Many studies reported reduced antioxidant capacity in the vasculature under hypertensive conditions. However, little is known about the effects of antihypertensive treatments on the regulation of vascular antioxidant enzymes. Thus, we hypothesized that antihypertensive treatments prevent the reduction of antioxidant enzyme activity and expression in the small vessels of angiotensin II-induced hypertensive rats (ANG). We observed the small mesenteric arteries and small renal vessels of normotensive rats (NORM), ANG, and ANG treated with a triple antihypertensive therapy of reserpine, hydrochlorothiazide, and hydralazine (ANG + TTx). Systolic blood pressure was increased in ANG, which was attenuated by 2 weeks of triple therapy (127, 191, and 143 mmHg for NORM, ANG, and ANG + TTx, respectively;
In many diseases, an imbalance between prooxidants and antioxidants leads to oxidative stress. Oxidative stress exerts many deleterious effects on the vasculature, including a decrease in nitric oxide (NO) levels, vascular remodeling, and/or vasoconstriction, thereby leading to vascular dysfunction (1). Oxidative stress has been implicated in the pathogenesis and complications of many cardiovascular diseases, including hypertension, hypercholesterolemia, atherosclerosis, diabetes, and heart failure (2). Increased reactive oxygen species (ROS) production during hypertension has been shown in human patients (3,4) and experimental animal models, such as spontaneously hypertensive rats (SHR) (5), deoxycorticosterone acetate-salt hypertensive rats (6), Dahl salt-sensitive rats (SS) (7), and angiotensin II-induced hypertensive rats (ANG) (8). Specifically, reduced antioxidant capacity appears to contribute to the overproduction of ROS in human patients with hypertension (9), SHR (10), SS (11), diabetic rats (12), and ANG (13).
Our laboratory has reported that the small arteries of ANG show increased oxidative stress and reduced NO signaling (14). Moreover, we also showed previously that an antihypertensive triple therapy with reserpine, hydrochlorothiazide, and hydralazine restored NO signaling (15). The activity and protein expression of antioxidant enzymes in small vessels have not been investigated as intensively as those in conduit vessels. Furthermore, very little information is available regarding the relationship between antihypertensive treatments and antioxidant enzyme regulation. Thus, we aimed to investigate the antioxidant capacity of small vessels under hypertensive conditions.
Superoxide dismutase (SOD) is an antioxidant enzyme that catalyzes the dismutation of O2− to H2O2. H2O2 is then converted to O2 and H2O by catalase. Three isoforms of SOD have been identified: intracellular copper zinc SOD (CuZn-SOD; SOD1), mitochondrial manganese SOD (Mn-SOD; SOD2), and extracellular CuZn-SOD (ecSOD; SOD3). We hypothesized that reduced antioxidant capacity in the small vessels of ANG will be restored by antihypertensive treatments. We used ANG in this study because we also used this rat model in our previous studies on small arteries (14,15). The activities of SOD isoforms and catalase as well as each protein expression were evaluated in the small mesenteric arteries and small renal vessels of normotensive rats (NORM), ANG, and ANG treated with triple antihypertensive therapy (reserpine, hydrochlorothiazide, and hydralazine) (ANG + TTx).
Angiotensin II was purchased from Phoenix Pharmaceuticals, Inc (Belmont, CA, USA). Mini-osmotic pumps were purchased from Alzet (Model 2002, Cupertino, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Male Sprague-Dawley rats weighing 200–250 g (Charles River Laboratories, Wilmington, MA, USA) were divided into 3 groups: NORM, ANG, and ANG + TTx (30 mg/kg/day hydralazine, 10 mg/kg/day hydrochlorothiazide, 0.5 mg/kg/day reserpine). The dose and route of administration of the triple therapy were previously shown to be efficacious (16–19). To establish angiotensin II-induced hypertension, an osmotic mini-pump containing angiotensin II was implanted subcutaneously into rats under isoflurane anesthesia (IsoFlo; Abbott Laboratories, North Chicago, IL, USA). Angiotensin II was induced chronically at the rate of 70 ng/min for 2 weeks. Triple therapy (TTx) was incorporated in the drinking water 3 days prior to and throughout the 2-week period of angiotensin II infusion. The TTx solution was changed every three days and modified each drug concentration according to the water intake of each rat. Systolic blood pressure was measured after 7 and 14 days of angiotensin II infusion by tail-cuff plethysmography, as previously described (20). Systolic blood pressure of each rat was determined in the average of 4 to 6 independent readings. After 2 weeks, the rats were anesthetized with sodium pentobarbital (Nembutal, 50 mg/kg IP; Abbott Laboratories), and the small mesenteric arteries and small renal vessels were isolated, as previously described (21,22). Briefly, the mesenteric bed, including arteries and veins, was cut away from the intestinal wall, and placed in a dissecting dish containing ice-cold physiological saline solution (PSS; pH = 7.4; 130 mmol/L NaCl, 4.7 mmol/L KCl, 1.18 mmol/L KH2PO4, 1.17mmol/L MgSO4·7H2O, 14.9 mmol/L NaHCO3, 5.5 mmol/L dextrose, 0.26 mmol/L EDTA, 1.6 mmol/L CaCl2). The fat was carefully pulled off from the mesenteric beds, and then the veins were removed using an Olympus dissecting microscope (Center Valley, PA, USA). For small renal vessels, kidneys were removed and immediately placed in a dissecting dish containing ice-cold physiological saline solution. After removal of the renal capsule, the kidney was placed between a circle sieve of 70-mm pore size (Biodesign, Carmel, NY, USA). Small renal vessels were immediately isolated by rapid and gentle grating under the microscope. Isolated small mesenteric arteries and small renal vessels were subsequently frozen in liquid nitrogen and stored at −80°C until further use. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved and monitored by the Augusta University Institutional Animal Care and Use Committee.
Small mesenteric arteries and small renal vessels were isolated in cold physiological saline solution (PSS; same composition as that in the previous section), as previously described (15,21,22). Isolated vascular beds were placed into ice-cold homogenization buffer (20.0 mmol/L Na-HEPES, pH = 7.4; 1.0 mmol/L EGTA; 210.0 mmol/L mannitol; 70.0 mmol/L sucrose) and homogenized on ice with a glass tissue grinder. Supernatants were obtained by centrifugation (1,500 ×
The small mesenteric arteries and small renal vessels were placed into ice-cold homogenization buffer (same composition as that in the previous sec tion) containing protease inhibitors (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitors (Pierce, Pockford, IL, USA), and homogenized on ice with a glass tissue grinder. Protein concentration was determined by standard Bradford assay and western blotting, as described previously (23). Briefly, after transferring the proteins onto a PVDF membrane, the membrane was blocked in a blocking buffer (LI-COR Biosciences, Lincoln, NE, USA). Two-color immunoblotting was performed using primary antibodies against SOD1 (Stressgen, Victoria, British Columbia Canada), SOD2 (Stressgen, Victoria, British Columbia Canada), SOD3 (generously provided by Dr. Tohru Fukai, Augusta University), catalase (Abcam, Cambridge, MA, USA), and β-actin (Sigma-Aldrich). Specific bands were detected using an Odyssey Infrared Imager (LI-COR Biosciences, Lincoln, NE, USA). IRDye800 (Rockland Immunochemicals, Limerick, PA, USA) and AlexaFluor 680 (Molecular Probes, Eugene, OR, USA) were used as secondary antibodies to detect two primary antibodies from different species. Relative densitometric units of the proteins were calculated via normalization to that of β-actin.
Data are expressed as mean ± SEM and were analyzed by ANOVA followed by Bonferroni’s correction for multiple comparisons using the Prism (GraphPad Software, La Jolla, CA, USA).
Systolic blood pressure was significantly higher in ANG than in NORM after one week of angiotensin II infusion, and this increased pressure was maintained at the second week of angiotensin II infusion (n = 7; Fig. 1). However, TTx blocked the increase in systolic blood pressure for 2 weeks of treatment.
Total SOD activity in the small mesenteric arteries of ANG was lower than that of NORM (Fig. 2A), and this reduction was also observed in ANG + TTx (Fig. 2A). Potassium cyanide inhibits SOD1 and SOD3 activity (24); thus, the vessels were incubated with potassium cyanide to measure SOD2 activity. SOD2 activity was similar between groups (black inserted bars in Fig. 2A), indicating that the activity of SOD1 and SOD3, not SOD2, in the small mesenteric arteries were affected by angiotensin II-induced hypertension. SOD1 expression was lower in ANG and ANG + TTx than in NORM (Fig. 2B), and this downregulation of SOD expression was correlated with reduced SOD activity. The expression of SOD2 and SOD3 was not statistically different between groups (Fig. 2C, 2D, respectively).
Total SOD activity in the small renal vessels of ANG was not different from that of NORM (Fig. 3A). Interestingly, a reduction of SOD activity was observed in ANG + TTx (Fig. 3A). SOD2 activity after potassium cyanide incubation was similar between groups (black inserted bars in Fig. 3A). The expression of all three SOD isoforms were comparable between groups (Fig. 3B–3D).
Both the enzymatic activity and protein expression of catalase in the small mesenteric arteries were comparable between groups (Fig. 4A, 4B). Similarly, there was no change in catalase activity or protein expression in the small renal vessels between groups (Fig. 5A, 5B).
We hypothesized that angiotensin II-induced hypertension reduces antioxidant capacity, and antihypertensive treatment with ANG will restore antioxidant capacity in small vessels. We found that TTx significantly blunted the rise in blood pressure. However, reduced SOD activity and SOD1 expression in the small mesenteric arteries of ANG were not improved by this treatment. There was no difference in total SOD activity and expression of three SOD isoforms in the small renal vessels between ANG and NORM. Unexpectedly, we observed a decrease in SOD activity in the small renal vessels of ANG + TTx. The activity and expression of catalase in either vascular bed were not different between groups. These data indicated that SOD1 expression, and most likely SOD activity as well, in the small mesenteric arteries is downregulated by angiotensin II, but not high blood pressure. In contrast, antioxidant enzymes in the small renal vessels appeared to be resistant to angiotensin II and hypertension.
Angiotensin II is a well-recognized activator of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is a major source of O2− in the vascular system (25,26). Enzymes such as NADPH oxidase and uncoupled nitric oxide synthase 3 (NOS3) produce O2− or H2O2 mainly within the cell (27); thus, SOD activity in the intracellular space would be critical for lowering oxidative stress during angiotensin II activation. SOD1 is localized in the cellular cytosolic compartment. Transgenic mice overexpressing SOD1 showed an improvement in angiotensin II-mediated endothelial function and a decrease in angiotensin II-induced O2− production (28). Moreover, the infusion of recombinant SOD1 reduced blood pressure in ANG (29). Consistent with the roles of SOD1 reported in other studies, reduced SOD activity in the small mesenteric arteries of ANG was correlated with reduced SOD1 expression. We previously reported that H2O2 is increased in the small mesenteric arteries of ANG (14). H2O2 has been suggested to inactivate CuZn SODs (SOD1 and SOD3) by reacting with the Cu center of the enzyme, leading to the formation of Cu-OH radical (30). Therefore, increased H2O2 production in the small mesenteric arteries of ANG may reduce the activity of SOD1 by negative feedback mechanism.
Our previous study also showed reduced NO/cGMP signaling in the small mesenteric arteries of ANG and that antihypertensive treatment restored NO/cGMP signaling, which correlated with an increased biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for NO production from NOS (15). Interestingly, the increased level of oxidized BH4, 7,8-dihydrobiopterin (BH2), is not affected by antihypertensive treatment in ANG (15). These results correlate with our present finding of continued suppression of SOD activity with antihypertensive treatment. Taken together, we suggest that NO/cGMP signaling in the small mesenteric arteries can be restored by antihypertensive treatment (15) by increasing BH4 biosynthesis, not restoring antioxidant capacity.
Our findings highlighted a difference in the regulation of SOD activity and SOD1 expression between the small mesenteric arteries and small renal vessels under the same angiotensin II-induced hypertensive conditions. Although this study appears to be the only one investigating the regulation of antioxidant enzymes in small renal vessels, others have shown distinct regulation of antioxidant enzymes between conduit and mesenteric arteries (31) or renal cortex and medulla (32,33). Specifically, Gongora
We observed no change in either the activity or protein expression of SOD2 in the small mesenteric arteries or small renal vessels between all groups. Other studies also showed no change in SOD2 in the aorta of 2K1C rats (37), and in the aorta and mesenteric arteries of angiotensin II-induced hypertensive mice (31,38), renal cortex, and renal medulla of angiotensin II-induced hypertensive mice (13,33). Accordingly, SOD2 may play a pivotal role in cell survival because SOD2 protects mitochondrial function by eliminating mitochondria-derived O2−, but not in response to an angiotensin II-mediated increase in oxidative stress in the vasculature.
Total SOD activity in the small renal vessels of ANG was not different from that of NORM. Intriguingly, SOD activity in the small renal vessels of ANG + TTx was lower than that of ANG. It is not clear why SOD activity in the small renal vessels was reduced by TTx, but one speculation is that the antioxidant effects of hydralazine (39) may contribute to the antioxidant system of renal vessels, thereby lowering the activity of residential SODs. Further investigation is necessary to reveal the specific mechanism. The protein expression of the three SODs in the small renal vessels was not different between groups, indicating that SODs in the renal vasculature were resistant to angiotensin II induction and high blood pressure.
Catalase is a peroxidase that eliminates H2O2 in the cells by converting it to water and oxygen. Reduced catalase activity can lead to the accumulation of H2O2 in the tissues. Our present data showed that both the activity and protein expression of catalase in both the small mesenteric arteries and small renal vessels were not different between experimental groups. Interestingly, we previously showed increased H2O2 production in the small mesenteric arteries of ANG, but not of NORM (14). Thus, we speculate that increased H2O2 in the small mesenteric arteries of ANG may be due to both the observed prooxidant enzymatic sources of H2O2 production, such as NOS-derived H2O2, and reduced SOD activity, regardless of intact catalase activity and expression.
Vascular ROS level is regulated by the balance between the production and elimination of ROS. Increased O2− production has gained attention in the study of endothelial dysfunction because of its ability to react with NO, resulting in ONOO− production. ONOO− is a potent oxidizing agent causing deleterious lipid peroxidation and membrane damage in vascular cells. Therefore, SODs may be critical to protect NO bioavailability and prevent ONOO− generation in the blood vessels. Our data showed that SOD1 activity and expression in the small mesenteric arteries was downregulated, which was likely attributed to angiotensin II-dependent vascular dysfunction. In contrast, SOD activity and expression in the small renal vessels remained unchanged. This differential regulation of SOD highlighted a distinct difference in the importance of antioxidant system in each vascular bed.
This research was supported by the National Institutes of Health (NIH P01 HL69999 and NIH P01 HL95499), and also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1A6A1A03007648) and the Ministry of Science, ICT & Future Planning (2013R1A1A1062132 and 2017R1A2B4005463).
The authors declare no conflict of interest.
Fig. 1. Systolic blood pressure of NORM, ANG, and ANG + TTx (n = 7 per group) before and after 1 and 2 weeks of angiotensin II infusion. *
Fig. 2. Superoxide dismutase (SOD) activity (A; n = 5–6) and protein expression of SOD1 (B; n = 4–5), SOD2 (C; n = 5–7), and SOD3 (D; n = 4–5) in the small mesenteric arteries of NORM, ANG, and ANG + TTx. (A) SOD activity was normalized to mg protein, and black inserted bars are SOD activity with potassium cyanide treatment. (B, C, D) Relative densitometric units of the enzymes were normalized to that of β-actin. *
Fig. 3. Superoxide dismutase (SOD) activity (A; n = 4–5) and protein expression of SOD1 (B; n = 5–6), SOD2 (C; n = 6), and SOD3 (D; n = 6) in the small renal vessels from NORM, ANG, and ANG + TTx. (A) SOD activity was normalized to mg protein, and black inserted bars are SOD activity with potassium cyanide treatment. (B, C, D) Relative densitometric unit units of the enzymes were normalized to that of β-actin. *
Fig. 4. Catalase activity (A; n = 5–6) and protein expression (B; n = 3–5) in the small mesenteric arteries of NORM, ANG, and ANG + TTx. (A) Catalase activity was normalized to mg protein. (B) Relative densitometric unit of catalase were normalized to that of β-actin.
Fig. 5. Catalase activity (A; n = 7) and protein expression (B; n = 6) in the small renal vessels of NORM, ANG, and ANG + TTx. (A) Catalase activity was normalized to mg protein. (B) Relative densitometric unit of catalase were normalized to that of β-actin.
Funding Information
National Institutes of Health(NIH) National Research Foundation of Korea(NRF) Ministry of Education(Ministry of Education of the Republic of Korea) Ministry of Science, ICT and Future Planning(MSIP)
