Effects of ON101 for Hard-to-Heal Diabetic Foot Ulcers in A Randomized Phase III Trial: A Post hoc Analysis.

OBJECTIVE: Hard-to-heal diabetic foot ulcers (DFUs) are associated with higher mortality rates and an increased medical burden for patients. ON101, a new topical cream, exhibited better healing efficacy than the control dressing in a Phase III trial. In this post-hoc analysis, we further identify whether ON101 can improve the healing of ulcers with hard-to-heal risk factors in this cohort of DFU patients. APPROACH: To compare the efficacy of ON101 with absorbent dressing among various hard-to-heal wounds in patients with DFU, a post hoc analysis of a randomized phase III trial included 276 DFU patients was performed by subgrouping those patients based on ulcer depth, location, size, duration, and patients' glycated hemoglobin (HbA1c) levels and body mass index (BMI). RESULTS: In the full analysis set, the proportion of patients achieving healing was 61.7% in the ON101 group and 37.0% in the comparator (P =0.0001). In sub-group analysis according to risk factors, ON101 demonstrated superior healing capacity on Wagner grade 2 ulcers (P < 0.0001); plantar ulcers (P = 0.0016), ulcers size ≥5 cm² (P = 0.0122), ulcers duration ≥3 months (P = 0.0043); for patients with HbA1c ≥9% (P = 0.0285); and patients with BMI ≥25 (P = 0.0005). INNOVATION: ON101, a novel therapeutic drug, can modulate the functions of macrophages and demonstrate superior healing rates to conventional absorbent dressing in patients with hard-to-heal DFUs. CONCLUSIONS: The results of this post hoc study suggest that ON101 is a better therapeutic option than conventional dressing used in treatment for DFU patients with higher HbA1c, BMI, or ulcers with complex conditions such as longer duration, deeper wounds, larger size, and plantar location.

Rare type of tracheal agenesis: Unexpected presentation and immediate consideration of emergent esophageal intubation in neonatal resuscitation program. Case reports and review of the literature.

BACKGROUND: Tracheal agenesis, or tracheal atresia, is a rare congenital anomaly. The presence of a tracheoesophageal fistula (TEF) can help with breathing for newborns with tracheal agenesis. In this article, we presented three unique cases and outcomes of neonates with tracheal agenesis along with a review of the literature. METHODS: This study consisted of a single center case series followed by a review of literature. Case reports were generated using both written and electronic medical records from a single hospital. We summarized three unique cases and outcomes of neonates with tracheal agenesis and performed a review of the literature. RESULTS: We identified three cases of tracheal agenesis presented with severe cyanosis without spontaneous crying upon birth. Experienced pediatricians attempted to intubate the babies but were unsuccessful. Endotracheal tubes were subsequently either accidentally or purposely placed into the esophagus, and oxygen saturation levels improved. This suggested tracheal agenesis with TEF. Two cases underwent surgical intervention after resuscitation with esophageal intubation. CONCLUSION: Esophageal intubation may be a life-sustaining ventilation support for patients with tracheal agenesis and TEF at initial resuscitation. Clinicians should suspect tracheal agenesis when a newborn presents with severe cyanosis and voiceless crying upon birth, and esophageal intubation should be immediately attempted.

Decreased junctional adhesion molecule 3 expression induces reactive oxygen species production and apoptosis in trophoblasts†.

Junctional adhesion molecule 3 (JAM3) is involved in epithelial cell junction, cell polarity, and motility. The molecular mechanisms underlying the role of JAM3 in placental dysfunction remain unclear. We hypothesized that JAM3 expression regulates trophoblast fusion, differentiation, proliferation, and apoptosis. Our results revealed that JAM3 was expressed in the cytotrophoblasts and syncytiotrophoblasts of first-trimester and term placental villi. JAM3 expression in cell-cell junctions decreased with the formation of syncytiotrophoblasts. Using trophoblasts as an in vitro model, we observed that forskolin and JAM3 knockdown significantly reduced JAM3 expression and increased syncytium formation. JAM3 knockdown additionally inhibited trophoblast proliferation and increased the number of trophoblasts in the sub-G1 and G2/M phases, indicating cell-cycle disturbance and apoptosis. Cell-cycle arrest was associated with the engagement of checkpoint kinase 2-cell division cycle 25C-cyclin-dependent kinase 1/cyclin B1 signaling. Increased expression of BIM, NOXA, XAF1, cytochrome c, and cleaved caspase-3 further indicated trophoblast apoptosis. Overexpression of JAM3 or recombinant JAM3 protein enhanced trophoblast adhesion and migration, which were inhibited by JAM3 knockdown. JAM3 knockdown induced reactive oxygen species and syncytin 2 expression in trophoblasts. Furthermore, H2O2-induced oxidative stress reduced JAM3 expression in trophoblasts and cell culture supernatants. H2O2 simultaneously induced trophoblast apoptosis. JAM3 expression was significantly decreased in the plasmas and placentas of patients with early-onset severe preeclampsia. Thus, our results show that JAM3 may not only be a structural component of trophoblast cell junctions but also regulates trophoblast fusion, differentiation, proliferation, apoptosis, and motility. Dysregulated trophoblast JAM3 expression is crucial in preeclampsia development.

TDP-43 is essential for Eph receptor-class-specific spinal motor axon trajectory into the limb.

During the neural circuit formation, neuronal growth cones must be guided precisely to their neuronal or muscle targets, which can be achieved by the activation of membrane-bound guidance receptors at the periphery. However, the mechanisms that regulate the temporal availability of these receptors remain largely unknown. TAR DNA binding protein-43 (TDP-43) has been proposed to bind with the mRNAs of guidance receptors, thus prompting us to investigate its role in axon guidance of the spinal lateral motor column (LMC) neurons into the limb mesenchyme. We first identified the TDP-43 expression in the LMC neurons at the stage of axons growth into the limb using in situ mRNA hybridization. The loss and gain of TDP-43 function in chick LMC neurons redirected their axon trajectory with opposite effects. In mice, a spinal motor neuron-specific TDP-43 deletion led to the misrouting of LMC axons. Further, ectopic TDP-43 expression increased EphB protein levels in LMC neurons, suggesting that TDP-43 mediates LMC pathfinding by regulating EphB expression. Finally, TDP-43 levels influenced the growth preference of LMC neurites against ephrin-B, but not Netrin-1 and Semaphorin ligands. Our results demonstrate that TDP-43 is essential for the ephrinB:EphB signaling-mediated axon trajectory selection of LMC subtypes into the limb.

Vav2 is required for Netrin-1 receptor-class-specific spinal motor axon guidance.

BACKGROUND: Proper guidance of neuronal axons to their targets is required to assemble neural circuits during the development of the nervous system. However, the mechanism by which the guidance of axonal growth cones is regulated by specific intermediaries activated by receptor signaling pathways to mediate cytoskeleton dynamics is unclear. Vav protein members have been proposed to mediate this process, prompting us to investigate their role in the limb selection of the axon trajectory of spinal lateral motor column (LMC) neurons. RESULTS: We found Vav2 and Vav3 expression in LMC neurons when motor axons grew into the limb. Vav2, but not Vav3, loss-of-function perturbed LMC pathfinding, while Vav2 gain-of-function exhibited the opposite effects, demonstrating that Vav2 plays an important role in motor axon growth. Vav2 knockdown also attenuated the redirectional phenotype of LMC axons induced by Dcc, but not EphA4, in vivo and lateral LMC neurite growth preference to Netrin-1 in vitro. This study showed that Vav2 knockdown and ectopic nonphosphorylable Vav2 mutant expression abolished the Src-induced stronger growth preference of lateral LMC neurites to Netrin-1, suggesting that Vav2 is downstream of Src in this context. CONCLUSIONS: Vav2 is essential for Netrin-1-regulated LMC motor axon pathfinding through Src interaction.

A proposed approach to confirm heroin administration - Regional differences in heroin purity is a major factor.

In forensic toxicology, a marker of street heroin use is urgent especially in the absence of urinary 6-monoacetylmorphine. ATM4G, the Glucuronide of Acetylated product of Thebaine compound 4 Metabolite (ATM4), arising from byproducts of street heroin synthesis has been considered as a useful marker in some European studies. However, whether ATM4G is a universal marker particularly in Southeast Asia due to 'street' heroin with high purity, it's still unclear. To investigate putative markers for different regions, ATM4G and other metabolites including the Acetylated product of Thebaine compound 3 Metabolite (ATM3) and thebaol, also originated from thebaine were detected in 552 urine samples from heroin users in Taiwan. Results were compared with that from samples collected in the UK and Germany. Only a sulfo-conjugate of ATM4, ATM4S, was detected in 28 Taiwanese users using a sensitive MS3 method whilst out of 351 samples from the UK and Germany, ATM4G was present in 91. Thebaol-glucuronide was first time detected in 118. No markers were detected in urine following herbal medicine use or poppy seed ingestion. The presence of ATM4S/ATM4G might be affected by ethnicities and heroin supplied in regions. Thebaol-glucuronide is another putative marker with ATM4G and ATM4S for street heroin use.

Paxillin Is Required for Proper Spinal Motor Axon Growth into the Limb.

To assemble the functional circuits of the nervous system, the neuronal axonal growth cones must be precisely guided to their proper targets, which can be achieved through cell-surface guidance receptor activation by ligand binding in the periphery. We investigated the function of paxillin, a focal adhesion protein, as an essential growth cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show paxillin expression in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Paxillin loss-of-function and gain-of-function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of paxillin in motor axon guidance. In addition, a neuron-specific paxillin deletion in mice led to LMC axon trajectory selection errors. We also show that knocking down paxillin attenuates the growth preference of LMC neurites against ephrins in vitro, and erythropoietin-producing human hepatocellular (Eph)-mediated retargeting of LMC axons in vivo, suggesting paxillin involvement in Eph-mediated LMC motor axon guidance. Finally, both paxillin knockdown and ectopic expression of a nonphosphorylable paxillin mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating paxillin as a Src target in Eph signal relay in this context. In summary, our findings demonstrate that paxillin is required for motor axon guidance and suggest its essential role in the ephrin-Eph signaling pathway resulting in motor axon trajectory selection.SIGNIFICANCE STATEMENT During the development of neural circuits, precise connections need to be established among neurons or between neurons and their muscle targets. A protein family found in neurons, Eph, is essential at different stages of neural circuit formation, including nerve outgrowth and pathfinding, and is proposed to mediate the onset and progression of several neurodegenerative diseases, such as Alzheimer's disease. To investigate how Ephs relay their signals to mediate nerve growth, we investigated the function of a molecule called paxillin and found it important for the development of spinal nerve growth toward their muscle targets, suggesting its role as an effector of Eph signals. Our work could thus provide new information on how neuromuscular connectivity is properly established during embryonic development.

Placental multipotent mesenchymal stromal cell-derived Slit2 may regulate macrophage motility during placental infection.

Slit proteins have been reported to act as axonal repellents in Drosophila; however, their role in the placental microenvironment has not been explored. In this study, we found that human placental multipotent mesenchymal stromal cells (hPMSCs) constitutively express Slit2. Therefore, we hypothesized that Slit2 expressed by hPMSCs could be involved in macrophage migration during placental inflammation through membrane cognate Roundabout (Robo) receptor signaling. In order to develop a preclinical in vitro mouse model of hPMSCs in treatment of perinatal infection, RAW 264.7 cells were used in this study. Slit2 interacted with Robo4 that was highly expressed in RAW 264.7 macrophages: their interaction increased the adhesive ability of RAW 264.7 cells and inhibited migration. Lipopolysaccharide (LPS)-induced CD11bCD18 expression could be inhibited by Slit2 and by hPMSC-conditioned medium (CM). LPS-induced activation of p38 and Rap1 was also attenuated by Slit2 and by hPMSC-CM. Noticeably, these inhibitory effects of hPMSC-CM decreased after depletion of Slit2 from the CM. Furthermore, we found that p38 siRNA inhibited LPS-induced Rap1 expression in RAW 264.7 cells, indicating that Rap1 functions downstream of p38 signaling. p38 siRNA increased cell adhesion and inhibited migration through reducing LPS-stimulated CD11bCD18 expression in RAW 264.7 cells. Thus, hPMSC-derived Slit2 may inhibit LPS-induced CD11bCD18 expression to decrease cell migration and increase adhesion through modulating the activity and motility of inflammatory macrophages in placenta. This may represent a novel mechanism for LPS-induced placental infection.

Nck2 is essential for limb trajectory selection by spinal motor axons.

BACKGROUND: The development of a functioning nervous system requires precise assembly of neuronal connections, which can be achieved by the guidance of axonal growth cones to their proper targets. How axons are guided by signals transmitted to the cytoskeleton through cell surface-expressed guidance receptors remains unclear. We investigated the function of Nck2 adaptor protein as an essential guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory into the limb. RESULTS: Nck2 mRNA and protein are preferentially expressed in the medial subgroups of chick LMC neurons during axon trajectory into the limb. Nck2 loss- and gain-of-function in LMC neurons using in ovo electroporation perturb LMC axon trajectory selection demonstrating an essential role of Nck2 in motor axon guidance. We also showed that Nck2 knockdown and overexpression perturb the growth preference of LMC neurites against ephrins in vitro and Eph-mediated redirection of LMC axons in vivo. Finally, the significant changes of LMC neurite growth preference against ephrins in the context of Nck2 and α2-chimaerin loss- and gain-of-function implicated Nck2 function to modulate α2-chimaerin activity. CONCLUSIONS: Here, we showed that Nck2 is required for Eph-mediated axon trajectory selection from spinal motor neurons through possible interaction with α2-chimaerin. Developmental Dynamics 247:1043-1056, 2018. © 2018 Wiley Periodicals, Inc.

Oxidative stress reduces trophoblast FOXO1 and integrin ß3 expression that inhibits cell motility.

Preeclampsia is a serious pregnancy complication associated with placental oxidative stress and impaired trophoblast migration. The mechanism of defective trophoblast migration remains unknown. Forkhead box O1 (FOXO1) is a transcription factor. Integrin ß3 is involved in cell motility. We hypothesized that FOXO1 mediates expression of trophoblast integrin ß3, which could be impaired by oxidative stress and have implications in preeclampsia. The expressions of FOXO1 and integrin ß3 were significantly reduced in preeclamptic placentas (n = 15) compared to that of controls (n = 15; p < 0.01). HTR-8/SVneo and JEG-3 trophoblasts were transfected to express wild-type FOXO1-WT or constitutively-expressed nuclear mutant form, FOXO1-AAA. The FOXO1 in HTR-8/SVneo and 3A-Sub-E trophoblasts was silenced by small interfering RNA. AKT-mediated phosphorylation inactivated FOXO1, but FOXO1-AAA was not phosphorylated. The expression of trophoblast integrin ß3 was significantly elevated by FOXO1 overexpression and inhibited by FOXO1 knockdown. FOXO1 regulates integrin ß3 at the transcriptional level via binding to the putative FOXO1 response element site between position -1154 to -1139 (TGAGATGTTTTGAAAG) in HTR-8/SVneo trophoblasts. The level of phosphorylated FOXO1 was decreased, and the FOXO1 level was increased in trophoblasts treated with AKT inhibitor MK2206, leading to upregulation of integrin ß3. The capabilities of trophoblast adhesion and migration were enhanced by FOXO1-overexpression or MK2206, and inhibited by silencing FOXO1 or oxidative stress with H2O2. These results suggest that FOXO1 enhances trophoblast integrin ß3 expression, and mediates cell adhesion and migration. By affecting the expression of FOXO1 and cell motility in trophoblasts, oxidative stress plays a role in the development of preeclampsia.

Ephexin1 Is Required for Eph-Mediated Limb Trajectory of Spinal Motor Axons.

The precise assembly of a functional nervous system relies on the guided migration of axonal growth cones, which is made possible by signals transmitted to the cytoskeleton by cell surface-expressed guidance receptors. We investigated the function of ephexin1, a Rho guanine nucleotide exchange factor, as an essential growth-cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show that ephexin1 is expressed in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Ephexin1 loss of function and gain of function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of ephexin1 in motor axon guidance. In addition, ephexin1 loss in mice of either sex led to LMC axon trajectory selection errors. We also show that ephexin1 knockdown attenuates the growth preference of LMC neurites against ephrins in vitro and Eph receptor-mediated retargeting of LMC axons in vivo, suggesting that ephexin1 is required in Eph-mediated LMC motor axon guidance. Finally, both ephexin1 knockdown and ectopic expression of nonphosphorylatable ephexin1 mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating ephexin1 as an Src target in Eph signal relay in this context. In summary, our findings demonstrate that ephexin1 is essential for motor axon guidance and suggest an important role in relaying ephrin:Eph signals that mediate motor axon trajectory selection.SIGNIFICANCE STATEMENT The proper development of functioning neural circuits requires precise nerve connections among neurons or between neurons and their muscle targets. The Eph tyrosine kinase receptors expressed in neurons are important in many contexts during neural-circuit formation, such as axon outgrowth, axon guidance, and synaptic formation, and have been suggested to be involved in neurodegenerative disorders, including amyotrophic lateral sclerosis and Alzheimer's disease. To dissect the mechanism of Eph signal relay, we studied ephexin1 gain of function and loss of function and found ephexin1 essential for the development of limb nerves toward their muscle targets, concluding that it functions as an intermediary to relay Eph signaling in this context. Our work could thus shed new light on the molecular mechanisms controlling neuromuscular connectivity during embryonic development.

Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling.

The objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.

The effect of conditioned medium derived from human placental multipotent mesenchymal stromal cells on neutrophils: possible implications for placental infection.

The role of human placental multipotent mesenchymal stromal cells (hPMSCs) in placental inflammation is unknown. We hypothesize that hPMSCs are involved in the early phases of placental infection. hPMSCs were isolated from term placentas and neutrophils from peripheral blood. The expression of toll-like receptors (TLRs) and cytokines by hPMSCs was determined by RT-PCR, flow cytometry and enzyme-linked immunosorbent assay. The effect of conditioned medium of hPMSCs with or without lipopolysaccharide (LPS) pretreatment on neutrophil functions: migration, apoptosis and production of reactive oxygen species (ROS) was assessed by flow cytometry and western blot. hPMSCs expressed TLR1, TLR3, TLR4, TLR6, TLR7 and TLR9. LPS stimulation increased the expression of TLR4 and the production of IL-6 and IL-8 by hPMSCs. Neutrophils exhibited chemotaxis to hPMSC-conditioned medium, which was inhibited by IL-8 depletion. Neutrophil CD11b activation was promoted by hPMSC-conditioned medium, which was further enhanced in media from hPMSCs pretreated with LPS. hPMSC-conditioned medium reduced neutrophil ROS production. Neutrophil phagocytosis was increased by LPS alone but not by hPMSC-conditioned medium with or without LPS stimulation. hPMSC-conditioned medium induced STAT3 activation in neutrophils, which was inhibited by neutralizing antibody to IL-6. hPMSC-conditioned medium rescued neutrophils from apoptosis, but this effect was significantly reduced in conditioned medium of hPMSCs with LPS pretreatment. Depletion of IL-6 from the conditioned medium further inhibited the anti-apoptotic effect on neutrophils. Our results demonstrate that hPMSCs can interact with peripheral blood neutrophils in response to inflammatory signals of the placenta. Cytokines produced by hPMSCs can induce neutrophil chemotaxis and reduce neutrophil apoptosis.

Elastic liposomes as carriers for oral delivery and the brain distribution of (+)-catechin.

The aim of this work was to investigate whether the oral bioavailability and brain regional distribution of (+)-catechin could be improved by utilizing elastic liposomes. Liposomes containing soy phosphatidylcholine, cholesterol, and Tween 80 in the presence of 15% ethanol were prepared by a thin-film method and subsequent sonication and extrusion. The size, zeta potential, and stability of the liposomes in simulated gastrointestinal (GI) media were characterized. The mean size of liposomes was 35-70 nm, which decreased with an increase in the Tween 80 concentration. The zeta potential of the system was about-15 mV. More than 80% of the (+)-catechin was entrapped in the aqueous core of liposomes produced with 1% Tween 80. Liposomes entrapping (+)-catechin remained stable in the presence of GI fluids, especially in simulated intestinal fluid. The liposomes showed suppressed and sustained release of (+)-catechin compared with that from an aqueous solution. The aqueous control and liposomes were orally administered to rats. The blood level of liposomal (+)-catechin was enhanced at a later stage after administration compared with the free control. In the experiment on the brain distribution, liposomes with elastic properties showed 2.9- and 2.7-fold higher (+)-catechin accumulations compared with the aqueous solution in the cerebral cortex and hippocampus, respectively. Greater compound accumulations with liposomes were also detected in the striatum and thalamus. The experimental results suggest that elastic liposomes may offer a promising strategy for improving (+)-catechin delivery via oral ingestion.

Paracrine factors from human placental multipotent mesenchymal stromal cells protect endothelium from oxidative injury via STAT3 and manganese superoxide dismutase activation.

Reactive oxygen species may cause oxidative damage in the placenta, yet some mechanisms must exist to reduce or prevent such damage. We investigated whether oxidative injury to placental endothelial cells is inhibited by activation of antioxidant enzymes by paracrine factors secreted by human placental multipotent mesenchymal stromal cells (hPMSC). hPMSC-conditioned medium and umbilical endothelial cells were assayed for cytokines and cytokine receptor expression by immunoassay and real-time PCR. Endothelial cell survival was evaluated by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay and caspase 3 activity assay. tert-Butyl hydroperoxide was used to induce oxidative injury in endothelial cells, with fluorescent microscopy and flow cytometry used to detect intracellular peroxides and cell apoptosis. Western blot, real-time PCR, STAT3 DNA-binding activity assay, and STAT3 siRNA were used to assess endothelial cell antioxidant enzymes. hPMSC-conditioned medium supported endothelial cell survival and reduced endothelial cell intracellular peroxides and apoptosis. hPMSCs expressed the transcripts of the interleukin (IL) 6 cytokine family, including IL6 and leukemia-inhibitory factor. hPMSC-conditioned medium activated STAT3 expression in endothelial cells, which was inhibited by neutralizing antibody to interleukin 6 signal transducer (IL6ST) but not to IL6 or leukemia-inhibitory factor. STAT3 siRNA or manganese superoxide dismutase (SOD2) siRNA transfected into endothelial cells inhibited the antiapoptotic effect of conditioned medium. SOD2 was significantly upregulated in endothelial cells by conditioned medium via STAT3 activation that, in turn, was inhibited by IL6ST-neutralizing antibody or STAT3 siRNA. Paracrine factors secreted by hPMSCs support endothelial cell survival. STAT3 activation and SOD2 production protect against oxidative stress-induced endothelial cell damage.

Angiogenesis in differentiated placental multipotent mesenchymal stromal cells is dependent on integrin alpha5beta1.

Human placental multipotent mesenchymal stromal cells (hPMSCs) can be isolated from term placenta, but their angiogenic ability and the regulatory pathways involved are not known. hPMSCs were shown to express integrins alpha(v), alpha(4), alpha(5), beta(1), beta(3), and beta(5) and could be induced to differentiate into cells expressing endothelial markers. Increases in cell surface integrins alpha(5) and beta(1), but not alpha(4), alpha(v)beta(3), or alpha(v)beta(5), accompanied endothelial differentiation. Vascular endothelial growth factor-A augmented the effect of fibronectin in enhancing adhesion and migration of differentiated hPMSC through integrin alpha(5)beta(1), but not alpha(v)beta(3) or alpha(v)beta(5). Formation of capillary-like structures in vitro from differentiated cells was inhibited by pre-treatment with function-blocking antibodies to integrins alpha(5) and beta(1). When hPMSCs were seeded onto chick chorioallantoic membranes (CAM), human von Willebrand factor-positive cells were observed to engraft in the chick endothelium. CAMs transplanted with differentiated hPMSCs had a greater number of vessels containing human cells and more incorporated cells per vessel compared to CAMs transplanted with undifferentiated hPMSCs, and overall angiogenesis was enhanced more by the differentiated cells. Function-blocking antibodies to integrins alpha(5) and beta(1) inhibited angiogenesis in the CAM assay. These results suggest that differentiated hPMSCs may contribute to blood vessel formation, and this activity depends on integrin alpha(5)beta(1).

Engraftment potential of human placenta-derived mesenchymal stem cells after in utero transplantation in rats.

BACKGROUND: Human placental mesenchymal stem cells (hPMCs) are thought to be multipotent, but their fate after in utero transplantation is not known. METHODS: hPMCs isolated from term placenta were assessed for their phenotype markers, mutilineage capacity, and immunomodulatory properties. Their engraftment potential was analyzed in a pregnant rat model after in utero transplantation at embryonic day 17. Immunohistochemistry, tracing of labeled cells, fluorescence in situ hybridization and real-time PCR were used to assess post-transplant chimerism. RESULTS: In vitro, lineage-negative, CD34-negative hPMCs differentiated into osteocytes, adipocytes, hepatocytes and endothelial cells with tube formation, and actively suppressed the rat lymphocyte proliferative response to allogeneic lymphocyte stimulation (P < 0.0001). After in utero transplantation into pregnant rats, a low level of engraftment was achieved in various fetal tissues. Engraftment occurred in more than 60% of the fetal rats. Cells persisted for at least 12 weeks after delivery and evidence was obtained to suggest differentiation into specific lineages, including hepatocytes and hematopoietic cells. However, a greater number of hPMCs migrated to the placenta than to the fetus, thus limiting the degree of cell engraftment in fetal organs. CONCLUSIONS: We conclude that hPMCs are mutipotent cells that can be engrafted long-term in immunocompetent rats after in utero transplantation.

Trafficking of multipotent mesenchymal stromal cells from maternal circulation through the placenta involves vascular endothelial growth factor receptor-1 and integrins.

Maternal cells can become engrafted in various fetal organs during pregnancy. The nature of the cells and the mechanisms of maternofetal cell trafficking are not clear. We demonstrate that human lineage-negative, CD34-negative (Lin(-)CD34(-)) multipotent mesenchymal stromal cells express alpha(2), alpha(4), alpha(5), and beta(1) integrins, which mediate their adhesion to endothelium, and vascular endothelial growth factor receptor-1 (VEGFR-1), which mediates their response to vascular endothelial growth factor A (VEGF-A). A maternal-fetal VEGF-A concentration gradient exists across the placental barrier, and cord blood plasma induces transendothelial and trans-Matrigel migration of stem cells in vitro. Migration is inhibited by a VEGF-A-neutralizing antibody or antibodies against VEGFR-1 or integrin alpha(2), alpha(4), alpha(5), or beta(1). When Lin(-)CD34(-) multipotent mesenchymal stromal cells are transferred to rat maternal venous blood, they traffic through the placenta, engraft in various fetal organs, and persist in offspring for at least 12 weeks. Cell proliferation ability is retained in the xenogeneic placenta. Maternofetal trafficking is significantly reduced by blocking antibodies against integrins alpha(2), alpha(4), alpha(5), and beta(1) or VEGFR-1. These results suggest that maternal microchimerism arises by the trafficking of multipotent mesenchymal stromal cells via VEGF-A- and integrin-dependent pathways across the hemochorial placenta to fetal tissues.

Mitogen-activated protein kinases potentiate thyroid hormone receptor transcriptional activity by stabilizing its protein.

Transcriptional regulation of downstream gene expression by thyroid hormone (T(3)) is mediated by the thyroid hormone receptor (TR). T(3) binding induces a complicated transition, where TR converts from a transcriptional repressor into a transcriptional activator and instigates downstream gene transcription. Binding of T(3) to TR also induces the degradation of TR, resulting in desensitization of the cells to further T(3) treatment. It has been shown that phosphorylation of TR plays a critical role in its activity and stability after T(3) binding. However, the kinases in control of phosphorylating TR in the nucleus have not been identified. In this study we demonstrate that MAPKs are possible candidates responsible for the nuclear phosphorylation of TR. Suppression of MAPKs with specific inhibitors repressed TR transcriptional activity and antagonized okadeic acid-induced TR transcriptional activity potentiation. Overexpression of the MAPK activator, MKK6, and its constitutively active mutant, MKK6EE, significantly increased TR activity and protected TR from degradation. Involvement of the 26S ubiquitin proteasome in hormone binding-induced TR degradation was also examined. We found that MAPKs enhanced the DNA binding affinity of TR. Our results suggest that MAPKs are the major kinases responsible for the nuclear phosphorylation of TR and are critical factors modulating the transcriptional activity and protein stability of TR subsequent to ligand binding.

Activation of antimetastatic Nm23-H1 gene expression by estrogen and its alpha-receptor.

Metastasis of various malignant cells is inversely related to the abundance of the Nm23-H1 protein. The role of estrogens in tumor metastasis has now been investigated by examining the effect of E2 on the expression of the Nm23-H1 gene. Three human breast carcinoma cell lines, in which endogenous ERalpha is expressed at different levels, were used as a tool to assess the role of ERalpha in Nm23-H1 gene-mediated metastasis. E2 induced time-dependent increases in the abundance of Nm23-H1 mRNA and protein, with the extent of these effects correlating with the level of expression of ERalpha. E2 induced a marked decrease in the invasive activity of MCF-7 and BT-474 cells but had no effect on BCM-1 cells, which had virtually no ERalpha. Consistent with these results, the ER-mediated Nm23-H1 promoter activity was inhibited 3-fold by the E2 antagonist, ICI 182,780. Deletion analysis of the promoter region of the Nm23-H1 gene identified a positive estrogen-responsive element located in -108/-94. ER protein bound specifically to the -108/-79 fragment with high avidity. These results indicate that E2, acting through ERalpha, activated transcription of the Nm23-H1 gene via a positive estrogen-responsive element in the promoter region of the gene. These results suggest that E2 could suppress tumor metastasis by activating the expression of the Nm23-H1 gene.