In vivo delivery and long-term tissue retention of nano-encapsulated sirolimus using a novel porous balloon angioplasty system.

AIMS: Among antirestenotic compounds, sirolimus displays a superior safety profile compared to paclitaxel, but its pharmacokinetic properties make it a challenging therapeutic candidate for single-time delivery. Herein we evaluate the feasibility of delivery, long-term retention and vascular effects of sirolimus nanoparticles delivered through a novel porous angioplasty balloon in normal porcine arteries and in a swine model of in-stent restenosis (ISR). METHODS AND RESULTS: Sirolimus nanoparticle formulation was delivered via porous balloon angioplasty to 753 coronary artery segments for pharmacokinetic studies and 26 segments for biological effect of sirolimus delivery in different clinical scenarios (de novo [n=8], ISR [n=6] and following stent implantation [n=12]). Sirolimus coronary artery concentrations were above the target therapeutic level of 1 ng/mg after 26 days, and were >100-fold higher in coronary artery treatment sites than in distal myocardium and remote tissues at all time points. At 28 days, reduction in percent stenosis in formulation-treated sites compared to balloon angioplasty treatment was noted in all three clinical scenarios, with the largest effect seen in the de novo study. CONCLUSIONS: Local coronary delivery of sirolimus nanoparticles in the porcine model using a novel porous balloon delivery system achieved therapeutic long-term intra-arterial drug levels without significant systemic residual exposure.

Dynamic changes in the microRNA expression profile reveal multiple regulatory mechanisms in the spinal nerve ligation model of neuropathic pain.

Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 3'-UTR of voltage-gated sodium channel Scn11a, alpha 2/delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain.

Monoacylglycerol lipase activity is a critical modulator of the tone and integrity of the endocannabinoid system.

Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.

Depression-like phenotype following chronic CB1 receptor antagonism.

Rimonabant was the first clinically marketed cannabinoid (CB)(1) receptor antagonist developed to treat obesity. Unfortunately, CB(1) receptor antagonism produced adverse psychiatric events in patients. To determine whether this occurs pre-clinically, we investigated the effects of rimonabant in rodent models of mood disorders. Chronic treatment with rimonabant increased immobility time in the rat forced swim test and reduced the consumption of sucrose-sweetened water in an assay postulated to model anhedonia. These responses were similar to the effects elicited by chronic mild stress in these behavioral models, which, taken together, are indicative of a depression-like phenotype. Additionally, chronic treatment with rimonabant produced decreases in frontal cortex serotonin levels, marked reductions in hippocampal cell proliferation, survival, and BDNF levels, and elevations in the concentrations of pro-inflammatory cytokines including interferon gamma and TNF alpha. These preclinical findings mimic clinical reports and implicate possible mechanisms responsible for the unfavorable psychiatric events reported following chronic rimonabant use.

Genetic and functional analysis of human P2X5 reveals a distinct pattern of exon 10 polymorphism with predominant expression of the nonfunctional receptor isoform.

P2X5 is a member of the P2X family of ATP-gated nonselective cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymorphism (SNP) at the 3' splice site of exon 10 of the human P2X5 gene. As reported previously, presence of a T at the SNP location results in inclusion of exon 10 in the mature transcript, whereas exon 10 is excluded when a G is present at this location. Our genotyping of human DNA samples reveals predominance of the G-bearing allele, which was exclusively present in DNA samples from white American, Middle Eastern, and Chinese donors. Samples from African American donors were polymorphic, with the G allele more frequent. Reverse transcription-polymerase chain reaction analysis of lymphocytes demonstrated a 100% positive correlation between genotype and P2X5 transcript. Immunostaining of P2X1/P2X5 stably coexpressing cell lines showed full-length P2X5 to be expressed at the cell surface and the exon 10-deleted isoform to be cytoplasmic. Fluorometric imaging-based pharmacological characterization indicated a ligand-dependent increase in intracellular calcium in 1321N1 astrocytoma cells transiently expressing full-length P2X5 but not the exon 10-deleted isoform. Likewise, electrophysiological analysis showed robust ATP-evoked currents when full-length but not the exon 10-deleted isoform of P2X5 was expressed. Taken together, our findings indicate that most humans express only a nonfunctional isoform of P2X5, which is in stark contrast to what is seen in other vertebrate species in which P2X5 has been studied, from which only the full-length isoform is known.

The molecular basis of pain and its clinical implications in rheumatology.

Nociceptive pain in response to peripheral noxious stimuli, and inflammatory pain resulting from tissue damage, serve as warnings that normal bodily function cannot resume until the stimulus abates or the tissue repairs. Stimuli cause numerous receptors, ion channels and other cellular machinery to respond, and propagate signals to the central nervous system, where this information is processed and perceived as pain. In healthy individuals, tissue damage results in physiologic--generally reparative--changes that lead to heightened sensory perception and, often, pain. In rheumatic diseases, the joint pain bears much in common with chronic inflammatory pain, but the underlying disease state is typically much more intricate and no reparative function is evident. Addressing the complex pains of rheumatic disease remains an ongoing challenge. Pain signaling pathways involve many molecular components that could potentially be targets for pharmacotherapeutic intervention, but the complexity of this system might also mean that multiple sites must be affected simultaneously to disrupt propagation of pain signals. In addition, to be therapeutically viable, pain drugs must be safe and not alter tactile sensory function, alertness or cognitive function. In this article we review the molecular functions in nociceptive, inflammatory and rheumatic pain pathways, and the therapeutic options they might offer.

Proapoptotic effects of NARC 1 (= PCSK9), the gene encoding a novel serine proteinase.

BACKGROUND: NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC 1 might be involved in the propagation of apoptotic signaling in neurons. METHODS: CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scanning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC 1 proapoptotic effect. RESULTS: Wild-type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF-sensitive component of cell death. NH(2)-terminal deletion constructs of NARC 1 had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAF. CONCLUSION: The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc 1 active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc 1 protein.

Laser scanning cytometry in the characterization of the proapoptotic effects of transiently transfected genes in cerebellar granule neurons.

BACKGROUND: Low transient transfection efficiency limits the ability to characterize putative proapoptotic gene function in neurons. Laser scanning cytometry (LSC), with its high capacity, medium throughput means of collecting fluorescent emissions from cultured cells, offers an effective technology for scoring cell death in neuronal transfectants. METHODS: Cerebellar granule neurons (CGNs) were transfected with EGFP-fusion constructs of Caspase-3 and Caspase-9 using a DNA-calcium phosphate coprecipitation method. CGNs were fixed, permeablized, and stained with propidium iodide (PI) nuclear dye. An LSC method, based on a combination of Long Red Max Pixel, Long Red Integral, and Green Integral fluorescence parameters was validated for the scoring of apoptotic cell death in CGNs. RESULTS: In Caspase-3 and Caspase-9 transfected CGNs, cell death was scored both in transfectants and nontransfected culture-mates. The cell death phenotype was found to be independent of transfection efficiency. LSC scoring of Caspase-9 transfectants was compared with visual scoring following Hoechst 33342 staining, yielding results that were similar qualitatively, but not quantitatively, likely owing to the greater sensitivity to green fluorescence of laser scanning compared to human vision. CONCLUSION: LSC scoring of transiently transfected CGNs offers a rapid and reliable means of characterizing proapoptotic gene effects.

Ischemia-stimulated neurogenesis is regulated by proliferation, migration, differentiation and caspase activation of hippocampal precursor cells.

A brief ischemic injury to the gerbil forebrain that caused selective damage in the CA1 region of the hippocampus also enhanced the production of new cells in the hippocampal neurogenic area. When evaluated 1 week after bromodeoxyuridine (BrdU) injection, approximately ten times more labeled cells were detected in the hippocampal dentate gyrus in ischemic animals than controls, indicating a stimulation of mitotic activity. To assess the temporal course of the survival and fate of these newborn cells, we monitored BrdU labeling and cell marker expression up to 60 days after ischemia (DAI). Loss of BrdU-positive cells was observed from both control and ischemic animals, but at 30 DAI and afterward, the ischemic group maintained more than 3 times as many BrdU-positive cells as the control group. In addition, ischemic injury also fostered the neuronal differentiation of these cells beyond the capacity observed in control animals and facilitated the migration of developing neurons to a neuronal cellular layer. The establishment of a temporal correlation between differentiation and migration provides evidence of the functional maturation of these cells. Surprisingly, we found that ischemic injury induced activation of caspase-3, not only in the CA1 region as expected, but also in the dentate subgranular zone (SGZ). Active caspase-3 immunoreactivity in the subgranular layer was co-localized with an early neuronal marker, suggesting that caspase-mediated apoptosis could mediate the loss of neurogenic cells in the SGZ. Inhibiting caspase-3 in the context of ischemia-induced neurogenesis might provide an opportunity for functional repair and a therapeutic outcome in the wake of ischemic injury.

Functional characterization of Narc 1, a novel proteinase related to proteinase K.

The NARC 1 gene encodes a novel proteinase K family proteinase. The domain structure of rat Narc 1 resembles that of the subtilisin-like proprotein convertases (SPCs), except that rNarc 1 lacks the canonical P-domain of SPCs, retaining only the RGD motif as part of what might be a cryptically functioning P-domain. Narc 1 undergoes autocatalytic intramolecular processing at the site LVFAQ/, resulting in the cleavage of its prosegment and the generation of an active proteinase with a broad alkaline pH optimum and no apparent calcium requirement for activity. Both primary and secondary structural determinants influence Narc 1 substrate recognition. Our functional characterization of Narc 1 reinforces the inference drawn from the analysis of its predicted structure that this enzyme is most closely related to representatives of the proteinase K family, but that it is also sufficiently different to warrant its possible classification in a separate sub-family.