Background and objective:Aging is characterized by a decline in tissue structure and function that may be explained by the development of cellular senescence. However, the acquisition of specific phenotypic responses in senescent gingival fibroblasts is still poorly understood. Here, we have analyzed whether proliferation of primary cultures of human gingival fibroblasts may affect different cell functions relevant to cellular senescence and tissue deterioration. Methods: Human gingival fibroblasts from five young donors were expanded until cellular senescence was achieved. Cellular senescence was evaluated by determining modifications in cell size, cell proliferation, p16 and p21 mRNA levels, H2Ax phosphorylation, cell viability, and senescence-associated beta-galactosidase staining. Inflammation was evaluated by analyzing the secretion of cytokines and nuclear translocation of NF-κB. Collagen remodeling was evaluated using a collagen gel contraction assay. Immunofluorescence and confocal microscopy were used to determine changes in the localization of the cytoskeletal proteins. Data analysis was performed to identify changes between cultures of the same donor at early and late passages using the paired sample t test or the Wilcoxon matched-pairs signed-rank test.Results: Late passage cells showed changes compatible with cellular senescence that included increased cell size, reduced cell proliferation, staining for SA-beta gal, phosphorylated H2Ax, and increased p16 and p21 mRNA levels. Late passage cells showed a decrease in collagen contraction and reduced co-localization between the cytoskeletal proteins actin and vinculin. Importantly, late passage cells neither demonstrated changes in the secretion of inflammatory cytokines nor NF-κB activation. Conclusion:Our results imply that cytoskeletal changes and inhibition of cell proliferation represent early modifications in the structure and function of senescent gingival fibroblasts that are not coupled with the acquisition of an inflammatory phenotype. Further studies are needed to clarify the impact of different senescence stages during aging of the periodontium. K E Y W O R D S aging, fibroblast, gingiva, senescence | 433 PÁEZ Et al. How to cite this article: Páez J, Hernández R, Espinoza J, et al. Uncoupled inflammatory, proliferative, and cytoskeletal responses in senescent human gingival fibroblasts. J Periodont Res. 2020;55:432-440. https ://doi.

Soluble methylglyoxal is a highly cytotoxic compound that induces cell death through apoptosis in gingival fibroblasts. TIMP-1 is induced in these cells upon direct exposure to methylglyoxal or after culture of gingival fibroblasts over methylglyoxal-treated collagen. As TIMP-1 has been implicated in cell survival and matrix remodeling, we propose that increased TIMP-1 protein levels may be part of a protective response of gingival connective tissue cells upon exposure to methylglyoxal or after the interaction with the collagen matrix that has been modified by this agent.

Background: Glucose‐derived metabolites may alter the structure and biologic properties of important proteins in periodontium, such as collagens. As a consequence, it is possible that collagen‐binding cells may change their phenotypic traits. Although the glucose‐derived product methylglyoxal (MGO) has been detected in periodontal lesions, the precise effect of collagen glycation on gingival connective tissue biology is not fully understood. The present study evaluates whether collagen glycation by MGO may affect phenotypic properties and remodeling capacity of human gingival fibroblasts (HGFs). Methods: Primary cultures of HGFs were grown on Type I collagen matrices previously treated with MGO. Cell cultures were tested for cell viability, apoptosis, α‐smooth muscle actin (SMA), fibronectin (FN) production, and collagen remodeling. Mechanical properties and morphology of MGO‐treated collagen gels were evaluated using rheometry and atomic force microscopy. Statistical analysis was performed by Kruskal‐Wallis and Mann‐Whitney U tests. Results: MGO‐treated collagen did not affect cell viability or apoptosis. In addition, MGO did not induce significant changes in morphology or mechanical properties of the collagen matrix. However, MGO‐treated collagen stimulated an increase in the myofibroblast marker α‐SMA, production and assembly of FN, and contraction of collagen matrices. Moreover, use of a triple‐helical peptide that reconstitutes the collagen‐binding domain for integrins GFOGER reverted the assembly of FN induced by MGO‐treated collagen. Conclusions: The present study shows that collagen glycation by MGO stimulates differentiation of myofibroblasts and production and assembly of FN. These responses may alter the homeostatic balance and wound‐healing response of gingival connective tissues affected by diabetes mellitus or aging.

2177 Background. Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We have recently demonstrated that chronic cocaine use is associated with endothelial dysfunction (Sáez et al. Thromb Res 2011; 128: 18), a key event in the onset and progression of atherosclerosis. There is growing evidence that the RhoA/Rho kinase (ROCK) pathway has an important pathophysiological role in vascular endothelial dysfunction. Accordingly, we hypothesized that cocaine use induces activation of RhoA/ROCK pathway. Objectives. The main aim of this work was to investigate the activation of RhoA/ROCK pathway through ex vivo, in vivo and in vitro studies. Methods. Ex vivo studies. We studied 13 cocaine dependent individuals (aged 19–52 years mean age 37 years) who met DSM-IV criteria for cocaine dependence, seeking treatment for cocaine abuse and age, sex-matched healthy controls (aged 20–49 years, mean age 35 years). Samples were obtained at admission, within 72 hours of drug exposure. Endothelial cell damage was determined by enumerating circulating endothelial cells (CECs). Rho-kinase activity was assessed by the levels of phosphorylated to total myosin light chain phosphatase 1 (MYPT1-P/T) in circulating leukocytes. In vivo studies. Adult male Sprague-Dawley rats were randomly assigned to receive either cocaine (30mg/kg, provided by NIDA, USA) or saline intraperitoneally once daily for 21 days. The levels of aortic phosphorylated MYPT1 (phospho-MYPT1) were assessed by western blot in aorta extracts. In vitro experiments. Human umbilical vein endothelial cells (HUVECs) were cultured under standard conditions and supplemented for 5 hours with plasma from chronic cocaine users, normal plasma, cocaine (10μM) or vehicle. After media removal, HUVECs were either lysed for determination of ROCK activity or co-cultured with resting platelets and immunostained for von Willebrand factor (FVW). Platelet adhesion was evaluated by immunofluorescence microsocopy. Experiments were conducted in the presence or absence of ROCK inhibitors, Y-27632 (10 μM) or atorvastatin (10μM). Results. Cocaine users showed significantly elevated number of CECs compared to the controls (65 ± 6.6 vs 14 ± 3.4 cells/mL, p: 0.0002). In the control subjects, leukocyte mean MYPT1-P/T ratio was 2.2 ± 0.8 whereas in cocaine addicts were significantly increased (9.8 ± 2.8; p 0.015). ROCK activity was higher by 100% (p: 0.019) in the aortic wall of the cocaine-treated rats compared to sham animals. HUVECs supplemented with plasma from cocaine users showed an increase in ROCK activity by 25% (p: 0.039), released significantly higher amount of FVW (p<0.05) and adhered a larger number of platelets (22.6±5 vs 7.9±3 platelets/cell, respectively; p: 0.006) compared with control plasma. Cocaine exposure induced a dramatically higher number of platelets adhered to HUVEC than in vehicle-treated cells (220±73 vs 10.2±1.1 platelets/cell, respectively; p>0.001).ROCK inhibitors, atorvastatin and Y-27632 reduced the release of FVW by HUVECs exposed to plasma from cocaine users by 65% (p: 0.004) and strongly inhibited platelet adhesion (by 75% in plasma-treated cells and by 90% in HUVECs exposed to cocaine, p:< 0.006). Conclusions. We found an increase in Rho kinase activity in peripheral leukocytes of cocaine abusers, in the vessel wall of rats exposed to cocaine and a marked positive effect of ROCK inhibitors on the cellular injury induced by cocaine or plasma from cocaine consumers on endothelial cells. Collectively, these data suggest that activation of RhoA/ROCK pathway plays a key role in cocaine-induced endothelial dysfunction. Inhibition of ROCK may provide therapeutic benefits in a comprehensive treatment for cocaine addiction. Disclosures: No relevant conflicts of interest to declare.

Objectives Myofibroblasts constitute a specific cell phenotype involved in connective tissue healing. Diabetes alters the wound healing response. However, it is not clear whether diabetes modifies the involvement of myofibroblasts in periodontal wounds. Materials and Methods Type I diabetes was induced in rats through streptozotocin injection, and periodontal wounds were performed. Wound healing was evaluated histologically at 2, 5, 7, and 15 days by measuring epithelial migration, neutrophil infiltration, and collagen and biofilm formation. Distribution of myofibroblasts was evaluated through immunofluorescence for α‐smooth muscle actin. Data analyses were performed using the Shapiro–Wilk, ANOVA, or Kruskal–Wallis tests. Results Diabetic wounds were characterized by delayed epithelial closure, increased neutrophil infiltration, biofilm formation, and reduced collagen formation. Quantification of the myofibroblasts showed a significant reduction at 5 and 7 days in wounds of diabetic rats and an increase at 15 days when compared to wounds of non‐diabetic rats. Conclusions Diabetic wound healing was associated with decreased epithelial and connective tissue healing, increased levels of inflammation, and biofilm formation. Myofibroblast differentiation was delayed in diabetic periodontal wounds at early time points. However, myofibroblasts persisted at later time points of healing. The present study suggests that diabetes alters the involvement of myofibroblasts during periodontal wound healing.