Expression Of Del-1 Research Articles

Exploration of MELK as a downstream of Del-1 and druggable targets in triple-negative breast cancer.

In our previous study, Developmental endothelial locus-1 (Del-1) was a promising predictive marker for breast cancer. However, the downstream targets of Del-1 remain unknown. Here, we sought to discover a druggable target downstream of Del-1 and investigate the mechanism by which it regulates the course of breast cancer. To investigate Del-1 downregulation effect on breast cancer, we performed transcriptome analysis using RNA sequencing of Del-1 knockdowned MDA-MB-231 cell line Plus, to investigate the expression of Del-1 and Maternal embryonic leucine zipper kinase (MELK), mRNA levels in eight different triple-Negative Breast Cancer (TNBC) cell lines were analyzed. High-throughput sequencing was performed on total RNA isolated. OTS167 was used for MELK inhibition. The effects of MELK on cell proliferation and invasion were determined using the MTT and Matrigel transwell assays. Furthermore, we examined MELK expression in breast cancer tissue. Del-1 and MELK mRNA expression levels were significantly higher in the TNBC cell lines, MDA-MB-468, HCC-1806, and MBA-MB-231. Knocking down Del-1 with siRNA in HCC-1806 and MBA-MB-231 cells significantly decreased MELK expression and thus suggested a possible relationship between Del-1 and MELK. In MDA-MB-468 cells, a basal-like 1 TNBC cell line, OTS167 significantly inhibited breast cancer cell proliferation and promoted cell apoptosis. To further investigate the relationship between Del-1 and MELK, dual inhibition of both Del-1 and MELK was performed, which significantly reduced the viability of MDA-MB-468 and MBA-MB-231 cells. We found that MELK acts downstream of Del-1 and is a promising druggable target, especially in basal-like and mesenchymal stem-like subtype.

MELK and Del-1 as druggable targets in TNBC.

e15100 Background: In our previous study, the researchers found that Del-1 was a promising predictive marker for breast cancer. However, the Del-1 downstream targets and biological effectors remain unknown and undruggable in breast cancer. We used RNA-seq to analyze the transcriptome and investigate the mechanism of Del-1 in regulating the course of breast cancer to uncover a druggable target. Methods: To evaluate the expression levels of Del-1 and MELK, mRNA levels in several breast cancer cell lines were analyzed. Due to the heterogeneity of TNBC, eight different TNBC cell lines were analyzed (BT20, TNBC; MDA-MB-468, basal-like 1; HCC-1806, basal-like 2; DU4475, Immunomodulatory; BT549, mesenchymal-like; MDA-MB-231, mesenchymal stem-like; HS578T, mesenchymal stem-like; MDA-MB-453, TNBC Luminal androgen receptor). Total RNA was isolated using RNAiso Plus (TaKaRa, Otsu, Shiga, Japan). Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit. High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA). OTS167 was treated for inhibition of MELK. The effects of MELK on cell proliferation and invasion were determined using MTT and Matrigel Transwell assays. To further investigate the relationship between DEL-1 and MELK, the researchers performed dual inhibition of DEL-1 & MELK. Results: Del-1 and MELK expression were significantly increased in TNBC cell lines compared to luminal or HER 2 positive breast cancer cell lines. Expression of Del-1 and MELK was dramatically enhanced in MB 468, HCC-1806, and MB 231 TNBC cell lines. Three TNBC cell lines were transfected with Del-1-specific siRNA to examine the effect of Del-1 downregulation on breast cancer. Del-1 knockdown HCC-1806 & MDA-MB 231 cells significantly decreased MELK expression, suggesting the possible relation between Del-1 and MELK. OTS 167, a MELK inhibitor, significantly inhibits breast cell proliferation and promotes cell apoptosis (MBA-MB-468, p < 0.001). Furthermore, when the OTS 167 was treated, the cell viability decreased as the dose of the MELK inhibitor increased, and the degree was the greatest in MBA-MB 468, basal-like 1 TNBC cell line. To further investigate the relationship between DEL-1 and MELK, the researchers inhibited DEL-1 and then MELK in MTT assay. Dual inhibition significantly reduced cell viability in MDA-MB468 and MDA-MB 231 cell lines, with the basal-like 1 cell lines showing the most significant effect. Conclusions: In this study, we sought to investigate the association between De1-1 and MELK in TNBC cell lines and tumor tissue. We found that MELK is downstream of del-1 and is a promising target, especially in the basal-like 1 subtype. Further research is warranted.

Resolvin D1 (RvD1) Regulates Porphyromonas Gingivalis Lipopolysaccharide-induced Del-1 and Cytokine Expressions in Human Gingival Fibroblasts

Purpose: To detect the effect of Resolvin D1 (RvD1) on Developmental endothelial locus-1 (Del-1) and cytokine expressions of human gingival fibroblast cells exposed to Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS).
 
 Material and Methods: The effect of RvD1 on cell viability of human gingival fibroblasts exposed to P. gingivalis-LPS were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Meanwhile, the effect of RvE1 on Del-1 and cytokine (IL-1β, IL-6, IL-8, IL-10, IL-17) expressions of human gingival fibroblasts exposed to P. gingivalis-LPS were studied by real-time PCR experiment, statistical analysis was performed using GraphPad Prism version 5 for Windows.
 
 Results: Cell viability assay results demonstrated that RvD1 concentrations upregulated cell number compared to control group at 24 and at 72 hours. While RvD1 reduced IL-6, IL-8, IL-17 mRNA expression, IL-10 and Del-1 mRNA expression increased as a time- and dose-dependent manner. Also, IL-1β was not affected by RvD1 treatments. 
 
 Conclusion: The increased expression of Del-1 and IL-10 by RvD1 down-regulated the pro-inflammatory cytokine expressions induced by P. gingivalis-LPS in gingival fibroblast. RvD1 displayed regulatory effects on gingival inflammation in P. gingivalis LPS-induced cell culture experiment. In particular, results of study display that Del-1 induced by RvD1 may have therapeutic potential to modulate periodontal inflammation.

Negative Regulators of Inflammation Response to the Dynamic Expression of Cytokines in DF-1 and MDCK Cells Infected by Avian Influenza Viruses.

The H5N1 and H9N2 avian influenza viruses (AIVs) seriously endanger the poultry industry and threaten human health. Characteristic inflammatory responses caused by H5N1 and H9N2 AIVs in birds and mammals result in unique clinical manifestations. The role of anti-inflammatory regulators, PTX3, Del-1, and GDF-15, in H5N1 and H9N2-AIV-mediated inflammation in birds and mammals has not yet been verified. Here, the expression of PTX3, Del-1, and GDF-15 in DF-1 and MDCK cells infected with H5N1 and H9N2 AIVs and their effect on inflammatory cytokines were analyzed. Infection with both AIVs increased PTX3, Del-1, and GDF-15 expression in DF-1 and MDCK cells. Infection with H9N2 or H5N1 AIV in DF-1 and MDCK cells with overexpression of all three factors, either alone or in combination, inhibited the expression of tested inflammatory cytokines. Furthermore, co-expression of PTX3, Del-1, and GDF-15 enhanced the inhibition, irrespective of the cell line. The findings from this study offer insight into the pathogenic differences between H5N1 and H9N2 AIVs in varied hosts. Moreover, our findings can be used to help screen for host-specific anti-inflammatory agents.

MELK is a downstream target of Del-1 and promotes breast cancer progression.

e12578 Background: Del-1 is a promising prognostic marker for breast cancer in our previous study. However, the downstream targets and biological effectors of Del-1 remain unclear and still untargetable in breast cancer. We performed transcriptome analysis using RNA-seq and explored the mechanism of Del-1 in regulating the progression of breast cancer to find druggable target. Methods: Total RNA was isolated using RNAiso Plus (TaKaRa, Otsu, Shiga, Japan), Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit. High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA). OTSSP167(OTS167) were treated for inhibition of MELK. The effects of MELK on cell proliferation, invasion were determined using MTT, Matrigel Transwell assays. The tumoral expression of Del-1 and MELK were determined based on tissue microarrays and immunohistochemistry results from 440 early breast cancer patients. Results: To investigate Del-1 downregulation effect on breast cancer, we performed RNA-seq of Del-1 knock downed MDA-MB-231 and MCF 7-Tamoxifen resistant (TamR) cell line. Compared with si-control, MELK gene were downregulated in both knock downed cell lines. While a high Del-1 and MELK mRNA expressions were found in all the breast cancer cell lines, both expressions were significantly higher in MDA MB-231. MELK inhibitor (OTS167) treatment to breast cancer cell line MDA-MB-231 and MCF 7-TamR showed similar results as Del-1 down regulation by si-RNA. Inhibition of MELK suppressed proliferation and invasion of breast cancer cell line. Tumoral MELK expression correlate with increased aggressiveness and poor clinical outcomes in 440 breast cancer patients. Conclusions: Our results indicate that Del-1 may regulate MELK expression which has a role in breast cancer progression. In conclusion, modulation of Del-1 status by targeting MELK may be a new therapeutic strategy for breast cancer patients.

Inhibition of p38/MK2 Signaling Prevents Vascular Invasion of Melanoma

Melanoma cells can switch between distinct gene expression profiles, resulting in proliferative or invasive phenotypes. Signaling pathways involved in this switch were analyzed by gene expression profiling of a cohort of 22 patient-derived melanoma cell lines. CDH1 negativity was identified as a surrogate marker for the invasive phenotype. CDH1 expression could be turned on and off by modulating activity of p38 or its downstream target MK2, suggesting that this pathway controls melanoma progression. Mechanistically, MK2 inhibition prevented melanoma-induced vascular barrier disruption, reduced the expression of PODXL and DEL-1, and prevented vascular dissemination invivo. PODXL and DEL-1 expression in patients with melanoma were associated with poor survival and thus can be used as prognostic markers. Downstream targets of MK2 may thus serve as candidate therapeutics.

Abstract 4892: Del-1 promotes the proliferation and migration of tamoxifen-resistant breast cancer

Abstract Background: We previously demonstrated a prognostic role of exosomal Del-1 with breast cancer patients. However, the mechanisms of Del-1 expression are barely understood. Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer. Accordingly, we investigated the function of Del-1 in tamoxifen-resistant (TAMR) breast cancer cell line and tumor specimens. Materials and Methods: Del-1 expression in MCF7 and TAMR MCF7 cells was performed by quantitative RT-PCR, western blot, and ELISA. The effects of Del-1 with RNA interference on proliferation, migration and invasion of TAMR MCF7 cells were observed by MTT, wound healing and Matrigel transwell assay. Meanwhile, the tumoral expression of Del-1 was determined immunohistochemistry (IHC) from 10 tamoxifen resistant-breast cancer patients. Tumor tissue before (tamoxifen sensitive) and after tamoxifen resistance were assessed. Results: Del-1 was highly expressed in TAMR MCF7 cells compared to MCF7 cells (p<0.001). And the expression of secretory del-1 was also increased in the culture media of TAMR MCF7 cells. Moreover, down-regulation of Del-1 significantly inhibited the proliferation and migration of TAMR MCF7 cells. There was no difference in the invasion of TAMR MCF7 cells. Increased Del-1 expression was preferentially observed in in TAMR tumor tissues when compared to tamoxifen sensitive or normal breast tissue. Conclusion: Prominent expression of Del-1 in TAMR MCF7 cells was associated with the proliferation and migration of TAMR MCF7 cells. And the tumoral expression of Del-1 was also significantly associated with tamoxifen resistance. These results suggest that the expression of Del-1 promote tamoxifen resistance in breast cancer cells and could be a novel target for anti-breast cancer treatment. Citation Format: Yee Soo Chae, Jong Gwang Kim, Jae-Hwan Jeong, Soo Jung Lee, Jeeyeon Lee, Jin Hyang Jung, Ho Yong Park, Moon-Chang Baek, Senten Richard J, Ji Young Park. Del-1 promotes the proliferation and migration of tamoxifen-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4892.

Del-1 Expression as a Potential Biomarker in Triple-Negative Early Breast Cancer

Objective: A differential diagnostic role for plasma Del-1 was proposed for early breast cancer (EBC) in our previous study. We examined tumoral Del-1 expression and analyzed its prognostic impact among patients with EBC. Methods: Del-1 mRNA expression was assessed in breast epithelial and cancer cells. Meanwhile, the tumoral expression of Del-1 was determined based on tissue microarrays and immunohistochemistry results from 440 patients. Results: While a high Del-1 mRNA expression was found in all the breast cancer cell lines, the expression was significantly higher in MDA-MB-231. Tumoral expression of Del-1 was also significantly associated with a negative expression of estrogen receptor or progesterone receptor, and low expression of Ki-67, particularly in the case of triple-negative breast cancer (TNBC) (p < 0.036). Furthermore, a correlation was found between Del-1 expression and an aggressive histological grade, nuclear mitosis, and polymorphism, suggesting a possible role in tumor progression. In the survival analysis, a worse distant disease-free survival trend was noted for the group overexpressing Del-1. Conclusion: While all the investigated breast cancer cell lines exhibited Del-1 expression, the expression rate and intensity were specifically prominent in TNBC. In addition, based on its relationship to an unfavorable histology and worse survival trend, Del-1 could act as a molecular target in TNBC patients.

Clinical association between chronic periodontitis and the leukocyte extravasation inhibitors developmental endothelial locus-1 and pentraxin-3.

This clinical study aimed to determine whether periodontal disease is associated with expression of developmental endothelial locus-1 (Del-1) and pentraxin-3 (PTX-3), endogenous inhibitors of leukocyte extravasation in humans. Expression of DEL1, PTX3, interleukin-17A (IL17A), and lymphocyte function-associated antigen-1 (LFA1) was determined, using RT-PCR and melting curve analysis, in biopsies of gingival tissues from 95 patients: 42 with moderate periodontitis; 40 with severe periodontitis; and 13 healthy controls. Relative expression of DEL1 and PTX3 was statistically significantly weaker in patients with periodontitis than in the control subjects. On the contrary, both IL17A and LFA1 showed statistically significant stronger expression in patients with periodontitis than in healthy controls. Correlation analysis, performed using Spearman's test, showed that expression of DEL1 was statistically significantly linked to periodontitis (ρ = -0.103) and to age (ρ = -0.134), but not to the gender of the patient, and that expression of PTX3 was significantly correlated with periodontitis (ρ = -0.354). Expression of neutrophil extravasation inhibitors DEL1 and PTX3 show significant, but weak, association with the clinical manifestation of chronic periodontitis.

Del-1 overexpression potentiates lung cancer cell proliferation and invasion

Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild type of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma.

Antagonistic effects of IL-17 and D-resolvins on endothelial Del-1 expression through a GSK-3β-C/EBPβ pathway.

Del-1 is an endothelial cell-secreted anti-inflammatory protein. In humans and mice, Del-1 expression is inversely related to that of IL-17, which inhibits Del-1 through hitherto unidentified mechanism(s). Here we show that IL-17 downregulates human endothelial cell expression of Del-1 by targeting a critical transcription factor, C/EBPβ. Specifically, IL-17 causes GSK-3β-dependent phosphorylation of C/EBPβ, which is associated with diminished C/EBPβ binding to the Del-1 promoter and suppressed Del-1 expression. This inhibitory action of IL-17 can be reversed at the GSK-3β level by PI3K/Akt signalling induced by D-resolvins. The biological relevance of this regulatory network is confirmed in a mouse model of inflammatory periodontitis. Intriguingly, resolvin-D1 (RvD1) confers protection against IL-17-driven periodontal bone loss in a Del-1-dependent manner, indicating an RvD1-Del-1 axis against IL-17-induced pathological inflammation. The dissection of signalling pathways regulating Del-1 expression provides potential targets to treat inflammatory diseases associated with diminished Del-1 expression, such as periodontitis and multiple sclerosis.

Crosstalk between bone marrow-derived mesenchymal stem cells and regulatory T cells through a glucocorticoid-induced leucine zipper/developmental endothelial locus-1-dependent mechanism.

Bone marrow is a reservoir for regulatory T (T(reg)) cells, but how T(reg) cells are regulated in that environment remains poorly understood. We show that expression of glucocorticoid (GC)-induced leucine zipper (GILZ) in bone marrow mesenchymal lineage cells or bone marrow-derived mesenchymal stem cells (BMSCs) increases the production of T(reg) cells via a mechanism involving the up-regulation of developmental endothelial locus-1 (Del-1), an endogenous leukocyte-endothelial adhesion inhibitor. We found that the expression of Del-1 is increased ∼4-fold in the bone tissues of GILZ transgenic (Tg) mice, and this increase is coupled with a significant increase in the production of IL-10 (2.80 vs. 0.83) and decrease in the production of IL-6 (0.80 vs. 2.33) and IL-12 (0.25 vs. 1.67). We also show that GILZ-expressing BMSCs present antigen in a way that favors T(reg) cells. These results indicate that GILZ plays a critical role mediating the crosstalk between BMSCs and T(reg) in the bone marrow microenvironment. These data, together with our previous findings that overexpression of GILZ in BMSCs antagonizes TNF-α-elicited inflammatory responses, suggest that GILZ plays important roles in bone-immune cell communication and BMSC immune suppressive functions.

Exercise before Scuba Diving Ameliorates Decompression-Induced Neutrophil Activation

The goals of this study were to investigate the difference in responses between a scuba dive preceded by aerobic exercise (EX) and a nonexercise control dive (CON) and to further evaluate the potential relation between venous gas emboli (VGE) and microparticles (MP). We hypothesized that exercise would alter the quantity and subtype of annexin V-positive MP and VGE. Nineteen divers performed two dives to 18 m seawater for 41 min separated by at least 3 d, one of which was preceded by 60 min of treadmill interval exercise. Blood was obtained before exercise, before diving, and 15 min, 2 h, 4 h, and 24 h after surfacing. Intravascular bubbles were quantified by transthoracic echocardiography at 15, 40, 80, and 120 min. The median VGE remained unchanged between the two dives; however, there was a significant increase in VGE in the exercise dive at 40 and 80 min at rest. MP were significantly elevated by approximately 2 times at all time points after CON compared with those after EX. Markers of neutrophil and platelet activation were elevated by both dives, and these elevations were attenuated in the EX dive. We conclude that some of the differences observed between the EX and CON related to MP and platelet and neutrophil activation provide additional insight into the potential protective benefits of exercise; however, further study is needed to understand the mechanism and true potential of these benefits.

Novel inflammatory pathways in periodontitis.

New insights into the biological mechanisms involved in modulating periodontal inflammation and alveolar bone loss are paving the way for novel therapeutic strategies for periodontitis. The neutrophil adhesion cascade for transmigration in response to infection or inflammation is a key paradigm in immunity. Developmental endothelial locus-1 (Del-1) is one of several newly identified endogenous inhibitors of the leukocyte adhesion cascade. Del-1 competes with intercellular adhesion molecule-1 (ICAM-1) on endothelial cells for binding to the LFA-1 integrin on neutrophils, thereby regulating neutrophil recruitment and local inflammation. In animal periodontitis models, Del-1 deficiency resulted in severe inflammation and alveolar bone loss, but local treatment with recombinant Del-1 prevented neutrophil infiltration and bone loss. The expression of Del-1 is inhibited by the pro-inflammatory cytokine IL-17. Nucleic-acid-receptor-mediated inflammatory responses may be important in periodontal disease pathogenesis. Bacterial nucleic acids released during inflammation are detected by host microbial DNA sensors, e.g., Toll-like receptor-9 (TLR-9), leading to the activation of pro- and/or anti-inflammatory signaling pathways. DNA from periodontitis-associated bacteria induced pro-inflammatory cytokine production in human macrophage-like cells through the TLR-9 and NF-κB signaling pathways, but had less effect on human osteoblasts. Inhibition of TLR-9 signaling in human macrophages reduced cytokine production in response to P. gingivalis DNA. Differential expression of a polymorphic site in the TLR-9 gene promoter region and increased TLR-9 gene and protein expression were reported in chronic periodontitis. Further research to confirm that periodontal bacterial DNA contributes to destructive inflammation in vivo could provide alternative therapeutic targets to control periodontitis.

Irradiation with a low-level diode laser induces the developmental endothelial locus-1 gene and reduces proinflammatory cytokines in epithelial cells

We demonstrated previously that low-level diode laser irradiation with an indocyanine green-loaded nanosphere coated with chitosan (ICG-Nano/c) had an antimicrobial effect, and thus could be used for periodontal antimicrobial photodynamic therapy (aPDT). Since little is known about the effects of aPDT on periodontal tissue, we here investigated the effect of low-level laser irradiation, with and without ICG-Nano/c, on cultured epithelial cells. Human oral epithelial cells were irradiated in a repeated pulse mode (duty cycle, 10 %; pulse width, 100 ms; peak power output, 5 W). The expression of the developmental endothelial locus 1 (Del-1), interleukin-6 (IL-6), IL-8, and the intercellular adhesion molecule-1 (ICAM-1) were evaluated in Ca9-22 cells stimulated by laser irradiation and Escherichia coli-derived lipopolysaccharide (LPS). A wound healing assay was carried out on SCC-25 cells irradiated by diode laser with or without ICG-Nano/c. The mRNA expression of Del-1, which is known to have anti-inflammatory activity, was significantly upregulated by laser irradiation (p < 0.01). Concurrently, LPS-induced IL-6 and IL-8 expression was significantly suppressed in the LPS + laser group (p < 0.01). ICAM-1 expression was significantly higher in the LPS + laser group than in the LPS only or control groups. Finally, compared with the control, the migration of epithelial cells was significantly increased by diode laser irradiation with or without ICG-Nano/c. These results suggest that, in addition to its antimicrobial effect, low-level diode laser irradiation, with or without ICG-Nano/c, can suppress excessive inflammatory responses via a mechanism involving Del-1, and assists in wound healing.

Expression and Function of the Homeostatic Molecule Del-1 in Endothelial Cells and the Periodontal Tissue

Developmental endothelial locus-1 (Del-1) is an endothelial cell-secreted protein that limits the recruitment of neutrophils by antagonizing the interaction between the LFA-1 integrin on neutrophils and the intercellular adhesion molecule (ICAM)-1 on endothelial cells. Mice with genetic or age-associated Del-1 deficiency exhibit increased neutrophil infiltration in the periodontium resulting in inflammatory bone loss. Here we investigated additional novel mechanisms whereby Del-1 could interfere with neutrophil recruitment and inflammation. Treatment of human endothelial cells with Del-1 did not affect the expression of endothelial molecules involved in the leukocyte adhesion cascade (ICAM-1, VCAM-1, and E-selectin). Moreover, genetic or age-associated Del-1 deficiency did not significantly alter the expression of these adhesion molecules in the murine periodontium, further ruling out altered adhesion molecule expression as a mechanism whereby Del-1 regulates leukocyte recruitment. Strikingly, Del-1 inhibited ICAM-1-dependent chemokine release (CXCL2, CCL3) by neutrophils. Therefore, Del-1 could potentially suppress the amplification of inflammatory cell recruitment mediated through chemokine release by infiltrating neutrophils. Interestingly, Del-1 was itself regulated by inflammatory stimuli, which generally exerted opposite effects on adhesion molecule expression. The reciprocal regulation between Del-1 and inflammation may contribute to optimally balance the protective and the potentially harmful effects of inflammatory cell recruitment.

The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss

Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.

Downregulation of developmentally regulated endothelial cell locus-1 inhibits the growth of colon cancer

Developmentally regulated endothelial cell locus-1 (Del1) is an embryonic angiogenic factor expressed in early embryonic endothelial cells, but recently has been found to be expressed in some forms of cancers including colon and breast cancers, and melanoma, and human cancer cell lines. Overexpression of Del1 accelerates tumor growth by enhancing vascular formation, implying Del1 may be a potential target for anti-angiogenic cancer therapy. The study aims to investigate whether downregulation of Del1 could inhibit the growth of tumors established in nude Balb/c mice by subcutaneous implantation of human LS-174T colon cancer cells. The shRNA expression vectors targeting human Del1, and vascular endothelial growth factor (VEGF) were constructed. Gene transfection of Del1-shRNA downregulated expression of Del1 in LS-174T cells in vivo and in vitro, but did not alter the proliferative or survival properties of cells in vitro. Gene transfection of VEGF-shRNA downregulated expression of both VEGF and Del1 in LS-174T cells in vivo and in vitro. Both Del1-shRNA and VEGF-shRNA gene therapies exhibited anti-tumor activities and they also showed a synergistic effect in suppressing growth of colon tumors by anti-angiogenesis and anti-proliferation. Although further investigation to clarify the mechanisms explaining the role of Del1 in tumor growth, and the interaction between VEGF and Del1, is required, the results indicate that downregulation of Del1 presents a potent therapeutic strategy to combat colon cancer.

Downregulation of developmentally regulated endothelial cell locus-1 inhibits the growth of colon cancer

Developmentally regulated endothelial cell locus-1 (Del1) is an embryonic angiogenic factor expressed in early embryonic endothelial cells, but recently has been found to be expressed in some forms of cancers including colon and breast cancers, and melanoma, and human cancer cell lines. Overexpression of Del1 accelerates tumor growth by enhancing vascular formation, implying Del1 may be a potential target for anti-angiogenic cancer therapy. The study aims to investigate whether downregulation of Del1 could inhibit the growth of tumors established in nude Balb/c mice by subcutaneous implantation of human LS-174T colon cancer cells. The shRNA expression vectors targeting human Del1, and vascular endothelial growth factor (VEGF) were constructed. Gene transfection of Del1-shRNA downregulated expression of Del1 in LS-174T cells in vivo and in vitro, but did not alter the proliferative or survival properties of cells in vitro. Gene transfection of VEGF-shRNA downregulated expression of both VEGF and Del1 in LS-174T cells in vivo and in vitro. Both Del1-shRNA and VEGF-shRNA gene therapies exhibited anti-tumor activities and they also showed a synergistic effect in suppressing growth of colon tumors by anti-angiogenesis and anti-proliferation. Although further investigation to clarify the mechanisms explaining the role of Del1 in tumor growth, and the interaction between VEGF and Del1, is required, the results indicate that downregulation of Del1 presents a potent therapeutic strategy to combat colon cancer.

Increased Rate of Hair Regrowth in Mice with Constitutive Overexpression of Del1

Developmental endothelial locus (Del)1 is a secreted extracellular matrix-associated protein that stimulates angiogenesis through integrin binding and is implicated in vasculogenesis. We hypothesized that increased expression of an angiogenic factor would lead to enhanced wound healing. Transgenic mice had Del1 cloned behind a keratin 14 promoter (K14-Del1) to drive constitutive expression in basal keratinocytes. Transgenic animals and wild-type litter mates underwent excisional wounding or depilation, and tissues were harvested at various time points. Wound healing and hair regrowth were assessed by photography, histology, and immunohistochemistry. For injection experiments, purified Del1 protein was injected in the flanks of wild-type mice with carrier on the contralateral flank as a control. Del1 expression during hair development was performed using transgenic mice with a LacZ cassette introduced downstream from the native promoter. K14-Del1 animals appeared normal and healed excisional wounds normally but demonstrated an increased rate of hair regrowth after wound healing. Using depilation experiments to specifically address hair follicle growth, we found increased hair regrowth was independent of wounding. This was confirmed by injection of purified Del1 protein. During normal hair anagenesis, Del1 is expressed in the root of the hair follicle. Constitutive expression of Del1 in skin does not affect skin vascularity or improve wound healing. Surprisingly, we found the primary effect of constitutive Del1 expression in the basal keratinocytes was increased hair growth following induction of anagenesis. During normal hair anagenesis, we see expression of Del1 in the root of the hair follicle suggesting it may function there to stimulate hair growth.