Bradley K. Taylor

Epidermal growth factor receptor inhibition attenuates liver fibrosis and development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most rapidly increasing cause of cancer‐related mortality in the United States. Because of the lack of viable treatment options for HCC, prevention in high‐risk patients has been proposed as an alternative strategy. The main risk factor for HCC is cirrhosis and several lines of evidence implicate epidermal growth factor (EGF) in the progression of cirrhosis and development of HCC. We therefore examined the effects of the EGF receptor (EGFR) inhibitor erlotinib on liver fibrogenesis and hepatocellular transformation in three different animal models of progressive cirrhosis: a rat model induced by repeated, low‐dose injections of diethylnitrosamine (DEN), a mouse model induced by carbon tetrachloride (CCl4), and a rat model induced by bile duct ligation (BDL). Erlotinib reduced EGFR phosphorylation in hepatic stellate cells (HSC) and reduced the total number of activated HSC. Erlotinib also decreased hepatocyte proliferation and liver injury. Consistent with all these findings, pharmacological inhibition of EGFR signaling effectively prevented the progression of cirrhosis and regressed fibrosis in some animals. Moreover, by alleviating the underlying liver disease, erlotinib blocked the development of HCC and its therapeutic efficacy could be monitored with a previously reported gene expression signature predictive of HCC risk in human cirrhosis patients. Conclusion: These data suggest that EGFR inhibition using Food and Drug Administration‐approved inhibitors provides a promising therapeutic approach for reduction of fibrogenesis and prevention of HCC in high‐risk cirrhosis patients who can be identified and monitored by gene expression signatures. (Hepatology 2014;59:1577‐1590)

Stimulation of dopamine D2 receptors in the nucleus accumbens inhibits inflammatory pain.

Previous studies suggest that dopamine in the nucleus accumbens links noxious or mesolimbic stimulation with the feedback inhibition of nociception. To test the hypothesis that pharmacological agonism at dopamine receptors in the nucleus accumbens elicits antinociception, we bilaterally microinjected dopamine D1- and D2-receptor subtype selective drugs, and then evaluated behavioral responses to noxious intraplantar formalin. While the D1-selective agonist SKF 38393 was without effect at a dose of 0.5 nmol/side, the D2-selective agonist quinpirole dose-dependently (0.05-5.0 nmol/side, bilateral) inhibited the persistent phase of formalin-induced nociception. This was blocked by pre-administration of a selective D2-dopaminergic antagonist raclopride (0.3 nmol/side, bilateral). Quinpirole did not produce overt behavioral effects and did not change rotarod latency. Our results indicate that quinpirole acts at dopamine D2 receptors in the nucleus accumbens to inhibit persistent nociception at doses that circumvent confounding non-specific motor deficits, namely, sedation and motor coordination.

Prognostic Gene Expression Signature for Patients With Hepatitis C–Related Early-Stage Cirrhosis

BACKGROUND & AIMS Cirrhosis affects 1% to 2% of the world population and is the major risk factor for hepatocellular carcinoma (HCC). Hepatitis C cirrhosis-related HCC is the most rapidly increasing cause of cancer death in the United States. Noninvasive methods have been developed to identify patients with asymptomatic early-stage cirrhosis, increasing the burden of HCC surveillance, but biomarkers are needed to identify patients with cirrhosis who are most in need of surveillance. We investigated whether a liver-derived 186-gene signature previously associated with outcomes of patients with HCC is prognostic for patients with newly diagnosed cirrhosis but without HCC. METHODS We performed gene expression profile analysis of formalin-fixed needle biopsy specimens from the livers of 216 patients with hepatitis C-related early-stage (Child-Pugh class A) cirrhosis who were prospectively followed up for a median of 10 years at an Italian center. We evaluated whether the 186-gene signature was associated with death, progression of cirrhosis, and development of HCC. RESULTS Fifty-five (25%), 101 (47%), and 60 (28%) patients were classified as having poor-, intermediate-, and good-prognosis signatures, respectively. In multivariable Cox regression modeling, the poor-prognosis signature was significantly associated with death (P = .004), progression to advanced cirrhosis (P < .001), and development of HCC (P = .009). The 10-year rates of survival were 63%, 74%, and 85% and the annual incidence of HCC was 5.8%, 2.2%, and 1.5% for patients with poor-, intermediate-, and good-prognosis signatures, respectively. CONCLUSIONS A 186-gene signature used to predict outcomes of patients with HCC is also associated with outcomes of patients with hepatitis C-related early-stage cirrhosis. This signature might be used to identify patients with cirrhosis in most need of surveillance and strategies to prevent the development of HCC.

Constitutive μ-Opioid Receptor Activity Leads to Long-Term Endogenous Analgesia and Dependence

Pain and Dependence The properties and functions of µ-opioid receptors have been studied intensively with respect to the binding of endogenous or exogenous ligands. However, much less is known about the constitutive, ligand-independent, activation of opioid receptors. Working in mice, Corder et al. (p. 1394) observed the prolonged constitutive activation of µ-opioid receptors in the spinal dorsal horn after transient peripheral inflammation. The results suggest that constitutive activation of µ-opioid receptors depresses nociception—the perception of pain—for long periods of time and induces cellular and physical dependence on endogenous opioid signaling. Transient inflammation can lead to prolonged activation of pain-relieving opioid receptors in the spinal cord. Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced μ-opioid receptor (MOR) constitutive activity (MORCA) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3′,5′-monophosphate overshoot and hyperalgesia) that required N-methyl-d-aspartate receptor activation of adenylyl cyclase type 1. Thus, MORCA initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MORCA suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA3 and HXB3). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB3 at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA3, or HXB3 evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA3 produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA3 correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA3 triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA3-evoked allodynia. These data indicate that spinal HXA3 is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

Noradrenergic neurons in the locus coeruleus contribute to neuropathic pain

Current theories of neuropathic hypersensitivity include an imbalance of supraspinal inhibition and facilitation. Our overall hypothesis is that the locus coeruleus (LC), classically interpreted as a source of pain inhibition, may paradoxically result in facilitation after tibial and common peroneal nerve transection (spared sural nerve injury--SNI). We first tested the hypothesis that non-noxious tactile hind paw stimulation of the spared sural innervation territory increases neuronal activity in the LC in male rats. We observed a bilateral increase in the stimulus-evoked expression of transcription factors Fos and phosphorylated CREB (pCREB) in LC after SNI but not sham surgery; these markers of neuronal activity correlated with the intensity of tactile allodynia. We next tested the hypothesis that noradrenergic neurons contribute to the development of neuropathic pain. To selectively destroy these neurons, we delivered antidopamine-beta-hydroxylase saporin (anti-DbetaH-saporin) into the i.c.v. space 2 weeks before SNI. We found that anti-DbetaH-saporin, but not an IgG-saporin control, reduced behavioral signs of tactile allodynia, mechanical hyperalgesia, and cold allodynia from 3 to 28 days. after SNI. Our final experiment tested the hypothesis that the LC contributes to the maintenance of neuropathic pain. We performed SNI, waited 2 weeks for maximal allodynia and hyperalgesia to develop, and then administered the local anesthetic lidocaine (4%) directly into the LC parenchyma. Lidocaine reduced all behavioral signs of neuropathic pain in a reversible manner, suggesting that the LC contributes to pain facilitation. We conclude that, in addition to its well-known inhibition of acute and inflammatory pain, the LC facilitates the development and maintenance of neuropathic pain in the SNI model. Further studies are needed to determine the facilitatory pathways emanating from the LC.

Antihyperalgesic effects of intrathecal neuropeptide Y during inflammation are mediated by Y1 receptors

&NA; Inflammation induces an up‐regulation of neuropeptide tyrosine (NPY) and its receptors in the dorsal horn, suggesting an important role in nociceptive transmission. Our initial studies revealed that NPY dose‐dependently increased hotplate response latency, and to a lesser degree, thermal paw withdrawal latency (PWL); these effects occurred at doses that affect neither motor coordination (as assessed by the rotarod test) nor paw skin temperature. We next evaluated the behavioral effects of intrathecal administration of NPY and NPY antagonists with the aim of assessing the contribution of NPY to correlates of persistent nociception associated with the unilateral plantar injection of carrageenan or complete Freunds adjuvant (CFA). NPY robustly and dose‐dependently increased PWL on the side ipsilateral to carrageenan injection, with only a small effect on the contralateral side. Similarly, NPY (30 &mgr;g) produced a large and long‐lasting increase in PWL on the side ipsilateral to CFA injection (140% change), with only a small effect on the contralateral side (25% change). The ipsilateral effect of NPY was completely inhibited with the potent Y1 antagonist, BIBO 3304 (3 &mgr;g), but not the Y2 antagonist, BIIE 0246. When administered alone, BIBO 3304 (but not BIIE 0246) slightly decreased thermal PWL on the side ipsilateral (25% change), but not contralateral, to CFA injection; this suggests that inflammation strengthens inhibitory NPY tone. We conclude that spinal Y1 receptors contribute to the inhibitory effects of NPY on thermal hypersensitivity in the awake rat. Further studies are necessary to determine whether enhanced release of NPY and Y1‐mediated inhibition of spinal nociceptive transmission ultimately results in a compensatory, adaptive inhibition of thermal hypersensitivity in the setting of inflammation.

Intrathecal Rosiglitazone Acts at Peroxisome Proliferator–Activated Receptor-γ to Rapidly Inhibit Neuropathic Pain in Rats

UNLABELLED In this report, we demonstrate the transcription, expression, and DNA-binding properties of the peroxisome proliferator-activated receptor (PPAR)-gamma subtype of the peroxisome proliferator-activated nuclear receptor family to the spinal cord with real-time PCR, Western blot, and electrophoretic mobility shift assay. To test the hypothesis that activation of spinal PPAR-gamma decreases nerve injury-induced allodynia, we intrathecally administered PPAR-gamma agonists and/or antagonists in rats after transection of the tibial and common peroneal branches of the sciatic nerve. Single injection of either a natural (15-deoxy-prostaglandin J2, 15d-PGJ2) or synthetic (rosiglitazone) PPAR-gamma agonist dose-dependently decreased mechanical and cold hypersensitivity. These effects were maximal at a dose of 100 microg and peaked at approximately 60 minutes after injection, a rapid time course suggestive of transcription-independent mechanisms of action. Concurrent administration of a PPAR-gamma antagonist (bisphenol A diglycidyl ether, BADGE) reversed the effects of 15d-PGJ2 and rosiglitazone, further indicating a receptor-mediated effect. In animals without nerve injury, rosiglitazone did not alter motor coordination, von Frey threshold, or withdrawal response to a cool stimulus. Intraperitoneal and intracerebroventricular administration of PPAR-gamma agonists (100 microg) did not decrease mechanical and cold hypersensitivity, arguing against effects subsequent to diffusion from the intrathecal space. We conclude that ligand-induced activation of spinal PPAR-gamma rapidly reverses nerve injury-induced mechanical allodynia. New or currently available drugs targeted at spinal PPAR-gamma may yield important therapeutic effects for the management of neuropathic pain. PERSPECTIVE PPAR-gamma receptor agonists such as rosiglitazone and pioglitazone are approved as insulin sensitizers by the United States Food and Drug Administration. We demonstrate PPAR-gamma expression in the spinal cord and report that activation of these receptors inhibits allodynia. BBB-permeant PPAR-gamma agonists may yield important therapeutic effects for the management of neuropathic pain.

Spinal mechanisms of NPY analgesia

We review previously published data, and present some new data, indicating that spinal application of neuropeptide Y (NPY) reduces behavioral and neurophysiological signs of acute and chronic pain. In models of acute pain, early behavioral studies showed that spinal (intrathecal) administration of NPY and Y2 receptor agonists decrease thermal nociception. Subsequent neurophysiological studies indicated that Y2-mediated inhibition of excitatory neurotransmitter release from primary afferent terminals in the substantia gelatinosa may contribute to the antinociceptive actions of NPY. As with acute pain, NPY reduced behavioral signs of inflammatory pain such as mechanical allodynia and thermal hyperalgesia; however, receptor antagonist studies indicate an important contribution of spinal Y1 rather than Y2 receptors. Interestingly, Y1 agonists suppress inhibitory synaptic events in dorsal horn neurons (indeed, well known mu-opioid analgesic drugs produce similar cellular actions). To resolve the behavioral and neurophysiological data, we propose that NPY/Y1 inhibits the spinal release of inhibitory neurotransmitters (GABA and glycine) onto inhibitory neurons, e.g. disinhibition of pain inhibition, resulting in hyporeflexia. The above mechanisms of Y1- and Y2-mediated analgesia may also operate in the setting of peripheral nerve injury, and new data indicate that NPY dose-dependently inhibits behavioral signs of neuropathic pain. Indeed, neurophysiological studies indicate that Y2-mediated inhibition of Ca(2+) channel currents in dorsal root ganglion neurons is actually increased after axotomy. We conclude that spinal delivery of Y1 agonists may be of use in the treatment of chronic inflammatory pain, and that the use of Y1 and Y2 agonists in neuropathic pain warrants further consideration.

Toward performance-diverse small-molecule libraries for cell-based phenotypic screening using multiplexed high-dimensional profiling

Significance A large compound screening collection is usually constructed to be tested in many distinct assays, each one designed to find modulators of a different biological process. However, it is generally not known to what extent a compound collection actually contains molecules with distinct biological effects (or even any effect) until it has been tested for a couple of years. This study explores a cost-effective way of rapidly assessing the biological performance diversity of a screening collection in a single assay. By simultaneously measuring a large number of cellular features, unbiased profiling assays can distinguish compound effects with high resolution and thus measure performance diversity. We show that this approach could be used as a filtering strategy to build effective screening collections. High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cell-based and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance.