1. Introduction
Hypertension, a chronic condition of elevation in blood pressure (BP), is a well-known risk factor for the development of cardiovascular diseases (CVDs), including heart failure, coronary heart diseases, myocardial fibrosis, and infarction [1], atherosclerosis [1,2], stroke [1,3], and kidney failure [4]. The pathogenesis of hypertension is multifaceted, and one of the well-characterized causes is endothelial dysfunction resulting from nitric oxide (NO) deficiency [5,6,7]. Circulatory NO, derived primarily from endothelial NO synthase (eNOS) in vascular endothelial cells [8], participates in BP regulation via maintenance of vascular tone through the classical NO/soluble guanyl cyclase/cyclic guanosine monophosphate signaling to decrease intracellular calcium in vascular smooth muscle cells and evoke vasodilatation [9,10]. In addition, NO regulates cardiovascular functions by exhibiting anti-inflammatory [11], anti-platelet aggregatory [12], and anti-proliferative [13] actions. Accordingly, deficiency in NO production and/or availability leads to impaired vascular relaxation, increased vascular resistance, augmented platelet aggregation, and vascular inflammation or proliferation; all of which have been demonstrated to underpin the pathophysiology of hypertension [6,14,15].
A rat model of hypertension induced by the systemic administration of Nω-Nitro-
The rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside [25], is one of the major sites of action in the brain stem where NO participates in the neural control of cardiovascular functions [6,26,27,28]. Emerging evidence from preclinical studies in animal models suggests that NO in RVLM exhibits a predominantly sympathoinhibitory action in the regulation of sympathetic nerve activity (SNA) under physiological conditions [28,29]. Deficiency in tissue NO availability under various cardiovascular conditions, including hypertension [29], chronic heart failure [30], and metabolic syndrome [31,32], may lead to an increase in SNA because of the blunted sympathoinhibitory action of NO in RVLM. Moreover, NO participates in the neural control of cardiovascular systems via interactions with reactive oxygen species (ROS), in particular superoxide anion (O2•−) [6,26,32]. Under physiological conditions, NO is generated from its substrate L-arginine through coupling of NOS [33]. This coupling capacity is reduced under hypertensive conditions, wherein NOS removes an electron from its cofactor, NADPH, and donates it to an oxygen molecule to generate O2•− rather than NO [32,33,34]. The causal role of ROS at RVLM in systemic
Epidemiological studies indicate aging is a predominant risk factor for CVDs. The prevalence of hypertension increases markedly with aging, attributed primarily to alterations in the structure, responsiveness, function, and rigidity of vessel walls [35], as well as dysregulation of the autonomic nervous system [36]. Several theories have so far been proposed to explain the etiology of biological aging [37]; among them, tissue oxidative stress was postulated to be a common denominator [38]. Indeed, a wide array of studies suggests the engagement of ROS in age-related CVDs, including hypertension, atherosclerosis, atrial fibrillation, and stroke [38]. Despite evidence suggesting causal links between oxidative stress, aging, and CVDs, there is a paucity in the literature on the molecular mechanisms that underlie age-related dysregulation of redox homeostasis and its contribution to the development of CVDs.
Redox signaling is critically involved in cardiovascular pathophysiology, and oxidative stress in RVLM has been demonstrated unambiguously to play a pivotal role in neural mechanisms of hypertension via the increase in neurogenic sympathetic outflow [6,14,26,27,28]. However, there is no current information on whether redox homeostasis in RVLM is affected by systemic
2. Materials and Methods
2.1. Animals
Experiments were carried out in young (4 weeks old, n = 51) and adult (12 weeks old, n = 63) male normotensive Wistar-Kyoto (WKY) rats purchased from BioLASCO, Taipei, Taiwan. They were housed in animal rooms under temperature control (24 ± 0.5 °C) and 12 h light/dark (5 am to 5 pm) cycle. Standard laboratory rat chow (PMI Nutrition International, Brentwood, MO, USA) and tap water were available ad libitum. All animals were allowed to acclimatize for 14 days (young group) or housed to the age of 20 weeks (adult group) in an AAALAC-International accredited animal holding facility in Kaohsiung Chang Gung Memorial Hospital, Taiwan, before experimental manipulations. All experiments were performed in accordance with the guidelines for animal experimentation approved by our institutional animal care and use committee (no. 2018051701) as adopted and promulgated by the U.S. National Institutes of Health.
2.2. Implantation of Osmotic Minipump
Implantation of osmotic minipump into the peritoneal cavity or the cisterna magna was carried out according to previously reported procedures [32]. Briefly, for intraperitoneal (i.p.) implantation, animals were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and a midline incision of the abdominal cavity was made, followed by implantation of an Alzet® osmotic minipump (model 2002, DURECT Co., Cupertino, CA, USA) into the peritoneal cavity. The incision was closed with layered sutures. Some animals also received implantation of an additional micro-osmotic minipump into the cistern magna. For intracisternal (i.c.) implantation, we performed a midline incision of the dorsal neck, and dissected the underneath muscle layers to expose the dura mater between the foramen magnum and C1 lamina. The dura mater was perforated with a 22-gauge steel needle, followed by insertion of a polyvinylchloride tubing into the cistern magna using an Alzet® brain infusion kit. Observation of presence of cerebrospinal fluid at the outer end of the catheter assured the patency of the implantation. Tissue glue was used to seal the catheter to the dura mater and the incision was closed with layered sutures. A micro-osmotic minipump (Alzet® 1007D) was positioned subcutaneously in the neck, and was connected to the outer end of the catheter. No obvious neurological signs were observed after minipump implantation. All animals received postoperative intramuscular injection of procaine penicillin (1000 IU). Only animals that showed recovery and progressive weight gain after the operation were used in subsequent experiments.
2.3. Blood Pressure and Heart Rate Measurement
Systolic blood pressure (SBP) and heart rate (HR) were determined in conscious animals with a tail-cuff sphygmomanometer (MK-2000; Momuroki Kikai Co., Tokyo, Japan). Baseline values were measured on days 3 and 1 prior to, on the day (day 0) before osmotic pump implantation, and then on days 1, 3, 5, 7, 9, 11, and 14 following i.p.
2.4. Evaluation of Sympathetic Vasomotor Activity
Sympathetic vasomotor activity was measured at the end of i.p.
2.5. Measurement of Plasma Norepinephrine
We measured plasma norepinephrine (NE) level by the o-phthalaldehyde (OPA) method using high-performance liquid chromatography (HPLC) with fluorescence detection. Plasma sample was mixed with ice-cold trichloroacetic acid, and centrifuged at room temperature. The supernatant was mixed with 4-fold methanol after being filtered through a syringe filter (0.22 µm; Chroma Technology Corp., Bellows Falls, VT, USA), centrifuged again at room temperature, and kept at −80 °C until analyses. NE was measured by HPLC as previously described [39], and comparing the area under the curve of each sample against standard NE solutions of known concentrations was used to compute the concentration (µg/µL). Each sample was analyzed in triplicates and the mean was used for statistical analyses.
2.6. Measurement of Serum Nitric Oxide
Serum NO concentration was assessed indirectly by measuring the levels of nitrate and nitrite, the stable end-product of NO, with a NO colorimetric assay kit (Arbor assay kit, Ann Arbor, MI, USA) according to manufacturer’s instructions. The absorbance of the solution was read on a microplate reader at 540 nm (ThermoFisher Scientific Inc., Waltham, MA, USA). Each sample was analyzed in triplicates and the mean was used for statistical analyses.
2.7. Measurement of Plasma Malondialdehyde
Assays for lipid peroxidation are commonly used for estimation of oxidative status. Levels of lipid peroxidation in plasma were measured by a malondialdehyde (MDA; a primary indicator of lipid peroxidation) assay kit (Biovision, Milpitas, CA, USA), following the protocol provided by the manufacturer. Briefly, plasma samples were reacted with thiobarbituric acid (TBA) at 95 °C for 60 min. A microplate spectrophotometer (ThermoFisher Scientific) was used to determine the level of MDA-TBA adduct with colorimetric absorbance read at 532 nm. Each sample was analyzed in triplicates and the mean was used for statistical analyses.
2.8. Measurement of Plasma Proinflammatory Cytokines
The levels of proinflammatory cytokines, including interleukin-1 β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), in plasma, were measured using anti-rat ELISA Kits (ThermoFisher Scientific) according to the manufacturer’s specification. Plasma was collected and centrifuged for 10 min at 4 °C. The supernatants were used immediately to measure the concentrations of proinflammatory cytokines. Positive and negative controls were included on each plate. The final concentration of the cytokines was calculated by converting the optical density readings against a standard curve. Each sample was analyzed in triplicate and the mean was used for statistical analyses.
2.9. Tissue Collection and Protein Extraction from Rostral Ventrolateral Medulla
Animals were deeply anesthetized at the end of the experiments with an overdose of pentobarbital sodium (100 mg/kg, i.p.), followed by intracardial infusion with 500 mL of warm (37 °C) normal saline. The brain stem was rapidly removed and immediately frozen on ice. Using a rodent brain matrix (World Precision Instruments, Sarasota, FL, USA) and based on the atlas of Watson and Paxinos [40], the medulla oblongata covering RVLM was blocked between 0.5 and 1.5 mm rostral to the obex. Tissue from bilateral RVLM was collected by micropunches, and was stored at −80 °C until use.
For total protein extraction from RVLM, tissue micropunches were homogenized using a Dounce grinder with a tight pestle in ice-cold lysis buffer mixed with a cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) to prevent protein degradation. Solubilized proteins were centrifuged at 20,000× g at 4 °C for 15 min, and the total protein in supernatant was quantified by the Bradford assay with a protein assay kit (Bio-Rad, Hercules, CA, USA).
2.10. Measurement of Reactive Oxygen Species in RVLM
RVLM tissues were homogenized in sodium phosphate buffer (20 mM), centrifuged and the supernatant was collected for ROS measurement by electron paramagnetic resonance (EPR) spin trapping technique, as described previously [41]. EPR spectra were captured using a Brucker EMXplus spectrometer (Bruker, Ettlingen, Germany). Typical parameters were set at microwave power: 20 mW, modulation frequency: 100 kHz; modulation amplitude: 2 G; time constant: 655.36 ms; conversion time: 656 ms; sweep time: 335.87 s. We added a membrane-permeable superoxide dismutase (SOD; 350 U/mL) into the incubation medium to determine ROS specificity. Spectra represented the average of 6 scans. Each sample was analyzed in triplicate and the mean was used for statistical analyses.
2.11. Measurement of Nitric Oxide in RVLM
For measurement of NO in RVLM tissue, tissue micropunches were homogenized in lysis buffer, centrifuged, and the supernatant was stored at −80 °C until use after being deproteinized using a Centricon-30 filtrator (Microcon YM-30, Bedford, MA, USA). Tissue NO levels were determined based on chemiluminescence reaction with the purge system of a NO analyzer (Sievers NOA 280TM, Boulder, CO, USA) [32]. Each sample was analyzed in triplicate and the mean was used for statistical analyses.
2.12. Measurement of Nitric Oxide Synthase Activity in RVLM
Tissue NOS activity in RVLM was measured according to previously reported procedures [32]. RVLM tissues were lysed and centrifuged to obtain the supernatants, which were then used for detection of the enzyme activity following the manufacturer’s instructions of a NOS activity assay kit (Merck KGaA, Darmstadt, Germany). After colorimetric reaction, the optical density was read using a microplate spectrophotometer (ThermoFisher Scientific) at an absorbance wavelength of 540 nm. Each sample was analyzed in triplicate and the mean was used for statistical analyses.
2.13. Measurement of NADPH Oxidase Activity in RVLM
NADPH oxidase activity of protein samples from RVLM was measured using the lucigenin-derived chemiluminescence method [32]. Briefly, the luminescence assay was performed in phosphate buffer with NADPH as the substrate. After dark adaptation, a tissue homogenate (100 µg protein) was added, and the chemiluminescence value was recorded. O2•− production was measured with the addition of NADPH, in the presence or absence of an NADPH oxidase, diphenyleneiodonium. All measurements were conducted in the dark room with temperature maintained at 22–24 °C. Light emission was recorded by a Sirius Luminometer (Berthold, Germany). Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories). Each sample was analyzed in triplicate and the mean was used for statistical analyses.
2.14. Measurement of Total Antioxidant Activity in RVLM
Tissue antioxidant activity in RVLM was measured by a total antioxidant capacity assay kit (Sigma-Aldrich), following the protocol provided by the manufacturer. RVLM tissues were homogenized in lysis buffer, centrifuged, and the supernatant was used for analysis. The reaction was based on Cu2+ reduction by the small molecule antioxidants and the reduced Cu+ ion chelates with a colorimetric probe that was read with a standard 96-well spectrophotometric microplate reader at 570 nm. Antioxidant capacity was determined by comparison with Trolox, a water-soluble vitamin E analog that serves as an antioxidant standard. Each sample was analyzed in triplicate and the mean was used for statistical analyses.
2.15. Measurement of Mitochondrial Respiratory Enzyme Activity in RVLM
The mitochondrial fraction from RVLM tissue was isolated following procedures reported previously [42]. Purity of the mitochondrial-rich fraction was verified by the expression of the mitochondrial cytochrome c oxidase. Activities of mitochondrial respiratory chain enzymes were measured immediately after mitochondrial isolation, according to procedures reported previously [42] using a thermostatically regulated spectrophotometer (ThermoFisher Scientific). Enzyme activity was expressed in nmol/mg protein/min.
For the measurement of nicotinamide adenine dinucleotide (NADH) cytochrome c reductase (NCCR; enzyme for electron transport between ETC Complex I and Complex III) activity, mitochondrial fraction was incubated in a mixture containing K2HPO4 buffer, KCN, β-NADH, and rotenone at 37 °C for 2 min. After the addition of cytochrome c (50 µM), the reduction of oxidized cytochrome c was measured as the difference in the presence or absence of rotenone at 550 nm for 3 min at 37 °C.
For the determination of succinate cytochrome c reductase (SCCR; enzyme for electron transport between ETC Complex II and Complex III) activity, mitochondrial fraction was performed in the same buffer solution supplemented with succinate. After a 5 min equilibration at 37 °C, cytochrome c (50 µM) was added and the reaction was monitored at 550 nm for 3 min at 37 °C.
For the determination of cytochrome c oxidase (CCO, marker enzyme for ETC Complex IV) activity, mitochondria fraction was pre-incubated at 30 °C for 5 min in K2HPO4 buffer, then 45 µM ferrocytochrome c was added to start the reaction, which was monitored at 550 nm for 3 min at 30 °C. In all measurements, experiments were performed in triplicate, and the mean was used for statistical analyses.
2.16. Measurement of ATP Levels in RVLM
RVLM tissues were centrifuged at 10,000× g for 10 min after homogenization in a protein extraction solution (Pierce, Rockford, IL, USA), ATP concentration in the supernatant was determined by an ATP colorimetric assay kit (AbCam, Waltham, MA, USA) using a microplate reader (ThermoFisher Scientific). The ATP level was normalized to the protein concentration of the sample. Each measurement was performed in triplicate and the mean was used for statistical analyses.
2.17. Western Blot Analysis
We determined the expression levels of gp91phox, p22phox, p67phox, and p47phox subunits of NADPH oxidase, manganese dismutase (SOD2), nuclear factor erythroid 2-related factor 2 (Nrf2), nNOS, iNOS, eNOS, NADPH oxidase activator 1 (Noxa1), uncoupling protein 3 (UCP3), and GAPDH in total protein extracted from RVLM by Western blot analysis [43]. In brief, 8–12% SDS-polyacrylamide gel electrophoresis was used for protein separation, and a Bio-Rad miniprotein-III wet transfer unit (Bio-Rad) was employed to transfer samples onto polyvinylidene difluoride transfer membranes (Immobilon-P membrane; Millipore, Bedford, MA) for 1.5 h at 4 °C. The transfer membranes were then incubated with a blocking solution (5% nonfat dried milk dissolved in Tris-buffered saline-Tween buffer) for 1 h at room temperature. Primary antisera used in this study included goat polyclonal, rabbit polyclonal or monoclonal, or mouse monoclonal antiserum against gp91phox (1:5000; BD Biosciences, Sparks, MD, USA), p22phox, p67phox, and p47phox (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), SOD2 (1:3000; Stressgen, San Deigo, CA, USA), Nrf2 (1:1000; Santa Cruz), nNOS, iNOS, and eNOS (1:1000; BD Biosciences), Noxa1 (1:1000, Santa Cruze), UCP3 (1:1000, Santa Cruz), and GAPDH (1:10,000; Merck). Membranes were subsequently washed three times with TBS-t buffer, followed sequentially by incubation with the secondary antibodies (1:10,000; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h and horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoResearch). An enhanced chemiluminescence Western blot detection system (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) was used to detect specific antibody–antigen complex. For the detection of eNOS or nNOS dimerization, nondenaturing, low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis was used [32,34]. During the electrophoresis process and transfer of proteins to nitrocellulose membrane, buffers were placed in an ice-water bath and the whole apparatus was kept at 4 °C. ImageJ software (NIH, Bethesda, MD, USA) was used to quantify the number of detected proteins, which was expressed as the ratio to loading control (GAPDH).
2.18. Generation of Lentiviral Vector
NOXa1 shRNA lentiviral particles (sc-150038-V, Santa Cruz) were used in gene silencing experiments. These transduction-ready viral particles contain a target-specific construct that encodes a 19–25 nt (plus hairpin) shRNA designed to knock down gene expression of Noxa1. Each vial contains 200 µL frozen stock of 1.0 × 106 infectious units of virus (IFU) in Dulbecco’s Modified Eagle’s Medium with HEPES pH 7.3 (25 mM). Control shRNA lentiviral particles (sc-108080, Santa Cruz) contain an shRNA construct that encodes a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA.
2.19. Microinjection of Lentiviral Vectors into RVLM
Microinjection of the Lv-Noxa1-shRNA, or scramble (Lv-scr-RNA), was carried out stereotaxically and sequentially into the bilateral RVLM of rats that were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Adequate anesthesia of animals was confirmed by observations of unresponsive to paw pinch and no corneal withdrawal reflex. The animals were placed into a stereotaxic head holder (Kopf, Tujunga, CA, USA) on a thermostatically controlled heating pad. Bilateral microinjection of the viral vectors was carried out, as described previously [32,44]. In brief, a glass micropipette (external tip diameter: 50–80 µm), connected to a 0.5-µL Hamilton microsyringe, was positioned into RVLM. A total of eight injections (4 on each side) of undiluted viral particles (200 nl total volume on each side) were made at two rostro-caudal levels at stereotaxic coordinates of 4.5–5.0 mm posterior to lambda, 1.8–2.1 mm lateral to the midline, and 8.0–8.5 mm below the dorsal surface of cerebellum. These coordinates cover the confines of RVLM within which sympathetic premotor neurons reside [25,40]. After the lentivirus injection, the wound was closed in layers, and animals were allowed to recover in individual cages with free access to food and water.
2.20. Reverse Transcription and quantitative Polymerase Chain Reaction
Total RNA from RVLM tissues was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. All RNA isolated was quantified by spectrophotometry and the optical density 260/280 nm ratio was determined. Reverse transcriptase (RT) reaction was performed using a SuperScript Preamplification System (Invitrogen) for the first-strand cDNA synthesis.
Noxa1 and Ucp3 mRNA levels were analyzed by quantitative polymerase chain reaction (qPCR) using SYBR Green and normalized to the GAPDH mRNA signal as described [44]. The following primers were used: Noxa1: 5′-TTA CTC TGC CCC TGA AGG TC-3′ (forward) and 5′-CTC GGG CTT TGT TGA AC-3′ (reverse); Ucp3: 5′-TTC CTG GGG GCC GGC ACT G-3′ (forward) and 5′-CAT GGT GGA TCC GAG CTC GGT AC-3′ (reverse) [45]; and GAPDH: 5′-AGA CAG CCG CAT CTT CTT GT-3′ (forward), 5′-CTT GCC GTG GGT AGA GTC AT-3′ (reverse). Noxa1 and Ucp3 mRNA were amplified under the following conditions: 95 °C for 3 min, followed by 50 cycles consisting of 95 °C for 10 s, 50 °C for 20 s, and 72 ° C for 2 s, and finally a 10 min extension at 40 °C. GAPDH was amplified under identical conditions, with the exception of a 55 °C primer annealing temperature. All samples were analyzed in triplicate. All qPCR reactions were followed by dissociation curve analysis. Relative quantification of gene expression was performed using the 2ΔΔCT method.
For amplification of oxidative stress-related mRNA, RT² Profiler PCR Arrays (Qiagen GmbH, Hilden, Germany) were employed following the manufacturer’s protocol. The microarrays include primer assays for 84 oxidative stress-focused genes, 5 housekeeping genes, a genomic DNA control, 3 wells containing reverse-transcription controls, and 3 wells containing a positive PCR control (Supplementary Figure S1). Total RNA from RVLM tissues was converted into first-strand cDNA using the RT2 First Strand Kit. The cDNA was next mixed with an appropriate RT2 SYBR® Green Mastermix. This mixture was then aliquoted into the wells of the RT2 Profiler PCR Array. PCR was performed, and the relative expression was determined using data from the real-time cycler and the 2∆∆CT method.
2.21. Experimental Design
Figure 1 illustrates the experimental design of the present study. The first group of young and adult rats (n = 6 per group) was used to evaluate the effect of i.p.
The protocol was repeated in a second group of young and adult animals to evaluate various treatments (n = 5 per group; see below) on hemodynamic and/or biochemical changes induced by i.p.
The third group of young and adult animals was used to identify candidate genes discriminately expressed in RVLM of adult animals (n = 3 per group), and the functional significance of the identified mRNA in susceptibility to hypertension induced by
Treatments employed in the present study included i.p. infusion of
2.22. Statistical Analysis
All data were presented as mean ± standard deviation (SD). The normality of the data distribution was checked before all the statistical analyses using Shapiro–Wilk test to confirm that the data complied with normal distribution. Differences in SBP and HR to various treatments were analyzed with a two-way analysis of variance (ANOVA) with repeated measures, followed by the Tukey multiple comparisons test using time and treatment group as the main factors. All the other differences in mean values were analyzed by one-way ANOVA with Tukey’s multiple comparisons tests. Statistical differences between experimental groups in young and adult animals were evaluated using unpaired Student’s t-tests. All the data were analyzed by GraphPad Prism software (version 6.0; GraphPad Software Inc., La Jolla, CA, USA). p < 0.05 was considered statistically significant.
3. Results
3.1. Age-Dependent Changes in Blood Pressure, Heart Rate, Sympathetic Vasomotor Activity, and Plasma NE Levels in Response to Systemic NO Deficiency
Our first set of experiments evaluated the age-dependent hemodynamic responses to systemic NO deficiency, a well-established animal model for the study of human hypertension [16]. In young (at age of 8 weeks) normotensive WKY rats, i.p. infusion of
3.2. Effect of Systemic
3.3. Differential Effect of Systemic
A series of studies from our laboratory [32,39,42,43] suggest that the LF component of SAP signals originates from the RVLM, and tissue oxidative stress in RVLM augments sympathetic vasomotor activity and BP [28,32,39,41,42,43,44]. We therefore investigated whether the differential effect of
Dysregulated redox homeostasis because of an imbalance in ROS production over degradation leads to tissue oxidative stress [6,26,28,33]. In RVLM, we reported previously that increases in the protein expression of NADPH oxidase subunits [43], and decreases in the expression of antioxidants [46], contribute to oxidative stress that results in sympathoexcitation and hypertension in spontaneously hypertensive rats and normotensive animals treated with angiotensin II (Ang II). We therefore examined the expression of NADPH oxidase subunits and antioxidants in RVLM of animals that were subjected to i.p.
Mitochondria are considered another important cellular source of ROS. In RVLM, impairment of enzyme activity of the mitochondrial electron transport chain (ETC) for oxidative phosphorylation contributes to cellular oxidative stress, leading to sympathetic hyperactivity and neurogenic hypertension [42]. In young normotensive rats, systemic
The increase in ROS production in RVLM could also result from NOS uncoupling [32]. In RVLM of the
We interpret our observations that tissue ROS levels, particularly after i.p.
3.4. Causal Involvement of Tissue Oxidative Stress in RVLM in Age-Dependent Exacerbation of Hemodynamic Responses to Systemic NO Deficiency
To ascertain a causal disparate role of NO deficiency and ROS production in RVLM in age-dependent augmentation of hemodynamic responses to i.p.
These results are interpreted to suggest that in response to systemic NO deficiency, a predominant increase in vasomotor tone because of vascular constriction and sympathetic outflow from RVLM may underline the increase in SBP of the
3.5. Identification of Additional Age-Dependent Redox Homeostasis-Related Genes in RVLM in the Systemic NO-Deficiency Model of Hypertension
Our observations that the increase in tissue ROS levels was greater in RVLM of the
3.6. Silencing Nox1a mRNA in RVLM Ameliorates Oxidative Stress and Attenuates Hemodynamic Responses to Systemic NO Deficiency in Adult Rats
Our final series of experiments was performed to validate the functional significance of the newly identified Noxa1 gene in RVLM on exacerbated hemodynamic responses in the
4. Discussion
The present study was designed to explore the role of oxidative stress in RVLM on age-dependent susceptibility to hypertension in response to systemic NO deficiency, and to decipher the underlying molecular mechanisms. There are four major findings. First, i.p. infusion of
NO deficiency is a well-characterized trait in human hypertension [5,6,7], and NOS inhibition by
To date, cardiovascular responses to
In addition to diminished eNOS/NO signaling, we found in the present study that systemic
Another major cellular source of ROS production is the mitochondrial ETC in association with oxidative phosphorylation for ATP synthesis [55]. The effects of NO on mitochondrial functions and metabolism are mediated mainly through their interactions at specific sites in the ETC enzyme complexes. In this regard, NO, at subnanomolar amounts, inhibits Complex IV via interactions with the ferrous heme iron or oxidized copper at the heme iron:copper binuclear center of the enzyme [56]. At high concentrations, NO inhibits Complex I via oxidation or S-nitrosation of specific thiols [57]. Accordingly, an increase in CCO activity observed in RVLM of
Under the condition of oxidative stress, NOS may remove an electron from NADPH and donate it to an oxygen molecule for generation of O2•− rather than NO [6,14,59]. In RVLM, tissue oxidative stress causes an uncoupling of eNOS during hypertension [28], further depleting the levels of NO and aggravating hypertension progression. In addition, a redox-sensitive feedforward mechanism of nNOS uncoupling in RVLM contributes to sympathoexcitation and hypertension associated with metabolic disorders [32]. In the present study, the ratio between dimmers over monomers of either eNOS or nNOS was not affected (cf. Figure 5B) by i.p.
Redox homeostasis depends on the balance between the production and degradation of the oxidants. At the same time, antioxidant treatments offset the development of
Aging is associated with an increase in ROS production, which together with a decline in antioxidant defense efficiency significantly contributes to the manifestation of an oxidative stress state [38]. Compared to young rats, we found in this study greater increases in NADPH oxidase activity and augmented ROS accumulation in RVLM of adult rats in response to systemic NO deficiency. These intriguing findings prompted us to search for additional candidate molecules that are associated with age-dependent oxidative stress in RVLM. Based on microarray analysis of redox signal-related genes, we identified four genes whose expression levels are at least two times up- or downregulated in RVLM of adult animals. We found that Noxa1 and Gpx2 mRNA were upregulated, whereas Duox2 and Ucp3 mRNA were downregulated. Among them, upregulation of the antioxidant Gpx2 could be an antioxidant defense mechanism to compensate for tissue oxidative stress, and Duox2 is a p22phox-independent isoform that is not important in cardiovascular pathophysiology [6]. We therefore focused on Noxa1 and Ucp3 mRNA to further interrogate their roles in the augmented ROS levels in RVLM of adult rats. First, we confirmed that expression of Noxa1 mRNA was higher, whereas Ucp3 mRNA was lower, in RVLM of adult rats. An age-dependent decrease in Ucp3 expression in male mice has recently been reported [63]. Second, expression of Noxa1, but not Ucp3, mRNA was upregulated by systemic
Treatments targeting NO and ROS signals in the periphery and RVLM were employed to further verify the differential roles of NO and ROS in RVLM on age-dependent cardiovascular responses induced by
There are several limitations to our study. First, the present findings were made from animals that were subjected to a low-dose
5. Conclusions
In conclusion, our findings reveal that disparate mechanisms underlie the increase in SNA and BP in rats subjected to systemic
Conceptualization, J.Y.H.C. and H.R.; methodology, Y.-M.C.; formal analysis, Y.-M.C. and J.Y.H.C.; investigation, Y.-M.C. and J.Y.H.C.; writing—original draft preparation, Y.-M.C. and J.Y.H.C.; writing—review and editing, H.R. and J.Y.H.C.; visualization, Y.-M.C. and J.Y.H.C.; funding acquisition, J.Y.H.C. All authors have read and agreed to the published version of the manuscript.
The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Chang Gung Memorial Hospital, Taiwan (protocol code: 2018051701, date of approval: 8 June 2018).
The data presented in this study are available on reasonable request from the corresponding author.
The authors thank Yen-Hua Hung for her technical assistance in this study and administrative support of the project.
The authors declare no conflict of interest.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Enlarge this image.Figure 1. Experimental design of the present study. The first group of young and adult animals (n = 6 per group) was used to assess systolic blood pressure (SBP), heart rate (HR), and power density of low-frequency component in the SBP spectrum at various time intervals (arrows) before and after i.p. infusion of l-NAME for 14 days. Nitric oxide (NO), reactive oxygen species (ROS), and/or proinflammatory cytokine in plasma and/or tissue of rostral ventrolateral medulla (RVLM) were measured at the end of the 14-day l-NAME treatment (double arrows). The protocol was repeated in a second group of young and adult rats (n = 5 per group) to evaluate the effect of various treatments, delivered via oral gavage or intracisternal infusion during days 7–14, on l-NAME-induced changes in SBP, HR, LF power, and NO and ROS levels in RVLM. The third group of adult animals (n = 6 per group) was used to examine the effect of gene silencing NADPH oxidase activator 1 (Noxa1) in RVLM on changes in SBP, HR, LF power, and NO and ROS levels in RVLM induced by systemic l-NAME. Lentiviral vector contains a target-specific construct that encodes a short hairpin RNA (shRNA) to knock down gene expression of Noxa1 (Lv-Noxa1-shRNA) or control scrambled shRNA (Lv-scr-shRNA) was microinjected into the bilateral RVLM (open arrow) on day 10 after l-NAME infusion.
Enlarge this image.Figure 2. Temporal changes in hemodynamic parameters and serum NO (nitrite and nitrate) levels in response to intraperitoneal infusion of l-NAME (10 mg/kg/day) for 14 days. Changes in (A) systolic blood pressure (SBP); (B) power density of low-frequency (LF) component of SBP signal; (C) plasma norepinephrine (NE) levels; (D) heart rate (HR); as well as (E) serum NO levels detected at different time points in the untreated group, or animals treated with i.p. infusion of saline or l-NAME. Data are presented as mean ± SD, n = 6 per group at each time interval. * p < 0.05 versus saline-treated group (pink or gray bars) in post hoc Tukey’s multiple-range test.
Enlarge this image.Figure 3. Effect of systemic l-NAME treatment on the expression of NOS isoforms and NOS activity in RVLM: (A) Representative gels (insets) and densitometric analysis of results from Western blot analysis showing changes in protein expression of nNOS, iNOS, and eNOS and (B) enzyme activity of NOS in RVLM 14 days after i.p. infusion of saline or l-NAME (10 mg/kg/day) in young (8 weeks, open circles or squares) or adult (20 weeks, filled circles or sqaures) rats. Data on protein expression were normalized to the respective saline control value, which is set to 1.0, and are presented as mean ± SD, n = 6 per group. * p < 0.05 versus corresponding saline-treated group in unpaired Student’s t-test.
Enlarge this image.Figure 4. Effect of systemic l-NAME treatment on reactive oxygen species (ROS) levels and expression of proteins involved in ROS production and degradation. Data showing ROS levels in RVLM (A) and MDA levels in plasma (B) of young (8 weeks, open circles or squares) or adult (20 weeks, filled circles or squares) rats after i.p. infusion of saline or l-NAME (10 mg/kg/day) for 14 days. Also shown are representative gels (insets) and densitometric analysis of results from Western blot changes in protein expression of pg91phox, p22phox, p47phox, and p67phox (C) or SOD2 and Nrf2 (D), as well as enzyme activity of NADPH oxidase and total antioxidants (E) in RVLM 14 days after saline or l-NAME treatment. Data on protein expression are normalized to the respective saline control value, which is set to 1.0. Data are presented as mean ± SD, n = 6 per group. * p < 0.05 versus corresponding saline-treated group in unpaired Student’s t-test.
Enlarge this image.Figure 5. Effect of systemic l-NAME treatment on mitochondrial electron transport chain enzyme activity and NO synthase (NOS) uncoupling in RVLM: (A) Enzyme activities of NADH cytochrome c reductase (NCCR, marker for coupling capacity between complexes I and III), succinate cytochrome c reductase (SCCR, marker for coupling capacity between complexes II and III), and cytochrome c oxidase (CCO, marker for complexes IV) in RVLM of young (8 weeks, open circles or squares) or adult (20 weeks, filled circles or squares) rats after i.p. infusion of saline or l-NAME (10 mg/kg/day). (B) Representative gels (insets) and densitometric analysis of results from Western blot showing changes in the ratio of dimers versus monomers of eNOS and nNOS protein in RVLM of young and adult rats after saline or l-NAME treatment. Data on protein expression were normalized to the respective saline control value, which is set to 100%. Data are presented as mean ± SD, n = 6 per group. * p < 0.05 versus corresponding saline-treated group, and # p < 0.05 versus saline-treated young rats in unpaired Student’s t-test.
Enlarge this image.Figure 6. Effect of NO donor and antioxidants on changes in SBP, and tissue ROS and NO levels in RVLM of young and adult rats induced by systemic l-NAME treatment. Temporal changes in SBP at different postinfusion time points after i.p. infusion of l-NAME, alone or with additional oral intake or i.c. infusion of various pharmacological treatments in young (A) and (B) adult rats. Also shown are tissue levels of ROS (C) and NO (D) in RVLM, measured at day 14 after i.p. infusion of l-NAME, alone or with additional i.c. infusion of pharmacological treatments. The pharmacological treatments included i.p. infusion of l-NAME (10 mg/kg/day), oral intake via gavage of L-arginine (2%), tempol (100 µmol/kg) or amlodipine (10 mg/kg), or i.c. infusion of L-arginine (2 µg/kg/day), tempol (1 μmol//h/μL), or mitoQ10 (2.5 μmol//h/μL). Control infusion of 0.9% saline (for i.p. or oral gavage treatment) or artificial CSF (aCSF; for i.c. infusion) served as the volume and vehicle control. Data are presented as mean ± SD, n = 5–6 per group, and * p < 0.05 versus the corresponding saline-treated group, and # p < 0.05 versus the l-NAME group in post hoc Tukey’s multiple comparisons tests or unpaired Student’s t-test. Data on saline and l-NAME treatments from Figure 2 are adopted for comparison.
Enlarge this image.Figure 7. Age-dependent expression of ROS-related proteins in RVLM and the effect of systemic l-NAME treatment on their expressions: (A) Relative expression of Noxa1 and Ucp3 mRNA quantified by RT-qPCR in RVLM tissues from young (open circules or squares) and adult (filled circles or squares) rats on 14 days after i.p. infusion of saline or l-NAME. (B) Representative gels (insets) and densitometric analysis of results from Western blot changes in protein expression of Noxa1 and UCP3 in RVLM of young and adult 14 days after systemic l-NAME treatment. Data on protein expression were normalized to the respective saline control value, which is set to 1.0. Data are presented as mean ± SD, n = 6 per group. * p < 0.05 versus corresponding saline-treated groups, and # p < 0.05 versus saline-treated young rats in unpaired Student’s t-test.
Enlarge this image.Figure 8. Effect of manipulations of Noxa1 gene in RVLM on mRNA and protein expression, and changes in cardiovascular responses and levels of ROS in RVLM and serum NO of adult rats induced by systemic l-NAME treatment: (A) Changes in mRNA transcription of Noxa1 and representative gels (insets) and densitometric analysis of results from Western blot showing changes in protein expression of Noxa1 in RVLM tissues 4 days after bilateral microinjection into RVLM of lentiviral vectors (Lv) containing short hairpin interfering RNA (shRNA) targeting the rat Noxa1 sequence (Lv-Noxa1-shRNA) or a scrambled (scr) control shRNA. (B) SBP, power density of the LF component of SBP signals, and plasma NE levels; and (C) tissue ROS and (D) serum NO levels, determined on day 14 after i.p. infusion of saline or l-NAME (10 mg/kg/day) in adult rats that received additional treatment with bilateral microinjection into RVLM of Lv-scr-RNA or Lv-Noxa1-shRNA on day 10 after l-NAME treatment. Data on mRNA transcription and protein expression are normalized to the respective saline control value, which is set to 1.0. Data are presented as mean ± SD, n = 6 per group. * p < 0.05 versus corresponding saline-treated groups, and # p < 0.05 versus Lv-scr-RNA-treated groups in unpaired Student’s t-test.
Changes in plasma levels of proinflammatory cytokines in young (8 weeks) and adult (20 weeks) rats in response to i.p. infusion of saline or
| Saline | ||||
|---|---|---|---|---|
| Young | Adult | Young | Adult | |
| IL-1β (ng/mL) | 0.58 ± 0.11 | 0.47 ± 0.21 | 0.69 ± 0.31 | 0.61 ± 0.34 |
| IL-6 (pg/mL) | 208 ± 42 | 186 ± 39 | 223 ± 68 | 217 ± 56 |
| TNF-α (pg/mL) | 89 ± 38 | 105 ± 43 | 93 ± 54 | 119 ± 72 |
Saline or
Supplementary Materials
The following supporting information can be downloaded at:
References
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