ABSTRACT
The transforming growth factor (TGF)-ß/Smad signal transduction pathway is closely associated with hypertrophic scar (HS) formation. Smad interacting protein 1 (SIP1) is a cytoplasmic protein that efficiently regulates Smad2-/3-dependent signaling within the TGF-ß1 pathway. SIP1 influences collagen synthesis in the HS through a heretofore unknown mechanism. This study investigated the role of the SIP1-mediated TGF-ß1/Smad signaling pathway in extracellular matrix (ECM) protein production and hypertrophic scarring. SIP1 expression was markedly lower in HS vs. normal skin (NS) tissue, and α-smooth muscle actin (α-SMA) content and collagen I/III (Col I/III) synthesis were inversely correlated with SIP1 expression. Furthermore, SIP1 inhibited Smad2/3 phosphorylation in vitro, and improved the collagen-based architecture of the scar while reducing collagen expression and overall scar formation in a rabbit ear model of HS. Based on these findings, we propose that SIP1 acts as a molecular modulator capable of altering Smad2-/3-facilitated signaling through the control of Smad phosphorylation, thus inhibiting α-SMA and collagen upregulation in fibroblasts and, ultimately, HS formation. The low SIP1 content in scar tissue also suggests that SIP1 (and positive regulation thereof) is a prospective target for selective HS drug therapy.
Subject(s)
Cicatrix, Hypertrophic/metabolism , Collagen Type I/metabolism , Signal Transduction , Transforming Growth Factor beta1/metabolism , Zinc Finger E-box Binding Homeobox 2/metabolism , Actins/genetics , Actins/metabolism , Animals , Cells, Cultured , Cicatrix, Hypertrophic/genetics , Collagen Type I/genetics , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression/drug effects , Humans , Phosphorylation/drug effects , Rabbits , Skin/metabolism , Skin/pathology , Smad Proteins/metabolism , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/pharmacology , Zinc Finger E-box Binding Homeobox 2/geneticsABSTRACT
We measured gene expression of paracrine regulators involved in adipocyte differentiation within the stromovascular fraction of abdominal subcutaneous adipose tissue from obese individuals with (n=30) and without (n=18) type 2 diabetes mellitus (T2DM). Despite similar adiposity by design, subjects with T2DM had larger adipocytes (0.92+/-0.28 vs. 0.75+/-0.17 microl, p<0.05) than controls. Gene expression of the adipogenic marker aP2 was lower (0.35+/-0.16 vs. 0.58+/-0.27 arbitrary units, p<0.05) whereas the expression of matricellular peptidase, MMP2 was higher (1.65+/-0.17 vs. 1.27+/-0.21, p=0.02) in T2DM vs. controls. The gene expression levels between the aP2 and MMP2 were inversely correlated (r=-0.32, p=0.03). We conclude that early steps of adipogenesis may be impaired in T2DM independently of obesity due, in part, to an upregulation of the MMP2 transcription.
Subject(s)
Adipocytes/metabolism , Adipocytes/pathology , Adipogenesis , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/pathology , Matrix Metalloproteinase 2/metabolism , Obesity/enzymology , Obesity/pathology , Adult , Diabetes Mellitus, Type 2/complications , Female , Humans , Male , Obesity/complicationsABSTRACT
Physical activity (PA) is known to decline with age; however, there is a paucity of data on activity in persons who are in their nineties and beyond. We used objective and reliable methods to measure PA in nonagenarians (>or=90 yr; n=98) and hypothesized that activity would be similar to that of aged (60-74 yr; n=58) subjects but less than in young (20-34 yr; n=53) volunteers. Total energy expenditure (TEE) was measured by doubly labeled water over 14 days and resting metabolic rate (RMR) by indirect calorimetry. Measures of PA included activity energy expenditure adjusted for body composition, TEE adjusted for RMR, physical activity level (PAL), and activity over 14 days by accelerometry expressed as average daily durations of light and moderate activity. RMR and TEE were lower with increasing age group (P<0.01); however, RMR was not different between aged and nonagenarian subjects after adjusting for fat-free mass, fat mass, and sex. Nonagenarians had a lower PAL and were more sedentary than the aged and young groups (P<0.01); however, the nonagenarians who were more active on a daily basis walked further during a timed test, indicating higher physical functionality. For all measures of activity, no differences were found between young and aged volunteers. PA was markedly lower in nonagenarians compared with young and aged adults. Interestingly, PA was similar between young volunteers and those who were in their 60s and 70s, likely due to the sedentary nature of our society, particularly in young adults.
Subject(s)
Aged, 80 and over/physiology , Aging/physiology , Motor Activity/physiology , Adolescent , Adult , Aged , Anthropometry , Body Composition/physiology , Female , Humans , Life Style , Male , Metabolism/physiology , Middle Aged , Walking/physiologyABSTRACT
OBJECTIVE: Our objective was to test the safety and metabolic effects of a novel beta(3)-adrenoreceptor agonist (TAK-677) in humans. DESIGN, SETTING, AND PARTICIPANTS: Sixty-five obese (body mass index = 33.9 +/- 2.1 kg/m2, mean +/- se) men and women (31.4 +/- 0.9 yr) participated in a double-blind placebo-controlled study at an institutional research center. INTERVENTION: Participants were randomized to 0.1 mg TAK-677 twice daily (BID) (n = 21), 0.5 mg TAK-677 BID (n = 22), or placebo BID (n = 22) for 29 d. OUTCOMES: Drug safety, 24-h respiratory quotient (RQ), 24-h energy expenditure (EE), body composition, fat distribution, and fasting plasma concentration of substrates and hormones were assessed. An acute-response study was also conducted. RESULTS: The drug was well tolerated by all participants; however, heart rate was elevated (9 +/- 2 beats per minute) with the 0.5-mg BID dose. After 28 d of treatment and when compared with placebo, there was no change in 24-h RQ with either 0.1-mg BID (P = 0.1) or 0.5-mg BID (P = 1.0) doses of TAK-677. However, TAK, 0.5 mg BID, resulted in a small increase in 24-h EE that was significantly different from placebo [change from baseline, 13 +/- 17 (0.5 mg BID) vs.-39 +/- 18 (placebo) kcal/d, P < 0.05]. Changes in weight, fat-free mass, and abdominal fat depots (visceral or sc) were not different between the three groups, nor were changes in fasting insulin, free fatty acid, or glucose concentrations. CONCLUSION: TAK-677 has no effect on 24-h RQ or fat oxidation but does slightly increase 24-h EE at the highest dose (0.5 mg BID). The acute studies showed large interindividual variability in plasma concentrations of TAK-677 indicating some possible problems with bioavailability and therefore efficacy.
Subject(s)
Acetates/administration & dosage , Adrenergic beta-3 Receptor Antagonists , Adrenergic beta-Antagonists/administration & dosage , Energy Metabolism/drug effects , Indoles/administration & dosage , Obesity/drug therapy , Obesity/metabolism , Abdominal Fat/drug effects , Adult , Body Weight/drug effects , Circadian Rhythm , Double-Blind Method , Fasting , Female , Homeostasis/drug effects , Humans , Longitudinal Studies , Male , Placebos , Treatment FailureABSTRACT
BACKGROUND: The aging process occurs at variable rates both among and within species and may be related to the variability in oxygen consumption and free radical production impacting oxidative stress. The current study was designed to test whether nonagenarians have a relatively low metabolic rate and whether it is associated with low levels of oxidative stress relative to age. METHODS: Resting metabolic rate (RMR) and markers of oxidative stress to lipids, proteins, and DNA were measured in three groups of individuals aged 20-34 (n=47), 60-74 (n=49), and>or=90 years (n=74). RESULTS: RMR, adjusted for fat-free mass, fat mass, and sex, was lower in both older groups when compared to the young group (pSubject(s)
Aging/physiology
, Basal Metabolism/physiology
, Oxidative Stress/physiology
, Adult
, Aged
, Aged, 80 and over
, Body Composition
, DNA/metabolism
, Female
, Humans
, Lipid Metabolism
, Male
, Middle Aged
, Proteins/metabolism
ABSTRACT
OBJECTIVE: To evaluate the effects and mechanism of indoleamine 2, 3-dioxygenase (IDO) modified rat bone marrow mesenchymal stem cells (BMSCs) in composite tissue allograft rejection. METHODS: BMSCs isolated from Brown Norway (BN) rats (aged, 4-6 weeks) were infected by IDO [green fluorescent protein (GFP)]-lentivirus. The high expression target gene and biological activity cell line (IDO-BMSCs) were screened. IDO mRNA and protein expressions were detected by RT-PCR and Western blot. The biological activity of IDO in supernatant was detected by measuring the amount of kynurenine generation. In mixed lymphocyte reaction system, different numbers of IDO-BMSCs mixed with responding cells (peripheral blood mononuclear cell isolated from 4-6-week-old LEWIS rats, as recipient) and stimulating cells (peripheral blood mononuclear cell isolated from BN rats, as donor), with the cells ratios of 1:5:5, 1:10:10, 1:50:50, and 1:100:100 (as experimental groups 1, 2, 3, and 4, respectively). Each reaction system was blocked by 1 mmol/L 1-methyl-tryptophan (1-MT) (IDO specific inhibitor): IDO-BMSCs mixed with responding cells (1:5) as the negative control group, responding cells mixed with stimulating cells (1:1) as positive control group; and IDO-BMSCs were cultured in RPM11640 medium alone as blank control group. MTT assay was used to detect the T lymphocytes proliferation at 5 days. Furthermore, GFP-BMSCs (group A), IDO-BMSCs (group B), and normal saline (group C) were infused via the tail vein of allogeneic limb transplantation rats, and graft survival time and rejection were observed in each group. RESULTS: The IDO expression of BMSCs after genetic modification was higher than that before genetic modification. IDO-BMSCs could significantly improved kynurenine concentration in culture medium supernatant when compared with GFP-BMSCs (P < 0.05). Before adding 1-MT, with the ratio of IDO-BMSCs to responding cells decreased, T lymphocytes proliferation rate increased in experimental groups 1, 2, and 3, showing significant differences between groups (P < 0.05); there was no significant difference between experimental group 4 and the positive control group (F > 0.05). After adding 1-MT, T lymphocytes proliferation rate was significantly higher than that before adding 1-MT in the other experimental groups (P < 0.05) except experimental group 4 (F > 0.05). In vivo, IDO-BMSCs could promote colonization in allograft, inhibit transplantation rejection, and prolong survival time of composite tissue allograft; the survival time of composite tissue allograft was (11.5 ± 0.6) days in group A, (14.5 ± 0.8) days in group B, and (9.0 ± 0.3) days in group C, and it was significantly longer in group B than in groups A and C, and in group A than in group C (P < 0.05). CONCLUSION: JDO-BMSCs can promote the survival of allogeneic composite tissue grafts in rats, and its mechanism may involve in inhibition of T lymphocytes proliferation and promotion their own colonization in allograft.
Subject(s)
Bone Marrow Cells/enzymology , Composite Tissue Allografts , Graft Rejection/prevention & control , Graft Survival/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Animals , Bone Marrow Cells/drug effects , Genetic Therapy/methods , Graft Rejection/enzymology , Graft Rejection/genetics , Graft Survival/drug effects , Graft Survival/immunology , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Leukocytes, Mononuclear , Mesenchymal Stem Cells , Rats , Rats, Inbred BN , Transplantation, HomologousABSTRACT
OBJECTIVE: To observe the level of intracellular reactive oxygen species (ROS) in rats with severe burn and pulmonary microvascular endothelial cells (PMVECs) treated with serum of rat with burn injury, and to investigate the relationship between ROS and apoptosis of PMVECs. METHODS: (1) Twenty-four SD rats were divided into sham injury group ( n = 3) and burn group (n = 21) according to the random number table (the same grouping method below). Rats in burn group were inflicted with 30% TBSA full-thickness scald on the back, and rats in sham injury group were sham injured. Blood samples were collected from abdominal aorta at post injury hour 6, 12, 24, 36, 48, 60, 72 respectively from 3 rats of burn group. The serum content of ROS was assayed by ELISA. The same determination was performed in rats of sham injury group. (2) Five rats were subjected to scald injury as above, and burn serum was prepared 24 hours after injury. Another 5 rats without receiving any treatment were used to prepare normal serum. (3) Marginal pulmonary tissue was harvested from 20 SD young rats. Cells were cultured with tissue block method and indentified with immunohistochemical staining. The third passage of PMVECs in logarithmic phase were inoculated in 6-well plates and 12-well plates. PMVECs in both plates were divided into 4 groups: normal serum group, burn serum group, normal serum + MnTBAP group, and burn serum + MnTBAP group, with 3 wells in each group. Cells in the former 2 groups were respectively cultured with special nutrient solution of endothelial cells without serum added with 15% healthy rat serum or 15% burn rat serum. Cells in the latter 2 groups were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 µmol/L MnTBAP in the nutrient solution. After being cultured for 24 h, the content of ROS in PMVECs in 6-well plates was detected with flow cytometry. The apoptosis of PMVECs in 12-well plates was observed with acridine orange-ethidium bromide staining, and the apoptosis rate was calculated. Data were processed with one-way analysis of variance and LSD-t test. RESULTS: (1) The serum contents of ROS in rats of burn group were respectively (187 ± 21), (235 ± 22), (231 ± 25), (291 ± 20), (315 ±23) nmol/mL at post injury hour 24, 36, 48, 60, 72, which were significantly higher than that in sham injury group [(141 ± 19) nmol/mL, with t values respectively 7. 86, 9. 57, 13. 87, 14.98, 18.40, P values below 0.01]. (2) Primary cells grew slowly and showed a cobblestone appearance. After passages, cells grew with orderly distribution. The positive rate of coagulation factor VIII of cells was (96 ± 5)% , and thus they were identified as PMVECs. (3) In normal serum group, burn serum group, normal serum + MnTBAP group, and burn serum + MnTBAP group, the contents of ROS in PMVECs were respectively 798 ± 40, 1 294 ± 84, 763 ± 59, 926 ± 42 ( F =93.01, P <0.01), and the apoptosis rates of PMVECs were respectively (6.2 ± 1.3)%, (57.3 ± 6. 7)%, (3.7 ± 0. 8)%, (28.7 ± 5. 7)% (F = 224.50, P <0.01) after being cultured for 24 h. Compared with those of normal serum group, the content of ROS and apoptosis rate of PMVECs in burn serum group increased significantly (with t values respectively 10.40 and 49.06, P values below 0.01). The content of ROS and apoptosis rate of PMVECs in burn serum + MnTBAP group were significantly lower than those in burn serum group (with t values respectively 7.48 and 23.94, P values below 0.01). CONCLUSIONS: Serum content of ROS was increased in severely burned rats. Burn rat serum stimulation on PMVECs can lead to the increase of the intracellular ROS and induce apoptosis. However application of MnTBAP can scavenge ROS and reduce the apoptosis induced by burn rat serum.
Subject(s)
Burns/blood , Endothelial Cells/pathology , Reactive Oxygen Species/blood , Serum/metabolism , Animals , Apoptosis , Burns/therapy , Enzyme-Linked Immunosorbent Assay , Lung/blood supply , Oxygen , RatsABSTRACT
OBJECTIVE: To investigate the effects of mouse adipose-derived stem cell conditioned medium (ADSC-CM) on apoptosis of keratinocytes (human epithelial cell line HaCaT) induced by thermal injury in vitro. METHODS: (1) Adipose-derived stem cells (ADSCs) from inguinal adipose tissue of 5 healthy BALB/c mice were isolated, cultured, and purified by collagenase digestion in vitro. The 3rd passage of cells were collected for morphologic observation, detection of expressions of surface markers CD31, CD34, CD45, CD90, and CD105 with flow cytometer, and identification of adipogenic and osteogenic differentiation. (2) HaCaT cells were incubated in water at 51.5 °C for 35 seconds to reproduce thermal injury model, and then the apoptosis rate was detected immediately after injury by flow cytometer. (3) Thermally injured HaCaT cells were divided into routine culture group (RC, cultured with DMEM containing 10% FBS), serum-free group (cultured with serum-free DMEM), 50%ADSC-CM group (cultured with DMEM containing 50%ADSC-CM), and 100%ADSC-CM group (cultured with 100%ADSC-CM) according to the random number table. After 24 hours, apoptosis of HaCaT cells was observed by acridine orange-ethidium bromide (AO-EB) staining; apoptotic rate was determined by flow cytometer; the mRNA and protein levels of Bcl-2 and caspase-3 were respectively determined by real-time fluorescent quantitative RT-PCR technique and Western blotting (protein level was denoted as gray value); the cell cycles were determined by flow cytometer. All above experiments were repeated for 3 times. Data were processed with one-way analysis of variance and LSD- t test. RESULTS: (1) The 3rd passage of cells proliferated well showing fusiform shape similar to fibroblasts. The positive expression rates of CD31, CD34, and CD45 were less than 10.0%, while those of CD90 and CD105 were above 90.0%. The cells could differentiate into adipocytes and osteoblasts. They were identified as ADSCs. (2) Immediately after injury, apoptotic rate of HaCaT cell was (9.8 ± 0.4)%. (3) The number of apoptotic cells was significantly higher in serum-free group than in the other three groups with AO-EB staining. The apoptotic rate of serum-free group [(8.1 ± 1.2)%] was significantly higher than that of 50%ADSC-CM group [(6.0 ± 0.8)%], group RC [(4.6 ± 0.8)%], or 100%ADSC-CM group [(3.1 ± 0.4)%], with t values respectively 3.02, 4.96, 6.60, P values below 0.01. There was no statistically significant difference in apoptotic rate between group RC and 100% ADSC-CM group (t = 1.50, P > 0.05), while statistically significant difference was found between 100% ADSC-CM group and 50%ADSC-CM group (t = 10.21, P < 0.01). (4) The mRNA level of Bcl-2 of serum-free group (0.34 ± 0.08) was significantly lower than that of group RC, 50%ADSC-CM group, and 100%ADSC-CM group (0.98 ± 0.04, 0.77 ± 0.05, 1.06 ± 0.04, with t values respectively 12.87, 8.07, 14.11, P values below 0.01). Compared with that of 100%ADSC-CM group, the mRNA level of Bcl-2 of group RC was slightly decreased (t = 0.08, P > 0.05) and that of 50%ADSC-CM group was significantly decreased (t = 8.08, P < 0.01). (5) The mRNA level of caspase-3 of serum-free group (1.15 ± 0.05) was obviously higher than that of 50%ADSC-CM group (0.72 ± 0.11), group RC (0.41 ± 0.03), or 100%ADSC-CM group (0.38 ± 0.11), with t values respectively 6.93, 13.97, 22.79, P values below 0.01. Compared with 100%ADSC-CM group, the mRNA level of caspase-3 was slightly increased in group RC (t = 0.05, P > 0.05) and significantly increased in 50%ADSC-CM group (t = 4.77, P < 0.01). (6) The protein level of Bcl-2 was significantly lower in serum-free group (0.93 ± 0.04) than in group RC, 50%ADSC-CM group, and 100%ADSC-CM group (1.74 ± 0.06, 1.32 ± 0.05, 1.90 ± 0.04, with t values respectively 20.45, 11.15, 31.38, P values below 0.01). Compared with that of 100%ADSC-CM group, the protein level of Bcl-2 of group RC was slightly decreased (t = 1.33, P > 0.05), but that of 50%ADSC-CM group was obviously decreased (t = 17.30, P < 0.01). (7) The protein level of caspase-3 was obviously higher in serum-free group (0.63 ± 0.08) than in 50%ADSC-CM group, group RC, and 100%ADSC-CM group (0.46 ± 0.03, 0.29 ± 0.08, 0.21 ± 0.03, with t values respectively 3.28, 5.05, 8.46, P values below 0.01). Compared with that of 100%ADSC-CM group, the protein level of caspase-3 of group RC was slightly increased (t = 0.08, P > 0.05), but that of 50%ADSC-CM group was significantly increased (t = 3.52, P < 0.05). (8) Compared with that of serum-free group, the percentage of cells in G2/M phase of each of the other 3 groups was significantly decreased (with t values respectively 6.88, 4.08, 7.28, P < 0.05 or P < 0.01). Compared with that in serum-free group, the percentage of cells in S phase was significantly increased in group RC and 100% ADSC-CM group (with t values respectively 2.67 and 2.40, P values below 0.05). There was no statistically significant difference in the percentage of cells in G0/G1 phase among all groups (F = 0.70, P > 0.05). CONCLUSIONS: 100% xenogeneic ADSC-CM can suppress apoptosis of HaCaT cells induced by thermal injury through regulating the expression of Bcl-2 and caspase-3, and accelerate cell cycle progression by ameliorating the retardation of cell growth in G2/M phase, and all these effects may give rise to some potential in the treatment of burn wounds at early stage.
Subject(s)
Apoptosis/physiology , Burns , Caspase 3/metabolism , Culture Media, Conditioned , Keratinocytes/metabolism , Stem Cells , Adipocytes , Adipose Tissue , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Fibroblasts , Humans , In Vitro Techniques , Keratinocytes/physiology , Mice , Mice, Inbred BALB C , Real-Time Polymerase Chain ReactionABSTRACT
BACKGROUND: The hypertrophic scar (HS) is a serious fibrotic skin condition and a major clinical problem. Interleukin-10 (IL-10) has been identified as a prospective scar-improving compound based on preclinical trials. Our previous work showed that IL-10 has anti-fibrotic effects in transforming growth factor (TGF)-ß1-stimulated fibroblasts, as well as potential therapeutic benefits for the prevention and reduction of scar formation. However, relatively little is known about the mechanisms underlying IL-10-mediated anti-fibrotic and scar-improvement actions. OBJECTIVE: To explore the expression of the IL-10 receptor in human HS tissue and primary HS fibroblasts (HSFs), and the molecular mechanisms contributing to the anti-fibrotic and scar-improvement capabilities of IL-10. METHODS: Expression of the IL-10 receptor was assessed in HS tissue and HSFs by immunohistochemistry, immunofluorescence microscopy, and polymerase chain reaction analysis. Primary HSFs were treated with IL-10, a specific phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) or a function-blocking antibody against the IL-10 receptor (IL-10RB). Next, Western blot analysis was used to evaluate changes in the phosphorylation status of AKT and signal transducers and activators of transcription (STAT) 3, as well as the expression levels of fibrosis-related proteins. RESULTS: HS tissue and primary HSFs were characterized by expression of the IL-10 receptor and by high expression of fibrotic markers relative to normal controls. Primary HSFs expressed the IL-10 receptor, while IL-10 induced AKT and STAT3 phosphorylation in these cells. In addition, LY294002 blocked AKT and STAT phosphorylation, and also up-regulated expression levels of type I and type III collagen (Col 1 and Col 3) and alpha-smooth muscle actin (α-SMA) in IL-10-treated cells. Similarly, IL-10RB reduced STAT3/AKT phosphorylation and blocked the IL-10-mediated mitigation of fibrosis in HSFs. CONCLUSION: IL-10 apparently inhibits fibrosis by activating AKT and STAT3 phosphorylation downstream of the IL-10 receptor, and by facilitating crosstalk between the PI3K/AKT and STAT3 signal transduction pathways.
Subject(s)
Cicatrix, Hypertrophic/drug therapy , Fibroblasts/drug effects , Interleukin-10/pharmacology , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Cicatrix, Hypertrophic/metabolism , Cicatrix, Hypertrophic/pathology , Enzyme Activation/drug effects , Fibroblasts/pathology , Fibrosis , Humans , Interleukin-10/therapeutic use , Receptors, Interleukin-10/metabolism , Skin/pathologyABSTRACT
CCN2 is a matricellular protein that appears to be important in scar formation. CCN2 mediates the pro-fibrotic effects in hypertrophic scars (HTSs) through an unknown mechanism. However, many activities of CCN2 protein are known to be mediated by direct binding to integrin receptors. In this study, we investigated the role of integrin α(ν)ß(3) in the differentiation of hypertrophic scar fibroblasts (HTSFs) induced by CCN2. The levels of integrin α(ν)ß(3) between normal skin and hypertrophic scar (HTS) tissues were compared, and integrin α(ν)ß(3) was found to be upregulated in HTS. CCN2 was shown to induce HTSF differentiation and collagen (COL) synthesis at the mRNA and protein levels. Based on these results, the expression of integrin α(ν)ß(3) was upregulated by CCN2 stimulation during HTSF differentiation. Blockade of integrin α(ν)ß(3) prevented CCN2-induced HTSF differentiation and COL synthesis. Furthermore, the CCN2-induced increase in contractility of the HTSF in COL lattices was inhibited by integrin α(ν)ß(3) blocking antibodies. HTSs were established in a rabbit ear model, and the inhibitor of integrin α(ν)ß(3) signiï¬cantly improved the architecture of the rabbit ear scar. Results of the present study showed that integrin α(ν)ß(3) contributes to pro-fibrotic CCN2 signaling. Blocking this pathway may therefore be beneficial for the treatment of HTS.
Subject(s)
Cell Differentiation/drug effects , Cicatrix, Hypertrophic/metabolism , Connective Tissue Growth Factor/metabolism , Fibroblasts/metabolism , Integrin alphaVbeta3/metabolism , Adult , Animals , Cicatrix, Hypertrophic/genetics , Collagen/biosynthesis , Connective Tissue Growth Factor/genetics , Disease Models, Animal , Female , Humans , Integrin alphaVbeta3/genetics , Male , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Up-Regulation , Young AdultABSTRACT
CCN2 plays an important role in the pathogenesis of hypertrophic scars (HTSs). Although CCN2 is involved in many fibroproliferative events, the CCN2 induction signaling pathway in HTSs is yet to be elucidated. Here, we first investigated the effect of the mitogen-activated protein kinases (MAPKs) on CCN2-induced α-smooth muscle actin (α-SMA) and collagen I expression in human HTS fibroblasts (HTSFs). Then, we established HTSs in a rabbit ear model and determined the effect of MAPKs on the pathogenesis of HTSs. MAPK pathways were activated by CCN2 in HTSFs. Extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors significantly inhibited CCN2-induced expression of α-SMA and collagen I in HTSFs. In the rabbit ear model of the HTS, JNK and ERK inhibitors significantly improved the architecture of the rabbit ear scar and reduced scar formation on the rabbit ear. Our results indicate that ERK and JNK mediate collagen I expression and scarring of the rabbit ear, and may be considered for specific drug therapy targets for HTSs.
Subject(s)
Cicatrix, Hypertrophic/enzymology , Connective Tissue Growth Factor/metabolism , Extracellular Matrix Proteins/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/enzymology , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System , Skin/enzymology , Actins/metabolism , Animals , Cells, Cultured , Cicatrix, Hypertrophic/drug therapy , Cicatrix, Hypertrophic/genetics , Cicatrix, Hypertrophic/pathology , Collagen Type I/metabolism , Disease Models, Animal , Enzyme Activation , Extracellular Matrix Proteins/genetics , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Fibroblasts/drug effects , Fibroblasts/pathology , Gene Expression Regulation , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , MAP Kinase Signaling System/drug effects , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Rabbits , Recombinant Proteins/metabolism , Skin/drug effects , Skin/pathology , Time Factors , Transforming Growth Factor beta1/metabolism , Wound HealingABSTRACT
This study assessed cardiovascular disease risk factors in three groups of human subjects aged 20-34 [young, 20 male (M)/33 female (F)], 60-74 (aged, 29M/29F), and > 90 years (nonagenarian, 47M/50F). Components of the metabolic syndrome, cardiovascular disease, and markers of inflammation and oxidative stress were assessed. Nonagenarians weighed less than the two other groups (P < 0.001); however, there was no difference in percent fat among the three groups. Aged individuals had the highest prevalence of the metabolic syndrome (P < 0.001) according to the Adult Treatment Panel III classification. Both fibrinogen and homocysteine concentrations were significantly higher in the nonagenarians compared to younger groups. However, there were no significant differences between groups in fasting insulin, high sensitive C-reactive protein, and plasminogen activator inhibitor 1 concentrations. There were also no relationships between inflammation/ oxidative stress and the metabolic syndrome or cardiovascular disease although nonagenarians appear to be protected from oxidative damage to DNA.
ABSTRACT
The size of adipocytes influences their function suggesting a differential responsiveness to intervention. We hypothesized that weight loss in patients with type 2 diabetes mellitus (T2DM) predominantly decreases the size of large and very-large adipocyte subfractions in parallel with beneficial changes in serum adipokines and improved insulin sensitivity. A total of 44 volunteers from the Look Action for Health in Diabetes trial, who lost weight after 1-year of intense lifestyle intervention, were included. Insulin sensitivity (hyperinsulinemic-euglycemic clamp), size of subcutaneous abdominal adipocytes (osmium fixation), and selected serum adipokines were measured. A 13% weight loss was accompanied by 46% improvement in insulin sensitivity (increased glucose disposal rate from 5.9+/-2.2 to 8.6+/-2.7 mg/min/kg fat-free mass, P<0.05) in parallel with a 36% increase in plasma adiponectin concentration (6.1+/-3.1 to 8.3+/-3.9 microg/ml, P<0.05], but no changes in the proinflammatory cytokines interleukin-6 and tumor necrosis factor-alpha. Change in adiponectin correlated with changes in glucose disposal rate (r=0.34, P<0.05). Mean adipocyte size decreased (0.84+/-0.25 to 0.64+/-0.23 microl, P<0.05), mainly due to changes in the large adipocyte subfraction (size 0.75-0.44 microl, relative number 19-26%; P<0.05). Our data suggest that change in the large adipocyte subfraction may contribute to the improvement in insulin sensitivity via an increase in serum adiponectin. Such a relationship, which does not imply cause and effect, could not be obtained by measuring only mean adipocyte size. These data provide support for the measures of adipocyte size distribution in concert with in vitro adipokine secretion and lipolysis in future studies.