Time Course and Contributors to Greater Glycocalyx Thickness and Integrity Following Western Diet Consumption

Abstract

The endothelial glycocalyx is a gel-like structure on the endothelium that plays a critical role in vasculature. We have shown that 12 weeks of a Western diet (WD) increases glycocalyx thickness and integrity. However, the time course and contributors to these adaptations are unknown. After 1, 2, 4, and 12 weeks of WD, we observed greater glycocalyx thickness and integrity at all time points in WD mice compared to age-matched control diet-fed mice. Hyaluronan is a major component of the glycocalyx that contributes to its structural and functional integrity. Acute hyaluronidase administration eliminated elevations in glycocalyx thickness in mice following one week of WD, while having no effect on glycocalyx thickness in control diet-fed mice. Hyaluronidase administration increased perfused boundary region (PBR), a glycocalyx integrity marker, in both WD and control diet-fed mice, but more so in WD mice, eliminating group differences in PBR. Lastly, WD blunted acetylcholine-mediated vasodilation in carotid arteries, indicating endothelial dysfunction. Interestingly, flow-mediated vasodilation was preserved at low flow rates in WD-fed mice, yet at the highest flow rate vasodilation was blunted. Greater glycocalyx thickness in WD mice may mechanotransduce more shear stress at a given flow rate, preserving flow-mediated vasodilation at lower flow rates, but prematurely blunting vasodilation as flow rate increases. Flow-mediated vasodilation was similarly blunted in the presence of intraluminal hyaluronidase in WD and control diet-fed mice. Taken together, these findings demonstrate that WD-induced elevations in glycocalyx properties occur after one week and appear to be dependent on hyaluronan content in the glycocalyx.

NEW & NOTEWORTHY

We observed greater glycocalyx thickness and integrity starting as early as one week after mice began a Western diet. Greater glycocalyx thickness was dependent on the hyaluronan content in the glycocalyx. Differences in glycocalyx integrity between control and Western diet-fed mice were eliminated by hyaluronidase. In isolated arteries, agonist-mediated vasodilation was impaired in Western diet-fed mice, while flow-mediated vasodilation was partially preserved, suggesting a thicker glycocalyx may compensate for endothelial dysfunction in Western diet-fed mice.

INTRODUCTION

Cardiovascular disease (CVD) is the leading cause of death in the United States (1). A major contributor to the CVD progression is vascular dysfunction that is believed to be preceded by a depleted or dysfunctional glycocalyx (2–4). The glycocalyx is a gel-like layer lining the luminal surface of endothelium (5). Hyaluronan is one of the primary glycosaminoglycans in the glycocalyx, which is critical to its structural and functional integrity (6, 7). Functionally, the glycocalyx modulates the vascular permeability, microcirculatory homeostasis, and flow homogeneity, while mechanotransducing shear stress from blood flow to endothelium to induce vasodilation (8–10). Thus, targeting the endothelial glycocalyx has become a therapeutic strategy to ameliorate vascular dysfunction and prevent CVD (11–13).

Western diet (WD), characterized by high fat and sugar content, is a prominent dietary risk factor for CVDs and diabetes (14). Glycocalyx depletion has been observed in patients with diabetes (15–17), as well as in mouse models of diabetes (18–20). However, there are conflicting results showing deterioration (21, 22), no change (23), or enhancement (24, 25) of the glycocalyx in response to a WD in mice. Previously, our laboratory demonstrated that 12 weeks of WD resulted in greater glycocalyx thickness and integrity compared to mice fed a control diet, indicating seemingly beneficial microcirculatory adaptations to WD (25), although vascular function was impaired in WD-fed mice (26). Currently, the time course and contributors to WD-induced elevations in glycocalyx thickness and integrity remain unknown. Thus, in the current study, we sought to determine the time course of adaptation in glycocalyx properties in response to a WD. There are several glycosaminoglycans in the glycocalyx such as hyaluronan, heparan sulfate, and chondroitin sulfate that contribute to its glycocalyx integrity, as well as its ability to mechanotransduce shear stress. Our group has primarily focused on hyaluronan (12, 27), and we have previously shown that age-related reductions in hyaluronan synthase 2 (Has2) are accompanied by glycocalyx depletion (28) and that supplementation with hyaluronan restores the glycocalyx in old mice (3). Thus, to further investigate the potential mechanisms underlying WD-induced elevations in glycocalyx thickness and integrity, we sought to determine the role of hyaluronan content in the glycocalyx of WD-fed mice.

METHODS

Ethical approval

All animal procedures conformed to the Guide to the Care and Use of Laboratory Animals: Eighth Edition (National Research Council. 2011) and were approved by the Florida State University Animal Care and Use Committee.

Animals

Male and female UM-HET3 mice were housed in standard mouse cages under a 12:12 light:dark cycle in a temperature-controlled environment. Upon weaning at ~21 days old, mice were fed standard normal chow diet (NC) (LabDiet #5001; protein: 28.5%, carbohydrate: 58.0%, fat: 13.5% by kcal). At 3 mo old, mice continued on NC or were fed a WD (LabDiet #5TJN; protein: 15.8%, carbohydrate: 45.1%, fat: 39.1% by kcal) for 1, 2, 4 or 12 weeks. Food and water were supplied ad libitum in group housed cages. Animals were studied in nonfasted conditions. Animals that were administered hyaluronidase (Sigma, type IV-S) received it via retro-orbital injection at a dose of 35 units in 100 μl 0.9% saline with measurements occurring 60 min after injection.

Intravital Microscopy

The mesenteric microcirculation was observed using intravital microscopy in mice anesthetized with isoflurane (3%) in room air at 100 ml/min flow rate, as described previously (3, 25, 28).

Glycocalyx Thickness and Integrity

Manual determination of glycocalyx thickness was performed by measuring the change in width of flowing red blood cell (RBC) column before and after the passage of a spontaneous white blood cell in individual capillaries, as described previously (29). Perfused boundary region (PBR) represents the depth of penetration of RBCs into the endothelial glycocalyx and is taken as a marker of glycocalyx integrity, with a larger PBR indicating worse glycocalyx integrity. PBR is calculated using the following equation:

$$\text{PBR}=\frac{\text{(maximal perfused diameter}-\text{median perfused diameter)}}{\text{2}}$$

Microvascular Density

The automated analysis algorithm identifies perfused microvessel segments as any 10 μm segment with sufficient contrast that contains RBCs in ≥50% of their length in the first frame of a video recording. From this, microvascular density is calculated using the following equation:

$$\text{microvascular density}=\frac{\text{(number of 4–25 μm perfused microvessel segments}\times 10\mu \text{m})}{\text{tissue area recorded}}$$

Microvascular density includes length of all perfused microvessel segments with diameters 4–25 μm, encompassing capillaries, arterioles, and venules, relative to the tissue area recorded, and is expressed as μm/mm².

Capillary Density

The automated analysis algorithm calculates capillary density similar to microvascular density using the following equation:

$$\text{capillary density}=\frac{(\text{number of 4–6 μm perfused microvessel segments}\times 10\mu \text{m})}{\text{tissue area recorded}}$$

Capillary density includes length of all perfused microvessel segments with diameters 4–6 μm, encompassing capillaries, relative to the tissue area recorded, and is expressed as μm/mm².

Red Blood Cell Velocity

RBC velocities are determined in individual vessel segments by cross correlation of longitudinal RBC intensity profiles between frames of recorded videos and a correlation coefficient of 0.80 or more was required to ensure accurate estimates of longitudinal RBC displacement (30). RBC velocity is determined using the following equation:

$$\text{RBC Velocity}=\frac{\text{RBC displacement}}{\text{Time between consecutive video frames}}$$

Ex Vivo Arterial Function

To assess endothelium-dependent dilation, carotid arteries were cannulated in the stage of a pressure myograph (DMT Inc, Hinnerup, Denmark), and perfused with physiological salt solution, as described previously (31). Arteries were submaximally pre-constricted with 2 μM phenylephrine and vasodilation was measured in response to the cumulative addition of acetylcholine (1×10⁻¹⁰ to 1×10⁻⁴ M) in the absence or presence of the nitric oxide (NO) synthase inhibitor, L-NAME (0.1 mM, 30 min), as described previously (32). Vasodilation was also measured in response to changes in intraluminal flow were obtained by exposing carotid arteries to graded increases in intraluminal flow without changes to intraluminal pressure, as previously described (33, 34). Diameters were measured in response to incremental pressure differences of 4, 10, 20, and 40 cm H²O in the absence or presence of L-NAME or hyaluronidase (intralumenal; 14 μg/ml). Endothelium-independent dilation was assessed in response to the cumulative addition of sodium nitroprusside (1×10⁻¹⁰ to 1×10⁻⁴ M). Luminal diameters were measured by VasoTracker software (35).

Statistical Analysis

Statistical tests were performed with GraphPad Prism (GraphPad Software Inc, San Diego, CA). Two- and three-way mixed model ANOVAs were used to evaluate the effect of Sex × Group or Sex × Group × Time/Concentration, respectively. When a significant ANOVA was present, the two-stage step-up method of Benjamini, Krieger, and Yekutieli was used to identify values that were significantly different. Statistical significance was set at P<0.05 for all analyses. Data are presented as means±SEM.

RESULTS

Time Course of Glycocalyx Adaptations to WD

At each time point across the 12-week dietary treatment, glycocalyx thickness was greater in WD compared to NC mice (Figure 1A; P<0.05 for all). WD mice also had lower PBR compared to NC mice at each time point across the 12-week dietary treatment (Figure 1B; P < 0.05 for all). There were no differences in PBR or glycocalyx thickness across the time points within the diet groups (P>0.05 for all). Microvascular density was not different between NC and WD groups (Table 1; P>0.05). While there was a tendency for microvascular density to decrease with advancing age in NC mice, reaching statistical significance in NC mice between weeks 1 and 12 (Table 1; P<0.05). Over the entire 12-week time period, WD mice had greater capillary density (4–6 μm diameter microvessel segments) compared to NC mice at each time point (Figure 1C; P<0.05 for all). Capillary density was lower with increasing age in both NC and WD mice. In NC mice, capillary density was lower at weeks 4 and 12 compared to week 1 and at week 12 compared to week 2, respectively (P<0.05 for all). In WD mice, capillary density was lower at weeks 4 and 12 compared to weeks 1 and 2 (P<0.05 for all). Microvascular density at each microvessel segment diameter in NC and WD are presented in Figure 1D (week 1), Figure 1E (week 2), Figure 1F (week 4), and Figure 1G (week 12). At each time point, density at 4, 5, and 6 μm was greater in WD compared to NC mice (P<0.05). Additionally, at week 12, density was also greater at 7, 8, 9, and 10 μm in WD compared to NC mice (P<0.05). RBC velocity was similar between NC and WD mice at each time point (Table 1; P>0.05 for all), although there was a statistical difference in NC mice between weeks 2 and 12 (P<0.05). There were no differences in nonfasted blood glucose at 1-week (NC: 175±6 vs. WD: 175±3 mg/dL; P>0.05).

Figure 1.

Comparison of normal chow (NC)- and Western diet (WD)-treated mice at 1-, 2-, 4-, and 12-week time points using one-way ANOVA to determine differences in glycocalyx thickness (A; n = 11–20 mice/group), perfused boundary region (PBR; B; n = 16 mice/group), and capillary density (4–6 μm; C; n = 16 mice/group). Three-way mixed model ANOVA to determine differences in microvascular density at 1- (D; n= 16 mice/group), 2- (E; n= 16 mice/group), 4- (F; n= 16 mice/group), and 12-week (G; n= 16 mice/group) timepoints in NC- and WD-treated mice. When a significant ANOVA was present, the two-stage step-up method of Benjamini, Krieger, and Yekutieli was used to identify group differences. *P<0.05 vs. NC within the same time point. †P<0.05 vs. Week 1 within the diet group. ‡P<0.05 vs. Week 2 within the same diet group. Data are individual values (males = circles, females = triangles) and/or means±SEM.

Table 1.

Animal Characteristics

	1 Week		2 Weeks		4 Weeks		12 Weeks
	NC	WD	NC	WD	NC	WD	NC	WD	Group
Male/Female	8/8	8/8	8/8	8/8	8/8	8/8	8/8	8/8
Age, mo	3.3±0.0	3.4±0.1	3.4±0.0	3.5±0.1	3.9±0.0†‡	3.9±0.1†‡	6.4±0.1†‡§	6.5±0.1†‡§	<0.001
Body mass, g	26.2±1.0	29.1±1.5	28.8±1.5	34.2±1.5	31.1±1.5	35.3±1.8†	30.3±1.4	40.6±2.8*†‡	<0.001
Microvascular density, μm/mm2	2191±215	2098±166	1763±166	1842±177	1440±88	1629±158	1321±87†	1758±122	0.001
RBC velocity, μm/s	102±5	107±5	113±6	105±5	105±4	101±4	92±5‡	94±3	0.030

Values are means±SEM. Data were analyzed using one-way ANOVA with the two-stage step-up method of Benjamini, Krieger, and Yekutieli to identify differences. NC, normal chow; RBC, red blood cell; WD, Western diet.

^*P<0.05 vs. NC within diet length group.

^†P<0.05 vs. 1 Weeks within diet group.

^‡P<0.05 vs. 2 Weeks within diet group.

^§P<0.05 vs. 4 Weeks within diet group.

Role of Hyaluronan on Glycocalyx Adaptations to Western Diet

Glycocalyx thickness was greater in WD compared to NC mice at baseline (Figure 2A; P<0.05), but not post-hyaluronidase (P>0.05). Hyaluronidase injection decreased glycocalyx thickness in WD mice (P<0.05), but did not change glycocalyx thickness in NC mice (P>0.05). PBR was lower in WD compared to NC mice at baseline (Figure 2B; P<0.05). Hyaluronidase injection increased PBR in both NC and WD mice compared to baseline (P<0.05 for both), although the increase was seemingly larger in WD mice, as PBR was not different between NC and WD mice post-hyaluronidase (P>0.05). Capillary density was greater in WD compared NC mice at baseline (Figure 2C; P<0.05), but not post-hyaluronidase (P>0.05). Microvascular density at each microvessel segment diameter between conditions are presented in Figure 2D (NC baseline vs. WD baseline), Figure 2E (NC post-hyaluronidase vs. WD post-hyaluronidase), Figure 2F (NC baseline vs. NC post-hyaluronidase), and Figure 2G (WD baseline vs. WD post-hyaluronidase). There were differences in density of the smaller microvessel segments between NC and WD mice at baseline (4 to 9 μm) and post-hyaluronidase (5 to 8 μm), respectively (P<0.05 for all). Hyaluronidase lowered the density of several microvessel segments in NC (5 to 15 μm) and WD (4 to 14 μm) mice (P<0.05 for all). RBC velocity was similar between NC and WD mice at baseline and post-hyaluronidase, although there was a tendency for RBC velocity to increase post-hyaluronidase, this difference did not reach statistical significance (NC baseline: 120±9; NC post-hyaluronidase: 140±10; WD baseline: 113±5; WD post-hyaluronidase: 128±7 μm/sec; P>0.05 for all).

Figure 2.

Comparison of normal chow (NC)- and Western diet (WD)-treated mice at baseline and 1-hour post-hyaluronidase (HAase) administration using two-way repeated measured ANOVA to determine differences in glycocalyx thickness (A; n = 8 mice/group), perfused boundary region (PBR; B; n = 12 mice/group), and capillary density (4–6 μm; C; n = 12 mice/group). Three-way mixed model ANOVA to determine differences in microvascular density between NC baseline vs. WD baseline (D; n= 12 mice/group), NC post-HAase vs. WD post-HAase (E; n= 12 mice/group), NC baseline vs. NC post-HAase (F; n= 12 mice/group), and WD baseline vs. NC post-HAase (G; n= 12 mice/group). When a significant ANOVA was present, the two-stage step-up method of Benjamini, Krieger, and Yekutieli was used to identify group differences. *P<0.05 vs. NC within the same time point. †P<0.05 vs. baseline within the same diet group. ‡P<0.05 vs. Week 2 within the diet group. Data are individual values (males = circles, females = triangles) and/or means±SEM.

Role of Hyaluronan on Endothelial Function

There was an effect of diet treatment on flow-mediated vasodilation, indicating greater vasodilation in NC compared to WD mice (Figure 3A; P<0.05). Indeed, flow-mediated vasodilation was greater at Δ40 cmH₂O pressure gradient in NC compared to WD mice (P<0.05), although vasodilation was similar between NC and WD mice at Δ4, Δ8, and Δ20 cmH₂O pressure gradients (P>0.05). Compared to basal condition, there was also an effect of hyaluronidase or L-NAME on flow-induced vasodilation in NC mice (P<0.05), while no difference was present between hyaluronidase or L-NAME treatments compared to basal condition in WD mice (P>0.05). There was an effect of diet treatment on acetylcholine-mediated vasodilation, indicating greater vasodilation in NC compared to WD mice (Figure 3B; P<0.05). Indeed, acetylcholine-mediated vasodilation was greater at 1×10⁻⁶, 1×10⁻⁵, and 1×10⁻⁴ M (P<0.05). There was also an effect of L-NAME on acetylcholine-mediated vasodilation in NC and WD mice (P<0.05). There were no differences in sodium nitroprusside-mediated vasodilation between groups (NC: 91±1 vs. WD: 91±2%, P>0.05).

Figure 3.

Three-way mixed model ANOVA to determine differences in carotid artery vasodilation to intraluminal flow at each pressure gradient (A; n = 6–8 mice/group) and vasodilation to acetylcholine (B; n = 7–10 mice/group) between (NC)- and Western diet (WD)-treated mice. When a significant ANOVA was present, the two-stage step-up method of Benjamini, Krieger, and Yekutieli was used to identify group differences. *P<0.05 vs. NC within the same pressure gradient or concentration of acetylcholine. Data are means±SEM.

DISCUSSION

In the present study, we identified that glycocalyx thickness becomes greater and less penetrable to RBCs (i.e., PBR) as early as 1 week after beginning a WD. Glycocalyx thickness and integrity in WD mice remain greater than NC mice at 2, 4, and 12 week timepoints. To investigate the contribution of hyaluronan in the WD-induced adaptations to the glycocalyx, we found that hyaluronidase administration lowered glycocalyx thickness and integrity in WD mice, abolishing differences in these outcomes between NC and WD mice. While hyaluronidase administration did not affect glycocalyx thickness in NC mice, it did worsen glycocalyx integrity. Lastly, we examined ex vivo arterial function in isolated carotid arteries and found that flow-mediated vasodilation was similar between NC and WD at lower flow rates, but as flow rate increased, flow-mediated vasodilation was impaired in WD-fed mice. In the presence of hyaluronidase or L-NAME, flow-mediated vasodilation was similarly blunted in NC and WD mice. Additionally, acetylcholine-mediated vasodilation was also blunted in WD compared to NC mice. Collectively, these findings suggest that WD rapidly augments glycocalyx properties by increasing hyaluronan in the glycocalyx. While acetylcholine-mediated vasodilation is blunted in WD-fed mice, flow-mediated vasodilation is only blunted at higher flow rates, suggesting that the apparent improvement in glycocalyx thickness and integrity may compensate for WD-induced endothelial dysfunction at lower flow rates.

Greater Glycocalyx Thickness and Integrity Occurs After 1 Week of Western Diet

We have previously shown that 12 weeks of WD results in greater glycocalyx thickness and integrity (25). In the present study, we sought to determine when these adaptations occur. To do so, we fed mice a WD for 1, 2, 4, or 12 weeks. We observed greater glycocalyx thickness and lower PBR in each WD group compared to age-matched NC mice. Glycocalyx thickness and PBR were similar between WD groups, indicating that changes in glycocalyx properties occur within 1 week of beginning WD and do not appear to change thereafter. In the few studies that have examined the adaptations of the glycocalyx to a WD there have been conflicting results showing deterioration (21, 22), no change (23), or enhancement (24, 25) of the glycocalyx in response to a WD in mice. Many of these studies used different types of WD and mouse strains, and each study examined the glycocalyx in different vascular beds. The glycocalyx is viewed as a dynamic and heterogeneous structure with various thickness in capillary, small arteries, and conduit arteries (36). In our previous study that demonstrated an age-related reduction in glycocalyx properties, there was a tendency for glycocalyx properties to differ between gastrocnemius and mesenteric microcirculations within the same animal (28). Thus, it is possible that tissue-specific differences in glycocalyx properties may exist, as a result of local tissue-specific adaptations to a given perturbation. Nevertheless, future studies are warranted to determine if different vascular beds have differing adaptations to a WD.

Hyaluronan Contributes to a Thicker and Less Penetrable Glycocalyx in Western Diet-Fed Mice

After determine the time course that glycocalyx adaptation occur in response to a WD, we next sought to examine the contribution of hyaluronan in the glycocalyx of WD-fed mice. Hyaluronan is one of the primary glycosaminoglycans in the glycocalyx (6, 7). The functionality of hyaluronan in the glycocalyx is dependent on its molecular weight, as high molecular weight-hyaluronan (HMW-HA) (>1 MDa) is considered favorable to the glycocalyx (12). We have shown that age-related glycocalyx degradation and arterial dysfunction are accompanied by lower arterial Has2 gene and protein expression (28, 37). HMW-HA in the glycocalyx can be degraded by acute infusion of hyaluronidase (6). Thus, we next examined glycocalyx properties in WD-fed mice before and after infusion of hyaluronidase. We found that hyaluronidase infusion lowered glycocalyx thickness in WD mice, but did not affect glycocalyx thickness in NC mice. On the contrary, hyaluronidase infusion caused a similar increase in PBR in NC and WD mice. Importantly, differences in glycocalyx thickness and PBR in WD mice were abolished after hyaluronidase treatment. It is unclear why hyaluronidase infusion increased PBR in both NC and WD mice, but only decreased glycocalyx thickness in WD mice. In the initial study that used hyaluronidase to degrade the glycocalyx (6), no difference in permeation of the glycocalyx to 580 and 2,000 kDa FITC-dextrans were present after hyaluronidase infusion in the hamster cremaster microcirculation. However, there was an increase in ability of 70 and 145 kDa FITC-dextrans to freely flow through the glycocalyx. Others have shown that hyaluronidase decreases hyaluronan in the glycocalyx (38), resulting in a reduction in glycocalyx volume in mice (39, 40). In the endothelial glycocalyx, hyaluronan is bound to CD44 and interweaved into other components like heparin sulfate and chondroitin sulfate (27, 41). In absence of a WD-induced increase in heparin sulfate or chondroitin sulfate, excess hyaluronan in the glycocalyx may protrude deeper into the vascular lumen. Thus, when hyaluronidase is infused, we observe a reduction in glycocalyx thickness and an increase in PBR in WD-fed mice. However, had there been an increase in heparin sulfate and chondroitin sulfate we may not have observed a reduction in glycocalyx thickness in WD-fed mice. Collectively, these findings indicate that hyaluronan plays a major role in glycocalyx integrity, but it is unclear whether it contributes to overall glycocalyx thickness. One possibility is that the WD used in our study results in excess hyaluronan integration in the glycocalyx, which increases glycocalyx thickness. Future studies are warranted to determine the precise role that hyaluronan plays in this apparent WD-induced increase in glycocalyx thickness as well as in other vascular beds.

Impact of WD-induced glycocalyx augmentation on endothelial function

The glycocalyx mechanotransduces blood flow-induced shear stress, stimulating endothelium-dependent vasodilation via endothelial nitric oxide synthase-produced nitric oxide (42–44). Several classic features of CVD, such as elevated oxidative stress and inflammation and reduced nitric oxide bioavailability are linked to a depleted glycocalyx (10, 45–47). In the present study, it appeared that the thicker glycocalyx of WD mice had greater hyaluronan content. Hyaluronan plays a critical role in the ability of the glycocalyx to stimulate flow-mediated vasodilation, as acute hyaluronidase incubation decreases nitric oxide production to increases in flow (42). It is important to note that hyaluronidase does not affect acetylcholine-mediated nitric oxide production (42). Therefore, to study the functional role of WD-induced increases in glycocalyx thickness and integrity on endothelial function, we examined flow- and acetylcholine-mediated vasodilation. Similar to our previous study (26), acetylcholine-mediated vasodilation was blunted in WD mice and was accompanied by lower nitric oxide bioavailability. Interestingly, while flow-mediated vasodilation was similar in NC and WD mice at lower flow rates, as the pressure differential increased, flow-mediated vasodilation was blunted in WD mice. We typically observe the greatest flow-mediated vasodilation at Δ4 to Δ10 cmH²O with a plateau or slight reduction in vasodilation at greater pressure differentials (34). We do not go beyond Δ40 cmH²O due to significant vasoconstriction that has been observed at Δ60 cmH²O in carotid arteries (unpublished observations). Carotid artery constriction to higher flow rates has been shown in rats (48). A blunting of flow-mediated vasodilation has also been observed in other arteries (49–51). Thus, it is possible that high flow-mediated constriction in the carotid artery is a protective mechanism to limit excess brain perfusion similar to observations of flow-mediated constriction in cerebral arteries (51, 52). In the present study, greater glycocalyx thickness in WD may allow for a greater mechanotransduction of shear stress to endothelial cells at a given flow rate. Because nitric oxide production has been shown to be linearly related to flow rate (42), the ability of a thicker glycocalyx to mechanotransduce a greater shear stress signal may compensate for WD-induced endothelial dysfunction by producing more nitric oxide at a given flow rate. However, there are likely competing mechanisms that govern the balance of vasodilator and vasoconstrictor response to increases in flow that are dependent on artery size and type. Indeed, nitric oxide production is similar in response to flow and acetylcholine (42), yet flow-mediated vasodilation is usually lower than acetylcholine-mediated vasodilation in isolated arteries (34, 53–57). Moreover, the disparity in flow- and acetylcholine-mediated vasodilation appears to be greater in larger arteries like the carotid artery (34, 53). It is possible that the protective mechanisms that limit flow-mediated vasodilation at the highest flow rates may be responsible for vasoconstriction observed in WD mice at Δ40 cmH²O, which did not occur in NC mice.

Perspectives

In the present study, changes in glycocalyx thickness were also accompanied by a leftward shift in microvascular density, demonstrated by an increase in density of perfused capillary segments. We did not observe any differences in microvascular density summed across the entire range measured (i.e., 4–25 μm microvessels) between diet groups. Thus, it appears that in WD mice there was a leftward shift toward increasing the density of the smallest perfused capillary segments, while maintaining a similar net microvascular density across the entire range measured. These data support the notion that WD increases glycocalyx thickness because assuming there were no changes in anatomical microvessel diameter, any increase in glycocalyx thickness would decrease the glycocalyx-free area of the lumen and lead to a leftward shift in microvascular density, as this measurement is dependent on the width of the flow. Indeed, there was a greater capillary density at each week of WD in the time course study where glycocalyx was also thicker. When glycocalyx thickness was lower with hyaluronidase infusion in WD mice, we also saw a reduction in capillary density. Manual measurement of glycocalyx thickness by measuring the change in RBC flow width before and after the passage of a spontaneous WBC is time intensive and inherently more subjective than outcomes determined using the GlycoCheck capture and analysis software (i.e., PBR). However, as stated above, there are likely limitations in the ability of the GlycoCheck to identify differences in PBR at the smallest microvessel segment diameter. Thus, it is possible that microvascular density in the smallest microvessel segments is an indicator of glycocalyx thickness, as the glycocalyx length is increased, there would be less area that can be perfused by RBCs leading to a leftward shift in microvascular density. Future studies that directly manipulate the glycocalyx and quantify its properties using more direct methods are warranted to test this hypothesis.

We also observed a time-dependent decrease in capillary densities in both NC and WD-fed mice across the 12-week dietary intervention, indicating a possible early age-related change in these mice from 3–6 months of age. Future studies go beyond 12-week dietary intervention are needed to investigate whether WD-induced increased capillary density would eventually disappear with advanced age, as a decrease in microvascular density with advancing age is observed in humans (58).

Limitations

There are several limitations in the present study. First, the ability of the GlycoCheck system to detect differences in PBR at the smallest microvessel segment diameters may be limited by the size of RBCs passing through them. Therefore, microvascular density in the smallest vessels may serve as an automated indicator of glycocalyx thickness, as increased glycocalyx thickness would reduce the perfused diameter and lead to a leftward shift in microvascular density. Future studies using direct methods to manipulate and quantify the glycocalyx are needed to validate this hypothesis. Additionally, the anatomical microvessel diameter is not measured by the GlycoCheck system, thus, we are not able to determine if any group differences are due to changes in the anatomical structure of the microvessels. While glycocalyx properties were measured in the mesenteric microcirculation, endothelial function was measured in carotid arteries. We also did not measure glycocalyx properties in carotid arteries. Future studies are warranted to determine if WD-induced changes in endothelial function and glycocalyx properties occur in mesenteric and carotid arteries, respectively. We previously demonstrated a difference in blood glucose between mice after 12 weeks of WD (25). In the current study, we only measured blood glucose levels in mice after 1 week of WD, demonstrating no group difference. Lastly, we only measured glycocalyx properties in the present study, but have previously reported time-course in vascular function to WD (26), demonstrating elevated systolic blood pressure in WD-fed mice as early as week 2 with continued elevation thereafter. Thus, it is likely that blood pressure increased in 2, 4 and 12 week WD mice in the present study, despite greater glycocalyx thickness. Future studies are warranted to test this relationship

Conclusion

In the present study, we found that glycocalyx thickness increases and becomes less penetrable to RBCs as early as 1 week into the WD dietary intervention and remains unchanged thereafter. We also found that greater glycocalyx thickness and integrity in WD-fed mice was at least in part, to be due to increases in hyaluronan in the glycocalyx. Lastly, while agonist-mediated vasodilation is impaired in WD mice, flow-mediated vasodilation, which is dependent on the glycocalyx to mechanotransduce shear stress to endothelial cells is preserved at lower flow rates but is impaired at higher flow rates. These findings raise the possibility that glycocalyx augmentation in WD-fed mice may serve a compensatory role in preserving endothelial function under certain conditions, such as low shear stress. However, this potential benefit appears limited, as vasodilation was still impaired at higher flow rates and in response to acetylcholine. Although it is unclear from this study what component of a WD augments glycocalyx properties, future studies are warranted to investigate that, as it may have therapeutic efficacy in CVD states that exhibit glycocalyx degradation.