Vincent Chi Hang Lui

Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut

Enteric neural crest cells (NCCs) migrate and colonize the entire gut and proliferate and differentiate into neurons and glia of the enteric nervous system in vertebrate embryos. We have investigated the mitogenic and morphogenic functions of Sonic hedgehog (Shh) on enteric NCCs in cell and organ culture. Enteric NCCs expressed Shh receptor Patched and transcripts encoding the Shh signal transducer (Gli1). Shh promoted the proliferation and inhibited the differentiation of NCCs. The pro-neurogenic effect of glial cell line–derived neurotrophic factor (GDNF) on NCCs was abolished by Shh. In gut explants, NCCs migrated from the explants onto the adjacent substratum if GDNF was added, whereas addition of Shh abolished this migration. Neuronal differentiation and coalescence of neural crest–derived cells into myenteric plexuses in explants was repressed by the addition of Shh. Our data suggest that Shh controls the proliferation and differentiation of NCCs and modulates the responsiveness of NCCs toward GDNF inductions.

Genome-wide association study identifies NRG1 as a susceptibility locus for Hirschsprung's disease

Hirschsprungs disease (HSCR), or aganglionic megacolon, is a congenital disorder characterized by the absence of enteric ganglia in variable portions of the distal intestine. RET is a well-established susceptibility locus, although existing evidence strongly suggests additional loci contributing to sporadic HSCR. To identify these additional genetic loci, we carried out a genome-wide association study using the Affymetrix 500K marker set. We successfully genotyped 293,836 SNPs in 181 Chinese subjects with sporadic HSCR and 346 ethnically matched control subjects. The SNPs most associated with HSCR were genotyped in an independent set of 190 HSCR and 510 control subjects. Aside from SNPs in RET, the strongest overall associations in plausible candidate genes were found for 2 SNPs located in intron 1 of the neuregulin1 gene (NRG1) on 8p12, with rs16879552 and rs7835688 yielding odds ratios of 1.68 [CI95%:(1.40, 2.00), P = 1.80 × 10−8] and 1.98 [CI95%:(1.59, 2.47), P = 1.12 × 10−9], respectively, for the heterozygous risk genotypes under an additive model. There was also a significant interaction between RET and NRG1 (P = 0.0095), increasing the odds ratio 2.3-fold to 19.53 for the RET rs2435357 risk genotype (TT) in the presence of the NRG1 rs7835688 heterozygote, indicating that NRG1 is a modifier of HSRC penetrance. Our highly significant association findings are backed-up by the important role of NRG1 as regulator of the development of the enteric ganglia precursors. The identification of NRG1 as an additional HSCR susceptibility locus not only opens unique fields of investigation into the mechanisms underlying the HSCR pathology, but also the mechanisms by which a discrete number of loci interact with each other to cause disease.

Silver Nanoparticles Mediate Differential Responses in Keratinocytes and Fibroblasts during Skin Wound Healing

With advances in nanotechnology, pure silver has been recently engineered into nanometer‐sized particles (diameter <100 nm) for use in the treatment of wounds. In conjunction with other studies, we previously demonstrated that the topical application of silver nanoparticles (AgNPs) can promote wound healing through the modulation of cytokines. Nonetheless, the question as to whether AgNPs can affect various skin cell types—keratinocytes and fibroblasts—during the wound‐healing process still remains. Therefore, the aim of this study was to focus on the cellular response and events of dermal contraction and epidermal re‐epithelialization during wound healing under the influence of AgNPs; for this we used a full‐thickness excisional wound model in mice. The wounds were treated with either AgNPs or control with silver sulfadiazine, and the proliferation and biological events of keratinocytes and fibroblasts during healing were studied. Our results confirm that AgNPs can increase the rate of wound closure. On one hand, this was achieved through the promotion of proliferation and migration of keratinocytes. On the other hand, AgNPs can drive the differentiation of fibroblasts into myofibroblasts, thereby promoting wound contraction. These findings further extend our current knowledge of AgNPs in biological and cellular events and also have significant implications for the treatment of wounds in the clinical setting.

HOXB5 expression is spatially and temporarily regulated in human embryonic gut during neural crest cell colonization and differentiation of enteric neuroblasts

HOX genes from paralogous groups 4 and 5 are particularly relevant to the gut neuromusculature development because these genes are expressed at the splanchnic mesoderm surrounding the gut diverticulum, and at the level of the neural tube from where the vagal neural crest cells (NCCs) originate. In this study, we examined the migration and differentiation of NCCs, and investigated the expression patterns of HOXB5 in human embryonic guts. Human embryos of gestational week‐4 to ‐8.5 were studied. Vagal NCCs enter the esophagus, migrate, and colonize the entire gut in a rostrocaudal manner between week‐4 and week‐7. The migrating NCCs in gut express HOXB5. Two separate and discontinuous mesenchymal expression domains of HOXB5 were detected in the gut: the distal domain preceding the migratory NCCs; and the proximal domain overlapping with the NCCs. The two expression domains shift caudally in parallel with the rostrocaudal migration of NCCs between week‐4 and week‐5. Neuron and glia differentiation of NCCs are concomitant with HOXB5 down‐regulation in NCCs and the mesenchyme. By week‐7, myenteric plexuses have formed; HOXB5 expression is switched on in the plexuses. We found that (1) the migratory route of NCCs in human embryonic gut was similar to that in mice and chicks; and (2) the expression pattern of HOXB5 correlated with the migration and differentiation of NCCs, suggesting a regulatory role of HOXB5 in the development of NCCs. Developmental Dynamics 228:1–10, 2003.

Identification of a HOXD13 mutation in a VACTERL patient

VACTERL acronym is assigned to a non‐random association of malformations in humans with poorly known etiology. It is comprised of vertebral defects (V), anal atresia (A), cardiac anomaly (C), tracheoesophageal fistula with esophageal atresia (TE), renal dysplasia (R) and limb lesions (L). Here, we report on, for the first time, a female patient with VACTERL association with a 21 base‐pair deletion in the exon 1 triplet repeats of HOXD13, a sonic hedgehog (SHH) downstream target. Our data provide the first piece of clinical evidence of the implication of the SHH pathway in VACTERL. Moreover, HOXD13 may not only be implicated in limb malformations but also in the development of gut and genitourinary structures, as predicted from the mouse models.

Genome-wide association study identifies a susceptibility locus for biliary atresia on 10q24.2

Biliary atresia (BA) is characterized by the progressive fibrosclerosing obliteration of the extrahepatic biliary system during the first few weeks of life. Despite early diagnosis and prompt surgical intervention, the disease progresses to cirrhosis in many patients. The current theory for the pathogenesis of BA proposes that during the perinatal period, a still unknown exogenous factor meets the innate immune system of a genetically predisposed individual and induces an uncontrollable and potentially self-limiting immune response, which becomes manifest in liver fibrosis and atresia of the extrahepatic bile ducts. Genetic factors that could account for the disease, let alone for its high incidence in Chinese, are to be investigated. To identify BA susceptibility loci, we carried out a genome-wide association study (GWAS) using the Affymetrix 5.0 and 500 K marker sets. We genotyped nearly 500 000 single-nucleotide polymorphisms (SNPs) in 200 Chinese BA patients and 481 ethnically matched control subjects. The 10 most BA-associated SNPs from the GWAS were genotyped in an independent set of 124 BA and 90 control subjects. The strongest overall association was found for rs17095355 on 10q24, downstream XPNPEP1, a gene involved in the metabolism of inflammatory mediators. Allelic chi-square test P-value for the meta-analysis of the GWAS and replication results was 6.94 x 10(-9). The identification of putative BA susceptibility loci not only opens new fields of investigation into the mechanisms underlying BA but may also provide new clues for the development of preventive and curative strategies.

Association study of PHOX2B as a candidate gene for Hirschsprung’s disease

Background: Hirschsprung’s disease (HSCR) is a congenital disorder characterised by an absence of ganglion cells in the nerve plexuses of the lower digestive tract. Manifestation of the disease has been linked to mutations in genes that encode the crucial signals for the development of the enteric nervous system—the RET and EDNRB signalling pathways. The Phox2b gene is involved in neurogenesis and regulates Ret expression in mice, in which disruption of the Phox2b results in a HSCR-like phenotype. Aims: To investigate the contribution of PHOX2B to the HSCR phenotype. Methods: Using polymerase chain reaction amplification and direct sequencing, we screened PHOX2B coding regions and intron/exon boundaries for mutations and polymorphisms in 91 patients with HSCR and 71 ethnically matched controls. Seventy five HSCR patients with no RET mutations were independently considered. Haplotype and genotype frequencies were compared using the standard case control statistic. Results: Sequence analysis revealed three new polymorphisms: two novel single nucleotide polymorphisms (A→G1364; A→C2607) and a 15 base pair deletion (DEL2609). Statistically significant differences were found for A→G1364. Genotypes comprising allele G were underrepresented in patients (19% v 36%; χ2=9.30; p=0.0095 and 22% v 36%; χ2=7.38; p=0.024 for patients with no RET mutations). Pairwise linkage disequilibrium (LD) analysis revealed no LD between physically close polymorphisms indicating a hot spot for recombination in exon 3. Conclusion: The PHOX2B A→G1364 polymorphism is associated with HSCR. Whether it directly contributes to disease susceptibility or represents a marker for a locus in LD with PHOX2B needs further investigation. Our findings are in accordance with the involvement of PHOX2B in the signalling pathways governing the development of enteric neurones.

Depletion of intestinal resident macrophages prevents ischaemia reperfusion injury in gut

Background and aims: The cellular and molecular events involved in ischaemia reperfusion (IR) injury are complex and not fully understood. Previous studies have implicated polymorphonuclear neutrophils (PMN) as major inflammatory cells in IR injury. However, anti-PMN antiserum treatment offers only limited protection, indicating that other inflammatory cells are involved. We have therefore investigated the contribution of resident macrophages in IR injury using an IR gut injury model. Methods: DA rats were divided into sham operation and IR groups. The superior mesenteric artery was clamped for 30, 45, or 60 minutes (ischaemia) followed by 60 minutes of reperfusion. IR injuries were evaluated by histological staining. Expression of early growth response factor 1 (Egr-1), myeloperoxidase (MPO), and proinflammatory cytokines was analysed by immunohistochemistry, reverse transcription-polymerase chain reaction, and western blotting analysis. The specific role of macrophages in IR gut injury was also evaluated in resident macrophage depleted rats. Results: Mucosal sloughing and villi destruction were seen in 45/60 minute and 60/60 minute IR guts. PMN infiltration at the damaged mucosal area was undetectable in 45/60 minute and 60/60 minute IR guts. PMN were localised around the capillaries at the base of the crypts in 60/60 minute IR gut. Obvious PMN infiltration was only observed in damaged villi after three hours of reperfusion. Elevated nuclear Egr-1 immunostaining was localised in resident macrophages at the damaged villi before histological appearance of mucosal damage. Furthermore, resident macrophages at the damaged site expressed MPO. Protein levels of the proinflammatory cytokines RANTES and MCP-1 were increased in IR gut. Depletion of resident macrophages by dichloromethylene bisphosphonate significantly reduced mucosal damage in rat guts after IR. Conclusion: Our findings indicate that resident macrophages play a role in early mucosal damage in IR gut injury. Therefore, macrophages should be treated as a prime target for therapeutic intervention for IR damage.

Chinese patients with sporadic Hirschsprung’s disease are predominantly represented by a single RET haplotype

Hirschsprung’s disease is a developmental disorder characterised by the absence of ganglion cells in the nerve plexuses of the lower digestive tract. The Hirschsprung phenotype is variable and can be classified into two groups: SSA, or short segment aganglionosis, which includes patients with aganglionosis as far as the rectosigmoid junction; and LSA, or long segment aganglionosis, which includes patients with aganglionosis beyond the rectosigmoid junction. The condition presents in the neonatal period as a failure to pass meconium, chronic severe constipation, colonic distension, secondary electrolyte disturbances, and sometimes enterocolitis and bowel perforation. The estimated population incidence is 1/5000 live births, although this is a representative value. The highest incidence is in Asian populations (2.8 per 10 000 life births) and the lowest in Hispanics (1 per 10 000 life births).1 The male to female (M:F) ratio is approximately 4:1 for SSA patients and approximately 1:1 for LSA patients.1 Approximately 20% of cases are familial. The recurrence risks for siblings of SSA variant probands ranges between 1.5% and 3.3%, while the risks for those of LSA variant probands varies from 2.9% to 17.6%.1 Hirschsprung’s disease is often associated with chromosomal abnormalities, with other neurodevelopmental disorders such as Waardenburg syndrome type 4, and with a variety of additional isolated anomalies and syndromes.1,2 The disease has a complex genetic aetiology, and many studies indicate the receptor tyrosine kinase gene ( RET ) as the major susceptibility gene for Hirschsprung’s disease.1–14 RET mutations are also associated with multiple endocrine neoplasia type 2 (MEN2),15 and medullary thyroid carcinoma.16 Papillary thyroid carcinoma is associated with RET somatic rearrangements.17 Mutations in the RET gene account for up to 50% of the familial cases and anywhere between 7% and 35% of the sporadic cases, and they lack genotype–phenotype correlation.5– …

Implications of Endocrine Gland–Derived Vascular Endothelial Growth Factor/Prokineticin-1 Signaling in Human Neuroblastoma Progression

Purpose: Neuroblastoma is a common pediatric tumor that is derived from improperly differentiated neural crest cells (NCC). We recently revealed that endocrine gland–derived vascular endothelial growth factor/prokineticin-1 (EG-VEGF/Prok-1) is a key factor mediating the growth and differentiation of enteric NCCs during development. In this report, we further elucidate its role in neuroblastoma progression. Experimental Design: We studied the expression and copy number of EG-VEGF/Prok-1 receptors (PK-R1 and PK-R2) in 26 neuroblastoma tumors by real-time reverse transcription-PCR and immunohistochemical analysis. Implication of EG-VEGF/Prok-1 signaling in neuroblastoma progression was further shown in a neuroblastoma cell line (SK-N-SH). Results: We found that all neuroblastoma samples from stages II to IV expressed both PK-R1 and PK-R2. Kruskall-Wallis signed rank tests revealed that the expression level of PK-R1 transcript is associated with the stages and metastasis of the neuroblastoma (P < 0.05), and PK-R2 is persistently higher in advanced-stage neuroblastoma samples. About 38% of the neuroblastoma tumors (10:26) possessed MYCN amplification, whereas no PK-R1 and PK-R2 amplifications were detected, suggesting that the overexpression of the receptors was not due to gene amplification. Subsequent functional studies showed that EG-VEGF/Prok-1 activates the Akt pathway to induce the proliferation of neuroblastoma cells. Targeted down-regulation studies revealed that EG-VEGF/Prok-1–mediated proliferation requires the presence of these two receptors, and that PK-R2 is essential for inhibiting apoptosis. In vitro migration and invasion assays also indicated that EG-VEGF/Prok-1 significantly enhances the cell migration/invasion of SK-N-SH. Conclusions: Our study has shown for the first time that aberrant EG-VEGF/Prok-1 signaling favors neuroblastoma progression and could be a potential target for future neuroblastoma treatment.