The weakness of senescent dermal fibroblasts.

Skin is the largest human organ with easily noticeable biophysical manifestations of aging. As human tissues age, there is chronological accumulation of biophysical changes due to internal and environmental factors. Skin aging leads to decreased elasticity and the loss of dermal matrix integrity via degradation. The mechanical properties of the dermal matrix are maintained by fibroblasts, which undergo replicative aging and may reach senescence. While the secretory phenotype of senescent fibroblasts is well studied, little is known about changes in the fibroblasts biophysical phenotype. Therefore, we compare biophysical properties of young versus proliferatively aged primary fibroblasts via fluorescence and traction force microscopy, single-cell atomic force spectroscopy, microfluidics, and microrheology of the cytoskeleton. Results show senescent fibroblasts have decreased cytoskeletal tension and myosin II regulatory light chain phosphorylation, in addition to significant loss of traction force. The alteration of cellular forces is harmful to extracellular matrix homeostasis, while decreased cytoskeletal tension can amplify epigenetic changes involved in senescence. Further exploration and detection of these mechanical phenomena provide possibilities for previously unexplored pharmaceutical targets against aging.

Peripheral monocytes are functionally altered and invade the CNS in ALS patients.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease affecting primarily the upper and lower motor neurons. A common feature of all ALS cases is a well-characterized neuroinflammatory reaction within the central nervous system (CNS). However, much less is known about the role of the peripheral immune system and its interplay with CNS resident immune cells in motor neuron degeneration. Here, we characterized peripheral monocytes in both temporal and spatial dimensions of ALS pathogenesis. We found the circulating monocytes to be deregulated in ALS regarding subtype constitution, function and gene expression. Moreover, we show that CNS infiltration of peripheral monocytes correlates with improved motor neuron survival in a genetic ALS mouse model. Furthermore, application of human immunoglobulins or fusion proteins containing only the human Fc, but not the Fab antibody fragment, increased CNS invasion of peripheral monocytes and delayed the disease onset. Our results underline the importance of peripheral monocytes in ALS pathogenesis and are in agreement with a protective role of monocytes in the early phase of the disease. The possibility to boost this beneficial function of peripheral monocytes by application of human immunoglobulins should be evaluated in clinical trials.

Genetically Encoded FRET-Based Tension Sensors.

Genetically encoded Förster resonance energy transfer (FRET)-based tension sensors measure piconewton-scale forces across individual molecules in living cells or whole organisms. These biosensors show comparably high FRET efficiencies in the absence of tension, but FRET quickly decreases when forces are applied. In this article, we describe how such biosensors can be generated for a specific protein of interest, and we discuss controls to confirm that the observed differences in FRET efficiency reflect changes in molecular tension. These FRET efficiency changes can be related to mechanical forces as the FRET-force relationship of the employed tension sensor modules are calibrated. We provide information on construct generation, expression in cells, and image acquisition using live-cell fluorescence lifetime imaging microscopy (FLIM). Moreover, we describe how to analyze, statistically evaluate, and interpret the resulting data sets. Together, these protocols should enable the reader to plan, execute, and interpret FRET-based tension sensor experiments. © 2019 by John Wiley & Sons, Inc.

Age Increases Monocyte Adhesion on Collagen.

Adhesion of monocytes to micro-injuries on arterial walls is an important early step in the occurrence and development of degenerative atherosclerotic lesions. At these injuries, collagen is exposed to the blood stream. We are interested whether age influences monocyte adhesion to collagen under flow, and hence influences the susceptibility to arteriosclerotic lesions. Therefore, we studied adhesion and rolling of human peripheral blood monocytes from old and young individuals on collagen type I coated surface under shear flow. We find that firm adhesion of monocytes to collagen type I is elevated in old individuals. Pre-stimulation by lipopolysaccharide increases the firm adhesion of monocytes homogeneously in older individuals, but heterogeneously in young individuals. Blocking integrin αx showed that adhesion of monocytes to collagen type I is specific to the main collagen binding integrin αxß2. Surprisingly, we find no significant age-dependent difference in gene expression of integrin αx or integrin ß2. However, if all integrins are activated from the outside, no differences exist between the age groups. Altered integrin activation therefore causes the increased adhesion. Our results show that the basal increase in integrin activation in monocytes from old individuals increases monocyte adhesion to collagen and therefore the risk for arteriosclerotic plaques.