Fibroblast Nox2 (NADPH Oxidase-2) Regulates ANG II (Angiotensin II)-Induced Vascular Remodeling and Hypertension via Paracrine Signaling to Vascular Smooth Muscle Cells.

OBJECTIVE: The superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)-induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (fibroblast-specific Nox2 knockout or Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II-stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell growth, which was inhibited by the short hairpin RNA (shRNA)-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II-treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro. CONCLUSIONS: These results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II-induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.

Relationship Between Strength, Athletic Performance, and Movement Skill in Adolescent Girls.

ABSTRACT: Sommerfield, LM, Harrison, CB, Whatman, CS, and Maulder, PS. Relationship between strength, athletic performance, and movement skill in adolescent girls. J Strength Cond Res 36(3): 674-679, 2022-Muscular strength in youth has been linked to health and physical benefits, enhanced movement skill, and an active lifestyle in adulthood. However, the relationship between maximum strength, athletic performance, and movement skill in youth females remains unclear. The purpose was to examine the relationship between maximum strength, athletic performance, and movement skill and determine whether differences exist between strong girls (SGs), average girls (AGs), and weak girls (WGs). One hundred four girls (age 14.0 ± 0.6 years, height 162.6 ± 5.9 cm, body mass 57.3 ± 9.7 cm) from a girls' secondary school performed an isometric midthigh pull (IMTP), double- and single-leg (right leg = R, left leg = L) countermovement jump, 10- and 20-m sprints, a drop vertical jump ,and the back squat assessment. Significance was set at p < 0.01 for correlations and p < 0.05 for one-way analysis of variance. Correlations revealed IMTP had significant small to large relationships with all performance variables (r = 0.27-0.62) except right-leg countermovement jump and left-leg countermovement jump height (r = 0.17-0.23). Relative IMTP had significant moderate to large relationships with all performance variables (r = 0.32-0.60). There were significant differences between strength groups for all performance measures. Strong girls had significantly faster sprint times than AGs. In addition, SGs and AGs performed significantly better than WGs in all assessments. The results of this study demonstrate the importance of strength for athletic performance and movement skill in adolescent girls.

Effects of Combined Resistance Training and Weightlifting on Injury Risk Factors and Resistance Training Skill of Adolescent Males.

ABSTRACT: Pichardo, AW, Oliver, JL, Harrison, CB, Maulder, PS, Lloyd, RS, and Kandoi, R. Effects of combined resistance training and weightlifting on injury risk factors and resistance training skill of adolescent males. J Strength Cond Res 35(12): 3370-3377, 2021-The purpose of this study was to investigate the effects of resistance training with or without weightlifting on risk factors for injury and resistance training skill in circa-peak height velocity boys. Sixty-seven boys (aged 12-14 years) from a local secondary school were divided into 3 groups: combined resistance training (CRT), combined resistance training with weightlifting movements (CRT&WL), or a control group (CON). Experimental groups completed twice-weekly training programs over the course of an academic year. The tuck jump assessment, asymmetry measures for single-leg horizontal jump, isometric midthigh pull, and the Star Excursion Balance Test, and resistance training skill were measured pre-, mid-, and post-intervention. Only the CRT group significantly improved tuck jump assessment score pre- to post-test (p = 0.006, -20.4%, d = -0.39) but there were no clear effects on asymmetry measures for any group. Both groups significantly improved resistance training skill from pre- to post-test (CRT&WL: p = 0.002, 17.6%, d = 1.00; CRT: p = 0.026, 9.2%, d = 0.53). This study suggests that a school-based CRT program may provide significant improvements in jump landing kinematics, whereas the inclusion of weightlifting movements may provide greater improvements in resistance training skill.

Effects of Combined Resistance Training and Weightlifting on Motor Skill Performance of Adolescent Male Athletes.

Pichardo, AW, Oliver, JL, Harrison, CB, Maulder, PS, Lloyd, RS, and Kandoi, R. Effects of combined resistance training and weightlifting on motor skill performance of adolescent male athletes. J Strength Cond Res 33(12): 3226-3235, 2019-Resistance training and weightlifting are regarded as safe and effective training methods for youth. However, no studies have examined the effects of a year-long resistance training program using weightlifting movements on strength, speed, or power. Therefore, the purpose of this study was to determine the long-term effects of combined resistance training (traditional strength training + plyometrics) with or without weightlifting movements on motor skill performance of adolescent males. Fifty-nine males aged 12-14 years were matched by maturity and allocated to a combined resistance training or a combined resistance training with weightlifting group. Each group completed 28 total weeks of training over an academic year. Pre-, mid- (14 weeks of training), and post-training (28 weeks of training) tests included the resistance training skills battery quotient (RTSQ), absolute isometric midthigh pull peak force (IMTPABS) and ratio-scaled isometric midthigh pull peak force (IMTPREL), countermovement jump, horizontal jump, and 10-, 20-, and 30-m sprint. Repeated-measure analysis of variance revealed that there were no significant between-group responses, but all variables improved significantly within-group. Both groups made small-moderate improvements in RTSQ, IMTPABS, and IMTPREL after the first 14 training weeks (d = 0.45-0.86), whereas small-moderate improvements in lower body power, upper body power, and speed were made after the second 14 training weeks (d = 0.30-0.95). Both groups made small-moderate improvements in all performance variables after 28 weeks of training. These findings highlight the importance of establishing movement competency and strength as a foundation for the subsequent development of power. Furthermore, these findings may help practitioners understand the time course of certain adaptations following a long-term periodized plan for adolescent males.

Small-sided games for young athletes: is game specificity influential?

This study aimed to quantify and compare the physiological, physical and technical demands of a sport-specific and non-sport-specific small-sided game (SSG) in young athletes. Ten male soccer players (mean ± SD: age, 13.0 ± 0.3 years, [Formula: see text]O2peak, 54.4 ± 4.9 ml · kg(-1) · min(-1)) completed 3 vs. 3 and 6 vs. 6 soccer and "bucketball" SSGs twice. Games lasted for 16 min and were performed in a randomised order, at least 48 h apart. Movement patterns and heart rate were measured using 4 Hz global positioning systems. Technical skill executions were assessed by video analysis and ratings of perceived exertion (RPE) using the Borg scale (RPEs, 6-20). Total distance (4.9 ± 4.1% and 8.3 ± 6.6%), distance at 7-12.9 km · h(-1) (14.5 ± 12.5% and 14.9 ± 16.1%), total possessions (14.7 ± 18.0% and 12.9 ± 18.9%) and percentage of successful passes (24.1 ± 11.7% and 30.1 ± 17.6%) were higher for bucketball compared to soccer (3 vs. 3 and 6 vs. 6, respectively). Total distance at 13-17.9 km · h(-1) was higher for 6 vs. 6 than 3 vs. 3 bucketball (32.3 ± 21.2%). There was likely substantial difference for %HRpeak (89.5 ± 3.1 vs. 87.4 ± 2.8 b · min(-1)) and time above 90% HRpeak (570 ± 288 s vs. 361 ± 288 s) between bucketball and soccer (3 vs. 3 and 6 vs. 6, respectively). Young players travelled further at higher overall speeds, experienced higher physiological workloads and performed more successful technical executions during the non-sport-specific SSG.

Quantification of physiological, movement, and technical outputs during a novel small-sided game in young team sport athletes.

The aim of this study was to quantify the physiological responses, time-motion characteristics, and technical executions associated with a novel non-sport-specific small-sided game (SSG) in young team sport players. On 6 separate occasions, 12 young male team sport athletes (mean ± SD: age, 13.0 ± 0.3 years; height, 157.4 ± 4.9 cm; body mass, 47.0 ± 5.0 kg; and V[Combining Dot Above]O2peak, 55.1 ± 4.6 ml·kg·min) completed various "bucketball" SSG formats (i.e., 3 vs. 3, 4 vs. 4, and 6 vs. 6) twice each. Heart rate (HR) was measured during each SSG at 5-second intervals. Time-motion characteristics were measured using global positioning systems. Ratings of perceived exertion (RPEs) were recorded immediately after the SSGs using the Borg scale (RPEs, 6-20). Technical skill executions were measured using a high-speed digital video camera. Analysis revealed a tendency for the 3 vs. 3 games to elicit higher HRs (88.3 ± 4.3) than either 4 vs. 4 (85.9 ± 4.9) or 6 vs. 6 formats (85.9 ± 3.2). Total distance traveled at 13-17.9 km·h was more during 6 vs. 6 than 3 vs. 3 games (very likely substantial true difference, 97%), and total possessions and number of catches, passes, and shots were all higher in 3 vs. 3 compared with 4 vs. 4 and 6 vs. 6 games. There was no difference in RPE between the game formats. The results of this study indicate that 3 vs. 3 non-sport-specific SSGs provide higher stimulus for aerobic fitness adaptation and technical improvement than 4 vs. 4 and 6 vs. 6 formats, and their use for training young team sport athletes is recommended.

Evidence that nitric oxide inhibits vascular inflammation and superoxide production via a p47phox-dependent mechanism in mice.

1. Regulation of vascular Nox2-containing NADPH oxidase by p47(phox) plays a pivotal role in the development of atherosclerotic lesions through the generation of superoxide. Reduced vascular nitric oxide (NO) bioavailability is a major contributing factor in the initiation of atherosclerosis because it leads to an increase in adhesion molecule expression for inflammatory cell recruitment into the vessel wall. 2. The aim of the present study was to examine whether the anti-oxidant and anti-inflammatory effects of endogenous NO involve inhibition of NADPH oxidase-dependent superoxide production. 3. To inhibit endogenous NO production, male C57Bl/6 wild-type (WT) mice or age-matched p47(phox-/-) mice were treated with N(G)-nitro-L-arginine methyl ester (L-NAME; 100 mg/kg per day for 4 weeks). Blood pressure was measured weekly via the tail-cuff method. Basal and phorbol dibutyrate (PDB)-stimulated aortic superoxide production was detected using lucigenin- and L-012-enhanced chemiluminescence, respectively. Aortic Nox2, p47(phox) and vascular cell adhesion molecule (VCAM)-1 expression were measured with western blotting. Plasma angiotensin (Ang) II levels were determined by radioimmunoassay. 4. Compared with vehicle (tap water)-treated WT mice (n = 4), L-NAME-treated WT mice had significantly higher systolic blood pressure (SBP; n = 6; P < 0.05) and basal and stimulated aortic extracellular superoxide production (n = 6-8; P < 0.05), but lower plasma AngII levels (P < 0.05). There was no change in Nox2 expression following l-NAME treatment of WT mice (n = 6); however, significant increases in both aortic p47(phox) (n = 6; P < 0.05) and VCAM-1 expression (n = 6; P < 0.05) were observed. In p47(phox-/-) mice, l-NAME treatment significantly increased SBP (n = 3-4; P < 0.05), but failed to increase aortic superoxide production and VCAM-1 expression. 5. In conclusion, endogenous NO suppresses vascular inflammation, via inhibition of p47(phox) expression, leading to attenuation of NADPH oxidase-dependent superoxide production.

A prospective study of sport injuries in youth females.

OBJECTIVES: To examine sports and physical education (PE) injury rates in youth females during a school year and to investigate if an association exists between injury and phase of the menstrual cycle. DESIGN: Prospective cohort. SETTING: An online questionnaire was used to record training and competition exposure and self-reported injuries for 30-weeks. PARTICIPANTS: 103 PE students (12-15 years) from a girls' secondary school. MAIN OUTCOME MEASURES: Injury rates and prevalence of acute, gradual onset/overuse and substantial injuries. RESULTS: On average, girls trained 3.4 h/week and competed 1 h/week. During the study, 74 participants reported 595 injuries. The average weekly prevalence of all injuries was 20.7% (95% CI: 20.0-21.3), of which 8.6% (95% CI: 8.3-9.0) were acute injuries and 12.0% (95% CI: 11.4-12.6) were gradual onset/overuse injuries. The overall rate of sport and PE injuries was 10.4 injuries/1000 h of exposure. The most common acute injury involved the ankle (35%) while the most common gradual onset/overuse injury involved the knee (51%). There was no significant association between the stage of the menstrual cycle and the likelihood of injury (P = 0.18). CONCLUSION: The high number of injuries in this population of girls suggests preventative measures, particularly targeting the lower extremity, are needed.

Nox2-containing NADPH oxidase and xanthine oxidase are sources of superoxide in mouse trachea.

1. Superoxide anion plays an important role in host defence against invading pathogens and in the inflammation that arises in lungs. The aim of the present study was to elucidate whether the two key candidate superoxide-producing enzymes in mammalian cells, namely Nox2-containing NADPH oxidase and xanthine oxidase, are responsible for superoxide production in mouse trachea. 2. Superoxide production by isolated trachea, as measured by L-012-dependent chemiluminescence, was markedly reduced by superoxide dismutase (300 U/mL) and the xanthine oxidase inhibitor allopurinol (100 micromol/L). Tracheas from Nox2(-/-) mice had significantly lower levels (~60%) of superoxide than control mice. 3. These novel findings suggest that superoxide production by mouse trachea is attributed to both Nox2-containing NADPH oxidase and xanthine oxidase.

The Influence of Maturity Offset, Strength, and Movement Competency on Motor Skill Performance in Adolescent Males.

This study aimed to examine the extent to which maturity offset, strength, and movement competency influences motor skill performance in adolescent boys. One hundred and eight secondary school boys completed anthropometric and physical testing on two non-consecutive days for the following variables: Maturity offset, isometric mid-thigh pull absolute (IMTPABS) and relative (IMTPREL) peak force, resistance training skills quotient, 10-, 20-, and 30-m sprint time, countermovement jump height, horizontal jump distance, anaerobic endurance performance, and seated medicine ball throw (SMBT). The IMTPREL displayed significant small to large correlations with all performance variables (r = 0.27-0.61), whereas maturity offset was significantly correlated with IMTPABS (r = 0.69), sprint (r = 0.29-0.33), jump (r = 0.23-0.34), and SMBT (r = 0.32). Absolute and relative strength were the strongest predictors of all performance variables and combined with maturity to explain 21%-76% of the variance. Low and average relative strength boys were nearly eight times (odds ratio: 7.80, confidence interval: 1.48-41.12, p < 0.05) and nearly four times (odds ratio: 3.86, confidence interval: 0.95-15.59, p < 0.05) more likely to be classified as lower competency compared to high relative strength boys. Relative strength has more influence on motor skill performance than maturity when compared with movement competency.

Development of Aerobic Fitness in Young Team Sport Athletes.

The importance of a high level of aerobic fitness for team sport players is well known. Previous research suggests that aerobic fitness can be effectively increased in adults using traditional aerobic conditioning methods, including high-intensity interval and moderate-intensity continuous training, or more recent game-based conditioning that involves movement and skill-specific tasks, e.g. small-sided games. However, aerobic fitness training for youth team sport players has received limited attention and is likely to differ from that for adults due to changes in maturation. Given young athletes experience different rates of maturation and technical skill development, the most appropriate aerobic fitness training modes and loading parameters are likely to be specific to the developmental stage of a player. Therefore, we analysed studies that investigated exercise protocols to enhance aerobic fitness in young athletes, relative to growth and maturation, to determine current best practice and limitations. Findings were subsequently used to guide an evidence-based model for aerobic fitness development. During the sampling stage (exploration of multiple sports), regular participation in moderate-intensity aerobic fitness training, integrated into sport-specific drills, activities and skill-based games, is recommended. During the specialisation stage (increased commitment to a chosen sport), high-intensity small-sided games should be prioritised to provide the simultaneous development of aerobic fitness and technical skills. Once players enter the investment stage (pursuit of proficiency in a chosen sport), a combination of small-sided games and high-intensity interval training is recommended.

NOX2ß: A novel splice variant of NOX2 that regulates NADPH oxidase activity in macrophages.

Nox2 oxidase is one isoform in a family of seven NADPH oxidases that generate reactive oxygen species (ROS) and thereby contribute to physiological and pathological processes including host defense, redox signaling and oxidative tissue damage. While alternative mRNA splicing has been shown to influence the activity of several Nox-family proteins, functionally relevant splice variants of Nox2 have not previously been identified. We immunoscreened several mouse tissues and cells for the presence of truncated Nox2 proteins and identified a 30 kDa protein in lung, spleen and macrophages. RT-PCR analysis of mRNA from primary and immortalised (RAW264.7) mouse macrophages, and from human alveolar macrophages, identified a truncated Nox2 transcript which, upon sequence analysis, was found to be a product of the 'exon skipping' mode of alternative splicing, lacking exons 4-10 of the Nox2 gene. The predicted protein is comparable in size to that identified by immunoscreening and contains two transmembrane helices and an extended cytosolic C-terminus with binding sites for NADPH and the Nox organiser protein p47phox. Importantly, selective siRNA-mediated knockdown of the transcript reduced expression of the 30 kDa protein in macrophages, and suppressed phorbol ester-stimulated ROS production by 50%. We thus provide the first evidence that Nox2 undergoes alternative mRNA splicing to yield a 30 kDa protein - herein termed Nox2ß - that regulates NADPH oxidase activity in macrophages from mice and humans. The discovery of Nox2ß paves the way for future examination of its role in physiological and pathological processes.

NADPH oxidases as regulators of tumor angiogenesis: current and emerging concepts.

SIGNIFICANCE: Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and peroxynitrite are generated ubiquitously by all mammalian cells and have been understood for many decades as inflicting cell damage and as causing cancer by oxidation and nitration of macromolecules, including DNA, RNA, proteins, and lipids. RECENT ADVANCES: A current concept suggests that ROS can also promote cell signaling pathways triggered by growth factors and transcription factors that ultimately regulate cell proliferation, differentiation, and apoptosis, all of which are important hallmarks of tumor cell proliferation and angiogenesis. Moreover, an emerging concept indicates that ROS regulate the functions of immune cells that infiltrate the tumor environment and stimulate angiogenesis, such as macrophages and specific regulatory T cells. CRITICAL ISSUES: In this article, we highlight that the NADPH oxidase family of ROS-generating enzymes are the key sources of ROS and, thus, play an important role in redox signaling within tumor, endothelial, and immune cells thereby promoting tumor angiogenesis. FUTURE DIRECTIONS: Knowledge of these intricate ROS signaling pathways and identification of the culprit NADPH oxidases is likely to reveal novel therapeutic opportunities to prevent angiogenesis that occurs during cancer and which is responsible for the revascularization after current antiangiogenic treatment.