Maternal and Neonatal Outcomes of Twin Pregnant Women With Anemia.

The aim of this study was to investigate the prevalence of anemia in twin pregnancies and the influence of anemia on maternal and neonatal outcomes. This retrospective study included twin pregnant women who delivered in a tertiary hospital in China from January 2018 to December 2018. Patients were divided by WHO criteria (hemoglobin <11.0 g/dL): the anemic and nonanemic groups. Patients with anemia were further classified as recovered or unrecovered subgroup after oral iron therapy. Maternal and neonatal outcomes in women carrying twins were compared using Student's t test and the chi-squared test or the Fisher exact test. Univariable and multivariable logistic regression models were used to determine the association of maternal and neonatal characteristics with anemia. Linear regression analysis was used to estimate mean birth weight and gestational week. The prevalence of anemia was 42.6% (182/427) in twin pregnancies. The anemic group had higher rates of low 1-minute Apgar score (4.4% vs. 1.8%, p = .028), perinatal death (1.9% vs. 0.2%, p = .012) and neonatal intensive care unit (NICU) admission (27.2% vs. 20.2%, p = .017; adjusted OR, 1.478; 95% CI [1.07, 2.044]). The recovered subgroup had lower NICU admission rate (13.5% vs. 30.3%, p = .006; OR, 0.388; 95% CI [0.186, 0.809]), higher gestational week and birth weight (ß, 0.954 week; 95% CI [0.114, 1.794] and ß, 171.01 g; 95% CI [9.894, 332.126] respectively). The prevalence of anemia in twin gestation is high. Anemia is associated with adverse neonatal outcomes, and correction of anemia significantly improved the pregnancy outcomes.

Comparison of Chemical Compositions and Antioxidant Activities for the Immature Fruits of Citrus changshan-huyou Y.B. Chang and Citrus aurantium L.

Quzhou Aurantii Fructus (QAF), the dried immature fruit of Citrus changshan-huyou Y.B. Chang, is similar to Aurantii Fructus (AF), the dried immature fruit of Citrus aurantium L. or its cultivars, in terms of composition, pharmacological action, and appearance. However, potential chemical markers to distinguish QAF from AF remain unknown owing to the lack of a comprehensive systematic chemical comparison aligned with discriminant analysis. To achieve a better understanding of the differences in their composition, this study aimed to identify the basic chemical compounds in QAF (n = 42) and AF (n = 8) using ultra-performance liquid chromatography coupled with electron spray ionization and quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and gas chromatography coupled with mass spectrometry (GC-MS). Principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and hierarchical clustering analysis (HCA) were used to further analyze, screen, and verify potential chemical markers; the antioxidant capacity was assayed in vitro. A total of 108 compounds were found in QAF and AF, including 25 flavonoids, 8 limonoids, 2 coumarins, and 73 volatile components. The chemometric analysis indicated that the main components in QAF and AF were very similar. Trace differential components, including 9 flavonoids, 2 coumarins, 5 limonoids, and 26 volatile compounds, were screened as potential chemical markers to distinguish between QAF and AF. Additionally, the antioxidant capacity of QAF was found to be greater than that of AF. This research provides insights into the quality control and clinical application of QAF.

Rapid identification of chemical compositions from three species of Siegesbeckiae Herba by ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry in combination with deoxyribonucleic acid barcoding.

Siegesbeckiae Herba, a traditional Chinese medicine, originates from Siegesbeckia orientalis, S. glabrescens, and S. pubescens in the Pharmacopoeia of the People's Republic of China. However, accurate identification of decoction pieces from the three plants remains a challenge. In this study, 26 batches of Siegesbeckiae Herba were identified by deoxyribonucleic acid barcoding, and their chemical compositions were determined using ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry. The results showed that the internal transcribed spacer 2 and internal transcribed spacer 1-5.8 S- internal transcribed spacer 2 sequences could distinguish three species. In total, 48 compounds were identified including 12 marker compounds screened for three species using the partial least square discriminant analysis. Among these, two diterpenoids 16-O-malonylkirenol and 15-O-malonylkirenol, and a novel diterpenoid 15,16-di-O-malonylkirenol were isolated and identified. A convenient method for the identification of Siegesbeckiae Herba was established using kirenol and 16-O-acetlydarutoside as control standards by thin-layer chromatography. Unexpectedly, none of the batches of S. orientalis contained kirenol, which did not meet the quality standards of Siegesbeckiae Herba, suggesting that the rationality of kirenol as a quality marker for S. orientalis should be further investigated. The results of this study will contribute to the quality control of Siegesbeckiae Herba.

Loss of transforming growth factor-ß1 in epithelium cells affects enamel formation in mice.

OBJECTIVES: In order to understand the specific in vivo function of transforming growth factor-beta1 (TGF-ß1), we successfully established aTGF-ß1 deficient mouse model using a conditional knockout method. In the present study, we aimed to further understand the potential role of TGF-ß1 in enamel formation. DESIGN: Transgenic mice withoutTGF-ß1 in epithelial cells were generated. Scanning electron microscopy and micro-computed tomography analysis were used to detect the dental appearance, enamel microstructure and tooth density. Histological analysis was used to examine the residual organic matrix of enamel. Quantitative real-time polymerase chain reaction was used to analyze the expressions of enamel matrix proteins at the mRNA level. RESULTS: The enamel of mandibular molars and incisors inTGF-ß1 conditional knockout mice displayed severe attrition and lower density compared with the wild-type littermates. A slender microstructure of enamel rod was observed, and enamel matrix proteins were retained in the enamel space at the maturation stage in conditional knockout mice. Moreover, the expressions of enamel matrix protein-encoding genes, such as amelogenin (Amelx), ameloblastin (Ambn), Enamelin (Enam) and matrix metalloproteinase-20 (Mmp-20), were increased in enamel organs of conditional knockout mice. On the other hand, the expressions of Amelotin (Amtn), kallikrein-related peptidase-4 (Klk4), C4orf26 and WD repeat-containing protein 72 (Wdr72) were dramatically decreased at the transition and maturation stages. CONCLUSIONS: TGF-ß1 played an important role in enamel mineralization through decreasing synthesis ofAmelx, Ambn and Enam and increasing synthesis of Klk4, Amtn, Corf26 and Wdr72.

Ablation of Runx2 in Ameloblasts Suppresses Enamel Maturation in Tooth Development.

Runt-related transcription factor 2 (Runx2) is involved in the early stage of tooth development. However, only few studies have reported the role of Runx2 in enamel development, which may be attributed to that Runx2 full knockout mice cannot survive after birth. In the present study, we successfully established a Runx2-deficient mouse model using a conditional knockout (cKO) method. We observed a significant reduction in the degree of mineralization and the decreased size of enamel rods in cKO mice. Histological analysis showed the retained enamel proteins in enamel layer at maturation stage in cKO molars. Further analysis by qRT-PCR revealed that the expressions of genes encoding enamel structure proteins, such as amelogenin (AMELX), ameloblastin (AMBN) and enamelin (ENAM), were increased in cKO enamel organs. On the other hand, the expression of kallikrein-related peptidase-4 (KLK4) at the mRNA and protein levels was dramatically decreased from late secretory stage to maturation stage in cKO enamel organs, while the expression of matrix metalloproteinase-20 (MMP-20) was not significantly altered. Finally, immunohistochemistry indicated that the uptake of amelogenins by ameloblasts was significantly decreased in cKO mice. Taken together, Runx2 played critical roles in controlling enamel maturation by increasing synthesis of KLK4 and decreasing synthesis of AMELX, AMBN and ENAM.

Therapeutic window of globular adiponectin against cerebral ischemia in diabetic mice: the role of dynamic alteration of adiponectin/adiponectin receptor expression.

Recent studies have demonstrated that adiponectin (APN) attenuates cerebral ischemic/reperfusion via globular adiponectin (gAD). However, the therapeutic role of gAD in cerebral ischemic injury in type 1 diabetes mellitus (T1DM) remains unclear. Our results showed that gAD improved neurological scores and reduced the infarct volumes in the 8-week T1DM (T1DM-8W) mice, but not in the 2-week T1DM (T1DM-2W) mice. Moreover, the ischemic penumbra APN levels increased and peaked in T1DM-2W mice, and reduced to normal in T1DM-8W mice, while the APN receptor 1 (AdipoR1) expression change was the opposite. Administration of rosiglitazone in T1DM-2W mice up-regulated the expression of AdipoR1 and restored the neuroprotection of gAD, while intracerebroventricular injection of AdipoR1 small interfering RNA (siRNA) in T1DM-8W mice reversed it. Furthermore, the expression of p-PERK, p-IRE1 and GRP78 were increased whereas the expressions of CHOP and cleaved caspase-12 as well as the number of apoptotic neurons were decreased after gAD treatment in T1DM-8W mice. These beneficial effects of gAD were reversed by pretreatment with AdipoR1 siRNA. These results demonstrated a dynamic dysfunction of APN/AdipoR1 accompanying T1DM progression. Interventions bolstering AdipoR1 expression during early stages and gAD supplementation during advanced stages may potentially reduce the cerebral ischemic injury in diabetic patients.

Electroacupuncture attenuates cerebral ischemia-reperfusion injury in diabetic mice through adiponectin receptor 1-mediated phosphorylation of GSK-3ß.

Diabetes mellitus substantially increases the risk of stroke and enhances brain's vulnerability to ischemia insult. Electroacupuncture (EA) pretreatment was proved to induce cerebral ischemic tolerance in normal stroke models. Whether EA could attenuate cerebral ischemia injury in diabetic mice and the possible underlying mechanism are still unrevealed. Male C57BL/6 mice were subjected to streptozotocin (STZ) for diabetic models. After inducing focal cerebral ischemia model, the levels of plasma and cerebral adiponectin (APN) were measured as well as the expression of cerebral adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2). The neurobehavioral score, infarction volume, and cellular apoptosis were evaluated with or without AdipoR1 short interfering RNA (siRNA). The role of phosphorylation of glycogen synthesis kinase 3 beta (GSK-3ß) at Ser-9 in the EA pretreatment was also assessed. EA pretreatment increased both plasma and cerebral APN levels and enhanced neuronal AdipoR1 in diabetic mice. In addition, EA reduced infarct size, improved neurological outcomes, and inhibited cell apoptosis after reperfusion. These beneficial effects were reversed by AdipoR1 knockdown. Furthermore, EA increased GSK-3ß phosphorylation (p-GSK-3ß) in the ipsilateral penumbra. Augmented p-GSK-3ß induced neuroprotective effects similar to those of EA pretreatment. In contrast, dampened p-GSK-3ß could reverse the neuroprotective effects of EA. In addition, the increase in p-GSK-3ß by EA was abolished by AdipoR1 knockdown. We conclude that EA pretreatment increases the production of APN, which induce protective effects against cerebral ischemia-reperfusion injury through neuronal AdipoR1-mediated phosphorylation of GSK-3ß in diabetic mice.

Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia.

We investigated the protective effect of electroacupuncture (EA) on cerebral ischemic injury in diabetic mice, and explored the role of NADPH oxidase-mediated oxidative stress. Male C57BL/6 mice were injected streptozotocin to induce diabetes. The mice were pretreated with EA at acupoint "Baihui" for 30 min. Two hours after the end of EA pretreatment, focal cerebral ischemia was induced following 24h reperfusion. The neurobehavioral scores and infarction volumes, malondialdehyde (MDA), reactive oxygen species (ROS), and activation of NADPH oxidase were determined in the presence or absence of the NADPH oxidase inhibitor apocynin or activator tetrabromocinnamic acid (TBCA). EA pretreatment reduced infarct size and improved neurological outcomes 24h after reperfusion in the diabetic mice. EA also decreased cerebral MDA and ROS levels compared with the control group, and inhibited the NADPH oxidase activation. The beneficial effects were abolished by TBCA while pretreatment with apocynin mimicked the neuroprotective and anti-oxidative effects of EA. Our results demonstrated that EA attenuated cerebral ischemic injury by inhibiting NAPDH oxidase-mediated oxidative damage in diabetic mice. These results suggest a novel mechanism of EA pretreatment-induced tolerance in diabetic cerebral ischemia.

[Effect of Actinidia chinensis planch polysaccharide on the growth and apoptosis, and p-p38 expression in human gastric cancer SGC-7901 cells].

OBJECTIVE: To investigate the effect of Actinidia chinensis Planch polysaccharide (ACPS) on the growth and apoptosis of human gastric cancer SGC-7901 cells, and to explore the effect of SGC-7901 cells on p-p38 expression. METHODS: The inhibition rates at different concentrations of ACPS on SGC-7901 cells at 24, 48, and 72 h were detected using CCK-8 method. Apoptosis ratios in SGC-7901 were determined by flow cytometry after 48-h treatment of different concentrations of ACPS. The expression of pro-caspase-9, PARP, and p-p38 in SGC-7901 cells after treated by different concentrations of ACPS was detected using Western blot. The expression of pro-caspase-9, PARP, and p-p38 was detected after SGC-7901 cells were pre-treated by p38 specific inhibitor. RESULTS: Compared with the control group, the optical density of SGC-7901 cells decreased after treated by 1, 2.5, 5, and 10 mg/mL ACPS (P < 0.05). Meanwhile, the longer the acting time, the lower the optic density (P < 0.01). IC50 was 7.43 mg/mL at 24 h; 3.88 mg/mL at 48 h, and 1.32 mg/mL at 72 h respectively. ACPS suppressed the protein expression of pro-caspase-9 (P < 0.01) and up-regulated the expression of PARP (89KD) (both P < 0.01). Further study showed that the protein expression of p-p38 was up-regulated in SGC-7901 cells treated by ACPS of different concentrations at 24 h (P < 0.05). The expression of phosphorylation p38 and the ACPS induced apoptosis of SGC-7901 cells could be inhibited after treated by specific inhibitor for 2 h. CONCLUSIONS: ACPS could inhibit the growth of SGC-7901 cells and induce apoptosis. The underlying mechanism of inducing apoptosis was partially due to activating the p38MAPK path and further activating Caspase9 and PARP, finally leading to cell death.

M9, a novel region of amino-Nogo-A, attenuates cerebral ischemic injury by inhibiting NADPH oxidase-derived superoxide production in mice.

AIMS: In acute stroke, neurological damage is due to oxidative stress and neuronal apoptotic death. This study investigated whether Nogo-A 290-562 residues region (M9), fused to the transduction domain of the HIV trans-activator (TAT) protein, is neuroprotective against cerebral ischemia and the mechanisms. METHODS: Transient focal cerebral ischemia was induced by middle cerebral artery occlusion in male C57BL/6J mice. TAT-M9, its mutation or vehicle was applied via intraperitoneal injection at the onset of reperfusion. The neurobehavioral scores, infarction volumes, neuronal apoptosis, and the ratio of Bax/Bcl-2 were evaluated. Malondialdehyde (MDA), reactive oxygen species (ROS) levels, and NADPH oxidase activation were measured in the presence or absence of the NADPH oxidase inhibitor apocynin or activator tetrabromocinnamic acid (TBCA). RESULTS: Immunofluorescence results confirmed that TAT-M9 was transduced into brain parenchyma, and it significantly improved neurological behavior, reduced infarct volumes, protected neuronal cells from apoptosis, inhibited activation of NADPH oxidase, and decreased MDA and ROS contents. Furthermore, apocynin imitated the beneficial effects of TAT-M9, while TBCA abolished them. CONCLUSIONS: Our results demonstrate that TAT-M9 administration attenuates cerebral ischemia by inhibiting NADPH oxidase-mediated oxidative damage and neuronal apoptosis in mice. TAT-M9 may be a potential treatment for cerebrovascular disease.

Ischemic postconditioning protects the spinal cord from ischemia-reperfusion injury via modulation of redox signaling.

BACKGROUND: It is well known that ischemic postconditioning reduces ischemic-reperfusion injury, but the underlying mechanism is not fully understood. The current study investigated the role of reactive oxygen species-mediated upregulation of endogenous antioxidant enzymes in the generation of a protective effect induced by ischemic postconditioning against spinal cord reperfusion injury in the rabbit. METHODS: New Zealand White rabbits were randomly allocated to sham, ischemia-reperfusion, and postconditioning groups (3 cycles of 30 seconds of reperfusion and 30 seconds of occlusion during the onset of reperfusion). Spinal cord ischemia was induced by clamping the infrarenal abdominal aorta for 20 minutes in the ischemia-reperfusion and postconditioning groups. Forty-eight hours after reperfusion, the neurologic status of the lower limbs was assessed. Blood samples were collected for analysis of serum neuron-specific enolase levels, and the lumbar spinal cord segments (L5-7) were harvested for histopathologic and antioxidant enzyme activities and mRNA analysis with or without administration of N-2-mercaptopropionylglycine (an effective oxygen free radical scavenger) given at different reperfusion times. RESULTS: Continuous administration of N-2-mercaptopropionylglycine for 13 minutes, starting at 10 minutes before (but not 10 minutes after) the beginning of reperfusion, attenuated the neuroprotective effect of postconditioning against spinal cord ischemia and reversed the increase in activity of the antioxidant enzymes superoxide dismutase and catalase in spinal cord tissue subjected to ischemic postconditioning. CONCLUSIONS: The results indicate that reactive oxygen species-triggered upregulation of endogenous antioxidant enzyme activities may be involved in the mechanism of neuroprotection of ischemic postconditioning.

TREK1 activation mediates spinal cord ischemic tolerance induced by isoflurane preconditioning in rats.

The aim of this study is to examine the role of one of the two-pore (2P) domain K(+) channels, TREK (TWIK-related K(+) channels, TREK)-1, mediated neuroprotection on spinal cord afforded by isoflurane preconditioning. In Experiment 1, male Sprague-Dawley rats were randomly assigned to control (Con) group, an isoflurane preconditioning (Iso) group, and sham group. Twenty-four hours after the last pretreatment, spinal cord ischemia was induced in Con and Iso groups. Neurobehavioral testing and histopathologic examination were performed after reperfusion. In Experiment 2, the expression of the TREK1 in the spinal cord was assessed by immunohistochemistry, Western blot and real-time polymerase chain reaction. In Experiment 3, Amiloride, a blocker of stretch-sensitive channels, was administered intraperitoneally immediately prior to each isoflurane preconditioning. Iso group showed a significant reductions in motor deficit index as well as increases in the number of normal neurons compared with the Con group. The expression of TREK1 protein and the level of mRNA after ischemia were higher in the rats of the Iso group than those in the Con group. Amiloride pretreatment abolished the protective effects of Iso preconditioning. These finding indicate that isoflurane preconditioning had a neuroprotective effect against spinal cord ischemia reperfusion injury. These effects may be mediated through the TREK1 pathway.

Inhibition of mitochondrial permeability transition pore opening contributes to the neuroprotective effects of ischemic postconditioning in rats.

Ischemic postconditioning (IPost) has been shown to attenuate cerebral ischemia-reperfusion injury. However, the mechanism remains elusive. Because opening of the mitochondrial permeability transition pore (MPTP) is a crucial determinant of cell death after ischemia-reperfusion, we hypothesized that the neuroprotective effect of IPost may be associated with inhibition of MPTP opening. In part 1 of this study, pentobarbital-anesthetized rats subjected to middle cerebral artery occlusion for 90 min, followed by reperfusion for 72 h, were assigned to receive one of the following treatments: three cycles of IPost (15s each), intracerebroventricular injection of saline (control), administration of the MPTP inhibitor cyclosporin A (CsA) (2 µmol/L, 15 µL) or its vehicle alcohol, administration of the MPTP opener atractyloside (Atr) (2 mmol/L, 15 µL), or IPost plus CsA/Atr treatment. Neurological deficit scores (NDS) and infarct volumes were assessed. Mitochondrial ultrastructure and swelling were also examined after reperfusion. In part 2, control and IPost groups underwent ischemia (90 min) and reperfusion (15 min). CsA and Atr groups were treated as described in part 1. Brain mitochondria were isolated after reperfusion and MPTP activity was evaluated. IPost or CsA treatment significantly improved NDS and reduced infarction volume, while Atr reversed the neuroprotective effects of IPost, and attenuated the decrease in mitochondrial swelling induced by IPost or CsA. Thus, inhibiting MPTP opening may play a crucial role in the neuroprotective effects of IPost, which may have potential clinical value against cerebral ischemia-reperfusion injury.

[Effect of sevoflurane postconditioning on spinal cord ischemia reperfusion injury via the release of oxygen free radicals in rabbits].

OBJECTIVE: To investigate whether postconditioning with sevoflurane could alleviate spinal cord ischemia reperfusion injury in rabbits, and whether the beneficial effect is dependent on oxygen free radicals. METHODS: In Experiment 1, 48 male New Zealand white rabbits were randomly assigned to six groups (n=8 each). Animals in the sham group only underwent sham-operation. Animals in the control group underwent spinal cord ischemia for 20 min without postconditioning. Animals in 02 postconditioning group (Group O2) inhaled 100% O2 from 5 min before reperfusion and lasted 13 min. Animals in sevoflurane postconditioning groups (Group Sevo0.5, Sevo1.0 and Sevo1.5) inhaled 0.5, 1.0, 1.5 minimum alveolar concentration (MAC) sevoflurane in 100% O2 from 5 min before reperfusion and lasted 10 min, then inhaled 100% O2 for 3 min to wash out the remaining sevoflurane. In Experiment 2, 36 male New Zealand White rabbits were randomly assigned to four groups (n=9 each). Animals in O2 and Sevo groups received normal saline (5 ml/kg intravenously) 1 h before postconditioning with 100% O2 and 1.0 MAC sevoflurane, respectively. In the DMTU + Sevo and DMTU + O2 groups, 5 ml/kg of 10% dimethylthiourea (DMTU, a potent oxygen free radical scavenger, dissolved in saline) was administered intravenously at the same time. Spinal cord ischemia was induced by an infrarenal aorta clamping for 20 min. Forty-eight hours after reperfusion, hind-limb motor function and histopathology of the spinal cord were examined in a blinded fashion. RESULTS: (1) The neurologic and histopathologic outcomes in the sevoflurane postconditioning groups were better than those in the control group (P < 0.05), the histopathologic outcomes in Sevo1.0 group were better than that in Sevo0.5 and Sevo1.5 groups (P < 0.05). (2) The neurologic and histopathologic outcomes in Sevo, DMTU + Sevo and DMTU + O2 groups were better than those in the O2 group (P < 0.05), the histopathologic outcomes in Sevo group were better than that in the DMTU + Sevo and DMTU + O2 groups (P < 0.05). CONCLUSIONS: Our study demonstrates that sevoflurane postconditioning against spinal cord ischemia injury via the release of oxygen free radicals in rabbits.

[Ischemic postconditioning induces neuroprotection via up-regulation of endogenous antioxidant enzyme activities: experiment with rabbits].

OBJECTIVE: To test the hypothesis that spinal cord protection induced by ischemic postconditioning is mediated by an increase of endogenous antioxidant enzyme activities during reperfusion phase in spinal cord. METHODS: Seventy-eight male New Zealand rabbits were randomly divided into 3 groups: Sham group (n = 18) undergoing sham operation without aortic occlusion; ischemia/reperfusion (I/R) group (n = 30) undergoing occlusion of the infrarenal abdominal aorta for 20 min, followed by reperfusion; and postconditioning (PostC) group (n = 30) undergoing occlusion of the infrarenal abdominal aorta for 20 min followed by 3 cycles of 30 s reperfusion/30 s ischemia just at the onset of reperfusion. 30 min, and 1, 3, 6, 24, and 48 h after reperfusion 5 rabbits from each group (and 3 from the Sham group) were killed with their spinal cords taken out, and spectrophotometric method was used to determine the antioxidant enzyme activity and malondialdehyde (MDA) content 6, 24, and 48 h after reperfusion motor function scoring of the hind limbs was conducted. RESULTS: (1) The motor function scores of the PostC group were significantly higher than those of the I/R group 6, 24, and 48 h after reperfusion (all P < 0.05). (2) The activities of superoxide dismutase (SOD) and catalase (CAT) in the spinal cord tissue of the PostC group 30 min-6 h after reperfusion were all significantly higher than those of the I/R group (all P < 0.05). There were no significant differences in the activity of glutathione peroxidase (GSH-px) at different time points between the PostC and I/R groups. (3) The MDA levels 24 h and 48 h after reperfusion of the PostC group were both significantly lower than that of the I/R group (both P < 0.01). CONCLUSION: Ischemic postconditioning shows effect against spinal cord ischemic-reperfusion injury mediated, at least partially, by up-regulating the activities of SOD and CAT in spinal cord during early reperfusion phase.

Level of endothelial cell apoptosis required for a significant decrease in microvessel density.

Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF(165)) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.

Diverse signaling pathways through the SDF-1/CXCR4 chemokine axis in prostate cancer cell lines leads to altered patterns of cytokine secretion and angiogenesis.

The establishment of metastatic bone lesions in prostate cancer (CaP) is a process partially dependent on angiogenesis. Previously we demonstrated that the stromal-derived factor-1 (SDF-1 or CXCL12)/CXCR4 chemokine axis is critical for CaP cell metastasis. In this investigation, cell lines were established in which CXCR4 expression was knocked down using siRNA technology. When CaP cells were co-transplanted with human vascular endothelial cells into SCID mice, significantly fewer human blood vessels were observed paralleling the reductions in CXCR4 levels. Likewise, the invasive behaviors of the CaP cells were inhibited in vitro. From these functional observations we explored angiogenic and signaling mechanisms generated following SDF-1 binding to CXCR4. Differential activation of the MEK/ERK and PI3K/AKT pathways that result in differential secretion IL-6, IL-8, TIMP-2 and VEGF were seen contingent on the cell type examined; VEGF and TIMP-2 expression in PC3 cells are dependent on AKT activation and ERK activation in LNCaP and LNCaP C4-2B cells leads to IL-6 or IL-8 secretion. At the same time, expression of angiostatin levels were inversely related to CXCR4 levels, and inhibited by SDF-1 stimulation. These data link the SDF-1/CXCR4 pathway to changes in angiogenic cytokines by different signaling mechanisms and, suggest that the delicate equilibrium between proangiogenic and antiangiogenic factors may be achieved by different signal transduction pathways to regulate the angiogenic phenotype of prostate cancers. Taken together, our results provide new information regarding expression of functional CXCR4 receptor-an essential role and potential mechanism of angiogenesis upon SDF-1 stimulation.

Expression of Fas ligand is not a main mechanism used by tumors to counteract antitumor immunity.

The role of Fas ligand (FasL) in tumor immune privilege is controversial. In this study, 22 human tumor cell lines reported to be FasL+ were reevaluated by Western blot analysis, ELISA, and a functional assay. None of the cells lines expressed FasL. To assess whether human tumors express FasL in vivo, susceptibility to FasL-mediated killing was evaluated. About 75% of the 22 tumors tested were sensitive to FasL-mediated apoptosis, suggesting, therefore, that only about 25% could possibly express FasL. To investigate whether "FasL+" human tumor cells could suppress the proinflammatory effects of FasL in vivo, FasL transfectants were generated from two prototype "FasL+" tumor cell lines. The transfectants expressing FasL were rejected by SCID mice. In contrast, all the mice inoculated with parental tumor cells developed large tumors. These results suggested that human tumor cells that express FasL and resist both FasL-mediated apoptosis and inflammation are rare or nonexistant. We concluded that FasL expression is not a main mechanism that tumors use to counteract antitumor immunity.

Effect of lipopolysaccharides on vascular endothelial growth factor expression in mouse pulp cells and macrophages.

Vascular endothelial growth factor (VEGF), a potent pro-angiogenic factor, might regulate the neovascularization observed in the pulp of teeth with deep caries. The purpose of this in vitro study was to evaluate the effect of bacterial lipopolysaccharides (LPS) on VEGF expression in dental pulp cells. Mouse odontoblast-like cells (MDPC-23) or undifferentiated pulp cells (OD-21) were exposed to 0-20 microg ml-1Escherichia coli LPS or 0-80 microg ml-1Prevotella intermedia LPS. As controls, mouse macrophages or gingival fibroblasts were exposed to LPS, since these cells are known to secrete VEGF. The VEGF expression was evaluated by reverse transcriptase polymerase chain reaction or enzyme-linked immunosorbent assay. The baseline expression levels of VEGF protein were higher in MDPC-23 and OD-21 than in fibroblasts or macrophages. Vascular endothelial growth factor protein expression was upregulated in MDPC-23 and macrophages exposed to E. coli LPS, but not in OD-21 cells or fibroblasts. Higher concentrations of P. intermedia LPS were required to induce VEGF expression in MDPC-23 cells. Treatment with LPS did not affect VEGF expression at the mRNA level in any of the cells evaluated. These results demonstrate that bacterial LPS upregulates VEGF expression in odontoblast-like cells and macrophages, and suggest that the regulation of VEGF expression occurs primarily at a post-transcriptional level.