Seprase is a homodimeric 170 kDa integral membrane gelatinase whose expression correlates with the invasiveness of the human melanoma cell line LOX. Here, we report the molecular cloning of a cDNA that encodes the 97 kDa subunit of seprase. Its deduced amino acid sequence predicts a type II integral membrane protein with a cytoplasmic tail of 6 amino acids, followed by a transmembrane domain of 20 amino acids and an extracellular domain of 734 amino acids. The carboxyl terminus contains a putative catalytic region (approximately 200 amino acids) which is homologous (68% identity) to that of the nonclassical serine protease dipeptidyl peptidase IV (DPPIV). The conserved serine protease motif G-X-S-X-G is present as G-W-S-Y-G. However, sequence analysis of seprase cDNA from LOX and other cell lines strongly suggests that seprase and human fibroblast activation protein alpha (FAP alpha) are products of the same gene. We propose that seprase/FAP alpha and DPPIV represent a new subfamily of serine integral membrane proteases (SIMP).

Dipeptidyl peptidase IV (DPP4/CD26) and seprase/fibroblast activation protein A are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH 2 terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo-and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices.

Abstract. In order to investigate the mechanism by which oligodendrogliomas cause neuronal damage, media conditioned by G26/24 oligodendroglioma cells, were fractionated into shed vesicles and vesicle-free supernatants, and added to primary cultures of rat fetal cortical neurons. After one night treatment with vesicles, a reproducible, dose-dependent, inhibitory effect on neurite outgrowth was already induced and, after 48-72 h of incubation, neuronal apoptosis was evident. Vesicle-free supernatants and vesicles shed by NIH-3T3 cells had no inhibitory effects on neurons. Western blot analyses showed that treated neurons expressed a decreased amount of neurofilament (NF), growth-associated protein (GAP-43) and microtubule-associated protein (MAP-2). Moreover procaspase-3 and -8 were activated while Bcl-2 expression was reduced. Vesicles were found positive for the proapoptotic molecule, Fas-ligand (Fas-L), and for the B isoform of Nogo protein, a myelin component with inhibitory effects on neurons. Nogo B involvement in the vesicle effects was analyzed both by testing the neutralizing capability of anti-Nogo antibodies and by removing the Nogo receptor from neurons by phospholipase C digestion. These treatments did not revert the vesicle effects. To test the role of Fas-L, vesicles were treated with functional anti-Fas-L monoclonals. Vesicle inhibitory and proapoptotic effects were reduced. Vesicles shed by ovarian carcinoma cells (OvCa), which are known to vehicle biologically active Fas-L, had similar effects on neurons to those of oligodendroglioma vesicles, and their inhibitory effects were also reduced by anti Fas-L antibodies. We therefore conclude that vesicles shed by G26/24 cells induce neuronal apoptosis at least partially by a Fas-L mediated mechanism.

Medical devices, such as silicone-based prostheses designed for soft tissue implantation, often induce a suboptimal foreign-body response which results in a hardened avascular fibrotic capsule around the device, often leading to patient discomfort or implant failure. Here, it is proposed that additive manufacturing techniques can be used to deposit durable coatings with multiscale porosity on soft tissue implant surfaces to promote optimal tissue integration. Specifically, the "liquid rope coil effect", is exploited via direct ink writing, to create a controlled macro open-pore architecture, including over highly curved surfaces, while adapting atomizing spray deposition of a silicone ink to create a microporous texture. The potential to tailor the degree of tissue integration and vascularization using these fabrication techniques is demonstrated through subdermal and submuscular implantation studies in rodent and porcine models respectively, illustrating the implant coating's potential applications in both traditional soft tissue prosthetics and active drug-eluting devices.