Risk factors for intracellular fatty accumulation in rotator cuff muscle: a histologic analysis.

BACKGROUND: Fatty accumulation in rotator cuff muscles has been associated with shoulder dysfunction, risk of repair failure, and poor postoperative outcomes. This study sought to assess risk factors associated with true fatty accumulation based on histologic analysis and determine whether preoperative function directly correlated with this fatty rotator cuff accumulation. METHODS: Supraspinatus muscle biopsy specimens obtained prospectively from patients undergoing arthroscopic rotator cuff repair were stained with LipidTOX to quantify lipid accumulation. Two-step cluster analysis with Goutallier classification was used to define the fatty and non-fatty rotator cuff groups. We further performed a receiver operating characteristic curve analysis to confirm the group cutoff values. RESULTS: In total, 51 patients (aged 60.1 ± 10.5 years) were included. There were 19 high-grade partial tears, 10 small tears, 7 medium tears, 10 large tears, and 5 massive tears. Both cluster and receiver operating characteristic curve analyses yielded a cutoff value of 30% LipidTOX/4',6-diamidino-2-phenylindole (DAPI) separating the fatty vs. non-fatty groups. In the univariate analysis, patients with fatty rotator cuffs were aged 63.2 years on average compared with 59.7 years in the non-fatty group (P = .038). Female patients made up 57.1% of the fatty cohort, which was statistically higher than the non-fatty group (P = .042). Massive and large tears were more likely to occur in the fatty group (P = .005). In the multivariate analysis, full tendon tears had the largest predictive status of falling into the fatty group (odds ratio, 15.4; P = .008), followed by female sex (odds ratio, 4.9; P = .036). Patients in the fatty group had significantly higher American Shoulder and Elbow Surgeons scores (P = .048) and lower visual analog scale scores (P = .002). DISCUSSION AND CONCLUSION: This prospective histologic assessment revealed that full-thickness rotator cuff tears and female sex were the largest risk factors for intracellular lipid accumulation. Although tear size correlated with fatty accumulation, the sex disparity is a noteworthy finding that warrants further research.

Radiation-induced bone loss in mice is ameliorated by inhibition of HIF-2α in skeletal progenitor cells.

Radiotherapy remains a common treatment modality for cancer despite skeletal complications. However, there are currently no effective treatments for radiation-induced bone loss, and the consequences of radiotherapy on skeletal progenitor cell (SPC) survival and function remain unclear. After radiation, leptin receptor-expressing cells, which include a population of SPCs, become localized to hypoxic regions of the bone and stabilize the transcription factor hypoxia-inducible factor-2α (HIF-2α), thus suggesting a role for HIF-2α in the skeletal response to radiation. Here, we conditionally knocked out HIF-2α in leptin receptor-expressing cells and their descendants in mice. Radiation therapy in littermate control mice reduced bone mass; however, HIF-2α conditional knockout mice maintained bone mass comparable to nonirradiated control animals. HIF-2α negatively regulated the number of SPCs, bone formation, and bone mineralization. To test whether blocking HIF-2α pharmacologically could reduce bone loss during radiation, we administered a selective HIF-2α inhibitor called PT2399 (a structural analog of which was recently FDA-approved) to wild-type mice before radiation exposure. Pharmacological inhibition of HIF-2α was sufficient to prevent radiation-induced bone loss in a single-limb irradiation mouse model. Given that ~90% of patients who receive a HIF-2α inhibitor develop anemia because of off-target effects, we developed a bone-targeting nanocarrier formulation to deliver the HIF-2α inhibitor to mouse bone, to increase on-target efficacy and reduce off-target toxicities. Nanocarrier-loaded PT2399 prevented radiation-induced bone loss in mice while reducing drug accumulation in the kidney. Targeted inhibition of HIF-2α may represent a therapeutic approach for protecting bone during radiation therapy.

Association Between Supraspinatus Tendon Retraction, Histologic Myofiber Size, and Supraspinatus Muscle Atrophy on MRI.

BACKGROUND: Atrophy of the rotator cuff is a negative prognostic indicator after rotator cuff repair. Although full-thickness rotator cuff tears accompanied by tendon retraction are commonly associated with decreased muscle cross-sectional area (CSA) on magnetic resonance imaging (MRI), it is unclear whether this is accompanied by histologic atrophy of rotator cuff myofibers. PURPOSE: To evaluate the effect of supraspinatus tendon retraction and myofiber size on supraspinatus atrophy on MRI. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: Supraspinatus muscle biopsy specimens were obtained from consecutive patients undergoing arthroscopic shoulder surgery. Rotator cuff tears were classified according to size. Preoperative MRI was used to measure tendon retraction and CSA of the supraspinatus muscle in the Y-shaped view. The occupation ratio of the supraspinatus was calculated by dividing the supraspinatus CSA by the supraspinatus fossa CSA. Muscle biopsy specimens were examined using laminin to quantify myofiber CSA. The association between supraspinatus tear size and measures of histologic and MRI muscle atrophy were compared using standard statistical tests. Multivariable logistic regression analysis was used to identify independent predictors of muscle atrophy on MRI. RESULTS: A total of 38 patients were included: 8 with no tear, 14 with a partial-thickness tear, and 16 with a full-thickness tear. Increasing tear size was associated with greater distance of tendon retraction (P < .001), smaller mean histologic myofiber size (P = .004), lower mean supraspinatus CSA on MRI (P < .001), and lower occupation ratio: 0.73 (control), 0.66 (partial tear), 0.53 (small to medium full-thickness tear), and 0.38 (large to massive full-thickness) (P < .001). On Pearson correlation analysis, tendon retraction demonstrated strong correlation with occupation ratio (-0.725; P < .001) and weak correlation with myofiber size (-0.437; P = .006), while occupation ratio showed moderate correlation with myofiber size (0.593; P < .001). Multivariable linear regression analysis demonstrated that increasing tendon retraction (P < .001), age (P = .034), and smaller histologic myofiber CSA (P = .047) were independently associated with greater supraspinatus atrophy on MRI. CONCLUSION: Supraspinatus muscle atrophy appreciated on MRI is independently associated with patient age, tendon retraction, and atrophy of the supraspinatus myofibers at the histologic level.

Interleukin-6 signaling mediates cartilage degradation and pain in posttraumatic osteoarthritis in a sex-specific manner.

Osteoarthritis (OA) and posttraumatic OA (PTOA) are caused by an imbalance in catabolic and anabolic processes in articular cartilage and proinflammatory changes throughout the joint, leading to joint degeneration and pain. We examined whether interleukin-6 (IL-6) signaling contributed to cartilage degradation and pain in PTOA. Genetic ablation of Il6 in male mice decreased PTOA-associated cartilage catabolism, innervation of the knee joint, and nociceptive signaling without improving PTOA-associated subchondral bone sclerosis or chondrocyte apoptosis. These effects were not observed in female Il6-/- mice. Compared with wild-type mice, the activation of the IL-6 downstream mediators STAT3 and ERK was reduced in the knees and dorsal root ganglia (DRG) of male Il6-/- mice after knee injury. Janus kinases (JAKs) were critical for STAT and ERK signaling in cartilage catabolism and DRG pain signaling in tissue explants. Whereas STAT3 signaling was important for cartilage catabolism, ERK signaling mediated neurite outgrowth and the activation of nociceptive neurons. These data demonstrate that IL-6 mediates both cartilage degradation and pain associated with PTOA in a sex-specific manner and identify tissue-specific contributions of downstream effectors of IL-6 signaling, which are potential therapeutic targets for disease-modifying OA drugs.

Histologic Differences in Human Rotator Cuff Muscle Based on Tear Characteristics.

BACKGROUND: Fatty accumulation in the rotator cuff is associated with shoulder dysfunction and a risk of failure of rotator cuff repair. The aims of this study were to (1) describe cellular findings in rotator cuff muscles in patients presenting with varying degrees of rotator cuff tendon pathology by examining fat content and myofiber cross-sectional area of rotator cuff muscles and (2) correlate histologic features to magnetic resonance imaging (MRI) grades derived with the Goutallier classification. METHODS: Rotator cuff muscle biopsies were performed in a consecutive series of patients undergoing arthroscopic shoulder surgery. Rotator cuffs were graded according to the Goutallier classification and labeled as either partial-thickness or full-thickness. Patients without a rotator cuff tear undergoing arthroscopic surgery served as controls. The biopsy specimens were examined using LipidTOX to visualize lipid accumulation. Laminin was used to quantify myofiber cross-sectional area. RESULTS: Twenty-seven patients with a rotator cuff tear and 12 without a tear (controls) were included. There were 24 males (62%). The mean age was 55 years. Patients in the control cohort were younger (mean, 46 years) than those in the treatment group (mean, 60 years, p < 0.01). Within the treatment group, 12 and 15 patients were recorded as having partial and full-thickness rotator cuff tears, respectively. Lipid accumulation visualized at the cellular level was fairly-to-moderately correlated with the Goutallier classification on MRI (R s = 0.705, 95% confidence interval [CI] = 0.513, 0.829). Muscle biopsy specimens with a Goutallier grade of 2+ had significantly more lipid accumulation than those with grade-0 (p < 0.01) or grade-1 (p < 0.01) fatty accumulation. Muscle biopsies at the sites of full-thickness tears showed significantly greater lipid accumulation than those associated with either partial (p < 0.01) or no (p < 0.01) tears. Partial-thickness rotator cuff tears had no difference in lipid accumulation in comparison to the control group. Muscle biopsy specimens from full-thickness tears had significantly smaller myofiber cross-sectional area when compared with partial-thickness tears (p = 0.02) and controls (p < 0.01). CONCLUSIONS: Cellular lipid accumulation correlates with the MRI Goutallier grade of fatty accumulation, thus verifying the Goutallier classification at the cellular level. Muscle biopsy specimens from partial-thickness tears are more similar to controls than to those from full-thickness tears, whereas full-thickness tears of all sizes showed significantly greater lipid content and smaller myofiber cross-sectional area compared with partial-thickness tears and controls. CLINICAL RELEVANCE: Our research confirms the utility of using the Goutallier classification to predict rotator cuff muscle quality and shows that tendon attachment, even if partially torn, protects the muscle from fatty accumulation.

Identification of distinct non-myogenic skeletal-muscle-resident mesenchymal cell populations.

Mesenchymal progenitors of the lateral plate mesoderm give rise to various cell fates within limbs, including a heterogeneous group of muscle-resident mesenchymal cells. Often described as fibro-adipogenic progenitors, these cells are key players in muscle development, disease, and regeneration. To further define this cell population(s), we perform lineage/reporter analysis, flow cytometry, single-cell RNA sequencing, immunofluorescent staining, and differentiation assays on normal and injured murine muscles. Here we identify six distinct Pdgfra+ non-myogenic muscle-resident mesenchymal cell populations that fit within a bipartite differentiation trajectory from a common progenitor. One branch of the trajectory gives rise to two populations of immune-responsive mesenchymal cells with strong adipogenic potential and the capability to respond to acute and chronic muscle injury, whereas the alternative branch contains two cell populations with limited adipogenic capacity and inherent mineralizing capabilities; one of the populations displays a unique neuromuscular junction association and an ability to respond to nerve injury.

Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development.

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow-associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; Rosa26fs-tdTomato and Col10a1Cre; Rosa26fs-tdTomato) in combination with a PDGFRaH2B-GFP transgenic line, single-cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow-associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte-derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

HES1 is a novel downstream modifier of the SHH-GLI3 Axis in the development of preaxial polydactyly.

Sonic Hedgehog/GLI3 signaling is critical in regulating digit number, such that Gli3-deficiency results in polydactyly and Shh-deficiency leads to digit number reductions. SHH/GLI3 signaling regulates cell cycle factors controlling mesenchymal cell proliferation, while simultaneously regulating Grem1 to coordinate BMP-induced chondrogenesis. SHH/GLI3 signaling also coordinates the expression of additional genes, however their importance in digit formation remain unknown. Utilizing genetic and molecular approaches, we identified HES1 as a downstream modifier of the SHH/GLI signaling axis capable of inducing preaxial polydactyly (PPD), required for Gli3-deficient PPD, and capable of overcoming digit number constraints of Shh-deficiency. Our data indicate that HES1, a direct SHH/GLI signaling target, induces mesenchymal cell proliferation via suppression of Cdkn1b, while inhibiting chondrogenic genes and the anterior autopod boundary regulator, Pax9. These findings establish HES1 as a critical downstream effector of SHH/GLI3 signaling in the development of PPD.

Insulin/IGF Signaling and Vitellogenin Provisioning Mediate Intergenerational Adaptation to Nutrient Stress.

The roundworm C. elegans reversibly arrests larval development during starvation [1], but extended early-life starvation reduces reproductive success [2, 3]. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success [4]. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell fate markers. We found that maternal DR and reduced maternal insulin/insulin-like growth factor (IGF) signaling (IIS) increase oocyte provisioning of vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability.