Protection from chronic stress- and depressive symptom-induced vascular endothelial dysfunction in female rats is abolished by preexisting metabolic disease

Abstract

While it is known that chronic stress and clinical depression are powerful predictors of poor cardiovascular outcomes, recent clinical evidence has identified correlations between the development of metabolic disease and depressive symptoms, creating a combined condition of severely elevated cardiovascular disease risk. In this study, we used the obese Zucker rat (OZRs) and the unpredictable chronic mild stress (UCMS) model to determine the impact of preexisting metabolic disease on the relationship between chronic stress/depressive symptoms and vascular function. Additionally, we determined the impact of metabolic syndrome on sex-based protection from chronic stress/depressive effects on vascular function in female lean Zucker rats (LZRs). In general, vasodilator reactivity was attenuated under control conditions in OZRs compared with LZRs. Although still impaired, conduit arterial and resistance arteriolar dilator reactivity under control conditions in female OZRs was superior to that in male or ovariectomized (OVX) female OZRs, largely because of better maintenance of vascular nitric oxide and prostacyclin levels. However, imposition of metabolic syndrome in combination with UCMS in OZRs further impaired dilator reactivity in both vessel subtypes to a similarly severe extent and abolished any protective effect in female rats compared with male or OVX female rats. The loss of vascular protection in female OZRs with UCMS was reflected in vasodilator metabolite levels, which closely matched those in male and OVX female OZRs subjected to UCMS. These results suggest that presentation of metabolic disease in combination with depressive symptoms can overwhelm the vasoprotection identified in female rats and, thereby, may reflect a severe impairment to normal endothelial function.

NEW & NOTEWORTHY This study addresses the protection from chronic stress- and depression-induced vascular dysfunction identified in female compared with male or ovariectomized female rats. We determined the impact of preexisting metabolic disease, a frequent comorbidity of clinical depression in humans, on that vascular protection. With preexisting metabolic syndrome, female rats lost all protection from chronic stress/depressive symptoms and became phenotypically similar to male and ovariectomized female rats, with comparably poor vasoactive dilator metabolite profiles.

INTRODUCTION

Clinical and population health studies have recognized that elevated cardiovascular disease risk can come from a wide variety of sources, spanning lifestyle (8, 13), diet (20, 36), environmental (22, 40, 48, 52), and genetic (43) influences. Among these, the presence of metabolic disease has been established as one of the more powerful risk factors for negative cardiovascular outcomes (38). The presentation of these conditions, including obesity, hypertension, impaired glycemic control, and dyslipidemia, with the associated systemic condition of a proinflammatory/prooxidant and prothrombotic phenotype, has long been termed “metabolic syndrome” (2).

More recently, the presence of chronic, irresolvable psychological stress in one’s environment, especially when it can result in clinical depression (24), has become recognized as a risk factor for poor cardiovascular health outcomes (16, 24). While the specific mechanisms linking chronic stress/depression with poor cardiovascular outcomes remain an active area of investigation, even after alterations in the hypothalamic-pituitary-adrenal axis (31, 49) and the sympathetic nervous system (1), the genesis of a chronic prooxidant and proinflammatory state (25, 32), and the profound changes to lifestyle that can result (29) are taken into account, clinical depression alone represents a major risk factor for poor cardiovascular and cerebrovascular outcomes. Additionally, given demonstrated links between metabolic disease, chronic stress/depression, and poor cardiovascular outcomes (34), a high risk state develops when these conditions are combined. The increasing prevalence of metabolic syndrome (34) and the high incidence of chronic stress/depression highlight the need to interrogate the concomitant effects of these conditions on vascular function.

Biological sex represents another variable in the pathophysiological relationship between chronic stress/depression and vascular disease. Premenopausal women exhibit a protection from cardiovascular and cerebrovascular pathologies compared with postmenopausal women and age-matched men, even in the face of comparable levels of risk (41). Furthermore, although the cognitive and behavioral symptoms of depression are more acutely experienced by premenopausal women than by men, premenopausal women manifest a protection from poor cardiovascular outcomes to these stressors (3, 27, 28). While the importance of normal sex hormone, particularly estrogen, levels has been clearly identified, with a number of putative mechanistic links being actively pursued (7), this remains an area of investigation in which much work remains.

In a companion study (9), we used male and female lean Zucker rats (LZRs) to determine the impact of depressive symptoms [via the unpredictable chronic mild stress (UCMS) model (50, 51)] on the function of conduit and cerebral resistance arteries. We reported that while the imposition of depressive symptoms in LZRs resulted in an impaired endothelial function in both vascular segments, largely reflecting a loss of vascular nitric oxide (NO) levels and a shift in arachidonic acid metabolism toward an increased production of thromboxane (Tx)A₂, the severity of this effect was reduced in female rats compared with male rats. Ovariectomy (OVX) of female rats before the imposition of UCMS resulted in a near-complete loss of the protective effect identified in intact female rats, and their vascular and endothelial responses to chronic stress and depressive symptoms became virtually identical to the responses of male rats.

Given established correlations between elevated cardiovascular disease risk stemming from metabolic syndrome and the evolution of depression/depressive symptoms in afflicted individuals (34), the purpose of the present study was to determine the impact of preexisting metabolic disease on the relationships determined in the companion study (9). We used the obese Zucker rat [OZR (fa/fa)] model of the metabolic syndrome and subjected the same groups of age-matched rats (male, female, and OVX female rats) to UCMS. OZRs, which have a mutation in their leptin receptor causing insensitivity and a blunted satiety reflex, develop the full metabolic syndrome due to chronic hyperphagia (21, 47). This study tested the hypothesis that the preexisting condition of the metabolic syndrome would compromise the protective effect against depressive symptom-induced vascular dysfunction demonstrated by female rats compared with male or OVX female rats.

MATERIALS AND METHODS

Animals

Male and female LZRs and OZRs (Harlan/Envigo) were purchased at ~8 wk of age and maintained on standard chow and drinking water ad libitum for the duration of the study. All animals were housed in an accredited animal care facility at the University of Western Ontario or the West Virginia University Health Sciences Center, and all procedures had received prior Institutional Animal Care and Use Committee approval. A separate cohort of female LZRs and OZRs (Harlan/Envigo) was acquired at the same age after OVX surgery, which was performed between 5 and 6 wk of age by the supplier, and similarly housed. At 9 wk of age, rats from each sex/condition were divided into the following two groups: control (all with normal handling, with the exception of the UCMS protocol) and UCMS (see below). After 8 wk under control or UCMS conditions, rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a carotid artery was cannulated for the determination of mean arterial pressure. From each animal, a venous blood sample was also acquired (venipuncture) for the determination of circulating endocrine, oxidant, and inflammatory biomarkers. Thereafter, rats received a low dose of heparin (100 IU/kg iv) to prevent the formation of blood clots during tissue harvest. All data for LZRs in the present study are identical to those in the companion study (9); no additional LZRs were used for collection of data specific to this study.

UCMS Protocol

All rats were singly housed, with the control group in a separate quiet room adjacent to the room used for UCMS treatments. The UCMS group was randomly exposed to the following environmental stressors daily throughout each 24-h period: 1) damp bedding (addition of 10 oz of water to each standard cage for the next 3 h); 2) water [removal of all bedding and addition of ~0.5 in. of ~30°C water to the empty cage for the next 3 h (room temperature was ~24°C)]; 3) cage tilted to 45° with or without bedding for 3 h; 4) social stress (rat switched into a cage of a neighboring rat for 3 h); 5) no bedding for 3 h or, on 2 occasions/wk, overnight; 6) succession of 30-min light-dark cycles throughout a 24-h period; and 7) exposure to predator smells (e.g., cat fur) and/or sounds (e.g., cat growling) for 8 h. After 8 wk, all rats were subjected to a series of behavioral tests to evaluate the outcomes of the UCMS procedures [for a full description of the UCMS protocol, see Frisbee et al. (23)].

Coat Status

Coat status was evaluated throughout the duration of the UCMS protocol, as previously described elsewhere (50). The total cumulative score was computed from an individual score of 0 (clean) or 1 (dirty) assigned to eight body parts (head, neck, dorsal coat, ventral coat, tail, forelimb, hindlimb, and genital region).

Sucrose Spray Test

The sucrose spray test was used to evaluate acute grooming behavior, defined as cleaning of the fur by licking or scratching (50). A 10% sucrose solution was sprayed on the dorsal coat of each rat, and grooming activity was recorded for 5 min. The viscosity of the sucrose solution will dirty the coat and induce grooming behavior, with depressive symptoms characterized by an increased latency (idle time between spray and initiation of grooming) and decreased frequency (number of times a particular body part is groomed).

Novelty Suppressed Feeding Test

The novelty suppressed feeding test was performed as previously described (46). At the conclusion of the UCMS period, all access to food was removed for 24 h (ad libitum access to water was continued). Subsequently, individual rats were placed in one corner of an empty cage (18 × 24 in.) with fresh bedding. One pellet of the normal chow was placed in the center of the cage, and the time from placement of the food pellet to the moment the rat actually began consuming (i.e., not just sniffing or handling) the food pellet was noted. At this time, the animal was removed from this cage and returned to its home cage, and normal access to food was restored.

Measurements of Vascular Reactivity

Conduit arteries.

After removal of the resistance arteriole in each rat, the thoracic aorta was removed, rinsed in physiological salt solution (PSS), cleared of surrounding tissue, and cut into 2- to 3-mm rings. Each ring was mounted in a myobath chamber between a fixed point and a force transducer (World Precision Instruments, Sarasota, FL) and set to 0.5 g of tension for 45 min to equilibrate. Organ baths contained PSS at 37°C and were aerated with 95% O₂-5% CO₂. Rings were preconditioned by treatment with 10⁻⁷ M phenylephrine for 5 min, at which time 10⁻⁵ M methacholine was added to the bath to assess endothelial integrity. Any ring that failed to demonstrate both a brisk constrictor response to phenylephrine and viable endothelial function was discarded. Subsequently, rings were treated with increasing concentrations of phenylephrine (10⁻¹⁰–10⁻⁴ M) to assess constrictor reactivity. For assessment of relaxation, rings were pretreated with 10⁻⁶ M phenylephrine and exposed to increasing concentrations of methacholine (10⁻¹⁰–10⁻⁴ M) and sodium nitroprusside (10⁻¹⁰–10⁻⁴ M). To assess the roles of NO, cyclooxygenase, and reactive oxidant stress in modulation of vascular responses to the agonist treatments, concentration-response curves were also conducted after pretreatment of the rings for 45–60 min with N-nitro-l-arginine methyl ester (l-NAME; 10⁻⁴ M), indomethacin (10⁻⁵ M), and tempol (10⁻⁴ M), respectively.

While deeply anesthetized, each rat was decapitated, and the brain was removed from the skull case and placed in cold (4°C) PSS. Subsequently, a middle cerebral artery (MCA) was dissected from its origin at the circle of Willis. Each MCA was doubly cannulated in a heated (37°C) chamber that allowed perfusion and superfusion of the lumen and exterior of the vessel, respectively, with PSS from separate reservoirs. PSS, which was equilibrated with 21% O₂-5% CO₂-74% N₂, had the following composition (in mM): 119 NaCl, 4.7 KCl, 1.17 MgSO₄, 1.6 CaCl₂, 1.18 NaH₂PO₄, 24 NaHCO₃, 0.026 EDTA, and 5.5 glucose. Any side branches were ligated using a single strand teased from 6-0 suture. Vessel diameter was measured using television microscopy and an on-screen video micrometer.

Isolated MCAs.

After cannulation, MCAs were extended to their in situ length and equilibrated at 80% of the animal's mean arterial pressure to approximate in vivo perfusion pressure (33). Any vessel that did not demonstrate significant active tone at the equilibration pressure was discarded. Active tone at the equilibration pressure was calculated as (ΔD/D_max) × 100, where ΔD is the diameter increase from rest in response to Ca²⁺-free PSS and D_max is the maximum diameter measured at the equilibration pressure in Ca²⁺-free PSS.

After equilibration, the dilator reactivity of the MCA was assessed in response to increasing concentrations (10⁻¹⁰–10⁻⁶ M) of acetylcholine (ACh). Vascular responses to ACh were also measured after acute (45–60 min) incubation with l-NAME (10⁻⁴ M), indomethacin (10⁻⁵ M), and/or tempol (10⁻⁴ M).

To determine myogenic activation, the vessel perfusate outflow line was clamped, stopping perfusate flow through the vessel, and the height of the perfusion reservoir was changed to vary intraluminal pressure in 20-mmHg increments between 40 and 160 mmHg. Vessel diameter was determined after 10–15 min at each pressure, and pressure levels were randomized for each myogenic activation curve. After completion of all procedures, the perfusate and superfusate were replaced with Ca²⁺-free PSS, and the passive diameter of the fully relaxed vessel was determined over the identical intraluminal pressure range.

Measurement of Vascular NO and H₂O₂ Levels

From each rat, pooled conduit arteries (e.g., carotid, femoral, iliac, distal aorta, and saphenous) were harvested for the determination of vasoactive metabolite levels. Vascular NO and H₂O₂ production was assessed using amperometric sensors (World Precision Instruments). Briefly, arteries were isolated, placed in a sealed reaction chamber, and incubated with warmed (37°C) PSS equilibrated with 95% O₂-5% CO₂. A NO sensor (model ISO-NOPF100, World Precision Instruments) and a H₂O₂ sensor (model ISO-HPO100, World Precision Instruments) were inserted into the chamber, and baseline levels of current were obtained. Subsequently, increasing concentrations (10⁻¹⁰–10⁻⁶ M) of methacholine were added to the bath, and changes in “NO” and “H₂O₂” currents were determined.

Determination of Vascular Metabolites of Arachidonic Acid

Vascular production of 6-keto-PGF_1α [the stable breakdown product of PGI₂ (37)] and 11-dehydro-TxB₂ [the stable plasma breakdown product of TxA₂ (14)] in response to challenge with reduced Po₂ was assessed using the pooled conduit arteries. Pooled arteries from each animal were incubated in microcentrifuge tubes in 1 ml PSS for 30 min under control conditions (21% O₂). The superfusate was removed, stored in a new microcentrifuge tube, and frozen in liquid N₂; a new aliquot of PSS was added to the vessels, and the equilibration gas was switched to 0% O₂ for the subsequent 30 min. After the second 30-min period, this new PSS was transferred to a fresh tube, frozen in liquid N₂, and stored at −80°C. Metabolite release by the vessels was determined using commercially available enzyme immunoassay kits for 6-keto-PGF_1α and 11-dehydro-TxB₂ (Cayman Chemical).

Biochemical Analyses

Plasma corticosterone and nitrotyrosine levels were determined using commercially available ELISA kits (Cayman Chemical). Sex hormone profiles were determined on a fee-for-service basis by a professional clinical laboratory using commercially available ELISA kits (MyBioSource, Cayman Chemical). All other plasma biomarkers (e.g., insulin and biomarkers of inflammation) were measured using commercially available kits and multiplexed bioassay systems (Meso Scale Diagnostics or Luminex/Thermo Fisher).

Data and Statistical Analyses

Mechanical responses after challenge with logarithmically increasing doses of methacholine/ACh were fit with the following three-parameter logistic equation:

$$y=\min +\left(\frac{\max -\min }{1+{10}^{\log {\text{ED}}_{50}-x}}\right)$$

where y is the vessel diameter, min and max are the lower (minimum) and upper (maximum) bounds, respectively, of the change in diameter or tension development with agonist concentration, x is the logarithm of the agonist concentration, and logED₅₀ is the logarithm of the agonist concentration (x) where the response (y) is halfway between the bounds. For the presentation of results, we focused on the changes in the bounds as a representation of vessel reactivity, as one bound will remain consistent between all groups (defined as the prechallenge diameter), and we did not determine a consistent or significant change in the logED₅₀ values between treatment groups. As a result of this approach, the other bound represents that statistically determined asymptote for the concentration-response relationship and does not assume that the vascular response at the highest used concentration of the agonist represents the maximum possible response. Rather, the sigmodal relationship of best fit to the data will predict the statistical bound of the response given the data points entered into the model. As such, the bound is frequently slightly larger than the dilator response of the vessel at the highest concentration of the agonist.

The myogenic properties of MCAs from each experimental group were plotted as mean diameter at each intraluminal pressure and fitted with a linear regression: y = α₀ + βx, where the slope coefficient (β) represents the degree of myogenic activation (δdiameter/δpressure). Increasingly negative values of β, therefore, represent a greater degree of myogenic activation in response to changes in intravascular pressure. A similar analysis was used to determine NO and H₂O₂ levels in response to increasing concentrations of methacholine, where β is the rate of change in NO or H₂O₂ released by the vessels in response to agonist challenge.

All calculations of passive arteriolar wall mechanics (used as indicators of structural alterations to the individual microvessel) are based on those previously used (4), with minor modification. Vessel wall thickness (WT; in μm) was calculated as follows:

$$\text{WT}=\frac{\left(\text{OD}-\text{ID}\right)}{2}$$

where OD and ID are arteriolar outer and inner diameters (in μm), respectively.

Incremental arteriolar distensibility (Dist_inc; in %change in arteriolar diameter/mmHg) was calculated as follows

$${\text{Dist}}_{\text{inc}}=\frac{\Delta \text{ID}}{\left(\text{ID}\times \Delta {\text{P}}_{\text{IL}}\right)}\times 100$$

where ΔID is the change in internal arteriolar diameter for each incremental change in intraluminal pressure (ΔP_IL).

For the calculation of circumferential stress, intraluminal pressure was converted from mmHg to N/m², where 1 mmHg = 1.334 × 10² N/m². Circumferential stress (σ) was then calculated as follows:

$$\sigma =\frac{\left({\text{P}}_{\text{IL}}\times \text{ID}\right)}{2\text{WT}}$$

Circumferential strain (ε) was calculated as follows:

$$\epsilon =\frac{\left(\text{ID}-{\text{ID}}_5\right)}{{\text{ID}}_{\text{5}}}$$

where ID₅ is the internal arteriolar diameter at the lowest intravascular pressure (i.e., 5 mmHg). The stress-strain relationship from each vessel was fit (ordinary least squares analysis, r² > 0.85) with the following exponential equation:

$$\sigma ={\sigma }_5{e}^{\beta \epsilon }$$

where σ₅ is circumferential stress at ID₅ and β is the slope coefficient describing arterial stiffness. Higher levels of β are indicative of increasing arterial stiffness (i.e., requiring a greater degree of distending pressure to achieve a given level of wall deformation).

Data and Statistical Analyses

Values are means ± SE. Differences in all calculated parameters or descriptive characteristics between the different experimental groups were assessed using ANOVA with a Student-Newman-Keuls test post hoc, as appropriate. In all cases, P < 0.05 was taken to reflect statistical significance.

RESULTS

Table 1 shows physical and biochemical data from all groups of animals at 17 wk of age. The comparison of LZRs with OZRs (regardless of condition) demonstrates the impact of the leptin resistance and chronic hyperphagia, with all markers of metabolic syndrome, including an elevated prooxidant and proinflammatory state, in OZRs compared with LZRs. These relationships were present between male, female, and OVX female rats. Imposition of UCMS on OZRs resulted in a worsening of specific indexes of metabolic syndrome, specifically those associated with glycemic control and arterial pressure, as well as the biomarkers for chronic stress (cortisol), a chronic prooxidant and proinflammatory environment. The most severe effects on the markers of metabolic syndrome and the chronic prooxidant/proinflammatory environment were identified in OZRs, although all differences between male, female, and OVX female rats were eliminated with the multipathology state.

Table 1.

Baseline characteristics

	Control						UCMS
	LZR			OZR			LZR			OZR
	Male	Female	OVX	Male	Female	OVX	Male	Female	OVX	Male	Female	OVX
Mass, g	391 ± 7	354 ± 5	342 ± 8	596 ± 12*	572 ± 11*	593 ± 9*	422 ± 12	344 ± 10	385 ± 10§	661 ± 9*†‡	535 ± 10*†‡	585 ± 11*†§
Mean arterial pressure, mmHg	105 ± 4	102 ± 3	106 ± 4	132 ± 6*	132 ± 5*	142 ± 5*	111 ± 4	114 ± 6	117 ± 5	137 ± 5*†	142 ± 6*†	145 ± 6*†
Glucose, mg/dl	123 ± 6	114 ± 5	190 ± 8§	166 ± 9*	131 ± 8	170 ± 9§	154 ± 11*	136 ± 6*	205 ± 12§	186 ± 12*†	158 ± 10*†‡	186 ± 9§
Insulin, ng/ml	1.4 ± 0.2	1.1 ± 0.3	1.6 ± 0.3	7.1 ± 1.0*	5.1 ± 0.6*	6.9 ± 0.5*	3.8 ± 0.3‡	2.5 ± 0.3§	3.1 ± 0.3	7.8 ± 1.0*†	7.4 ± 0.8*†‡	7.9 ± 1.1*†
Triglycerides, mg/dl	77 ± 8	60 ± 10	62 ± 7	368 ± 40*	264 ± 36*	303 ± 43*	72 ± 8	78 ± 6	62 ± 9	404 ± 41*†	393 ± 28*†‡	382 ± 14*†
Total cholesterol, mg/dl	104 ± 10	96 ± 11	127 ± 9	138 ± 9*	122 ± 10*	150 ± 14	98 ± 9	88 ± 11	130 ± 10§	141 ± 12*†	135 ± 11*†	206 ± 20*†§
Nitrotyrosine, pg/ml	14 ± 3	12 ± 4	17 ± 4	54 ± 5*	40 ± 6*	52 ± 7*	28 ± 5	21 ± 4	30 ± 5	60 ± 8*†	55 ± 7*†	59 ± 9*†
Corticosterone, ng/ml	87 ± 7	161 ± 10	149 ± 10	111 ± 11	146 ± 9	151 ± 11	129 ± 14	250 ± 20	170 ± 11§	253 ± 19*†‡	292 ± 23‡	282 ± 17*†‡
TNF-α, pg/ml	2.9 ± 0.3	2.9 ± 0.2	4.0 ± 0.4	6.0 ± 0.5*	5.7 ± 0.4*	9.1 ± 0.5*§	5.3 ± 0.5	6.4 ± 0.6	7.1 ± 0.5	6.1 ± 0.7	8.8 ± 0.4*†‡	7.9 ± 0.7

Values are means ± SE. LZR, lean Zucker rats; OZR, obese Zucker rats; OVX, ovariectomy.

^*P < 0.05 vs. LZR under control conditions (for that condition);

^†P < 0.05 vs. LZR subjected to the unpredictable chronic mild stress (UCMS) model;

^‡P < 0.05 vs. OZR under control conditions;

^§P < 0.05 vs. matched female rats.

Table 2 shows data describing the sex hormone profiles for male and female LZRs and OZRs at 17 wk under control conditions and after the UCMS protocol. UCMS reduced testosterone levels in LZRs and OZRs, with lower levels in OZRs than in LZRs. Obesity tended to increase estrogen levels in female OZRs compared with female LZRs, although this response was not statistically significant. Progesterone levels, low in male and OVX female rats and elevated in intact female rats, were largely unaffected by UCMS or metabolic syndrome. Luteinizing hormone and follicle-stimulating hormone levels were relatively low in male and female rats but were increased with OVX in LZRs and OZRs.

Table 2.

Sex hormone profiles

	Control						UCMS
	LZR			OZR			LZR			OZR
	Male	Female	OVX	Male	Female	OVX	Male	Female	OVX	Male	Female	OVX
Testosterone, ng/ml	2.7 ± 0.3	0.2 ± 0.1	0.3 ± 0.1	2.1 ± 0.2	0.2 ± 0.2	0.2 ± 0.1	1.7 ± 0.3*	0.1 ± 0.1	0.2 ± 0.1	1.6 ± 0.3*	0.3 ± 0.2	0.3 ± 0.1
Estradiol, pg/ml	20.2 ± 4.1	70.8 ± 5.7	16.0 ± 3.9§	28.1 ± 3.9	76.3 ± 5.2	23.7 ± 4.4§	17.7 ± 4.6	61.8 ± 5.6	12.2 ± 4.1‡§	20.4 ± 5.0	73.2 ± 6.3	24.0 ± 3.5§
Progesterone, ng/ml	5.9 ± 1.9	31.3 ± 5.0	7.2 ± 1.8§	4.9 ± 1.6	35.3 ± 4.2	3.5 ± 1.0§	3.9 ± 1.1	28.4 ± 5.6	5.3 ± 2.2§	4.0 ± 1.1	32.2 ± 3.9	6.1 ± 0.9§
Follicle-stimulating hormone, ng/ml	6.8 ± 1.6	8.9 ± 2.9	26.4 ± 5.2§	5.5 ± 0.8	10.5 ± 2.2	30.1 ± 4.6§	4.2 ± 1.1	10.9 ± 2.7	34.4 ± 5.6§	6.0 ± 1.1	9.2 ± 2.1	28.6 ± 5.4§
Luteinizing hormone, ng/ml	5.2 ± 0.9	7.2 ± 2.0	32.4 ± 5.7§	4.1 ± 0.6	5.9 ± 1.1	28.4 ± 4.2§	2.9 ± 0.8	8.4 ± 1.7	29.1 ± 4.9§	4.5 ± 0.7	6.0 ± 1.2	27.0 ± 3.9§

Values are means ± SE. LZR, lean Zucker rats; OZR, obese Zucker rats; OVX, ovariectomy.

^*P < 0.05 vs. LZR under control conditions (for that condition);

^†P < 0.05 vs. LZR subjected to the unpredictable chronic mild stress (UCMS) model;

^‡P < 0.05 vs. OZR under control conditions;

^§P < 0.05 vs. matched female rats.

Behavioral Responses to UCMS and Metabolic Syndrome

Figure 1 shows behavioral data and severity of depressive symptoms after UCMS in LZRs and OZRs. Throughout the UCMS protocol, OZRs demonstrated a consistently higher coat score than matched LZRs; grooming coat scores were lower for UCMS OZRs (male, female, and OVX female) than LZRs (Fig. 1A). Results from the novelty suppressed feeding test showed a similar pattern in OZRs and LZRs; UCMS delayed time to consumption in all groups, although the hyperphagic behavior inherent in OZRs only allows for meaningful comparison of control with UCMS OZRs and not with LZRs (Fig. 1B). In terms of latency to groom (Fig. 1C) and frequency of grooming (Fig. 1D), metabolic syndrome or UCMS impaired responses to varied extents between male and female rats; however, UCMS and metabolic syndrome in combination had a greater impact than either condition alone for both sexes, and UCMS resulted in no significant differences between male and female OZRs.

Fig. 1.

Behavioral responses of male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). Data are presented for coat score (A), novelty suppressed feeding response (B), and latency to start grooming (C) and frequency of subsequent grooming (D) after sucrose spray. Values are means ± SE; n = 6 animals/group. *P < 0.05 vs. LZR control (for that condition); †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray bars) are shown to facilitate comparisons.

Reactivity of Ex Vivo Aortic Rings

Data describing the methacholine-induced reactivity of aortic rings from male and female LZRs and OZRs under control conditions and after UCMS are shown in Fig. 2. An increase in agonist concentration resulted in a relaxation that was attenuated in control male and female OZRs compared with the respective LZRs, although the reactivity was better maintained in control female OZRs than male OZRs (responses in control OVX female OZRs were very similar to those in control male OZRs). UCMS not only increased the severity of the vascular dysfunction in all groups of OZRs but also abolished any differences in methacholine-induced reactivity between female and male OZRs, such that both sexes (and OVX female OZRs) demonstrated a comparable abrogation of relaxation.

Fig. 2.

Relaxation of ex vivo aortic rings in response to increasing concentrations of methacholine in male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). Values are means ± SE; n = 6 animals/group. *P < 0.05 vs. LZR control (for that condition); †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray symbols) are shown facilitate comparisons.

Figure 3 shows the impact of targeted pharmacological inhibition against two fundamental mechanisms underlying methacholine-induced vasorelaxation of aortic rings: inhibition of NO production using l-NAME and inhibition of PGI₂ production using indomethacin. In response to treatment with l-NAME, vessels from all control OZRs (male, female, and OVX female) exhibited a significant reduction in reactivity, although this effect was greatest in female OZRs (Fig. 3A). With the imposition of UCMS in OZRs, the impact of l-NAME on methacholine-induced relaxation was significantly reduced and assumed a level that was not different between groups of OZRs. Treatment with indomethacin resulted in a modest but significant impairment to relaxation to methacholine across all OZR groups under control and UCMS conditions (Fig. 3B), whereas responses to l-NAME + indomethacin resulted largely in an additive effect of both agents alone (Fig. 3C). Treatment with the antioxidant tempol improved methacholine-induced relaxation in the three groups of OZRs under control conditions, and this effect was increased to a similar extent under combined conditions of metabolic disease and UCMS in all groups of OZRs (Fig. 3D).

Fig. 3.

Relaxation of ex vivo aortic rings in response to increasing concentrations of methacholine in male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). Data are presented as differences in the lower bound of methacholine-induced reactivity for aortic rings under control (untreated) conditions and after acute inhibition of nitric oxide synthase with N-nitro-l-arginine methyl ester (l-NAME; A), acute inhibition of cyclooxygenase with indomethacin (INDO; B) and l-NAME + INDO (C), and pretreatment with the antioxidant tempol (D). Values are means ± SE; n = 6 animals/group. *P < 0.05 vs. LZR control (for that condition); †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray bars) are shown to facilitate comparisons.

The mechanical characteristics of isolated MCAs at the equilibration pressures across the groups are shown in Table 3. With the exception of the impact of the combination of metabolic syndrome and UCMS in OZRs for a reduction in the passive diameter of the MCA, there were no consistent differences between groups that achieved statistical significance.

Table 3.

Baseline characteristics of isolated middle cerebral arteries at the equilibration pressures

	Control						UCMS
	LZR l			OZR			LZR			OZR
	Male	Female	OVX	Male	Female	OVX	Male	Female	OVX	Male	Female	OVX
Equilibration pressure, mmHg	84 ± 4	81 ± 3	82 ± 4	106 ± 4*	105 ± 4*	113 ± 3*	89 ± 5	83 ± 3	90 ± 5	110 ± 4*†	114 ± 3*†	117 ± 4*†
Active inner diameter, μm	118 ± 4	114 ± 5	115 ± 4	112 ± 3	115 ± 4	114 ± 4	108 ± 5	114 ± 5	110 ± 4	108 ± 4	108 ± 4	109 ± 3
Passive inner diameter, μm	190 ± 4	188 ± 4	186 ± 5	185 ± 4	186 ± 5	187 ± 4	182 ± 5	186 ± 6	178 ± 5	170 ± 4*†‡	176 ± 5	172 ± 4‡
Active tone, %	37 ± 3	39 ± 4	38 ± 4	39 ± 2	38 ± 3	39 ± 3	40 ± 3	38 ± 4	38 ± 3	36 ± 3	38 ± 3	37 ± 2

Values are means ± SE. LZR, lean Zucker rats; OZR, obese Zucker rats; OVX, ovariectomy. Equilibration pressure is defined as 80% of the animal’s mean arterial pressure.

^*P < 0.05 vs. LZR under control conditions (for that condition);

^†P < 0.05 vs. LZR subjected to the unpredictable chronic mild stress (UCMS) model;

^‡P < 0.05 vs. OZR under control conditions.

Reactivity of Isolated MCAs

The dilator reactivity of isolated MCAs from OZRs under control conditions and in response to UCMS is shown in Fig. 4. In agreement with our observations in conduit arteries, MCAs from OZRs under control conditions demonstrated an impaired vasodilator reactivity to ACh compared with MCAs from LZRs, with less impaired responses in vessels from control female OZRs than control male or control OVX female OZRs. In all OZR groups, UCMS abolished any differences in MCA dilation to ACh; vessels from all three groups of OZRs (male, female, and OVX female) subjected to UCMS presented similar degrees of very poor dilator reactivity.

Fig. 4.

Dilation of isolated middle cerebral arteries (MCAs) in response to increasing concentrations of acetylcholine in male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). Values are means ± SE; n = 6 animals/group. *P < 0.05 vs. LZR control (for that condition); †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray symbols) are shown to facilitate comparisons.

The impact of acute pharmacological intervention on the ACh-induced dilation of MCAs from male, female, and OVX female OZRs under control conditions and after 8 wk of UCMS is shown in Fig. 5. Pretreatment of MCAs with l-NAME resulted in a significant reduction in reactivity in OZRs of both sexes and after OVX in female rats under control conditions, although this effect was greatest in MCAs from intact female rats (Fig. 5A). The impact of l-NAME on MCA dilation in OZRs after UCMS was significantly reduced, and there were no differences between sexes. These relationships were not substantially impacted by l-NAME + indomethacin (Fig. 5C), and indomethacin alone (Fig. 5B) had minimal impact on the reactivity of MCAs to increasing concentrations of ACh in OZRs under control conditions or after 8 wk of UCMS. Pretreatment of MCAs with the antioxidant tempol increased reactivity of vessels from all groups of OZRs under control conditions, with the greatest effect in male and OVX female OZRs (Fig. 5D). The magnitude of the effect was similar between control and UCMS conditions in male and OVX female OZRs but was significantly increased in MCAs from female OZRs.

Fig. 5.

Dilation of isolated middle cerebral arteries (MCAs) in response to increasing concentrations of acetylcholine in male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). Data are presented as differences in the upper bound of acetylcholine-induced reactivity for MCAs under control (untreated) conditions and after acute inhibition of nitric oxide synthase with N-nitro-l-arginine methyl ester (l-NAME; A), acute inhibition of cyclooxygenase with indomethacin (INDO; B) and l-NAME + INDO (C), and pretreatment with the antioxidant tempol (D). Values are means ± SE; n = 6 animals/group. *P < 0.05 vs. LZR control (for that condition); †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray bars) are shown to facilitate comparisons.

The myogenic activation of MCAs in response to increasing intravascular pressure is shown in Fig. 6. While metabolic syndrome resulted in an increase in myogenic activation of MCAs from male and OVX female OZRs, metabolic syndrome alone had no significant impact on myogenic activation of MCAs from intact female OZRs. However, while the combination of metabolic syndrome and UCMS had no significant additive effect compared with metabolic syndrome alone in male and OVX female OZRs, myogenic activation of MCAs from intact female OZRs after UCMS was not different from that in the other groups.

Fig. 6.

A: myogenic activation of isolated middle cerebral arteries (MCAs) in response to increasing intravascular pressure in male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). B: slope coefficient (β) from a linear regression fit describing myogenic activation of MCAs from each group. Values are means ± SE; n = 6 animals/group. *P < 0.05 vs. LZR control (for that condition); †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray symbols and bars) are shown to facilitate comparisons.

Data describing the effects of metabolic disease and UCMS on the mechanics of the MCA wall in male, female, and OVX female OZRs are shown as the slope coefficient (β) from the stress-strain relationship in Fig. 7. Metabolic syndrome alone had minimal impact on β from the stress-strain relationship in MCAs of any of the groups of OZRs. However, the combination of metabolic syndrome and UCMS resulted in a significant increase in β (a leftward shift of the stress-strain relationship) in male and both groups of female OZRs compared with responses in any other group. There were no differences in β between male and female (intact or OVX) OZRs after the imposition of UCMS.

Fig. 7.

Slope coefficient (β) describing the stress-strain relationship for isolated middle cerebral arteries (MCAs) in response to increasing intravascular pressure under passive conditions in male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). Values are means ± SE; n = 6 animals/group. †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray bars) are shown to facilitate comparisons.

Alterations to Vasoactive Metabolite Levels

The vascular metabolism of arachidonic acid to PGI₂ or TxA₂ under the conditions of the present study is shown in Fig. 8. Compared with responses in LZR groups, OZR groups demonstrated decreased production of PGI₂ (from levels of 6-keto-PGF_1α), with corresponding increases in the production of TxA₂ (from 11-dehydro-TxB₂). This effect was exacerbated by imposition of UCMS in the setting of metabolic syndrome, wherein PGI₂ production was nearly abolished and TxA₂ production was further increased. In vessels from OVX female OZRs, all responses under control or UCMS conditions were comparable to those in vessels from male OZRs under the respective conditions.

Fig. 8.

Vascular production of PGI₂ (A) and thromboxane (Tx)A₂ (B) in male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). Data are presented as production of 6-keto-PGF_1α and 11-dehydro-TxB₂, stable breakdown products of PGI₂ and TxA₂, respectively. Values are means ± SE; n = 6 animals/group. *P < 0.05 vs. LZR control (for that condition); †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray bars) are shown to facilitate comparisons.

Figure 9 shows vascular NO and H₂O₂ levels in OZRs under control conditions and with imposition of the UCMS protocol. Comparable to the pattern in the data determined for PGI₂ and TxA₂, vascular NO levels were reduced (and H₂O₂ production was increased) in all groups of OZRs compared with LZRs under control conditions. After imposition of the UCMS protocol, the decrease in NO levels and increase in H₂O₂ levels were exacerbated compared with control OZRs. Female OZRs were somewhat protected from these changes, with higher NO levels and lower H₂O₂ production than male or OVX female OZRs, although the addition of UCMS abolished any differences in results between the three groups of OZRs.

Fig. 9.

Vascular production of nitric oxide (NO; A) and H₂O₂ (B) in male, female, and ovariectomized (OVX) female lean and obese Zucker rats (LZRs and OZRs, respectively) under control conditions and after 8 wk of unpredictable chronic mild stress (UCMS). Data are presented as the slope of NO or H₂O₂ production in response to increasing concentrations of methacholine (MCh). Values are means ± SE; n = 6 animals/group. *P < 0.05 vs. LZR control (for that condition); †P < 0.05 vs. LZR UCMS (for that condition); ‡P < 0.05 vs. OZR control (for that condition). Data from LZR (gray bars) are shown to facilitate comparisons.

DISCUSSION

In the present study, we used the OZR model of metabolic syndrome to address the impact of preexisting metabolic disease on the effects of chronic stress and depression for vascular health. Given the increasing prevalence of clinical depression in society (52), which parallels the prevalence of metabolic diseases (38), with clear correlations (34), the impact of their combined presentation on cardiovascular disease risk and vascular health warrants investigation. We also evaluated the impact of preexisting metabolic disease on the vascular protection from UCMS identified in female LZRs compared with male or OVX female LZRs (9).

A major observation of the present study is that the detrimental effect of chronic stress and elevated depressive symptoms on vascular reactivity in control, otherwise healthy rats that was identified in the companion study (9) was enhanced in conduit arteries and cerebral resistance arterioles of OZRs, such that responses to methacholine or ACh, respectively, were further abrogated beyond the extent of UCMS or metabolic syndrome alone (5, 12, 19, 39). The combination of UCMS and metabolic syndrome results in elevated cardiovascular/cerebrovascular disease risk, and this appears to represent a condition wherein normal endothelial function is attenuated, with minimal levels of NO and PGI₂ and high levels of vascular TxA₂ production. While this outcome appears to stem from the prooxidant and proinflammatory environment associated with metabolic syndrome and chronic stress/depressive symptoms, cardiovascular disease risk will be elevated through multiple means, including vasomotor control, development of proatherosclerotic (18) and prothrombotic (35) phenotypes, and, potentially, significant effects on venular and venous function through elevated leukocyte adhesion/rolling (44) and fluid balance (42).

The combined impact of UCMS and metabolic syndrome did not alter myogenic activation of MCAs beyond that previously determined for metabolic syndrome alone (12, 39), supportive of previous reports of constrictor reactivity of resistance vessels in mouse models (17, 19, 26, 45). This has several implications, including the possibility that UCMS does not directly impact vascular smooth muscle function but largely works through alterations in endothelial function or neurohumoral pathways that may not be discernible when ex vivo preparations are used. However, the increased myogenic activation in MCAs from female OZRs after UCMS suggests that this may have contributed to the further loss of dilator reactivity in those animals with the additional presence of depressive symptoms.

It has been suggested that the cerebral resistance circulation may be somewhat protected, compared with the peripheral circulation, from changes to vascular wall mechanics that are associated with metabolic syndrome (12, 39), although this outcome can be variable (10). However, the present results suggest that combined imposition of metabolic syndrome and UCMS attenuated this protection and resulted in a decreased distensibility (or increased stiffness) of the MCA wall of OZRs. This shifted stress-strain relationship for MCAs from OZRs with UCMS may lead to poor cerebrovascular outcomes through an increased likelihood that pressure waves from the cardiac cycle could penetrate into the cerebral microcirculation and, thus, impact hemodynamics and microvascular wall structures in the more distal microcirculation. Future investigation into the mechanisms of this remodeling effect and its potential for blunting or reversibility via intervention may be justified.

The results from the present study suggest that the ability of healthy LZRs to tolerate chronic stress/depressive symptoms or OZRs to tolerate metabolic syndrome and maintain moderate vascular function is overwhelmed by the combined presentation of metabolic disease and UCMS. As evidenced by the data shown in Tables 1–3 and Figs. 8 and 9, the profile of vasoactive metabolites is further altered by the combined presentation of both conditions, such that the evolving prooxidant and proinflammatory environment within the vasculature nearly abolished NO and PGI₂ and further elevated TxA₂ production. While vascular H₂O₂ production is elevated in OZRs with UCMS, potentially as a compensatory mechanism in the setting of low NO levels (6), its effectiveness may be limited, as overall vascular reactivity remains attenuated.

The second major aspect of this study was to determine the impact of preexisting metabolic disease in OZRs on the sex-based protection from UCMS identified in otherwise healthy female LZRs (9). Based on the present results, it seems apparent that female LZRs can somewhat buffer the impact of UCMS and that female OZRs can somewhat buffer the impact of metabolic syndrome, while better maintaining vascular reactivity than male LZRs with UCMS or male OZRs, respectively. Thereby, female rats may be better able to maintain endothelial function, despite the challenged environment, manifested through improved vascular NO and PGI₂ levels and maintenance of low TxA₂ production. This partial protection appears to be associated with normal levels of sex hormones, as early OVX of OZRs in the present study or of LZRs in the companion study (9) abolished the vasoprotective effects associated with chronic stress/depressive symptoms or metabolic disease, respectively, and this was reflected in the shifts in endothelial function and the profile of vasoactive metabolite levels. It is important to recognize that, especially in female OZRs, the combined presentation of metabolic syndrome and UCMS increased the severity of both hypertension and insulin resistance. As such, the well-documented impact of these conditions alone on vascular reactivity must be taken into consideration when the results of the present study are interpreted.

As OVX of female OZRs at a very early age and before onset of the UCMS protocol resulted in outcomes that were extremely similar to those in male OZRs after UCMS, the results of the present study support the broader concept of the importance of circulating estrogen and, potentially, progesterone levels in maintaining vascular health and conferring vascular protection (15). However, in OZRs of either sex and with OVX, estrogen levels were comparable to and, in some cases, higher than those determined in matched LZRs, regardless of control or UCMS condition. This suggests that the beneficial effects of circulating estrogen, even if supplemented by nonovarian-based production of estrogen due to the increased adiposity in OZRs (11), are insufficient to maintain significant vascular protection under the conditions of the present study. Caution should be used in interpreting results from the early (at 5–6 wk of age) OVX of female rats in the present study, as this is more relevant to a condition wherein circulating estrogen levels were never truly established, rather than a condition defined by the loss of postpubertal estrogen levels. Additional experiments are required to determine whether the results of the present study are comparable to a condition wherein sex hormone levels had been established and then were abolished. Given the importance of estrogen in terms of maintaining vascular endothelial function through multiple pathways (15, 30), the mechanistic failure of the vascular environment and how the combination of metabolic disease and UCMS can overwhelm the protective effects of estrogen are critical areas of future study.

GRANTS

This study was supported by American Heart Association Grants IRG 14330015, PRE 16850005, and EIA 0740129N; National Institutes of Health Grant RR-2865AR; and Canadian Natural Sciences and Engineering Research Council Grant R4081A03.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

S.D.B., S.H., P.D.C., K.C.L., S.F., D.N.J., and J.C.F. conceived and designed research; S.D.B. and J.C.F. performed experiments; S.D.B., S.H., P.D.C., S.M., K.C.L., S.F., J.K.S., D.N.J., and J.C.F. interpreted results of experiments; S.D.B., S.M., K.C.L., S.F., D.N.J., and J.C.F. drafted manuscript; S.D.B., S.H., P.D.C., S.M., K.C.L., S.F., J.K.S., D.N.J., and J.C.F. edited and revised manuscript; S.D.B., S.H., P.D.C., S.M., K.C.L., S.F., J.K.S., D.N.J., and J.C.F. approved final version of manuscript; S.F. and J.C.F. analyzed data; J.C.F. prepared figures.