Given the need for robust and cost‐efficient in vitro models to study angiogenesis and reproducibly analyze potential pro‐ and antiangiogenic compounds in preclinical studies, we developed a 3‐dimensional in vitro angiogenesis assay that is based on collagen gel‐embedded, size‐defined spheroids generated from cultured human umbilical vein endothelial cells (HUVECs). Despite its wide distribution, limitations, sensitivity, robustness, and improvements, the capacity of this assay for functional screening purposes has not been elucidated thus far. By using time‐lapse video microscopy, we show that tip cells lead the formation of capillary‐like and partially lumenized sprouts originating from the spheroids. Angiogenic sprouting from spheroids generated from 5 different primary cultured human endothelial cell types was induced by physiologic concentrations of vascular endothelial cell growth factor 165. Based on this assay system, we determined the capacity of 880 approved drugs to interfere with or boost angiogenic sprouting, thereby assessing their putative angiogenesis‐related side effects or novel applications. However, although this assay allowed for a rapid and reproducible determination of functional IC ₅₀ values of individual compounds, the sprouting results were partially affected by the HUVEC passage number and donor variability. To overcome this limitation, immortalized HUVECs (iHUVECs) showing a more homogenous response in terms of proliferation and sprouting over multiple population doublings were used in the course of this study. Collectively, the spheroid‐based angiogenesis assay provides a sensitive and versatile tool to study the impact of pro‐ and antiangiogenic determinants on multiple steps of the angiogenic cascade. It is compatible with different endothelial cell types and allows use of iHUVECs to improve its overall robustness.—Heiss, M., Hellström, M., Kalén, M., May, T., Weber, H., Hecker, M., Augustin, H. G., Korff, T. Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro. FASEB J . 29, 3076‐3084 (2015). www.fasebj.org

The stiffness of a tendon, which influences muscular force transfer to the skeleton and increases during childhood, is dependent on its material properties and dimensions, both of which are influenced by chronic loading. The aims of this study were to: (i) determine the independent contributions of body mass, force production capabilities and tendon dimensions to tendon stiffness during childhood; and (ii) descriptively document age‐related changes in tendon mechanical properties and dimensions. Achilles tendon mechanical and material properties were determined in 52 children (5–12 years) and 19 adults. Tendon stiffness and Young’s modulus (YM) were calculated as the slopes of the force–elongation and stress‐strain curves, respectively. Relationships between stiffness vs. age, mass and force, and between YM vs. age, mass and stress were determined by means of polynomial fits and multiple regression analyses. Mass was found to be the best predictor of stiffness, whilst stress was best related to YM (< 75 and 51% explained variance, respectively). Combined, mass and force accounted for up to 78% of stiffness variation. Up to 61% of YM variability could be explained using a combination of mass, stress and age. These results demonstrate that age‐related increases in tendon stiffness are largely attributable to increased tendon loading from weight‐bearing tasks and increased plantarflexor force production, as well as tendon growth. Moreover, our results suggest that chronic increases in tendon loading during childhood result in microstructural changes which increase the tendon’s YM. Regarding the second aim, peak stress increased from childhood to adulthood due to greater increases in strength than tendon cross‐sectional area. Peak strain remained constant as a result of parallel increases in tendon length and peak elongation. The differences in Achilles tendon properties found between adults and children are likely to influence force production, and ultimately movement characteristics, which should be explicitly examined in future research.

Effective pedaling technique leads to greater efficiency. Thus, the purpose of this study was to determine the effect of different pedaling techniques on mechanical effectiveness and gross efficiency during steady-state cycling. Methods: Eight male cyclists exercised on a cycle ergometer at 90 rpm and 200 W using four different pedaling techniques: preferred pedaling; pedaling in circles; emphasizing the pull during the upstroke; and emphasizing the push during the downstroke. Each exercise bout lasted 6 min and was interspersed with 6 min of passive rest. We obtained mechanical effectiveness and gross efficiency using pedal-reaction forces and respiratory measures, respectively. Results: When the participants were instructed to pull on the pedal during the upstroke, mechanical effectiveness was greater (index of force effectiveness = 62.4 ± 9.8%) and gross efficiency was lower (gross efficiency = 19.0 ± 0.7%) compared with the other pedaling conditions (index of force effectiveness = 48.2 ± 5.1% and gross efficiency = 20.2 ± 0.6%; means and standard deviations collapsed across preferred, circling, and pushing conditions). Mechanical effectiveness and gross efficiency during the circling and pushing conditions did not differ significantly from the preferred pedaling condition. Conclusions: Mechanical effectiveness is not indicative of gross efficiency across pedaling techniques. These results thereby provide coaches and athletes with useful information for interpreting measures of mechanical effectiveness....

SUMMARY: Maximum power production during multi-joint tasks increases as children grow older. Previous research suggests that in adults, maximum power production in jumping is related to lower limb stiffness. In a developmental context, the question arises as to whether the relationship between maximum power production and lower limb stiffness is age-dependent. The purpose of this study was to investigate the relationship between lower limb stiffness and peak power production in adolescents (AD) and pre-adolescents (PA). With institutional approval, two groups of pre-adults (pre-adolescents: 11–13 years of age, N=43; adolescents: 16–18 years of age, N=30) performed 30 two-legged hops at their preferred frequency and three maximum counter-movement jumps. AD produced significantly greater peak power during the counter-movement jump than PA(t71=–5.28, P<0.001) even when body mass was accounted for. Lower limb stiffness was significantly correlated with peak power production during the counter-movement jump in AD (R=0.62, P<0.001) but not in PA (R=0.26, P=0.10). When normalised to body mass, the relationship between lower limb stiffness and peak power also differed between the two age groups (R=0.30, P=0.11 for AD and R=0.02, P=0.88 for PA). In addition, we found that during hopping, both PA and AD behaved like a simple spring-mass system. Our findings highlight the importance of lower limb stiffness in the context of muscular power production during multi-joint tasks. They let us speculate that during adolescence, children acquire the ability to take greater advantage of elastic energy storage in the musculotendinous system when performing maximum counter-movement jumps.

Background: Nuclear factor of activated T‐cells 5 ( NFAT 5) has recently been described to control the phenotype of vascular smooth muscle cells ( VSMC s). Although an increase in wall stress or stretch (eg, elicited by hypertension) is a prototypic determinant of VSMC activation, the impact of this biomechanical force on the activity of NFAT 5 is unknown. This study intended to reveal the function of NFAT 5 and to explore potential signal transduction pathways leading to its activation in stretch‐stimulated VSMC s. Methods and Results: Human arterial VSMC s were exposed to biomechanical stretch and subjected to immunofluorescence and protein‐biochemical analyses. Stretch promoted the translocation of NFAT 5 to the nucleus within 24 hours. While the protein abundance of NFAT 5 was regulated through activation of c‐Jun N‐terminal kinase under these conditions, its translocation required prior activation of palmitoyltransferases. DNA microarray and ChiP analyses identified the matrix molecule tenascin‐C as a prominent transcriptional target of NFAT 5 under these conditions that stimulates migration of VSMC s. Analyses of isolated mouse femoral arteries exposed to hypertensive perfusion conditions verified that NFAT 5 translocation to the nucleus is followed by an increase in tenascin‐C abundance in the vessel wall. Conclusions: Collectively, our data suggest that biomechanical stretch is sufficient to activate NFAT 5 both in native and cultured VSMC s where it regulates the expression of tenascin‐C. This may contribute to an improved migratory activity of VSMC s and thus promote maladaptive vascular remodeling processes such as hypertension‐induced arterial stiffening.