Mark Beveridge

Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta

Concentrative absorption of glutamate by the developing placenta is critical for proper fetal development. The expression of GLAST1, GLT1, EAAC1, and EAAT4, known to be capable ofd-aspartate-inhibitable and Na+-coupled glutamate transport (system [Formula: see text]), was evaluated in day 14 vs. day 20 rat chorioallantoic placenta. Steady-state mRNA levels were greater at day 20 for all transporters. Immunohistochemistry determined that the expression of GLAST1, GLT1, and EAAC1 was greater throughout the day 20 placenta and was asymmetric with respect to cellular localization. EAAT4 protein was not detected. System[Formula: see text] activity was responsible for most of the Na+-dependent glutamate uptake and was greater in day 20 than in day 14apical and basal membrane subdomains of the labyrinth syncytiotrophoblast. Greater quantities of EAAC1 and GLAST1 protein were identified on day 20, and quantities were greater in basal than in apical membranes. GLT1 expression, unchanged in apical membranes, was decreased in basal membranes. These data correlate transporter mRNA and protein content with transport activity and demonstrate an increasing capacity for glutamate absorption by the developing placenta.

Ontogeny of amino acid transport system A in rat placenta

Amino acid transport System A has previously been demonstrated in apical membranes derived from rat placenta, as well as in apical and basal membranes derived from human placenta. We have studied Na(+)-dependent alpha-(methylamino)isobutyric acid (MeAIB) transport in apical and basal predominant membrane fractions prepared from 14 and 20 day gestation rat placenta. Marker enzyme recoveries did not differ significantly between age groups. Markers for intracellular organelles were also found to be comparable. Na(+)-dependent MeAIB transport was not sensitive to freezing and could be found in all membrane components tested. Kinetic parameters were studied--Km = 852 +/- 215 microM, Vmax = 718 +/- 126 pmol/5 sec/mg protein--20 day apical; Km = 748 +/- 269 microM, Vmax = 610 +/- 176 pmol/5 sec/mg protein--20 day basal-predominant; Km 614 +/- 261 microM, Vmax = 123 +/- 45 pmol/5 sec/mg protein-14 day apical. Kinetic parameters could not be determined in the 14 day gestation basal-predominant fraction because of the small amount of uptake present. We conclude that System A like activity is found in both apical and basal predominant membrane fractions derived from rat placenta, and that this activity increases over the last one third of gestation.

Demonstration of system y+L activity on the basal plasma membrane surface of rat placenta and developmentally regulated expression of 4F2HC mRNA.

Na(+)-independent cationic amino acid transport in the rat placenta occurs by leucine-sensitive and leucine-insensitive pathways. The ontogeny of these transport mechanisms within the rat placenta has been described recently. To assign the leucine-inhibitable portion of uptake definitively the uptake of [3H]arginine was studied in the presence of both BCH (to inhibit system Bo,+) and varied concentrations of leucine. Uptake of arginine into basal-enriched membrane vesicles derived from rat placenta was, in the presence of sodium, inhibited by micromolar concentrations of leucine, consistent with assignment of this activity to system y+L. In contrast, the majority of arginine uptake into apical-enriched membrane vesicles was leucine insensitive. Messenger RNA derived from rat placenta at days 14, 16, 18 and 20 of gestation was hybridized with full-length rat cDNA probes against NBAT and 4F2HC (thought to encode proteins associated with system bo,+ and y+L activities, respectively). No NBAT mRNA was detected, whereas 4F2HC mRNA was present at all gestational stages, increasing 12-fold over the last third of gestation. It is concluded that system y+L is present in the basal plasma membrane of the rat placenta syncytium and is subject to developmental regulation by a mechanism that alters the steady content of 4F2HC mRNA.

Glutamine transport in human and rat placenta

Glutamine plays an important role in fetal nutrition. This study explored the transport of [3H]glutamine into apical and basal predominant membrane vesicles derived from rat and human placenta. Na+-dependent glutamine transport was present in both apical and basal predominant vesicles derived from 20- and, to a lesser degree, 14-day gestation rat placenta. Amino-acid transport systems A, ASC-like, B(o,+) (in apical membrane vesicles) and, perhaps, y+L were involved in Na+-dependent glutamine transport. Na+-dependent glutamine uptake into human placental microvillus and basolateral membrane vesicles also occurred via several distinct transport activities. Glutamine transport via system N was not detected in either rat or human placental preparations. Na+-dependent glutamine transport in the rat was more pronounced in basal as compared to apical membrane vesicles. Conversely, in the human preparations, activity was significantly higher in microvillus as compared to basolateral membrane vesicles. It is concluded that Na+-dependent glutamine transport occurs through a variety of transport agencies in both the rat and human placenta. Transport varies with ontogeny and between species.

Effect of chronic cocaine administration on amino acid uptake in rat placental membrane vesicles

This study evaluated the effects of chronic exposure to cocaine during pregnancy on amino acid uptake in placental membrane vesicles. Pregnant rats received 62 mg/kg of cocaine hydrochloride by intraperitoneal (IP) injection as a divided daily dose on gestation days 8-19 inclusive. Fetal body weights were significantly decreased by 19% in the cocaine group, while placental weights were unchanged. Placental apical membrane vesicles were prepared from control and cocaine-treated animals, and marker enzyme enrichments for alkaline phosphatase and [3H]-dihydroalprenolol binding did not differ between cocaine and control groups. Rates of uptake (10 sec) of selected radiolabeled amino acids were measured utilizing a rapid filtration technique. Na(+)-dependent apical membrane [3H]-glutamine transport (50 microM) was reduced by 95% (p < 0.05) in cocaine-treated compared to control placentas. Uptake of 50 microM [3H]-methyl aminoisobutyric acid (MeAIB) into apical membranes was also decreased by 43% (p < 0.05) in cocaine membranes. Na(+)-independent [3H]-arginine transport (10 microM), however, did not differ between control or cocaine-treated groups. In summary, chronic cocaine administration selectively inhibited the transport of glutamine and MeAIB into apical membrane vesicles, but had minimal effect on arginine transport. We postulate that this diminution in uptake may contribute to the fetal growth retardation noted in our model.

In Situ Transplantation of Alginate Bioencapsulated Adipose Tissues Derived Stem Cells (ADSCs) via Hepatic Injection in a Mouse Model

Objective Adipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model. Methods The ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two). Results Our results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.

The pancreatic tumor microenvironment drives changes in miRNA expression that promote cytokine production and inhibit migration by the tumor associated stroma

The pancreatic adenocarcinoma (PDAC) microenvironment is largely comprised of fibrotic tumor associated stroma (TAS) that contributes to the lethal biology of PDAC. microRNA (miRNA) are small non-coding RNAs that regulate gene expression. We hypothesized that interactions between PDAC cells and TAS cells within the microenvironment modulate miRNA expression and thus, tumor biology. We observed that miR-205 and members of the miR-200 family (miR-200a, -200b, -200c, -141 and miR-429) were exclusively expressed in PDAC cells, consistent with an epithelial miRNA signature, while miR-145 and miR-199 family members (miR-199a and -199b) were solely expressed in TAS cells, consistent with a stromal miRNA signature. This finding was confirmed by qRT-PCR of RNA obtained by laser-capture microdissection of surgical specimens. Using an in vitro co-culture model, we further demonstrated regulation of miRNA expression by cell-cell contact. Forced expression in TAS cells of miR-200b/-200c and miR-205 to mimic these observed changes in miRNA concentrations induced secretion of GM-CSF and IP10, and notably inhibited migration. These data suggest interactions within the tumor microenvironment alter miRNA expression, which in turn have a functional impact on TAS.

Intestinal microbiota enhances pancreatic carcinogenesis in preclinical models

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States yet data are scant regarding host factors influencing pancreatic carcinogenesis. Increasing evidence support the role of the host microbiota in carcinogenesis but its role in PDAC is not well established. Herein, we report that antibiotic-mediated microbial depletion of KrasG12D/PTENlox/+ mice showed a decreased proportion of poorly differentiated tumors compared to microbiota-intact KrasG12D/PTENlox/+ mice. Subsequent 16S rRNA PCR showed that ~50% of KrasG12D/PTENlox/+ mice with PDAC harbored intrapancreatic bacteria. To determine if a similar observation in humans correlates with presence of PDAC, benign and malignant human pancreatic surgical specimens demonstrated a microbiota by 16S bacterial sequencing and culture confirmation. However, the microbial composition did not differentiate PDAC from non-PDAC tissue. Furthermore, murine pancreas did not naturally acquire a pancreatic microbiota, as germ-free mice transferred to specific pathogen-free housing failed to acquire intrapancreatic bacteria over time, which was not augmented by a murine model of colitis. Finally, antibiotic-mediated microbial depletion of Nod-SCID mice, compared to microbiota-intact, showed increased time to PDAC xenograft formation, smaller tumors, and attenuated growth. Interestingly, both xenograft cohorts were devoid of intratumoral bacteria by 16S rRNA PCR, suggesting that intrapancreatic/intratumoral microbiota is not the sole driver of PDAC acceleration. Xenografts from microbiota-intact mice demonstrated innate immune suppression by immunohistochemistry and differential regulation of oncogenic pathways as determined by RNA sequencing. Our work supports a long-distance role of the intestinal microbiota on PDAC progression and opens new research avenues regarding pancreatic carcinogenesis.

Abstract 4322: Exosomal delivery of stroma-derived miR-145 inhibits pancreatic cancer cell proliferation

We previously reported that within the pancreatic ductal adenocarcinoma (PDAC) microenvironment, miR-145 and miR-199a are exclusively expressed in tumor-associated stroma (TAS) cells, but these miRNAs are present in PDAC cells following co-culture with TAS cells. We hypothesized that miRNAs function as paracrine signals via exosomal exchange between TAS cells and adjacent PDAC cells. Primary cultures of human TAS and PDAC cells were employed. Membrane-bound microparticles were isolated from TAS conditioned, serum-free culture media by sequential ultracentrifugation followed by ultrafiltration. Exosomes and microvesicles were then assayed for particle size distribution using nanoparticle tracking analysis and electronic microscopy. miRNA expression levels were determined using quantitative PCR. miRNA transfection was performed with RNAiMax reagents. Cell viability was measured by Alamar Blue. Statistics were performed using Prism 6 software. Following transfection of human TAS cells with cel-miR-39, a nonhuman miRNA, we demonstrated that miRNA exchanges occurred between TAS cells and neighboring PDAC cells via a process that is not dependent upon cell-cell contact. We next confirmed the presence and enrichment of miR-145-5p in TAS-cell-derived exosomes (8-fold higher concentrations in exosomes than parental cells, p Taken together, our data suggest that stroma derived exosomes deliver miRNAs to adjacent PDAC cells and may function as tumor-suppressing paracrine signals in the case of miR-145. This finding provides a potential explanation for the observation that stroma depletion paradoxically accelerates PDAC progression in murine models. Citation Format: Song Han, Sayali Belsare, DongYu Zhang, Mark Beveridge, Carlos Rinaldi, Jose G. Trevino, Thomas D. Schmittgen, Steven J. Hughes. Exosomal delivery of stroma-derived miR-145 inhibits pancreatic cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2017-4322