Perivascular adipose tissue (PVAT), as a mechanical support, has been reported to systemically regulate vascular physiology by secreting adipokines and cytokines. How PVAT spatially and locally changes as atherosclerosis progresses is not known, however. We aimed to reveal the molecular changes in PVAT in advanced atherosclerosis based on multimodal nonlinear optical (MNLO) imaging. First, using an atherogenic apolipoprotein E knockout mouse model, we precisely assessed the browning level of thoracic PVAT via a correlative analysis between the size and number of lipid droplets (LDs) of label-free MNLO images. We also biochemically demonstrated the increased level of brown fat markers in the PVAT of atherosclerosis. In the initial stage of atherosclerosis, the PVAT showed a highly activated brown fat feature due to the increased energy expenditure; however, in the advanced stage, only the PVAT in the regions of the atherosclerotic plaques, not that in the nonplaque regions, showed site-specific changes. We found that p-smad2/3 and TGF-β signaling enhanced the increase in collagen to penetrate the PVAT and the agglomeration of LDs only at the sites of atherosclerotic plaques. Moreover, atherosclerotic thoracic PVAT (tPVAT) was an increased inflammatory response. Taken together, our findings show that PVAT changes differentially from the initial stages to advanced stages of atherosclerosis and undergoes spatial impairment focused on atherosclerotic plaques. Our study may provide insight into the local control of PVAT as a therapeutic target.

The outer epidermal skin is a primary barrier that protects the body from extrinsic factors, such as ultraviolet (UV) radiation, chemicals and pollutants. The complete epithelialization of a wound by keratinocytes is essential for restoring the barrier function of the skin. However, age-related alterations predispose the elderly to impaired wound healing. Therefore, wound-healing efficacy could be also considered as a potent function of an anti-aging reagent. Here, we examine the epidermal wound-healing efficacy of the fourth-generation retinoid, seletinoid G, using HaCaT keratinocytes and skin tissues. We found that seletinoid G promoted the proliferation and migration of keratinocytes in scratch assays and time-lapse imaging. It also increased the gene expression levels of several keratinocyte proliferation-regulating factors. In human skin equivalents, seletinoid G accelerated epidermal wound closure, as assessed using optical coherence tomography (OCT) imaging. Moreover, second harmonic generation (SHG) imaging revealed that seletinoid G recovered the reduced dermal collagen deposition seen in ultraviolet B (UVB)-irradiated human skin equivalents. Taken together, these results indicate that seletinoid G protects the skin barrier by accelerating wound healing in the epidermis and by repairing collagen deficiency in the dermis. Thus, seletinoid G could be a potent anti-aging agent for protecting the skin barrier.

Background: Fibrosis plays an important role in both normal physiological and pathological phenomena as fibroblasts differentiate to myofibroblasts. The activation of fibroblasts is determined through interactions with the surrounding extracellular matrix (ECM). However, how this fibroblast-to-myofibroblast transition (FMT) is regulated and affected by elastin concentration in a three-dimensional (3D) microenvironment has not been investigated. Methods: We developed an insoluble elastin-gradient 3D hydrogel system for long-lasting cell culture and studied the molecular mechanisms of the FMT in embedded cells by nanoflow LC–MS/MS analysis along with validation through real-time PCR and immunofluorescence staining. Results: By optimizing pH and temperature, four 3D hydrogels containing fibroblasts were successfully fabricated having elastin concentrations of 0, 20, 50, and 80% in collagen. At the low elastin level (20%), fibroblast proliferation was significantly increased compared to others, and in particular, the FMT was clearly observed in this condition. Moreover, through mass spectrometry of the hydrogel environment, it was confirmed that differentiation proceeded in two stages. In the early stage, calcium-dependent proteins including calmodulin and S100A4 were highly associated. On the other hand, in the late stage after several passages of cells, distinct markers of myofibroblasts were presented such as morphological changes, increased production of ECM, and increased α-SMA expression. We also demonstrated that the low level of elastin concentration induced some cancer-associated fibroblast (CAF) markers, including PDGFR-β, and fibrosis-related disease markers, including THY-1. Conclusion: Using our developed 3D elastin-gradient hydrogel system, we evaluated the effect of different elastin concentrations on the FMT. The FMT was induced even at a low concentration of elastin with increasing CAF level via calcium signaling. With this system, we were able to analyze varying protein expressions in the overall FMT process over several cellular passages. Our results suggest that the elastin-gradient system employing nonlinear optics imaging provides a good platform to study activated fibroblasts interacting with the microenvironment, where the ECM plays a pivotal role. Graphical

Precise measurement of particulate matter (PM) on skin is important for managing and preventing PM-related skin diseases. This study aims to directly visualize the deposition and penetration of PM into human skin using a multimodal nonlinear optical (MNLO) imaging system. We successfully obtained PM particle signals by merging two different sources, C–C vibrational frequency and autofluorescence, while simultaneously visualizing the anatomical features of the skin via keratin, collagen, and elastin. As a result, we found morphologically dependent PM deposition, as well as increased deposition following disruption of the skin barrier via tape-stripping. Furthermore, PM penetrated more and deeper into the skin with an increase in the number of tape-strippings, causing a significant increase in the secretion of pro-inflammatory cytokines. Our results suggest that MNLO imaging could be a useful technique for visualizing and quantifying the spatial distribution of PM in ex vivo human skin tissues.