR-2 and TLR-4 cartilage expression. Biochim
Biophys Acta. 2011;1812(9):1170-1181.
53. Pauloin T, Dutot M, Joly F, Warnet JM, Rat P. High molecu-
lar weight hyaluronan decreases UVB-induced apoptosis
and inflammation in human epithelial corneal cells. Mol
Vis. 2009;15:577-583.
54. Pauloin T, Dutot M, Liang H, Chavinier E, Warnet JM, Rat
P. Corneal protection with high-molecular-weight hyal-
uronan against in vitro and in vivo sodium lauryl sulfate-
induced toxic effects. Cornea. 2009;28(9):1032-1041.
55. Pauloin T, Dutot M, Warnet JM, Rat P. In vitro modulation
of preservative toxicity: high molecular weight hyaluro-
nan decreases apoptosis and oxidative stress induced
by benzalkonium chloride. Eur J Pharm Sci. 2008;34(4-
5):263-273.
56. Macri A, Scanarotti C, Bassi AM, Giuffrida S, Sangalli G,
Traverso CE et al. Evaluation of oxidative stress levels in
the conjunctival epithelium of patients with or without
dry eye, and dry eye patients treated with preservative-
Vol. 28, No. 3 March 2016 87
 Litwiniuk et al

free hyaluronic acid 0.15 % and vitamin B12 eye drops.
Graefes Arch Clin Exp Ophthalmol. 2014;253(3):425-
430.
57. Ye J, Zhang H, Wu H, et al. Cytoprotective effect of hy-
aluronic acid and hydroxypropyl methylcellulose
against DNA damage induced by thimerosal in Chang
conjunctival cells. Graefes Arch Clin Exp Ophthalmol.
2012;250(10):1459-1466.
2000; 275(36):27641-27649.
69. Mo W, Yang C, Liu Y, He Y, Wang Y, Gao F. The influence of
hyaluronic acid on vascular endothelial cell proliferation
and the relationship with ezrin/merlin expression. Acta
Biochim Biphys Sin. 2011;43(12):930-939.
70. Fuchs K, Hippe A, Schmaus A, Homey B, Sleeman JP,
Orian-Rousseau V. Opposing effects of high- and low-mo-
lecular weight hyaluronan on CXCL12-induced CXCR4
signaling depend on CD44. Cell Death Dis. 2013;4:e819.

E
58. Halicka HD, Mitlitski V, Heeter J, Balazs EA, Darzynkie-
wicz Z. Attenuation of the oxidative burst-induced DNA DOI: 10.1038/cddis.2013.364.
damage in human leukocytes by hyaluronan. Int J Mol 71. Yang C, Cao M, Liu H, et al. The high and low molecu-

Med. 2009;23(5):695-699.
59. Miki Y,Teramura T,Tomiyama T, et al. Hyaluronan reversed
proteoglycan synthesis inhibited by mechanical stress:
possible involvement of antioxidant effect. Inflamm
AT
lar weight forms of hyaluronan have distinct effects on
CD44 clustering. J Biol Chem. 2012; 287(51):43094-
43107.
72. Neuman MG, Nanau RM, Oruña-Sanchez L, Coto G. Hy-

Res. 2010;59(6):471-477.
60. Yu CJ, Ko CJ, Hsieh CH, et al. Proteomic analysis of os-
teoarthritic chondrocyte reveals the hyaluronic acid-
regulated proteins involved in chondroprotective effect
under oxidative stress. J Proteomics. 2014;99:40-53.
PL
61. Ke C, Sun L, Qiao D, Wang D, Zeng X. Antioxidant activity
of low molecular weight hyaluronic acid. Food Chem
U
Toxicol. 2011;49(10):2670-2675.
62. Najafi A, Najafi M, Zanganeh Z, Sharafi M, Martinez-Pastor
D
IC
aluronic acid and wound healing. J Pharm Pharm Sci.
2015;18(1):53-60.
73. Litwiniuk M, Grzela T. Amniotic membrane: new con-
cepts for an old dressing. Wound Repair Regen. 2014;
22(4):451-456.
74. Litwiniuk M, Bikowska B, Niderla-Bielińska J, et al. High
molecular weight hyaluronan and stroma-embedded fac-
tors of radiation-sterylized amniotic membrane stimu-
late proliferation of HaCaT cell line in vitro. Cent Eur J

F, Adeldust H. Cryopreservation of ram semen in extend- Immunol. 2011;36(4):205-211.

ers containing soybean lecithin as cryoprotectant and
hyaluronic acid as antioxidant. Reprod Domest Anim.
T

2014;49(6):934-940.
63. Toole BP. Hyaluronan: from extracellular glue to pericel-
O

lular cue. Nat Rev Cancer. 2004;4(7):528-539.

64. Slevin M, Krupinski J, Gaffney J, et al. Hyaluronan-mediat-
ed angiogenesis in vascular disease: uncovering RHAMM
N

and CD44 receptor signaling pathways. Matrix Biol.

2007;26(1):58-68.
65. Savani RC, Cao G, Pooler PM, Zaman A, Zhou Z, DeLisser
O

HM. Differential involvement of the hyaluronan (HA) re-

ceptors CD44 and receptor for HA-mediated motility in
endothelial cell function and angiogenesis. J Biol Chem.
D

2001;276(39):36770-36780.
66. Cao G, Savani RC, Fehrenbach M, et al. Involvement of en-
dothelial CD44 during in vivo angiogenesis. A J Pathol.
2006;169(1):325-336.
67. Hamilton S, Fard S, Paiwand F, et al. The hyaluronan re-
ceptors CD44 and RHAMM (CD168) form complexes
with ERK 1,2 that sustain high basal motility in breast
cancer cells. J Biol Chem. 2007;282(22):16667-16680.
68. Lokeshwar V, Selzer M. Differences in hyaluronic acid-
mediated functions and signaling in arterial, microvessel
and vein-derived human endothelial cells. J Biol Chem.

88 WOUNDS® www.woundsresearch.com

View publication stats