Testosterone exposure in prenatal life disrupts epithelial nuclear morphology, smooth muscle layer pattern, and FGF10 and Shh expression in prostate.

The aim of this study was to evaluate whether high levels of exogenous testosterone (T) interfere in prostate morphogenesis. Pregnant females were exposed to subcutaneous injections of T cypionate (500 µg/animal) at gestational days 20 and 22. Male and female pups were euthanized at postnatal days 1 and 15. 15-day-old males had only fibroblast growth factor 10 (FGF10) immunostaining and nuclear form factor altered by the treatment, whereas treated females (T1 and T15) had almost all analyzed parameters changed. T1 females showed an increased anogenital distance (AGD), whereas T15 females had both AGD and ovary weight increased. T1 females had a higher number of epithelial buds emerging from the urethral and vaginal epithelium. We observed ectopic prostatic tissue surrounding the vagina in both T1 and T15 females. Moreover, the ectopic acini of T15 females showed delayed luminal formation, and there was a thickening of the periacinar smooth muscle layer (SML). Finally, FGF10 immunostaining intensity decreased in both T15 male and female prostates. Indeed, Sonic hedgehog (Shh) was upregulated in T15 female prostates, whereas no difference was observed between the male groups. These data showed that exogenous T changed the nuclear morphology of prostate epithelial cells in both males and females. Surprisingly, smooth muscle hyperplasia was also observed in the ectopic female prostate. Moreover, T downregulated FGF10 in both male and female prostates. Interestingly, the results suggest that FGF10 downregulation is mediated by the upregulation of Shh in females. In conclusion, exogenous T disrupts prostate development, particularly, affecting, the female.

Apical ectodermal ridge regulates three principal axes of the developing limb.

Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.

miR-199 family contributes to regulation of sonic hedgehog expression during craniofacial development.

BACKGROUND: The frontonasal ectodermal zone (FEZ) is a signaling center that regulates patterned development of the upper jaw, and Sonic hedgehog (SHH) mediates FEZ activity. Induction of SHH expression in the FEZ results from SHH-dependent signals from the brain and neural crest cells. Given the role of miRNAs in modulating gene expression, we investigated the extent to which miRNAs regulate SHH expression and FEZ signaling. RESULTS: In the FEZ, the miR-199 family appears to be regulated by SHH-dependent signals from the brain; expression of this family increased from HH18 to HH22, and upon activation of SHH signaling in the brain. However, the miR-199 family is more broadly expressed in the mesenchyme of the frontonasal process and adjacent neuroepithelium. Downregulating the miR-199 genes expanded SHH expression in the FEZ, resulting in wider faces, while upregulating miR-199 genes resulted in decreased SHH expression and narrow faces. Hypoxia inducible factor 1 alpha (HIF1A) and mitogen-activated protein kinase kinase kinase 4 (MAP3K4) appear to be potential targets of miR-199b. Reduction of MAP3K4 altered beak development but increased apoptosis, while reducing HIF1A reduced expression of SHH in the FEZ and produced malformations independent of apoptosis. CONCLUSIONS: Our results demonstrate that this miRNA family appears to participate in regulating SHH expression in the FEZ; however, specific molecular mechanisms remain unknown.

Subverting Hedgehog Protein Autoprocessing by Chemical Induction of Paracatalysis.

Hedgehog proteins, a family of vital cell signaling factors, are expressed in precursor form, which requires specialized autoprocessing, called cholesterolysis, for full biological activity. Cholesterolysis occurs in cis through the action of the precursor's C-terminal enzymatic domain, HhC. In this work, we describe HhC activator compounds (HACs), a novel class of noncovalent modulators that induce autoprocessing infidelity, diminishing native cholesterolysis in favor of precursor autoproteolysis, an otherwise minor and apparently nonphysiological side reaction. HAC-induced autoproteolysis generates hedgehog protein that is cholesterol free and hence signaling deficient. The most effective HAC has an AC50 of 9 µM, accelerates HhC autoproteolytic activity by 225-fold, and functions in the presence and absence of cholesterol, the native substrate. HACs join a rare class of "antagonists" that suppress native enzymatic activity by subverting mechanistic fidelity.

Sonic hedgehog expression in zebrafish forebrain identifies the teleostean pallidal signaling center and shows preglomerular complex and posterior tubercular dopamine cells to arise from shh cells.

Ventralization, a major patterning process in the developing vertebrate neural tube (central nervous system, CNS), depends on Sonic hedgehog (SHH) as a main signaling morphogen. We studied the CNS of late larval and young adult zebrafish in a transgenic shh-GFP line revealing increased neuroanatomical detail due to the progressed differentiation state compared to earlier stages. Some major findings emerge from the present study. (a) shh -GFP is still expressed along the adult zebrafish CNS neuraxis in most locations seen in larvae. (b) We newly identify a ventroposterior shh pallidal domain representing the basal telencephalic signaling center important for basal ganglia development known in other vertebrates (i.e., the anterior entopeduncular area-basal medial ganglionic eminence of mammals). (c) We further show late-emerging shh-GFP positive radial glia cells in the medial zone of the dorsal telencephalon (i.e., the teleostan pallial amygdala). (d) Immunostains for tyrosine hydroxylase demonstrate that there is selective colocalization in adult dopamine cells with shh-GFP in the posterior tuberculum, including in projection cells to striatum, which represents a striking parallel to amniote mesodiencephalic dopamine cell origin from shh expressing floor plate cells. (e) There is no colocalization of shh and islet1 as shown by respective shh-GFP and islet1-GFP lines. (f) The only radially far migrated shh-GFP cells are located in the preglomerular area. (g) There are no adult cerebellar and tectal shh-GFP cells confirming their exclusive role during early development as previously reported by our laboratory.

Sonic Hedgehog Protein is Frequently Up-Regulated in Pancreatic Cancer Compared to Colorectal Cancer.

Sonic hedgehog (SHH) is a secreted protein which functions in autocrine or paracrine fashion on target cells to activate hedgehog (HH) signalling cascade responsible for growth and proliferation. This study is an attempt to understand the expression dynamics of SHH protein in colon, rectal and pancreatic cancers. Protein expression of SHH was studied by Western Blotting in the histologically confirmed colon, rectum and pancreatic cancer tissue samples along with their adjacent normal tissues. Only 31.4% (11 of 35) and 26.9% (7 of 26) of colon and rectal cancer cases respectively showed an increase in SHH expression in tumours compared to 72.7% (24 of 33) of the pancreatic cancer cases when compared with their adjacent normal tissues. Our results suggest that SHH may have a strong role in the predisposition of Pancreatic cancer and could possibly be used as a diagnostic or prognostic biomarker.

SHH signaling mediated by a prechordal and brain enhancer controls forebrain organization.

Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.

Lentiviral­mediated Shh reverses the adverse effects of high glucose on osteoblast function and promotes bone formation via Sonic hedgehog signaling.

Patients with diabetes tend to have an increased incidence of osteoporosis, which may be associated with hyperglycemia; however, the pathogenic mechanisms governing this interaction remain unknown. The present study sought to investigate whether elevated extracellular glucose levels of bone mesenchymal stem cells (BMSCs) could influence osteoblastic differentiation and whether the intracellular Sonic hedgehog (Shh) pathway could adjust the effects. Furthermore, to verify the results in vivo, a rat tooth extraction model was constructed. BMSCs were incubated in eight types of culture medium, including low glucose (LG), LG + lentivirus (Lenti), LG + Lenti­small interfering RNA (Lenti­siRNA), LG + Lenti­Shh, high glucose (HG), HG + Lenti, HG + Lenti­siRNA and HG + Lenti­Shh. The lentiviral transfection efficiency was observed using a fluorescence microscope; protein and mRNA expression was detected by western blotting and reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The matrix mineralization and alkaline phosphatase (ALP) activity of BMSCs were examined by Alizarin red staining and ALP activity assays, respectively. The expression of osteogenesis­related genes in BMSCs were quantified by RT­qPCR. The alveolar ridge reduction was measured and histological sections were used to evaluate new bone formation in the tooth socket. With high concentrations of glucose, Shh expression, matrix mineralization nodules formation, ALP activity and the levels of bone morphogenic protein 4 (BMP4), bone sialoprotein (BSP) and osteopontin (OPN) expression were greatly reduced compared with LG and corresponding control groups. Whereas activated Shh signaling via Lenti­Shh could increase the number of matrix mineralization nodules, ALP activity, and the expression levels of BMP4, BSP and OPN in BMSCs. Additionally, in vivo assays demonstrated that Lenti­Shh induced additional bone formation. Collectively, the results of the present study indicated that HG inhibited the Shh pathway in osteoblasts and resulted in patterning defects during osteoblastic differentiation and bone formation, while the activation of Shh signaling could suppress these deleterious effects.

Genetic detection of Sonic hedgehog (Shh) expression and cellular response in the progression of acute through chronic demyelination and remyelination.

Multiple sclerosis is a demyelinating disease in which neurological deficits result from damage to myelin, axons, and neuron cell bodies. Prolonged or repeated episodes of demyelination impair remyelination. We hypothesized that augmenting Sonic hedgehog (Shh) signaling in chronically demyelinated lesions could enhance oligodendrogenesis and remyelination. Shh regulates oligodendrocyte development during postnatal myelination, and maintains adult neural stem cells. We used genetic approaches to detect Shh expression and Shh responding cells in vivo. ShhCreERT2 or Gli1CreERT2 mice were crossed to reporter mice for genetic fate-labeling of cells actively transcribing Shh or Gli1, an effective readout of canonical Shh signaling. Tamoxifen induction enabled temporal control of recombination at distinct stages of acute and chronic cuprizone demyelination of the corpus callosum. Gli1 fate-labeled cells were rarely found in the corpus callosum with tamoxifen given during acute demyelination stages to examine activated microglia, reactive astrocytes, or remyelinating cells. Gli1 fate-labeled cells, mainly reactive astrocytes, were observed in the corpus callosum with tamoxifen given after chronic demyelination. However, Shh expressing cells were not detected in the corpus callosum during acute or chronic demyelination. Finally, SAG, an agonist of both canonical and type II non-canonical Hedgehog signaling pathways, was microinjected into the corpus callosum after chronic demyelination. Significantly, SAG delivery increased proliferation and enhanced remyelination. SAG did not increase Gli1 fate-labeled cells in the corpus callosum, which may indicate signaling through the non-canonical Hedgehog pathway. These studies demonstrate that Hedgehog pathway interventions may have therapeutic potential to modulate astrogliosis and to promote remyelination after chronic demyelination.

WIF-1 and Ihh Expression and Clinical Significance in Patients With Lung Squamous Cell Carcinoma and Adenocarcinoma.

This study investigated the expression of wingless-type inhibitory factor-1 (WIF-1) and Ihh protein in tumor tissues and their clinical significance in patients with lung squamous cell carcinoma and adenocarcinoma. The expression of WIF-1 and Ihh protein in 74 squamous cell carcinomas and 76 adenocarcinomas was measured by immunohistochemistry. The percentage of positive WIF-1 protein expression was significantly higher, while positive Ihh protein expression was significantly lower in patients with well-differentiated lung squamous cell carcinoma and adenocarcinoma, tumor node metastasis (TNM) stage I disease, and lymph node metastasis than that in patients with poorly differentiated tumor, TNM stage III disease, and lymph node metastasis (P<0.05, <0.01). Kaplan-Meier survival analysis showed that TNM stage and lymph node metastasis were significantly associated with the mean overall survival of patients with lung squamous cell carcinoma and adenocarcinoma (P<0.05 or <0.01). Patients with lung squamous cell carcinoma (P=0.037) and adenocarcinoma (P=0.001) with positive Ihh protein expression survived significantly shorter than patients with negative Ihh protein expression. In contrast, no significant difference in mean survival was observed in patients with lung squamous cell carcinoma and adenocarcinoma with positive and negative WIF-1 protein expression (P>0.05). Ihh is a marker for poor prognosis in patients with lung squamous cell carcinoma and adenocarcinoma. WIF-1 is not a predictive marker for lung cancer.

Keratinocyte Sonic Hedgehog Upregulation Drives the Development of Giant Congenital Nevi via Paracrine Endothelin-1 Secretion.

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.

Hedgehog signaling pathway as a potential target in the treatment of advanced gastric cancer.

The sonic hedgehog signaling pathway is critical for cell proliferation, cell differentiation, and organ development during embryogenesis. Aberrant activation of the sonic hedgehog pathway has been associated with a variety of human cancers. Currently, there is no target molecular drug that clinically affects sonic hedgehog activation in patients with gastric cancer. In this review, we will focus on the current clinical treatment options for advanced gastric cancer and discuss the sonic hedgehog signaling pathway. We also review our understanding of the role of sonic hedgehog signaling in advanced gastric cancer progression. Finally, we will describe current known molecular pathways that crosstalk with the sonic hedgehog pathway in cancer stem cells.

CGRP attenuates hyperoxia-induced oxidative stress-related injury to alveolar epithelial type II cells via the activation of the Sonic hedgehog pathway.

The aim of this study was to examine the effect of calcitonin gene-related peptide (CGRP) on primary alveolar epithelial type II (AECII) cells and expression of Sonic hedgehog (SHH) signaling pathway components following exposure to hyperoxia. The AECII cells were isolated and purified from premature rats and exposed to air (21% oxygen), air + CGRP, hyperoxia (95% oxygen) or hyperoxia + CGRP. The production of intracellular reactive oxygen species (ROS) was determined using the 2',7'-dichlorofluorescin diacetate molecular probe. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the culture supernatant were detected by spectrophotometry. The apoptosis of AECII cells was assayed by flow cytometry, and the mRNA and protein expression levels of Shh and Ptc1 in the AECII cells were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunofluorescence, respectively. The cellular pathological changes partly improved and apoptosis was markedly decreased upon treatment with CGRP under hyperoxic conditions. The levels of ROS in the hyperoxia + CGRP group were significantly lower than thoe in the hyperoxia group. In addition, the hyperoxia-induced increase in MDA levels and the decrease in SOD activity in the culture supernatant of the AECII cells were attenuated by CGRP. Compared with the cells exposed to air, hyperoxia markedly inhibited the mRNA and protein expression levels of Shh and Ptc1 in the AECII cells; however, this inhibition was partly attenuated by treatment with CGRP. On the whole, our data suggest that CGRP can partly protect AECII cells from hyperoxia-induced injury, and the upregulation of CGRP may be a potential therapeutic approach with which to combat hyperoxia-induced lung injury, which may be associated with the activation of the SHH signaling pathway.

In smokers, Sonic hedgehog modulates pulmonary endothelial function through vascular endothelial growth factor.

BACKGROUND: Tobacco-induced pulmonary vascular disease is partly driven by endothelial dysfunction. The Sonic hedgehog (SHH) pathway is involved in vascular physiology. We sought to establish whether the SHH pathway has a role in pulmonary endothelial dysfunction in smokers. METHODS: The ex vivo endothelium-dependent relaxation of pulmonary artery rings in response to acetylcholine (Ach) was compared in 34 current or ex-smokers and 8 never-smokers. The results were expressed as a percentage of the contraction with phenylephrine. We tested the effects of SHH inhibitors (GANT61 and cyclopamine), an SHH activator (SAG) and recombinant VEGF on the Ach-induced relaxation. The level of VEGF protein in the pulmonary artery ring was measured in an ELISA. SHH pathway gene expression was quantified in reverse transcriptase-quantitative polymerase chain reactions. RESULTS: Ach-induced relaxation was much less intense in smokers than in never-smokers (respectively 24 ± 6% and 50 ± 7% with 10-4M Ach; p = 0.028). All SHH pathway genes were expressed in pulmonary artery rings from smokers. SHH inhibition by GANT61 reduced Ach-induced relaxation and VEGF gene expression in the pulmonary artery ring. Recombinant VEGF restored the ring's endothelial function. VEGF gene and protein expression levels in the pulmonary artery rings were positively correlated with the degree of Ach-induced relaxation and negatively correlated with the number of pack-years. CONCLUSION: SHH pathway genes and proteins are expressed in pulmonary artery rings from smokers, where they modulate endothelial function through VEGF.

Molecular Profiling of the Developing Lacrimal Gland Reveals Putative Role of Notch Signaling in Branching Morphogenesis.

Purpose: Although normal function of the lacrimal gland is essential for vision (and thus for human well-being), the lacrimal gland remains rather poorly understood at a molecular level. The purpose of this study was to identify new genes and signaling cascades involved in lacrimal gland development. Methods: To identify these genes, we used microarray analysis to compare the gene expression profiles of developing (embryonic) and adult lacrimal glands. Differential data were validated by quantitative RT-PCR, and several corresponding proteins were confirmed by immunohistochemistry and Western blot analysis. To evaluate the role of NOTCH signaling in lacrimal gland (LG) development, we used the NOTCH inhibitor DAPT and conditional Notch1 knockouts. Results: Our microarray data and an in silico reconstruction of cellular networks revealed significant changes in the expression patterns of genes from the NOTCH, WNT, TGFß, and Hedgehog pathways, all of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Our study also revealed new putative lacrimal gland stem cell/progenitor markers. We found that inhibiting Notch signaling both increases the average number of lacrimal gland lobules and reduces the size of each lobule. Conclusions: Our findings suggest that NOTCH-, WNT-, TGFß-, and Hedgehog-regulated EMT transition are critical mechanisms in lacrimal gland development and morphogenesis. Our data also supports the hypothesis that NOTCH signaling regulates branching morphogenesis in the developing lacrimal gland by suppressing cleft formation.

Expression and role of anion exchanger 1 in esophageal squamous cell carcinoma.

Recent studies have described important roles for the anion exchanger (AE) in epithelial carcinogenesis and tumor behavior. The objectives of the present study were to investigate the role of AE1 in the regulation of genes involved in tumor progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC). An immunohistochemical analysis was performed on 61 primary tumor samples obtained from ESCC patients who underwent esophagectomy. AE1 was primarily located in the cell membranes or cytoplasm of carcinoma cells, and its distribution pattern was related to the histological degree of the differentiation of SCC or the pT category. Among patients with pT2-3 ESCC, the 5-year survival rate of patients with diffuse AE1 expression (40.2%) was significantly lower than that of patients with focal expression (74.0%). AE1 was strongly expressed in KYSE150 and TE8 human ESCC cells. The depletion of AE1 using siRNA inhibited cell proliferation, migration, and invasion and induced apoptosis. The results of the microarray analysis revealed that MAPK and Hedgehog signaling pathway-related genes, such as DHH, and GLI1, were down-regulated in AE1-depleted KYSE150 cells. In conclusions, the results of the present study suggest that the diffuse expression of AE1 is related to a worse prognosis in patients with advanced ESCC, and that it regulates tumor progression by affecting MAPK and Hedgehog signaling pathways. These results provide an insight into the role of AE1 as a mediator of and/or a biomarker for ESCC.

High variability of expression profiles of homeologous genes for Wnt, Hh, Notch, and Hippo signaling pathways in Xenopus laevis.

Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization.

Tetramethylpyrazine attenuates sinusoidal angiogenesis via inhibition of hedgehog signaling in liver fibrosis.

Accumulating evidence indicates that hedgehog signaling plays a pivotal role in pathological angiogenesis and is involved in wound-healing responses in a number of adult tissues, including the liver. We previously demonstrated that hedgehog signaling promoted proliferation and inhibited apoptosis in hepatic stellate cells. This study was aimed to evaluate the effect of tetramethylpyrazine (TMP) on hedgehog signaling and to further examine the molecular mechanisms of TMP-induced antiangiogenesic effects in liver fibrosis. We found that TMP ameliorated the expression of proangiogenic markers vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor 2 (VEGF-R2), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor-ß receptor (PDGF-ßR) and hypoxia inducible factor 1α (HIF-1α), concomitant with reduced abundance of endothelial markers platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), CD34 and von willebrand factor in vivo and in vitro. Interestingly, TMP attenuated the abundance of sonic hedgehog, smoothened (Smo) and glioblastoma but increased the expression of hedgehog-interacting protein in liver sinusoidal endothelial cells, which was underlying mechanism for the antiangiogenesic activity of TMP. Downregulation of Smo activity, using selective Smo inhibitor cyclopamine, lead to a synergistic effect with TMP, whereas Smo overexpression plasmid impaired the induction of antiangiogenesic effects of TMP. Overall, these results provide novel implications to reveal the molecular mechanism of TMP-inhibited liver sinusoidal angiogenesis, by which points to the possibility of using TMP-based antiangiogenic drugs for the treatment of liver fibrosis. © 2017 IUBMB Life, 69(2):115-127, 2017.

Resveratrol Pretreatment Decreases Ischemic Injury and Improves Neurological Function Via Sonic Hedgehog Signaling After Stroke in Rats.

Resveratrol has neuroprotective effects for ischemic cerebral stroke. However, its neuroprotective mechanism for stroke is less well understood. Beneficial actions of the activated Sonic hedgehog (Shh) signaling pathway in stroke, such as improving neurological function, promoting neurogenesis, anti-oxidative, anti-apoptotic, and pro-angiogenic effects, have been noted, but relatively little is known about the role of Shh signaling in resveratrol-reduced cerebral ischemic injury after stroke. The present study tests whether the Shh pathway mediates resveratrol to decrease cerebral ischemic injury and improve neurological function after stroke. We observed that resveratrol pretreatment significantly improved neurological function, decreased infarct volume, enhanced vitality, and reduced apoptosis of neurons in vivo and vitro after stroke. Meanwhile, expression levels of Shh, Ptc-1, Smo, and Gli-1 mRNAs were significantly upregulated and Gli-1 was relocated to the nucleus. Intriguingly, in vivo and in vitro inhibition of the Shh signaling pathway with cyclopamine, a Smo inhibitor, completely reversed the above effects of resveratrol. These results suggest that decreased cerebral ischemic injury and improved neurological function by resveratrol may be mediated by the Shh signaling pathway.

Increased expression of the sonic hedgehog and vascular endothelial growth factor with co-localization in varicocele veins.

Objectives Varicocele is characterized by dilatation and tortuosity of the internal spermatic vein. Sonic hedgehog plays an important role in angiogenesis and vascular remodeling under hypoxic stress. We studied the relationship and distribution of SHH and vascular endothelial growth factor in internal spermatic vein in patients diagnosed with varicocele. Methods Specimens of 1 cm were taken from the internal spermatic vein during left varicocele repair (N = 20). The control samples of ISV were obtained from eight male patients who underwent left inguinal herniorrhaphy. We analyzed the sonic hedgehog and vascular endothelial growth factor expression and distribution by immunoblotting, immunohistochemistry, immunofluorescent staining, and confocal laser scanning microscopy. The data were analyzed using the Student's t test. Results Immunoblotting showed higher expression of sonic hedgehog and vascular endothelial growth factor proteins in varicocele veins than in the control group ( P < 0.05) which located over muscle layer and endothelium was demonstrated by immunohistochemical staining. Both proteins with co-localization in the muscle layer and especially distributed in endothelium of varicocele veins were revealed under confocal microscopy. Conclusions These findings showed the upexpression of sonic hedgehog and vascular endothelial growth factor with co-localization in varicocele veins which imply that the reducing hypoxia or using sonic hedgehog antagonists may be helpful for this vascular disease.