Kuo-Hsing Ma

siRNA-targeting transforming growth factor-β type I receptor reduces wound scarring and extracellular matrix deposition of scar tissue.

Hypertrophic scarring is related to persistent activation of transforming growth factor-β (TGF-β)/Smad signaling. In the TGF-β/Smad signaling cascade, the TGF-β type I receptor (TGFBRI) phosphorylates Smad proteins to induce fibroblast proliferation and extracellular matrix deposition. In this study, we inhibited TGFBRI gene expression via TGFBRI small interfering RNA (siRNA) to reduce fibroblast proliferation and extracellular matrix deposition. Our results demonstrate that downregulating TGFBRI expression in cultured human hypertrophic scar fibroblasts significantly suppressed cell proliferation and reduced type I collagen, type III collagen, fibronectin, and connective tissue growth factor (CTGF) mRNA, and type I collagen and fibronectin protein expression. In addition, we applied TGFBRI siRNA to wound granulation tissue in a rabbit model of hypertrophic scarring. Downregulating TGFBRI expression reduced wound scarring, the extracellular matrix deposition of scar tissue, and decreased CTGF and α-smooth muscle actin mRNA expression in vivo. These results suggest that TGFBRI siRNA could be applied clinically to prevent hypertrophic scarring.

Phasic dysfunction of dopamine transmission in Tourette's syndrome evaluated with 99mTc TRODAT-1 imaging

This study investigated the complex dysregulation of the dopaminergic neurotransmitter system in Tourettes syndrome (TS) patients challenged with methylphenidate (MPH). Eight drug-naïve male patients (aged 21-25 years) who met DSM-IV criteria for TS and had a mean disease severity of 25 on the Yale Global Tic Severity Scale were recruited. Brain (99m)TC TRODAT-1 dopamine transporter (DAT) single photon emission computed tomography (SPECT) was performed 5 days before, and 2 h after 10 mg of orally administered MPH. Eight age-matched healthy males served as controls. Repeated measures analysis of variance was used to measure differences in DAT-binding ratios before and after MPH challenge between the TS patients and controls. The DAT-binding ratios decreased significantly after MPH treatment in both groups. However, a significant interaction between group and MPH effects was found only in the right caudate, which was mainly due to a smaller decline of the DAT-binding ratio after MPH in the TS group than in the controls. Such a distinction was not found in the other striatal sub-regions in the two groups. No correlation, however, was observed between the tic severity score and DAT-binding ratio measured from the whole striatum or its sub-regions. The observed change in the DAT-binding ratio might indicate a functional abnormality of the dopaminergic system in the right caudate nucleus of TS patients. Future studies exploring dopamine transmission are thus needed to understand the pathophysiology of TS.

Validation of 4-[18F]-ADAM as a SERT imaging agent using micro-PET and autoradiography.

Serotonin transporters (SERTs) have been implicated in various neuropsychiatric disorders. We aim to validate 4-[(18)F]-ADAM (N,N-dimethyl-2-(2-amino-4-[(18)F]fluorophenylthio)benzylamine) as a SERT imaging agent in rats using micro-positron emission tomography (micro-PET) and autoradiography. Sixty to ninety min after injecting 4-[(18)F]-ADAM, specific uptake ratios (SURs) were determined by micro-PET measurements in various brain regions of normal control rats. For n=3, the SUR in the midbrain was 4.94+/-0.16, for the hypothalamus it was 4.39+/-0.031 and for the caudate it was 4.18+/-0.53. The retention of 4-[(18)F]-ADAM in the hypothalamus and midbrain regions increased rapidly between 5 to 10 min after injection and declined thereafter. The SURs determined by autoradiography were: 9.31+/-1.41 for the midbrain, 7.15+/-1.45 for the hypothalamus and 5.22+/-1.14 for the caudate putamen. Both micro-PET and autoradiography studies revealed a dose-dependent progressive inhibition of radioligand uptake in the frontal cortex, caudate putamen and hypothalamus in rats treated with 0.01 to 0.25 mg/kg paroxetine. A decrease in 4-[(18)F]-ADAM uptake of approximately 84% was observed in the midbrain of rats pretreated with 0.25 mg/kg paroxetine as compared to controls (4.94+/-0.16 versus 0.80+/-0.17, n=3). Both 5,7-dihydroxytryptamine and p-chloroamphetamine-treated rats showed pronounced reduction in 4-[(18)F]-ADAM binding when compared to normal controls. Rats pretreated with p-chloroamphetamine exhibited significant inhibition of 4-[(18)F]-ADAM uptake in brain regions rich in SERT over a period of four weeks. Thus, 4-[(18)F]-ADAM is a SERT-specific radioligand that may be useful for evaluating neuropsychiatric conditions involving serotonergic dysfunction.

Variations of the cystic artery in Chinese adults.

The origin and course of the cystic artery related to the Calot triangle were studied in 72 autopsies. The cystic artery arises from many possible origins; the right hepatic artery is the most common origin (76.6%). The Calot triangle (hepatocystic triangle), which is an important imaginary referent area for biliary surgery, is bounded by the common hepatic duct (CHD), the cystic duct, and the cystic artery. Of all the cystic arteries, 86.1% coursed through the Calot triangle, and 100% of the cystic arteries originating from the right hepatic artery coursed through the Calot triangle. However, only 54% of the cystic arteries that originated from the left, bifurcation, proper, and common hepatic arteries ran through the triangle. None of the cystic arteries that originated from the gastroduodenal, celiac, superior mesentery, or superior pancreaticoduodenal arteries passed through the triangle. Furthermore, 72.7% of the cystic arteries that originated from the right hepatic artery ran beneath the CHD as they entered the Calot triangle; the others ran anterior to the CHD. Of the cystic arteries that arose from locations other than the right hepatic artery, 29.4% ran posterior to the CHD, and 11.8% ran anterior to the CHD. The current study provides detailed information about anatomic variance in Chinese adults that may help avoid injury during open or laparoscopic cholecystectomies.

Characterization of 4-[ 18F]-ADAM as an imaging agent for SERT in non-human primate brain using PET: A dynamic study

INTRODUCTION Serotonin transporter (SERT) has been associated with many psychiatric diseases. This study investigated the biodistribution of a serotonin transporter imaging agent, N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)benzylamine (4-[(18)F]-ADAM), in nonhuman primate brain using positron emission tomography (PET). METHODS Six and four Macaca cyclopis monkeys were used to determine the transit time (i.e., time necessary to reach biodistribution equilibrium) and the reproducibility of 4-[(18)F]-ADAM biodistribution in the brain, respectively. The sensitivity and specificity of 4-[(18)F]-ADAM binding to SERT were evaluated in one monkey challenged with different doses of fluoxetine and one monkey treated with 3,4-methylendioxymethamphetamine (MDMA). Dynamic PET imaging was performed for 3 h after 4-[(18)F]-ADAM intravenous bolus injection. The specific uptake ratios (SURs) in the midbrain (MB), thalamus (TH), striatum (ST) and frontal cortex (FC) were calculated. RESULTS The distribution of 4-[(18)F]-ADAM reached equilibrium 120-150 min after injection. The mean SURs were 2.49 ± 0.13 in MB, 1.59 ± 0.17 in TH, 1.35 ± 0.06 in ST and 0.34 ± 0.03 in FC, and the minimum variability was shown 120-150 min after 4-[(18)F]-ADAM injection. Using SURs and intraclass coefficient of correlation, the test/retest variability was under 8% and above 0.8, respectively, in SERT-rich areas. Challenge with fluoxetin (0.75-2 mg) dose-dependently inhibited the SURs in various brain regions. 4-[(18)F]-ADAM binding was markedly reduced in the brain of an MDMA-treated monkey compared to that in brains of normal controls. CONCLUSION 4-[(18)F]-ADAM appears to be a highly selective radioligand for imaging SERT in monkey brain.

Autophagy activation is involved in 3,4-methylenedioxymethamphetamine ('ecstasy')--induced neurotoxicity in cultured cortical neurons.

Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.

Association study of serotonin transporter availability and SLC6A4 gene polymorphisms in patients with major depression.

The serotonin transporter (SERT) is hypothesized to be an important component of the pathophysiology of major depression (MD). The aim of this study was to use [(123)I]ADAM single-photon emission computed tomography (SPECT) to explore whether SERT availability in four regions of the brain (striatum, thalamus, midbrain and pons) is different in patients with MD and healthy individuals. The effects of three genetic variants (rs25531, rs6354 and STin2) of the serotonin transporter gene (SLC6A4) on SERT availability were also investigated. This study included 40 MD patients and 12 controls. The mean specific uptake ratio (SUR) values in the thalamus differed significantly between MD patients and controls. Genetic variants of SLC6A4, age, gender, severity of depression, and smoking behavior did not influence SERT availability. SERT availability might be a useful biomarker of the development of MD; however, a larger sample size is needed to provide more concrete evidence.

Effects of interleukin-15 on neuronal differentiation of neural stem cells.

Interleukin-15 (IL-15) signaling has pleiotropic actions in many cell types during development and has been best studied in cells of immune system lineage, where IL-15 stimulates proliferation of cytotoxic T cells and induces maturation of natural killer cells. A few reports have indicated that IL-15 and the IL-15 receptor are expressed in central nervous system tissues and neuronal cell lines. Because this aspect of IL-15 action is poorly studied, we used cultured rat neural stem cells (NSCs) to study IL-15 signal transduction and activity. Primary cultures of rat NSCs in culture will form neurospheres and will differentiate into neuron, astrocyte, and oligodendrocyte progenitors under permissive conditions. We found by immunofluorescence that the IL-15Ralpha subunit of the IL-15 receptor was expressed in NSCs and differentiating neurons, but not astrocyte or oligodendrocyte progenitors. We also showed that IL-15 treatment reduced MAP-2 protein levels in neurons and could reduce neurite outgrowth in differentiating neurons but did not affect NSC proliferation, and cell proportions and viability of the corresponding lineage cells. In the presence of a STAT3 inhibitor, Stattic, IL-15 no longer reduced MAP-2 protein levels. IL-15 treatment caused STAT3 phosphorylation. Furthermore, using anti-IL-15Ralpha antibody to block IL-15 signaling completely inhibited IL-15-induced phosphorylation of STAT3 and prevented IL-15 from decreasing neurite outgrowth. In conclusion, IL-15 may influence neural cell differentiation through a signal transduction pathway involving IL-15Ralpha and STAT3. This signal transduction modifies MAP-2 protein levels and, consequently, the differentiation of neurons from NSCs, as evidenced by reduced neurite outgrowth.

Involvement of SHP2 in focal adhesion, migration and differentiation of neural stem cells

OBJECTIVES SHP2 (Src-homology-2 domain-containing protein tyrosine phosphatase) plays an important role in cell adhesion, migration and cell signaling. However, its role in focal adhesion, differentiation and migration of neural stem cells is still unclear. METHODS In this study, rat neurospheres were cultured in suspension and differentiated neural stem cells were cultured on collagen-coated surfaces. RESULTS The results showed that p-SHP2 co-localized with focal adhesion kinase (FAK) and paxillin in neurospheres and in differentiated neural precursor cells, astrocytes, neurons, and oligodendrocytes. Suppression of SHP2 activity by PTP4 or siRNA-mediated SHP2 silencing caused reduction in the cell migration and neurite outgrowth, and thinning of glial cell processes. Differentiation-induced activation of FAK, Src, paxillin, ERK1/2, and RhoA was decreased by SHP2 inactivation. CONCLUSIONS These results indicate that SHP2 is recruited in focal adhesions of neural stem cells and regulates focal adhesion formation. SHP2-mediated regulation of neural differentiation and migration may be related to formation of focal adhesions and RhoA and ERK1/2 activation.

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca2+ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G0/G1 phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.