Novel MicroRNA Regulators of Atrial Natriuretic Peptide Production

Atrial natriuretic peptide (ANP) has a central role in regulating blood pressure in humans. Recently, microRNA 425 (miR-425) was found to regulate ANP production by binding to the mRNA of NPPA, the gene encoding ANP. mRNAs typically contain multiple predicted microRNA (miRNA)-binding sites, and binding of different miRNAs may independently or coordinately regulate the expression of any given mRNA. We used a multifaceted screening strategy that integrates bioinformatics, next-generation sequencing data, human genetic association data, and cellular models to identify additional functional NPPA-targeting miRNAs. Two novel miRNAs, miR-155 and miR-105, were found to modulate ANP production in human cardiomyocytes and target genetic variants whose minor alleles are associated with higher human plasma ANP levels. Both miR-15 and miR-105 repressed NPPA mRNA in an allele-specific manner, with the minor allele of each respective variant conferrin resistance to the miRNA either by disruption of miRNA base pairing or by creation of wobble base pairing. Moreover, miR-15 enhanced the repressive effects of miR-425 on ANP production in human cardiomyocytes. Our study combines computational genomic, and cellular tools to identify novel miRNA regulators of ANP production that could be targeted to raise ANP levels which may have applications for the treatment of hypertension or heart failure

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Beige fibro-adipogenic progenitor transplantation reduces muscle degeneration and improves function in a mouse model of delayed repair of rotator cuff tears

BackgroundMuscle atrophy and fatty infiltration (FI) are common occurrences following rotator cuff (RC) tears. Tears of all sizes are subject to muscle degeneration. The degree of muscle degeneration following RC tears is highly correlated with repair success and functional outcomes. We have recently discovered that muscle fibro-adipogenic progenitors (FAPs) can differentiate into uncoupling protein 1 (UCP-1)-expressing beige adipocytes and induce muscle regeneration. This study evaluated the potential of local cell transplantation of beige adipose FAPs (BAT-FAPs) to treat RC muscle degeneration in a murine model of RC repair.MethodsBAT-FAPs were isolated from muscle in UCP-1 reporter mice by flow cytometry as UCP-1+/Sca1+/PDGFR+/CD31-/CD45-/integrin α7-. C57/BL6J mice underwent supraspinatus tendon tear with suprascapular nerve transection followed by repair 2 or 6 weeks after the initial injury. At the time of repair, mice received either no additional treatment, phosphate-buffered saline injection, or BAT-FAP injection. Functional outcomes were assessed by gait analysis. Mice were humanely killed at 6 weeks after cell transplantation. Supraspinatus muscle FI, fibrosis, muscle fiber size, and vascularity were analyzed and quantified via ImageJ. Analysis of variance with post hoc Tukey test and P <.05 was used to determine statistical significance.ResultsCell transplantation diminished fibrosis, FI, and atrophy and enhanced vascularization in both delayed repair models. Cell transplantation resulted in improved shoulder function as assessed with gait analysis in both the delayed repair models.ConclusionsBAT-FAPs significantly reduced muscle degeneration and improved shoulder function after RC repair. BAT-FAPs hold significant promise as a therapeutic adjunct to repair for patients with advanced RC pathology

Beige FAPs Transplantation Improves Muscle Quality and Shoulder Function After Massive Rotator Cuff Tears

Rotator cuff (RC) tears are a common cause of upper extremity disability. Any tear size can result in subsequent muscle atrophy and fatty infiltration (FI). Preoperative muscle degeneration can predict repair and postoperative functional outcomes. Muscle residential fibro-adipogenic progenitors (FAPs) are found to be capable of differentiating into beige adipocytes that release factors to promote muscle growth. This study evaluated the regenerative potential of local cell transplantation of beige FAPs to mitigate muscle degeneration in a murine massive RC tear model. Beige FAPs were isolated from muscle in UCP-1 reporter mice by flow cytometry as UCP-1+ /Sca1+ /PDGFR+ /CD31- /CD45- /integrin α7- . C57/BL6J mice undergoing supraspinatus tendon tear with suprascapular nerve transection (TT + DN) received either no additional treatment, phosphate-buffered saline injection, or beige FAP injection 2 weeks after the initial injury. Forelimb gait analysis was used to assess shoulder function with DigiGait. Mice were sacrificed 6 weeks after cell transplantation. FI, fibrosis, fiber size, vascularity were analyzed and quantified via ImageJ. Our results showed that beige FAP transplantation significantly decreased fibrosis, FI, and atrophy, enhanced vascularization compared with saline injection and non-treatment groups. Beige FAP transplantation also significantly improved shoulder function as measured by gait analysis. This study suggests that beige-differentiated FAPs may serve as a treatment option for RC muscle atrophy and FI, thus improving shoulder function in patients with massive RC tendon tears. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1159-1166, 2020

Human Rotator Cuff Tears Have an Endogenous, Inducible Stem Cell Source Capable of Improving Muscle Quality and Function After Rotator Cuff Repair.

BackgroundThe muscle quality of the rotator cuff (RC), measured by atrophy and fatty infiltration (FI), is a key determinant of outcomes in RC injury and repair. The ability to regenerate muscle after repair has been shown to be limited.PurposeTo determine if there is a source of resident endogenous stem cells, fibroadipogenic progenitor cells (FAPs), within RC injury patients, and if these cells are capable of adipogenic, fibrogenic, and pro-myogenic differentiation.Study designControlled laboratory study.MethodsA total of 20 patients between the ages of 40 and 75 years with partial- or full-thickness RC tears of the supraspinatus and evidence of atrophy and FI Goutallier grade 1, 2, or 3 were selected from 2 surgeons at an orthopaedic center. During the surgical repair procedure, supraspinatus muscle biopsy specimens were obtained for analysis as were deltoid muscle biopsy specimens to serve as the control. FAPs and satellite cells were quantified using fluorescence-activated cell sorting. Muscle FI and fibrosis was quantified using Oil Red O and Masson trichrome staining. FAP differentiation and gene expression profiles were compared across tear sizes after culture in adipogenic, fibrogenic, and beta-3 agonist (amibegron) conditions. Analysis of variance was used for statistical comparisons between groups, with P < .05 as statistically significant.ResultsHistologic analysis confirmed the presence of fat in biopsy specimens from patients with full-thickness tears. There were more FAPs in the full-thickness tear group compared with the partial-thickness tear group (9.43% ± 4.25% vs 3.84% ± 2.54%; P < .01). Full-thickness tears were divided by tear size, with patients with larger tears having significantly more FAPs than those with smaller tears. FAPs from muscles with full-thickness tendon tears had more adipogenic and fibrogenic potential than those with partial tears. Induction of a beige adipose tissue (BAT) phenotype in FAPs was possible, as demonstrated by increased expression of BAT markers and pro-myogenic genes including insulin-like growth factor 1 and follistatin.ConclusionEndogenous FAPs are present within the RC and likely are the source of FI. These FAPs were increased in muscles with in larger tears but are capable of adopting a pro-myogenic BAT phenotype that could be utilized to improve muscle quality and patient function after RC repair

Current Perceptions of Diversity Among Head Team Physicians and Head Athletic Trainers: Results Across US Professional Sports Leagues.

BackgroundDiscrepancies in race, ethnicity, and sex among health care providers and their patients have been shown to affect the patient-provider relationship as well as the quality of care. Currently, minority and female representation among orthopaedic surgeons remains low. Given the large proportion of minority athletes and their degree of public visibility, professional sports serves as an important arena within which to analyze the diversity of health care providers.PurposeTo describe and evaluate the current level of diversity of head team physicians (HTPs) and head athletic trainers (ATCs), primarily in terms of race and sex, within men's professional sports leagues in the United States.Study designCross-sectional study.MethodsFive major US professional sports leagues were evaluated: National Basketball Association, National Football League, National Hockey League, Major League Soccer, and Major League Baseball. Publicly available data were collected to identify the HTPs and head ATCs for each team within these leagues. Two independent observers analyzed photographs and names of these individuals to determine his or her perceived race and sex, with disagreements being resolved by a third independent observer. Other physician data collected included graduate degree(s), specialty, and number of years in practice. Kappa coefficients (κ) were employed to evaluate interobserver reliability. Chi-square, Fisher exact, and t tests were used for statistical comparisons across leagues.ResultsThe κ values for perceived race were 0.85 for HTPs and 0.89 for head ATCs, representing near-perfect interobserver agreement. Minorities comprised 15.5% of HTPs and 20.7% of ATCs (P = .24). Women comprised 3.9% of HTPs and 1.3% of head ATCs (P = .017). The majority of HTPs were orthopaedic surgeons with medical doctorates. Female HTPs had significantly fewer years in practice compared with male HTPs (15.0 ± 4.9 vs 23.1 ± 9.6; P = .04).ConclusionThe lead physicians and athletic training providers for men's professional sports teams demonstrated low rates of minority and female representation, denoting a highly visible area for discussing the role of increased diversity in health care

Rotator Cuff Fibro‐Adipogenic Progenitors Demonstrate Highest Concentration, Proliferative Capacity, and Adipogenic Potential Across Muscle Groups

Fatty infiltration (FI) of rotator cuff (RC) muscles is common in patients with RC tears. Studies have demonstrated that fibro-adipogenic progenitors (FAPs), a population of resident muscle stem cells, are the main contributors of FI, which adversely affects muscle quality and RC repair success. Although FI is common in RC injuries, it is not frequently reported after other musculotendinous injuries. Additionally, studies have shown the development of different pathology patterns across muscle groups suggestive of intrinsic differences in cellular composition and behavior. This study evaluates FAP distribution and differentiation properties across anatomic locations in mice. Muscles from seven different anatomic locations were harvested from PDGFRα-eGFP FAP reporter mice. FAPs were quantified using histology and FACS sorting with BD Aria II with CD31(−)/CD45(−)/Integrinα7(−)/Sca-1(+) and PDGFRα reporter signal (n = 3 per muscle). The cells were analyzed for adipogenesis using immunocytochemistry and for proliferation properties with Brdu-Ki67 staining. In a separate group of mice, RC and tibialis anterior muscles received glycerol injection and were harvested after 2 weeks for FI quantification (n = 4). One-way analysis of variance was used for statistical comparisons among groups, with significance at p < 0.05. FAPs from the RC, masseter, and paraspinal muscles were more numerous and demonstrated greater proliferative capacity and adipogenic potency than those from the tibialis anterior and gastrocnemius. The RC demonstrated significantly greater levels of FI than the tibialis anterior after glycerol-injection injury. Clinical Significance: This study suggests differences in FAP distribution and differentiation characteristics may account for the propensity to develop FI in RC tears as compared with other musculotendinous injuries

Surgical Stabilization of Shoulder Instability in Patients With or Without a History of Seizure: A Comparative Analysis.

PurposeTo compare patients from a large multicenter cohort with a history of seizure and those without a history of seizure regarding preoperative and intraoperative findings and surgical procedures performed.MethodsPatients undergoing shoulder stabilization from 2011 to 2018 at 11 orthopaedic centers were prospectively enrolled. Those with a history of seizure were identified and compared with non-seizure controls. Preoperative demographic, history, physical examination, and imaging findings were collected. Intraoperative findings and surgical procedures performed were recorded. The Mann-Whitney test, χ2 test, and logistic regression analysis were used to examine differences between the groups and define independent risk factors. Owing to the number of statistical tests performed, the false discovery method was used to determine adjusted P values to achieve α &lt; .05.ResultsDuring enrollment, 25 of 1,298 shoulder stabilization patients (1.9%) had a history of seizure. The sex ratio and age were similar between groups, as was posterior instability incidence (23.2% in control group vs 28.0% in seizure group). Seizure patients more frequently had more than 5 dislocations in the year preceding surgery (P&nbsp;= .016) and had increased preoperative radiographic evidence of bone loss (P &lt; .001). Intraoperatively, seizure patients had a higher prevalence of reverse Hill-Sachs lesions (P &lt; .001) and large (&gt;30% of glenoid fossa) bony Bankart lesions (P &lt; .001). Arthroscopic Bankart repair was the most common procedure in both groups. However, open procedures were performed in 15.6% of controls and 40.0% of seizure patients (P&nbsp;= .001). These procedures were most commonly bony procedures.ConclusionsSeizure patients had more prior dislocations, had more preoperative bone loss, and underwent more open stabilization procedures than controls because of bone loss. Studies examining recurrence after stabilization will help establish appropriate management practices in this population.Level of evidenceLevel III, retrospective review of prospectively collected cohort

Gender and Racial Diversity Among the Head Medical and Athletic Training Staff of Women’s Professional Sports Leagues

BackgroundDespite increased awareness for promoting diversity, orthopaedics remains one of the least diverse specialties. Studying health care providers in women's professional sports provides a unique opportunity to analyze gender and racial diversity.HypothesesThere would be low female and minority representation across the various women's professional sports leagues. There would be an increased number of female head certified athletic trainers (ATCs) when compared with head team physicians (HTPs).Study designCross-sectional study.MethodsWe evaluated the perceived race and sex of designated HTPs and ATCs in the Women's National Basketball Association, National Women's Soccer League, and National Women's Hockey League. Type of doctorate degree, specialty, and years in practice were also collected. Kappa (κ) coefficient measurements were used to determine interobserver agreement on race. Categorical and continuous variables were analyzed using chi-square and t tests, respectively.ResultsThere were significantly more female ATCs than female HTPs (74.1% vs 37.5%; P = .01). Minority representation between HTPs and ATCs was not significantly different (20.8% vs 40.7%; P = .13). Black HTPs (12.5%) and Black ATCs (22.2%) composed the largest proportion among the minority groups. There was high interobserver agreement of perceived race across HTPs (κ = 1.0) and ATCs (κ = 0.95).ConclusionAlthough there were more female ATCs than HTPs in women's professional sports leagues, both cohorts lack perceived racial diversity. These data suggest an opportunity for diversification in medical and training staff of women's professional sports

A novel mouse model of hindlimb joint contracture with 3D‐printed casts

Stiff joints formed after trauma, surgery or immobilization are frustrating for surgeons, therapists and patients alike. Unfortunately, the study of contracture is limited by available animal model systems, which focus on the utilization of larger mammals and joint trauma. Here we describe a novel mouse-based model system for the generation of joint contracture using 3D-printed clamshell casts. With this model system we are able to generate both reversible and irreversible contractures of the knee and ankle. Four- or 8-month-old female mice were casted for either 2 or 3 weeks before liberation. All groups formed measurable contractures of the knee and ankle. Younger mice immobilized for less time formed reversible contractures of the knee and ankle. We were able to generate irreversible contracture with either longer immobilization time or the utilization of older mice. The contracture formation translated into differences in gait, which were detectable using the DigiGait® analysis system. This novel model system provides a higher throughput, lower cost and more powerful tool in studying the molecular and cellular mechanisms considering the large existing pool of transgenic/knockout murine strains