Age-associated reduction of cell spreading induces mitochondrial DNA common deletion by oxidative stress in human skin dermal fibroblasts: implication for human skin connective tissue aging

Abstract Background Reduced cell spreading is a prominent feature of aged dermal fibroblasts in human skin in vivo. Mitochondrial DNA (mtDNA) common deletion has been reported to play a role in the human aging process, however the relationship between age-related reduced cell spreading and mtDNA common deletion has not yet been reported. Results To examine mtDNA common deletion in the dermis of aged human skin, the epidermis was removed from full-thickness human skin samples using cryostat. mtDNA common deletion was significantly elevated in the dermis of both naturally aged and photoaged human skin in vivo. To examine the relationship between age-related reduced cell spreading and mtDNA common deletion, we modulated the shape of dermal fibroblasts by disrupting the actin cytoskeleton. Reduced cell spreading was associated with a higher level of mtDNA common deletion and was also accompanied by elevated levels of endogenous reactive oxygen species (ROS). Boosting cellular antioxidant capacity by using antioxidants was found to be protective against mtDNA common deletion associated with reduced cell spreading. Conclusion mtDNA common deletion is highly prevalent in the dermis of both naturally aged and photoaged human skin in vivo. mtDNA common deletion in response to reduced cell spreading is mediated, at least in part, by elevated oxidative stress in human dermal fibroblasts. These data extend current understanding of the mitochondrial theory of aging by identifying the connection between mtDNA common deletion and age-related reduction of cell spreading.http://deepblue.lib.umich.edu/bitstream/2027.42/112063/1/12929_2015_Article_167.pd

Smad3-dependent CCN2 mediates fibronectin expression in human skin dermal fibroblasts.

The potential involvement of connective tissue growth factor (CCN2/CTGF) in extracellular matrix (ECM) production is recognized. However, the role CCN2 in fibronectin (FN) gene expression has remained incompletely understood and even controversial. Here we report that CCN2 is absolutely necessary for FN expression in primary human skin dermal fibroblasts, the major cells responsible for ECM production in skin. Gain- and loss-of-function approaches demonstrate that CCN2 is an essential component of FN expression in both basal and stimulation by TGF-β signaling, the major regulator of FN expression. CCN2 is significantly induced by Smad3, a critical mediator of TGF-β signaling. CCN2 acts as a downstream mediator of TGF-β/Smad signaling and acting synergistically with TGF-β to regulate FN gene expression. Finally, we observed that CCN2 and FN predominantly expressed in the dermis of normal human skin, stromal tissues of skin squamous cell carcinoma (SCC), and simultaneously induced in wounded human skin in vivo. These findings provide evidence that CCN2 is responsible for mediating the stimulatory effects of TGF-β/Smad on FN gene expression, and attenuation of CCN2 expression may benefit to reduce fibrotic ECM microenvironment in disease skin

CCN2 and FN expression are primarily regulated by Smad3 in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with the indicated siRNAs (20nM). Total RNA and whole cell extract were prepared 48 hours after transfection. mRNA levels were quantified by real-time RT-PCR. Protein levels were determined by ProteinSimple capillary electrophoresis immunoassay (B, C) and Western blots (E). mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative digital images (B, C) and Western blots (E). (A) Smad2 and Smad3 mRNA levels. (B) CCN2, FN, and Smad3 protein levels. (C) CCN2, FN, and Smad2 protein levels. (D) CCN2 and FN mRNA levels. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. (E) CCN2, FN, Smad2, and Smad3 protein levels. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. Data are expressed as mean±SEM, N = 3, *p<0.05 vs control.</p

FN expression is regulated by CCN2 in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with non-specific control siRNA or CCN2 siRNAs (20nM) (A, B, C), or control vector (pCDNA3.1, 2μg) or increasing amounts of CCN2 vector (0.5, 1, and 2μg) (D, E). Total RNA and whole cell extract were prepared 48 hours after transfection. (A) CCN2 mRNA levels. (B) FN mRNA levels. (C) CCN and FN Protein levels. (D) FN mRNA levels. (E) CCN and FN protein levels. mRNA levels were quantified by real-time RT-PCR. Protein levels were determined by ProteinSimple capillary electrophoresis immunoassay (C) and Western analysis (E). mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative digital images (C) and Western blots (E). Data are expressed as mean±SEM, N = 3–5, *p<0.05.</p

CCN2 and FN are primarily expressed in the dermis of normal human skin, stromal tissues of skin SCC, and wounded human skin.

<p>(A, B) Epidermis and dermis were captured by LCM. Total RNA was extracted from captured tissue, and mRNA levels were quantified by real-time RT-PCR. CCN2 (A) and FN (B) mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Data are relative levels to 36B4 (mean±SEM), N = 6, *p<0.05. (C) Double immunostaining for CCN2 and FN in normal human skin. OCT-embedded normal human skin sections (7μm) were co-immunofluorescence stained with CCN2 and FN. Representative of six individuals. Bar = 50μm. (D) Expression of CCN2 and FN in the stromal tissues of SCC was determined by immunohistology. Arrow heads indicate tumor islands. Representative of five SCC. Bar = 100μm. (E) Double immunostaining for CCN2 and FN. Representative of six individuals. Bar = 50μm. (F) Partial thickness wounds were made in forearm skin of healthy adult individuals by CO₂ laser (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173191#sec002" target="_blank">Methods</a> for details). Skin samples were obtained at indicated times, and mRNA levels were quantified by real-time RT-PCR. CCN2 and FN mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Mean±SEM, N = 6, *p<0.05 vs control.</p

CCN2 functions primary mediator in Smad3-dependent expression of FN in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with the indicated siRNAs (20nM) and vectors (1μg). Total RNA and whole cell extract were prepared 48 hours after transfection. mRNA and protein levels were quantified by real-time RT-PCR and Western analysis, respectively. mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative Western blots. (A, C) CCN2 and FN mRNA levels. (B, D) CCN2, FN, Smad2, and Smad3 protein levels. (E) The ability of CCN2 to regulate FN expression is dependent on intact TGF-β signaling. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. Data are expressed as mean±SEM, N = 3, *p<0.05 vs control.</p

Cost-Effectiveness of Mobile Health–Based Integrated Care for Atrial Fibrillation: Model Development and Data Analysis

BackgroundMobile health (mHealth) technology is increasingly used in disease management. Using mHealth tools to integrate and streamline care has improved clinical outcomes of patients with atrial fibrillation (AF). ObjectiveThe aim of this study was to investigate the potential clinical and health economic outcomes of mHealth-based integrated care for AF from the perspective of a public health care provider in China. MethodsA Markov model was designed to compare outcomes of mHealth-based care and usual care in a hypothetical cohort of patients with AF in China. The time horizon was 30 years with monthly cycles. Model outcomes measured were direct medical cost, quality-adjusted life years (QALYs), and incremental cost-effectiveness ratio (ICER). Sensitivity analyses were performed to examine the robustness of the base-case results. ResultsIn the base-case analysis, mHealth-based care gained higher QALYs of 0.0730 with an incurred cost of US $1090. Using US$33,438 per QALY (three times the gross domestic product) as the willingness-to-pay threshold, mHealth-based care was cost-effective, with an ICER of US $14,936 per QALY. In one-way sensitivity analysis, no influential factor with a threshold value was identified. In probabilistic sensitivity analysis, mHealth-based care was accepted as cost-effective in 92.33% of 10,000 iterations. ConclusionsThis study assessed the expected cost-effectiveness of applying mHealth-based integrated care for AF according to a model-based health economic evaluation. The exploration suggested the potential cost-effective use of mHealth apps in streamlining and integrating care via the Atrial fibrillation Better Care (ABC) pathway for AF in China. Future economic evaluation alongside randomized clinical trials is highly warranted to verify the suggestion and investigate affecting factors such as geographical variations in patient characteristics, identification of subgroups, and constraints on local implementation

Additional file 1 of Challenge of ending TB in China: tuberculosis control in primary healthcare sectors under integrated TB control model–a systematic review and meta-analysis

Supplementary Material