Ya Gao

Propranolol induces regression of hemangioma cells via the down-regulation of the PI3K/Akt/eNOS/VEGF pathway.

Infantile hemangioma (IH) is a benign vascular neoplasm resulting from the abnormal proliferation of endothelial cells and pericytes in infants. Propranolol, a non‐selective β‐adrenergic blocker, has recently emerged as an effective therapy for IH, causing regression. However, its potential therapeutic mechanism remains largely unknown.

Propranolol represses infantile hemangioma cell growth through the β2-adrenergic receptor in a HIF-1α-dependent manner

Propranolol, as a non-selective blocker of the β-adrenergic receptor (AR), is utilised as the first-line treatment for infantile hemangiomas. However, the underlying mechanism remains poorly understood. The present study was designed to investigate the molecular basis of propranolol on the regression of infantile hemangiomas using a proliferating infantile hemangioma-derived endothelial cell line. In infantile hemangioma patients, we found that propranolol significantly decreased the expression levels of the hypoxia inducible factor (HIF)-1α in serum and urine, as well as in hemangioma tissues. In vitro analysis revealed that propranolol reduces the expression of HIF-1α in hemangioma cells in a dose- and time-dependent manner, mainly by acting on β2-AR. Interestingly, it was observed that overexpression of HIF-1α apparently abrogated the inhibitory effects of propranolol on vascular endothelial growth factor (VEGF) expression and cell growth. Our data further demonstrated that propranolol inhibited the signal transducer and activator of transcription 3 (STAT3), a critical oncogenic signaling molecule, and the anti-apoptotic protein Bcl-2. Additionally, overexpression of HIF-1α significantly reversed the inhibitory effects of propranolol on STAT3 signaling. In a mouse xenograft hemangioma model, overexpression of HIF-1α significantly attenuated the therapeutic effects of propranolol and inhibited propranolol-induced hemangioma cell apoptosis. Moreover, the protein levels of VEGF, phosphorylated STAT3, total STAT3 and Bcl-2 were significantly upregulated by HIF-1α overexpression in propranolol-treated nude mice bearing hemangiomas. Collectively, our data provide evidence that propranolol may regress infantile hemangiomas by suppressing VEGF and STAT3 signaling pathways in an HIF-1α-dependent manner.

Combination of basic fibroblast growth factor and epidermal growth factor enhances proliferation and neuronal/glial differential of postnatal human enteric neurosphere cells in vitro.

Human enteric neural stem cells (hENSCs) proliferate and differentiate into neurons and glial cells in response to a complex network of neurotrophic factors to form the enteric nervous system. The primary aim of this study was to determine the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on in-vitro expansion and differentiation of postnatal hENSCs-containing enteric neurosphere cells. Enteric neurosphere cells were isolated from rectal polyp specimens of 75 children (age, 1–13 years) and conditioned with bFGF, EGF, bFGF+EGF, or plain culture media. Proliferation of enteric neurosphere cells was examined using the methyl thiazolyl tetrazolium colorimetric assay over 7 days of culture. Fetal bovine serum (10%) was added to induce the differentiation of parental enteric neurosphere cells, and differentiated offspring cells were immunophenotyped against p75 neutrophin receptor (neural stem cells), peripherin (neuronal cells), and glial fibrillary acidic protein (glial cells). Combining bFGF and EGF significantly improved the proliferation of enteric neurosphere cells compared with bFGF or EGF alone (both P<0.01) throughout 7 days of culture. The addition of bFGF drove a significantly greater proportion of enteric neurosphere cells to differentiate into neuronal cells than that of EGF (P<0.01), whereas addition of EGF resulted in significantly more glial differentiation compared with addition of bFGF (P<0.01). Combining bFGF and EGF drove enteric neurosphere cells to differentiate into neuronal cells in a proportion similar to glial cells. Our results showed that the combination of bFGF and EGF significantly enhanced the proliferation and differentiation of postnatal hENSCs-containing enteric neurosphere cells in vitro.

Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro

A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprungs disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8 was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin(+) cells decreased whilst the percentage of GFAP(+) cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies.

Nontoxicity of lentiviral vector infection to viability, migration, apoptosis, and differentiation of postnatal rat enteric neural crest-derived cells.

Lentiviral vector infection of enhanced green fluorescent protein fluorescence reporter genes in enteric neural crest-derived cells maintained efficient, stable, long-term labeling and the infected enteric neural crest-derived cells could survive, proliferate, and express fluorescent reporter genes. However, the method does not show whether there is some defined or undefined toxicity to the enteric neural crest-derived cells, which may affect enteric neural crest-derived cells’ properties. Here, we evaluated the enteric neural crest-derived cells properties under the influence of lentivirus infection of enhanced green fluorescent protein fluorescence reporter genes. This study used the cell count kit-8 for measurement of vitality, transwell for cell migration, immunocytochemistry for cell count and identification, and tested the apoptosis of the enteric neural crest-derived cells with flow cytometry. The enteric neural crest-derived cells with or without lentivirus and their derivative enteric neural crest-derived cells could form characteristic neurospheres, and maintain their level of fluorescent label steady. When cultured under inducing conditions, enteric neural crest-derived cells differentiated into neurons and glia. The results showed that the enteric neural crest-derived cells with or without lentivirus showed no significant difference in viability, migration, apoptosis, neuronal, and glial ratio. The study identified that lentivirus can be used in a nontoxic manner for infection of enhanced green fluorescent protein fluorescence reporter genes into enteric neural crest-derived cells.

Clinical observations on the treatment of infantile hemangiomas with topical imiquimod 5% cream

Abstract Objective To observe the efficacy and safety of topical imiquimod 5% cream in the treatment of uncomplicated infantile hemangiomas (IHs). Methods A total of 68 IHs were treated with topical imiquimod 5% cream. Among them, 36 were superficial, 22 were mixed, and 10 were deep. The size of IHs ranged from 1.0 cm, × 1.5 cm to an area of a whole forearm. All the hemangiomas were in a proliferative stage. Imiquimod was applied 3 times weekly in 44 patients and 5 times weekly in 24 patients for up to 36 weeks. Results All superficial IHs improved, and 18 achieved complete clinical resolution, 10 had excellent improvement, 5 showed moderate improvements, and 3 patients displayed minimal improvement. Two mixed IHs showed excellent improvement, 3 showed moderate improvement and 5 manifested minimal improvements. The remaining 12 mixed IHs and all deep IHs did not respond to the therapy. The total incidence of local adverse events was 58.82%(40/68), which included erythema or edema, local itching, incrustation or peeling, erosion or ulceration, although most of these were mild to moderate reactions and did not affect the treatment. Scarring occurred in 2 mixed IHs. No systemic side effects developed. Conclusion Imiquimod 5% cream may be a safe and effective alternative for the treatment of superficial IHs and some mixed IHs in which the superficial component predominates. An appropriate treatment duration for proliferative IHs treated with this therapy may be 24 weeks. Some local adverse events, such as crusting and erosion with possible scarring potential may occur and should be addressed by prompt, but temporary, discontinuation of the imiquimod. Topical imiquimod 5% cream can be prudently used in the treatment of IHs larger than 5.0 cm, × 5.0 cm in newborns and infants less than 6 months of age. To our knowledge, this is the largest IH group treated with imiquimod that has been reported in the literature to date.

Identifying key genes associated with Hirschsprung’s disease based on bioinformatics analysis of RNA-sequencing data

BackgroundHirschsprung’s disease (HSCR) is a type of megacolon induced by deficiency or dysfunction of ganglion cells in the distal intestine and is associated with developmental disorders of the enteric nervous system. To explore the mechanisms of HSCR, we analyzed the RNA-sequencing data of the expansion and the narrow segments of colon tissues separated from children with HSCR.MethodsRNA-sequencing of the expansion segments and the narrow segments of colon tissues isolated from children with HSCR was performed. After differentially expressed genes (DEGs) were identified using the edgeR package in R, functional and pathway enrichment analyses of DEGs were carried out using DAVID software. To further screen the key genes, protein-protein interaction (PPI) network and module analyses were conducted separately using Cytoscape software.ResultsA total of 117 DEGs were identified in the expansion segment samples, including 47 up-regulated and 70 down-regulated genes. Functional enrichment analysis suggested that FOS and DUSP1 were implicated in response to endogenous stimulus. In the PPI network analysis, FOS (degree=20), EGR1 (degree=16), ATF3 (degree=9), NOS1 (degree=8), CCL5 (degree=8), DUSP1 (degree=7), CXCL3 (degree=6), VIP (degree=6), FOSB (degree=5), and NOS2 (degree=4) had higher degrees, which could interact with other genes. In addition, two significant modules (module 1 and module 2) were identified from the PPI network.ConclusionsSeveral genes (including FOS, EGR1, ATF3, NOS1, CCL5, DUSP1, CXCL3, VIP, FOSB, and NOS2) might be involved in the development of HSCR through their effect on the nervous system.

Combination of exogenous cell transplantation and 5-HT4 receptor agonism induce endogenous enteric neural crest-derived cells in a rat hypoganglionosis model

ABSTRACT Enteric neural crest‐derived cells (ENCCs) can migrate into endogenous ganglia and differentiate into progeny cells, and have even partially rescued bowel function; however, poor reliability and limited functional recovery after ENCC transplantation have yet to be addressed. Here, we investigated the induction of endogenous ENCCs by combining exogenous ENCC transplantation with a 5‐HT4 receptor agonist mosapride in a rat model of hypoganglionosis, established by benzalkonium chloride treatment. ENCCs, isolated from the gut of newborn rats, were labeled with a lentiviral eGFP reporter. ENCCs and rats were treated with the 5‐HT4 receptor agonist/antagonist. The labeled ENCCs were then transplanted into the muscular layer of benzalkonium chloride‐treated colons. At given days post‐intervention, colonic tissue samples were removed for histological analysis. ENCCs and neurons were detected by eGFP expression and immunoreactivity to p75NTR and peripherin, respectively. eGFP‐positive ENCCs and neurons could survive and maintain levels of fluorescence after transplantation. With longer times post‐intervention, the number of peripherin‐positive cells gradually increased in all groups. Significantly more peripherin‐positive cells were found following ENCCs plus mosapride treatment, compared with the other groups. These results show that exogenous ENCCs combined with the 5‐HT4 receptor agonist effectively induced endogenous ENCCs proliferation and differentiation in a rat hypoganglionosis model. HIGHLIGHTSSurvival and differentiation of exogenous ENCCs in treated colons.With longer times post‐intervention, the number of ENCCs and their progeny cells gradually increased.Exogenous ENCCs combined with the 5‐HT4 receptor agonist ffectively induced ENCCs proliferation and differentiation.

A Time-Limited and Partially Reversible Model of Hypoganglionosis Induced by Benzalkonium Chloride Treatment

Serosal application of benzalkonium chloride (BAC) has been previously applied to produce a model of aganglionosis; however, confusion remains regarding the extent of chemical ablation of enteric myenteric plexus after BAC treatment. The time sequence of BAC-induced effects on the myenteric plexus of the rat colon was determined and followed the morphologic changes. After sacrifice of animals 7, 14, 28, 56, 84 or 168xa0days postintervention, colonic tissue samples were removed, fixed in formalin, and cut into 5-μm longitudinal sections for histological analysis. The neural analysis was used to re-evaluate BAC treatments for the appropriate model. Compared with rats in sham groups, rats in 0.1xa0%-30-min BAC group maintained only 15.27xa0±xa04.80xa0% of ganglia per section in a 1-cm/5-μm slice and 11.76xa0±xa02.30xa0% of ganglionic cells after 28xa0days, the lower and stable number of ganglionic cells between Day 7 and 84 (from 11.67xa0±xa02.10 to 19.05xa0±xa05.10xa0%). Although an increase, ganglionic cell numbers did not recover at Day168 when compared with the numbers in sham groups. The results showed that characteristics of rats in the 0.1xa0%-30-min BAC group between Day 7 and 84 most closely kept in stable state, suggesting that these treatment parameters are ideal for producing a hypoganglia model of hypoganglionosis.

Detection of autophagy in Hirschsprung's disease: implication for its role in aganglionosis.

Hirschsprung’s disease (HD) is a common congenital gastrointestinal malformation, characterized by the lack of ganglion cells from the distal rectum to the proximal bowel, but the pathogenesis is not well understood. This paper evaluates the effects of autophagy in HD. Using electron microscopy, the autophagosomes were detected in three segments: narrow segment (NS), transitional segment (TS), and dilated segment (DS). Typical autophagosome structures are found in the Auerbach plexus of both NS and TS. Real-time PCR results showed that Beclin1 (NS vs. TS, P<0.01) and LC3 (NS vs. TS, P<0.05) mRNA were the highest in the NS, but p75 (NS vs. TS, P<0.01) was the highest in the DS. Correlation analysis results showed a positive correlation between Beclin1 and LC3 mRNA levels (R=0.736, P=0.000), whereas inverse correlations were found between p75 and Beclin1/LC3 mRNA levels (p75 vs. Beclin1: R=−0.714, P=0.000; p75 vs. LC3: R=−0.619, P=0.000). Immunohistochemistry analyses indicated a consistent result with mRNA levels, by increased Beclin1-positive and LC3-positive neurons, but reduced p75-positive neurons in the Auerbach plexus of TS compared with DS. These findings indicated that autophagy exists in the bowel of patients with HD. On the basis of the detection of the highest expression of the autophagy genes in NS, autophagy may additionally cause the lack of neurons.