Development of Noninvasive in Vivo Approach to Assess Vascular Permeability in Inflammation Using Fluorescence Imaging.

INTRODUCTION: In vivo fluorescence imaging can quantify vascular permeability without requiring sacrifice of animals. However, use of this noninvasive approach for vascular permeability assessment in remote organ injury caused by systemic inflammatory disease has not been reported. METHODS: Evans blue (EB) and Genhance 750 fluorescent dye were mixed and injected into mice. The lung as a remote organ and the footpad as a noninvasive observational site were assessed in a cecal ligation and puncture (CLP)-induced systemic inflammation mouse model and compared with sham and hydrocortisone pretreated (CLP + HC) mouse models. Extraction of EB in harvested tissues was assessed as a conventional indicator of vascular permeability. Fluorescent intensities in the footpad or harvested lung were assessed and their correlation was analyzed to investigate this novel, noninvasive approach for estimation of lung vascular permeability. RESULTS: Fluorescent intensity in the footpad and harvested lung in the CLP group was significantly higher than in the other groups (footpad, sham vs. CLP, P < 0.0001; CLP vs. CLP + HC, P = 0.0004; sham vs. CLP + HC, P = 0.058; lung, sham vs. CLP, P < 0.0001; CLP vs. CLP + HC, P < 0.0001; sham vs. CLP + HC, P = 0.060). The fluorescent intensity in the footpad was strongly correlated with that in the lung (r = 0.95). CONCLUSIONS: This fluorescent technique may be useful for vascular permeability assessment based on EB quantification. Footpad fluorescent intensity was strongly correlated with that in the lung, and may be a suitable indicator in noninvasive estimation of lung vascular permeability.

Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.

Role of Prickle1 and Prickle2 in neurite outgrowth in murine neuroblastoma cells.

Murine Prickle2 but not Prickle1 gene expression was induced in C1300 neuroblastoma cell line during neurite-like process formation induced by all trans-retinoic acid (RA). Overexpression of Prickle1 or Prickle2 in C1300 cells induced striking neurite-like process formation without RA. Prickle1 and Prickle2 associate with Dishevelled1 (Dvl1) and overexpression of Prickle1 or Prickle2 resulted in the reduction of Dvl1 protein in C1300 cells. Overexpression of Dvl1 in C1300 cells prevented the neurite-like process formation induced by Prickle1 or Prickle2 overexpression. Prickle1 and Prickle2 promote neurite-like process formation of C1300 cells via the Dvl1-dependent mechanism.