*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 <sup>μ</sup>L 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 RT<sup>2</sup> First Strand Kit. The cDNA was next mixed with an appropriate RT<sup>2</sup> 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 2ΔΔCT 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. L-NAME on SBP, HR, LF component of SAP signals, plasma NE and serum NO levels, plasma MAD and proinflammatory cytokine, and tissue ROS levels and expression of proteins for the production and degradation of ROS in RVLM. The hemodynamic parameters were recorded on days 3 and 1 prior to, and on the day (day 0) before, osmotic pump implantation and days 1, 3, 5, 7, 9, 11, and 14 following i.p. infusion of L-NAME or 0.9% saline. NE and NO were measured on day 3 before, and days 0, 7, and 14 following L-NAME treatment. At the end of the 14-day infusion, the power density of the LF component of SAP signals was determined before animals were killed to collect blood for proinflammatory cytokine measurements, and collect RVLM tissue for measurements of ROS and protein expressions.

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. L-NAME infusion. The pharmacological manipulations, including oral gavage or i.c. infusion, were performed from days 7 to 14 during the 14-day L-NAME treatment period. BP was recorded on day 3 before, and on days 0, 3, 7, 9, 11, and 14 following L-NAME infusion, and power density of LF component, tissue levels of ROS and NO in RVLM were determined at the end of the treatment period.

**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.

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 L-NAME (*n* = 6 per group) in adult animals. Gene manipulation was carried out via bilateral microinjection of lentiviral vectors into RVLM on day 10 following L-NAME infusion, and mRNA and protein expressions, as well as SBP were measured at the end of the 14-day infusion.

Treatments employed in the present study included i.p. infusion of L-NAME (10 mg/kg/day; Sigma-Aldrich, MA, USA), oral intake via gavage of L-arginine (2%, Sigma-Aldrich), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol; 100 μmol/kg; Sigma-Aldrich), or amlodipine (10 mg/kg; Sigma-Aldrich); or i.c. infusion of L-arginine (2 μg/kg/day), tempol (1 μmol//h/μL), or mitoQ10 (2.5 μmol//h/μL; Sigma-Aldrich); or microinjection bilaterally into RVLM of Lv-Noxa1-shRNA (1 × 105 IFU per animal) or Lv-scr-shRNA (1 × 105 IFU per animal). 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. The composition of aCSF was (mM): NaCl 117, NaHCO3 25, Glucose 11, KCl 4.7, CaCl2 2.5, MgCl2 1.2, and NaH2PO4.

#### *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 L-NAME (10 mg/kg/day) for 14 days resulted in gradual increases in SBP, power density of the LF component of the SAP signals, our experimental index for neurogenic sympathetic vasomotor activity [32,43], and plasma NE levels, but not HR, which became statistically significant on postinfusion days 7−14 (Figure 2A−D). Similar cardiovascular responses were observed in adult animals (at age of 20 weeks). Of note, baseline SBP (105 ± 4.4 versus 90.7 ± 5.4 mmHg, *n* = 6, *p* < 0.05), as well as temporal increases in SBP (139.8 ± 3.5 versus 125.5 ± 6.5 mmHg, *n* = 6; *p* < 0.05), LF power density (2.76 ± 0.26 versus 2.39 ± 0.24 mmHg2, *<sup>n</sup>* = 6, *<sup>p</sup>* < 0.05), and plasma NE levels (7.16 ± 0.33 versus 6.40 ± 0.46 ng/mL, *n* = 6, *p* < 0.05) measured on day 14 postinfusion, were significantly greater in adult animals when compared to young rats. L-NAME infusion evoked similar decreases in serum NO (nitrite and nitrate) levels, detected on days 7 and 14 postinfusion in both age groups (Figure 2E). The same treatment, on the other hand, exerted no effect on plasma Il-1β, IL-6, and TNF-α levels measured at the end of L-NAME infusion (Table 1). These data suggest an age-dependent vulnerability in hemodynamic changes associated with systemic NO deficiency.

**Table 1.** 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 L-NAME.


Saline or L-NAME (10 mg/kg/day) was infused into the peritoneal cavity for 14 days. Data are presented as mean ± SD, *n* = 6 per group. No significant difference exists between groups in One-Way ANOVA. IL-1β, interleukin 1-β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α.

#### *3.2. Effect of Systemic* L*-NAME Treatment on Expression of NOS Isoforms and Activity in RVLM*

L-NAME reportedly crosses the BBB to alter NOS expression and inhibit NOS activity in brain [22–24]. At the end of 14-day i.p. infusion of L-NAME, protein expression of eNOS, but not nNOS or iNOS, isoform in RVLM was significantly decreased (Figure 3A), alongside a notable suppression of NOS activity (Figure 3B) in both young and adult animals. Of note, systemic L-NAME treatment resulted in comparable suppression in eNOS expression and total NOS activity in RVLM of both age groups.

**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.

**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.

## *3.3. Differential Effect of Systemic* L*-NAME Treatment on Tissue ROS Levels in RVLM of Animals at Different Ages*

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 [28,32,39,41–44]. We therefore investigated whether the differential effect of L-NAME infusion on hemodynamic parameters at different ages is the consequence of disparate tissue oxidative stress in RVLM. As shown in Figure 4A, baseline tissue ROS levels were higher, albeit statistically insignificant, in RVLM of adult animals. Moreover, i.p. L-NAME (10 mg/kg/day) infusion for 14 days resulted in further increases in tissue ROS levels in RVLM of adult, but not young rats. On the other hand, there were no detectable increases in plasma MDA levels (a biomarker of oxidative stress) in both groups (Figure 4B).

**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.

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. L-NAME infusion. In RVLM of WKY rats at age of 8 weeks, the protein expression of pg91phox and p22phox, but not p47phox or p67phox subunit of NADPH oxidase (Figure 4C), determined on day 14 following i.p. infusion of L-NAME, was appreciably increased. Interestingly, the protein expression of two key antioxidants, SOD2 and Nrf2 (Figure 4D), was also increased at the same postinfusion time point. Similar results were found in RVLM of L-NAMEtreated animals at the age of 20 weeks. As shown in Figure 4E, systemic L-NAME treatment also significantly augmented the enzyme activity of NADPH oxidase and total antioxidant capacity in RVLM, measured on day 14 postinfusion in both age groups. Notably, the increase in NADPH oxidase activity in RVLM was significantly greater in the adult animals (+66.5 ± 13.8% versus +30.5 ± 10.2 %, *n* = 6, *p* < 0.05).

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 L-NAME treatment increased the enzyme activity of CCO (electron transport in Complex IV), but not NCCR (enzyme for electron transport between Complexes I and III) or SCCR (enzyme for electron transport between Complexes II and III) in RVLM (Figure 5A), accompanied by a mild increase in tissue ATP content (15.8 ± 2.1 versus 17.8 ± 1.3 pmol/μg, *n* = 6, *p* = 0.071). On the other hand, the enzyme activity of both NCCR and CCO was notably depressed in RVLM of control adult rats, and remained reduced following L-NAME treatment; together with a moderate decrease in tissue ATP content (15.4 ± 1.3 versus 13.2 ± 1.3 pmol/μg, *n* = 6, *p* < 0.05).

**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.

The increase in ROS production in RVLM could also result from NOS uncoupling [32]. In RVLM of the L-NAME-treated animals, the ratio of eNOS monomer over dimer, an experimental index of NOS uncoupling, remained unchanged in both age groups (Figure 5B); although, eNOS expression, NOS activity, and tissue NOx levels were suppressed. Similarly, the same treatment did not affect nNOS coupling, which has been reported to promote tissue oxidative stress in RVLM [32], in both age groups.

We interpret our observations that tissue ROS levels, particularly after i.p. L-NAME infusion, were significantly augmented in RVLM of adult rats to suggest an active role of ROS in RVLM in age-dependent exacerbation of hemodynamic responses in this NO deficiency model of hypertension. The elevated protein expression and enzyme capacity of the antioxidants may explain why redox homeostasis in RVLM of young rats is maintained despite the increase in both protein expression and enzyme activity of NADPH oxidase after L-NAME treatment. On the other hand, the enhanced augmentation of NADPH oxidase activity may account for the further increase in tissue ROS levels observed in RVLM of adult animals. The reduced mitochondrial bioenergetics because of impaired enzyme activity of NCCR and CCO may also give rise to the heightened ROS levels in RVLM of adult animals after systemic L-NAME treatment.

## *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. L-NAME infusion, young and adult animals were randomly divided into six groups to receive, respectively, an oral intake or i.c. infusion of L-arginine, a NOS precursor; tempol, a ROS scavenger; mitoQ10, a mitochondrial-targeted SOD mimetic, or amlodipine, a vasodilator of dihydropyridine class of calcium channel blockers; from days 7 to 14 following i.p. infusion of L-NAME (10 mg/kg/day). In young rats, oral intake of L-arginine (2%) or amlodipine (10 mg/kg), or i.c. infusion of L-arginine (2 μg/kg/day), but not oral intake of tempol (100 μmol/kg) or i.c. infusion of tempol (1 μmol//h/μL) or mitoQ10 (2.5 μmol//h/μL), significantly attenuated the increase in SBP in the L-NAME-treated animals (Figure 6A). On the other hand, when L-arginine, tempol, or mitoQ was microinfused into the cisterna magna of adult animals, one week after i.p. L-NAME infusion, the treatments discernibly diminished the increases in SBP induced by systemic L-NAME treatment (Figure 6B). Microinfusion of tempol or mitoQ10, but not L-arginine, into the cisterna magna, at the same time, restored tissue ROS in RVLM of the L-NAME-treated rats to saline-control levels (Figure 6C). Infusion of tempol or mitoQ10 into the cisternal magna had no apparent effect on tissue NO levels in RVLM (Figure 6D), and i.c. infusion of tempol or mitoQ10 infusion did not affect the reduced serum NO levels following L-NAME infusion.

**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 salinetreated 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.

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 L-NAME-treated young animals. When animals become older, tissue oxidative stress in RVLM is actuated to further increase sympathetic vasomotor activity and promote greater hemodynamic responses in the systemic L-NAME treatment model of hypertension.

## *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 L-NAME-treated adult animals (cf. Figure 4E) prompted the speculation that additional oxidative stress-related genes on top of those reported previously are upregulated in this brain stem site. To address this issue, we performed a whole genome microarray analysis of RVLM tissue using a Qiagen RT2 ProfilerTM PCR array (Qiagen). As shown in the supplementary data (Supplementary Figure S1), four differentially expressed genes, whose expression levels are at least two times different from young rats, were identified in RVLM of adult animals. These included two upregulated genes, NADPH oxidase activator 1 (*Noxa1*) and glutathione peroxidase 2 (*GPx2*), and two downregulated genes, dual oxidase 2 (*Duox2*) and uncoupling protein 3 (*Ucp3*). Given the limitation in the RVLM sample volume, we decided to verify the accuracy of our microarray analysis by real-time qPCR only on the candidate transcriptomes that exhibited the highest (Noxa1) and lowerest (*Ucp3*) changes. Our RT-qPCR results confirmed the microarray data for *Noxa1* and *Ucp3* mRNA in RVLM (Figure 7A). Moreover, their expressions were further upregulated in the L-NAME-treated animals in both age groups. Similar patterns were found in protein expression of Noxa1 and UCP3 in RVLM of both groups of animals (Figure 7B). Of note, the upregulation of *Noxa1* (+1.9 ± 0.2 versus +1.1 ± 0.5 fold, *n* = 6, *p* < 0.05), but not a change in *Ucp3* mRNA (+1.7 ± 0.4 versus +1.8 ± 0.3 fold, *n* = 6, *p* > 0.05), was significantly greater in RVLM of adult animals when compared to young rats. Together these results suggest that age-dependent alterations in oxidative stress-related gene transcription, including the upregulation of a ROS-producing enzyme, *Nox1a*, and the downregulation of an antioxidant, *Ucp3*, may contribute to the elevated ROS levels in RVLM following i.p. L-NAME infusion.

#### *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 L-NAME-treated adult animals. In situ gene silencing via bilateral microinjection into RVLM of lentiviral vectors encoding shRNA targeting *Noxa1* (Lv-Noxa1-shRNA; 1 × 105 IFU) was performed on day 10 after the onset of i.p. L-NAME infusion. Effective transduction of the viral vectors into RVLM was confirmed by qPCR, which showed a significant decrease in *Noxa1* mRNA and protein, measured on day 4 after vector transduction (Figure 8A). Compared to their scramble shRNA control, silencing *Nox1a* in RVLM with its shRNA resulted in notable attenuation of hypertension as well as the increase in sympathetic vasomotor activity and plasma NE levels (Figure 8B) in the L-NAME-treated adult animals. Gene silencing of *Noxa1* also significantly alleviated the elevated ROS levels (Figure 8C) in RVLM, but not the reduced plasma NO levels (Figure 8D). These data provide evidence to suggest an active role of *Noxa1* in RVLM on age-dependent susceptibility to hypertension induced by systemic L-NAME treatment in adult rats.

**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 <sup>±</sup> SD, *<sup>n</sup>* = 6 per group. \* *<sup>p</sup>* < 0.05 versus corresponding saline-treated groups, and # *<sup>p</sup>* < 0.05 versus saline-treated young rats in unpaired Student's *t*-test.

**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.

#### **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 L-NAME evoked oxidative stress in RVLM in adult, but not young, normotensive rats, accompanied by augmented enzyme activity of NADPH oxidase and reduced mitochondrial NCCR and CCO enzyme activities. Second, treatment with Larginine via oral gavage or infusion into the cistern magna, but not i.c. tempol or mitoQ10, significantly offset the L-NAME-induced hypertension in young rats. On the other hand, all treatments appreciably reduced L-NAME-induced hypertension in adult rats. Third, four genes involved in ROS production and clearance were differentially expressed in RVLM in an age-related manner. Of them, *Noxa1* and *GPx2* were upregulated and *Duox2* and

*Ucp3* were down-regulated. Systemic L-NAME treatment caused greater upregulation of *Noxa1*, but not *Ucp3*, mRNA expression in RVLM of adult rats. Fourth, gene silencing of *Noxa1* in RVLM effectively alleviated oxidative stress and protected adult rats against L-NAME-induced hypertension. These data together suggest that hypertension induced by systemic L-NAME treatment in young rats is mediated primarily by NO deficiency that occurs both in vascular smooth muscle cells and RVLM. On the other hand, enhanced augmentation of oxidative stress in RVLM contributes to a heightened susceptibility of adult rats to hypertension induced by systemic L-NAME treatment.

NO deficiency is a well-characterized trait in human hypertension [5–7], and NOS inhibition by L-NAME is commonly used to establish NO deficiency in animal models of human hypertension [16]. L-NAME exerts various pharmacological effects on cardiovascular functions and molecular activities that are dependent on dose (1–50 mg/kg/day), route (oral, subcutaneous, i.p., or cerebral ventricle), and mode (acute, daily bolus, or continuous infusion) of administration, as well as the duration of treatment (minutes, hours, days, or weeks). In this study, we employed a relatively low dose for i.p. infusion of L-NAME (10 mg/kg/day) for 2 weeks to establish the expected hemodynamic responses, with minimal concomitant oxidative and inflammatory actions (cf. Table 1) to avoid their confounding influences on hypertension development. At a higher dosage and/or longer duration of L-NAME treatment, ROS production in vascular smooth muscle cells [47], kidney [48], and heart [21], as well as pro-inflammatory cytokines in kidney [49] and heart [21], have been demonstrated to mediate hypertension and associated cardiovascular complications such as cardiac hypertrophy and renal injury. The findings that the plasma level of NE was increased and serum NO level was reduced, and that oral intake of L-arginine and amlodipine (cf. Figure 6A,B) conferred protection against L-NAME-induced hypertension suggest that an increase in SNA and vasoconstriction may contribute to the observed hemodynamic changes. We did not find significant changes in HR following L-NAME administration; although, other studies demonstrated reduction [50] or augmentation [18] in HR. The exact reason behind these diverse findings is not clear, but might simply be the consequence of differences in dose and duration of L-NAME treatment. In this regard, L-NAME given at 1.5 times our dose for 2 weeks decreases [50], and at 4 times our dose for 5 weeks increases, HR [18].

To date, cardiovascular responses to L-NAME treatment have primarily focused on its effects on NOS expression and NO bioavailability in the vasculature, with little attention on the role of L-NAME in the central nervous system. As such, one of the major findings of the present study is the identification of the suppressive effects of systemic L-NAME treatment on eNOS protein expression, NOS activity, and tissue NO level in RVLM of both young and adult rats. L-NAME has been reported to cross the BBB to reach brain tissues [22–24]. All three NOS isoforms are constitutively present in RVLM [51], and their roles in RVLM on neural control of cardiovascular functions, have been extensively reviewed in the literature [26]. In the present study, we found that the expression of eNOS, but not nNOS or iNOS, protein in RVLM was suppressed by i.p. L-NAME infusion. Since constitutive eNOS tonically inhibits RVLM neuronal activity and sympathetic outflow [52], a diminished eNOS expression and eNOS-derived NO availability in RVLM may therefore contribute to the observed increases in sympathetic vasomotor activity and plasma NE levels in rats subjected to systemic L-NAME treatment. These findings are in concordance with the observations that inhibition of eNOS by L-NAME evokes central sympathoexcitation, leading to increased SNA in experimental animals [19] and healthy men [53]. In addition, diminished eNOS expression and eNOS-derived NO bioavailability in the hypothalamic paraventricular nucleus (PVN) by L-NAME contributes to sympathoexcitation and hypertension associated with heart failure [54]. We reported previously a concentration-dependent action of NO in RVLM on neural control of cardiovascular functions. Whereas nNOS-derived NO is responsible for sympathoexcitation, iNOS-induced NO elicits sympathoinhibition [51]. In contrast, a sympathoinhibitory action of NO derived from nNOS in RVLM has also been reported [29]. In the present study, neither nNOS nor iNOS expression in RVLM was

affected by systemic L-NAME treatment. These results are at variance with a previous study [24] that shows nNOS mRNA in RVLM is downregulated in young (4 weeks), but upregulated in adult (10 weeks) rats, following oral intake of L-NAME (50 mg/kg/day) for 6 weeks. Such discrepancies may again reflect differences in dose, route, and duration of L-NAME treatment.

In addition to diminished eNOS/NO signaling, we found in the present study that systemic L-NAME treatment also affected proteins involved in ROS production and clearance in RVLM. Of the major sources for the production of ROS, we found the protein expression of gp91phox and p22phox subunits was increased in both age groups, alongside elevated enzyme activity of the NADPH oxidase. In addition, the enzyme activity of mitochondrial CCO was increased in young, but decreased in adult, rats, and NCCR activity was also decreased in adult rats. On the contrary, NOS uncoupling, which is considered a secondary source of ROS production [6], was not affected by systemic L-NAME treatment. The NADPH oxidase family is the most important enzymatic source of ROS in the cardiovascular system [6,26]. In RVLM, augmented ROS production resulting from increases in pg91phox and P22phox subunits initiates a series of molecular events, leading to tissue oxidative stress and sympathoexcitation that contribute to the neural mechanism of hypertension [43]. Expression of p47phox and p67phox proteins, the other two NADPH oxidase subunits that play active roles in the redox-associated neural mechanism of hypertension [43], on the other hand, were not affected by L-NAME treatment; this might be related to the susceptibility of individual subunits to NO deficiency. The mechanisms underpinning the increase in gp91phox and p22phox protein expressions induced by L-NAME are not immediately clear, but might be the consequence of transcriptional upregulation of these subunits [14,24].

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 L-NAME-treated young rats could result from the withdrawal of an inhibitory effect of NO on Complex IV. On the other hand, the mechanism that underlies the lower NCCR and CCO activity in RVLM of adult rats and their reduced responsiveness to NO deficiency is unclear. Nonetheless, it is noteworthy that aging selectively downregulates genes encoding Complex I and III of the mitochondria ETC both in rat and human hearts [58]. Functionally, impairment of both NCCR and CCO activity in RVLM has been reported to increase mitochondrial ROS production that contributes to hypertension in SHR or Ang II treatment in normotensive rats [46].

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. L-NAME infusion, suggesting a negligible role of NOS uncoupling in ROS production in RVLM in response to systemic NO deficiency.

Redox homeostasis depends on the balance between the production and degradation of the oxidants. At the same time, antioxidant treatments offset the development of L-NAME-induced hypertension by a reduction in ROS production during NOS inhibition [50]. In young and adult rats subjected to systemic L-NAME treatment, we found that the protein expression of two key antioxidants, SOD2 and Nrf2, was significantly increased, alongside an increase in total antioxidant activity in RVLM. SOD2, or manganese SOD, is one of the

most well-characterized antioxidant defensive mechanisms for the elimination of cellular oxidants, particularly O2 •−. Transcribed from *sod2* and synthesized in the cytoplasm, SOD2 is subsequently relocated to the mitochondrial matrix, endowed with the responsibility to scavenge O2 •− produced by the mitochondrial ETC [60]. In RVLM, transcriptional upregulation of *sod2* protects against mitochondrial oxidative stress and hypertension in Ang II treatment in normotensive rats [61]. SOD2 also participates in the protection again hypertension and cardiovascular complications conferred by the mitochondriatarget antioxidants [62]. Nrf2 is the master regulator of antioxidant genes and, hence, of antioxidant status. Nrf2 has been demonstrated to be a key to redox homeostasis in RVLM; targeted ablation of Nrf2 in RVLM leads to hypertension [41]. The increase in the expression of these antioxidants may explain why redox homeostasis in RVLM of young rats was maintained despite significant elevations in ROS production by mitochondria and NADPH oxidase induced by systemic NO deficiency. These data, at the same time, suggest that the oxidant responsiveness to systemic L-NAME treatment is well tolerated in RVLM of young rats but may turn remitted when animals become older.

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 L-NAME treatment, suggesting that transcriptional regulation of ROS signal-related genes as a consequence of tissue NO deficiency is target specific. Third, gene silencing of *Nox1* appreciably alleviated the augmented ROS levels in RVLM, indicating the agedependent accumulation of ROS in RVLM may be attributed to an upregulation of *Noxa1* transcription. Finally, the functional significance of the newly identified *Noxa1* mRNA in RVLM on age-dependent susceptibility of cardiovascular responses to tissue NO deficiency is validated by our findings that bilateral microinjection into RVLM of Lv-Noxa1-shRNA appreciably ameliorated hypertension, the exaggerated sympathetic vasomotor activity, and plasma NE levels evoked by L-NAME treatment in adult animals. Noxa1 is a critical functional homolog of p67phox for NADPH oxidase activation in vascular smooth muscle cells [64]. NOX1 (a p22phox-dependent oxidase) interacts with p67phox homolog Noxa1, causing constitutive production of O2 •− [65]. Conversely, genetic deletion of *Noxa1* reduces basal and Ang II-induced hypertension and renal oxidative stress [66]. The observations of comparable changes in Ucp3 expression in RVLM of young and adult animals to systemic L-NAME treatment (cf. Figure 7) are interpreted to suggest a minor role of mitochondrial Ucp3 in RVLM on age-related susceptibility to hypertension in adult rats to systemic NO deficiency. This suggestion, nonetheless, waits for further validation.

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 L-NAME. In young rats, both oral intake and i.c. infusion of L-arginine, but not i.c. application of tempol or mitoQ10, significantly reduced hypertension induced by systemic NO deficiency. These results indicate that L-NAME-induced

cardiovascular responses in young animals may mainly be the result of the NO deficiency that occurs both in the smooth muscle cells and RVLM. The engagement of tissue oxidative stress in RVLM on cardiovascular responses to L-NAME in adult animals was unveiled by observations that apart from L-arginine, i.c. infusion of tempol and mitoQ10 significantly diminished hypertension. It is noteworthy that i.c. infusion of tempol or mitoQ10 had no effect on the reduced NO levels in RVLM, indicating the protective actions of tempol and mitoQ10 are not secondary to changes in tissue NO contents. Moreover, the results that i.c. infusion of L-arginine had no effect on the augmented ROS levels in RVLM of adult rats (cf. Figure 6C) suggesting that aging-associated oxidative stress may be related to changes in ROS signals but not NOS activity or tissue NO bioavailability in RVLM. This notion of a minor role of NO in ROS production in RVLM of adult animals is further supported by findings that L-NAME had no effect on the reduced enzyme activity of mitochondrial NCCR and CCO in RVLM of adult rats (cf. Figure 5A). Finally, the observations that oral intake of L-arginine and amlodipine protected both young and adult rats from L-NAMEinduced hypertension suggest that the observed cardiovascular changes are likely the final outcomes of vasoconstriction in response to systemic NO deficiency.

There are several limitations to our study. First, the present findings were made from animals that were subjected to a low-dose L-NAME treatment. As discussed above, in view of the disparity of cardiovascular responses that are dependent on doses of L-NAME, the notion of an interplay between NOS and ROS in the pathogenesis of hypertension induced by systemic NO deficiency should be taken with caution. Second, since the present study focused only on RVLM, the roles of NOS and ROS in other "pre-autonomic" neurons, such as the nucleus tractus solitarii (NTS) and PVN, in neural mechanisms of the L-NAME-induced cardiovascular complications remain to be delineated. In this regard, both NOS and ROS signaling in the NTS and PVN have been reported to play pivotal roles in hypertension induced by systemic L-NAME treatment [6,20,26,30,31,67]. Third, an increase in Ang II release along with depressed NO production is considered the principal culprit in hemodynamic and structural alterations in L-NAME-treated rats [68]. In RVLM, Ang II induces ROS production via activation of NADPH oxidase [6,26,32]. In addition, NO deficiency differentially affects the expression of Ang II receptors in RVLM of young versus adult rats [24]. Therefore, it would be of interest to further investigate the role of Ang II in RVLM in the interplay between NOS and ROS on age-dependent susceptibility to hypertension induced by systemic L-NAME treatment. Fourth, in the present study, we used a commercially available microarray kit to screen the oxidative stress-related genes that are differentially expressed in RVLM of young versus adult rats. The genes provided in the kit are far from complete; consequently, the identified genes could be underestimated. In a recent study, out of 47 genes that are involved in ROS metabolism, 39 are downregulated and 8 upregulated in the aged (24 months) versus adult (6 months) rat heart [58].

#### **5. Conclusions**

In conclusion, our findings reveal that disparate mechanisms underlie the increase in SNA and BP in rats subjected to systemic L-NAME treatment in an age-dependent manner. In young rats, cardiovascular responses to L-NAME are mediated mainly by NO deficiency, both in the vascular smooth muscle cells and RVLM. When animals become older, additional ROS generation from both mitochondrial (reduction in enzyme activity of NCCR and CCO) and extra-mitochondrial (transcriptional upregulation of Noxa1) pathways may contribute to the enhanced susceptibility to sympathoexcitation and hypertension induced by systemic L-NAME treatment. This information provides novel insights into potential targets involved in the responsiveness to systemic NO deficiency during aging that could be manipulated to prevent age-associated deterioration in cardiovascular functions. Moreover, recognizing the functional significance of aging on the transcription of genes encoding ROS signaling molecules may help to identify novel targets that can be selectively intervened to prevent aging-associated hypertension and cardiovascular complications.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10092232/s1, Figure S1: Differentially expressed oxidative stress-related genes in RVLM of adult animals.

**Author Contributions:** 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.

**Funding:** This research was funded in part by the Ministry of Science and Technology, Taiwan (grant number: MOST108-2923-B-182A-001-MY3), Chang Gung Medical Foundation, Taiwan (grant number: OMRPG80011), and International Joint Project of GACR, Czech Republic and MOST, Taiwan: 19-08260J.

**Institutional Review Board Statement:** 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).

**Data Availability Statement:** The data presented in this study are available on reasonable request from the corresponding author.

**Acknowledgments:** The authors thank Yen-Hua Hung for her technical assistance in this study and administrative support of the project.

**Conflicts of Interest:** The authors declare no conflict of interest.
