Periostin and CCN2 Scaffolds Promote the Wound Healing Response in the Skin of Diabetic Mice.

IMPACT STATEMENT: Nonhealing skin wounds remain a significant burden on health care systems, with diabetic patients 20 times as likely to undergo a lower extremity amputation due to impaired healing. Novel treatments that suppress the proinflammatory signature and induce the proliferative and remodeling phases are needed clinically. We demonstrate that the addition of periostin and CCN2 in a scaffold form increases closure rates of full-thickness skin wounds in diabetic mice, concomitant with enhanced angiogenesis. Our results demonstrate the efficacy of periostin- and CCN2-containing biomaterials to stimulate wound closure, which could represent a novel method for the treatment of diabetic skin wounds.

Fabricating surfaces with distinct geometries and different combinations of cell adhesion proteins.

A step-by-step procedure is described for functionalizing the surface of a glass coverslip so that a single cell contacts distinct patterns of extracellular matrix and cell-cell adhesion proteins. This dual-micropatterned substratum is accomplished through a two-step process. First, extracellular matrix (ECM) is microcontact-printed onto a silanized glass surface using electron beam lithography, etched resist-coated wafers, and Polydimethylsiloxane (PDMS) stamps of differing geometries. Then, non-ECM-coated surfaces are incubated sequentially with biotin, NeutrAvidin, and biotinylated Protein A to attach Fc-cadherin fusion proteins, Fc, or PEG. Cells are seeded at low density onto the functionalized surface for single-cell analysis of protein recruitment/turnover and cellular motility.

99mTc-labeled tricarbonyl his-CNA35 as an imaging agent for the detection of tumor vasculature.

UNLABELLED: Given the importance of angiogenesis for a tumor's survival and growth, several therapeutic strategies rely on the selective inhibition of angiogenesis and the destruction of existing tumor vasculature. These strategies raise the need for a noninvasive tool to evaluate tumor vasculature. We describe the radiosynthesis and evaluation of an imaging tracer that specifically binds tumor subendothelial collagen and thereby images tumor vasculature. METHODS: (99m)Tc-tricarbonyl was prepared and labeled with His-collagen-binding adhesion protein 35 (CNA35). After in vitro specificity testing, in vivo biodistribution and dosimetric studies were performed in healthy nude mice via planar imaging. (99m)Tc-(CO)(3) His-CNA35 was evaluated for in vivo imaging of tumor vasculature in a HT29 colorectal carcinoma xenograft. RESULTS: The labeling procedure yielded a compound with 95%-99% radiochemical purity and good in vitro stability. An in vitro binding test confirmed specificity and functionality. (99m)Tc-(CO)(3) His-CNA35 rapidly cleared from the blood and predominantly accumulated in the kidneys and liver. The effective dose for a proposed single injection of 500 MBq of (99m)Tc-(CO)(3) His-CNA35 is 3.70 mSv per organ or 2.01 mSv/g of tissue. Tumors were successfully visualized, and uptake correlated with ex vivo immunohistochemical staining of tumor vasculature. CONCLUSION: (99m)Tc-(CO)(3) His-CNA35 may be a useful radioligand for the in vivo detection of tumor vasculature through subendothelial collagen binding. A noninvasive method of imaging tumor vasculature that could provide a reliable assessment of tumor vasculature would allow evaluation of the effectiveness of commonly used antiangiogenic therapies and determination of their optimal dosing and scheduling.

Molecular design of bioconjugated cell adhesion peptide with a water-soluble polymeric modifier for enhancement of antimetastatic effect.

The adhesive interaction of tumor cells with various components of the extracellular matrix (ECM), such as laminin and fibronectin appears to play a crucial role in tumor metastasis. It has been reported that adhesive peptides, such as Tyr-Ile-Gly-Ser-Arg (YIGSR) in laminin and Arg-Gly-Asp (RGD), inhibited adhesion and invasion of various tumor cells to ECM in vitro, and exhibited inhibitory effects on pulmonary metastasis of B16-BL6 melanoma cells in mice. However, large doses of these peptides were required for significant anti-metastatic effects in vivo, probably due to their rapid degradation by various peptidases and their rapid excretion from the blood into the urine. To overcome these problems, the development of an appropriate drug delivery system (DDS) is required to improve in vivo stability and prolong plasma half-lives. Several strategies such as peptide-cyclization and D-amino acid substation have been reported to improve stability in blood by inhibiting enzymatic degradation. However, even these approaches have proven insufficient to overcome rapid renal clearance from the circulation. On the other hand, bioconjugation with water-soluble polymeric modifiers could markedly prolong the plasma half-lives by not only increasing peptidase resistance but also impeding renal excretion. In addition, it is possible to strictly control the in vivo pharmacokinetics of a peptide by introducing functional molecules with targeting or slow release capacities to the polymeric modifier. In this review we demonstrate with reference to our recent studies that bioconjugation of adhesive peptides with the appropriate polymeric modifier can enhance antimetastatic activity and may facilitate therapeutic use.

Synthesis, conformational analysis and biological activities of lanthionine analogs of a cell adhesion modulator.

Cell adhesion is critical for many biological processes, such as hemostasis, wound healing, tumor metastasis and inflammation. Integrins are important mediators of cell adhesion. The integrin alpha4beta1, also known as VLA-4, is a cell surface receptor involved in inflammation. A cyclic peptide, 1-FCA-Arg-c[Cys-Asp-Thz-Cys]-OH, is a potent antagonist to VLA-4 with an IC50 of 2.4 nM. In the current study, we synthesized the lanthionine analogs of 1-FCA-Arg-c[Cys-Asp-Thz-Cys]-OH and determined the conformations of both the parent compound and its lanthionine analog in solution by NMR and computer simulations. The lanthionine analog retains its selectivity to VLA-4 with high nanomolar potency. Both molecules adopt similar topological arrangements in their conformations, while some important differences remain in the sulfur bridge region, which may cause the difference in potency.

Inhibition of intercellular adhesion molecule 1-dependent biological activities by a synthetic peptide analog.

We have used a combination of hydropathy analysis of the intercellular adhesion molecule 1 (ICAM-1) sequence and dot-matrix comparison of the sequence with the homologous, but functionally distinct, protein myelin-associated glycoprotein to identify a putative functional binding region. One polar, and presumably surface-exposed, region of ICAM-1 showed no significant identity with myelin-associated glycoprotein. A synthetic peptide analog based on the sequence of this region (JF9) mimicked the inhibitory effects of the anti-ICAM-1 monoclonal antibody WEHI-CAM-1. These included inhibition of ICAM-1-dependent homotypic aggregation of Raji Burkitt lymphoma and phorbol-ester treated U937 cells at concentrations as low as 80 micrograms/ml (24 microM). In addition, at a concentration of 100 micrograms/ml, the peptide analog effectively inhibited cytotoxic cell activity, an ICAM-1-dependent effector function of the immune response. This simple method of sequence analysis may have general applicability to the identification of functional domains in homologous, but functionally distinct, proteins such as the translated products of gene families.