The Delayed Presentation of Achilles Tendon Ruptures Is Associated With Marked Alterations in the Gene Expression of COL1A1, MMPs, TIMPs, and IL-6.

BACKGROUND: Both acute and chronic Achilles tendon ruptures are affected by alterations in the extracellular matrix during the healing process of the tendon. Yet, these alterations in gene expression patterns are not well characterized. PURPOSE: To characterize temporal and spatial differences in gene expression patterns after an Achilles tendon rupture and to evaluate if cells from chronic Achilles tendon ruptures have the same ability to form new tendon tissue (tendon constructs) as healthy tendon cells. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 35 patients with surgically treated Achilles tendon ruptures were included in the study and divided into 3 groups: acute (<4 weeks), short-term chronic (1-6 months), and long-term chronic (>6 months). Biopsy specimens were collected during surgical repair and were used to analyze the gene expression within the different groups and to compare mRNA levels in the proximal and distal tendon ends. A complementary in vitro experiment was performed to evaluate if cells from chronic Achilles tendon ruptures can form tendon constructs. RESULTS: The mRNA levels for COL1A1 and COL3A1 were significantly higher in the short-term chronic group compared with the acute group (P < .05). Both MMP-1 and MMP-13 had the highest mRNA levels in the acute group (P < .01) compared with the long-term chronic group, while MMP-2 had the highest mRNA level in the short-term chronic group. Significant differences between the proximal and distal tendon ends were only detected for the monocyte and macrophage marker CD163 (P < .05), which was more expressed proximally. Cells extracted from chronic Achilles tendon ruptures displayed a similar ability and effectiveness to form tendon constructs as healthy tendon cells. CONCLUSION: A high collagenase gene activity after an Achilles tendon rupture indicated possible rapid matrix degradation in the acute phase. Chronic ruptures appeared to initiate the healing process even before treatment, indicated by the higher expression of collagen in the short-term chronic group. Cells from chronic Achilles tendon ruptures also displayed an ability to form new tendon tissue in vitro. CLINICAL RELEVANCE: The study shows a rapid increase in collagenase gene expression, which could lead to matrix degradation that continues for months after an Achilles tendon rupture.

Muscle fascicle and sarcomere adaptation in response to Achilles tendon elongation in an animal model.

Permanent loss of muscle function seen after an Achilles tendon rupture may partly be explained by tendon elongation and accompanying shortening of the muscle. Muscle fascicle length shortens, serial sarcomere number is reduced, and the sarcomere length is unchanged after Achilles tendon transection (ATT), and these changes are mitigated with suturing. The method involved in this study was a controlled laboratory study. Two groups of rats underwent ATT on one side with a contralateral control (CTRL): A) ATT with 3 mm removal of the Achilles tendon and no suturing (substantial tendon elongation), and B) ATT with suture repair (minimal tendon elongation). The operated limb was immobilized for 2 wk to reduce load. Four weeks after surgery the rats were euthanized, and hindlimbs were analyzed for tendon length, gastrocnemius medialis (GM) muscle mass, length, fascicle length, sarcomere number and length. No differences were observed between the groups, and in both groups the Achilles tendon length was longer (15.2%, P < 0.001), GM muscle mass was smaller (17.5%, P < 0.001), and muscle length was shorter (8.2%, P < 0.001) on the ATT compared with CTRL side. GM fascicle length was shorter (11.2%, P < 0.001), and sarcomere number was lower (13.8%, P < 0.001) on the ATT side in all regions. Sarcomere length was greater in the proximal (5.8%, P < 0.001) and mid (4.2%, P = 0.003), but not distal region on the ATT side. In this animal model, regardless of suturing, ATT resulted in tendon elongation, loss of muscle mass and length, and reduced serial sarcomere number, which resulted in an "overshoot" lengthening of the sarcomeres.NEW & NOTEWORTHY Following acute Achilles tendon rupture, patients are often left with functional deficits. The specific reason remains largely unknown. The shortened muscle leads to reduced fascicle length, in turn leading to adaptation by reduced serial sarcomere numbers. Surprisingly, this adaptation appears to "overshoot" and lead to increased sarcomere length. The present animal model advances understanding of how muscle sarcomeres, which are difficult to measure in humans, are affected when undue elongation takes place after tendon rupture.

Elastin levels are higher in healing tendons than in intact tendons and influence tissue compliance.

Elastic fibers containing elastin play an important role in tendon functionality, but the knowledge on presence and function of elastin during tendon healing is limited. The aim of this study was to investigate elastin content and distribution in intact and healing Achilles tendons and to understand how elastin influence the viscoelastic properties of tendons. The right Achilles tendon was completely transected in 81 Sprague-Dawley rats. Elastin content was quantified in intact and healing tendons (7, 14, and 28 days post-surgery) and elastin distribution was visualized by immunohistochemistry at 14 days post-surgery. Degradation of elastin by elastase incubation was used to study the role of elastin on viscoelastic properties. Mechanical testing was either performed as a cyclic test (20× 10 N) or as a creep test. We found significantly higher levels of elastin in healing tendons at all time-points compared to intact tendons (4% in healing tendons 28 days post-surgery vs 2% in intact tendons). The elastin was more widely distributed throughout the extracellular matrix in the healing tendons in contrast to the intact tendon where the distribution was not so pronounced. Elastase incubation reduced the elastin levels by approximately 30% and led to a 40%-50% reduction in creep. This reduction was seen in both intact and healing tendons. Our results show that healing tendons contain more elastin and is more compliable than intact tendons. The role of elastin in tendon healing and tissue compliance indicates a protective role of elastic fibers to prevent re-injuries during early tendon healing. PLAIN LANGUAGE SUMMARY: Tendons transfer high loads from muscles to bones during locomotion. They are primarily made by the protein collagen, a protein that provide strength to the tissues. Besides collagen, tendons also contain other building blocks such as, for example, elastic fibers. Elastic fibers contain elastin and elastin is important for the extensibility of the tendon. When a tendon is injured and ruptured the tissue heals through scar formation. This scar tissue is different from a normal intact tendon and it is important to understand how the tendons heal. Little is known about the presence and function of elastin during healing of tendon injuries. We have shown, in animal experiments, that healing tendons have higher amounts of elastin compared to intact tendons. The elastin is also spread throughout the tissue. When we reduced the levels of this protein, we discovered altered mechanical properties of the tendon. The healing tendon can normally extend quite a lot, but after elastin removal this extensibility was less obvious. The ability of the healing tissue to extend is probably important to protect the tendon from re-injuries during the first months after rupture. We therefore propose that the tendons heal with a large amount of elastin to prevent re-ruptures during early locomotion.

Heterotopic Ossification After an Achilles Tendon Rupture Cannot Be Prevented by Early Functional Rehabilitation: A Cohort Study.

BACKGROUND: Tendon loading might play a role in the development of heterotopic ossification after Achilles tendon ruptures. Early heavy loading on a healing tendon in animals has been shown to prolong the proinflammatory response, and inflammatory cells are thought to drive heterotopic ossification formation. Taken together, this suggests that early rehabilitation might influence heterotopic ossification development. QUESTIONS/PURPOSES: The purposes of this study were to investigate (1) whether the presence of heterotopic ossification after Achilles tendon ruptures influences clinical outcome and (2) whether early mobilization or weightbearing prevents the development of heterotopic ossification. METHODS: This was a retrospective analysis of 69 patients from a previous clinical trial. All patients were treated surgically, but with three different early rehabilitation protocols after surgery: late weightbearing and ankle immobilization, late weightbearing and ankle mobilization, and early weightbearing and ankle mobilization. Plain radiographs taken 2, 6, 12, 26, and 52 weeks postoperatively were analyzed for heterotopic ossification, which was detected in 19% of patients (13 of 69) at 52 weeks. Heterotopic ossification was measured, scored, and correlated to clinical outcomes; heel-raise index (HRI), ankle joint ROM, tendon strain, Achilles tendon rupture score (ATRS), and Victorian Institute of Sport Assessment-Achilles (VISA-A) questionnaire scores at 26 and 52 weeks postoperatively. RESULTS: Heterotopic ossification had no adverse effects on patient-reported outcomes (ATRS or VISA-A), tendon strain, or ROM. In fact, patients with heterotopic ossification tended to have a better HRI at 52 weeks compared with patients without (mean difference 14% [95% CI -0.2 to 27]; p = 0.053). Neither the occurrence (heterotopic ossification/no heterotopic ossification) nor the heterotopic ossification severity (ossification score) differed between the three rehabilitation groups. Seventeen percent of the patients (four of 24) with early functional rehabilitation (early weightbearing and ankle joint mobilization exercise) had heterotopic ossification (score, 2-3) while late weightbearing and immobilization resulted in heterotopic ossification in 13% of the patients (score, 3-4). CONCLUSIONS: Heterotopic ossification occurs relatively frequently after Achilles tendon ruptures but appears to have no adverse effects on functional outcomes. Furthermore, heterotopic ossification develops during the first 6 weeks after rupture, and weightbearing or ankle-joint mobilization does not prevent this from occurring. LEVEL OF EVIDENCE: Level III, prognostic study.

Putative Role of Nuclear Factor-Kappa B But Not Hypoxia-Inducible Factor-1α in Hypoxia-Dependent Regulation of Oxidative Stress in Hematopoietic Stem and Progenitor Cells.

Aims: Adaptation to low oxygen of hematopoietic stem cells (HSCs) in the bone marrow has been demonstrated to depend on the activation of hypoxia-inducible factor (HIF)-1α as well as the limited production of reactive oxygen species (ROS). In this study, we aimed at determining whether HIF-1α is involved in protecting HSCs from ROS. Results: Oxidative stress was induced by DL-buthionine-(S,R)-sulfoximine (BSO)-treatment, which increases the mitochondrial ROS level. Hypoxia rescued Lineage-Sca-1+c-kit+ (LSK) cells from BSO-induced apoptosis, whereas cells succumbed to apoptosis in normoxia. Apoptosis in normoxia was inhibited with the antioxidant N-acetyl-L-cysteine or by overexpression of anti-apoptotic BCL-2. Moreover, stabilized expression of oxygen-insensitive HIFs could not protect LSK cells from oxidative stress-induced apoptosis at normoxia, neither could short hairpin RNA to Hif-1α inhibit the protective effects by hypoxia in LSK cells. Likewise, BSO treatment of LSK cells from Hif-1α knockout mice did not suppress the effects seen in hypoxia. Microarray analysis identified the nuclear factor-kappa B (NF-κB) pathway as a pathway induced by hypoxia. By using NF-κB lentiviral construct and DNA-binding assay, we found increased NF-κB activity in cells cultured in hypoxia compared with normoxia. Using an inhibitor against NF-κB activation, we could confirm the involvement of NF-κB signaling as BSO-mediated cell death was significantly increased in hypoxia after adding the inhibitor. Innovation: HIF-1α is not involved in protecting HSCs and progenitors to elevated levels of ROS on glutathione depletion during hypoxic conditions. Conclusion: The study proposes a putative role of NF-κB signaling as a hypoxia-induced regulator in early hematopoietic cells.

Depletion of cytotoxic (CD8+) T cells impairs implant fixation in rat cancellous bone.

As cytotoxic (CD8+ ) T cells seem to impair shaft fracture healing, we hypothesized that depletion of CD8+ cells would instead improve healing of cancellous bone. Additionally, we also tested if CD8-depletion would influence the healing of ruptured Achilles tendons. Rats received a single injection of either anti-CD8 antibodies or saline and put through surgery 24 h later. Three different surgical interventions were performed as follows: (1) a drill hole in the proximal tibia with microCT (BV/TV) to assess bone formation; (2) a screw in the proximal tibia with mechanical evaluation (pull-out force) to assess fracture healing; (3) Achilles tendon transection with mechanical evaluation (force-at-failure) to assess tendon healing. Furthermore, CD8-depletion was confirmed with flow cytometry on peripheral blood. Flow cytometric analysis confirmed depletion of CD8+ cells (p < 0.001). Contrary to our hypothesis, depletion of CD8+ cells reduced the implant pull-out force by 19% (p < 0.05) and stiffness by 34% (p < 0.01), although the bone formation in the drill holes was the same as in the controls. Tendon healing was unaffected by CD8-depletion. Our results suggest that CD8+ cells have an important part in cancellous bone healing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

The Ruptured Achilles Tendon Elongates for 6 Months After Surgical Repair Regardless of Early or Late Weightbearing in Combination With Ankle Mobilization: A Randomized Clinical Trial.

BACKGROUND: Treatment strategies for Achilles tendon rupture vary considerably, and clinical outcome may depend on the magnitude of tendon elongation after surgical repair. The aim of this project was to examine whether tendon elongation, mechanical properties, and functional outcomes during rehabilitation of surgically repaired acute Achilles tendon ruptures were influenced by different rehabilitation regimens during the early postsurgical period. HYPOTHESIS: Restricted early weightbearing that permits only limited motion about the ankle in the early phase of tendon healing limits tendon elongation and improves functional outcome. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: 75 consecutive patients with an acute Achilles tendon rupture were included. They underwent surgical repair, and tantalum beads were placed in the distal and proximal parts of the tendon; thereafter, the patients were randomized into 3 groups. The first group was completely restricted from weightbearing until week 7. The second group was completely restricted from weightbearing until week 7 but performed ankle joint mobilization exercises. The first and second groups were allowed full weightbearing after week 8. The third group was allowed partial weightbearing from day 1 and full weightbearing from week 5. All patients received the same instructions in home exercise guidelines starting from week 9. RESULTS: The rehabilitation regimen in the initial 8 weeks did not significantly influence any of the measured outcomes including tendon elongation. Achilles tendon elongation and tendon compliance continued for up to 6 months after surgery, and muscle strength, muscle endurance, and patient-reported functional scores did not reach normal values at 12 months. CONCLUSION: Differences in rehabilitation loading pattern in the initial 8 weeks after the repair of an Achilles tendon rupture did not measurably alter the outcome. The time to recover full function after an Achilles tendon rupture is at least 12 months. Registration: NCT02422004 ( ClinicalTrials.gov identifier).

Different gene response to mechanical loading during early and late phases of rat Achilles tendon healing.

Mechanical loading stimulates tendon healing both when applied in the inflammatory phase and in the early remodeling phase of the process, although not necessarily via the same mechanisms. We investigated the gene response to mechanical loading in these two phases of tendon healing. The right Achilles tendon in rats was transected, and the hindlimbs were unloaded by tail suspension. The rats were exposed to 5 min of treadmill running 3 or 14 days after tendon transection. Thereafter, they were resuspended for 15 min or 3 h until euthanasia. The controls were suspended continuously. Gene analysis was first performed by microarray analysis followed by quantitative RT-PCR on selected genes, focusing on inflammation. Fifteen minutes after loading, the most important genes seemed to be the transcription factors EGR1 and C-FOS, regardless of healing phase. These transcription factors might promote tendon cell proliferation and differentiation, stimulate collagen production, and regulate inflammation. Three hours after loading on day 3, inflammation was strongly affected. Seven inflammation-related genes were upregulated according to PCR: CCL20, CCL7, IL-6, NFIL3, PTX3, SOCS1, and TLR2. These genes can be connected to macrophages, T cells, and recruitment of leukocytes. According to Ingenuity Pathway Analysis, the recruitment of leukocytes was increased by loading on day 3, which also was confirmed by histology. This inflammation-related gene response was not seen on day 14 Our results suggest that the immediate gene response after mechanical loading is similar in the early and late phases of healing but the late gene response is different.NEW & NOTEWORTHY This study investigates the direct effect of mechanical loading on gene expression during different healing phases in tendon healing. One isolated episode of mechanical loading was studied in otherwise unloaded healing tendons. This enabled us to study a time sequence, i.e., which genes were the first ones to be regulated after the loading episode.

Pyruvate dehydrogenase kinase 1 is essential for transplantable mouse bone marrow hematopoietic stem cell and progenitor function.

BACKGROUND: Accumulating evidence suggests that hypoxic areas in the bone marrow are crucial for maintenance of hematopoietic stem cells (HSCs) by supporting a quiescent state of cell cycle and regulating the transplantation capacity of long-term (LT)-HSCs. In addition, HSCs seem to express a metabolic profile of energy production away from mitochondrial oxidative phosphorylation in favor of glycolysis. At oxygen deprivation, hypoxia inducible factor 1α (HIF-1α) is known to induce glycolytic enzymes as well as suppressing mitochondrial energy production by inducing pyruvate dehydrogenase kinase 1 (Pdk1) in most cell types. It has not been established whether PDK1 is essential for HSC function and mediates hypoxia-adapting functions in HSCs. While the Pdk gene family contains four members (Pdk1-4), it was recently shown that Pdk2 and Pdk4 have an important role in regulating LT-HSCs. PRINCIPLE FINDINGS: Here we demonstrate that PDK1 activity is crucial for transplantable HSC function. Whereas Pdkl, Pdk2, and Pdk3 transcripts were expressed at higher levels in different subtypes of HSCs compared to differentiated cells, we could not detect any major differences in expression between LT-HSCs and more short-term HSCs and multipotent progenitors. When studying HIF-1α-mediated regulation of Pdk activity in vitro, Pdk1 was the most robust target regulated by hypoxia, whereas Pdk2, Pdk3, and Pdk4 were not affected. Contrary, genetic ablation in a cre-inducible Hif-1α knockout mouse did not support a link between HIF-1α and Pdk1. Silencing of Pdk1 by shRNA lentiviral gene transfer partially impaired progenitor colony formation in vitro and had a strong negative effect on both long-term and short-term engraftment in mice. CONCLUSIONS: Our study demonstrates that PDK1 has broad effects in hematopoiesis and is a critical factor for engraftment of both HSCs and multipotent progenitors upon transplantation to recipient mice. While Pdk1 was a robust hypoxia-inducible gene mediated by HIF-1α in vitro, we could not find evidence of any in vivo links between Pdk1 and HIF-1α.

Ruptured human Achilles tendon has elevated metabolic activity up to 1 year after repair.

PURPOSE: Following Achilles tendon rupture, running is often allowed after 6 months. However, tendon healing is slow and the metabolic status of the tendon at this point is unknown. The purpose of this study was to investigate tendon metabolism (glucose uptake) and vascularization at 3, 6 and 12 months after Achilles tendon rupture as measured using PET and power Doppler ultrasonography (PDUS). METHODS: The study group comprised 23 patients with surgically repaired Achilles tendon rupture who were investigated at 3 months (n = 7), 6 months (n = 7) and 12 months (n = 9) after surgery. The triceps surae complex was loaded over 20 min of slow treadmill walking while a radioactive tracer ((18)F-FDG) was administered prior to PET. Vascularization was measured in terms of PDUS flow activity, and patient-reported outcomes were scored using the Achilles tendon rupture score (ATRS) and sports assessment (VISA-A) questionnaire. RESULTS: Relative glucose uptake ((18)F-FDG) was higher in repaired tendons than in intact tendons at all time-points (6, 3 and 1.6 times higher at 3, 6 and 12 months, respectively; P ≤ 0.001), and was also higher in the tendon core than in the periphery at 3 and 6 months (P ≤ 0.02), but lower at 12 months (P = 0.06). Relative glucose uptake was negatively related to ATRS at 6 months after repair (r = -0.89, P ≤ 0.01). PDUS flow activity was higher in repaired tendons than in intact tendons at 3 and 6 months (P < 0.05 for both), but had normalized by 12 months. CONCLUSION: These data demonstrate that the healing process as determined by metabolic activity and vascularization continues for 6 months after injury when large loads are typically allowed on the tendon. Indeed, metabolic activity remained elevated for more than 1 year after injury despite normalized vascularization. The robust negative correlation between tendon metabolism and patient-reported outcome suggests that a high metabolic activity 6 months after the injury may be related to a poor clinical healing outcome.

Modulation of glutamine metabolism by the PI(3)K-PKB-FOXO network regulates autophagy.

The PI(3)K-PKB-FOXO signalling network provides a major intracellular hub for the regulation of cell proliferation, survival and stress resistance. Here we report an unexpected role for FOXO transcription factors in regulating autophagy by modulating intracellular glutamine levels. To identify transcriptional targets of this network, we performed global transcriptional analyses after conditional activation of the key components PI(3)K, PKB/Akt, FOXO3 and FOXO4. Using this pathway approach, we identified glutamine synthetase as being transcriptionally regulated by PI(3)K-PKB-FOXO signalling. Conditional activation of FOXO also led to an increased level of glutamine production. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes in a glutamine-synthetase-dependent manner. This resulted in an increased level of autophagy as measured by LC3 lipidation, p62 degradation and fluorescent imaging of multiple autophagosomal markers. Inhibition of FOXO3-mediated autophagy increased the level of apoptosis, suggesting that the induction of autophagy by FOXO3-mediated glutamine synthetase expression is important for cellular survival. These findings reveal a growth-factor-responsive network that can directly modulate autophagy through the regulation of glutamine metabolism.

Etanercept does not impair healing in rat models of tendon or metaphyseal bone injury.

BACKGROUND AND PURPOSE: Should blockade of TNF-α be avoided after orthopedic surgery? Healing of injuries in soft tissues and bone starts with a brief inflammatory phase. Modulation of inflammatory signaling might therefore interfere with healing. For example, Cox inhibitors impair healing in animal models of tendon, ligament, and bone injury, as well as in fracture patients. TNF-α is expressed locally at increased levels during early healing of these tissues. We therefore investigated whether blocking of TNF-α with etanercept influences the healing process in established rat models of injury of tendons and metaphyseal bone. METHODS: Rats were injected with etanercept, 3.5 mg/kg 3 times a week. Healing of transected Achilles tendons and bone healing around screws implanted in the tibial metaphysis were estimated by mechanical testing. Tendons were allowed to heal either with or without mechanical loading. Ectopic bone induction following intramuscular BMP-2 implants has previously been shown to be stimulated by etanercept in rodents. This was now tested as a positive control. RESULTS: Tendon peak force after 10 days was not significantly influenced by etanercept. Changes exceeding 29% could be excluded with 95% confidence. Likewise, screw pull-out force was not significantly influenced. More than 25% decrease or 18% increase could be excluded with 95% confidence. However, etanercept treatment increased the amount of bone induced by intramuscular BMP-2 implants, as estimated by blind histological scoring. INTERPRETATION: Etanercept does not appear to impair tendon or metaphyseal bone healing to any substantial degree.

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.

Irreversible pan-ERBB inhibitor canertinib elicits anti-leukaemic effects and induces the regression of FLT3-ITD transformed cells in mice.

Recent findings have indicated that tyrosine kinase inhibitors (TKIs) targeting the ERBB receptor family display anti-leukaemic effects, despite the lack of receptor expression on human leukaemic cells. The occurrence of activating mutations in the gene encoding FMS-like tyrosine kinase 3 (FLT3) in patients with acute myeloid leukaemia (AML) has rendered inhibition of this receptor a promising therapeutic target. Due to possibility of cross-reactivity, we investigated the effect of the irreversible pan-ERBB inhibitor canertinib (CI-1033) on leukaemic cells expressing FLT3. The drug had anti-proliferative and apoptotic effects on primary AML cells and human leukaemic cell lines expressing mutated FLT3. In several AML patient samples, a blast cell population expressing FLT3-internal tandem duplication (ITD) was eradicated by canertinib. Canertinib inhibited receptor autophosphorylation and kinase activity of both mutated and FLT3 ligand stimulated wildtype FLT3, leading to inhibition of the PI3-kinase and MAP kinase pathways. Apoptotic induction was dependent on pro-apoptotic BH3-only protein BCL2L11/BIM because siRNA silencing attenuated apoptosis. Moreover, the drug induced regression of cells expressing FLT3-ITD in a murine in vivo-transplantation model at previously described tolerated doses. These results indicate that canertinib, as an irreversible TKI, could constitute a novel treatment regimen in patients with mutated or overexpressed FLT3.

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.

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.

Tissue memory in healing tendons: short loading episodes stimulate healing.

Intact tendons adapt slowly to changes in mechanical loading, whereas in healing tendons the effect of mechanical loading or its absence is dramatic. The longevity of the response to a single loading episode is, however, unknown. We hypothesized that the tissue has a "memory" of loading episodes and that therefore short loadings are sufficient to elicit improved healing. The Achilles tendon of 70 female rats was transected and unloaded by tail suspension for 12 days (suspension started on day 2 after surgery). Each day, the rats were let down from suspension for short daily training episodes according to different regimes: 15 min of cage activity or treadmill running for 15, 30, 60, or 2x15 min. Rats with transected Achilles tendons and full-time cage activity served as controls. The results demonstrated that full-time cage activity increased the peak force over three times compared with unloading. Short daily loading episodes (treadmill running) increased the peak force about half as much as full-time activity. Prolongation of treadmill running above 15 min or dividing the daily training in two separate episodes had minimal further effect. This mechanical stimulation increased the cross-sectional area but had no effect on the mechanical properties of the repair tissue. The findings indicate that once the tissue had received information from a certain loading type and level, this is "memorized" and leads to a response lasting many hours. This suggests that patients might be allowed early short loading episodes following, e.g., an Achilles tendon rupture for a better outcome.

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.

Mechanical load and BMP signaling during tendon repair: a role for follistatin?

Healing of the rat Achilles tendon is sensitive to mechanical loading, and the callus strength is reduced by 3/4 after 14 days, if loading is prevented. Exogenous GDFs stimulate tendon healing. This response is influenced by loading: without loading, cartilage and bone formation is initiated. This implies BMP signaling is crucial during tendon healing and influenced by mechanical loading. We therefore asked if mechanical loading influences the gene expression of the BMP signaling system in intact and healing tendons, and how the BMP signaling system changes during healing. The genes were four BMPs (OP-1/BMP-7, GDF-5/CDMP-1/BMP-14, GDF-6/CDMP-2/BMP-13, and GDF-7/CDMP-3/BMP-12), two receptors (BMPR1b and BMPR2), and the antagonists follistatin and noggin. The Achilles tendon was transected in rats and left to heal. Half of the rats had one Achilles tendon unloaded by injection of Botox in the calf muscles. Ten tendons were analyzed before transection and for each of four time points. All genes except noggin were expressed at all time points, but followed different patterns during healing. Loading strongly decreased the expression of follistatin, which could lead to increased signaling. The BMP system appears involved in tendon maintenance and healing, and may respond to mechanical loading.