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. 2010 Dec 2;110(2):520–527. doi: 10.1152/japplphysiol.00786.2009

Reactive oxygen species cause endothelial dysfunction in chronic flow overload

X Lu 1, X Guo 1, C D Wassall 2, M D Kemple 2, J L Unthank 3,4, G S Kassab 1,3–,4,5,✉
PMCID: PMC3043782  PMID: 21127212

Abstract

Although elevation of shear stress increases production of vascular reactive oxygen species (ROS), the role of ROS in chronic flow overload (CFO) has not been well investigated. We hypothesize that CFO increases ROS production mediated in part by NADPH oxidase, which leads to endothelial dysfunction. In six swine, CFO in carotid arteries was induced by contralateral ligation for 1 wk. In an additional group, six swine received apocynin (NADPH oxidase blocker and anti-oxidant) treatment in conjunction with CFO for 1 wk. The blood flow in carotid arteries increased from 189.2 ± 25.3 ml/min (control) to 369.6 ± 61.9 ml/min (CFO), and the arterial diameter increased by 8.6%. The expressions of endothelial nitric oxide synthase (eNOS), p22/p47phox, and NOX2/NOX4 were upregulated. ROS production increased threefold in response to CFO. The endothelium-dependent vasorelaxation was compromised in the CFO group. Treatment with apocynin significantly reduced ROS production in the vessel wall, preserved endothelial function, and inhibited expressions of p22/p47phox and NOX2/NOX4. Although the process of CFO remodeling to restore the wall shear stress has been thought of as a physiological response, the present data implicate NADPH oxidase-produced ROS and eNOS uncoupling in endothelial dysfunction at 1 wk of CFO.

Keywords: reactive oxygen species, NADPH oxidase, chronic flow overload, endothelial dysfunction

flow overload (FO) occurs when the blood flow rate in blood vessels is elevated over its basal physiological level. FO may occur physiologically during exercise or pregnancy and pathophysiologically in arteriovenous fistula, contralateral stenosis, or cardiac hypertrophy (1, 6, 8, 15, 19, 48). Since flow-induced shear stress on the endothelium regulates vascular function and dictates structural homeostasis of the blood vessel wall, the blood flow rate is closely regulated. FO in an artery elicits an acute endothelium-derived vasodilatation mediated by endothelial nitric oxide (NO), prostacyclin (PGI2), endothelium-derived hyperpolarizing factor (EDHF) (3, 9, 37, 42), and subsequent vascular remodeling (8, 18, 29, 48). In addition to NO, reactive oxygen species (ROS) have been reported in FO. For instance, flow-mediated superoxide (O2−) generation was observed in arteries of normal conduit artery of rabbits (22), FO carotid arteries of mice (6), and coronary resistance arteries of humans (23).

ROS are involved in physiological and pathological processes. Recently, ROS (particularly O2−) have been recognized as signaling molecules to mediate specific cellular responses in the vasculature, including activating matrix metalloproteinases (MMPs), vascular remodeling, vascular smooth muscle cell (VSMC) hypertrophy, and cellular apoptosis (6, 28, 34, 43, 47, 51,). Furthermore, O2− production may lead to hydrogen peroxide (H2O2) formation, which is thought to regulate potassium channels (23). In the vasculature, there may be several sources of O2− such as NADPH oxidase, xanthine oxidase, mitochondria, and endothelial NO synthase (eNOS) when uncoupled (30).

The increase in ROS generation in hypertension, diabetes, hypercholesterolemia, atherosclerosis, and ischemia is considered deleterious to the vasculature (2, 5, 11, 12, 20, 25, 27, 33, 44). NADPH oxidase has been identified as a major source of ROS in blood vessels in response to chemical (e.g., hypercholesterolemia and diabetes) or physical (e.g., hypertension and oscillatory shear stress) stimuli (5, 7, 17, 20, 28, 44). The increase in ROS production from NADPH oxidase, as occurs in hypertension, atherosclerosis, and diabetes, attenuates endothelium-dependent vasodilatation and causes endothelial dysfunction (5, 20, 32, 41, 49, 53).

Although the increase in ROS production in CFO has been established in both conduit artery and various small arteries (6, 22, 23, 47) and endothelial dysfunction in CFO has also been found in mesenteric artery of obese rats (4), the effect of CFO on endothelial function of elastic (carotid) artery of large animal is unknown. Hence, we hypothesize that 1) the ROS increase compromises endothelial function in CFO and 2) the source of ROS is, in part, due to NADPH oxidase. To test these hypotheses, an intact porcine carotid artery was exposed to CFO for 1 wk by a contralateral ligation. ROS was detected with the spin trap N-tert-butyl-α-phenylnitrone (PBN) by electron paramagnetic resonance (EPR) spectroscopy. The production of ROS was also assayed with ethidium fluorescence analysis and luminol-enhanced chemiluminescence. The expressions of eNOS and NADPH oxidase were measured. Endothelial function was examined through endothelium-dependent relaxation in response to acetylcholine (ACh). Uncoupled eNOS in CFO was found with acute administration of eNOS cofactor tetrahydrobiopterin (BH4). In one additional group of swine, the animals with CFO carotid arteries were orally fed apocynin (4-hydroxy-3-methoxy-acetophenone) to determine the effect of NADPH oxidase in the vessel wall. The results support our hypotheses and demonstrate that CFO causes endothelial dysfunction in conduit vessels.

MATERIALS AND METHODS

Twelve male Duroc swine weighing 34 ± 4 kg (range 30–39 kg) were randomly divided into two groups. In group I, the right carotid artery was exposed to CFO for 1 wk by ligation of the contralateral carotid artery. In group II, the right carotid artery was exposed to CFO for 1 wk while animals were orally fed apocynin at the dose of 60 mg·kg−1·day−1 from the postoperative first day to the termination (CFO+A). The left carotid artery served as a control for each group and was harvested at the time of ligation. All experiments were performed in accordance with national and local ethical guidelines, including the Institute of Laboratory Animal Research (ILAR) Guide, Public Health Service policies, Animal Welfare Act, and an approved Indiana University School of Medicine IACUC protocol.

Animal Preparation

Surgical anesthesia was induced with ketamine (20 mg/kg im) and atropine (0.04 mg/kg im) and maintained with isofluorane (1–2%). Blood gas values were measured, and ventilation was adjusted to maintain normal values of Po2 and Pco2. In preliminary experiments, we measured the flow rates and external diameters to confirm that the left and right common carotid arteries are equivalent to ensure that one can serve as control for the other. Furthermore, the flow rates and external diameter of the right carotid artery were immediately measured after the left carotid artery was excised to quantify the immediate FO.

Subsequent to a left cervicotomy, the left common carotid artery was exposed gently to avoid vasomotion by dissection, and the in vivo external diameter was measured with the aid of a stereo microscope. The vessel was further dissected to place a flow probe (TS420 Transonic System). After data collection, the artery was ligated by suture and excised (length of ∼3 cm); the right carotid artery was not exposed to any surgical trauma. This measurement was taken on day 0 to avoid intervention on the experimental vessel. The incision was closed, and the animal was recovered. The animals in group II were fed apocynin (60 mg·kg−1·day−1) orally mixed in food. After 1 wk and following a right cervicotomy, the in vivo external diameter and flow rate of right carotid artery were measured (data points for week 1), and the vessel segment was excised. The excised vessels from control and experimental groups were immediately stored in 4°C HEPES physiological salt solution (HEPES-PSS, pH 7.4, mM: 142 NaCl, 4.7 KCl, 2.7 sodium HEPES, 3 HEPES acid, 1.17 MgSO4, 2.79 CaCl, 5.5 glucose) and divided into segments for various measurements after dissection of adjacent tissue. HEPES and HEPES salt were purchased from Sigma, whereas other chemicals were purchased from Fisher Scientific.

Vasoactivity

An isovolumic myograph, which retains the physiological loading of a pressure myograph with sensitivity of a wire myograph, was used to evaluate the vasoactivity of the artery (24). Briefly, the carotid vessel was cannulated on the connectors, which were fixed in a bath containing HEPES-PSS. The vessel was incubated in the bath at 37°C for 40 min. The vessel was stretched to in situ length with the aid of a digital caliper (resolution of 0.1 mm) and preloaded at a physiological pressure of 80 mmHg. Contraction or relaxation was pharmacologically induced with both ends closed. The contraction or relaxation of the vessel wall induces an increase or decrease in pressure, respectively. The diameter of the carotid artery remained approximately constant during vasoreactivity. The pressure and the external diameter were measured with a pressure transducer (Mikro-Tip SPR-524, Millar Instruments) and with a dimensional tracer (DiamTrak 3+, Australia; 10-μm resolution), respectively.

All arterial segments were precontracted (increase in pressure) to an approximate transluminal pressure (170 ± 20 mmHg) with phenylephrine (10−8 to 10−6 M), and thereafter endothelium-dependent relaxation (decrease in pressure) was assessed with a series of doses of acetylcholine (Ach, 10−9 to 10−6 M). The effect of eNOS uncoupling on endothelium-dependent relaxation was detected by incubation of the vessel segments in CFO with l-arginine (10−5 M) and eNOS cofactor BH4 (10−5 M) for 40 min. The circumferential tension of the vessel at every dose was calculated by Laplace's law, tension = pressure × radius. The % decrease in tension was calculated by the equation: %Tension = (Td − Ti)/(Tmax − Ti) × 100. where, Td, Ti, and Tmax are the tension at every dose (Td), physiological level (Ti), and maximum tension (Tmax) at submaximal concentration of phenylephrine, respectively. Non-receptor-dependent contraction to potassium (KCl, 60 mM) was used to verify identical contractility of VSMC in CFO. Endothelium-independent vasorelaxation to sodium nitroprusside (SNP, 10−5 M) was measured to verify the sensitivity of VSMC in response to NO.

ROS Detection

We detected ROS with paramagnetic resonance spectroscopy, ethidium fluorescence assay, and luminol derivative (L-012) enhanced chemiluminescence analysis as described below.

Electron paramagnetic resonance spectroscopy.

Immediately after the vessel was divided, the vessel ring for electron paramagnetic resonance (EPR) was videotaped from the side (5 mm in length) and cross-views under stereo microscope. The volume of the segment was calculated based on the product of cross-sectional area and axial length. After measurement by EPR as described below, ROS generation was expressed as mole per unit of volume.

ROS concentration in tissue samples was determined from the EPR spectra obtained by incubating the tissue samples with the spin trapping agent N-tert-butyl-α-phenylnitrone (PBN; Sigma) at 190 mM in HEPES-PSS for 30 min at 37°C in the dark. A ring incubated with 4-Hydroxy-TEMPO (a superoxide dismutase mimic) served as the control for ROS measurement. The tissue was subsequently inserted into a syringe along with the supernatant, immediately frozen in liquid nitrogen, and stored at −80°C until EPR analysis was performed. To avoid ROS produced during freezing and thawing of samples, the sample was quickly removed while in its frozen state from the syringe and placed in a Dewar containing liquid nitrogen. The Dewar was then inserted into the microwave cavity of the EPR spectrometer. The sample remained at liquid nitrogen temperature throughout the EPR analysis (1, 21).

The EPR equipment and settings were as follows. A Bruker ESP X-band spectrometer equipped with a TE102 cavity was utilized to detect signals. Parameters for the spectra were 9.4-GHz microwave frequency, 25.2-mW microwave power, 4.0-G modulation amplitude, 1×105 receiver gain, 5.24-s time constant, 3,330-G center magnetic field, and 100-G magnetic field sweep width. All experiments were completed at liquid nitrogen temperature.

Four EPR scans were taken per tissue sample and analyzed with Bruker WINEPR software (version 2.11) based on the spectral intensity and line width. ROS concentrations were determined with 2,2,6,6-tetramethylpiperidine 1-oxyl, TEMPO, solution (0.1 μM, Sigma) used as a concentration standard. All EPR parameters and conditions were applied to both standard and experimental samples.

Chemiluminescence.

A highly sensitive chemiluminescence probe, luminol derivative L-012 (Wako Chemicals) was used to detect ROS in the vessel tissue. Three arterial segments, 2–3 mm in length, were incubated in 96-well plates with HEPES-PSS at 37°C for 1 h. NADPH (0.3 M) and L-012 (500 μM) were administered in wells (36). Some of the segments were incubated with 1 mM oxyopurinol (a xanthine oxidase inhibitor), 50 μM rotenone (a mitochondria inhibitor), or 1 μM l-NAME (Nω-nitro-l-arginine methyl ester; an eNOS inhibitor) to verify the involvement of xanthine oxidase, mitochondria, and eNOS. Light emission was detected by ultra-sensitive photon counter (Wallac EnVision, 2104 Multilabel Reader, PerkinElmer). Counts were obtained at 1-min intervals for 40 min. ROS levels were reported as relative light units after subtracting background luminescence and were normalized to dry tissue weight.

Ethidium fluorescent assay.

A 5-mm ring was cut from the segment of carotid artery and incubated with nitrogen-bubbled HEPES-PSS (to remove possible ROS) containing dihydroethidium (DHE, Sigma) at optimal concentration (determined in pilot experiments) of 0.2 μM for 30 min at 37°C in the dark (47). Another ring that served as a control for ROS measurement was first incubated with 4-hydroxy-TEMPO (Tempol, Sigma), an O2− scavenger, for 30 min at 37°C, and then with DHE for 30 min at 37°C. An additional ring without DHE incubation was used as the control for autofluorescence of the vessel tissue. A 4′-6-diamidino-2-phenylindole (DAPI) staining was performed to determine nuclear area, which was a reference for analysis of ethidium fluorescence. After being washed five times with saline, the rings were sliced at 20-μm-thick transverse sections by a cryostat (Leica CM 1850). Confocal microscopy (LSM 510 META, Zeiss) was used to visualize the fluorescence (excitation/emission: 518/605 nm, 63 × oil objective). A gray scale analysis was carried out to determine the fluorescence area fraction. The autofluorescence of vessel tissue was subtracted to remove the background.

Protein Expression

The segment for Western blotting was homogenized in a lysis buffer and then incubated on ice for 1 h. The sample was centrifuged at 1,000 g for 15 min at 1°C, and the supernatant was drawn off. The total value of protein was measured by a BCA kit (Bio-Rad). Equal amounts of protein (25 μg) were loaded, electrophoresed in 10% SDS-PAGE gel, and transferred onto a ployvinylidene difluoride membrane. After blocking the sample for 2 h in 8% dried milk in TBS-Tween buffer, the membrane was incubated overnight at 4°C with specific primary antibody with either anti-eNOS (1:1,000 dilution in blocking buffer, BD transduction laboratory), anti-p22phox (1:1,000, Santa Cruz Biotech) anti-p47phox (1:500, Santa Cruz Biotech), anti-NOX2 (1:250, Santa Cruz Biotech), or anti-NOX4 (1:250, Santa Cruz Biotech). The membrane was then rinsed and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotech) for 2 h (eNOS: goat anti-mouse 1:3,000 dilution in blocking buffer; p22phox and p47phox: goat anti-rabbit 1:5,000, NOX2 and NOX4: bovine anti-goat 1:5,000). The specific protein was detected by enhanced chemiluminescence (ECL; Amersham) and evaluated by densitometry (Sigma Scan). All samples from each group were simultaneously probed with anti-β-actin, a mouse monoclonal antibody (primary antibody 1:1,000 dilution in blocking buffer, Santa Cruz Biotech), to correct for sample loading.

Statistical Analysis

All data given in the text and figures are expressed as means ± SD. Student's t-test (two-tailed distribution, two-sample unequal variance) and Duncan's test following ANOVA were used to detect differences between groups. For all analyses, P < 0.05 was used to indicate statistical significance.

RESULTS

The blood flow in carotid arteries was significantly increased in CFO and CFO+A after contralateral ligation (189.2 ± 25.3, 369.6 ± 61.9, and 382.3 ± 62.5 ml/min in the control, CFO, and CFO+A groups, respectively; P < 0.001). The flow rate in the apocynin treatment group was not significantly different than the untreated group. Figure 1 represents a typical tracing of a control and a carotid artery exposed to CFO. The mean and peak of the blood flow doubled in CFO (Fig. 1A). After normalization of pulsatile blood flow with mean flow rate, the two curves with respect to their mean values largely overlapped (Fig. 1B), which suggests that the oscillatory components of pulsatile blood flow were not significantly changed in the CFO model.

Fig. 1.

Fig. 1.

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A typical waveform of blood flow in the carotid artery at baseline and flow overload (CFO). A: the real time recordings. B: the normalized tracing by the mean flow rate. The normalized flow tracing at chronic flow overload (CFO) approximately overlaps the normalized baseline.

The outer diameter of the vessel significantly increased by 8.6% (4.60 ± 0.41 to 4.98 ± 0.46 mm) after 1 wk of exposure to CFO (P < 0.05) and did not change significantly after treatment with apocynin (4.98 ± 0.46 to 5.06 ± 0.48 mm). We did not observe a significant change of arterial wall thickness in either the CFO or the CFO+A group compared with the control group (data not shown). The systemic blood pressure measured at the femoral artery did not change after either surgical ligation of the carotid artery or after treatment with apocynin compared with the pressure before ligation (88 ± 12 vs. 86 ± 11 mmHg). The wall shear stress (WSS; WSS ∼ QD−3, where Q and D represent flow rate and inner diameter, respectively) was found to remain elevated by ∼50% after 1 wk of the CFO and CFO+A groups.

Endothelial Function

The endothelial function was quantified by the % decrease in tension in response to ACh (Fig. 2). The dose curves indicated that endothelial function was compromised in the CFO group (P < 0.05) but was preserved with apocynin treatment. This result implied that endothelial dysfunction in CFO was related to ROS upregulation. Furthermore, we found that the endothelial dysfunction of the vessels in CFO were completely reserved after acute incubation with BH4 and l-arginine, which suggests that eNOS uncoupling may play a role in CFO. The endothelium-independent vasorelaxation in response to SNP did not show differences in groups, which implied that the VSMC did not develop resistance to nitric oxide (103.5 ± 15.5, 102.6 ± 17.2, and 103.6 ± 21.1% for control, CFO, and CFO+A, respectively). The potassium-induced contraction, which is not receptor-dependent, did not reveal differences of VSMC contractility (tension) in the three groups (49.6 ± 6.98, 51.3 ± 7.66, and 51.1 ± 7.96 mN/mm for control, CFO, and CFO+A, respectively).

Fig. 2.

Fig. 2.

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Endothelium-dependent vasorelaxation of carotid arteries. The arteries were precontracted to approximate tension with phenylephrine (PE), and dose-responsive vasorelaxation was induced with acetylcholine (ACh). C, control group; CFO, chronic flow overload group; CFO+A, chronic flow overload group treated with apocynin; CFO+BH4, vessel segment in CFO acute incubation with tetrahydrobiopterin (BH4). *Significant difference between groups (P < 0.05; ANOVA followed by Duncan's test for multiple groups).

ROS Generation

The EPR measurements are presented in Fig. 3. ROS generation significantly increased in the CFO group. Apocynin treatment restored ROS generation to the control value. Since PBN is lipophilic and forms stable lipid-derived, spin adducts (radical bound to spin trap), it provides reliable overall concentration measurements of ROS in biological tissue under physiological conditions without lipid radical adduct extraction that may involve artifacts such as failure to retrieve all of the adducts from the biological sample and additional ROS generation during centrifugation. Since PBN is indeed able to trap NO in vascular tissue, we verified that NO was not significantly produced in our experimental preparation with DAF-2DA (NO probe) and l-NAME (an eNOS inhibitor).

Fig. 3.

Fig. 3.

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A: representative samples of the EPR spectra. The concentration of ROS is proportional to the signal intensity. B: ROS concentration by EPR spectroscopy using the PBN as a spin trap in HEPE-PSS. *Significant difference compared with control (P < 0.05). #Significant difference compared with CFO (P < 0.05).

Luminol derivative L-012 is highly sensitive at physiological range of pH (7.5) and reacts with various types of ROS generated from various biological tissues. L-012 enhanced chemiluminescence increased in the vessel tissue in CFO after addition of NADPH (Fig. 4). A 1-wk treatment of apocynin decreased the level of ROS. The inhibitors of xanthine oxidase, mitochondria, and eNOS did not cause any change of chemiluminescence (data not shown), which indicates that those ROS sources were not involved in ROS production in CFO.

Fig. 4.

Fig. 4.

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ROS production determined by chemiluminescence analysis in arterial segments. A luminol derivative L-012 was used to enhance chemiluminescence by ROS in arterial segments. A: representative samples of L-012 enhanced-chemiluminescence. B: ROS production measured by chemiluminescence analysis. *Significant difference compared with control (P < 0.05). #Significant difference compared with CFO (P < 0.05).

Confocal microscopic images of ethidium fluorescence showed that ROS increased in all three wall layers in CFO group (Fig. 5A). The fraction area of fluorescent assay in the carotid vessels exposed to CFO was more than three times higher than that of control segments (Fig. 5B). With the treatment of apocynin, the production of ROS was significantly lower than that in the CFO group (Fig. 5A). When the tissue from either the control or the CFO group was first incubated with Tempol, the DHE assay was almost completely eliminated by Tempol (Fig. 5A).

Fig. 5.

Fig. 5.

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The fluorescent detection of ROS by dihydroethidium (DHE). A: photographs from the confocal microscopy, where the bright red spots indicate ROS production. The autofluorescence from ECM (AF) of tissue was evaluated by visualizing the tissue without DHE incubation (blank DHE) using the confocal microscopy. To mimimize autofluorescence, the parameters of confocal microscopy were maintained the same as the autofluorescent data but at lower laser power. There were significant spots indicating basal production of ROS in all three layers (intima, media, and adventitia). Treated with apocynin, the spots were smaller and less intense than those of CFO but still larger and brighter than those of control (C). TT, vessel segment treated with Tempol (Tempol is a cell-permeable SOD and catalase mimetic). With pre-incubation with Tempol, the confocal microscopic spots were drastically decreased in the tissue segment. AD, adventitial layer; ME, medial layer; IN, intimal layer. B: the fractional area of fluorescent assay of the arterial sections based on the measurement of the confocal microscopic images. *Significant difference compared with control (P < 0.05). #Significant difference compared with CFO (P < 0.05).

Expression of eNOS and NADPH Oxidase

The expression of eNOS was significantly upregulated in the CFO group and unaffected by apocynin treatment (Fig. 6). The subunits p22phox and p47phox are well known to be expressed ubiquitously in endothelial and VSMC. The protein expression of cytosolic assembling subunits of NADPH oxidase (p22phox and p47phox) were significantly elevated in carotid segments exposed to CFO but not in CFO+A (Fig. 6). NOX2 and NOX4 (NADPH oxidase 2 and 4) were found to be upregulated in CFO and CFO+A.

Fig. 6.

Fig. 6.

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The proteins expression evaluated with Western blot. A: Western blotting bands. The molecular weight was confirmed as eNOS, 120; p22phox, 22; p47phox, 47; NOX2, 91; and NOX4: 55. B: ratios of total pixels of the bands were measured by use of imaging software. *Significant difference in comparison between control and CFO or CFO+A (P < 0.05). †Significant difference in comparison between CFO and CFO+A (P < 0.05).

DISCUSSION

The major findings are that chronic shear stress elevation of ∼50% increases ROS production mediated by NADPH oxidase and induces endothelial dysfunction in swine carotid artery. Recent observations suggest that NADPH oxidase is directly involved in O2− production in mouse carotid arteries where the flow was increased by a factor of three to five by construction of an arteriovenous fistula (6). The present study demonstrates a similar finding in conduit artery of the porcine model in response to a more modest increase in WSS. Moreover, we show for the first time endothelial dysfunction as a consequence of increased oxidative stress in conduit artery where eNOS uncoupling may play a role during CFO.

The porcine carotid artery was exposed to approximately twice the physiological flow rate for 1 wk in this study. Although the change of flow rate was relatively small compared with a typical arteriovenous fistula (over fivefold increase in flow rate) (18), the objective was to study a model with minimal disturbance since surgery elicits inflammatory response and consequent oxidative stress. The diameter enlargement of the artery was accordingly small (8.6%). Such a change in flow and diameter in a conduit artery may occur under physiological conditions, such as in exercise (15, 50). Interestingly, ROS generation increased by onefold in response to the relatively small changes in flow or WSS (56% increase) as shown in Fig. 3. Although mechanical stimulations, including oscillatory WSS and cyclic stretch, have been shown to increase ROS production in the vasculature (7, 14, 17, 28), this is the first report of increased ROS generation in response to CFO in a conduit vessel of a large animal.

The diameter increase during CFO is attributed to a physiological response to restore the homeostatic shear stress on the endothelium. It is well known that acute vasodilatation is stimulated by WSS elevation (16). Following acute vasodilatation, chronic remodeling occurs and further increases the diameter (6, 8). Interestingly, the complete restoration of WSS requires a relatively long period in an arteriovenous fistula; e.g., over 4 wk in carotid arteries of rabbits (47), over 3 wk in the carotid arteries of mice (6), and 6 mo in the carotid arteries of dogs (18). In the present study, the WSS was not restored and remained elevated after 1 wk. In fact, a conduit artery possesses the capacity to acutely increase its diameter up to 15% in response to a 10- to 20-fold increase in flow (8, 40). Even in the 1-wk study, the diameter may enlarge by 15–20% in response to a three- to fivefold increase in flow rate (6, 18). Hence, acute diameter enlargement in response to CFO depends on the magnitude of WSS and the subsequent remodeling duration since the restoration of WSS may be very long. Since vasodilatation increases vascular stretch, which can activate NADPH oxidase in blood vessel wall (31), the stretch may play a role in CFO. The elevated stretch results from diameter increase (flow-induced vasodilatation) and vascular wall softening due to NO-induced vascular tone reduction. Both effects increase the magnitude of stretch at pulsatile blood pressure.

The expansive remodeling of the blood vessel wall in response to an increase in flow rate has been previously viewed as a physiological adaptation to restore the WSS (18, 52). Here, we show that the remodeling response leads to compromised endothelial function (over a 1-wk period). The endothelium-dependent vasodilatation in the present study (Fig. 2) is significantly decreased in response to a modest flow increase in a relatively short period. In resistance arteries, Pourageaud et al. observed that CFO increased the endothelium-dependent vasodilatation to ACh, and flow-induced vasodilatation was slightly (35) but not statistically attenuated. In diabetic rats, CFO results in significant endothelial dysfunction in mesenteric arteries (4). The mechanism involved in the different endothelial responses in large conduit arteries and small resistance arteries to CFO is unclear. It may be that resistance arteries are exposed to more significant variations of blood flow under physiological conditions due to lateral-contralateral adjustment of microcirculation and adapt to larger variation of blood flow compared with large conduit arteries.

NADPH oxidase (NOX) has been recognized as a major source of O2− in the vasculature in response to mechanical stimulation (17, 28). It is known that p22phox is an important trans-membrane protein that combines with NOX to assemble NADPH oxidase complex and is known to be upregulated by WSS (39). The p47phox, as cytosolic subunit of NADPH oxidase, may also affect the activity of NOX. NOX4 is a p22phox-dependent enzyme (2) and does not require cytosolic proteins (p47phox, p67phox) for its activity (10, 26, 38). Apocynin is an inhibitor of NADPH oxidase under in vivo condition where H2O2 and myeloperoxidase is present and is suggested to inhibit the translocation of cytoplasmic subunits (45). It is also possible that treatment of apocynin in this study shifted the balance of oxidative stress in vascular tissue through nonspecific antioxidant effects (13, 36). Since the expression of NOX isoforms may be located in endothelial and VSMC, we cannot separate the effect of WSS on endothelium and circumferential stretch acting throughout the vessel wall on NOX isoforms. The elucidation of the role of NOX in endothelial and VSMC during CFO requires further study.

Ethidium fluorescence visualized by confocal microscopy indicates that ROS increases not only in endothelium but also in the media and in the adventitia. This is interesting because WSS acts directly on the luminal surface of endothelium; i.e., WSS is not likely to directly stimulate the media and the adventitia. A fundamental question involves the mechanism by which elevated WSS sensed by the endothelium transmits its effect on more remote regions of the wall. A possible mechanism may be similar to hypertension via the NADPH oxidase pathway (44, 45, 49). The common factor in CFO and hypertension is the increase in circumferential stretch and stress. Hypertension increases circumferential stress and strain by an increase in blood pressure, whereas flow-induced vasodilatation increases vessel stretch similarly through an increase in diameter. The circumferential stress or strain, mediated by mechanotransduction in CFO, may activate NADPH oxidase and elicit ROS generation.

In summary, ROS production increases in porcine carotid arteries in response to a onefold increase in the blood flow rate, which leads to endothelial dysfunction. NOX2 and NOX4 oxidase and p22phox and p47phox are upregulated in CFO, and NADPH oxidase is likely involved in the increase in oxidative stress. The chronic use of apocynin prevents the elevation of ROS levels, even though NOX2 and NOX4 are upregulated, and preserves endothelial function. The mechanisms by which apocynin prevents the upregulation of p22phox and p47phox but not NOX2 and 4 remain unclear. Although the process of CFO-induced remodeling to restore WSS has previously been thought of as a physiological response, the present data suggest that CFO mediated by ROS causes endothelial dysfunction, which may result from eNOS uncoupling in the first week of outward vascular remodeling.

GRANTS

This research was supported in part by National Heart, Lung, and Blood Institute Grants R01 HL055554-11 and R01-HL-084529.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

ACKNOWLEDGMENTS

The authors acknowledge Hans Mooij, Charles Dang, and Carlos Linares for excellent technical assistance.

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