A novel indole derivative, 2-{3-[1-(benzylsulfonyl)piperidin-4-yl]-2-methyl-1H-indol-1-yl}-1-(pyrrolidin-1-yl)ethenone, suppresses hedgehog signaling and drug-resistant tumor growth.

The Hedgehog (Hh) signaling pathway plays important roles in various physiological functions. Several malignancies, such as basal cell carcinoma (BCC) and medulloblastoma (MB), have been linked to the aberrant activation of Hh signaling. Although therapeutic drugs have been developed to inhibit Hh pathway-dependent cancer growth, drug resistance remains a major obstacle in cancer treatment. Here, we show that the newly identified, 2-{3-[1-(benzylsulfonyl)-1,2,3,6-tetrahydropyridin-4-yl]-2-methyl-1H-indol-1-yl}-1-(pyrrolidin-1-yl)ethenone analog (LKD1214) exhibits comparable potency to vismodegib in suppressing the Hh pathway activation. LKD1214 represses Smoothened (SMO) activity by blocking its ciliary translocation. Interestingly, we also identified that it has a distinctive binding interface with SMO compared with other SMO-regulating chemicals. Notably, it maintains an inhibitory activity against the SmoD477H mutant, as observed in a patient with vismodegib-resistant BCC. Furthermore, LKD1214 inhibits tumor growth in the mouse model of MB. Collectively, these findings suggest that LKD1214 has the therapeutic potential to overcome drug-resistance in Hh-dependent cancers.

Staphylococcal enterotoxin B exposed to pregnant rats inhibits the hedgehog signaling pathway in thymic T lymphocytes of the offspring.

The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 µg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.

Reappraisal of the Roles of the Sonic Hedgehog Signaling Pathway in Hepatocellular Carcinoma.

HCC remains one of the leading causes of cancer-related death globally. The main challenges in treatments of hepatocellular carcinoma (HCC) primarily arise from high rates of postoperative recurrence and the limited efficacy in treating advanced-stage patients. Various signaling pathways involved in HCC have been reported. Among them, the Sonic hedgehog (SHH) signaling pathway is crucial. The presence of SHH ligands is identified in approximately 60% of HCC tumor tissues, including tumor nests. PTCH-1 and GLI-1 are detected in more than half of HCC tissues, while GLI-2 is found in over 84% of HCC tissues. The SHH signaling pathway (including canonical and non-canonical) is involved in different aspects of HCC, including hepatocarcinogenesis, tumor growth, tumor invasiveness, progression, and migration. The SHH signaling pathway also contributes to recurrence, metastasis, modulation of the cancer microenvironment, and sustaining cancer stem cells. It also affects the resistance of HCC cells to chemotherapy, target therapy, and radiotherapy. Reappraisal of the roles of the SHH signaling pathway in HCC may trigger some novel therapies for HCC.

Role of Hedgehog Signaling Pathways in Multipotent Mesenchymal Stem Cells Differentiation.

Multipotent mesenchymal stem cells (MSCs) have high self-renewal and multi-lineage differentiation potentials and low immunogenicity, so they have attracted much attention in the field of regenerative medicine and have a promising clinical application. MSCs originate from the mesoderm and can differentiate not only into osteoblasts, cartilage, adipocytes, and muscle cells but also into ectodermal and endodermal cell lineages across embryonic layers. To design cell therapy for replacement of damaged tissues, it is essential to understand the signaling pathways, which have a major impact on MSC differentiation, as this will help to integrate the signaling inputs to initiate a specific lineage. Hedgehog (Hh) signaling plays a vital role in the development of various tissues and organs in the embryo. As a morphogen, Hh not only regulates the survival and proliferation of tissue progenitor and stem populations but also is a critical moderator of MSC differentiation, involving tri-lineage and across embryonic layer differentiation of MSCs. This review summarizes the role of Hh signaling pathway in the differentiation of MSCs to mesodermal, endodermal, and ectodermal cells.

IGFBP5 Promotes Neuronal Apoptosis in a 6-OHDA-Toxicant Model of Parkinson's Disease by Inhibiting the Sonic Hedgehog Signaling Pathway.

INTRODUCTION: Parkinson's disease (PD) is the most common neurodegenerative disease worldwide. Studies have shown that insulin-like growth factor-binding protein 5 (IGFBP5) may contribute to methamphetamine-induced neurotoxicity and neuronal apoptosis in PC-12 cells and rat striatum. Here, we studied the expression and role of IGFBP5 in the 6-OHDA-toxicant model of PD. METHODS: PC-12 and SH-SY5Y cells were exposed to 50 µm 6-OHDA for 24 h. qRT-PCR, western blotting, CCK-8 assay, EdU staining, annexin V staining, and immunofluorescence were performed to study the effects of IGFBP5-specific siRNAs. The effects of IGFBP5 on a rat 6-OHDA model of PD were confirmed by performing behavioral tests, tyrosine hydroxylase (TH) immunofluorescence staining, and western blotting. RESULTS: In the GSE7621 dataset, IGFBP5 was highly expressed in the substantia nigra tissues of PD patients compared to healthy controls. In PC-12 and SH-SY5Y cells, IGFBP5 was upregulated following 6-OHDA exposure in a dose-dependent manner. Silencing of IGFBP5 promoted PC-12 and SH-SY5Y proliferation and inhibited apoptosis under 6-OHDA stimulation. Silencing of IGFBP5 relieved 6-OHDA-induced TH-positive neuron loss. Hedgehog signaling pathway was predicted as a downstream signaling pathway of IGFBP5. Negative regulation between IGFBP5 and sonic hedgehog (SHH) signaling pathway was confirmed in vitro. The effects of IGFBP5 silencing on SH-SY5Y cells were partially reversed using cyclopamine, a direct inhibitor of the SHH signaling pathway. In addition, silencing of IGFBP5 attenuated motor deficits and neuronal damage in 6-OHDA-induced PD rats. CONCLUSION: Elevated IGFBP5 expression may be involved in 6-OHDA-induced neurotoxicity through regulation of the SHH signaling pathway.

EHMT2 Suppresses ARRB1 Transcription and Activates the Hedgehog Signaling to Promote Malignant Phenotype and Stem Cell Property in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) represents the primary subtype of head and neck squamous cell carcinoma (HNSCC), characterized by a high morbidity and mortality rate. Although previous studies have established specific correlations between euchromatic histone lysine methyltransferase 2 (EHMT2), a histone lysine methyltransferase, and the malignant phenotype of OSCC cells, its biological functions in OSCC remain largely unknown. This study, grounded in bioinformatics predictions, aims to clarify the influence of EHMT2 on the malignant behavior of OSCC cells and delve into the underlying mechanisms. EHMT2 exhibited high expression in OSCC tissues and demonstrated an association with poor patient outcomes. Artificial EHMT2 silencing in OSCC cells, achieved through lentiviral vector infection, significantly inhibited colony formation, migration, invasion, and cell survival. Regarding the mechanism, EHMT2 was found to bind the promoter of arrestin beta 1 (ARRB1), thereby suppressing its transcription through H3K9me2 modification. ARRB1, in turn, was identified as a negative regulator of the Hedgehog pathway, leading to a reduction in the proteins GLI1 and PTCH1. Cancer stem cells (CSCs) were enriched through repeated sphere formation assays in two OSCC cell lines. EHMT2 was found to activate the Hedgehog pathway, thus promoting sphere formation, migration and invasion, survival, and tumorigenic activity of the OSCC-CSCs. Notably, these effects were counteracted by the additional overexpression of ARRB1. In conclusion, this study provides novel evidence suggesting that EHMT2 plays specific roles in enhancing stem cell properties in OSCC by modulating the ARRB1-Hedgehog signaling cascade.

USP5 promotes tumorigenesis by activating Hedgehog/Gli1 signaling pathway in osteosarcoma.

Changes in protein ubiquitination have been linked to cancer. Deubiquitinating enzymes (DUBs) counteract E3 ligase activities and have emerged as promising targets for cancer treatment. Ubiquitin-specific peptidase 5 (USP5) is a member of the DUBs family and has been implicated in promoting tumorigenesis in numerous cancers. However, the clinical significance and biological function of USP5 in osteosarcoma (OS) remains unclear. Here, we found elevated USP5 expression in OS tissues compared with normal bone tissues. Furthermore, we observed significant associations of elevated USP5 levels with increased mortality and more malignant phenotypes in OS patients. Moreover, our results revealed that USP5 could facilitate metastasis and cell progression in OS by activating the hedgehog (Hh) signaling pathway using cultured cells and animal tumor models. Mechanistically, USP5 appeared to stabilize and deubiquitinate Gli1, a key mediator of the Hh signaling pathway. Additionally, the oncogenic effect of USP5 in OS was dependent on Gli1 stability. Our findings support the model where USP5 contributes to OS pathogenesis by activating the Hh/Gli1 signaling pathway, making USP5 a potential diagnostic and therapeutic target for OS.

Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling.

Hedgehog (Hh) signaling is crucial in cardiovascular development and maintenance. However, the biological role of Patched1 (Ptch1), an inhibitory receptor of the Hh signaling pathway, remains elusive. In this study, a Ptch1 ortholog was characterized in Nile tilapia (Oreochromis niloticus), and its function was investigated through CRISPR/Cas9 gene knockout. When one-cell embryos were injected with CRISPR/Cas9 targeting ptch1, the mutation efficiency exceeded 70%. During 0-3 days post fertilization (dpf), no significant differences were observed between the ptch1 mutant group and the control group; at 4 dpf (0 day after hatching), about 10% of the larvae showed an angiogenesis defect and absence of blood flow; from 5 dpf, most larvae exhibited an elongated heart, large pericardial cavity, and blood leakage and coagulation, ultimately dying during the 6-8 dpf period due to the lack of blood circulation. Consistently, multiple differentially expressed genes related to angiogenesis, blood coagulation, and heart development were enriched in the ptch1 mutants. Furthermore, Smoothened (Smo) antagonist (cyclopamine) treatment of the ptch1 mutants greatly rescued the cardiovascular disorders. Collectively, our study suggests that Ptch1 is required for cardiovascular development and vascular integrity via Smo signaling, and excessive Hh signaling is detrimental to cardiovascular development.

Beyond cyclopamine: Targeting Hedgehog signaling for cancer intervention.

Hedgehog (Hh) signaling plays a significant role in embryogenesis and several physiological processes, such as wound healing and organ homeostasis. In a pathological setting, it is associated with oncogenesis and is responsible for disease progression and poor clinical outcomes. Hedgehog signaling mediates downstream actions via Glioma Associated Oncogene Homolog (GLI) transcription factors. Inhibiting Hh signaling is an important oncological strategy in which inhibitors of the ligands SMO or GLI have been looked at. This review briefly narrates the Hh ligands, signal transduction, the target genes involved and comprehensively describes the numerous inhibitors that have been evaluated for use in various neoplastic settings.

IFT27 regulates the long-term maintenance of photoreceptor outer segments in zebrafish.

Approximately a quarter of Retinitis Pigmentosa (RP) is caused by mutations in transport-related genes in cilia. IFT27 (Intraflagellar Transport 27), a core component of the ciliary intraflagellar transport (IFT) system, has been implicated as a significant pathogenic gene in RP. The pathogenic mechanisms and subsequent pathology related to IFT27 mutations in RP are largely obscure. Here, we utilized TALEN technology to create an ift27 knockout (ift27-/-) zebrafish model. Electroretinography (ERG) detection showed impaired vision in this model. Histopathological examinations disclosed that ift27 mutations cause progressive degeneration of photoreceptors in zebrafish, and this degeneration was late-onset. Immunofluorescence labeling of outer segments showed that rods degenerated before cones, aligning with the conventional characterization of RP. In cultured human retinal pigment epithelial cells, we found that IFT27 was involved in maintaining ciliary morphology. Furthermore, decreased IFT27 expression resulted in the inhibition of the Hedgehog (Hh) signaling pathway, including decreased expression of key factors in the Hh pathway and abnormal localization of the ciliary mediator Gli2. In summary, we generated an ift27-/- zebrafish line with retinal degeneration which mimicked the symptoms of RP patients, highlighting IFT27's integral role in the long-term maintenance of cilia via the Hh signaling pathway. This work may furnish new insights into the treatment or delay of RP caused by IFT27 mutations.

Identification of rare variants in PTCH2 associated with non-syndromic orofacial clefts.

Orofacial clefts (OFCs) represent the most prevalent congenital craniofacial anomalies, significantly impacting patients' appearance, oral function, and psychological well-being. Among these, non-syndromic OFCs (NSOFCs) are the most predominant type, with the etiology attributed to a combination of genetic and environmental factors. Rare variants of key genes involved in craniofacial development-related signaling pathway are crucial in the occurrence of NSOFCs, and our recent studies have identified PTCH1, a receptor-coding gene in the Hedgehog signaling pathway, as a causative gene for NSOFCs. However, the role of PTCH2, the paralog of PTCH1, in pathogenesis of NSOFCs remains unclear. Here, we perform whole-exome sequencing to explore the genetic basis of 144 sporadic NSOFC patients. We identify five heterozygous variants of PTCH2 in four patients: p.L104P, p.A131G, p.R557H, p.I927S, and p.V978D, with the latter two co-occurring in a single patient. These variants, all proven to be rare through multiple genomic databases, with p.I927S and p.V978D being novel variants and previously unreported. Sequence alignment suggests that these affected amino acids are evolutionarily conserved across vertebrates. Utilizing predictive structural modeling tools such as AlphaFold and SWISS-MODEL, we propose that these variants may disrupt the protein's structure and function. In summary, our findings suggest that PTCH2 may be a novel candidate gene predicted to be associated with NSOFCs, thereby broadening the spectrum of causative genes implicated in the craniofacial anomalies.

Current Status of Hedgehog Signaling Inhibitors.

The Hedgehog (Hh) signaling pathway plays a crucial role in diverse biological processes such as cell differentiation, proliferation, senescence, tumorigenesis, malignant transformation, and drug resistance. Aberrant Hh signaling, resulting from mutations and excessive activation, can contribute to the development of various diseases during different stages of biogenesis and development. Moreover, it has been linked to unfavorable outcomes in several human cancers, including basal cell carcinoma (BCC), multiple myeloma (MM), melanoma, and breast cancer. Hence, the presence of mutations and excessive activation of the Hh pathway presents obstacles and constraints in the realm of cancer treatment. Extant research has demonstrated that small molecule inhibitors are regarded as the most effective therapeutic approaches for targeting the Hh pathway in contrast to traditional chemotherapy and radiotherapy. Consequently, this review focuses on the present repertoire of small molecule inhibitors that target various components of the Hh pathway, including Hh ligands, Ptch receptors, Smo transmembrane proteins, and Gli nuclear transcription factors. This study provides a comprehensive analysis of small molecules' structural and functional aspects in the preclinical and clinical management of cancer. Additionally, it elucidates the obstacles encountered in targeting the Hh pathway for human cancer therapy and proposes potential therapeutic approaches.

Endoplasmic reticulum aminopeptidase 2 regulates CD4+ T cells pyroptosis in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease with a complex pathogenesis that has not yet been fully elucidated, and T-cell pyroptosis is an important pathogenetic factor in RA. This study aimed to investigate the role of endoplasmic reticulum aminopeptidase 2 (ERAP2) in the pyroptosis of CD4+ T cells in RA and the specific molecular mechanism. METHODS: Peripheral venous blood was collected from human subjects, and CD4+ T cells were isolated and activated to measure the level of pyroptosis and ERAP2 expression. Pyroptosis levels were assessed using immunofluorescence, flow cytometry, qRT-PCR, and Western blotting. Changes in pyroptosis levels were observed upon knockdown or overexpression of ERAP2. To detect activated Caspase-1 in tissues, chimeric mice were engrafted with human synovial tissue and reconstituted with human CD4+ T cells. CD4 + T cells were treated with GLI1 antagonists and SMO receptor agonists to detect changes in pyroptosis levels. RESULTS: CD4+ T cell levels undergoing pyroptosis were found to be elevated in the blood and synovium of RA patients. The gene and protein expression of ERAP2 were significantly higher in CD4+ T cells from RA patients. Deletion of ERAP2 suppressed pyroptosis of these cells, attenuated the activation of Caspase-1 in tissue T cells, and reduced tissue inflammatory responses. Reciprocally, overexpression of ERAP2 triggered inflammasome assembly, activated Caspase-1, and induced pyroptosis in CD4+ T cells. Mechanistically, ERAP2 inhibits the Hedgehog signaling pathway and upregulates the expression of nucleotide-binding oligomerization segment-like receptor family 3(NLRP3), cleaved Caspase-1, and Gasdermin D to promote pyroptosis in CD4+ T cells. CONCLUSIONS: Taken together, our results identify a novel mechanism by which ERAP2 regulates RA development and document the effect of the ERAP2/Hedgehog signaling axis on pyroptosis of CD4+ T cells from RA patients.

Identification of Fasudil as a collaborator to promote the anti-tumor effect of lenvatinib in hepatocellular carcinoma by inhibiting GLI2-mediated hedgehog signaling pathway.

Lenvatinib is a frontline tyrosine kinase inhibitor for patients with advanced hepatocellular carcinoma (HCC). However, just 25% of patients benefit from the treatment, and acquired resistance always develops. To date, there are neither effective medications to combat lenvatinib resistance nor accurate markers that might predict how well a patient would respond to the lenvatinib treatment. Thus, novel strategies to recognize and deal with lenvatinib resistance are desperately needed. In the current study, a robust Lenvatinib Resistance index (LRi) model to predict lenvatinib response status in HCC was first established. Subsequently, five candidate drugs (Mercaptopurine, AACOCF3, NU1025, Fasudil, and Exisulind) that were capable of reversing lenvatinib resistance signature were initially selected by performing the connectivity map (CMap) analysis, and fasudil finally stood out by conducting a series of cellular functional assays in vitro and xenograft mouse model. Transcriptomics revealed that the co-administration of lenvatinib and fasudil overcame lenvatinib resistance by remodeling the hedgehog signaling pathway. Mechanistically, the feedback activation of EGFR by lenvatinib led to the activation of the GLI2-ABCC1 pathway, which supported the HCC cell's survival and proliferation. Notably, co-administration of lenvatinib and fasudil significantly inhibited IHH, the upstream switch of the hedgehog pathway, to counteract GLI2 activation and finally enhance the effectiveness of lenvatinib. These findings elucidated a novel EGFR-mediated mechanism of lenvatinib resistance and provided a practical approach to overcoming drug resistance in HCC through meaningful drug repurposing strategies.

Pharmacology of Veratrum californicum Alkaloids as Hedgehog Pathway Antagonists.

Veratrum californicum contains steroidal alkaloids that function as inhibitors of hedgehog (Hh) signaling, a pathway involved in the growth and differentiation of cells and normal tissue development. This same Hh pathway is abnormally active for cell proliferation in more than 20 types of cancer. In this current study, alkaloids have been extracted from the root and rhizome of V. californicum, followed by their separation into five fractions using high performance liquid chromatography. Mass spectrometry was used to identify the presence of twenty-five alkaloids, nine more than are commonly cited in literature reports, and the Bruker Compass Data Analysis software was used to predict the molecular formula for every detected alkaloid. The Gli activity of the raw extract and each fraction were compared to 0.1 µM cyclopamine, and fractions 1, 2, and 4 showed increased bioactivity through suppression of the Hh signaling pathway. Fractions 2 and 4 had enhanced bioactivity, but fraction 1 was most effective in inhibiting Hh signaling. The composition of fraction 1 consisted of veratrosine, cycloposine, and potential isomers of each.

Role of the Hedgehog signaling pathway in hepatocellular carcinoma and its tumor microenvironment / 临床肝胆病杂志

The Hedgehog （Hh） signaling pathway plays an important role in the development and progression of hepatocellular carcinoma and its tumor microenvironment， and abnormal activation of Hh signal can accelerate the growth of tumor. The crosstalk between the Hh signaling pathway and TME is closely associated with tumor growth and the formation of inhibitory tumor microenvironment. Evidence shows that inhibition of Hh signal plays an important role in inhibiting the growth of hepatocellular carcinoma. This article reviews the current research status of the role， mechanism， and potential therapeutic significance of abnormal activation of Hh signal in hepatocellular carcinoma and its tumor microenvironment， so as to provide new ideas for the treatment of hepatocellular carcinoma.

Itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of hemangioma endothelial cells by downregulating the hedgehog signaling pathway.

Infantile hemangioma (IH) is among the most prevalent benign vascular tumours in infants. The pathogenesis of IH mainly involves abnormal proliferation of vascular endothelial cells and the formation of new vessels. Itraconazole was shown to be effective in treating IH; however, the mechanism underlying its action is still unclear. The purpose of this study was to examine the effects of itraconazole on the proliferation, apoptosis, and angiogenesis of hemangioma endothelial cells (HemECs); human umbilical vein endothelial cells served as the control group. The expression of genes involved in the hedgehog (HH) signaling pathway (SHH, PTCH1, SMO, and GLI1) was determined using real-time quantitative polymerase chain reaction. Western blotting was used to determine the expression of related proteins. In this study, itraconazole significantly dose- and time-dependently inhibited the viability of HemECs. Itraconazole suppressed the expression of PCNA, Ki67, and vascular endothelial growth factor (VEGF), demonstrating that this treatment inhibited cell proliferation and angiogenesis. Moreover, itraconazole induced apoptosis of HemECs by activating the expression of BAX and inhibiting the expression of BCL2. Itraconazole inhibited SHH, PTCH1, SMO, and GLI1 expression. Activation of the HH pathway by recombinant human sonic hedgehog (rhSHH) protein attenuated the effect of itraconazole on HemECs. In conclusion, itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of HemECs via the downregulation of the HH signaling pathway. Therefore, itraconazole may be an alternative choice for the treatment of IH.

The emerging role of the hedgehog signaling pathway in immunity response and autoimmune diseases.

The Hedgehog (Hh) family is a prototypical morphogen involved in embryonic patterning, multi-lineage differentiation, self-renewal, morphogenesis, and regeneration. There are studies that have demonstrated that the Hh signaling pathway differentiates developing T cells into MHC-restricted self-antigen tolerant T cells in a concentration-dependent manner in the thymus. Whereas Hh signaling pathway is not required in the differentiation of B cells but is indispensable in maintaining the regeneration of hematopoietic stem cells (HSCs) and the viability of germinal centers (GCs) B cells. The Hh signaling pathway exerts both positive and negative effects on immune responses, which involves activating human peripheral CD4+ T cells, regulating the accumulation of natural killer T (NKT) cells, recruiting and activating macrophages, increasing CD4+Foxp3+ regulatory T cells in the inflammation sites to sustain homeostasis. Hedgehog signaling is involved in the patterning of the embryo, as well as homeostasis of adult tissues. Therefore, this review aims to highlight evidence for Hh signaling in the differentiation, function of immune cells and autoimmune disease. Targeting Hh signaling promises to be a novel, alternative or adjunct approach to treating tumors and autoimmune diseases.

Dl-3-n-butylphthalide promotes angiogenesis in ischemic stroke mice through upregulating autocrine and paracrine sonic hedgehog.

Dl-3-n-butylphthalide (NBP) is a small-molecule drug used in the treatment of ischemic stroke in China, which is proven to ameliorate the symptoms of ischemic stroke and improve the prognosis of patients. Previous studies have shown that NBP accelerates recovery after stroke by promoting angiogenesis. In this study, we investigated the mechanisms underlying the angiogenesis-promoting effects of NBP in ischemic stroke models in vitro and in vivo. OGD/R model was established in human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs), while the tMCAO model was established in mice. The cells were pretreated with NBP (10, 50, 100 µM); the mice were administered NBP (4, 8 mg/kg, i.v.) twice after tMCAO. We showed that NBP treatment significantly stimulated angiogenesis by inducing massive production of angiogenic growth factors VEGFA and CD31 in both in vitro and in vivo models of ischemic stroke. NBP also increased the tubule formation rate and migration capability of HUVECs in vitro. By conducting the weighted gene co-expression network analysis, we found that these effects were achieved by upregulating the expression of a hedgehog signaling pathway. We demonstrated that NBP treatment not only changed the levels of regulators of the hedgehog signaling pathway but also activated the transcription factor Gli1. The pro-angiogenesis effect of NBP was abolished when the hedgehog signaling pathway was inhibited by GDC-0449 in HUVECs, by Sonic Hedgehog(Shh) knockdown in HUVECs, or by intracerebroventricular injection of AAV-shRNA(shh)-CMV in tMCAO mice. Furthermore, we found that HUVECs produced a pro-angiogenic response not only to autocrine Shh, but also to paracrine Shh secreted by astrocytes. Together, we demonstrate that NBP promotes angiogenesis via upregulating the hedgehog signaling pathway. Our results provide an experimental basis for the clinical use of NBP.

Inhibition of CK2α accelerates skin wound healing by promoting endothelial cell proliferation through the Hedgehog signaling pathway.

Diabetes is a chronic disease characterized by perturbed glucose and lipid metabolism, resulting in high blood glucose levels. Many complications induced by endothelial dysfunction can cause disability and even death of diabetic patients. Here, we found that the protein level of casein kinase 2α (CK2α) was increased in the endothelium of mice with type I diabetes (T1D) induced by streptozotocin (STZ) injection. Although a potential correlation between the protein level of CK2α and endothelial dysfunction in diabetes was established, the contribution of CK2α to the progression of endothelial dysfunction in diabetes remained largely unknown. By using CX4945 (a selective CK2α antagonist) and Si-csnk2a1 (small interfering RNA targeting CK2α), we found that inhibition of CK2α accelerated skin wound healing in T1D mice by promoting proliferation of endothelial cells. Administration of CX4945 or Si-csnk2a1 rescued the impaired Hedgehog signaling pathway in high glucose-treated human umbilical vein endothelial cells (HUVECs). Exploration of the underlying molecular mechanism revealed that the protective effect of CK2α inhibition on angiogenesis, which contributes to skin wound healing in diabetic mice, was blocked by administration of GANT61 (an inhibitor targeting the Hedgehog signaling pathway). Our findings establish CK2α as a regulator of endothelial dysfunction in diabetes and demonstrate that inhibition of CK2α accelerates skin wound healing in T1D mice by promoting endothelial cell proliferation via the Hedgehog signaling pathway.