Temporal and spatial expression of tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and -2) in the bovine corpus luteum

The matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), may mediate the dramatic structural and functional changes in the corpus luteum (CL) over the course of its life span. In addition to regulating MMP activity, TIMPs are also involved in a variety of cellular processes, including cell proliferation and steroidogenesis. In a series of initial studies, we determined that matrix metalloproteinase inhibitory activity was present in protein extracts from early (4 days old, estrus = day 0), mid (10–12 days old) and late (16 days old) CL (n = 3 for each stage). Reverse zymography revealed four metalloproteinase inhibitory protein bands with relative Molecular masses that are consistent with those reported for TIMP-1 to -4. In order to gain a better understanding of TIMPs and their role in luteal function, we further characterized this inhibitory activity with a particular focus on the temporal and spatial expression of TIMP-1 and TIMP-2 in the bovine CL. Northern blotting revealed that the TIMP-1 transcript (0.9 kb) was expressed at a higher (p \u3c 0.05) level in early and mid cycle CL than in the late stage. In contrast, two TIMP-2 mRNA species, one major 1 kb species and one minor 3.5 kb species, were significantly (p \u3c 0.05) increased in the mid and late cycle CL than in the early. Western blotting analyses demonstrated no differences in TIMP-1 (29 kDa) protein levels between early and mid stages, while its levels decreased (p \u3c 0.05) from the mid to late stage CL. Conversely, TIMP-2 (22 kDa) protein was detected at a low level in the early CL, but significantly (p \u3c 0.05) increased in the mid and late stages. Immunohistochemistry revealed that both TIMP-1 and -2 were localized to large luteal cells from all three ages of CL. TIMP-1 was also localized in capillary smooth muscle cells, while TIMP-2 was restricted to the endothelial cells in the capillary compartment. In conclusion, the different temporal expression patterns of TIMP-1 and TIMP-2 suggest that TIMP-1 may be important for luteal formation and development, while TIMP-2 may play significant roles during luteal development and maintenance. Furthermore, the distinct localization of these two inhibitors in the vascular compartment indicates that they may serve diverse physiological functions during different stages of luteal angiogenesis

Cdc42-Dependent Transfer of mir301 from Breast Cancer-Derived Extracellular Vesicles Regulates the Matrix Modulating Ability of Astrocytes at the Blood–Brain Barrier

Breast cancer brain metastasis is a major clinical challenge and is associated with a dismal prognosis. Understanding the mechanisms underlying the early stages of brain metastasis can provide opportunities to develop efficient diagnostics and therapeutics for this significant clinical challenge. We have previously reported that breast cancer-derived extracellular vesicles (EVs) breach the blood–brain barrier (BBB) via transcytosis and can promote brain metastasis. Here, we elucidate the functional consequences of EV transport across the BBB. We demonstrate that brain metastasis-promoting EVs can be internalized by astrocytes and modulate the behavior of these cells to promote extracellular matrix remodeling in vivo. We have identified protein and miRNA signatures in these EVs that can lead to the interaction of EVs with astrocytes and, as such, have the potential to serve as targets for development of diagnostics and therapeutics for early detection and therapeutic intervention in breast cancer brain metastasis

Lipocalin 2 expression is associated with aggressive features of endometrial cancer

Background: Increased expression of lipocalin 2 (LCN2) has been observed in several cancers. The aim of the present study was to investigate LCN2 in endometrial cancer in relation to clinico-pathologic phenotype, angiogenesis, markers of epithelial-mesenchymal transition (EMT), and patient survival. Methods: Immunohistochemical staining was performed using a human LCN2 antibody on a population-based series of endometrial cancer patients collected in Hordaland County (Norway) during 1981-1990 (n = 256). Patients were followed from the time of primary surgery until death or last follow-up in 2007. The median follow-up time for survivors was 17 years. Gene expression data from a prospectively collected endometrial cancer series (n = 76) and a publicly available endometrial cancer series (n = 111) was used for gene correlation studies. Results: Expression of LCN2 protein, found in 49% of the cases, was associated with non-endometrioid histologic type (p = 0.001), nuclear grade 3 (p = 0.001), >50% solid tumor growth (p = 0.001), ER and PR negativity (p = 0.028 and 0.006), and positive EZH2 expression (p < 0.001). LCN2 expression was significantly associated with expression of VEGF-A (p = 0.021), although not with other angiogenesis markers examined (vascular proliferation index, glomeruloid microvascular proliferation, VEGF-C, VEGF-D or bFGF2 expression). Further, LCN2 was not associated with several EMT-related markers (E-cadherin, N-cadherin, P-cadherin, β-catenin), nor with vascular invasion (tumor cells invading lymphatic or blood vessels). Notably, LCN2 was significantly associated with distant tumor recurrences, as well as with the S100A family of metastasis related genes. Patients with tumors showing no LCN2 expression had the best outcome with 81% 5-year survival, compared to 73% for intermediate and 38% for the small subgroup with strong LCN2 staining (p = 0.007). In multivariate analysis, LCN2 expression was an independent prognostic factor in addition to histologic grade and FIGO stage. Conclusion: Increased LCN2 expression is associated with aggressive features and poor prognosis in endometrial cancer

Nanoparticle elasticity directs tumor uptake

To date, the role of elasticity in drug delivery remains elusive due to the inability to measure microscale mechanics and alter rheology without affecting chemistry. Herein, we describe the in vitro cellular uptake and in vivo tumor uptake of nanolipogels (NLGs). NLGs are composed of identical lipid bilayers encapsulating an alginate core, with tunable elasticity. The elasticity of NLGs was evaluated by atomic force microscopy, which demonstrated that they exhibit Young’s moduli ranging from 45 ± 9 to 19,000 ± 5 kPa. Neoplastic and non-neoplastic cells exhibited significantly greater uptake of soft NLGs (Young’s modulus 13.8 MPa). In an orthotopic breast tumor model, soft NLGs accumulated significantly more in tumors, whereas elastic NLGs preferentially accumulated in the liver. Our findings demonstrate that particle elasticity directs tumor accumulation, suggesting that it may be a design parameter to enhance tumor delivery efficiency

Host non-inflammatory neutrophils mediate the engraftment of bioengineered vascular networks

Notwithstanding remarkable progress in vascular network engineering, implanted bioengineered microvessels largely fail to form anastomoses with the host vasculature. Here, we demonstrate that implants containing assembled human vascular networks (A-Grafts) fail to engraft due to their inability to engage non-inflammatory host neutrophils upon implantation into mice. In contrast, unassembled vascular cells (U-Grafts) readily engage alternatively polarized neutrophils, which in turn serve as indispensable mediators of vascular assembly and anastomosis. The depletion of host neutrophils abrogated vascularization in U-Grafts, whereas an adoptive transfer of neutrophils fully restored vascularization in myeloid-depleted mice. Neutrophil engagement was regulated by secreted factors and was progressively silenced as the vasculature matured. Exogenous addition of factors from U-Grafts reengaged neutrophils and enhanced revascularization in A-Grafts, a process that was recapitulated by blocking Notch signaling. Our data suggest that the pro-vascularization potential of neutrophils can be harnessed to improve the engraftment of bioengineered tissues

Inhibiting Metastatic Breast Cancer Cell Migration via the Synergy of Targeted, pH-triggered siRNA Delivery and Chemokine Axis Blockade

Because breast cancer patient survival inversely correlates with metastasis, we engineered vehicles to inhibit both the C-X-C chemokine receptor type 4 (CXCR4) and lipocalin-2 (Lcn2) mediated migratory pathways. pH-responsive liposomes were designed to protect and trigger the release of Lcn2 siRNA. Liposomes were modified with anti-CXCR4 antibodies to target metastatic breast cancer (MBC) cells and block migration along the CXCR4-CXCL12 axis. This synergistic approach—coupling the CXCR4 axis blockade with Lcn2 silencing—significantly reduced migration in triple-negative human breast cancer cells (88% for MDA-MB-436 and 92% for MDA-MB-231). The results suggested that drug delivery vehicles engineered to attack multiple migratory pathways may effectively slow progression of MBC