Background: Progressive nephropathy represents a substantial source of morbidity and mortality in type 1 diabetes. Increasing albuminuria over time is a strong predictor of progressive renal dysfunction and heightened cardiovascular risk. Early albuminuria likely reflects vascular endothelial dysfunction, which may be mediated in part by chronic inflammation.

Methods: We measured baseline levels of four inflammatory biomarkers (high sensitivity C-reactive protein [hsCRP], soluble intercellular adhesion molecule-1 [sICAM-1], soluble vascular cell adhesion molecule-1 [sVCAM-1], and soluble tumor necrosis factor alpha receptor-1 [sTNF-R1]) in stored blood samples from the 1441 participants of the Diabetes Control and Complication Trial (DCCT). We used mixed effects regression models to determine the average change in urinary albumin excretion (AER) by tertiles of each biomarker. We also used Cox proportional hazards models to estimate the relative risk of incident sustained microalbuminuria according to levels of each biomarker.

Results:  After adjustment for baseline age, sex, duration of diabetes, hemoglobin A1c%, and randomized treatment assignment, we observed a significantly higher 5.9 mcg/min/year increase in AER among those in the highest compared to the lowest tertile of baseline sICAM-1 (p=0.04). Those in the highest tertile of sICAM-1 had an adjusted relative risk of 1.67 (95% CI, 0.96 to 2.92) of developing incident sustained albuminuria (p-for-trend=0.03). No significant associations were noted between AER and the other inflammatory markers, although a pattern of steeper increases in AER over time was observed for those in the highest tertiles of VCAM and TNFR-1. 

Conclusions:  Higher baseline sICAM-1 levels predicted an increased risk of progressive nephropathy in type 1 diabetes and may represent an early risk marker that reflects the important role of vascular endothelial dysfunction in this long-term complication. 

Zili Zhang, MD, PhD, Oregon Health & Science University, Portland, OR, USA; Wenwei Zhong, MD, Oregon Health & Science University, Portland, OR, USA; Doran Spencer, PhD student, Oregon Health & Science University, Portland, OR, USA

T cell-mediated uveitis is strongly associated with systemic inflammatory disorders including inflammatory bowel disease, sarcoidosis, ankylosing spondylitis, and juvenile idiopathic arthritis. Recent studies have discovered a novel subpopulation of T cells, called Th17 cells. These cells have a characteristic of producing interleukin (IL)-17, and are highly pathogenic. In this study, we employed DO11.10 mice to investigate the role of IL-17 in the pathogenesis of uveitis. CD4+ T cells in DO11.10 mice are genetically engineered to react with ovalbumin (OVA). First, DO11.10 splenocytes were isolated to assess their response to OVA in vitro. Uveitis was induced by intravitrealinjection of OVA. At 24 hours after OVA injection, ocular inflammation was evaluated by intravital microscopy. Then, the mice were sacrificed and the eyes were harvested for total RNA.  ELISPOT assay revealed that 24-hour OVA challenge significantly induced IL-17 production.in DO11.10 splenocytes. Intravitreally injected OVA elicited marked neutrophil-predominant inflammatory cell infiltration in the eyes. This leukocyte influx appeared to be mediated by CD4+ lymphocytes as evidenced by the fact that depletion of CD4+ cells by GK1.5 antibody significantly inhibited the ocular inflammation. Compared to control mice receiving vehicle alone, real time-PCR showed that OVA treatment induced an array of IL-17-related gene expression. Moreover, intraocular injection of anti-IL-17 antibody markedly reduced OVA-mediated inflammatory cell infiltration in the eyes. Finally, anti-IL-17 antibody attenuated ocular expression of CXCL2 and CXCL5, two well documented neutrophil chemotactic cytokines. These results indicate that we have established a uveitis model to study the contribution of Th-17 cells. Furthermore, our study suggests that IL-17 is implicated in the pathogenesis of T cell-mediated uveitis in part through neutrophil chemotaxis as its downstream effect.

Vandana Menon, MD , Tufts-New England Medical Center, Boston, MA, USA; Lijun Li, MD, Tufts-New England Medical Cetner, Boston, MA, USA; Tom Greene, PhD, University of Utah, Salt Lake City, UT, USA; Xuelei Wang, MS, Cleveland Clinic Foundation, Cleveland, OH, USA; Vaidyanathapuram Balakrishnan, MD, Tufts-New England Medical Center, Boston, MA, USA; Gerald Beck, PhD, Cleveland Clinic Foundation, Cleveland, OH, USA; John Kusek, PhD, NIDDK, Bethesda, MD, USA; Allan Collins, MD, Hennepin Faculty Associates, Minneapolis, MN, USA; Andrew Levey, MD, Tufts-New England Medical Cetner, Boston, MA, USA; Mark Sarnak, MD, Tufts-New England Medical Center, Boston, MA, USA

Adiponectin is an adipocyte derived peptide with putative anti-atherogenic properties. Data regarding adiponectin and cardiovascular disease (CVD) outcomes in the general population, diabetics, and in patients with heart failure have been inconsistent. Limited data exist on the relationship between adiponectin and mortality in chronic kidney disease (CKD). 

The Modification of Diet in Renal Disease Study was a randomized controlled trial conducted 1989-1993 to study the effects of strict blood pressure control and dietary protein restriction on the progression of predominantly non-diabetic, stage 3-4 CKD. Adiponectin was measured in frozen serum samples obtained at baseline (N=820). Survival status and cause of death, up to December 31, 2000, were obtained from the National Death Index.

The mean age was 52years and 85% were white. Mean±SD glomerular filtration rate (GFR) was 33±12 ml/min/1.73m2 and adiponectin was 12.8±8.0 mg/mL. Triglycerides, glucose, body mass index, GFR, and albumin were inversely related, and proteinuria and HDL cholesterol were directly related, to adiponectin. All-cause mortality was 22% (n=169) and CVD mortality was 13% (n=96). In Cox models adjusting for demographic, CVD, and kidney disease risk factors, the highest tertile of adiponectin was associated with increased risk of all-cause (HR, 95% CI=1.60, 1.02-2.53) and CVD (2.56, 1.42-4.63) mortality. In linear analyses, the adjusted HR per 1mg/mL increase in adiponectin was 1.03, 1.01-1.05 for all-cause and 1.06, 1.03-1.09 for CVD mortality.

High rather than low adiponectin is associated with increased mortality in this cohort of patients with stage 3-4 non-diabetic CKD. High adiponectin mayreflect malnutrition, reduced GFR, or a parallel pathologic process that accompanies reduced kidney function. Further studies are needed to confirm this association and to elucidate the underlying mechanisms.

Terumi Midoro-Horiuti, MD, PhD, University of Texas Medical Branch, Galveston, TX, USA; Ruby Tiwari, PhD, University of Texas Medical Branch, Galveston, TX, USA; Randall Goldblum, MD, University of Texas Medical Branch, Galveston, TX, USA

Background: The prevalence and morbidity of asthma and other allergic diseases in industrial countries have increased dramatically over the last two-three decades. Asthma has become the most common chronic disease of childhood and asthma at any age is likely to originate in childhood or earlier. While the causes of the increase in asthma have not been fully elucidated, exposures to various environmental pollutants and contaminants are likely contributors. Given that indicators of allergic sensitization are often present at birth and that most cases of asthma develop in early childhood, environmental exposures in utero and during the first years of life are likely to be very important.  Objective: We recently reported that estradiol and various environmental estrogens induce mast cell degranulation and enhance IgE-mediated release of allergic mediators in cultured mast cells and basophils. We hypothesized that these agents would enhance allergic sensitization as well as responsiveness to allergens. To test this hypothesis, mice were exposed to the common environmental estrogen BPA during fetal and early postnatal periods by loading their mothers. The response of the pups to allergic sensitization and airway challenges were assessed.  Methods: Female BALB/c mice received 10 microg/ml BPA in their drinking water from one week prior to impregnation to the end of the study. Infant mice were given one intraperitoneal injection of 5 microg of ovalbumin (OVA) on day 4 and 3% OVA by nebulization for 10 min on days 13, 14 and 15. On day 17, serum IgE antibodies to OVA were assessed by ELISA, and airway inflammation and hyperresponsiveness (AHR) by enumerating eosinophils (Eo) in bronchoalveolar lavage (BAL) fluid and whole body barometric plethysmography.  Results: Infants from BPA-exposed mother responded to this sensitization with higher serum IgE anti-OVA concentrations, compared to infants from unexposed mothers (p<0.05).  Similarly Eos in their airways were significantly greater (p<0.05). Finally, AHR of the OVA-sensitized infants from BPA-treated mothers was enhanced relative to that of infants from mothers that were not exposed to BPA (p<0.05).  Conclusions: Perinatal exposure to the environmental estrogen BPA enhances allergic sensitization and bronchial inflammation and responsiveness in a susceptible animal model of asthma. Whether there is a critical period / burden for this exposure remains to be elucidated.

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Tzung Hsiai, MD/PhD, USC, Los Angeles, CA, USA; Mahsa Rouhanizadeh, PhD, USC, Los Angeles , CA, USA; Wakako Takabe, PhD, USC, Los Angeles, CA, USA

Objective- Mitochondrial inner membrane potential (ΔΨm) is an indicator of mitochondrial energy state and reactive oxygen species (mtROS). Mitochondrial dysfunction is intimately related to cardiovascular diseases. We hypothesized that shear stress regulates ΔΨm via and ·NO signaling. Methods: We developed a biophysical method to quantify mitochondrial ΔΨm using the mitochondrial membrane potential probe, Tetramethyl rhodamine methyl ester (TMRM+), in relation to the plasma membrane potential (ΔΨp) in endothelial cells based on the Nerst principle. We established the dynamic changes of TMRM+ fluorescence in response to metabolic inhibitors and membrane uncouplers to characterize membrane potential using the flow cytometry (FACS) analysis. Confluent human (HAEC) were subjected to pulsatile (PSS) and oscillatory shear stress (OSS). Results: Both PSS and OSS hyperpolarize membrane potential (static: 140 mV±7.5 OSS: 152±5.3 mV; PSS: 173.5±8.9 mV, P <0.01, n=4). PSS up-regulated Mn-SOD expression by 3.7-fold with a concomitant decrease in MitoSox Red signal, a probe to assess mitochondrial O2·- production (mtROS), and an increase in carboxy-H2DCFDA intensity, a dye to assess cellular H2O2 production (n=5, P < 0.05). Silencing Mn-SOD abrogated the PSS-mediated decrease in mtROS level. To simulate shear stress-mediated NO production, we added EDTA-NO, which resulted in a dose dependent increase in ΔΨm. Addition of L-NAME attenuated EDTA-NO-induced hyperpolarization of ΔΨm. Treating HAEC with eNOS siRNA did not alter ΔΨm and PSS hyperpolarized ΔΨm in eNOS siRNA-treated EC. Addition of Mn-SOD mimetic also mimicked PSS-induced hyperpolarization in ΔΨm. Conclusion: We demonstrated a biophysical approach to monitor the dynamic changes in ΔΨm. Our findings indicate that ·NO generation and Mn-SOD expression influence ΔΨm in response to pulsatile shear stress.

John Kao, MD, University of Michigan, Ann Arbor, MI, USA; Min Zhang, MD, University of Michigan, Ann Arbor, MI, USA; Bradford Berndt, BS, University of Michigan, Ann Arbor , MI, USA; Siva Rathinavelu, PhD, University of Michigan, Ann Arbor, MI, USA

Background: We previously found that the adoptive transfer of H. pylori-pulsed bone marrow-derived conventional dendritic cells (cDCs) induced pathogen specific helper T cells response with concurrent induction of a regulatory T cell (Treg) response but a modest reduction in H. pylori colonization. Aims: to determine the effect of depleting regulatory T cells using anti-CD25 antibodies on H. pylori-pulsed DC vaccine efficacy. Methods: Bone marrow cells from C57BL/6 mice were cultured in the presence of GM-CSF and IL-4 (5ng/mL) for 6 days and cDCs were enriched using Optiprep. C57BL/6 mice were adoptively transferred with H. pylori-pulsed cDC (106 cells i.p. on day 0 and day 14). Mice were pretreated with PBS (n=4) or neutralizing CD25 antibody (n=5) purified from the PC61 cell line (1 mg i.p. on day 0). Depletion of CD4+CD25+ Treg population was confirmed by FACS. All mice were challenged with live H. pylori SS1 on day 21 and mice were sacrificed on day 35. CD4+ T cells isolated from spleen of mice were ex vivo stimulated with H. pylori SS1 sonicate and unstimulated bone marrow derived DCs as stimulator cells. H. pylori-specific IFN-gamma released by MACS microbeads sorted splenic CD4+ T cells were compared between PBS or PC61 treated groups. H. pylori colonization was quantified with qPCR using H. pylori-specific 16s rRNA primers. Results: Pretreatment with PC61 (anti-CD25 antibodies) significantly reduced the percent of CD4+CD25+ Tregs in mice on day 35 compared to PBS treated mice (PBS=1.74±0.08, PC61=0.67±0.15, P< 0.05). Production of IFN-γ by H. pylori-specific CD4+ splenic T cells was higher in the PC61 pretreated group compared to the PBS treated group (PBS=1063±26.5, PC61=1242±10.51, P < 0.05). H. pylori colonization measured by qPCR showed a significant reduction in colonization in the PC61 treated group compared to the PBS treated group P < 0.05).  No significant difference in gastritis scores was found between the two groups. Conclusion: Depletion of CD4+CD25+ Tregs may enhance anti-H. pylori immunity by promoting unrestrained Th1 response critical for H. pylori sterilization.

Arno Tilles, MD, Massachusetts General Hospital, Boston, MA, USA; Jaesung Park, PhD, Postech, Pohang, Kyung Buk, NA, Korea, South; Francois Berthiaume, PhD, Massachusetts General Hospital, Boston, MA, USA; Mehmet Toner, PhD, Massachusetts General Hospital, Boston, MA, USA; Martin Yarmush, MD/PhD, Massachusetts General Hospital, Boston, MA, USA

Over 30,000 patients die annually in the United States from liver failure. In fulminant hepatic failure, a clinical syndrome associated with high mortality, orthotopic liver transplantation is the primary therapeutic option for patients not responding to supportive therapy. However, the persistent scarcity of donor organs has limited this therapeutic modality resulting in a continued increase in the number of patients who die waiting for a donor liver. An extracorporeal bioartificial liver (BAL) device containing fully functioning hepatocytes could provide vital support to a liver failure patient until a donor liver was available or until the patient’s own liver regenerated. Our goal was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow, high cell-density bioreactor. Photolithographic techniques were used to microfabricate surface features (i.e., grooves) onto the underlying glass substrates. The grooves, perpendicular to the radial flow direction, protected the seeded hepatocytes from the high shear stresses caused by volumetric flow rates of medium necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor creating a channel height of 100 µm between each substrate. The cell density in the bioreactor was as high as 40×106 hepatocytes in 5 mL. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of hepatocytes remained stable over 5 days of perfusion and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves.  This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.

Samuel Shelburne, MD, Baylor College of Medicine, Houston, TX, USA; David Keith, B.S., Baylor College of Medicine, Houston, TX, USA; Michael Davenport, B.A., Baylor College of Medicine, Houston, TX, USA; Richard Brennan, PhD, MD Anderson Cancer Center, H, TX, USA; James Musser, MD, PhD, The Methodist Hospital Research Institute, Houston, TX, USA

Background: The molecular mechanisms by which bacterial pathogens regulate virulence factor production in response to the host environment are poorly understood. Previous genome-wide in vivo and ex vivo analyses have demonstrated that the major human pathogen group A Streptococcus (GAS) has high transcript levels of genes encoding proteins involved in virulence factor production and complex carbohydrate utilization during host-pathogen interaction. These data led us to hypothesize that GAS may directly link the expression of virulence factors and genes involved in complex carbohydrate utilization through a shared regulatory system. We specifically focused on catabolite control protein A (CcpA) because, in Bacillus species, CcpA alters carbohydrate utilization gene expression in response to changes in environmental carbohydrate levels. Methods: Real-time quantitative reverse transcription PCR was used to compare GAS gene transcript levels between organisms grown in human saliva and nutrient-rich medium. Genetic inactivation of GAS ccpA was performed via a non-polar insertional mutagenesis technique. Expression microarray analysis was used to define the CcpA transcriptome. A mouse infection model was used to investigate the contribution of CcpA to invasive and pharyngeal GAS disease. Binding of purified, recombinant GAS CcpA to DNA of selected promoter regions was analyzed using fluorescence spectroscopy in the presence and absence of the key CcpA co-factor HPr-Ser46-P. Results: Compared to growth in a standard laboratory medium, growth of GAS in human saliva resulted in significantly different transcript levels of 59 of 78 tested genes, including ccpA. Transcriptome analysis during growth in nutrient-rich medium revealed that CcpA significantly influenced the transcript levels of many carbohydrate utilization genes as well as numerous well characterized GAS virulence factors, including the potent cytotoxin streptolysin S. When grown in human saliva, a nutrient-poor medium, CcpA influenced production of several key virulence factors not influenced during growth in nutrient-rich medium, including the cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Compared with the wild-type parental strain, the ccpA isogenic mutant strain was significantly less virulent in a mouse model of invasive infection. Also, the ccpA isogenic mutant strain was significantly impaired in ability to colonize the mouse oropharynx. Purified, recombinant CcpA specifically bound to the promoter region of the genes encoding streptolyin S, a key virulence factor. Conclusion: We have discovered that CcpA directly links environmental carbohydrate concentrations with virulence factor production in GAS. Given that several other major Gram-positive pathogens encode proteins with a high-degree of similarity to GAS CcpA, such a pathway may be a fundamental aspect of pathogenesis for a wide variety of microbes.

Madhumathi Rao, MD, FRCP(E), Tufts-New England Medical Center, Boston, MA, USA; Kinjalika Sathi, BS, Tufts University School of Medicine, Boston, MA, USA; Vered levy, PhD, Tufts University School of Medicine, Boston, MA, USA; Smitha Padala, MD, Tufts-New England Medical Center, Boston , MA, USA; Caren Demello, MD, Tufts-New England Medical Center, Boston, MA, USA; Sarah Haigh, PhD, Tufts University School of Medicine, Boston, MA, USA; Vaidyanathapuram Balakrishnan, MD,PhD, Tufts-New England Medical Center, Boston, MA, USA; Orian Shirihai, MD PhD, Tufts University School of Medicine, Boston, MA, USA

The uremic syndrome is characterized by abnormalities in energy metabolism, including insulin resistance, dyslipidemia and protein energy malnutrition. Mitochondria are integral to these metabolic processes; measurement of the Mitochondrial Membrane Potential (MMP) is an established approach to assess mitochondrial functional integrity, a reduced MMP indicating mitochondrial dysfunction. We used the Live Cell Array (Molecular Cytomics ™) to measure MMP in peripheral blood mononuclear cells (PBMC) from patients with chronic kidney disease (CKD) including subjects on dialysis (end stage renal disease, ESRD) and healthy volunteers (HV). The array facilitates the study of a heterogenous cell population and allows the performance of functional studies at the resolution of a single cell, each cell in the array having a designated address. The objective of the present study was to develop and standardize the best protocol to study these parameters in CKD/ESRD patients and draw comparisons to cells obtained from HV. A total of 25 subjects were studied, 18 with CKD/ESRD (age range 45-87) and 7 HV. PBMC’s were isolated from a blood sample and incubated with verapamil and the fluorescent dyes, tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) and MitoTracker Green FM (MTG) and then loaded onto a Live Cell Array slide. After basal bright field, MTG and TMRE images were taken, these were then repeated after incubation with 5mM oligomycin, an inhibitor of F1F0ATP synthase that hyperpolarizes the inner mitochondrial membrane. Images were analyzed before and after adding oligomycin; the fluorescense intensity obtained after taking the ratio of TMRE to MTG images was used as a measure of MMP. For statistical comparisons the parameters of the cell distribution obtained for each patient were used as a summary measure including a measure of variability. Paired comparisons accounting for within patient clustering were made for pre-post oligomycin measurements. Empiric distributions for the cell population and the ? following oligomycin allowed assessment of the proportion of cells at extremes of the distribution. Preliminary results showed that MMP had greater variability within patients compared to controls under both basal and post-oligomycin conditions. After adding oligomycin, both controls and subjects showed a significant increase in MMP. The mean MMP was lower at baseline among patients compared to controls but higher after addition of oligomycin; the interaction between pre-post oligomycin difference in MMP with subject category (CKD vs control) was significant at p<0.001. Even with limited data, certain trends were apparent, including greater heterogeneity among cells and an exaggerated response to oligomycin among patients.Use of the Live Cell Array to study MMP at single cell level offers insight into pathophysiology and variable cell responses in disease states.

Shenandoah Robinson, MD, Case Western Reserve University, Cleveland, OH, USA; Qing Li, MD, Case Western Reserve University, Cleveland, OH, USA

The incidence of preterm births in the USA is 12.7%, an increase of 20% during the past 15 years. Although a marked decline in mortality for these infants has occurred, especially for those born before 28 weeks gestation, there has not been a concomitant decline in neurological morbidity. Infants who were born preterm are at risk for chronic deficits including cerebral palsy, epilepsy, cognitive delay and behavioral problems. The burden of these deficits affects the children, their families, and society. These deficits arise from loss of oligodendrocytes and neurons due to persistent apoptosis well after the insult itself. Intrauterine processes that predispose to brain injury and preterm birth are typically subclinical, hindering prenatal interventions. More effective neonatal treatments to improve the outcomes are essential. Erythropoietin (EPO), a pleiotrophic cytokine, is a potential neuroprotectant, but its mechanism in the developing brain is unclear. EPO is essential for central nervous system development, and regulates neural cell survival and differentiation. When EPO binds its cognate receptor, survival pathways are activated; while unbound EPO receptors trigger apoptosis. Developing neurons and oligodendrocytes express EPO receptors, and the expression is upregulated by injury. We hypothesized that prenatal injury upregulates EPOR expression without a proportionate increase in EPO, leaving developing neural cells prone to apoptosis. Methods: A model of prenatal systemic hypoxia-ischemia on embryonic E18 in rats was used with in vivo and in vitro analyses. EPO or saline was administered postnatally to mimic delayed administration in humans. Biochemical, histological and functional analyses were performed. Means for control versus insult were compared with a two-tailed t test, and for groups with one-way ANOVA with Tukey's HSD correction. A p value <0.05 was considered significant. Results: After the prenatal injury EPOR expression was upregulated in both the gray and white matter, and immunocytochemistry showed increased EPOR expression on neurons, developing oligodendrocytes, and microglia, while EPO expression was unaffected. Exogenous EPO rescued neural cells from apoptosis after glutamate toxicity in vitro in a dose dependent manner, and after hypoxia-ischemia in vivo. Exogenous EPO administrered neonatally after prenatal injury restored myelin basic protein production and cortical parvalbumin+ neuron numbers to control levels. Adult functional improvement was evidenced by improved performance on the horizontal ladder test (p=0.003), and seizure threshold restoration (p<0.05). Conclusions: Together, these results suggest exogenous EPO can rescue developing neural cells from apoptosis after prenatal injury, with lasting functional improvement in adults. Moreover, these results suggest a mechanism to explain EPO's efficacy, and thus support the potential use of EPO in human infants after preterm birth.

Natalie Torok, MD, UC Davis, Sacramento, CA, USA; Joy Jiang, MD, UC Davis, Sacramento, CA, USA; Kornelia Baghy, MSc, UC Davis, Sacramento, CA, USA

We have previously shown that hepatic stellate cells (HSC) are activated by the phagocytosis of apoptotic bodies (AB) from hepatocytes. Phagocytosis induces NADPH oxidase (NOX) resulting in production of superoxide (O2-) and procollagen alpha 1(I) upregulation. As NOXs are central to the fibrogenic process, and NOX2 is described as the phagocytic NOX, our hypothesis is that NOX2 activation plays a role in phagocytosis of AB and subsequent O2- production. Furthermore, the small GTP-ase Rac1, a regulatory element of NOXs could also be involved in phagocytosis of AB, and in downstream signaling leading to HSC activation. Methods: Primary rat HSC were transfected with NOX2-specific siRNA, exposed to AB, and the phagocytic rate, O2- production (lucigenin assay) and the procollagen alpha 1(I) expression (real-time PCR) were tested. NOX-dependent Rac1 activation was evaluated by the Rac-GTP pull-down assay after exposing HSC to AB in the presence or absence of 100 uM apocynin (NOX inhibitor), or 600 nM LY29002 (PI3K inhibitor). LX-2 cells were transfected with the constitutively active Rac1 (Q63L), then exposed to AB and the rate of phagocytosis, and changes in the actin cytoskeleton (phalloidin staining) were evaluated. Results: The rate of phagocytosis in NOX2 siRNA-treated cells decreased to 0.7-fold (±0.2), compared to scrambled siRNA-transfected cells. The O2- production of NOX2 siRNA transfected-cells decreased to 0.33-fold (± ) and the procollagen α1(I) expression to 0.67-fold (± ). Phagocytosis of AB induced Rac1 GTP-ase activation, and this was inhibited by apocynin suggesting that Rac1 activation occurs in concert with NOX2 activity. Inhibition of PI3K did not affect Rac1 activity. To determine if Rac1 is directly involved in the phagocytosis of AB, HSC were transfected with Rac1-Q63L, and we found that this increased the phagocytic rate by 1.65-fold (±0.16), when compared to cells transfected with the empty vector. There was also a significant reorganization in the actin cytoskeleton surrounding the phagosomes suggesting that Rac1 mediates its effects on phagocytosis via regulation of the actin cytoskeleton. In conclusion, NOX2 is involved in phagocytosis of AB by HSC and is responsible for O2- production and upregulation of procollagen alpha 1(I) expression. NOX-dependent Rac1 activation leads to an increase in phagocytosis with reorganization of the actin cytoskeleton in HSC, perpetuating liver fibrogenesis.