Cerium dioxide, a Jekyll and Hyde nanomaterial, can increase basal and decrease elevated inflammation and oxidative stress.

It was hypothesized that the catalyst nanoceria can increase inflammation/oxidative stress from the basal and reduce it from the elevated state. Macrophages clear nanoceria. To test the hypothesis, M0 (non-polarized), M1- (classically activated, pro-inflammatory), and M2-like (alternatively activated, regulatory phenotype) RAW 264.7 macrophages were nanoceria exposed. Inflammatory responses were quantified by IL-1ß level, arginase activity, and RT-qPCR and metabolic changes and oxidative stress by the mito and glycolysis stress tests (MST and GST). Morphology was determined by light microscopy, macrophage phenotype marker expression, and a novel three-dimensional immunohistochemical method. Nanoceria blocked IL-1ß and arginase effects, increased M0 cell OCR and GST toward the M2 phenotype and altered multiple M1- and M2-like cell endpoints toward the M0 level. M1-like cells had greater volume and less circularity/roundness. M2-like cells had greater volume than M0 macrophages. The results are overall consistent with the hypothesis.

Exercise-Induced Changes in Glucose Metabolism Promote Physiological Cardiac Growth.

BACKGROUND: Exercise promotes metabolic remodeling in the heart, which is associated with physiological cardiac growth; however, it is not known whether or how physical activity-induced changes in cardiac metabolism cause myocardial remodeling. In this study, we tested whether exercise-mediated changes in cardiomyocyte glucose metabolism are important for physiological cardiac growth. METHODS: We used radiometric, immunologic, metabolomic, and biochemical assays to measure changes in myocardial glucose metabolism in mice subjected to acute and chronic treadmill exercise. To assess the relevance of changes in glycolytic activity, we determined how cardiac-specific expression of mutant forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase affect cardiac structure, function, metabolism, and gene programs relevant to cardiac remodeling. Metabolomic and transcriptomic screenings were used to identify metabolic pathways and gene sets regulated by glycolytic activity in the heart. RESULTS: Exercise acutely decreased glucose utilization via glycolysis by modulating circulating substrates and reducing phosphofructokinase activity; however, in the recovered state following exercise adaptation, there was an increase in myocardial phosphofructokinase activity and glycolysis. In mice, cardiac-specific expression of a kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transgene (GlycoLo mice) lowered glycolytic rate and regulated the expression of genes known to promote cardiac growth. Hearts of GlycoLo mice had larger myocytes, enhanced cardiac function, and higher capillary-to-myocyte ratios. Expression of phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in the heart (GlycoHi mice) increased glucose utilization and promoted a more pathological form of hypertrophy devoid of transcriptional activation of the physiological cardiac growth program. Modulation of phosphofructokinase activity was sufficient to regulate the glucose-fatty acid cycle in the heart; however, metabolic inflexibility caused by invariantly low or high phosphofructokinase activity caused modest mitochondrial damage. Transcriptomic analyses showed that glycolysis regulates the expression of key genes involved in cardiac metabolism and remodeling. CONCLUSIONS: Exercise-induced decreases in glycolytic activity stimulate physiological cardiac remodeling, and metabolic flexibility is important for maintaining mitochondrial health in the heart.

Analytical High-resolution Electron Microscopy Reveals Organ-specific Nanoceria Bioprocessing.

This is the first utilization of advanced analytical electron microscopy methods, including high-resolution transmission electron microscopy, high-angle annular dark field scanning transmission electron microscopy, electron energy loss spectroscopy, and energy-dispersive X-ray spectroscopy mapping to characterize the organ-specific bioprocessing of a relatively inert nanomaterial (nanoceria). Liver and spleen samples from rats given a single intravenous infusion of nanoceria were obtained after prolonged (90 days) in vivo exposure. These advanced analytical electron microscopy methods were applied to elucidate the organ-specific cellular and subcellular fate of nanoceria after its uptake. Nanoceria is bioprocessed differently in the spleen than in the liver.

From Dose to Response: In Vivo Nanoparticle Processing and Potential Toxicity.

Adverse human health impacts due to occupational and environmental exposures to manufactured nanoparticles are of concern and pose a potential threat to the continued industrial use and integration of nanomaterials into commercial products. This chapter addresses the inter-relationship between dose and response and will elucidate on how the dynamic chemical and physical transformation and breakdown of the nanoparticles at the cellular and subcellular levels can lead to the in vivo formation of new reaction products. The dose-response relationship is complicated by the continuous physicochemical transformations in the nanoparticles induced by the dynamics of the biological system, where dose, bio-processing, and response are related in a non-linear manner. Nanoscale alterations are monitored using high-resolution imaging combined with in situ elemental analysis and emphasis is placed on the importance of the precision of characterization. The result is an in-depth understanding of the starting particles, the particle transformation in a biological environment, and the physiological response.

Subchronic toxicity and cardiovascular responses in spontaneously hypertensive rats after exposure to multiwalled carbon nanotubes by intratracheal instillation.

The tremendous demand of the market for carbon nanotubes has led to their massive production that presents an increasing risk through occupational exposure. Lung deposition of carbon nanotubes is known to cause acute localized pulmonary adverse effects. However, systemic cardiovascular damages associated with acute pulmonary lesion have not been thoroughly addressed. Four kinds of multiwalled carbon nanotubes (MWCNTs) with different lengths and/or iron contents were used to explore the potential subchronic toxicological effects in spontaneously hypertensive (SH) rats and normotensive control Wistar-Kyoto (WKY) rats after intratracheal instillation. MWCNTs penetrated the lung blood-gas barrier and accumulated in the liver, kidneys, and spleen but not in the heart and aorta of SH rats. The pulmonary toxicity and cardiovascular effects were assessed at 7 and 30 days postexposure. Compared to the WKY rats, transient influences on blood pressure and up to 30 days persistent decrease in the heart rate of SH rats were found by electrocardiogram monitoring. The subchronic toxicity, especially the sustained inflammation of the pulmonary and cardiovascular system, was revealed at days 7 and 30 in both SH and WKY rat models. Histopathological results showed obvious morphological lesions in abdominal arteries of SH rats 30 days after exposure. Our results suggest that more attention should be paid to the long-term toxic effects of MWCNTs, and particularly, occupationally exposed workers with preexisting cardiovascular diseases should be monitored more thoroughly.

Persistent hepatic structural alterations following nanoceria vascular infusion in the rat.

Understanding the long-term effects and possible toxicity of nanoceria, a widely utilized commercial metal oxide, is of particular importance as it is poised for development as a therapeutic agent based on its autocatalytic redox behavior. We show here evidence of acute and subacute adverse hepatic responses, after a single infusion of an aqueous dispersion of 85 mg/kg, 30 nm nanoceria into Sprague Dawley rats. Light and electron microscopic evidence of avid uptake of nanoceria by Kupffer cells was detected as early as 1 hr after infusion. Biopersistent nanoceria stimulated cluster of differentiation 3(+) lymphocyte proliferation that intermingled with nanoceria-containing Kupffer cells to form granulomata that were observed between days 30 and 90. Ultrastructural tracking of ceria nanoparticles revealed aggregated nanoceria in phagolysosomes. An increased formation of small nanoceria over time observed in the latter suggests possible dissolution and precipitation of nanoceria. However, the pathway for nanoceria metabolism/secretion remains unclear. Although frank hepatic necrosis was not observed, the retention of nanoceria increased hepatic apoptosis acutely, this persisted to day 90. These findings, together with our earlier reports of 5-nm ceria-induced liver toxicity, provide additional guidance for nanoceria development as a therapeutic agent and for its risk assessment.

Biodistribution and biopersistence of ceria engineered nanomaterials: size dependence.

The aims were to determine the biodistribution, translocation, and persistence of nanoceria in the brain and selected peripheral organs. Nanoceria is being studied as an anti-oxidant therapeutic. Five, 15, 30, or 55 nm ceria was iv infused into rats which were terminated 1, 20, or 720 h later. Cerium was determined in blood, brain, liver, and spleen. Liver and spleen contained a large percentage of the dose, from which there was no significant clearance over 720 h, associated with adverse changes. Very little nanoceria entered brain parenchyma. The results suggest brain delivery of nanoceria will be a challenge. FROM THE CLINICAL EDITOR: This team of investigators revealed that nanoceria, which is being studied as an anti-oxidant, has very limited uptake by the brain regardless of the range of sizes studied, suggesting major challenges in the application of this novel approach in the central nervous system.

Macrophage Polarization Status Impacts Nanoceria Cellular Distribution but Not Its Biotransformation or Ferritin Effects.

The innate immune system is the first line of defense against external threats through the initiation and regulation of inflammation. Macrophage differentiation into functional phenotypes influences the fate of nanomaterials taken up by these immune cells. High-resolution electron microscopy was used to investigate the uptake, distribution, and biotransformation of nanoceria in human and murine M1 and M2 macrophages in unprecedented detail. We found that M1 and M2 macrophages internalize nanoceria differently. M1-type macrophages predominantly sequester nanoceria near the plasma membrane, whereas nanoceria are more uniformly distributed throughout M2 macrophage cytoplasm. In contrast, both macrophage phenotypes show identical nanoceria biotransformation to cerium phosphate nanoneedles and simultaneous nanoceria with ferritin co-precipitation within the cells. Ferritin biomineralization is a direct response to nanoparticle uptake inside both macrophage phenotypes. We also found that the same ferritin biomineralization mechanism occurs after the uptake of Ce-ions into polarized macrophages and into unpolarized human monocytes and murine RAW 264.7 cells. These findings emphasize the need for evaluating ferritin biomineralization in studies that involve the internalization of nano objects, ranging from particles to viruses to biomolecules, to gain greater mechanistic insights into the overall immune responses to nano objects.

Alteration of hepatic structure and oxidative stress induced by intravenous nanoceria.

Beyond the traditional use of ceria as an abrasive, the scope of nanoceria applications now extends into fuel cell manufacturing, diesel fuel additives, and for therapeutic intervention as a putative antioxidant. However, the biological effects of nanoceria exposure have yet to be fully defined, which gave us the impetus to examine its systemic biodistribution and biological responses. An extensively characterized nanoceria (5 nm) dispersion was vascularly infused into rats, which were terminated 1 h, 20 h or 30 days later. Light and electron microscopic tissue characterization was conducted and hepatic oxidative stress parameters determined. We observed acute ceria nanoparticle sequestration by Kupffer cells with subsequent bioretention in parenchymal cells as well. The internalized ceria nanoparticles appeared as spherical agglomerates of varying dimension without specific organelle penetration. In hepatocytes, the agglomerated nanoceria frequently localized to the plasma membrane facing bile canaliculi. Hepatic stellate cells also sequestered nanoceria. Within the sinusoids, sustained nanoceria bioretention was associated with granuloma formations comprised of Kupffer cells and intermingling CD3⁺ T cells. A statistically significant elevation of serum aspartate aminotransferase (AST) level was seen at 1 and 20 h, but subsided by 30 days after ceria administration. Further, elevated apoptosis was observed on day 30. These findings, together with increased hepatic protein carbonyl levels on day 30, indicate ceria-induced hepatic injury and oxidative stress, respectively. Such observations suggest a single vascular infusion of nanoceria can lead to persistent hepatic retention of particles with possible implications for occupational and therapeutic exposures.

Opening of epithelial tight junctions and enhancement of paracellular permeation by chitosan: microscopic, ultrastructural, and computed-tomographic observations.

This study investigates the effects of chitosan (CS) on the opening of epithelial tight junctions (TJs) and paracellular transport at microscopic, ultrastructural, and computed-tomographic levels in Caco-2 cell monolayers and animal models. Using immunofluorescence staining, CS treatment was observed to be associated with the translocation of JAM-1 (a trans-membrane TJ protein), resulting in the disruption of TJs; the removal of CS was accompanied by the recovery of JAM-1. Ultrastructural observations by TEM reveal that CS treatment slightly opened the apical intercellular space, allowing lanthanum (an electron-dense tracer) to stain the intercellular surface immediately beneath the TJs, suggesting the opening of TJs. Following the removal of CS, the TJs were completely recovered. Similar microscopic and ultrastructural findings were obtained in animal studies. CS nanoparticles were prepared as an insulin carrier. The in vivo fluorescence-microscopic results demonstrate that insulin could be absorbed into the systemic circulation, while most CS was retained in the microvilli scaffolds. These observations were verified in a biodistribution study following the oral administration of isotope-labeled nanoparticles by single-photon emission computed tomography. Above results reveal that CS is a safe permeation enhancer and is an effective carrier for oral protein delivery.

Colon Adenocarcinoma Metastasis Through Ileocolic Fistula to Small Bowel in the Setting of Crohn's Disease.

Patients with Crohn's disease are at higher risk of developing colorectal cancer and gastrointestinal fistula. Few cases in the past described colorectal cancer metastasized within the gastrointestinal tract through a fistula. We report a case of sigmoid colon adenocarcinoma in a patient with Crohn's disease that metastasized to the ileum through an ileocolic fistula tract. In addition to presenting a unique pathological phenomenon in these patients, this case raises awareness of the importance of regular follow-up and early initiation of inflammatory bowel disease therapies.

Cardiopulmonary Bypass Video Is an Effective Learning Tool.

Background: Surgical procedures require the collaboration of medical personnel with multiple skill sets who have different levels of training. Someone new to surgical procedures, such as a medical student, faces a steep learning curve. Studies have shown that video-assisted learning is associated with improved learning of surgical procedures. Methods: During their surgical rotation orientation, third-year medical students were invited via email to participate in a learning study featuring a cardiopulmonary bypass video. Study participants took a pretest, reviewed the locally developed video, and took a posttest and an attitudinal questionnaire after viewing the video. Results: A convenience sample of 31 third-year medical students participated in the study. Overall knowledge scores improved from pretest to posttest (36.9% vs 79.6%, P<0.001). In the posttest attitudinal questionnaire, students reported that they preferred video-assisted learning to reading written protocols (90.3% strongly agree/agree) and that they were more knowledgeable about the function of the cardiopulmonary bypass machine (80.7% strongly agree/agree) after viewing the video. Students also reported that the video would be useful during their surgical clerkships (90.4% strongly agree/agree). Conclusion: Video-assisted learning was associated with comprehension of the material immediately after viewing the video, and medical students considered it to be appropriate and useful. This educational video may benefit other learners who are entering the cardiopulmonary bypass operating room for the first time.

Nanoceria distribution and effects are mouse-strain dependent.

Prior studies showed nanoparticle clearance was different in C57BL/6 versus BALB/c mice, strains prone to Th1 and Th2 immune responses, respectively. Objective: Assess nanoceria (cerium oxide, CeO2 nanoparticle) uptake time course and organ distribution, cellular and oxidative stress, and bioprocessing as a function of mouse strain. Methods: C57BL/6 and BALB/c female mice were i.p. injected with 10 mg/kg nanoceria or vehicle and terminated 0.5 to 24 h later. Organs were collected for cerium analysis; light and electron microscopy with elemental mapping; and protein carbonyl, IL-1ß, and caspase-1 determination. Results: Peripheral organ cerium significantly increased, generally more in C57BL/6 mice. Caspase-1 was significantly elevated in the liver at 6 h, to a greater extent in BALB/c mice, suggesting inflammasome pathway activation. Light microscopy revealed greater liver vacuolation in C57BL/6 mice and a nanoceria-induced decrease in BALB/c but not C57BL/6 mice vacuolation. Nanoceria increased spleen lymphoid white pulp cell density in BALB/c but not C57BL/6 mice. Electron microscopy showed intracellular nanoceria particles bioprocessed to form crystalline cerium phosphate nanoneedles. Ferritin accumulation was greatly increased proximal to the nanoceria, forming core-shell-like structures in C57BL/6 but even distribution in BALB/c mice. Conclusions: BALB/c mice were more responsive to nanoceria-induced effects, e.g. liver caspase-1 activation, reduced liver vacuolation, and increased spleen cell density. Nanoceria uptake, initiation of bioprocessing, and crystalline cerium phosphate nanoneedle formation were rapid. Ferritin greatly increased with a macrophage phenotype-dependent distribution. Further study will be needed to understand the mechanisms underlying the observed differences.

Steroidogenic acute regulatory-related lipid transfer domain protein 5 localization and regulation in renal tubules.

STARD5 is a cytosolic sterol transport protein that is predominantly expressed in liver and kidney. This study provides the first report on STARD5 protein expression and distribution in mouse kidney. Immunohistochemical analysis of C57BL/6J mouse kidney sections revealed that STARD5 is expressed in tubular cells within the renal cortex and medullar regions with no detectable staining within the glomeruli. Within the epithelial cells of proximal renal tubules, STARD5 is present in the cytoplasm with high staining intensity along the apical brush-border membrane. Transmission electron microscopy of a renal proximal tubule revealed STARD5 is abundant at the basal domain of the microvilli and localizes mainly in the rough endoplasmic reticulum (ER) with undetectable staining in the Golgi apparatus and mitochondria. Confocal microscopy of STARD5 distribution in HK-2 human proximal tubule cells showed a diffuse punctuate pattern that is distinct from the early endosome marker EEA1 but similar to the ER membrane marker GRP78. Treatment of HK-2 cells with inducers of ER stress increased STARD5 mRNA expression and resulted in redistribution of STARD5 protein to the perinuclear and cell periphery regions. Since recent reports show elevated ER stress response gene expression and increased lipid levels in kidneys from diabetic rodent models, we tested STARD5 and cholesterol levels in kidneys from the OVE26 type I diabetic mouse model. Stard5 mRNA and protein levels are increased 2.8- and 1.5-fold, respectively, in OVE26 diabetic kidneys relative to FVB control kidneys. Renal free cholesterol levels are 44% elevated in the OVE26 mice. Together, our data support STARD5 functioning in kidney, specifically within proximal tubule cells, and suggest a role in ER-associated cholesterol transport.

Intracellular adenosine triphosphate delivery enhanced skin wound healing in rabbits.

Small unilamellar lipid vesicles were used to encapsulate adenosine triphosphate (ATP-vesicles) for intracellular energy delivery. This technique was tested in full-thickness skin wounds in 16 adult rabbits. One ear was rendered ischemic by using a minimally invasive surgery. The other ear served as a normal control. Four circular full-thickness wounds were created on the ventral side of each ear. ATP-vesicles or saline was used and the wounds were covered with Tegaderm (3M, St. Paul, MN). Dressing was changed and digital photos were taken daily until all the wounds were healed. The mean healing times of ATP-vesicles-treated wounds were significantly shorter than that of saline-treated wounds on ischemic and nonischemic ears. Histologic study indicated better-developed granular tissue and reepithelialization in the ATP-vesicles-treated wounds. The wounds treated by ATP-vesicles exhibited extremely fast granular tissue growth. More CD31 positive cells were seen in the ATP-vesicles-treated wounds. This preliminary study shows that direct intracellular delivery of ATP can accelerate the healing process of skin wounds on ischemic and nonischemic rabbit ears. The extremely fast granular tissue growth was something never seen or reported in the past.

Application of Electron Microscopes in Nanotoxicity Assessment.

In this chapter, we highlight the applications of electron microscopes (EMs) in nanotoxicity assessment. EMs can provide detailed information about the size and morphology of nanomaterials (NMs), their localization in cells and tissues, the nano-bio interactions, as well as the ultrastructural changes induced by NMs exposure. Here, we share with the readers how we prepare the tissue sample, and the different types of EMs used among the nanotoxicologists. It is possible to deploy conventional EMs along or in combination with other analytical techniques, such as electron energy loss spectroscopy (EELS), energy dispersive X-ray spectroscopy (EDS or EDX), and TEM-assisted scanning transmission X-ray microscopy (STXM), toward further elemental and chemical characterization. Appropriate images are inserted to illustrate throughout this chapter.

Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells.

The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

In-vivo efficacy of novel paclitaxel nanoparticles in paclitaxel-resistant human colorectal tumors.

Colloidal carriers have been shown to improve tumor therapy by increased drug delivery into tumor sites resulting directly from the enhanced permeability and retention effect (EPR). However, the clinical outcome of tumor therapy is often limited due to multidrug resistance. Several different types of resistance exist with expression of p-glycoprotein being the most commonly described. Paclitaxel entrapped in emulsifying wax nanoparticles (PX NPs) was shown to overcome drug resistance in a human colon adenocarcinoma cell line (HCT-15). In the present studies, the in-vivo efficacy of PX NPs in a HCT-15 mouse xenograft model was demonstrated. Significant inhibition in tumor growth was observed in mice receiving PX NPs treatment. Additionally, mice dosed with Taxol also demonstrated slower tumor growth; however, the efficacy of the Taxol treatment was shown in the in-vitro HUVEC model to be due to the antiangiogenic effect of paclitaxel. It was concluded that the enhanced efficacy of PX NPs over Taxol in the xenograft model was due to both overcoming paclitaxel resistance and an antiangiogenic effect.

Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery.

INTRODUCTION/OBJECTIVES: Adventitial application of FeCl(3) causes endothelial injury, platelet aggregation, and a rapid onset of thrombus formation. The transmigration pathway of the ferric ion has not been definitively identified. Using a combination of TEM and X-ray elemental analysis, this study aims to elucidate the endothelial pathway of ferric ion migration in carotid artery. METHODS AND RESULTS: Vascular injury was induced by placing a Whatman #1 filter paper strip saturated with 10% FeCl(3) over the common carotid artery in male C57BL/6 mice for 3 min. After rinsing in saline, the mice were terminated at 10 or 30 min. The FeCL(3) exposed segments of the common carotid artery were dissected, and processed for TEM. Thrombus formation was observed in all cases. Endothelial and smooth muscle injuries were observed in segments of the vessel in direct contact with the oxidant. The endothelial injury ranged from minimal damage to total denudation. The basal endothelial surface adjacent to the internal elastic lamina showed accumulation of electron opaque vesicles. The membrane enclosed particles transmigrated across the endothelium and exocytosed into the lumen. The nature of the particles shown by STEM/EDS was rich in ferric ion. Elemental analysis also showed that some ferric oxide aggregates formed near the developing thrombus in the vascular lumen. CONCLUSION: Our results showed the ferric ion permeated the endothelial basal lamina before entering the arterial lumen via endocytic-exocytic pathway. This study provides an ultrastructural framework for future analysis of the adluminal and luminal injuries in this model.

Enhancement of cell adhesion, retention, and survival of HUVEC/cbMSC aggregates that are transplanted in ischemic tissues by concurrent delivery of an antioxidant for therapeutic angiogenesis.

A recurring obstacle in cell-base strategies for treating ischemic diseases is the significant loss of viable cells that is caused by the elevated levels of regional reactive oxygen species (ROS), which ultimately limits therapeutic capacity. In this study, aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs), which are capable of inducing therapeutic angiogenesis, are prepared. We hypothesize that the concurrent delivery of an antioxidant N-acetylcysteine (NAC) may significantly increase cell retention following the transplantation of HUVEC/cbMSC aggregates in a mouse model with hindlimb ischemia. Our in vitro results demonstrate that the antioxidant NAC can restore ROS-impaired cell adhesion and recover the reduced angiogenic potential of HUVEC/cbMSC aggregates under oxidative stress. In the animal study, we found that by scavenging the ROS generated in ischemic tissues, NAC is likely to be able to establish a receptive cell environment in the early stage of cell transplantation, promoting the adhesion, retention, and survival of cells of engrafted aggregates. Therapeutic angiogenesis is therefore enhanced and blood flow recovery and limb salvage are ultimately achieved. The combinatory strategy that uses an antioxidant and HUVEC/cbMSC aggregates may provide a new means of boosting the therapeutic efficacy of cell aggregates for the treatment of ischemic diseases.