Stellate ganglion block ameliorates vascular calcification by inhibiting endoplasmic reticulum stress
Abstract
Aims: Vascular calcification (VC) underlies substantial cardiovascular morbidity and mortality. No clinically therapies have emerged presently. Stellate ganglion block (SGB) is one of the most often used sympathetic blockade procedure, and regulates vascular dilation. However, the effect of SGB on VC is still unknown. Therefore, we aimed to identify the ameliorative effect of SGB on VC.
Key finding: In vivo VC was induced in rats by administering vitamin D3 plus nicotine (VDN), and in vitro calcification of rat aortic vascular smooth muscle cells (VSMC) was induced by β-glycerophosphate. In VDN rats, alkaline phosphatase (ALP) activity and Calcium contents were higher than that in control rats. The transformation of VSMC from contractile to osteoblast-like phenotype was observed in calcified aorta. SGB ameliorated the increase of ALP activity and Calcium content, and the transformation of VSMC in calcified aorta. The stimulation of endoplasmic reticulum stress (ERS) in calcified aorta was also attenuated by SGB treatment. The inducer of ERS, tunicamycin could block the beneficial effect of SGB on VC, and the ERS inhibitor, 4-PBA could mimic the amelioration of SGB. Furthermore, SGB attenuated the increased plasma levels of norepinephrine in VDN rats. In vitro experiments, norepinephrine exaggerated VSMC calcification, phenotype transformation and ERS.Significance: These results demonstrate that SGB could inhibit sympathetic nervous activity, and then prevent the activation of ERS followed by ameliorating VC. Sympathetic over-activation might play critical role in the pathogenesis of VC, which provides new strategy and target for therapy and prevention of VC.
Introduction
Vascular calcification (VC) is an ectopic deposition of hydroxyapatite with a high degree of crystallization in the media (medial calcification) or intima (intimal calcification) of the arterial wall. Medial calcification usually occurs in patients with hypertension, type II diabetes, chronic kidney disease, and aging degeneration [19]. This pathology underlies substantial cardiovascular morbidity and mortality. The rigid deposits of hydroxyapatite crystallization increase stiffness and fragility, resulting in impairment of vasomotion, leading to complications such as heart failure, myocardial infarction, and stroke [6]. However, no clinically effective therapies for VC have emerged to date. Recently, VC is considered as an actively regulated biological process which is similar as bone formation. Transformation of vascular smooth muscle cells (VSMC) from contractile to osteoblast-like phynotype plays a key role in pathophysiology of VC [10]. The realization that calcification is results from active cellular processes offers hope of treatment and reverse for the disease.
Endoplasmic reticulum stress (ERS) is triggered by a loss of homeostasis in the endoplasmic reticulum (ER), such as accumulation of misfolded/unfolded proteins in the ER lumen. To restore the homeostasis of ER, ERS activates a series of adaptive mechanisms known as unfolded protein response (UPR), including translational attenuation, up-regulation of ER chaperones and related proteins, and degradation of unfolded proteins by quality-control system. However, when ER function is severely impaired, ERS elicits apoptotic signals, which has been implicated in numerous human diseases including VC [20]. Previous studies have demonstrated that the PERK-eIF2α-ATF4-CHOP signaling pathway, an important apoptosis pathway in ERS contributes to progression of VC [8, 9, 25, 26]. Some factors also improved VC by inhibiting ERS [3, 22], and other factors conversely exacerbate VC through activating ERS [21]. These results confirmed the key role of ERS in pathophysiology of VC.
The stellate ganglion is a part of the cervical sympathetic trunk, and is located just anterior to the transverse process of C7vertebrae. Stellate ganglion block (SGB) is achieved by injecting an anesthetic such as lidocaine around the stellate ganglion, which has been used since 1930s for treatment of sympathetically mediated cervical pain and vascular insufficiency syndromes. SGB is generally safe, and does not carry systemic side effects [16]. Determined by Doppler ultrasonography, SGB increases distal radial arteries and internal mammary arteries diameters. Therefore, SGB is considered as an alternative to topical and systemic vasodilators [7, 14]. In spontaneously hypertensive rats (SHR), SGB could significantly decrease blood pressure by up-regulated expression of eNOS, and reduce apoptosis of myocardial cells [4, 5]. Considering the protective effect of SGB on cardiovascular system, we hypothesized that SGB might ameliorate VC. In this article, used by the VC model of rats and cultured vascular smooth muscle cells (VSMC), effect of SGB on VC was detected, and the mechanism was further investigated.
6 weeks old male Sprague–Dawley (SD) rats (180~200 g) were from the Animal Center, Hebei Medical University (Shijiazhuang, China), and were housed understandard conditions (room temperature 20 ± 8 ℃, humidity 60 ± 10 %, lights from 6:00 to 18:00) and they were given standard rodent chow and water freely. All animal procedures were complied with the Animal Management Rule of the Ministry ofHealth, People’s Republic of China (documentation No. 55, 2001) and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978), and approved by the Animal Care Committee of Hebei Medical University.Tunicamycin (Tm) was from Cayman Chemical (Ann Arbor, USA). ALP and Ca kit was from Biosino Bio-technology and Science Inc (Beijing, CN). Antibodies against calponin, RUNX2 and GRP78 were from Epitomic Inc (Burlingame, USA). Antibodies against SM22α, BMP2, active-caspase12 and beta-actin were from GeneTex Inc (Irvine, USA). Antibodies against CHOP were from Affinity Biosciences Inc (Cincinnati, USA). The second antibodies were from Kirkegaard & Perry Laboratories Inc (Gaithersburg, USA). Potent ECL kit was from MultiSciences Biotech Co (Hangzhou, CN). Other chemicals and reagents were of analytical grade.VC model in ratsVC was induced by administration of vitamin D3 plus nicotine (VDN) as described [2, 27]. Modified, male SD rats were given vitamin D3 (300,000 IU/kg, intramuscularly) simultaneously with nicotine (25 mg/kg in 5 ml peanut oil, intragastrically) at 8:00 on the 1st day. The nicotine administration was repeated at 20:00. At the 2nd and 15th day, rats were retreated with vitamin D3.
Rats in the control group received normal saline intramuscularly and 2 gavages of peanut oil without nicotine (5 ml/kg). The aorta tissues of all rats were harvested after 28 days of nicotine treatment. Euthanasia was achieved with an intraperitoneal injection of 20 % urethane (1 g/kg).SGB in ratsAt the 2nd day after nicotine treated, the SGB was performed using a lateral percutaneous approach as reported [15], and modified minor. The animal was placed in the right lateral decubitus position. The lower part of the cervical vertebrae was fixed between the first and third fingers of the physician’s left hand while palpating the C7 process with the second finger. A short beveled needle (25-gauge 5/8” needle) attached with a 1 mL syringe was inserted laterally towards the vertebral body surface. When contact was made, the injectate (0.3 mL 0.5% lidocaine for the SGB group or0.2 mL saline for controls) was given. All rats were observed for ptosis on the left side as an indicator of successful SGB. The SGB was operated once a day for 4 weeks.As described, calcium levels in aortic vessel were determined by colorimetry through a reaction with o-cresolphtalein complexon, and ALP activity in aortic vessel was measured by the use of an ALP Colorimetric assay kit following the kit’s instruction.
All experiments were repeated at least 3 times.Western blot analysisloaded on 10% or 12% SDS gels and then transferred to a nitrocellulose membrane. Nonspecific proteins were blocked with 5% nonfat dried milk for 1 h. Membraneswere incubated with the primary antibodies overnight at 4℃, and with secondary antibody (HRP-conjugated anti-goat or anti-rabbit IgG) for 1 h. The reaction was visualized by ECL, and autoradiograph was scanned. Protein expression was analyzedby NIH image software and normalized to that of β-actin. All experiments were repeated at least 3 times.The plasma levels of norepinephrine were assessed by high-performance liquid chromatography with electrochemical detection according to the protocol reported by Weicker H et al [34].Male SD rats (150 ± 10 g) were sacrificed by cervical dislocation. Thoracic aortas were removed of endothelium and adventitia, and then cut into small pieces. The small pieces were placed in Dulbecco’s modified Eagle’s medium (DMEM)involving 20% fetal bovine serum (FBS), and incubated at 37 ℃ in an incubator possessing 5% CO2. After migrating from explants, VSMC were collected and maintained incubating in DMEM involving 10% FBS. VSMC at passages from five toeight were utilized for investigation. To induce calcification, the VSMC were administrated with 5 mmol/L β-glycerophosphate (Sigma, St. Louis, USA) and 2.5 mmol/L CaCl2.Statistical analysis was performed by the Graphpad software (GraphPad Prism v5.00 for Windows; GraphPad Software Inc., San Diego, CA, USA). Comparisons among more than 2 groups were analyzed by one-way ANOVA followed by Tukey test. Data are expressed as mean ± SD. A P < 0.05 was considered statistically significant. Results Compared with control rats, ALP activity and Ca content of aortal tissue in VDN rats was significantly increased (Fig 1a and b, both P < 0.05). Detected by Western blot, the protein level of molecular markers of VSMC contractile phenotype, SM22α and calponin, was significantly down-regulated. Conversely, that of VSMC osteoblast-like phenotype, BMP2 and RUNX2, was significantly up-regulated (Fig 1c~g, all P < 0.05).After SGB treatment, the increase of aortal tissue ALP activity and Ca content in VDN rats was decreased (Fig 1a and b, both P < 0.05). In VDN rats with SGB, the down-regulation of SM22α and calponin was reversed, and the up-regulation of BMP2 and RUNX2 was simultaneously decreased (Fig 1c~g, all P < 0.05).The inhibited effect of SGB on ERS in aortal tissue of VDN ratsCompared with control rats, protein levels of ERS markers, including GRP78, active caspase-12 and CHOP, were all significantly up-regulated in VDN rats (Fig 2, all P < 0.05). However, this up-regulation of GRP78, active caspase-12, and CHOP was reversed by SGB treatment (Fig 2, all P < 0.05).ERS mediated the ameliorated effect of SGB on VC in VDN ratsThe ERS inducer (tunicamycin, Tm, 1 μg/kg, intraperitoneal injected, 30 min before SGB), and inhibitor (4-phenylbutyric acid, PBA, 50 mg/rat, intraperitoneal injected, 1 per day) was used to investigate role of ERS in the ameliorative effect of SGB. Tm could block the ameliorated effect of SGB on VDN rats. Tm increased the ALP activity and Ca content (Fig 1a and b, both P < 0.05), down-regulated the protein level of SM22α and calponin, up-regulated the protein level of BMP2 and RUNX2 in aorta of VDN rats with SGB treatment (Fig 1 c~g, all P < 0.05). The protein level of GRP78, active-caspase12, and CHOP was also induced by Tm (Fig 2, P < 0.05). However, there was no difference between VDN rats and VDN plus Tm rats (P >0.05).Effect of PBA on VC in VDN rats was similar as that of SGB. PBA treatment also could decrease ALP activity and Ca content in calcified aorta (Fig 1a and b, P < 0.05). In VDN plus PBA rats, the down-regulation of SM22α and calponin was reversed, and the up-regulation of BMP2 and RUNX2 was simultaneously decreased (Fig 1c~g, P < 0.05). PBA treatment attenuated the increased protein level of GPR78, active-caspase12 and CHOP in calcified aorta (Fig 2, P < 0.05).SGB inhibited increased plasma levels of norepinephrine in rats with VCCompared with control rats, plasma levels of norepinephrine significantly were decreased in rats with SGB treatment, and were increased in VC rats (P < 0.05, Fig 3). Interestingly, SGB dramatically attenuated the elevated plasma levels of norepinephrine in VDN rats (P < 0.05, Fig 3).Calcification of cultured VSMC induced by β-glycerophosphate was used to detect effect of norepinephrine on VC. Compared with control group, Ca content and ALP activity significantly increased in calcification group (P < 0.05, Fig 4a and b), which were further promoted by norepinephrine (1 μmol/L) treatment (P < 0.05, Fig 4a and b).Detected by Western blot, protein levels of molecular markers of VSMC contractile phenotype, SM22α and calponin, were significantly down-regulated in calcification group compared with that in control group. Conversely, that of VSMC osteoblast-like phenotype, BMP2 and RUNX2, was significantly up-regulated (all P < 0.05, Fig 4c~g). Norepinephrine treatment further promoted the downregulation of SM22α and calponin, and the upregulation of BMP2 and RUNX2 in calcified VSMC (P < 0.05, Fig 4c~g).Norepinephrine exaggerated calcification-induced ERS activationDetected by Western blot, protein levels of molecular markers of ERS, GRP78, active caspase-12, and CHOP, were significantly up-regulated in calcification group compared with that in control group (P < 0.05, Fig 5), which were further promoted by norepinephrine treatment (P < 0.05, Fig 5). Discussion VDN rats. SGB could ameliorate amplification of VC, transformation of VSMC phenotype, and activation of ERS. Furthermore, Tm (a general ERS inducer) blocked the ameliorated effect of SGB on VC. ERS inhibitor, e.g. PBA had similar improved effect on calcified aorta compared with SGB. The plasma of norepinephrine was elevated in VDN rats, which could be decreased by SGB treatment. Furthermore, norepinephrine promoted VSMC calcification and ERS activation in calcified VSMC in vitro.VC has been considered as an actively regulated biological process which is similar as bone formation. Transformation of VSMC plays a key role in pathophysiology of VC. Here, we observed that the Ca content and ALP activity were both increased in vascular tissue of VDN rats, suggested successful induction of VC. Furthermore, protein level of VSMC contractile phenotype marker, SM22α and calponin, was decreased in VDN rats. Conversely, that of osteoblast-like phenotype marker, BMP2 and RUNX2, was increased. It suggested that VSMC transforms from contractile phenotype to osteoblast-like phenotype in VDN rats, which are in accordance with previous articles [1, 2, 38, 39].A large amount of signaling pathway, such as ERS, has been suggested to contribute VC formation. In two rat models of VC, vitamin D3 plus nicotine (VDN) and rapid calcification, activation of ERS followed by apoptosis in calcified artery were proved. ERS was also induced in aortas of 5/6 nephrectomized ApoE-/- mice, which was associated with VC. Furthermore, inhibition of ERS by knockdown of ATF4 or CHOP, key factors mediated ERS-induced apoptosis significantly decrease apoptosis of VSMC and ameliorate VC [8, 9, 25, 26]. Presently, we also showed that protein level of GRP78, active-caspase 12, and CHOP in calcified aorta was up-regulated, suggested ERS activation in VC. The VC could be ameliorated by inhibiting ERS [3, 22], and over-activation of ERS conversely exacerbated amplification of calcification in vascular tissue [21]. These results confirm that ERS is involved in pathophysiology of VC, and might be a new target for VC treatment. SGB is one of the most often used sympathetic blockade procedure, and provides a valuable therapeutic benefit to relieve pain syndromes [18]. Furthermore, SGB is considered as an alternative vasodilators [7, 14], and can decrease blood pressure in SHR [4, 5]. Here, we firstly show that SGB significantly prevents the increase of ALP activity and Ca content in calcified vascular of VDN rats, which suggested that beneficial effect of SGB on VC.We further demonstrate that inhibition of ERS by SGB mediates the beneficial effect of SGB on VC. On one hand, SGB down-regulates the up-regulation of GRP78, active-caspase12, and CHOP in calcified aorta, demonstrated that SGB inhibits activation of ERS accompanied with amelioration of VC. On the other hand, ERS inhibitor, Tm blockes the beneficial effect of SGB on VC. These results demonstrate that SGB could ameliorate VC through inhibiting ERS. The detailed mechanism and intracellular signaling pathway of inhibiting ERS by SGB should be further investigated. Besides of ERS, SGB also might inhibit cellular apoptosis [5] and the activation of immune response [31, 36], through which calcified vessel also could be improved [12, 28-31]. Of course, the beneficial effect and mechanism of SGB on VC should be further confirmed and investigated in future. Although mechanism of SGB still remains controversy, it has been depicted as including peripheral vasodilatation and elevating blood flow due to sympathetic block [11, 37]. SGB may function by inhibition of sympathetic activity, and reduction of norepinephrine release [17, 33, 36]. To detect whether SGB ameliorates VC through blockade of sympathetic nerves, plasma levels of norepinephrine, an indicator of sympathetic activity is assessed. Our results demonstrate that plasma levels of norepinephrine elevate in rats with VC, which can be attenuated by SGB treatment. These data suggest that SGB maybe ameliorate VC through reducing norepinephrine release.Series of articles have reported exacerbated effect of norepinerphine on ERS activation [23, 24, 35]. Our results also confirm that norepinerhrine further promotes ERS activation in calcified VSMC. Although phenylephrine exaggerates cardiomyocyte calcification through α-adrenoceptor activation [13], effect of norepinephrine on vascular calcification remains unknown. To confirm direct effect of norepinephrine on vascular calcification, in vitro model of VSMC calcification induced by β-glycerophosphate was used. Our results demonstrate that norepinephrine exaggerates VSMC calcification and phenotype transformation. Taken together, we show that norepinephrine exacerbates VC through activation of ERS, and SGB maybe ameliorate VC through inhibition of sympathetic activation and norepinephrine release. Conclusion In conclusion, our results demonstrate that SGB ameliorates aortal calcification through inhibiting ERS in VDN rats, which is mediated by inhibiting sympathetic nervous acitivity and norepinephrine release (Fig 6). Sympathetic over-activation maybe play critical role in pathogenesis of VC, which provides new strategy and target for therapy and prevention of VC. SGB could be used as a clinically effective treatment for VC in β-Glycerophosphate future.