Boyden Chamber Migration Assay Research Articles

Inhibition of microRNA-181c-5p rescues diabetes-impaired angiogenesis through activation of key mediators of angiogenesis, cell survival and novel genes, elmo3 and trib1

Abstract Introduction Patients with diabetes have impaired angiogenesis and poor coronary collateral vessel formation post-myocardial infarction (MI), which associates with higher mortality. There is a significant unmet clinical need for new agents that stimulate angiogenesis in response to ischaemia in diabetes. We have identified that miR-181c-5p has anti-angiogenic properties, but it’s role in diabetes remains unknown. Aim To elucidate the role of miR-181c-5p in diabetes-impaired angiogenesis. Methods Human coronary artery endothelial cells were transfected with a miR-181c-5p inhibitor (antimiR-181c-5p) or negative control (antimiR-Neg), exposed to glucose (5–25mM, 48h) then underwent Matrigel tubulogenesis assay or Boyden Chamber migration assay. Levels of proteins important for angiogenesis (e.g., VEGFA, p-ERK2, p-eNOS) were determined by Western Blot. Whole transcriptome sequencing was performed in vitro to identify novel gene targets of miR-181c-5p. In vivo, diabetic mice underwent hindlimb ischaemia or wound healing surgery and were injected with antimiR-181c-5p or antimiR-Neg. Tissues were extracted early (day 3) and late (days 10-14) post-surgery. Hindlimb blood-flow reperfusion was measured by Laser Doppler imaging. Hindlimb apoptosis was assessed by TUNEL and necrotic area was assessed by H&E. Wound area was calculated daily. Neovascularisation was assessed by CD31 (capillaries) and α-actin (arterioles) immunostaining. Results Inhibition of miR-181c-5p increased endothelial tubule number (+28%, P<0.01), tubule length (+12%, P<0.01) and cell migration (+67%, P<0.05). This associated with increased VEGFA (+21%, P<0.05) and p-ERK2 (+32%, P<0.05). Whole transcriptome and pathway analysis revealed changes to angiogenesis pathways and identified a first-time involvement of genes Elmo3 and Trib1 in the pro-angiogenic action of antimiR-181c-5p in diabetes. In vivo, inhibition of miR-181c-5p increased blood flow reperfusion to ischaemic hindlimbs (+30%, P<0.001) and arteriolar density (+45%, P<0.05) in diabetic mice. Mechanistically, this was associated with early changes to mediators of angiogenesis, Erk2 mRNA (+35%, P<0.05), p-ERK2 (+35%, P<0.05) and Trib1 mRNA (+80%, P<0.05); cell survival, Bcl-2 mRNA (+44%, P<0.05); and late apoptotic clearance, Elmo3 mRNA (+57%, P<0.001). Furthermore, this was also associated with an increase in late stage hindlimb apoptosis (+94%, P<0.05) and reduced necrotic area (-90%, P<0.05) in diabetic mice. Inhibition of miR-181c-5p increased diabetic wound closure (+22%, P<0.01), wound capillaries (+61%, P<0.05), Bcl-2 mRNA (+52%, P<0.05) and Elmo3 mRNA (+50%, P<0.05) in diabetic wounds. Conclusions Inhibition of miR-181c-5p rescues diabetes-impaired angiogenesis by activation of angiogenesis and cell survival mediators, and through novel genes, Trib1 and Elmo3. Our findings have implications for a novel miRNA-based strategy that improves myocardial neovascularisation and the prognosis of diabetic patients post-MI.

The ancestral type of the R-RAS protein has oncogenic potential.

The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

Altered Phenotypes of Breast Epithelial × Breast Cancer Hybrids after ZEB1 Knock-Out.

ZEB1 plays a pivotal role in epithelial-to-mesenchymal transition (EMT), (cancer) cell stemness and cancer therapy resistance. The M13HS tumor hybrids, which were derived from spontaneous fusion events between the M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg breast cancer cells, express ZEB1 and exhibit prospective cancer stem cell properties. To explore a possible correlation between the ZEB1 and stemness/ EMT-related properties in M13HS tumor hybrids, ZEB1 was knocked-out by CRISPR/Cas9. Colony formation, mammosphere formation, cell migration, invasion assays, flow cytometry and Western blot analyses were performed for the characterization of ZEB1 knock-out cells. The ZEB1 knock-out in M13HS tumor cells was not correlated with the down-regulation of the EMT-related markers N-CADHERIN (CDH2) and VIMENTIN and up-regulation of miR-200c-3p. Nonetheless, both the colony formation and mammosphere formation capacities of the M13HS ZEB1 knock-out cells were markedly reduced. Interestingly, the M13HS-2 ZEB1-KO cells harbored a markedly higher fraction of ALDH1-positive cells. The Transwell/ Boyden chamber migration assay data indicated a reduced migratory activity of the M13HS ZEB1-knock-out tumor hybrids, whereas in scratch/ wound-healing assays only the M13SH-8 ZEB1-knock-out cells possessed a reduced locomotory activity. Similarly, only the M13HS-8 ZEB1-knock-out tumor hybrids showed a reduced invasion capacity. Although the ZEB1 knock-out resulted in only moderate phenotypic changes, our data support the role of ZEB1 in EMT and stemness.

Gintonin-Induced Wound-Healing-Related Responses Involve Epidermal-Growth-Factor-like Effects in Keratinocytes.

Epidermal growth factor (EGF) receptor activation and related downstream signaling pathways are known to be one of the major mechanisms of the proliferation and migration of keratinocytes. The heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors and stimulates keratinocyte proliferation and migration. Gintonin, a novel ginseng compound, is a lysophosphatidic acid (LPA) receptor ligand. Gintonin has skin-wound-healing effects. However, the underlying mechanisms for these gintonin actions remain unclear. In this study, we aimed to elucidate the involvement of EGFRs in gintonin-induced wound repair in HaCaT keratinocytes. In this study, a water-soluble tetrazolium salt-based assay, a modified Boyden chamber migration assay, and immunoblotting were performed. Gintonin increased EGF receptor activation in HaCaT cells. However, the gintonin-induced phosphorylation of the EGF receptor was markedly reduced via treatment with the LPA inhibitor Ki16425 or the EGF receptor inhibitor erlotinib. Gintonin-enhanced proliferation and migration were blocked by the EGF receptor inhibitors (erlotinib and AG1478). Additionally, gintonin stimulated the expression and release of HB-EGF in HaCaT cells. EGF receptor inhibitors blocked gintonin-enhanced HB-EGF expression. These results indicate that the wound-healing effects of gintonin are closely related to the collaboration between EGF receptor activation and HB-EGF release-mediated downstream signaling pathways.

Abstract P3156: Activated Fibroblast-Derived Exosomal Mir-216a-5p Promotes Endothelial Dysfunction By Targeting PGM5 In Diabetic Heart

Background: Diabetic patients are more susceptible to heart failure (HF). High blood glucose level in diabetic patients eventually triggers the body's inflammatory response and causes cardiac fibroblast activation, endothelial cell dysfunction (ECD), and, ultimately, HF. Previously, we have shown that activated fibroblast-mediated ECD leads to HF. However, the molecular mechanism of fibroblast-induced ECD in diabetics is not yet well defined. Therefore, we hypothesized that “myofibroblast in the diabetic heart secretes exosomes loaded with antiangiogenic/profibrotic factors, which impair EC function.” Methods: Exosomes were isolated from diabetic mice plasma and fibroblast conditioned media by ultracentrifugation and characterized by nanosight & electron microscopy. We cultured mouse primary heart endothelial cells in EC growth media. ECs were treated with exosomes derived from fibroblasts (treated with either 25mM glucose, 500nM Angiotensin II (AngII) or both) for 48 hr. Mannitol (25mM) served as control. Pathway-based miRNA array was screened with exosome-derived miR, and targets were predicted bioinformatically for selected miR. Results: Glu-AngII cotreatment significantly activated fibroblasts as shown by qPCR (Col1α, and FN expression) and western blot (pSmad2/Smad2, p-p38/p38). Interestingly, exosomes derived from activated fibroblasts significantly induced ECD (eNOS, VEGF, CD31 genes, and proteins expression). We further checked the effect of diabetic mice plasma exosomes on ECs function. We found significantly impaired endothelial function, as shown by Matrigel tube formation and Boyden chamber migration assays. Micro RNA array and qPCR data showed that miR-216a-5p was highly upregulated in exosomes derived from FBs cotreated with Glu-AngII. Pathway-based analysis revealed that PGM5 could be a potential target in miR-216-induced ECD. Interestingly, miR-216a-5p inhibition significantly rescued diabetes-induced PGM5 inhibition and improved endothelial function Conclusions: Taken together, this study demonstrates that fibroblast in the diabetic heart releases miR-216a-5p through exosomes, which promote endothelial dysfunction via the miR-216a-5p/PGM5 axis.

Paeonol Promotes Reendothelialization After Vascular Injury Through Activation of c-Myc/VEGFR2 Signaling Pathway.

Dysfunction of endothelium is associated with multiple pathological vascular diseases. However, how to regulate reendothelialization after vascular injury is not well defined. This study aims to determine whether and how Paeonol controls reendothelialization following artery injury. The endothelium of murine carotid artery was denuded by catheter guide wires injury. H&E staining and IF staining were performed to determine whether Paeonol is critical for reendothelialization. BRDU Incorporation Assay, Boyden Chamber Migration Assay, Tube Formation Assay, and Spheroid Sprouting Assay were used to investigate whether Paeonol is involved in regulating proliferation and migration of endothelial cells. The underlying mechanism of how Paeonol regulates reendothelialization was determined by Molecular docking simulation and CO-IP Assay. Paeonol treatment significantly inhibits neointima formation in carotid artery ligation model by promoting proliferation and migration of endothelial cells. Mechanistically, Paeonol enhances c-Myc expression, consequently interacts with VEGFR2 results in activating VEGF signaling pathway, and eventually promotes reendothelialization after vascular injury. Our data demonstrated that Paeonol plays a critical role in regulating vascular reendothelialization, which may be therapeutically used for treatment of pathological vascular diseases.

Andrographolide suppresses hypoxia-induced embryonic hyaloid vascular system development through HIF-1a/VEGFR2 signaling pathway.

Ocular abnormalities and the development of retinal vasculature may cause postnatal retinopathy. In the past decade, tremendous progress has been made in identifying the mechanisms that regulate retina vasculature. However, the means of regulating embryonic hyaloid vasculature development is largely unknown. This study aims to determine whether and how andrographolide regulates embryonic hyaloid vasculature development. Murine embryonic retinas were used in this study. Whole mount isolectin B4 (IB4) staining, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and immunofluorescence staining (IF) were performed to determine whether andrographolide is critical for embryonic hyaloid vasculature development. BrdU incorporation assay, Boyden chamber migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay were performed to evaluate whether andrographolide regulates the proliferation and migration of vascular endothelial cells. Molecular docking simulation and Co-immunoprecipitation assay were used to observe protein interaction. Hypoxia conditions exist in murine embryonic retinas. Hypoxia induces HIF-1a expression; high-expressed HIF-1a interacts with VEGFR2, resulting in the activation of the VEGF signaling pathway. Andrographolide suppresses hypoxia-induced HIF-1a expression and, at least in part, interrupts the interaction between HIF-1a and VEGFR2, causing inhibiting endothelial proliferation and migration, eventually inhibiting embryonic hyaloid vasculature development. Our data demonstrated that andrographolide plays a critical role in regulating embryonic hyaloid vasculature development.

Andrographolide inhibits murine embryonic neuronal development through PFKFB3-mediated glycolytic pathway

Dysregulation of neuronal development may cause neurodevelopmental disorders. However, how to regulate embryonic neuronal development and whether this regulation can be medical interrupted are largely unknown. This study aimed to investigate whether and how andrographolide (ANP) regulates embryonic neuronal development. The pregnant mice at embryonic day 10.5 (E10.5) were administrated with ANP, and the embryonic brains were harvested at E17.5 or E18.5. Immunofluorescence (IF), Immunohistochemistry (IHC) performed to determine whether ANP is critical in regulating neuronal development. Real-time quantitative PCR, western blotting, cell counting kit-8 assay, Flow Cytometry assay, Boyden Chamber Migration assay carried out to evaluate whether ANP regulates neuronal proliferation and migration. Protein-protein interaction, CO-immunoprecipitation and IF staining carried out to evaluate whether ANP regulates the interaction between PFKFB3, NeuN and TBR1. Knockdown or overexpression of PFKFB3 by adenovirus infection were used to determine whether ANP inhibits neuronal development through PFKFB3 mediated glycolytic pathway. Our data indicated that ANP inhibited the maturation of embryonic neurons characterized by suppressing neuronal proliferation and migration. ANP regulated the interaction between PFKFB3, NeuN, and TBR1. Knockdown of PFKFB3 aggravated ANP mediated inhibition of neuronal proliferation and migration, while overexpression of PFKFB3 attenuated ANP mediated neuronal developmental suppression. In summary, ANP suppressed the expression of PFKFB3, and interrupted the interaction between TRB1 and NeuN, resulting in suppressing neuronal proliferation, migration and maturation and eventually inhibiting murine embryonic neuronal development.

Modulation of Autophagy is a Potential Strategy for Enhancing the Anti-Tumor Effect of Mebendazole in Glioblastoma Cells.

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has been suggested as a repositioning candidate for the treatment of brain tumors. However, the efficacy of MBZ needs further study to improve the beneficial effect on the survival of those patients. In this study, we explored a novel strategy to improve MBZ efficacy using a drug combination. When glioblastoma cells were treated with MBZ, cell proliferation was dose-dependently inhibited with an IC50 of less than 1 μM. MBZ treatment also inhibited glioblastoma cell migration with an IC50 of less than 3 μM in the Boyden chamber migration assay. MBZ induced G2-M cell cycle arrest in U87 and U373 cells within 24 h. Then, at 72 h of treatment, it mainly caused cell death in U87 cells with an increased sub-G1 fraction, whereas polyploidy was seen in U373 cells. However, MBZ treatment did not affect ERK1/2 activation stimulated by growth factors. The marked induction of autophagy by MBZ was observed, without any increased expression of autophagy-related genes ATG5/7 and Beclin 1. Co-treatment with MBZ and the autophagy inhibitor chloroquine (CQ) markedly enhanced the anti-proliferative effects of MBZ in the cells. Triple combination treatment with temozolomide (TMZ) (another autophagy inducer) further enhanced the anti-proliferative effect of MBZ and CQ. The combination of MBZ and CQ also showed an enhanced effect in TMZ-resistant glioblastoma cells. Therefore, we suggest that the modulation of protective autophagy could be an efficient strategy for enhancing the anti-tumor efficacy of MBZ in glioblastoma cells.

G protein–coupled receptor 21 in macrophages: An in vitro study

GPR21 is an orphan and constitutively active receptor belonging to the superfamily of G-Protein Coupled Receptors (GPCRs). GPR21 couples to the Gq family of G proteins and is expressed in macrophages. Studies of GPR21 knock-out mice indicated that GPR21 may be involved in promoting macrophage migration. The aim of this study was to evaluate the role of GPR21 in human macrophages, analyzing (i) its involvement in cell migration and cytokine release and (ii) the consequence of its pharmacological inhibition by using the inverse agonist GRA2. THP-1 cells were activated and differentiated into either M1 or M2 macrophages. GPR21 expression was evaluated at gene and protein level, the signalling pathway was investigated by an IP1 assay, and cytokine release by ELISA. Cell migration was detected by the Boyden chamber migration assay, performed on macrophages derived from both the THP-1 cell line and human peripheral blood monocytes. In addition, we compared the effect of the pharmacological inhibition of GPR21 with the effect of the treatment with a specific GPR21 siRNA to downregulate the receptor expression, thus confirming that GRA2 acts as an inverse agonist of GPR21. GRA2 does not affect cell viability at the tested concentrations, but significantly reduces the release of TNF-α and IL-1β from M1 macrophages. The analysis of the migratory ability highlighted opposite effects of GRA2 on M1 and M2 macrophages since it decreased M1, while it promoted M2 cell migration. Therefore, the pharmacological inhibition of GPR21 could be of interest for pathological conditions characterized by low grade chronic inflammation.

Andrographolide Promotes Interaction Between Endothelin-Dependent EDNRA/EDNRB and Myocardin-SRF to Regulate Pathological Vascular Remodeling.

IntroductionPathological vascular remodeling is a hallmark of various vascular diseases. Smooth muscle cell (SMC) phenotypic switching plays a pivotal role during pathological vascular remodeling. The mechanism of how to regulate SMC phenotypic switching still needs to be defined. This study aims to investigate the effect of Andrographolide, a key principle isolated from Andrographis paniculate, on pathological vascular remodeling and its underlying mechanism.MethodsA C57/BL6 mouse left carotid artery complete ligation model and rat SMCs were used to determine whether Andrographolide is critical in regulating SMC phenotypic switching. Quantitative real-time PCR, a CCK8 cell proliferation assay, BRDU incorporation assay, Boyden chamber migration assay, and spheroid sprouting assay were performed to evaluate whether Andrographolide suppresses SMC proliferation and migration. Immunohistochemistry staining, immunofluorescence staining, and protein co-immunoprecipitation were used to observe the interaction between EDNRA, EDNRB, and Myocardin-SRF.ResultsAndrographolide inhibits neointimal hyperplasia in the left carotid artery complete ligation model. Andrographolide regulates SMC phenotypic switching characterized by suppressing proliferation and migration. Andrographolide activates the endothelin signaling pathway exhibited by dramatically inducing EDNRA and EDNRB expression. The interaction between EDNRA/EDNRB and Myocardin-SRF resulted in promoting SMC differentiation marker gene expression.ConclusionAndrographolide plays a critical role in regulating pathological vascular remodeling.

Wound Healing Effect of Gintonin Involves Lysophosphatidic Acid Receptor/Vascular Endothelial Growth Factor Signaling Pathway in Keratinocytes.

Gintonin, a novel compound of ginseng, is a ligand of the lysophosphatidic acid (LPA) receptor. The in vitro and in vivo skin wound healing effects of gintonin remain unknown. Therefore, the objective of this study was to investigate the effects of gintonin on wound healing-linked responses, especially migration and proliferation, in skin keratinocytes HaCaT. In this study, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, Boyden chamber migration assay, scratch wound healing assay, and Western blot assay were performed. A tail wound mouse model was used for the in vivo test. Gintonin increased proliferation, migration, and scratch closure in HaCaT cells. It also increased the release of vascular endothelial growth factor (VEGF) in HaCaT cells. However, these increases, induced by gintonin, were markedly blocked by treatment with Ki16425, an LPA inhibitor, PD98059, an ERK inhibitor, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), a calcium chelator, and U73122, a PLC inhibitor. The VEGF receptor inhibitor axitinib also attenuated gintonin-enhanced HaCaT cell proliferation. Gintonin increased the phosphorylation of AKT and ERK1/2 in HaCaT cells. In addition, gintonin improved tail wound healing in mice. These results indicate that gintonin may promote wound healing through LPA receptor activation and/or VEGF release-mediated downstream signaling pathways. Thus, gintonin could be a beneficial substance to facilitate skin wound healing.

Abstract P374: Functional Impairment Of Cardiac Endothelial Cells In Diabetic Failing Heart Via Dysregulated Exosomal Mirnas

Introduction: Cardiac endothelial cells (ECs) and fibroblasts (FBs) together maintain cardiac homeostasis. Their functional impairment aggravates complications in the heart. In diabetes, acute inflammation leads to cardiac FBs activation, which predisposes the diabetic myocardium to severe fibrosis. Further, inflammation-related vascular dysfunction is a major end-organ complication in diabetics. However, it is not known, whether myofibroblast regulates ECs function in a diseased diabetic heart. Therefore, we hypothesized that “myofibroblast in diabetic heart secretes exosomes packed with antiangiogenic/profibrotic factors, which impede EC function and exaggerate pathological remodelling in pressure-overloaded (PO) myocardium. Methods: Exosomes were isolated from diabetic mice plasma and FB condition media by ultracentrifugation and characterized by nanosight & electron microscopy. We cultured mouse primary heart ECs in growth media and treated with exosomes derived from FBs (treated with 25mM glucose or 500nM Angiotensin II (AngII) or both) for 48 hr. Mannitol (25mM) served as control. Results: Ang II and glucose significantly activate FBs as shown by qPCR (fibronectin, collagenase1α1) and western blot (pSmad2, p-p38). Exosomes derived from diabetic Ang II treated FBs significantly impaired ECs function as shown by Matrigel tube formation and Boyden chamber migration assays. Interestingly, ECs markers (eNOS, VEGF, CD31) genes and proteins expression were significantly inhibited in ECs treated with exosomes-derived from glucose and Ang II treated FBs. We, further, checked the effect of diabetic mice plasma exosomes on ECs function and found significantly impaired as shown by tube formation and migration data. Finally, microRNA (miR) array and qPCR analysis revealed that miR-216a-5p, miR-26a-5p and miR7a-5p were highly upregulated in exosomes derived from FBs co-treated with glucose and AngII. Conclusions: Taken together, this study demonstrates that glucose and AngII co-treated FBs-derived exosomes are enriched in pro-fibrotic factors and can lead to EC dysfunction and promotes cardiac fibrosis in PO myocardium. In future studies, we will modulate the target miRs in diabetic FBs to see whether it rescue reparative function of ECs and inhibits fibrosis in failing heart.

Functional Properties of a Purified Reconstituted Bilayer Matrix Design Support Natural Wound Healing Activities.

Background:Biomaterial engineering has produced numerous matrices for use in tissue repair, utilizing various materials and processing methods, which can impact the ability of the products to encourage wound healing. Recently, we observed favorable clinical outcomes, using a novel purified reconstituted bilayer matrix (PRBM; Geistlich Derma-Gide) to treat chronic diabetic foot ulcers.Methods:Evaluations of the structural and functional characteristics of PRBM in vitro were performed to assess how this biomaterial may affect the favorable clinical results observed by influencing the wound environment and key physiologic mechanisms necessary for the healing process. Investigations included scanning electron microscopy, cell culture analyses, gene expression assays, matrix metalloproteinase activity assessment, and pH measurement.Results:Cross-sectional scanning electron microscopy demonstrated a distinct bilayer structure with porous and compact layers. The PRBM structure allowed cell types involved in wound healing to bind and proliferate. Expression analysis of growth factor-responsive genes demonstrated binding and preservation of bioactive growth factors TGF-β1, bFGF, and VEGF by PRBM. Boyden chamber migration assays revealed increased cellular migration compared with controls. In the presence of PRBM, the activity of MMP-1, MMP-2, and MMP-9 was significantly lower compared with control samples. pH of the PRBM in solution was slightly acidic.Conclusions:Based on in vitro evaluations, it appears that the PRBM processing without deleterious chemical crosslinking results in a suitable ECM possessing characteristics to aid natural wound healing, including cell attachment, migration, proliferation, differentiation, and angiogenesis. These in vitro data support the promising healing rate observed clinically when chronic DFUs are treated with PRBM.

PELP-1 regulates adverse responses to endocrine therapy in Estrogen Receptor (ER) positive breast cancer

Introduction: Endocrine therapy has played an important role in the management of ER positive breast cancer over recent decades. Despite this, not all patients respond equally to endocrine intervention, which can lead to resistance, associated disease relapse and progression. Previous reports suggest that endocrine agents themselves may induce an invasive phenotype in ER positive breast cancers with low/aberrant expression of E-cadherin. Here we investigate this phenomenon further and provide data supporting a role for the ER co-receptor, PELP-1, in mediating an adverse response to endocrine agents.Materials and Methods: The effects of tamoxifen, fulvestrant and estrogen withdrawal (as a model for aromatase inhibitor therapy) on the invasive and migratory capacity of endocrine-sensitive MCF-7 and T47D cells, in the presence or absence of functional E-cadherin and/or PELP-1 (using siRNA knockdown), was assessed via Matrigel invasion and Boyden chamber migration assays. The effects of these endocrine therapies alongside E-cadherin/PELP-1 modulation on cell proliferation were further assessed by MTT assay. Western blotting using phospho-specific antibodies was performed to investigate signalling pathway changes associated with endocrine-induced changes in invasion and migration.Results: Both tamoxifen and fulvestrant induced a pro-invasive and pro-migratory phenotype in ER positive breast cancer cells displaying a high basal expression of PELP-1, which was augmented in the context of poor cell-cell contact. This process occurred in a Src-dependent manner with Src inhibition reversing endocrine induced invasion/migration. While this adverse response was observed using both tamoxifen and fulvestrant therapy, it was not observed under conditions of estrogen withdrawal.Conclusions: Our data confirms previous reports that anti-estrogens induce an adverse cell phenotype in ER+ breast cancer, particularly in the absence of homotypic cell contact. These results implicate E-cadherin and PELP-1 as potential biomarkers when deciding upon optimum adjuvant endocrine therapy, whereby tumours with high PELP-1/low E-cadherin expression may benefit from estrogen withdrawal therapy via aromatase inhibition, as opposed to ER modulation/antagonism.

Krill oil extract inhibits the migration of human colorectal cancer cells and down-regulates EGFR signalling and PD-L1 expression

BackgroundThe currently available treatments for colorectal cancer (CRC) are often associated with serious side-effects. Therefore, the development of a novel nutraceutical agent may provide an alternative complementary therapy for CRC. Overexpression of the epidermal growth factor receptor (EGFR) associates with a range of cancers while downregulation of EGFR signalling can inhibit cancer growth. Our previous studies have shown that the free fatty acid extract (FFAE) of krill oil exhibits anti-proliferative and pro-apoptotic properties. This study determines the effects of krill oil extract on the migration of human CRC cells, and its potential role in modulating EGFR signalling pathway and the expression of programmed death ligand 1 (PD-L1).MethodsHuman CRC cells, DLD-1 and HT-29 were treated with FFAE of KO at 0.03 and 0.12 μL/100 μL for 8 or 24 h. Cell migration was determined by Boyden chamber migration assay. The expression of EGFR, phosphorylated EGFR (pEGFR), protein kinase B (AKT), phosphorylated AKT (pAKT), extracellular signal regulated kinase (ERK1/2), phosphorylated ERK1/2 (pERK1/2) as well as PD-L1 were assessed by western blotting and immunohistochemistry.ResultsThe FFAE of krill oil significantly inhibited cell migration compared to ethanol-treated (vehicle control) cells (P < 0.01 to P < 0.001). At the molecular level, krill oil extract reduced the expression of EGFR, pEGFR (P < 0.001 for both) and their downstream signalling, pERK1/2 and pAKT (P < 0.01 to P < 0.001) without altering total ERK 1/2 and AKT levels. In addition, the expression of PD-L1 was reduced by 67 to 72% (P < 0.001) following the treatment with krill oil extract.ConclusionThis study has demonstrated that krill oil may be a potential therapeutic/adjunctive agent for CRC attributed to its anti-migratory effects.. The potential anti-cancer properties of krill oil are likely to be associated with the downregulation of EGFR, pEGFR and their downstream pERK/ERK1/2 and pAKT/AKT signalling pathways along with the downregulation of PD-L1.

Recombinant Tissue Plasminogen Activator (r-tPA) Induces In-Vitro Human Neutrophil Migration via Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1).

Thrombolysis is the gold standard treatment for acute ischemic stroke. Besides its fibrinolytic role, recombinant tissue plasminogen activator (r-tPA) holds several non-fibrinolytic functions. Here, we investigated the potential role of r-tPA on human primary neutrophil migration in vitro. By means of modified Boyden chamber migration assay and checkerboard analysis we showed a dose-dependent chemotactic effect of r-TPA with a maximum effect reached by 0.03 mg/mL (0.003–1 mg/mL). Pre-incubation with MAP kinases inhibitors allowed the identification of PI3K/Akt, but not ERK1/2 as the intracellular pathway mediating the observed effects. Furthermore, by means of real-time PCR, immunocytochemistry and cytofluorimetry we demonstrated that the r-tPA receptor low density lipoprotein receptor-related protein 1 (LRP-1) is synthetized and expressed by neutrophils in response to r-tPA and TNF-α. Inhibition of LRP-1 by receptor-associated protein (RAP), prevented r-tPA-mediated F-actin polymerization, migration and signal through Akt but not ERK1/2. Lastly, also neutrophil degranulation in response to r-tPA seems to be mediated by LRP-1 under adhesion conditions. In conclusion, we show that r-tPA induces neutrophil chemotaxis through LRP-1/Akt pathway. Blunting r-tPA-mediated neutrophil activation might be beneficial as an adjuvant therapy to thrombolysis in this setting.

Abstract B35: Characterization of exosomes extracted from primary tumor samples of HPV+/- oropharyngeal cancer

Abstract Objective: To determine if distinctions in exosomal cargo contribute to differences in EMT between HPV-related and HPV-unrelated oropharyngeal disease and can provide a signature for prediction of tumor aggressiveness and metastatic potential. Subjects and Methods: Specimen from patients with HPV+/- OPSCC were collected according to an IRB-approved protocol including primary tumor, lymph node metastasis, distant metastasis, and serum when available. Exosomes isolated from HPV+ and HPV- tumors were analyzed with miARma-seq and proteomics. These same exosomes were cocultured with well-characterized HPV+ and HPV- cell lines as well as fibroblasts and normal keratinocytes. Tumor aggressiveness, EMT, and potential for metastasis were measured utilizing wound healing scratch assays and transwell invasion assay techniques based on the Boyden Chamber migration assay. Confirmation of epithelial to mesenchymal transition was evaluated by Western blot and qPCR using a panel of well-established EMT markers (e-cadherin, n-cadherin, vimentin, snail, slug, twist, ZEB1/2). Results: Tumors from HPV+ patients demonstrate exosomal content enriched in EMT markers compared to HPV-negative patient cultures. Exosomes extracted from primary tumor and lymph node metastasis of the same patient demonstrate differential expression of markers of EMT. Exosomes extracted from lymph node and cocultured with fibroblasts demonstrate increased invasion and migration. Conclusions: Exosomes can be extracted from primary patient tumor cultures of both primary tumor and lymph node metastasis. These exosomes demonstrate differential expression of markers of EMT. Exosomal contents can be exploited for biomarker discovery to aide in prognostication. Citation Format: Vivian F. Wu, Xiang Guo Dai, Michael Chopp. Characterization of exosomes extracted from primary tumor samples of HPV+/- oropharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B35.

Obesity and Breast Cancer: Unraveling the Role of Adipocyte‐Derived Exosomes

Excessive adiposity strongly influences risk, prognosis and progression of breast cancer. The revised concept of adipose tissue from an inert depot for body energy to an endocrine and immunologically active organ placed growing interest on the potential role of adipocyte secreted soluble factors and vesicles, particularly exosomes, as important players in cancer biology. In the last decades, several reports demonstrated that exosomes, small membrane‐derived vesicles, are able to modify target cell phenotype, by transferring bioactive molecules such as proteins, mRNAs, microRNAs, DNAs, lipids and transcriptional factors. However, whether adipose tissue‐derived exosomes, acting as a mode of cell‐to‐cell communication, might have a role in mediating the adverse effects of obesity on breast cancer is still not completely understood.Here, we evaluated the effects of exosomes derived from differentiated 3T3‐L1 adipocytes (3T3‐L1 A), a well recognized ‘in vitro’ model of white adipocytes, on breast cancer phenotype, using as experimental models both estrogen receptor α (ERα)‐positive MCF‐7 and triple negative MDA‐MB‐231 breast cancer cells.Exosomes from 3T3‐L1 A were isolated by sequential ultracentrifugation method, and characterized by Nanoparticle Tracking Analysis, Transmission Electron Microscopy and Immunoblotting analyses. 3T3‐L1 A‐ derived exosome uptake in both breast cancer cell lines was tested by monitoring internalization of exosomes labeled with PKH‐26. Treatment of MCF‐7 and MDA‐MB‐231 breast cancer cells with adipocyte‐derived exosomes did not modify anchorage‐dependent cell growth, while it significantly increased the ability of cells to form colonies in anchorage‐independent soft agar growth assays. In addition, we found that both breast cancer cells treated with adipocyte‐derived exosomes displayed increased migration and invasion capabilities as evidenced by wound healing, and Boyden Chamber migration and invasion assays. Accordingly, adipocyte‐derived exosomes significantly induce the expression of genes involved in Epithelial to Mesenchymal Transition (EMT) in both cell lines. Since migratory potential and EMT represent important features for breast cancer stem cells (BCSCs), we also explored the effect of 3T3L‐1 A‐derived exosomes on MCF‐7 and MDA‐MB‐231 BCSC activity. Interestingly, we found that adipocyte‐derived exosomes were able to increase Mammosphere Forming Efficiency and Self Renewal in breast cancer cells. In conclusion, our data highlight how adipocyte‐derived exosomes may contribute to a more aggressive breast cancer phenotype, providing novel insight in the link between obesity and breast cancer. This may open a new molecular window for preventive and therapeutic strategies to fight breast cancer in obese patients.Support or Funding InformationThis research was funded by Fondazione AIRC: IG #11595 to SA and IG #21414 to SC, and PRIN2017 #2017EKMFTN to SC; PRIN2017 #2017WNKSLR to IB

The inhibition of AGTR1 and Aromatase as a new potential therapeutic strategy for Glioblastoma treatment.

IntroductionGlioblastoma multiforme (GBM) is the most malignant form of glioma causing 3–4% of all cancer‐related deaths. Specific cancer treatment for GBM includes surgery, radiotherapy and chemotherapy with temozolomide. Despite the continuous development of new clinical therapies, the prognosis and survival of GBM patients remain dismal. Thus, the deregulated growth, the migratory and invasive capabilities of glioma cells remain the most important obstacles to the development of effective GBM treatments. Angiotensin II (Ang II)/Ang II receptor 1 (AGTR1) axis is expressed in normal and tumoral tissues, including human GBM. It has been reported in different experimental models that Ang II is able to stimulate aromatase expression through the AGTR1 activation. Different studies have demonstrated that aromatase enzyme is expressed in GBM cells and local estrogen production may act as an autocrine or paracrine factor in enhancing glioblastoma growth and progression. In the present study, we investigated if Ang II/AGTR1 signalling may modulate aromatase expression and consequently local estrogen production, thus influencing glioblastoma survival and growth.MethodsThe Aromatase expression role under treatment with Ang II has been assessed by RT‐PCR, Western blotting, Aromatase activity assay, Transient transfection assays, ELISA assay, cell proliferation assay (Thymidine incorporation), Boyden chamber migration and invasion assays, in the GBM cell line U‐87 MG.ResultsWe identified in U‐87 MG cells that the Ang II up‐regulates the aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. In order to evaluate if Ang II may activate aromatase at transcriptional level we transiently transfected U‐87 MG cells with a vector containing human aromatase promoter I.4, broadly distributed around the brain and widely expressed in glioblastoma cells. Ang II treatment induces an increased aromatase promoter transcriptional activity. Mutagenesis studies and chromatin immunoprecipitation analysis reveal that the integrity of specificity protein 1 (SP1) and interferon‐gamma activation site (GAS) binding sites, within the aromatase promoter is required for Ang II modulation of human aromatase I.4 promoter activity in U87‐MG cells. The enhanced aromatase activity was paralleled by an higher estradiol production concomitant with an increase in cell proliferation, migration and invasion in U‐87 MG cells. These effects are reversed by treatment with an AGTR1 antagonist and/or an aromatase inhibitor, such as Losartan and Anastrozole respectively.ConclusionsAng II/AGTR1 signalling through Aromatase enhances local estrogen production which in turn promotes tumor growth and invasiveness. Therefore, their combined inhibitory targeting may prospectively provide a novel therapeutic strategy for Glioblastoma treatment.