ABSTRACT
Objective
Endothelial dysfunction is a critical initiating factor in the development of hypertension and related complications. Follistatin-like 1 (FSTL1) can promote endothelial cell function and stimulates revascularization in response to ischemic insult. However, it is unclear whether FSTL1 has an effect on ameliorating endothelial dysfunction in spontaneously hypertensive rats (SHRs).
Methods
Wistar Kyoto (WKY) and SHRs were treated with a tail vein injection of vehicle (1 mL/day) or recombinant FSTL1 (100 μg/kg body weight/day) for 4 weeks. Blood pressure was measured by tail-cuff plethysmograph, and vascular reactivity in mesenteric arteries was measured using wire myography.
Results
We found that treatment with FSTL1 reversed impaired endothelium-dependent relaxation (EDR) in mesenteric arteries and lowered blood pressure of SHRs. Decreased AMP-activated protein kinase (AMPK) phosphorylation, elevated endoplasmic reticulum (ER) stress markers, increased reactive oxygen species (ROS), and reduction of nitric oxide (NO) production in mesenteric arteries of SHRs were also reversed by FSTL1 treatment. Ex vivo treatment with FSTL1 improved the impaired EDR in mesenteric arteries from SHRs and reversed tunicamycin (ER stress inducer)-induced ER stress and the impairment of EDR in mesenteric arteries from WKY rats. The effects of FSTL1 were abolished by cotreatment of compound C (AMPK inhibitor).
Conclusions
These results suggest that FSTL1 prevents endothelial dysfunction in mesenteric arteries of SHRs through inhibiting ER stress and ROS and increasing NO production via activation of AMPK signaling.
Hypertension is one of the most common diseases and its incidence is increasing dramatically worldwide, which will account for 10% of the population by 2025 (Citation1). Hypertension can lead to a series of complications, including chronic renal insufficiency, stroke, coronary heart disease, etc., which are initially manifested as endothelial dysfunction (Citation2). Thus, a strategy to protect endothelial function could be useful as an adjunct therapy for lowering blood pressure and preventing the end-organ damage associated with essential hypertension (Citation3).
Follistatin-like 1 (FSTL1), also known as TSC-36, is a secreted glycoprotein that belongs to the follistatin family of proteins that exists abundantly in human tissue including heart, kidney, skeletal muscle, and vascular endothelium, etc. (Citation4). Circulating FSTL1 levels in a healthy individual are reported to be roughly 1.2–40 pg/mL, and are significantly increased after aerobic training (Citation5). Moreover, several studies report that FSTL1 is a cardiokine upregulated by various cardiac stresses including myocardial infarction and heart failure (Citation6,Citation7). Current data suggest that the increased plasma FSTL1 is a useful biomarker of heart failure and is related with cardiovascular events and mortality (Citation7). Remarkably, FSTL1 can promote and stimulate revascularization in response to ischemic insult through a nitric-oxide synthase-dependent mechanism (Citation8). However, it is unclear whether FSTL1 has any direct effect on improving the impaired vascular endothelial function in hypertensive animal models.
Accumulating evidence indicates that impaired vascular endothelial function is associated with increased endoplasmic reticulum (ER) stress (Citation9). Previous studies have shown that FSTL1 acts an AMP-activated protein kinase (AMPK) activator to prevent myocardial ischemia/reperfusion injury by inhibiting apoptosis and inflammatory response (Citation6). Furthermore, AMPK has been widely reported to increase nitric oxide (NO) bioavailability and improve endothelial function by inhibiting ER stress in diet-induced obese mice (Citation10) and the offspring of dams with diabetes (Citation11). Therefore, we hypothesize that FSTL1 improves endothelial dysfunction in spontaneously hypertensive rats (SHRs) through restoration of AMPK signaling, leading to the inhibition of ER stress and superoxide generation and thus increasing NO bioavailability in vascular endothelium.
Materials and methods
Animal protocols
Male Wistar Kyoto (WKY) and SHRs (220–250 g) at the age of 16 weeks were purchased from the Chinese Academy of Medical Sciences (Beijing, China). This study was approved by the Committee on Animal Care of the Ganzhou Municipal Hospital and was carried out in accordance with the NIH guidelines for the care and use of laboratory animals. All the rats were maintained at a constant temperature at 21°C with a 12-h light cycle and had access to food and water ad libitium. WKY and SHRs were randomly assigned to control group and FSTL1 group. Control rats received a tail vein injection of vehicle (1 mL/day) for 4 weeks, and FSTL1 group received a tail vein injection of recombinant FSTL1 (100 μg/kg body weight/day, Sino Biological Inc., Beijing, China) for 4 weeks.
Blood pressure measurement
After a week of adaptation, indirect blood pressures of WKY and SHRs were measured and then every 1 week until 20 weeks of age using the tail-cuff method (BP-98A; Softron, Tokyo, Japan). Direct blood pressures were measured through the left carotid artery cannulation at 20 weeks of age. The rats from these separate groups were euthanized using CO2 inhalation for function and molecular mechanism study.
Mesenteric artery preparation and functional study
The reactivity of mesenteric arteries was determined as previously described (Citation12). Briefly, the third-order branches of the superior mesenteric artery were dissected and cut into several ring segments (~2 mm in length). The rings were mounted on 40 μm stainless-steel wires in a myograph (DMT, Aarhus, Denmark) for isometric tension recording. Rings were maintained in physiological saline solution at 37°C and supplied with carbogen (95% oxygen, 5% carbon dioxide). After a 30-min equilibration period, rings were stretched to an optimal baseline tension. Each ring was then stimulated with increasing concentrations of phenylephrine (PE, 10−9-10−5M; Sigma-Aldrich). Once a sustained tension was reached, either acetylcholine chloride (ACh, 3 nM-10 μM, Sigma-Aldrich) or sodium nitroprusside (SNP, 1 nM-10 μM, Sigma-Aldrich) was added cumulatively to evoke endothelium-dependent or endothelium-independent relaxations, respectively. The possible role of endothelial nitric oxide synthase (eNOS) in ACh-mediated endothelium-dependent relaxation (EDR) was investigated by preincubation with L-NAME (100 µM, Sigma-Aldrich) for 30 min.
Ex vivo culture of rat mesenteric artery rings
To determine whether FSTL1 exerts a direct effect on EDR, we cultured the rat mesenteric artery rings ex vivo. The mesenteric artery rings were cut into several ring segments (~2 mm in length) and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Gaithersburg, MD, USA). FSTL1 (100 µg/mL; Sino Biological Inc), compound C (AMPK antagonist, 5 µmol/L, Sigma-Aldrich) was added into the culture medium that bathed mesenteric artery rings in incubator at 37°C for 24 hours. After the incubation, rings were transferred to fresh Krebs solution for functional studies in myograph.
ROS measurement
Reactive oxygen species (ROS) levels in mesenteric artery rings were determined using an ROS assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, the mesenteric artery rings were incubated with the oxidative fluorescent dye dihydroethidium (DHE, 5 µmol/L) for 15 min at 37°C. After washing with phosphate-buffered saline (PBS), the fluorescence intensity was determined via the Olympus Fluoview FV1000 laser scanning confocal system.
Measurement of NO metabolites
The mesenteric artery rings were stimulated with ACh (10−6 mol/L) for 5 min, and incubation solution was assayed for the stable end products of NO, i.e., nitrate (NO3-) and nitrite (NO2-) using nitrate reductase method (Nanjing Jianchen Bioengineering Institute, Nanjing, China).
Western blot analysis
Western blotting was performed according to standard protocols described previously (Citation13). Briefly, mesenteric artery rings were homogenized using ice-cold RIPA lysis buffer. The proteins lysates were separated by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and transferred onto nitrocellulose membranes. The blots were blocked with 8% fat-free milk in TBS (Tris-buffered saline) with 0.5% Tween-20 for 90 min at room temperature. Following blocking, the membranes were probed with primary antibodies against phosphorylated AMPKα at Thr172, phosphorylated eNOS at Ser1177, phosphorylated PERK (1:500; Cell Signaling Technology, Danvers, MA, USA), t-eNOS, t-AMPKα, t-PERK and CHOP (1:1000; Cell Signaling Technology), and GAPDH (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies at 4°C overnight. After washing blots, the membranes were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:10000, Li-Cor Bioscience, Bad Homburg, Germany).
Statistical analysis
The data were expressed as mean ± SEM. The relaxation was presented as percentage reduction of the phenylephrine contraction. Concentration-response curves were analyzed using the GraphPad Prism 5.0 software. In the experiments comparing multiple time points, separate t-tests were used for each time point. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc tests whenever appropriate. Value of P < .05 was considered significant.
Results
FSTL1 lowers blood pressure in SHRs independent of body weight and heart rate
Indirect blood pressure of WKY and SHRs was monitored from 17 to 20 weeks of age using the tail-cuff method. FSTL1 decreased systolic blood pressure (SBP) in SHRs (). Blood pressure was measured directly through the left carotid artery cannulation at 20 weeks of age, which was also significantly decreased in SHRs with FSTL1 treatment (). By contrast, in WKY rats, FSTL1 had no effect on SBP. Furthermore, there were no obvious differences in body weight and heart rate between vehicle treatment and FSTL1 treatment ().
Figure 1. Effect of FSTL1 on blood pressure, body weight, and heart rate in SHRs. (a) Indirect blood pressure was measured from 17 to 20 weeks of age by tail-cuff plethysmography.*P < .05 vs WKY+Vehicle (n = 6); #P < .05 vs SHR+Vehicle. Direct blood pressure (b) was measured at 20 weeks of age. *P < .05 vs others (n = 6). Heart rate (c) and body weight (d) were measured at 20 weeks of age. *P < .05 vs WKY+Vehicle (n = 6).
FSTL1 improves endothelial function in SHRs
To determine if FSTL1 had an effect on vasorelaxation in WKY and SHRs, after preconstricted with PE, mesenteric resistance vessels were treated with cumulative dose of ACh (10−8.5–10−5mol/L). The present study revealed that FSTL1 augmented ACh-induced EDR in mesenteric arteries from SHRs, but not WKY rats (). Moreover, pretreatment with L-NAME (100 µM) inhibited the ACh-induced relaxation and the remaining relaxations were similar in SHRs (). The NO donor, SNP (10−9–10−5mol/L), was used to induce endothelium-independent relaxations. We found that there was no difference in the SNP-induced relaxation in these arteries from WKY and SHRs (). Furthermore, we also found that FSTL1 could attenuate PE-induced vasoconstriction in SHRs but no WKY rats (). Treatment with L-NAME augmented PE-induced contractions in arteries from SHRs with FSTL1 treatment (), suggesting that FSTL1 treatment had an increased NO production.
Figure 2. Effect of FSTL1 on vascular function in WKY and SHRs. The WKY and SHRs were treated with vehicle or FST1 (100 μg/kg body weight/day) for 4 weeks. The vascular function included ACh-induced relaxation (a), ACh-induced relaxation in the presence of L-NAME (b), SNP-induced relaxation (c), PHE-induced contraction (d), PE-induced contraction in the presence of L-NAME (f) were tested by multi-myograph system in mesenteric arteries from WKY and SHRs. *P < .05 vs others (n = 6).
FSTL1 promotes NO production via inhibiting ER stress in SHRs
The reduction of NO production is closely related to vascular endothelial dysfunction in SHRs (Citation14). However, less is known about the role of FSTL1 in NO production in mesenteric arteries of SHRs. We evaluated NO production by measuring the nitrate/nitrite level and the phosphorylation of eNOS at Ser1177. Our present study showed that compared with WKY rats, NO production and the phosphorylation of eNOS at Ser1177 were decreased in SHRs, which were increased after FSTL1 treatment (). Previous studies have shown that ER stress initiates a burst of oxidative stress in the ER lumen which triggers the elevation of ROS production and reduces NO production (Citation15). Our present study found that the expressions of ER stress markers such as p-PERK and CHOP were increased in SHRs compared to WKY rats, which were reduced by treatment with FSTL1 (). Treatment with FSTL1 for 4 weeks reduced the increased ROS production in SHRs but not WKY rats ().
Figure 3. FSTL1 increased NO production and eNOS phosphorylation in mesenteric arteries in SHRs. The WKY and SHRs were treated with vehicle or FST1 (100 μg/kg body weight/day) for 4 weeks. (a) The mesenteric artery rings were stimulated with ACh (10−6 mol/L) for 5 min. NO metabolites in endothelium-intact mesenteric arteries were measured via nitrate reductase method. (b) The level of phosphorylated eNOS in mesenteric arteries was measured by western blot. *P < .05 vs others (n = 6).
Figure 4. FSTL1 prevented the increased ER stress ROS production in mesenteric arteries from SHRs. The WKY and SHRs were treated with vehicle or FST1 (100 μg/kg body weight/day) for 4 weeks. (a) The protein expressions of ER stress markers, such as p-PERK and CHOP in mesenteric arteries were analyzed by western blotting. (b) ROS in mesenteric arteries was assessed with DHE intensity via fluorescence microscope. *P < .05 vs others (n = 6).
FSTL1 inhibits ER stress dependent of AMPK signaling in SHRs
Previous reports suggested that AMPK signaling activation leads to the inhibition of ER stress in the aortas of obese mice (Citation10). However, little is known about the effect of FSTL1 on ER stress. Our present study showed that AMPKα phosphorylation at Thr172 were downregulated in mesenteric arteries from SHRs. Treatment with FSTL1 increased AMPKα phosphorylation, and reduced the expression of ER stress proteins. However, the effect of FSTL1 was inhibited by AMPK antagonist compound C in the process (), indicating a protective role for FSTL1 against increased ER stress via the AMPK signaling in SHRs. Moreover, a positive role of ER stress in endothelial dysfunction was demonstrated by ex vivo exposure to tunicamycin (ER stress inducer, 2 µg/mL) for 24 h in mesenteric arteries from WKY rats. Coincubation with FSTL1 decreased tunicanmycin-induced the upregulation of ER markers, such as p-PERK and CHOP, in mesenteric arteries from WKY rats, which were inhibited by AMPK antagonist compound C (). These results strongly suggested that FSTL1 has a direct inhibitory effect on ER stress through the AMPK signaling.
Figure 5. FSTL1 reduced ER stress via activation of AMPK signaling. (a,b) Representative bands and densitometry of western blots showing the levels of protein expressions in AMPKα phosphorylation and ER stress markers such as p-PERK and CHOP in mesenteric arteries from WKY and SHRs incubated with FSTL1 (100 µg/mL) or compound C (5 µmol/L) for 24 h. (c–f) The mesenteric arteries from WKY rats were incubated with tunicamycin (tuni, 2 µg/mL), FSTL1 (100 µg/mL) or compound C (5 µmol/L) for 24 h. The protein expressions of ER stress markers, such as p-PERK and CHOP, in mesenteric arteries were analyzed by western blotting. *P < .05 vs WKY and SHR+FSTL1 (n = 6).
AMPK contributes to the beneficial effect of FSTL1 on endothelial function in SHRs
We wondered whether the beneficial effect of FSTL1 on endothelial function was AMPK signaling dependent. Coincubation with FSTL1 markedly improved the relaxation responses of mesenteric arteries from SHRs, which was inhibited by AMPK antagonist compound C (). Moreover, a positive role of ER stress in endothelial dysfunction was demonstrated by ex vivo exposure to tunicamycin (ER stress inducer, 2 µg/mL) for 24 h in mesenteric arteries from WKY rats. ACh-induced EDR was impaired by tunicamycin, which was reversed by the co-incubation with FSTL1. AMPK antagonist compound C inhibited FSTL1-induced improvement of EDR in tunicamycin-treated mesenteric arteries from WKY rats (), suggesting that the beneficial effect of FSTL1 on endothelial function was dependent of AMPK signaling.
Figure 6. Role of the AMPK signaling in the vascular benefit of FSTL1. (a) ACh-induced relaxation in mesenteric arteries from WKY and SHRs incubated with FSTL1 (100 µg/mL) or compound C (5 µmol/L) for 24 h were tested by multi myograph system. (b) The mesenteric arteries from WKY rats were incubated with tunicamycin (tuni, 2 µg/mL), FSTL1 (100 µg/mL) or compound C (5 µmol/L) for 24 h and ACh-induced relaxation was tested by multi myograph system. *P < .05 vs others (n = 6).
Discussion
Endothelial dysfunction is thought to be a critical initiating factor in the development of hypertension and its associated complications (Citation16). Previous studies in humans have shown that plasma FSTL1 levels are independently and positively associated with adverse cardiovascular events and mortality in patients with acute coronary syndrome (Citation17) and heart failure (Citation7). However, few studies have investigated the effect of FSTL1 on endothelial function in SHRs. The present study demonstrates that FSTL1 exerts an endothelial protective effect in SHRs, at least in part, through decreasing ER stress, inhibiting ROS generation, and restoring NO production, via activating AMPK signaling pathway.
Alterations to the function in resistance artery have profound effects on the development of hypertension (Citation18). Indeed, the impairment of vascular functions has been recently investigated in models of hypertension including spontaneously (Citation19), obese (Citation20) and age-related (Citation21). These studies have evidenced a vascular dysfunction mainly related to impaired endothelial function through the assessment of the NO pathway. FSTL1 has been demonstrated to play crucial roles in revascularization and modulation of inflammatory responses (Citation22). Moreover, FSTL1 can activate NO synthase pathway to improve differentiation and migration of endothelial cells (Citation23). In the present study, we found that FSTL1 resulted in an attenuation of the PHE-induced vasocontraction response in SHRs and the effect was reversed by L-NAME, indicating PHE-induced enhanced contraction response was associated with a decreased endothelial NO production. It is well established that the reduction of NO production is a dominant contributing factor in the development of endothelial dysfunction in SHRs (Citation24). In the present study, we found that FSTL1 improved ACh-induced EDR by increasing NO production. Some studies have reported that SNP-induced endothelium-independent relaxation is weakened in SHRs compared with WKY rats (Citation25), which is inconsistent with our findings. The reason for this discrepancy is not clear. However, the different age of rats used to determine the SNP-induced endothelium-independent relaxation could be the reason of such discrepancy.
Emerging evidence suggests that ER stress is upregulated in aortas of SHRs (Citation26) and obese mice (Citation27), which is involved in vascular endothelial dysfunction. In the present study, we observed that ER stress was upregulated and EDR was impaired in mesenteric arteries from SHRs, which in accordance with previous reports (Citation28). FSTL1 treatment prevented the increase of ER stress and normalized impaired EDR, and reduced blood pressure in SHRs, indicating that ER stress is involved in FSTL1-induced improvement of vascular endothelial dysfunction in SHRs. To determine a causal link between ER stress and vascular endothelial dysfunction in SHRs, ER stress was induced by tunicamycin in mesenteric arteries from WKY rats. We showed that induction of ER stress directly impaired endothelial function accompanied by elevated the expression of ER stress marker in mesenteric arteries. More importantly, FSTL1 in vivo rescued endothelial function of mesenteric arteries treated with tunicamycin. These results suggest that FSTL1 has a protective effect on endothelial function through alleviation of ER stress.
In addition to its beneficial metabolic effect, more evidence suggests that AMPK is an important regulator of vascular homeostasis (Citation29). AMPK activation is reported to suppress vascular smooth muscle cells proliferation, migration, and exert a vasorelaxation effect in isolated aortic rings (Citation30,Citation31). Extensive studies have shown that activation of AMPK exerts therapeutic effects by suppressing ER stress, promoting eNOS activity, and improving vascular endothelial functions in diabetes mellitus (Citation32) and hypertension (Citation33). AMPK is reported to be essential in maintaining ER homeostasis, and AMPK inhibition or AMPKα deletion causes aberrant ER stress, and increased vascular contractility and hypertension (Citation34). Previous studies have found that basal AMPK activation is blunted in aortic rings of SHRs versus WKY rats, which is associated with decreased NO bioavailability (Citation35). Indeed, we found that AMPKα phosphorylation was downregulated in mesenteric arteries from SHRs and that AMPK activation was able to improve vascular endothelial functions in SHRs and rescue ER stress-induced endothelial dysfunction.
The phosphorylation of eNOS plays an important role in hypertension (Citation36). The mechanisms, regarding to how AMPK regulates eNOS phosphorylation, are complicated. Some reports show that there is a direct linkage between AMPK and eNOS, and AMPK can directly induce phosphorylation/activation of eNOS and increase NO production (Citation37). However, other reports show that some signaling pathways are engaged in interaction between AMPK and eNOS, including AMPK/Akt and AMPK/PPARδ signaling pathways. Our present study showed that downregulation of AMPK signaling cascade in SHRs increased ER stress, which reduced eNOS phosphorylation and diminished the NO production.
In conclusion, the present study showed that FSTL1 protected endothelial dysfunction through upregulation of AMPK signaling with a subsequent reduction of ER stress and ROS production, as well as an increase in eNOS activity in SHRs. Activation of AMPK in the vasculature may be a potential strategy for improving endothelial dysfunction in hypertension.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Additional information
Funding
References
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