Hypoxia-Induced Changes in the Bioactivity of Cytotrophoblast-Derived Exosomes

Migration of extravillous trophoblasts (EVT) into decidua and myometrium is a critical process in the conversion of maternal spiral arterioles and establishing placenta perfusion. EVT migration is affected by cell-to-cell communication and oxygen tension. While the release of exosomes from placental cells has been identified as a significant pathway in materno-fetal communication, the role of placental-derived exosomes in placentation has yet to be established. The aim of this study was to establish the effect of oxygen tension on the release and bioactivity of cytotrophoblast (CT)-derived exosomes on EVT invasion and proliferation. CT were isolated from first trimester fetal tissue (n = 12) using a trypsin-deoxyribonuclease-dispase/Percoll method. CT were cultured under 8%, 3% or 1% O2 for 48 h. Exosomes from CT-conditioned media were isolated by differential and buoyant density centrifugation. The effect of oxygen tension on exosome release (µg exosomal protein/10⁶cells/48 h) and bioactivity were established. HTR-8/SVneo (EVT) were used as target cells to establish the effect (bioactivity) of exosomes on invasion and proliferation as assessed by real-time, live-cell imaging (Incucyte™). The release and bioactivity of CT-derived exosomes were inversely correlated with oxygen tension (p<0.001). Under low oxygen tensions (i.e. 1% O2), CT-derived exosomes promoted EVT invasion and proliferation. Proteomic analysis of exosomes identified oxygen-dependent changes in protein content. We propose that in response to changes in oxygen tension, CTs modify the bioactivity of exosomes, thereby, regulating EVT phenotype. Exosomal induction of EVT migration may represent a normal process of placentation and/or an adaptive response to placental hypoxia.     

CEP Biomarkers as Potential Tools for Monitoring Therapeutics

Background

Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids that are elevated in ocular tissues and plasma in age-related macular degeneration (AMD) and in rodents exposed to intense light. The goal of this study was to determine whether light-induced CEP adducts and autoantibodies are modulated by pretreatment with AL-8309A under conditions that prevent photo-oxidative damage of rat retina. AL-8309A is a serotonin 5-HT_1A receptor agonist.

Methods

Albino rats were dark adapted prior to blue light exposure. Control rats were maintained in normal cyclic light. Rats were injected subcutaneously 3x with 10 mg/kg AL-8309A (2 days, 1 day and 0 hours) before light exposure for 6 h (3.1 mW/cm², λ=450 nm). Animals were sacrificed immediately following light exposure and eyes, retinas and plasma were collected. CEP adducts and autoantibodies were quantified by Western analysis or ELISA.

Results

ANOVA supported significant differences in mean amounts of CEP adducts and autoantibodies among the light + vehicle, light + drug and dark control groups from both retina and plasma. Light-induced CEP adducts in retina were reduced ~20% following pretreatment with AL-8309A (n = 62 rats, p = 0.006) and retinal CEP immunoreactivity was less intense by immunohistochemistry. Plasma levels of light-induced CEP adducts were reduced at least 30% (n = 15 rats, p = 0.004) by drug pretreatment. Following drug treatment, average CEP autoantibody titer in light exposed rats (n = 22) was unchanged from dark control levels, and ~20% (p = 0.046) lower than in vehicle-treated rats.

Conclusions

Light-induced CEP adducts in rat retina and plasma were significantly decreased by pretreatment with AL-8309A. These results are consistent with and extend previous studies showing AL-8309A reduces light-induced retinal lesions in rats and support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics.     

Loss of the SV2-like Protein SVOP Produces No Apparent Deficits in Laboratory Mice

Neurons express two families of transporter-like proteins − Synaptic Vesicle protein 2 (SV2A, B, and C) and SV2-related proteins (SVOP and SVOPL). Both families share structural similarity with the Major Facilitator (MF) family of transporters. SV2 is present in all neurons and endocrine cells, consistent with it playing a key role in regulated exocytosis. Like SV2, SVOP is expressed in all brain regions, with highest levels in cerebellum, hindbrain and pineal gland. Furthermore, SVOP is expressed earlier in development than SV2 and is one of the neuronal proteins whose expression declines most during aging. Although SV2 is essential for survival, it is not required for development. Because significant levels of neurotransmission remain in the absence of SV2 it has been proposed that SVOP performs a function similar to that of SV2 that mitigates the phenotype of SV2 knockout mice. To test this, we generated SVOP knockout mice and SVOP/SV2A/SV2B triple knockout mice. Mice lacking SVOP are viable, fertile and phenotypically normal. Measures of neurotransmission and behaviors dependent on the cerebellum and pineal gland revealed no measurable phenotype. SVOP/SV2A/SV2B triple knockout mice did not display a phenotype more severe than mice harboring the SV2A/SV2B gene deletions. These findings support the interpretation that SVOP performs a unique, though subtle, function that is not necessary for survival under normal conditions.     

Partial Restoration of Macrophage Alteration from Diet-Induced Obesity in Response to Porphyromonas gingivalis Infection

Obesity is a chronic inflammatory disease that weakens macrophage innate immune response to infections. Since M1 polarization is crucial during acute infectious diseases, we hypothesized that diet-induced obesity inhibits M1 polarization of macrophages in the response to bacterial infections. Bone marrow macrophages (BMMΦ) from lean and obese mice were exposed to live Porphyromonas gingivalis (P. gingivalis) for three incubation times (1 h, 4 h and 24 h). Flow cytometry analysis revealed that the M1 polarization was inhibited after P. gingivalis exposure in BMMΦ from obese mice when compared with BMMΦ from lean counterparts. Using a computational approach in conjunction with microarray data, we identified switching genes that may differentially control the behavior of response pathways in macrophages from lean and obese mice. The two most prominent switching genes were thrombospondin 1 and arginase 1. Protein expression levels of both genes were higher in obese BMMΦ than in lean BMMΦ after exposure to P. gingivalis. Inhibition of either thrombospondin 1 or arginase 1 by specific inhibitors recovered the M1 polarization of BMMΦ from obese mice after P. gingivalis exposure. These data indicate that thrombospondin 1 and arginase 1 are important bacterial response genes, whose regulation is altered in macrophages from obese mice.     

Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation

Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ?K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ?K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (?K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ?K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ?K107 or ?K226.     

Population genetics structure of glyphosate‐resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance

Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate‐resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K‐means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) encoding gene from glyphosate‐resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target‐site resistance mechanism.     

Antileishmanial activity and cytotoxicity of ent-beyerene diterpenoids

We describe the in vitro activity of two natural isomeric ent-beyerene diterpenes, several derivatives and synthetic intermediates. Beyerenols 1 and 2 showed EC₅₀ of 4.6?±?9.4 and 5.3?±?9.4?μg/mL against amastigotes of L. (V) brazilensis, with SI of 5.1 and 7.7, respectively. Beyerenol 1 was synthesized from stevioside. In vivo experiments with bereyenols showed cure in 50% of hamsters infected with L. (V) brazilensis topically applied as Cream I (beyerenol 1, 0.81%, w/w) and Cream III (beyerenol 2, 1.96%, w/w). These results suggest that beyerenols are potential candidates for cutaneous leishmaniasis chemotherapy by topical application. In vitro assays of amastigotes of L. (V) brazilensis showed EC₅₀ of 1.1?±?0.1 and 1.3?±?0.04?μg/mL, with SI of 3.1 and 3.5 for hydrazone intermediates 10 and 11, respectively.