c-Myc Drives inflammation of the maternal-fetal interface, and neonatal lung remodeling induced by intra-amniotic inflammation.

Background: Intra-amniotic inflammation (IAI) is associated with increased risk of preterm birth and bronchopulmonary dysplasia (BPD), but the mechanisms by which IAI leads to preterm birth and BPD are poorly understood, and there are no effective therapies for preterm birth and BPD. The transcription factor c-Myc regulates various biological processes like cell growth, apoptosis, and inflammation. We hypothesized that c-Myc modulates inflammation at the maternal-fetal interface, and neonatal lung remodeling. The objectives of our study were 1) to determine the kinetics of c-Myc in the placenta, fetal membranes and neonatal lungs exposed to IAI, and 2) to determine the role of c-Myc in modulating inflammation at the maternal-fetal interface, and neonatal lung remodeling induced by IAI. Methods: Pregnant Sprague-Dawley rats were randomized into three groups: 1) Intra-amniotic saline injections only (control), 2) Intra-amniotic lipopolysaccharide (LPS) injections only, and 3) Intra-amniotic LPS injections with c-Myc inhibitor 10058-F4. c-Myc expression, markers of inflammation, angiogenesis, immunohistochemistry, and transcriptomic analyses were performed on placenta and fetal membranes, and neonatal lungs to determine kinetics of c-Myc expression in response to IAI, and effects of prenatal systemic c-Myc inhibition on lung remodeling at postnatal day 14. Results: c-Myc was upregulated in the placenta, fetal membranes, and neonatal lungs exposed to IAI. IAI caused neutrophil infiltration and neutrophil extracellular trap (NET) formation in the placenta and fetal membranes, and neonatal lung remodeling with pulmonary hypertension consistent with a BPD phenotype. Prenatal inhibition of c-Myc with 10058-F4 in IAI decreased neutrophil infiltration and NET formation, and improved neonatal lung remodeling induced by LPS, with improved alveolarization, increased angiogenesis, and decreased pulmonary vascular remodeling. Discussion: In a rat model of IAI, c-Myc regulates neutrophil recruitment and NET formation in the placenta and fetal membranes. c-Myc also participates in neonatal lung remodeling induced by IAI. Further studies are needed to investigate c-Myc as a potential therapeutic target for IAI and IAI-associated BPD.

Anti-inflammatory Properties of the Alpha-Melanocyte-Stimulating Hormone in Models of Granulomatous Inflammation.

PURPOSE: Alpha-melanocyte stimulating hormone (α-MSH) is known to have anti-inflammatory effects. However, the anti-inflammatory properties of α-MSH on normal bronchial epithelial cells are largely unknown, especially in the context of in vitro sarcoidosis models. METHODS: We evaluated the anti-inflammatory effects of α-MSH on two different in vitro sarcoidosis models (lung-on-membrane model; LOMM and three-dimensional biochip pulmonary sarcoidosis model; 3D-BSGM) generated from NBECs and an in vivo sarcoidosis mouse model. RESULTS: Treatment with α-MSH decreased inflammatory cytokine levels and downregulated type I interferon pathway genes and related proteins in LOMM and 3D-BSGM models. Treatment with α-MSH also significantly decreased macrophages and cytotoxic T-cells counts in a sarcoidosis mice model. CONCLUSION: Our results confirm the direct role of type I IFNs in the pathogenesis of sarcoid lung granulomas and highlight α-MSH as a potential novel therapeutic agent for treating pulmonary sarcoidosis.

Activity of the growth hormone-releasing hormone antagonist MIA602 and its underlying mechanisms of action in sarcoidosis-like granuloma.

OBJECTIVES: Growth hormone-releasing hormone (GHRH) is a potent stimulator of growth hormone (GH) secretion from the pituitary gland. Although GHRH is essential for the growth of immune cells, the regulatory effects of its antagonist in granulomatous disease remain unknown. METHODS: Here, we report expression of GHRH receptor (R) in human tissue with sarcoidosis granuloma and demonstrate the anti-inflammatory effects of MIA602 (a GHRH antagonist) in two in vitro human granuloma models and an in vivo granuloma model using different methods including ELISA, immunohistochemistry, RNA-seq analysis and flow cytometry. RESULTS: MIA602 decreases the levels of IL-2, IL-2R, IL-7, IL-12, IL-17A and TNF-α in an in vitro granuloma model. Further, we show that the anti-inflammatory effect of MIA602 appears to be mediated by a reduction in CD45+CD68+ cells in granulomatous tissue and upregulation in PD-1 expression in macrophages. Analysis of the expression of proteins involved in the mitochondrial stage of apoptosis showed that MIA602 increases the levels of caspase-3, BCL-xL/BAK dimer and MCl-1/Bak dimer in the granuloma. These findings indicate that MIA602 may not induce apoptosis. CONCLUSIONS: Our findings further suggest that GHRH-R is potentially a clinical target for the treatment of granulomatous disease and that MIA602 may be used as a novel therapeutic agent for sarcoidosis.

Novel three-dimensional biochip pulmonary sarcoidosis model.

Sarcoidosis is a multi-system disorder of granulomatous inflammation which most commonly affects the lungs. Its etiology and pathogenesis are not well defined in part due to the lack of reliable modeling. Here, we present the development of an in vitro three-dimensional lung-on-chip biochip designed to mimic granuloma formation. A lung on chip fluidic macrodevice was developed and added to our previously developed a lung-on-membrane model (LOMM). Granulomas were cultured from blood samples of patients with sarcoidosis and then inserted in the air-lung-interface of the microchip to create a three-dimensional biochip pulmonary sarcoidosis model (3D BSGM). Cytokines were measured after 48 hours. ELISA testing was performed to measure cytokine response difference between LOMM with 3D BSGM. There were statistically significant differences in IL-1ß (P = 0.001953), IL-6 (P = 0.001953), GM-CSF (P = 0.001953), and INF-γ expressions (P = 0.09375) between two groups. The current model represents the first 3D biochip sarcoidosis model created by adding a microfluidics system to a dual-chambered lung on membrane model and introducing developed sarcoid-granuloma to its air-lung-interface.

Nicotine, smoking, podocytes, and diabetic nephropathy.

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. Besides glycemic and blood pressure control, environmental factors such as cigarette smoking (CS) adversely affect the progression of DN. The effects of CS on DN progression have been attributed to combustion-generated molecules without consideration to the role of nicotine (NIC), responsible for the addictive properties of both CS and electronic cigarettes (ECs). Podocytes are essential to preserve the structure and function of the glomerular filtration barrier, and strong evidence indicates that early podocyte loss promotes DN progression. We performed experiments in human podocytes and in a mouse model of diabetes that develops nephropathy resembling human DN. We determined that NIC binding to podocytes in concentrations achieved with CS and ECs activated NADPH oxidase, which sets in motion a dysfunctional molecular network integrated by cyclooxygenase 2, known to induce podocyte injury; downregulation of AMP-activated protein kinase, important for maintaining cellular energy stores and antioxidation; and upregulation of CD36, which increased lipid uptake and promoted apoptosis. In diabetic mice, NIC increased proteinuria, a recognized marker of chronic kidney disease progression, accompanied by reduced glomerular podocyte synaptopodin, a crucial stabilizer of the podocyte cytoskeleton, and increased fibronectin expression. This novel study critically implicates NIC itself as a contributor to DN progression in CS and EC users.NEW & NOTEWORTHY In this study, we demonstrate that nicotine increases the production of reactive oxygen species, increases cyclooxygenase-2 expression, and upregulates Cd36 while inducing downregulation of AMP-activated protein kinase. In vivo nicotine increases proteinuria and fibronectin expression in diabetic mice. This study demonstrates that effects of nicotine on podocytes are responsible, at least in part, for the deleterious effects of smoking in the progression of chronic kidney disease, including diabetic nephropathy.

The effect of estrogen on diabetic wound healing is mediated through increasing the function of various bone marrow-derived progenitor cells.

OBJECTIVE: Endothelial progenitor cells (EPCs) are the key cells of postnatal neovascularization, and mesenchymal stem cells (MSCs) possess pluripotent differentiation capacity and contribute to tissue regeneration and wound healing. Both EPCs and MSCs are critical to the wound repair process, which is hindered in diabetes mellitus. Diabetes has been shown to decrease the function of these progenitor cells, whereas estrogen has beneficial wound healing effects. However, the role of estrogen in modulating EPC and MSC biology in diabetes is unknown. We investigated the effect of estrogen on improving bone marrow (BM)-derived EPC and MSC function using a murine diabetic wound healing model. METHODS: Female diabetic db+/db+ and nondiabetic control mice were wounded cutaneously and treated with topical estrogen or placebo cream. On day 5 after wounding, BM cells were harvested to quantify EPC number and colony-forming units of EPCs and MSCs. Wound healing rate was concurrently studied. Vessel density and scar density were then quantified using whole body perfusion and laser confocal microscopy. EPC recruitment was documented by immunohistochemistry to identify CD34- and vascular endothelial growth factor receptor 2-positive cells in the vessel wall. Data were analyzed by analysis of variance. RESULTS: Topical estrogen significantly increased colony-forming units of both EPCs and MSCs compared with placebo treatment, indicating improved viability and proliferative ability of these cells. Consistently, increased recruitment of EPCs to diabetic wounds and higher vessel density were observed in estrogen-treated compared with placebo-treated mice. Consequently, topical estrogen significantly accelerated wound healing as early as day 6 after wounding. In addition, scar density resulting from collagen deposition was increased in the estrogen-treated group, reflecting increased MSC activity and differentiation. CONCLUSIONS: Estrogen treatment increases wound healing and wound neovascularization in diabetic mice. Our data implicate that these beneficial effects may be mediated through improving the function of BM-derived EPCs and MSCs.

Oral nicotine aggravates endothelial dysfunction and vascular inflammation in diet-induced obese rats: Role of macrophage TNFα.

Obesity and cigarette smoke are major cardiovascular (CV) risk factors and, when coexisting in the same individuals, have additive/synergistic effects upon CVD. We studied the mechanisms involved in nicotine enhancement of CVD in Sprague Dawley rats with diet-induced obesity. The rats were fed either a high fat (HFD) or normal rat chow diet with or without nicotine (100 mg/L in drinking water) for 20 weeks. HFD rats developed central obesity, increased systolic blood pressure (SBP), aortic superoxide (O2-) production, and impaired endothelial nitric oxide synthase (eNOS) and endothelium-dependent relaxation to acetylcholine (EDR). Nicotine further increased SBP, O2- and impaired eNOS and EDR in obese rats. In the peritoneal macrophages from obese rats, tumor necrosis factor (TNF) α, interleukin 1ß and CD36 were increased, and were further increased in nicotine-treated obese rats. Using PCR array we found that 3 of 84 target proinflammatory genes were increased by 2-4 fold in the aorta of obese rats, 11 of the target genes were further increased in nicotine-treated obese rats. HUVECs, incubated with conditioned medium from the peritoneal macrophages of nicotine treated-obese rats, exhibited reduced eNOS and increased NADPH oxidase subunits gp91phox and p22phox expression. Those effects were partially prevented by adding anti-TNFα antibody to the conditioned medium. Our results suggest that nicotine aggravates the CV effects of diet-induced obesity including the oxidative stress, vascular inflammation and endothelial dysfunction. The underlying mechanisms may involve in targeting endothelium by enhancement of macrophage-derived TNFα.

SDF-1α-induced dual pairs of E-selectin/ligand mediate endothelial progenitor cell homing to critical ischemia.

Homing of endothelial progenitor cells (EPC) to the ischemic tissues is a key event in neovascularization and tissue regeneration. In response to ischemic insult, injured tissues secrete several chemo-cytokines, including stromal cell-derived factor-1α (SDF-1α), which triggers mobilization and homing of bone marrow-derived EPC (BMD-EPC). We previously reported that SDF-1α-induced EPC homing is mediated by a panel of adhesion molecules highly or selectively expressed on the activated endothelium in ischemic tissues, including E-selectin. Elevated E-selectin on wound vasculature serve as docking sites for circulating EPC, which express counterpart E-selectin ligands. Here, we show that SDF-1α presented in wound tissue and released into circulation can act both locally and remotely to induce ischemic tissue endothelium and BMD-EPC to express both E-selectin and its ligands. By performing BM transplantation using E-selectin-/- and E-selectin+/+ mice as the donors and recipients respectively, we demonstrate that upregulated dual E-selectin/ligand pairs reciprocally expressed on ischemic tissue endothelium and BMD-EPC act as double-locks to secure targeted EPC- endothelium interactions by which to facilitate EPC homing and promote neovascularization and tissue repair. These findings describe a novel mechanism for BMD-EPC homing and indicate that dual E-selectin/ligand pairs may be effective targets/tools for therapeutic neovascularization and targeted cell delivery.

Directing and Potentiating Stem Cell-Mediated Angiogenesis and Tissue Repair by Cell Surface E-Selectin Coating.

Stem cell therapy has emerged as a promising approach for treatment of a number of diseases, including delayed and non-healing wounds. However, targeted systemic delivery of therapeutic cells to the dysfunctional tissues remains one formidable challenge. Herein, we present a targeted nanocarrier-mediated cell delivery method by coating the surface of the cell to be delivered with dendrimer nanocarriers modified with adhesion molecules. Infused nanocarrier-coated cells reach to destination via recognition and association with the counterpart adhesion molecules highly or selectively expressed on the activated endothelium in diseased tissues. Once anchored on the activated endothelium, nanocarriers-coated transporting cells undergo transendothelial migration, extravasation and homing to the targeted tissues to execute their therapeutic role. We now demonstrate feasibility, efficacy and safety of our targeted nanocarrier for delivery of bone marrow cells (BMC) to cutaneous wound tissues and grafted corneas and its advantages over conventional BMC transplantation in mouse models for wound healing and neovascularization. This versatile platform is suited for targeted systemic delivery of virtually any type of therapeutic cell.

Combination therapy of amlodipine and atorvastatin has more beneficial vascular effects than monotherapy in salt-sensitive hypertension.

BACKGROUND: Current treatment for the secondary prevention of cardiovascular diseases frequently involves the prescription of several combination therapies, particularly antihypertensive medications and HMG-CoA reductase inhibitor. We have previously shown that in salt-sensitive hypertension either a statin or the calcium channel blocker amlodipine (Aml) have vasoprotective effects. Here, we investigated in aortas from Dahl salt-sensitive (DS) rats the effects of Aml, the statin atorvastatin (AT), and their combination on endothelial function, superoxide (O2 (-)) production, and the expression of endothelial nitric oxide synthase (eNOS), chemokine monocyte chemoattractant protein-1 (MCP-1), and lectin-like oxidized LDL receptor-1 (LOX-1). METHODS: Groups of DS rats were fed either normal-salt (NS, 0.5% NaCl) or high-salt (HS, 4% NaCl) diet or a HS diet with AT (15mg/kg/day), Aml (5mg/kg/day) or combination of AT/Aml for 6 weeks. RESULTS: Rats on the HS diet developed hypertension, aortic hypertrophy, accompanied by increased plasma C-reactive protein (CRP), aortic O2 (-), MCP-1 (80%), and LOX-1 (55%) expression and reduced eNOS and endothelial-dependent relaxation to acetylcholine (EDR). Aml reduced systolic blood pressure (SBP), aortic hypertrophy, plasma CRP, vascular O2 (-), and MCP-1 expression and improved eNOS and EDR. AT reduced aortic hypertrophy and plasma CRP, improved EDR, and normalized vascular O2 (-), eNOS, and proinflammatory gene expression with mild reduction in SBP. Combination therapy further reduced the SBP and normalized aortic hypertrophy, EDR, and plasma CRP. CONCLUSIONS: The combination therapy of Aml/AT has an additive beneficial effect on the vasculature. These novel findings may provide scientific basis for the combination therapy of statins with antihypertensive agents to reduce and prevent cardiovascular diseases.

A novel autologous cell-based therapy to promote diabetic wound healing.

OBJECTIVES: We have previously shown that stromal cell-derived factor-1α (SDF-1α) is downregulated within diabetic cutaneous wounds, and that direct application of recombinant SDF-1α increases wound closure rates, neovascularization, and endothelial progenitor cell (EPC) recruitment. However, increased wound levels of exogenous SDF-1α results in elevated systemic levels of this proangiogenic chemokine that raises concerns for tumorigenesis and inflammation. We now seek to test the efficacy of a novel, safer cell-based therapy (CBT) employing ex vivo primed bone marrow-derived stem cells (BMDSC) with SDF-1α. We also elucidate the mechanism of action of this new approach for accelerating diabetic wound healing. METHODS: Unfractionated BMDSC from diabetic Lepr mice were incubated for 20 hours with SDF-1α (100 ng/mL) or bovine serum albumin (control). Pretreated BMDSC (1 × 10) were injected subcutaneously into full-thickness skin wounds in Lepr mice (n = 8 per group). Wound closure rates, capillary density, and the recruitment of EPC were assessed with serial photography, DiI perfusion, confocal microscopy, and immunohistochemistry. The expression of molecular targets, which may mediate prohealing/proangiogenic effects of SDF-1α-primed BMDSC was evaluated by polymerase chain reaction array and immunoblotting assay. The biological function of a potential mediator was tested in a mouse wound-healing model. Serum SDF-1α levels were measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: SDF-1α-primed BMDSC significantly promote wound healing (P < 0.0001), neovascularization (P = 0.0028), and EPC recruitment (P = 0.0059). Gene/protein expression studies demonstrate upregulation of Ephrin Receptor B4 and plasminogen as downstream targets potentially mediating the prohealing and proangiogenic responses. Ex vivo BMDSC activation and the subsequent inoculation of cells into wounds does not increase systemic SDF-1α levels. CONCLUSIONS: We report a novel CBT that is highly effective in promoting healing and neovascularization in a murine model of type 2 diabetes. Furthermore, we identify new molecular targets that may be important for advancing the field of wound healing.

Inhibition of tumor angiogenesis and melanoma growth by targeting vascular E-selectin.

OBJECTIVES: Aggressive human melanomas express, C-X-C chemokine receptor 4 (CXCR4), the receptor for the chemokine, stromal cell-derived factor-1alpha (SDF-1α). The CXCR4-SDF-1α axis has been postulated to increase melanoma invasiveness. We discovered that SDF-1α specifically upregulates E-selectin on endothelial cells, thus tethering circulating endothelial progenitor cells (EPC) and facilitating homing. We investigated the hypothesis that small interfering ribonucleic acid (siRNA)-mediated E-selectin blockade inhibits melanoma angiogenesis and tumor growth. METHODS: Human melanoma cells overexpressing SDF-1α were xenografted on severe combined immunodeficiency (SCID) mice. SDF-1α expression in cells was measured by enzyme-linked immunosorbent assay (ELISA). In vitro melanoma cell growth was examined by cell proliferation assay. In vivo vascular E-selectin knockdown was achieved by administration of high-volume E-selectin siRNA (100 pmol/180 µL/week × 3 times) and inhibition was validated by immunostaining (N = 6/group, E-Selectin siRNA vs control siRNA). Tumor angiogenesis was quantified (DiI-perfusion and LASER confocal microscopy). EPC homing to tumor vasculature was detected by immunostaining. Explanted in vivo tumor size and weight were measured. RESULTS: Three melanoma cells tested expressed undetectable levels of SDF-1α. Additional enforced overexpression of SDF-1α (by Lenti-SDF-1α) increased melanoma cell growth both in vitro and in vivo, enhanced EPC homing to tumor tissue, and increased tumor angiogenesis. Knocking-down vascular E-selectin significantly inhibited SDF-1α-induced EPC homing, tumor angiogenesis, and decreased melanoma growth in vivo. CONCLUSIONS: Downregulation of vascular E-selectin profoundly inhibits EPC homing, tumor angiogenesis, and tumor growth in human melanoma xenograft murine model, potentially by suppression of E-selectin-mediated EPC-endothelial cells interactions/homing. These findings identify E-selectin as a novel target for inhibition of melanoma angiogenesis and tumor growth.

Identification of E-selectin as a novel target for the regulation of postnatal neovascularization: implications for diabetic wound healing.

OBJECTIVES: We previously reported that stromal cell-derived factor-1α (SDF-1α, a homing signal for recruiting endothelial progenitor cells (EPC) to areas of neovascularization), is down-regulated in diabetic wounds (Gallagher et al, J Clin Invest. 2007;117:1249-1259). We now investigate signals whereby mature endothelial cells (EC) and circulating EPC achieve SDF-1α-mediated EPC homing. METHODS: SDF-1α in diabetic wounds were therapeutically increased by injection of SDF-1α-engineered bone marrow-derived fibroblasts versus control cells (N = 48 [20, non-obese diabetic (NOD)], [28, streptozotocin-C57]). Polymerase chain reaction-array gene expression differences were validated by Western blotting and immunohistochemistry. The role of adhesion molecule(s) in mediating SDF-1α-induced EPC homing, and wound healing was furthered studied using antagonists in vitro and in vivo. RESULTS: Increasing wound SDF-1α via cell-based therapy promotes healing in diabetic mice (∼20% increase in healing rates by day 3, P = 0.006). SDF-1α increased EC-EPC adhesion and specifically upregulated E-selectin expression in human microvascular EC (2.3-fold increase, P < 0.01). This effect was also significant in blood vessels of the experimental mice and resulted in increased wound neovascularization. The regulatory effects of SDF-1α on EC-EPC adhesion and EPC homing were specifically mediated by E-selectin, as the application of E-selectin antagonists significantly inhibited SDF-1α-induced EC-EPC adhesion, EPC homing, wound neovascularization, and wound healing. CONCLUSIONS: SDF-1α-engineered cell-based therapy promotes diabetic wound healing in mice by specifically upregulating E-selectin expression in mature EC leading to increase EC-EPC adhesion, EPC homing, and increased wound neovascularization. These findings provide novel insight into the signals underlying the biological effect of SDF-1α on EPC homing and point to E-selectin as a new potential target for therapeutic manipulation of EPC trafficking in diabetic wound healing.

Nicotine augments glomerular injury in a rat model of acute nephritis.

BACKGROUND/AIMS: Epidemiologic studies suggest that cigarette smoke worsens the progression of renal injury in patients with glomerular diseases. The mechanisms involved have not been elucidated. These studies were designed to determine whether nicotine worsens markers of inflammation including glomerular cell proliferation and fibronectin deposition in an in vivo model of glomerular injury. METHODS: Sprague-Dawley rats were injected with anti-Thy1 antibody and given either tap water or nicotine in the drinking water until sacrifice at day 14. Fibronectin expression was measured by Western blot and immunohistochemistry. COX-2 expression was also determined by Western blot in the kidney cortex of rats treated with nicotine and in cultured human mesangial cells treated with nicotine. RESULTS: Anti-Thy1 antibody administration resulted in a significant increase in the number of cells per glomerulus that was further increased by the administration of nicotine. In nephritic rats, the administration of nicotine significantly increased fibronectin and COX-2 expression. In cultured human mesangial cells we also demonstrated that nicotine increases COX-2 expression and activity and that COX-2 mediates mesangial cell proliferation in response to nicotine. CONCLUSION: Either in vivo or in vitro treatment with nicotine leads to activation of inflammatory mediators and hallmarks of glomerular injury, which may explain the mechanisms involved in the deleterious effects of cigarette smoking on renal disease.

Angiotensin II increases the expression of the transcription factor ETS-1 in mesangial cells.

Maladaptive activation of the renin-angiotensin system (RAS) has been shown to play a critical role in the pathogenesis of chronic kidney disease. Reactive oxygen species (ROS) are critical signals for many of the nonhemodynamic effects of angiotensin II (ANG II). We have demonstrated that ANG II increases mesangial and cortical cyclooxygenase-2 (COX-2) expression and activity via NADPH oxidase-derived ROS. The transcription factor ETS-1 (E26 transformation-specific sequence) has been identified as a critical regulator of growth-related responses and inflammation. The present studies were designed to determine: 1) whether ANG II induces ETS-1 expression in vitro in cultured rat mesangial cells and in vivo in rats infused with ANG II; and 2) whether ROS and COX-2 are mediators of ETS-1 induction in response to ANG II. Mesangial cells stimulated with ANG II (10(-7) M) exhibited a significant increase in ETS-1 expression that was prevented by the angiotensin type 1 receptor blocker candesartan. NADPH oxidase inhibition with dyphenilene iodinium or apocynin also prevented ETS-1 induction, establishing the role of ROS as mediators of ETS-1 expression in response to ANG II. COX-2 inhibition prevented ETS-1 expression in response to ANG II, suggesting that COX-2 is required for ETS-1 induction. By utilizing short interfering RNAs against ETS-1, we have also determined that ETS-1 is required to induce the production of fibronectin in response to ANG II. Furthermore, rats infused with ANG II manifested increased glomerular expression of ETS-1. These studies unveil novel pathways that may play an important role in the pathogenesis of renal injury when RAS is activated.

Nicotine: the link between cigarette smoking and the progression of renal injury?

Cigarette smoke (CS) is the most important source of preventable morbidity and mortality in the United States. Recent clinical studies have suggested that, in addition to being a major cardiovascular risk factor, CS promotes the progression of kidney disease. The mechanisms by which CS promotes the progression of chronic kidney disease have not been elucidated. Here we demonstrate for the first time that human mesangial cells (MCs) are endowed with the nicotinic ACh receptors (nAChRs) alpha4, alpha5, alpha7, beta2, beta3, and beta4. Studies performed in other cell types have shown that these nAChRs are ionotropic receptors that function as agonist-regulated Ca(2+) channels. Nicotine induced MC proliferation in a dose-dependent manner. At 10 (-7) M, a concentration found in the plasma of active smokers, nicotine induced MC proliferation [control, 1,328 +/- 50 vs. nicotine, 2,761 +/- 90 counts/minute (cpm); P < 0.05] and increased the synthesis of fibronectin (50%), a critical matrix component involved in the progression of chronic kidney disease. We and others have shown that, in response to PKC activation, MC synthesize reactive oxygen species (ROS) via NADPH oxidase. In the current studies we demonstrate that PKC inhibition as well as diphenyleneiodonium and apocynin, two inhibitors of NADPH oxidase, prevented the effects of nicotine on MC proliferation and fibronectin production, hence establishing ROS as second messengers of the actions of nicotine. Furthermore, nicotine increased the production of ROS as assessed by 2',7'-dichlorofluorescein diacetate fluorescence [control, 184.4 +/- 26 vs. nicotine, 281.5 +/- 26 arbitrary fluorescence units (AFU); n = 5 experiments, P < 0.05]. These studies unveil previously unrecognized mechanisms that indict nicotine, a component of CS, as an agent that may accelerate and promote the progression of kidney disease.

Stable compounds of cigarette smoke induce endothelial superoxide anion production via NADPH oxidase activation.

OBJECTIVE: Endothelial dysfunction is an early manifestation of cigarette smoke (CS) toxicity. We have previously demonstrated that CS impairs nitric oxide (NO)-mediated endothelial function via increased generation of superoxide anion (O2*). In these studies, we investigated whether stable compounds present in CS activate specific pathways responsible for the increased endothelial O2* production. METHODS AND RESULTS: Short exposure of bovine pulmonary artery endothelial cells (BPAECs), human pulmonary artery endothelial cells, and rat pulmonary arteries to CS extracts (CSEs) resulted in a large increase in O2* production (20-fold, 3-fold, and 2-fold increase, respectively; P<0.05 versus control), which was inhibited by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenyleneiodinium, apocynin, and gp91 docking sequence-tat peptide but not by oxypurinol, the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester, or the mitochondrial respiration inhibitor rotenone. Exposure of BPAECs to acrolein, a stable thiol-reactive agent found in CS, increased O2* production 5-fold, which was prevented by prior inhibition of NADPH oxidase. CONCLUSIONS: These studies demonstrate that thiol-reactive stable compounds in CS can activate NADPH oxidase and increase endothelial O2* production, thereby reducing NO bioactivity and resulting in endothelial dysfunction. Clinically, these studies may contribute to the development of agents able to mitigate CS-mediated vascular toxicity.