Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA.

BACKGROUND: Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA. METHODS: The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. RESULTS: The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. CONCLUSIONS: This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Weak SARS-CoV-2-specific responses of TIGIT-expressing CD8 + T cells in people living with HIV after a third dose of a SARS-CoV-2 inactivated vaccine.

BACKGROUND: T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), an inhibitory receptor expressed on T cells, plays a dysfunctional role in antiviral infection and antitumor activity. However, it is unknown whether TIGIT expression on T cells influences the immunological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated vaccines. METHODS: Forty-five people living with HIV (PLWH) on antiretroviral therapy (ART) for more than two years and 31 healthy controls (HCs), all received a third dose of a SARS-CoV-2 inactivated vaccine, were enrolled in this study. The amounts, activation, proportion of cell subsets, and magnitude of the SARS-CoV-2-specific immune response of TIGIT + CD4 + and TIGIT + CD8 + T cells were investigated before the third dose but 6 months after the second vaccine dose (0W), 4 weeks (4W) and 12 weeks (12W) after the third dose. RESULTS: Compared to that in HCs, the frequency of TIGIT + CD8 + T cells in the peripheral blood of PLWH increased at 12W after the third dose of the inactivated vaccine, and the immune activation of TIGIT + CD8 + T cells also increased. A decrease in the ratio of both T naïve (T N ) and central memory (T CM ) cells among TIGIT + CD8 + T cells and an increase in the ratio of the effector memory (T EM ) subpopulation were observed at 12W in PLWH. Interestingly, particularly at 12W, a higher proportion of TIGIT + CD8 + T cells expressing CD137 and CD69 simultaneously was observed in HCs than in PLWH based on the activation-induced marker assay. Compared with 0W, SARS-CoV-2-specific TIGIT + CD8 + T-cell responses in PLWH were not enhanced at 12W but were enhanced in HCs. Additionally, at all time points, the SARS-CoV-2-specific responses of TIGIT + CD8 + T cells in PLWH were significantly weaker than those of TIGIT - CD8 + T cells. However, in HCs, the difference in the SARS-CoV-2-specific responses induced between TIGIT + CD8 + T cells and TIGIT - CD8 + T cells was insignificant at 4W and 12W, except at 0W. CONCLUSIONS: TIGIT expression on CD8 + T cells may hinder the T-cell immune response to a booster dose of an inactivated SARS-CoV-2 vaccine, suggesting weakened resistance to SARS-CoV-2 infection, especially in PLWH. Furthermore, TIGIT may be used as a potential target to increase the production of SARS-CoV-2-specific CD8 + T cells, thereby enhancing the effectiveness of vaccination.

Changing roles of CD3 + CD8 low T cells in combating HIV-1 infection.

BACKGROUND: Cluster of differentiation 8 (CD8 T) cells play critical roles in eradicating human immunodeficiency virus (HIV)-1 infection, but little is known about the effects of T cells expressing CD8 at low levels (CD8 low ) or high levels (CD8 high ) on HIV-1 replication inhibition after HIV-1 invasion into individual. METHODS: Nineteen patients who had been acutely infected with HIV-1 (AHI) and 20 patients with chronic infection (CHI) for ≥2 years were enrolled in this study to investigate the dynamics of the quantity, activation, and immune responses of CD3 + CD8 low T cells and their counterpart CD3 + CD8 high T cells at different stages of HIV-1 infection. RESULTS: Compared with healthy donors, CD3 + CD8 low T cells expanded in HIV-1-infected individuals at different stages of infection. As HIV-1 infection progressed, CD3 + CD8 low T cells gradually decreased. Simultaneously, CD3 + CD8 high T cells was significantly reduced in the first month of AHI and then increased gradually as HIV-1 infection progressed. The classical activation of CD3 + CD8 low T cells was highest in the first month of AHI and then reduced as HIV-1 infection progressed and entered the chronic stage. Meanwhile, activated CD38 - HLA-DR + CD8 low T cells did not increase in the first month of AHI, and the number of these cells was inversely associated with viral load ( r = -0.664, P = 0.004) but positively associated with the CD4 T-cell count ( r = 0.586, P = 0.014). Increased programmed cell death protein 1 (PD-1) abundance on CD3 + CD8 low T cells was observed from the 1st month of AHI but did not continue to be enhanced, while a significant T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) abundance increase was observed in the 12th month of infection. Furthermore, increased PD-1 and TIGIT abundance on CD3 + CD8 low T cells was associated with a low CD4 T-cell count (PD-1: r = -0.456, P = 0.043; TIGIT: r = -0.488, P = 0.029) in CHI. Nonetheless, the nonincrease in PD-1 expression on classically activated CD3 + CD8 low T cells was inversely associated with HIV-1 viremia in the first month of AHI ( r = -0.578, P = 0.015). Notably, in the first month of AHI, few CD3 + CD8 low T cells, but comparable amounts of CD3 + CD8 high T cells, responded to Gag peptides. Then, weaker HIV-1-specific T-cell responses were induced in CD3 + CD8 low T cells than CD3 + CD8 high T cells at the 3rd and 12th months of AHI and in CHI. CONCLUSIONS: Our findings suggest that CD3 + CD8 low T cells play an anti-HIV role in the first month of infection due to their abundance but induce a weak HIV-1-specific immune response. Subsequently, CD3 + CD8 low T-cell number decreased gradually as infection persisted, and their anti-HIV functions were inferior to those of CD3 + CD8 high T cells.

T-cell responses to SARS-CoV-2 Omicron spike epitopes with mutations after the third booster dose of an inactivated vaccine.

The rapidly spreading severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant contains more than 30 mutations that mediate escape from antibody responses elicited by prior infection or current vaccines. Fortunately, T-cell responses are highly conserved in most individuals, but the impacts of mutations are not clear. Here, we showed that the T-cell responses of individuals who underwent booster vaccination with CoronaVac were largely protective against the SARS-CoV-2 Omicron spike protein. To specifically estimate the impact of Omicron mutations on vaccinated participants, 16 peptides derived from the spike protein of the ancestral virus or Omicron strain with mutations were used to stimulate peripheral blood mononuclear cells (PBMCs) from the volunteers. Compared with the administration of two doses of vaccine, booster vaccination substantially enhanced T-cell activation in response to both the ancestral and Omicron epitopes, although the enhancement was slightly weakened by the Omicron mutations. Then, the peptides derived from these spike proteins were used separately to stimulate PBMCs. Interestingly, compared with the ancestral peptides, only the peptides with the G339D or N440K mutation were detected to significantly destabilize the T-cell response. Although more participants need to be evaluated to confirm this conclusion, our study nonetheless estimates the impacts of mutations on T-cell responses to the SARS-CoV-2 Omicron variant.

Generation of human blastocyst-like structures from pluripotent stem cells.

Human blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm, all of which are essential for early development and organ formation. However, due to ethical concerns and restricted access to human blastocysts, a comprehensive understanding of early human embryogenesis is still lacking. To bridge this knowledge gap, a reliable model system that recapitulates early stages of human embryogenesis is needed. Here we developed a three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids are similar with human embryos that were cultured for 8 or 10 days in vitro, in terms of embryonic structures, cell lineages and transcriptomic profiles. In conclusion, we developed a scalable system to mimic human blastocyst development, which can potentially facilitate the study of early implantation failure that induced by developmental defects at early stage.

A lncRNA-miRNA-mRNA network for human primed, naive and extended pluripotent stem cells.

Human pluripotent stem cells (hPSCs) represent a promising platform for studying embryonic development, and different states of pluripotency reflect the different stages of embryo development. Here, we successfully converted three in-house-derived primed hPSC lines (H10, H24, and iPS) to a naive state and an expanded pluripotent stem cell (EPS) state. Primed, naive and EPS cells displayed state-specific morphologies and expressed pluripotent markers. The expression of SSEA4 and TRA-1-60 was downregulated in the conversion process. The H3K27me3 expression level also decreased, indicating that global methylation was reduced and that the X chromosome started to reactivate. RNA-sequencing analysis results revealed that differentially expressed genes (DEGs) were significantly enriched in both naive hPSCs and EPS cells when compared to the primed state. However, imprinted gene expression barely changed before and after state reversion. Gene ontology (GO) analyses showed that the upregulated DEGs were mostly enriched in RNA processing, DNA replication and repair, and regulation of cell cycle process, while downregulated DEGs were related to extracellular adhesion and various tissue developmental processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that EPS cells were enriched in the PI3K-Akt and Wnt signaling pathways. Analysis of the lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network between primed, naive hPSCs and EPS cells revealed that hsa-miR-424-5p, has-miR-16-5p, has-miR-27b-3p, has-miR-29c-3p, and KCNQ1OT1 were crucial nodes with high degrees of connectivity. Our work may represent new insight into the intrinsic molecular features of different hPSC states.

Transplanted mouse liver stem cells at different stages of differentiation ameliorate concanavalin A-induced acute liver injury by modulating Tregs and Th17 cells in mice.

Acute liver failure (ALF) is a disease with a considerably high mortality rate that still lacks a safe and effective treatment. Transplantation of liver stem cells (LSCs) has been considered to be a promising therapeutic alternative for ALF since LSCs have been shown to be involved in immunomodulation and functional reconstruction of the liver. Our present study evaluated and compared the protective effects of the two mouse LSC lines, YE and R5, as well as those of adult mouse hepatocyte (HC), on concanavalin A (ConA)-induced acute liver injury. YE and R5 cells were analyzed by microscopy, functional assays, and gene expression. We confirmed that YE and R5 cells were undifferentiated cells that had partial hepatocytic functions and a potential to differentiate into hepatocytes. YE cells has characteristics of LSCs at the early stage of differentiation, whereas the differentiation stage of R5 cells was later than that of YE cells. Subsequently, YE, R5, and HC cells were intraperitoneally transplanted into three groups of mice, followed by injection of ConA through the tail vein of each mouse at 12 h later. Blood tests, histology, flow cytometry, and quantitative PCR were then used to evaluate the therapeutic effects of the cell transplantations at 24 h after ConA injections. Compared with that of the ConA control group, YE, R5, and HC cells reduced the expression of alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in serum and alleviated the degree of hepatic necrosis. Moreover, transplantation of these cells induced more regulatory T cells (Tregs) and less T-helper 17 (Th17) cells in the liver and spleen, and also promoted the expression of forkhead box protein 3 (Foxp3) and interleukin (IL)-10; in contrast, these transplantations induced various degrees of inhibition in the expression of retinoic acid-related orphan receptor γt (RORγt), IL-17A, IL-17F, and tumor necrosis factor-α (TNF-α). The protective effects of YE and R5 cells were significantly stronger than those of HC cells, and YE cells at the earlier differentiation stage than that of R5 cells exhibited the strongest protective effects. These results demonstrate that mouse LSCs at different stages of differentiation alleviate ConA-induced acute liver injury in mice by modulating Tregs, Th17 cells, and cytokine secretion.

Cell survival controlled by lens-derived Sema3A-Nrp1 is vital on caffeine-suppressed corneal innervation during chick organogenesis.

In this study, we investigated the effect of caffeine overexposure on corneal innervation in the early chicken embryo. Caffeine administration restricted corneal innervation by affecting trigeminal nerve development. Immunohistochemistry for phospho-Histone3 (pHIS3) and C-caspase3 revealed that cell survival was repressed by caffeine administration. Whole-mount in situ hybridization against semaphorin 3A (Sema3A) and neuropilin-1 (Nrp1) showed that both caffeine and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH, a free radical generator) administration upregulates the expression of both Sema3A and Nrp1. Next, we demonstrated that lens ablation in the developing chicken embryos significantly affected NF-labeled periocular nerve fascicles and innervation to the central eye region. Subsequently, we used a neuroblastoma cell line to investigate in vitro whether or not Sema3A-Nrp1 signaling exerts a key role on the caffeine-suppressed neuron survival. Knocking-down Sema3A through transfection with Sema3A-siRNA dramatically decreased the responsiveness of cells to caffeine administration, as well as cell apoptosis. We suggest that Sema3A-Nrp1 signaling regulates Trp53 and Cdkn1a through Slit2-Robo1 and Ephb2. Taken together, we speculate here that caffeine-enhanced reactive oxygen species upregulates Sema3A-Nrp1 expression in the lens and periocular tissues, resulting in corneal cell apoptosis, accompanied by its chemorepellent role on the invasion of the developing cornea by trigeminal sensory fibers.

Targeted elimination of mutant mitochondrial DNA in MELAS-iPSCs by mitoTALENs.

Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). We engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) and successfully eliminated the m.3243A>G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Furthermore, we successfully achieved reduction in the human m.3243A>G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

Exposure to Excess Phenobarbital Negatively Influences the Osteogenesis of Chick Embryos.

Phenobarbital is an antiepileptic drug that is widely used to treat epilepsy in a clinical setting. However, a long term of phenobarbital administration in pregnant women may produce side effects on embryonic skeletogenesis. In this study, we aim to investigate the mechanism by which phenobarbital treatment induces developmental defects in long bones. We first determined that phenobarbital treatment decreased chondrogenesis and inhibited the proliferation of chondrocytes in chick embryos. Phenobarbital treatment also suppressed mineralization in both in vivo and in vitro long bone models. Next, we established that phenobarbital treatment delayed blood vessel invasion in a cartilage template, and this finding was supported by the down-regulation of vascular endothelial growth factor in the hypertrophic zone following phenobarbital treatment. Phenobarbital treatment inhibited tube formation and the migration of human umbilical vein endothelial cells. In addition, it impaired angiogenesis in chick yolk sac membrane model and chorioallantoic membrane model. In summary, phenobarbital exposure led to shortened lengths of long bones during embryogenesis, which might result from inhibiting mesenchyme differentiation, chondrocyte proliferation, and delaying mineralization by impairing vascular invasion.

Effects of oxidative stress on hyperglycaemia-induced brain malformations in a diabetes mouse model.

Pregestational diabetes mellitus (PGDM) enhances the risk of fetal neurodevelopmental defects. However, the mechanism of hyperglycaemia-induced neurodevelopmental defects is not fully understood. In this study, several typical neurodevelopmental defects were identified in the streptozotocin-induced diabetes mouse model. The neuron-specific class III beta-tubulin/forkhead box P1-labelled neuronal differentiation was suppressed and glial fibrillary acidic protein-labelled glial cell lineage differentiation was slightly promoted in pregestational diabetes mellitus (PGDM) mice. Various concentrations of glucose did not change the U87 cell viability, but glial cell line-derived neurotrophic factor expression was altered with varying glucose concentrations. Mouse maternal hyperglycaemia significantly increased Tunel(+) apoptosis but did not dramatically affect PCNA(+) cell proliferation in the process. To determine the cause of increased apoptosis, we determined the SOD activity, the expression of Nrf2 as well as its downstream anti-oxidative factors NQO1 and HO1, and found that all of them significantly increased in PGDM fetal brains compared with controls. However, Nrf2 expression in U87 cells was not significantly changed by different glucose concentrations. In mouse telencephalon, we observed the co-localization of Tuj-1 and Nrf2 expression in neurons, and down-regulating of Nrf2 in SH-SY5Y cells altered the viability of SH-SY5Y cells exposed to high glucose concentrations. Taken together, the data suggest that Nrf2-modulated antioxidant stress plays a crucial role in maternal hyperglycaemia-induced neurodevelopmental defects.

Nrf2 signalling and autophagy are involved in diabetes mellitus-induced defects in the development of mouse placenta.

It is widely accepted that diabetes mellitus impairs placental development, but the mechanism by which the disease operates to impair development remains controversial. In this study, we demonstrated that pregestational diabetes mellitus (PGDM)-induced defects in placental development in mice are mainly characterized by the changes of morphological structure of placenta. The alteration of differentiation-related gene expressions in trophoblast cells rather than cell proliferation/apoptosis is responsible for the phenotypes found in mouse placenta. Meanwhile, excess reactive oxygen species (ROS) production and activated nuclear factor erythroid2-related factor 2 (Nrf2) signalling were observed in the placenta of mice suffering from PGDM. Using BeWo cells, we also demonstrated that excess ROS was produced and Nrf2 signalling molecules were activated in settings characterized by a high concentration of glucose. More interestingly, differentiation-related gene expressions in trophoblast cells were altered when endogenous Nrf2 expression is manipulated by transfecting Nrf2-wt or Nrf2-shRNA. In addition, PGDM interferes with autophagy in both mouse placenta and BeWo cells, implying that autophagy is also involved, directly or indirectly, in PGDM-induced placental phenotypes. Therefore, we revealed that dysfunctional oxidative stress-activated Nrf2 signalling and autophagy are probably responsible for PGDM-induced defects in the placental development of mice. The mechanism was through the interference with differentiation-related gene expression in trophoblast cells.

Proper autophagy is indispensable for angiogenesis during chick embryo development.

People have known that autophagy plays a very important role in many physiological and pathological events. But the role of autophagy on embryonic angiogenesis still remains obscure. In this study, we demonstrated that Atg7, Atg8 and Beclin1 were expressed in the plexus vessels of angiogenesis at chick yolk sac membrane and chorioallantoic membrane. Interfering in autophagy with autophagy inducer or inhibitor could restrict the angiogenesis in vivo, which might be driven by the disorder of angiogenesis-related gene expressions, and also lead to embryonic hemorrhage, which was due to imperfection cell junctions in endothelial cells including abnormal expressions of tight junction, adheren junction and desmosome genes. Using HUVECs, we revealed that cell viability and migration ability changed with the alteration of cell autophagy exposed to RAPA or 3-MA. Interestingly, tube formation assay showed that HUVECs ability of tube formation altered with the change of Atg5, Atg7 and Atg8 manipulated by the transfection of their corresponding siRNA or plasmids. Moreover, the lost cell polarity labeled by F-actin and the absenced ß-catenin in RAPA-treated and 3-MA-treated cell membrane implied intracellular cytoskeleton alteration was induced by the activation and depression of autophagy. Taken together, our current experimental data reveal that autophagy is really involved in regulating angiogenesis during embryo development.

Ethanol exposure represses osteogenesis in the developing chick embryo.

It is known that excess alcohol consumption during pregnancy can increase the risk of fetal alcohol spectrum disorder (FASD). However, the effect of ethanol exposure on bone morphogenesis in fetus is largely unknown. In this study, we demonstrated that ethanol treatment of gastrulating chick embryos could inhibit long bone (humerus, radius and ulna) development. Histological examination revealed that ethanol exposure reduced the width of the proliferation and hypertrophic zones. In addition, cell proliferation and alkaline phosphatase activities were repressed. We also investigated the effect on chondrogenesis and chondrogenesis was inhibited. Ethanol exposure also induced excess reactive oxygen species (ROS) production and altered the expression of osteogenesis-related genes. The inhibiting effect on flat bone (sclerotic ossicle) and the generation of cranial neural crest cells (progenitors of craniofacial bones) was also presented. In conclusion, ethanol exposure during the embryonic period retards bone development through excess ROS production and altered bone-associated gene expression.

Dexamethasone Exposure Accelerates Endochondral Ossification of Chick Embryos Via Angiogenesis.

Dexamethasone (Dex) is widely used to treat chronic inflammatory diseases in the clinic. Increasingly, there is more attention being paid to the side effect of Dex. In this study, we investigated the involvement and mechanism of Dex exposure in accelerating mineralization during long bone formation. We first determined that Dex exposure could accelerate long bone mineralization in vivo, but there was no apparent difference between control and Dex-treated in the phalanges model in vitro. Next, we established that Dex exposure promoted angiogenesis in the chick yolk sac membrane model. In addition, it increased human umbilical vein endothelial cell proliferation and migration in culture. We found that Dex could enhance angiogenesis when phalanges were cultured on chick chorioallantoic membrane and correspondingly increased the expression of angiogenesis-related genes in the phalanges. Furthermore, we also revealed that Dex exposure reduced the number of osteoblasts and simultaneously increased the number of osteocytes in ex vivo-cultured phalanges. Runx-2 and Col10α1 expressions were up-regulated by Dex exposure, indicating that Dex exposure accelerated the terminal differentiation of osteoblasts. Lastly, we demonstrated that MC3T3-E1 cells cultured in the presence of Dex accelerated their mineralization. In summary, we have shown that the ability of Dex to initiate angiogenesis is the mechanism that allows it to accelerate mineralization during long bone formation.

Acute and sub-chronic toxicity of tetrandrine in intravenously exposed female BALB/c mice.

OBJECTIVE: To evaluate the acute and sub-chronic toxicity of intravenously administered tetrandrine (TET) in female BALB/c mice. METHODS: The median lethal dose (LD50) of intravenously administered TET was calculated in mice using Dixon's up-and-down method. In the acute toxicity study, mice were intravenously administered with TET at a single dose of 20, 100, 180, 260 and 340 mg/kg, respectively and were evaluated at 14 days after administration. In the sub-acute toxicity study, mice were intravenously administered various doses of TET (30, 90 and 150 mg/kg) each day for 14 consecutive days. Clinical symptoms, mortality, body weight, serum biochemistry, organ weight and histopathology were examined at the end of the experiment, as well as after a 1-week recovery period. RESULT: LD50 was found to be 444.67±35.76 mg/kg. In the acute toxicity study, no statistically signifificant differences in body weight, blood biochemistry, or organ histology were observed between the administration and control groups when mice were intravenously administered with single dose at 20, 100, 180, 260 and 340 mg/kg of TET (P >0.05). In the sub-acute toxicity study, no signifificant changes in body weight, biochemistry and organ histology were observed with up to 90 mg/kg of TET compared with the control group (P >0.05), however, in the 150 mg/kg administered group, TET induced transient toxicity to liver, lungs and kidneys, but withdrawal of TET can lead to reversal of the pathological conditions. CONCLUSIONS: The overall fifindings of this study indicate that TET is relatively non-toxic from a single dose of 20, 100, 180, 260 or 340 mg/kg, and that up to 90 mg/kg daily for 14 consecutive days can be considered a safe application dose.

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.

Investigating the effect of excess caffeine exposure on placental angiogenesis using chicken 'functional' placental blood vessel network.

It is now known that over-consumption of caffeine by pregnant mothers could have detrimental effects on normal fetal development. However, it remains obscure how caffeine's harmful effect impacts directly or indirectly on the developing embryo/fetus through damaging placenta development. In this study, we demonstrated the morphological similarities between the yolk sac and chorioallantoic membranes (CAM) of chick embryos and the villi of the mammalian placenta. Using the chick yolk sac and the CAM as a model, we found that 5-15 µmol per egg of caffeine exposure inhibited angiogenesis. Under the same condition, cell proliferation in extraembryonic mesoderm was reduced while apoptosis was enhanced. Semi-quantitative RT-PCR analysis revealed that caffeine treatment down-regulated VEGF, VEGFR2, PIGF, IGF2 and NRP1 expression, but up-regulated Ang1 and Ang2 expression. We performed in situ hybridization to show VE-cadherin expression and as to demonstrate the blood vessels in the CAM and yolk sac membranes. This distribution of the VE-cadherin(+) blood vessels was determined to be reduced after caffeine treatment. Furthermore, MDA activity was induced after caffeine exposure, but GSH-PX activity was inhibited after caffeine exposure; SOD activity was unchanged as compared with the control. In summary, our results suggest that caffeine exposure could negatively impact on angiogenesis in the chick yolk sac and CAM by targeting angiogenesis-related genes. Some of these genes are also involved in regulating excess ROS generation. The results implied that the negative impact of caffeine on fetal development was partly attributed to impaired placental angiogenesis.

High glucose environment inhibits cranial neural crest survival by activating excessive autophagy in the chick embryo.

High glucose levels induced by maternal diabetes could lead to defects in neural crest development during embryogenesis, but the cellular mechanism is still not understood. In this study, we observed a defect in chick cranial skeleton, especially parietal bone development in the presence of high glucose levels, which is derived from cranial neural crest cells (CNCC). In early chick embryo, we found that inducing high glucose levels could inhibit the development of CNCC, however, cell proliferation was not significantly involved. Nevertheless, apoptotic CNCC increased in the presence of high levels of glucose. In addition, the expression of apoptosis and autophagy relevant genes were elevated by high glucose treatment. Next, the application of beads soaked in either an autophagy stimulator (Tunicamycin) or inhibitor (Hydroxychloroquine) functionally proved that autophagy was involved in regulating the production of CNCC in the presence of high glucose levels. Our observations suggest that the ERK pathway, rather than the mTOR pathway, most likely participates in mediating the autophagy induced by high glucose. Taken together, our observations indicated that exposure to high levels of glucose could inhibit the survival of CNCC by affecting cell apoptosis, which might result from the dysregulation of the autophagic process.

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.