Development of a novel bioartificial cornea using 3D bioprinting based on electrospun micro-nanofibrous decellularized extracellular matrix.

Corneal damage contributes to blindness in millions of people. Simulating natural corneas with artificial corneas is challenging due to material and manufacturing limitations, including poor mechanical properties, complex manufacturing processes, and ocular histocompatibility. In this study, electrospun micro-nanofibrous decellularized extracellular matrix (dECM) is combined with digital light processing 3D bioprinting and validated as a bioartificial cornea for the first time. Electrospinning gives the material a controllable shape, and the electrospun micro-nanofibrous dECM, with preserved inherent biochemical components, can better mimic the natural ECM native microenvironment. An efficient platform can be developed for creating novel structural materials, when combined with intelligent manufacturing. Artificial biological corneas developed using this method showed five-fold improvements in mechanical properties (248.5 ± 35.67 kPa vs. 56.91 ± 3.68 kPa,p< 0.001), superior guidance for cell organization and adhesion, and better maintenance of the cellular phenotype of keratocytes. In animal studies,in vivotransplantation of this artificial cornea showed better regeneration, which accelerated corneal epithelialization and maintained corneal transparency. This method has potential for biomedical applications, and bioartificial corneas manufactured by this method have ideal properties as an alternative to lamellar keratoplasty, with promise for clinical transformation.

3D bioprinting of corneal decellularized extracellular matrix: GelMA composite hydrogel for corneal stroma engineering.

Millions of individuals across the world suffer from corneal stromal diseases that impair vision. Fortunately, three-dimensional (3D) bioprinting technology which has revolutionized the field of regenerative tissue engineering makes it feasible to create personalized corneas. In this study, an artificial cornea with a high degree of precision, smoothness, and programmable curvature was prepared by using digital light processing (DLP) 3D bioprinting in one piece with no support structure, and the construct was then confirmed by optical coherence tomography (OCT). On the basis of this approach, we developed a novel corneal decellularized extracellular matrix/gelatin methacryloyl (CECM-GelMA) bioink that can produce complex microenvironments with highly tunable mechanical properties while retaining high optical transmittance. Furthermore, the composite hydrogel was loaded with human corneal fibroblasts (hCFs), and in vitro experiments showed that the hydrogel maintained high cell viability and expressed core proteins. In vivo tests revealed that the hydrogel might promote epithelial regeneration, keep the matrix aligned, and restore clarity. This demonstrates how crucial a role CECM plays in establishing a favorable environment that encourages the transformation of cell function. Therefore, artificial corneas that can be rapidly customized have a huge potential in the development of in vitro corneal matrix analogs.

Effect of Anti-Allergic Therapy on Sleep Quality of Children with Allergic Conjunctivitis and Their Parents.

PURPOSE: To assess the effect of anti-allergic therapy on sleep quality of children with allergic conjunctivitis (AC) and their parents. METHODS: This prospective single-arm intervention study included 54 AC child-parent dyads. Chinese versions of the Children's Sleep Habits Questionnaire (CSHQ) and Pittsburgh Sleep Quality Index (PSQI) were used to assess the sleep quality of children and their parents, respectively. RESULTS: CSHQ and PSQI total scores were significantly decreased after treatment, with fewer children and parents reporting poor sleep quality. Part impaired sleep behaviors of children and parents can recover to the normal levels. Sleep quality improved greater in children with a severe type of AC, those with worse baseline signs, and without other allergic diseases. For both children and parents, greater improvements in sleep quality were associated with longer treatment duration and with worse baseline sleep quality. CONCLUSION: Successful management of AC improves sleep quality for both children and their parents.

[Mechanism of electroacupuncture penetration needling for relieving synovial inflammation of knee osteoarthritis through TLR4/MyD88/NF-κB signal pathway].

OBJECTIVE: To observe the effects of electroacupuncture (EA) penetration needling on Toll-like receptors 4/myeloid differentiation factor 88/nuclear factor-kappa B (TLR4/MyD88/NF-κB) signaling pathway in rat synovium and the serum-related inflammatory factors, so as to explore the mechanism of EA penetration needling on synovial inflammation in rats with knee osteoarthritis (KOA). METHODS: SD male rats were randomly divided into sham-operation group, model group, EA+penetration needling group, and conventional EA group, with 16 rats in each group. The rats model was prepared by anterior cruciate ligment transection and these rats were forced to exercise for 8 weeks after operation. After successful modeling, in the EA+penetration needling group, the needles were inserted at "Dubi" (ST35) "Neixiyan" (EX-LE4), and at "Xuehai"(SP10) "Liangqiu"(ST34) on the right hind limb, towards each other, 5-8 mm in depth, respectively. In the conventional EA group, the needles were inserted at ST35 and EX-LE4 on the right hind limb, obliquely, at 30° angle to the skin, 3-5 mm in depth; and were inserted at SP10 and ST34 on the right hind limb perpendicularly, 3-5 mm in depth. In these two groups, electric stimulation was operated with dense-disperse wave, 2 Hz/10 Hz in frequency and 0.5-1.5 mA in intensity, retained for 20 min in each treatment. The treatment was given once daily, 10 days as 1 course of treatment, and 2 courses were required at the interval of 2 days. After the intervention, the knee joint effusion was observed by musculoskeletal ultrasound; the contents of IL-1ß, IL-6 and TNF-α in serum were determined by ELISA; the morphological changes in the synovium were observed after H.E. staining; the positive expression of NF-κB p65 in the synovial membrane was detected by immunohistochemical method; the expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 proteins in the synovial membrane were determined by Western blot. RESULTS: Compared with the sham-operation group, in the model group, the knee joint effusion was obviously increased, the synovial lining cells were distributed irregularly, the cells were disarranged, the pannus was formed largely, and a great number of the inflammatory cells were infiltrated; the contents of serum IL-1ß, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were increased (P<0.05). Compared with the model group, the knee joint effusion was reduced, the synovial lining cells were proliferated, a small number of the inflammatory cells were infiltrated, and the pannus was formed lightly; the contents of serum IL-1ß, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were lower (P<0.05) in the EA+penetration needling group and the conventional EA group. In the conventional EA group, the knee joint effusion was increased, the synovial lining cells were proliferated, the inflammatory cells were infiltrated largely, and the pannus was formed increasingly; the contents of serum IL-1ß, IL-6 and TNF-α, and the protein expression levels of TLR4, MyD88 and NF-κB p65 in the synovial tissue were increased when compared with the EA+penetration needling group (P<0.05). CONCLUSION: The EA+penetration needling can significantly relieve the synovial inflammatory reaction and the knee joint effusion in KOA rats. The mechanism is probably related to down-regulating the downstream inflammatory cascade through inhibiting the transduction of TLR4/MyD88/NF-κB signaling pathway.

Fecal calprotectin as an indicator in risk stratification of pouchitis following ileal pouch-anal anastomosis for ulcerative colitis.

BACKGROUND: Pouchitis is the most common complication following restorative proctocolectomy and ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). Fecal calprotectin (FC) is a noninvasive indicator of the intestinal inflammatory status. This study was conducted to evaluate the clinical value of the FC concentration for the diagnosis and risk assessment of pouchitis. PATIENTS AND METHODS: This retrospective study involved patients who underwent IPAA for UC at Tianjin Medical University General Hospital from January 2015 to January 2019. The patients were categorized into pouchitis and non-pouchitis groups based on their Pouchitis Disease Activity Index (PDAI) score. Laboratory indicators, including the FC concentration, were collected from both groups. RESULTS: Sixty-six patients with UC after IPAA were included in the study and divided into the non-pouchitis group (n = 40) and pouchitis group (n = 26). The correlation coefficient between the FC concentration and the PDAI score was 0.651 (p < 0.001). Receiver operating characteristic analysis showed that the FC cut-off value for predicting pouchitis was 579.60 µg/g (area under the curve, 0.938). The patients were then divided into three subgroups according to their PDAI score (0-2, 3-6, and ≥7), and significant differences in the FC concentration were found among the three subgroups. The best FC cut-off value for predicting a high risk of pouchitis (PDAI score of 3-6) was 143.25 µg/g (area under the curve, 0.876). CONCLUSIONS: FC is a useful biomarker in patients with pouchitis. Patients are advised to regularly undergo FC measurement to monitor for pouchitis. An FC concentration in the range of 143.25-579.60 µg/g is predictive of a high risk for pouchitis, and further examination and preventive treatment are necessary in such patients.KEY MESSAGESFecal calprotectin can be used to quantify pouch inflammation.Fecal calprotectin can be used to predict a high risk of pouchitis.

Effect of Anti-allergic Therapy on Quality of Life in Children with Allergic Conjunctivitis and Their Parents.

PURPOSE: To evaluate the effect of anti-allergic therapy on quality of life (QoL) in children with allergic conjunctivitis (AC) and their parents. METHODS: Prospective single-arm intervention study including 55 AC child-parent pairs. The endpoint was that AC was successfully controlled after anti-allergic therapies. The primary outcome was the change in QoL of children from baseline to endpoint as measured by the total Pediatric Quality of Life Inventory (PesdQL) score. RESULTS: The total PedsQL scores of children and parents were improved after treatment (P < .001). QoL improved greater in children with vernal keratoconjunctivitis/atopic keratoconjunctivitis, those without other allergy outside the eye, and those with lower baseline total PedsQL score. Greater improvement of parents' QoL was associated with longer treating duration, greater improvement of children's QoL, lower baseline parents' total PedsQL score, and more severe baseline children's signs. CONCLUSION: Both pediatric AC patients' and their parents 'reduced QoL were improved after anti-allergic therapy. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trial Registry Identifier: ChiCTR2000037866.

Impaired sleep quality in children with allergic conjunctivitis and their parents.

OBJECTIVES: To investigate the sleep quality in children with allergic conjunctivitis (AC) and their parents. METHODS: Prospective, case-controlled study. Zhongshan Ophthalmic Center, a tertiary referral centre. Participants comprised 73 children aged 4-12 years with AC and their parents, and 81 healthy, age-matched children who served as controls and their parents. General information was recorded and ocular manifestations of children with AC were scored. Sleep quality of the children and parents were assessed using Children's Sleep Habits Questionnaire (CSHQ) and Pittsburgh Sleep Quality Index (PSQI). RESULTS: Children with AC and their parents had reduced sleep quality (Children's CSHQ: 48.3 ± 6.55 vs. 38.8 ± 4.63; Parental PSQI: 5.62 ± 2.12 vs. 3.40 ± 1.90, both p < 0.001) and significantly higher prevalence of poor sleep quality (CSHQ ≥ 41 in Children: 89.0% vs. 23.5%; PSQI > 7 in Parents: 18.5% vs. 1.23%, both p < 0.001). Children with AC scored worse on subcomponents of CSHQ including sleep onset delay, sleep duration, parasomnia, sleep-disordered breathing, and daytime sleepiness. Parents scored worse on subscores of PSQI including sleep duration, sleep disturbances, use of sleeping medication, and daytime sleepiness. Poor sleep quality in children with AC was associated with follicle formation (OR:3.95; 95% CI: 1.88-8.31, p < 0.001) and keratitis (OR:6.03; 95% CI: 1.29-28.3, p = 0.028). Parental poor sleep quality was associated with follicle formation (OR:7.14; 95% CI: 2.06-24.8, p = 0.002) and keratitis (OR:4.49; 95% CI: 1.27-15.9, p = 0.020) in children. CONCLUSIONS: AC has a negative association with sleep quality in children and their parents, especially in those children with severe follicle formation and keratitis. STATE THE DETAILS OF CLINICAL TRIALS: Chictr.org.cn, https://www.chictr.org.cn/showproj.aspx?proj=43511 , ChiCTR1900027486. STATEMENT OF SIGNIFICANCE: Allergic conjunctivitis is a frequently encountered problem diagnosed and managed by ophthalmologists, paediatricians, allergists, and primary care physicians and has become a major public health issue. Sleep is crucial for learning and effective development in children. Our study discovered a strong association between these two conditions. This is the first study to evaluate the association of allergic conjunctivitis and sleep quality in children and their parents. This case-controlled study found that allergic conjunctivitis had a negative impact on sleep quality not only for children but also for their parents. The findings of this study suggest a multifaceted impact of AC with sleep quality; detailed assessment of sleep quality for improved care of paediatric patients with allergic conjunctivitis would be useful.

Botany, traditional uses, phytochemistry and pharmacological activity of Crataegus pinnatifida (Chinese hawthorn): a review.

OBJECTIVES: Crataegus pinnatifida (C. pinnatifida), including C. pinnatifida Bge. and its variant C. pinnatifida Bge. var. major N, E. Br., has traditionally been used as a homologous plant for traditional medicine and food in ethnic medical systems in China. Crataegus pinnatifida, especially its fruit, has been used for more than 2000 years to treat indigestion, stagnation of meat, hyperlipidemia, blood stasis, heart tingling, sores, etc. This review aimed to provide a systematic summary on the botany, traditional uses, phytochemistry, pharmacology and clinical applications of C. pinnatifida. KEY FINDINGS: This plant contains flavonoids, phenylpropanoids, terpenoids, organic acids, saccharides and essential oils. Experimental studies showed that it has hypolipidemic, antimyocardial, anti-ischemia, antithrombotic, anti-atherosclerotic, anti-inflammatory, antineoplastic neuroprotective activity, etc. Importantly, it has good effects in treating diseases of the digestive system and cardiovascular and cerebrovascular systems. SUMMARY: There is convincing evidence from both in vitro and in vivo studies supporting the traditional uses of C. pinnatifida. However, multitarget network pharmacology and molecular docking technology should be used to study the interaction between the active ingredients and targets of C. pinnatifida. Furthermore, exploring the synergy of C. pinnatifida with other Chinese medicines to provide new understanding of complex diseases may be a promising strategy.

Case Report: An Unusual Case of Pulmonary Metastatic Adenocarcinoma From Low-Grade Appendiceal Mucinous Neoplasms.

Background: Low-grade appendiceal mucinous neoplasms (LAMNs) are indolent tumors with low-grade cytology. Although peritoneal dissemination is common due to tumor rupture and mucinous deposits on the visceral peritoneal surface, distant involvement, such as lung, is rarely seen due to lack of invasiveness. Case Presentation: A 70-year-old woman presented to the hospital due to continuously elevated carcinoembryonic antigen (CEA) levels for 10 months without any symptoms. PET/CT revealed two lesions located in the left lung and appendix. The postoperative pathology results revealed pulmonary mucinous adenocarcinoma and LAMN. Then we performed next-generation sequencing (NGS) to clarify the relationship between the two tumors. The sequencing result showed that both tumors harbored the common tumor mutations, KRAS (p.G12D), GNAS (p.R201H), and BRAF (p.R735Q), which indicated that the pulmonary tumor was a metastasis of LAMN. Conclusion: This case is unusual in that the primary LAMN and the pulmonary metastasis are present at the time of diagnosis. This study reported the first pulmonary metastasis from LAMN verified by NGS.

Association of Allergic Conjunctivitis With Health-Related Quality of Life in Children and Their Parents.

IMPORTANCE: Allergic conjunctivitis (AC) is one of the most common allergic diseases and is especially problematic in children and adolescents. The course of AC is generally prolonged and often recurs. Understanding the health-related quality of life (QOL) of both children with AC and their parents would be useful. OBJECTIVE: To evaluate the association between AC and health-related QOL in children and their parents. DESIGN, SETTING, AND PARTICIPANTS: A prospective case-control study was conducted at Zhongshan Ophthalmic Center, a single tertiary referral center, from November 16, 2019, through January 20, 2020. Participants comprised 92 children aged 5 to 18 years with AC and their parents and 96 healthy, age-matched children who served as controls and their parents. The 92 children in the AC group were subdivided into cohorts with vernal keratoconjunctivitis (VKC) (23 [25.0%]) or atopic keratoconjunctivitis (AKC) (7 [7.6%]) and seasonal allergic conjunctivitis (SAC) (26 [28.3%]) or perennial allergic conjunctivitis (PAC) (36 [39.1%]). EXPOSURES: Allergic conjunctivitis. MAIN OUTCOMES AND MEASURES: Pediatric Quality of Life Inventory, version 4.0 (PedsQL), scores for children and their parents. Scores range from 0 to 100, with higher scores indicating better health-related QOL and fewer negative aspects. RESULTS: In the AC group, 77 of 92 (83.7%) participants were boys, and 67 (72.8%) of the parents were women. Of the individuals in the control group, 55 of 96 (57.3%) of the children were girls and 76 (79.2%) of the parents were women. Median total PedsQL scores were reduced in both children with AC (69.6 [interquartile range [IQR], 66.3-72.8 vs 96.7; IQR, 92.7-98.9; P < .001) and their parents (68.8; IQR, 63.9-71.4 vs 96.5; IQR, 95.1-97.9; P < .001). The reduction in health-related QOL was more severe in children with VKC/AKC than in those with SAC/PAC (difference, -3.3; 95% CI, -5.4 to -1.1; P = .004) and their parents (difference, -4.3; 95% CI, -7.1 to -2.1; P < .001). In the AC group, a higher corneal fluorescein staining score was associated with lower QOL in children (ß, -1.16; 95% CI, -1.80 to -0.52; P = .001); higher corneal fluorescein staining scores (ß, -1.12; 95% CI, -1.74 to -0.50; P = .001) and multiple clinical consultations (ß, -3.96; 95% CI, -7.34 to -0.57; P = .02) were associated with lower QOL in parents. The parents' QOL scores were correlated with their children's QOL scores (correlation coefficient, r = 0.59; P < .001). CONCLUSIONS AND RELEVANCE: These findings suggest AC has a negative association with health-related QOL for children and their parents, especially in children with VKC/AKC or higher corneal fluorescein staining scores.

N-Acetylcysteine Suppresses LPS-Induced Pathological Angiogenesis.

BACKGROUND/AIMS: Angiogenesis is a key feature during embryo development but is also part of the pathogenesis of cancer in adult life. Angiogenesis might be modulated by inflammation. METHODS: We established an angiogenesis model in chick chorioallantoic membrane (CAM) induced by the exposure of lipopolysaccharide (LPS), and analyzed the effects of the antioxidant N-acetylcysteine (NAC) on angiogenesis in this model as well as on the expression of key genes known to involved in the regulation of angiogenesis. RESULTS: Treatment with NAC was able to normalize LPS induced angiogenesis and restore the LPS-induced damage of vascular epithelium in chick CAM. Using quantitative PCR, we showed that NAC administration normalized the LPS induced expression of Keap1-Nrf2 signaling and oxidative stress key enzyme gene expressions (SOD, GPx and YAP1). CONCLUSION: We established a LPS-induced angiogenesis model in chick CAM. NAC administration could effectively inhibit LPS-induced angiogenesis and restore the integrity of endothelium on chick CAM. LPS exposure caused an increased expression of genes involved in oxidative stress in chick CAM. NAC administration could abolish this effect.

Lipopolysaccharides (LPS) Induced Angiogenesis During Chicken Embryogenesis is Abolished by Combined ETA/ETB Receptor Blockade.

BACKGROUND/AIMS: Angiogenesis plays a key role during embryonic development. The vascular endothelin (ET) system is involved in the regulation of angiogenesis. Lipopolysaccharides (LPS) could induce angiogenesis. The effects of ET blockers on baseline and LPS-stimulated angiogenesis during embryonic development remain unknown so far. METHODS: The blood vessel density (BVD) of chorioallantoic membranes (CAMs), which were treated with saline (control), LPS, and/or BQ123 and the ETB blocker BQ788, were quantified and analyzed using an IPP 6.0 image analysis program. Moreover, the expressions of ET-1, ET-2, ET3, ET receptor A (ETRA), ET receptor B (ETRB) and VEGFR2 mRNA during embryogenesis were analyzed by semi-quantitative RT-PCR. RESULTS: All components of the ET system are detectable during chicken embryogenesis. LPS increased angiogenesis substantially. This process was completely blocked by the treatment of a combination of the ETA receptor blockers-BQ123 and the ETB receptor blocker BQ788. This effect was accompanied by a decrease in ETRA, ETRB, and VEGFR2 gene expression. However, the baseline angiogenesis was not affected by combined ETA/ETB receptor blockade. CONCLUSION: During chicken embryogenesis, the LPS-stimulated angiogenesis, but not baseline angiogenesis, is sensitive to combined ETA/ETB receptor blockade.

Excess Imidacloprid Exposure Causes the Heart Tube Malformation of Chick Embryos.

As a neonicotinoid pesticide, imidacloprid is widely used to control sucking insects on agricultural planting and fleas on domestic animals. However, the extent to which imidacloprid exposure has an influence on cardiogensis in early embryogenesis is still poorly understood. In vertebrates, the heart is the first organ to be formed. In this study, to address whether imidacloprid exposure affects early heart development, the early chick embryo has been used as an experimental model because of its accessibility at its early developmental stage. The results demonstrate that exposure of the early chick embryo to imidacloprid caused malformation of heart tube. Furthermore, the data reveal that down-regulation of GATA4, NKX2.5, and BMP4 and up-regulation of Wnt3a led to aberrant cardiomyocyte differentiation. In addition, imidacloprid exposure interfered with basement membrane breakdown, E-cadherin/laminin expression, and mesoderm formation during the epithelial-mesenchymal transition (EMT) in gastrula chick embryos. Finally, the DiI-labeled cell migration trajectory indicated that imidacloprid restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. A similar observation was also obtained from the cell migration assay of scratch wounds in vitro. Additionally, imidacloprid exposure negatively affected the cytoskeleton structure and expression of corresponding adhesion molecules. Taken together, these results reveal that the improper EMT, cardiac progenitor migration, and differentiation are responsible for imidacloprid exposure-induced malformation of heart tube during chick embryo development.

From the Cover: Exposing Imidacloprid Interferes With Neurogenesis Through Impacting on Chick Neural Tube Cell Survival.

As a neonicotinoid pesticide, imidacloprid is widely used to control insects in agriculture and fleas on domestic animals. However, it is not known whether imidacloprid exposure negatively affects neurogenesis during embryonic development. In this study, using a chick embryo model, we investigated the effects of imidacloprid exposure on neurogenesis at the earliest stage and during late-stage embryo development. Exposing HH0 chick embryos to imidacloprid in EC culture caused neural tube defects (NTDs) and neuronal differentiation dysplasia as determined by NF/Tuj1 labeling. Furthermore, we found that F-actin accumulation on the apical side of the neural tube was suppressed by exposure to imidacloprid, and the expression of BMP4 and Shh on the dorsal and ventral sides of the neural tubes, respectively, were also reduced, which in turn affects the dorsolateral hinge points during bending of the neural plate. In addition, exposure to imidacloprid reduced cell proliferation and increased cell apoptosis, as determined by pHIS3 labeling and TUNEL staining, respectively, also contributing to the malformation. We obtained similar results in late-stage embryos exposed to imidacloprid. Finally, a bioinformatics analysis was employed to determine which genes identified in this study were involved in NTDs. The experimental evidence and bioinformatics analysis suggested that imidacloprid exposure during chick embryo development could increase the risk of NTDs and neural dysplasia.

Angiogenesis is repressed by ethanol exposure during chick embryonic development.

It is now known that excess alcohol consumption during pregnancy can cause fetal alcohol syndrome to develop. However, it is not known whether excess ethanol exposure could directly affect angiogenesis in the embryo or angiogenesis being indirectly affected because of ethanol-induced fetal alcohol syndrome. Using the chick yolk sac membrane (YSM) model, we demonstrated that ethanol exposure dramatically inhibited angiogenesis in the YSM of 9-day-old chick embryos, in a dose-dependent manner. Likewise, the anti-angiogenesis effect of ethanol could be seen in the developing vessel plexus (at the same extra-embryonic regions) during earlier stages of embryo development. The anti-angiogenic effect of ethanol was found associated with excess reactive oxygen species (ROS) production; as glutathione peroxidase activity increased while superoxide dismutase 1 and 2 activities decreased in the YSMs. We further validated this observation by exposing chick embryos to 2,2'-azobis-amidinopropane dihydrochloride (a ROS inducer) and obtained a similar anti-angiogenesis effect as ethanol treatment. Semiquantitative reverse transcription-polymerase chain reaction analysis of the experimental YSMs revealed that expression of angiogenesis-related genes, vascular endothelial growth factor and its receptor, fibroblast growth factor 2 and hypoxia-inducible factor, were all repressed following ethanol and 2,2'-azobis-amidinopropane dihydrochloride treatment. In summary, our results suggest that excess ethanol exposure inhibits embryonic angiogenesis through promoting superfluous ROS production during embryo development.

The impact of high-salt exposure on cardiovascular development in the early chick embryo.

In this study, we show that high-salt exposure dramatically increases chick mortality during embryo development. As embryonic mortality at early stages mainly results from defects in cardiovascular development, we focused on heart formation and angiogenesis. We found that high-salt exposure enhanced the risk of abnormal heart tube looping and blood congestion in the heart chamber. In the presence of high salt, both ventricular cell proliferation and apoptosis increased. The high osmolarity induced by high salt in the ventricular cardiomyocytes resulted in incomplete differentiation, which might be due to reduced expression of Nkx2.5 and GATA4. Blood vessel density and diameter were suppressed by exposure to high salt in both the yolk sac membrane (YSM) and chorioallantoic membrane models. In addition, high-salt-induced suppression of angiogenesis occurred even at the vasculogenesis stage, as blood island formation was also inhibited by high-salt exposure. At the same time, cell proliferation was repressed and cell apoptosis was enhanced by high-salt exposure in YSM tissue. Moreover, the reduction in expression of HIF2 and FGF2 genes might cause high-salt-suppressed angiogenesis. Interestingly, we show that high-salt exposure causes excess generation of reactive oxygen species (ROS) in the heart and YSM tissues, which could be partially rescued through the addition of antioxidants. In total, our study suggests that excess generation of ROS might play an important role in high-salt-induced defects in heart and angiogenesis.