Hyaluronan in Cardiac Disease: Implications for the Extracellular Matrix Beyond Collagen

Abstract

The structure of the heart changes in response to physiological cues. Such intrinsic plasticity of the heart involves changes to the extracellular matrix (ECM) and supports the adaptation to physiologic excursions such as athletic performance and pregnancy. Likewise, the ability of the ECM is critical for wound healing in the heart following myocardial infarction. Considering the involvement of the matrix in health and disease, it follows that a deep understanding of its composition and regulation would accelerate our pace of developing therapeutic interventions for the heart. Yet, we understand little of the composition of cardiac ECM and how its regulated. One such matrix component, hyaluronan (HA), has received scant attention in the heart despite its abundance rivaling that of collagen. This review addresses the biological origins of HA and its impact on the heart. HA production is linked to intermediary metabolism and is produced in response to injury. Much remains to be resolved regarding its production and degradation. And, although there are insights from other fields, the extent to which HA impacts the function of potential target cells is far from resolved. This review will also address techniques and tools available to assess cardiac HA and highlight areas of opportunity in the field along with some limitations in literature.

Graphical Abstract

Overview

The extracellular matrix (ECM) is in a state of continuous flux.(1) Components are formed and degraded, and during homeostatic conditions, balance is maintained. There are, however, physiological conditions in which the ECM of an organ, such as the heart, changes to adapt to exceptional physiological demands. Conditions of physiologic hypertrophy—such as post-natal cardiac growth, maternal heart growth during pregnancy, and exercise-induced conditioning—require changes to the ECM to accommodate changes (increases) in cardiomyocyte size and changes in vascularity.(1-4) Yet, even more acute and striking changes to the ECM occur in the context of disease in the heart.(5-7) In the realm of pathology, most attention has been directed toward collagen; however, collagen is but one important component of the ECM. Indeed, hyaluronan (HA) abundance rivals collagen during disease.(7) And, even more than collagen, HA can instigate biologic responses.

Homeostatic conditions require HA synthesis to be tightly coupled with its degradation to preserve a stable cardiac microenvironment (Figure 1); however, various diseases disrupt this balance by favoring excess HA production and suppressing its degradation, leading to pathological HA accumulation. Understanding how HA is produced, degraded, and interacts with its cellular targets is critical for interpretating its role in cardiovascular diseases. This review posits that HA functions as a context-dependent immunomodulator in the heart, and understanding its size-, source-, and tissue-specific effects is essential to elucidate its vital role in cardiac physiology and disease.

Figure 1. HA biosynthesis and degradation in the heart.

HA is produced at the plasma membrane by three hyaluronan synthases (HAS1–3), which use GlcNAc and GlcA (from UDP-GlcNAc and UDP-GlcA, respectively) to generate polymers ranging from low (HA^LMW) to high (HA^HMW) molecular weight HA. HAS2 (encoded by Has2) produces HA^HMW, whereas HAS3 (encoded by Has3) favors HA^LMW, contributing to isoform-specific biological functions. HA can be broken down enzymatically at the cell surface (HYAL2, TMEM2) or in lysosomes (HYAL1). Depolymerized HA can generate substrate for intermediary metabolism or be recycled for de novo HA synthesis, though this has not been validated in the heart. HA and its fragments interact with other cells and ECM components by binding to various receptors and hyalectins. HA-receptor interaction could also signal cell trafficking and proliferation of fibroblasts and immune cells. Together, these pathways maintain HA homeostasis and shape the size-dependent signaling cues that influence cardiac disease.

Hyaluronan Biosynthesis

HA is a linear, non-branching, repeating, anionic disaccharide, which, when newly formed, can be several MDa in size.(8, 9) In mammals, there is little evidence of subsequent chemical modification or additional glycan modification after HA is produced.(10) The two sugar units of HA are N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) via UDP-GlcNAc and UDP-GlcA. UDP-GlcNAc is formed by the hexosamine biosynthetic pathway.(11) This pathway has classically been viewed as a nutrient-sensitive pathway as excess glucose entry into many cell types provokes an increase in UDP-GlcNAc production.(8, 11, 12) Others have also shown an important role for the hexosamine biosynthetic pathway in stress responsiveness and cell survival.(13) UDP-GlcNAc can also arise from an epimerase reaction.(14) UDP-GlcA, whose formation is less biologically complex, is most frequently associated with glucuronidation and detoxification of products within cells.(14, 15)

HA is synthesized at the cell membrane through the action of hyaluronan synthases, which are encoded by Has1, Has2, and Has3 genes.(16-18) Among these, Has2 is most frequently implicated as a source for HA and produces high molecular weight HA (HA^HMW), which is typically accepted to be HA of at least 0.5 MDa in size.(7, 19) Has1 has generally lower activity and expression in the cardiovascular system; however, there is evidence for its inducibility and responsiveness during inflammation.(20) Has1 is thought to produce a wide range of HA sizes between 0.2–2 MDa.(21) Interestingly, Has3 produces low molecular weight HA (HA^LMW), which is HA below 0.5 MDa in size.(22) The biological redundancy of three Has isoforms remains unresolved and constantly debated; however, certain aspects of cardiovascular development and diseases rely on their non-overlapping functions as discussed later.

Given that formation of these two sugar-donors is derived from fundamental metabolic substrate (i.e., glucose), it is not surprising that their formation is linked to glucose metabolism.(23, 24) Vigetti et al showed that one consequence of increased flux through the hexosamine biosynthetic pathway—increased O-GlcNAc (O-linked N-acetylglucosamine on Ser/Thr residues) protein modification—increases production of HA.(23) They identified a specific amino acid in hyaluronan synthase 2 (HAS2) that is modified by O-GlcNAc. Hence, increased flux through the hexosamine biosynthetic pathway can be viewed as a feed-forward mechanism to increase production of HA via HAS2. Although this was shown to occur in cancer cells, the extent to which this happens in the cardiovascular system is less clear. It is clear, however, that HAS2 is important in cardiovascular health and disease, which is addressed below.

Hyaluronan Degradation

Balanced HA degradation is just as important as its production. Hyaluronan degradation may occur through both non-enzymatic and enzymatic routes.(25-27) Given the enormous size HA^HMW, it should not be surprising that it can be degraded by mechanical action, such as in striated muscle where the process of contraction and relaxation could contribute to breakdown of HA.(27) There is evidence that this can occur, and such effects would be important in the heart and active skeletal muscle. In joints, where the compressive forces may be much higher than in striated muscle, mechanical degradation is perhaps more plausible.(27, 28) Regardless, the extent to which mechanical degradation occurs in vivo in the heart remains to be established. Other data support a role for oxidative stress in the cleavage of HA into lower molecular weight forms.(29) Like mechanical degradation, the extent to which this occurs in vivo is not clear.

Enzymatic degradation has been well documented in vivo.(26, 30) There are six members of the classical hyaluronidase gene family; however, only three have appreciable expression and/or activity.(26) Of these, Hyal1 and Hyal2 have the most widespread expression(31-33); Hyal3 expression is more restricted, and its importance may be most notable in the acrosome of sperm where it may facilitate access to the ovum.(34) At the cellular level, expression of HYAL1 is intracellular, typically in association with lysosomes while HYAL2 is more associated with extracellular expression at the cell surface.(25) In this regard, HYAL2 may catabolize HA^HMW into HA^LMW.(26, 35) Once it is at the stage of HA^LMW, it can undergo receptor-mediated endocytosis and be further catabolized into dimeric units of HA by HYAL1.(35) Dimeric HA and the monomeric subunits are reincorporated into an oligosaccharide or otherwise used in an intermediary metabolism pathway.

There has been, however, some disagreement over the extracellular cleavage of HA^HMW.(36-38) There is ample evidence that HYAL2 degrades HA^HMW; however, not all studies agree.(38) More importantly, other candidate hyaluronidases have been proposed, and these may have even greater capacity for degradation of HA^HMW than HYAL2. One such candidate is TMEM2, which is encoded by Cemip2.(37) Several recent studies have shown that TMEM2 is a functional hyaluronidase, though the conclusion is not yet universally accepted.(37, 38) There is some data that also indicate that some hyaluronidases may serve a scaffolding/binding function in addition to (or instead of) their catalytic function.(39) Clearly, more study is needed in this area.

Hyaluronan Receptors and Binding Proteins

Once produced and processed, HA can be bound by receptors and other non-signaling binding proteins. Although there is a seemingly ever-increasing list of receptors, some of the most common include CD44, LYVE-1(lymphatic vessel endothelial hyaluronan receptor 1), and HMMR/RHAMM (hyaluronan-mediated mobility receptor; also known as, CD168 or RHAMM, receptor for hyaluronan-mediated mobility).(40-42) CD44, also known as hyaluronan receptor, is widely expressed.(41) Its functions are varied and complicated owing to its cell-dependent functions. Moreover, it also presents as variants(43); however, the role of CD44 variants in cardiovascular disease has received much less attention than in the cancer and immunology fields. LYVE-1 is found on lymphatic endothelial cells among several other cell types; it has recently been used as a marker for subpopulations of macrophages.(44, 45) HMMR/RHAMM has been shown to be important for development and regeneration.(46)

In addition to these traditional HA receptors, a host of other receptors—such as toll-like receptors, (e.g., TLR2 and TLR4), along with layilin and HARE (hyaluronan receptor for endocytosis, also known as stabilin-2)—may have secondary roles as HA receptors.(47-49) There is also a family of HA binding proteins found in the matrix, sometimes referred to as hyalectins. Among this family members are common constituents in the matrix, such as versican, aggrecan, and hyaluronan binding protein.(50-53) More comprehensive discussions of HA receptors, particularly in the context of cancer and inflammation, can be found elsewhere.(54) There are fascinating interactions that occur among hyaluronan, glycosaminoglycans, and other proteins in the ECM. Nevertheless, the roles of these HA binding proteins have received little attention in the heart, though important roles have been identified, as in the example of hapln1a (paralog of Hapln1 in mammals) in zebrafish.(55) Hence, their roles remain largely enigmatic.

Tools for Studying Hyaluronan

Interrogating HA biology in the heart is challenging due to its wide range of molecular sizes, dynamic turnover, and context-dependent functions. Multiple techniques are available to assess HA abundance, function, and spatial organization, but with distinct advantages and limitations. The choice of experimental method used to study HA biology in the heart must be guided by specific questions.

One of the most widely used approaches for detecting HA in biological tissues is HA affinity staining.(7, 56-58) This method involves incubating tissue sections with biotinylated hyaladherins such as HABP, followed by detection with fluorophore- or HRP-conjugated antibodies. This straightforward technique provides spatial localization of HA, allowing visualization of its distribution within the tissue; however, this technique provides no information on HA size or cellular source. Fluorophore-assisted carbohydrate electrophoresis (FACE) or conventional agarose gel electrophoresis can otherwise be employed to interrogate HA size distribution.(7, 56, 59) Yet, this method requires laborious HA extraction from tissue samples and is insensitive to HA^LMW. It is often best practice to complement these techniques with commercially validated enzyme-linked immunoassays (ELISA).(56, 59) This sensitive technique is extremely useful for detecting small and subtle changes in total HA content. ELISA should not be used on its own due to its limitation in providing size-dependent biological effects. Nevertheless, discrepancies between HA detection methods are not uncommon. For example, strong HABP staining may occur in localized regions without an increase in total HA by ELISA in certain conditions like focal myocarditis. This reflects regional accumulation or matrix retention. Conversely, ELISA and gel analysis may show increased total HA with minimal histological change if HA is diffusely present. This is likely to occur in early stages of tissue remodeling, when HA increase throughout interstitium without forming dense accumulations. Interpreting HA in these settings requires careful consideration of size, localization, and matrix binding.

Some studies also rely on indirect quantitation such as CEST imaging(20), qPCR or transcriptomic analysis of HA-related genes(20, 60, 61) and Alcian blue staining.(61) Whilst these methods can be useful for initial estimation of HA content and hypothesis generation, they are inadequate for rigorous understanding of HA biology in the heart. Alcian blue staining detects all glycosaminoglycan and mucopolysaccharides in fixed tissue sections.(61) This technique lacks specificity and results in overestimation of HA content without proper technical controls such as hyaluronidase treatment.(9) These indirect methods hint at whether HA biology is perturbed but lacks reliability in unraveling how HA size, source and abundance shapes cardiac development and diseases.

Genetically engineered mice are indispensable for understanding the size and source-specific contribution of HA in cardiac diseases. Gain of function models such as nmrHas2 overexpression allow context-specific interrogation of HA biosynthesis.(19) Loss of function models, such as Has1^−/−, Has3^−/−, and Has1/3 double knockouts are useful for HA depletion independent of Has2.(20, 22, 62) Germline deletion of Has2 results in embryonic lethality in mice(63), but availability of Has2^fl/fl mice allows generation of conditional global and cell-specific Has2^−/−.(7, 20) Additionally, genetic deletion of HA degrading enzymes such as HYAL2 provides insight into how aberrant HA degradation contributes to cardiac pathology.(32, 33) Receptor-specific insights can be drawn from Cd44^−/− mice(56, 64), but the broad expression and promiscuity of CD44 necessitate careful interpretation.

HA biology in the heart can also be studied via several pharmacological approaches. Because HA synthesis relies on the availability of UDP-glucuronic acid and UDP-N-acetylglucosamine, 4-methylumbelliferone (4-MU), which depletes cellular UDP-glucuronic acid is frequently used to inhibit HA synthesis.(20, 65) 4-MU is approved for treating biliary spasm in Europe and Asia(66); however, one major caveat of 4-MU is that a large dose of this agent is needed to effectively suppress HA synthesis in rodents.(67) Although some studies report significant inhibition of HA synthesis with 1-to-2-week administration of 4-MU chow(20, 67), others do not.(68) It is also important to note that treatment with 4-MU has a global effect on UDP-glucuronic acid availability which is essential for glucuronidation. For this reason, 4-MU is known to have off target effects which should be considered carefully when interpreting data. For this same reason, 4-MU should inhibit total HA synthesis making it difficult to tease apart the role of different size fragments in vivo. Aside from 4-MU, exogenous hyaluronidases have been used to enzymatically degrade HA in vivo. PEGylated recombinant human hyaluronidase (PEGPH20) which offers enhanced stability is available.(69)

Hyaluronan in Cardiac Development

HA in Cardiac Development:

HA is essential for cardiac morphogenesis as reviewed before, providing permissive matrix for early cell migration, structural scaffolding for cardiac jelly and cushions, and coordinating signaling pathways that drive mesenchymal cell formation, valve maturation and ventricular trabeculation.(70, 71)

Early Embryogenesis:

HAS1 and HAS2 are expressed as early as gastrulation in mouse embryos(72), though only HAS2 remains strongly expressed beyond E8.5.(63, 72) Affinity staining studies also indicated early presence of HA in the yolk sac and basement membranes of primitive ectoderm and primitive endoderm.(73, 74) One of the key morphogenetic events during gastrulation is the migration of epiblast cells through the primitive streak. HA is likely essential during this stage, to create a hydrated, loosely packed matrix that enables epiblast detachment, egression and migration.(75) As cardiac progenitor cells emerge, migrate and form the bilateral field of cells, a gelatinous HA-rich matrix known as cardiac jelly fills the space between endocardium and myocardium. Germline deletion of Has2, and not Has1 or Has3 results in embryonic lethality at E9.5-10 with severe cardiovascular abnormalities, including absence of cardiac jelly, suggesting that Has2-mediated synthesis of HA is crucial for cardiac jelly formation.(63, 76)

Endocardial Cushions:

As the heart tube loops to allow septation and chamber formation, cardiac jelly expands to form endocardial cushions at the atrioventricular junction and outflow tract—precursors for the valves and septa. Endocardial cells then migrate into these cushions to give rise to mesenchymal cells. Notably, hyaluronidase treatment prevented the formation of these endocardial cushions.(77) Atrioventricular explants isolated from Has2-deficient mouse embryos also failed to exhibit robust endocardial cell migration unless exogenous HA was present.(76) These observations collectively suggest that HA is not only a structural component of cardiac jelly but also actively regulates formation and cellularization of endocardial cushions. Mechanistically, HA is needed for activation of endocardial ErbB2 and ErbB3 receptor tyrosine kinases-dependent PI3K signaling that promote mesenchymal cell formation and migration into the cushion matrix.(78-80) It also creates a permissive environment for cushion cellularization by acting as reservoir for signaling molecules needed for mesenchymal cell formation such as BMP2 and TGFβ.(81-84)

Maturation and Trabeculation:

Post-cellularization, endocardial cushions remodel into mature valve leaflets. During this process, HA interacts with various signaling molecules, including TGFβ, ErbB, and Wnt/β-Catenin to facilitate cell elongation and refine matrix remodeling.(80, 85-88) Over time, HA is progressively cleared to allow fibrous ECM formation necessary for proper valve function. Loss of Hyal2 causes aberrant HA accumulation and thickening of all heart valves.(32, 33) In humans, abnormal accumulation of HA is associated with aortic valve disease, myxomatous mitral valves and valve diseases related to Marfan syndrome.(89, 90) Beyond valve morphogenesis, HA is also needed for ventricular trabeculation. Has2^−/− embryos exhibit severely underdeveloped right ventricle that lack trabeculations and display abnormally thin ventricular walls.(63, 76)

Hyaluronan in Cardiac Diseases

Mirroring its importance in cardiac development, HA has been implicated in various cardiac diseases (Table 1). Aberrant HA production, degradation, and receptor interactions drive cardiac inflammation, fibrosis, remodeling and arrhythmogenesis. Despite the robust body of literature discussed below, it should be noted that many of the key enzymes responsible for HA production, degradation, and recognition may go somewhat unnoticed in transcriptional profiling efforts.(91)

Table 1.

In vivo evidence linking HA to cardiac diseases.

Disease	HA manipulation	HA measurement	Key outcomes	Mechanistic insight	Reference
Ischemic injury and remodeling	Genetic deletion (Has1/Has2/Has3/Cd44) or with 4-MU	HA affinity staining, ELISA, gel, CEST imaging, mRNA transcripts	HA accumulates early post-injury; Has2 deletion and 4-MU worsen function; Has3 loss increases scar size; and Cd44 loss alters immune response	HA promotes myofibroblast activation and macrophage recruitment likely via CD44	(7,20,22,64,60,97)
Cardiac hypertrophy	Adamts5 deletion and with 4-MU	HA affinity staining, ELISA, mRNA transcripts	Early HA accumulation in hypertrophy; 4-MU reduces hypertrophy & fibrosis; whereas Adamts5 loss increases HA and worsens function	HA influences fibroblast activation, inflammation, and matrix turnover; whereas hyaladherin degradation controls HA accumulation	(99,58,65)
Myocarditis	Pharmacologically with 4-MU and HAHMW injection	HA affinity staining, ELISA	4-MU worsens fibrosis, while HAHMW reduces fibrosis	B10 cell-derived HA is antifibrotic	(101)
Cardiac Arrhythmias	Anti-CD44 blocking antibody, Cd44 gene deletion	HA affinity staining, ELISA	HA accumulates in atria; CD44 blockade reduces fibrosis and atrial fibrillation; Cd44 deletion reduces ventricular arrhythmias	HA-CD44 signaling promotes arrhythmia substrate and calcium mishandling	(57,104)
Metabolic cardiomyopathy	Exogenous PEGPH20 to breakdown HA	ELISA, HA affinity staining	No change in cardiac HA with diabetogenic diet but PEGPH20 reduces HA in obese mice and improves insulin resistance in these animals	HA drives matrix expansion and inflammation in obesity, but not necessarily in early insulin resistance	(59,69)

Ischemic heart diseases:

HA has been studied in the context of ischemic injury and post-infarction remodeling. In the context of the infarcted heart, early work in the field suggested that administration of hyaluronidase to degrade accumulating HA reduced signs of myocardial necrosis and infarct size.(92-94) Experimentally, HA accumulates in the infarct zone as early as 24 hours after infarction in various animal models.(60, 95, 96) This accumulation persists up to 28 days in mice with myocardial infarction.(7) Several studies show that HA colocalizes with CD44, collagen as well as Mac-2 macrophages, indicating HA biosynthesis correlates with collagen deposition and macrophage infiltration post MI.(7, 20, 60) HA gel analysis indicates that the HA accumulating post MI is predominantly HA^HMW.(7) Has1 and Has2 mRNA are robustly induced as early as 6 hours post ischemia-reperfusion (I/R) in mice, yet conditional deletion of Has2 but not Has1 exacerbates scar formation and severely impairs cardiac function post I/R injury.(20) Inhibiting HA synthesis pharmacologically with 4-MU also impairs cardiac function post I/R injury.(20) Although cardiac Has3 expression is not induced early in this context, it has been shown that Has3-deficient mice also exhibit increased scar size and collagen deposition that accompanies impaired cardiac function up to 21 days post-I/R.(22) Meanwhile, Cd44^−/− mice also exhibit aggravated neutrophil and macrophage response after I/R but show attenuated fibrotic response.(64) These findings suggest that HA^HMW-dependent matrix stabilization and inflammatory signaling is required for early remodeling, but sustained HA accumulation likely promotes fibrosis through CD44-dependent immune cell recruitment and fibroblast activation. It is currently unclear how hyaluronidases (HYAL1, HYAL2, or HYAL3) or TMEM2 influence cardiac I/R as well as post-infarction remodeling; however, a recent study showed that loss of hyaluronan binding protein (KIAA1199/CEMIP) function led to reduced fibrosis and improved cardiac function 28 days post-MI in mice.(97)

Cardiac hypertrophy:

HA accumulation is also a hallmark of hypertrophic remodeling.(98, 99) In humans with hypertrophic cardiomyopathy (HCM) and left ventricular outflow tract obstruction, an intense HA affinity staining was observed in the intercellular space, in fibrous septa, around blood vessels and areas of focal fibrosis.(98) Similarly, in rats, abdominal aorta ligation-induced pressure overload triggers early upregulation of Has1 and Has2, driving HA deposition in perivascular and interstitial regions.(99) Unlike humans, HA accumulation in rats with pressure overload was also accompanied by transient CD44 upregulation that normalized by week 6.(99) HAS1 and HAS2 remain modestly elevated.(99) It appears that HA may have a dual role in this context. It may be initially produced to buffer mechanical stress, support compensatory growth and coordinate early inflammatory responses along with CD44; however, continuous HA biosynthesis and impaired matrix degradation may sustain HA accumulation later in the remodeling heart driving maladaptive cardiac dysfunction.(58) In mice lacking catalytic domain of the matrix degrading enzyme, ADAMTS5, angiotensin II infusion led to robust HA accumulation that coincided with reduced ejection fraction and impaired global longitudinal strain.(58) More recently, 4-MU, which has been suggested to inhibit HA synthesis, attenuated myocardial remodeling in a mouse model of pressure overload.(65) Male mice fed continuously with 4-MU had lower myocardial HA content along with improvement in fractional shortening and attenuated myocardial immune cell expansion post transverse aortic constriction surgery(65), suggesting HA synthesis drives pro-inflammatory responses in cardiac hypertrophy.

Myocarditis:

Evidence linking HA to acute myocarditis dates to 1993 when Waldenström and his colleagues demonstrated that progressive accumulation of HA occurred in endomysium of BALB/c mice inoculated with Coxsackie B3 virus.(100) HA affinity staining revealed more pronounced HA accumulation in regions with focal inflammatory infiltrates.(100) Recently, it was also reported that experimental autoimmune myocarditis in mice is also regulated by HA secretion from B10 cells.(101) HA abundance increased after experimental autoimmune myocarditis in these animals and correlated with IL-10 producing B10 cells. Contrary to previous reports, this study showed that 4-MU administration exacerbated myocardial fibrosis in mice with experimental autoimmune myocarditis, which persisted despite daily 4-MU administration.(101) Conversely, injection of HA^HMW attenuated experimental autoimmune myocarditis-driven fibrosis, suggesting that HA may be antifibrotic in this context.(101) HA may be protective in some myocarditis contexts, likely anti-inflammatory signaling mediated by HA^HMW, whereas dysregulated HA turnover may promote fibroblast activation.

Cardiac arrhythmias:

HA is also intricately linked to arrhythmogenesis, especially in the context of atrial fibrillation.(61, 102) Human atrial biopsies revealed that ECM remodeling occurs early before incident atrial fibrillation and predicts the persistence of arrhythmic events.(102) Furthermore, histochemical analysis revealed that glycosaminoglycan and collagen accumulate in the atrial tissues of patients with atrial fibrillation.(61) HA abundance in coronary sinuses is also associated with persistence of atrial fibrillation in humans.(103) In rodents, global overexpression of TGFβ resulted in remarkable atrial fibrosis without overt ventricular remodeling, and this coincided with HA accumulation.(57) These animals were more prone to pacing-induced atrial fibrillation as compared to controls.(57) Blocking CD44 signaling led to diminished atrial fibrosis and reduced atrial fibrillation inducibility, supporting a role for CD44 receptor-dependent mechanisms(57) Interestingly, this study also reported that atrial fibroblasts are more sensitive to TGFβ-stimulated HA production than ventricular fibroblasts.(57) HA-CD44 interactions might also be relevant for ventricular arrhythmias, considering Cd44^−/− mice were less prone to isoproterenol-induced triggered events and ventricular arrhythmias.(104)

Cardiometabolic diseases:

Limited evidence is currently available regarding local HA accumulation in cardiometabolic disorders such as diabetic cardiomyopathy and HFpEF, although HA has been systemically implicated in diabetes and obesity.(105-107) In mice fed on diabetogenic diet for 11 weeks, cardiac HA levels remained comparable to the normal chow-fed controls.(59) Notably, cardiac fibroblasts isolated from these animals showed no difference in HA production as compared to the controls, both at basal and stimulated conditions.(59) Hyperglycemia on its own failed to elicit increased HA synthesis in cardiac fibroblasts in vitro.(59) Meanwhile, our study recently showed that glucose availability significantly influences HA^HMW production in cardiac fibroblast isolated from naïve animals.(7) Nonetheless, interpretation of these conflicting observations is difficult considering cardiac fibroblasts are extremely sensitive to culture conditions and in vitro observations may understate the consequences of chronic metabolic perturbations involving multiple organ systems in cardiometabolic disorders. One recent study also evaluated whether PEGylated human recombinant hyaluronidase (PEGPH20) improves cardiac insulin resistance in obese mice.(69) Male mice fed on high fat diet for 16 weeks developed cardiac insulin resistance with HA and collagen deposition and this was attenuated by PEGPH20 treatment.(69) This suggest that HA-driven matrix expansion and inflammation may contribute to metabolic cardiac dysfunction.

HA likely contributes to various cardiac diseases through both structural matrix effects and receptor-mediated signaling that promotes inflammation and fibrosis. CD44 is frequently implicated in HA-driven inflammation and fibrosis. Toll-like receptors (TLR2 and TLR4) may also mediate pro-inflammatory effects. Mechanistically, HA fragments and polymers can engage these receptors through HA binding sites to regulate signaling molecules involved in inflammation, fibrosis and remodeling. It is also often suggested that HA may serve as a scaffold that concentrates cytokines and growth factors in the local microenvironment. Future studies that further define HA size distribution and role of receptor-specific-loss-of-function models in various cardiac diseases could establish causality and distinguish these mechanisms.

Hyaluronan as an Immunomodulator

HA is not inert but acts as a potent immunomodulatory signal driving cardiac disease. It can shape macrophage activation and T cell responses, yet these effects are highly context-dependent, influenced by HA size, cellular source, and the local tissue environment. This area of investigation also involves some of the more conflicting findings.

Studies of isolated immune cells:

It is widely held that HA size dictates its immunomodulatory activity, with HA^LMW generally considered pro-inflammatory and HA^HMW largely anti-inflammatory. In a study where alveolar macrophages were stimulated with HA fragments and their full-size counterparts, only HA fragments augmented expression of inflammatory genes such as Ccl3, Ccl4, Ccl5, and Cxcl15.(108) Similarly, murine macrophages exposed to HA^LMW adopt classically activated gene profile, i.e., Nos2, Cd80, and Tnfa, whereas HA^HMW promotes a more alternatively activated gene profile, i.e., Arg1 and Il10.(109) This push towards an alternatively activated phenotype was maintained even when cells were treated with a pro-inflammatory stimulus. This agrees with a growing body of data suggesting that HA^HMW acts as an anti-inflammatory mediator by physically blocking the binding of pro-inflammatory stimuli to cell surface receptors. In another study, Streptococcus equi-derived hyaluronan was degraded by acid hydrolysis and used to stimulate human blood phagocytes. “Low” (52 kDa), “medium” (250 kDa), and “high” (970 kDa) molecular weight HA all stimulated ROS production, surface expression of pro-inflammatory markers (CD11b, CD35, and CD66), and TNFα production. While all forms of HA stimulated a pro-inflammatory response, HA^LMW had the most pronounced effect. Interestingly, low/medium molecular weight HA increased PMA (phorbol myristate acetate)-activated ROS production, while HA^HMW significantly decreased it, again supporting the idea that HA^HMW may, under certain conditions, inhibit pro-inflammatory stimuli.(110) This idea is further supported by work done in the cancer field where it was shown that methods of reducing HA can increase the binding and efficacy of anti-cancer drugs.(111, 112)

HA as a physical barrier:

Previous work also has shown that HA^HMW can specifically inhibit macrophage phagocytosis in a dose and size dependent manner; however, in both studies, extensive washing after HA incubation led to loss of the inhibition suggesting that HA^HMW needs to be physically present to inhibit phagocytes from interacting with their targets.(113, 114) We showed that HA^HMW inhibited Fc-mediated phagocytosis in murine bone marrow-derived macrophages while an equal mass of HA^LMW had no effect, which is suggestive of a barrier function of HA^HMW.(56) Further supporting this notion, macrophages appear to have no appreciable production of HA in vitro, though they have receptors to recognize and respond to HA.(7, 56, 91, 115) Hence, it is likely that fibroblasts (at least in the heart) are the primary sources of HA, and fibroblast-derived HA impacts the function of other cells, such as macrophages.

Impact of HA source:

Apart from HA size, recent work also suggests that HA source and preparation influences the immunomodulatory role of HA.(116, 117) For instance, HA^HMW (from rooster comb) induced robust TNFα production in whole blood samples and macrophages in vitro; however, endotoxin contamination was observed in the animal derived HA samples.(116) Another study tested the effect of HA fragments and HA^HMW from various sources in mouse bone marrow derived macrophages and found that only HA-derived from human umbilical cord, bovine testes, and Streptomyces hyaluronlyticus stimulated pro-inflammatory cytokines in macrophages and dendritic cells.(117) Surprisingly, pharmaceutical grade HA and HA fragments had no effect on macrophage activation in vitro or leukocyte recruitment in vivo.(117) Contrary to these reports, BMDMs treated with endotoxin-free HA^HMW secreted more pro-inflammatory cytokines (IL-2, IL-17, and IP-10), while treatment with an equal mass of HA^LMW led to a significant reduction in MIP-3.(56) In part, this response was dependent on Cd44 expression, further highlighting that the impact of HA is dependent on fragment size and which cellular receptors are present.(56) Although different sources of HA could include coincident HA binding proteins, it is important to test for endotoxin in commercial preparations of HA, especially those derived from prokaryotes. Ultimately, the impact of HA on inflammation in vivo is more complicated due to dynamic synthesis and degradation, which result in a variety of sizes co-existing in the same environment (vide supra). Without fully understanding the composition of the ECM, it is difficult to determine whether HA accumulation after injury is protective or maladaptive.

Inflammation in non-cardiac organs:

In a model of bleomycin induced lung injury, an HA blocking peptide (Pep-1) resulted in decreased neutrophil recruitment to the lung suggesting that binding of HA fragments promotes recruitment of inflammatory cells consistent with the idea that HA^LMW is pro-inflammatory; however, this treatment also increased the number of apoptotic cells found in the lung. When Has2 overexpression was used to increase the synthesis of HA^HMW, the number of apoptotic cells was reduced, and survival was improved. Together this suggests that HA fragments do promote an inflammatory response, but HA provides an overall protective signal that promotes survival.(118) In a model of PM_2.5 indued lung inflammation, intratracheal instillation of HA^HMW reduced neutrophil infiltration, TNFα secretion, oxidative stress, and lung edema again demonstrating a protective role for HA^HMW.(119) Administration of HA prior to LPS stimulation to induce septic shock resulted in improved behavior and decreased serum Il-6 and TNFα levels. This response was Cd44 dependent and supports the idea that HA binding to CD44 inhibits stimulating factors such as LPS from binding to target receptors such as TLRs.(120) Alternatively, use of the HA synthesis inhibitor 4-MU suppressed LPS induced lung inflammation and reduced signs of lung damage suggesting that the HA that is internally produced following LPS exposure promotes a harmful pro-inflammatory response.(121) In another model treatment with 4-MU was shown to improve kidney function following ischemic injury. Significant decreases in macrophage and neutrophil infiltration accompanied by a reduction in fibrosis suggests that endogenous HA accumulation promotes a maladaptive inflammatory response following kidney injury.(122) The idea that administration of exogenous HA could be beneficial while endogenous HA could be maladaptive could potentially be explained by differences in HA size and metabolic turnover rates in tissue. Better understanding the dynamic metabolism of HA in different states of injury and disease will allow us to gain a clearer understanding of its role in regulating inflammation.

Atherosclerosis, an inflammatory prelude to MI:

Atherosclerosis is a chronic inflammatory condition that precedes myocardial infarction and ischemic cardiomyopathy. Because several reviews have extensively discussed the role of HA in initiation and progression of atherosclerosis, readers are recommended to read these articles for deeper understanding of the role of HA in these conditions.(123-125) Some of the key previous observations include but are not limited to: Has3 loss in Apoe^−/− mice leads to less macrophage-driven inflammation and atherosclerosis(126); HAS3-mediated low molecular weight HA synthesis supports neointimal hyperplasia(127); and 4-MU treatment in Apoe^−/− mice paradoxically led to increased atherosclerosis with partial loss of glycocalyx(128). These observations support a prominent role for Has3 but no other isoforms in pathogenesis of atherosclerosis.

Myocardial inflammation:

CD44, a principal receptor for HA, has been shown to play an important role in inflammation following ischemia-reperfusion injury of the heart. Because Cd44 deficient mice exhibited an enhanced and prolonged inflammatory response, the CD44-HA axis may support pro-resolving actions of HA that have been observed after injury.(64) Whole-body deletion of Has2 prior to ischemia-reperfusion injury to inhibit HA^HMW synthesis increased scar size, caused apoptosis of cardiac macrophages, and inhibited fibroblast activation. This suggests that HA synthesis is important for proper macrophage and fibroblast responses following injury.(129) Yet, the non-selective approach of Has2 deletion in that study left some questions unanswered. We recently used a more selective approach for Has2 deletion (i.e., in fibroblasts via Col1a2-Cre) and found that deletion of fibroblast Has2 in a non-reperfused MI model exacerbated heart failure in male, but not female, mice.(115) Hence, it is possible that Has2 may have different impacts in different cell types; however, fibroblasts are at least one critical source in heart failure, if not many forms of cardiovascular disease. Finally, Has3 deletion, which should limit HA^LMW production, decreased CD4⁺ T cells, reduced ejection fraction, and increased scar size in an ischemia-reperfusion model. Has3 deletion appeared to inhibit T cell activation and increase apoptosis suggesting that Has3 mediated HA synthesis supports the adaptive immune response following MI.(22) Others have shown that intravenous administration of HA oligosaccharides following ischemia-reperfusion injury reduced infarct size and improved cardiac function. An increase in CD206⁺ cells (M2-like macrophages) and a decrease in Gr1⁺ cells (neutrophils) in these hearts suggests that this treatment promotes a more pro-resolving phenotype earlier after injury. Additionally, CD31 and Ki-67 staining was increased in tissues indicating increased angiogenesis. In vitro experiments demonstrated that treatment of peritoneal macrophages with HA oligosaccharides enhanced migration and invasion capabilities and increased VEGF production which could help to support the increase in angiogenesis. This suggests that administration of HA oligosaccharides following injury inhibits the pro-inflammatory period that is typically observed immediately following MI and promotes angiogenesis.(130)

In summary, it seems that HA supports myocardial infarct healing; however, it is important to note that many of these described models induce global changes, which can make cell-specific interpretations more difficult. Nevertheless, across these disease contexts, a unifying theme is that HA actively shapes immune responses in the heart and vasculature that dictate functional outcomes, which necessitates further examination.

Concluding Remarks

Many outstanding questions remain (Table 2). Collectively, HA appears to be far more than a structural component of the cardiac ECM—it is a dynamic regulator of cardiac biology from development to disease. HA synthesis, degradation and receptor-mediated interactions are tightly regulated under physiological conditions, but diseases disrupt this homeostasis. Aberrant HA turnover is involved in cardiac repair and remodeling after ischemic insults, inflammation, fibrosis, hypertrophy, and arrhythmogenesis. Each of these areas has fundamental physiological differences making it difficult to generate a clear statement about the role of HA in cardiovascular disease. While some of the observations made may seem to be conflicting, it is important to consider that the impact of HA on the primary endpoints being investigated may be different. The role of HA in the heart is highly context-dependent where molecular size, cellular source and local tissue environment (surrounding cells, binding partners, etc.) determine whether HA is protective or detrimental to the heart.

Table 2.

Areas requiring additional investigation in the field.

Area of Research	Outstanding Questions and Objectives
Hyaluronan Synthesis	What factors regulate HA production in the heart? To what extent does the synthesis of HA further stimulate its production in the heart?
Hyaluronan Degradation	Does mechanical degradation of HA occur in the heart, and what are the implications of this degradation? To what extent does oxidative degradation of HA occur in the heart, and what are the implications of this degradation? Contextually define the enzymes responsible for degradation of HA in the heart. Do hyaluronidases serve a scaffolding/binding function in the heart? If so, which ones? Is this in addition to or in place of their catalytic activity?
Hyaluronan Binding Proteins	How do hyaluronan binding proteins contribute to the extracellular matrix structure and composition in the heart? Do hyaluronan binding proteins play a role in cardiovascular disease?
Tools for Studying Hyaluronan	Enhance methods to quantify the abundance of different size HA fragments.
Cardiovascular Disease	Ischemic Heart Disease: How do hyaluronidases influence HA abundance and impact ventricular remodeling? Cardiac Hypertrophy: Does dysregulated HA biosynthesis drive cardiac dysfunction? Cardiometabolic Diseases: Limited evidence regarding the presence and function of HA in diabetic cardiomyopathy and HFpEF.
Impact on Immune Cells	Given the divergent literature on the impact of HA on immune cells, careful studies should be conducted on their interaction, especially in the intact heart.
General Pathology	Consider composition of the ECM and the dynamic synthesis and degradation of HA. How do size and source dependent effects of HA translate in humans? Does targeting HA accumulation have a translational benefit in the clinic?

Several critical gaps remain, including how HA dynamics are regulated in chronic cardiometabolic diseases, and how size and source-dependent effects translate in humans. Although 4-MU and hyaluronidases have been used to pharmacologically target aberrant HA turnover, these approaches lack specificity in tackling the context-dependent role of HA in cardiac diseases. It is also unknown whether targeting HA accumulation has a translational benefit in the clinic. There are ample opportunities to further explore how HA is regulated in more timely clinical conditions such as HFpEF considering cardiac remodeling, arrhythmias, inflammation and fibrosis determine clinical outcomes in patients with HFpEF.