Page 1 of 32 Diabetes
Hyaluronidase 1 deficiency preserves endothelial function and glycocalyx
integrity in early streptozotocin-induced diabetes
Sophie Dogné,1 Géraldine Rath,2 François Jouret,3 Nathalie Caron,1 Chantal Dessy,2* and
Bruno Flamion,1*
1 Molecular Physiology Research Unit, NARILIS, University of Namur, Namur,
Belgium;
2 Pole of Pharmacology and Therapeutics, IREC, Université catholique de Louvain,
Brussels, Belgium;
3 Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA), Cardiovascular
Sciences, University of Liège, Liège, Belgium.
* These authors contributed equally to the study
Corresponding author: Sophie Dogné, Laboratory of Physiology & Pharmacology, University
of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium; Phone: +3281725660; Fax:
+3281724329; [email protected]
Short running title: HYAL1 deficiency protects the endothelium
Word count of main text (max 4000 words): 4052
Number of Tables: 1
Number of Figures: 7
Diabetes Publish Ahead of Print, published online May 31, 2016 Diabetes Page 2 of 32
ABSTRACT (max. 200 words): 193
Hyaluronic acid (HA) is a major component of the glycocalyx involved in vascular wall and endothelial glomerular permeability barrier. Endocytosed hyaluronidase HYAL1 is known to degrade HA into small fragments in different cell types including endothelial cells. In diabetes, the size and permeability of the glycocalyx are altered. In addition, type 1 diabetic patients present increased plasma levels of both HA and HYAL1.
To investigate the potential implication of HYAL1 in the development of diabetes induced endothelium dysfunction, we measured endothelial markers, endothelium dependent vasodilation, arteriolar glycocalyx size, and glomerular barrier properties in wild type and
HYAL1 knockout (KO) mice with or without streptozotocin induced diabetes. We observed that, 4 weeks after streptozotocin injections, the lack of HYAL1: 1) prevents diabetes induced increases in soluble P selectin concentrations and limits the impact of the disease on endothelium dependent hyperpolarization (EDH) mediated vasorelaxation; 2) increases glycocalyx thickness and maintains glycocalyx structure and HA content during diabetes; 3) prevents diabetes induced glomerular barrier dysfunction assessed using urinary albumin/creatinine ratio and urinary 70/40 kDa dextran ratio. Our findings suggest that
HYAL1 contributes to endothelial and glycocalyx dysfunction induced by diabetes. HYAL1 inhibitors could be explored as a new therapeutic approach to prevent vascular complications in diabetes.
INTRODUCTION
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 Page 3 of 32 Diabetes
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 NO release (7). The glycocalyx also acts as a regulator of vascular
permeability, a reservoir for various antithrombotic factors, and an anti adhesive 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 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
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
diabetic population. Glycocalyx defects may play a central role in diabetes pathogenesis by Diabetes Page 4 of 32
contributing to the pro inflammatory 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, both Type 1 and Type 2 diabetic patients 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 and HYAL1 KO mice using streptozotocin (STZ) injections, and endothelial dependent vasorelaxation, circulating endothelial markers, and the size and HA content of the glycocalyx were measured.
RESEARCH DESIGN AND METHODS
Animals. All experiments were performed on 7 to 9 week old male C57Bl/6 (WT) mice and
B6.129X1 Hyal1tm1Stn/Mmcd (Hyal1-/- or KO) mice obtained from MMRRC (Mutant Mouse
Regional Resource Centers, USA) backcrossed onto a C57Bl/6 genetic background for 9 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 5 daily intraperitoneal injections of 55 mg/kg STZ in 10mM 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 diabetic groups for the experiments. Mean arterial blood pressure was measured using a Page 5 of 32 Diabetes
noninvasive computerized tail cuff method (CODA, Kent Scientific, Torrington, CT) in non
anesthetized mice after acclimation (17).
Chemical assays. 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.
Preparation of aortic samples. Aortas were isolated, cleaned of fat on ice, frozen in liquid
nitrogen, and stored at 80°C. They were then lyophilized during 16h and treated with Pronase
(at 3mg/ml in 100 mM ammonia/formic acid buffer, pH 7 8) for 24h 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 (2ml) by
holding their extremity on the needle using pliers.
Hyaluronidase activity. Plasma HYAL1 activity was measured using two different
approaches: a) zymography in renatured and native conditions, as described previously (4); b)
gel electrophoresis (19) followed by quantification of oligosaccharide bands using image J
(public domain, NIH).
Glycocalyx staining in myocardial arterioles. The method followed a procedure previously
described (20). Briefly, the aorta of anesthetized mice was retrogradely cannulated and the
vena cava transsected. The following solutions were infused at a flow rate of 0.4 ml/min and a
pressure of 33±5 mmHg: a cardioplegic solution during 3 min; a phosphate buffered 4%
paraformaldehyde – 1% glutaraldehyde (pH 7.4) fixative solution for 2 min, and finally, the Diabetes Page 6 of 32
same solution containing 0.05% Alcian Blue 8GX (Sigma Aldrich) during 30 min. The left ventricular wall was cut in 2 mm segments, fixed for 1h in the fixative solution, and postfixed in 1% osmium tetroxide and 1% lanthanum nitrate for 1h, then processed for transmission electronic microscopy using a standard procedure. Sections were visualized with a FEI Tecnai microscope and photomicrographs analyzed using image J. The glycocalyx thickness of cardiac arterioles was calculated by dividing the surface area by the underlying endothelium length.
Dextran excretion. Anesthetized mice were intravenously injected with a mixture of 10 mg/ml Texas Red 40kDa neutral dextran and 2.5 mg/ml FITC 70kDa anionic dextran
(Molecular Probes, Eugene, OR). Urine was collected during 30 min and fluorescence was measured to determine glomerular permselectivity based on the 70kDa to 40 kDa dextran ratio. The urinary albumin/creatinine (A/C) concentration ratio was also measured.
Endothelium-dependent vasodilation. 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, then cut into <2mm rings and mounted in a wire myograph (model 610M DMT, Danish Myo Technology A/S, Aarhus, Denmark) as previously described (21). After 45 min stabilization in Tyrode solution containing 10 5M indomethacin, tension normalization, and 60 min equilibration, vessels were contracted using
100mM KCl. Then, cumulative concentrations of ACh (10 8M to 3.10 5M) were added to induce endothelium dependent relaxation. After washout and stabilization, vessels were again contracted using 3.10 6M phenylephrine in the absence or the presence of 10 4M 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 Page 7 of 32 Diabetes
indomethacin and L NAME. Vessels were pre contracted with 10 7M U46,619 (Sigma
Aldrich), a thromboxane A2 agonist, instead of phenylephrine to obtain a stable contractile
state. Cumulative amounts of CYPPA (3.10 7M – 6.10 5M) were then added.
mRNA expression. Total RNA was isolated from microdissected mesenteric arteries using
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 (Applied Biosystems 7300 Real Time PCR System, Warrington, Cheshire,
UK) with SYBR green detection. mRNA levels were calculated using the 2 ddCT method.
Immunohistology. Mesenteric arteries were fixed in alcoholic Bouin’s solution for 72h and
embedded in paraffin. Detection of SK3 and von Willebrand factor (vWF) in 6 m sections
were 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, Denmark) followed by biotinylated secondary antibodies and streptavidin peroxidase.
Quantification of the SK3 immunostaining was carried out using image J.
Statistical methods. Two way Analysis of Variance (ANOVA) followed by Bonferroni post
hoc tests was performed to compare the four groups of mice in each experiment.
RESULTS
Effects of diabetes on health conditions of WT and Hyal1-/- mice
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). Diabetes Page 8 of 32
Effect of diabetes on HA and hyaluronidase activity in WT and Hyal1-/- mice
A twofold increase in circulating HA levels was observed in diabetic vs non diabetic WT mice (Fig. 2A). In KO mice, baseline HA concentrations were 4 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 around 80 kDa in both denaturing and native conditions, corresponding to the activity of the HYAL1 precursor, which did not increase following diabetes induction (Fig. 2B&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 non diabetic 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.
HA on the luminal side of vessels
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 not difference in the amount of HA in the unflushed aortic segments between any of the groups (Fig. 3A).
However, while 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.
Glycocalyx thickness and integrity Page 9 of 32 Diabetes
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
the Materials and Methods section (Fig. 3B C). As summarized in Fig. 3D, glycocalyx
thickness was >3 fold higher in KO than WT mice, both in diabetic and non diabetic 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.
Glycocalyx barrier function
The relative permeability of the glomerular endothelial glycocalyx was then evaluated by
measuring the concentration ratio of 70 kDa to 40 kDa fluorescent dextran recovered in the
urine following intravenous injection. As shown in Fig. 4, the 70/40 kDa excretion ratio
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 albumin/creatinine ratio 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.
Markers of endothelium dysfunction
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 sVCAM1were
lower in KO than WT mice, suggesting a healthier endothelial status in the absence of
HYAL1.
Endothelial-dependent vasorelaxation Diabetes Page 10 of 32
Endothelial function was investigated ex vivo in small diameter, second branch mesenteric arteries, allowing assessment of both nitric oxide (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.
Exploration of the EDH pathway components
The mRNA expression level of several components of the EDH pathway (22) in mesenteric arteries was screened using real time RT PCR. No difference amongst experimental groups was detected for connexins 37, 40, 43, and 45; small conductance potassium (SK) channels
SK1 and SK2; intermediate conductance potassium (IK) 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 than in WT vessels independently of diabetes (Fig. 7C).
To determine whether the activity of SK3 was increased in KO vs 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 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.
Page 11 of 32 Diabetes
DISCUSSION
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 present study confirms
a slight increase in serum hyaluronidase activity, which is likely due to HYAL1, in early
diabetic WT mice (Fig. 1H). 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.
Lack of HYAL1 prevents 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 are 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 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. Diabetes Page 12 of 32
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 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 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.
Lack of HYAL1 maintains glycocalyx structure and prevents HA shedding Page 13 of 32 Diabetes
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 post ischemic
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). Diabetes Page 14 of 32
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 remodelling (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.
Lack of HYAL1 prevents glomerular barrier dysfunction
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 A/C ratio and 70/40 kDa dextran ratio, 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 A/C and 70/40 kDa dextran ratios were similar in WT and HYAL1 deficient mice.
Page 15 of 32 Diabetes
Mechanisms of endothelial protection in the absence of HYAL1
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 maintain 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 vs 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.
Conclusion and perspectives Diabetes Page 16 of 32
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 micro vascular 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 impact glomerular permeability and glycocalyx alterations are reported in diabetic mice 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 down regulates 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
Type 2 diabetic patients (50) where it may have potential deleterious effects, our study fails to demonstrate any HA accumulation in the aorta of congenital HYAL1 deficient mice, neither before nor after induction of diabetes. Page 17 of 32 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
Author contributions: SD designed and performed the experiments, contributed to discussion
and wrote the manuscript; GR and FJ designed and performed experiments; NC contributed to
the discussion; CD and BF designed and supervised the experiments, contributed to the
discussion and reviewed/edited the manuscript. Our sincere thanks to Laurence Jadin for
reviewing the manuscript.
SD 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. No
potential conflicts of interest relevant to this article were reported.
Parts of the study were presented in poster form at the Experimental Biology scientific
meeting, San Diego, CA, USA, 26 30 April 2014 and at the 10th International Conference on
Hyaluronan, Florence, Italy, 7 11 June 2015.
This research used resources of the Electron Microscopy Service located at the University of
Namur. This Service is member of the “Plateforme Technologique Morphologie – Imagerie”.
CD is a senior research associate of the Fonds National de la recherche Scientifique (FNRS).
FJ is an MD postdoctoral fellow of the FNRS.
REFERENCES
1. Hascall V, Esko JD. Hyaluronan. In: Essentials of Glycobiology. 2nd ed. Edited by Varki A, Cummings RD, Esko JD, et al.. Cold Spring Harbor (NY), 2009. 2. Csoka AB, Frost GI, Stern R. The six hyaluronidase like genes in the human and mouse genomes. Matrix Biol 2001;20:499 508. 3. Fiszer Szafarz B, Litynska A, Zou L. Human hyaluronidases: electrophoretic multiple forms in somatic tissues and body fluids. Evidence for conserved hyaluronidase Diabetes Page 18 of 32
potential N glycosylation sites in different mammalian species. J Biochem Biophys Methods 2000;45:103 116. 4. Puissant E, Gilis F, Dogne S, Flamion B, Jadot M, Boonen M. Subcellular trafficking and activity of Hyal 1 and its processed forms in murine macrophages. Traffic 2014;15:500 515. 5. Natowicz MR, Short MP, Wang Y, Dickersin GR, Gebhardt MC, Rosenthal DI, et al. Clinical and biochemical manifestations of hyaluronidase deficiency. N Engl J Med 1996;335:1029 1033. 6. Martin DC, Atmuri V, Hemming RJ, Farley J, Mort JS, Byers S, et al. A mouse model of human mucopolysaccharidosis IX exhibits osteoarthritis. Hum Mol Genet 2008;17:1904 1915. 7. Tarbell JM, Pahakis MY. Mechanotransduction and the glycocalyx. J Intern Med 2006;259:339 350. 8. Mulivor AW, Lipowsky HH. Role of glycocalyx in leukocyte endothelial cell adhesion. Am J Physiol Heart Circ Physiol 2002;283:H1282 1291. 9. Henry CB, Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan. Am J Physiol 1999;277:H508 514. 10. Kelly R, Ruane O'Hora T, Noble MI, Drake Holland AJ, Snow HM. Differential inhibition by hyperglycaemia of shear stress but not acetylcholine mediated dilatation in the iliac artery of the anaesthetized pig. J Physiol 2006;573:133 145. 11. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA, et al. Role of hyaluronic acid glycosaminoglycans in shear induced endothelium derived nitric oxide release. Am J Physiol Heart Circ Physiol 2003;285:H722 726. 12. Shakya S, Wang Y, Mack JA, Maytin EV. Hyperglycemia induced changes in hyaluronan contribute to impaired skin wound healing in diabetes: Review and perspective. Int J Cell Biol 2015;2015:701738. 13. Nieuwdorp M, van Haeften TW, Gouverneur MC, Mooij HL, van Lieshout MH, Levi M, et al. Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo. Diabetes 2006;55:480 486. 14. Broekhuizen LN, Lemkes BA, Mooij HL, Meuwese MC, Verberne H, Holleman F, et al. Effect of sulodexide on endothelial glycocalyx and vascular permeability in patients with type 2 diabetes mellitus. Diabetologia 2010;53:2646 2655. 15. Nieuwdorp M, Mooij HL, Kroon J, Atasever B, Spaan JA, Ince C, et al. Endothelial glycocalyx damage coincides with microalbuminuria in type 1 diabetes. Diabetes 2006;55:1127 1132. 16. Ceriello A, Giugliano D, Dello Russo P, Passariello N, Saccomanno F, Sgambato S. Glycosaminoglycans in human diabetes. Diabete Metab 1983;9:32 34. 17. Krege JH, Hodgin JB, Hagaman JR, Smithies O. A noninvasive computerized tail cuff system for measuring blood pressure in mice. Hypertension 1995;25:1111 1115. 18. Haserodt S, Aytekin M, Dweik RA. A comparison of the sensitivity, specificity, and molecular weight accuracy of three different commercially available Hyaluronan ELISA like assays. Glycobiology 2011;21:175 183. 19. Ikegami Kawai M, Takahashi T. Microanalysis of hyaluronan oligosaccharides by polyacrylamide gel electrophoresis and its application to assay of hyaluronidase activity. Anal Biochem 2002;311:157 165. 20. van den Berg BM, Vink H, Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ Res 2003;92:592 594. 21. Ghisdal P, Godfraind T, Morel N. Effect of nitro L arginine on electrical and mechanical responses to acetylcholine in the superior mesenteric artery from stroke prone hypertensive rat. Br J Pharmacol 1999;128:1513 1523 Page 19 of 32 Diabetes
22. Ding H, Hashem M, Wiehler WB, Lau W, Martin J, Reid J, et al. Endothelial dysfunction in the streptozotocin induced diabetic apoE deficient mouse. Br J Pharmacol 2005;146:1110 1118. 23. Hougaard C, Eriksen BL, Jorgensen S, Johansen TH, Dyhring T, Madsen LS, et al. Selective positive modulation of the SK3 and SK2 subtypes of small conductance Ca2+ activated K+ channels. Br J Pharmacol 2007;151:655 665. 24. Ikegami Kawai M, Suzuki A, Karita I, Takahashi T. Increased hyaluronidase activity in the kidney of streptozotocin induced diabetic rats. J Biochem 2003;134:875 880. 25. Gustavsson C, Agardh CD, Zetterqvist AV, Nilsson J, Agardh E, Gomez MF. Vascular cellular adhesion molecule 1 (VCAM 1) expression in mice retinal vessels is affected by both hyperglycemia and hyperlipidemia. PLoS One 2010;5:e12699. 26. De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoutte PM. Endothelial dysfunction in diabetes. Br J Pharmacol 2000;130:963 974. 27. Matsumoto T, Miyamori K, Kobayashi T, Kamata K. Specific impairment of endothelium derived hyperpolarizing factor type relaxation in mesenteric arteries from streptozotocin induced diabetic mice. Vascul Pharmacol 2006;44:450 460. 28. De Vriese AS, Van de Voorde J, Blom HJ, Vanhoutte PM, Verbeke M, Lameire NH. The impaired renal vasodilator response attributed to endothelium derived hyperpolarizing factor in streptozotocin induced diabetic rats is restored by 5 methyltetrahydrofolate. Diabetologia 2000;43:1116 1125. 29. Colombaro V, Jadot I, Decleves AE, Voisin V, Giordano L, Habsch I, et al. Lack of hyaluronidases exacerbates renal post ischemic injury, inflammation, and fibrosis. Kidney Int 2015. 30. Taylor MS, Bonev AD, Gross TP, Eckman DM, Brayden JE, Bond CT, et al. Altered expression of small conductance Ca2+ activated K+ (SK3) channels modulates arterial tone and blood pressure. Circ Res 2003;93:124 131. 31. Brahler S, Kaistha A, Schmidt VJ, Wolfle SE, Busch C, Kaistha BP, et al. Genetic deficit of SK3 and IK1 channels disrupts the endothelium derived hyperpolarizing factor vasodilator pathway and causes hypertension. Circulation 2009;119:2323 2332. 32. Zhu JH, Jia RP, Xu LW, Wu JP, Wang ZZ, Wang SK, et al. Reduced expression of SK3 and IK1 channel proteins in the cavernous tissue of diabetic rats. Asian J Androl 2010;12:599 604. 33. Zuurbier CJ, Demirci C, Koeman A, Vink H, Ince C. Short term hyperglycemia increases endothelial glycocalyx permeability and acutely decreases lineal density of capillaries with flowing red blood cells. J Appl Physiol (1985) 2005;99:1471 1476. 34. Gouverneur M, Spaan JA, Pannekoek H, Fontijn RD, Vink H. Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx. Am J Physiol Heart Circ Physiol 2006;290:H458 452. 35. Rubio Gayosso I, Platts SH, Duling BR. Reactive oxygen species mediate modification of glycocalyx during ischemia reperfusion injury. Am J Physiol Heart Circ Physiol 2006;290:H2247 2256. 36. Chappell D, Hofmann Kiefer K, Jacob M, Rehm M, Briegel J, Welsch U, et al. TNF alpha induced shedding of the endothelial glycocalyx is prevented by hydrocortisone and antithrombin. Basic Res Cardiol 2009;104:78 89. 37. Yung S, Chau MK, Zhang Q, Zhang CZ, Chan TM. Sulodexide decreases albuminuria and regulates matrix protein accumulation in C57BL/6 mice with streptozotocin induced type I diabetic nephropathy. PLoS One 2013;8:e54501. 38. Packham DK, Wolfe R, Reutens AT, Berl T, Heerspink HL, Rohde R, et al. Sulodexide fails to demonstrate renoprotection in overt type 2 diabetic nephropathy. J Am Soc Nephrol 2012;23:123 130. Diabetes Page 20 of 32
39. Becker BF, Jacob M, Leipert S, Salmon AH, Chappell D. Degradation of the endothelial glycocalyx in clinical settings: searching for the sheddases. Br J Clin Pharmacol 2015;80:389 402. 40. Pohl U, Herlan K, Huang A, Bassenge E. EDRF mediated shear induced dilation opposes myogenic vasoconstriction in small rabbit arteries. Am J Physiol 1991;261:H2016 2023. 41. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, Tarbell JM. Heparan sulfate proteoglycan is a mechanosensor on endothelial cells. Circ Res 2003;93:e136 142. 42. Salmon AH, Satchell SC. Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability. J Pathol 2012;226:562 574. 43. Eskens BJ, Zuurbier CJ, van Haare J, Vink H, van Teeffelen JW. Effects of two weeks of metformin treatment on whole body glycocalyx barrier properties in db/db mice. Cardiovasc Diabetol 2013;12:175. 44. Bourguignon V, Flamion B. Respective roles of hyaluronidases 1 and 2 in endogenous hyaluronan turnover. FASEB J 2016 (in press). 45. Nagy N, Freudenberger T, Melchior Becker A, Rock K, Ter Braak M, Jastrow H, et al. Inhibition of hyaluronan synthesis accelerates murine atherosclerosis: novel insights into the role of hyaluronan synthesis. Circulation 2010;122:2313 2322. 46. Nagasu H, Satoh M, Kiyokage E, Kidokoro K, Toida K, Channon KM, et al. Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice. Lab Invest 2016;96:25 36. 47. Kumase F, Morizane Y, Mohri S, Takasu I, Ohtsuka A, Ohtsuki H. Glycocalyx degradation in retinal and choroidal capillary endothelium in rats with diabetes and hypertension. Acta Med Okayama 2010;64:277 283. 48. Nakazawa T, Kaneko Y, Mori A, Saito M, Sakamoto K, Nakahara T, et al. Attenuation of nitric oxide and prostaglandin independent vasodilation of retinal arterioles induced by acetylcholine in streptozotocin treated rats. Vascul Pharmacol 2007;46:153 159. 49. Liao YJ, Ueno M, Nakagawa T, Huang C, Kanenishi K, Onodera M, et al. Oxidative damage in cerebral vessels of diabetic db/db mice. Diabetes Metab Res Rev 2005;21:554 559. 50. Heickendorff L, Ledet T, Rasmussen LM. Glycosaminoglycans in the human aorta in diabetes mellitus: a study of tunica media from areas with and without atherosclerotic plaque. Diabetologia 1994;37:286 292.
TABLES
Table 1. Beneficial effects observed in HYAL1 KO mice exposed to streptozotocin induced diabetes as compared with WT mice.
Diabetic Diabetic Likely mechanism of protection in absence of
WT KO HYAL1 Effect of diabetes on: a) endothelium
Plasma P selectin (↑) Endothelium is less activated Page 21 of 32 Diabetes
Plasma ICAM1 (↑) No protection *
Plasma VCAM1 ( ) No effect of diabetes *
EDH vasorelaxation (↓) Higher baseline SK3 or thicker glycocalyx
SK3 ( ) No effect of diabetes *
b) glycocalyx
Glycocalyx HA (↓) Glycocalyx HA is more adherent
Glycocalyx size ( ) No effect of diabetes *
Syndecan1 shedding ( ) No effect of diabetes *
c) Fonctionnal glomerular barrier
Urinary A/C ratio (↑) Preserved integrity of glomerular glycocalyx
Urinary 70/40kDa dextran ratio (↑) Preserved integrity of glomerular glycocalyx
WT, wild type; KO, knockout; ICAM1, intercellular cell adhesion molecule 1; VCAM1, vascular cell adhesion
molecule 1; EDH, endothelium derived hyperpolarization; SK3, small conductance potassium channel 3; A/C,
albumin/creatinine.
* Soluble ICAM1, VCAM1 were lower, while SK3 expression was higher and glycocalyx was thicker, in HYAL1 KO
mice than in WT mice.
(↑) indicates that a significant increase in this parameter was measured 4 weeks after streptozotocin induced diabetes;
(↓), a significant decrease; and ( ), the absence of change. Diabetes induced injuries in WT mice are indicated by a
black rectangle, and the absences of change are indicated by a hatched rectangle. A black rectangle staying black in
the « KO » column means no benefit of HYAL1 deficiency; a black rectangle becoming white in the « KO » column
means a significant protection afforded by HYAL1 deficiency.
FIGURE LEGENDS
Figure 1. Effects of diabetes on glycemia, body weight and arterial pressure. A. Fasting
glycemia (box and whisker plots) during diabetes induction (streptozotocin [STZ] injections)
vs control conditions (citrate buffer injections). N= 7 to 36 in each group of wild type (WT)
and HYAL1 knockout (KO) mice without or with diabetes. Diabetic mice include only those
with fasting glycemia ≥300 mg/dL. B. Body weight tracking before, during, and 28 days after Diabetes Page 22 of 32
STZ or citrate buffer injections (n= 16 to 36 in each group; means ± SEM). C. Mean arterial pressure (means ± SEM) 28 days after STZ or citrate buffer treatment (n= 3 to 5 in each group). In all data sets, statistical significance was calculated using 2 way ANOVA and
Bonferroni post tests (# p<0.05, ### p<0.001, #### p<0.0001 within genotype).
Figure 2. Plasma hyaluronan (HA) concentration and hyaluronidase activity. A. Plasma
HA concentrations of WT and KO mice without or with STZ induced diabetes (n= 4 or 5 in each group). B D. Serum (B C) and aortic wall (D) hyaluronidase activity measured using zymography under native (B & D) or denaturing (C) conditions in each experimental group.
Contrary to denaturing conditions, the native procedure does not allow to determine the molecular weight of the HA degrading enzyme. E H. Measurement of HA oligosaccharide production by serum hyaluronidase activity using polyacrylamide gel electrophoresis. E.
Rooster comb HA (5 µg) was incubated for 5 h with 1 µl of serum obtained from 2 animals of each group (lanes 1 8). The serum of KO mice (lanes 5 8) and undigested HA (lane 10) served as negative controls. HA incubated with 5U bovine testes hyaluronidase during 2 h
(lane 9) served as positive control. F. A gradient of bovine testes hyaluronidase at concentrations ranging of from 0 (lane 1) to 5 U (lane 5), incubated for 2 h with the same amount of HA, was also performed for comparison. G H. Quantification of serum hyaluronidase activity: Polyacrylamide gel electrophoresis of HA samples incubated with serum of WT mice without (lanes 1 4) and with (lanes 5 8) diabetes and used for quantification of hyaluronidase activity. The negative and positive controls used in G are similar to those used in E. (H) Quantification of hyaluronidase activity: signal intensity measurements for successive and sufficiently distinct oligosaccharide bands (i.e. below the dashed line in G). Statistical analysis uses 2 way ANOVA (# and §, p<0.05; **, p<0.01; §§§§ and ****, p<0.0001). Page 23 of 32 Diabetes
Figure 3. Flushable HA in aortas of diabetic and non-diabetic WT and Hyal1 KO mice
and structural evaluation of the endothelial glycocalyx. A. HA content of aortic
homogenates (n= 5 to 7 in each group) standardized to dry weight before (white columns) and
after (black columns) saline flushing. Statistical analysis was performed using 2 way
ANOVA and Bonferroni post tests to compare HA content before vs after flushing in each
group (####, p<0.0001). B C. Representative transmission electronic microscopy (TEM)
images of myocardial arterioles of WT(B) and KO (C) mice without or with STZ induced
diabetes following perfusion of the heart with Alcian Blue 8GX, in which the thickness of the
glycocalyx can be appreciated. Bars correspond to 1µm. D. Evaluation of glycocalyx
thickness obtained by dividing the glycocalyx surface area by the endothelium length
measured using ImageJ. N= 3 to 4 mice in each group, corresponding to approximately 50
TEM images of microvessels (≥8µm in diameter) in each group. Data are means ± SEM of
each experimental group. Statistical analysis was performed using 2 way ANOVA (**,
p<0.01). E. Plasma syndecan 1 levels in each experimental group (mean ± SEM, N = 7 10 in
each group). Statistical analysis was performed using 2 way ANOVA (N.S).
Figure 4. Functional status of endothelial glycocalyx. A: Ratio of 70/40 kDa dextran in
urine 30 min after intrajugular injection of a 1:4 mixture of 40 and 70 kDa dextrans in WT
(n=17) and KO (n=8) mice without or with diabetes. Statistical analysis was performed using
2 way ANOVA (*, p<0.05) and Bonferroni post tests within genotypes (##, p<0.01). B:
Albumin/creatinine ratio measured in the same urine samples. Statistical analysis was
performed using 2 way ANOVA (*, p<0.05) and Bonferroni post tests within genotypes (#,
p<0.05) or across genotypes (§, p<0.05).
Diabetes Page 24 of 32
Figure 5. Endothelial injury markers. Evaluation of three markers of endothelial damage in
WT and KO mice before and after STZ induced diabetes (n=5 to 8 in each group). A. Serum soluble P selectin (sP selectin) levels. B. Plasma soluble ICAM1 (sICAM1) levels. C. Plasma soluble VCAM1 (sVCAM1) levels. Data are means ± SEM. Statistical analysis was performed using 2 way ANOVA (*, p<0.05; **, p<0.01; ****, p<0.0001) and Bonferroni post tests inside genotype (#, p<0.05; ##, p<0.01; ###, p<0.001) or across genotype (§, p<0.05).
Figure 6. Endothelium-derived hyperpolarization (EDH)-mediated vasodilation.
Mesenteric arteriolar rings of WT and KO mice without or with STZ induced diabetes (n=24 to 26 rings in each group) were pre constricted with 3.10 6 M phenylephrine in the presence of
10 4 M L NAME and 10 5 M indomethacin. Vasodilatation was then induced using increasing concentrations (10 8 M to 3.10 5 M) of acetylcholine. Data are means ± SEM. Statistical analysis, 2 way ANOVA (****, p<0.0001).
Figure 7. Expression and activity of SK3 channels in mesenteric arteries. A: SK3 relative mRNA expression in mesenteric arteries of WT and KO mice, without or with STZ induced diabetes (normalized to β actin, n=8 to 10 in each group). Data are means ± SEM. Statistical analysis was performed on ∆CT values using 2 way ANOVA (***, p<0.001) and Bonferroni post tests across genotypes (§, p<0.05; §§, p<0.01). B: SK3 and von Willebrand factor (vWF) immunostaining in mesenteric arteries of WT and KO mice without and with STZ induced diabetes. C: Quantification of SK3 immunostaining using ImageJ (n=9 to 12 vessel sections in each group). Data are means ± SEM. Statistical analysis was performed using 2 way ANOVA
(***, p<0.01) and Bonferroni post tests across genotypes (§, p<0.05; §§, p<0.01). D: Residual contraction after CYPPA activation of SK3 channels. Mesenteric rings of WT and KO mice Page 25 of 32 Diabetes
without or with STZ induced diabetes (n=10 to 12 in each group) were pre constricted with