Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications

High-Throughput Screening of Surface Marker Expression on Undifferentiated and Differentiated Human Adipose-Derived Stromal Cells

Zadatak ovog rada je provesti numerički proračun naprezanja i deformacija u postojećem konstrukcijskom rješenju za vertikalni spremnik za vino. Numerička analiza čvrstoće provedena je u računalnom paketu Abaqus koji je temeljen na metodi konačnih elementa. U uvodu je općenito opisan predmet analize (vertikalni spremnik za vino) i materijal od kojeg je predmet izrađen. U drugom poglavlju opisan je vertikalni spremnik za vino sa svojim dijelovima i sadržajem spremnika. U trećem poglavlju napravljen je analitički proračun svakog važnijeg dijela vertikalnog spremnika pomoću metoda linearne analize konstrukcija. U četvrtom poglavlju opisana je metoda konačnih elemenata i računalni program Abaqus. Pojedini tipovi konačnih elemenata, korišteni u ovome radu, su detaljnije razrađeni. Za primjere u kojima je poznato analitičko rješenje, izvršena je provjera učinkovitosti pojedinih konačnih elemenata i odabir prikladnih tehnika modeliranja. Prikazan je također tok numeričkog proračuna zadanog vertikalnog spremnika. Razmatrana su tri modela, ljuskasti model, osnosimetrični model i trodimenzijski model. Prikazani su rezultati naprezanja i pomaka kao i usporedba tih rezultata s analitičkim rješenjem.The task of this work is to conduct a control of a numerical analisys of stresses and strains in the existing design for the vertical wine container. Numerical strength analysis was performed in the computer package Abaqus which is based on the finite element method. The introduction generally describes the object of analysis (vertical wine container) and the material from which the object is made. The second chapter describes a vertical wine container with its parts and the contents of the container. In the third chapter, an analytical calculation of each important part of the vertical tank is made using the method of linear analysis of structures. The fourth chapter describes the finite element method and the Abaqus computer program. Certain types of finite elements, used in this work, are elaborated in more detail. For examples where an analytical solution is known, the efficiency of individual finite elements is checked and appropriate modeling techniques are selected. The flow of the numerical calculation of the given vertical tank is also shown. Three models were considered, the shell model, the axisymmetric model, and the three-dimensional model. The results of stresses and displacements are presented as well as a comparison of these results with the analytical solution

shPHD-2 Suppresses PHD-2 and Upregulates HIF-1α and Downstream Angiogenic Genes In Vitro.

<p>Cells for all experiments were cultured under hypoxic conditions of 5% oxygen. (a) Schematics of shPHD-2 and shScr constructs. (b) qRT-PCR showed decreased PHD-2 expression <i>in vitro</i> from diabetic mouse fibroblasts transfected with shPHD-2 compared to fibroblasts transfected with shScr (<i>****p</i><0.0001). (c) Western blot revealed higher levels of HIF-1α protein from shPHD-2 fibroblasts compared to shScr fibroblasts. (d) qRT-PCR additionally showed that SDF-1, an angiogenic chemokine downstream of HIF-1α, was more highly expressed in the shPHD-2 group than in shScr <i>in vitro</i> (****<i>p</i><0.0001), as was (e) VEGFα (*<i>p</i><0.05).</p

shPHD-2 Promotes Accelerated Wound Healing and Increased Vascular Density <i>In Vivo</i>.

<p>(a,b) Diabetic wounds treated with shPHD-2 healed significantly faster than wounds treated with shScr, (c) closing in an average of 14.33 ± 0.45 days compared to 19 ± 0.33 days (*****<i>p</i><0.00001). (d,e) CD31 staining revealed enhanced vascular density in wound beds treated with shPHD-2 versus shScr (***<i>p</i><0.001). Scale bar = 100μm.</p

shPHD-2 Promotes Angiogenesis <i>In Vivo</i>.

<p>(a) BLI confirmed uptake of shPHD-2 plasmid <i>in vivo</i> as indicated by expression of firefly luciferase (right) compared to a non-injected mouse (left). (b) RNA derived from the tissue surrounding the wound beds five days after injection showed decreased PHD-2 transcript as a result of shPHD-2 plasmid treatment compared to shScr (**<i>p</i><0.01). (c) Protein derived from the same tissue samples congruously showed higher levels of HIFα protein in the shPHD-2 group. (d) qRT-PCR further demonstrated the upregulation of angiogenic gene PDGFα in the shPHD-2 treatment group (*<i>p</i><0.05).</p