Intervertebral disc herniation effects on multifidus muscle composition and resident stem cell populations.
ABSTRACT: Background:Paraspinal muscles are crucial for vertebral stabilization and movement. These muscles are prone to develop fatty infiltration (FI), fibrosis, and atrophy in many spine conditions. Fibro-adipogenic progenitors (FAPs), a resident muscle stem cell population, are the main contributors of muscle fibrosis and FI. FAPs are involved in a complex interplay with satellite cells (SCs), the primary myogenic progenitor cells within muscle. Little is known about the stem cell composition of the multifidus. The aim of this study is to examine FAPs and SCs in the multifidus in disc herniation patients. Multifidus muscle samples were collected from 10 patients undergoing decompressive spine surgery for lumbar disc herniation. Hamstring muscle was collected from four patients undergoing hamstring autograft ACL reconstruction as an appendicular control. Multifidus tissue was analyzed for FI and fibrosis using Oil-Red-O and Masson's trichrome staining. FAPs and SCs were visualized using immunostaining and quantified with fluorescence-activated cell sorting (FACS) sorting. Gene expression of these cells from the multifidus were analyzed with reverse transcription-polymerase chain reaction and compared to those from hamstring muscle. FI and fibrosis accounted for 14.2%± 7.4% and 14.8%±4.2% of multifidus muscle, respectively. The multifidus contained more FAPs (11.7%±1.9% vs 1.4%±0.2%; P<.001) and more SCs (3.4%±1.6% vs 0.08%±0.02%; P=.002) than the hamstring. FAPs had greater ? Smooth Muscle Actin (?SMA) and adipogenic gene expression than FAPs from the hamstring. SCs from the multifidus displayed upregulated expression of stem, proliferation, and differentiation genes. Conclusion:The multifidus in patients with disc herniation contains large percentages of FAPs and SCs with different gene expression profiles compared to those in the hamstring. These results may help explain the tendency for the multifidus to atrophy and form FI and fibrosis as well as elucidate potential approaches for mitigating these degenerative changes by leveraging these muscle stem cell populations.
Project description:Growing evidence suggests an association of lumbar paraspinal muscle morphology with low back pain (LBP) and lumbar pathologies. Unilateral spinal disorders provide unique models to study this association, with implications for diagnosis, prognosis, and management. Statistical shape analysis is a technique that can identify signature shape variations related to phenotypes but has never been employed in studying paraspinal muscle morphology. We present the first investigation using this technique to reveal disease-related paraspinal muscle asymmetry, using MRIs of patients with a single posterolateral disc herniation at the L5-S1 spinal level and unilateral leg pain. Statistical shape analysis was conducted to reveal disease- and phenotype-related morphological variations in the multifidus and erector spinae muscles at the level of herniation and the one below. With the analysis, shape variations associated with disc herniation were identified in the multifidus on the painful side at the level below the pathology while no pathology-related asymmetry in cross-sectional area (CSA) and fatty infiltration was found in either muscle. The results demonstrate higher sensitivity and spatial specificity for the technique than typical CSA and fatty infiltration measures. Statistical shape analysis holds promise in studying paraspinal muscle morphology to improve our understanding of LBP and various lumbar pathologies.
Project description:<h4>Background</h4>Individual study results have demonstrated unclear relationships between neurocompressive disorders and paraspinal muscle morphology. This systematic review aimed to synthesize current evidence regarding the relationship lumbar neurocompressive disorders may have with lumbar paraspinal muscle morphology.<h4>Methods</h4>Searches were conducted in seven databases from inception through October 2017. Observational studies with control or comparison groups comparing herniations, facet degeneration, or canal stenosis to changes in imaging or biopsy-identified lumbar paraspinal muscle morphology were included. Data extraction and risk of bias assessment were performed by review author pairs independent of one another. Morphological differences between individuals with and without neurocompressive disorders were compared qualitatively, and where possible, standardised mean differences were obtained.<h4>Results</h4>Twenty-eight studies were included. Lumbar multifidus fiber diameter was smaller on the side of and below herniation for type I [SMD: -0.40 (95% CI = -0.70, -0.09) and type II fibers [SMD: -0.38 (95% CI = -0.69, -0.06)] compared to the unaffected side. The distribution of type I fibers was greater on the herniation side [SMD: 0.43 (95% CI = 0.03, 0.82)]. Qualitatively, two studies assessing small angular fiber frequency and fiber type groupings demonstrated increases in these parameters below the herniation level. For diagnostic imaging meta-analyses, there were no consistent differences across the various assessment types for any paraspinal muscle groups when patients with herniation served as their own control. However, qualitative synthesis of between-group comparisons reported greater multifidus and erector spinae muscle atrophy or fat infiltration among patients with disc herniation and radiculopathy in four of six studies, and increased fatty infiltration in paraspinal muscles with higher grades of facet joint degeneration in four of five studies. Conflicting outcomes and variations in study methodology precluded a clear conclusion for canal stenosis.<h4>Conclusions</h4>Based on mixed levels of risk of bias data, in patients with chronic radiculopathy, disc herniation and severe facet degeneration were associated with altered paraspinal muscle morphology at or below the pathology level. As the variability of study quality and heterogeneous approaches utilized to assess muscle morphology challenged comparison across studies, we provide recommendations to promote uniform measurement techniques for future studies.<h4>Trial registration</h4>PROSPERO 2015: CRD42015012985.
Project description:Fibro-adipogenic progenitors (FAPs) are typically activated in response to muscle injury, and establish functional interactions with inflammatory and muscle stem cells (MuSCs) to promote muscle repair. We found that denervation causes progressive accumulation of FAPs, without concomitant infiltration of macrophages and MuSC-mediated regeneration. Denervation-activated FAPs exhibited persistent STAT3 activation and secreted elevated levels of IL-6, which promoted muscle atrophy and fibrosis. FAPs with aberrant activation of STAT3-IL-6 signalling were also found in mouse models of spinal cord injury, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS) and in muscles of ALS patients. Inactivation of STAT3-IL-6 signalling in FAPs effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and ALS (SODG93A mice). Activation of pathogenic FAPs following loss of integrity of neuromuscular junctions further illustrates the functional versatility of FAPs in response to homeostatic perturbations and suggests their potential contribution to the pathogenesis of neuromuscular diseases.
Project description:Still little is known about how spinopelvic alignment affects spinal load distribution. Musculoskeletal modeling can potentially help to discover associations between spine alignment and risk factors of spinal disorders (e.g. disc herniation, vertebral fracture, spondylolisthesis, low back pain). The present study exploited the AnyBody full-body musculoskeletal model to assess the relation between lumbar loads and spinopelvic alignment in the sagittal plane. The model was evaluated in the standing position. The simulated postures were set using spinopelvic parameters gleaned from the literature and characterizing the healthy adult population. The parameters were: sagittal vertical axis, Roussouly lumbar type, sacral slope, and pelvic incidence. A total of 2772 configurations were simulated based on the following measurements: compression force and anterior shear at levels L4L5 and L5S1; multifidus, longissimus spinae, and rectus abdominis muscle forces. Changes in global sagittal alignment, lumbar typology, and sacral inclination, but not in pelvic incidence, were found to affect intervertebral loads in the lumbar spine and spinal muscle activation. Considering these changes would be advantageous for clinical evaluation, due to the recognized relation between altered loads and risk of disc herniation, vertebral fracture, spondylolisthesis, and low back pain. Musculoskeletal modeling proved to be a valuable biomechanical tool to non-invasively investigate the relation between internal loads and anatomical parameters.
Project description:<h4>Background</h4>A longstanding goal in regenerative medicine is to reconstitute functional tissues or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised.<h4>Methods</h4>We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®.<h4>Results</h4>We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.<h4>Conclusions</h4>Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.
Project description:Fibrosis in skeletal muscle develops after injury or in response to chronic kidney disease (CKD), but the origin of cells becoming fibrous tissue and the initiating and sustaining mechanisms causing muscle fibrosis are unclear. We identified muscle fibro/adipogenic progenitor cells (FAPs) that potentially differentiate into adipose tissues or fibrosis. We also demonstrated that CKD stimulates myostatin production in muscle. Therefore, we tested whether CKD induces myostatin, which stimulates fibrotic differentiation of FAPs leading to fibrosis in skeletal muscles. We isolated FAPs from mouse muscles and found that myostatin stimulates their proliferation and conversion into fibrocytes. In vivo, FAPs isolated from EGFP-transgenic mice (FAPs-EGFP) were transplanted into muscles of mice with CKD or into mouse muscles that were treated with myostatin. CKD or myostatin stimulated FAPs-EGFP proliferation in muscle and increased ?-smooth muscle actin expression in FAP-EGFP cells. When myostatin was inhibited with a neutralizing peptibody (a chimeric peptide-Fc fusion protein), the FAP proliferation and muscle fibrosis induced by CKD were both suppressed. Knocking down Smad3 in cultured FAPs interrupted their conversion into fibrocytes, indicating that myostatin directly converts FAPs into fibrocytes. Thus, counteracting myostatin may be a strategy for preventing the development of fibrosis in skeletal muscles of patients with CKD.
Project description:<h4>Background</h4>Chronic muscle injury is characteristics of fatty infiltration and fibrosis. Recently, fibro/adipogenic progenitors (FAPs) were found to be indispensable for muscular regeneration while were also responsible for fibrosis and fatty infiltration in muscle injury. Many myokines have been proven to regulate the adipose or cell proliferation. Because the fate of FAPs is largely dependent on microenvironment and the regulation of myokines on FAPs is still unclear. We screened the potential myokines and found Interleukin-15 (IL-15) may regulate the fatty infiltration in muscle injury. In this study, we investigated how IL-15 regulated FAPs in muscle injury and the effect on muscle regeneration.<h4>Methods</h4>Cell proliferation assay, western blots, qRT-PCR, immunohistochemistry, flow cytometric analysis were performed to investigate the effect of IL-15 on proliferation and adipogensis of FAPs. Acute muscle injury was induced by injection of glycerol or cardiotoxin to analyze how IL-15 effected on FAPs in vivo and its function on fatty infiltration or muscle regeneration.<h4>Results</h4>We identified that the expression of IL-15 in injured muscle was negatively associated with fatty infiltration. IL-15 can stimulate the proliferation of FAPs and prevent the adipogenesis of FAPs in vitro and in vivo. The growth of FAPs caused by IL-15 was mediated through JAK-STAT pathway. In addition, desert hedgehog pathway may participate in IL-15 inhibiting adipogenesis of FAPs. Our study showed IL-15 can cause the fibrosis after muscle damage and promote the myofiber regeneration. Finally, the expression of IL-15 was positively associated with severity of fibrosis and number of FAPs in patients with chronic rotator cuff tear.<h4>Conclusions</h4>These findings supported the potential role of IL-15 as a modulator on fate of FAPs in injured muscle and as a novel therapy for chronic muscle injury.
Project description:We show that extracellular vesicles (EVs) released by mesenchymal cells (i.e., fibro-adipogenic progenitors-FAPs) mediate microRNA (miR) transfer to muscle stem cells (MuSCs) and that exposure of dystrophic FAPs to HDAC inhibitors (HDACis) increases the intra-EV levels of a subset of miRs, which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis, and inflammation. Increased levels of miR-206 in EVs released by FAPs of muscles from Duchenne muscular dystrophy (DMD) patients or mdx mice exposed to HDACi are associated with enhanced regeneration and decreased fibrosis. Consistently, EVs from HDACi-treated dystrophic FAPs can stimulate MuSC activation and expansion ex vivo, and promote regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation in mdx mice, in vivo. AntagomiR-mediated blockade of individual miRs reveals a specific requirement of miR-206 for EV-induced expansion of MuSCs and regeneration of dystrophic muscles, and indicates that cooperative activity of HDACi-induced miRs accounts for the net biological effect of these EVs. These data point to pharmacological modulation of EV content as novel strategy for therapeutic interventions in muscular dystrophies.
Project description:Platelet-derived growth factor receptor ? (PDGFR?) exhibits divergent effects in skeletal muscle. At physiological levels, signalling through this receptor promotes muscle development in growing embryos and angiogenesis in regenerating adult muscle. However, both increased PDGF ligand abundance and enhanced PDGFR? pathway activity cause pathological fibrosis. This excessive collagen deposition, which is seen in aged and diseased muscle, interferes with muscle function and limits the effectiveness of gene- and cell-based therapies for muscle disorders. Although compelling evidence exists for the role of PDGFR? in fibrosis, little is known about the cells through which this pathway acts. Here we show in mice that PDGFR? signalling regulates a population of muscle-resident fibro/adipogenic progenitors (FAPs) that play a supportive role in muscle regeneration but may also cause fibrosis when aberrantly regulated. We found that FAPs produce multiple transcriptional variants of Pdgfra with different polyadenylation sites, including an intronic variant that codes for a protein isoform containing a truncated kinase domain. This variant, upregulated during regeneration, acts as a decoy to inhibit PDGF signalling and to prevent FAP over-activation. Moreover, increasing the expression of this isoform limits fibrosis in vivo in mice, suggesting both biological relevance and therapeutic potential of modulating polyadenylation patterns in stem-cell populations.
Project description:Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.