Microcirculatory and glycocalyx properties are lowered by high-salt diet but augmented by Western diet in genetically heterogeneous mice

Abstract

Many individuals in industrialized societies consume a high-salt, Western diet(WD); however, the effects of this diet on microcirculatory properties and glycocalyx barrier function are unknown. Young genetically heterogeneous male and female mice underwent 12 wk of normal chow (NC) diet, NC diet with 4% salt (NC4%), Western diet (WD), or WD with 4% salt (WD4%). Microcirculatory properties and glycocalyx barrier function were evaluated in the mesenteric microcirculation, using an intravital microscope equipped with an automated capture and analysis system. Total microvascular density summed across 4- to 25-μm microvessel segment diameters was lower in NC4% than in NC and WD (P < 0.05). Perfused boundary region (PBR), a marker of glycocalyx barrier function, averaged across 4- to 25-μm microvessel segment diameters was similar between NC and NC4%, as well as between WD and WD4% (P > 0.05). PBR was lower in WD and WD4% than in NC and NC4% (P < 0.05), indicating augmented glycocalyx barrier function in WD and WD4%. There were strong, inverse relationships between PBR and adiposity and blood glucose (r = −0.44 to −0.61, P < 0.05). In summary, NC4% induces deleterious effects on microvascular density, whereas WD augments glycocalyx barrier function. Interestingly, the combination of high-salt, Western diet in WD4% resulted in lower total microvascular density like NC4% and augmented glycocalyx barrier function like WD. These data suggest distinct microcirculatory adaptations to high-salt and Western diets that coincide when these diets are combined in young genetically heterogeneous male and female mice.

NEW & NOTEWORTHY Many individuals in industrialized societies consume a combination of high-salt and Western diet; however, the effects of this diet on microcirculatory and glycocalyx properties are unknown. This study reveals that a high-salt diet lowers microcirculatory and glycocalyx properties, whereas a Western diet augments glycocalyx barrier function and thickness. Taken together, these data indicate that there are distinct microcirculatory adaptations to high-salt and Western diets that coincide when high-salt and Western diets are combined.

INTRODUCTION

Cardiovascular disease (CVD) is the leading cause of mortality in industrialized societies, and age is a major predictor of CVD. A prominent feature of CVD is age-related microcirculatory dysfunction, characterized by low capillary density, impaired perfusion, and a deteriorated glycocalyx (1, 2). The glycocalyx is a negatively charged gel-like structure that lines the luminal surface of the vascular endothelium (3). Within the microvasculature, key functions of the glycocalyx are to modulate flow resistance to maintain microcirculatory blood flow homogeneity (4), modulating vascular permeability (5), and buffering endothelial cells from plasma sodium (6), oxidants (7), cytokines (8), and circulating immune cells (9). Although a deteriorated glycocalyx is present in advanced age (2), and in age-related chronic diseases, such as CVD (10) and type-2 diabetes (11), it is not well understood what initiates glycocalyx deterioration.

In most industrialized societies, individuals consume a high-salt diet in combination with a Western diet (WD), characterized by excessive consumption of fat and sugar. High-salt and Western diets have been shown to increase the risk of CVD (12). However, very few studies have investigated the effects of a combined high-salt, Western diet on cardiovascular health. In the present study, we sought to determine the effects of high-salt and/or Western diet and hypothesized that these diets would lower microcirculatory and glycocalyx properties, with the greatest effect occurring in mice that consumed a combined high-salt, Western diet. To test this hypothesis, we examined mesenteric microcirculatory and glycocalyx properties after 12 wk of dietary intervention in genetically heterogeneous young mice.

METHODS

Ethical Approval

All animal procedures conformed to the Guide to the Care and Use of Laboratory Animals (8th ed.; 13) and were approved by the Florida State University Animal Care and Use Committees.

Animals

Young male and female UM-HET3 mice used in this study were first-generation offspring of CByB6F1/J and C3D2F1/J parents, which were obtained from Jackson Laboratory, as previously described (14). All mice were housed in standard mouse cages under a 12-h:12-h light/dark cycle in a temperature-controlled environment. At 3 mo of age, mice were randomized into one of four dietary interventions for 12 wk: normal chow (NC) diet (LabDiet No. 5001; protein: 28.5%, carbohydrate: 58.0%, fat: 13.5% by kcal), NC diet with 4% salt (NC4%; LabDiet No. 5G21; protein: 27.5%, carbohydrate: 59.5%, fat: 13.0% by kcal), Western diet (WD; LabDiet No. 5TJN; protein: 15.8%, carbohydrate: 45.1%, fat: 39.1% by kcal), or WD with 4% salt (WD4%; LabDiet No. 5GDL; protein: 16.3%, carbohydrate: 43.3%, fat: 40.4% by kcal). Food and water were supplied ad libitum in group-housed cages. Body mass and blood glucose were measured in the postprandial state on the day of euthanasia.

Intravital Microscopy

The mesenteric microcirculation was observed using intravital microscopy, as previously described (2). Briefly, mice were anesthetized with isoflurane (3%) in room air at 100 mL/min flow rate and placed in the supine position on a heated platform (37°C). Subsequently, an incision was made along the linea alba, and the intestines were mobilized, gently exteriorized, and placed into a warm isotonic 0.9% saline bath (37°C). The cecum was identified and positioned for intravital microscopy. Intravital microscopy was performed with a handheld video capillary microscope (CapiScope HVCS, KK Technology, Honiton, UK) to view the mesenteric microcirculation. The intravital microscope is equipped with a sidestream dark-field (SDF) camera that uses green light-emitting stroboscopic diodes (540 nm). The green light is primarily absorbed by hemoglobin in red blood cells (RBCs), which allows RBCs to be viewed in contrast to the background as they flow through the microcirculation.

Video of the mesenteric microcirculation was recorded and analyzed using an automated capture and analysis system (GlycoCheck, MicroVascular Health Solutions LLC, Alpine, UT). An image captured by the SDF camera with analysis overlay is available in Supplemental Fig. S1 (see https://doi.org/10.6084/m9.figshare.17707478). In each mouse, 24 trials are acquired with each trial consisting of several ∼2-s video recordings that capture ∼5,000 microvessel segments that are 10 µm in length and range from 4 to 25 µm in perfused diameter. After two to three trials are obtained in an area, the microscope is moved to a different area of the cecum to counterbalance the spatial heterogeneity within the cecal mesenteric microcirculation. After 24 trials are acquired, the automated analysis system determines functional outcomes in perfused microvessel segments.

Microcirculatory Density and Hemodynamics

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

$$\text{microvascular density}=\frac{\text{number of perfused microvessel segments }\times \mathrm{ }\text{10}}{\text{tissue area recorded}}.$$

RBC velocities are determined by dividing RBC displacement by the time between consecutive video frames, and microvascular blood flow was determined in 9- to 11-µm-diameter microvessels (15).

Glycocalyx Barrier Function and Thickness

Perfused boundary region (PBR) represents the depth of penetration of RBCs into the endothelial glycocalyx and is taken as a marker of glycocalyx barrier function, with a larger PBR indicating greater perturbation of the glycocalyx. PBR is calculated using the following equation:

$$\mathrm{P}\mathrm{B}\mathrm{R}=\frac{(\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{f}\mathrm{u}\mathrm{s}\mathrm{e}\mathrm{d}\mathrm{ }\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{ }-\mathrm{ }\mathrm{m}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{n}\mathrm{ }\mathrm{R}\mathrm{B}\mathrm{C}\mathrm{ }\mathrm{c}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{n}\mathrm{ }\mathrm{w}\mathrm{i}\mathrm{d}\mathrm{t}\mathrm{h})}{2}.$$

To adjust PBR for flow-dependent variability, the linear regression between PBR and RBC velocity at microvessels ≥ 10-μm diameter was used to calculate the PBR when RBC velocity equals 0 µm/s (15). Manual determination of glycocalyx thickness was performed by measuring the change in width of flowing RBC column before and after the passage of a spontaneous leukocyte in individual capillaries, as previously described (16).

Statistical Analysis

Statistical tests were performed with GraphPad Prism (GraphPad Software, Inc., San Diego, CA). One-way and two-way ANOVAs were used to evaluate diet groups, as well as interactions between diet groups and sex (diet × sex), respectively. A two-way, mixed-model ANOVA was used to evaluate interactions between diet groups and microvessel diameter (diet × diameter). When a significant ANOVA was present, Tukey’s post hoc test was used to identify values that were significantly different. Bivariate correlations were used to determine relations between dependent variables. Statistical significance was set at P < 0.05 for all analyses. Data are presented as means ± SE.

RESULTS

Animal Characteristics

Animal characteristics are presented in Table 1. WD and WD4% had greater body mass, visceral adipose tissue mass, and blood glucose than NC and NC4% (P < 0.05). In general, tissue weights tended to be higher in WD and WD4% than in NC and NC4%. Visceral adipose tissue mass relative to body mass was also higher in WD and WD4% than in NC and NC4% (P < 0.05), whereas other tissue weights relative to body mass tended to be lower in WD and WD4% than in NC and NC4%. There appeared to be differences in body mass and some tissue masses between male and female mice, these data were not specifically analyzed for sex-related differences and are not discussed because we observed no sex-related differences in our primary outcomes. However, these data, separated by sex, are available as a reference in the Supplemental Tables S1 and S2.

Table 1.

Animal characteristics

	NC	NC4%	WD	WD4%	Group
Male/Female	13/15	10/9	10/9	11/7
Age, mo	6.6 ± 0.0	6.5 ± 0.1	6.7 ± 0.0	6.6 ± 0.0	0.066
Body mass, g	31.3 ± 1.0	29.9 ± 1.3	42.7 ± 1.8*†	40.4 ± 1.8*†	<0.001
Visceral adipose tissue, mg	360 ± 54	293 ± 58	1,149 ± 143*†	924 ± 56*†	<0.001
Visceral adipose tissue/body mass, mg/g	10.8 ± 1.4	8.9 ± 1.3	26.5 ± 3.2*†	22.5 ± 1.2*†	<0.001
Heart, mg	152 ± 5	149 ± 6	164 ± 7	168 ± 6	0.087
Heart/body mass, mg/g	4.9 ± 0.1	5.0 ± 0.1	3.9 ± 0.2*†	4.2 ± 0.1*†	<0.001
Liver, mg	1,547 ± 47	1,474 ± 59	2,065 ± 185*†	1,714 ± 96	<0.001
Liver/body mass, mg/g	49.8 ± 0.9	49.7 ± 1.5	47.6 ± 2.8	42.5 ± 1.5*†	0.014
Spleen, mg	81 ± 3	82 ± 3	95 ± 5*	89 ± 4	0.022
Spleen/body mass, mg/g	2.7 ± 0.1	2.8 ± 0.2	2.3 ± 0.1†	2.3 ± 0.2†	0.011
Quadricep, mg	217 ± 8	229 ± 14	213 ± 8	218 ± 12	0.714
Quadricep/body mass, mg/g	7.0 ± 0.3	7.7 ± 0.4	5.1 ± 0.3*†	5.5 ± 0.3*†	<0.001
Gastrocnemius, mg	154 ± 5	147 ± 7	147 ± 8	168 ± 7	0.143
Gastrocnemius/body mass, mg/g	5.0 ± 0.1	5.0 ± 0.2	3.5 ± 0.2*†	4.2 ± 0.1*†‡	<0.001
Soleus, mg	10 ± 0	11 ± 1	10 ± 1	12 ± 1*	0.024
Soleus/body mass, mg/g	0.3 ± 0.0	0.4 ± 0.0*	0.2 ± 0.0*†	0.3 ± 0.0†‡	<0.001
Plantaris, mg	23 ± 1	22 ± 2	21 ± 1	26 ± 1‡	0.030
Plantaris/body mass, mg/g	0.7 ± 0.0	0.7 ± 0.0	0.5 ± 0.0*†	0.6 ± 0.0‡	<0.001
Kidney, mg	253 ± 11	246 ± 25	250 ± 15	266 ± 14	0.848
Kidney/body mass, mg/g	8.2 ± 0.2	8.3 ± 0.8	6.0 ± 0.3*†	6.6 ± 0.2*†	<0.001
Blood glucose, mg/dL	162 ± 3	162 ± 3	183 ± 4*†	187 ± 5*†	<0.001

Values are means ± SE. Data were analyzed using one-way ANOVA with Tukey’s post hoc test to identify differences in animal characteristics. *P < 0.05 vs. NC. †P < 0.05 vs. NC4%. ‡P < 0.05 vs. WD. NC, normal chow; NC4%, NC with 4% salt; WD, Western diet; WD4%, WD with 4% salt.

Microcirculatory Density and Hemodynamics

Microvascular density at each microvessel segment diameter is presented in Fig. 1A. There were no sex-related differences in microvascular density (Supplemental Fig. S2; P > 0.05). Microvascular density summed across 4- to 25-µm microvessel segment diameters was lower in NC4% than in NC and WD (Fig. 1B; P < 0.05). After microvessels segments were separated into capillaries (4–6 µm diameter) and large microvessels (10–25 µm diameter), capillary density was lower in NC4% than in NC, WD, and WD4% (Fig. 1C; P < 0.05). Large microvessel density similar among groups (Fig. 1D; P > 0.05), although there was a trend for lower density in NC4% than in NC (P = 0.05). RBC velocity was higher in WD4% than in NC and NC4% (Fig. 1E; P < 0.05). There was a trend for higher RBC velocity in WD than in NC and NC4% (P = 0.09 and 0.07, respectively). The microvascular flow was similar among diet groups (Fig. 1F; P > 0.05).

Figure 1.

Group-by-microvessel diameter and group comparisons in normal chow (NC), NC with 4% salt (NC4%), Western diet (WD), and WD with 4% salt (WD4%) diet-treated male and female mice. Data were analyzed using a two-way, mixed-model ANOVA with Tukey’s post hoc test to identify differences in microvascular density at each microvessel segment diameter (A). Data were analyzed using a one-way ANOVA with Tukey’s post hoc test to identify differences in total microvascular density (4–25 µm diameter; B), capillary density (4–6 µm diameter; C), large microvessel density (10–25 µm diameter; D), red blood cell (RBC) velocity (E), and microvascular flow (F). *P < 0.05 vs. NC. †P < 0.05 vs. NC4%. Data are means ± SE. n = 18–28 mice/group.

Glycocalyx Barrier Function and Thickness

PBR at each microvessel segment diameter is presented in Fig. 2A. There were no sex-related differences in PBR (Supplemental Fig. S3; P > 0.05). PBR averaged across 4- to 25-µm microvessel segment diameters was similar between NC and NC4%, as well as between WD and WD4% (Fig. 2B; P > 0.05). However, PBR was lower in WD and WD4% than in NC and NC4% (P < 0.05). Capillary PBR was lower in WD than in NC4% (Fig. 2C; P < 0.05). Large microvessel PBR was lower in WD and WD4% than in NC and NC4% (Fig. 2D; P < 0.05). Flow-corrected PBR, which adjusts for RBC velocity, remained lower in WD and WD4% than in NC and NC4% (Fig. 2E; P < 0.05). Manually measured glycocalyx thickness was lower in NC4% than in NC, WD, and WD4% (Fig. 2F; P < 0.05). Although glycocalyx thickness in WD was higher than in NC (P < 0.05), glycocalyx thickness was similar between NC and WD4% (P > 0.05). Lumen diameters in capillaries that were used for manual glycocalyx thickness analysis were similar (NC: 5.7 ± 0.2, NC4%: 5.6 ± 0.1, WD: 5.5 ± 0.1, WD4%: 5.4 ± 0.1 µm; P > 0.05).

Figure 2.

Group-by-microvessel diameter and group comparisons in normal chow (NC), NC with 4% salt (NC4%), Western diet (WD), and WD with 4% salt (WD4%) diet-treated male and female mice. Data were analyzed using a two-way, mixed-model ANOVA with Tukey’s post hoc test to identify differences in perfused boundary region (PBR) at each microvessel segment diameter (A). Data were analyzed using one-way ANOVA with Tukey’s post hoc test to identify differences in PBR (4–25 µm diameter; B), capillary PBR (4–6 µm diameter; C), large microvessel PBR (10–25 µm diameter; D), flow-corrected PBR (E), and glycocalyx thickness measured in ∼5-µm-diameter capillaries (F). *P < 0.05 vs. NC. †P < 0.05 vs. NC4%. Data are means ± SE. n = 18–28 mice/group.

Correlational Analysis

Bivariate correlations were determined between PBR and selected variables: body mass (Fig. 3A), visceral adipose tissues mass (Fig. 3B), blood glucose (Fig. 3C), and glycocalyx thickness (Fig. 3D). There were strong inverse relationships that ranged from r = −0.28 to −0.61 between PBR and each variable (P < 0.05).

Figure 3.

Bivariate correlational analysis was used to determine relations between perfused boundary region (PBR) and body mass (A), visceral adipose tissue mass (B), blood glucose (C), and glycocalyx thickness (D) in normal chow (NC), NC with 4% salt (NC4%), Western diet (WD), and WD with 4% salt (WD4%) diet-treated male and female mice. n = 18–28 mice/group.

DISCUSSION

In the present study, we observed alterations in microcirculatory and glycocalyx properties in young, genetically heterogeneous mice after 12 wk of NC4%, WD, or WD4% diets. When compared with NC, microvascular density was lower in NC4%, although there was a differential effect of Western diets that resulted in elevated density in 4-µm microvessels and tendency for lower density in larger microvessels. Microvascular hemodynamics were also altered, with a tendency for higher RBC velocity in WD and WD4%. There was a higher PBR in 4-µm microvessels and ∼26% lower glycocalyx thickness in NC4% than in NC. Surprisingly, PBR was ∼14%–16% lower in WD and WD4% than in NC and NC4%, indicating greater glycocalyx barrier function in mice that consumed Western diets. Annually measured glycocalyx thickness was higher in WD than in NC and NC4%, whereas glycocalyx thickness in NC and WD4% were similar. Last, there was a strong relationship between glycocalyx barrier function and adiposity, blood glucose, and glycocalyx thickness. Taken together, these findings indicate that high-salt diets induce deleterious effects on microcirculatory and glycocalyx properties, whereas Western diets augment microcirculatory hemodynamics and glycocalyx properties. The distinct microcirculatory adaptations to high-salt and Western diets coincide when these diets were combined.

High-Salt Diet Lowers Microcirculatory and Glycocalyx Properties

Total microvascular density was ∼29% lower in NC4% than in NC, and this reduction in density was observed mostly in smaller microvessel diameters. There was a ∼31% lower capillary density and tendency for lower large microvessel density in NC4% than in NC, indicating the deleterious effects of a high-salt diet across the microcirculation. Reductions in microvascular density have been observed in humans and rats, following high-salt diets (17, 18), although in humans salt sensitivity levels seem to play a larger role (19). There was a negligible effect of high-salt diets on glycocalyx barrier function with a tendency for elevated capillary PBR in NC4% compared with NC. Given that RBC diameter in mice is ∼5.8 µm, we had not anticipated any differences in PBR at the capillary level (20), thus, even a trend at this level may have physiological significance. We also manually measured capillary glycocalyx thickness, which revealed that NC4% had a ∼26% lower glycocalyx thickness than NC. Others have shown that increases sodium concentrations lower glycocalyx thickness in vitro endothelial cells (6) and human umbilical arteries (21). Although we are aware of no studies that have investigated the effects of a high-salt diet on glycocalyx properties, glycocalyx damage is present 24 h after hypertonic saline gavage in young mice (22). Thus, these data provide the first evidence of the detrimental effects of a high-salt diet on glycocalyx properties.

Western Diet Augments Capillary Density and Glycocalyx Properties

Total microvascular density was similar in NC and WD, however, there was a differential effect of WD that shifted toward a greater capillary density and lower large microvessel density. Although microvascular flow was similar between groups, there was a tendency for RBC velocity to be elevated in WD. Contrary to our hypothesis, there was a robust ∼16% decrease in PBR in WD, compared with NC, indicating greater glycocalyx barrier function in WD that was largely dependent on microvessel size, with the greatest elevation in the large microvessel segments. There was also a positive effect of WD on capillary glycocalyx thickness, resulting in a ∼26% higher glycocalyx thickness in WD than in NC. Increased capillary density in WD may be related to augmented glycocalyx properties, as a thicker, less penetrable glycocalyx would result in a luminal narrowing across the microcirculation, leading to a smaller functional diameter that RBCs can travel through. This would shift the perfused microvessel population toward a greater density of smaller microvessels. Not many studies have investigated the effects of Western-type diets on glycocalyx properties, thus, there is minimal consensus. Although others that have used a Western-type diet observed a deteriorated (23) or no change (24) in glycocalyx thickness in resistance (i.e., mesenteric) and large (i.e., carotid) arteries, medium- and high-fat diets have been shown to augment glycocalyx thickness in the brain microcirculation (25). Eskens et al. (26) observed a deteriorated glycocalyx after 6 wk of Western diet in young C57BL/6J mice; however, these effects may have been more pronounced because of a lack of genetic diversity, increasing susceptibility to a Western-type diet. Indeed, fasted blood glucose of >200 mg/dL was attained 1 wk after the start of Western diet in C57BL/6J mice. In contrast, unfasted blood glucose in our genetically heterogeneous mice was ∼183 mg/dL. Others have shown no effect of Western-type diet on fasting blood glucose in UM-HET3 mice, although insulin sensitivity and glucose tolerance are impaired (27). Thus, it is possible that the genetically heterogeneous mice are more resilient to the deleterious metabolic effects of WD in inbred strains. Although acute hyperglycemia of 300 mg/dL sustained over 6 h in young, healthy human subjects has been shown to deteriorate the glycocalyx (28), in nonexperimental conditions, blood glucose levels this high would only be observed in people with diabetes. Thus, less is understood regarding the impact of mild elevations in blood glucose on glycocalyx health. Importantly, we also observed a strong, inverse relationship between PBR and body mass, visceral adipose tissue, and blood glucose, providing further evidence that there may not be a detrimental effect of WD-induced obesity and mild elevations in blood glucose on glycocalyx properties in young mice. From a translational perspective, it is tenable, as young humans who consume a Western-type diet are unlikely to become diabetic or even prediabetic for decades. Nevertheless, elevated blood glucose, excessive fat, and sugar consumption are risk factors for diabetes, and individuals with type-2 diabetes are known to have lower glycocalyx barrier function (11). Thus, the role of the glycocalyx in diabetes is unclear. It is possible that elevated glycocalyx barrier function induces dysfunctional cardiovascular adaptations. Future study is warranted to identify the role of the glycocalyx in the progression of type-2 diabetes, and its influence on cardiovascular physiology.

Combined High-Salt, Western Diet Lowers Microvascular Density, While Augmenting Glycocalyx Properties

In comparison to NC, the combined high-salt, Western diet in WD4% resulted in lower microvascular density in medium-size microvessels such as NC4%, and a simultaneous elevation in smaller-sized microvessels such as WD. In further disagreement with our hypothesis, there was a ∼14%–15% reduction in PBR in WD4%, compared with NC and NC4%, and no difference in PBR compared with WD. Thus, these data in WD4% further support the negligible effect of high-salt diets on glycocalyx barrier function, while again demonstrating the beneficial effect of Western diets on glycocalyx health. Like WD, augmented glycocalyx barrier function in WD4% was greatest in large microvessel segments. Although we did not observe a difference in capillary PBR between NC, WD, and WD4%, PBR in both Western-diet groups was lower than NC4%. Given the similarity in diameter of RBCs to capillaries, it is possible that an increase in capillary PBR beyond what was observed in NC is not physiologically possible or necessary. There was a tendency for lower glycocalyx thickness in WD4% than in WD, which provides support to the finding that high-salt lowered glycocalyx thickness in NC4% than in NC. Indeed, glycocalyx thickness in WD4% remained like NC, but higher than NC4%. From these data, we conclude that high-salt diets lower capillary glycocalyx barrier function and decrease glycocalyx thickness. Still, the benefits of a Western diet in WD4% appear to offset the deteriorating effects that high-salt diet has on glycocalyx properties. Taken together, these data indicate that there may be a minimum glycocalyx thickness required to achieve maximal glycocalyx barrier function at least in the setting of high-salt and Western-diet consumption.

There are several strengths of this study that can be emphasized. We used a large sample of genetically heterogeneous male and female mice. This approach required a greater sample size to account for increased variability, but significant findings under these conditions strengthen the translational value of this experimental design. In addition, measurements of microcirculatory and glycocalyx barrier function were performed using an automated capture and analysis system, reducing the possibility of inter- and intrarater variability. Last, our experimental diets were formulated using a reductionist approach to determine the distinct roles of high-salt or Western diets on their own and combined on microcirculatory and glycocalyx properties. Considering that a combined high-salt, Western diet is widely consumed among many individuals in industrialized societies, this study possesses translational importance, although it is not without limitations. The findings in this study are observational, thus, the lack of direct, mechanistic evidence is a weakness. Moreover, our findings that WD and WD4% augment glycocalyx barrier function should not be perceived as an improvement. Others have demonstrated lower glycocalyx barrier function in various disease states, but this does not mean that a thicker glycocalyx with greater barrier function is healthy. Indeed, it may induce dysfunction. Thus, future studies are warranted to determine the direct mechanisms by which Western diets augment glycocalyx properties, and determine the role of the glycocalyx on adverse cardiovascular adaptations to these types of diet.

Conclusions

In line with our hypothesis, high-salt diets lower microcirculatory and glycocalyx properties. On the contrary, Western diets had a differential effect on microcirculatory properties, while augmenting glycocalyx barrier function and thickness. Taken together, these data indicate that there are distinct microcirculatory adaptations to high-salt and Western diets that appear to coincide when these two diets are combined in genetically heterogeneous young mice.

SUPPLEMENTAL DATA

Supplemental Tables S1and S2 and Supplemental Figs. S1–S3: https://doi.org/10.6084/m9.figshare.17707478.

GRANTS

This study was funded in part by National Center for Complementary and Integrative Health Grant R00 AT010017.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.R.M. conceived and designed research; X.Z., C.D., A.J.K., and D.R.M. performed experiments; X.Z., C.D., A.J.K., and D.R.M. analyzed data; D.R.M. interpreted results of experiments; D.R.M. prepared figures; X.Z., C.D., A.J.K., and D.R.M. drafted manuscript; X.Z., C.D., A.J.K., and D.R.M. edited and revised manuscript; X.Z., C.D., A.J.K., and D.R.M. approved final version of manuscript.