Efforts to improve bone response to biomaterials have focused on ligands that bind ␣5␤1 integrins. However, antibodies to ␣5␤1 reduce osteoblast proliferation but do not affect differentiation when cells are grown on titanium (Ti). ␤1-silencing blocks the differentiation stimulus of Ti microtopography, suggesting that other ␤1 partners are important. Stably ␣2-silenced MG63 human osteoblast-like cells were used to test whether ␣2␤1 specifically mediates osteoblast response to Ti surface micron-scale structure and energy. WT and ␣2-silenced MG63 cells were cultured on tissue culture polystyrene (TCPS) and Ti disks with different surface microtopographies: machined pretreatment (PT) surfaces [mean peak to valley roughness (R a) < 0.02 m], PT surfaces that were grit-blasted and acid-etched (SLA; R a ‫؍‬ 4 m), and SLA with high surface energy (modSLA). Alkaline phosphatase (ALP), ␣2 and ␤1 mRNA, but not ␣5, ␣v, ␤3, type-I collagen, or osteocalcin, increased on SLA and modSLA at 6 days. ␣2 increased at 8 days on TCPS and PT, but remained unchanged on SLA and modSLA. ␣2-protein was reduced 70% in ␣2-siRNA cells, whereas ␣5-mRNA and protein were unaffected. ␣2-knockdown blocked surface-dependent increases in ␤1 and osteocalcin and decreases in cell number and increases in ALP and local factors typical of MG63 cells grown on SLA and modSLA [e.g., prostaglandin E 2, osteoprotegerin, latent and active TGF-␤1, and stimulatory effects of 1␣,25(OH)2D3 on these parameters]. This finding indicates that ␣2␤1 signaling is required for osteoblastic differentiation caused by Ti microstructure and surface energy, suggesting that conclusions based on cell behavior on TCPS are not predictive of behavior on other substrates or the mechanisms involved.␣-2 integrin siRNA ͉ MG63 human osteoblasts ͉ titanium surface roughness T itanium (Ti) and Ti alloys are commonly used as biomaterials because their surface properties provide a biocompatible interface with peri-implant tissues. Strategies for modifying the nature of this interface frequently involve changes to the surface, thereby affecting protein adsorption, cell-substrate interactions, and tissue development (1). A common modification has been to create micron-scale and submicron scale roughness. Preclinical and clinical studies (2-12) show that these surfaces support greater bone-to-implant contact than smooth surfaces.How surface microstructure promotes an osteogenic response is an important question, because bone-forming osteoblasts preferentially colonize bone surfaces that have been preconditioned by bone-resorbing osteoclasts (13), resulting in complex micron-scale and submicron-scale morphologies (14). In vitro experiments using model surfaces indicate that migration, growth, and colony morphology of rat bone marrow cells (15) and osteoblasts (16-18) are sensitive to microstructure. These observations suggest that structural elements can modulate the spatial organization of cells and their ECM.The topography of osteoclast resorption pits in bone can be modeled by using Ti subs...

Mesenchymal stem cells (MSCs) express an osteoblastic phenotype when treated with BMP-2, and BMP-2 is used clinically to induce bone formation although high doses are required. Pulsed electromagnetic fields (PEMF) also promote osteogenesis in vivo, in part through direct action on osteoblasts. We tested the hypothesis that PEMF enhances osteogenesis of MSCs in the presence of an inductive stimulus like BMP-2. Confluent cultures of human MSCs were grown on calcium phosphate disks and were treated with osteogenic media (OM), OM containing 40 ng/mL rhBMP-2, OM þ PEMF (8 h/day), or OM þ BMP-2 þ PEMF. MSCs demonstrated minor increases in alkaline phosphatase (ALP) during 24 days in culture and no change in osteocalcin. OM increased ALP and osteocalcin by day 6, but PEMF had no additional effect at any time. BMP-2 was stimulatory over OM, and PEMF þ BMP-2 synergistically increased ALP and osteocalcin. PEMF also enhanced the effects of BMP-2 on PGE2, latent and active TGF-b1, and osteoprotegerin. Effects of PEMF on BMP-2-treated cells were greatest at days 12 to 20. These results demonstrate that PEMF enhances osteogenic effects of BMP-2 on MSCs cultured on calcium phosphate substrates, suggesting that PEMF will improve MSC response to BMP-2 in vivo in a bone environment. ß

A pro-angiogenic role for Jagged-dependent activation of Notch signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of DLL-class and JAG-class ligand/receptor interactions, and developed Notch decoys that function as ligand-specific Notch inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro and retinal angiogenesis, demonstrating JAG-dependent Notch signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG/NOTCH signaling uniquely inhibited expression of anti-angiogenic sVEFGFR-1/sFlt-1. N11-13 decoy interfered with DLL1/DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis and in tumors. Thus, blockade of JAG- or DLL-mediated Notch signaling inhibits angiogenesis by distinct mechanisms. JAG/Notch signaling positively regulates angiogenesis by suppressing sVEGFR-1/sFlt-1 and promoting mural/endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth.

Background: ER␣36 is present in ER␣-negative breast cancer and mediates rapid responses. Results: Estrogen promoted cell survival and increased metastatic factors in breast cancer through membrane ER␣36. Conclusion: ER␣36 plays a major role in estrogen responses of ER␣-negative breast cancers. Significance: Examining the role of ER␣36 in ER␣-negative breast cancer is essential for understanding the negative effects of estrogen in breast cancer.Protein kinase C (PKC) signaling can be activated rapidly by 17␤-estradiol (E 2 ) via nontraditional signaling in ER␣-positive MCF7 and ER␣-negative HCC38 breast cancer cells and is associated with tumorigenicity. Additionally, E 2 has been shown to elicit anti-apoptotic effects in cancer cells counteracting pro-apoptotic effects of chemotherapeutics. Supporting evidence suggests the existence of a membraneassociated ER that differs from the traditional receptors, ER␣ and ER␤. Our aim was to identify the ER responsible for rapid PKC activation and to evaluate downstream effects, such as proliferation, apoptosis, and metastasis. RT-PCR, Western blot, and immunofluorescence were used to determine the presence of ER splice variants in multiple cell lines. E 2 effects on PKC activity were measured with and without ER-blocking antibodies. Cell proliferation was determined by [ 3 H]thymidine incorporation, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, (MTT) whereas apoptosis was determined by DNA fragmentation and TUNEL. Quantitative RT-PCR and sandwich ELISA were used to determine the effects on metastatic factors. The role of membrane-dependent signaling in cancer cell invasiveness was examined using an in vitro assay. The results indicate the presence of an ER␣ splice variant, ER␣36, in ER␣-positive MCF7 and ER␣-negative HCC38 breast cancer cells, which localized to plasma membranes and rapidly activated PKC in response to E 2 , leading to deleterious effects such as enhancement of proliferation, protection against apoptosis, and enhancement of metastatic factors. These findings propose ER␣36 as a novel target for the development of therapies that can prevent progression of breast cancer in the primary tumor as well as during metastasis.The complexities of breast cancer growth and metastasis present several problems in development of treatments for patients. The main screening process in determining the treatment and prognosis of breast cancer patients is receptor status. Growth of estrogen receptor (ER) 2 -positive breast cancers is typically enhanced by estrogen, but ER interactions with DNA are not necessary for this growth to occur (1, 2), suggesting that non-nuclear actions of ERs may play a role. Triple negative breast cancers, which are ER-negative, progesterone receptornegative, and human epidermal growth factor receptor 2 (HER2)-negative, are typically characterized as more aggressive and less responsive to hormone treatments (3). These tumors are also less responsive to treatments such as tamoxifen, a commonly used ...