Interleukin-17 induces hypertension but does not impair cerebrovascular function in pregnant rats.

Abstract

Preeclampsia affects 5–8% of pregnancies and is characterized by hypertension, placental ischemia, neurological impairment, and an increase in circulating inflammatory cytokines, including Interleukin-17 (IL17). While placental ischemia has also been shown to impair cerebrovascular function, it is not known which placental-associated factor(s) drive this effect. The purpose of this study was to examine the effects of IL17 on cerebrovascular function during pregnancy. To achieve this goal, pregnant rats were infused with either IL17 (150 pg/day, 5 days, osmotic minipump), or vehicle (saline/0.7% BSA osmotic minipump) starting at gestational day (GD) 14. On GD 19, the cerebral blood flow (CBF) response to increases in mean arterial pressure (MAP) was measured in vivo, and myogenic constrictor responses of the middle cerebral artery (MCA) were assessed ex vivo. IL17 increased MAP but impaired CBF responses only at the highest arterial pressure measured (190 mmHg). Myogenic constrictor responses overall were mostly unaffected by IL17 infusion; however, the intraluminal pressure at which peak myogenic tone was generated was lower in the IL17 infused group (120 vs 165 mm Hg), suggesting maximal tone is exerted at lower intraluminal pressures in IL17-treated pregnant rats. Consistent with the lack of substantial change in overall myogenic responsiveness, there was no difference in cerebral vessel expression of putative mechanosensitive protein βENaC, but a tendency towards a decrease in ASIC2 (p=0.067) in IL17 rats. This study suggests that infusion of IL17 independent of other placental ischemia-associated factors is insufficient to recapitulate the features of impaired cerebrovascular function during placental ischemia. Further studies to examine of the role of other pro-inflammatory cytokines, individually or a combination, are necessary to determine mechanisms of cerebral vascular dysfunction during preeclampsia.

INTRODUCTION

Preeclamptic patients often experience neurological complications and cerebrovascular events are reported to account for 40% of all preeclampsia/eclampsia-related deaths [1,2]. Placental ischemia induces the release of several inflammatory and anti-angiogenic factors which are considered to have a central role in the pathophysiology of preeclampsia. Placental ischemia in human patients, and in a rodent model of reduced uterine perfusion pressure (RUPP), causes 1) an increase in circulating pro-inflammatory cytokines, including Interleukin-17 (IL17), 2) impaired myogenic constriction, and 3) impaired cerebral blood flow (CBF) autoregulation [1,3–8]. CBF autoregulation is defined as the ability of the cerebral vasculature to regulate blood flow according to local tissue demands. Myogenic constriction is an important mechanism that contributes to CBF autoregulation. Increased perfusion pressure to small arteries and arterioles causes the vessel wall to stretch, resulting in an initial increase in vascular diameter. The increased diameter lengthens vascular smooth muscle cells (VSMC) wrapped circumferentially around the vessel, initiating a reflexive shortening and vasoconstriction [9]. Vasoconstriction in response to an increase in perfusion pressure prevents tissue hyperperfusion. In contrast, loss of myogenic constriction may result in hyperperfusion which is associated with increases blood-brain-barrier permeability, cerebral edema, neurological complications and brain injury, all features observed in preeclampsia [10–12]. While there are a number of vascular proteins involved in the mechanotransduction of the myogenic response in vessels, both βENaC and ASIC2 have been shown to play an integral role in sensing smooth muscle stretch [9,13–15].

Evidence suggests that circulating IL17 is increased in preeclamptic women and also in placental ischemic rats caused by RUPP, and that this contributes to the elevated blood pressure [7,8,16,17]. Furthermore, IL17 plays an integral role in vascular dysfunction [18,19]. These findings suggest an association between placental ischemia, cerebrovascular function, and IL17 (pro-inflammatory cytokine). However, it is unknown whether IL17 has a direct effect, independent of the ischemic placenta, on the impairment of cerebrovascular function during pregnancy. Therefore, the goal of this study is to test the hypothesis that IL17 directly impairs CBF autoregulation and myogenic constriction during pregnancy. To determine if there was an association between cerebrovascular function and cerebrovascular expression of 2 putative mechanosensing proteins involved in the myogenic response, cerebrovascular βENaC and ASIC2 was assessed [9,13–15]. The results of this study demonstrate that IL17 infusion superimposed on a normal pregnancy is insufficient to recapitulate features of impaired cerebral vascular function associated with placental ischemia, i.e. loss of myogenic control and blood flow autoregulation.

METHODS

Animals.

Timed-pregnant Sprague-Dawley (CD) rats (Charles Rivers Laboratories) arrived at the Lab Animal Facilities at the University of Mississippi Medical Center (UMMC) on gestational day (GD) 9–13. All animal procedures are conducted at UMMC with approval by the Institutional Animal Care and Use Committee. Rats are randomly assigned to groups and maintained on a 12-hour light/dark cycle with access to food and water ad libitum.

IL17 infusion.

On GD 14, osmotic minipumps containing recombinant mouse IL17A (R & D Biosystems; Cat# 421-ML) are surgically implanted intraperitoneally [20]. GD 14 coincides with surgical implantation of vascular clips in our rat model of reduced uterine perfusion. Pumps are loaded with either IL17 reconstituted in sterile saline with 0.7% BSA or saline/0.7% BSA alone (vehicle control) and delivered at a constant rate of 150 pg/24 hr. Cerebral vessels and plasma samples from dams are collected on GD 19, froze in liquid nitrogen, and stored at -80°C for later use.

Blood Pressure Measurements.

On GD 18, the right carotid artery of pregnant rats is catheterized to measure arterial pressure on GD 19. Blood pressure is recorded in conscious animals after 45 min of acclimation to restrainer cages for a minimum of 30 minutes using LabChart (AD Instruments) [20].

Measurement of Cerebral Blood Flow.

Immediately following blood pressure measurement, vehicle (n=8) and IL17-infused (n=8) pregnant rats are anesthetized using 50 mg/kg Inactin (i.p.) and 30 mg/kg ketamine (i.m.) on GD 19. The trachea is cannulated using PE-200 tubing and connected to a ventilator to maintain constant peak expired CO2, an indicator of blood CO₂ [4,21]. The head is secured in a stereotaxic frame and an incision made to expose the skull. Two closed, 4 mm x 4 mm, cranial windows are drilled at approximately 3–4 mm lateral and 2 mm distal to Bregma [4]. Probe retainers are affixed over dorsal branches of the middle cerebral artery and blood flow is measured with a Perimed 5000 dual channel laser Doppler Flowmeter. Arterial pressure is measured by a carotid artery catheter surgically implanted on GD 18 in all rats as described in the previous paragraph. CBF is recorded continuously while MAP is increased at 20 mm Hg intervals from approximately 100 to 190 mm Hg by graded infusion of phenylephrine (50 μg/mL) via the left femoral vein catheter. MAP and peak expired CO₂ are recorded throughout using. Expired PCO₂ is maintained below 50 mm Hg for each experiment. CBF recordings are averaged between the hemispheres. In cases where a probe experienced technical difficulties acquiring laser Doppler reading, CBF recordings were taken exclusively from the remaining functioning probe.

Pressure-, KCl-, and α-adrenergic receptor-induced vascular reactivity.

To determine if changes in vascular reactivity might account for changes in cerebral blood flow responses, we evaluated in vitro vascular reactivity in middle cerebral artery (MCA) segments in a separate group of vehicle and IL17-infused animals (n=6/group). For these experiments, rats are decapitated, brains immediately removed and placed in ice cold physiological salt solution (PSS) composed of (in mM: 130.0 NaCl, 4.0 KCl, 1.8 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 6.0 glucose, 4.0 NaHCO₃, 10 HEPES, and 0.03 EDTA), then equilibrated with a gas mixture of 95% O₂ and 5% CO₂, at pH ~7.4. MCA segments, approximately 1 mm in length, are carefully dissected under a stereo microscope and mounted on glass cannulas in a Living Systems CH1 chamber. Pressure is initially equilibrated at 0 mm Hg for 30 min, then set to 75 mm Hg for 15 min at 37°C. Inflow pressure is controlled using a pressure servo-control system. The inner and outer diameters are measured by microscopy using MetaMorph software (Universal Imaging, Downingtown, PA).

Increasing concentrations of KCl (from 4 to 20, 40, and 80 mM) are used to test the depolarization-induced constriction of the vessels following equilibration to 75 mm Hg. Phenylephrine (PE; 10⁻⁷–10⁻⁴ M) is used to test a adrenergenic-receptor agonist-induced constriction to ensure the viability of the segments at the end of the experiment. Any segments that fail to constrict by less than 50% to KCl are excluded from the study. Agonist-induced constrictor responses are assessed at 75 mm Hg.

Measurement of myogenic constrictor responses.

Myogenic constriction is determined by recording diameter responses to by 25 mm Hg stepwise increases in intraluminal pressure from 25 to 175 mm Hg in Ca²⁺ containing PSS to assess active tone. An image is collected after 5 min at each pressure step. The protocol is repeated after a 30 min incubation in Ca²⁺-free PSS to assess passive tone and the stepwise increase in intraluminal pressure response repeated. The Ca²⁺ free solution is identical to the Ca2+ containing solution except CaCl2 is replaced with 2.0 mM/L EGTA [3,13]. Pressure-induced constrictor responses are calculated as myogenic tone (%) using the following formula: ((D_P-D_A)/D_P) x 100, where D_P is passive diameter and D_A is active diameter of the vessels at a given intraluminal pressure value. Circumferential strain of the vessel wall is calculated using the following formula: (D_P–D₁₅)/D₁₅), where D_P is the passive diameter at a given intraluminal pressure and D₁₅ is the passive diameter at 25 mm Hg under Ca2⁺-free conditions. Circumferential stress is calculated using the following formula: P × D_P/(2 × WT), where D_P is passive diameter, WT is wall thickness and P is intraluminal pressure (where 1 mm Hg=1.334 × 102 N/m²) under Ca^2+-free conditions.

Measurement of cerebral vascular putative mechano-signaling proteins βENaC and ASIC2.

To determine if IL17 infusion altered the expression of βENaC and ASIC2, we used western blot to study changes in protein expression in surface cerebral vessels of vehicle and IL17-infused animals (n=10–12/group). For these experiments, vessels were collected and snap frozen in liquid nitrogen. Samples were homogenized in using Biovision kit #268 and isolated into soluble and total membrane associated fractions. The lysates were collected and stored at -70º C before use. Approximately 15 μg of soluble lysate were separated on 7 and 10% gels (Biorad Criterion), transferred to nitrocellulose then probed with rabbit anti βENaC_C-term (1:2000) or ASIC2_C-term (1:2000), antibodies developed and extensively characterized by our laboratory [13,22,23], followed by labeling with mouse anti-β-actin (1:10,000) and mouse anti-vinculin (1:1000) to control for loading. Total membrane associated fractions provided inconsistent results due to difficulty disrupting protein-protein interactions. Thus, we used soluble fractions to assess βENaC and ASIC2 expression. Membranes were then labeled with donkey anti-rabbit IgG conjugated to IR700 and donkey anti-mouse IgG conjugated to IR800 (1:20,000, Odyssey). Protein labeling was visualized using the Odyssey LiCor Scanner.

Statistical Analysis.

CBF values are plotted as percent change from baseline versus absolute MAP. Differences in values measured at each MAP interval are determined. For CBF and myogenic studies (Figures 2, 3, and 4A-C), groups are compared using repeated measures two-way ANOVA followed by two stage linear step-up procedure of Benjamini, Krieger and Yekutieli Post-hoc analysis (to correct for multiple comparisons). A two-tailed t-test was used for morphometric and vascular parameters (Tables 1 and 2, Figure 4D) and conscious mean blood pressure (Figure 1). A one-tailed t-test was used on Western blot data because the direction of change was predicted a priori. All statistical analyses are performed using Prism software and data are presented as Mean ± SEM. A value of p≤0.05 is statistically significant. Result “trends” are also reported to avoid erroneous true/false conclusions based on bright-line rules. Certain p values are provided to demonstrate confidence.

Figure 2. IL17 impairs CBF responses at high perfusion pressures.

CBF responses to stepwise increases of MAP are recorded. (A) IL17 (n=8) increased CBF at pressures at 190 mm Hg compared to vehicle (n=8) pregnant rats. (B) Exhaled CO₂ is consistently maintained throughout the experiments. There was a significant effect of MAP on CBF (p<0.0001), but no effect of IL17 treatment (p=0.341), and a trend towards interaction between MAP and treatment (p=0.0855) using a repeated measures two-way ANOVA. A two stage linear step-up procedure of Benjamini, Krieger and Yekutieli Post-hoc analysis identified a difference between IL17 and control groups only at 190 mm Hg. Data are presented as Mean ± SEM.

*Significantly different from vehicle at p<0.05.

Figure 3. Vasoconstrictor responses of isolated middle cerebral arteries (MCAs) to depolarizing and α-adrenoreceptor agonist agents are identical to vehicle control in IL17-infused rats.

(A) Vasoconstriction responses of isolated MCAs from IL17 (n=6) and vehicle (n=6) rats to KCl are identical. Two-way repeated measures ANOVA showed an effect of concentration (p<0.0001), but no effect of treatment (p=0.597), and trend towards interaction of treatment and concentration (p=0.0712) on vasoconstriction. (B) Normalized vasoconstriction responses of isolated MCAs are similar to absolute responses shown in Panel A. ( C) Vasoconstriction responses of isolated MCAs from IL17 and vehicle rats to PE are similar. Two-way repeated measures ANOVA showed an effect of concentration (p<0.0001), but no effect of treatment (p=0.945), or interaction of treatment and concentration (p=0.988) on vasoconstriction.

*Significantly different from vehicle at p<0.05.

Figure 4. IL17 has minor effect on myogenic vasoconstriction of isolated middle cerebral arteries (MCAs) at higher intraluminal pressures.

Myogenic constriction of isolated MCAs from IL17 and vehicle rats in response to stepwise increases in intraluminal pressure under active (Ca²⁺-containing, Panel A) and passive (Ca ²⁺-free, Panel B) conditions are similar. Two-way repeated-measures ANOVA showed an effect of pressure (p<0.0001), but no effect of IL17 (p=0.8264) and interaction of IL17 and pressure (p=0.222) under active conditions. Two-way repeated measures ANOVA showed an effect of pressure (p<0.0001), but no effect of IL17 (p=0.905) and interaction of IL17 and pressure (p=0.7634) under passive conditions. (C) The relationship between intraluminal pressure and calculated myogenic tone was similar in vehicle and IL treated animals. There is a significant effect of MAP on myogenic constrictor responses (p<0.0001) revealed by repeated measures two-way ANOVA, but not for treatment (p=0.7035) or their interaction (p=0.3205). (D) The intraluminal pressure at which maximal myogenic tone is developed is reduced in IL17-infused rats (p=0.0019). These data are analyzed using a two-tailed, independent t-test in IL (n=6) and vehicle (n=6) infused rats. All data presented as Mean ± SEM.

**Significantly different from vehicle at p<0.001.

Table 1.

General animal characteristics at gestational day 19. Maternal weight, number of live pups, organ weight and percent of resorbed (non-viable) pups are measured in pregnant rats. Comparisons aie made using a two-tailed independent t-test. Data are presented as Mean ± SEM; p values aie provided to demonstrate confidence.

Characteristics	Control (n)	IL-17 (n)	p values
Maternal Weight (g)	319 ± 9 (9)	315 ± 8 (11)	0.554
Maternal Heart Weight (g)	0.93 ± 0.05 (11)	0.98 ± 0.06 (10)	0.530
Pup Weight (g)	2.64 ± 0.08 (11)	2.68 ± 0.06 (11)	0.671
Placental Weight (g)	0.52 ± 0.03 (11)	0.58 ± 0.02 (11)	0.100
Number of Live Pups	12.9 ± 0.6 (11)	12.6 ± 0.8 (11)	0.720
% Resorptions	1 ± 0 (11)	1 ± 0 (10)	0.530

Table 2.

Phenotypic characteristics of the MCA in pregnant rats. The inner and outer diameter (μm), wall thickness (μm), and wall to lumen ratio of the MCA are examined in pregnant rats. Comparisons made using a two-tailed independent t-test. Data are presented as Mean ± SEM; p values are provided to demonstrate confidence.

Characteristics	Control (n = 6)	IL-17 (n = 6)	p values
Inner diameter (μm)	108.84 ± 6.83	120.09 ± 4.3	0.19
Outer diameter (μm)	129.18 ± 6.7	137.81 ± 4.2	0.29
Wall thickness (μm)	20.34 ± 1.09	17.72 ± 0.78	0.08
Wall-to-lumen ratio	0.18 ± 0.02	0.15 ± 0.02	0.26

Figure 1. IL17 increases MAP in pregnant rats.

Mean arterial pressure was assessed on GD 19. IL17 increased MAP compared to vehicle pregnant rats. Comparisons made using a student t-test. Data are presented as Mean ± SEM.

*Significantly different from control at p<0.05.

RESULTS

IL17 infusion on maternal and fetal aspects.

On GD 19, maternal weight, maternal heart weight, pup weight, number of live pups, and % pup resorptions were not different between vehicle treated and IL17 treated animals. There was a tendency for increase placental weight (0.58±0.02 vs. 0.52±0.03 g; p=0.100; Table 1) in the IL17 infused animals. MAP is significantly increased from 100±3 mm Hg in control pregnant rats to 112±3 mm Hg in IL17-infused pregnant rats (p=0.004; Figure 1).

IL17 has minimal effect on cerebral blood flow control.

CBF autoregulation is measured as a percent change in CBF from baseline 100 mm Hg and at MAPs from 120–190 mm Hg. While we did find a significant effect of MAP on CBF (p<0.0001), we did not find a significant effect of IL17 alone on % CBF (p=0.341) nor an interaction between MAP and treatment (p=0.086) using a repeated measures two-way ANOVA. Post hoc analyses indicated CBF responses are significantly increased in IL17-infused rats only at the highest pressure of 190 mm Hg (215±18% vs. 160±22; p=0.0469). These findings suggest IL17 has a minimal effect on CBF exerted outside the autoregulatory range (Figure 2A). There is no significant difference in peak expiration CO₂ levels at any MAP, indicating that arterial CO₂ levels are unlikely to affect CBF responses (Figure 2B).

Myogenic constriction of the MCA is minimally affected by IL17.

Changes in the MCA inner diameter in response to KCl (4–80 mM) and phenylephrine (PE, 10⁻⁷-10⁻³ M) are shown in Figure 3A/B and C, respectively. Vasoconstriction responses to KCl are marginally different between IL17 and vehicle rats. Two-way repeated measures ANOVA on absolute inner diameter responses showed an effect of concentration (p<0.0001), but no effect of treatment (p=0.597) and a trend towards interaction of treatment and concentration (p=0.0712) on vasoconstriction (Fig 3A). Post hoc analysis shows a significant effect of treatment only at 40 mM KCl (p=0.0497). Responses to normalized data reveal a potential interaction between IL17 and depolarization induced constrictor responses. A two way repeated measures ANOVA shows a significant effect of KCl concentration (p<0.0001), a trend towards an effect of treatment (p=0.0715) and significant interaction between treatment and KCl concentration (p=0.0372). Vasoconstriction responses to PE are identical between IL17 and vehicle rats (10⁻³ M, 58.5±5.9 vs 61.1±4.8%, Fig 3C). Two-way repeated measures ANOVA showed an effect of concentration (p<0.0001), but no effect of treatment (p=0.945), and interaction of treatment and concentration (p=0.988) on vasoconstriction. These findings suggest IL17 did not alter vasoconstriction per se in the MCA during pregnancy.

Myogenic vasoconstriction responses, calculated myogenic tone, and intraluminal pressure at peak tone of isolated MCAs from IL17 and vehicle rats are shown in Figure 4. The active (Ca²⁺-containing) and passive (Ca²⁺-free) inner diameters of MCAs from IL17 and vehicle rats are shown in Panels A and B, respectively. There is no difference in active or passive responses between IL17 and vehicle pregnant rats found with a repeated measures two-way ANOVA. Calculated myogenic tone is shown in Panel C. There is a significant effect of MAP on myogenic tone (p<0.0001), but not for treatment (p=0.704) or their interaction (p=0.320, Two-way repeated measures ANOVA). We noticed a subtle difference in the intraluminal pressure-myogenic tone relationship where the myogenic tone seemed to peak, suggesting the intraluminal pressure versus myogenic tone relationship might be altered in IL17-infused animals. To address this possibility, we analyzed the intraluminal pressure at peak myogenic tone for each animal and found myogenic tone peaks at 167±6 mm Hg in vehicle MCAs, but peaks at 121±8 mm Hg (p=0.002) in IL17-infused animals (Fig 4D). These findings suggest that IL17 has a minor effect on myogenic constrictor responses in MCAs.

To determine whether differences in mechanical and morphological properties might contribute to altered myogenic constrictor responses, we calculated the circumferential wall strain and stress under Ca²⁺-free conditions, and the mean vessel wall thickness (Table 2). Circumferential wall stress and strain, wall thickness, and wall-to-lumen ratios are identical between IL17 and vehicle rats (data not shown), suggesting that the mechanical and morphological properties of isolated MCAs were also unchanged in the IL17 rats.

Expression of cerebral vascular βENaC and ASIC2.

Consistent with the functional studies on myogenic responsiveness and cerebral blood flow regulation, expression of the putative mechano-signaling proteins βENaC/β-actin (88 ± 15% of vehicle, p=0.277, Fig. 5) and ASIC2/β-actin (decreased to 62 ± 12% of vehicle, p=0.066, Fig. 6) in cerebral vessel homogenates were either unchanged or tended to be inhibited by IL17 infusion. βENaC was detected near 250 kDa and ASIC2 near 60 kDa. We have observed higher than predicted molecular weight for βENaC in vessel homogenates [24]. Normalization to vinculin, a protein closer in molecular weight to βENaC and ASIC2, did not alter the outcome. Despite the larger sample sizes, n=10–12, substantial variability in the western blot signal contributed to reduced statistical power associated with the analysis.

Figure 5. IL17 has no significant effect on rat cerebral vessel βENaC expression.

(A) Representative western blot of 15 μg total protein from isolated cerebral vessels (n=2 trials) labeled with rabbit (R) anti-βENaC, followed by mouse (M) anti-β-actin and mouse anti-vinculin. (B) Group data of βENaC expression is normalized to β-actin and vinculin. Cerebral vessels from vehicle (n=12) and IL17 (n=11) treated animals. Data are presented as Mean ± SEM and analyzed using independent, one-tailed t-test, p values = 0.277 and 0.17,?ENaC normalized to β-actin and vinculin, respectively.

Figure 6. IL17 has minor effect on ASIC2 expression in rat cerebral vessels.

(A) Representative western blot of 15 μg total protein from isolated cerebral vessels (n=2 trials) labeled with rabbit (R) anti-ASIC2, followed by mouse (M) anti-b-actin and mouse anti-vinculin. (B) Group data of ASIC2 expression normalized to β-actin and vinculin. Cerebral vessels from vehicle (n=12) and IL17 (n=10) treated animals. Data are Mean ± SEM and analyzed using independent, one-tailed t-test, p values=0.067 and 0.0659 for ASIC2 normalized to β-actin and vinculin, respectively.

DISCUSSION

Cerebrovascular-associated encephalopathies frequently occur in preeclampsia/eclampsia, with extreme cases leading to life-long disability or maternal death [25–28]. Disruption of appropriate CBF autoregulation due to impaired myogenic constriction of the cerebral arterial vasculature may contribute to cerebrovascular dysfunction and neurophysiological impairments in preeclampsia/eclampsia [12,25,28]. In experimental models, placental ischemia impairs cerebrovascular myogenic constriction and CBF autoregulation. Loss of autoregulatory function is also associated with increased BBB permeability and brain water content [3,4]. While widespread systemic vascular impairment is a clinical feature of preeclampsia, the processes driving altered CBF autoregulation are unclear. IL17 is a pro-inflammatory cytokine synthesized by T cells invading the ischemic placenta [17]. It is elevated in preeclamptic patients and rodent models and thus may be a contributing factor cerebrovascular dysfunction [7,8,16,17]. While IL17 is associated with endothelial dysfunction, vascular inflammation, and arterial stiffening in humans and animal models with inflammatory disorders, it is unclear whether chronic increases in circulating IL17 during pregnancy impairs cerebrovascular function [19,29–33].

Our main findings suggest that IL17 infusion, superimposed on normal pregnancy, is insufficient to recapitulate loss of cerebrovascular function associated with placental ischemic pregnancies. IL17 did not significantly impair CBF autoregulation or cerebrovascular myogenic tone within the normal autoregulatory range (~50–150 mm Hg), suggesting modest elevations in plasma IL17 alone do not fully account for placental ischemia-induced CBF impairment observed previously (53). Placental-ischemia alters the expression of multiple inflammatory cytokines. Increases in IL17 alone may not be sufficient to cause dramatic vascular responses at these pressures. Thus, IL17 may act synergistically with other cytokines to disrupt cerebrovascular function. Further studies addressing the importance of other placental-derived factors, alone and in combination with IL17, are needed to address this possibility.

A limitation of our CBF autoregulation approach is that we can measure relative, but not absolute, changes in cerebral blood flow in response to increases in pressure. Therefore, we are unable to determine if IL17 alters baseline CBF. We did observe a dysfunction in CBF regulation only observed at the highest perfusion pressure examined (190 mm Hg). While this pressure is outside the autoregulatory range, our finding is potentially meaningful because it is consistent with an increased susceptibility to neurological complications (e.g. seizures) in preeclampic patients experiencing either sustained increases in cerebral perfusion pressure, or in patients who might experience transient blood pressure spikes. The loss of CBF regulation in the current study is similar than those found in our previous studies with infusion of the 1) pro-inflammatory cytokine TNF-α, and 2) agonistic antibody to the AT1 receptor in pregnant rats [22,34]. While TNFα, AT1 auto antibody, and IL17 alone do not impair CBF to the same extent as placental ischemia itself, understanding the roles of specific cytokines could yield new information into how placental ischemia impairs cerebrovascular function in placental ischemia.

Myogenic constriction is the primary mechanism responsible for maintaining pressure-flow relationships at perfusion pressures above the set-point/baseline pressure and is suppressed in placental ischemic rats. Thus the myogenic response plays a prominent role in protecting the parenchyma from hyper-perfusion during transient increases in blood pressure, such as those observed in preeclampic patients [35–37]. We wanted to determine if myogenic constriction might be compromised by IL17 infusion. Evaluation of myogenic responses in MCAs of IL17-infused animals suggests the peak myogenic tone development was largely intact and we only noted that peak myogenic tone developed at lower intraluminal pressures following IL17. It is plausible that IL17, via hypertension-mediated effects, through indirect effects on vascular signaling mechanisms (e.g. ROS), or direct regulation of myogenic proteins may impair the normal mechanoregulatory function of cerebral vessels. During chronic hypertension, the myogenic response tends to adapt and shift towards a higher pressure range [38,39]. The lack of shift may be due to the short duration of hypertension. Alternatively, IL17 may disrupt this hypertensive pregnancy-induced myogenic adaptive response, suggesting that IL17 may alter expression/regulation of proteins involved in transducing the stimulus (stretch/pressure)-response (vasoconstriction) relationship in cerebral VSMCs/vessels.

To address the possibility that IL17 may alter expression of proteins mediating transduction of VSMC stretch, we evaluated expression of the putative mechanosensing proteins βENaC and ASIC2. βENaC and ASIC2 are evolutionarily related to a family of ion channels referred to as degenerins, known mechanosensory proteins that mediate transduction of mechanical stimuli in sensory neurons and muscle in the nematode and fly models. Several studies from our laboratory demonstrate that myogenic responses in cerebral vessels are abolished or attenuated in animal models lacking normal expression of βENaC and ASIC2, respectively [9,13]. In the current study, we found IL17 had no effect on expression of cerebral vascular βENaC, but tended to decrease ASIC2 protein. The reduction in ASIC2 expression in IL17 treated pregnant rats may contribute to the subtle changes in high pressure cerebral blood flow responses and the intraluminal pressure-peak myogenic tone relationship of the MCA in pregnant rats.

IL17 did not substantially change the contractility per se (depolarization- and α-adrenergic-induced contraction), mechanical properties (stress and strain) or morphological aspects of the MCA. Similar to placental ischemic rats, we observed an increase in MAP in IL17-infused rats, but no change in active/passive diameter or wall thickness between either pregnant group [3]. These findings suggest that elevation of plasma IL17 during pregnancy is insufficient to induce the substantial loss cerebral vascular function (myogenic constriction and CBF autoregulation) observed in placental ischemic pregnancies [40–42].

This study highlights the role of the inflammatory cytokine, IL17, on cerebrovascular function during pregnancy. IL17 may have a direct and/or indirect (through inflammation/hypertension/oxidative stress) modulatory effect on cerebrovascular properties during pregnancy and thus may be a likely contributor of neurological impairment in preeclampsia. These findings suggest a need for further investigation of the role of other placental ischemia-derived factors, or a combination thereof, on cerebrovascular function in preeclampsia. It is possible that multiple placental factors act in concert to impair CBF autoregulation in placental ischemia.

Highlights.

• IL17, a potent pro-inflammatory cytokine, increases blood pressure in pregnant rats.

• Cerebral Blood Flow is largely unaffected by IL17, except at high arterial pressures.

• Myogenic Tone of the Middle Cerebral Artery is unaffected by IL17 during pregnancy.

• IL17 causes peak myogenic tone to be achieved at lower arterial pressures.

• Cerebrovascular protein expression of ASIC2, a mechanosensing degenerin, may be involved in IL17-mediated cerebrovascular functional changes.