Effect of HO-1 transgene delivery on wounds.

<p>A – Effect of HO-1 transgene delivery on wound closure in the db/db diabetic mice. Adenoviral vectors (2.3×10⁷ IU in 100 µL of PBS) were injected subcutaneously near the wound immediately after injury. Control animal were injected with the same amount of AdGFP carriers. Each bar represents mean+SD; N = 5–8 animals per group. * P<0.05 in comparison to control, AdGFP treated mice. B – representative pictures showing blood vessels in the wounded skin of db/db mice injected with AdHO-1 or AdGFP vectors. CD31 staining of the skin cross-section. Scale bar = 100 µm. C – Number of vessels in wounded skin in the db/db mice injected with AdHO-1 or AdGFP, on the 3^rd and 14^th days after wounding. Analysis of specimens stained for CD31 to visualize endothelial cells. Each bar represents mean+SD values for 5–8 animals. * P<0.05 in comparison to control, AdGFP injected animals.</p

Effect of SnPPIX (10 µmol/L) on time of gap closure by HaCaT cells cultured <i>in vitro</i> in the presence of hydroxyurea (10 mmol/L).

<p>Scratch assay. A – Quantitative data. Each point represents mean±SD of 3 experiments done in duplicates. * P<0.05 in comparison to control. B – representative pictures. Scale bar = 200 µm.</p

Activity of primary murine keratinocytes isolated from HO-1^+/+ and HO-1^Tg newborns and cultured <i>in vitro.</i>

<p>A – Migration of cells measured by time of gap closure in the presence of hydroxyurea (10 mmol/L). Scratch assay. B – Spontaneous proliferation of cells cultured for 48 h. BrdU incorporation assay. C – Viability of cells cultured in hypoxia (1% O₂) for 24 h. MTT reduction assay. D – Concentration of VEGF in media harvested from cell cultures after a 24 h incubation. Each bar represents mean+SD of 3–5 experiments. * P<0.05, ** P<0.01 in comparison to HO-1^+/+.</p

Closure of cuteneous wounds in the HO-1^+/+ (WT), HO-1^+/− (HT), or HO-1^−/− (KO) C57BLxFVB mice.

<p>A – 3-month old animals. B – 6-month old animals. Each point represents individual animal (N = 4–5), lines connect the median values. Crossed points represent animals subjected to euthanasia. * P<0.05, ** P<0.01 in comparison to WT. C – representative pictures showing the wounds in 6-month old animals immediately after wounding and on day 10^th. Scale bar = 5 mm.</p

A – Closure of cutaneous wounds in the HO-1^+/+ wild type and HO-1^Tg mice.

<p>Each bar represents mean+SD. N = 10 animals per group. * P<0.05, ** P<0.01, *** P<0.001 in comparison to HO-1^+/+ mice. B – Representative pictures demonstrating CD31 staining of endothelial cells in the wounded skin (3 days after wounding) in the 3-month old mice of different genotypes. Scale bar = 100 µm. C – Number of vessels in wounded skin (3 days after wounding, CD31 staining) in the 3-month old mice of different genotypes. Each bar represents analysis of samples from 5–8 animals. Data are presented as mean+SD. * P<0.05 in comparison to HO-1^+/+ animals.</p

HO-1 overexpression in murine keratinocytes.

<p>A – Expression of human HO-1 mRNA in the skin of HO-1^+/+ and transgenic HO-1^Tg mice C57BL mice. Electrophoresis of RT-PCR products (2% agarose gel). EF2 was used as a housekeeping gene. One of 4 similar analyses. B – Representative pictures of immunocytofluorescent staining for HO-1 in primary murine keratinocytes isolated from newborns and cultured <i>in vitro</i>. Scale bar = 100 µm. C – Concentration of HO-1 in lysates of primary murine keratinocytes isolated from newborns and cultured in vitro. ELISA. Each bar represents mean+SD of 6 measurements. *** P<0.001 in comparison to HO-1^+/+.</p

List of genes differently expressed in animals with pharmacologically or genetically inhibited HO-1, 3 days after wounding.

<p>Presence of proteins in the serum of mice was analyzed using RayBiotech arrays followed by densitometric measurements. Data are shown as semiquantitative estimations of signals: + indicates OD>0.1 of value measured for positive control.</p

A - Expression of HO-1 protein in healthy and wounded skin.

<p>H - healthy skin, PC - positive control (HaCaT cells stimulated with 10 µmol/L of hemin for 24 h). Western blot analysis. Tubulin was used as a housekeeping gene to control the protein loading. One of 3 similar blots. B - Effect of SnPPIX (45 µmol/kg of body weight) injected subcutaneously (once a day for 10 days) on wound closure in C57BL mice. C – Effect of SnPPIX (45 µmol/kg of body weight), injected intraperitoneally (once a day for 10 days) on wound closure in C57BL mice. Each bar represents mean+SD; N = 10 animals per group. * P<0.05, ** P<0.01 in comparison to control, vehicle injected animals. D – representative pictures taken immediately after wounding, and on the 3^rd and 7^th days. Scale bar = 1 mm.</p