Fructose Consumption Affects Placental Production of H2S: Impact on Preeclampsia-Related Parameters.

H2S, a gasotransmitter that can be produced both via the transsulfuration pathway and non-enzymatically, plays a key role in vasodilation and angiogenesis during pregnancy. In fact, the involvement of H2S production on plasma levels of sFLT1, PGF, and other molecules related to preeclampsia has been demonstrated. Interestingly, we have found that maternal fructose intake (a common component of the Western diet) affects tissular H2S production. However, its consumption is allowed during pregnancy. Thus, (1) to study whether maternal fructose intake affects placental production of H2S in the offspring, when pregnant; and (2) to study if fructose consumption during pregnancy can increase the risk of preeclampsia, pregnant rats from fructose-fed mothers (10% w/v) subjected (FF) or not (FC) to a fructose supplementation were studied and compared to pregnant control rats (CC). Placental gene expression, H2S production, plasma sFLT1, and PGF were determined. Descendants of fructose-fed mothers (FC) presented an increase in H2S production. However, if they consumed fructose during their own gestation (FF), this effect was reversed so that the increase disappeared. Curiously, placental synthesis of H2S was mainly non-enzymatic. Related to this, placental expression of Cys dioxygenase, an enzyme involved in Cys catabolism (a molecule required for non-enzymatic H2S synthesis), was significantly decreased in FC rats. Related to preeclampsia, gene expression of sFLT1 (a molecule with antiangiogenic properties) was augmented in both FF and FC dams, although these differences were not reflected in their plasma levels. Furthermore, placental expression of PGF (a molecule with angiogenic properties) was decreased in both FC and FF dams, becoming significantly diminished in plasma of FC versus control dams. Both fructose consumption and maternal fructose intake induce changes in molecules that contribute to increasing the risk of preeclampsia, and these effects are not always mediated by changes in H2S production.

sFlt-1-enriched exosomes induced endothelial cell dysfunction and a preeclampsia-like phenotype in mice.

Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by maternal endothelial dysfunction and end-organ damage. Our previous work demonstrated that PE patient-derived exosomes contained higher levels of soluble FMS-like tyrosine kinase-1 (sFlt-1) and significantly induced endothelial dysfunction and PE development. However, the mechanisms underlying the effect of sFlt-1-enriched exosomes (sFlt-1-Exo) on PE development are poorly characterized. Here, we revealed that trophoblast-derived sFlt-1-Exo treatment induced significant inhibition of human umbilical vein endothelial cell (HUVEC) migration and tube formation, as well as an increase in sFlt-1 secretion. Mechanistically, we found that the increased sFlt-1 secretion in the cell culture medium was attributed to enhanced transcription of sFlt-1 in HUVECs. Importantly, we observed that treating pregnant mice with sFlt-1-Exo or recombinant mouse sFlt-1 triggered a preeclampsia-like phenotype, characterized by elevated blood pressure, proteinuria, increased plasma sFlt-1 and adverse pregnancy outcomes. These results strongly suggested that sFlt-1-Exo-induced endothelial dysfunction could be partially attributed to the upregulation of sFlt-1 in endothelial cells, potentially leading to the development of a preeclampsia-like phenotype in mice.

Vitamins and their derivatives synergistically promote hair shaft elongation ex vivo via PlGF/VEGFR-1 signalling activation.

BACKGROUND: Although vitamins or their derivatives (Vits), such as panthenyl ethyl ether, tocopherol acetate, and pyridoxine, have been widely used in topical hair care products, their efficacy and mode of action have been insufficiently studied. OBJECTIVE: To elucidate the biological influence of Vits on hair follicles and determine the underlying mechanisms. METHODS: A mouse vibrissa hair follicle organ culture model was utilized to evaluate the effects of Vits on hair shaft elongation. Gene and protein expression analyses and histological investigations were conducted to elucidate the responsible mechanisms. A human hair follicle cell culture was used to assess the clinical relevance. RESULTS: In organ culture models, the combination of panthenyl ethyl ether, tocopherol acetate, and pyridoxine (namely, PPT) supplementation significantly promoted hair shaft elongation. PPT treatment enhanced hair matrix cell proliferation by 1.9-fold compared to controls, as demonstrated by Ki67-positive immunoreactivity. PPT-treated mouse dermal papillae exhibited upregulated Placental growth factor (Plgf) by 1.6-fold compared to controls. Importantly, the addition of PlGF neutralizing antibodies to the ex vivo culture diminished the promotive effect on hair growth and increase in VEGFR-1 phosphorylation achieved by PPT. A VEGFR-1 inhibitor also inhibited the promotion of hair growth. Microarray analysis suggested synergistic summation of individual Vits' bioactivity, putatively explaining the effect of PPT. Moreover, PPT increased PlGF secretion in cultured human dermal papilla cells. CONCLUSION: Our findings suggested that PPT promoted hair shaft elongation by activating PlGF/VEGFR-1 signalling. The current study can shed light on the previously underrepresented advantage of utilizing Vits in hair care products.

The effect of genistein on IGF-1, PlGF, sFLT-1 and fetoplacental development.

The mechanisms by which genistein, a phytoestrogen, affects fetoplacental development adversely are still poorly understood. It is reported that genistein ingestion modulates thyroid functions, leptin hormone, C-reactive protein, and thyroxin kinase activities. In this study, we evaluated changes in serum and placental insulin-like growth factor-I (IGF-1), placental growth factor (PIGF), and soluble fms-like tyrosine kinase-1 (sFLT-1) in pregnant rats exposed to genistein using ELISA. According to the treatments, Rats were divided into control, 2 mg genistein, and 4 mg genistein groups. Genistein groups were administered with the doses orally from gestational day (GD) one onwards until sacrifice, while the control group received an equal volume of distilled water the vehicle. At GD-12, GD-16, and GD-20, serum samples and placenta homogenates were prepared from maternal blood samples and the placenta and were analysed to determine the concentration of IGF-1, sFLT-1, and PIGF. Serum IGF-1 and PIGF were both increased in all genistein groups at GD-12 and GD-16, and at GD-20 in the 4 mg group. However, serum IGF-1and PIGF levels were decreased in the placenta from all genistein groups at GD-20. Placenta sFLT-1 levels increased at both GD-16 and GD-20 in genistein-treated rat serum. An initial decrease in placental sFLT-1 at GD-12 was followed by an increase at GD-16 and finally a decrease at GD-20 in all genistein-treated rats. The sFL-1/PlGF ratio in placenta samples of genistein-exposed rats was decreased at GD-16 and increased at GD-20, while the reverse was recorded in the serum sample at the same gestational periods. The fetoplacental growth disruption mechanism of genistein can be partly explained by its interference with placental growth factor signalling.

Maternal cadmium exposure impairs placental angiogenesis in preeclampsia through disturbing thyroid hormone receptor signaling.

Cadmium is a ubiquitous environmental pollutant, which can increase the risk of preeclampsia. This study was designed to determine the mechanism of cadmium exposure during pregnancy impaired placental angiogenesis that was associated with the occurrence of preeclampsia. The effects of cadmium exposure on placental thyroid hormone receptor signaling were explored. JEG3 cells were treated with CdCl2 (20 µM) and the Dio2 inhibitor, IOP (100 µM). Cadmium levels in maternal blood and placentae were increased in preeclampsia group. Placental angiogenesis of preeclampsia was decreased with decreased expression of PLGF and VEGF and increased expression of sFlt1. Meanwhile, the expression and nuclear translocation of thyroid hormone receptor α were decreased in preeclampsia placenta, as well as the expression of Dio2, but not the expression and nuclear translocation of thyroid hormone receptor ß. Furthermore, we found that cadmium exposure downregulated the expression of thyroid hormone receptor α and Dio2, but not the expression of thyroid hormone receptor ß in JEG3 cells. Also, we found that cadmium exposure decreased the expression of PLGF and VEGF and increased the expression of sFlt1 in JEG3 cells. IOP pretreatment decreased the expression of PLGF and increased the expression of sFlt1. In conclusion, our results elucidated that cadmium exposure would impair placental angiogenesis in preeclampsia through disturbing thyroid hormone receptor signaling.

Mitochondrial-targeting antioxidant MitoQ modulates angiogenesis and promotes functional recovery after spinal cord injury.

BACKGROUND: In traumatic spinal cord injury (SCI), secondary injuries, including cellular death, mitochondrial dysfunction, and vascular injury, have been considered as important causes of impaired functional recovery after SCI. Postinjury angiogenesis has been considered to be a potential strategy for SCI treatment. New-born vessels may play a key role in nerve regeneration, which indicates the importance of angiogenesis in nerve regeneration. Recent studies have revealed the crosstalk between reactive oxygen species (ROS) and angiogenesis. As the main source of cellular ROS, mitochondria have been proven to be essential to the angiogenesis process. METHODS: SCI was established in a T10 clip-compression animal model. Then, the animals received an intraperitoneal injection of MitoQ (5 mg/kg/d) on Days 0, 1, and 2 after surgery. The Basso Mouse Scale (BMS) score and footprint analysis (CatWalk analysis) were performed to evaluate functional recovery after SCI. Immunofluorescence and fluorescence assays (LEL-FITC/CD31/Iba-1/Neurofilament) were performed to evaluate angiogenesis, microglia activation and neural regeneration. RT-qPCR (VEGFR-1, VEGFR-2 and VEGFA) was performed to evaluate angiogenesis-related factor in injured spinal cord. ATP production assay and western-blotting assay (Mfn-1 and Drp-1) were performed to evaluate mitochondrial function in the injured spinal cord. BV2 cells were used as in vitro cell model. After receiving TBHP or TBHP-MitoQ treatment, ELISA and immunofluorescence assays were used to evaluate the level of VEGFA secretion from BV2 cells. A coculture system of HUVECs and BV2 cells was established. Tube formation assays and immunofluorescence assays (CD31) were performed on HUVECs in a coculture system to evaluate angiogenesis promotion. ATP production assays were performed to evaluate mitochondrial function in BV2 cells. MitoSOX Red and DCFH-DA staining were performed to evaluate mitochondrial and cellular ROS. RESULTS: In vitro MitoQ promoted the secretion of VEGFA from BV2 cells, which was verified through ELISA and immunofluorescence assays. The angiogenic promotion of MitoQ-treated BV2 cells was evaluated by tube formation and immunofluorescence assays (CD31) in a coculture system of BV2 cells and HUVECs. MitoQ inhibited cellular and mitochondrial-derived ROS in TBHP-treated BV2 cells. ATP production was increased in MitoQ-treated BV2 cells. To verify MitoQ's effect in vivo, a T10 clip-compression animal model was established successfully. MitoQ significantly promoted functional recovery, as shown by the BMS assay and gait analysis. The promotion of neural regeneration was identified through immunofluorescence assay of neurofilament. Immunofluorescence and fluorescence assays (LEL-FITC/CD31/Iba-1) and RT-qPCR (VEGFR-1, VEGFR-2 and VEGFA) indicated that MitoQ could promote angiogenesis and inhibit macrophage/microglia activation in lesion-site after SCI. Enhanced ATP production and increased Mfn-1 with decreased Drp-1 protein expression showed MitoQ could promote mitochondrial function in SCI. CONCLUSION: The mitochondrial-specific antioxidant MitoQ promotes functional recovery and tissue preservation through the enhancement of angiogenesis with the modification of mitochondrial function after SCI.

A Hypoxia-Regulated Retinal Pigment Epithelium-Specific Gene Therapy Vector Reduces Choroidal Neovascularization in a Mouse Model.

BACKGROUND: Wet age-related macular degeneration (wAMD) is characterized by the presence of choroidal neovascularization (CNV). Although there are some clinical drugs targeting vascular endothelial growth factor (VEGF) and inhibiting CNV, two major side effects limit their application, including the excessive activity of anti-VEGF and frequent intraocular injections. To explore better treatment strategies, researchers developed a hypoxic modulator retinal pigment epithelium (RPE)- specific adeno-associated virus (AAV) vector expressing endostatin to inhibit CNV. However, the mechanism of endostatin is complex. Instead, soluble fms-like tyrosine kinase-1 (sFlt-1) can inhibit VEGF-induced angiogenesis through two simple and clear mechanisms, giving rise to sequestration of VEGF and forming an inactive heterodimer with the membrane-spanning isoforms of the VEGF receptor Flt-1 and kinase insert domain-containing receptor. OBJECTIVE: In this study, we chose sFlt-1 as a safer substitute to treat wAMD by inhibiting VEGFinduced angiogenesis. METHODS: The AAV2/8-Y733F-REG-RPE-sFlt-1 vector was delivered by intravitreal injection to the eyes of mice. AAV2/8-Y733F vector is a mutant of the AAV2/8 vector, and the REG-RPE promoter is a hypoxia-regulated RPE-specific promoter. Two animal models were used to evaluate the function of the vector. RESULTS: In the cobalt chloride-induced hypoxia model, the results demonstrated that the AAV2/8- Y733F-REG-RPE-sFlt-1 vector induced the expression of the sFlt-1 gene in RPE cells through hypoxia. In the laser-induced CNV model, the results demonstrated that the AAV2/8-Y733F-REG-RPE-sFlt- 1 vector reduced laser-induced CNV. CONCLUSION: Hypoxia regulated, RPE-specific AAV vector-mediated sFlt-1 gene is a hypoxiaregulated antiangiogenic vector for wAMD.

Targeting Vascular endothelial growth factor A with soluble vascular endothelial growth factor receptor 1 ameliorates nerve injury-induced neuropathic pain.

Neuropathic pain is a distressing medical condition with few effective treatments. The role of Vascular endothelial growth factor A (VEGFA) in inflammation pain has been confirmed in many researches. However, the mechanism of VEGFA affects neuropathic pain remains unclear. In this study, we demonstrated that VEGFA plays an important role in spare nerve injury (SNI)-induced neuropathic pain, which is mediated by enhanced expression and colocalized of VEGFA, p-AKT and TRPV1 in SNI-induced neuropathic pain model. Soluble VEGFR1 (sFlt1) not only relieved mechanical hyperalgesia and the expression of inflammatory markers, but ameliorated the expression of VEGFA, VEGFR2, p-AKT, and TRPV1 in spinal cord. However, these effects of sFlt1 can be blocked by rpVEGFA and by 740 Y-P. Therefore, our study indication that targeting VEGFA with sFlt1 reduces neuropathic pain development via the AKT/TRPV1 pathway in SNI-induced nerve injury. This study elucidates a new therapeutic target for neuropathic pain.

Targeted Antagonism of Vascular Endothelial Growth Factor Reduces Mortality of Mice with Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is associated with a mortality of 45%. Our previous research indicated that anti-vascular endothelial growth factor (VEGF) could maintain the normal structure and function of the respiratory barrier. However, systemic application of VEGF antagonists would lead to animal death. This study attempts to study the targeted drug delivery for ARDS. In this study, we used soluble fms-like tyrosine kinase-1 (sFlt)-targeted ultrasound microbubbles to antagonize the effect of VEGF on lung tissue. Ninety male BALB/c mice were randomly assigned to 6 groups: phosphate buffer saline (PBS) group (PBS+PBS); blank group (PBS+empty microbubbles); lipopolysaccharide (LPS) group (LPS+PBS); ARDS group (LPS+empty microbubbles); control group (PBS+sFlt microbubbles); and treatment group (LPS+sFlt microbubbles). After administration of LPS or PBS in the corresponding groups, the sFlt-targeted microbubbles or empty microbubbles were injected into the blood circulation. Then the lungs were irradiated with ultrasound, which ruptured the drug-loaded microbubbles and helped release drugs to the lung tissues targeted. The lung injury score, lung wet/dry ratio (W/D), liver and kidney functions, and the mortality of the mice in all groups were investigated at the predetermined time point. The difference in mortality between groups was examined by Fisher test. Other data were analyzed by one-way analysis of variance (ANOVA). A value of P<0.05 indicates that the difference was significant. The results showed that the PaO2 levels were normal in the PBS group, the blank group, and the control group. The LPS group and ARDS group showed significant hypoxia. PaO2 was improved significantly in the treatment group. The lung injury score and W/D were normal in the PBS group, the blank group, and the control group. The lung injury score and W/D increased significantly in the LPS group and ARDS group and decreased in the treatment group (P<0.05). The mortality rate of the ARDS model was 60% (95% confidence interval 47.5%-72.5%), and that with sFlt-targeted microbubbles was significantly lower at only 40% (95% confidence interval 27.5%-52.5%, P<0.05). It was concluded that anti-VEGF with sFlt targeted ultrasound microbubbles attenuated the lung injury and ultimately reduced the 7-day mortality effectively. It might be a suitable therapeutic tool for the treatment of ARDS.

Vascular endothelial growth factor increased the permeability of respiratory barrier in acute respiratory distress syndrome model in mice.

BACKGROUND: Acute respiratory distress syndrome is associated with a mortality of 45%. The authors investigated the possible mechanisms and effect of vascular endothelial growth factor on alveolar epithelial barrier permeability in acute respiratory distress syndrome mice model. METHODS: Eighty Male BALB/c mice were randomly assigned to four group: PBS group, LPS group, sFlt group, or LPS + sFlt group. The levels of vascular endothelial growth factor and total protein in bronchoalveolar lavage fluid were compared, together with lung injury score and the histopathology of alveolar epithelial barrier. The expressions of vascular endothelial growth factor and tight junction proteins mRNA in lung tissue were also studied. RESULTS: Lipopolysaccharide (LPS) inhaling was accompanied with increasing lung vascular endothelial growth factor (VEGF) expression. Anti-VEGF with soluble fms-like tyrosine kinase-1 (sFlt-1) attenuated the lung injury effectively. CONCLUSIONS: Our data indicate that anti-vascular endothelial growth factor with soluble fms-like tyrosine kinase-1 could maintain the normal structure and function of respiratory membrane in acute respiratory distress syndrome mice model and might be a suitable therapeutic tool for the treatment of acute respiratory distress syndrome.

Placental growth factor reverses decreased vascular and uteroplacental MMP-2 and MMP-9 and increased MMP-1 and MMP-7 and collagen types I and IV in hypertensive pregnancy.

Preeclampsia is a complication of pregnancy manifested as maternal hypertension (HTN) and fetal intrauterine growth restriction, with unclear mechanisms. Placental ischemia increases antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) relative to angiogenic placental growth factor (PlGF); however, the molecular targets are unclear. To test the hypothesis that placental ischemia-induced changes in sFlt-1 and PlGF target vascular and uteroplacental matrix metalloproteinases (MMPs), we tested whether raising the sFlt-1-to-PlGF ratio by infusing sFlt-1 (10 µg·kg-1·day-1) in pregnant (Preg) rats increases blood pressure (BP) and alters MMPs and whether correcting sFlt-1/PlGF by infusing PlGF (20 µg·kg-1·day-1) in Preg rats with reduced uterine perfusion pressure (RUPP) improves BP and reverses the changes in MMPs. On gestational day 19, BP was higher and the litter size and uterine, placenta, and pup weight were less in Preg + sFlt-1 and RUPP than Preg rats and restored in RUPP + PlGF versus RUPP rats. Gelatin and casein zymography and Western blots revealed decreases in MMP-2 and MMP-9 and increases in MMP-1 and MMP-7 in the aorta, uterine artery, uterus, and placenta of Preg + sFlt-1 and RUPP versus Preg rats, which were reversed in RUPP + PlGF versus RUPP rats. Collagen types I and IV were more abundant in Preg + sFlt-1 and RUPP versus Preg rats and were reversed in RUPP + PlGF versus RUPP rats. Thus, PlGF reverses decreased vascular and uteroplacental MMP-2 and MMP-9 and increased MMP-1, MMP-7, and collagen types I and IV induced by placental ischemia and sFlt-1 in HTN in pregnancy. Angiogenic factors and MMP modulators could rectify changes in MMPs and collagen, restore vascular and uteroplacental remodeling, and improve HTN and intrauterine growth restriction in preeclampsia. NEW & NOTEWORTHY Understanding the mechanisms of preeclampsia could help in its prevention and management. This study shows that correcting soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) imbalance by infusing PlGF reverses the decreases in vascular and uteroplacental matrix metalloproteinase (MMP)-2 and MMP-9 and the increases in MMP-1, MMP-7, and collagen types I and IV induced by placental ischemia and antiangiogenic sFlt-1 in hypertension in pregnancy. Angiogenic factors and MMP modulators could rectify changes in vascular and uteroplacental MMPs and collagen content and ameliorate hypertension and intrauterine growth restriction in preeclampsia.

Soluble Flt-1 Has Cytotoxic Effects on BeWo Choriocarcinoma Cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis and is overexpressed in many kinds of malignant tumors. Soluble VEGF receptor 1 (sVEGFR-1/ soluble fms-like tyrosine kinase 1 [sFlt-1]) plays a role as an inhibitor of VEGF, and an antitumor effect has been shown in several studies using sFlt-1. Recently, in addition to its antiangiogenic effect, it was reported that sFlt-1 has direct cytotoxicity. MATERIALS AND METHODS: Transfection of sFlt-1 plasmid DNA was performed in the BeWo choriocarcinoma cell line. Overexpression of sFlt-1 in BeWo cells was confirmed by ELISA. In order to evaluate cell proliferation, cell counting and BrdU uptake assay were performed. Cytotoxicity was tested by LDH assay. TdT-Mediated dUTP Nick end Labeling (TUNEL) staining and quantitative analysis of caspase-cleaved keratin 18 (ccK18) level were done to evaluate cell apoptosis. RESULTS: The cell number was significantly less, and the ratio of cytotoxicity was significantly higher in sFlt-1 group compared to the control group. TUNEL staining and ccK18 level suggested nonapoptotic cell death. CONCLUSION: Soluble Flt-1 showed a cytotoxic effect on BeWo cells. Our results suggest that sFLT-1 could be therapeutic for malignant tumors.

Dual targeting of vascular endothelial growth factor and bone morphogenetic protein-9/10 impairs tumor growth through inhibition of angiogenesis.

Clinical development of anti-angiogenic agents has been a major landmark in cancer therapy for several types of cancers. Signals mediated by both vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-9 and 10 have been implicated in tumor angiogenesis. However, previous studies have shown that targeting the individual signals was not sufficiently effective in retarding tumor growth in certain preclinical and clinical conditions. In the present study, we developed a novel decoy chimeric receptor that traps both VEGF and BMP-9/10. Single targeting of either VEGF or BMP-9/10 signals significantly reduced the formation of tumor vessels in a mouse xenograft model of human pancreatic cancer; however, it did not show significant therapeutic effects on tumor growth. In contrast, dual targeting of the angiogenic signals resulted in more significant inhibition of tumor angiogenesis, leading to delay of tumor growth. Our findings suggest that simultaneous blockade of VEGF and BMP-9/10 signals is a promising therapeutic strategy for the cancers that are resistant to anti-VEGF and BMP-9/10 therapies.

Soluble VEGFR1 reverses BMP2 inhibition of intramembranous ossification during healing of cortical bone defects.

BMP2 is widely used for promotion of bone repair and regeneration. However, bone formation induced by BMP2 is quite variable. Bone forming progenitor cells in different locations appear to respond to BMP2 in different ways, and repair outcomes can vary as a consequence of modulating effects by other factors. In this study, we have examined the effects of VEGF on BMP2-induced repair of a cortical bone defect, a 1 mm diameter drill hole, in the proximal tibia of mice. Treatment of the defect with either a bolus of PBS or soluble VEGFR1 (sVEGFR1), a decoy receptor for VEGF, had the same effects on bone formation via intramembranous ossification in the defect and cartilage formation and injured periosteum, during the healing process. In contrast, treatment with BMP2 inhibited intramembranous bone formation in the defect while it promoted cartilage and endochondral bone formation in the injured periosteum compared with mice treated with PBS or sVEGFR1. The inhibitory effect of BMP2 on bone formation was unlikely due to increased osteoclast activity and decreased invasion of blood vessels in the defect. Most importantly, co-delivery of BMP2 and sVEGFR1 reversed the inhibition of intramembranous bone formation by BMP2. Furthermore, the decreased accumulation of collagen and production of bone matrix proteins in the defect of groups with BMP2 treatment could also be prevented by co-delivery of BMP2 and sVEGFR1. Our data indicate that introducing a VEGF-binding protein, such as sVEGFR1, to reduce levels of extracellular VEGF, may enhance the effects of BMP2 on intramembranous bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1461-1469, 2017.

Bone Marrow-Derived Endothelial Progenitor Cells Reduce Recurrent Miscarriage in Gestation.

Bone marrow-derived endothelial progenitor cells (EPCs) have been shown to contribute to not only angiogenesis in ischemic tissue but also neovascularization in uterine endometrium formation. Reduced neovascularization and elevation of serum soluble Flt1, a functional blockage of VEGF, in the development of placenta is thought to be one of the major causes of repeated miscarriages in gestation. We then examined whether transfusion of VEGF-expressing extrinsic EPCs prevented frequent miscarriage via its promotional effect on neovascularization with a VEGFeNOS signaling pathway in a mouse miscarriage model. The results showed that systemic EPC transfusion significantly reduced the rate of miscarriage, and EPCs were frequently observed in the miscarriage placenta. In contrast, only a few EPCs were detected in the placenta of normal gestation. The vascular pattern was irregular, and vessel size was small in the miscarriage placenta compared with that of normal gestation. The placental vascular pattern in miscarriage tended to be normalized with increased vessel size up to a similar level as normal gestation by EPC recruitment. For the mechanistic insight, since soluble Flt1 inhibits EPC functions, it was suggested that the increased soluble Flt1 could suppress the recruited EPC functional activity in the miscarriage placenta. In vitro experiments by soluble Flt1 treatment in cultured EPCs suggested that the vascular abnormality could be partly due to the inhibition of eNOS expression by the increased amounts of soluble Flt1. These findings from animal experiments indicated that autologous EPC therapy may be a novel therapy to prevent miscarriage in high-risk pregnancies, such as preeclampsia.

The role of high-mobility group box protein 1 in collagen antibody-induced arthritis is dependent on vascular endothelial growth factor.

High-mobility group box 1 (HMGB1) has been implicated in angiogenesis and rheumatoid arthritis (RA). The aim of this study was to define more clearly the role of HMGB1 in the synovial angiogenesis and pathogenesis of an immune model of arthritis. BALB/c mice were injected with monoclonal anti-collagen antibody cocktail followed by lipopolysaccharide to induce arthritis. HMGB1 and vascular endothelial growth factor (VEGF) were over-expressed in the areas of the synovium where more inflammation and neoangiogenesis were present. The selective blockade of HMGB1 or VEGF resulted alternatively in a lower severity of arthritis evaluated by the arthritis index. Furthermore, exogenous HMGB1 administration caused a worsening of arthritis, associated with VEGF up-regulation and increased synovial angiogenesis. The selective inhibition of VEGF also resulted in no induction of arthritis in mice receiving exogenous HMGB1. Cytokine enzyme-linked immunosorbent assay (ELISA) analyses performed on peripheral blood and synovial fluid demonstrated a significant reduction of interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α in mice where HMGB1 and VEGF pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the peripheral IL-17A concentration. The development of arthritis mediated by HMGB1 and the synovial angiogenesis can be blocked by inhibiting the VEGF activity. The proinflammatory and proangiogenic cytokine IL-17A was increased when HMGB1 is inhibited, but the synovial angiogenesis was nevertheless reduced in this model of arthritis. Taken together, these findings shed new light on the role of this nuclear protein in the pathogenesis of arthritis in an RA-like model.

Hypertension in an Animal Model of HELLP Syndrome is Associated With Activation of Endothelin 1.

Women with hypertensive forms of pregnancy such as hemolysis-elevated liver enzymes-low platelet syndrome have increased circulating endothelin 1; however, the relationship between hypertension and endothelin 1 has not been studied. Using an animal model, we sought to determine whether there was an increased activation/dysfunction of endothelin 1, the effect of endothelin 1 receptor-A blockade on hypertension and other manifestations of hemolysis, elevated liver enzymes, and low platelets syndrome. On gestational day 12, timed-pregnant rats were infused with soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEndoglin; 4.7 and 7 µg/kg) via mini-osmotic pumps for 8 days. A subset of rats were treated with receptor-A antagonist (ABT-627, 5mg/kg) for 8 days. Rats with hemolysis-elevated liver enzymes-low platelet syndrome had significantly increased hypertension (P = .0001), circulating endothelin 1 (P = .03), and a significant 3.3- and 7.2-fold increase in preproendothelin messenger RNA (mRNA) expression in the placenta and liver (P = .01 and .04). Urinary protein:creatinine ratio was significantly increased in these animals (P = .0007), and circulating factors from these rats stimulated a significant increase in endothelial cell secretion of endothelin 1 (P = .001) in an in vitro assay. Blockade of the endothelin 1 receptor A significantly decreased hypertension (P = .001), circulating endothelin 1, and interleukin 17 (P = .004 and .003), placental preproendothelin mRNA expression (P = .016), and urinary protein:creatinine ratio (P = .007) in rats with hemolysis-elevated liver enzymes-low platelet syndrome. Blockade of the endothelin 1 receptor A significantly decreased hemolysis (P = .009), liver enzymes (P = .011), and significantly increased platelet levels (P = .03) and decreased circulating CD4+ and CD8+ T lymphocytes (P = .0004 and .0001) in rats infused with sFlt-1 and sEndoglin. These data support the hypothesis that endothelin 1 activation has a critical role in pathophysiology of as hemolysis-elevated liver enzymes-low platelet syndrome.

Placental-Specific sFLT-1 e15a Protein Is Increased in Preeclampsia, Antagonizes Vascular Endothelial Growth Factor Signaling, and Has Antiangiogenic Activity.

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a-specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a.

Comparison of Different Cytokine Conditions Reveals Resveratrol as a New Molecule for Ex Vivo Cultivation of Cord Blood-Derived Hematopoietic Stem Cells.

UNLABELLED: Human cord blood (CB)-derived hematopoietic stem cells (HSCs) are an interesting source for HSC transplantation. However, the number of collected CB-HSCs is often too low for one transplantation; therefore, ex vivo expansion of CB-HSCs is desirable. Current expansion protocols are based on the use of cytokine combinations, including insulin-like growth factor-binding protein 2 (IGFBP2) and angiopoietin-like proteins, or combinations with "small molecules" such as stemregenin-1. The aim of our project was to compare the potential of different CB-HSC expansion strategies side-by-side by phenotypical analysis in vitro and serial engraftment properties in NOD/SCID/IL2rg-/- (NSG) immunodeficient mice. We further identified resveratrol, a naturally occurring polyphenol, as a new, alternative small molecule combined with cytokines to facilitate serum-free ex vivo expansion of human CB-HSCs. The cultivation in resveratrol preserved the CB-HSC phenotype in vitro most efficiently and was ∼2 times more potent than commonly used cytokine conditions (including stem cell factor, thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-6) and the recently established serum-free culture, including IGFBP2 and angiopoietin-like 5. Serial transplantation studies further confirmed resveratrol to support robust multilineage engraftment in primary and secondary NSG recipients. Therefore, our work proposes resveratrol as a new small molecule for improved ex vivo culture and modification of human HSCs based on an efficient ex vivo propagation of the HSC fate. SIGNIFICANCE: Human cord blood (CB)-derived hematopoietic stem cells (HSCs) are an important source for HSC transplantations but restricted in their usage because of their low numbers. In gene therapy, modifications of HSCs relies on their ex vivo modification without losing their stemness properties. Therefore, ex vivo cultivation and expansion of CB-HSCs is important for their effective application in HSC transplantation and gene therapy. Several promising protocols for serum-free cultivation of HSCs using different combinations of cytokines or so-called small molecules are described. A direct comparison was performed of three described serum-free cytokine conditions, demonstrating that the natural occurring polyphenol resveratrol is able to support ex vivo cultivation of CB-HSCs. The results show that resveratrol is an additional candidate for improving ex vivo cultures of HSCs for transplantation and gene therapeutic applications in the future.

Ginsenoside Rh2 Suppresses Neovascularization in Xenograft Psoriasis Model.

BACKGROUND/AIMS: Psoriasis is a common inflammatory skin disease of undetermined etiology and poor prognosis. The current therapies have focused on direct inhibition of local inflammation, e.g. through hormone treatments. However, neovascularization plays a critical role in the development of psoriasis but so far no therapies have been developed to suppress psoriasis-associated neovascularization. METHODS: We treated AGR129 mice that had received human PN skin grafts with different doses of Ginsenoside Rh2 (GRh2). The acanthosis and papillomatosis index were evaluated. The percentage of T lymphocytes in the grafts was quantified by flow cytometry. The levels of vascularization in the grafts were quantified based on CD31-positive area. We examined the levels of VEGF-A in the skin treated with GRh2. We treated AGR129 mice that had received human PN skin grafts with different doses of soluble Flt-1 (sFlt1) and then evaluated the effects on the acanthosis and papillomatosis index, T lymphocyte percentage and vessel density. RESULTS: GRh2 dose-dependently decreased the acanthosis and papillomatosis index, T lymphocyte percentage and vessel density in PN skin grafts in mice. GRh2 inhibited VEGF-A levels in the PN skin grafts. Treatment with sFlt1 mimicked the effects of GRh2 on the acanthosis and papillomatosis index, T lymphocyte percentage and vessel density in PN skin grafts in mice. CONCLUSIONS: GRh2 may have an anti-psoriasis effect through neovascularization suppression.