Enhanced potency of cell-based therapy for ischemic tissue repair using an injectable bioactive epitope presenting nanofiber support matrix.

The translation of cell-based therapies for ischemic tissue repair remains limited by several factors, including poor cell survival and limited target site retention. Advances in nanotechnology enable the development of specifically designed delivery matrices to address these limitations and thereby improve the efficacy of cell-based therapies. Given the relevance of integrin signaling for cellular homeostasis, we developed an injectable, bioactive peptide-based nanofiber matrix that presents an integrin-binding epitope derived from fibronectin, and evaluated its feasibility as a supportive artificial matrix for bone marrow-derived pro-angiogenic cells (BMPACs) used as a therapy in ischemic tissue repair. Incubation of BMPACs with these peptide nanofibers in vitro significantly attenuated apoptosis while enhancing proliferation and adhesion. Pro-angiogenic function was enhanced, as cells readily formed tubes. These effects were, in part, mediated via p38, and p44/p42 MAP kinases, which are downstream pathways of focal adhesion kinase. In a murine model of hind limb ischemia, an intramuscular injection of BMPACs within this bioactive peptide nanofiber matrix resulted in greater retention of cells, enhanced capillary density, increased limb perfusion, reduced necrosis/amputation, and preserved function of the ischemic limb compared to treatment with cells alone. This self-assembling, bioactive peptide nanofiber matrix presenting an integrin-binding domain of fibronectin improves regenerative efficacy of cell-based strategies in ischemic tissue by enhancing cell survival, retention, and reparative functions.

Bone marrow progenitor cell therapy-mediated paracrine regulation of cardiac miRNA-155 modulates fibrotic response in diabetic hearts.

Diabetes is associated with a higher incidence of myocardial infarction (MI) and increased risk for adverse vascular and fibrogenic events post-MI. Bone marrow-derived progenitor cell (BMPC) therapy has been shown to promote neovascularization, decrease infarct area and attenuate left ventricular (LV) dysfunction after MI. Unlike vascular effects, the anti-fibrosis mechanisms of BMPC, specifically under diabetic conditions, are poorly understood. We demonstrated that intramyocardial delivery of BMPCs in infarcted diabetic db/db mice significantly down-regulates profibrotic miRNA-155 in the myocardium and improves LV remodeling and function. Furthermore, inhibition of paracrine factor hepatocyte growth factor (HGF) signaling in vivo suppressed the BMPC-mediated inhibition of miR-155 expression and the associated protective effect on cardiac fibrosis and function. In vitro studies confirmed that the conditioned media of BMPC inhibited miR-155 expression and profibrotic signaling in mouse cardiac fibroblasts under diabetic conditions. However, neutralizing antibodies directed against HGF blocked these effects. Furthermore, miR-155 over-expression in mouse cardiac fibroblasts inhibited antifibrotic Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene, non-Alu-containing (SnoN) signaling and abrogated antifibrogenic response of HGF. Together, our data demonstrates that paracrine regulation of cardiac miRNAs by transplanted BMPCs contributes to the antifibrotic effects of BMPC therapy. BMPCs release HGF, which inhibits miR-155-mediated profibrosis signaling, thereby preventing cardiac fibrosis. These data suggest that targeting miR-155 might serve as a potential therapy against cardiac fibrosis in the diabetic heart.

Topical administration of allogeneic mesenchymal stromal cells seeded in a collagen scaffold augments wound healing and increases angiogenesis in the diabetic rabbit ulcer.

There is a critical clinical need to develop therapies for nonhealing diabetic foot ulcers. Topically applied mesenchymal stromal cells (MSCs) provide a novel treatment to augment diabetic wound healing. A central pathological factor in nonhealing diabetic ulcers is an impaired blood supply. It was hypothesized that topically applied allogeneic MSCs would improve wound healing by augmenting angiogenesis. Allogeneic nondiabetic bone-marrow derived MSCs were seeded in a collagen scaffold. The cells were applied to a full-thickness cutaneous wound in the alloxan-induced diabetic rabbit ear ulcer model in a dose escalation fashion. Percentage wound closure and angiogenesis at 1 week was assessed using wound tracings and stereology, respectively. The topical application of 1,000,000 MSCs on a collagen scaffold demonstrated increased percentage wound closure when compared with lower doses. The collagen and collagen seeded with MSCs treatments result in increased angiogenesis when compared with untreated wounds. An improvement in wound healing as assessed by percentage wound closure was observed only at the highest cell dose. This cell-based therapy provides a novel therapeutic strategy for increasing wound closure and augmenting angiogenesis, which is a central pathophysiological deficit in the nonhealing diabetic foot ulcer.

Growth differentiation factor-5 enhances in vitro mesenchymal stromal cell chondrogenesis and hypertrophy.

The regenerative potential for adult bone marrow-derived mesenchymal stromal cells (MSCs) has been extensively investigated in the setting of arthritic disease and focal cartilage defects. In vitro chondrogenic differentiation of MSCs is regularly accomplished by the widely used pellet culture system where MSCs are maintained in high-density pellets to mimic mesenchymal condensation during development. Supplementation of chondrogenic MSC pellet cultures with growth differentiation factor-5 (GDF-5), a highly regulated gene in the chondrogenic phase of endochondral ossification (EO), was investigated here under the hypothesis that GDF-5 will enhance the chondrogenic differentiation of MSCs, thereby supporting their entry into ossification. The supplementation of chondrogenic MSC pellets with the recombinant human GDF-5 protein significantly enhanced MSC chondrogenic differentiation, as demonstrated by enhanced collagen type II and sulfated glycosaminoglycan (GAG) incorporation into the extracellular matrix. Increased P-SMADs 1-5-8 were observed in pellets treated with GDF-5 and transforming growth factor (TGF)-ß 3 when compared to the pellets treated with TGF-ß 3 alone, demonstrated by immunostaining and western blot analysis of the chondrogenic pellet extract. A concurrent increase in alkaline phosphatase, collagen types I and X, and osteopontin secretion indicated a transition of these cultures to hypertrophy. Together, these data support the application of GDF-5 to enhance MSC chondrogenic differentiation and hypertrophy as a precursor to EO.

Autologous circulating angiogenic cells treated with osteopontin and delivered via a collagen scaffold enhance wound healing in the alloxan-induced diabetic rabbit ear ulcer model.

INTRODUCTION: Diabetic foot ulceration is the leading cause of amputation in people with diabetes mellitus. Peripheral vascular disease is present in the majority of patients with diabetic foot ulcers. Despite standard treatments there exists a high amputation rate. Circulating angiogenic cells previously known as early endothelial progenitor cells are derived from peripheral blood and support angiogenesis and vasculogenesis, providing a potential topical treatment for non-healing diabetic foot ulcers. METHODS: A scaffold fabricated from Type 1 collagen facilitates topical cell delivery to a diabetic wound. Osteopontin is a matricellular protein involved in wound healing and increases the angiogenic potential of circulating angiogenic cells. A collagen scaffold seeded with circulating angiogenic cells was developed. Subsequently the effect of autologous circulating angiogenic cells that were seeded in a collagen scaffold and topically delivered to a hyperglycemic cutaneous wound was assessed. The alloxan-induced diabetic rabbit ear ulcer model was used to determine healing in response to the following treatments: collagen seeded with autologous circulating angiogenic cells exposed to osteopontin, collagen seeded with autologous circulating angiogenic cells, collagen alone and untreated wound. Stereology was used to assess angiogenesis in wounds. RESULTS: The cells exposed to osteopontin and seeded on collagen increased percentage wound closure as compared to other groups. Increased angiogenesis was observed with the treatment of collagen and collagen seeded with circulating angiogenic cells. CONCLUSIONS: These results demonstrate that topical treatment of full thickness cutaneous ulcers with autologous circulating angiogenic cells increases wound healing. Cells exposed to the matricellular protein osteopontin result in superior wound healing. The wound healing benefit is associated with a more efficient vascular network. This topical therapy provides a potential novel therapy for the treatment of non-healing diabetic foot ulcers in humans.

Isolation of circulating angiogenic cells.

Endothelial progenitor cells (EPCs) were first identified by Ashara et al. in 1997 (Asahara et al. Science 275:964-967, 1997) and were thought to contribute to angiogenesis and vasculogenesis. Since their discovery, circulating levels of EPCs were found to serve as biomarkers as low levels correlate with increased cardiovascular events and death from cardiovascular causes (Werner et al. N Engl J Med 353:999-1007, 2005; Fadini et al. J Am Coll Cardiol 45:1449-1457, 2005; Hill et al. N Engl J Med 348:593-600, 2003; Schmidt-Lucke et al. Circulation 111:2981-2987, 2005). Additionally, EPC dysfunction has been associated with diabetes mellitus and other disease states. However, recently there has been a great deal of controversy in the field over the exact definition and function of an EPC. To help classify EPCs, they have been divided into two distinct groups (1) circulating angiogenic cells (also referred to as early EPCs) and (2) endothelial colony forming cells (also referred to as late outgrowth EPCs). Circulating angiogenic cells are believed to represent a cell population enriched in monocytes and exert their angiogenic effects via paracrine and signaling mechanisms whereas endothelial colony forming cells are true EPCs and may enhance angiogenesis and vasculogenesis by incorporating into the newly forming vessels. Here the isolation and identification of circulating angiogenic cells are described.

Comparison of viral and nonviral vectors for gene transfer to human endothelial progenitor cells.

BACKGROUND/AIMS: The ability of endothelial progenitor cells (EPCs) to home to sites of neoangiogenesis makes them attractive candidates for use in the field of gene therapy. The efficacy of this approach depends on the efficiency of the vector used for transgene delivery. METHODS/RESULTS: In this study, we have compared the efficiency of adenovirus, five serotypes of AAV2, VSVG-pseudotyped lentivirus, and nonviral plasmid/liposome DNA vectors to deliver the green fluorescence protein reporter gene to human early EPCs to determine efficacy and vector-related cell toxicity. Adenovirus proved most effective with efficiencies of up to 80% with low levels of cell death. Lower levels of expression were seen with other vectors. Electroporation proved unsuitable at the parameters tested. We have also identified at least two distinct subpopulations that exist in the heterogeneous parent EPC culture, one of which is amenable to transduction with adenovirus and one that is not. In addition, adenoviral transduction did not disrupt the ability of the cells to incorporate into endothelial structures in vitro. CONCLUSION: We have found adenovirus to be the most efficient of the vector systems tested for gene delivery to EPCs, an effect that is mediated almost entirely by one of two identified subpopulations.

Microtubule acetylation through HDAC6 inhibition results in increased transfection efficiency.

The success of viral and nonviral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm and reach the nucleus. We, as well as other researchers, have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While disruption of microtubules with nocodazole was shown to greatly inhibit cytoplasmic plasmid trafficking, it did not abolish it. It has been demonstrated that a pool of stabilized post-translationally acetylated microtubules exists in cells, and that this acetylation may play a role in protein trafficking. In order to determine whether this modification could account for the residual DNA trafficking in nocodazole-treated cells, we inhibited or knocked down the levels of the tubulin deacetylase, histone deacetylase 6 (HDAC6), thereby generating higher levels of acetylated microtubules. Electroporation of plasmids into cells with inhibited or silenced HDAC6 resulted in increased gene transfer. This increased transfection efficiency was not because of increased transcriptional activity, but rather, because of increased cytoplasmic trafficking. When plasmids were cytoplasmically microinjected into HDAC6-deficient cells, they entered the nucleus within 5 minutes of injection, almost 10 times faster than in wild-type cells. Taken together, these results suggest that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.

Intracellular trafficking of plasmids for gene therapy: mechanisms of cytoplasmic movement and nuclear import.

Under physiologically relevant conditions, the levels of non-viral gene transfer are low at best. The reason for this is that many barriers exist for the efficient transfer of genes to cells, even before any gene expression can occur. While many transfection strategies focus on DNA condensation and overcoming the plasma membrane, events associated with the intracellular trafficking of the DNA complexes have not been as extensively studied. Once internalized, plasmids must travel potentially long distances through the cytoplasm to reach their next barrier, the nuclear envelope. This review summarizes the current progress on the cytoplasmic trafficking and nuclear transport of plasmids used for gene therapy applications. Both of these processes utilize specific and defined mechanisms to facilitate movement of DNA complexes through the cell. The continued elucidation and exploitation of these mechanisms will lead to improved strategies for transfection and successful gene therapy.

Intracellular trafficking of plasmids during transfection is mediated by microtubules.

Little is known about how plasmids move through the cytoplasm to the nucleus. It has been suggested that the dense latticework of the cytoskeleton impedes free diffusion of large macromolecules, including DNA. However, since transfections do work, there must be mechanisms by which DNA circumvents cytoplasmic obstacles. One possibility is that plasmids become cargo on cytoskeletal motors, much like viruses do, and move to the nucleus in a directed fashion. Using microinjection and electroporation approaches in the presence of drugs that alter the dynamics and organization of the cytoskeleton, we show that microtubules are involved in plasmid trafficking to the nucleus. Further, by co-injecting inhibitory antibodies, we find that dynein likely facilitates this movement. These results were confirmed using an in vitro spin-down assay that demonstrated that plasmids bind to microtubules through adaptor proteins provided by cytoplasmic extracts. Taken together, these results suggest that plasmids, like most viruses, utilize the microtubule network and its associated motor proteins to traffic through the cytoplasm to the nucleus.

Bacitracin: substantiation and elimination of contaminating proteolytic activity and use as an affinity chromatography ligand to purify a siderophore-degrading enzyme.

Bacitracin is a commercial general peptidase inhibitor that may be used to purify proteases. Significant protease contamination of a commercial bacitracin preparation was noted and four procedures were developed to overcome the contamination. Dialysis, gel-filtration chromatography, molecular weight cutoff filters, and heat inactivation were effective, resulting in the diminution or elimination of proteolysis while maintaining the inhibitory effect of bacitracin. Attachment of bacitracin to an affinity chromatography resin did not immobilize a siderophore-degrading enzyme, as has been noted with peptidases. It did, however, result in its partial purification from some of the contaminating proteins originally present.