May-Jywan Tsai

Acid fibroblast growth factor and peripheral nerve grafts regulate Th2 cytokine expression, macrophage activation, polyamine synthesis, and neurotrophin expression in transected rat spinal cords.

Spinal cord injury elicits an inflammatory response that recruits macrophages to the injured spinal cord. Quantitative real-time PCR results have shown that a repair strategy combining peripheral nerve grafts with acidic fibroblast growth factor (aFGF) induced higher interleukin-4 (IL-4), IL-10, and IL-13 levels in the graft areas of rat spinal cords compared with transected spinal cords at 10 and 14 d. This led to higher arginase I-positive alternatively activated macrophage (M2 macrophage) responses. The gene expression of several enzymes involved in polyamine biosynthesis pathways was also upregulated in the graft areas of repaired spinal cords. The treatment induced a twofold upregulation of polyamine levels at 14 d, as confirmed by HPLC. Polyamines are important for the repair process, as demonstrated by the observation that treatment with inhibitors of arginase I and ornithine decarboxylase attenuates the functional recoveries of repaired rats. After 14 d, the treatment also induced the expression of neurotrophin nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as M2 macrophages within grafted nerves expressing BDNF. IL-4 was upregulated in the injury sites of transected rats that received aFGF alone compared with those that received nerve grafts alone at 10 d. Conversely, nerve graft treatment induced NGF and BDNF expression at 14 d. Macrophages expressing polyamines and BDNF may benefit axonal regeneration at 14 d. These results indicate that aFGF and nerve grafts regulate different macrophage responses, and M2 macrophages may play an important role in axonal regeneration after spinal cord injury in rats.

Selective Augmentation of Prostacyclin Production by Combined Prostacyclin Synthase and Cyclooxygenase-1 Gene Transfer

BackgroundWe tested the hypothesis that combined cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) gene transfer selectively augments prostacyclin production without a concurrent overproduction of other prostanoids. Methods and ResultsECV304 cells were transfected with bicistronic pCOX-1/PGIS versus pCOX-1 or pPGIS, and prostanoids were analyzed. Contrary to the high prostaglandin E2 synthesis in pCOX-1 transfected cells, selective prostacyclin formation was noted with bicistronic plasmid transfection. Next, we determined the optimal ratio of Ad-COX-1 to Ad-PGIS by transfecting human umbilical vein endothelial cells with various titers of these 2 adenoviral constructs and determined the level of protein expression and prostanoid synthesis. Our results show that optimal ratios of adenoviral titers to achieve a large prostacyclin augmentation without overproduction of prostaglandin E2 or F2&agr; were 50 to 100 plaque forming units (pfu) of Ad-COX-1 to 50 pfu of Ad-PGIS per cell. A higher Ad-PGIS to Ad-COX-1 ratio caused a paradoxical decline in prostacyclin synthesis. ConclusionsProstacyclin synthesis can be selectively augmented by cotransfecting endothelial cells with an optimal ratio of COX-1 to PGIS. Combined COX-1 and PGIS gene transfer has the potential for therapeutic augmentation of prostacyclin.

Recovery of neurological function of ischemic stroke by application of conditioned medium of bone marrow mesenchymal stem cells derived from normal and cerebral ischemia rats

BackgroundSeveral lines of evidence have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSC) release bioactive factors and provide neuroprotection for CNS injury. However, it remains elusive whether BM-MSC derived from healthy donors or stroke patients provides equal therapeutic potential. The present work aims to characterize BM-MSC prepared from normal healthy rats (NormBM-MSC) and cerebral ischemia rats (IschBM-MSC), and examine the effects of their conditioned medium (Cm) on ischemic stroke animal model.ResultsIsolated NormBM-MSC or IschBM-MSC formed fibroblastic like morphology and expressed CD29, CD90 and CD44 but failed to express the hematopoietic marker CD34. The number of colony formation of BM-MSC was more abundant in IschBM-MSC than in NormBM-MSC. This is in contrast to the amount of Ficoll-fractionated mononuclear cells from normal donor and ischemic rats. The effect of cm of BM-MSC was further examined in cultures and in middle cerebral artery occlusion (MCAo) animal model. Both NormBM-MSC Cm and IschBM-MSC Cm effectively increased neuronal connection and survival in mixed neuron-glial cultures. In vivo, intravenous infusion of NormBM-MSC Cm and IschBM-MSC Cm after stroke onset remarkably improved functional recovery. Furthermore, NormBM-MSC Cm and IschBM-MSC Cm increased neurogenesis and attenuated microglia/ macrophage infiltration in MCAo rat brains.ConclusionsOur data suggest equal effectiveness of BM-MSC Cm derived from ischemic animals or from a normal population. Our results thus revealed the potential of BM-MSC Cm on treatment of ischemic stroke.

Highly Sensitive Ammonia Sensor with Organic Vertical Nanojunctions for Noninvasive Detection of Hepatic Injury

We successfully demonstrate the first solid-state sensor to have reliable responses to breath ammonia of rat. For thioacetamide (TAA)-induced hepatopathy rats, we observe that the proposed sensor can detect liver that undergoes acute-moderate hepatopathy with a p-value less than 0.05. The proposed sensor is an organic diode with vertical nanojunctions produced by using low-cost colloidal lithography. Its simple structure and low production cost facilitates the development of point-of-care technology. We also anticipate that the study is a starting point for investigating sophisticated breath-ammonia-related disease models.

Toona sinensis Roem (Meliaceae) leaf extract alleviates hyperglycemia via altering adipose glucose transporter 4

In this study we tested the effects of Toona sinensis leaf extracted with water (TSL1) on alloxan-induced (50 mg/kgBwt, i.v.) diabetic Long-Even rats. Diabetic rats given TS leaf with water (TSL1), with 50% alcohol (TSL3) or with H2O extract (TSL5) showed lower levels of plasma glucose. Normal rats given Glibenclamide (GC) had lower levels of plasma glucose, but TSL1 administration showed no significant effect on plasma glucose. By contrast, TSL1 or GC given to alloxan-induced diabetic rats showed a 40% reduction in plasma glucose compared to diabetic rats. Diabetic rats had lower levels of insulin. Interestingly, TSL1 or GC given to diabetic rats showed improvements in plasma insulin levels. Diabetic rats had lower expressions of glucose transporter 4 (GLUT4) mRNA (RT-PCR) and GLUT4 protein (Western blot) in brown and white adipose tissues; in contrast, diabetic rats given TSL1 or GC showed a significant increase in both GLUT4 mRNA and protein levels. Moreover, the expressions of GLUT4 mRNA in red and white muscles were not significantly different among diabetic rats, diabetic rats given TSL1 or GC, and the normal rats. Compared to diabetic rats, diabetic rats given TSL1 or GC had lower levels of GLUT4 protein in white muscle but not in red muscle. Conclusively, T. sinensis Roem (Meliaceae) leaf possesses the hypoglycemia effect underlying an increment of insulin to mediate the adipose glucose transporter 4 mechanism.

Dual effect of adenovirus‐mediated transfer of BMP7 in mixed neuron‐glial cultures: Neuroprotection and cellular differentiation

Bone morphogenetic proteins (BMPs), members of the TGF‐β superfamily, have been implicated in nervous system development and in response to injury. Previous studies have shown that recombinant BMP7 can enhance dendritic growth and protect cultured neurons from oxidative stress. Because of the presence of extracellular BMP antagonists, BMP7 seems to act locally. Therefore, the present study uses BMP7 overexpression using adenovirus (Ad)‐mediated gene transfer to examine its effect in mixed neuronal cultures. Enhanced BMP7 expression selectively induces neuronal CGRP expression in a time‐dependent manner. BMP7 overexpression not only significantly protects cultures from H2O2 toxicity but reduces lipopolysaccharide (LPS) stimulation. Concurrently, it profoundly reduces microglial numbers, but increases oligodendroglial and endothelial cells. Together, low‐dose and continuously expressed BMP7 is both neuroprotective and differentiation‐inductive.

Antroquinonol Targets FAK-Signaling Pathway Suppressed Cell Migration, Invasion, and Tumor Growth of C6 Glioma.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine that is overexpressed in many types of tumors and plays a pivotal role in multiple cell signaling pathways involved in cell survival, migration, and proliferation. This study attempts to determine the effect of synthesized antroquinonol on the modulation of FAK signaling pathways and explore their underlying mechanisms. Antroquinonol significantly inhibits cell viability with an MTT assay in both N18 neuroblastoma and C6 glioma cell lines, which exhibits sub G1 phase cell cycle, and further induction of apoptosis is confirmed by a TUNEL assay. Antroquinonol decreases anti-apoptotic proteins, whereas it increases p53 and pro-apoptotic proteins. Alterations of cell morphology are observed after treatment by atomic force microscopy. Molecular docking results reveal that antroquinonol has an H-bond with the Arg 86 residue of FAK. The protein levels of Src, pSrc, FAK, pFAK, Rac1, and cdc42 are decreased after antroquinonol treatment. Additionally, antroquinonol also regulates the expression of epithelial to mesenchymal transition (EMT) proteins. Furthermore, antroquinonol suppresses the C6 glioma growth in xenograft studies. Together, these results suggest that antroquinonol is a potential anti-tumorigenesis and anti-metastasis inhibitor of FAK.

Neuronal morphological change of size-sieved stem cells induced by neurotrophic stimuli.

Size-sieved stem cells (SSCs) derived from human bone marrow have the ability to differentiate into bone, fat and cartilage. SSCs can differentiate into active neural cells after exposure to antioxidant agents. The aim of the present study is to understand if SSCs can be stimulated to differentiate into neurons in response to neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), pituitary adenylate cyclase-activating polypeptide (PACAP) and dibutyryl cAMP (dbcAMP). SSCs in a serum-free medium transform from a fibroblastic-like form to a multipolar morphology. Treatment of SSCs with GDNF, PACAP, and dbcAMP increased the production of neurofilament light protein (NF-L) and a cytoskeleton protein-alpha-tubulin. Examination of a vesicle protein-synapsin-1 or a neuronal progenitor marker-internexin in SSCs indicated that treatment with GDNF, PACAP, and dbcAMP further elongated cell processes and increased process branching. The findings indicate that neurotrophic signaling and cAMP-dependent signaling might promote the neuronal differentiation of SSCs.

Resveratrol inhibits glucose-induced migration of vascular smooth muscle cells mediated by focal adhesion kinase.

SCOPE Diabetes is a critical factor for atherosclerosis, as hyperglycemia induces vascular smooth muscle cell (VSMC) proliferation and migration and subsequently contributes to the formation of atherosclerotic lesions. This study investigates whether resveratrol plays a regulatory role in the proliferation and migration of VSMCs under high glucose induction to imitate a hyperglycemic condition. METHODS AND RESULTS Resveratrol inhibited the migration of VSMCs in the wound-healing assay and the formation of lamellipodia and filopodia as assessed by atomic force microscopy scanning. Resveratrol suppressed the mRNA expression of c-Src, Rac1, cdc42, IRS-1, MEKK1, MEKK4, and mitogen-activated protein kinase along with the protein levels of c-Src, p-Src, and cdc42 in VSMCs. Resveratrol decreased the level of p-FAK protein under normal glucose conditions. Resveratrol could inhibit the activities of matrix metalloproteinase (MMP) 2 and MMP 9 as shown by zymography. Moreover, resveratrol also regulated the mitogen-activated protein kinase pathway and MMP activities of VSMC migration under the high glucose condition. CONCLUSION The antimigratory effects of resveratrol by reduced MMP expression through the inhibition of Rac1, p-FAK, and lamellipodia formation and the activation of p-AKT and p-ERK1/2 suggest that resveratrol is a potential compound for the treatment of vascular diseases via the regulation of VSMC migration.

Adenoviral gene transfer of bone morphogenetic protein-7 enhances functional recovery after sciatic nerve injury in rats

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β subfamily, function as instructive signals for neuronal lineage commitment and promote neuronal differentiation. However, the mechanism of BMP7 action in vivo after peripheral nerve injury is poorly understood. This study examines the efficacy of gene transfer of adenoviral (Ad) BMP7 on peripheral neuropathy. Transgene expression was found in both Ad-infected sciatic nerves and their respective remote neurons, indicating Ad transduction by a retrograde transport. After AdBMP7 infection to nerves, the sciatic nerves were crushed or transected. Hind limb functional behavior, including rotarod test and sciatic functional index, were conducted in rats weekly after nerve injury. Interestingly, enhanced BMP7 expression significantly improved hind limb functional recovery in AdBMP7-transduced rats when compared with AdGFP-transduced nerve-crushed or transected rats. Furthermore, AdBMP7 transduction reduced injury-induced macrophage activation, nerve demyelination and axonal degeneration. By contrast, AdBMP7 infection did not affect the hyperalgesia paw-withdrawal latency after nerve injury. We further examined the effect of AdBMP7 infection on sciatic nerve explant and Schwann cell cultures. Enhanced cell proliferation was significantly increased by AdBMP7 transduction in both cultures. Taken together, BMP7 overexpression by Ad gene transfer was beneficial in both nerves and Schwann cells on functional recovery after sciatic nerve injury in rats.