Identification of Genomic Predictors of Muscle Fiber Size.

The greater muscle fiber cross-sectional area (CSA) is associated with greater skeletal muscle mass and strength, whereas muscle fiber atrophy is considered a major feature of sarcopenia. Muscle fiber size is a polygenic trait influenced by both environmental and genetic factors. However, the genetic variants underlying inter-individual differences in muscle fiber size remain largely unknown. The aim of our study was to determine whether 1535 genetic variants previously identified in a genome-wide association study of appendicular lean mass are associated with the CSA of fast-twitch muscle fibers (which better predict muscle strength) in the m. vastus lateralis of 148 physically active individuals (19 power-trained and 28 endurance-trained females, age 28.0 ± 1.1; 28 power-trained and 73 endurance-trained males, age 31.1 ± 0.8). Fifty-seven single-nucleotide polymorphisms (SNPs) were identified as having an association with muscle fiber size (p < 0.05). Of these 57 SNPs, 31 variants were also associated with handgrip strength in the UK Biobank cohort (n = 359,729). Furthermore, using East Asian and East European athletic (n = 731) and non-athletic (n = 515) cohorts, we identified 16 SNPs associated with athlete statuses (sprinter, wrestler, strength, and speed-strength athlete) and weightlifting performance. All SNPs had the same direction of association, i.e., the lean mass-increasing allele was positively associated with the CSA of muscle fibers, handgrip strength, weightlifting performance, and power athlete status. In conclusion, we identified 57 genetic variants associated with both appendicular lean mass and fast-twitch muscle fiber size of m. vastus lateralis that may, in part, contribute to a greater predisposition to power sports.

A Biosafety Study of Human Umbilical Cord Blood Mononuclear Cells Transduced with Adenoviral Vector Carrying Human Vascular Endothelial Growth Factor cDNA In Vitro.

The biosafety of gene therapy remains a crucial issue for both the direct and cell-mediated delivery of recombinant cDNA encoding biologically active molecules for the pathogenetic correction of congenital or acquired disorders. The diversity of vector systems and cell carriers for the delivery of therapeutic genes revealed the difficulty of developing and implementing a safe and effective drug containing artificial genetic material for the treatment of human diseases in practical medicine. Therefore, in this study we assessed changes in the transcriptome and secretome of umbilical cord blood mononuclear cells (UCB-MCs) genetically modified using adenoviral vector (Ad5) carrying cDNA encoding human vascular endothelial growth factor (VEGF165) or reporter green fluorescent protein (GFP). A preliminary analysis of UCB-MCs transduced with Ad5-VEGF165 and Ad5-GFP with MOI of 10 showed efficient transgene expression in gene-modified UCB-MCs at mRNA and protein levels. The whole transcriptome sequencing of native UCB-MCs, UCB-MC+Ad5-VEGF165, and UCB-MC+Ad5-GFP demonstrated individual sample variability rather than the effect of Ad5 or the expression of recombinant vegf165 on UCB-MC transcriptomes. A multiplex secretome analysis indicated that neither the transduction of UCB-MCs with Ad5-GFP nor with Ad5-VEGF165 affects the secretion of the studied cytokines, chemokines, and growth factors by gene-modified cells. Here, we show that UCB-MCs transduced with Ad5 carrying cDNA encoding human VEGF165 efficiently express transgenes and preserve transcriptome and secretome patterns. This data demonstrates the biosafety of using UCB-MCs as cell carriers of therapeutic genes.

Induction of Angiogenesis by Genetically Modified Human Umbilical Cord Blood Mononuclear Cells.

Stimulating the process of angiogenesis in treating ischemia-related diseases is an urgent task for modern medicine, which can be achieved through the use of different cell types. Umbilical cord blood (UCB) continues to be one of the attractive cell sources for transplantation. The goal of this study was to investigate the role and therapeutic potential of gene-engineered umbilical cord blood mononuclear cells (UCB-MC) as a forward-looking strategy for the activation of angiogenesis. Adenovirus constructs Ad-VEGF, Ad-FGF2, Ad-SDF1α, and Ad-EGFP were synthesized and used for cell modification. UCB-MCs were isolated from UCB and transduced with adenoviral vectors. As part of our in vitro experiments, we evaluated the efficiency of transfection, the expression of recombinant genes, and the secretome profile. Later, we applied an in vivo Matrigel plug assay to assess engineered UCB-MC's angiogenic potential. We conclude that hUCB-MCs can be efficiently modified simultaneously with several adenoviral vectors. Modified UCB-MCs overexpress recombinant genes and proteins. Genetic modification of cells with recombinant adenoviruses does not affect the profile of secreted pro- and anti-inflammatory cytokines, chemokines, and growth factors, except for an increase in the synthesis of recombinant proteins. hUCB-MCs genetically modified with therapeutic genes induced the formation of new vessels. An increase in the expression of endothelial cells marker (CD31) was revealed, which correlated with the data of visual examination and histological analysis. The present study demonstrates that gene-engineered UCB-MC can be used to stimulate angiogenesis and possibly treat cardiovascular disease and diabetic cardiomyopathy.

Influence of Recombinant Codon-Optimized Plasmid DNA Encoding VEGF and FGF2 on Co-Induction of Angiogenesis.

Several methods for the stimulation of skin wound repair have been proposed over the last few decades. The most promising among them are gene and stem cell therapy. Our present experiments combined several approaches via the application of human umbilical cord blood mononuclear cells (hUCB-MC) that were transfected with pBud-VEGF165-FGF2 plasmid (gene-cell therapy) and direct gene therapy using pBud-VEGF165-FGF2 plasmid to enhance healing of full thickness skin wounds in rats. The dual expression cassette plasmid pBud-VEGF165-FGF2 encodes both VEGF and FGF2 therapeutic genes, expressing pro-angiogenic growth factors. Our results showed that, with two weeks post-transplantation, some transplanted cells still retained expression of the stem cell and hematopoietic markers C-kit and CD34. Other transplanted cells were found among keratinocytes, hair follicle cells, endothelial cells, and in the derma. PCNA expression studies revealed that transplantation of transfected cells terminated proliferative processes in regenerating wounds earlier than transplantation of untransfected cells. In the direct gene therapy group, four days post-operatively, the processes of flap revascularization, while using Easy LDI Microcirculation Camera, was higher than in control wounded skin. We concluded that hUCB-MC can be used for the treatment of skin wounds and transfection these cells with VEGF and FGF2 genes enhances their regenerative abilities. We also concluded that the application of pBud-VEGF165-FGF2 plasmids is efficient for the direct gene therapy of skin wounds by stimulation of wound revascularization.

Evaluation of Direct and Cell-Mediated Lactoferrin Gene Therapy for the Maxillofacial Area Abscesses in Rats.

Resistance to antibacterial therapy requires the discovery of new methods for the treatment of infectious diseases. Lactoferrin (LTF) is a well-known naïve first-line defense protein. In the present study, we suggested the use of an adenoviral vector (Ad5) carrying the human gene encoding LTF for direct and cell-mediated gene therapy of maxillofacial area phlegmon in rats. Abscesses were developed by injection of the purulent peritoneal exudate in the molar region of the medial surface of the mandible. At 3-4 days after phlegmon maturation, all rats received ceftriaxone and afterward were subcutaneously injected around the phlegmon with: (1) Ad5 carrying reporter gfp gene encoding green fluorescent protein (Ad5-GFP control group), (2) Ad5 carrying LTF gene (Ad5-LTF group), (3) human umbilical cord blood mononuclear cells (UCBC) transduced with Ad5-GFP (UCBC + Ad5-GFP group), and (4) UCBC transduced with Ad5-LTF (UCBC + Ad5-LTF group). Control rats developed symptoms considered to be related to systemic inflammation and were euthanized at 4-5 days from the beginning of the treatment. Rats from therapeutic groups demonstrated wound healing and recovery from the fifth to seventh day based on the type of therapy. Histological investigation of cervical lymph nodes revealed purulent lymphadenitis in control rats and activated lymphatic tissue in rats from the UCBC + Ad5-LTF group. Our results propose that both approaches of LTF gene delivery are efficient for maxillofacial area phlegmon recovery in rats. However, earlier wound healing and better outcomes in cervical lymph node remodeling in the UCBC + Ad5-LTF group, as well as the lack of direct exposure of the viral vector to the organism, which may cause toxic and immunogenic effects, suggest the benefit of cell-mediated gene therapy.

Genetically modified human umbilical cord blood cells expressing vascular endothelial growth factor and fibroblast growth factor 2 differentiate into glial cells after transplantation into amyotrophic lateral sclerosis transgenic mice.

Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF(165)) and human fibroblast growth factor 2 (FGF(2)) (pBud-VEGF-FGF(2)). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models ((G)93(A) mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal ß(3)-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF(2). Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific ß(3)-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF(2), hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF(2) on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.