T lymphocytes are not required for the development of fatty degeneration after rotator cuff tear

Rotator cuff tears are among the most common and debilitating upper extremity injuries. Chronic cuff tears result in atrophy and an infiltration of fat into the muscle, a condition commonly referred to as ‘fatty degeneration’. While stem cell therapies hold promise for the treatment of cuff tears, a suitable immunodeficient animal model that could be used to study human or other xenograft-based therapies for the treatment of rotator cuff injuries had not previously been identified.  A full-thickness, massive supraspinatus and infraspinatus tear was induced in adult T-cell deficient rats. We hypothesised that, compared with controls, 28 days after inducing a tear we would observe a decrease in muscle force production, an accumulation of type IIB fibres, and an upregulation in the expression of genes involved with muscle atrophy, fibrosis and inflammation.  Chronic cuff tears in nude rats resulted in a 30% to 40% decrease in muscle mass, a 23% reduction in production of muscle force, and an induction of genes that regulate atrophy, fibrosis, lipid accumulation, inflammation and macrophage recruitment. Marked large lipid droplet accumulation was also present.  The extent of degenerative changes in nude rats was similar to what was observed in T-cell competent rats. T cells may not play an important role in regulating muscle degeneration following chronic muscle unloading. The general similarities between nude and T-cell competent rats suggest the nude rat is likely an appropriate preclinical model for the study of xenografts that have the potential to enhance the treatment of chronically torn rotator cuff muscles.

Related papers:

title: National trends in rotator cuff repair.
abstract: Recent publications suggest that arthroscopic and open rotator cuff repairs have had comparable clinical results, although each technique has distinct advantages and disadvantages. National hospital and ambulatory surgery databases were reviewed to identify practice patterns for rotator cuff repair.



The rates of medical visits for rotator cuff pathology, and the rates of open and arthroscopic rotator cuff repair, were examined for the years 1996 and 2006 in the United States. The national incidence of rotator cuff repairs and related data were obtained from inpatient (National Hospital Discharge Survey, NHDS) and ambulatory surgery (National Survey of Ambulatory Surgery, NSAS) databases. These databases were queried with use of International Classification of Diseases, Ninth Revision (ICD-9) procedure codes for arthroscopic (ICD-9 codes 83.63 and 80.21) and open (code 83.63 without code 80.21) rotator cuff repair. We also examined where the surgery was performed (inpatient versus ambulatory surgery center) and characteristics of the patients, including age, sex, and comorbidities.



The unadjusted volume of all rotator cuff repairs increased 141% in the decade from 1996 to 2006. The unadjusted number of arthroscopic procedures increased by 600% while open repairs increased by only 34% during this time interval. There was a significant shift from inpatient to outpatient surgery (p < 0.001).



The increase in national rates of rotator cuff repair over the last decade has been dramatic, particularly for arthroscopic assisted repair.

title: Massive Tears of the Rotator Cuff
abstract: Even though it may seem simple to classify rotator cuff tears only by size, clinical studies tend to confirm that treatment outcomes are directly related to it. Tear size was identified as the single most important factor determining final active motion, strength, satisfaction and need for revision in a series of 105 patients who underwent open rotator cuff repair [11]. When comparing repairs performed in groups of large and massive tears with groups including small and medium sized ones there is a definite tendency to poorer outcomes in those with the bigger defects independent of the surgical technique used [22]. In this unfavourable scenario treatment protocols should be well-established. The surgeon finds himself facing a wide range of options with confusing data supporting each one. There are reports of successful non-operative management; debridement and decompression has been shown to help others; structurally-failed repairs are not unheard of with good clinical outcomes; and partial repairs can restore function [4, 27]. It is only by reviewing pertinent information and matching it with personal experience that the surgeon can make appropriate treatment decisions.

title: Fatty muscle degeneration in cuff ruptures. Pre- and postoperative evaluation by CT scan.
abstract: A preoperative computed tomography (CT) scan grading muscular fatty degeneration in five stages was done in 63 patients scheduled for repair of a torn rotator cuff. The results were compared with postoperative evaluation done after a mean of 17.7 months in 57 patients. Postoperative arthrographies were also performed in 56 patients. Preoperative CT scans demonstrated that infraspinatus fatty degeneration can occur in the presence of large anterosuperior tears even when the infraspinatus tendon is not torn; it worsens with time. The subscapularis rarely degenerates, and when it does it degenerates moderately, even when its tendon is not torn. After an effective surgical repair, moderate supraspinatus degeneration regressed in six of 14 patients; that of the infraspinatus never regressed but rather, increased, in three patients. One of these deteriorations, involving both supra- and infraspinatus, could probably be attributed to a partial subscapular nerve injury. Infraspinatus degeneration was correlated with functional pre- and postoperative impairment of active external rotation. Recurrence of infraspinatus tear was never observed, but recurrence occurred in 25% of supraspinatus repairs. Infraspinatus degeneration had a highly negative influence on the outcome of supraspinatus repairs. It seems preferable to operate on wide tears before irreversible muscular damage takes place.

title: Fatty Infiltration and Atrophy of the Rotator Cuff do not Improve after Rotator Cuff Repair and Correlate with Poor Functional Outcome
abstract: The role of degenerative changes in rotator cuff musculature with respect to the functional outcomes of rotator cuff repair have only recently been recognized and are still not well understood. In addition, the reversibility of these changes with repair of the tendons is questionable. Hypothesis Poorer preoperative muscle quality negatively affects outcome, and a successful outcome (in terms of a healed repair) might demonstrate improvements in fatty infiltration and muscle atrophy.   Thirty-eight patients (mean age, 62 years) were prospectively evaluated with preoperative and 1-year postoperative clinical examination and appropriate magnetic resonance image sequencing to determine grades of muscle atrophy and fatty infiltration of the supraspinatus and infraspinatus muscles. American Shoulder and Elbow Society (ASES), Constant, and pain scores were determined as well as strength measurements. The retear rate and progression of muscle degeneration were also evaluated. Independent predictors of outcome measurements and cuff integrity were determined.  The overall clinical outcome, including ASES, Constant, and pain scores, improved significantly (P < .0001). Strength in forward elevation improved significantly (P < .006), while external rotation strength did not. There was a strongly negative correlation between muscle quality and outcome results in most cases. When the results were adjusted for multivariate effect, muscle atrophy and fatty infiltration of the infraspinatus muscle were the only independent predictors of ASES and Constant scores (P < .03). Tear size and rotator cuff healing did not play an independent role. Tear size, however, was the only independent predictor of ultimate cuff integrity (P = .002). Both atrophy and fatty infiltration progressed significantly over the course of the study. In cases in which the tendon had re-torn, the progression was found to be more significant than when the repair proved successful (P < .003).  Muscle atrophy and fatty infiltration of the rotator cuff muscles, particularly of the infraspinatus, play a significant role in determining functional outcome after cuff repair. Tear size appears to have the most influential effect on repair integrity. A successful repair did not lead to improvement or reversal of muscle degeneration and a failed repair resulted in significantly more progression. In general, healed repairs demonstrated minimal progression. These findings suggest that repairs should be performed, if possible, before more significant deterioration in the cuff musculature in order to optimize outcomes, and that understanding the degree of muscle atrophy and fatty infiltration before surgery can help guide patient expectations.

title: The Inflammatory Response to Skeletal Muscle Injury
abstract: Injury of skeletal muscle, and especially mechanically induced damage such as contusion injury, frequently occurs in contact sports, as well as in accidental contact sports, such as hockey and squash. The large variations with regard to injury severity and affected muscle group, as well as nonspecificity of reported symptoms, complicate research aimed at finding suitable treatments. Therefore, in order to increase the chances of finding a successful treatment, it is important to understand the underlying mechanisms inherent to this type of skeletal muscle injury and the cellular processes involved in muscle healing following a contusion injury.Arguably the most important of these processes is inflammation since it is a consistent and lasting response. The inflammatory response is dependent on two factors, namely the extent of actual physical damage and the degree of muscle vascularization at the time of injury. However, long-term antiinflammatory treatment is not necessarily effective in promoting healing, as indicated by various studies on NSAID treatment. Because of the factors named earlier, human studies on the inflammatory response to contusion injury are limited, but several experimental animal models have been designed to study muscle damage and regeneration.The early recovery phase is characterized by the overlapping processes of inflammation and occurrence of secondary damage. Although neutrophil infiltration has been named as a contributor to the latter, no clear evidence exists to support this claim. Macrophages, although forming part of the inflammatory response, have been shown to have a role in recovery, rather than in exacerbating secondary damage. Several probable roles for this cell type in the second phase of recovery, involving resolution processes, have been identified and include the following: (i) phagocytosis to remove cellular debris; (ii) switching from a pro- to anti-inflammatory phenotype in regenerating muscle; (iii) preventing muscle cells from undergoing apoptosis; (iv) releasing factors to promote muscle precursor cell activation and growth; and (v) secretion of cytokines and growth factors to facilitate vascular and muscle fibre repair. These many different roles suggest that a single treatment with one specific target cell population (e.g. neutrophils, macrophages or satellite cells) may not be equally effective in all phases of the post-injury response.To find the optimal targeted, but time-course-dependent, treatments requires substantial further investigations. However, the techniques currently used to induce mechanical injury vary considerably in terms of invasiveness, tools used to induce injury, muscle group selected for injury and contractile status of the muscle, all of which have an influence on the immune and/or cytokine responses. This makes interpretation of the complex responses more difficult. After our review of the literature, we propose that a standardized non-invasive contusion injury is the ideal model for investigations into the immune responses to mechanical skeletal muscle injury. Despite its suitability as a model, the currently available literature with respect to the inflammatory response to injury using contusion models is largely inadequate.Therefore, it may be premature to investigate highly targeted therapies, which may ultimately prove more effective in decreasing athlete recovery time than current therapies that are either not phase-specific, or not administered in a phase-specific fashion.

title: Regulatory interactions between muscle and the immune system during muscle regeneration.
abstract: Recent discoveries reveal complex interactions between skeletal muscle and the immune system that regulate muscle regeneration. In this review, we evaluate evidence that indicates that the response of myeloid cells to muscle injury promotes muscle regeneration and growth. Acute perturbations of muscle activate a sequence of interactions between muscle and inflammatory cells. The initial inflammatory response is a characteristic Th1 inflammatory response, first dominated by neutrophils and subsequently by CD68(+) M1 macrophages. M1 macrophages can propagate the Th1 response by releasing proinflammatory cytokines and cause further tissue damage through the release of nitric oxide. Myeloid cells in the early Th1 response stimulate the proliferative phase of myogenesis through mechanisms mediated by TNF-alpha and IL-6; experimental prolongation of their presence is associated with delayed transition to the early differentiation stage of myogenesis. Subsequent invasion by CD163(+)/CD206(+) M2 macrophages attenuates M1 populations through the release of anti-inflammatory cytokines, including IL-10. M2 macrophages play a major role in promoting growth and regeneration; their absence greatly slows muscle growth following injury or modified use and inhibits muscle differentiation and regeneration. Chronic muscle injury leads to profiles of macrophage invasion and function that differ from acute injuries. For example, mdx muscular dystrophy yields invasion of muscle by M1 macrophages, but their early invasion is accompanied by a subpopulation of M2a macrophages. M2a macrophages are IL-4 receptor(+)/CD206(+) cells that reduce cytotoxicity of M1 macrophages. Subsequent invasion of dystrophic muscle by M2c macrophages is associated with progression of the regenerative phase in pathophysiology. Together, these findings show that transitions in macrophage phenotype are an essential component of muscle regeneration in vivo following acute or chronic muscle damage.

title: Aberrant repair and fibrosis development in skeletal muscle
abstract: The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells) is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.

title: Definitive engagement of cytotoxic CD8 T cells in C protein-induced myositis, a murine model of polymyositis.
abstract: To substantiate a pathogenic role of cytotoxic CD8 T cells in the development of a murine polymyositis model, C protein-induced myositis (CIM).



Beta(2)-microglobulin-null mutant, perforin-null mutant, and wild-type (WT) C57BL/6 mice were immunized with skeletal muscle C protein fragments to provoke CIM. Regional lymph node CD8 or CD4 T cells stimulated with C protein-pulsed dendritic cells were transferred adoptively to naive mice. Inflammation and damage of the muscle tissues were evaluated histologically.



The incidence of myositis development was significantly lower in β₂-microglobulin-null and perforin-null mutant mice compared with WT mice. Inflammation was less severe in mutant mice, and the incidence of muscle injury was reduced significantly. Adoptive transfer of lymph node T cells from mice with CIM induced myositis in naive recipient mice. The CD8 T cell-induced muscle injuries were significantly more severe than the CD4 T cell-induced muscle injuries.



Perforin-mediated cytotoxicity by CD8 T cells is definitively responsible for muscle injury in CIM.

title: Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation.
abstract: The phenotype of macrophages in atherosclerotic lesions can vary dramatically, from a large lipid laden foam cell to a small inflammatory cell. Classically, the concept of macrophage heterogeneity discriminates between two extremes called either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Polarisation of plaque macrophages is predominantly determined by the local micro-environment present in the atherosclerotic lesion and is rather more complex than typically described by the M1/M2 paradigm. In this review we will discuss the role of various polarising factors in regulating the phenotypical state of plaque macrophages. We will focus on two main levels of phenotype regulation, one determined by differentiation factors produced in the lesion and the other determined by T-cell-derived polarising cytokines. With foam cell formation being a key characteristic of macrophages during atherosclerosis initiation and progression, these polarisation factors will also be linked to lipid handling of macrophages.

title: Macrophages, inflammation, and insulin resistance.
abstract: Obesity induces an insulin-resistant state in adipose tissue, liver, and muscle and is a strong risk factor for the development of type 2 diabetes mellitus. Insulin resistance in the setting of obesity results from a combination of altered functions of insulin target cells and the accumulation of macrophages that secrete proinflammatory mediators. At the molecular level, insulin resistance is promoted by a transition in macrophage polarization from an alternative M2 activation state maintained by STAT6 and PPARs to a classical M1 activation state driven by NF-kappaB, AP1, and other signal-dependent transcription factors that play crucial roles in innate immunity. Strategies focused on inhibiting the inflammation/insulin resistance axis that otherwise preserve essential innate immune functions may hold promise for therapeutic intervention.

title: Soft tissue rheumatic lesions and HIV infection in Zambians.
abstract: To explore the relationship between human immunodeficiency virus (HIV) infection and soft tissue rheumatic lesions in HIV-positive black Zambians.



We performed a prospective study of all patients over 18 years of age attending a rheumatic clinic in a teaching hospital. All patients underwent routine blood tests, and radiographs were performed when indicated. HIV status was determined by ELISA, and clinical staging was determined by World Health Organization criteria. Patients with isolated sacroiliac pain, enthesitis, or a soft tissue lesion were selected for analysis. For HIV-positive patients, only those in clinical stage 1 (asymptomatic or persistent generalized lymphadenopathy) were selected.



Our study cohort comprised 120 patients (41 men, 79 women, age 23-70 yrs). Diagnosis and number (% HIV positive) were distributed as follows: sacroiliitis, 14 (100%); heel pain, 14 (100%); costochondritis, 3 (100%); polyenthesitis (> or = 4 sites), 20 (100%); carpal tunnel syndrome, 8 (63%); rotator cuff syndrome, 18 (30%); tendinitis, 8 (25%); sciatica/cervical spondylosis, 12 (16%); sacroiliac strain, 7 (0%); and de Quervain's tenosynovitis, 16 (0%). HIV seroprevalence was 54% overall, 74% in those under 45 years of age, and 17% in those over 45 years of age. Population prevalence of HIV in Lusaka is about 30% in the 30-40-year age range. Mean erythrocyte sedimentation rate (ESR) in 65 patients positive for HIV was 80 mm/h and in 55 patients negative for HIV, 18 mm/h. Within each subgroup the mean ESR was significantly higher in HIV-positive patients.



A young age and a raised ESR are both good indications of HIV infection in Zambian patients with soft tissue lesions. Enthesitis is a distinct HIV-related phenomenon, either an early form or a forme fruste of HIV-related spondyloarthropathy.

title: The athymic nude rat. I. Morphology of lymphoid and endocrine organs.
abstract: Lymphoid and endocrine organs and salivary gland of 3-, 7-, and 10-week-old congenital athymic ( rnu rnu ) rats and thymus-bearing heterozygous (+/rnu) littermates were histologicaily examined. The thymus rudiment of 3-week-old nudes consisted of undifferentiated epithelial cells and small cysts or ducts. In older animals, the epithelium differentiated into serous or mucous acini while cysts increased in size. In popliteal and mesenteric lymph nodes of nude rats, the thymus-dependent paracortex was virtually free of lymphocytes, whereas postcapillary venules were thin walled. Germinal centers were only incidentally seen in the follicles. The depletion of the thymus-dependent area in the spleen of nude rats was less pronounced when compared with effects in the lymph nodes. No alterations were observed in the pituitary gland, thyroid, adrenals, ovaries, or testes. The absence of morphological effects on the reproductive organs may explain that nude rats of both sexes are fertile; and that the normal sexual difference in body weight is still retained. The normal sexual dimorphism in the salivary glands was less pronounced in the nude rats. The absence of alterations in the endocrine organs of nude rats up to an age of 10 weeks indicates that, by using young adult nude rats for immunological studies, differences in reaction pattern are due to the deficiency of the thymus-dependent immunity.

title: Allograft rejection in athymic nude rats by transferred T-cell subsets. I. The response of naive CD4+ and CD8+ thoracic duct lymphocytes to complete allogeneic incompatibilities.
abstract: PVG.rnu/rnu nude rats were pre-grafted with two allogeneic skin grafts, AO(RTlu) and BN(RTln), 6-14 days in advance of cell transfer. Cellular requirements for rejection were established by transferring graded numbers of B cell-depleted (Ig-) thoracic duct lymphocytes (TDL) or purified W3/25+ (CD4+) or OX8+ (CD8+) TDL subsets. Allografts were rejected by 10(5) to 5 x 10(6) Ig- TDL in a dose-dependent fashion. A similar dose-response relationship was found by transferring 5 x 10(5) to 5 x 10(6) Ig- OX8- TDL (purified by depletion of B cells and OX8+ cells). Larger numbers of Ig- OX8- TDL (10-30 x 10(6)) did not significantly accelerate rejection. W3/25+ TDL alone (10(5)), highly purified by fluorescence-activated cell sorting (FACS), were sufficient to induce allograft rejection in this athymic nude rat model. In contrast, 10 times more FACS purified OX8+ TDL (10(6)) were unable to initiate skin graft rejection despite the complete class I and class II MHC incompatibilities. Furthermore, the addition of 10(6) OX8+ cells did not accelerate or retard the rejection induced by 10(5) W3/25+ cells alone. Pre-grafted nude recipients, irradiated (500 R) 2 hr before W3/25+ TDL injection, in order to eliminate putative nude T cells, rejected allografts on the same day as unirradiated controls. We conclude that when confronted with complete MHC disparities, CD4+ T cells are necessary and sufficient to induce skin allograft rejection whereas CD8+ T cells do not appear to contribute.

title: Biologic approaches to enhance rotator cuff healing after injury.
abstract: Despite the advances in surgical procedures to repair the rotator cuff, there is a high incidence of failure. Biologic approaches, such as growth factor delivery and stem cell and gene therapy, are potential targets for optimization to improve the outcome of rotator cuff therapies and reduce rates of reinjury. This article outlines the current evidence for growth factor and stem cell therapy in tendon healing and the augmentation of rotator cuff repair.



Literature on the PubMed-National Center for Biotechnology Information database was searched using the keywords growth factor, factor, gene therapy, stem cell, mesenchymal, or bone marrow in combination with rotator cuff, supraspinatus, or infraspinatus. Articles that studied growth factors or stem cells alone in rotator cuff repair were selected. Only 3 records showed use of stem cells in rotator cuff repair; thus, we expanded our search to include selected studies on stem cells and Achilles or patellar tendon repairs. Bibliographies and proceedings of meetings were searched to include additional applicable studies. We also included hitherto unpublished data by our group on the use of stem cell transplantation for rotator cuff therapy.



More than 70 articles are summarized, with focus on recent original research papers and significant reviews that summarized earlier records.



Use of growth factors, stem cell therapy, and other tissue-engineering means serve to augment classical surgical rotator cuff repair procedures. The combination of stem cells and growth factors resulted in enhanced repair that emulated uninjured tissue, but the literature search reflected paucity of research in this field. Preclinical evidence from gene therapy and stem cell studies can be used as a start to move therapy from the experimental phase to clinical translation in patients.

title: Adult mesenchymal stem cells for tissue engineering versus regenerative medicine
abstract: Adult mesenchymal stem cells (MSCs) can be isolated from bone marrow or marrow aspirates and because they are culture‐dish adherent, they can be expanded in culture while maintaining their multipotency. The MSCs have been used in preclinical models for tissue engineering of bone, cartilage, muscle, marrow stroma, tendon, fat, and other connective tissues. These tissue‐engineered materials show considerable promise for use in rebuilding damaged or diseased mesenchymal tissues. Unanticipated is the realization that the MSCs secrete a large spectrum of bioactive molecules. These molecules are immunosuppressive, especially for T‐cells and, thus, allogeneic MSCs can be considered for therapeutic use. In this context, the secreted bioactive molecules provide a regenerative microenvironment for a variety of injured adult tissues to limit the area of damage and to mount a self‐regulated regenerative response. This regenerative microenvironment is referred to as trophic activity and, therefore, MSCs appear to be valuable mediators for tissue repair and regeneration. The natural titers of MSCs that are drawn to sites of tissue injury can be augmented by allogeneic MSCs delivered via the bloodstream. Indeed, human clinical trials are now under way to use allogeneic MSCs for treatment of myocardial infarcts, graft‐versus‐host disease, Crohn's Disease, cartilage and meniscus repair, stroke, and spinal cord injury. This review summarizes the biological basis for the in vivo functioning of MSCs through development and aging.  2007.

title: Immunodeficient mouse models to study human stem cell-mediated tissue repair.
abstract: Hematopoietic stem cell transplantation has traditionally been used to reconstitute blood cell lineages that had formed abnormally because of genetic mutations, or that had been eradicated to treat a disease such as leukemia. However, in recent years, much attention has been paid to the new concept of "stem cell plasticity," and the hope that stem cells could be used to repair damaged tissues generated immense excitement. The field is now in a more realistic and critical period of intense investigation and the concept of cell fusion to explain some of the observed effects has been shown after specific types of damage in liver and muscle, both organs that contain a high number of multinucleate cells. The field is still an extremely exciting one, and many questions remain to be answered before stem cell therapy for tissue repair can be used effectively in the clinic. Immune deficient mouse models of tissue damage provide a system in which human stem cell migration to sites of damage and subsequent contribution to repair can be carefully evaluated. This chapter gives detailed instructions for methods to study human stem cell contribution to damaged liver and to promote repair of damaged vasculature in immune deficient mouse models.

title: Rotator cuff tear reduces muscle fiber specific force production and induces macrophage accumulation and autophagy
abstract: Full‐thickness tears to the rotator cuff can cause severe pain and disability. Untreated tears progress in size and are associated with muscle atrophy and an infiltration of fat to the area, a condition known as “fatty degeneration.” To improve the treatment of rotator cuff tears, a greater understanding of the changes in the contractile properties of muscle fibers and the molecular regulation of fatty degeneration is essential. Using a rat model of rotator cuff injury, we measured the force generating capacity of individual muscle fibers and determined changes in muscle fiber type distribution that develop after a full thickness rotator cuff tear. We also measured the expression of mRNA and miRNA transcripts involved in muscle atrophy, lipid accumulation, and matrix synthesis. We hypothesized that a decrease in specific force of rotator cuff muscle fibers, an accumulation of type IIb fibers, and an upregulation in fibrogenic, adipogenic, and inflammatory gene expression occur in torn rotator cuff muscles. Thirty days following rotator cuff tear, we observed a reduction in muscle fiber force production, an induction of fibrogenic, adipogenic, and autophagocytic mRNA and miRNA molecules, and a dramatic accumulation of macrophages in areas of fat accumulation.

title: A rat model of massive rotator cuff tears
abstract: Rotator cuff tears (RCTs) are the most common tendon injury seen in orthopedic patients. Massive RCT does not heal spontaneously and results in poor clinical outcomes. Muscle atrophy and fatty infiltration in rotator cuff muscles are major complications of chronic massive RCT and are thought to be the key factors responsible for the failure of attempted massive RCT repair. However, the pathophysiology of rotator cuff muscle atrophy and fat infiltration remains largely unknown, and no small animal model has been shown to reproduce the histologic and molecular changes seen in massive RCT. In this article, we report a novel rat massive RCT model, in which significant and consistent muscle atrophy and fat infiltration were observed in the rotator cuff muscles after rotator cuff tendon transection and denervation. The supraspinatus and infraspinatus muscle lost 25.4% and 28.9% of their wet weight 2 weeks after complete tendon transection, respectively. Six weeks after surgery, the average wet weight of supraspinatus and infraspinatus muscles decreased 13.2% and 28.3%, respectively. Significant fat infiltration was only observed in infraspinatus 6 weeks after tendon transection.

title: Development and use of an animal model for investigations on rotator cuff disease.
abstract: Although both intrinsic and extrinsic factors have been implicated in the cause of rotator cuff disease, previous studies have not been designed to test hypotheses of this disease, partly because of the lack of an appropriate animal model. Thirty-three animals were evaluated according to a 34 item checklist of criteria to determine their appropriateness as an animal model for investigations on the rotator cuff. Only the rat shoulder satisfactorily fulfilled all criteria, with a prominent supraspinatus tendon passing under an enclosed arch. Once the rat was identified, 36 rats were randomized to three experimental groups. One group (n = 12) was treated with an intratendinous injection of bacterial collagenase simulating an acute intrinsic injury, another group (n = 12) was treated with an acromial alteration to reduce the subacromial space simulating an external compression, and the third group (n = 12) was treated with a combination of both modifications. Significant increases in cellularity, number of fibroblasts, and collagen disorganization were seen in all experimental tendons compared with a contralateral control group. Semiquantitative grading of histologic sections revealed marked changes in all groups at 4 and 8 weeks. Injuries appeared to be healing at 12 weeks except in the combination group, which seemed to demonstrate persistent alterations. This study supports the rat as an appropriate model for investigating rotator cuff disease. In addition, this study demonstrates that both intrinsic and extrinsic alterations can induce changes in the supraspinatus tendon, which may have similarities to human tendon disease.

title: Aging-associated exacerbation in fatty degeneration and infiltration after rotator cuff tear.
abstract: Rotator cuff tears are one of the most common musculoskeletal complaints and a substantial source of morbidity in elderly patients. Chronic cuff tears are associated with muscle atrophy and an infiltration of fat to the area, a condition known as "fatty degeneration." To improve the treatment of cuff tears in elderly patients, a greater understanding of the changes in the contractile properties of muscle fibers and the molecular regulation of fatty degeneration is essential.



Using a full-thickness, massive supraspinatus and infraspinatus tear model in elderly rats, we measured fiber contractility and determined changes in fiber type distribution that develop 30 days after tear. We also measured the expression of messenger RNA and micro-RNA transcripts involved in muscle atrophy, lipid accumulation, and matrix synthesis. We hypothesized that a decrease in specific force of muscle fibers, an accumulation of type IIb fibers, and an upregulation in atrophic, fibrogenic, and inflammatory gene expression would occur in torn cuff muscles.



Thirty days after the tear, we observed a reduction in muscle fiber force and an induction of RNA molecules that regulate atrophy, fibrosis, lipid accumulation, inflammation, and macrophage recruitment. A marked accumulation of advanced glycation end products and a significant accretion of macrophages in areas of fat accumulation were observed.



The extent of degenerative changes in old rats was greater than that observed in adults. In addition, we identified that the ectopic fat accumulation that occurs in chronic cuff tears does not occur by activation of canonical intramyocellular lipid storage and synthesis pathways.

title: Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation.
abstract: Myostatin (MSTN) is a member of the transforming growth factor-β superfamily of cytokines and is a negative regulator of skeletal muscle mass. Compared with MSTN(+/+) mice, the extensor digitorum longus muscles of MSTN(-/-) mice exhibit hypertrophy, hyperplasia, and greater maximum isometric force production (F(o)), but decreased specific maximum isometric force (sF(o); F(o) normalized by muscle cross-sectional area). The reason for the reduction in sF(o) was not known. Studies in myotubes indicate that inhibiting myostatin may increase muscle mass by decreasing the expression of the E3 ubiquitin ligase atrogin-1, which could impact the force-generating capacity and size of muscle fibers. To gain a greater understanding of the influence of myostatin on muscle contractility, we determined the impact of myostatin deficiency on the contractility of permeabilized muscle fibers and on the levels of atrogin-1 and ubiquitinated myosin heavy chain in whole muscle. We hypothesized that single fibers from MSTN(-/-) mice have a greater F(o), but no difference in sF(o), and a decrease in atrogin-1 and ubiquitin-tagged myosin heavy chain levels. The results indicated that fibers from MSTN(-/-) mice have a greater cross-sectional area, but do not have a greater F(o) and have a sF(o) that is significantly lower than fibers from MSTN(+/+) mice. The extensor digitorum longus muscles from MSTN(-/-) mice also have reduced levels of atrogin-1 and ubiquitinated myosin heavy chain. These findings suggest that myostatin inhibition in otherwise healthy muscle increases the size of muscle fibers and decreases atrogin-1 levels, but does not increase the force production of individual muscle fibers.

title: Molecular diversity of myofibrillar proteins: gene regulation and functional significance.
abstract: Myofibrillar proteins exist as multiple isoforms that derive from multigene (isogene) families. Additional isoforms, including products of tropomyosin, myosin light chain 1 fast, troponin T, titin, and nebulin genes, can be generated from the same gene through alternative splicing or use of alternative promoters. Myofibrillar protein isogenes are differentially expressed in various muscle types and fiber types but can be coexpressed within the same fiber. Isogenes are regulated by transcriptional and posttranscriptional mechanisms; however, specific regulatory sequences and transcriptional factors have not yet been identified. The pattern of isogene expression varies during muscle development in relation to the different origin of myogenic cells and primary/secondary fiber generations and is affected by neural and hormonal influences. The variable expression of myofibrillar protein isoforms is a major determinant of the contractile properties of skeletal muscle fibers. The diversity among isomyosins is related to the differences in the parameters of chemomechanical transduction as ATP hydrolysis rate and shortening velocity. Troponin and tropomyosin isoforms determine the variable sensitivity to calcium, whereas titin isoforms dictate the elastic properties of muscle fibers at rest. Both myosin and troponin isoforms contribute to the differences in the resistance to fatigue of muscle fibers.

title: Role of cyclooxygenase‐1 and ‐2 in satellite cell proliferation, differentiation, and fusion
abstract: Skeletal muscle satellite cells play an important role in muscle regeneration. Previous work has suggested that nonsteroidal anti‐inflammatory drugs may inhibit their activity. We cultured skeletal muscle satellite cells from 9‐month‐old Sprague‐Dawley rats and exposed them to naproxen sodium (a nonselective cyclooxygenase inhibitor), NS‐398 (a selective cyclooxygenase‐2 inhibitor), and SC‐560 (a selective cyclooxygenase‐1 inhibitor) for 96 h. Cyclooxygenase‐2 inhibition alone resulted in decreased satellite cell proliferation, and inhibition of both cyclooxygenase‐1 and cyclooxygenase‐2 resulted in decreased satellite cell differentiation and fusion. This study suggests that the cyclooxygenase enzymes appear to play an important part in satellite cell proliferation, differentiation, and fusion and that nonsteroidal anti‐inflammatory medication may have an adverse effect on muscle regeneration following injury. The use of a selective cyclooxygenase‐2 inhibitor over nonspecific cyclooxygenase inhibitors in the treatment of muscle injuries is not supported. Muscle Nerve 30: 497–500, 2004

title: Old and new generation lipid mediators in acute inflammation and resolution.
abstract: Originally regarded as just membrane constituents and energy storing molecules, lipids are now recognised as potent signalling molecules that regulate a multitude of cellular responses via receptor-mediated pathways, including cell growth and death, and inflammation/infection. Derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), each lipid displays unique properties, thus making their role in inflammation distinct from that of other lipids derived from the same PUFA. The diversity of their actions arises because such metabolites are synthesised via discrete enzymatic pathways and because they elicit their response via different receptors. This review will collate the bioactive lipid research to date and summarise the findings in terms of the major pathways involved in their biosynthesis and their role in inflammation and its resolution. It will include lipids derived from AA (prostanoids, leukotrienes, 5-oxo-6,8,11,14-eicosatetraenoic acid, lipoxins and epoxyeicosatrienoic acids), EPA (E-series resolvins), and DHA (D-series resolvins, protectins and maresins).

title: NS-398, a cyclooxygenase-2-specific inhibitor, delays skeletal muscle healing by decreasing regeneration and promoting fibrosis.
abstract: Nonsteroidal anti-inflammatory drugs are often prescribed after muscle injury. However, the effect of nonsteroidal anti-inflammatory drugs on muscle healing remains primarily controversial. To further examine the validity of using these drugs after muscle injury, we investigated the working mechanism of NS-398, a cyclooxygenase-2-specific inhibitor. In vitro experiments showed that NS-398 inhibited the proliferation and maturation of differentiated myogenic precursor cells, suggesting a detrimental effect on skeletal muscle healing. Using a mouse laceration model, we analyzed the in vivo effect of NS-398 on skeletal muscle healing at time points up to 4 weeks after injury. The in vivo results revealed delayed muscle regeneration at early time points after injury in the NS-398-treated mice. Compared to controls, lacerated muscles treated with NS-398 expressed higher levels of transforming growth factor-beta1, which corresponded with increased fibrosis. In addition, transforming growth factor-beta1 co-localized with myostatin, a negative regulator of skeletal muscle growth. We also found reduced neutrophil and macrophage infiltration in treated muscles, indicating that the delayed skeletal muscle healing observed after NS-398 treatment could be influenced by the anti-inflammatory effect of NS-398. Our findings suggest that the use of cyclooxygenase-2-specific inhibitors to treat skeletal muscle injuries warrants caution because they may interfere with muscle healing.

title: Signaling in muscle atrophy and hypertrophy.
abstract: Muscle performance is influenced by turnover of contractile proteins. Production of new myofibrils and degradation of existing proteins is a delicate balance, which, depending on the condition, can promote muscle growth or loss. Protein synthesis and protein degradation are coordinately regulated by pathways that are influenced by mechanical stress, physical activity, availability of nutrients, and growth factors. Understanding the signaling that regulates muscle mass may provide potential therapeutic targets for the prevention and treatment of muscle wasting in metabolic and neuromuscular diseases.

title: Evaluation of Akt/mTOR activity in muscle atrophy after rotator cuff tears in a rat model
abstract: Atrophy of the rotator cuff muscles is a factor that complicates the treatment of a massive rotator cuff tear (RCT). However, the molecular mechanisms that govern the development of muscle atrophy after RCTs have not been well defined. The Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a central role in maintaining muscle mass in response to mechanical loading. The role of this pathway in the development of muscle atrophy after a massive RCT remains unknown. The purpose of this study was to investigate the regulation of the Akt/mTOR pathway in the development of muscle atrophy after a RCT and suprascapular nerve (SSN) injury. We evaluated the activity of the Akt/mTOR signaling pathway and how this pathway interacts with two atrophy‐related genes, MuRF‐1 and MAFbx, in supraspinatus muscles of rats that underwent unilateral complete rotator cuff tendon transection or SSN transection. Akt/mTOR activity was significantly reduced after tendon rupture, but increased after nerve injury. MuRF‐1 and MAFbx were only up‐regulated following denervation. These results suggest that tendon transection leads to a decrease in protein synthesis with down‐regulation of the Akt/mTOR signaling pathway, whereas denervation leads to an increase in protein degradation via up‐regulation of expression of MuRF‐1 and MAFbx.

title: Translational Suppression of Atrophic Regulators by MicroRNA-23a Integrates Resistance to Skeletal Muscle Atrophy*
abstract: Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3′-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.

title: Role of miRNAs in muscle stem cell biology: proliferation, differentiation and death.
abstract: miRNAs are small non-coding RNAs that regulate post-transcriptionally gene expression by degradation or translational repression of specific target mRNAs. In the 90s, lin-4 and let-7 were firstly identified as small regulatory RNAs able to control C. elegans larval development, by specifically targeting the 3'UTR of lin-14 and lin-28, respectively. These findings have introduced a novel and wide layer of complexity in the regulation of mRNA and protein expression. Lin-4 and let-7 are now considered the founding members of an abundant class of small fine-tuned RNAs, called microRNAs (miRNAs), in viruses, green algae, plants, flies, worms, and in mammals. In humans, the estimated number of genes encoding for miRNAs is as high as 1000 and around 30% of the protein-coding genes are post-transcriptionally controlled by miRNAs. This article reviews the role of miRNAs in regulating several biological responses in muscle cells, ranging from proliferation, differentiation and adaptation to stress cues. Cardiac and skeletal muscles are powerful examples to summarize the activity of miRNAs in cell fate specification, lineage differentiation and metabolic pathways. Indeed, specific miRNAs control the number of proliferating muscle progenitors to guarantee the proper formation of the heart and muscle fibers and to assure the self-renewal of muscle progenitors during adult tissue regeneration. On the other side, several other miRNAs promote the differentiation of muscle progenitors into skeletal myofibers or into cardiomyocytes, where metabolic activity, survival and remodeling process in response to stress, injury and chronic diseases are also fine-tuned by miRNAs.

title: MMP inhibition as a potential method to augment the healing of skeletal muscle and tendon extracellular matrix.
abstract: The extracellular matrix (ECM) of skeletal muscle and tendon is composed of different types of collagen molecules that play important roles in the transmission of forces throughout the body, and in the repair and regeneration of injured tissues. Fibroblasts are the primary cells in muscle and tendon that maintain, repair, and modify the ECM in response to mechanical loading, injury, and inactivity. Matrix metalloproteinases (MMPs) are enzymes that digest collagen and other structural molecules, which are synthesized and excreted by fibroblasts. MMPs are required for baseline ECM homeostasis, but disruption of MMP regulation due to injury or disease can alter the normal ECM architecture and prevent proper force transmission. Chronic injuries and diseases of muscles and tendons can be severely debilitating, and current therapeutic modalities to enhance healing are quite limited. This review will discuss the mechanobiology of MMPs, and the potential use of MMP inhibitors to improve the treatment of injured and diseased skeletal muscle and tendon tissue.

title: Hyaluronic acid, HAS1, and HAS2 are significantly upregulated during muscle hypertrophy.
abstract: Hyaluronic acid (HA) is a component of the extracellular matrix (ECM) in most vertebrate tissues and is thought to play a significant role during development, wound healing, and regeneration. In vitro studies have shown that HA enhances muscle progenitor cell recruitment and inhibits premature myotube fusion, implicating a role for this glycosaminoglycan in functional repair. However, the spatiotemporal distribution of HA during muscle growth and repair was unknown. We hypothesized that inducing hypertrophy via synergist ablation would increase the expression of HA and the HA synthases (HAS1-HAS3). We found that HA and HAS1-HAS3 were significantly upregulated within the plantaris muscle in response to Achilles tenectomy. HA concentration significantly increased 2.8-fold after 2 days but decreased towards levels comparable to age-matched controls by 14 days. Using immunohistochemistry, we found the colocalization of HAS1-HAS3 with macrophages, blood vessel epithelia, and fibroblasts varied in response to time and/or tenectomy. At the level of gene expression, only HAS1 and HAS2 significantly increased with respect to both time and tenectomy. The profiles of additional genes that influence ECM composition during muscle repair, tenascin-C, type I collagen, the HA-degrading hyaluronidases (Hyal) and matrix metalloproteinases (MMP) were also investigated. Hyal1 and Hyal2 were highly expressed in skeletal muscle but did not change after tenectomy; however, indicators of hypertrophy, MMP-2 and MMP-14, were significantly upregulated from 2 to 14 days. These results indicate that HA levels dynamically change in response to a hypertrophic stimulus and various cells may participate in this mechanism of skeletal muscle adaptation.

title: Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis
abstract: Efficient tissue regeneration is dependent on the coordinated responses of multiple cell types. Here, we describe a new subpopulation of fibro/adipogenic progenitors (FAPs) resident in muscle tissue but arising from a distinct developmental lineage. Transplantation of purified FAPs results in the generation of ectopic white fat when delivered subcutaneously or intramuscularly in a model of fatty infiltration, but not in healthy muscle, suggesting that the environment controls their engraftment. These cells are quiescent in intact muscle but proliferate efficiently in response to damage. FAPs do not generate myofibres, but enhance the rate of differentiation of primary myogenic progenitors in co-cultivation experiments. In summary, FAPs expand upon damage to provide a transient source of pro-differentiation signals for proliferating myogenic progenitors.

title: Regulation of Myocardial Fibrosis by MicroRNAs
abstract: Interstitial and perivascular fibrosis is a hallmark of adverse cardiac remodeling in response to stress such as hypertension, valve disease, or myocardial infarction. The cross talk between fibroblasts and cardiomyocytes seems to be a major determinant of the hypertrophic response, and fibroblasts may prove to be essential regulators of cardiac remodeling. The present review summarizes current knowledge on the modulation of myocardial fibrosis by microRNAs (miRNAs), single-stranded molecules consisting of approximately 22 noncoding nucleotides that regulate a variety of target genes involved in cardiovascular (patho)physiology. Dissection of miRNA-mediated mechanisms on myocardial and cellular and subcellular levels will provide insights into the impact of miRNAs for cardiac structural changes induced by different stressors and also expand our understanding of the interdependence of different cell types in the heart with regard to extracellular matrix formation during healing and remodeling after myocardial infarction or in response to pressure overload. The first successful treatment of fibrosis and failure in a murine pressure overload model by application of miRNA antagonists such as antagomirs in vivo raises the hope that manipulating miRNAs may emerge as a novel treatment strategy for fibrotic changes not only in the heart but also in other organs.

title: MicroRNA-221/222 upregulation indicates the activation of stellate cells and the progression of liver fibrosis
abstract: MicroRNAs (miRNAs) are important in hepatic pathophysiology and the development of liver cancer.  To explore miRNAs that are regulated with the progression of liver fibrosis caused by chronic liver disease.  The regulated miRNAs in human livers infected with hepatitis C virus were identified by microarray analysis. Their expression in human livers with non-alcoholic steatohepatitis, mouse livers from two fibrosis models and cultured stellate cells was validated by real-time RT-PCR. The regulation of miR-222 expression in stellate cells by nuclear factor kappa B (NF-κB) was assayed. Finally, the effects of an miR-222 precursor or inhibitor on the expression of cyclin-dependent kinase inhibitor 1B (CDKN1B) and the growth of LX-2 cells were determined.  It was found that miR-199a-5p/199a-3p and miR-221/222 were upregulated in the human liver in a fibrosis progression–dependent manner. Among these miRNAs, miR-221/222 were upregulated in LX-2 cells and increased during the course of culture-dependent activation of mouse primary stellate cells, in a manner similar to the expression of α1(I) collagen and α-smooth muscle actin mRNAs. The expression of miR-221/222 increased in mouse models of liver fibrosis. In contrast, an NF-κB inhibitor significantly suppressed the miR-222 induction that was stimulated in culture by transforming growth factor α or tumour necrosis factor α. Although overexpression or downregulation of miR-222 failed to regulate the growth of LX-2 cells, miR-222 bound to the CDKN1B 3′UTR and regulated the expression of the corresponding protein. Conclusion miR-221/222 may be new markers for stellate cell activation and liver fibrosis progression.

title: Induction of microRNA-214-5p in human and rodent liver fibrosis
abstract: BackgroundmiRNAs are non-coding RNAs that regulate gene expression in a wide range of biological contexts, including a variety of diseases. The present study clarified the role of miR-214-5p in hepatic fibrogenesis using human clinical tissue samples, livers from rodent models, and cultured hepatic stellate cells.MethodsThe expression of miR-214-5p and genes that are involved in liver fibrosis were analyzed in hepatitis C virus-infected human livers, rodent fibrotic livers, a human stellate cell line (LX-2), and the cells from intact mouse livers using real-time PCR. The effect of miR-214-5p overexpression in LX-2 cells on cell function was investigated. Twist-1 expression in the liver tissues of mouse models and primary-cultured stellate cells was also analyzed.ResultsmiR-214-5p was upregulated in human and mouse livers in a fibrosis progression–dependent manner. miR-214-5p expression increased during the culture-dependent activation of mouse primary stellate cells and was significantly higher in stellate cells than in hepatocytes. The overexpression of miR-214-5p in LX-2 cells increased the expression of fibrosis-related genes, such as matrix metalloproteinase (MMP)-2, MMP-9, α-smooth muscle actin, and transforming growth factor (TGF)-β1. TGF-β stimulation induced miR-214-5p in LX-2 cells. Twist-1 was increased in fibrotic mouse livers and induced during mouse stellate cell activation.ConclusionmiR-214-5p may play crucial roles in the activation of stellate cells and the progression of liver fibrosis. Twist-1 may regulate miR-214-5p expression in the liver, particularly in stellate cells.

title: miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARgamma expression.
abstract: microRNAs (miRNAs) are non-coding small RNAs regulating gene expression, cell growth, and differentiation. Although several miRNAs have been implicated in cell growth and differentiation, it is barely understood their roles in adipocyte differentiation. In the present study, we reveal that miR-27a is involved in adipocyte differentiation by binding to the PPARgamma 3'-UTR whose sequence motifs are highly conserved in mammals. During adipogenesis, the expression level of miR-27a was inversely correlated with that of adipogenic marker genes such as PPARgamma and adiponectin. In white adipose tissue, miR-27a was more abundantly expressed in stromal vascular cell fraction than in mature adipocyte fraction. Ectopic expression of miR-27a in 3T3-L1 pre-adipocytes repressed adipocyte differentiation by reducing PPARgamma expression. Interestingly, the level of miR-27a in mature adipocyte fraction of obese mice was down-regulated than that of lean mice. Together, these results suggest that miR-27a would suppress adipocyte differentiation through targeting PPARgamma and thereby down-regulation of miR-27a might be associated with adipose tissue dysregulation in obesity.

title: Lipid droplets: A dynamic organelle moves into focus
abstract: Lipid droplets (LDs) were perceived as static storage deposits, which passively participate in the energy homeostasis of both cells and entire organisms. However, this view has changed recently after the realization of a complex and highly dynamic LD proteome. The proteome contains key components of the fat mobilization system and proteins that suggest LD interactions with a variety of cell organelles, including the endoplasmic reticulum, mitochondria and peroxisomes. The study of LD cell biology, including cross‐talk with other organelles, the trafficking of LDs in the cell and regulatory events involving the LD coat proteins is now on the verge of leaving its infancy and unfolds that LDs are highly dynamic cellular organelles.

title: Lipid droplets in inflammation and cancer.
abstract: Accumulation of lipid droplets (also known as lipid bodies or adiposomes) within leukocytes, epithelial cells, hepatocytes and other non-adipocytic cells is a frequently observed phenotype in infectious, neoplastic and other inflammatory conditions. Lipid droplet biogenesis is a regulated cellular process that culminates in the compartmentalization of lipids and of an array of enzymes, protein kinases and other proteins, suggesting that lipid droplets are inducible organelles with roles in cell signaling, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Enzymes involved in eicosanoid synthesis are localized at lipid droplets and lipid droplets are sites for eicosanoid generation in cells during inflammation and cancer. In this review, we discuss the current evidence related to the biogenesis and function of lipid droplets in cell metabolism and signaling in inflammation and cancer. Moreover, the potential of lipid droplets as markers of disease and targets for novel anti-inflammatory and antineoplastic therapies will be discussed.

title: Adipocyte differentiation-related protein and OXPAT in rat and human skeletal muscle: involvement in lipid accumulation and type 2 diabetes mellitus.
abstract: A disordered lipid metabolism is implicated in the development of skeletal muscle insulin resistance. Lipid droplet proteins of the PAT [perilipin, adipocyte differentiation-related protein (ADRP), and TIP47] family have been shown to regulate lipid accumulation and intracellular metabolism in other tissues.



This study aimed to explore the role of the PAT proteins OXPAT and ADRP in skeletal muscle lipid metabolism and their putative role in modulating insulin sensitivity.



Muscle OXPAT and ADRP protein content was examined during the development of insulin resistance in Zucker diabetic fatty (ZDF) rats and in type 2 diabetes patients and BMI-matched control subjects. Furthermore, we examined the effect of 8 wk of insulin sensitizing by rosiglitazone on muscle OXPAT and ADRP content.



OXPAT and ADRP protein expression is muscle fiber type specific in humans and rats, with highest protein content in fibers containing most intramyocellular lipids (IMCL). Muscle OXPAT and ADRP protein content was 2- to 3-fold higher in ZDF rats during the progression of type 2 diabetes than in lean normoglycemic control rats, which was paralleled by high IMCL levels. Muscle OXPAT and ADRP content, as well as IMCL level, was not different between type 2 diabetes patients and control subjects. ADRP content was negatively associated with insulin-stimulated glucose uptake (r = -0.50; P = 0.017). Interestingly, rosiglitazone treatment decreased muscle OXPAT (-29%) and ADRP (-28%) content in diabetes patients, without affecting IMCL.



These results indicate involvement of OXPAT and ADRP in muscular lipid accumulation and type 2 diabetes.

title: Lipid metabolism in skeletal muscle: generation of adaptive and maladaptive intracellular signals for cellular function.
abstract: Fatty acids derived from adipose tissue lipolysis, intramyocellular triacylglycerol lipolysis, or de novo lipogenesis serve a variety of functions in skeletal muscle. The two major fates of fatty acids are mitochondrial oxidation to provide energy for the myocyte and storage within a variety of lipids, where they are stored primarily in discrete lipid droplets or serve as important structural components of membranes. In this review, we provide a brief overview of skeletal muscle fatty acid metabolism and highlight recent notable advances in the field. We then 1) discuss how lipids are stored in and mobilized from various subcellular locations to provide adaptive or maladaptive signals in the myocyte and 2) outline how lipid metabolites or metabolic byproducts derived from the actions of triacylglycerol metabolism or β-oxidation act as positive and negative regulators of insulin action. We have placed an emphasis on recent developments in the lipid biology field with respect to understanding skeletal muscle physiology and discuss unanswered questions and technical limitations for assessing lipid signaling in skeletal muscle.

title: The potential for novel anti-inflammatory therapies for coronary artery disease
abstract: Although drugs that lead to cholesterol and lipid lowering have proved to have significant effects in lowering cardiovascular morbidity and mortality, coronary artery disease remains a principal cause of death worldwide. There is a clear need to discover further therapeutic approaches to control this disease adequately. This review focuses on the mechanisms that have been implicated in the recruitment, activation and differentiation of inflammatory monocytes/macrophages in nascent vascular lesions into lipid-laden foam cells. These mechanisms might provide attractive targets for novel therapies for coronary artery disease.

title: microRNAs in the Regulation of Adipogenesis and Obesity
abstract: Worldwide obesity is a growing health problem, associated with increased risk of chronic disease. Understanding the molecular basis of adipogenesis and fat cell development in obesity is essential to identify new biomarkers and therapeutic targets for the development of anti-obesity drugs. microRNAs (miRNAs) appear to play regulatory roles in many biological processes associated with obesity, including adipocyte differentiation, insulin action and fat metabolism. Recent studies show miRNAs are dysregulated in obese adipose tissue. During adipogenesis miRNAs can accelerate or inhibit adipocyte differentiation and hence regulate fat cell development. In addition miRNAs may regulate adipogenic lineage commitment in multipotent stem cells and hence govern fat cell numbers. Recent findings suggest miR-519d may be associated with human obesity, but larger case-control studies are needed. Few miRNA targets have been experimentally validated in adipocytes but interestingly both miR-27 and miR-519d target PPAR family members, which are well established regulators of fat cell development. In this review recent advances in our understanding of the role of miRNAs in fat cell development and obesity are discussed. The potential of miRNA based therapeutics targeting obesity is highlighted as well as recommendations for future research which could lead to a breakthrough in the treatment of obesity.

title: The effect of tear size and nerve injury on rotator cuff muscle fatty degeneration in a rodent animal model.
abstract: Irreversible muscle changes after rotator cuff tears is a well-known negative prognostic factor after shoulder surgery. Currently, little is known about the pathomechanism of fatty degeneration of the rotator cuff muscles after chronic cuff tears. The purposes of this study were to (1) develop a rodent animal model of chronic rotator cuff tears that can reproduce fatty degeneration of the cuff muscles seen clinically, (2) describe the effects of tear size and concomitant nerve injury on muscle degeneration, and (3) evaluate the changes in gene expression of relevant myogenic and adipogenic factors after rotator cuff tears using the animal model.



Rotator cuff tears were created in rodents with and without transection of the suprascapular nerve. The supraspinatus and infraspinatus muscles were examined at 2, 8, and 16 weeks after injury for histologic evidence of fatty degeneration and expression of myogenic and adipogenic genes.



Histologic analysis revealed adipocytes, intramuscular fat globules, and intramyocellular fat droplets in the tenotomized and neurotomized supraspinatus and infraspinatus muscles. Changes increased with time and were most severe in the muscles with combined tenotomy and neurotomy. Adipogenic and myogenic transcription factors and markers were upregulated in muscles treated with tenotomy or tenotomy combined with neurotomy compared with normal muscles.



The rodent animal model described in this study produces fatty degeneration of the rotator cuff muscles similar to human muscles after chronic cuff tears. The severity of changes was associated with tear size and concomitant nerve injury.

title: Identification of lipid droplet-associated proteins in the formation of macrophage-derived foam cells using microarrays.
abstract: A large number of macrophage-derived foam cells stores excessive neutral lipids in intracellular droplets, and plays a major role during the development of atherosclerosis. The formation and catabolism of intracellular lipid droplets (LDs) are regulated by LD-associated proteins, a group of proteins which are located on the surface of LDs and regulate the formation, morphology and lipolysis of LDs. In order to illustrate the function of LD-associated proteins during the process of atherosclerosis, the foam cell model is induced by oxidized low-density lipoprotein (ox-LDL) in macrophages originated from the THP-1 cell line, and cDNA microarrays are used to monitor the gene expression profiles of LD-associated proteins. Gene expression data show that 2% of changed genes are lipid binding genes during the transformation of foam cells. The major candidate genes, the cell death-inducing DFF45-like effector (CIDE) family and Perilipin, Adipophilin, and TIP47 (PAT) family, have different alterations during the formation of foam cells. CIDEB, CIDEC, Adipophilin, S3-12 and LSDP5 were up-regulated, while TIP47 was down-regulated. There was no significant change in CIDEA and Perilipin. These results were confirmed by real-time PCR and immunoblotting. This study presents a comprehensive analysis of the gene expression of LD-associated proteins during the differentiation of human foam cells, which may play an important role in the process of atherosclerosis.

title: PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL.
abstract: The formation of foam cells from macrophages in the arterial wall is characterized by dramatic changes in lipid metabolism, including increased expression of scavenger receptors and the uptake of oxidized low-density lipoprotein (oxLDL). We demonstrate here that the nuclear receptor PPARgamma is induced in human monocytes following exposure to oxLDL and is expressed at high levels in the foam cells of atherosclerotic lesions. Ligand activation of the PPARgamma:RXRalpha heterodimer in myelomonocytic cell lines induces changes characteristic of monocytic differentiation and promotes uptake of oxLDL through transcriptional induction of the scavenger receptor CD36. These results reveal a novel signaling pathway controlling differentiation and lipid metabolism in monocytic cells, and suggest that endogenous PPARgamma ligands may be important regulators of gene expression during atherogenesis.

title: Force‐velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.
abstract: 1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force‐velocity curves of sixty‐seven fibres were obtained by load‐clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross‐sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack‐test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I‐IIA and IIA‐IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty‐one fibres in the range 12‐22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol‐1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12‐17 degrees C range, but less dependent between 17 and 22 degrees C.