PDK1 is essential for hematopoietic reconstitution.

<p>(<b>A</b>) Experimental design of mice transplanted with LSK cells with silenced <i>Pdk1</i> expression using two different shRNA lentiviruses. (<b>B</b>-<b>C)</b> CD45.2 mice were transplanted with 7 x 10⁴ GFP⁺ LSK cells from CD45.1 mice transduced with non-targeting shRNA-scramble control (n = 11) or two different shRNA-<i>Pdk1</i> (n = 14) along with 2 x 10⁵ BM supporter cells. Total percent of GFP⁺ donor cells (<b>B</b>) or myeloid (Gr1⁺Mac-1⁺) and lymphoid (CD19⁺B220⁺) cells (<b>C</b>) in peripheral blood after transplantation was determined by flow cytometry after 4, 8 and 16 weeks. Each dot represents one mouse, and the data are presented as mean (horizontal line). Statistical analysis was performed using Mann-Whitney <i>U</i> test. **, <i>P</i> < .01; ***, <i>P</i> < .001.</p

PDK1 is essential for both long- and short-term hematopoietic reconstitution.

<p>(<b>A</b>) MPPs silenced for <i>Pdk1</i> were plated after cell sorting in semi-solid methylcellulose media supplemented with cytokines and incubated in parallel plates either in hypoxia and normoxia. After 8 days the numbers of colonies were scored. Data are presented as mean ± SD (n = 6–8). Statistical analysis was performed using a student`s t-test. (<b>B</b>) Experimental design of mice transplanted with MPPs or LT-HSCs with silenced <i>Pdk1</i> expression using two different shRNA lentiviruses. (<b>C-D</b>) Freshly isolated MPPs and LT-HSCs from CD45.1 mice were transduced with shRNA-scramble control (n = 11) or two shRNAs to <i>Pdk1</i>. 7 x 10⁴ GFP⁺ MPP cells (<b>C</b>) or 4.3 x 10³ GFP⁺ LT-HSCs (<b>D</b>) were transplanted into CD45.2 mice along with 2 x 10⁵ BM supporter cells (n = 5-10/group). Total percent of GFP⁺ donor cells (upper panels) or the distribution between myeloid (Gr1⁺Mac-1⁺) versus lymphoid (CD19⁺B220⁺) cells (lower panels) in peripheral blood after transplantation was determined by flow cytometry after 3 and 6 weeks. Each dot represents one mouse, and the data are presented as mean (horizontal line). Statistical analysis was performed using Mann-Whitney <i>U</i> test. scr: scramble control. *, <i>P</i> < .05; **, <i>P</i> < .01; ***, <i>P</i> < .001.</p

Primitive HSCs express higher levels of <i>Pdk1</i> compared to more differentiated cells.

<p>(<b>A</b>) Example dot plots of flow cytometry gating strategy for LSK cells, LT-HSCs (CD34^-FLT3^-), ST-HSCs (CD34⁺FLT3^-), and MPPs (CD34⁺FLT3⁺). 7AAD^-Lineage^- cells were positively selected for Sca-1 and c-kit to sort out LSK cells. (<b>B</b>) <i>Pdk1-3</i> expression in sorted LT-HSCs, ST-HSCs, MPPs cells, and differentiated c-kit⁺ or c-kit^- cells by qRT-PCR. Data were normalized to <i>Hprt</i> expression (n = 3, in triplicates). Each dot represents one sample, and data are presented as mean (horizontal line) ± SD. Statistical analysis was performed using a student’s t-test. N: normoxia, H: hypoxia. *, <i>P</i> < .05; **, <i>P</i> < .01; ***, <i>P</i> < .001.</p

<i>Pdk1</i> is a major HIF-1 target in HSCs.

<p>(<b>A</b>) <i>Pdk1-4</i> expression in LSK cells transduced with <i>caHif-1α</i> or with empty vector, 24 hours after culture in normoxia. LSK cells were sorted for GFP expression 2 days after transduction. Data were normalized to <i>β-actin</i> x 10⁻³ expression (n = 3, in triplicates). Data are presented as mean (horizontal line) ± SD. Statistical analysis was performed using a student’s t-test. <i>Pdk4</i> expression was under the detection limit (not shown). (<b>B</b>) Experimental design of conditional gene targeting by pIpC-induced knockout in control or <i>Mx1-Cre</i>:<i>Hif-1α</i>^{<i>flox/flox</i>} mice. (<b>C</b>) qRT-PCR analysis of <i>Pdk1</i> RNA expression <i>in vivo</i> in FACS-sorted LT-HSCs, ST-HSCs, MPPs, and differentiated c-kit⁺ or c-kit^- cells from BM of <i>Hif-1α</i>^<i>+/+</i> or <i>Hif-1α</i>^<i>Δ/Δ</i> mice. Data were normalized to the expression of <i>Hprt</i> and collected from three individual experiments analyzed in triplicate qRT-PCRs. Data are presented as mean ± SD. Statistical analysis was performed using a student’s t-test. (<b>D</b>) qRT-PCR analysis of <i>Pdk1-4</i> expression in <i>Hif-1α</i>^<i>+/+</i> or <i>Hif-1α</i>^<i>Δ/Δ</i> in colony forming unit-granulocyte/monocyte (CFU-GM) after 10 days in methylcellulose and normoxia before 48 hours in normoxia or hypoxia. Data were normalized to <i>Hprt</i> expression and collected from 16 (<i>Hif-1α</i>^<i>+/+</i>) or 14 (<i>Hif-1α</i>^<i>Δ/Δ</i>) individual colonies analyzed in triplicates by qRT-PCR. Data represent median and ranges. Statistical analysis was performed using a Mann-Whitney <i>U</i> test. <i>Pdk4</i> expression was undetected (not shown). N: normoxia, H: hypoxia. *, <i>P</i> < .05; **, <i>P</i> < .01; ***, <i>P</i> < .001.</p

Different mechanisms activated by mild versus strong loading in rat Achilles tendon healing

<div><p>Background</p><p>Mechanical loading stimulates Achilles tendon healing. However, various degrees of loading appear to have different effects on the mechanical properties of the healing tendon, and strong loading might create microdamage in the tissue. This suggests that different mechanisms might be activated depending on the magnitude of loading. The aim of this study was to investigate these mechanisms further.</p><p>Methods</p><p>Female rats had their right Achilles tendon cut transversely and divided into three groups: 1) unloading (calf muscle paralysis by Botox injections, combined with joint fixation by a steel-orthosis), 2) mild loading (Botox only), 3) strong loading (free cage activity). Gene expression was analyzed by PCR, 5 days post-injury, and mechanical testing 8 days post-injury. The occurrence of microdamage was analyzed 3, 5, or 14 days post-injury, by measuring leakage of injected fluorescence-labelled albumin in the healing tendon tissue.</p><p>Results</p><p>Peak force, peak stress, and elastic modulus of the healing tendons gradually improved with increased loading as well as the expression of extracellular matrix genes. In contrast, only strong loading increased transverse area and affected inflammation genes. Strong loading led to higher fluorescence (as a sign of microdamage) compared to mild loading at 3 and 5 days post-injury, but not at 14 days.</p><p>Discussion</p><p>Our results show that strong loading improves both the quality and quantity of the healing tendon, while mild loading only improves the quality. Strong loading also induces microdamage and alters the inflammatory response. This suggests that mild loading exert its effect via mechanotransduction mechanisms, while strong loading exert its effect both via mechanotransduction and the creation of microdamage.</p><p>Conclusion</p><p>In conclusion, mild loading is enough to increase the quality of the healing tendon without inducing microdamage and alter the inflammation in the tissue. This supports the general conception that early mobilization of a ruptured tendon in patients is advantageous.</p></div

Mechanical properties of rat Achilles tendons, 8 days post-injury.

<p>Mechanical properties of rat Achilles tendons, 8 days post-injury.</p

Raw data from fluorescence measurements.

<p>Raw data from fluorescence measurements.</p

Overview of the fluorescence value measured.

<p>Overview of the fluorescence value measured.</p

Results from gene expression and microdamage analyses.

<p><b>A)</b> Gene expression for extracellular matrix genes, 5 days after tendon injury. The fold change of genes for <i>Collagen I</i> (<i>COL1A1</i>), <i>collagen III</i> (<i>COL3A1</i>), <i>collagen V</i> (<i>COL5A1</i>), and <i>lysyl oxidase</i> (<i>LOX</i>) are shown. Three different loading conditions were tested; unloading (Botox and steel-orthosis), mild loading (Botox), and strong loading (free cage activity), N = 11–12. <b>B)</b> Leakage of fluorescent protein (BSA-FITC) as a sign of bleeding and microdamage: 3, 5, or 14 days after tendon injury. The result describes the fraction of the fluorescence in the tendon tissue compared to the fluorescence in the blood plasma (ratio of (counts per second / mg specimen) / (counts per second / mg blood plasma)). Two different loading conditions were tested: mild (Botox) and strong loading (N = 12). The rats were intravenously injected with BSA-FITC 1 hour before euthanasia. The fluorescence detected in the tendon tissue was normalized to the tissue weight and the fluorescence detected in the blood plasma. * p < 0.001.</p

Gene expression, 5 days post-injury.

<p>Gene expression, 5 days post-injury.</p