Piezo1 Activation Drives Enhanced Collagen Synthesis in Aged Animal Skin Induced by Poly L-Lactic Acid Fillers.

Poly L-lactic acid (PLLA) fillers stimulate collagen synthesis by activating various immune cells and fibroblasts. Piezo1, an ion channel, responds to mechanical stimuli, including changes in extracellular matrix stiffness, by mediating Ca2+ influx. Given that elevated intracellular Ca2+ levels trigger signaling pathways associated with fibroblast proliferation, Piezo1 is a pivotal regulator of collagen synthesis and tissue fibrosis. The aim of the present study was to investigate the impact of PLLA on dermal collagen synthesis by activating Piezo1 in both an H2O2-induced cellular senescence model in vitro and aged animal skin in vivo. PLLA elevated intracellular Ca2+ levels in senescent fibroblasts, which was attenuated by the Piezo1 inhibitor GsMTx4. Furthermore, PLLA treatment increased the expression of phosphorylated ERK1/2 to total ERK1/2 (pERK1/2/ERK1/2) and phosphorylated AKT to total AKT (pAKT/AKT), indicating enhanced pathway activation. This was accompanied by upregulation of cell cycle-regulating proteins (CDK4 and cyclin D1), promoting the proliferation of senescent fibroblasts. Additionally, PLLA promoted the expression of phosphorylated mTOR/S6K1/4EBP1, TGF-ß, and Collagen I/III in senescent fibroblasts, with GsMTx4 treatment mitigating these effects. In aged skin, PLLA treatment similarly upregulated the expression of pERK1/2/ERK1/2, pAKT/AKT, CDK4, cyclin D1, mTOR/S6K1/4EBP1, TGF-ß, and Collagen I/III. In summary, our findings suggest Piezo1's involvement in PLLA-induced collagen synthesis, mediated by heightened activation of cell proliferation signaling pathways such as pERK1/2/ERK1/2, pAKT/AKT, and phosphorylated mTOR/S6K1/4EBP1, underscoring the therapeutic potential of PLLA in tissue regeneration.

High-Intensity Focused Ultrasound Increases Facial Adipogenesis in a Swine Model via Modulation of Adipose-Derived Stem Cell Cilia.

Decreased medial cheek fat volume during aging leads to loss of a youthful facial shape. Increasing facial volume by methods such as adipose-derived stem cell (ASC) injection can produce facial rejuvenation. High-intensity focused ultrasound (HIFU) can increase adipogenesis in subcutaneous fat by modulating cilia on ASCs, which is accompanied by increased HSP70 and decreased NF-κB expression. Thus, we evaluated the effect of HIFU on increasing facial adipogenesis in swine (n = 2) via modulation of ASC cilia. Expression of CD166, an ASC marker, differed by subcutaneous adipose tissue location. CD166 expression in the zygomatic arch (ZA) was significantly higher than that in the subcutaneous adipose tissue in the mandible or lateral temporal areas. HIFU was applied only on the right side of the face, which was compared with the left side, where HIFU was not applied, as a control. HIFU produced a significant increase in HSP70 expression, decreased expression of NF-κB and a cilia disassembly factor (AURKA), and increased expression of a cilia increasing factor (ARL13B) and PPARG and CEBPA, which are the main regulators of adipogenesis. All of these changes were most prominent at the ZA. Facial adipose tissue thickness was also increased by HIFU. Adipose tissue volume, evaluated by magnetic resonance imaging, was increased by HIFU, most prominently in the ZA. In conclusion, HIFU increased ASC marker expression, accompanied by increased HSP70 and decreased NF-κB expression. Additionally, changes in cilia disassembly and length and expression of adipogenesis were observed. These results suggest that HIFU could be used to increase facial volume by modulating adipogenesis.

Population pharmacokinetic modeling of sufentanil in adult Korean patients undergoing cardiopulmonary bypass surgery.

Sufentanil is frequently used as an anesthetic agent in cardiac surgery owing to its cardiovascular safety and favorable pharmacokinetics. However, the pharmacokinetics profiles of sufentanil in patients undergoing cardiopulmonary bypass (CPB) surgery remain less understood, which is crucial for achieving the desired level of anesthesia and mitigating surgical complications. Therefore, this study aimed to develop a population pharmacokinetic model of sufentanil in patients undergoing CPB surgery and elucidate the clinical factors affecting its pharmacokinetic profile. Adult patients who underwent cardiac surgery with CPB and were administered sufentanil for anesthesia were enrolled. Arterial blood samples were collected to quantify plasma concentrations of sufentanil and clinical laboratory parameters, including inflammatory cytokines. A population pharmacokinetic model was established using nonlinear mixed-effects modeling. Simulations were performed using the pharmacokinetic parameters of the final model. Overall, 20 patients were included in the final analysis. Sufentanil pharmacokinetics were modeled using a two-compartment model, accounting for CPB effects. Sufentanil clearance increased 2.80-fold during CPB and warming phases, while the central compartment volume increased 2.74-fold during CPB. CPB was a significant covariate affecting drug clearance and distribution volume. No other significant covariates were identified despite increased levels of the inflammatory cytokines, including IL-6, IL-8, and TNF-α during CPB. The simulation indicated a 30 µg loading dose and 40 µg/h maintenance infusion for target-controlled infusion. Additionally, a bolus dose of 60 µg was added at CPB initiation to adjust for exposure changes during this phase. Considering the target sufentanil concentrations, a uniform dosing regimen was acceptable for effective analgesia.

The Modulation of Respiratory Epithelial Cell Differentiation by the Thickness of an Electrospun Poly-ε-Carprolactone Mesh Mimicking the Basement Membrane.

The topology of the basement membrane (BM) affects cell physiology and pathology, and BM thickening is associated with various chronic lung diseases. In addition, the topology of commercially available poly (ethylene terephthalate) (PET) membranes, which are used in preclinical in vitro models, differs from that of the human BM, which has a fibrous and elastic structure. In this study, we verified the effect of BM thickness on the differentiation of normal human bronchial epithelial (NHBE) cells. To evaluate whether the thickness of poly-ε-carprolactone (PCL) mesh affects the differentiation of NHBE cells, cells were grown on thin- (6-layer) and thick-layer (80-layer) meshes consisting of electrospun PCL nanofibers using an air-liquid interface (ALI) cell culture system. It was found that the NHBE cells formed a normal pseudostratified epithelium composed of ciliated, goblet, and basal cells on the thin-layer PCL mesh; however, goblet cell hyperplasia was observed on the thick-layer PCL mesh. Differentiated NHBE cells cultured on the thick-layer PCL mesh also demonstrated increased epithelial-mesenchymal transition (EMT) compared to those cultured on the thin-layer PCL mesh. In addition, expression of Sox9, nuclear factor (NF)-κB, and oxidative stress-related markers, which are also associated with goblet cell hyperplasia, was increased in the differentiated NHBE cells cultured on the thick-layer PCL mesh. Thus, the use of thick electrospun PCL mesh led to NHBE cells differentiating into hyperplastic goblet cells via EMT and the oxidative stress-related signaling pathway. Therefore, the topology of the BM, for example, thickness, may affect the differentiation direction of human bronchial epithelial cells.

Extracellular Vesicles from Ecklonia cava and Phlorotannin Promote Rejuvenation in Aged Skin.

Plant-derived extracellular vesicles (EVs) elicit diverse biological effects, including promoting skin health. EVs isolated from Ecklonia cava (EV-EC) carry heat shock protein 70 (HSP70), which inhibits key regulators such as TNF-α, MAPKs, and NF-κB, consequently downregulating matrix metalloproteinases (MMPs). Aging exacerbates oxidative stress, upregulating MAPK and NF-κB signaling and worsening extracellular matrix degradation in the skin. E. cava-derived phlorotannin (PT) mitigates MAPK and NF-κB signaling. We evaluated the impact of EV-EC and PT on skin rejuvenation using an in vitro keratinocyte senescence model and an in vivo aged-mouse model. Western blotting confirmed the presence of HSP70 in EV-EC. Treatment with EV-EC and PT in senescent keratinocytes increased HSP70 expression and decreased the expression of TNF-α, MAPK, NF-κB, activator protein-1 (AP-1), and MMPs. Oxidative stress was also reduced. Sequential treatment with PT and EV-EC (PT/EV-EC) yielded more significant results compared to individual treatments. The administration of PT/EV-EC to the back skin of aged mice mirrored the in vitro findings, resulting in increased collagen fiber accumulation and improved elasticity in the aged skin. Therefore, PT/EV-EC holds promise in promoting skin rejuvenation by increasing HSP70 expression, decreasing the expression of MMPs, and reducing oxidative stress in aged skin.

Radiofrequency Treatment Attenuates Age-Related Changes in Dermal-Epidermal Junctions of Animal Skin.

The dermal-epidermal junction (DEJ) is essential for maintaining skin structural integrity and regulating cell survival and proliferation. Thus, DEJ rejuvenation is key for skin revitalization, particularly in age-related DEJ deterioration. Radiofrequency (RF) treatment, known for its ability to enhance collagen fiber production through thermal mechanisms and increase heat shock protein (HSP) expression, has emerged as a promising method for skin rejuvenation. Additionally, RF activates Piezo1, an ion channel implicated in macrophage polarization toward an M2 phenotype and enhanced TGF-ß production. This study investigated the impact of RF treatment on HSP47 and HSP90 expression, known stimulators of DEJ protein expression. Furthermore, using in vitro and aged animal skin models, we assessed whether RF-induced Piezo1 activation and the subsequent M2 polarization could counter age-related DEJ changes. The RF treatment of H2O2-induced senescent keratinocytes upregulated the expression of HSP47, HSP90, TGF-ß, and DEJ proteins, including collagen XVII. Similarly, the RF treatment of senescent macrophages increased Piezo1 and CD206 (M2 marker) expression. Conditioned media from RF-treated senescent macrophages enhanced the expression of TGF-ß and DEJ proteins, such as nidogen and collagen IV, in senescent fibroblasts. In aged animal skin, RF treatment increased the expression of HSP47, HSP90, Piezo1, markers associated with M2 polarization, IL-10, and TGF-ß. Additionally, RF treatment enhanced DEJ protein expression. Moreover, RF reduced lamina densa replication, disrupted lesions, promoted hemidesmosome formation, and increased epidermal thickness. Overall, RF treatment effectively enhanced DEJ protein expression and mitigated age-related DEJ structural changes by increasing HSP levels and activating Piezo1.

Co-Treatment with Phlorotannin and Extracellular Vesicles from Ecklonia cava Inhibits UV-Induced Melanogenesis.

Hyperpigmentation due to ultraviolet (UV)-induced melanogenesis causes various esthetic problems. Phlorotannin (PT) and extracellular vesicles (EVs) derived from various plants suppress melanogenesis pathways. We used UV-exposed keratinocytes and animal skin to determine if co-treatment with PT and EVs from Ecklonia cava (EVE) could inhibit melanogenesis by reducing UV-induced oxidative stress and the expression of the thioredoxin-interacting protein (TXNIP)/nucleotide-binding oligomerization domain-like receptor family pyrin domain containing the 3 (NLRP3)/interleukin-18 (IL-18) pathway, which are upstream signals of the microphthalmia-associated transcription factor. UV exposure increased oxidative stress in keratinocytes and animal skin, as evaluated by 8-OHdG expression, and this effect was reduced by co-treatment with PT and EVE. UV also increased binding between NLRP3 and TXNIP, which increased NLRP3 inflammasome activation and IL-18 secretion, and this effect was reduced by co-treatment with PT and EVE in keratinocytes and animal skin. In melanocytes, conditioned media (CM) from UV-exposed keratinocytes increased the expression of melanogenesis-related pathways; however, these effects were reduced with CM from UV-exposed keratinocytes treated with PT and EVE. Similarly, PT and EVE treatment reduced melanogenesis-related signals, melanin content, and increased basement membrane (BM) components in UV-exposed animal skin. Thus, co-treatment with PT and EVE reduced melanogenesis and restored the BM structure by reducing oxidative stress and TXNIP/NLRP3/IL-18 pathway expression.

Diesel exhaust particle exposure exacerbates ciliary and epithelial barrier dysfunction in the multiciliated bronchial epithelium models.

Airway epithelium, the first defense barrier of the respiratory system, facilitates mucociliary clearance against inflammatory stimuli, such as pathogens and particulates inhaled into the airway and lung. Inhaled particulate matter 2.5 (PM2.5) can penetrate the alveolar region of the lung, and it can develop and exacerbate respiratory diseases. Although the pathophysiological effects of PM2.5 in the respiratory system are well known, its impact on mucociliary clearance of airway epithelium has yet to be clearly defined. In this study, we used two different 3D in vitro airway models, namely the EpiAirway-full-thickness (FT) model and a normal human bronchial epithelial cell (NHBE)-based air-liquid interface (ALI) system, to investigate the effect of diesel exhaust particles (DEPs) belonging to PM2.5 on mucociliary clearance. RNA-sequencing (RNA-Seq) analyses of EpiAirway-FT exposed to DEPs indicated that DEP-induced differentially expressed genes (DEGs) are related to ciliary and microtubule function and inflammatory-related pathways. The exposure to DEPs significantly decreased the number of ciliated cells and shortened ciliary length. It reduced the expression of cilium-related genes such as acetylated α-tubulin, ARL13B, DNAH5, and DNAL1 in the NHBEs cultured in the ALI system. Furthermore, DEPs significantly increased the expression of MUC5AC, whereas they decreased the expression of epithelial junction proteins, namely, ZO1, Occludin, and E-cadherin. Impairment of mucociliary clearance by DEPs significantly improved the release of epithelial-derived inflammatory and fibrotic mediators such as IL-1ß, IL-6, IL-8, GM-CSF, MMP-1, VEGF, and S100A9. Taken together, it can be speculated that DEPs can cause ciliary dysfunction, hyperplasia of goblet cells, and the disruption of the epithelial barrier, resulting in the hyperproduction of lung injury mediators. Our data strongly suggest that PM2.5 exposure is directly associated with ciliary and epithelial barrier dysfunction and may exacerbate lung injury.

3D-Printed Auxetic Skin Scaffold for Decreasing Burn Wound Contractures at Joints.

For patients with severe burns that consist of contractures induced by fibrous scar tissue formation, a graft must adhere completely to the wound bed to enable wound healing and neovascularization. However, currently available grafts are insufficient for scar suppression owing to their nonuniform pressure distribution in the wound area. Therefore, considering the characteristics of human skin, which is omnidirectionally stretched via uniaxial stretching, we proposed an auxetic skin scaffold with a negative Poisson's ratio (NPR) for tight adherence to the skin scaffold on the wound bed site. Briefly, a skin scaffold with the NPR effect was fabricated by creating a fine pattern through 3D printing. Electrospun layers were also added to improve adhesion to the wound bed. Fabricated skin scaffolds displayed NPR characteristics (-0.5 to -0.1) based on pulling simulation and experiment. Finger bending motion tests verified the decreased marginal forces (<50%) and deformation (<60%) of the NPR scaffold. In addition, the filling of human dermal fibroblasts in most areas (>95%) of the scaffold comprising rarely dead cells and their spindle-shaped morphologies revealed the high cytocompatibility of the developed scaffold. Overall, the developed skin scaffold may help reduce wound strictures in the joints of patients with burns as it exerts less pressure on the wound margin.

High-Intensity Focused Ultrasound Increases Collagen and Elastin Fiber Synthesis by Modulating Caveolin-1 in Aging Skin.

Caveolin-1 (Cav-1) induces cellular senescence by reducing extracellular signal-regulated kinase (ERK)1/2 phosphorylation and activating p53 via inhibition of mouse double minute 2 homolog (MDM2) and sirtuin 1 (Sirt1), promoting cell cycle arrest and decreasing fibroblast proliferation and collagen synthesis. High-intensity focused ultrasound (HIFU) treatment increases collagen synthesis, rejuvenating skin. Using H2O2-induced senescent fibroblasts and the skin of 12-month-old mice, we tested the hypothesis that HIFU increases collagen production through Cav-1 modulation. HIFU was administered at 0.3, 0.5, or 0.7 J in the LINEAR and DOT modes. In both models, HIFU administration decreased Cav-1 levels, increased ERK1/2 phosphorylation, and decreased the binding of Cav-1 with both MDM2 and Sirt1. HIFU administration decreased p53 activation (acetylated p53) and p21 levels and increased cyclin D1, cyclin-dependent kinase 2, and proliferating cell nuclear antigen levels in both models. HIFU treatment increased collagen and elastin expression, collagen fiber accumulation, and elastin fiber density in aging skin, with 0.5 J in LINEAR mode resulting in the most prominent effects. HIFU treatment increased collagen synthesis to levels similar to those in Cav-1-silenced senescent fibroblasts. Our results suggest that HIFU administration increases dermal collagen and elastin fibers in aging skin via Cav-1 modulation and reduced p53 activity.

Nutritional indices for screening sarcopenia before adult cardiac surgery.

Background: Malnutrition can increase and exacerbate sarcopenia, and preoperative nutritional indices could have potential use as screening tools for sarcopenia in all patients, not only those with limited activity. Muscle strengths, such as grip strength, chair stand test, are used to screen for sarcopenia, but these measurements are time-consuming and cannot be applied to all patients. This retrospective study was conducted to determine whether nutritional indices can predict the presence of sarcopenia before adult cardiac surgery. Methods: The study subjects were 499 patients aged ≥18 who had undergone cardiac surgery using a cardiopulmonary bypass (CPB). Bilateral psoas muscle mass areas at the top level of the iliac crest were measured by abdominal computed tomography. Preoperative nutritional statuses were evaluated using COntrolling NUTritional status (CONUT) score, Prognostic Nutritional Index (PNI), and Nutritional Risk Index (NRI). Receiver operating characteristic (ROC) curve analysis was used to identify the nutritional index that best predicted the presence of sarcopenia. Results: The 124 patients (24.8%) in the sarcopenic group were older (69.0 vs. 62.0 years; P<0.001), and had a lower mean body weight (58.90 vs. 65.70 kg; P<0.001) and body mass index (BMI) (2.22 vs. 2.49 kg/m2; P<0.001), and a poorer nutritional status than the 375 patients in the non-sarcopenic group. ROC curve analysis showed that NRI [area under the curve (AUC) 0.716, confidence intervals (CI): 0.664-0.768] better predicted the presence of sarcopenia than CONUT score (AUC 0.607, CI: 0.549-0.665) or PNI (AUC 0.574, CI: 0.515-0.633). The optimal NRI cut-off value was 105.25, which provided a sensitivity of 67.7% and a specificity of 65.1% for the prevalence of sarcopenia. The median durations of mechanical support (17 vs. 16 hours; P=0.008) and intensive care unit stay (3 vs. 2 days; P=0.001) were significantly longer in the sarcopenic group. Conclusions: NRI offers a more straightforward, faster, and reproducible screening tool than muscle strength or mass measurement for identifying sarcopenia, and an alternative means of assessment in patients with limited activity before adult cardiac surgery.

Rice Germ Attenuates Chronic Unpredictable Mild Stress-Induced Muscle Atrophy.

Chronic stress leads to hypothalamic-pituitary-adrenal axis dysfunction, increasing cortisol levels. Glucocorticoids (GCs) promote muscle degradation and inhibit muscle synthesis, eventually causing muscle atrophy. In this study, we aimed to evaluate whether rice germ supplemented with 30% γ-aminobutyric acid (RG) attenuates muscle atrophy in an animal model of chronic unpredictable mild stress (CUMS). We observed that CUMS raised the adrenal gland weight and serum adrenocorticotropic hormone (ACTH) and cortisol levels, and these effects were reversed by RG. CUMS also enhanced the expression of the GC receptor (GR) and GC-GR binding in the gastrocnemius muscle, which were attenuated by RG. The expression levels of muscle degradation-related signaling pathways, such as the Klf15, Redd-1, FoxO3a, Atrogin-1, and MuRF1 pathways, were enhanced by CUMS and attenuated by RG. Muscle synthesis-related signaling pathways, such as the IGF-1/AKT/mTOR/s6k/4E-BP1 pathway, were reduced by CUMS and enhanced by RG. Moreover, CUMS raised oxidative stress by enhancing the levels of iNOS and acetylated p53, which are involved in cell cycle arrest, whereas RG attenuated both iNOS and acetylated p53 levels. Cell proliferation in the gastrocnemius muscle was reduced by CUMS and enhanced by RG. The muscle weight, muscle fiber cross-sectional area, and grip strength were reduced by CUMS and enhanced by RG. Therefore, RG attenuated ACTH levels and cortisol-related muscle atrophy in CUMS animals.

Exogenous 8-Hydroxydeoxyguanosine Attenuates PM2.5-Induced Inflammation in Human Bronchial Epithelial Cells by Decreasing NLRP3 Inflammasome Activation.

Particulate matter 2.5 (PM2.5) induces lung injury by increasing the generation of reactive oxygen species (ROS) and inflammation. ROS aggravates NLRP3 inflammasome activation, which activates caspase-1, IL-1ß, and IL-18 and induces pyroptosis; these factors propagate inflammation. In contrast, treatment with exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases RAC1 activity and eventually decreases dinucleotide phosphate oxidase (NOX) and ROS generation. To establish modalities that would mitigate PM2.5-induced lung injury, we evaluated whether 8-OHdG decreased PM2.5-induced ROS generation and NLRP3 inflammasome activation in BEAS-2B cells. CCK-8 and lactate dehydrogenase assays were used to determine the treatment concentration. Fluorescence intensity, Western blotting, enzyme-linked immunosorbent assay, and immunoblotting assays were also performed. Treatment with 80 µg/mL PM2.5 increased ROS generation, RAC1 activity, NOX1 expression, NLRP3 inflammasome (NLRP3, ASC, and caspase-1) activity, and IL-1ß and IL-18 levels in cells; treatment with 10 µg/mL 8-OHdG significantly attenuated these effects. Furthermore, similar results, such as reduced expression of NOX1, NLRP3, ASC, and caspase-1, were observed in PM2.5-treated BEAS-2B cells when treated with an RAC1 inhibitor. These results show that 8-OHdG mitigates ROS generation and NLRP3 inflammation by inhibiting RAC1 activity and NOX1 expression in respiratory cells exposed to PM2.5.

Poly-D,L-Lactic Acid Filler Increases Extracellular Matrix by Modulating Macrophages and Adipose-Derived Stem Cells in Aged Animal Skin.

Poly-D,L-lactic acid (PDLLA) filler corrects soft tissue volume loss by increasing collagen synthesis in the dermis; however, the mechanism is not fully understood. Adipose-derived stem cells (ASCs) are known to attenuate the decrease in fibroblast collagen synthesis that occurs during aging, and nuclear factor (erythroid-derived 2)-like-2 factor (NRF2) increases ASCs survival by inducing M2 macrophage polarization and IL-10 expression. We evaluated the ability of PDLLA to induce collagen synthesis in fibroblasts by modulating macrophages and ASCs in a H2O2-induced cellular senescence model and aged animal skin. PDLLA increased M2 polarization and NRF2 and IL-10 expression in senescence-induced macrophages. Conditioned media from senescent macrophages treated with PDLLA (PDLLA-CMMΦ) reduced senescence and increased proliferation and expression of transforming growth factor-ß (TGF-ß) and fibroblast growth factor (FGF) 2 in senescence-induced ASCs. Conditioned media from senescent ASCs treated with PDLLA-CMMΦ (PDLLA-CMASCs) increased the expression of collagen 1a1 and collagen 3a1 and reduced the expression of NF-κB and MMP2/3/9 in senescence-induced fibroblasts. Injection of PDLLA in aged animal skin resulted in increased expression of NRF2, IL-10, collagen 1a1, and collagen 3a1 and increased ASCs proliferation in aged animal skin. These results suggest that PDLLA increases collagen synthesis by modulating macrophages to increase NRF2 expression, which stimulates ASCs proliferation and secretion of TGF-ß and FGF2. This leads to increased collagen synthesis, which can attenuate aging-induced soft tissue volume loss.

Poly-D,L-Lactic Acid Stimulates Angiogenesis and Collagen Synthesis in Aged Animal Skin.

Angiogenesis promotes rejuvenation in multiple organs, including the skin. Heat shock protein 90 (HSP90), hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF) are proangiogenic factors that stimulate the activities of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal-regulated kinase 1/2 (ERK1/2). Poly-D,L-lactic acid (PDLLA), polynucleotide (PN), and calcium hydroxyapatite (CaHA) are dermal fillers that stimulate the synthesis of dermal collagen. However, it is not yet known whether these compounds promote angiogenesis, which leads to skin rejuvenation. Here, we evaluated whether PDLLA, PN, and CaHA stimulate angiogenesis and skin rejuvenation using H2O2-treated senescent macrophages and endothelial cells as an in vitro model for skin aging, and we used young and aged C57BL/6 mice as an in vivo model. Angiogenesis was evaluated via endothelial cell migration length, proliferation, and tube formation after conditioned media (CM) from senescent macrophages was treated with PDLLA, PN, or CaHA. Western blot showed decreased expression levels of HSP90, HIF-1α, and VEGF in senescent macrophages, but higher expression levels of these factors were found after treatment with PDLLA, PN, or CaHA. In addition, after exposure to CM from senescent macrophages treated with PDLLA, PN, or CaHA, senescent endothelial cells expressed higher levels of VEGF receptor 2 (VEGFR2), PI3K, phosphorylated AKT (pAKT), and phosphorylated ERK1/2 (pERK1/2) and demonstrated greater capacities for cell migration, cell proliferation, and tube formation. Based on the levels of 4-hydroxy-2-nonenal, the oxidative stress level was lower in the skin of aged mice injected with PDLLA, PN, or CaHA, while the tumor growth factor (TGF)-ß1, TGF-ß2, and TGF-ß3 expression levels; the density of collagen fibers; and the skin elasticity were higher in the skin of aged mice injected with PDLLA, PN, or CaHA. These effects were greater in PDLLA than in PN or CaHA. In conclusion, our results are consistent with the hypothesis that PDLLA stimulates angiogenesis, leading to the rejuvenation of aged skin. Our study is the first to show that PDLLA, PN, or CaHA can result in angiogenesis in the aged skin, possibly by increasing the levels of HSP90, HIF-1α, and VEGF and increasing collagen synthesis.

Poly-L-Lactic Acid Fillers Improved Dermal Collagen Synthesis by Modulating M2 Macrophage Polarization in Aged Animal Skin.

Poly-L-lactic acid (PLLA) fillers correct cutaneous volume loss by stimulating fibroblasts to synthesize collagen and by augmenting the volume. PLLA triggers the macrophage-induced activation of fibroblasts that secrete transforming growth factor-ß (TGF-ß). However, whether M2 macrophage polarization is involved in PLLA-induced collagen synthesis via fibroblast activation in aged skin is not known. Therefore, we evaluated the effect of PLLA on dermal collagen synthesis via M2 polarization in an H2O2-induced cellular senescence model and aged animal skin. H2O2-treated macrophages had increased expression levels of the M1 marker CD80 and decreased expression levels of the M2 marker CD163, which were reversed by PLLA. The expression levels of interleukin (IL)-4 and IL-13, which mediate M2 polarization, were decreased in H2O2-treated macrophages and increased upon the PLLA treatment. CD163, IL-4, and IL-13 expression levels were decreased in aged skin, but increased after the PLLA treatment. The expression levels of TGF-ß, pSMAD2/SMAD2, connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), collagen type 1A1 (COL1A1), and COL3A1 were also decreased in aged skin, but increased after the PLLA treatment. Moreover, PLLA upregulated phosphatidylinositol 3-kinase p85α (PI3-kinase p85α)/protein kinase B (AKT) signaling, leading to fibroblast proliferation. PLLA decreased the expression of matrix metalloproteinase (MMP) 2 and MMP3, which destroy collagen and elastin fibers in aged skin. The amount of collagen and elastin fibers in aged skin increased following the PLLA treatment. In conclusion, PLLA causes M2 polarization by increasing IL-4 and IL-13 levels and upregulating TGF-ß expression and collagen synthesis in aged skin.

Radiofrequency Irradiation Attenuated UVB-Induced Skin Pigmentation by Modulating ATP Release and CD39 Expression.

Hyperpigmentation stimulated by ultraviolet (UV)-induced melanin overproduction causes various cosmetic problems. UV radiation's activation of the cyclic adenosine monophosphate (cAMP)-mediated cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein (CREB)/microphthalmia-associated transcription factor (MITF) pathway is the main pathway for melanogenesis. However, the secretion of adenosine triphosphate (ATP) from keratinocytes due to UV radiation also leads to melanogenesis. Adenosine, converted from ATP by CD39 and CD73, can activate adenylate cyclase (AC) activity and increase intracellular cAMP expression. cAMP-mediated PKA activation results in dynamic mitochondrial changes that affect melanogenesis via ERK. We evaluated whether radiofrequency (RF) irradiation could decrease ATP release from keratinocytes and suppress the expression of CD39, CD73, and A2A/A2B adenosine receptors (ARs) and the activity of AC and downregulate the PKA/CREB/MITF pathway, which would eventually decrease melanogenesis in vitro in UV-irradiated cells and animal skin. Our results indicate that RF decreased ATP release from UVB-irradiated keratinocytes. When conditioned media (CM) from UVB-irradiated keratinocytes (CM-UVB) were administered to melanocytes, the expressions of CD39, CD73, A2A/A2BARs, cAMP, and PKA increased. However, the expression of these factors decreased when CM from UVB and RF-irradiated keratinocytes (CM-UVB/RF) was administered to melanocytes. The phosphorylation of DRP1 at Ser637, which inhibits mitochondrial fission, increased in UVB-irradiated animal skin and was decreased by RF irradiation. The expression of ERK1/2, which can degrade MITF, was increased using RF treatment in UVB-irradiated animal skin. Tyrosinase activity and melanin levels in melanocytes increased following CM-UVB administration, and these increases were reversed after CD39 silencing. Tyrosinase activity and melanin levels in melanocytes were decreased by CM-UVB/RF irradiation. In conclusion, RF irradiation decreased ATP release from keratinocytes and the expressions of CD39, CD73, and A2A/A2BARs, which decreased AC activity in melanocytes. RF irradiation downregulated the cAMP-mediated PKA/CREB/MITF pathway and tyrosinase activity, and these inhibitory effects can be mediated via CD39 inhibition.

The Extracellular Matrix Vitalizer RATM Increased Skin Elasticity by Modulating Mitochondrial Function in Aged Animal Skin.

Oxidative stress-induced cellular senescence and mitochondrial dysfunction result in skin aging by increasing ECM levels-degrading proteins such as MMPs, and decreasing collagen synthesis. MMPs also destroy the basement membrane, which is involved in skin elasticity. The extracellular matrix vitalizer RATM (RA) contains various antioxidants and sodium hyaluronate, which lead to skin rejuvenation. We evaluated whether RA decreases oxidative stress and mitochondrial dysfunction, eventually increasing skin elasticity in aged animals. Oxidative stress was assessed by assaying NADPH oxidase activity, which is involved in ROS generation, and the expression of SOD, which removes ROS. NADPH oxidase activity was increased in aged skin and decreased by RA injection. SOD expression was decreased in aged skin and increased by RA injection. Damage to mitochondrial DNA and mitochondrial fusion markers was increased in aged skin and decreased by RA. The levels of mitochondrial biogenesis markers and fission markers were decreased in aged skin and increased by RA. The levels of NF-κB/AP-1 and MMP1/2/3/9 were increased in aged skin and decreased by RA. The levels of TGF-ß, CTGF, and collagen I/III were decreased in aged skin and increased by RA. The expression of laminin and nidogen and basement membrane density were decreased in aged skin and increased by RA. RA increased collagen fiber accumulation and elasticity in aged skin. In conclusion, RA improves skin rejuvenation by decreasing oxidative stress and mitochondrial dysfunction in aged skin.