Microvascular tissue as a platform technology to modify the local microenvironment and influence the healing cascade.

Aims: Profiling of microvascular tissue allows identification of components that stimulate wound healing. Here we study those elements for biological effect and establish clinical proof-of-concept using a microvascular tissue graft (mVASC®) in chronic refractory wounds. Methods: mVASC was characterized for tissue fragments and protein composition, evaluated for angiogenic potential in preclinical models, and applied clinically to a series of nonhealing wounds with compromised vascularity of different etiologies. Results: mVASC increased endothelial cell migration in vitro and angiogenesis in mouse ingrowth and hindlimb ischemia models. Clinically, mVASC stimulated wound neovascularization, granulation and epithelialization, and complete and durable healing. Conclusion: Microvascular tissue contains elements relevant to tissue repair and can be clinically applied to enable or accelerate the closure of challenging wounds.

Evaluation of a collagen-coated, resorbable fiber scaffold loaded with a peptide basic fibroblast growth factor mimetic in a sheep model of rotator cuff repair.

BACKGROUND: A new scaffold design combined with a peptide growth factor was tested prospectively for safety and for improved tendon healing in sheep. METHODS: The infraspinatus tendon was detached and then surgically repaired to the humerus using sutures and anchors in 50 adult sheep. The repairs in 40 of these sheep were reinforced with a scaffold containing F2A, a peptide mimetic of basic fibroblast growth factor. The sheep were examined after 8 or 26 weeks with magnetic resonance imaging, full necropsy, and histopathologic analysis. A second cohort of 30 sheep underwent surgical repair--20 with scaffolds containing F2A. The 30 shoulders were tested mechanically after 8 weeks. RESULTS: The scaffold and F2A showed no toxicity. Scaffold-repaired tendons were 31% thicker than surgically repaired controls (P = .037) at 8 weeks. There was more new bone formed at the tendon footprint in sheep treated with F2A. Surgically repaired tendons delaminated from the humerus across 14% of the footprint area. The extent of delamination decreased to 1.3% with increasing doses of F2A (P = .004). More of the repair tissue at the footprint was tendon-like in the peptide-treated sheep. On mechanical testing, only 7 shoulders tore at the repair site. The repairs in the other 23 shoulders were already stronger than the midsubstance tendon at 8 weeks. CONCLUSIONS: The new scaffold and peptide safely improved tendon healing.

Glioma cell integrin expression and their interactions with integrin antagonists: Research Article.

A panel of human glioma cell explants was screened for integrin expression by flow cytometry using α(ν)ß-specific antibodies. A lower percentage of the glioma cells were positive for the α(ν)ß3 (mean % positive = 20.8%) integrin, whereas higher percentages were positive for the ανß5 (mean % positive = 72.7%), VLA5α (mean % positive = 87%) and VLAß1 (mean % positive = 41.7%) integrins. A series of RGD peptides was designed, synthesized and tested for binding to integrin receptors. Based on the results of the binding to the isolated integrin receptors and the expression of integrins on glioma cell lines, a peptide that binds potently to the α(ν)ß3, α(ν)ß5 and α(5)ß(1) was selected for further investigations with regards to its effect on glioma cells. The peptide, Ac-c[(Pen)-Tyr(Me)-Ala-Arg-Gly-Asp-Asn-Tic-Cys]NH(2) (RGD peptide), exhibited high potential for use in clinical intracranial administration since it had good stability in rat brain cell homogenates placed into artificial cerebrospinal fluid. Using an HPLC method for quantification of peptides in rat brain cell homogenates, we could demonstrate the half-life of the RGD peptide approximated 20 hr. Relative to a scrambled peptide control (non-RGD sequence, same amino acids), the experimental RGD peptide significantly decreased glioma cell proliferation of the entire panel of rat and human glioma cells tested. Adhesion of recently passaged glioma cells to glioma-derived extracellular matrix protein-coated plates was inhibited significantly by the RGD peptide. The peptide also reversed attachment of plated glioma cells. The RGD peptide caused some, but not substantial, glioma cell injury, as evidenced by a quantitative in vitro nuclear DNA morphologic assay and by a flow cytometric assay employing 7-amino actinomycin D (7AAD). We histologically monitored for toxicity caused by various doses of the RGD peptide infused repeatedly into normal cannulated rat brain. At safe doses, the experimental RGD peptide-treated brains did not show significant differences from those infused with scrambled peptide or buffer-treated controls. In tumor-bearing brains, slightly smaller tumor areas were measured with a higher necrotic-to-tumor index in the RGD peptide treated relative to the scrambled peptide-treated controls. This was obtained with intracranial peptide administrations or combined intracranial and intraperitoneal injections. From this in vitro work, we conclude that the anti-glioma effects of the RGD peptide tested resulted from lowered glioma proliferation and adhesion/mobility, rather than from significant glioma cell injury in the timeframe analyzed. Although other mechanisms not discerned from our limited histopathological observations may be operational, from our in vivo work, we conclude that repeated administration of RGD peptide into brain is safe but that better delivery of the peptides to infiltrating tumor cells is necessary.

Incorporation of thioether building blocks into an alphavbeta3-specific RGD peptide: synthesis and biological activity.

We report the design, synthesis, and binding affinities of a family of thioether analogues of the alpha(v)beta(3)-specific compound c[(Mpa)RGDD(tBuG)C]-NH(2). The synthesis of the thioether building blocks is scalable and produced the desired products in good yields. The linear peptides were synthesized on solid supports, followed by cyclization in solution. Our analogues demonstrate interesting binding data to the isolated receptors. In particular, the peptide c[NH-Arg-Gly-Asp-Asp-(tBuG)-Cys(S-CH(2)-CO)]NH(2) (1) exhibits differences in binding when compared to the parent compound and demonstrates potent affinity to the alpha(v)beta(3) and alpha(5)beta(1) receptors while having reduced binding to the alpha(IIb)beta(3) receptor. This result combined with the replacement of the disulfide with a thioether makes this compound interesting for further development.

Incorporation of (2S,3S) and (2S,3R) beta-methyl aspartic acid into RGD-containing peptides.

We report the synthesis and biological activity of a series of side-chain-constrained RGD peptides containing the (2S,3R) or (2S,3S) beta-methyl aspartic acid within the RGD sequence. These compounds have been assayed for binding to the integrin receptors alpha(IIb)beta3 and alpha(v)beta3 and the results demonstrate the importance of the side-chain orientation of this particular residue within the RGD sequence. Based on our findings, the (2S,3S) beta-methylated analogues of our RGD sequences maintain their binding potency to the integrin receptors while the (2S,3R) beta-methylated analogues exhibit a drastically reduced binding affinity. Our studies demonstrate that the three-dimensional orientation of the aspartyl side chain is a very important parameter for integrin binding and that small changes that affect the side-chain orientations give rise to drastic changes in binding affinity. These results provide important information for the design of more potent RGD mimetics.

Receptor-bound conformation of an alpha(5)beta(1) integrin antagonist by (15)N-edited 2D transferred nuclear overhauser effects.

We report the results of (15)N-edited 2D transferred NOE experiments of the partially (15)N-labeled alpha(5)beta(1) antagonist c[Mpa(15)N-Arg-(15)N-Gly-(15)N-Asp-(15)N-Asp-(15)N-Val-Cys]-NH(2) (Mpa denotes mercaptopropionic acid) in the presence of the native alpha(5)beta(1) receptor. The alpha(5)beta(1) integrin receptor is believed to be involved in tumor metastasis and the rational design of alpha(5)beta(1) integrin antagonist is therefore of considerable interest. Our experiments provide insight into the alpha(5)beta(1) receptor-bound conformation of the antagonist c[MpaRGDDVC]-NH2 and will be important for the design of novel antagonists.

Studies of the receptor-bound conformation of alphaIIbbeta3 antagonists by 15N-edited NMR spectroscopy.

We report the results of NMR studies and computer simulations of potent antagonists reflective of the alpha(IIb)beta(3) receptor-bound conformations. The peptides c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Phe(4)-(15)N-Arg(5)-Cys]-NH(2) (Phe-Arg analog) (Mpa: 3-mercaptopropionic acid) and c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Asp(4)-(15)N-Val(5)-Cys]-NH(2) (Asp-Val analog) were subjected to (15)N-edited NMR experiments to study the conformations of these peptides in the absence and in the presence of alpha(IIb)beta(3) receptor. The NMR studies of the Phe-Arg analog, a selective alpha(IIb)beta(3) antagonist, resulted in distinctly different experimental data in the presence and absence of the receptor. The computer simulations for this peptide resulted in one large family of structures consistent with the experimental data. This conformation suggests a type I beta-turn spanning residues Arg(1) and Gly(2) when bound to the receptor and we were able to establish a model for the three dimensional arrangement of the pharmacophores. The studies on the Asp-Val analog, an alpha(v)beta(3) antagonist that binds to the alpha(IIb)beta(3) with moderate affinity, resulted in conformations that are not as well defined as those for the Phe-Arg analog but are consistent with the model established for this analog. These results are important for the design of novel alpha(IIb)beta(3) antagonists.