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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 (HXA(3) and HXB(3)). 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 HXB(3) 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, HXA(3), or HXB(3) evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA(3) produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA(3) correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA(3) 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 HXA(3)-evoked allodynia. These data indicate that spinal HXA(3) is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

We discuss and present new data regarding the physiological and molecular mechanisms of nuclear receptor activation in pain control, with a particular emphasis on non-genomic effects of ligands at peroxisome proliferator-activated receptor (PPAR), GPR30, and classical estrogen receptors. PPARalpha agonists rapidly reduce both acute and chronic pain in a number of pain assays. These effects precede transcriptional anti-inflammatory actions, and are mediated in part by IK(ca) and BK(ca) channels on DRG neurons. In contrast to the peripheral site of action of PPARalpha ligands, the dorsal horn supports the expression of PPARgamma. Intrathecal administration of PPARgamma ligands rapidly (< or =5 min) attenuated mechanical and thermal hypersensitivity associated with nerve injury in a dose-dependent manner that could be blocked with PPARgamma antagonists. By contrast, a PPARgamma antagonist itself rapidly increased the mechanical allodynia associated with nerve injury. These data suggest that ligand-dependent, non-genomic activation of spinal PPARgamma decreases behavioral signs of inflammatory and neuropathic pain. We also report that the GPR30 is expressed on cultured sensory neurons, that activation of the receptor elicits signaling to increase calcium accumulation. This signaling may contribute to increased neuronal sensitivity as treatment with the GPR30 agonist induces hyperalgesia. Finally, application of the membrane-impermeable 17beta-E(2)-BSA rapidly (within 15 min) enhanced BK-stimulated inositol phosphate (IP) accumulation and PGE(2)-mediated cAMP accumulation in trigeminal ganglion cultures. We conclude that nuclear receptor ligands may operate through rapid, non-genomic mechanisms to modulate inflammatory and neuropathic pain.

Mesenchymal stem cells (MSCs) can be used in adult stem cell-based gene therapy for vascular diseases. To test the hypothesis that MSCs alone or endothelial nitric oxide synthase (eNOS)-modified MSCs can be used for treatment of erectile dysfunction (ED), syngeneic rat MSCs (rMSCs) were isolated, ex vivo expanded, transduced with adenovirus containing eNOS, and injected into the penis of aged rats. Histological analysis demonstrated that rMSCs survived for at least 21 days in corporal tissue after intracavernous injection, and an inflammatory response was not induced. Intracavernous administration of eNOS-modified rMSCs improved the erectile response in aged rats at 7 and 21 days after injection. The increase in erectile function was associated with increased eNOS protein, NOS activity, and cGMP levels, rMSCs alone increased erectile function of aged rats at day 21, but not at day 7, with the transplanted cells exhibiting positive immunostaining for several endothelial and smooth muscle cell markers. This change in rMSC phenotype was accompanied by upregulation of penile eNOS protein expression/activity and elevated cGMP levels. These findings demonstrate that an adenovirus can be used to transduce ex vivo expanded rMSCs to express eNOS and that eNOS-modified rMSCs improve erectile function in the aged rat. Intracavernous injection of unmodified wildtype rMSCs improved erectile function 21 days after injection through mechanisms involving improved endothelium-derived NO/cGMP signaling and rMSC differentiation into penile cells expressing endothelial and smooth muscle markers. These data highlight the potential clinical use of adult stem cell-based therapy for the treatment of ED. endothelium; gene therapy; cGMP; phosphodiesterase type 5; neuronal nitric oxide synthase

The administration of mesenchymal stem cells (MSCs) has been proposed for the treatment of pulmonary hypertension. However, the effect of intratracheally administered MSCs on the pulmonary vascular bed in monocrotaline-treated rats has not been determined. In the present study, the effect of intratracheal administration of rat MSCs (rMSCs) on monocrotaline-induced pulmonary hypertension and impaired endothelium-dependent responses were investigated in the rat. Intravenous injection of monocrotaline increased pulmonary arterial pressure and vascular resistance and decreased pulmonary vascular responses to acetylcholine without altering responses to sodium nitroprusside and without altering systemic responses to the vasodilator agents when responses were evaluated at 5 wk. The intratracheal injection of 3 x [10.sup.6] rMSCs 2 wk after administration of monocrotaline attenuated the rise in pulmonary arterial pressure and pulmonary vascular resistance and restored pulmonary responses to acetylcholine toward values measured in control rats. Treatment with rMSCs decreased the right ventricular hypertrophy induced by monocrotaline. Immunohistochemical studies showed widespread distribution of lacZ-labeled rMSCs in lung parenchyma surrounding airways in monocrotaline-treated rats. Immunofluorescence studies revealed that transplanted rMSCs retained expression of von Willebrand factor and smooth muscle actin markers specific for endothelial and smooth muscle phenotypes. However, immunolabeled cells were not detected in the wall of pulmonary vessels. These data suggest that the decrease in pulmonary vascular resistance and improvement in response to acetylcholine an endothelium-dependent vasodilator in monocrotaline-treated rats may result from a paracrine effect of the transplanted rMSCs in lung parenchyma, which improves vascular endothelial function in the monocrotaline-injured lung. marrow stromal cells