Lidocaine potentiates the chondrotoxicity of methylprednisolone.

PURPOSE: This study examined the viability of bovine articular chondrocytes after exposure to methylprednisolone, methylprednisolone with lidocaine, and methylprednisolone in a simulated inflammatory environment. METHODS: Bovine articular chondrocytes were suspended in alginate beads and cultured in Dulbecco's modified Eagle's medium/F-12 for 1 week before experimentation. Suspended chondrocytes were exposed to 0.9% saline solution (negative control), methylprednisolone (4, 8, and 16 mg/mL), methylprednisolone (8 mg/mL) with 1% lidocaine, or methylprednisolone (8 mg/mL) and saline solution in a simulated inflammatory environment (interleukin [IL] 1beta exposure, 10 ng/mL) for 15, 30, and 60 minutes. Flow cytometry was performed 1 day, 4 days, and 7 days after exposure by use of annexin V and propidium iodide to assess chondrocyte viability. RESULTS: Chondrocyte viability decreased from 84% in saline solution to 62%, 38%, and 2.4% 1 day after 60 minutes of exposure to 4, 8, and 16 mg/mL of methylprednisolone, respectively (n = 7, P < .05). Chondrotoxicity increased with increasing time of exposure to methylprednisolone and with increasing time after exposure. In IL-1beta-activated chondrocytes, viability decreased from 76% in saline solution to 2.9% after 60 minutes of methylprednisolone exposure (8 mg/mL) (n = 4, P < .05). The combination of 8 mg/mL of methylprednisolone and 1% lidocaine further reduced viability to 1.0% after 60 minutes (n = 4, P < .05). CONCLUSIONS: These results show a dose- and time-dependent decrease in chondrocyte viability after exposure to clinically relevant doses of methylprednisolone. The combination of methylprednisolone and lidocaine was toxic, with virtually no cells surviving after treatment. In addition, methylprednisolone did not mitigate the inflammatory effects of IL-1beta; rather, it further potentiated the chondrotoxicity. CLINICAL RELEVANCE: Intra-articular injections of corticosteroids and local anesthetics are widely used in clinical practice. This in vitro study provides information on the potential effects of these drugs on articular cartilage.

Antioxidant effects of green tea and its polyphenols on bladder cells.

Genitourinary tract inflammation/ailments affect the quality of life and health of a large segment of society. In recent years, studies have demonstrated strong antioxidant effects of green tea and its associated polyphenols in inflammatory states. This in vitro study examined the antioxidant capabilities (and putative mechanisms of action) of green tea extract (GTE), polyphenon-60 (PP-60, 60% pure polyphenols), (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) in normal/malignant human bladder cells following catechin treatment+/-1 mM H2O2 (oxidative agent). Cell viability, apoptosis and reactive oxygen species (ROS) formation were evaluated. Our results showed that H2O2 exposure significantly reduced normal (UROtsa) and high-grade (TCCSUP, T24) bladder cancer (BlCa) cell viability compared with control-treated cells (p<0.001). No affect on low-grade RT4 and SW780 BlCa cell viability was observed with exposure to H2O2. Compared to H2O2-treated UROtsa, treatment with PP-60, ECG and EGCG in the presence of H2O2 significantly improved UROtsa viability (p<0.01), with strongest effects evoked by ECG. Additionally, though not as effective as in UROtsa cells, viability of both high-grade TCCSUP and T24 BlCa cells, in comparison to H2O2-treated cells, was significantly improved (p<0.01) by treatment with PP-60, ECG, and EGCG in the presence of H2O2. Overall, our findings demonstrate that urothelium cell death via H2O2-induced oxidative stress is mediated, in part, through superoxide (O2-.;), and potentially, direct H2O2 mechanisms, suggesting that green tea polyphenols can protect against oxidative stress/damage and bladder cell death.

Reactive oxygen and nitrogen species: implications for cardiovascular device engineering.

The development of medical devices for cardiovascular applications has suffered due to lack of understanding of why vascular wall cells act nonphysiologically when exposed to biomaterials. One possible reason might be the chemical environment associated with cardiovascular disease. An improved understanding of cellular and subcellular mechanisms may assist in future device design to account for the disease environment. Reactive oxygen and nitrogen species (ROS/RNS) are produced through normal cellular metabolism and are rendered harmless by enzymatic systems. However, during a disease process, these systems may act aberrantly, and either fail to convert ROS and RNS to harmless substances or by producing more oxidants. There is indirect evidence that the implantation of biomedical materials may also be responsible for the triggering of these aberrant pathways that may lead to the eventual failure of the device. The understanding of how the vascular environment may be changed at the subcellular level by the presence of a biomaterial is critical. In the following pages, we hope to review the current thinking within vascular biology regarding ROS and RNS, how they are measured, and how they may impact vascular cells.

Endothelial cell seeding onto various biomaterials causes superoxide-induced cell death.

The seeding and/or in-growth of endothelial cells on a number of blood-contacting implants are a concern for both biomaterials and tissue engineering. While endothelialization has been viewed positively, owing to their ability to regulate both smooth muscle and blood, there is evidence which suggests that endothelial cells on a nonoptimized surface may be counterproductive. The present study describes the experimentation designed to elucidate the effect of culture substrate on intracellular superoxide (SO) levels, a marker for endothelial cell dysfunction. The adaptation of the use of dihydroethidium under physiologically relevant shearing conditions is also reported. The present study describes a standardized method for the use of dihydroethidium as a marker for intracellular oxidative stress under physiologic shear. Levels of hydrogen peroxide (oxidative stress producing agent) are optimized to a minimum of 60 microM (under static conditions) to allow for the detection of SO within the free radical scavenging environment. A flow rate of 24.4 mL/min is applied and found to produce physiologically relevant shear stress (8.2 dynes/cm(2)) within the system under study. Dihydroethidium is a useful marker for assessing intracellular oxidative stress in studies that require shear.

Mechanisms of H2O2-induced oxidative stress in endothelial cells.

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H(2)O(2) induces production of O(2)*(-) by activating NADPH oxidase. However, the mechanisms whereby H(2)O(2) induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H(2)O(2) on O(2)*(-) levels on porcine aortic endothelial cells (PAEC). Treatment with 60 micromol/L H(2)O(2) markedly increased intracellular O(2)*(-) levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O(2)*(-) levels and attenuated cytotoxicity resulting from treatment with H(2)O(2). L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O(2)*(-) levels in PAEC treated with H(2)O(2), suggesting that both NOS and NADPH oxidase contribute to H(2)O(2)-induced O(2)*(-) in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O(2)*(-) levels in PAEC treated with H(2)O(2) to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H(2)O(2) produces oxidative stress in endothelial cells by increasing intracellular O(2)*(-) levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H(2)O(2) and oxidant-generating enzymes that may contribute to endothelial dysfunction.

Early diagnosis to enable early treatment of pre-osteoarthritis.

Osteoarthritis is a prevalent and disabling disease affecting an increasingly large swathe of the world population. While clinical osteoarthritis is a late-stage condition for which disease-modifying opportunities are limited, osteoarthritis typically develops over decades, offering a long window of time to potentially alter its course. The etiology of osteoarthritis is multifactorial, showing strong associations with highly modifiable risk factors of mechanical overload, obesity and joint injury. As such, characterization of pre-osteoarthritic disease states will be critical to support a paradigm shift from palliation of late disease towards prevention, through early diagnosis and early treatment of joint injury and degeneration to reduce osteoarthritis risk. Joint trauma accelerates development of osteoarthritis from a known point in time. Human joint injury cohorts therefore provide a unique opportunity for evaluation of pre-osteoarthritic conditions and potential interventions from the earliest stages of degeneration. This review focuses on recent advances in imaging and biochemical biomarkers suitable for characterization of the pre-osteoarthritic joint as well as implications for development of effective early treatment strategies.

Serum CTXii Correlates With Articular Cartilage Degeneration After Anterior Cruciate Ligament Transection or Arthrotomy Followed by Standardized Exercise.

BACKGROUND: Anterior cruciate ligament injury increases risk for accelerated development of osteoarthritis. The effect of exercise on articular cartilage following joint injury is not well understood. Biochemical biomarkers of collagen degradation and proteoglycan turnover are potential indicators for early articular cartilage degeneration. HYPOTHESIS: This study tests the hypothesis that serum concentrations of CS846 and CTXii correlate with structural changes to articular cartilage following joint injury in exercised animals. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty-four Sprague-Dawley rats underwent either arthrotomy alone (sham surgery) or anterior cruciate ligament transection (ACLT). Animals were recovered for 3 weeks and then exercised on a treadmill at 18 m per minute, 1 hour per day, 5 days per week, until sacrifice either 6 or 12 weeks later. Articular cartilage was assessed grossly, and histology was graded using modified Mankin, toluidine blue, and modified David-Vaudey scales. Serum collected preoperatively and at sacrifice was assayed by ELISA for CTXii and CS846. RESULTS: At 6 weeks, gross grades (P < 0.01), modified Mankin scores (P < 0.03), and toluidine blue scores (P < 0.04) were higher, reflecting increased degeneration in ACLT animals compared with sham surgery animals. Serum CS846 increased after 6 weeks in ACLT animals (P < 0.05). Serum CTXii levels strongly correlated with Mankin degenerative scores (coefficient = 0.81, P < 0.01) and David-Vaudey histology grades (coefficient = 0.73, P < 0.01) at 6 weeks. While gross grades remained higher at 12 weeks in ACLT animals (P < 0.04), no differences were seen in serum CS846 and CTXii. Histology scores also showed no differences between ACLT and sham due to increasing degeneration in the sham surgery group. CONCLUSION: The strong correlation between serum CTXii and microstructural changes to articular cartilage following joint injury demonstrates potential use of serum biomarkers for early detection of cartilage degeneration. Increasing cartilage degeneration in exercised sham-surgery animals suggests that early loading may have negative effects on articular cartilage due to either mechanical injury or hemarthrosis after arthrotomy. CLINICAL RELEVANCE: Patients with anterior cruciate ligament injury are at increased risk for development of posttraumatic osteoarthritis. CTXii may be useful for early detection of joint degeneration. Further study on the effects of exercise after injury is important to postinjury and postoperative rehabilitation.

Optical coherence tomography grading correlates with MRI T2 mapping and extracellular matrix content.

Optical coherence tomography (OCT) and T2 mapping are emerging clinical imaging technologies with potential to detect subsurface changes in cartilage retaining a macroscopically intact articular surface. This study tests the hypothesis that OCT correlates with magnetic resonance imaging (MRI) T2 values, and that OCT signal is sensitive to cartilage matrix degeneration. Forty-five osteochondral cores were harvested from five human tibial plateau explants after MRI T2 mapping. Cores underwent OCT imaging and were graded as follows: A, obvious birefringence; B, no birefringence; C, subsurface voids and/or irregular surface. Extracellular matrix content was determined and cores underwent histologic and polarized light microscopy (PLM) evaluation. Grade B and C cores had 25% higher superficial T2 values (p = 0.047) and 50% higher deep T2 values (p = 0.012) than grade A cores. Grade B and C cores had 36% higher glycosaminoglycan (GAG) content compared to grade A cores (p = 0.009). Histology and PLM demonstrated increased surface irregularity and structural disorganization with increasing OCT grade. OCT grade and T2 value increased with increasing collagen disorganization, suggesting that MRI T2 mapping and OCT are sensitive to changes in collagen structure. Our results demonstrate the ability of OCT and T2 mapping to detect early cartilage degeneration in clinically normal appearing cartilage.

Optical coherence tomography detection of subclinical traumatic cartilage injury.

OBJECTIVES: Posttraumatic arthritis is a major cause of disability. Current clinical imaging modalities are unable to reliably evaluate articular cartilage damage before surface breakdown, when potentially reversible changes are occurring. Optical coherence tomography (OCT) is a nondestructive imaging technology that can detect degenerative changes in articular cartilage with an intact surface. This study tests the hypothesis that OCT detects acute articular cartilage injury after impact at energy levels resulting in chondrocyte death and microstructural changes, but insufficient to produce macroscopic surface damage. METHODS: Bovine osteochondral cores underwent OCT imaging and were divided into a control with no impact or were subjected to low (0.175 J) or moderate (0.35 J) energy impact. Cores were reimaged with OCT after impact and the OCT signal intensity quantified. A ratio of the superficial to deep layer intensities was calculated and compared before and after impact. Chondrocyte viability was determined 1 day after impact followed by histology and polarized microscopy. RESULTS: Macroscopic changes to the articular surface were not observed after low and moderate impact. The OCT signal intensity ratio demonstrated a 27% increase (P = 0.006) after low impact and a 38% increase (P = 0.001) after moderate impact. Cell death increased by 150% (P < 0.001) and 200% (P < 0.001) after low and moderate energy impacts, respectively. When compared with unimpacted controls, both Mankin histology and David-Vaudey polarized microscopy scores increased (P = 0.036 and P = 0.002, respectively) after moderate energy impact. CONCLUSIONS: This study shows that OCT detects acute cartilage changes after impact injury at levels insufficient to cause visible damage to the articular surface but sufficient to cause chondrocyte death and microscopic matrix damage. This finding supports the use of OCT to detect microstructural subsurface cartilage damage that is poorly visualized with conventional imaging.

In vivo effects of single intra-articular injection of 0.5% bupivacaine on articular cartilage.

BACKGROUND: Single intra-articular injections of local anesthetics are commonly used clinically. Recent in vitro studies have demonstrated chondrotoxic effects of local anesthetics, with the greatest emphasis on bupivacaine toxicity. This in vivo study was conducted to determine whether a single intra-articular injection of 0.5% bupivacaine results in chondrocyte morbidity and rapid chondrolysis. METHODS: Forty-eight Sprague-Dawley rats received a 100-microL injection of sterile 0.9% saline solution (negative control) into one stifle joint and 100 microL of either preservative-free 0.5% bupivacaine (experimental group) or 0.6 mg/mL monoiodoacetate (positive control) into the contralateral joint. The rats were killed at one week, four weeks, twelve weeks, or six months. Live and dead cells were quantified with use of three-dimensional confocal reconstructions of fluorescent-stained tissues at standardized locations on the distal part of the femur. Histological findings were graded with use of a modified Mankin score, and cell density was quantified with use of custom image-analysis software. RESULTS: In the specimens injected with bupivacaine, the chondral surfaces remained intact as seen with gross and histological examination. No differences in superficial chondrocyte viability or modified Mankin scores were observed between the saline-solution and bupivacaine groups at any location or time point (p > 0.05). Quantitative histological analysis of the bupivacaine-treated knees at six months revealed an up to 50% reduction in chondrocyte density compared with that of the saline-solution-treated knees (p < or = 0.01). Monoiodoacetate injection resulted in death of up to 87% of the superficial chondrocyte cells at one week and chondrolysis at six months. Despite severe histological abnormalities by four weeks after monoiodoacetate injection, cartilage injury was not evident on gross inspection until six months. CONCLUSIONS: This in vivo study showing reduced chondrocyte density without cartilage tissue loss six months after a single intra-articular injection of 0.5% bupivacaine suggests bupivacaine toxicity. The effects of bupivacaine were milder than those of an injection of 0.6% monoiodoacetate, which resulted in chondrolysis over the same time period.

Sustained hypoxia enhances chondrocyte matrix synthesis.

Articular cartilage is an avascular tissue with chondrocytes in the deeper zones existing under conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether sustained hypoxia enhances the production of cartilage matrix proteins. Freshly isolated primary bovine articular chondrocytes were encapsulated in three-dimensional alginate beads and maintained at 2% oxygen with media changes using media pre-equilibrated to 2% oxygen. Immunolocalization of HIF-1alpha was performed to verify hypoxic conditions. Sustained hypoxia resulted in an increase in proteoglycan synthesis after only 1 day, as measured by 35S-sulfate incorporation. This increase was maintained for the duration of the 17 day study. After 17 days of hypoxic culture, increases in total type II collagen and COL2A1 gene expression were probed by indirect immunofluorescence, type II collagen ELISA, and real-time qPCR; in addition, increased glycosaminoglycan deposition was observed as determined by chemical analysis. These studies show that sustained hypoxia enhances articular chondrocyte matrix synthesis and viability in three-dimensional alginate culture.

Induction of apoptosis in human bladder cancer cells by green tea catechins.

Cell culture and animal studies have demonstrated strong chemopreventative effects of green tea and its associated polyphenols in multiple cancers, though the exact mechanisms of action are not well understood. This in vitro study examined the antiproliferative/pro-apoptotic potential of green tea extract (GTE), polyphenon-60 (PP-60), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) in both normal and malignant human bladder cells. Cell growth (proliferation/apoptosis) was measured in UROtsa (normal), SW780 (tumorigenic; low-grade), and TCCSUP (tumorigenic; high-grade) human bladder urothelial cells by cell proliferation (XTT) assay after treatment with 0-80 microg/mL of GTE, PP-60, ECG and EGCG for 72 h. Molecular signaling pathways of catechin-induced apoptosis were analyzed using Human signal transduction RT(2) Profiler PCR array (SuperArray). Compared to control-treated cells, treatment with catechin agents significantly suppressed cell growth in a dose-dependent fashion (P < 0.01), with strongest effects evoked by ECG and EGCG in UROtsa cells, ECG in low-grade RT4 and SW780 cells, and PP-60 and EGCG in high-grade TCCSUP and T24 cells. Microarray analysis indicated distinct differences in mRNA gene expression regarding growth signaling pathway activation induced by EGCG in normal/tumorigenic human bladder cell lines, providing a rationale for the putative therapeutic usage of green tea polyphenols against bladder disease.

Progressive chondrocyte death after impact injury indicates a need for chondroprotective therapy.

BACKGROUND: Impact injury to articular cartilage can lead to posttraumatic osteoarthritis. HYPOTHESES: This study tests the hypotheses that (1) chondrocyte injury occurs after impact at energies insufficient to fracture the cartilage surface, and that (2) cartilage injury patterns vary with impact energy, time after injury, and cartilage thickness. STUDY DESIGN: Controlled laboratory study. METHODS: Fresh bovine osteochondral cores were randomly divided into 5 groups: (1) control, (2) 0.35 J, (3) 0.71 J, (4) 1.07 J, and (5) 1.43 J impact energies. Cores were subjected to computer-controlled impact loading and full-thickness sections were then prepared and incubated in Dulbecco's Modified Eagle's Medium/F12 at 37 degrees C. Adjacent sections were harvested 1 and 4 days after impact for viability staining and fluorescent imaging. The area of dead and living chondrocytes was quantified using custom image analysis software and reported as a percentage of total cartilage area. RESULTS: The highest impact energy fractured the cartilage in all cores (1.43 J, n = 17). Seventy-three percent and 64% of the osteochondral cores remained intact after lower energy impacts of 0.71 J and 1.07 J, respectively. At lower energy levels, fractured cores were thinner (P <.01) than those remaining intact. In cores remaining intact after impact injury, chondrocyte death increased with increasing impact energy (P <.05) and with greater time after impact (P <.05). A progressive increase in dead cells near the bone/cartilage interface and at the articular surface was observed. CONCLUSION: These data showing progressive chondrocyte death after impact injury at energies insufficient to fracture the cartilage surface demonstrate a potential need for early chondroprotective therapy. CLINICAL RELEVANCE: These data show that efforts to reduce chondrocyte morbidity after joint injury may be a useful strategy to delay or prevent the onset of posttraumatic osteoarthritis.

Functional and immunohistochemical characterization of CB1 and CB2 receptors in rat bladder.

OBJECTIVES: To determined the localization of CB(1) and CB(2) receptors in rat bladder and investigate the effect of a mixed CB(1)/CB(2) receptor agonist, ajulemic acid (AJA), on chemically evoked release of the sensory neuropeptide calcitonin gene-related peptide (CGRP). METHODS: Whole rat bladders were incubated in a series of tissue baths containing physiologic salt solution to measure baseline CGRP release by enzyme immunoassay. Capsaicin (30 nM) and adenosine triphosphate (10 muM) were used to provoke CGRP release in the presence or absence of AJA. Specificity of AJA for CB(1) and CB(2) receptors was determined using antagonists. Localization was determined by immunofluorescence for CB(1) and CB(2) receptors in fixed bladders. RESULTS: Immunofluorescence showed the localization of CB(1) and CB(2) receptors in the bladder. Mean baseline CGRP release was 605 +/- 62 pg/g of bladder weight, and AJA had no effect on CGRP release. The addition of adenosine triphosphate/capsaicin significantly increased the CGRP release over baseline, by 44% (P < .05), and AJA application significantly decreased CGRP release, by 29% compared with controls (P < .05). The CB(1) and CB(2) antagonists AM 251 and AM 630, respectively, reversed the blunting effect of AJA on evoked CGRP release, resulting in an increase of 40% and 38% over baseline, respectively. CONCLUSIONS: CB(1) and CB(2) receptors are localized in the urothelium of rat bladder, and application of AJA inhibits the evoked release of CGRP by acting on CB(1) and CB(2) receptors. These findings identify a potential new pathway for study in the evaluation and treatment of painful bladder syndrome/interstitial cystitis.

Optimizing CO2 normalizes pH and enhances chondrocyte viability during cold storage.

Fresh osteochondral allografts are an important treatment option for the repair of full-thickness articular cartilage defects. Viable chondrocytes within the transplanted tissue are considered important to maintaining matrix integrity. The purpose of this study is to determine whether an increase in pH decreases chondrocyte viability during cold storage and whether equilibration of Dulbecco's modified Eagle's medium (DMEM) in 5% CO(2) normalizes pH and increases chondrocyte survival during storage at 4 degrees C. Freshly isolated bovine articular chondrocytes cultured in alginate beads were stored for up to 5 days at 4 degrees C or 37 degrees C in DMEM exposed to ambient air or in DMEM equilibrated with 5% CO(2). Chondrocyte viability was determined by flow cytometry. Physiologic pH was maintained when DMEM was equilibrated with 5% CO(2), while pH increased in ambient air. After 5 days of storage at 4 degrees C, chondrocyte necrosis was higher when stored in ambient air than if equilibrated with 5% CO(2). No decrease in chondrocyte viability was observed with storage at 37 degrees C. In addition, chondrocyte viability in bovine cartilage osteochondral cores was examined after storage for 14 days at 4 degrees C in DMEM with and without HEPES, and with and without 5% CO(2). Under these conditions, the superficial layer of chondrocytes was more viable when stored in DMEM with HEPES or DMEM equilibrated with 5% CO(2) than when stored in DMEM in ambient air. This data shows that an increase in pH decreased bovine chondrocyte viability when refrigerated at 4 degrees C in DMEM, and that optimization of CO(2) normalized pH and improved chondrocyte viability during cold storage in DMEM.

Mechanisms of H2O2-induced oxidative stress in endothelial cells exposed to physiologic shear stress.

Hydrogen peroxide (H2O2) is produced by inflammatory and vascular cells and induces oxidative stress, which may contribute to vascular disease and endothelial cell dysfunction. In smooth muscle cells, H2O2 induces production of O2 by activating NADPH oxidase. However, the mechanisms whereby H2O2 induces oxidative stress in endothelial cells are not well understood, although O2 may play a role. Recent studies have documented increased O2 in endothelial cells exposed to H2O2 via uncoupled nitric oxide synthase (NOS) and NADPH oxidase under static conditions. To assess responses to H2O2 in porcine aortic endothelial cells (PAEC) under shearing conditions, a constant flow rate of 24. 4 ml/min was applied to produce physiologically relevant shear stress (8. 2 dynes/cm). Here we demonstrate that treatment with 100 muM H2O2 increases intracellular O2 levels in PAEC. In addition, we demonstrate that l-NAME, an inhibitor of NOS, and apocynin, an inhibitor of NADPH oxidase, reduced O2 levels in PAEC treated with H2O2 under physiologic shear suggesting that both NOS and NADPH oxidase contribute to H2O2-induced O2 in PAEC. Co-inhibition of NOS and NADPH oxidase also reduced intracellular O2 levels under shear. We conclude that H2O2-induced oxidative stress in endothelial cells exhibits increased intracellular O2 levels through NOS and NADPH oxidase under shear. The inhibition of NOS and NADPH with H2O2 exposure is nonlinear, suggesting some interdependent or compensating system within endothelial cells. These findings suggest a complex interaction between H2O2 and oxidant-generating enzymes that may contribute to endothelial dysfunction in cardiovascular diseases.