Gangrene, revascularization, and limb function improved with E-selectin/adeno-associated virus gene therapy.

OBJECTIVE: Novel therapeutic angiogenic concepts for critical limb ischemia are still needed for limb salvage. E-selectin, a cell-adhesion molecule, is vital for recruitment of the stem/progenitor cells necessary for neovascularization in ischemic tissues. We hypothesized that priming ischemic limb tissue with E-selectin/adeno-associated virus (AAV) gene therapy, in a murine hindlimb ischemia and gangrene model, would increase therapeutic angiogenesis and improve gangrene. METHODS: FVB/NJ mice were given intramuscular hindlimb injections of either E-selectin/AAV or LacZ/AAV and then underwent induction of gangrene via femoral artery ligation and concomitant systemic injections of the nitric oxide synthesis inhibitor L-NAME (L-NG-Nitro arginine methyl ester; 40 mg/kg). Gangrene was evaluated via the Faber hindlimb appearance score. The rate of ischemic limb reperfusion and ischemic tissue angiogenesis were evaluated using laser Doppler perfusion imaging and DiI perfusion with confocal laser scanning microscopy of the ischemic footpads, respectively. The treadmill exhaustion test was performed on postoperative day (POD) 8 to determine hindlimb functionality. RESULTS: The E-selectin/AAV-treated mice (n = 10) had decreased Faber ischemia scores compared with those of the LacZ/AAV-treated mice (n = 7) at both PODs 7 and 14 (P < .05 and P < .01, respectively), improved laser Doppler perfusion imaging reperfusion indexes by POD 14 (P < .01), and greater gangrene footpad capillary density (P < .001). E-selectin/AAV-treated mice also had improved exercise tolerance (P < .05) and lower relative muscular atrophy (P < .01). CONCLUSION: We surmised that E-selectin/AAV gene therapy would significantly promote hindlimb angiogenesis, reperfusion, and limb functionality in mice with hindlimb ischemia and gangrene. Our findings highlight the reported novel gene therapy approach to critical limb ischemia as a potential therapeutic option for future clinical studies.

The effect of estrogen on diabetic wound healing is mediated through increasing the function of various bone marrow-derived progenitor cells.

OBJECTIVE: Endothelial progenitor cells (EPCs) are the key cells of postnatal neovascularization, and mesenchymal stem cells (MSCs) possess pluripotent differentiation capacity and contribute to tissue regeneration and wound healing. Both EPCs and MSCs are critical to the wound repair process, which is hindered in diabetes mellitus. Diabetes has been shown to decrease the function of these progenitor cells, whereas estrogen has beneficial wound healing effects. However, the role of estrogen in modulating EPC and MSC biology in diabetes is unknown. We investigated the effect of estrogen on improving bone marrow (BM)-derived EPC and MSC function using a murine diabetic wound healing model. METHODS: Female diabetic db+/db+ and nondiabetic control mice were wounded cutaneously and treated with topical estrogen or placebo cream. On day 5 after wounding, BM cells were harvested to quantify EPC number and colony-forming units of EPCs and MSCs. Wound healing rate was concurrently studied. Vessel density and scar density were then quantified using whole body perfusion and laser confocal microscopy. EPC recruitment was documented by immunohistochemistry to identify CD34- and vascular endothelial growth factor receptor 2-positive cells in the vessel wall. Data were analyzed by analysis of variance. RESULTS: Topical estrogen significantly increased colony-forming units of both EPCs and MSCs compared with placebo treatment, indicating improved viability and proliferative ability of these cells. Consistently, increased recruitment of EPCs to diabetic wounds and higher vessel density were observed in estrogen-treated compared with placebo-treated mice. Consequently, topical estrogen significantly accelerated wound healing as early as day 6 after wounding. In addition, scar density resulting from collagen deposition was increased in the estrogen-treated group, reflecting increased MSC activity and differentiation. CONCLUSIONS: Estrogen treatment increases wound healing and wound neovascularization in diabetic mice. Our data implicate that these beneficial effects may be mediated through improving the function of BM-derived EPCs and MSCs.

Intramuscular E-selectin/adeno-associated virus gene therapy promotes wound healing in an ischemic mouse model.

BACKGROUND: Poor wound healing in critical limb ischemia (CLI) is attributed to impaired neovascularization and reperfusion. Optimizing the ischemic wound with adhesion molecules that enhance stem cell homing may revolutionize treatment. The purpose of this study is to test the efficacy of adhesion molecule E-selectin on wound healing in an ischemic mouse wound. METHODS: Adult FVB/NJ mice underwent unilateral femoral artery and vein ligation to induce CLI. A 4-mm punch biopsy wound was created on the anterior thigh to simulate ischemic wounds. Intramuscular injection of adeno-associated virus (AAV) carrying either E-selectin (E-selectin/AAV, n = 11) or LacZ as control (LacZ/AAV, n = 10) was performed. Gross wound size was measured for 10 d postoperatively. Ischemic hindlimb reperfusion was quantified using laser Doppler imaging. Wound tissue neovascularization was visualized using DiI perfusion and confocal microscopy. E-selectin expression in wounds was verified by immunofluorescence. RESULTS: Immunofluorescence confirmed E-selectin/AAV delivery in treatment versus control limbs. Wounds from E-selectin/AAV mice versus controls revealed surface area healing of 54% versus 20% (P < 0.01) on postoperative day (POD) 1, 78% versus 51% on POD 4 (P < 0.01), and 97% versus 84% on POD 10 (P < 0.01). Laser Doppler imaging revealed greater reperfusion in E-selectin/AAV mice versus controls by POD 10 (0.49 versus 0.27, P < 0.05). DiI perfused ligated hindlimb in E-selectin/AAV versus control mice revealed mean neovascularization intensity score of 30 versus 18 (P < 0.05) on POD 10. CONCLUSIONS: Intramuscularly injected E-selectin/AAV gene therapy in mice with CLI significantly increases wound angiogenesis and limb reperfusion, expediting overall wound healing.

Solute carrier family 2 member 1 is involved in the development of nonalcoholic fatty liver disease.

UNLABELLED: Susceptibility to develop nonalcoholic fatty liver disease (NAFLD) has genetic bases, but the associated variants are uncertain. The aim of the present study was to identify genetic variants that could help to prognose and further understand the genetics and development of NAFLD. Allele frequencies of 3,072 single-nucleotide polymorphisms (SNPs) in 92 genes were characterized in 69 NAFLD patients and 217 healthy individuals. The markers that showed significant allele-frequency differences in the pilot groups were subsequently studied in 451 NAFLD patients and 304 healthy controls. Besides this, 4,414 type 2 diabetes mellitus (T2DM) cases and 4,567 controls were genotyped. Liver expression of the associated gene was measured and the effect of its potential role was studied by silencing the gene in vitro. Whole genome expression, oxidative stress (OS), and the consequences of oleic acid (OA)-enriched medium on lipid accumulation in siSLC2A1-THLE2 cells were studied by gene-expression analysis, dihydroethidium staining, BODIPY, and quantification of intracellular triglyceride content, respectively. Several SNPs of SLC2A1 (solute carrier family 2 [facilitated glucose transporter] member 1) showed association with NAFLD, but not with T2DM, being the haplotype containing the minor allele of SLC2A1 sequence related to the susceptibility to develop NAFLD. Gene-expression analysis demonstrated a significant down-regulation of SLC2A1 in NAFLD livers. Enrichment functional analyses of transcriptome profiles drove us to demonstrate that in vitro silencing of SLC2A1 induces an increased OS activity and a higher lipid accumulation under OA treatment. CONCLUSIONS: Genetic variants of SLC2A1 are associated with NAFLD, and in vitro down-regulation of this gene promotes lipid accumulation. Moreover, the oxidative response detected in siSLC2A1-THLE2 cells corroborated the antioxidant properties previously related to this gene and linked the most representative clinical characteristics of NAFLD patients: oxidative injury and increased lipid storage.

SNP-PHAGE: high-throughput SNP discovery pipeline.

High-throughput genotyping technologies have become popular in studies that aim to reveal the genetics behind polygenic traits such as complex disease and the diverse response to some drug treatments. These technologies utilize bioinformatics tools to define strategies, analyze data, and estimate the final associations between certain genetic markers and traits. The strategy followed for an association study depends on its efficiency and cost. The efficiency is based on the assumed characteristics of the polymorphisms' allele frequencies and linkage disequilibrium for putative casual alleles. Statistically significant markers (single mutations or haplotypes) that cause a human disorder should be validated and their biological function elucidated. The aim of this chapter is to present a subset of bioinformatics tools for haplotype inference, tag SNP selection, and genome-wide association studies using a high-throughput generated SNP data set.

Delineating genetic relationships among the Maya.

By 250 AD, the Classic Maya had become the most advanced civilization within the New World, possessing the only well-developed hieroglyphic writing system of the time and an advanced knowledge of mathematics, astronomy and architecture. Though only ruins of the empire remain, 7.5 million Mayan descendants still occupy areas of Mexico, Guatemala, Belize, El Salvador, and Honduras. Although they inhabit distant and distinct territories, speak more than 28 languages, and have been historically divided by warfare and a city-state-like political system, and they share characteristics such as rituals, artistic, architectural motifs that distinguish them as unequivocally Maya. This study was undertaken to determine whether these similarities among Mayan communities mirror genetic affinities or are merely a reflection of their common culture. Four Mayan populations were investigated (i.e., the K'iche and Kakchikel from Guatemala and the Campeche and Yucatan from Mexico) and compared with previously published populations across 15 autosomal STR loci. As a whole, the Maya emerge as a distinct group within Mesoamerica, indicating that they are more similar to each other than to other Mesoamerican groups. The data suggest that although geographic and political boundaries existed among Mayan communities, genetic exchanges between the different Mayan groups have occurred, supporting theories of extensive trading throughout the empire.