Pernilla Eliasson

The hematopoietic stem cell niche: low in oxygen but a nice place to be.

The enormous regenerative capacity of the blood system to sustain functionally mature cells are generated from highly proliferative, short‐lived progenitors, which in turn arise from a rare population of pluripotent and self‐renewing hematopoietic stem cells (HSC). In the bone marrow, these stem cells are kept in a low proliferative, relatively quiescent state in close proximity to stromal cells and osteoblasts, forming specialized niches. The interaction in particular to bone is crucial to prevent exhaustion of HSCs from uncontrolled cell‐cycle entry and to excessive proliferation. In addition, the niche and its components protect stem cells from stress, such as accumulation of reactive oxygen species and DNA damage. One of the key issues is to identify conditions to increase the number of HSCs, either in vivo or during ex vivo growth cultures. This task has been very difficult to resolve and most attempts have been unsuccessful. However, the mechanistic insights to HSC self‐renewal and preservation are gradually increasing and there is now hope that future research will enable scientists and clinicians to modulate the process. In this review, we will focus on the molecular mechanisms of self‐renewal and HSC maintenance in the light of novel findings that HSCs reside at the lowest end of an oxygen gradient. Hypoxia appears to regulate hematopoiesis in the bone marrow by maintaining important HSC functions, such as cell cycle control, survival, metabolism, and protection against oxidative stress. To improve the therapeutic expansion of HSCs we need to learn more about the molecular mechanisms of hypoxia‐mediated regulation. J. Cell. Physiol. 222:17–22, 2010.

Hypoxia mediates low cell-cycle activity and increases the proportion of long-term–reconstituting hematopoietic stem cells during in vitro culture

OBJECTIVE Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia-inducible factor (HIF)-1alpha. MATERIALS AND METHODS HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O(2), and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1alpha was studied using methods of protein stabilization and gene silencing. RESULTS When long-term FLT3(-)CD34(-) Lin(-)Sca-1(+)c-Kit(+) (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G(0). Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21(cip1), p27(Kip1), and p57(Kip2) increased in LSK cells by hypoxia, only p21(cip1) was upregulated in FLT3(-)CD34(-)LSK cells. We could demonstrate that expression of p27(Kip1) and p57(Kip2) was dependent of HIF-1alpha. Surprisingly, overexpression of constitutively active HIF-1alpha or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability. CONCLUSIONS Our results imply that hypoxia, in part via HIF-1alpha, maintains HSCs by decreasing proliferation and favoring quiescence.

Rat Achilles tendon healing: mechanical loading and gene expression

Injured tendons require mechanical tension for optimal healing, but it is unclear which genes are upregulated and responsible for this effect. We unloaded one Achilles tendon in rats by Botox injections in the calf muscles. The tendon was then transected and left to heal. We studied mechanical properties of the tendon calluses, as well as mRNA expression, and compared them with loaded controls. Tendon calluses were studied 3, 8, 14, and 21 days after transection. Intact tendons were studied similarly for comparison. Altogether 110 rats were used. The genes were chosen for proteins marking inflammation, growth, extracellular matrix, and tendon specificity. In intact tendons, procollagen III and tenascin-C were more expressed in loaded than unloaded tendons, but none of the other genes was affected. In healing tendons, loading status had small effects on the selected genes. However, TNF-alpha, transforming growth factor-beta1, and procollagens I and III were less expressed in loaded callus tissue at day 3. At day 8 procollagens I and III, lysyl oxidase, and scleraxis had a lower expression in loaded calluses. However, by days 14 and 21, procollagen I, cartilage oligomeric matrix protein, tenascin-C, tenomodulin, and scleraxis were all more expressed in loaded calluses. In healing tendons, the transverse area was larger in loaded samples, but material properties were unaffected, or even impaired. Thus mechanical loading is important for growth of the callus but not its mechanical quality. The main effect of loading during healing might thereby be sought among growth stimulators. In the late phase of healing, tendon-specific genes (scleraxis and tenomodulin) were upregulated with loading, and the healing tissue might to some extent represent a regenerate rather than a scar.

Achilles tendon healing in rats is improved by intermittent mechanical loading during the inflammatory phase.

Tendons adapt to changes in mechanical loading, and numerous animal studies show that immobilization of a healing tendon is detrimental to the healing process. The present study addresses whether the effects of a few episodes of mechanical loading are different during different phases of healing. Fifty female rats underwent Achilles tendon transection, and their hind limbs were unloaded by tail suspension on the day after surgery. One group of 10 rats was taken down from suspension to run on a treadmill for 30 min/day, on days 2–5 after transection. They were euthanized on day 8. Another group underwent similar treadmill running on days 8–11 and was euthanized on day 14. Continuously unloaded groups were euthanized on days 8 and 14. Tendon specimens were then evaluated mechanically. The results showed that just four loading episodes increased the strength of the healing tendon. This was evident irrespective of the time point when loading was applied (early or late). The positive effect on early healing was unexpected, considering that the mechanical stimulation was applied during the inflammatory phase, when the calluses were small and fragile. A histological study of additional groups with early loading also showed some increased bleeding in the loaded calluses. Our results indicate that a short episodes of early loading may improve the outcome of tendon healing. This could be of interest to clinical practice.

Achilles tendon rupture – treatment and complications: A systematic review

Achilles tendon rupture is a frequent injury with an increasing incidence. Until now, there is no consensus regarding optimal treatment. The aim of this review was to illuminate and summarize randomized controlled trials comparing surgical and non‐surgical treatment of Achilles tendon ruptures during the last 10 years. Seven articles were found and they were all acceptable according to international quality assessment guidelines. Primary outcomes were re‐ruptures, other complications, and functional outcomes. There was no significant difference in re‐ruptures between the two treatments, but a tendency to favoring surgical treatment. Further, one study found an increased risk of soft‐tissue‐related complications after surgery. Patient satisfaction and time to return to work were significantly different in favor of surgery in one study, and there was also better functional outcome after surgery in some studies. These seven studies indicate that surgical patients have a faster rehabilitation. However, the differences between surgical and non‐surgical treatment appear to be subtle and it could mean that rehabilitation is more important, rather than the actual initial treatment. Therefore, further studies will be needed in regard to understanding the interplay between acute surgical or non‐surgical treatment, and the rehabilitation regimen for the overall outcome after Achilles tendon ruptures.

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells

Background: Lysyl oxidase catalyzes collagen cross-link formation, which is essential for mechanically strong collagen fibrils. Results: LOX inhibition stops early mechanical development of tendon constructs and leads to irregularly shaped collagen fibrils. Conclusion: Collagen cross-linking is essential for successful fibrillogenesis and regulates fibril shape. Significance: LOX activity is required in the control of collagen fibril architecture by a mechanism that remains to be explained. Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation.

BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor-induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3.

Constitutively activating internal tandem duplications (ITD) of FLT3 (FMS-like tyrosine kinase 3) are the most common mutations in acute myeloid leukemia (AML) and correlate with poor prognosis. Receptor tyrosine kinase inhibitors targeting FLT3 have developed as attractive treatment options. Because relapses occur after initial responses, identification of FLT3-ITD-mediated signaling events are important to facilitate novel therapeutic interventions. Here, we have determined the growth-inhibitory and proapoptotic mechanisms of 2 small molecule inhibitors of FLT3, AG1295 or PKC412, in hematopoietic progenitor cells, human leukemic cell lines, and primary AML cells expressing FLT3-ITD. Inactivation of the PI3-kinase pathway, but not of Ras-mitogen-activated protein (MAP) kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced up-regulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from gene-targeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor, where loss of Puma only provided transient protection from apoptosis, but loss of Bim preserved clonal survival upon FLT3-ITD inhibition.

Influence of a single loading episode on gene expression in healing rat Achilles tendons

Mechanical loading stimulates tendon healing via mechanisms that are largely unknown. Genes will be differently regulated in loaded healing tendons, compared with unloaded, just because of the fact that healing processes have been changed. To avoid such secondary effects and study the effect of loading per se, we therefore studied the gene expression response shortly after a single loading episode in otherwise unloaded healing tendons. The Achilles tendon was transected in 30 tail-suspended rats. The animals were let down from the suspension to load their tendons on a treadmill for 30 min once, 5 days after tendon transection. Gene expression was studied by Affymetrix microarray before and 3, 12, 24, and 48 h after loading. The strongest response in gene expression was seen 3 h after loading, when 150 genes were up- or downregulated (fold change ≥2, P ≤ 0.05). Twelve hours after loading, only three genes were upregulated, whereas 38 were downregulated. Fewer than seven genes were regulated after 24 and 48 h. Genes involved in the inflammatory response were strongly regulated at 3 and 12 h after loading; this included upregulation of iNOS, PGE synthase, and IL-1β. Also genes involved in wound healing/coagulation, angiogenesis, and production of reactive oxygen species were strongly regulated by loading. Microarray results were confirmed for 16 selected genes in a repeat experiment (N = 30 rats) using real-time PCR. It was also confirmed that a single loading episode on day 5 increased the strength of the healing tendon on day 12. In conclusion, the fact that there were hardly any regulated genes 24 h after loading suggests that optimal stimulation of healing requires a mechanical loading stimulus every day.

Primary gene response to mechanical loading in healing rat Achilles tendons

Loading can stimulate tendon healing. In healing rat Achilles tendons, we have found more than 150 genes upregulated or downregulated 3 h after one loading episode. We hypothesized that these changes were preceded by a smaller number of regulatory genes and thus performed a microarray 15 min after a short loading episode, to capture the primary response to loading. We transected the Achilles tendon of 54 rats and allowed them to heal. The hind limbs were unloaded by tail-suspension during the entire experiment, except during the loading episode. The healing tendon tissue was analyzed by mechanical testing, microarray, and quantitative real-time polymerase chain reaction (qRT-PCR). Mechanical testing showed that 5 min of loading each day for 4 days created stronger tissue. The microarray analysis after one loading episode identified 15 regulated genes. Ten genes were analyzed in a repeat experiment with new rats using qRT-PCR. This confirmed the increased expression of four genes: early growth response 2 (Egr2), c-Fos, FosB, and regulation of G protein signaling 1 (Rgs1). The other genes were unaltered. We also analyzed the expression of early growth response 1 (Egr1), which is often co-regulated with c-Fos or Egr2, and found that this was also increased after loading. Egr1, Egr2, c-Fos, and FosB are transcription factors that can be triggered by numerous stimuli. However, Egr1 and Egr2 are necessary for normal tendon development, and can induce ectopic expression of tendon markers. The five regulated genes appear to constitute a general activation machinery. The further development of gene regulation might depend on the tissue context.

Elevation of systemic matrix metalloproteinases 2 and 7 and tissue inhibitor of metalloproteinase 2 in patients with a history of Achilles tendon rupture: pilot study

Objectives In this study, serum levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) between patients with a history of Achilles tendon rupture and blood donor controls were compared, and their relation to mechanical properties of the tendons during healing were studied. Methods More than 3 years after injury, serum levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9 and MMP-13, TIMP-1 and TIMP-2 in eight patients who had Achilles tendon rupture were measured. Twelve blood donors served as controls. During the early phase of healing, the tendon modulus of elasticity was calculated from radiostereometric data and tendon cross-sectional area. Results Patients with a history of Achilles tendon rupture had increased levels of MMP-2 (median difference 10%, p=0.01), MMP-7 (median difference 15%, p=0.02) and TIMP-2 (median difference 36%, p=0.02), compared with controls. Levels of MMP-7, measured 3 years after injury, correlated inversely to tendon modulus of elasticity (rs=20.83, p=0.02) and positively to tendon elongation (rs=0.74, p=0.05) during the early phase of healing. There was a trend towards positive correlation between MMP-7 and cross-sectional area during the early phase of healing (rs=0.67, p=0.08). Conclusions Patients with a history of Achilles tendon rupture appear to have elevated levels of MMP-2, MMP-7 and TIMP-2 in serum. In these pilot data, the view that the MMP–TIMP system is involved in tendinopathy is supported and that disturbances in proteolytic control might be generalised are suggested.