Exposure to environmental chemicals adds to the burden of disease in humans and wildlife to a degree that is difficult to estimate and, thus, mitigate. The ability to assess the impact of existing ...chemicals for which little to no toxicity data are available or to foresee such effects during early stages of chemical development and use, and before potential exposure occurs, is a pressing need. However, the capacity of the current toxicity evaluation approaches to meet this demand is limited by low throughput and high costs. In the context of EPA’s ToxCast project, we have evaluated a novel cellular biosensor system (Factorial) that enables rapid, high-content assessment of a compound’s impact on gene regulatory networks. The Factorial biosensors combined libraries of cis- and trans-regulated transcription factor reporter constructs with a highly homogeneous method of detection enabling simultaneous evaluation of multiplexed transcription factor activities. Here, we demonstrate the application of the technology toward determining bioactivity profiles by quantitatively evaluating the effects of 309 environmental chemicals on 25 nuclear receptors and 48 transcription factor response elements. We demonstrate coherent transcription factor activity across nuclear receptors and their response elements and that Nrf2 activity, a marker of oxidative stress, is highly correlated to the overall promiscuity of a chemical. Additionally, as part of the ToxCast program, we identify molecular targets that associate with in vivo end points and represent modes of action that can serve as potential toxicity pathway biomarkers and inputs for predictive modeling of in vivo toxicity.
We developed a high-content reporter system that allows quantitative assessment of activities of multiple transcription factors (TFs) in a eukaryotic cell. The system comprises a library of reporter ...constructs that are evaluated according to their transcription rates. All reporters produce essentially identical messages that are subjected to 'processing', which generates a spectrum of distinguishable fragments that are analyzed quantitatively. The homogeneity of the reporter library afforded inherently uniform detection conditions for all reporters and provided repeatability, accuracy and robustness of assessment. We showed that this technology can be used to identify pathways transmitting cell responses to inducers, and that the profile of TF activities generated using this system represents a stable and sustained cell signature that clearly distinguishes different cell types and pathological conditions. This technology provides a framework for functional characterization of signal transduction networks through profiling activities of multiple TFs.
The Slit-Robo pathway is a key regulator of angiogenesis and cellular function in experimental models. Slit3 proteins exhibit both proangiogenic and antiangiogenic properties, but the exact mechanism ...remains unclear. It is theorized that Slit3 may be a potential treatment for vascular diseases and cancer.
Slit3 labeled with I-125 was encapsulated in microbeads composed of low-viscosity alginate of high-glucuronic acid content, first coated with poly-L-ornithine for various durations and finally with low-viscosity high mannuronic acid. Gamma counter was used to measure microbead encapsulation efficiency and Slit3 release. Markers of angiogenesis were assessed with Boyden chamber, scratch wound, and Matrigel tube formation assays using human umbilical vein and mouse endothelial cells.
On incubation of Slit3-loaded microbeads, there was an initial burst phase release of Slit3 for the first 24 h followed by sustained release for 6 to 12 d. Microbead composition determined encapsulation efficiency and rate of release; Slit3 encapsulation was most efficient in microbeads with lower low-viscosity alginate of high-glucuronic acid content concentrations (1.5%) and no poly-L-ornithine coating. Compared with controls (media alone), Slit3 microbeads significantly inhibited in vitro cellular migration, endothelial cell migration for wound closure at 24 and 48 h and endothelial tube formation (P < 0.001, respectively).
Slit3 can be effectively encapsulated and delivered via a controlled release pattern using alginate microbeads. Microbead encapsulation reduces in vitro endothelial tube formation and inhibits cellular migration to impair angiogenesis. Thus, Slit3 microparticles may be explored as a therapeutic option to mitigate tumor proliferation.
•Slit3 regulates angiogenesis and cell proliferation in many types of cancer.•Slit3 delivery via alginate microbeads inhibits cell migration and tube formation.•Alginate microbeads permit delivery of full length Slit3 protein.•Composition of microbead influences Slit3 encapsulation and release.•Sustained release Slit3 may have a therapeutic role in cancer and anti-angiogenesis.
Pulmonary hypertension is a highly morbid disease with no cure. Available treatments are limited by systemic adverse effects due to non‐specific biodistribution. Self‐assembled peptide amphiphile ...(PA) nanofibers are biocompatible nanomaterials that can be modified to recognize specific biological markers to provide targeted drug delivery and reduce off‐target toxicity. Here, PA nanofibers that target the angiotensin I‐converting enzyme and the receptor for advanced glycation end‐products (RAGE) are developed, as both proteins are overexpressed in the lung with pulmonary hypertension. It is demonstrated that intravenous delivery of RAGE‐targeted nanofibers containing the targeting epitope LVFFAED (LVFF) significantly accumulated within the lung in a chronic hypoxia‐induced pulmonary hypertension mouse model. Using 3D light sheet fluorescence microscopy, it is shown that LVFF nanofiber localization is specific to the diseased pulmonary tissue with immunofluorescence analysis demonstrating colocalization of the targeted nanofiber to RAGE in the hypoxic lung. Furthermore, biodistribution studies show that significantly more LVFF nanofibers localized to the lung compared to major off‐target organs. Targeted nanofibers are retained within the pulmonary tissue for 24 h after injection. Collectively, these data demonstrate the potential of a RAGE‐targeted nanomaterial as a drug delivery platform to treat pulmonary hypertension.
Peptide amphiphile nanofibers targeted to the receptor for advanced glycation end‐products (RAGE) significantly accumulate within diseased lungs of mice with hypoxia‐induced pulmonary hypertension following intravascular delivery. The targeted nanofibers remain localized to the pulmonary tissue for up to 24 h after injection. PA nanofibers may be effective delivery vehicles to provide targeted therapies for pulmonary hypertension.
The beta sub(3)-adrenergic receptor ( beta sub(3)AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of beta sub(3)AR ...activation is the substantial elevation in interleukin-6 (IL-6) levels. Although the beta sub(3)AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific beta sub(3)AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of beta sub(3)ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the G sub(s)-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-kappaB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block beta sub(3)AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the beta sub(3)AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the beta sub(3)AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.
The β
3-adrenergic receptor (β
3AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of β
3AR activation is the substantial ...elevation in interleukin-6 (IL-6) levels. Although the β
3AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific β
3AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of β
3ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated
via the G
s-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-κB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block β
3AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the β
3AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the β
3AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.
The beta(3)-adrenergic receptor (beta(3)AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of beta(3)AR activation is the ...substantial elevation in interleukin-6 (IL-6) levels. Although the beta(3)AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific beta(3)AR agonist CL316243 in adipocytes, cells that exhibit the highest expression of beta(3)ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the G(s)-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-kappaB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block beta(3)AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the beta(3)AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the beta(3)AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways.
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