We report for the first time sustained levels of circulating hGH after intramuscular naked DNA administration and, consequently, a highly significant weight increase of dwarf 'little' mice.
Mesenchymal stem cells (MSCs) possess pleiotropic properties that include immunomodulation, inhibition of apoptosis, fibrosis and oxidative stress, secretion of trophic factors, and enhancement of angiogenesis. These properties provide a broad spectrum for their potential in a wide range of injuries and diseases, including diabetic nephropathy (DN). MSCs are characterized by adherence to plastic, expression of the surface molecules CD73, CD90, and CD105 in the absence of CD34, CD45, HLA-DR, and CD14 or CD11b and CD79a or CD19 surface molecules, and multidifferentiation capacity in vitro. MSCs can be derived from many tissue sources, consistent with their broad, possibly ubiquitous distribution. This article reviews the existing literature and knowledge of MSC therapy in DN, as well as the most appropriate rodent models to verify the therapeutic potential of MSCs in DN setting. Some preclinical relevant studies are highlighted and new perspectives of combined therapies for decreasing DN progression are discussed. Hence, improved comprehension and interpretation of experimental data will accelerate the progress towards clinical trials that should assess the feasibility and safety of this therapeutic approach in humans. Therefore, MSC-based therapies may bring substantial benefit for patients suffering from DN.
The signal transducer and activator of transcription 5 (STAT5) is a transcription factor recruited by numerous cytokines. STAT5 is important for several physiological functions, including body and tissue growth, mammary gland development, immune system and lipid metabolism. However, the role of STAT5 signaling for brain functions is still poorly investigated, especially regarding cognitive aspects. Therefore, the objective of the present study was to investigate whether brain STAT5 signaling modulates learning and memory formation. For this purpose, brain-specific STAT5 knockout (STAT5 KO) mice were studied in well-established memory tests. Initially, we confirmed a robust reduction in STAT5a and STAT5b mRNA levels in different brain structures of STAT5 KO mice. STAT5 KO mice showed no significant alterations in metabolism, growth, somatotropic axis and spontaneous locomotor activity. In contrast, brain-specific STAT5 ablation impaired learning and memory formation in the novel object recognition, Barnes maze and contextual fear conditioning tests. To unravel possible mechanisms that might underlie the memory deficits of STAT5 KO mice, we assessed neurogenesis in the hippocampus, but no significant differences were observed between groups. On the other hand, reduced insulin-like growth factor-1 (IGF-1) mRNA expression was found in the hippocampus and hypothalamus of STAT5 KO mice. These findings collectively indicate that brain STAT5 signaling is required to attain normal learning and memory. Therefore, STAT5 is an important downstream cellular mechanism shared by several cytokines to regulate cognitive functions.
The possibilities for non-viral GH gene therapy are studied in immunocompetent dwarf mice (lit/lit). As expression vector we used a plasmid previously employed in immunodeficient dwarf mice (pUBI-hGH-gDNA) by replacing the human GH gene with the genomic sequence of mouse-GH DNA (pUBI-mGH-gDNA). HEK-293 human cells transfected with pUBI-mGH-gDNA produced 3.0 µg mGH/10(6) cells/day compared to 3.7 µg hGH/10(6) cells/day for pUBIhGH- gDNA transfected cells. The weight of lit/lit mice treated with the same two plasmids (50 µg DNA/mouse) by electrotransfer into the quadriceps muscle was followed for 3 months. The weight increase up to 15 days for mGH, hGH and saline treated mice were 0.130, 0.112 and 0.027 g/mouse/day. Most sera from hGH-treated mice contained anti-hGH antibodies already on day 15, with the highest titers on day 45, while no significant anti-mGH antibodies were observed in mGH-treated mice. At the end of 3 months, the weight increase for mGH-treated mice was 34.3%, while the nose-to-tail and femur lengths increased 9.5% and 24.3%. Mouse-GH and hGH circulating levels were 4-5 ng/mL 15 days after treatment, versus control levels of ~0.7 ng GH/mL (P<0.001). In mGH-treated mice, mIGF-I determined on days 15, 45 and 94 were 1.5- to 3-fold higher than the control and 1.2- to 1.6-fold higher than hGH-treated mice. The described homologous model represents an important progress forming the basis for preclinical testing of non-viral gene therapy for GH deficiency.
In previous work, sustained levels of circulating human growth hormone (hGH) and a highly significant weight increase were observed after electrotransfer of naked plasmid DNA (hGH-DNA) into the muscle of immunodeficient dwarf mice (lit/scid). In the present study, the efficacy of this in vivo gene therapy strategy is compared to daily injections (5 μg/twice a day) of recombinant hGH (r-hGH) protein, as assessed on the basis of several growth parameters. The slopes of the two growth curves were found to be similar (P > 0.05): 0.095 g/mouse/d for protein and 0.094 g/mouse/d for DNA injection. In contrast, the weight increases averaged 35.5% (P < 0.001) and 23.1% (P < 0.01) for protein and DNA administration, respectively, a difference possibly related to the electroporation methodology. The nose-to-tail linear growth increases were 15% and 9.6% for the protein and DNA treatments, respectively, but mouse insulin-like growth factor I (mIGF-I) showed a greater increase over the control with DNA (5- to 7-fold) than with protein (3- to 4-fold) administration. The weight increases of several organs and tissues (kidneys, spleen, liver, heart, quadriceps and gastrocnemius muscles) were 1.3- to 4.6-fold greater for protein than for DNA administration, which gave a generally more proportional growth. Glucose levels were apparently unaffected, suggesting the absence of effects on glucose tolerance. A gene transfer strategy based on a single hGH-DNA administration thus appears to be comparable to repeated hormone injections for promoting growth and may represent a feasible alternative for the treatment of growth hormone deficiency.
These results show that hGH-DNA administration into non-exposed tibialis muscle followed by the new HV/LV electrotransfer protocol was an equally efficient, less traumatic treatment, much more suitable for pre-clinical testing than administration into exposed quadriceps.
and injected with lipase inhibitor, further verifying that this genetic approach markedly diminished hepatic lipid synthesis. In BAT, Fgf21 transfer induced a "super-brown" phenotypic change, which was accompanied by elevated expression of a panel of vital genes for adaptive thermogenesis, such as Ucp1, Pgc1-alpha, Cidea, Dio2 and Elovl3. Collectively, our findings unveil that the triglyceride-lowering effect of cold exposure can be fully recapitulated by liver-specific delivery of the Fgf21 using a hydrodynamics-based procedure, indicating that this new approach may provide an alternative and more acceptable option for hyperlipidemia therapy in humans.
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