Hyaluronidase 1 Deficiency Preserves Endothelial Function and Glycocalyx Integrity in Early Streptozotocin-Induced Diabetes

The glycosaminoglycan hyaluronic acid (HA), or hyaluronan, is a major component of the extracellular matrix. HA mediates cell-cell and cell-matrix interactions and plays key roles in cell migration, tumor growth and progression, inflammation, and wound healing (1). HA is synthesized at the plasma membrane by different HA synthases and degraded by a family of endoglucosaminidases named hyaluronidases, mainly HYAL1 and HYAL2 in somatic tissues (2). HYAL1 is the only hyaluronidase present in human and mouse plasma (3). In all cell types, its enzymatic activity occurs at pH levels <4.0, which requires the enzyme to undergo endocytosis (4).

HYAL1 deficiency in humans is a rare disease; it is associated with bone erosions, synovitis, and polyarthritis together with high plasma HA levels (5). A mouse model of HYAL1 deficiency showed HA accumulation in serum without gross abnormalities except for a loss of proteoglycans in knee joints (6).

In the vascular network, HA is a major component of the endothelial glycocalyx alongside heparan sulfate– and chondroitin sulfate–containing proteoglycans (7). In the glycocalyx, HA binds to its receptor CD44 but has no covalent linkage and may freely exchange with the bloodstream. The glycocalyx is recognized as a major factor in vascular physiology and pathology; it contributes to shear force sensing and transduces these forces into intracellular responses, such as nitric oxide (NO) release (7). The glycocalyx also acts as a regulator of vascular permeability, a reservoir for various antithrombotic factors, and an antiadhesive barrier for leukocytes (8).

Through in vivo perfusion of hyaluronidase, which removes all HA in the endothelial surface layer, HA has been found to be essential to maintain glycocalyx integrity and the functional barrier (9). Hyaluronidase infusion also abolishes the NO-dependent response to increased shear stress in segments of pig iliac artery or dog coronary arteries but not the acetylcholine (ACh)-induced NO production (10,11).

In patients with type 1 and type 2 diabetes, endothelial dysfunction appears to be a consistent finding underlying the pathophysiology of macro- and microvascular complications, and therefore contributes to the increased mortality rates observed in the population with diabetes. Glycocalyx defects may play a central role in diabetes pathogenesis by contributing to the proinflammatory state implicated in impaired skin wound healing and atherosclerosis (12). Indeed, the glycocalyx itself is disturbed during both acute (13) and chronic hyperglycemia in man (14,15). In addition, patients with type 1 and type 2 diabetes have increased plasma HA levels (15,16) and hyaluronidase activity (14,15).

To date, the implication of elevated plasma HYAL1 and/or HA levels in the pathogenesis of diabetes remains unexplored. As plasma HYAL1 is endocytosed into endothelial cells and could therefore modulate their function possibly through glycocalyx regulation, we decided to investigate the potential role of HYAL1 in the development of diabetes-induced endothelial dysfunction. To this aim, diabetes was induced in wild-type (WT) and HYAL1 knockout (KO) mice using streptozotocin (STZ) injections, and endothelial-dependent vasorelaxation, circulating endothelial markers, and the size and HA content of the glycocalyx were measured.

All experiments were performed on 7–9-week-old male C57Bl/6 (WT) mice and B6.129 × 1-Hyal1^tm1Stn/Mmcd (Hyal1^−/− or KO) mice obtained from Mutant Mouse Regional Resource Centers (MMRRC) backcrossed onto a C57Bl/6 genetic background for nine generations. The animals were fed regular chow and tap water ad libitum. All experiments were approved by the local animal ethics committees of the University of Namur and the Université Catholique de Louvain (2012/UCL/MD/004).

Type 1 diabetes was induced by five daily intraperitoneal injections of 55 mg/kg STZ in 10 mmol/L citrate buffer, pH 4.5. Control mice received buffer alone. Four weeks after treatment, glycemia was measured using One Touch Vita test strips (LifeScan Europe, Zug, Switzerland; limited to an upper value of 600 mg/dL). Animals with glycemia ≥300 mg/dL were assigned to the groups with diabetes for the experiments. Mean arterial blood pressure was measured using a noninvasive computerized tail-cuff method (CODA; Kent Scientific, Torrington, CT) in nonanesthetized mice after acclimation (17).

Blood was collected through cardiac puncture into 0.2% EDTA tubes. Soluble intercellular cell adhesion molecule-1 (sICAM1), vascular cell adhesion molecule-1 (sVCAM1), and P-selectin (sP-selectin) were quantified using ELISA kits, and HA using an ELISA-like assay that allows detection of HA molecules ≥15 kDa (18), all obtained from R&D Systems (Minneapolis, MN). Syndecan-1 was measured using an ELISA kit from Diaclone (Besançon, France). Albumin and creatinine concentrations were measured in urine samples using Albuwell (Exocell, Philadelphia, PA) and creatinine (Enzo Life Sciences, Lausen, Switzerland) kits, respectively.

Aortas were isolated, cleaned of fat on ice, frozen in liquid nitrogen, and stored at −80°C. They were then lyophilized during 16 h and treated with Pronase (at 3 mg/mL in 100 mmol/L ammonia/formic acid buffer, pH 7–8) for 24 h at 55°C. After relyophilization, the samples were resuspended in water to allow HA measurement. In some experiments, aortas were first flushed on ice with a strong injection of cold PBS (2 mL) by holding their extremity on the needle using pliers.

Plasma HYAL1 activity was measured using two different approaches: 1) zymography in renatured and native conditions, as described previously (4), and 2) gel electrophoresis (19) followed by quantification of oligosaccharide bands using ImageJ (public domain, NIH).

The method followed a procedure previously described (20). In brief, the aorta of anesthetized mice was retrogradely cannulated and the vena cava transected. The following solutions were infused at a flow rate of 0.4 mL/min and a pressure of 33 ± 5 mmHg: a cardioplegic solution for 3 min, a phosphate-buffered 4% paraformaldehyde/1% glutaraldehyde (pH 7.4) fixative solution for 2 min, and finally, the same solution containing 0.05% Alcian Blue 8GX (Sigma-Aldrich) for 30 min. The left ventricular wall was cut in 2-mm segments, fixed for 1 h in the fixative solution, postfixed in 1% osmium tetroxide and 1% lanthanum nitrate for 1 h, and then processed for transmission electronic microscopy (TEM) using a standard procedure. Sections were visualized with a FEI Tecnai microscope and photomicrographs analyzed using ImageJ. The glycocalyx thickness of cardiac arterioles was calculated by dividing the surface area by the underlying endothelium length.

Anesthetized mice were intravenously injected with a mixture of 10 mg/mL Texas Red/40-kDa neutral dextran and 2.5 mg/mL FITC/70-kDa anionic dextran (Molecular Probes, Eugene, OR). Urine was collected for 30 min and fluorescence was measured to determine glomerular permselectivity based on the ratio of 70- to 40-kDa dextran. The urinary albumin-to-creatinine ratio (ACR) was also measured.

Second-order mesenteric arteries were isolated from animals under terminal anesthesia and placed in ice-cold Tyrode solution. Arteries were cleared of fat and connective tissue and then cut into <2-mm rings and mounted in a wire myograph (model 610M-DMT; Danish Myo Technology A/S, Aarhus, Denmark), as previously described (21). After a 45-min stabilization in Tyrode solution containing 10⁻⁵ mol/L indomethacin, tension normalization, and 60-min equilibration, vessels were contracted using 100 mmol/L KCl. Then, cumulative concentrations of ACh (10⁻⁸ to 3 × 10⁻⁵ mol/L) were added to induce endothelium-dependent relaxation. After washout and stabilization, vessels were again contracted using 3 × 10⁻⁶ mol/L phenylephrine in the absence or presence of 10⁻⁴ mol/L N-nitro-l-arginine methyl ester (l-NAME). Cumulative amounts of ACh were again added and the percentage of residual contraction was calculated. To test small conductance potassium channel-3 (SK3) activity, an SK3 opener, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CYPPA; Sigma-Aldrich), was used in the presence of indomethacin and l-NAME. Vessels were precontracted with 10⁻⁷ mol/L U46,619 (Sigma-Aldrich), a thromboxane A2 agonist, instead of phenylephrine to obtain a stable contractile state. Cumulative amounts of CYPPA (3 × 10⁻⁷ to 6 × 10⁻⁵ mol/L) were then added.

Total RNA was isolated from microdissected mesenteric arteries using the RNeasy Micro Kit (Qiagen, Hilden, Germany) and treated with DNAse. Reverse transcription was performed using random hexamers and Superscript II MMLV reverse transcriptase (Invitrogen, Carlsbad, CA). The levels of expression of several genes were determined using real-time PCR (7300 Real Time PCR System; Applied Biosystems, Cheshire, U.K.) with SYBR Green detection. mRNA levels were calculated using the 2^-ddCT method.

Mesenteric arteries were fixed in alcoholic Bouin solution for 72 h and embedded in paraffin. Detection of SK3 and von Willebrand factor (vWF) in 6-μm sections was performed with an anti-SK3 rabbit polyclonal antibody (Sc28621; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-vWF rabbit polyclonal antibody (A0082; Dako A/S, Glostrup, Denmark) followed by biotinylated secondary antibodies and streptavidin-peroxidase. Quantification of the SK3 immunostaining was performed using ImageJ.

Two-way ANOVA followed by Bonferroni post hoc tests was performed to compare the four groups of mice in each experiment.

Baseline fasting glycemia was similar in WT and KO mice and significantly increased, comparably in both genotypes, 28 days post–STZ treatment (Fig. 1A). Baseline body weights of 8-week-old KO mice were not significantly lower than those of WT mice. Furthermore, the growth-arresting impact of diabetes was similar in both genotypes (Fig. 1B). There was no effect of either genotype or diabetes on mean blood pressure (Fig. 1C).

A twofold increase in circulating HA levels was observed in diabetic versus nondiabetic WT mice (Fig. 2A). In KO mice, baseline HA concentrations were four times higher than in WT mice and did not further increase after induction of diabetes.

Zymography of serum samples revealed a single band of hyaluronidase activity at ∼80 kDa in both denaturing and native conditions, corresponding to the activity of the HYAL1 precursor, which did not increase after diabetes induction (Fig. 2B and C). In aortic wall homogenates, zymography in native conditions showed two bands, corresponding to the precursor and cleaved forms of HYAL1 (4), again with no increase in hyaluronidase activity during diabetes (Fig. 2D). Gel electrophoresis of HA solutions incubated with sera of nondiabetic and diabetic WT mice allowed a more accurate measurement of serum hyaluronidase activity and revealed a slight but significant increase in the amount of smaller oligosaccharides produced using hyperglycemic sera (Fig. 2E–H), suggesting an increased HYAL1 activity (3) in diabetic WT mice. The same analysis performed on HYAL1 KO mouse serum (Fig. 2E) confirmed the assay specificity.

To determine whether increased plasma HA concentrations are accompanied by changes in glycocalyx HA content, we measured the amount of HA in flushed segments of the aortic wall, which almost completely removes the endothelium while leaving the underlying aortic wall intact, and compared it with that of unflushed segments. There was no difference in the amount of HA in the unflushed aortic segments between any of the groups (Fig. 3A). However, whereas the flushable/glycocalyx HA accounted for approximately one-quarter of the total aortic HA in healthy WT and KO mice, it was nearly absent in diabetic WT mice but was preserved in diabetic KO mice.

Since HA is not the only component of glycocalyx, we examined its structure in small vessels (ventricular arterioles) using a well-established electron microscopic method as described in 2research design and methods (Fig. 3B and C). As summarized in Fig. 3D, glycocalyx thickness was greater than threefold higher in KO than WT mice, both in diabetic and nondiabetic mice. Diabetes did not induce any significant change in glycocalyx thickness in either genotype. Plasma syndecan-1 concentration (Fig. 3E), measured to investigate glycocalyx shedding, was not impacted by either genotype or diabetes.

The relative permeability of the glomerular endothelial glycocalyx was then evaluated by measuring the concentration ratio of 70- to 40-kDa fluorescent dextran recovered in the urine after intravenous injection. As shown in Fig. 4, the ratio of 70- to 40-kDa excretion increased during diabetes in WT but not in KO mice, suggesting that glomerular permselectivity to high-molecular-weight dextran is altered in diabetic WT mice but not in diabetic KO mice. Similarly, the urinary ACR increased in diabetic WT but not KO mice. This suggests that the absence of HYAL1 protects the glomerular endothelial glycocalyx against diabetes-induced functional damage.

In order to detect early signs of endothelial dysfunction, we measured the levels of circulating adhesion molecules. The level of sICAM1 was significantly upregulated during diabetes in both genotypes (Fig. 5B). The level of sP-selectin was also upregulated during diabetes but only in WT mice (Fig. 5A). The concentration of sVCAM1 was not affected by diabetes in any genotype (Fig. 5C). The baseline plasma concentrations of sICAM1 and sVCAM1 were lower in KO than WT mice, suggesting a healthier endothelial status in the absence of HYAL1.

Endothelial function was investigated ex vivo in small-diameter, second-branch mesenteric arteries, allowing assessment of both NO­- and endothelium-dependent hyperpolarization (EDH)–mediated relaxation. NO-dependent ACh-induced vasodilation was similar in all groups of mice (data not shown). EDH-dependent ACh-induced vasodilation, on the other hand, was severely altered in diabetic WT mice, with only a remaining 34% EDH-dependent vasodilation, while partially preserved in diabetic KO mice, with a remaining 56% EDH-dependent vasodilation (Fig. 6). The difference between diabetic WT and KO mice was highly significant (P < 0.0001). There was no difference between healthy WT and KO mice.

The mRNA expression level of several components of the EDH pathway (22) in mesenteric arteries was screened using real-time RT-PCR. No difference among experimental groups was detected for connexins 37, 40, 43, and 45; SK channels SK1 and SK2; intermediate conductance potassium channel IK1; transient receptor potential (TRP) channels TRPV4 and TRPC1; and caveolin-1 (data not shown). Conversely, SK3 mRNA expression was significantly upregulated in KO mice independently of diabetes (Fig. 7A). Immunohistochemistry allowed the detection of SK3 along the endothelium in a pattern similar to that of vWF (Fig. 7B). Quantification of the staining confirmed increased expression of SK3 in KO versus WT vessels independently of diabetes (Fig. 7C).

To determine whether the activity of SK3 was increased in KO versus WT mice, mesenteric artery segments were mounted on wire myographs, contracted with U46,619, and exposed to increasing concentrations of the CYPPA SK3 opener (23). SK3-dependent relaxation was more efficient in KO than in WT mice, whether the animals were healthy or diabetic (Fig. 7D). SK3 overexpression may thus explain the preservation of the EDH-dependent vasodilation observed in diabetic KO mice.

Increased serum hyaluronidase activity in diabetes has been reported in man (15) and rats (24), but the implication of these observations has been poorly studied. The current study confirms a slight increase in serum hyaluronidase activity, which is likely due to HYAL1, in early diabetic WT mice (Fig. 2H). Furthermore, experiments using HYAL1-deficient mice at a relatively early stage of type 1 diabetes (4 weeks after STZ injections) show, for the first time, that HYAL1 may have a pathogenic role in diabetes-induced endothelial dysfunction.

Endothelial dysfunction in murine diabetes can be evaluated by measuring plasma levels of sP-selectin, sICAM1, and sVCAM1, as well as ACh-dependent vasorelaxation. We showed that 4 weeks of STZ treatment in WT mice is sufficient to increase sP-selectin and sICAM1 but not sVCAM1, suggesting that the latter may associate with a later stage in diabetes development, as suggested previously (25).

Alteration in ACh-dependent vasorelaxation is another reliable marker of endothelial dysfunction, at least in rat models of type 1 and type 2 diabetes (26). In mice, loss of endothelium-dependent relaxation appears only after 10 weeks of hyperglycemia in STZ-induced diabetes (27). Concordant with these data and previous observations in diabetic rats (28), we found no defect in the overall ACh-dependent vasorelaxation after 4 weeks of STZ injections. Still, we demonstrated a clear reduction in EDH-dependent relaxation.

Furthermore, HYAL1 deficiency may prevent the diabetes-induced increase in sP-selectin, but not in sICAM1, and limit the impact of the disease on EDH-dependent vasorelaxation. The data on sP-selectin and EDH-dependent vasorelaxation suggest functionally important cardiovascular-protective effects of HYAL1 deficiency during diabetes.

Interestingly, the absence of HYAL1 also modified the baseline levels of circulating markers of endothelial damage, two of which (sICAM1 and sVCAM1) were lower in KO than in WT mice. This suggests that HYAL1-deficient endothelia may be less attractant to leukocytes. This hypothesis could be tested by measuring endothelial chemoattraction and diapedesis. Of note, HYAL1 deficiency did not prevent renal neutrophil and macrophage infiltration in a murine model of severe ischemia reperfusion injury (29), but this is a complex acute lesion model in which multiple factors could modulate the phenotype.

Through a deeper analysis of several key endothelial proteins, we observed increased expression of SK3 in HYAL1-deficient endothelium. This endothelial potassium channel is reportedly a fundamental determinant of vascular tone and blood pressure (30) and a mainspring of the EDH pathway (31). In the absence of diabetes, however, the EDH-mediated vasodilation measured in mesenteric arteries was not enhanced by lack of HYAL1. This suggests baseline endothelial SK3 levels are not rate limiting for EDH-induced vasodilation in normal physiological conditions but could become so when the EDH pathway needs to be activated, e.g., during early diabetes. SK3 downregulation was previously observed in STZ-induced diabetic ApoE-deficient mice (22) or in the cavernous tissues of diabetic rats (32) but not in C57Bl/6 diabetic mice (27). In line with the latter results, SK3 was not downregulated by diabetes in our study. The long-term benefits of the HYAL1 deficiency–associated increase in SK3 expression in diabetes remain to be demonstrated. The absence of impact of diabetes and genotype on other EDH components at the RNA level does not preclude an effect at the protein level. The small size of vascular samples has prevented us from thoroughly examining this hypothesis in the current study.

The HA content of the glycocalyx is reportedly crucial for endothelial barrier function (9). A detrimental effect of diabetes or acute hyperglycemia on endothelial glycocalyx has been demonstrated in several studies in man (13,15) and mice (33). We postulated that the mechanism of endothelial protection in HYAL1-deficient mice is linked to a stronger glycocalyx. In our study, 4-week STZ-induced diabetes did not reduce the size of the endothelial glycocalyx in WT mice, as measured with a sensitive electron microscopic technique. Nevertheless, diabetes-exposed glycocalyx became HA depleted and thus potentially more vulnerable. HA seems to be incorporated within the glycocalyx in a shear stress–dependent way (34). It was therefore highly relevant to observe that HYAL1-deficient endothelial surfaces, contrary to WT endothelia, maintained their HA content during diabetes (demonstrated in Fig. 3).

Furthermore, the absence of HYAL1 dramatically increased the thickness of the glycocalyx (in both healthy and diabetic glycocalyx). However, aortic flushes failed to demonstrate a higher HA content as the reason for the increased size of glycocalyx. This may be due to insufficient sensitivity of the methods and/or additional factors. One possibility is a better anchoring of HA into the glycocalyx and a longer stretching of the HA chains in the absence of HYAL1. This could lead to an apparent increased thickness of the glycocalyx without HA accumulation.

Glycocalyx shedding under severe inflammatory conditions, such as postischemic reperfusion, can be prevented using various treatments, e.g., hydrocortisone, antithrombin, or heparin (35,36). Sulodexide, a mix of heparan sulfate and dermatan sulfate, increases glycocalyx thickness in type 2 diabetes (14). However, although sulodexide had global beneficial effects on renal manifestations of experimental diabetes in C57Bl/6 mice (37), it failed to demonstrate renoprotection in overt type 2 diabetic nephropathy in man (38).

To our knowledge, such an increase in glycocalyx size and resistance as observed in our study using HYAL1 KO mice has never been described as a result of therapeutic intervention or genetic manipulation (39). A thicker glycocalyx may thus correspond to reduced access of circulating inflammatory cells to the endothelium (8) as well as more efficient shear stress–induced signals. The glycocalyx has indeed been suggested to mediate shear stress– or flow-induced NO production and vascular remodeling (40,41), although, perhaps surprisingly, its role in EDH-mediated vasorelaxation has not been explored to date. A thicker glycocalyx could also explain the higher baseline expression of SK3 observed in our study.

Finally, although a thicker glycocalyx correlated with beneficial effects on diabetes-induced endothelial dysfunction in the current study, this was only observed in the absence of HYAL1. Thus, caution is warranted before the cardiovascular consequences of a thicker baseline glycocalyx can be described as indisputably favorable.

Diabetes is a well-known aggressor of the glomerular glycocalyx and endothelial barrier, resulting in loss of permselectivity and ultrafiltration of albumin (42). Glycocalyx damage coincides with microalbuminuria in type 1 diabetes (15). Obese diabetic db/db mice have an altered glycocalyx with higher access of a 70-kDa dextran tracer to the vessel wall and higher clearance of this tracer (43). In the current study, 4-week STZ-induced diabetes significantly increased the urinary ACR and ratio of 70- to 40-kDa dextran, confirming altered glomerular permselectivity. These effects were completely prevented in diabetic HYAL1 KO mice, bringing further arguments for a functionally preserved glycocalyx and endothelial barrier when diabetes develops in the absence of HYAL1. Baseline ratios of albumin to creatinine and 70- to 40-kDa dextran were similar in WT and HYAL1-deficient mice.

Table 1 summarizes the beneficial effects observed in HYAL1 KO mice exposed to STZ-induced diabetes as compared with WT mice. The main protective effects include lower release of P-selectin into the circulation, lower shedding of HA from the endothelial surface, preserved integrity of the glomerular endothelial glycocalyx, and conservation of EDH-mediated vasorelaxation.

As for the mechanisms involved in HYAL1-mediated endothelial protection, our main hypothesis is that the absence of HYAL1 prevents glycocalyx HA shedding during diabetes and, from there, affords protection against diabetes-induced vascular damage and maintains a higher level of SK3 expression. In turn, this would preserve EDH relaxation during diabetes, when SK3 expression becomes rate limiting.

Another hypothesis is that elevated plasma HA levels in HYAL1 KO mice facilitate its reincorporation into the glycocalyx during diabetic injury. Previous studies have shown that the glycocalyx is quickly (30 min) restored after hyaluronidase treatment if HA and chondroitin sulfate are infused in sufficiently high amounts, i.e., >100 times the baseline plasma HA concentration (9). However, the difference in plasma HA concentrations between diabetic WT mice and diabetic HYAL1 KO mice in our study was only moderate (+58% in the latter) and unlikely to explain the large difference in glycocalyx HA content during exposure of WT versus KO mice to hyperglycemia. Additionally, we have shown similar molecular size profiles of circulating HA in HYAL1 KO and WT mice (44). Finally, the hypothesis that higher baseline expression of SK3 would explain a thicker glycocalyx seems unlikely based on the known function of SK3.

HYAL1 deficiency had several protective effects against early diabetes-induced endothelial and glycocalyx damage. Most, if not all, of these effects may result from a thicker and sturdier glycocalyx (with, e.g., a higher level of endothelial SK3 channels). This stronger glycocalyx may, in the long run, protect against macro- and microvascular complications of diabetes, both of which have been linked to glycocalyx impairment. In ApoE-deficient atheromatosis-prone mice, for instance, HA synthesis inhibition using 4-methylumbelliferone led to a loss of glycocalyx and enhanced atherosclerosis (45). HYAL1 inhibition may have an opposite, protective effect. In addition, the main diabetes-associated microvascular complications, i.e., retinopathy, neuropathy, and glomerular capillary injury, have been linked to a loss of glycocalyx. For instance, early diabetes impacts glomerular permeability and glycocalyx alterations are reported in diabetic mice even before the development of albuminuria (46). In early diabetic retinopathy in rats and mice, both glycocalyx- and EDH-mediated vasodilation of retinal vessels are altered (47,48). Diabetic neuropathy in mice is accompanied by glycocalyx alteration in brain microvessels (49). Finally, diabetes significantly reduces EDH-mediated vasorelaxation in human penile resistance arteries and downregulates SK3 and IK1 channels in rat corpus cavernosum tissues (32). HYAL1 inhibition could thus be a first step to prevent loss of glycocalyx during the development of diabetic nephropathy, retinal microangiopathy, blood-brain barrier damage, and erectile dysfunction.

Interestingly, even though HA has been shown to accumulate in the aortic tunica media of patients with type 2 diabetes (50), where it may have potential deleterious effects, our study fails to demonstrate any HA accumulation in the aorta of congenital HYAL1-deficient mice, either before or after induction of diabetes.

Overall, this report suggests that HYAL1 inhibitors could potentially be explored as a new therapeutic approach to prevent endothelial dysfunction in diabetes. As a caveat, the beneficial effects of HYAL1 deficiency were observed in early diabetes and should be confirmed over the course of the disease.

Acknowledgments. The authors offer sincere thanks to Laurence Jadin (University of Namur, Belgium) for reviewing the manuscript. This research used resources of the Electron Microscopy Service located at the University of Namur. This Service is a member of the Plateforme Technologique Morphologie-Imagerie.

Funding. C.D. is a senior research associate of the Fonds National de la Recherche Scientifique (FNRS). F.J. is an MD postdoctoral fellow of the FNRS.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. S.D. designed and performed the experiments, contributed to the discussion, and wrote the manuscript. G.R. and F.J. designed and performed the experiments. N.C. contributed to the discussion. C.D. and B.F. designed and supervised the experiments, contributed to the discussion, and reviewed and edited the manuscript. S.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in poster form at the 2014 Experimental Biology scientific meeting, San Diego, CA, 26–30 April 2014, and at the 10th International Conference on Hyaluronan, Florence, Italy, 7–11 June 2015.