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The Role of Hyaluronic Acid in Atherosclerosis and Intimal Hyperplasia

The Role of Hyaluronic Acid in Atherosclerosis and Intimal Hyperplasia

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Journal of Surgical Research 173, e63–e72 (2012) doi:10.1016/j.jss.2011.09.


RESEARCH REVIEW The Role of Hyaluronic Acid in Atherosclerosis and Intimal Hyperplasia
Benjamin Sadowitz, M.D.,*,† Keri Seymour, D.O.,*,† Vivian Gahtan, M.D.,*,† and Kristopher G. Maier, Ph.D.*,†,1
*SUNY Upstate Medical University, Division of Vascular Surgery and Endovascular Services, Syracuse, New York; and †Department of Veterans Affairs VA Healthcare Network Upstate New York at Syracuse, Syracuse, New York Originally submitted June 3, 2011; accepted for publication September 14, 2011

Atherosclerosis is a chronic inflammatory condition of the blood vessel wall that can lead to arterial narrowing and subsequent vascular compromise. Although there are a variety of open and endovascular procedures used to alleviate the obstructions caused by atherosclerotic plaque, blood vessel instrumentation itself can lead to renarrowing of the vessel lumen through intimal hyperplasia, wound contracture, or a combination of the two. While the cell types involved in both atherosclerosis and vessel renarrowing after surgical intervention are largely characterized, current research has shown that components of the extracellular matrix are also important in the pathogenesis of the aforementioned processes. One such component is hyaluronic acid (HA). The objective of this review, therefore, is to examine the involvement of HA in these pathologic processes. Literature on the structure and function of HA was reviewed, with particular attention given to the role of HA in the processes of atherogenesis, intimal hyperplasia, and wound contracture after blood vessel instrumentation. HA interacts with vascular smooth muscle cells (VSMCs), endothelial cells (ECs), and platelets to promote atherogenesis. In particular, VSMCs manufacture large amounts of HA that form ‘‘cable-like’’ structures important for leukocyte adhesion and rolling. Additionally, transmigration of leukocytes across the EC layer is mediated by HA. Platelets cleave large molecules of HA into fragments that up-regulate leukocyte production of chemokines and cytokines. HA also has a role in both intimal hyperplasia and wound contracture, the two processes most responsible for vessel renarrowing after vascular instrumentation.

HA has a complex, and sometimes conflicting, role in the pathologic processes of atherogenesis and vessel wall renarrowing after surgical intervention. Ó 2012
Elsevier Inc. All rights reserved.

Key Words: hyaluronic acid; CD44; RHAMM; extracellular matrix; atherosclerosis; restenosis; vascular smooth muscle cells; endothelial cells; platelets; leukocytes.


1 To whom correspondence and reprint requests should be addressed at SUNY Upstate Medical University, Division of Vascular Surgery and Endovascular Services, 750 East Adams Street, Syracuse, NY 13210. E-mail: maierk@upstate.edu.

Atherosclerosis is a chronic inflammatory disease of the blood vessel wall [1–3]. Disease progression eventually leads to plaque formation and narrowing of the vessel lumen [1, 2]. Medical therapy and lifestyle modification are the mainstays of treatment for atherosclerosis, but some atherosclerotic lesions progress to the point where surgical intervention is required [1]. Unfortunately, both percutaneous interventions and open surgical procedures also injure the vessel wall and can precipitate renarrowing of the vessel lumen secondary to intimal hyperplasia (IH), wound contracture, or both [4]. Early research intent on determining the pathogenesis of both atherosclerosis and renarrowing of the vessel lumen after surgical intervention mainly focused on the role of the platelets and cellular components in these processes, including vascular smooth muscle cells (VSMCs), macrophages, and endothelial cells (ECs) [1]. However, biologically active elements of the extracellular matrix (ECM) are also important in the vascular remodeling that takes place in both atherosclerosis and renarrowing of the vessel lumen after instrumentation [2, 5]. One such element of the ECM that is important in both of these processes is hyaluronic acid (HA), otherwise known as hyaluronan [2]. HA is a negatively-charged glycosaminoglycan (GAG) present in abundance in the


0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved.



body, especially in the skin, articular joints, vitreous humor of the eye, and the vasculature (Fig. 1A) [6]. Originally thought to be a mere ‘‘space-filler’’ in the ECM, it is now known that HA has important roles both structurally and as a signaling molecule [7]. Specifically, HA regulates cell adhesion, migration, and differentiation, and is involved in a host of cellular processes including morphogenesis, regeneration, and wound healing [5, 7, 8]. With regard to the pathogenesis of atherosclerosis and renarrowing of the vessel lumen after vascular instrumentation, HA is a key component of the lesions associated with both processes [4, 9]. Additionally, HA appears to interact with many cellular components involved in both atherosclerosis and vessel renarrowing after surgical intervention, most importantly VSMCs and leukocytes [2]. The purpose of this review, therefore, is to examine the role of HA in the pathogenesis of both ath-

erosclerosis and the processes causing vessel renarrowing after surgical instrumentation.

HA is a straight-chain GAG of the ECM composed of repeating disaccharides of glucuronic acid and N-acetyl glucosamine linked by alternating b1-3 and b1-4 linkages (Fig. 2) [10, 11]. Interestingly, HA undergoes little chemical modification as no sulfated, acetylated, or phosphorylated variants of this molecule exist [10, 11]. The simple structure of HA is stable, allowing for rapid recovery after mechanical distortion or compression [10]. Furthermore, the structure of HA has osmotic properties important for maintenance of fluid volume in the joint space [12]. HA exists as both high molecular weight (3000–4000 kDa) and low molecular weight fragments (20 kDa) [13]. HA is synthesized in the high molecular weight form and the low molecular weight form is generated by degradation of the parent molecule (reviewed below) [13].

Unlike other GAGs that are synthesized at the Golgi apparatus and endoplasmic reticulum around a protein core, HA is synthesized on the cytoplasmic surface of the plasma membrane as a free linear polymer [7, 11, 14, 15]. The three known HA synthases (HAS)— HAS-1, HAS-2, and HAS-3— produce HA polymers of varying lengths [8, 11, 16], and expression of these three isoforms is cell- and tissue-specific [11]. The HA synthases are integral membrane proteins and they are able to translocate the growing HA polymer out of the cell as it is being synthesized [11, 17]. The energy expenditure for synthesizing an average length HA chain (about 10,000 disaccharide repeats) is significant—50,000 ATP molecules, 20,000 NAD cofactors, and 10,000 acetylCoA groups are required [7].

Overall, the total body turnover of HA is on the order of several grams per day [15, 18, 19]. HA catabolism
FIG. 1. (A) Immunohistochemistry staining showing the presence of HA in an uninjured carotid artery of a male Sprague Dawley rat. HA was detected with a polyclonal sheep primary antibody (1:200) and stained brown in color. HA (arrows) is present in the endothelial and smooth muscle cell layer but is most prominent in the connective tissue of the adventitia (340). (B) Immunohistochemical staining showing the presence of HA after arterial injury by balloon catheter in a male Sprague Dawley rat. At 44 d after arterial injury, the area of intimal hyperplasia is pronounced at the lumen surface (solid black line). There is strong HA staining (arrowheads) now localized to the area of IH, with patchy distribution near smooth muscle cells in the medial layer (340).

FIG. 2. Hyaluronic acid structure. Hyaluronic acid is composed of repeating disaccharides of glucuronic acid and N-acetyl glucosamine linked by alternating b1-3 and b1-4 linkages. UDP-GlcA ¼ uridine diphosphate glucuronic acid; UDP-GlcNaC ¼ uridine diphosphate Nacetyl glucosamine.



occurs through three distinct pathways [11]. First, local cellular turnover of HA is mediated through its main receptors: CD44 and the receptor for HA-mediated motility (RHAMM). HA binds these receptors and is degraded both extracellularly and intracellulary [20–22]. At the tissue level, HA is released into the circulatory and lymphatic systems where it is cleared and degraded in the liver and lymph nodes through the action of unique receptors, such as the lymphatic vessel endothelial hyaluronic acid receptor (LYVE) – 1 and the HA receptor for endocytosis (HARE) [18, 23]. Lastly, HA is also degraded by free radicals under oxidative conditions [24, 25]. Receptor-mediated degradation of HA is accomplished through specialized enzymes known as hyaluronidases (HYAL) [19]. The two hyaluronidases that perform the bulk of HA turnover are HYAL1 and HYAL2 [19]. HYAL1 is a lysosomal enzyme with the ability to degrade HA molecules of any size and usually generates tetrasaccharides [26]. HYAL2, on the other hand, has a glycosylphosphatidylinositol tail that anchors this enzyme on the external side of the plasma membrane [27]. HYAL2 cleaves large HA polymers into high and low molecular weight fragments [13].

HA is currently known to interact with four different cell surface receptors with varying distributions throughout the body. A summary of these receptors, their locations, and their functional relationships with HA are presented in Table 1.

disaccharide subunits on the HA chain [21]. This lowaffinity binding of HA is shared by the LYVE-1 receptor as well [15, 30]. Specifically, the CD44 receptor on resting leukocytes is functionally ‘‘off’’ and only displays a high affinity for HA when activated [31, 32]; this activation appears to primarily occur under the direction of cytokines and/or growth factors during proinflammatory conditions [33–35]. However, some studies suggest that CD44 signaling may be more complex than a simple all or none response. CD44 exerts its effects on the cell through regulation of the actin cytoskeleton, which is mediated by the interaction of CD44 ezrin/radixin/moesin proteins that are recruited to the CD44/HA complex depending on which HA isoform is bound to CD44 [36, 37]. HA exists in both high and low molecular weight isoforms, and the signaling pathways impacted by CD44 depends on which isoform binds the receptor. In inflammation, hyaluronidases are up-regulated and mediate an increase in low molecular weight HA, which tends to promote cellular proliferation [38]. Thus, hyaluronidases and low molecular weight HA may be important in inflammatory states such as diabetes and vascular remodeling. In addition, studies from our laboratory have shown that antibodies to CD44 block the ability of HA or thrombospondin-1 (TSP-1) to induce VSMC chemotaxis [39]. Further, inhibitor studies demonstrated that HA and TSP-1-induced VSMC chemotaxis were dependent on phosphatidylinositol-3 kinase (PI3K) and src family proteins [39]. Thus, HA and TSP-1 share intracellular signaling pathways that are dependent on CD44 activation [39].

The CD44 family of transmembrane glycoproteins are actually splice variants encoded for by a single gene [14]. They are present on a variety of cells including T-cells, dendritic cells, monocytes, macrophages, VSMCs, and ECs [6, 28, 29]. A wide range of biologic processes are regulated by the CD44 receptor including organ development, neuronal axon guidance, lymphocyte homing and rolling, and hematopoiesis [20]. The CD44 receptor appears to facilitate these processes through a combination of its molecular actions; these actions include ligand binding, mediation of receptor tyrosine kinase signaling, and serving as a link between the plasma membrane and actin cytoskeleton [20, 28]. Although CD44 interacts with a variety of ligands in the ECM, HA appears to be its major ligand [6]. Furthermore, all isoforms of CD44 have a HA-binding site as part of their extracellular domain [28]. CD44 is unique from other HA receptors in that CD44 binding to HA involves a multitude of CD44 molecules in close proximity to each other interacting with multiple

Like CD44, RHAMM is coded for by a single gene [14]. RHAMM is found in a variety of cell types including macrophages, fibroblasts, and VSMCs [40]. Furthermore, splice variants of RHAMM are found in a multitude of places, including the cell surface, cytoskeleton, mitochondria, and the nucleus [28]. RHAMM interacts with HA in both the extracellular and intracellular compartments. For example, extracellular RHAMM has a crucial role in mediating cell motility in response to HA through activation of c-src, focal adhesion kinase (FAK), PI3K, and Ras-extracellular signal-regulated kinase (ERK) pathways [14, 40–42]. In addition, RHAMM acts as a functional extracellular HA receptor in human ECs. Specifically, Lokeshwar and Selzer demonstrated that HA-induced activation of FAK, paxillin, and ERK in both human pulmonary ECs and human microvessel ECs from the lung was blocked by an anti-RHAMM antibody [43]. Additionally, intracellular RHAMM is closely associated with



TABLE 1 Hyaluronic Acid Receptors, Their Locations, and Known Functional Relationships With Hyaluronic Acid
Receptor CD44 [81, 84, 92] Ubiquitous Location Known functional relationships with hyaluronic Acid

RHAMM [14, 40–42, 45]


LYVE-1 [15, 23, 30, 47]

Lymphatic system

HARE [49–53]

Liver, lymph nodes, spleen, corneal and lens epithelium, heart valve mesenchymal cells, ependymal cells lining the ventricles in the brain, epithelial cells covering the renal papillae 

Facilitates HA-mediated contraction of collagen by VSMCs and adventitial fibroblasts  Facilitates HA-induced VSMC proliferation  Mediates cell motility in response to HA through activation of c-Src, FAK, PI3-kinase, and Ras-ERK pathways  Is a functional HA receptor in ECs – induces activation of FAK, paxillin, and ERK  HA may interact with intracellular RHAMM to assist with RHAMMmediated spindle pole formation and stability  Appears to facilitate transport of HA into either lymphatic endothelial cells for degradation or into afferent lymphatics for eventual degradation by the lymph nodes  May mediate cell adhesion/migration in the lymphatic system through its interaction with HA  Scavenger receptor that clears HA from the circulation

HA ¼ hyaluronic acid; FAK ¼ focal adhesion kinase; PI3 kinase ¼ phosphatidylinositol-3-kinase; ERK ¼ extracellular signal-regulated kinase; VSMC ¼ vascular smooth muscle cell; RHAMM ¼ receptor for hyaluronic acid-mediated motility; LYVE-1 ¼ lymphatic vessel endothelial hyaluronic acid receptor.

microtubules and is thus important for cell cycle control and mitotic spindle formation and stability [44, 45]. Interestingly, Evanko et al. demonstrated that intracellular HA distribution parallels microtubule distribution in human arterial VSMCs [46]. In addition, they showed a close association between HA, tubulin, and RHAMM in the mitotic spindle [46]. These observations suggest a role for HA in RHAMM-mediated mitotic spindle formation and stability.

LYVE-1 is an integral membrane receptor with a high specificity for HA [23, 47]. Interestingly, although it is a homologue of CD44, the LYVE-1 receptor is almost exclusively expressed in the lymphatic system [23, 47]. Currently, the role of the LYVE-1 receptor in HA homeostasis remains unclear [15, 47]. Possibly,

the LYVE-1 receptor facilitates transport of HA into either lymphatic ECs for degradation or into afferent lymphatics for eventual degradation in the lymph nodes [15, 30]. Alternatively, the LYVE-1 receptor may mediate cell adhesion and migration in the lymphatic system using HA as an adjunct [15, 30]. It is interesting to note that the HA binding activity of the LYVE-1 receptor is quite low in native, intact lymphatic tissue [23, 47]. In addition, indentifying activators of the LYVE-1 receptor has proven difficult. Johnson et al. studied 20 different growth factors and cytokines, including tumor necrosis factor-a, transforming growth factor b1, and variety of interleukins for their ability to activate the LYVE-1 receptor on human dermal lymphatic ECs, but none of these compounds was able to change the binding affinity of LYVE-1 for HA [48]. Furthermore, TNF-a actually caused rapid internalization and degradation of the



LYVE-1 receptor [48]. Clearly, more research is needed to elucidate activators of the LYVE-1 receptor and define the functional relationship with HA.

cytes exposed to platelet-cleaved HA preparations upregulated production of the cytokines IL-6 and IL-8 [56]. IL-6 stimulates VSMC proliferation and is found in atherosclerotic lesions [57], whereas IL-8 promotes monocyte adherence [58–60].
Vascular Smooth Muscle Cells

HARE, also known as stabilin-2, was first identified by Zhou et al. in 2000 [18]. It is expressed as both a 315 kDa protein and a 190 kDa protein that is cleaved from the 315 kDa isoform [49]. HARE is largely found in the sinusoidal endothelium of the liver, lymph nodes, and spleen, where it acts as a scavenger receptor responsible for clearing glycosaminoglycans such as HA, chondroitin sulfates, and heparin [50–52]. Additionally, HARE is found in specialized structures of the eye, heart, brain, and kidney, including corneal and lens epithelium, heart valve mesenchymal cells, ependymal cells lining the ventricles in the brain, and epithelial cells covering the renal papillae [53].

Atherosclerosis is a chronic inflammatory state that targets the blood vessel wall [1]. Platelets, VSMCs, ECs, and macrophages are all important for atherogenesis, but equally important in the atherogenic process are components of the ECM, including HA, and their interaction with platelets and the aforementioned cell types. The following section, therefore, details some of the known interactions between HA, platelets, and the cellular components involved in atherogenesis and vascular inflammation.

Platelets are instrumental in the early stages of atherogenesis as they adhere to exposed collagen at areas of injured endothelium as well as to macrophages [1]. Once activated, platelets release the contents of their intracellular granules; these contents include cytokines and growth factors that promote chemotaxis and proliferation of VSMCs and monocytes [54]. In addition, increased platelet activity (assessed by flow cytometry and platelet aggregation) has a positive correlation with the severity of peripheral atherosclerotic disease [55]. Along with the aforementioned interactions, the interplay between platelets and HA is a key element in the atherogenic process as well. Platelets are unique compared with other cells and tissues in the body as they appear to only have HYAL2 and not HYAL1 [56]. HYAL2 allows platelets to cleave large molecules of HA into fragments that induce local leukocytes to secrete inflammatory chemokines and cytokines [56]. In particular, de la Motte et al. demonstrated that mono-

VSMCs are a functionally important part of the arterial wall as they maintain vascular tone and manufacture the ECM that gives structural support to the artery itself [1, 2]. In their natural state, VSMCs are located in the tunica media, but during the early stages of atherosclerosis these cells migrate to the tunica intima [61]. Once in the intima, these VSMCs begin manufacturing large amounts of ECM components, including HA [62]. Although HAS-2 is the HAS synthase isoform most responsible for HA production in VSMCs, all three isoforms affect the HA composition of the ECM in different ways [63]. Wilkinson et al. used a retroviral transduction system to overexpress each of the three HAS isoforms in murine VSMCs [63]. They determined that HAS-1 transduced VSMCs produced an ECM with a greater percentage of HA than either HAS-2 or HAS3 [63). Additionally, the HA secreted by HAS-1 transduced cells formed intercellular, ‘‘cable-like’’ structures in the ECM [63]. Interestingly, studies have demonstrated that these cable-like structures bind leukocytes and are involved in leukocyte adhesion and rolling [64–66]. These observations implicate VSMC-produced HAS-1 as important in the early proinflammatory stage of atherosclerosis, where leukocytes migrate across the endothelium and into the vessel wall. Despite evidence for its proatherogenic nature, it is important to note that high concentrations of HA actually reduces the number of VSMCs grown in culture in a dose-dependent manner [67]. It is speculated that the bulk of this in vitro inhibition is the result of saturation of cell surface binding sites by exogenous HA; this saturation would prevent VSMCs from binding HA in the ECM and receiving the appropriate stimuli for growth [67].
ECs and Leukocytes

ECs perform a myriad of functions that are essential for maintaining blood vessel wall integrity and circulatory function [68]. In addition, the EC layer acts as an interface between the underlying VSMCs and components of circulating blood [68]. Maintaining the structural stability of this layer is of paramount importance as any disruption can serve as a nidus for the generation of atherosclerotic plaque [1]. Once the EC layer is compromised, it displays increased adhesiveness of,



and permeability to, leukocytes at the site of injury [1]. In particular, macrophages and T lymphocytes traverse the EC layer at these areas of injury and accumulate in the interstitium of the blood vessel wall where, over time, they may become incorporated into a mature atherosclerotic plaque [69]. HA is a critical mediator of the interactions between ECs and leukocytes that are important in the pathogenesis of atherosclerosis. For example, Taylor et al. demonstrated that human dermal microvascular ECs cultured in vitro will dramatically up-regulate transcription of IL-8 when exposed to soluble HA fragments [58]. IL-8 recruits neutrophils to areas of endothelial injury and is a potent stimulator of lymphocyte migration [58–60].

However, evidence exists that contracture of the vessel wall itself plays the larger role in lumen narrowing compared with IH after PTA [75–80]. Alternatively, IH remains the chief reason for vessel renarrowing after PTA with stenting and bypasses using prosthetic grafts. With regard to stenting, the rigid framework of the stent itself is able to prevent the vessel wall narrowing that was previously described, so any restenosis that occurs is most likely due to IH. Additionally, prosthetic graft revascularization is still plagued by IH, making long-term patency of these grafts a challenging clinical problem. The sum total of these observations, therefore, makes it clear that both IH and arterial wall contraction are important in the process of vessel wall renarrowing after surgical intervention.
HA is Involved in Wound Contracture

In addition, HA appears to be a mediator for the transmigration of leukocytes across the EC layer and into the interstitium of the blood vessel wall, a critical step in the formation of atherogenic plaque. Specifically, active CD44 on T lymphocytes recognizes and binds HA presented by CD44 on ECs, allowing for extravasation of T lymphocytes at areas of endothelial injury and inflammation [66, 70, 71]. Furthermore, HA fragment concentration increases in injured tissue and promotes up-regulation of inflammation. HA in its native form, however, does not appear to possess any pro-inflammatory properties [29].

Chronic hyperglycemia can initiate an inflammatory cascade that mediates up-regulation of several proinflammatory proteins. Indeed, one study found that hyperglycemia increased all three HAS isoforms and HA production in VSMCs [72]. Studies in our laboratory have shown that TSP-1, which is known to be increased by hyperglycemia, increases the expression of HAS-2 in VSMCs [73]. Thus, HAS isoforms and HA production may have an important role in hyperglycemic-induced vascular remodeling in diseases such as diabetes.
HA AND VESSEL RENARROWING AFTER INTERVENTION Vessel Renarrowing is Largely Secondary to Intimal Hyperplasia and/or Wound Contracture

Interestingly, HA is a key component of wound contracture. With regard to the overall wound healing process, HA has a conflicting role depending on its size. The observed effects of HA on tissue contraction appear conflicting as well. Specifically, an in vitro study performed by Travis et al. demonstrated that HA increased ECM contraction of both arterial VSMCs and adventitial fibroblasts through a CD44-dependent mechanism [81]. Alternatively, De Vries et al. showed that HA-treated collagen matrices demonstrated a tendency to reduce wound contraction in a human punch biopsy wound model [82]. Taken together, these varying observations highlight the need for clearly identifying how native HA and HA fragments regulate wound healing and contracture in the arteries.
HA is Intimately Involved in IH

Although the pathologic consequences of vessel wall renarrowing are clear, the actual reason for the loss of arterial lumen caliber remains debatable and probably depends on the exact procedure performed. For example, loss of lumen caliber after percutaneous transluminal angioplasty (PTA) was initially attributed to the amount of IH present in the restenotic lesion [74].

After endothelial injury, the process of IH involves the release of growth factors and ECM proteins. These growth factors and ECM proteins promote the phenotypic transformation of VSMCs in the media from a contractile to a migratory state. Additionally, these VSMCs secrete large amounts of ECM, of which HA is a major component. In vitro studies have demonstrated that HA (at modest concentrations) mediates VSMC migration via both the CD44 and RHAMM receptors and VSMC proliferation via the CD44 receptor [39, 83, 84]. RHAMM expression is increased as early as 1 h after vascular injury and is localized to VSMCs at the edge of injury [83], while CD44 mRNA is up-regulated in VSMCs as late as 2 d after arterial injury [84]. Furthermore, the presence of HA has been confirmed by histologic analysis of both human restenotic arteries and the neointima of experimentally injured animals months after injury (Fig. 1B) [4, 85]. In human



restenotic lesions in particular, HA appears to be concentrated in the intima and adventitia, with only patchy areas of HA present in the media [4]. In addition, the loose, myxoid, fibroproliferative tissue characteristic of restenotic lesions in humans contains stellate-appearing VSMCs surrounded by HA [4, 86]. These observations indicate that HA has an integral and early role in the development and structure of restenotic lesions.
Diabetes and IH

of leukocyte adherence and migration and reinforces the importance of the interaction between HA and activated leukocytes during acute inflammation.
Age Affects the Pattern of HA Distribution in Stenotic Lesions After Acute Arterial Injury

Diabetes is associated with more advanced IH and subsequent restenosis after vascular instrumentation in humans [87], but the role of HA production and distribution in diabetic vascular remodeling is not well understood. Using rats with streptozocin-induced diabetes (characterized by hyperglycemia, hypoinslunemia and hypercholesterolemia), Chajara et al. employed their aortic balloon injury model to determine the effect of insulin treatment on HA production in the neointima. Their study demonstrated that (1) nondiabetic rats had marked HA expression in the neointima near the aortic lumen; (2) untreated diabetic rats had diffuse HA expression throughout the neointima; and (3) diabetic rats treated with insulin showed HA distribution similar to the euglycemic, nondiabetic rats [88]. In a similar study with hyperinsulinemic and hypertriglycerolemic rats, Chajara et al. demonstrated an increase in HA and hyaluronidase production as well as an increase in HA degradation after aortic injury compared with control rats [89]. Although it may not be clearly defined at this point in time, these studies provide evidence that HA has a role of importance in the vascular remodeling process associated with diabetes and insulin treatment.
High and Low Molecular Weight Fragments of HA Limit IH

HA accumulates in the intima of uninjured/aging arterial vessels over time, as evidenced in both human and animal studies [9, 91]. Interestingly, age appears to impart a distinct pattern of IH and HA expression within stenotic lesions produced by acute arterial injury as well. For example, in a rodent model of aortic balloon injury with resultant IH, Chajara et al. demonstrated that IH formation was more exaggerated in young rats (2–3 mo old) compared with older rats (18–24 mo) both 2- and 4 wk post-injury. Additionally, 2 wk post-injury HA content and hyaluronidase activity were markedly increased (148% and 116%, respectively) in younger rats, while older rats displayed only a slight increase (23% and 15%, respectively). At 28 d, both HA content and hyaluronidase activity continued to increase for both groups, but this increase was still more pronounced for the group of young rats [91]. Older rats did have an increased ratio of hyaluronidase to HA at all time points [91], likely representing a role for HA and hyaluronidase in the aging vessel.

Hyaluronidase activity leads to HA fragments of high and low molecular weight at the area of IH. Savani and Turley were able to demonstrate this relationship in an in vivo model; specifically, after balloon injury of rat carotid arteries and exposure to high concentrations of high molecular weight HA, they found that neointimal formation was significantly reduced [90]. Chajara et al. demonstrated similar findings in their rat aortic injury model using high exogenous concentrations of low molecular weight HA fragments [67]. Specifically, neointimal area (at 2 wk) was significantly decreased in those rats treated with HA fragments compared with control rats [67]. These studies show the potential benefit of high concentrations of HA to reduce IH when given at injury and for a period of time that covers the acute inflammatory reaction. This may be due to the inhibition

Atherosclerosis and vessel renarrowing after surgical instrumentation remain significant clinical problems, and HA has emerged as a key component of both of these pathologic processes. Specifically, HA has proatherogenic effects on platelets, ECs, and leukocytes. Furthermore, HA is up-regulated in areas of vascular injury and has been shown to increase VSMC migration and proliferation, key events in the progression of atherosclerosis and vascular renarrowing after surgical intervention. Paradoxically, however, high concentrations of HA appear to offer a protective effect against IH induced by acute arterial injury. Exogenous administration of HA, therefore, may potentially be utilized to prevent vessel renarrowing after surgical intervention for atherosclerosis. In the studies examined in this review, it appears that administration of HA during the early inflammatory phase (within wk 1 or 2) after arterial instrumentation provides a measure of benefit. Further study is needed, however, to determine the optimal timing, dosage, and HA fragment size that will produce durable results in animal models, and potentially in human subjects. Ultimately, the complex interplay of HA and its fragments with the ECM, platelets, and aforementioned cell types warrants careful analysis and further study to definitively determine the


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role of HA in both atherosclerosis and vascular renarrowing after surgical intervention.
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