Hitesh Peshavariya

Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase

All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 μM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 ± 6% and Nox4 siRNA 83 ± 7% of control) and lucigenin chemiluminescence (Nox2; 54 ± 6% and Nox4 74 ± 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.

Important Role of Nox4 Type NADPH Oxidase in Angiogenic Responses in Human Microvascular Endothelial Cells In Vitro

Objective—Redox signaling mediated by Nox2-containing NADPH oxidase has been implicated in angiogenic responses both in vitro and in vivo. Because Nox4 type NADPH oxidase is also highly expressed in endothelial cells, we studied the role of Nox4 in angiogenic responses in human endothelial cells in culture. Methods and Results—Inhibition of Nox4 expression by small interfering RNA reduced angiogenic responses as assessed by the tube formation and wound healing assays, in both human microvascular and umbilical vein endothelial cells. Overexpression of wild-type Nox4 enhanced, whereas expression of a dominant negative form of Nox4 suppressed the angiogenic responses in endothelial cells. These effects were mimicked by exogenous H2O2 and the antioxidant compound ebselen, respectively. Overexpression of Nox4 enhanced receptor tyrosine kinase phosphorylation and the activation of extracellular signal-regulated kinase (Erk). Inhibition of the Erk pathway reduced the endothelial angiogenic responses. Nox4 expression also promotes proliferation and migration of endothelial cells, and reduced serum deprivation–induced apoptosis. Conclusions—Nox4 type NADPH oxidase promotes endothelial angiogenic responses, at least partly, via enhanced activation of receptor tyrosine kinases and the downstream Erk pathway.

Regulation of cell proliferation by NADPH oxidase-mediated signaling: Potential roles in tissue repair, regenerative medicine and tissue engineering

The superoxide generating enzyme NADPH oxidase has received much attention as a major cause of oxidative stress underlying vascular disease. However, there is increasing evidence that oxidant signaling involving NADPH oxidase has other important roles in cell biology. Nox family proteins are the catalytic, electron-transporting subunits of the NADPH oxidase enzyme complex. It is now clear that reactive oxygen species (ROS) generated by NADPH oxidase participate in intracellular signaling processes that regulate cell differentiation and proliferation. These mechanisms are important in tissue repair and tumorigenesis, diverse conditions where cell proliferation is required, but when poorly controlled the generation of ROS is obviously detrimental. Indeed, NADPH oxidase-mediated cell proliferation has been observed in a wide range of cell types including those found in blood vessels, kidney, liver, skeletal muscle precursors, neonatal cardiac myocytes, lung epithelial cells, gastric mucosa, brain microglia, and a variety of cancer cells. NADPH oxidases act not as isolated elements downstream of a particular pathway, but rather may amplify multiple receptor tyrosine kinase-mediated processes by inhibiting protein tyrosine phosphatases. Therefore, NADPH oxidase-mediated redox signaling may represent a unique intracellular amplifier of diverse signaling pathways involved in tissue repair processes such as cell proliferation, wound healing, angiogenesis and fibrosis. Recent studies also suggest that NADPH oxidase is involved in differentiation of stem cells. As occurs in unresolved inflammation, however, hyperactivity of this enzyme system leads to tissue injury. Thus modulating NADPH oxidase may have significant impacts on regenerative medicine and tissue engineering, such as growing heart muscle.

NADPH oxidase isoform selective regulation of endothelial cell proliferation and survival.

Proliferation and apoptosis of endothelial cells are crucial angiogenic processes that contribute to carcinogenesis and tumor progression. Emerging evidence implicates the regulation of proliferation and apoptosis by reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2). In the present study, we investigated the roles of the ROS-generating Nox4- and Nox2-containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in proliferation of human endothelial cells by examining the impact of these enzyme systems on (1) specific proliferative and tumorigenic kinases, extracellular regulated kinase1/2 (ERK1/2) and Akt, (2) cytoskeletal organization, and (3) the mechanisms that influence cellular apoptosis. ROS production and the expression of NADPH oxidase subunit Nox4, but not Nox2, were markedly higher in proliferating than in quiescent endothelial cells. Addition of the H2O2 scavenger catalase or downregulation of Nox4 protein with specific siRNA reduced ROS levels, cell proliferation, and ERK1/2 phosphorylation but had no effect on either cell morphology or caspase 3/7 activity. Although downregulation of Nox2 protein with siRNA also reduced ROS production and cell proliferation, it caused an increase in caspase 3/7 activity, reduced Akt phosphorylation, and caused cytoskeletal disorganization. Therefore, in endothelial cells, Nox4-derived H2O2 activates ERK1/2 to promote proliferation, whereas Nox2-containing NADPH oxidase maintains the cytoskeleton and prevents apoptosis to support cell survival. Our study provides a new understanding of the molecular mechanisms that underpin endothelial cell survival and a rationale for the combined suppression of Nox4- and Nox2-containing NADPH oxidases for unwanted angiogenesis in cancer.

Differentiation of Human Adipose-Derived Stem Cells into Fat Involves Reactive Oxygen Species and Forkhead Box O1 Mediated Upregulation of Antioxidant Enzymes

Both reactive oxygen species (ROS) and Forkhead box O (FOXO) family transcription factors are involved in the regulation of adipogenic differentiation of preadipocytes and stem cells. While FOXO has a pivotal role in maintaining cellular redox homeostasis, the interactions between ROS and FOXO during adipogenesis are not clear. Here we examined how ROS and FOXO regulate adipogenesis in human adipose-derived stem cells (hASC). The identity of isolated cells was confirmed by their surface marker expression pattern typical for human mesenchymal stem cells (positive for CD29, CD44, CD73, CD90, and CD105, negative for CD45 and CD31). Using a standard adipogenic cocktail consisting of insulin, dexamethasone, indomethacin, and 3-Isobutyl-1-methylanxthine (IDII), adipogenesis was induced in hASC, which was accompanied by ROS generation. Scavenging ROS production with N-acetyl-L-cysteine or EUK-8, a catalytic mimetic of superoxide dismutase (SOD) and catalase, inhibited IDII-induced adipogenesis. We then mimicked IDII-induced oxidative stress through a lentiviral overexpression of Nox4 and an exogenous application of hydrogen peroxide in hASC and both manipulations significantly enhanced adipogenesis without changing the adipogenic differentiation rate. These data suggest that ROS promoted lipid accumulation in hASC undergoing adipogenesis. Antioxidant enzymes, including SOD2, catalase, and glutathione peroxidase were upregulated by IDII during adipogenesis, and these effects were blunted by FOXO1 silencing, which also suppressed significantly IDII-induced adipogenesis. Our findings demonstrated a balance of ROS generation and endogenous antioxidants in cells undergoing adipogenesis. Approaches targeting ROS and/or FOXO1 in adipocytes may bring new strategies to prevent and treat obesity and metabolic syndrome.

Hypoxic Preconditioning Enhances Survival of Human Adipose-Derived Stem Cells and Conditions Endothelial Cells In Vitro

To grow more robust cardiac tissue for implantation in vivo, strategies to improve survival of implanted stem cells are required. Here we report the protective effects of hypoxic preconditioning (HPC) and identify mechanisms for improving survival of adipose-derived stem cells (ASC) in vitro. Human ASC were preconditioned for 24 h with hypoxia and then exposed to simulated ischemia for a further 24 h. HPC significantly increased ASC viability, and reduced cell injury and apoptosis compared with non-preconditioned cells under ischemic conditions, as shown by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase-release, and caspase activity assays. Preconditioned ASC increased levels of hypoxia-inducible factor-1 alpha and secreted significantly more of the downstream target vascular endothelial growth factor (VEGF-A; 13-fold) compared with control during the 24 h. Exogenous VEGF (50 ng/mL) increased phosphorylation of Akt without affecting ERK1/2, JNK, or p38 MAPK protein levels. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned ASC, an effect that may be mediated via VEGF-A. Importantly, the protective effects of HPC were abolished by a neutralizing antibody against VEGF-A and the phosphoinositol 3-kinase inhibitor LY294002, demonstrating the importance of VEGF-A and Akt in hypoxia-induced ASC survival. Importantly, we showed that media derived from hypoxic preconditioned ASC support endothelial cell survival and endothelial tube formation in vitro. Our in vitro findings indicate that HPC may be a promising strategy to improve survival of ASC and promote angiogenesis in ischemic environments.

Hypoxic Conditioning Enhances the Angiogenic Paracrine Activity of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (ASCs) secrete cytokines and growth factors that can be harnessed in a paracrine fashion for promotion of angiogenesis, cell survival, and activation of endogenous stem cells. We recently showed that hypoxia is a powerful stimulus for an angiogenic activity from ASCs in vitro and here we investigate the biological significance of this paracrine activity in an in vivo angiogenesis model. A single in vitro exposure of ASCs to severe hypoxia (<0.1% O2) significantly increased both the transcriptional and translational level of the vascular endothelial growth factor-A (VEGF-A) and angiogenin (ANG). The angiogenicity of the ASC-conditioned medium (ASC(CM)) was assessed by implanting ASC(CM)-treated polyvinyl alcohol sponges subcutaneously for 2 weeks in mice. The morphometric analysis of anti-CD31-immunolabeled sponge sections demonstrated an increased angiogenesis with hypoxic ASC(CM) treatment compared to normoxic control ASC(CM) treatment (percentage vascular volume; 6.0%±0.5% in the hypoxic ASC(CM) vs. 4.1%±0.7% in the normoxic ASC(CM), P<0.05). Reduction of VEGF-A and ANG levels in the ASC(CM) with respective neutralizing antibodies before sponge implantation showed a significantly diminished angiogenic response (3.5%±0.5% in anti-VEGF-A treated, 3.2%±0.7% in anti-ANG treated, and 3.5%±0.6% in anti-VEGF-A/ANG treated). Further, both the normoxic and hypoxic ASC(CM) were able to sustain in vivo lymphangiogenesis in sponges. Collectively, the model demonstrated that the increased paracrine production of the VEGF-A and ANG in hypoxic-conditioned ASCs in vitro translated to an in vivo effect with a favorable biological significance. These results further illustrate the potential for utilization of an in vitro optimized ASC(CM) for in vivo angiogenesis-related applications as an effective cell-free technology.

Nox4 modulates collagen production stimulated by transforming growth factor β1 in vivo and in vitro.

The synthesis of extracellular matrix including collagen during wound healing responses involves signaling via reactive oxygen species (ROS). We hypothesized that NADPH oxidase isoform Nox4 facilitates the stimulatory effects of the profibrotic cytokine transforming growth factor (TGF) β(1) on collagen production in vitro and in vivo. TGFβ(1) stimulated collagen synthesis and hydrogen peroxide generation in mouse cardiac fibroblasts, and both responses were attenuated by a scavenger of superoxide and hydrogen peroxide (EUK-134). Furthermore, by expressing a dominant negative form of Nox4 (Adv-Nox4(ΔNADPH)) in fibroblasts, TGFβ(1)-induced hydrogen peroxide production and collagen production were abrogated, suggesting that Nox4-dependent ROS are important for TGFβ(1) signaling in collagen production. This was confirmed by the inhibitory effect of an adenovirus carrying siRNA targeting Nox4 (Adv-Nox4i) on TGFβ(1)-induced collagen synthesis and expression of activated myofibroblasts marker smooth muscle alpha actin. Finally we used a mouse model of subcutaneous sponge implant to examine the role of Nox4 in the local stimulatory effects of TGFβ(1) on collagen accumulation in vivo. TGFβ(1)-induced collagen accumulation was significantly reduced when the sponges were instilled with Adv-Nox4(ΔNADPH). In conclusion, Nox4 acts as an intermediary in the signaling of TGFβ(1) to facilitate collagen synthesis.

Transforming growth factor-β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo.

Angiogenesis, the formation of new blood vessels, is a key physiological event in organ development and tissue responses to hypoxia but is also involved in pathophysiologies such as tumour growth and retinopathies. Understanding the molecular mechanisms involved is important to design strategies for therapeutic intervention. One important regulator of angiogenesis is transforming growth factor‐β1 (TGF‐β1). In addition, reactive oxygen species (ROS) and the ROS‐forming NADPH oxidase type 4 (Nox4) have been implicated as additional regulators such as during hypoxia. Here, we show that both processes are indeed mechanistically linked. TGF‐β1‐stimulated Nox4 expression and ROS formation in endothelial cells. In cells from Nox4‐deficient mice, TGF‐β1‐induced cell proliferation, migration and tube formation were abolished. In vivo, TGF‐β1 stimulated growth of blood vessels into sponges implanted subcutaneously, and this angiogenesis was markedly reduced in Nox4 knockout mice. Thus, endothelial cells are regulated by a TGF‐β1 signalling pathway involving Nox4‐derived ROS to promote angiogenesis. In order to abrogate pathological angiogenesis triggered by a multitude of factors, such as TGF‐β1 and hypoxia, Nox4 may thus be an ideal therapeutic target.

Prostacyclin receptor suppresses cardiac fibrosis: Role of CREB phosphorylation

Cardiac fibrosis is a consequence of many cardiovascular diseases and contributes to impaired ventricular function. Activation of the prostacyclin receptor (IP) protects against cardiac fibrosis, but the molecular mechanisms are not totally understood. Using mouse cardiac fibroblasts, we found that IP activation with cicaprost suppressed expression of collagen I and other target genes of transforming growth factor-beta. This effect of cicaprost was unlikely to be mediated by inhibition of the Smad2/3 or mitogen-activated protein kinase (MAPK) activities, but was associated with cAMP elevation and phosphorylation of the transcription factor cAMP response element binding protein (CREB). Expression of a non-phosphorylated CREB mutant suppressed the inhibitory effect of cicaprost. It appears that phosphorylated CREB binds to and sequestrates the transcription coactivator CBP/p300 from binding to Smad. Inhibition of the intrinsic histone acetyl-transferase activity of CBP/p300 with garcinol significantly suppressed collagen I expression in fibroblasts. Using apolipoprotein E and IP double knockout mouse, we demonstrated that endogenous prostacyclin/IP signaling had an inhibitory effect on angiotensin II-induced cardiac fibrosis under hypercholesterolemic conditions. Taken together, our results suggest that the prostacyclin/IP pathway suppresses cardiac fibrosis, at least partly, by inducing CREB phosphorylation.