Use of a fibrin-based system for enhancing angiogenesis and modulating inflammation in the treatment of hyperglycemic wounds.

The complex pathophysiology of chronic ulceration in diabetic patients is poorly understood; diabetes-related lower limb amputation is a major health issue, which has limited effective treatment regimes in the clinic. This study attempted to understand the complex pathology of hyperglycemic wound healing by showing profound changes in gene expression profiles in wounded human keratinocytes in hyperglycemic conditions compared to normal glucose conditions. In the hyper-secretory wound microenvironment of hyperglycemia, Rab18, a secretory control molecule, was found to be significantly downregulated. Using a biomaterial platform for dual therapy targeting the two distinct pathways, this study aimed to resolve the major dysregulated pathways in hyperglycemic wound healing. To complement Rab18, and promote angiogenesis eNOS was also targeted, and this novel Rab18-eNOS therapy via a dynamically controlled 'fibrin-in-fibrin' delivery system, demonstrated enhanced wound closure, by increasing functional angiogenesis and reducing inflammation, in an alloxan-induced hyperglycemic preclinical ear ulcer model of compromised wound healing.

Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced endothelialization but does not reduce restenosis in a hypercholesterolemic model.

Although successful, drug-eluting stents require significant periods of dual anti-platelet therapy with a persistent risk of late stent thrombosis due to inhibition of re-endothelialization. Endothelial regeneration is desirable to protect against in-stent thrombosis. Gene-eluting stents may be an alternative allowing inhibition of neointima and regenerating endothelium. We have shown that adenoviral endothelial nitric oxide synthase (eNOS) delivery can result in significantly decreased neointimal formation and enhanced re-endothelialization. Here, we examined non-viral reporter and therapeutic gene delivery from a stent. We coated lipoplexes directly onto the surface of stents. These lipostents were then deployed in the injured external iliac artery of either normal or hypercholesterolemic New Zealand White rabbits and recovered after 28 days. Lipoplexes composed of lipofectin and a reporter lacZ gene or therapeutic eNOS gene were used. We demonstrated efficient gene delivery at 28 days post-deployment in the media (21.3±7.5%) and neointima (26.8±11.2%). Liposomal delivery resulted in expression in macrophages between the stent struts. This resulted in improved re-endothelialization as detected by two independent measures compared with vector and stent controls (P<0.05 for both). However, in contrast to viral delivery of eNOS, liposomal eNOS does not reduce restenosis rates. The differing cell populations targeted by lipoplexes compared with adenoviral vectors may explain their ability to enhance re-endothelialization without affecting restenosis. Liposome-mediated gene delivery can result in prolonged and localized transgene expression in the blood vessel wall in vivo. Furthermore, lipoeNOS delivery to the blood vessel wall results in accelerated re-endothelialization; however, it does not reduce neointimal formation.

An antibody fragment functionalized dendritic PEGylated poly(2-(dimethylamino)ethyl diacrylate) as a vehicle of exogenous microRNA.

The translation of interfering RNA to the clinic requires more effective delivery agents to enable safe and efficient delivery. The aim of this work was to create a multi-functional delivery agent using deactivation enhanced ATRP synthesis of poly(dimethylamino)ethyl methacrylate (pDMAEMA)-co-PEGMEA/PEGDA (pD-b-P/DA) with linear pDMAEMA as a macro-initiator. The pD-b-P/DA was characterized for its potential to bind synthetic microRNA mimics to form structures and reacted with antibody-derived fragments (Fabs) using Michael-type addition. Conjugation of antibody fragments was verified using SDS-PAGE. Functional delivery of these interfering RNA complexes was proven using a dual luciferase reporter assay. Functional silencing of a reporter gene was improved by complexation of microRNA mimics with pD-b-P/DA alone and with Fab-decorated pD-b-P/DA. The improved silencing with Fab-decorated pD-b-P/DA was evident at 48 h but disappeared at 96 h. The resultant agent enables complexation of nucleic acid (microRNA mimic) and facile conjugation of antibody fragments via a Michael-type addition. In conclusion, this platform is effective at silencing in this reporter system and has potential as an effective delivery system of interfering RNA.

Gene transfer into rat mesenchymal stem cells: a comparative study of viral and nonviral vectors.

Mesenchymal stem cells (MSCs) have been proposed for use in combinatorial gene and cell therapy protocols for the treatment of disease and promotion of repair. The efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression with minimal cell death. The study was carried out on bone-marrow derived rat MSCs, and a range of vectors was tested on the same stem cell preparation. Adenovirus, adeno-associated virus (AAV; serotypes 1, 2, 4, 5, and 6), lentivirus, and nonviral vectors were compared. Lentivirus proved to be most effective with transduction efficiencies of up to 95%, concurrent with low levels of cell toxicity. Adenovirus also proved effective, but a significant increase in cell death was seen with increasing viral titer. Rat MSCs remained refractory to transduction by all AAV serotypes, in contrast to rabbit MSCs tested at the same time. Lipofection of plasmid DNA gave moderate transfection levels but was also accompanied by cell death. Electroporative gene transfer proved ineffective at the parameters tested and resulted in high cell death. High and moderate levels of cell transduction using lentivirus vectors did not affect the ability of the cells to differentiate down the adipogenic pathway.

Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s).

Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (alpha EGFR ior egf/r3) against the human EGF receptor and gangliosides which are known to possess antiproliferative and anti-tyrosine kinase activity. The moderate growth-inhibitory effect of alpha EGFR ior egf/r3 was strongly potentiated by the addition of monosialoganglioside GM3. Likewise, the combination of alpha EGFR ior egf/r3 and GM3 inhibited EGF receptor autophosphorylation activity in H125 cells more strongly than either agent alone. A synergistic inhibition of EGF receptor autophosphorylation by alpha EGFR ior egf/r3 and GM3 was also observed in the human epidermoid carcinoma cell line A431. In both cell lines, the inhibition of EGF receptor autophosphorylation by GM3 was prevented by pretreatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases). Also, GM3 accelerated EGF receptor dephosphorylation in isolated A431 cell membranes. These findings indicate that GM3 has the capacity to activate EGF receptor-directed PTPase activity and suggest a novel possible mechanism for the regulation of cellular PTPases.

Association of SH2 domain protein tyrosine phosphatases with the epidermal growth factor receptor in human tumor cells. Phosphatidic acid activates receptor dephosphorylation by PTP1C.

The SH2 domain protein tyrosine phosphatases (PTPases) PTP1C and PTP1D were found associated with epidermal growth factor (EGF) receptor which was purified from A431 cell membranes by several steps of chromatography. Both PTPases also associated with the EGF receptor upon exposure of immunoprecipitated receptor to lysates of MCF7 mammary carcinoma cells. The associated PTPases had little activity toward the bound receptor when it was autophosphorylated in vitro. Receptor dephosphorylation could, however, be initiated by treatment of the receptor-PTPase complex with phosphatidic acid (PA). When autophosphorylated EGF receptor was exposed to lysates of PTP1C or PTP1D overexpressing 293 cells, the association of PTP1C but not of PTP1D was enhanced in the presence of PA. In intact A431 cells, an association of PTP1C and PTP1D with the EGF receptor was detectable by coimmunoprecipitation experiments. PA treatment reduced the phosphorylation state of ligand activated EGF receptors in A431 cells and in 293 cells overexpressing EGF receptors together with PTP1C but not in 293 cells overexpressing EGF receptors alone or together with PTP1D. We conclude that PTP1C but not PTP1D participates in dephosphorylation of activated EGF receptors. A possible role of PA for physiological modulation of EGF receptor signaling is discussed.