MS1 Cells Research Articles

A biomimetic skin microtissue biosensor for the detection of fish parvalbumin

In this paper, a biomimetic skin microtissue biosensor was developed based on three-dimensional (3D) bioprinting to precisely and accurately determine fish parvalbumin (FV). Based on the principle that allergens stimulate cells to produce ONOO− (peroxynitrite anion), a screen-printed electrode for the detection nanomolar level ONOO− was innovatively prepared to indirectly detect FV based on the level of ONOO− release. Gelatin methacryloyl (GelMA), RBL-2H3 cells, and MS1 cells were used as bio-ink for 3D bioprinting. The high-throughput and standardized preparation of skin microtissue was achieved using stereolithography 3D bioprinting technology. The printed skin microtissues were put into the self-designed 3D platform that integrated cell culture and electrochemical detection. The experimental results showed that the sensor could effectively detect FV when the optimized ratio of RBL-2H3 to MS1 cells and allergen stimulation time were 2:8 and 2 h, respectively. The linear detection range was 0.125–3.0 μg/mL, and the calculated lowest detection limit was 0.122 μg/mL. In addition, the sensor had excellent selectivity, specificity, stability, and reliability. Thus, this study successfully constructed a biomimetic skin microtissue electrochemical sensor for PV detection.

Synthesis and Evaluation of Clinically Translatable Targeted Microbubbles Using a Microfluidic Device for In Vivo Ultrasound Molecular Imaging.

The main aim of this study is to synthesize contrast microbubbles (MB) functionalized with engineered protein ligands using a microfluidic device to target breast cancer specific vascular B7-H3 receptor in vivo for diagnostic ultrasound imaging. We used a high-affinity affibody (ABY) selected against human/mouse B7-H3 receptor for engineering targeted MBs (TMBs). We introduced a C-terminal cysteine residue to this ABY ligand for facilitating site-specific conjugation to DSPE-PEG-2K-maleimide (M. Wt = 2.9416 kDa) phospholipid for MB formulation. We optimized the reaction conditions of bioconjugations and applied it for microfluidic based synthesis of TMBs using DSPE-PEG-ABY and DPPC liposomes (5:95 mole %). The binding affinity of TMBs to B7-H3 (MBB7-H3) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1B7-H3) by flow chamber assay, and by ex vivo in the mammary tumors of a transgenic mouse model (FVB/N-Tg (MMTV-PyMT)634Mul/J), expressing murine B7-H3 in the vascular endothelial cells by immunostaining analyses. We successfully optimized the conditions needed for generating TMBs using a microfluidic system. The synthesized MBs showed higher affinity to MS1 cells engineered to express higher level of hB7-H3, and in the endothelial cells of mouse tumor tissue upon injecting TMBs in a live animal. The average number (mean ± SD) of MBB7-H3 binding to MS1B7-H3 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1WT; 36.2 ± 7.5/FOV). The non-targeted MBs did not show any selective binding affinity to both the cells (37.7 ± 7.8/FOV for MS1B7-H3 and 28.3 ± 6.7/FOV for MS1WT cells). The fluorescently labeled MBB7-H3 upon systemic injection in vivo co-localized to tumor vessels, expressing B7-H3 receptor, as validated by ex vivo immunofluorescence analyses. We have successfully synthesized a novel MBB7-H3 via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MBB7-H3 showed significant binding affinity to vascular endothelial cells expressing B7-H3 both in vitro and in vivo, which shows its potential for clinical translation as a molecular ultrasound contrast agent for human applications.

Dapagliflozin improves pancreatic islet function by attenuating microvascular endothelial dysfunction in type 2 diabetes.

Sodium-glucose co-transporter 2 inhibitors, including dapagliflozin, improve ß cell function in type 2 diabetic individuals. Whether dapagliflozin can protect islet microvascular endothelial cells (IMECs) and thus contribute to the improvement of ß cell function remains unknown. The db/db mice were treated with dapagliflozin or vehicle for 6weeks. ß cell function, islet capillaries and the levels of inflammatory chemokines in IMECs were detected. The mouse IMEC cell line MS-1 cells were incubated with palmitate and/or dapagliflozin for 24h. Angiogenesis and inflammatory chemokine levels were evaluated, and the involved signalling pathways were analysed. The mouse ß cell line MIN6 cells, in the presence or absence of co-culture with MS-1 cells, were treated with palmitate and/or dapagliflozin for 24h. The expression of ß cell specific markers and insulin secretion in MIN6 cells were determined. Dapagliflozin significantly improved ß cell function, increased islet capillaries and decreased the levels of inflammatory chemokines of IMECs in db/db mice. In the palmitate-treated MS-1 cells, angiogenesis was enhanced and the levels of inflammatory chemokines were downregulated by dapagliflozin. Either a PI3K inhibitor or mTOR inhibitor eliminated the dapagliflozin-mediated effects. Importantly, dapagliflozin attenuated the palmitate-induced downregulation of ß cell function-related gene expression and insulin secretion in MIN6 cells co-cultured with MS-1 cells but not in those on mono-culture. Dapagliflozin restores islet vascularisation and attenuates the inflammation of IMECs in type 2 diabetic mice. The dapagliflozin-induced improvement of ß cell function is at least partially accounted for by its beneficial effects on IMECs in a PI3K/Akt-mTOR-dependent manner.

RNA binding protein HuD mediates the crosstalk between β cells and islet endothelial cells by the regulation of Endostatin and Serpin E1 expression

RNA binding protein HuD plays essential roles in gene expression by regulating RNA metabolism, and its dysregulation is involved in the pathogenesis of several diseases, including tumors, neurodegenerative diseases, and diabetes. Here, we explored HuD-mediated differential expression of secretory proteins in mouse insulinoma βTC6 cells using a cytokine array. Endostatin and Serpin E1 that play anti-angiogenic roles were identified as differentially expressed proteins by HuD. HuD knockdown increased the expression of α chain of collagen XVIII (Col18a1), a precursor form of endostatin, and Serpin E1 by associating with the 3′-untranslated regions (UTRs) of Col18a1 and Serpin E1 mRNAs. Reporter analysis revealed that HuD knockdown increased the translation of EGFP reporters containing 3′UTRs of Col18a1 and Serpin E1 mRNAs, which suggests the role of HuD as a translational repressor. Co-cultures of βTC6 cells and pancreatic islet endothelial MS1 cells were used to assess the crosstalk between β cells and islet endothelial cells, and the results showed that HuD downregulation in βTC6 cells inhibited the growth and migration of MS1 cells. Ectopic expression of HuD decreased Col18a1 and Serpin E1 expression, while increasing the markers of islet vascular cells in the pancreas of db/db mice. Taken together, these results suggest that HuD has the potential to regulate the crosstalk between β cells and islet endothelial cells by regulating Endostatin and Serpin E1 expression, thereby contributing to the maintenance of homeostasis in the islet microenvironment.

Attenuation of Pancreatic Cancer In Vitro and In Vivo via Modulation of Nrf2 and NF-κB Signaling Pathways by Natural Compounds.

Pancreatic cancer is a disease in which deregulation of signaling pathways plays a key role, thus searching for their novel modulators is a promising therapeutic strategy. Hence, in this study, the effect of phytochemical combinations on the canonical and non-canonical activation of Nrf2 and its interaction with the NF-κB pathway was evaluated in extensively proliferating pancreatic cancer cell line, PSN-1, in comparison to non-cancerous MS1 cells. The activation of Nrf2 and NF-κB, expression of their target genes, and effect on cell survival were assessed in PSN-1 cells. The tumor burden was evaluated in mice carrying xenografts. PSN-1 cells were more sensitive to the tested compounds as compared to the MS1 cell line. Combination of xanthohumol and phenethyl isothiocyanate was more effective than single compounds at decreasing the canonical and non-canonical activation of Nrf2 in PSN-1 cancer cells. Decreased activation of NF-κB, and subsequent reduced cytosolic COX-2 and nuclear STAT3 level indicated their anti-inflammatory and pro-apoptotic activities. In vivo studies showed the partial response in groups treated with xanthohumol or the combination of xanthohumol and phenethyl isothiocyanate. Overall, these results suggest that the combination of xanthohumol and phenethyl isothiocyanate may be a promising therapeutic candidate against pancreatic cancer.

1238-P: The Proinflammatory but Not Cytotoxic Effect of Aggregated IAPP on Islet Endothelial Cells Requires TLR2/4 Signaling

We have found that aggregation of the β-cell peptide human islet amyloid polypeptide (hIAPP) causes inflammation and toxicity in islet endothelial cells (IECs), which likely contributes to the islet capillary abnormalities seen in T2D. Based on data from other cell types where hIAPP stimulates the innate immune response, we hypothesized that TLR2/4 signaling is required for the effect of hIAPP on IECs. To test this, we treated MS-1 cells (an immortalized murine IEC cell line) with vehicle control, hIAPP ± the TLR2/4 inhibitor OxPAPc, or, in parallel, the TLR2/TLR4 agonists PAM3CSK4 or LPS for 24h. Cell viability and gene expression were determined. hIAPP resulted in increased levels of inflammatory markers and loss of cell viability (Table). Treatment with the TLR2/4 inhibitor blocked upregulation of Il6 and Vcam1 but not Edn1 expression and did not improve cell viability (Table). Conversely, treatment of IECs with TLR2/4 ligands led to upregulation of Il6 and Vcam1 but not Edn1 expression and did not recapitulate the decrease in cell viability induced by hIAPP (Table). We conclude that TLR2/4 signaling is necessary for the hIAPP-induced proinflammatory response in IECs. In contrast, hIAPP-induced cell death does not require TLR2/4 activation but may contribute to the innate immune response by stimulating release of damage associated molecular patterns and, thereby, inducing TLR signaling.View largeDownload slideView largeDownload slide DisclosureJ. J. Castillo: None. A. Aplin: None. M. F. Hogan: None. A. T. Templin: None. N. Esser: None. R. Akter: None. R. L. Hull: None.FundingNational Institutes of Health (R01DK088082); U.S. Department of Veterans Affairs (BX004063)

TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-β), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-β-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-β-induced EndMT. Using these techniques, we provide evidence that TGF-β2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-β-Induced Endothelial to Mesenchymal Transition Is Determined by a Balance Between SNAIL and ID Factors.

Endothelial-to-mesenchymal transition (EndMT) plays an important role in embryonic development and disease progression. Yet, how different members of the transforming growth factor-β (TGF-β) family regulate EndMT is not well understood. In the current study, we report that TGF-β2, but not bone morphogenetic protein (BMP)9, triggers EndMT in murine endothelial MS-1 and 2H11 cells. TGF-β2 strongly upregulates the transcription factor SNAIL, and the depletion of Snail is sufficient to abrogate TGF-β2-triggered mesenchymal-like cell morphology acquisition and EndMT-related molecular changes. Although SLUG is not regulated by TGF-β2, knocking out Slug also partly inhibits TGF-β2-induced EndMT in 2H11 cells. Interestingly, in addition to SNAIL and SLUG, BMP9 stimulates inhibitor of DNA binding (ID) proteins. The suppression of Id1, Id2, or Id3 expression facilitated BMP9 in inducing EndMT and, in contrast, ectopic expression of ID1, ID2, or ID3 abrogated TGF-β2-mediated EndMT. Altogether, our results show that SNAIL is critical and indispensable for TGF-β2-mediated EndMT. Although SLUG is also involved in the EndMT process, it plays less of a crucial role in it. In contrast, ID proteins are essential for maintaining endothelial traits and repressing the function of SNAIL and SLUG during the EndMT process. These data suggest that the control over endothelial vs. mesenchymal cell states is determined, at least in part, by a balance between the expression of SNAIL/SLUG and ID proteins.

Controlled 3D co-culture of beta cells and endothelial cells in a micropatterned collagen sheet for reproducible construction of an improved pancreatic pseudo-tissue.

The co-culture of beta cells and endothelial cells in constructing a pancreatic pseudo-tissue can provide a functional advancement for in vitro diabetic-related drug testing and biological studies or in vivo transplantation. In order to mimic the pancreatic tissue more similar to in vivo, it is necessary to control the microenvironment, including cell–cell and cell–extracellular matrix interactions. In this study, we report a geometrically controlled three-dimensional (3D) pancreatic model where MIN6 and MS1 cells are co-cultured within a micropatterned collagen sheet. In 4–10 days, depending on the cell seeding concentration, the MIN6 cells formed islet-like clusters surrounded by an endothelial MS1 cell monolayer. The MS1 cells also formed monolayers at the edge of the micropatterns connecting between the clusters, resulting in a blood vessel-like structure in which no cells were found. It was confirmed that the 3D co-culture structure was not formed in a non-patterned sheet and the structure also helped insulin secretion of MIN6 cells. By simply embedding the cell mixture and the hexagonal micropattern into the collagen sheet, we were also able to achieve the highly reproducible fabrication of a 3D pancreatic pseudo-tissue construct for in vivo and in vitro applications.

Toward the Clinical Development and Validation of a Thy1-Targeted Ultrasound Contrast Agent for the Early Detection of Pancreatic Ductal Adenocarcinoma.

We recombinantly expressed the Thy1-scFv with a carboxy-terminus cysteine residue to facilitate its thioether conjugation to the PEGylated MBs presenting with maleimide functional groups. After the scFv-MB conjugations, we tested binding activity of the MBThy1-scFv to MS1 cells overexpressing human Thy1 (MS1Thy1) under liquid shear stress conditions in vitro using a flow chamber setup at 0.6 mL/min flow rate, corresponding to a wall shear stress rate of 100 seconds, similar to that in tumor capillaries. For in vivo Thy1 US molecular imaging, MBThy1-scFv was tested in the transgenic mouse model (C57BL/6J - Pdx1-Cre; KRas; Ink4a/Arf) of PDAC and in control mice (C57BL/6J) with L-arginine-induced pancreatitis or normal pancreas. To facilitate its clinical feasibility, we further produced Thy1-scFv without the bacterial fusion tags and confirmed its recognition of human Thy1 in cell lines by flow cytometry and in patient PDAC frozen tissue sections of different clinical grades by immunofluorescence staining. Under shear stress flow conditions in vitro, MBThy1-scFv bound to MS1Thy1 cells at significantly higher numbers (3.0 ± 0.8 MB/cell; P < 0.01) compared with MBNontargeted (0.5 ± 0.5 MB/cell). In vivo, MBThy1-scFv (5.3 ± 1.9 arbitrary units [a.u.]) but not the MBNontargeted (1.2 ± 1.0 a.u.) produced high US molecular imaging signal (4.4-fold vs MBNontargeted; n = 8; P < 0.01) in the transgenic mice with spontaneous PDAC tumors (2-6 mm). Imaging signal from mice with L-arginine-induced pancreatitis (n = 8) or normal pancreas (n = 3) were not significantly different between the two MB constructs and were significantly lower than PDAC Thy1 molecular signal. Clinical-grade scFv conjugated to Alexa Fluor 647 dye recognized MS1Thy1 cells but not the parental wild-type cells as evaluated by flow cytometry. More importantly, scFv showed highly specific binding to VEGFR2-positive vasculature and fibroblast-like stromal components surrounding the ducts of human PDAC tissues as evaluated by confocal microscopy. Our findings summarize the development and validation of a clinically relevant Thy1-targeted US contrast agent for the early detection of human PDAC by US molecular imaging.

2043-P: Hyaluronan Produced by Inflamed Islet Endothelial Cells Contributes to HIAPP Aggregation

In type 2 diabetes (T2D), aggregation of human islet amyloid polypeptide (hIAPP) occurs within the islet extracellular matrix (ECM) and is toxic to β cells. We recently showed that hIAPP aggregation also induces a pro-inflammatory response in islet endothelial cells (ECs). In most tissues, this inflammatory response in ECs is mediated by upregulating production of ECM components, such as hyaluronan (HA). Indeed, we find that HA accumulates in the islet ECM of hIAPP transgenic mice, where it was colocalized with amyloid deposits, while no HA accumulation occurred in non-transgenic littermate controls (which do not develop islet amyloid). Therefore, we hypothesized that hIAPP aggregation results in HA production from islet ECs, and that this HA production exacerbates hIAPP deposition/toxicity. To test this hypothesis, we utilized immortalized murine islet ECs (MS-1 cells), which under normal conditions express low levels of HA synthase (Has isoforms 2 and 3). Treatment of these cells for 48 h with hIAPP (20 µM) increased Has2 and Has3 mRNA levels (23±4 and 8±1 fold over vehicle; n=4, p&amp;lt;0.05 for both), which was abrogated upon co-incubation with Congo red, an inhibitor of hIAPP aggregation (1.4±0.3 and 1.4±0.2 for Has2 and Has3, respectively; n=4). Next, we used a thioflavin T assay, wherein fluorescence is proportional to fibril formation from amyloidogenic peptides, to determine the kinetics of hIAPP aggregation ± HA. We found co-incubation of HA with synthetic hIAPP peptide resulted in a marked acceleration (log time of 1 vs. 40 hours) and increase in maximal (greater than 2-fold) hIAPP accretion. In sum, exposure of islet ECs to aggregated hIAPP increases HA synthesis by these cells, which in turn further exacerbates aggregation of hIAPP to form amyloid. Thus, interventions targeting endothelial cell inflammation and/or HA production may be useful in blocking amyloid formation and its toxic effects in T2D. Disclosure J.J. Castillo: None. M.F. Hogan: None. A. Aplin: None. N. Esser: None. A.T. Templin: None. R.L. Hull: None. Funding National Institutes of Health (T32HL007028)

Molecular mechanism of β-1, 4-galactosyltransferase 6 in regulating lymphocyte migration

Objective To study the molecular mechanism of β-1, 4-galactosyltransferase 6 (β4galt6) in regulating lymphocyte migration under inflammatory conditions. Methods CRISPR/Cas9 system was used to knock out the β4galt6 gene of mouse islet vascular endothelial cells (MS1). Adhesion assay was performed to compare the adhesion ability of lymphocytes to wild-type cells and gene knockout cells. Expression of adhesion molecules on the surface of wild-type cells and gene knockout cells were compared using RT-PCR and flow cytometry. Transwell model was used to compare the transmigration ability of lymphocytes across wild-type cells and gene knockout cells. Results The β4galt6 gene knockout cell line, β4galt6 KO, was successfully constructed. The percentage of lymphocytes adhereing to wild-type MS1 cells was significantly higher than that to β4galt6 KO cells under inflammatory conditions. The expression of adhesion molecules including intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1) and P-selectin on the surface of wild-type MS1 cells was significantly higher than that on β4galt6 KO cells. Moreover, the percentage of lymphocytes passing through wild-type MS1 cells was significantly higher than that through β4galt6 KO cells. Conclusion Under inflammatory conditions, β4galt6 could promote the migration of lymphocytes. Key words: β-1, 4-galactosyltransferase 6; Lymphocyte; Migration; Inflammation

Transcriptome Analysis of the Ammonia-Oxidizing Bacterium Nitrosomonas mobilis Ms1 Reveals Division of Labor between Aggregates and Free-living Cells.

Bacteria change their metabolic states to increase survival by forming aggregates. Ammonia-oxidizing bacteria also form aggregates in response to environmental stresses. Nitrosomonas mobilis, an ammonia-oxidizing bacterium with high stress tolerance, often forms aggregates mainly in wastewater treatment systems. Despite the high frequency of aggregate formation by N. mobilis, its relationship with survival currently remains unclear. In the present study, aggregates were formed in the late stage of culture with the accumulation of nitrite as a growth inhibitor. To clarify the significance of aggregate formation in N. mobilis Ms1, a transcriptome analysis was performed. Comparisons of the early and late stages of culture revealed that the expression of stress response genes (chaperones and proteases) increased in the early stage. Aggregate formation may lead to stress avoidance because stress response genes were not up-regulated in the late stage of culture during which aggregates formed. Furthermore, comparisons of free-living cells with aggregates in the early stage of culture showed differences in gene expression related to biosynthesis (ATP synthase and ribosomal proteins) and motility and adhesion (flagella, pilus, and chemotaxis). Biosynthesis genes for growth were up-regulated in free-living cells, while motility and adhesion genes for adaptation were up-regulated in aggregates. These results indicate that N. mobilis Ms1 cells adapt to an unfavorable environment and grow through the division of labor between aggregates and free-living cells.

Liraglutide ameliorates palmitate-induced oxidative injury in islet microvascular endothelial cells through GLP-1 receptor/PKA and GTPCH1/eNOS signaling pathways

In type 2 diabetes, lipotoxicity damages islet microvascular endothelial cells (IMECs), leading to pancreatic islet β cell dysfunction directly or indirectly. Glucagon-like peptide-1 (GLP-1) and its analogs have beneficial roles in endothelial cells. However, the protective effects of GLP-1 agents on IMECs and their potential mechanism remained obscure. In this study, exposure of MS-1 (a cell line derived from mouse IMECs) to different concentrations of palmitic acid (PA) was used to establish an injury model. The cells exposed to PA (0.25 mmol/L) were treated with a GLP-1 analog liraglutide (3, 10, 30, and 100 nmol/L). Reactive oxygen species (ROS) generation, apoptosis-related protein level, and endothelin-1 production were detected. The protein levels of signaling molecules were analyzed and specific inhibitors or blockers were used to identify involvement of signaling pathways in the effects of liraglutide. Results showed that PA significantly increased ROS generation and the levels of pro-apoptotic protein Bax, and decreased the levels of anti-apoptotic protein Bcl-2 and the mRNA expression and secretion of endothelin-1. Meanwhile, PA downregulated the protein levels of GLP-1 receptor (GLP-1R), phosphorylated protein kinase A (PKA), guanosine 5′-triphosphate cyclohydrolase 1 (GTPCH1), and endothelial nitric oxide synthase (eNOS). Furthermore, liraglutide ameliorated all these effects of PA in a dose-dependent manner. Importantly, GLP-1R antagonist exendin (9–39), PKA inhibitor H89, GTPCH1 inhibitor 2,4-diamino-6-hydroxypyrimidine, or NOS inhibitor N-nitro-l-arginine-methyl ester abolished the liraglutide-mediated amelioration in PA-impaired MS-1 cells. In conclusion, liraglutide ameliorates the PA-induced oxidative stress, apoptosis, and endothelin-1 secretion dysfunction in mouse IMECs through GLP-1R/PKA and GTPCH1/eNOS signaling pathways.

Leukemia-Associated HSC Vascular Niche Is Negatively Regulated By PERK of Unfolded Protein Response (UPR)

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived from early T cell progenitors. Diffuse infiltration of the bone marrow by T-ALL is associated with worse prognosis. We previously reported that actively proliferating leukemia cells inhibit normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. We found that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. However, the underlying molecular mechanism that leads to the suppression of hematopoiesis and decreased HSPC in the vascular niche is unclear. It has been demonstrated that rapid cellular proliferation associated with oncogenic activity such as MYC in T-ALL leads to a global increase in protein synthesis and an increase in misfolded/unfolded polypeptides in the endoplasmic reticulum (ER), referred to as unfolded protein response (UPR) or ER stress. Elevated ER stress leads to activation of at least three types of ER stress transducers through the release of inhibitory binding by glucose-regulated chaperone protein (GRP78/BIP): the protein kinase RNA-like ER kinase (PERK), the inositol-requiring enzyme 1 (IRE1), and the activating transcription factor 6 (ATF6). Activation of PERK phosphorylates eIF2 to repress global translation with the exception of a small number of proteins including ATF4 (activating transcription factor-4). ATF4 regulates genes involved in restoring ER homeostasis and genes in apoptosis. Here, we studied the role of UPR in the regulation of HSC niche function in the setting of T-ALL progression. Using in vitro assays in which T-ALL leukemia cells driven by activated Notch1 (ICN1) were co-cultured with endothelial cells (MILE SVEN 1, MS1), and in vivo ICN1-driven T-ALL model, we found that PERK-eIF2a-ATF4 pathway was activated in both MS1 cells and BM endothelial cells isolated from T-ALL mice, while IRE1 and ATF6 pathways were only mildly altered. The activation of PERK was accompanied with the increased expression of Jagged1 and suppressed expression of CXCL12 in both cultured endothelial cells and bone marrow endothelial cells from leukemia mice. PERK inhibitor (GSK2606414) treatment of co-cultured cells largely restored CXCL12 expression, which was also negatively regulated by Jagged1, and accelerated the leukemia cell apoptosis as indicated by the enhanced annexin staining. These findings suggest that PERK is the upstream regulator of Jagged1 and CXCL12 in the endothelial cells; however, the function of cell-autonomous PERK on leukemia cell survival needs to be further clarified. To understand the role of PERK in bone marrow endothelium during leukemia development in vivo, we examined T-ALL leukemia progression and its effect on vascular niche function in VE-CadherinERT2/PERKF/F mice in which Perk was specifically deleted in endothelial cells. Consistent with in vitro findings, T-ALL development induced endothelial PERK-eIF2a-ATF4 activation, while up-regulated Jagged1 and down-regulated CXCL12 were also identified in isolated BM endothelial cells. Compared to the wild type mice, VE-CadherinERT2/PERKF/F mice showed attenuated leukemia progression, increased HSPC (Lin-Sca-1+c-kit+) frequency, and improved survival. Taken together, our findings suggest that PERK activation in BM endothelial cells is a key regulator of the leukemia vascular niche to promote leukemia progression and to suppress normal hematopoiesis. Therefore, targeting PERK may offer an effective strategy in restoring normal HSPC homeostasis and limiting leukemia progression. Disclosures No relevant conflicts of interest to declare.

2132-P: Islet Amyloid Deposition Is Cytotoxic to Islet Endothelial Cells

Islet endothelial cells [ECs] produce numerous factors that support normal β-cell function and survival. Under diabetic conditions, islet ECs develop an activated (pro-inflammatory, pro-adhesive) phenotype which contributes to β-cell failure. In human type 2 diabetes [T2D], aggregation of the β-cell product, islet amyloid polypeptide [IAPP], results in amyloid deposition in the islet perivascular space. The toxic effects of IAPP aggregation on the β-cell are well established, however, whether islet ECs are also susceptible to IAPP toxicity remains unknown. We hypothesized that aggregation of amyloidogenic human IAPP (hIAPP) contributes to islet EC activation in T2D. To test this hypothesis, we performed 24h treatments of primary rat islet ECs with hIAPP [0-20 µM] for 24h, along with non-amyloidogenic rat IAPP [rIAPP; 20 µM] as a control, and, subsequently, the MS1 islet EC line with hIAPP [0-20 µM] ± the amyloid inhibitor Congo Red [CR; 100 µM]. In primary islet ECs, hIAPP, but not rIAPP, reduced cell viability and increased activation markers (interleukin-6, endothelin-1, vascular cell adhesion molecule; Table). A similar effect was seen in MS1 cells, and this was abrogated with CR (Table). Our data suggest that islet ECs are susceptible to hIAPP-induced toxicity. The resulting EC activation may interrupt the cross talk between the EC and β-cell, and thereby contribute to the β-cell toxicity of IAPP in T2D. Disclosure J.J. Castillo: None. M.F. Hogan: None. A. Aplin: None. D.J. Hackney: None. R.L. Hull: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK088082); National Heart, Lung, and Blood Institute (T32HL007028); U.S. Department of Veterans Affairs (BX004063)

MicroRNA-127 inhibits cell proliferation via targeting Kif3b in pancreatic β cells.

MicroRNAs (miRNAs) have been implicated in β cells dysfunction. Previous studies indicated that miR-127 was specifically abundant in β cells and one of its target genes, Kif3b, promoted cell proliferation. However, the impact of the miR-127-Kif3b axis on β cells remains unknown. In this study, we revealed that miR-127 level was declined both in islets from the mice with a high-fat diet and in MIN6 cells with elevated glucose treatment. The elevated level of miR-127 attenuated β cell proliferation by repressing Kif3b expression without affecting apoptosis and cell cycle, and it dampened insulin secretion. Moreover, β cell-derived miR-127 could also affect the islet endothelial cell-line, MS1, in vitro via the transfer of extracellular vesicles (EVs). Treating MS1 cells with the EVs secreted by MIN6 cells exhibited a higher ability in cell migration and tube formation. However, this effect was abolished by the miR-127 inhibitor co-cultured with EVs-treated MS1 cells. Thus, we define that miR-127 is a crucial regulator of insulin secretion and cell proliferation in pancreatic β cells as well as a potential functional regulation factor in islet endothelial cells.

Role of carboxypeptidase E in promoting the migration of lymphocytes through vascular endothelial cells

Objective To study the mechanism of carboxypeptidase E (CPE) in promoting the migration of lymphocytes and their subsets through vascular endothelial cells. Methods CRISPR/Cas9 technology was used to prepare cpe gene-knockout MS1 (Cpe-/-MS1) cells. Adhesion ability of lymphocytes to MS1 and Cpe-/-MS1 cells was analyzed with adhesion assay. Expression of adhesion molecules on these cells were detected by RT-PCR and flow cytometry. Transwell model was used to compare the difference in the transmigration of lymphocytes and their subsets through MS1 and Cpe-/-MS1 cells. Results Cpe-/-MS1 cells were successfully obtained. Under the stimulation of TNF-α, the adhesion ability of lymphocytes to MS1 cells was much better than that of Cpe-/-MS1 cells. Moreover, adhesion molecules expressed on MS1 cells were significantly more than those on Cpe-/-MS1 cells. The percentages of lymphocytes and their subsets that transmigrated through MS1 cells were significantly higher than those through Cpe-/-MS1 cells. Conclusion CPE involved in the adhesion of lymphocytes to vascular endothelial cells and the transmigration of them through vascular endothelial cells, which was of great significance for understanding the migration of lymphocytes across vascular endothelial cells to peripheral lymph nodes. Key words: Carboxypeptidase E; Lymphocyte; Vascular endothelial cell; Transmigration

Nuclear FAK and its kinase activity regulate VEGFR2 transcription in angiogenesis of adult mice

Focal adhesion kinase (FAK) is essential in embryonic angiogenesis by regulating endothelial cell (EC) survival and barrier functions through its kinase-independent and –dependent activities. Here, we generated EC-specific tamoxifen-inducible FAK knockout and FAK kinase-defective (KD) mutant knockin mice to investigate the role of FAK and its kinase activity in angiogenesis of adult animals. Unlike previous observations of their differential defects in embryonic vascular development, both FAK ablation and inactivation of its kinase activity resulted in deficient angiogenesis in wound-healing as well as retinal angiogenesis models. Consistent with these phenotypes, loss of FAK or its kinase activity decreased EC proliferation and migration to similar extents, suggesting FAK primarily acts as a kinase for the regulation of adult EC-mediated angiogenesis. Further mechanistic analyses were carried out using an established mouse EC line MS1 cells. Interestingly, we found that FAK regulated the expression of VEGFR2, a central mediator of various EC functions and angiogenesis, which requires both FAK kinase activity and its translocation into the nucleus. Moreover, nuclear FAK was detected in the RNA polymerase II complex associated with VEGFR2 promoter, suggesting its direct participation in the transcriptional regulation of VEGFR2. Together, our results provide significant insights into the signaling mechanisms of FAK in angiogenesis that may contribute to future design of more effective angiogenesis related therapy.

Bioconverted Orostachys japonicas Extracts Suppress Angiogenic Activity of Ms-1 Endothelial Cells.

Orostachys japonicus A. Berger (), known as Wa-song in Korea, has been reported to exert various biological effects, such as anti-tumor, anti-oxidant, and anti-febrile effects. However, the anti-angiogenic effects of O. japonicus extracts remain to be investigated. In the present study, we demonstrated the anti-angiogenic effects of bioconverted O. japonicus extract (BOE) in Ms-1 mouse endothelial cells and compared them with the bioactivities of O. japonicus extract (OE). BOE, but not OE, were found to exert anti-angiogenic effects, including inhibition of cell migration, cell adhesion, tube formation of Ms-1 cells, and blood vessel formation of matrigel plug assay in vivo. Furthermore, protein levels of phosphorylated Src kinase were lower in BOE-treated cells than in OE-treated cells. Treatment with OE or BOE did not influence cell viability during the experimental period. Bioconverted extract of O. japonicus have anti-angiogenic effects in vitro and vivo, but non-bioconverted extract do not. We suggest that these observed anti-angiogenic effects are caused by the changes in the composition of bioactive compounds in the extracts as a result of biological conversion.