KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A Predictive Model to Identify the Effects of Transcutaneous Sacral Nerve Stimulation With Pelvic Floor Exercises in Fecal Incontinence After Surgery for Anorectal Malformation.

INTRODUCTION: Although the combination of transcutaneous sacral nerve stimulation (tSNS) and pelvic floor exercises (PFEs) has shown significant effectiveness in treating fecal incontinence (FI) after surgery for congenital anorectal malformation (CARM), not all patients achieve satisfactory continence. Therefore, identifying which individuals will benefit from this method is crucial. METHODS: A prospective cohort study enrolled 92 children with FI. All patients underwent tSNS with PFE treatment, and an improved outcome was defined as a Wexner score ≤4. A predictive model to identify the effects of tSNS with PFEs in FI was developed based on the analysis of magnetic resonance imaging and high-resolution anorectal manometry with area under the receiver-operating characteristic curve to evaluate the predictive value of external anal sphincter (EAS) thickness index and anal squeezing pressure (ASP). RESULTS: tSNS with PFEs improved outcomes in 72 patients and led to poor outcomes in 20 (4 had their rectums deviate from the puborectalis muscle center or puborectal muscle ruptures while 16 lacked EAS with a lower ASP). The areas under the receiver-operating characteristic curve for EAS thickness index and ASP in predicting the effects of tSNS with PFEs were 0.915 (95% confidence interval 0.846-0.983, P = 0.000) and 0.886 (95% confidence interval 0.819-0.952, P = 0.000), respectively. By applying cutoff values of 0.076 for EAS thickness index and 21.95 mm Hg for ASP, tSNS with PFEs was found to be ineffective. DISCUSSION: tSNS with PFEs is effective for most patients with FI after CARM surgery, except when the rectum deviates from the puborectal muscle center, puborectal muscle rupture occurs, or EAS is absent with a low ASP.

Neuroendocrine neoplasm with metastasis to the thyroid: a case report and literature review.

Thyroid cancer can be divided into two types according to its cellular origin, i.e., malignant tumors originating from thyroid cells and cancers that metastasize to the thyroid from other sites, the latter of which are, clinically rare. This article reports the diagnosis and treatment of a rectal neuroendocrine neoplasm metastasis to the thyroid. No similar cases have been reported before. This case suggests that when evaluating thyroid tumors, clinicians should not only carefully identify the clinical features of the tumor but also pay special attention to the patient's history of tumors, especially neuroendocrine neoplasms. For definite secondary thyroid malignancies, neck surgery is feasible if the thyroid is the only site of metastasis; otherwise, the subsequent diagnosis and treatment plan should be determined after a comprehensive evaluation of the primary tumor and patient's general condition.

AKT serine/threonine kinase 2-mediated phosphorylation of fascin threonine 403 regulates esophageal cancer progression.

Fascin is the main actin-bundling protein in filopodia and is highly expressed in metastatic tumor cells. The overexpression of Fascin has been associated with poor clinical prognosis and metastatic progression. Post-translational modifications of Fascin, such as phosphorylation, can affect the proliferation and invasion of tumor cells by regulating the actin-bundling activity of Fascin. However, the phosphorylation sites of Fascin and their corresponding kinases require further exploration. In the current study, we identified novel phosphorylation of Fascin Threonine 403 (Fascin-T403) mediated by AKT serine/threonine kinase 2 (AKT2), which was studied using mass spectrometry data from esophageal cancer tissues (iProX database: IPX0002501000). A molecular dynamics simulation revealed that Fascin-Threonine 403 phosphorylation (Fascin-T403D) had a distinct spatial structure and correlation of amino acid residues, which was different from that of the wild type (Fascin-WT). Low-speed centrifugation assay results showed that Fascin-T403D affected actin cross-linking. To investigate whether Fascin-T403D affected the function of esophageal cancer cells, either Fascin-WT or Fascin-T403D were rescued in Fascin-knockout or siRNA cell lines. We observed that Fascin-T403D could suppress the biological behavior of esophageal cancer cells, including filopodia formation, cell proliferation, and migration. Co-immunoprecipitation (Co-IP) and Duolink in situ proximity ligation assay (PLA) were performed to measure the interaction between Fascin and AKT2. Using in vitro and in vivo kinase assays, we confirmed that AKT2, but not AKT1 or AKT3, is an upstream kinase of Fascin Threonine 403. Taken together, the AKT2-catalyzed phosphorylation of Fascin Threonine 403 suppressed esophageal cancer cell behavior, actin-bundling activity, and filopodia formation.

Normal development of hindgut and anorectum in human embryo.

PURPOSE: The aim of the present analysis is to examine the morphological changes, the spatiotemporal distribution of apoptosis/proliferation in the human embryonic anorectum, to reveal the normal development of human anorectum, and investigate the possible roles of apoptosis/proliferation during anorectal development. MATERIALS AND METHODS: The embryos were sectioned serially and sagittally, stained with hematoxylin and eosin (H & E) between the third and eighth week of gestation, TdT-mediated dUTP-digoxigenin nick end-labeling (TUNEL) and proliferative cell-specific nuclear antigen (PCNA) immunohistochemical staining from the sixth to the eighth week. RESULTS: From the fourth to the seventh week, with the growth of the mesenchyme around the cloaca, the cloaca was remolded, subsequently, the cloacal membrane (CM) moved perpendicularly then horizontally. The dorsal cloaca gradually descended to the tail groove, the urorectal septum (URS) and the CM approximated; however, the fusion of URS with the dorsal CM was never observed. During the eighth week, the URS shifted ventrally and finally fused with the ventral CM. Moreover, from the sixth to the eighth week, the apoptotic cells were concentrated in the CM, the mesenchyme of terminal rectum, and the dorsal rectum. Meanwhile, the proliferative cells could be observed in the ventral mesenchyme around the cloaca, the CM, the fused tissue between the URS, and the ventral CM. CONCLUSIONS: During the development of human anorectum, it was intriguing to reveal that the URS never fused with the dorsal CM before dorsal CM disintegration, the normal anorectal development may depend on the dorsal cloaca and the dorsal CM; furthermore, the distribution of apoptosis and proliferation in the anorectum and ventral cloacal mesenchyme played a pivotal role in the formation of the anorectum.

A Novel Scoring System for Predicting the Metastases of Posterior Right Recurrent Laryngeal Nerve Lymph Node Involvement in Patients With Papillary Thyroid Carcinoma by Preoperative Ultrasound.

Objective: Our goal was to investigate the correlation between papillary thyroid carcinoma (PTC) characteristics on ultrasonography and metastases of lymph nodes posterior to the right recurrent laryngeal nerve (LN-prRLN). There is still no good method for clinicians to judge whether a patient needs LN-prRLN resection before surgery, and we also wanted to establish a new scoring system to determine whether patients with papillary thyroid carcinoma require LN-prRLN resection before surgery. Patients and Methods: There were 482 patients with right or bilateral PTC who underwent thyroid gland resection from December 2015 to December 2017 recruited as study subjects. The relationship between the PTC characteristics on ultrasonography and the metastases of LN-prRLN was analyzed by univariate and logistic regression analyses. Based on the risk factors identified in univariate and logistic regression analysis, a nomogram-based LN-prRLN prediction model was established. Result: LN-prRLN were removed from all patients, of which 79 had LN-prRLN metastasis, with a metastasis rate of 16.39%. Multivariate logistic regression analysis revealed that LN-prRLN metastasis was closely related to sex, age, blood supply, larger tumors (> 1 cm) and capsular invasion. A risk prediction model has been established and fully verified. The calibration curve used to evaluate the nomogram shows that the consistency index was 0.75 ± 0.065. Conclusion: Preoperative clinical data, such as sex, age, abundant blood supply, larger tumor (> 1 cm) and capsular invasion, are positively correlated with LN-prRLN metastasis. Our scoring system can help surgeons non-invasively determine which patients should undergo LN-prRLN resection before surgery. We recommend that LN-prRLN resection should be performed when the score is above 103.1.

P300/CBP-associated factor (PCAF)-mediated acetylation of Fascin at lysine 471 inhibits its actin-bundling activity and tumor metastasis in esophageal cancer.

BACKGROUND: Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post-translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. METHODS: Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. RESULTS: Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti-AcK471-Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin-K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin-K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471-acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin-bundling activity. Clinically, high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients. CONCLUSIONS: Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.

In-vitro GLP-1 Release Assay Using STC-1 Cells.

Enteroendocrine cells (EECs) are known chemosensors in the gastrointestinal (GI) epithelium. They release a diversity of gut hormones in response to various stimuli. Here, we report an in-vitro assay to measure GLP-1 release from cultured murine EEC's under fatty acid stimulation.

Spatiotemporal pattern analysis of transcription factor 4 in the developing anorectum of the rat embryo with anorectal malformations.

PURPOSE: As a member of the transcription factors family, transcription factor 4(Tcf4) is known to influence gene expression in endodermally derived tissues including lung, liver, pancreas, stomach, and intestine. However, it remained unknown if this capability is active during anorectal development in the normal and anorectal malformations (ARM) rat embryos. MATERIALS AND METHODS: In this study, ethylenethiourea (ETU)-induced ARM model was introduced to investigate the expression pattern of Tcf4 during anorectal development using immunohistochemical staining, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: Immunostaining revealed that Tcf4 expression showed space-dependent changes in the developing anorectum: in normal embryos, Tcf4 protein is initially expressed in the dorsal endoderm of urorectal septum (URS) and hindgut on embryonic day 13 (E13). Additionally, separate expression domain develops intensively on the dorsal CM on E14. On E15, positive cells are then detected in the fused tissue of URS, and prominently in the anal membrane. In the ARM embryos, however, the epithelium of the cloaca, URS, and anorectum was negative or faint for Tcf4. In Western blot and RT-PCR, time-dependent changes of Tcf4 protein and mRNA expression were remarkable during the anorectal development: on E14, E14.5, and E15, the expression level reached the peak; after E16, Tcf4 expression gradually decreased. In contrast, in ARM embryos, spatiotemporal expression of Tcf4 was imbalanced during the anorectal morphogenesis from E13 to E16. CONCLUSIONS: These data implied that the downregulation of Tcf4 at the time of cloacal separation into rectum and urethra might be related to the development of ARM.

Chromosome 13q deletion syndrome involving 13q31­qter: A case report.

Partial deletions on the long arm of chromosome 13 lead to a number of different phenotypes depending on the size and position of the deleted region. The present study investigated 2 patients with 13q terminal (13qter) deletion syndrome, which manifested as anal atresia with rectoperineal fistula, complex type congenital heart disease, esophageal hiatus hernia with gastroesophageal reflux, facial anomalies and developmental and mental retardation. Array comparative genomic hybridization identified 2 regions of deletion on chromosome 13q31­qter; 20.38 Mb in 13q31.3­qter and 12.99 Mb in 13q33.1­qter in patients 1 and 2, respectively. Comparisons between the results observed in the present study and those obtained from patients in previous studies indicate that the gene encoding ephrin B2 (EFNB2) located in the 13q33.3­q34 region, and the gene coding for endothelin receptor type B, in the 13q22.1­31.3 region, may be suitable candidate genes for the observed urogenital/anorectal anomalies. In addition, the microRNA­17­92a­1 cluster host gene and the glypican 6 gene in the 13q31.3 region, as well as EFNB2 and the collagen type IV a1 chain (COL4A1) and COL4A2 genes in the 13q33.1­q34 region may together contribute to cardiovascular disease development. It is therefore possible that these genes may be involved in the pathogenesis of complex type congenital heart disease in patients with 13q deletion syndrome.

Increased B-type-natriuretic peptide promotes myocardial cell apoptosis via the B-type-natriuretic peptide/long non-coding RNA LSINCT5/caspase-1/interleukin 1ß signaling pathway.

Chronic heart failure (CHF) is the final stage of various heart diseases, and is increasingly recognized as a major health problem in the elderly. Previous studies demonstrated that B­type­natriuretic peptide (BNP) is an established biomarker of CHF. Furthermore, BNP also regulates cell proliferation, differentiation and apoptosis. Recent evidence has revealed that BNP affects myocardial cell apoptosis during myocardial ischemia­reperfusion injury. Long non­coding RNAs (lncRNAs) are emerging as novel molecular compounds involved in gene regulation, and have important roles in numerous human diseases. However, the mechanism underlying the BNP and lncRNA-induced regulation of myocardial cell apoptosis remains to be elucidated. The present study reported that lncRNA LSINCT5, upregulated by BNP, is able to regulate myocardial cell apoptosis via the activation of the caspase­1/interleukin (IL)­1ß signaling pathway. BNP-induced apoptosis of HCM cells was observed using flow cytometry, and involved caspase­1. In addition, expression profiling using a human lncRNA polymerase chain reaction array revealed that LSINCT5 was highly expressed in BNP-treated myocardial cells, as compared with untreated cells. The role of lncRNA LSINCT5 in HCM cell apoptosis was also investigated. The results of the present study indicated that LSINCT5 silencing by small interfering RNA inhibits caspase­1/IL­1ß signaling, and suppresses apoptosis in BNP-treated HCM cells. Therefore, high expression levels of BNP promote the apoptosis of myocardial cells through the lncRNA LSINCT5 mediator, which activates the caspase­1/IL­1ß signaling pathway. These findings uncovered a novel pathogenic mechanism, and provided a potential therapeutic target for CHF.

Effect of heat shock pretreatment on apoptosis and metallothionein expression in rat cardiomyocytes.

To investigate the effect of heat shock pretreatment on apoptosis and mitochondrial metallothionein (MT) expression in rat cardiomyocytes. In vitro cultured H9C2 cells were randomly divided into three groups: control, hydrogen peroxide (H2O2) injury, and H2O2 injury after heat shock pretreatment (n = 6 per group). Cardiomyocyte apoptosis and caspase-3 activity were assayed after treatment. Mitochondrial cytochrome (cyt) c and MT expression was assayed by Western blotting. Compared with the control group, the H2O2 injury group had a growing number of apoptotic cardiomyocytes (P < 0.01) and significantly elevated caspase-3 activity (P < 0.01) with markedly increased mitochondrial cyt c and MT expression (P < 0.01). After heat shock pretreatment, the numbers of apoptotic and necrotic cardiomyocytes (P < 0.01) and the caspase-3 activity significantly declined (P < 0.01), while mitochondrial cyt c and MT expression continued to increase (P < 0.01) compared with the H2O2 injury group. Heat shock pretreatment inhibits cardiomyocyte apoptosis, which may have a protective effect on cardiomyocytes by increasing the expression of myocardial protective MT and reducing the release of mitochondrial cyt c.

[Mutation and expression of WNT8b gene and SHH gene in Hirschsprung disease].

OBJECTIVE: To investigate the relationship of WNT8b and SHH genes mutation and Hirschsprung disease(HSCR) in Chinese children. METHODS: Preoperative whole blood preparations in 72 children with sporadic HSCR from northeast China were collected(study group). Seventy-two healthy children were used as controls(matched for sex and age). Genomic DNA was obtained from peripheral blood. Exon 1 of WNT8b gene and the exon 1 of SHH gene were analyzed for gene mutation. The mutation products were automatically sequenced. The levels of WNT8b and SHH mRNA were detected by quantitative real-time PCR(qRT-PCR) in blood samples. RESULTS: On sequencing, 13 out of 72 children with HSCR had WNT8b gene mutation in the coding area, including heterozygosity deletion in 8 cases (11.1%) and base replacement in 5(6.9%). Eleven children with HSCR had SHH gene mutation in the coding area including heterozygosity deletion in 7 cases(9.7%) and base replacement in 4(5.6%). No mutations in WNT8b and SHH genes were found in the control group. The WNT8b and SHH mRNA levels were different between the study group and the control group(30.01±1.13 vs. 17.33±0.62, and 28.25±1.27 vs. 18.94±0.31, P<0.05). CONCLUSIONS: WNT8b and SHH mutations and abnormal expressions are present in the peripheral blood of children with sporadic HSCR. These two genes may be related to the development of sporadic HSCR in children in the northeastern China.

Embryonic development of the internal anal sphincter in rats with anorectal malformations.

BACKGROUND/PURPOSE: The embryogenesis of the internal anal sphincter (IAS) in anorectal malformations (ARMs) remains unclear. This study aimed to investigate the development of the smooth muscle in the terminus of the digestive tract in normal and abnormal rats. METHODS: Rat embryos with ARMs were generated by administration of ethylenethiourea to pregnant rats. The normal rat embryos and embryos with ARMs from E13.5 to E21 were serially sectioned in the sagittal plane and stained immunohistochemically using specific antibody to α-smooth muscle actin (SMA). Temporospatial study was carried out on circular muscle of the distal portion of the hindgut. RESULTS: α-Smooth muscle actin immunolabeling cells could not be observed in the hindgut on E13.5, E14, and E14.5. On E15, there were α-SMA immunolabeling circular muscle cells in the hindgut; and the distal portion of the circular muscle was not thickened in the normal and ARMs rats. From E16 onward, the smooth muscle with slight dilated terminus, which was characterized by the features of IAS, could be noted in the primitive anorectum. In the normal group, the circular muscle in the distal portion of the hindgut thickened slightly and became the musculature with shutter-like bundles. In the ARMs group, the α-SMA immunolabeling myogenic precursors of the smooth muscle could be observed in the primitive anorectum as well. The musculature was similar to that in the normal group. On E15 and E16, there was no significant difference in the development of the circular muscle in the 2 groups. Moreover, the terminus of the circular muscle in the hindgut did not reach the orificium fistulae in ARMs rats. From E17 onward, in ARMs rats, the funnel-shaped distal hindgut communicated the genitourinary tract with a narrow fistula; the dilated musculature at this portion thinned gradually and formed an acute angled extremity in the ARMs group rather than formed blunt extremity in the normal group; the terminus circular muscle in the dorsal hindgut reached the orificium fistulae. During the following gestational days, the circular muscle of the hindgut in both normal and ARMs rats continued its own tendency. CONCLUSION: The IAS primordium started to appear at the terminus of the hindgut on E15 in the 2 groups. The IAS in the ARMs group failed to develop as well as that in the normal group. The IAS dysplasia occurred in the late embryonic development (E17-E21).

Temporal and spatial expression of caudal-type homeobox gene-1 in the development of anorectal malformations in rat embryos.

PURPOSE: The aim of this study was to determine caudal-type homeobox gene-1 (Cdx1) expressions during anorectal development in normal and anorectal malformation (ARMs) embryos and investigate the possible role of Cdx1 in the pathogenesis of ARM. MATERIALS AND METHODS: Anorectal malformation was induced by ethylenethiourea on the 10th gestational day (GD10) in rat embryos. Cesarean deliveries were performed to harvest embryos from GD13 to GD21. The temporal and spatial expression of Cdx1 was evaluated in normal rat embryos (n = 334) and ARM embryos (n = 328) from GD13 to GD20 using immunohistochemistry staining, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: Immunostaining revealed that in normal embryos, on GD13.5, Cdx1 expression was mainly located on the epithelium of the dorsal urorectal septum (URS), cloacal membrane, and the hindgut. On GD15, increased positive tissue staining was noted on the fused tissue of URS, especially in the very thin anal membrane. In the ARM embryos, however, the epithelium of the cloaca, URS, and anorectum was negative or faint for Cdx1. In Western blot and RT-PCR, in the normal group, Cdx1 protein and Cdx1 messenger RNA expression showed time-dependent changes in the developing hindgut, on GD14, GD14.5, and GD15. The expression level reached a peak when the anus was forming. Once the anus was open, Cdx1 expression gradually decreased. In addition, the expression level of Cdx1 in the ARM group from GD13 to GD16 was significant lower than that of the normal group (P < .05). CONCLUSIONS: In ARM embryos, an imbalance of spatiotemporal expression of Cdx1 was noted during anorectal morphogenesis from GD13 to GD16. This suggests that downregulation of Cdx1 at the time of cloacal separation into rectum and urethra might be related to the development of ARM.

Expression of EphB2 in the development of anorectal malformations in fetal rats.

PURPOSE: The receptor tyrosine kinase of the Eph family is a large group of highly conserved molecules that function in diverse intercellular recognition events. It has been reported that EphB2 is related to caudal remodeling events. The aim of this study is to investigate EphB2 expression in anorectal development in normal and rat embryos with anorectal malformations (ARMs) and attempt to define its role in anorectal morphogenesis. METHODS: The ethylenethiourea (ETU) rat model of the ARMs was used in this study. Immunohistochemical analyses and real time quantitative polymerase chain reaction (PCR) were carried out to investigate EphB2 protein localizations and messenger RNA (mRNA) expression levels. (1) Rat embryos with ARMs were obtained by treating pregnant rats (n = 24) with administration of ETU on gestation day (Gd) 10. Normal rat embryos (n = 111) and embryos treated by ETU without ARMs (n = 90) were the control groups, and embryos with ARMs (n = 108) from Gd13 to Gd16 were divided according to the sections taken from specimens. (2) Embryos were sequentially sectioned in the sagittal and transversal planes before staining with a specific antibody to EphB2. Spatiotemporal study was carried out on EphB2 expression. (3) Individual frozen sections were used to manually microdissect the cloaca and anorectal specimens for total RNA extraction. EphB2 expression was evaluated by real time quantitative PCR. RESULTS: On the immunologic labeling study, EphB2 expression was confined to the cloaca in control groups, whereas EphB2 expression was mainly located at the urorectal septum (URS) and cloacal membrane on Gd13 and Gd14. The increased positive expression was observed in the fused tissue of the URS and cloacal membrane on Gd15. On Gd16, the anal membrane broke down, and the rectum was able to be in contact with the anus, and EphB2 expression was then noted in mucous membrane of rectum. EphB2 expression was seen in the cloacal and anorectal tissues of embryos with ARMs. By integrated optical density (IOD) measurement, IOD value of EphB2 protein was significantly lower in the ARM group than that in the control groups on Gd13 to Gd16 (P < .05), respectively. As shown by real time quantitative PCR, EphB2 expression was detected in 3 groups. EphB2 mRNA level increased on Gd13 to Gd16 but gradually decreased after Gd16. The expression level of EphB2 mRNA in the ARM embryos was lower on Gd13 to Gd16 than that in control groups (P < .05). CONCLUSIONS: EphB2 expression decreased in the ARM embryos and was confined to URS and cloaca, whereas it was higher in control group. Our data thus indicated that EphB2 molecules possibly contributed to the anorectal morphogenesis and the decreased expression of EphB2 might be related to the development of ARMs.

Embryonic development of the striated muscle complex in rats with anorectal malformations.

PURPOSE: Many patients with anorectal malformations (ARMs) continue to have postoperative anal dysfunction. The striated muscle complex (SMC) is one of the most important factors that influence defecation function. To explore the development of SMC in ARMs, the authors investigated the pelvic muscle development in rat embryos affected with ARMs. METHODS: Anorectal malformation embryos were induced by ethylenethiourea on the 10th gestational day (E10). Normal rat embryos and embryos with ARMs from E13 to E21 were serial-sectioned in the sagittal, transverse, and coronal planes, stained with H&E and immunohistochemistry staining using specific antibodies to myogenin. Temporal and spatial sequence was carried out on SMC. RESULTS: On E16, in normal group, SMC appeared fibroid structure in normal rats; SMC arose from bulbocavernosus muscle and ran backward, parallel to the perineal skin, and loosely surrounded the anal canal and urethra. Although in ARM rats the rectum was absent, the location and appearance of SMC were similar to the normal group. On E18, in normal group, SMC musculature became much thicker than on E16 and SMC gave off 2 branches outside anterior to the rectum. Striated muscle complex surrounded the rectum more tightly. However, in ARM rats, obvious changes of SMC could be noted. In detail, SMC in ARMs were characterized by abnormal location, appearance, and path. Striated muscle complex shifted obviously cephalad, ventrally, and medianward and converged inferior to the rectal terminus and posterior to the urethra. The distance between SMC musculature and the perineal skin increased. This structure surrounded the fiberlike tissue posterior to the urethra. Under high-power view, there was connective tissue among intermuscular bundles, and the structure was disordered. During the following gestational days, SMC in normal and ARM groups continued their own tendency, respectively. CONCLUSIONS: This study illustrated the development of the SMC in normal and ARM rats. On E16, the location and appearance of SMC in ARM rats were similar to the normal rats, and at this time, the ectopic rectal orifice could be noted. From E18 on, the maldevelopment of SMC could be observed in ARM rats. These observations suggested that the morphological changes of SMC take place after the occurrence of abnormal anorectum.