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Tissue integrity signals communicated by high-molecular weight hyaluronan and the resolution of inflammation

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The extracellular matrix polysaccharide hyaluronan (HA) exerts size-dependent effects on leukocyte behavior. Low-molecular weight HA is abundant at sites of active tissue catabolism and promotes inflammation via effects on Toll-like receptor signaling. Conversely, high-molecular weight HA is prevalent in uninjured tissues and is anti-inflammatory. We propose that the ability of high-molecular weight but not low-molecular weight HA to cross-link CD44 functions as a novel form of pattern recognition that recognizes intact tissues and communicates "tissue integrity signals" that promote resolution of local immune responses.
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IMMUNOLOGY AT STANFORD UNIVERSITY
Tissue integrity signals communicated by high-
molecular weight hyaluronan and the resolution
of inflammation
S. M. Ruppert T. R. Hawn A. Arrigoni
T. N. Wight P. L. Bollyky
ÓSpringer Science+Business Media New York 2014
Abstract The extracellular matrix polysaccharide hyaluronan (HA) exerts size-dependent effects on leukocyte behavior.
Low-molecular weight HA is abundant at sites of active tissue catabolism and promotes inflammation via effects on Toll-
like receptor signaling. Conversely, high-molecular weight HA is prevalent in uninjured tissues and is anti-inflammatory.
We propose that the ability of high-molecular weight but not low-molecular weight HA to cross-link CD44 functions as a
novel form of pattern recognition that recognizes intact tissues and communicates ‘‘tissue integrity signals’’ that promote
resolution of local immune responses.
Keywords Hyaluronan Danger signals DAMP Integrity signal CD44 ECM
Abbreviations
HMW-HA High-molecular weight hyaluronan
LMW-HA Low-molecular weight hyaluronan
HA Hyaluronan
PAMPs Pathogen-associated molecular patterns
DAMPs Damage-associated molecular patterns
TLR Toll-like receptor
APC Antigen-presenting cell
DC Dendritic cell
Hyaluronan and the tissue response to injury
Hyaluronan (HA) is an extracellular matrix (ECM) glycos-
aminoglycan that is abundant in the ECM of inflamed tissues.
HA is a long, non-branching disaccharide made of glucu-
ronic acid and N-acetyl-glucosamine with diverse effects on
tissue structure and function (reviewed in [13]).
Both the size and the amount of HA are tightly regulated
during progression through the stages of an injury
response. Immediately upon injury, local HA production
increases substantially [2,3]. The three HA synthases
responsible for this production generate predominantly
high-molecular weight HA (HMW-HA) (defined here as
[5910
5
Da) [1,46]. During inflammation, this HA is
rapidly catabolized by a diverse group of host and (if
infection is present) microbial hyaluronidases (HA’ases),
mechanical forces, and oxidation [7,8], resulting in frag-
mentary, low-molecular weight HA (defined here as
\200 kDa) that are cleared via CD44-mediated
endocytosis. Upon the resolution of inflammation,
both the amount and size of HA return to basal levels.
However, in chronically inflamed tissues, shorter HA
polymers predominate. In light of these associations,
HA size has been termed a natural biosensor for the
state of tissue integrity [9,10].
S. M. Ruppert A. Arrigoni P. L. Bollyky (&)
Division of Infectious Diseases, Stanford University School
of Medicine, 300 Pasteur Drive, Rm. L-133, Stanford,
CA 94305-5107, USA
e-mail: pbollyky@stanford.edu
T. R. Hawn
Division of Allergy and Infectious Diseases, University of
Washington Medical Center, 1959 NE Pacific Ave, Seattle,
WA 98195, USA
T. N. Wight
Matrix Biology Division, Benaroya Research Institute, 1201 9th
Ave, Seattle, WA 98101, USA
P. L. Bollyky
123
Immunol Res
DOI 10.1007/s12026-014-8495-2
Here, we propose that the receptors that discriminate
between HMW-HA and low-molecular weight hyaluronan
(LMW-HA) together constitute an integrated system of
pattern recognition capable of communicating the presence
of either intact or fragmented ECM and, furthermore, that
the resulting contextual cues are relevant for integrating
wound healing with the local immune response to injury.
LMW-HA-mediated danger signals
Pattern recognition allows for efficient, choreographed
responses to environmental stimuli. During infection,
pathogen-associated molecular patterns (PAMPs) such as
lipopolysaccharide (LPS), instigate rapid, programmatic
responses that engender appropriately polarized immuno-
logic responses. Endogenous markers of inflammation,
termed danger-associated molecular patterns (DAMPs),
function in an analogous manner to microbial PAMPs and
trigger many of the same receptors [11,12]. DAMPs share
with PAMPs the properties of being small, structurally
repetitive molecules. However, unlike PAMPs, DAMPs are
also present in sterile inflammation. Examples of DAMPs
include heat-shock proteins [13,14], urate crystals [4,15],
and fragmentary components of the ECM [16,17].
LMW-HA is an ECM molecule that functions as a pro-
inflammatory DAMP [3,1824]. LMW-HA promotes the
activation and maturation of dendritic cells (DC) [1,25],
drives the release of pro-inflammatory cytokines such as
IL-1ß, TNF-a, IL-6, and IL-12 by multiple cell types [6,
2628], drives chemokine expression and cell trafficking
[2931], and promotes proliferation [3234] (Fig. 1).
These signals may be particularly relevant in settings of
sterile inflammation.
Many of the pro-inflammatory effects of LMW-HA are
attributed to interactions with the pattern recognition
receptors Toll-like receptor 2 (TLR2) or Toll-like receptor
4 (TLR4). LMW-HA promotes TLR-mediated phosphor-
ylation of MAPK, nuclear translocation of NF-jB, and
TNF-aproduction (reviewed in [3,35]). While HA mole-
cules of all sizes share the same repeating disaccharide
structure, only LMW-HA can signal through TLR2 or
TLR4 [1,4,6,36]. Therefore, only products of HA
catabolism, indicative of active inflammation, promote
TLR signaling.
HMW-HA-mediated tissue integrity signals
HMW-HA predominates in healthy tissues and typically
inhibits inflammation. Specifically, HMW-HA prevents
cell growth and differentiation [7,37], diminishes the
production of inflammatory cytokines by multiple cell
types [9,38], and impairs phagocytosis by macrophages
[11,39]. Recently, HMW-HA has been implicated in the
inhibition of tumor progression [13,25]. Administration of
HMW-HA is anti-inflammatory in lung injury models [4,
40], collagen-induced arthritis [16,41], and a variety of
other in vivo model systems [18,20,2224,42].
Most of these anti-inflammatory properties are attribut-
able to interactions of HMW-HA and CD44, the major cell-
surface HA-binding transmembrane glycoprotein. CD44 is
thought to translate cues from the ECM, including HA, into
signals that may influence growth, survival, activation, and
differentiation [25,43,44]. Consistent with this, CD44
-/-
mice are unable to efficiently resolve inflammation. Ini-
tially, this was demonstrated in a bleomycin-induced lung
injury model where CD44
-/-
mice have impaired clear-
ance of apoptotic neutrophils, persistent accumulation of
LMW-HA, and impaired activation of TGF-b1. Upon
reconstitution with CD44
?
leukocytes, the inflammation is
resolved [6,40,43]. Similar defects were subsequently
demonstrated in other injury models including bacterial
pneumonia [29,39]. Myocardial infarcts in CD44
-/-
mice
are associated with reduced collagen deposition and fewer
fibroblasts [32,45], suggesting that the loss of CD44
impairs the effectiveness and efficiency of wound repair.
In addition to these anti-inflammatory effects, CD44 is
reported to contribute to immune homeostasis via the
maintenance of type 1 helper T (Th1) memory cells.
Baaten and colleagues find that CD44 can counteract Fas-
mediated apoptosis of these cells [35,46]. These data
Fig. 1 Pro-inflammatory actions of LMW-HA and TLR signaling.
LMW-HA characterizes inflamed tissues with active matrix catabo-
lism. LMW-HA is an agonist of TLR signaling through interactions
with TLR2 and/or TLR4 and communicates ‘‘danger signals’’ to
infiltrating leukocytes. LMW-HA promotes leukocyte homing, pro-
liferation and production of pro-inflammatory cytokine production as
well as DC maturation and antigen presentation. LMW-HA also
induces turnover of CD44, potentially limiting the likelihood of CD44
cross-linking by intact ECM
Stanford Immunology
123
suggest that despite the multiple roles for CD44 in leuko-
cyte activation, survival, and costimulation, CD44 pri-
marily promotes homeostasis and the resolution, rather
than the propagation, of inflammation.
However, CD44 is not exclusively anti-inflammatory.
CD44 interacts with a large number of ECM components,
growth factors, and cytokines, and many of these factors,
including osteopontin, are pro-inflammatory [36,47].
Indeed, CD44 contributes to leukocyte trafficking [37,48]
and antibodies against CD44 that inhibit this trafficking
have been proposed as a therapy for inflammatory and
autoimmune diseases [38,49]. CD44 is also implicated in
TLR signaling in certain contexts [39,50,51]. We propose
that CD44 is anti-inflammatory in those contexts in which
it is cross-linked by HMW-HA and potentially other ECM
ligands.
How HMW-HA and CD44 inhibit inflammation
Most downstream effects of CD44-binding HA and other
ligands are mediated either via cellular re-organization (i.e.,
cytoskeleton or lipid raft architecture) or through receptor
complexes that CD44 partners with (reviewed in [25,52]).
Several such mechanisms are highlighted in Fig. 2.
HMW-HA negatively regulates pro-inflammatory TLR
signaling. Mice treated with HMW-HA prior to LPS
exposure have greatly decreased serum IL-6 and TNFa
levels and are protected from symptoms of sepsis [36,40]. In
other studies, oral administration of HMW-HA likewise
suppresses inflammation in a TLR4-dependent manner [41,
53] and actively downregulates LPS-induced NF-jB trans-
location [42,54]. Consistent with these findings, both
MyD88 expression levels and nuclear translocation of NF-
jB are increased in CD44
-/-
mice [43,44,55,56]. These
mice also have diminished levels of several negative regu-
lators of TLR signaling, including A20 and IRAK-M [35,40,
43], suggesting that CD44 could govern expression of these
molecules. In addition to inducing effects on TLR signaling
downstream, CD44 physically associates with TLR4 and co-
expressed MD-2 and facilitates the binding of LMW-HA to
this complex [39,57], presumably in the absence of HMW-
HA. Together, these data suggest that HMW-HA and CD44
modulate TLR signaling at multiple points.
CD44 cross-linking may disproportionately impact cell
types that are important for immune suppression, such as
regulatory T cells (Tregs). The suppressive capacity of
Tregs correlates with expression levels of the transcription
factor, Foxp3 [45]. Foxp3
?
Tregs are a specialized sub-
population of CD4
?
T cells that maintain immune
homeostasis via production of IL-10, an anti-inflammatory
cytokine critical for both immune regulation and for wound
healing [58]. Depletion of Tregs leads to multi-systemic
Fig. 2 Anti-inflammatory actions of HMW-HA and CD44 cross-
linking. Several mechanisms of anti-inflammatory activities propa-
gated via HA and CD44 have been reported. 1. HMW-HA and CD44
negatively regulate pro-inflammatory TLR signaling at multiple
levels. 2. CD44 is thought to play an important role in clearance of
pro-inflammatory LMW-HA. 3. HMW-HA promotes the function and
phenotypic stability of regulatory T-cell populations, including
Foxp3
?
Treg, TR1, and NKT cells. 4. CD44 cross-linking promotes
production of anti-inflammatory cytokines. 5. HA is a potent
antioxidant that limits the damage caused by free radicals generated
at sites of inflammation. HA length probably does not impact this
anti-oxidant property but is likely to contribute to its longevity. In
light of these anti-inflammatory roles, we propose that HMW-HA
functions as a tissue integrity signal that dampens inflammation at
sites of intact tissues
Stanford Immunology
123
autoimmunity in both mice and humans [46], but can be
prevented or ablated upon Treg adoptive transfer [47,48].
HMW-HA promotes the function and phenotypic sta-
bility of Tregs. Firan and colleagues were the first to
demonstrate an association between HMW-HA binding
and Foxp3 expression [49,59]. We subsequently reported
that HMW-HA enhanced the function and viability of
Tregs, particularly in low IL-2 environments [50,51,60].
Moreover, HMW-HA promotes Treg function via
increased Foxp3 expression and production of IL-10 [36,
51,52,61]. Along similar lines, we find that HMW-HA, in
the context of a TCR signal, induces conventional T cells
to produce IL-10 and behave like type 1 regulatory cells
(TR1), a subset of Tregs [36,52]. Similarly, stimulation of
CD44 through Ab cross-linking or HMW-HA causes
immunoregulatory natural killer T cells (NKT) to secrete
cytokines, up-regulate activation markers, and resist acti-
vation-induced cell death [50,51,53].
Another way CD44 may impact regulatory cell popu-
lations is through effects on antigenic responses. CD44
cross-linking is known to impact formation of TCR com-
plexes [53,54] and immune synapse function [55,56,62].
Therefore, CD44 may magnify low-level antigenic
responses and provide tonic signals to these cells. As Treg
and NKT cells both constitutively express high levels of
CD44 receptor, they are poised to respond to such signals.
Memory T cells are another group that is constitutively
CD44
high
and have impaired homeostasis in the absence of
CD44 [35,63].
CD44 cross-linking by HMW-HA as a novel form
of pattern recognition
The ability of HA to bind to CD44 is dependent upon
interactions with multiple CD44 molecules. This is because
the interaction between HA and CD44 is mediated by low-
affinity hydrogen bonds [25,57] such that interactions with
multiple receptors or increasing amounts of HA are
required for efficient binding. In a study in which HA
molecules bound to lipid nanoparticles were used to probe
a CD44-coated surface, it was demonstrated that only HA
molecules of [700 kDa could bind CD44 reliably [6,59].
HA binding is further influenced by the density of CD44
receptors on the cell surface and their relative affinity for
HA [60,64]. While the precise size cutoffs vary between
model systems, these data suggest that efficient HA binding
to CD44 is predicated upon CD44 cross-linking.
The ability of HMW-HA to cross-link spatially isolated
CD44 molecules and the inability of LMW-HA to do so
may explain how leukocytes discriminate between HMW-
HA and shorter HA polymers. Consistent with this, the
anti-inflammatory effects attributed to HMW-HA in mul-
tiple systems can be recapitulated using antibody-mediated
CD44 cross-linking [36,51,52,61,65]. We propose that
CD44 cross-linking functions as a form of pattern recog-
nition that distinguishes between tissue microenvironments
that are actively inflamed, characterized by LMW-HA, and
those that are healing or uninjured, characterized by HMW-
HA. This model does not exclude other roles for CD44 but
attributes the homeostatic and anti-inflammatory properties
of HMW-HA to CD44 cross-linking. Consistent with this
view, cross-linking is implicated in CD44-mediated effects
on IL-10 production [36,52,66], Treg homeostasis [50,51,
67], NKT cell function [53], neutrophil inhibition [62], and
cell survival [63]. CD44 interacts with multiple other cell-
surface receptors whose signaling influences leukocyte
proliferation, maturation, activation, and trafficking [25],
and we speculate that the contribution of HMW-HA and
CD44 cross-linking to these pathways will prove to pro-
mote homeostasis and the resolution of inflammation.
Of note, HMW-HA and CD44 also reduce inflammation
in ways that do not necessitate cross-linking. CD44 medi-
ates clearance of HA fragments such that, in the absence of
CD44, these fragments accumulate and drive inflammation
through interactions with TLR [6]. Further, HA acts as an
antioxidant and the greater the longevity of HMW-HA
could translate into a prolonged ability to mitigate oxida-
tive damage.
The integration of HA contextual cues
We propose a model whereby HA size-specific interactions
with TLR2/TLR4 and CD44 together constitute an inte-
grated system of pattern recognition that discriminates
between actively inflamed and healing tissues based on the
predominant size of HA. In this model, LMW-HA, typical
of actively inflamed tissues, communicates pro-inflamma-
tory ‘‘danger’’ signals via TLR signaling while HMW-HA,
typical of post-inflammatory or uninflamed tissues, com-
municates ‘‘tissue integrity’’ signals via CD44 cross-link-
ing. Together, these HA-mediated contextual signals
constitute an integrated system for sensing changes in the
inflammatory milieu and coordinating appropriate respon-
ses (Fig. 3). By ‘‘contextual signals,’’ we mean the infor-
mation gained from the immune environment, specifically
in the form of factors that engage co-stimulatory receptors.
Mechanisms must exist for the integration of these sig-
nals, given that HA molecules of disparate size are likely to
coexist in injured tissues. Since HA molecules of different
sizes may compete for binding to CD44, another variable
that might impact competitive binding interactions is the
molar predominance of LMW-HA generated from a single
HMW-HA molecule. The relative expression of HA
receptors may also be impacted by the HA molecules
themselves. For example, LMW-HA is known to induce
Stanford Immunology
123
CD44 cleavage [64], and this could make less CD44
available for cross-linking. Additionally, HA may regulate
its own catabolism; the enzymatic activity of the primary
extracellular hyaluronidase, HYAL-2, is dependent on
CD44 [65] along with other factors [66].
Other ECM components are likely to influence both HA
integrity as well as interactions with CD44. In vivo, a diverse
group of HA-binding molecules, called hyaladherins, con-
tribute to particular HA structural architectures and may also
contribute to CD44 cross-linking [67]. Hyaladherins and the
nature and extent of HA superstructures may be essential to
the anti-inflammatory properties of HMW-HA, an idea first
proposed by Day and De La Motte [68].
An example of a hyaladherin with established roles in
the cross-linking of CD44 is tumor necrosis a-stimulated
gene 6 (TSG-6). TSG-6 catalyzes the covalent transfer of
heavy chains of inter-a-inhibitor (IaI) present in serum to
HA polymers, forming complex, cross-linked HA net-
works, promoting further interactions of HA with CD44
[69]. Such structures form the basis of provisional wound
matrix, the scaffold that is a crucial early component of
healing tissue. Additionally, CD44 binds heavy chain
bound HA with greater avidity than unbound HA, pro-
moting more efficient wound healing [70]. TSG-6 prevents
HA degradation and inhibits enzymes involved in ECM
catabolism, including HA’ases [68][71]. TSG-6 levels are
diminished at sites of chronic inflammation [72,73], and,
indeed, treatment with TSG-6 has been shown to be of
benefit in some inflammatory disorders, particularly in the
context of autoimmunity [74,75]. Additionally, there are
other hyaladherins, such as pro-inflammatory versican [76],
that can inhibit HA–CD44 interactions.
Conclusions
The integration of wound healing and immune regulation is
critical for the resolution of inflammatory responses but is
poorly understood. Here, we have proposed that tissue
repair and the immune response to tissue damage are
synchronized by contextual cues that reflect the local
integrity of the ECM, in particular the size of HA. More-
over, we have proposed that the receptors that discriminate
between HA molecules of different sizes together consti-
tute an integrated system of pattern recognition. This sys-
tem of HA size-specific pattern recognition offers a
powerful model for how tissue repair and immune regu-
lation are integrated in injured and healing tissues.
This model also suggests that catabolic HA at sites of
sterile inflammation may drive autoimmunity and predicts
that it may be possible to impact HA integrity in vivo in ways
that promote wound healing and immune tolerance. Indeed,
delivery of exogenous HMW-HA is beneficial in both the
amelioration of airway inflammation [77] and in limiting
scar formation in dermatologic wounds [78]. Supplementa-
tion with TSG-6 to promote HA integrity is likewise a
strategy for immunotherapy with known benefit in autoim-
mune arthritis [74] and other inflammatory settings.
However, many questions remain. For example, are
there additional fragmentary ECM components, that are
also CD44 ligands, that function in an analogous manner to
HA? How are CD44 and TLR-mediated signals integrated?
How do other HA receptors (e.g., RHAMM, LYVE-1,
HARE) and the various sizes of HA polymers fit within this
model of pattern recognition? Do clinical preparations of
HMW-HA used to prevent abdominal adhesions (e.g., Se-
prafilm) or reduce joint inflammation (e.g., Synvisc) in fact
provide tissue integrity signals? These questions and others
warrant continued investigation into the role of HA in
immune regulation.
Acknowledgments This work was supported by National Institutes
of Health grants T32 AI07290 (to SMR); R01 DK096087-01, R01
HL113294-01A1, and U01 AI101984 (to PLB); and HL018645 and a
BIRT supplement AR037296 (to TNW). The authors declare that they
have no conflict of interest.
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... In general, the length of HA molecules can range from oligomers to extremely long forms, reaching up to millions of Daltons in molecular weight, and the HA biological functions are closely associated with its molecular mass. High-molecular weight HA contributes to tissue homeostasis [20], displaying anti-inflammatory [21] and antioxidant properties [22]. Under physiological conditions, most extracellular HAs exhibit a relatively high molecular weight. ...
... In general, the length of HA molecules can range from oligomers to extremely long forms, reaching up to millions of Daltons in molecular weight, and the HA biological functions are closely associated with its molecular mass. High-molecular weight HA contributes to tissue homeostasis [20], displaying antiinflammatory [21] and antioxidant properties [22]. Under physiological conditions, most extracellular HAs exhibit a relatively high molecular weight. ...
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Hyaluronan (HA) is one of the crucial components of the extracellular matrix in vertebrates and is synthesized by three hyaluronan synthases (HASs), namely HAS1, HAS2, and HAS3. The low expression level of HASs in normal keratinocytes and other various types of cells presents a recognized challenge, impeding biological and pathological research on their localization. In this study, the human proteins HAS1, HAS2, and HAS3 with fused maltose-binding protein (MBP) tags were successfully expressed at high levels and purified for the first time in HEK293F cells. The enzymatic properties of the three HAS proteins were further characterized and compared. A pulse-field gel electrophoresis analysis of the size distribution of the hyaluronan generated in vitro by the membrane proteins demonstrated that the three HAS isoforms generate HA polymer chains at different molecular masses. Kinetic studies demonstrated that the three HAS proteins differed in their catalytic efficiency and apparent Km values for the two substrates, UDP-GlcA and UDP-GlcNAc. Furthermore, the cellular hyaluronan secretion by the three isoenzymes was evaluated and quantified in the HEK 293T cells transfected with GFP-tagged HAS1-GFP, HAS2-GFP, and HAS3-GFP using an ELISA assay. These findings enhance our understanding of the membrane protein HASs in mammalian cells.
... By observing how immune responses and mechanisms of tissue damage are spatially related, researchers can also accurately assess how well immunomodulatory treatment strategies block specific pathomechanisms. This dual focus is crucial for the development of optimized therapeutic approaches that increasingly aim to alleviate systemic inflammatory mechanisms while preserving or restoring tissue integrity [31,32]. ...
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This study demonstrates the effectiveness of propidium iodide as a reliable marker for detecting dead or dying cells in frozen liver tissue sections. By comparing propidium iodide staining with the widely used Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, both methods showed consistent results in disease models such as alcohol-induced fibrosis and Western diet-induced fatty liver. Additionally, propidium iodide was successfully co-stained with other fluorescent markers, like phalloidin (for actin filaments) and antibodies targeting collagen, enabling detailed spatial analysis of dying cells within tissue. This multiplex approach allows for a deeper understanding of tissue organization and cell death localization, particularly in complex conditions like liver fibrosis. Moreover, our results suggest that propidium iodide staining can be applied beyond current models, offering a more accessible and cost-effective alternative to traditional methods, like TUNEL. Furthermore, its integration with other markers enables simultaneous analysis of immune responses and tissue damage, making it a powerful tool for future studies on liver disease and other inflammatory conditions. This technique has the potential to advance research into disease mechanisms and improve the evaluation of novel therapeutic strategies targeting tissue regeneration and inflammation control.
... Some properties of the different molecular weights of hyaluronic acid; Figure S1: Chemical structure of Gel-TA and HA-TA conjugates of different molecular weights and hydrogel formation in the presence of HRP and H 2 O 2 as enzymatic crosslinking agents; Figure S2: Sketch of the PTFE mold used for hydrogel formation; Figure S3: Formed hydrogels of hyaluronic acid with low, medium, and high molecular weights and gelatin functionalized with tyramine having different degrees of substitution (DS); Figure S4: Morphology of BPCs in cell-laden hydrogels; and Figure S5: Immunohistochemistry staining of collagen type II for 2.5% w/v hydrogels encapsulated with chondrocytes after culturing for 13 days in chondrogenic medium. References [39][40][41][42][43][44][45][46][47][48][49] ...
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Injectable hydrogels are promising materials for cartilage regeneration in tissue engineering due to their tunable crosslinking rates, mechanical properties, and biodegradation profiles. This study investigates the chondrogenic potential of hyaluronic acid (HA) hydrogels crosslinked via tyramine (TA) moieties, with and without gelatin modified with TA (Gel-TA). Incorporating Gel-TA improved cell viability, spreading, and cartilage matrix deposition, particularly in medium and high molecular weight (MMW and HMW) HA-TA/Gel-TA hydrogels. Although the hydrogels’ molecular weight did not significantly alter stiffness, MMW and HMW HA-TA/Gel-TA formulations exhibited enhanced functional properties such as slower degradation and superior cartilage matrix deposition. These attributes, coupled with Gel-TA’s effects, underscore the importance of both molecular weight and biofunctional components in hydrogel design for cartilage regeneration. While low molecular weight (LMW) HA-TA hydrogels offered excellent injectability and supported high cell viability, they degraded rapidly and exhibited reduced cartilage matrix formation. Gel-TA enhanced cell adhesion and spreading by providing integrin-binding sites and promoted collagen type II deposition, crucial for cartilage regeneration. Moreover, the increased stiffness of MMW and HMW HA-TA/Gel-TA hydrogels facilitated extracellular matrix production. These findings show the potential of Gel-TA-modified HA-TA hydrogels for cartilage tissue engineering, with the opportunity for further optimization through the incorporation of bioactive components.
... It is also crucial to underscore that high molecular weight (>500 kDa) HA exerts anti-inflammatory actions and is predominantly associated with tissue integrity. Conversely, low molecular weight (<200 kDa) HA has been linked to pro-inflammatory effects and is more closely related to pathogenesis [170,171]. Accordingly, a 1000 kDa molecular weight form of HA was employed in our study. ...
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Despite the promising potential of cell-based therapies developed using tissue engineering techniques to treat a wide range of diseases, including limbal stem cell deficiency (LSCD), which leads to corneal blindness, their commercialization remains constrained. This is primarily attributable to the limited cell sources, the use of non-standardizable, unscalable, and unsustainable techniques, and the extended manufacturing processes required to produce transplantable tissue-like surrogates. Herein, we present the first demonstration of the potential of a novel approach combining collagen films (CF), hyaluronic acid (HA), human telomerase-immortalized limbal epithelial stem cells (T-LESCs), and macromolecular crowding (MMC) to develop innovative biomimetic substrates for limbal epithelial stem cells (LESCs). The initial step involved the fabrication and characterization of CF and CF enriched with HA (CF-HA). Subsequently, T-LESCs were seeded on CF, CF-HA, and tissue culture plastic (TCP). Thereafter, the effect of these matrices on basic cellular function and tissue-specific extracellular matrix (ECM) deposition with or without MMC was evaluated. The viability and metabolic activity of cells cultured on CF, CF-HA, and TCP were found to be similar, while CF-HA induced the highest (p < 0.05) cell proliferation. It is notable that CF and HA induced cell growth, whereas MMC increased (p < 0.05) the deposition of collagen IV, fibronectin, and laminin in the T-LESC culture. The data highlight the potential of, in particular, immortalized cells and MMC for the development of biomimetic cell culture substrates, which could be utilized in ocular surface reconstruction following further in vitro, in vivo, and clinical validation of the approach.
... ГК, находящаяся в кровотоке, быстро разрушается, в то время как ГК, связанная с белками и внедренная в ткани, такие как суставы, базальные мембраны и стекловидное тело глаза, живет дольше [28]. Более длинные полимеры высокомолекулярной ГК обычно преобладают в большинстве тканей в стационарных условиях, в то время как более короткие полимеры LMW-ГК преобладают в очагах активного воспаления [29]. В свете этих ассоциаций размер LMW-ГК был назван естественным биосенсором состояния целостности тканей [30]. ...
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Hymecromone (4-MU) is a recognized agent currently used in clinical practice. Since 1960, hymecromone has been used in many countries as a choleretic and cholespasmolytic, a drug approved for use in humans with biliary tract disorders. The review presents both traditional European and Russian studies of the selective antispasmodic and choleretic properties of hymecromone, due to which hymecromone is the drug of choice for the treatment of biliary tract diseases, as well as new fundamental and clinical studies of numerous pleiotropic effects of 4-MU associated with inhibition of hyaluronic acid and many other properties of this exciting molecule. These include antibacterial, antiviral, and nonspecific anti-inflammatory effects. Positive results have been demonstrated in carbohydrate and lipid metabolism disorders, autoimmune diseases, as well as liver, heart, and kidney diseases. Numerous in vitro and in vivo studies have been presented in pancreatic, prostate, skin, esophagus, breast, liver, ovary, bone cancers, metastatic lesions, leukemia, autoimmune and inflammatory diseases. Hymecromone is indicated not only as a choleretic and cholespasmolytic but also as a choleseptic in cholangitis and chronic cholecystitis, including opisthorchiasis, which does not disagree with its label. Odecromone® (hymecromone, tablets 200 mg) is available on the Russian market; it replaced the originator drug and is its fully equivalent generic.
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Hyaluronan (HA) is a ubiquitous macromolecule in the human body with remarkable structure and function. HA presents a key role in several biological processes in mammals. The synthesis/catabolism of HA is critical in several pathologies and has been used as a marker for the prognosis of cancers. Among its physiological roles, HA is used for wound healing applications. This review reports many of the latest developments of hyaluronan and its derivatives in research, preclinical, and published clinical trials for wound healing. An adequate physico-chemical characterization and identification of selected physico-chemical properties of the prepared material are mandatory. Moreover, cytotoxicity and evaluation of biological effects in vitro using standardized protocols are required as preclinical. Finally, to choose adequate in vivo models for testing efficacy is requested. Unfortunately, the biological role of HA is still not well understood. Therefore, an overview of several HA-based products is provided and discussed. Several ways of HA chemical modification were evaluated. Finally, this review focuses on products containing HA, novel developments, gaps, and limitations of the current state of the art.
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The naked mole rat (Heterocephalus glaber) displays exceptional longevity, with a maximum lifespan exceeding 30 years. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years. In addition to their longevity, naked mole rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer. Here we identify a mechanism responsible for the naked mole rat's cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This high-molecular-mass HA accumulates abundantly in naked mole-rat tissues owing to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high-molecular-mass HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, HYAL2, naked mole-rat cells become susceptible to malignant transformation and readily form tumours in mice. We speculate that naked mole rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species.
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Genetic and immunological screening for type 1 diabetes has led to the possibility of preventing disease in susceptible individuals. Here, we show that human Mesenchymal Stem/Stromal Cells (hMSCs) and TNF-α-stimulated gene 6 (TSG-6), a protein produced by hMSCs in response to signals from injured tissues, delayed the onset of spontaneous autoimmune diabetes in NOD mice by inhibiting insulitis and augmenting regulatory T cells (Tregs) within the pancreas. Importantly, hMSCs with a knockdown of tsg-6 were ineffective at delaying insulitis and the onset of diabetes in mice. TSG-6 inhibited the activation of both T cell and antigen presenting cells (APCs) in a CD44-dependent manner. Moreover, multiple treatments of TSG-6 rendered APCs more tolerogenic, capable of enhancing Treg generation and delaying diabetes in an adoptive transfer model. Therefore, these results could provide the basis for a novel therapy for the prevention of type 1 diabetes.
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Background: The midgestational fetus is capable of regenerative healing. We have recently demonstrated a novel role for the anti-inflammatory cytokine interleukin 10 (IL-10) as a regulator of hyaluronan (HA) in the extracellular matrix. The signaling pathway of IL-10 has been studied in monocytes but is unknown in dermal fibroblasts. We hypothesized IL-10 signals through its primary receptor, IL-10R1, to activate STAT3, resulting in HA synthesis. Methods: Murine midgestational (E14.5) fetal fibroblasts were evaluated in vitro. Pericellular matrix was quantified using a particle exclusion assay. STAT3 levels and cellular localization were evaluated by Western blot/band densitometry and immunocytochemistry/confocal microscopy. HA levels were quantified by enzyme-linked immunosorbent assay. The effects of IL-10R1 signal blockade by a neutralizing antibody and STAT3 inhibition were evaluated. An ex vivo midgestation fetal forearm culture incisional wound model in control and transgenic IL-10-/- mice was used to evaluate the role of STAT3 on the extracellular matrix. Results: Fetal fibroblasts produce a robust hyaluronan-rich pericellular matrix that is IL-10R1 and STAT3 dependent. Inhibition of IL-10R1 signaling results in decreased phosphorylated STAT3 levels and inhibition of nuclear localization. Inhibition of STAT3 results in decreased HA production. At day 3, midgestation fetal wounds have efficient re-epithelialization, which is significantly slowed in IL-10-/- wounds at the same gestation and with inhibition of STAT3. Conclusions: Our data demonstrate that IL-10 regulates HA synthesis through its primary receptor IL-10R1 and STAT3 activation. This supports a novel nonimmunoregulatory mechanism of IL-10 in its role in fetal regenerative wound healing.
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Hyaluronan is a major component of synovial tissue and fluid as well as other soft connective tissues. It is a high-Mr polysaccharide which forms entangled networks already at dilute concentrations (< 1 mg/mL) and endows its solutions with unique rheological properties. Physiological functions of hyaluronan (lubrication, water homeostasis. macromolecular filtering, exclusion, etc.) have been ascribed to the properties of these networks. Recently a number of specific interactions between hyaluronan and a group of proteins named hyaladherins have also pointed towards a role of hyaluronan in recognition and the regulation of cellular activities. Many more or less well documented hypotheses have been proposed for the function of hyaluronan in joints, for example, that it should lubricate, protect cartilage surfaces, scavenge free radicals and debris, keep the joint cavities open, form flow barriers in the synovium and prevent capillary growth.
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To investigate the toxic effects of ethylenediaminetetraacetic acid disodium salt (EDTA), a corneal penetration enhancer in topical ophthalmic formulations, on DNA in human corneal epithelial cells (HCEs), and to investigate whether the effect induced by EDTA can be inhibited by high molecular weight hyaluronan (HA). Cells were exposed to EDTA in concentrations ranging from 0.00001 to 0.01% for 60 min, or 30 min high molecular weight HA pretreatment followed by EDTA treatment. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. The DNA single- and double-strand breaks of HCEs were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci, respectively. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2', 7'-dichlorodihydrofluorescein diacetate. EDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in human corneal epithelial cells at concentrations lower than 0.01%. However, a significant increase of DNA single- and double-strand breaks was observed in a dose-dependent manner with all the concentrations of EDTA tested in HCEs. In addition, EDTA treatment led to elevated ROS generation. Moreover, 30 min preincubation with high molecular weight HA significantly decreased EDTA-induced ROS generation and DNA damage. EDTA could induce DNA damage in HCEs, probably through oxidative stress. Furthermore, high molecular weight HA was an effective protective agent that had antioxidant properties and decreased DNA damage induced by EDTA.
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J. Neurochem. (2012) 122, 344–355. Toll-like receptor-4 (TLR4) signaling has been implicated in microglial activation and propagation of inflammation following spinal cord injury (SCI). As such, modulating microglial activation through TLR4 represents an attractive therapeutic approach to treat SCI. High molecular weight hyaluronan (HMW-HA), a polymer with multiple therapeutic uses, has been previously shown to modulate TLR4 activation in macrophages and has shown early promise as a therapeutic agent in SCI. However, the mechanism associated with HMW-HA has not been fully elucidated or tested in microglia, a similar cell type. In the current study, we sought to determine the effects of HMW-HA on TLR4 activation in microglia and to gain insights into the mechanism of action. Rat primary microglial cultures were exposed to lipopolysaccharides (LPS) and HMW-HA, and the extent and mechanisms of inflammation were studied. HMW-HA decreased LPS mediated IL-1β, IL-6, and Tumor necrosis factor-α gene expression and IL-6 and nitric oxide production. This decrease was associated with a reduction in ERK 1/2 and p38 phosphorylation, was dependent on the continued presence of HMW-HA, and activation of Akt and A20 protein expression was reduced by HMW-HA. Together, our results show that HMW-HA can reduce LPS-mediated inflammatory signaling in microglia. We suggest that HA possibly mediates its effects by blocking the induction of inflammatory signaling through an extracellular mechanism.
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Hyaluronic acid (HA) is a polysaccharide common to most species and is found in many sites in the human body, including the skin and soft tissue. A systematic review of the literature and meta-analysis was performed to identify randomized controlled trials, evaluating the use of HA derivatives in healing burns, epithelial surgical, and chronic wounds. Nine studies were identified, which met the search criteria and clinical endpoints of complete healing and percent wound size reduction when using HA vs. either an active or passive comparator. It was found in the vast majority of randomized controlled trials (eight of nine) that HA derivatives significantly improved the healing of wounds vs. traditional therapies or placebo (either via complete healing or a significant reduction in wound size) occurring from burns, venous insufficiency, diabetes, neuropathic insufficiency, and surgical removal of the epithelial layer (for tattoo removal). In the other remaining trial, one formulation of HA was compared with another, with the higher concentration showing improved application characteristics. Further, it was found in a meta-analysis in subsets of patients with diabetic foot ulcers (neuropathic) that HA derivatives healed these types of wounds significantly faster than standard of care. These studies in aggregate show that HA derivatives accelerate the healing process in burns, epithelial surgical wounds, and chronic wounds.
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Regulatory T cells (Tregs) are long-lived cells that suppress immune responses in vivo in a dominant and antigen-specific manner. Therefore, therapeutic application of Tregs to control unwanted immune responses is an active area of investigation. Tregs can confer long-term protection against auto-inflammatory diseases in mouse models. They have also been shown to be effective in suppressing alloimmunity in models of graft-versus-host disease and organ transplantation. Building on extensive research in Treg biology and preclinical testing of therapeutic efficacy over the past decade, we are now at the point of evaluating the safety and efficacy of Treg therapy in humans. This review focuses on developing therapy for transplantation using CD4+Foxp3+ Tregs, with an emphasis on the studies that have informed clinical approaches that aim to maximize the benefits while overcoming the challenges and risks of Treg cell therapy.