Impact of history of periodontitis on gene expression of bone-related factors in young patients

Abstract Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-β and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.

Positive outcomes of phosphodiesterase type 5 inhibitor on histopathologic and biochemical changes induced by ureteral obstruction

SUMMARY OBJECTIVES: We examined the effects of tadalafil, one of the phosphodiesterase type 5 (PDE5) inhibitors, in a rat model of with partial and complete unilateral ureteral obstruction (UUO). METHODS: The rats were divided into 5 groups: sham (n=6), partial unilateral ureteral obstruction (PUUO, n=6), PUUO with tadalafil treatment (PUUO+T; Cialis, 10 mg/72 h, intragastric; Lilly, Indianapolis, Indiana, USA), complete unilateral ureteral obstruction (CUUO, n=6), and CUUO with tadalafil treatment (CUUO+T). RESULTS: Fifteen days after the UUO, the ureter presented changes in the layers of urothelium and significant infiltration of inflammatory cells in the PUUO and CUUO groups. Compared with the sham, PUUO and CUUO groups had severe increased inflammatory cell infiltration. The urothelial epithelium exhibited cell degeneration and loss because of the swollen, atrophic, and denuded epithelial cells in the PUUO and CUUO groups. In the PUUO+T and CUUO+T groups, the urothelium revealed less epithelial cell degeneration and loss. The expressions of α-smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β) exhibited up-regulation in the PUUO and CUUO groups. The expression of TGF-β decreased positively correlated with that of α-SMA in the tadalafil therapy groups, PUUO+T and CUUO+T. CONCLUSION: The phosphodiesterase type 5 inhibitor's tadalafil reduced expressions of α-SMA and TGF-β in the obstructed ureters, measured by biochemical examinations. In addition, tadalafil decreased urothelium degeneration due to the decreased epithelial cell loss and inflammatory cell infiltration. Our results show that tadalafil prevents or slows down the onset of ureter inflammation and urothelial degeneration in rats with UUO.

RESUMO OBJETIVOS: Examinamos os efeitos do tadalafil em um dos inibidores da fosfodiesterase tipo 5 (PDE5) em um modelo de rato com obstrução ureteral unilateral parcial e completa (UUO). MÉTODOS: Os ratos foram divididos em cinco grupos: sham (n = 6), obstrução ureteral unilateral parcial (PUUO, n = 6), PUUO com tadalafil (PUUO T; Cialis, 10 mg/72 h, intragástrica; Lilly, Indianapolis, Indiana, EUA), completa obstrução ureteral unilateral (CUUO, n = 6) e CUUO com tratamento com tadalafil (CUUO T). RESULTADOS: Quinze dias após a UUO, o ureter apresentou alterações nas camadas de urotélio e infiltração significativa de células inflamatórias nos grupos PUUO e CUUO. Em comparação com os grupos sham, PUUO e CUUO, houve um aumento grave da infiltração de células inflamatórias. O epitélio urotelial exibiu degeneração e perda celular devido às células epiteliais inchadas, atróficas e desnudas nos grupos PUUO e CUUO. Nos grupos PUUO T e CUUO T, o urotélio revelou menor degeneração e perda de células epiteliais. Nós mostramos que a expressão da actina do músculo liso-α (α-SMA) e do fator de crescimento transformador-β (TGF-β) foram exibidas como sub-regulação nos grupos PUUO e CUUO. A expressão do TGF-β foi diminuída positivamente correlacionada com a da α-SMA nos grupos de terapia com tadalafil, PUUO T e CUUO T. CONCLUSÃO: O tadalafil do inibidor da fosfodiesterase tipo 5 reduziu as expressões α-SMA e TGF-β nos ureteres obstruídos, medidos por exames bioquímicos. Além disso, o tadalafil diminuiu a degeneração do urotélio devido à diminuição da perda de células epiteliais e da infiltração de células inflamatórias. Nossos resultados mostram que o tadalafil previne ou retarda o início da inflamação do ureter e degeneração urotelial em ratos com UUO.

Cytokines and chemokines associated with Treg/Th17 response in chronic inflammatory periapical disease

Abstract Cytokines and chemokines have a fundamental role in the maintenance of inflammation and bone response, which culminate in the development of chronic periapical lesions. Regulatory (Treg) and Th17 cytokines play a key role in regulating the immune response involved in this process. The aim of this study was to investigate the role of Treg and Th17 cells in chronic inflammatory periapical disease, by comparing the expression of the immunoregulatory mediators TGF-β, IL-10, CCL4, and the proinflammatory IL-17 and CCL20 in the periapical tissue of teeth with pulp necrosis, with and without associated chronic lesions. Eighty-six periapical tissue samples were obtained from human teeth. The samples were divided into three groups: pulp necrosis with a periapical lesion (n=26); pulp necrosis without a periapical lesion (n=30), and control (n=30). All samples were submitted to histopathological analysis and cytokine and chemokine measurement through ELISA. Statistical analyses were done with Kruskal-Wallis and Mann-Whitney tests and Spearman correlation. The group with pulp necrosis and a periapical lesion showed a higher expression of CCL4 and TGF-β in comparison with pulp necrosis without a lesion. CCL20 was higher in the group with a periapical lesion when compared to the control. In all groups there was a weak positive correlation between IL-17/CCL20, IL-10/CCL4, and IL-17/TGF-β. Both types of cytokines, pro-inflammatory and immunoregulatory, occur simultaneously in periapical tissue. However, a rise in immunosuppressive cytokines and chemokines (CCL4 and TGF-β) in periapical lesions suggests a role of these cytokines in stable periapical disease.

Effect of topical ozonetherapy on gingival wound healing in pigs: histological and immuno-histochemical analysis

Abstract In this study, the effects of ozonetherapy on secondary wound healing were evaluated histologically and immuno-histochemically. Material and Methods: 8 healthy pigs were used in this study. Six wounds with 10 mm in diameter were created through the punch technique on the palatinal gingiva of each pig. Ozone gas was applied on only 3 wounds (test group) and the remaining 3 were left to natural healing (control group). Biopsy samples were taken from one of the wounds in each group on the third day, from another wound of each group on the seventh day, and from another one on the tenth day. Routine histological analysis and immuno-histochemical staining were performed to investigate transforming growth factor-beta (TGF-β) and (VEGF) expressions. Results: No statistical difference was found between the test and control groups in terms of collagen fibers, epithelial formation and inflammation scores. A VEGF expression found in the test group was statistically higher than control group samples taken on the 3rd and 7th day. There was no statistical difference between the test and control groups in terms of TGF-β expression on any of the sampling days. Conclusion: The topical application of ozone gas could be effective in the early stages of wound healing by increasing the amount of VEGF expression. Clinical Relevance: Topical application of ozone gas may be effective in the early stages of oral wound healing.

Evaluating clinical and laboratory effects of ozone in non-surgical periodontal treatment: a randomized controlled trial

Abstract Objective: This study aims to evaluate the clinical and biochemical (oxidative stress and pro-inflammatory mediators) effects of the gaseous ozone use accompanied by scaling and root planning (SRP) in periodontal treatment. Material and Methods: The study population consisted of 40 patients with chronic periodontitis (CP) randomly sorted into two groups of 20. The experimental group received SRP plus 3 watts gaseous ozone in two separate applications five days apart, whereas the control group received SRP plus placebo. Clinical periodontal parameters were assayed and saliva samples were taken before the initial and one month after the second treatment. Periodontal examination assessed plaque index (PI), gingival index (GI), probing depth, and clinical attachment level (CAL). Total antioxidant status (TAS), total oxidant status (TOS), nitric oxide (NO), 8-hydroxy-2'-deoxyguanosine (8-OHdG), myeloperoxidase (MPO), glutathione (GSH), malondialdehyde (MDA), and transforming growth factor-beta (TGF-β) levels were evaluated from saliva samples. Results: Changes following treatment in PI, GI, probing depth, and CAL scores were similar for both groups (p>0.05). Of note, TGF-β levels were observed to be higher in the treatment group than in controls (p<0.05). Changes in 8-OHdG, TAS, TOS, NO, MPO, GSH and MDA levels, however, were not significantly different between groups (p>0.05). Conclusion: The findings of this study indicate that SRP plus gaseous ozone versus SRP alone does not correlate to a significant improvement in periodontal recovery.

Avaliação da eficácia de três marcadores imunoistoquímicos envolvidos nos diferentes tempos da cicatrização da ferida cirúrgica / Comparative efficacy of immunohistochemical markers in surgical healing

RESUMO Objetivo: avaliar a eficácia de três marcadores imunoistoquímicos envolvidos no processo de cicatrização de ferida cirúrgica. Métodos: estudo experimental em 40 ratos da raça Wistar, dos marcadores metaloproteinases e metaloproteinase da matriz 9 (MMP-9), fator de transformação do crescimento beta (TGF-β) e miofibroblasto e alfa actina de músculo liso (α-AML), estudados a partir de fragmentos de cicatriz cirúrgica de incisão abdominal envolvendo pele, aponeurose e peritônio. Os animais foram distribuídos em quatro subgrupos de dez de acordo com o dia da morte, programada em três, sete, 14 e 21 dias. Resultados: na expressão da MMP-9 ocorreu aumento progressivo de sua concentração, mais evidente do 7º ao 14º dias variando a imuno-expressão tecidual entre 2,65% e 11,50%.TGF- β mostrou expressão em nível alto no 3º dia, caiu no 7º, voltando a subir no 14º, com pequena queda no 21º dia variando a imuno-expressão tecidual entre 0,03% e 2,92%. A α-AML apresentou níveis com pouca variação e discreto aumento variando a imuno-expressão tecidual entre 0,88% e 3,23%. Conclusão: a MMP-9 se apresentou como melhor marcador, seguido pela TGF-β. Já o α-AML não se mostrou um bom sinalizador da evolução da reparação tissular.

ABSTRACT Objective: to evaluate the efficacy of three immunohistochemical markers involved in the wound healing process. Methods: experimental study of 40 Wistar rats of the markers metalloproteinases and matrix metalloproteinase 9 (MMP-9), beta transforming growth factor (TGF-β) and myofibroblasts and smooth muscle actin alpha (α-MLA) markers, studied from fragments of surgical scar of abdominal incision involving skin, aponeurosis and peritoneum. The animals were divided into four subgroups of ten according to the day of death, scheduled in three, seven, 14 and 21 days. Results: MMP-9 expression showed a progressive increase of its concentration, more evident from 7th to 14th days, varying the tissue immunoexpression between 2.65% and 11.50% . TGF- β showed expression at high level on the 3rd day, fell in the 7th, rising again in the 14th, with a small decrease in the 21st day, varying the tissue immunoexpression between 0.03% and 2.92%. The α-AML presented levels with little variation and a slight increase, varying the tissue immunoexpression between 0.88% and 3.23%. Conclusion: MMP-9 presented as the best marker, followed by TGF-β. However, α-AML was not a good indicator of the evolution of tissue repair.

Effects of grape seed extract on periodontal disease: an experimental study in rats

Abstract Natural compounds capable of modulating the host response have received considerable attention, and herbal products are suggested as adjunctive agents in periodontal disease treatment. Objective This study aimed to demonstrate the effect of grape seed extract (GSE) on periodontitis. Material and Methods Ligature induced periodontitis was created in 40 rats and they were assigned to four equal groups. One group was fed laboratory diet (group A) while three groups received GSE additionally. Silk ligatures were placed around the cervical area of the mandibular first molars for four weeks to induce periodontitis. The GSE groups were reallocated regarding GSE consumption as: for two weeks before ligation (group B; totally eight weeks), from ligation to two weeks after removal of the ligature (group C; totally six weeks), and for two weeks from ligature removal (group D; totally two weeks). Sections were assessed histologically and immunohistochemically. Inflammatory cell number (ICN), connective tissue attachment level (CAL), osteoclast density (OD), IL-10 and TGF-β stainings in gingival epithelium (GE), connective tissue (GC), and periodontal ligament (PL) were used as the study parameters. Results Lower ICN, higher CAL, and lower OD were observed in the GSE groups (p<0.05). IL-10 was more intensive in the GSE groups and in the GEs (p<0.05). Group B showed the highest IL-10 for PL (p<0.05). TGF-ß was higher in the GEs of all groups (p<0.017). Conclusions The results suggest anti-inflammatory activities of GSE, but further investigations are needed for clarification of these activities.

Women with recurrent spontaneous abortion have decreased 25(OH) vitamin D and VDR at the fetal-maternal interface

Immunological mechanisms have been proposed to underlie the pathogenesis of recurrent spontaneous abortion (RSA). Vitamin D has a potent immunomodulatory effect, which may affect pregnancy outcome. The objective of this study was to investigate 25-hydroxyvitamin D [25(OH) D] concentration and vitamin D receptor (VDR) expression in the decidual tissues of RSA patients. Thirty women with RSA (RSA group) and thirty women undergoing elective abortion (control group) were recruited during 2016 from gynecology outpatient clinics. We measured 25(OH) D, interleukin (IL)-17, IL-23, transforming growth factor β (TGF-β), VDR and 1-α-hydroxylase (CYP27B1) in decidual tissues collected during the abortion procedure. In the RSA group, 25(OH) D and TGF-β were significantly decreased while IL-17 and IL-23 were significantly increased compared with the control group. VDR expression was significantly decreased in the RSA group compared with the control group. Logistic regression analysis showed a significant negative correlation between 25(OH) D in decidual tissues and RSA. These results indicated that vitamin D concentrations in the decidua are associated with inflammatory cytokine production, suggesting that vitamin D and VDR may play a role in the etiology of RSA.

Effects of platelet rich plasma on fascial healing in rats with fecal peritonitis

ABSTRACT PURPOSE : To evaluate the effects of platelet rich plasma (PRP) on the healing of fascia wherein peritonitis has been created. METHODS: Twenty eight Wistar Albino rats were divided into four groups. Only a primary fascial repair following laparotomy was performed on Group 1, a primary fascial repair performed and PRP treatment applied following laparotomy on Group 2, and a fecal peritonitis created following laparotomy and a primary fascial repair carried out on Group 3. A fecal peritonitis was created following laparotomy and primary fascial repair and PRP treatment on the fascia was carried out on Group 4. RESULTS: TNF-α was found to be significantly lower in the control group (Group 1). It was detected at the highest level in the group in which fecal peritonitis was created and PRP applied (Group 4). TGF-β was determined as being significantly higher only in Group 4. Histopathologically, the differences between the groups in terms of cell infiltration and collagen deposition were not found to be significant. CONCLUSION: When platelet rich plasma was given histologically and biochemicaly as wound healing parameters cellular infiltration, collagen accumulation, and tissue hydroxyiproline levels were not increased but neovascularization, fibroblast activation and TNF Alfa levels were increased and PRP accelerated wound healing.

Superoxide overproduction and kidney fibrosis: a new animal model / Produção aumentada de superóxido e fibrose renal: novo modelo animal

Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis. .

Objetivo Estabelecer se a mutação no gene Immp2L induz à fibrose renal e se o envelhecimento exacerba a morfologia renal em camundongos. Métodos Foram usadas fêmeas de camundongos mutantes para proteína semelhante à peptidase 2 da camada interna da mitocôndria, com 3 e 18 meses de idade. Para analisar a fibrose renal, foram usados o escore clássico de fibrose, a coloração com tricrômio de Masson, e a análise de marcadores profibróticos, por meio da reação em cadeia de polimerase em tempo real (superóxido dismutase 1, metalonoproteinase-9, eritropoietina e fator transformador de crescimento beta), e a imunocoloração (fibroblastos e colágeno IV). Marcadores de estresse oxidativo foram determinados por imuno-histoquímica. O número de células apoptóticas renais foi analisado. A função renal foi estimada por creatinina sérica. Resultados Camundongos mutantes jovens apresentaram glomeruloesclerose em quantidade significativamente maior que animais da mesma idade (p=0,034). Os mutantes mostraram maior formação de cilindros tubulares (p=0,025), deposição de colágeno (p=0,019) e maior expressão de colágeno do tipo IV (p<0,001). A expressão de superóxido dismutase 1 foi maior em mutantes jovens (p=0,038). Mutantes idosas exibiram maior expressão dos marcadores de fibroblastos e macrófagos (p=0,007 e p=0,012, respectivamente). As reações da cadeia de polimerase em tempo real da metalanoproteinase-9 e da eritropoietina estavam aumentadas em 2,5 e 6 vezes, respectivamente, em mutantes idosas. A creatinina sérica foi significantemente maior em animais idosos mutantes (p<0,001). Conclusão Essa mutação alterou a arquitetura renal pelo aumento da deposição de matriz extracelular, estresse oxidativo e inflamação, sugerindo papel de proteção de Immp2L contra a fibrose renal. .

The TLR7 agonist Imiquimod promote the immunogenicity of msenchymal stem cells

BACKGROUND: Mesenchymal stem cells (MSCs) are considered the best candidate in stem cells therapy due to their multipotent differentiation ability, low expression of co-stimulatory molecules (CD80, CD86, CD34 and HLA-II) and immunosuppression effects on in vivo immune responses. MSCs were now widely used in clinical trials but received no encourage results. The major problem was the fate of engrafted MSCs in vivo could not be defined. Some studies indicated that MSCs could induce immune response and result in the damage and rejection of MSCs. As toll like receptors (TLRs) are important in inducing of immune responses, in this study we study the role of TLR7 in mediating the immune status of MSCs isolated from umbilical cord. RESULTS: Our results indicated that TLR7 agonist Imiquimod could increase the proliferation of PBMC isolated from healthy human volunteers and release of lactate dehydrogenase (LDH) in supernatant from PBMC-UCMSCs co-culture system. Flow cytometry and quantitative PCR also confirmed the regulated expression of surface co-stimulatory molecules and pro-inflammatory genes (IL-6, IL-8, IL-12, TGF-β and TNF-α). And the down-regulation expression of stem cell markers also confirmed the loss of stemness of UCMSCs. We also found that the osteo-differentiation ability of UCMSCs was enhanced in the presence of Imiquimod. CONCLUSION: To our knowledge, this is the first report that activation of TLR7 pathway increases the immunogenicity of UCMSCs. Extensive researches have now been conducted to study whether the change of immune status will be help in tumor rejection based on the tumor-tropism of MSCs.

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world’s population is latently infected (LTBI) with Mycobacterium tuberculosis (Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.

Tratamento da fibrose induzida pela infecção por Trypanosoma cruzi com composto inibidor da via de TGF-beta em modelo tridimensional de cultivo de células cardíacas / Treatment of t. cruzi-induced fibrosis with a tgf-β signaling inhibitor compound using a three-dimensional model of cardiac cells culture

A doença de Chagas é a principal causa de lesões cardíacas em jovens adultos economicamente produtivos em áreas endêmicas da América Latina. A cardiopatia chagásica (CC) se caracteriza como uma doença progressivamente debilitante, na qual o TGF-beta desempenha papel fundamental para o desenvolvimento da fibrose e hipertrofia cardíacas, através da regulação de componentes da matriz extracelular (MEC), tais como a fibronectina, as metaloproteases (MMPs) e os inibidores teciduais das MMPs (TIMPs). No presente estudo, foi verificada a capacidade do inibidor farmacológico da via de TGF-beta, SB-431542, em restaurar o equilíbrio da MEC, rompido pela infecção por T. cruzi, e os prováveis mecanismos envolvidos neste processo. Para tal, utilizamos um modelo tridimensional (3D) de cultivo de células cardíacas (denominados esferóides cardíacos), capaz de mimetizar aspectos da arquitetura e fisiologia do tecido cardíaco. O tratamento dos esferóides cardíacos infectados por T. cruzi com SB-431542 resultou na redução parcial da hipertrofia e fibrose dos esferóides cardíacos, por mecanismos envolvendo a redução na expressão de TIMP-1, o aumento na atividade das MMPs 2 e 9, a redução na expressão da fibronectina e a redução da carga parasitáriaAtravés de uma abordagem proteômica, conseguimos ainda identificar outras proteínas possivelmente envolvidas no processo de reversão da fibrose e hipertrofia dos esferóides cardíacos a partir do tratamento com SB-431542, como as integrinas, fibulinas, proteínas de ligação ao selênio, a titina, entre outras. Além disso, as análises proteômicas nos permitiram identificar alguns dos componentes moleculares envolvidos no processo de remodelamento do tecido cardíaco disparado pela infecção por T. cruzi, auxiliando assim, na compreensão dos mecanismos associados à progressão da hipertrofia e da fibrose ao longo do desenvolvimento da CC...

Chagas disease represents the leading cause of cardiac lesions in economicallyproductive adults in endemic areas of Latin America. Chagasic cardiomyopathy (CC)is a progressive dysfunctional illness, in which TGF-beta plays a central role indevelopment of fibrosis and hypertrophy through regulation of extracellular matrix(ECM) components, such as fibronectin, matrix metalloproteinases (MMPs) and tissueinhibitors of metalloproteinases (TIMPs). In the present study we tested the efficacy ofa pharmacological inhibitor of TGF-beta signaling pathway, SB-431542, in restoring ECMbalance disrupted by T. cruzi infection and the possible mechanisms involved in thisprocess. For that, we used a three-dimensional (3D) model of cardiac cells culture(named cardiac spheroids) that can mimic aspects of architecture and physiology ofliving cardiac tissues better than conventional two-dimensional (2D) models.Treatment of T. cruzi-infected-cardiac spheroids with SB-431542 resulted in areduction of spheroids hypertrophy and fibrosis by mechanisms involving a decreasein the expression of TIMP-1, an increase in the activities of MMP-2 and MMP-9, areduction in the expression of fibronectin and a reduction of parasite load. Moreover,we identified through a proteomic approach, other proteins possibly involved inreversion of fibrosis and hypertrophy of cardiac spheroids after treatment with SB-431542, such as integrins, fibulins, selenium binding proteins and titin, among others.In addition, the proteomic analysis allowed us to identify molecular componentsinvolved in tissue remodeling generated by T. cruzi infection, assisting in thecomprehension of mechanisms related to fibrosis and hypertrophy progression duringCC’s development...

Transforming growth factor-β/Smad signaling function in the aortopathies / Fator transformador de crescimento-β/Smad como via de sinalização em aortopatias

OBJECTIVES: Transforming growth factor (TGF)-β/Smad signaling pathway in aortic dissection patients and normal subjects has not been previously described. The present study was designed to evaluate the TGF-β/Smad signaling expressions in the patients with acute type A aortic dissection in comparison with those in the patients with thoracic aortic aneurysm and with coronary artery disease, and (or) the healthy subjects. METHODS: Consecutive surgical patients for acute type A aortic dissection (20 patients), aortic aneurysm (nine patients) or coronary artery disease (20 patients) were selected into this study. Blood samples (4 ml) were obtained from the right radial arterial indwelling catheter after systemic heparinization prior to the start of cardiopulmonary bypass in the operating room. Twenty-one young healthy volunteers without underlying health issues who donated forearm venous blood samples (4 ml) were taken as control. The surgical specimens of the aortic tissues were obtained immediately after they were severed during the operations of the replacement of the aorta in the patients with aortic dissection or aortic aneurysm. In patients receiving coronary artery bypass grafting, the tiny aortic tissues were taken when the punch holes of the proximal anastomosis on the anterior wall of the ascending aorta were made. The aortic tissues were for RNA, protein, or supernatant preparations until detection of TGF-β1 mRNA by quantitative real-time reverse transcription polymerase chain reaction, of TGF-β1, TGF-β receptor I, Smad2/3, Smad4 and Smad7 by Western blot, and of TGF-β1 by enzyme-linked immunosorbent assay, respectively. In particular, the linear correlations of the relative grayscales between different proteins of each group, and those correlations between the quantitative TGF-β1 by enzyme-linked immunosorbent assay and the time interval from the onset to surgery or the maximal dimensions of the ...

OBJETIVOS: Fator transformador de crescimento (TGF) -β/ Smad como via de sinalização em casos de dissecção aórtica e indivíduos normais não foi descrito anteriormente. O presente estudo foi elaborado para avaliar as expressões TGF-β/Smad como via de sinalização nos pacientes com dissecção aguda da aorta, em comparação com que nos pacientes com aneurisma da aorta torácica e com doença arterial coronariana, e (ou) com indivíduos saudáveis. MÉTODOS: Pacientes cirúrgicos consecutivos para o tipo A de dissecção aguda da aorta (20 pacientes), aneurisma da aorta (nove pacientes) ou doença arterial coronária (20 pacientes) foram selecionados para este estudo. Amostras de sangue (4 ml) foram obtidas a partir do cateter arterial radial direito após heparinização sistêmica antes do início da circulação extracorpórea na sala de cirurgia. Vinte e um voluntários jovens e saudáveis, sem problemas de saúde subjacentes que doaram amostras de sangue venoso do antebraço (4 ml) foram tomados como controle. Os espécimes cirúrgicos de tecidos aórtico foram obtidos imediatamente após terem sido cortados durante as operações da substituição da aorta nos pacientes com dissecção aórtica ou aneurisma da aorta. Em pacientes que foram submetidos à cirurgia de revascularização miocárdica, os tecidos da aorta minúsculos foram obtidos quando os orifícios da anastomose proximal na parede anterior da aorta ascendente foram feitos. Os tecidos da aorta foram para a RNA, proteínas ou preparações sobrenadantes até a detecção de TGF-β1 mRNA pela reação de transcrição reversa quantitativa em tempo real em cadeia da polimerase, de TGF-β1, receptor I de TGF-β, Smad2/3, Smad4 e Smad7 por Western Blot, e de TGF-β1 pelo teste de ELISA, respectivamente. Em particular, as correlações lineares dos tons de cinza relativo entre diferentes proteínas de cada grupo, e aquelas correlações entre os quantitativos TGF-β1 pelo teste de ELISA e o intervalo de ...

Effect of ProRoot MTA, Portland cement, and amalgam on the expression of fibronectin, collagen I, and TGFβ by human periodontal ligament fibroblasts in vitro.

Context: Today many materials have been introduced for root-end filling materials. One of them is mineral trioxide aggregate (MTA) that is mentioned as a gold standard. Aims: The purpose of this in vitro study was to evaluate the reaction of human periodontal ligament fibroblasts to the root-end filling materials, such as ProRoot MTA, Portland cement, and amalgam. Settings and Design: Eight impacted teeth were extracted in aseptic condition. The tissues around the roots were used to obtain fibroblast cells. After cell proliferation, they were cultured in the chamber slides and the extracts of the materials were added to the wells. Materials and Methods: Immunocytochemical method for measuring the expression of Fibronectin, collagen I and transforming growth factor beta (TGF®) was performed by Olysia Bioreport Imaging Software. Statistical Analysis Used: The results were analyzed by SPSS 13.0 and Tukey post hoc test with P<0.05 as the limit of significance. Results: Collagen expression in MTA specimens was higher than the other groups in 24 h significantly. After 48 h, the Portland cement group showed the most expression of collagen significantly and after 1 week, Portland cement and MTA groups had the most expression of collagen but there was no significant difference between these 2 groups. After 1 week, the Portland cement group demonstrated a higher amount of TGF® and fibronectin. Conclusions: The results suggest that Portland cement can be used as a less expensive root filling material with low toxicity. It has better effects than amalgam on the fibroblasts.

Transforming growth factor-β in graft vessels: histology and immunohistochemistry

OBJECTIVES: The biological functions of transforming growth factor-β signaling that involves Smad proteins have not been previously investigated with respect to coronary artery bypass grafts. The aim of the present study was to observe the immunostaining of proteins that are related to this signaling pathway. METHODS: Fifteen remnants of coronary artery bypass grafts, including nine saphenous veins, three radial arteries and three mammary arteries, were collected from 12 patients who were undergoing coronary artery bypass. Hematoxylin and eosin, Masson's trichrome, and immunohistochemical staining of transforming growth factor-β1, type I receptor of transforming growth factor-β, Smad2/3, Smad4, and Smad7 were performed. RESULTS: The saphenous veins showed more severe intimal degeneration, more severe smooth muscle cell proliferation and more collagen deposition than the arterial grafts, as evidenced by hematoxylin and eosin and Masson's trichrome stainings. Immunohistochemical assays demonstrated that the majority of the transforming growth factor-β1 signaling cytokines were primarily localized in the cytoplasm in the medial layers of all three types of grafts, whereas ectopic transforming growth factor-β1, type I receptor of transforming growth factor-β, and Smad7 overexpressions in the interstices were observed particularly in the saphenous vein and radial arterial grafts. CONCLUSION: Enhanced transforming growth factor-β1 signal transduction with medial smooth muscle cell proliferation and ectopic transforming growth factor-β1, the presence of the type I receptor of transforming growth factor-β, and Smad7 overexpressions in the extracellular matrix may provide primary evidence for early or late graft failure.

Menor expresión de la enzima convertidora de angiotensinaI omóloga (ECA2) se asocia a mayor remodelamiento de la pared aórtica de ratas con niveles geneticamente elevados de angiotensina II / Decreased expression of homologous angiotensin I converting enzyme (ACE2) is associated to increased remodeling of the aortic arterial wall in rats with genetically elevated levels of angiotensin II

Introducción: El polimorfismo de la enzima convertidora de angiotensina I (ECA) determina mayor actividadde ECA y niveles de angiotensina (Ang) II en ratas Brown Norway (BN) y menor actividad de ECA y niveles de Ang II en ratas Lewis (L). La interregulación entre ECA, ECA2 y su relación con remodelamiento aórtico hipertensivo no ha sido explorada Objetivo: Determinar la expresión de ECA y ECA2 y los parámetros de remodelamiento vascular hipertensivo en la aorta de ratas con niveles genéticamente determinados de ECA. Métodos: A ratas macho homocigotas de 150 grs BN y LL, se les indujo HTA por 6 semanas por el procedimiento Goldblatt (GB, 2 K-1clip). Ratas pseudo-operadas se usaron como controles (sham). Se determinó la presión arterial sistólica (PAS), el grosor de la túnica media (GTM), área de la TM (ATM), expresión génica deECA, ECA2 ,TGF-beta, PAI-1 y MCP-1 por RT-PCR y también proteica de ECA y ECA2 por Western Blot. Resultados: La masa cardiaca relativa y la PAS aumentaron significativamente en los grupos GB respecto a sus controles Sham, sin diferencias por efecto del polimorfismo de la ECA. En condiciones de normotensión las ratas BN mostraron que la pared aórtica expresa mayores niveles génicos y proteicos de la ECA(60% y 134%, respectivamente) y menores de ECA2 (74% y 73%, respectivamente) respecto de las ratas L(p<0.05). Estos resultados se asociaron con mayores GTM y ATM como en los niveles de mRNA de TGF-beta y, PAI-1 en las aortas de ratas BN respecto de las ratas L (p<0,05). En respuesta a un estrés hipertensivo las ratas con mayores niveles de ECA y menores niveles de ECA2 mostraron mayor GTM (p<0,05, respecto de GB-L), sin diferencias en los otros parámetros evaluados...

Background: Angiotensin I converting enzyme (ACE) polymorphism determines increased ACE and Ang IIlevels in Brown Norway rats (BN) and decreased ACE and Ang II levels in Lewis (L) rats. The interactionbetween ACE and ACE2 in relation to aortic remodeling associated to hypertension has not been explored. Aim: to determine the expression of ACE and ACE2 along with parameters of remodeling in hypertensive rats with genetically determined levels of ACE. Methods: BN and L rats weighing 150 g were made hypertensive by the Goldblatt procedure (GB, 2K-1 clip). Sham operated rats were used as controls. Systolic blood pressure (SBP), media thickness (MT), and MT area were measured. RT-PCR was used to determine the genetic expression of ACE, ACE2, TGF-beta, PAI-1 and MCP-1. Western Blot was used to measure the protein fraction of ACE and ACE2 Results: Relative cardiac mass and SBP increases significantly in GB rats compared to controls; ACE polymorphism did not influence this effect. The aortic wall of normotensive BN rats expressed increased genic and protein levels of ACE (60% and 134%, respectively) and decreased levels of ACE2 (74% and 73%, respectively) compared to L rats (p<0.05). These findings were associated to increased MT and MT area as well as increased mRNA for TGF-beta and PAI 1 in BN rats compared to L rats (p<0.05). In response to hypertensive stress, rats with increased ACE and decreased ACE2 levels developed increased MT compared to GB-L rats; other parameters of remodeling were not affected...

Serum hyaluronic acid level: Correlation with quantitative measurement of hepatic fibrosis in a cirrhotic rat model / 대한간학회지

BACKGROUNDS/AIMS: The serum level of hyaluronic acid (HA) has been suggested as a useful serologic marker for hepatic fibrosis. However, the relationship between serum HA levels and quantitative markers of fibrosis from liver tissue has not been reported. The aim of this study was to determine the correlation between serum HA level and quantitative measurement of hepatic fibrosis in a cirrhotic rat model. METHODS: Cirrhosis was produced by common bile duct ligation (BDL) in adult Sprague-Dawley rats. The animals were classified into four groups: (1) G1, sham operated (n=5); (2) G2, BDL for 2 weeks (n=6); (3) G3, BDL for 3 weeks (n=6); and (4) G4, BDL for 4 weeks (n=6). Hepatic fibrosis was analyzed histomorphologically using the Batts and Ludwig scoring system. Serum HA level and hepatic hydroxyproline content were quantified. The gene expressions in the liver of procollagen, collagen, and transforming growth factor-beta (TGF-beta) were measured by reverse transcriptase-polymerase chain reaction. RESULTS: In groups G1, G2, G3, and G4, the Batts and Ludwig scores (mean+/-SD) were 0, 1.3+/-0.5, 2.6+/-0.5, and 3.4+/-0.5, respectively (P<0.05), serum HA levels were 12.5+/-3.2, 30.0+/-4.3, 228.6+/-157.7, and 391.3+/-207.7 ng/mL (P<0.05), and the concentration of hydroxyproline was 12.4+/-2.8, 17.6+/-3.8, 17.9+/-2.4, and 33.4+/-3.4 microgram/g liver tissue, and it was significantly higher in group G4 than in the other groups (P<0.05). The gene expressions of collagen, procollagen, and TGF-beta1 in the liver were also significantly higher in group G4 compared with the other groups (P<0.05). Direct linear correlations were observed between serum HA level and hepatic hydroxyproline content, hepatic gene expressions of collagen, procollagen, TGF-beta1, and histomorphological grade of hepatic fibrosis (P<0.001). CONCLUSIONS: These results indicate that serum HA is a useful and noninvasive serologic marker for the evaluation of advanced hepatic fibrosis.

TGF-beta and new bone formation / TGF- ß y Neoformación Ósea

Este trabajo tiene como objetivo presentar, por medio de una revisión de la literatura, las principales características de la TGF-beta en la regulación de la neoformación ósea.

The aim of this work is to present, by a literature review, the principal characteristics of TGF-beta, in the regulation and new bone formation.

Pioglitazone reduces urinary protein and urinary transforming growth factor-beta excretion in patients with type 2 diabetes and overt nephropathy.

OBJECTIVE: Increased urinary excretion of protein and transforming growth factor-beta (TGF-beta) are associated with progression of diabetic nephropathy (DN). Thiazolidinediones (TZD) could reduce urinary protein excretion in patients with microalbuminuric DN. There is little data of patients with macroalbuminuric DN. Also, there are no available clinical data regarding the effect of TZD on TGF-beta and type IV collagen in clinical DN. The present study was carried out to evaluate the effect of pioglitazone (PGZ), a member of TZD, on urinary protein, urinary TGF-beta, and urinary type IV collagen excretion in type 2 diabetic patients with macroalbuminuric DN. MATERIAL AND METHOD: Forty patients with type 2 diabetes and overt nephropathy, proteinuria more than 500 mg/day, were randomly assigned to receive PGZ (30 mg/day, n = 24) or placebo (control group, n = 16), for 12 weeks. Blood pressure, plasma glucose, glycated hemoglobin, lipid profile, 24-hour proteinuria, urinary TGF-beta and urinary type IV collagen were determined and compared. RESULTS: Glycemic control and blood pressure in both groups were not significant different. At baseline, the levels of proteinuria, urinary TGF-beta, and type IV collagen were not significant different between both groups. The geometric mean of urinary protein excretion in the PGZ group was progressively reduced from 1.64 to 0.98 gram/day (g/d), or 40.1% decrease which was significantly different (p < 0.05) from the 4.3% increase (from 1.72 to 1.80 g/d) in the control group. Urinary TGF-beta excretion in the PGZ group was decreased by 47.8% which significantly differed from the 59.7% increase in the control group (p < 0.05). Urinary type IV collagen levels in the PGZ group were decreased by 35% which was slightly, but not significantly, different from the 51.6% elevation in the control group (p = 0.06). CONCLUSION: Besides the effectiveness in blood sugar control, pioglitazone could salutarily reduce proteinuria and synthesis of TGF-beta as well as type IV collagen. These beneficial effects of pioglitazone on diabetic nephropathy are comparable to angiotensin converting enzyme inhibitors and angiotensin receptor blockers