The Shh/Gli1 signaling pathway regulates regeneration via transcription factor Olig1 expression after focal cerebral ischemia in rats.

OBJECTIVE: Ischemic stroke is a major cause of death in the global population, with a high disability and mortality rate. Lack of regenerative ability is considered to be the fundamental cause. This study aims to determine the effect of Shh pathway, which mediates regenerative signaling in response to CNS injury, on myelin repair and Olig1 expression in focal ischemic lesions in the rat. METHODS: A model of middle cerebral artery occlusion (MCAO) was established using the intraluminal suture method where the middle cerebral artery (MCA) was restricted for 120 min. Cyclopamine, a specific inhibitor of Shh, or saline was administered 12h after MCAO surgery and lasted for 7d. After MCA occlusion, male Sprague-Dawley rats were randomly allocated to cyclopamine- or saline-treated groups. A group of no-injection animals after MCAO were used as control. The Shh signaling pathway, myelinogenesis-related factor MBP and Olig1 were tested using immunohistochemistry and RT-PCR assay. RESULTS: The levels of Shh and its component Gli1 were elevated from 1d up to 14d following ischemia, indicating that the Shh-Gli1 axis was broadly reactivated. Treatment with cyclopamine can partially block the Shh signaling pathway, prevent myelin repair, and decrease the Olig1 expression following ischemic stroke. CONCLUSION: That blockade of Shh signaling concurrently with the creation of a lesion aggravated ischemic myelin damage, probably via its downstream effects on Olig1 transcription. Shh plays a contributory role during regeneration in the CNS, thereby providing promising new therapeutic strategies to assist in recovery from ischemic stroke.

DL-3-n-butylphthalide suppressed autophagy and promoted angiogenesis in rats with vascular dementia by activating the Shh/Ptch1 signaling pathway.

DL-3-n-butylphthalide (NBP) has neuroprotective effect on chronic cerebral hypoperfusion animals. Here, we explored the role and underlying mechanism of NBP on autophagy and angiogenesis in rats with vascular dementia (VD). Adult male Sprague-Dawley (SD) rats were subjected to permanent bilateral occlusion of the common carotid arteries (2VO) to establish VD model. These rats were randomly divided into five groups: sham, model, NBP120 (120 mg/kg), Shh siRNA (50 nM), and NBP120 + Shh siRNA groups. Our results showed that NBP treatment attenuated memory damage in rats with VD, as demonstrated by Morris water maze tests. Immunofluorescence (IF) assay revealed that NBP induced neuronal process length and neuronal activity in hippocampus, which were reversed by Shh silencing. Furthermore, NBP treatment also reduced the expression of autophagy marker proteins B-cell lymphoma-2 interacting protein 1 (Beclin 1) and microtubule-associated protein 1 light chain 3 (LC3), which were further enhanced by Shh silencing. Meanwhile, NBP promoted the angiogenesis, which was accompanied by upregulated vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-1, and Angiopoietin (Ang) expression in the hippocampus. And Shh siRNA co-treatment blocked the angiogenesis induced by NBP. Altogether, our results established that NBP treatment suppressed autophagy and improved angiogenesis and neurobehavioral recovery in VD rats partly by activating the Shh/Ptch1 signaling pathway.

Enhancing the in vitro and in vivo activity of itraconazole against breast cancer using miltefosine-modified lipid nanocapsules.

Itraconazole (ITC), a well-tolerated antifungal drug, exerts multiple anticancer effects which justified its preclinical and clinical investigation as potential anti-cancer agent with reduced side effects. Enhancement of ITC anti-cancer efficacy would bring valuable benefits to patients. We propose herein lipid nanocapsules (LNCs) modified with a subtherapeutic dose of miltefosine (MFS) as a membrane bioactive amphiphilic additive (M-ITC-LNC) for the development of an ITC nanoformulation with enhanced anticancer activity compared with ITC solution (ITC-sol) and unmodified ITC-LNC. Both LNC formulations showed a relatively small size (43-46 nm) and high entrapment efficiency (>97%), though ITC release was more sustained by M-ITC-LNC. Cytotoxicity studies revealed significantly greater anticancer activity and selectivity of M-ITC-LNC for MCF-7 breast cancer cells compared with ITC-sol and ITC-LNC. This trend was substantiated by in vivo findings following a 14 day-treatment of murine mammary pad Ehrlich tumors. M-ITC-LNC showed the greatest enhancement of the ITC-induced tumor growth inhibition, proliferation, and necrosis. At the molecular level, the tumor content of Gli 1, caspase-3, and vascular endothelial growth factor verified superiority of M-ITC-LNC in enhancing the ITC antiangiogenic, apoptotic, and Hedgehog pathway inhibitory effects. Finally, histopathological and biochemical analysis indicated greater reduction of ITC systemic toxicity by M-ITC-LNC. Superior performance of M-ITC-LNC was attributed to the effect of MFS on the structural and release properties of LNC coupled with its distinct bioactivities. In conclusion, MFS-modified LNC provides a simple nanoplatform integrating the potentials of LNC and MFS for enhancing the chemotherapeutic efficacy of ITC and possibly other oncology drugs.

Interaction of tacrolimus through hedgehog signaling pathway: An in vitro evaluation using rat hepatocytes.

Tacrolimus (TAC) is the drug of choice in immunosuppressive therapy for organ transplantation; however, adverse effects are still a major concern. The current study aims to decipher the short-term exposure of TAC on rat hepatocytes in relation to activation of hedgehog (HH) signaling pathway. Time dependent study was conducted using primary rat hepatocytes treated with TAC (36 µM) for 6, 12, 24 and 48 h. Western blot analysis was performed using cell lysate in order to analyze the regulation of HH pathway proteins including HHIP, SMO, PTCH, IHH, SHH, and GLI transcription factors. The study revealed change in protein expression of HH signaling molecules with activation of HH pathway, due to downregulation of HHIP, and enrichment of HH ligands with activation of SMO and GLI transcription factors. It is therefore, concluded that short term TAC exposure leads to upregulation of HH pathway in liver, which may initially act to repair the liver damage but can worsen the condition in case of prolonged immunosuppressive therapy. This insight could lead to understand association of off target effects of immunosuppressive drugs and occurrence of other liver diseases in transplant patients when it comes to long term immunosuppressive therapy. These findings also illuminate a novel direction that use of HH inhibitor might provide a therapeutic strategy for immune suppression related liver disorders.

Neo-adjuvant Vismodegib followed by radiation in locally advanced basal cell carcinoma.

Basal cell carcinomas occur in up to 39% of Caucasian men and 28% of women. Rarely it can present a management dilemma in patients with neglected locally advanced disease of large dimension or involvement of critical structures. The Hedgehog pathway is constitutively active in almost all basal cell carcinomas and patients with Naevoid Basal Cell Carcinoma Syndrome have germline mutations in the Patched tumor suppressor gene, a Hedgehog pathway component, on chromosome 9q. This case describes an elderly patient with an untreated sporadic Basal cell carcinoma whose dimensions precluded local management approaches. The Hedgehog pathway inhibitor Vismodegib had a dramatic response allowing definitive treatment to be pursued.

Targeting of the Hedgehog/GLI and mTOR pathways in advanced pancreatic cancer, a phase 1 trial of Vismodegib and Sirolimus combination.

BACKGROUND/OBJECTIVES: Preclinical data indicated a functional and molecular interaction between Hedgehog (HH)/GLI and PI3K-AKT-mTOR pathways promoting pancreatic ductal adenocarcinoma (PDAC). A phase I study was conducted of Vismodegib and Sirolimus combination to evaluate maximum tolerated dose (MTD) and preliminary anti-tumor efficacy. METHODS: Cohort I included advanced solid tumors patients following a traditional 3 + 3 design. Vismodegib was orally administered at 150 mg daily with Sirolimus starting at 3 mg daily, increasing to 6 mg daily at dose level 2. Cohort II included only metastatic PDAC patients. Anti-tumor efficacy was evaluated every two cycles and target assessment at pre-treatment and after a single cycle. RESULTS: Nine patient were enrolled in cohort I and 22 patients in cohort II. Twenty-eight patients were evaluated for dose-limiting toxicities (DLTs). One DLT was observed in each cohort, consisting of grade 2 mucositis and grade 3 thrombocytopenia. The MTD for Vismodegib and Sirolimus were 150 mg daily and 6 mg daily, respectively. The most common grade 3-4 toxicities were fatigue, thrombocytopenia, dehydration, and infections. A total of 6 patients had stable disease. No partial or complete responses were observed. Paired biopsy analysis before and after the first cycle in cohort II consistently demonstrated reduced GLI1 expression. Conversely, GLI and mTOR downstream targets were not significantly affected. CONCLUSIONS: The combination of Vismodegib and Sirolimus was well tolerated. Clinical benefit was limited to stable disease in a subgroup of patients. Targeting efficacy demonstrated consistent partial decreases in HH/GLI signaling with limited impact on mTOR signaling. These findings conflict with pre-clinical models and warrant further investigations.

Effects of Apatinib on the "Stemness" of Non-Small-Cell Lung Cancer Cells In Vivo and Its Related Mechanisms.

Objective: To investigate the effects of Apatinib on the "stemness" of lung cancer cells in vivo and to explore its related mechanisms. Methods: A xenograft model of lung cancer cells A549 was established in nude mice and randomized into a control group (n = 4) and an Apatinib group (n = 4). Tumor tissues were harvested after 2 weeks, and mRNA was extracted to detect changes in stemness-related genes (CD133, EPCAM, CD13, CD90, ALDH1, CD44, CD45, SOX2, NANOG, and OCT4) and Wnt/ß-catenin, Hedgehog, and Hippo signal pathways. Results: Compared with the control group, the volume and weight of nude mice treated with Apatinib were different and had statistical significance. Apatinib inhibited the expressions of ABCG2, CD24, ICAM-1, OCT4, and SOX2 and upregulated the expressions of CD44, CD13, and FOXD3. Apatinib treatment also inhibited the Wnt/ß-catenin, Hedgehog, and Hippo signaling pathways. Conclusion: Apatinib suppressed the growth of non-small-cell lung cancer cells by repressing the stemness of lung cancer through the inhibition of the Hedgehog, Hippo, and Wnt signaling pathways.

Cyclopamine sensitizes glioblastoma cells to temozolomide treatment through Sonic hedgehog pathway.

AIM: Glioblastoma is an extremely aggressive glioma, resistant to radio and chemotherapy usually performed with temozolomide. One of the main reasons for glioblastoma resistance to conventional therapies is due to the presence of cancer stem-like cells. These cells could recapitulate some signaling pathways important for embryonic development, such as Sonic hedgehog. Here, we investigated if the inhibitor of the Sonic hedgehog pathway, cyclopamine, could potentiate the temozolomide effect in cancer stem-like cells and glioblastoma cell lines in vitro. MAIN METHODS: The viability of glioblastoma cells exposed to cyclopamine and temozolomide treatment was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while the induction of apoptosis was assessed by western blot. The stemness properties of glioma cells were verified by clonogenic and differentiation assay and the expression of stem cell markers were measured by fluorescence microscopy and western blot. KEY FINDINGS: The glioblastoma viability was reduced by cyclopamine treatment. Cyclopamine potentiated temozolomide treatment in glioblastoma cell lines by inducing apoptosis through activation of caspase-3 cleaved. Conversely, the combined treatment of cyclopamine and temozolomide potentiated the stemness properties of glioblastoma cells by inducing the expression of SOX-2 and OCT-4. SIGNIFICANCE: Cyclopamine plays an effect on glioblastoma cell lines but also sensibilize them to temozolomide treatment. Thus, first-line treatment with Sonic hedgehog inhibitor followed by temozolomide could be used as a new therapeutic strategy for glioblastoma patients.

Ethanol and Cannabinoids Regulate Zebrafish GABAergic Neuron Development and Behavior in a Sonic Hedgehog and Fibroblast Growth Factor-Dependent Mechanism.

BACKGROUND: Ethanol (EtOH) has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that EtOH and cannabinoids may interact to broadly disrupt neuronal function during brain development. METHODS: Zebrafish embryos were exposed to a range of EtOH and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk-taking behavior. RESULTS: Combined acute subthreshold EtOH and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the EtOH and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the EtOH- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by EtOH and cannabinoids. CONCLUSIONS: These studies provide evidence that forebrain GABAergic neuron development and zebrafish risk-taking behavior are sensitive to both EtOH and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of EtOH and cannabinoids in FASD.

A Light-Inducible Hedgehog Signaling Activator Modulates Proliferation and Differentiation of Neural Cells.

The Hedgehog signaling pathway shapes our body by regulating the proliferation and differentiation of cells. The spatial and temporal distribution pattern of its ligands finely controls the activity of the Hedgehog pathway during development. To model the control of Hedgehog signaling activities in vitro, we developed a light-inducible Hedgehog signaling activator 6-nitroveratryloxy-carbonyl Smoothened agonist (NVOC-SAG). NVOC-SAG controls the proliferation of mouse cerebellar granule neuron precursor cells and ventral and neural differentiation of human iPS cells in a light dependent manner. The compound provides a new method to control Hedgehog signaling activities.

Phenformin Inhibits Hedgehog-Dependent Tumor Growth through a Complex I-Independent Redox/Corepressor Module.

The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.

Silica Perturbs Primary Cilia and Causes Myofibroblast Differentiation during Silicosis by Reduction of the KIF3A-Repressor GLI3 Complex.

The purpose of this study was to determine the effects of Kinesin family member 3A (KIF3A) on primary cilia and myofibroblast differentiation during silicosis by regulating Sonic hedgehog (SHH) signalling. Methods: Changes in primary cilia during silicosis and myofibroblast differentiation were detected in silicotic patients, experimental silicotic rats, and a myofibroblast differentiation model induced by SiO2. We also explored the mechanisms underlying KIF3A regulation of Glioma-associated oncogene homologs (GLIs) involved in myofibroblast differentiation. Results: Primary cilia (marked by ARL13B and Ac-α-Tub) and ciliary-related proteins (IFT 88 and KIF3A) were increased initially and then decreased as silicosis progressed. Loss and shedding of primary cilia were also found during silicosis. Treatment of MRC-5 fibroblasts with silica and then transfection of KIF3A-siRNA blocked activation of SHH signalling, but increased GLI2FL as a transcriptional activator of SRF, and reduced the inhibitory effect of GLI3R on ACTA2. Conclusion: Our findings indicate that primary cilia are markedly altered during silicosis and the loss of KIF3A may promote myofibroblast differentiation induced by SiO2.

A large-scale drug screen identifies selective inhibitors of class I HDACs as a potential therapeutic option for SHH medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is one of the most frequent malignant brain tumors of children, and a large set of these tumors is characterized by aberrant activation of the sonic hedgehog (SHH) pathway. While some tumors initially respond to inhibition of the SHH pathway component Smoothened (SMO), tumors ultimately recur due to downstream resistance mechanisms, indicating a need for novel therapeutic options. METHODS: Here we performed a targeted small-molecule screen on a stable, SHH-dependent murine MB cell line (SMB21). Comprehensive isotype profiling of histone deacetylase (HDAC) inhibitors was performed, and effects of HDAC inhibition were evaluated in cell lines both sensitive and resistant to SMO inhibition. Lastly, distinct mouse models of SHH MB were used to demonstrate pharmacologic efficacy in vivo. RESULTS: A subset of the HDAC inhibitors tested significantly inhibit tumor growth of SMB21 cells by preventing SHH pathway activation. Isotype profiling of HDAC inhibitors, together with genetic approaches suggested that concerted inhibition of multiple class I HDACs is necessary to achieve pathway inhibition. Of note, class I HDAC inhibitors were also efficacious in suppressing growth of diverse SMO inhibitor‒resistant clones of SMB21 cells. Finally, we show that the novel HDAC inhibitor quisinostat targets multiple class I HDACs, is well tolerated in mouse models, and robustly inhibits growth of SHH MB cells in vivo as well as in vitro. CONCLUSIONS: Our data provide strong evidence that quisinostat or other class I HDAC inhibitors might be therapeutically useful for patients with SHH MB, including those resistant to SMO inhibition.

Metformin alleviates hyperglycemia-induced endothelial impairment by downregulating autophagy via the Hedgehog pathway.

Studies regarding macroautophagic/autophagic regulation in endothelial cells (ECs) under diabetic conditions are very limited. Clinical evidence establishes an endothelial protective effect of metformin, but the underlying mechanisms remain unclear. We aimed to investigate whether metformin exerts its protective role against hyperglycemia-induced endothelial impairment through the autophagy machinery. db/db mice were treated with intravitreal metformin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of metformin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered autophagosome synthesis in both the diabetic retinal vasculature and cultured HUVECs by metformin, along with restoration of hyperglycemia-impaired Hedgehog (Hh) pathway activity. Specifically, deletion of ATG7 in retinal vascular ECs of db/db mice and cultured HUVECs indicated a detrimental role of autophagy in hyperglycemia-induced endothelial dysfunction. Pretreatment with GANT61, a Hh pathway inhibitor, abolished the metformin-mediated downregulation of autophagy and endothelial protective action. Furthermore, GLI-family (transcription factors of the Hh pathway) knockdown in HUVECs and retinal vasculature revealed that downregulation of hyperglycemia-activated autophagy by the metformin-mediated Hh pathway activation was GLI1 dependent. Mechanistically, GLI1 knockdown-triggered autophagy was related to upregulation of BNIP3, which subsequently disrupted the association of BECN1/Beclin 1 and BCL2. The role of BNIP3 in BECN1 dissociation from BCL2 was further confirmed by BNIP3 overexpression or BNIP3 RNAi. Taken together, the endothelial protective effect of metformin under hyperglycemia conditions could be partly attributed to its role in downregulating autophagy via Hh pathway activation. Abbreviations: 3-MA = 3-methyladenine; 8×GLI BS-FL = 8×GLI-binding site firefly luciferase; AAV = adeno-associated virus; AAV-Cdh5-sh-Atg7 = AAV vectors carrying shRNA against murine Atg7 under control of murine Cdh5 promoter; AAV-Cdh5-sh-Gli1 = AAV vectors carrying shRNA against murine Gli1 under control of murine Cdh5 promoter; AAV-Cdh5-Gli1 = AAV vectors carrying murine Gli1 cDNA under the control of murine Cdh5 core promoter; ACAC = acetyl-CoA carboxylase; Ad-BNIP3 = adenoviruses harboring human BNIP3`; Ad-GLI1 = adenoviruses harboring human GLI1; Ad-sh-ATG7 = adenoviruses harboring shRNA against human ATG7; Ad-sh-BNIP3 = adenoviruses harboring shRNA against human BNIP3; Ad-sh-GLI = adenoviruses harboring shRNA against human GLI; AGEs = advanced glycation end products; ATG = autophagy-related; atg7flox/flox mice = mice bearing an Atg7flox allele, in which exon 14 of the Atg7 gene is flanked by 2 loxP sites; BafA1 = bafilomycin A1; BECN1 = beclin 1; CDH5/VE-cadherin = cadherin 5; CASP3 = caspase 3; CASP8 = caspase 8; CASP9 = caspase 9; ECs = endothelial cells; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GCL = ganglion cell layer; GFP-LC3B = green fluorescent protein labelled LC3B; HG = high glucose; Hh = Hedgehog; HHIP = hedgehog interacting protein; HUVECs = human umbilical vein endothelial cells; IB4 = isolectin B4; INL = inner nuclear layer; i.p. = intraperitoneal; MAP1LC3/LC3 = microtubule-associated protein 1 light chain 3; MAN = mannitol; MET = metformin; NG = normal glucose; ONL = outer nuclear layer; p-ACAC = phosphorylated acetyl-CoA carboxylase; PECAM1/CD31= platelet/endothelial cell adhesion molecule 1; PRKAA1/2 = protein kinase AMP-activated catalytic subunits alpha 1/2; p-PRKAA1/2 = phosphorylated PRKAA1/2; PTCH1 = patched 1; RAPA = rapamycin; RL = Renilla luciferase; SHH = sonic hedgehog; shRNA = short hairpin RNA; sh-PRKAA1/2 = short hairpin RNA against human PRKAA1/2; scrambled shRNA = the scrambled short hairpin RNA serves as a negative control for the target-specific short hairpin RNA, which has the same nucleotide composition as the input sequence and has no match with any mRNA of the selected organism database; SMO = smoothened, frizzled class receptor; sqRT-PCR = semi-quantitative RT-PCR; TEK/Tie2 = TEK receptor tyrosine kinase; Tek-Cre (+) mice = a mouse strain expressing Cre recombinase under the control of the promoter/enhancer of Tek, in a pan-endothelial fashion; TUNEL = terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling.

Induced lesion and inhibited Ihh-PTHrP signalling pathway activity in the articular cartilage of rats caused by T-2 toxin.

This study aims to investigate the role of Ihh-PTHrP signalling pathway in the articular cartilage injury of rats caused by T-2 toxin. Sixty male Wistar rats were randomly divided into four groups: group A, normal diet; group B, normal diet plus the dissolvent (0.9% sodium chloride sterile aqueous solution containing ethanol); group C, normal diet plus low T-2 toxin (0.1 mg/kg BW/day) and group D, normal diet plus high T-2 toxin (0.2 mg/kg BW/day) by intragastric administration daily for 4 weeks. Histological changes in articular cartilage were assessed by HE staining and scanning electron microscopy. The expression of Ihh and PTHrP in cartilage was assessed by immunohistochemistry. There is a significant difference in average weight gain between group A and group D P < 0.01, groups A and D P < 0.001, respectively. The result of scanning electron microscopy and HE staining showed that the damage of articular cartilage was much severe with the increase of T-2 toxin. Immunohistochemical analysis indicated that the expression of Ihh in group A and group B was higher than that of group C and group D (P < 0.05, <0.01, respectively). However, the expression of PTHrP was lower in group A and group B than that of group C and group D (P < 0.001, <0.001, respectively). These results indicated that T-2 toxin can cause the damage to articular cartilage and weight loss in rats. The effect of T-2 toxin on articular cartilage of rat may be related to the Ihh-PTHrP pathway.

Tissue Plasminogen Activator Causes Brain Microvascular Endothelial Cell Injury After Oxygen Glucose Deprivation by Inhibiting Sonic Hedgehog Signaling.

The thrombolytic activity of tissue plasminogen activator (tPA) has undisputed benefits. However, the documented neurotoxicity of tPA raises important issues. Currently, common treatments for stroke might not be optimum if exogenous tPA can pass through the blood-brain barrier and enter the brain, thus adding to the deleterious effects of tPA within the cerebral parenchyma. Here, we determined whether tPA could damage brain microvascular endothelial cells (BMECs) during cerebral ischemia. We showed that treatment of BMECs with tPA decreased trans-endothelial electrical resistance and cell proliferation, and blocked the cell cycle at the G0-G1 phase. In addition, the Sonic hedgehog (Shh) signaling pathway was involved in tPA-induced BMECs dysfunction. However, tPA-enhanced oxygen glucose deprivation-induced BMECs dysfunction was eliminated by Shh administration and the effects could be reversed by Shh inhibitors. Taken together, these results demonstrate that tPA administration might result in damage to the endothelial barrier owing to blocked Shh signaling pathway.

Icariin Reduces Cartilage Degeneration in a Mouse Model of Osteoarthritis and is Associated with the Changes in Expression of Indian Hedgehog and Parathyroid Hormone-Related Protein.

BACKGROUND The aim of this study was to determine the role of icariin, a Chinese traditional herbal medicine extracted from Epimedium, in osteoarthritis (OA), using the murine anterior cruciate ligament transection (ACLT)-induced model of OA and micromass culture of murine chondrocytes. MATERIAL AND METHODS Twenty-four three-month-old C57/6J mice were randomly divided into three groups: the sham group (no surgery and joint injection with normal saline) (N=8); the ACLT + ICA group (ACLT surgery and icariin treatment) (N=8); and the ACLT group (ACLT surgery and joint injection with normal saline) (N=8). At 12 weeks after ACLT surgery, murine articular cartilage was harvested from all mice for histological evaluation of any differences in cartilage degeneration. In vitro micromass culture of mouse chondrocytes was used to study the effects of icariin on chondrocyte differentiation and growth from the three mouse groups. RESULTS Icariin treatment (mice in the ACLT + ICA group) significantly reduced degeneration of cartilage in OA with increased cartilage thickness, associated with increased expression of collagen type II alpha 1 (COL2A1), decreased chondrocyte hypertrophy, and decreased expression of collagen type X (ColX) and matrix metalloproteinase 13 (MMP13). In vitro, icariin promoted chondrocyte differentiation by upregulating the expression of agrrecan, Sox9 and parathyroid hormone-related protein (PHrP) and down-regulation of Indian hedgehog (Ihh) and genes regulated by Ihh. CONCLUSIONS In a mouse model of OA icariin treatment reduced destruction of cartilage, promoted chondrocyte differentiation, upregulated expression of PHrP and down-regulated the expression of Ihh.

Sonic hedgehog signalling regulates the self-renewal and proliferation of skin-derived precursor cells in mice.

OBJECTIVES: The sonic hedgehog (Shh) signalling pathway has an important role in the maintenance of various stem cells and organogenesis during development. However, the effect of Shh in skin-derived precursors (SKPs), which have the capacity for multipotency and self-renewal, is not yet clear. The present study investigated the effects of the Shh signalling pathway on the proliferation and self-renewal of murine SKPs (mSKPs). METHODS: The Shh signalling pathway was activated by treatment with purmorphamine (Shh agonist) or recombinant Shh in mSKPs. Cyclopamine (Shh antagonist) or GANT-61 (Gli inhibitor) was used to inhibit the pathway. Western blot, qPCR, and immunofluorescence were used to analyse the expression of genes related to self-renewal, stemness, epithelial-mesenchymal transition (EMT) and the Shh signalling pathway. In addition, cell proliferation and apoptosis were examined. RESULTS: Inhibiting the Shh signalling pathway reduced mSKP proliferation and sphere formation, but increased apoptosis. Activating this signalling pathway produced opposite results. The Shh signalling pathway also controlled the EMT phenotype in mSKPs. Moreover, purmorphamine recovered the self-renewal and proliferation of aged mSKPs. CONCLUSION: Our results suggest that the Shh signalling pathway has an important role in the proliferation, self-renewal and apoptosis of mSKPs. These findings also provide a better understanding of the cellular mechanisms underlying SKP self-renewal and apoptosis that allow more efficient expansion of SKPs.