Tumor-Derived Exosomal miR-143-3p Induces Macrophage M2 Polarization to Cause Radiation Resistance in Locally Advanced Esophageal Squamous Cell Carcinoma.

We aimed to determine whether monitoring tumor-derived exosomal microRNAs (miRNAs) could be used to assess radiotherapeutic sensitivity in patients with locally advanced esophageal squamous cell carcinoma (ESCC). RNA sequencing was employed to conduct a comparative analysis of miRNA expression levels during radiotherapy, focusing on identifying miRNAs associated with progression. Electron microscopy confirmed the existence of exosomes, and co-cultivation assays and immunofluorescence validated their capacity to infiltrate macrophages. To determine the mechanism by which exosomal miR-143-3p regulates the interplay between ESCC cells and M2 macrophages, ESCC cell-derived exosomes were co-cultured with macrophages. Serum miR-143-3p and miR-223-3p were elevated during radiotherapy, suggesting resistance to radiation and an unfavorable prognosis for ESCC. Increased levels of both miRNAs independently predicted shorter progression-free survival (p = 0.015). We developed a diagnostic model for ESCC using serum microRNAs, resulting in an area under the curve of 0.751. Radiotherapy enhanced the release of miR-143-3p from ESCC cell-derived exosomes. Immune cell infiltration analysis at the Cancer Genome Atlas (TCGA) database revealed that ESCC cell-derived miR-143-3p triggered M2 macrophage polarization. Mechanistically, miR-143-3p upregulation affected chemokine activity and cytokine signaling pathways. Furthermore, ESCC cell exosomal miR-143-3p could be transferred to macrophages, thereby promoting their polarization. Serum miR-143-3p and miR-223-3p could represent diagnostic and prognostic markers for patients with ESCC undergoing radiotherapy. Unfavorable prognosis could be linked to the increased levels of ESCC cell-derived exosomal miR-143-3p, which might promote tumor progression by interacting with macrophages.

Minimal residual disease profiling predicts pathological complete response in esophageal squamous cell carcinoma.

Accurate presurgical prediction of pathological complete response (pCR) can guide treatment decisions, potentially avoiding unnecessary surgeries and improving the quality of life for cancer patients. We developed a minimal residual disease (MRD) profiling approach with enhanced sensitivity and specificity for detecting minimal tumor DNA from cell-free DNA (cfDNA). The approach was validated in two independent esophageal squamous cell carcinoma (ESCC) cohorts. In a cohort undergoing neoadjuvant, surgical, and adjuvant therapy (NAT cohort), presurgical MRD status precisely predicted pCR. All MRD-negative cases (10/10) were confirmed as pCR by pathological evaluation on the resected tissues. In contrast, MRD-positive cases included all the 27 non-pCR cases and only one pCR case (10/10 vs 1/28, P < 0.0001, Fisher's exact test). In a definitive radiotherapy cohort (dRT cohort), post-dRT MRD status was closely correlated with patient prognosis. All MRD-negative patients (25/25) remained progression-free during the follow-up period, while 23 of the 26 MRD-positive patients experienced disease progression (25/25 vs 3/26, P < 0.0001, Fisher's exact test; progression-free survival, P < 0.0001, log-rank test). The MRD profiling approach effectively predicted the ESCC patients who would achieve pCR with surgery and those likely to remain progression-free without surgery. This suggests that the cancer cells in these MRD-negative patients have been effectively eliminated and they could be suitable candidates for a watch-and-wait strategy, potentially avoiding unnecessary surgery.

Adaptive ultra-hypofractionated whole-pelvic radiotherapy in high-risk and very high-risk prostate cancer on 1.5-Tesla MR-Linac: Estimated delivered dose and early toxicity results.

Background: Magnetic resonance (MR)-guided ultra-hypofractionated radiotherapy with whole-pelvic irradiation (UHF-WPRT) is a novel approach to radiotherapy for patients with high-risk (HR) and very high-risk (VHR) prostate cancer (PCa). However, the inherent complexity of adaptive UHF-WPRT might inevitably result in longer on-couch time. We aimed to estimate the delivered dose, study the feasibility and safety of adaptive UHF-WPRT on a 1.5-Tesla MR-Linac. Methods: Ten patients with clinical stage T3a-4N0-1M0-1c PCa, who consecutively received UHF-WPRT, were enrolled prospectively. The contours of the target and organ-at-risks on the position verification-MR (PV-MR), beam-on 3D-MR(Bn-MR), and post-MR (after radiotherapy delivery) were derived from the pre-MR data by deformable image registration. The physician then manually adjusted them, and dose recalculation was performed accordingly. GraphPad Prism 9 (GraphPad Prism Software Inc.) was utilized for conducting statistical analyses. Results: In total, we collected 188 MR scans (50 pre-MR, 50 PV-MR, 44 Bn-MR, and 44 post-MR scans). With median 59 min, the mean prostate clinical target volume (CTV)-V100% was 98.59% ± 2.74%, and the mean pelvic CTVp-V100% relative percentages of all scans was 99.60% ± 1.18%. The median V29 Gy change in the rectal wall was -2% (-18% to 20%). With a median follow-up of 9 months, no patient had acute Common Terminology Criteria for Adverse Events (CTCAE) grade 2 or more severe genitourinary (GU) or gastrointestinal (GI) toxicities (0%). Conclusion: UHF-RT to the prostate and the whole pelvis with concomitant boost to positive nodes using an Adapt-To-Shape (ATS) workflow was technically feasible for patients with HR and VHR PCa, presenting only mild GU and GI toxicities. The estimated target dose during the beam-on phase was clinically acceptable based on the 3D-MR-based dosimetry analysis. Clinical trial registration: Chinese Clinical Trial Registry ChiCTR2000033382.

Treatment outcome, toxicity, and quality of life of patients with bronchus-associated lymphoid tissue lymphoma.

The disease failure patterns and optimal treatment of bronchus-associated lymphoid tissue (BALT) lymphoma are unknown. This retrospective study involved 71 patients with primary BALT lymphoma who had received radiotherapy (RT), surgery, immunochemotherapy (IC), or observation. The median follow-up time was 66 months. The 5-year overall survival and lymphoma-specific survival were 91.2% and 96.1%, respectively, and were not significantly different among treatments. The 5-year cumulative incidence of overall failure for RT, surgery, IC, and observation was 0%, 9.7% (p = .160), 30.8% (p = .017), and 31.3% (p = .039). There was no grade ≥3 toxicity in RT group according to the CTCAE 5.0 reporting system. Quality of life (QoL) was at similarly good levels among the treatment groups. BALT lymphoma had a favorable prognosis but persistent risk of relapse after IC or observation. Given the very low disease failure risk and good QoL, RT remains an effective initial treatment for BALT lymphoma.

BALT lymphoma has a favorable prognosis but a persistent progression and relapse risk.Radiotherapy is associated with lower failure of disease progression and relapse, low toxicity and good quality of life.

Multicenter phase II study of moderate low-dose radiotherapy in indolent non-Hodgkin lymphoma: CLCG-iNHL-01 protocol.

Background: Radiotherapy is an effective treatment for indolent non-Hodgkin lymphoma (iNHL); however, the optimal radiotherapy dose remains to be determined. We hypothesize that a suitable dose may exist between 4 and 24 Gy. Methods: This prospective multicenter phase II trial intends to recruit 73 sites of iNHL patients, who will receive involved-site radiotherapy of 12 Gy in four fractions. The primary objective is the 6-month clinical complete response rate. Tumor tissue, blood and conjunctival specimens will be collected to identify potential predictive biomarkers. Discussion: The CLCG-iNHL-01 trial will evaluate the efficacy and toxicity of 12 Gy in patients with iNHL and provide information on a novel hypofractionation regimen of low-dose radiotherapy. Clinical Trial Registration: NCT05543070 (ClinicalTrials.gov).

Determination of internal target volume with Cine-MRI sequence for prostate MRI-Guided radiotherapy.

BACKGROUND: In prostate radiotherapy, the intrafractional target motion negatively affects treatment accuracy. Generating internal target volume (ITV) using four-dimensional (4D) images may resolve the issue of intrafractional target motion induced by bladder filling and bowel movement. However, no 4D imaging techniques suitable for the prostate are currently available in clinical practice. PURPOSE: This study aimed to determine the ITV based on cine magnetic resonance imaging (MRI) sequence for intrafractional target motion management in prostate MRI-guided radiotherapy. MATERIALS AND METHODS: A reference ITV was generated in simulation process. Then, the reference ITV was adapted with cine MRI sequence before online planning in each fraction. Finally, the reference ITV was updated with the cine MRI sequence acquired during beam delivery after each fraction. Cine MRI sequences and positioning three-dimensional (3D) MRI from 35 patients were retrospectively collected. Clinical target volume (CTV) coverage was computed according to the two-dimensional contour of CTV and ITV on cine MRI images. Relative target size was calculated as the ratio of the volume of ITV and CTV. Isotropic planning target volume (PTV; 5 mm margin) and anisotropic PTV (3 mm margin in the posterior direction and 5 mm margin in other directions) were generated for comparison. RESULTS: The CTV coverage rate of the proposed ITV had a mean value of 98.61% ± 0.51%, whereas the CTV coverage rates of the isotropic and anisotropic PTVs were 97.43% ± 0.41% and 96.58% ± 0.73%, respectively. The proposed ITV had a relative target size of 1.79 ± 0.17, whereas the anisotropic and isotropic PTVs had relative target sizes of 1.92 ± 0.12 and 2.21 ± 0.19, respectively. For both the CTV coverage rate and target relative size, significant differences were observed between the proposed ITV and the other two PTVs (p < 0.05). CONCLUSION: The ITV achieved higher CTV coverage with smaller size than conventional isotropic and anisotropic PTVs, indicating that it can effectively deal with the intrafractional movement of the prostate.

Assessment of delivered dose in prostate cancer patients treated with ultra-hypofractionated radiotherapy on 1.5-Tesla MR-Linac.

Objective: To quantitatively characterize the dosimetric effects of long on-couch time in prostate cancer patients treated with adaptive ultra-hypofractionated radiotherapy (UHF-RT) on 1.5-Tesla magnetic resonance (MR)-linac. Materials and methods: Seventeen patients consecutively treated with UHF-RT on a 1.5-T MR-linac were recruited. A 36.25 Gy dose in five fractions was delivered every other day with a boost of 40 Gy to the whole prostate. We collected data for the following stages: pre-MR, position verification-MR (PV-MR) in the Adapt-To-Shape (ATS) workflow, and 3D-MR during the beam-on phase (Bn-MR) and at the end of RT (post-MR). The target and organ-at-risk contours in the PV-MR, Bn-MR, and post-MR stages were projected from the pre-MR data by deformable image registration and manually adapted by the physician, followed by dose recalculation for the ATS plan. Results: Overall, 290 MR scans were collected (85 pre-MR, 85 PV-MR, 49 Bn-MR and 71 post-MR scans). With a median on-couch time of 49 minutes, the mean planning target volume (PTV)-V95% of all scans was 97.83 ± 0.13%. The corresponding mean clinical target volume (CTV)-V100% was 99.93 ± 0.30%, 99.32 ± 1.20%, 98.59 ± 1.84%, and 98.69 ± 1.85%. With excellent prostate-V100% dose coverage, the main reason for lower CTV-V100% was slight underdosing of seminal vesicles (SVs). The median V29 Gy change in the rectal wall was -1% (-20%-17%). The V29 Gy of the rectal wall increased by >15% was observed in one scan. A slight increase in the high dose of bladder wall was noted due to gradual bladder growth during the workflow. Conclusions: This 3D-MR-based dosimetry analysis demonstrated clinically acceptable estimated dose coverage of target volumes during the beam-on period with adaptive ATS workflow on 1.5-T MR-linac, albeit with a relatively long on-couch time. The 3-mm CTV-PTV margin was adequate for prostate irradiation but occasionally insufficient for SVs. More attention should be paid to restricting high-dose RT to the rectal wall when optimizing the ATS plan.

Radiation Dose-Effect Relation in Patients with Esophageal Squamous Cell Carcinoma: A National Cancer Center Data and Literature-Based Analysis.

Introduction: Despite receiving definitive chemoradiotherapy (dCRT) with radiation dose (RTD) of 50.4 Gy, survival of esophageal carcinoma was dismal. The effect of RTD in cancer control and radiotoxicity, and the extent to which local-regional control (LRC) influenced survival remain vague. This study aimed at evaluating RTD-effect relationship in esophageal squamous cell carcinoma (ESCC). Methods: 1440 dRT/CRT-treated ESCC patients were enrolled. Restricted cubic spline regression model was applied to reveal nonlinear relationship between RTD and survival/radiotoxicity. Linear regression analysis (LRA) was performed to evaluate correlations between LRC and overall survival (OS) or progression-free survival (PFS). Results: For 1440 dRT/CRT-treated ESCC patients, with RTD escalating, hazard ratios (HRs) of OS, PFS, LRC declined until RTD exceeded 60 Gy, then increased. HR of treatment-related mortality was stable until RTD exceeded 60 Gy, then increased. HR of LRC was lower for majority of patients treated with RTD≥60 Gy, except for those with KPS<80, T1-2 lesion, or without lymph node metastasis. LRA revealed strong correlations between LRC and OS/PFS. 45.5% and 44.9% of OS and PFS improvements were owing to improved LRC. Conclusions: RTD of 60 Gy was well tolerated, with favorable survival resulted of LRC improvement in local-advanced ESCC. Further stratification analyses based on radiation sensitivity will be helpful to determine potential beneficiaries of RTD escalation.

Survival benefit of surgery in patients with clinical T4 esophageal cancer who achieved complete or partial response after neoadjuvant chemoradiotherapy or radiotherapy.

Objective: This study aimed to determine the long-term survival of patients with cT4 esophageal cancer (EC) and whether neoadjuvant chemoradiotherapy/radiotherapy plus surgery (nCRT/RT + S) is superior to definitive CRT(dCRT)/RT in terms of survival in cT4 EC downstaged after nCRT/RT. Summary background data: Treatment options for cT4 EC include dCRT/RT and nCRT/RT + S, but it is not clear whether the latter provides survival benefit in patients downstaged after nCRT/RT. Methods: From 2002 to 2017, 726 patients with cT4 esophageal squamous cell carcinoma (ESCC) were retrospectively analyzed. Patients achieving clinical complete response (cCR) or partial response (PR) after 4-week RT (median dose, 40.7 Gy) and considered fit for surgery were offered esophagectomy. Of the 726 patients, 308 (42.4%) achieved cCR/PR, while 74 patients received subsequent surgery (nCRT/RT + S group), 234 patients received dCRT/RT. Results: Median follow-up was 58 months. The 3-year overall survival (OS) and progression-free survival (PFS) rates for all patients were 33.3% and 35.6%, respectively. The corresponding OS and PFS rates were 54.8% and 48.5% in the nCRT/RT + S group versus 30.0% and 22.1% in the dCRT/RT group (both p < 0.0001). After adjusting the confounding variables with inverse probability of treatment weighting, the adjusted 3-year OS rates were 50.4% in the nCRT/RT + S group versus 50.8% in the dCRT/RT group (p = 0.15). However, the adjusted 3-year PFS rates were significantly different between the two groups (49.0% and versus 38.3%, p = 0.004). Postoperative complications occurred in 18 (24.3%) patients. Conclusion: The long-term survival of cT4 ESCC was improved after the use of three-dimensional CRT. In cT4, EC responded to nCRT/RT, surgery improves PFS but not OS.

Concurrent chemoradiotherapy versus radiotherapy alone for patients with locally advanced esophageal squamous cell carcinoma in the era of intensity modulated radiotherapy: a propensity score-matched analysis.

BACKGROUND: To investigate the survival benefit of concurrent chemoradiotherapy (CCRT) for patients with locally advanced esophageal squamous cell carcinoma (ESCC) during the years of intensity-modulated radiotherapy (IMRT). METHODS: Medical records of 1089 patients with ESCC who received IMRT from January 2005 to December 2017 were retrospectively reviewed. A total of 617 patients received CCRT, 472 patients received radiotherapy (RT) alone. Propensity score matching (PSM) method was used to eliminate baseline differences between the two groups. Survival and toxicity profile were evaluated afterward. RESULTS: After a median follow-up time of 47.9 months (3.2-149.8 months), both overall survival (OS) and progression-free survival (PFS) of the CCRT group were better than those of the RT alone group, either before or after PSM. After PSM, the 1-, 3-, and 5-year OS of RT alone and CCRT groups were 59.0% versus 70.2%, 27.7% versus 40.5% and 20.3% versus 33.1%, respectively (p < 0.001). The 1-, 3-, and 5-year PFS were 39.4% versus 49.0%, 18.3% versus 30.4% and 10.5% versus 25.0%, respectively (p < 0.001). The rates of ≥ grade 3 leukopenia and radiation esophagitis in the CCRT group were higher than that of RT alone group (p < 0.05). There was no significant difference in the probability of radiation pneumonitis between the two groups (p = 0.167). Multivariate Cox analysis indicated that female, EQD2 ≥60 Gy and concurrent chemotherapy were favorable prognostic factors for both OS and PFS. CONCLUSIONS: Concurrent chemotherapy can bring survival benefits to patients with locally advanced ESCC receiving IMRT. For patients who cannot tolerate concurrent chemotherapy, RT alone is an effective alternative with promising results.

Comparison of Two Major Staging Systems in Predicting Survival and Recommendation of Postoperative Radiotherapy Based on the 11th Japanese Classification for Esophageal Carcinoma After Curative Resection: A Propensity Score-Matched Analysis.

OBJECTIVE: The aim of this study was to compare the prognostic predictive power of the 11th Japan Esophageal Society (JES) staging system with the 8th edition of the American Joint Committee on Cancer (AJCC) staging system in patients with thoracic esophageal squamous cell carcinoma (TESCC), and to estimate the survival benefits of postoperative radiotherapy (PORT) based on a substage of the JES staging system. METHODS: Area under the curve (AUC) values of the receiver operating characteristic curve were calculated to evaluate prognostic efficacy. Propensity score matching (PSM) analysis was conducted to balance the two groups (surgery only [S group] or surgery plus PORT [S+RT group]) across substages of the 11th JES staging system according to independent prognostic factors for overall survival (OS) identified using Cox proportional hazards regression. RESULTS: A total of 2960 patients were eligible. The 5-year OS AUC for the 8th AJCC staging system was significantly higher than that for the 11th JES staging system (0.701 vs. 0.675, p < 0.001). Before PSM, PORT significantly improved 5-year OS rates for patients in stage III and IVA by 9.1% (p < 0.001) and 21.1% (p < 0.001), respectively. After PSM, the 5-year OS rates in stage II, III, and IVA of the S+RT group were significantly higher than those in the S group (70.9%, 39.7%, and 35.1% vs. 57.8%, 27.2%, and 10.3%, respectively; p < 0.001). CONCLUSION: The 11th JES staging system was less capable of predicting prognosis than the 8th AJCC staging system and patients in stage III of the JES staging system were highly recommended to undergo PORT.

Proteogenomics Study of Blastobotrys adeninivorans TMCC 70007-A Dominant Yeast in the Fermentation Process of Pu-erh Tea.

Blastobotrys adeninivorans plays an essential role in pile-fermenting of Pu-erh tea. Its ability to assimilate various carbon and nitrogen sources makes it available for application in a wide range of industry sectors. The genome of B. adeninivorans TMCC 70007 isolated from pile-fermented Pu-erh tea was sequenced and assembled. Proteomics analysis indicated that 4900 proteins in TMCC 70007 were expressed under various culture conditions. Proteogenomics mapping revealed 48 previously unknown genes and corrected 118 gene models predicted by GeneMark-ES. Ortho-proteogenomics analysis identified 17 previously unidentified genes in B. adeninivorans LS3, the first strain with a sequenced genome among the genus Blastobotrys as well. More importantly, five species specific genes were identified from TMCC 70007, which could serve as a barcode for strain typing and were applicable for fermentation process protection of this industrial species. The datasets generated from tea aqueous extract culture not only increased the proteome coverage and accuracy but also contributed to the identification of proteins related to polyphenols and caffeine, which were considered to change greatly during the microbial fermentation of Pu-erh tea. This study provides a proteome perspective on TMCC 70007, which was considered to be an important strain in the production of Pu-erh tea. The systematic proteogenomics analysis not only made a better annotation on the genome of B. adeninivorans TMCC 70007 as previous proteogenomics study but also provided solution for fermentation process protection on valuable industrial species with species specific genes uniquely identified from proteogenomics study.

Salvage chemoradiation therapy for recurrence after radical surgery or palliative surgery in esophageal cancer patients: a prospective, multicenter clinical trial protocol.

BACKGROUND: Currently, adjuvant therapy is not recommended for patients with thoracic esophageal squamous cell cancer (TESCC) after radical surgery, and a proportion of these patients go on to develop locoregional recurrence (LRR) within 2 years. Besides, there is no evidence for salvage chemoradiation therapy (CRT) in patients with residual tumor after esophagectomy (R1/R2 resection). In addition, factors like different failure patterns and relationship with normal organs influence the decision for salvage strategy. Here, we aimed to design a modularized salvage CRT strategy for patients without a chance of salvage surgery according to different failure patterns (including R1/R2 resection), and further evaluated its efficacy and safety. METHODS: Our study was designed as a one arm, multicenter, prospective clinical trial. All enrolled patients were stratified in a stepwise manner based on the nature of surgery (R0 or R1/2), recurrent lesion diameter, involved regions, and time-to-recurrence, and were further assigned to undergo either elective nodal irradiation or involved field irradiation. Then, radiation technique and dose prescription were modified according to the distance from the recurrent lesion to the thoracic stomach or intestine. Ultimately, four treatment plans were established. DISCUSSION: This prospective study provided high-level evidence for clinical salvage management in patients with TESCC who developed LRR after radical surgery or those who underwent R1/R2 resection. TRIAL REGISTRATION: Prospectively Registered. ClinicalTrials.gov NCT03731442 , Registered November 6, 2018.

A multicenter prospective phase III clinical randomized study of simultaneous integrated boost intensity-modulated radiotherapy with or without concurrent chemotherapy in patients with esophageal cancer: 3JECROG P-02 study protocol.

BACKGROUND: Since the development of three-dimensional conformal radiotherapy and intensity-modulated radiotherapy (IMRT), no prospective study has investigated whether concurrent chemoradiotherapy (SIB-IMRT with 60 Gy) remains superior to radiotherapy (SIB-IMRT) alone for unresectable esophageal cancer (EC). Furthermore, the optimal therapeutic regimen for patients who cannot tolerate concurrent chemoradiotherapy is unclear. We recently completed a phase I/II radiation dose-escalation trial using simultaneous integrated boost (SIB), elective nodal irradiation, and concurrent chemotherapy for unresectable EC. We now intend to conduct a prospective, phase III, randomized study of SIB-IMRT with or without concurrent chemotherapy. We aim to find a safe, practical, and effective therapeutic regimen to replace the conventional segmentation (1.8-2.0 Gy) treatment mode (radiotherapy ± chemotherapy) for unresectable EC. METHODS: This two-arm, open, randomized, multicenter, phase III trial will recruit esophageal squamous cell carcinoma patients (stage IIA-IVB [UICC 2002]; IVB only with metastasis to the supraclavicular or celiac lymph nodes). In all, 164 patients will be randomized using a 1:1 allocation ratio, and stratified by study site and disease stage, especially the extent of lymph node metastasis. Patients in the SIB arm will receive definitive SIB radiotherapy (95% planning target volume/planning gross tumor volume, 50.4 Gy/59.92 Gy/28 f, equivalent dose in 2-Gy fractions = 60.62 Gy). Patients in the SIB + concurrent chemotherapy arm will receive definitive SIB radiotherapy with weekly paclitaxel and a platinum-based drug (5-6 weeks). Four cycles of consolidated chemoradiotherapy will also be recommended. The primary objective is to compare the 1-year, 2-year, and 3-year overall survival of the SIB + chemotherapy group and SIB groups. Secondary objectives include progression-free survival, local recurrence-free rate, completion rate, and adverse events. Detailed radiotherapy protocol and quality-assurance procedures have been incorporated into this trial. DISCUSSION: In unresectable, locally advanced EC, a safe and effective total radiotherapy dose and reasonable segmentation doses are required for the clinical application of SIB-IMRT + two-drug chemotherapy. Whether this protocol will replace the standard treatment regimen will be prospectively investigated. The effects of SIB-IMRT in patients with poor physical condition who cannot tolerate definitive chemoradiotherapy will also be investigated. TRIAL REGISTRATION: clinicaltrials.gov ( NCT03308552 , November 1, 2017).

High salt-induced excess reactive oxygen species production resulted in heart tube malformation during gastrulation.

An association has been proved between high salt consumption and cardiovascular mortality. In vertebrates, the heart is the first functional organ to be formed. However, it is not clear whether high-salt exposure has an adverse impact on cardiogenesis. Here we report high-salt exposure inhibited basement membrane breakdown by affecting RhoA, thus disturbing the expression of Slug/E-cadherin/N-cadherin/Laminin and interfering with mesoderm formation during the epithelial-mesenchymal transition(EMT). Furthermore, the DiI+ cell migration trajectory in vivo and scratch wound assays in vitro indicated that high-salt exposure restricted cell migration of cardiac progenitors, which was caused by the weaker cytoskeleton structure and unaltered corresponding adhesion junctions at HH7. Besides, down-regulation of GATA4/5/6, Nkx2.5, TBX5, and Mef2c and up-regulation of Wnt3a/ß-catenin caused aberrant cardiomyocyte differentiation at HH7 and HH10. High-salt exposure also inhibited cell proliferation and promoted apoptosis. Most importantly, our study revealed that excessive reactive oxygen species(ROS)generated by high salt disturbed the expression of cardiac-related genes, detrimentally affecting the above process including EMT, cell migration, differentiation, cell proliferation and apoptosis, which is the major cause of malformation of heart tubes.

Baicalin administration attenuates hyperglycemia-induced malformation of cardiovascular system.

In this study, the effects of Baicalin on the hyperglycemia-induced cardiovascular malformation during embryo development were investigated. Using early chick embryos, an optimal concentration of Baicalin (6 µM) was identified which could prevent hyperglycemia-induced cardiovascular malformation of embryos. Hyperglycemia-enhanced cell apoptosis was reduced in embryos and HUVECs in the presence of Baicalin. Hyperglycemia-induced excessive ROS production was inhibited when Baicalin was administered. Analyses of SOD, GSH-Px, MQAE and GABAA suggested Baicalin plays an antioxidant role in chick embryos possibly through suppression of outwardly rectifying Cl(-) in the high-glucose microenvironment. In addition, hyperglycemia-enhanced autophagy fell in the presence of Baicalin, through affecting the ubiquitin of p62 and accelerating autophagy flux. Both Baicalin and Vitamin C could decrease apoptosis, but CQ did not, suggesting autophagy to be a protective function on the cell survival. In mice, Baicalin reduced the elevated blood glucose level caused by streptozotocin (STZ). Taken together, these data suggest that hyperglycemia-induced embryonic cardiovascular malformation can be attenuated by Baicalin administration through suppressing the excessive production of ROS and autophagy. Baicalin could be a potential candidate drug for women suffering from gestational diabetes mellitus.

Gut microbiota-derived endotoxin enhanced the incidence of cardia bifida during cardiogenesis.

Cytotoxicity and inflammation-associated toxic responses could be induced by bacterial lipopolysaccharides (LPS) in vitro and in vivo, respectively. However, the mechanism involved in LPS-induced cardiac malformation in prenatal fetus is still unknown. In this study, we demonstrated that LPS was induced in gut microbiota imbalance mice, and next, LPS exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Gene transfection and tissue transplantation trajectory indicated that LPS exposure restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. In vitro explant allograft of GFP-labeled anterior primitive streak demonstrated that LPS treatments could inhibit cell migration. A similar observation was also obtained from the cell migration assay of scratch wounds using primary culture of cardiomyocytes or H9c2 cells. In the embryos exposed to LPS, expressions of Nkx2.5 and GATA5 were disturbed. These genes are associated with cardiomyocyte differentiation when heart tube fusion occurs. Furthermore, pHIS3, C-caspase3 immunohistological staining indicated that cell proliferation decreased, cell apoptosis increased in the heart tube of chick embryo. Meanwhile, in vivo, pHIS3 immunohistological staining and Hochest/PI staining also draw the similar conclusions. The LPS exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. At last, we showed that LPS-induced cardia bifida could be partially rescued through the addition of antioxidants. Together, these results reveal that excess ROS generation is involved in the LPS-induced defects in heart tube during chick embryo development.

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7+ neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7+ and HNK-1+ cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, ß-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1+ neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU+ neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

Ethanol exposure leads to disorder of blood island formation in early chick embryo.

Ethanol's effect on embryonic vasculogenesis and its underlying mechanism is obscure. Using VE-cadherin in situ hybridization, we found blood islands formation was inhibited in area opaca, but abnormal VE-cadherin+ cells were seen in area pellucida. We hypothesise ethanol may affect blood island progenitor cell migration and differentiation. DiI and in vitro experiments revealed ethanol inhibited cell migration, Quantitative PCR analysis revealed that ethanol exposure enhanced cell differentiation in area pellucida of HH5 chick embryos and repressed cell differentiation in area pellucida of HH8 chick embryos. By exposing to 2,2'-azobis-amidinopropane dihydrochloride, a ROS inducer, which gave a similar anti-vasculogenesis effect as ethanol and this anti-vasculogenesis effect could be reversed by vitamin C. Overall, exposing early chick embryos to ethanol represses blood island progenitor cell migration but disturbed differentiation at a different stage, so that the disorder of blood island formation occurs through excess ROS production and altered vascular-associated gene expression.