Soluble HLA-G and TGF-ß in couples attending assisted reproduction - A possible role of TGF-ß isoforms in semen?

Soluble isoforms of the non-classical Human Leukocyte Antigen (HLA)-G as well as Transforming Growth Factor (TGF)-ß is expressed in seminal plasma possibly influencing the pregnancy potential. We wanted to examine the association of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGFß3 with pregnancy success in a cohort of 127 couples and 4 single women attending fertility treatment with the use of assisted reproduction technologies (ART). Soluble HLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in seminal plasma did not fluctuate significantly over time. We did not find any impact of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 on time-to-pregnancy measured as number of treatment cycles. There was a significant association between concentrations of seminal plasma sHLA-G and HLA-G variations in the 3'untranslated region (3'UTR) of the HLA-G gene, supporting and extending previous findings. Furthermore, by comparing seminal plasma concentrations of sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in male subjects with reduced semen quality, male subjects with normal semen quality, and sperm donors, we found that TGF-ß2 was significantly lower, and TGF-ß3 was significantly higher, in seminal plasma from sperm donors. These findings suggest that TGF-ß isoforms may influence semen quality and fertility.

Healing effect of andiroba-based emulsion in cutaneous wound healing via modulation of inflammation and transforming growth factor beta 31.

PURPOSE: To evaluate the effects and mechanisms of andiroba-based emulsion (ABE) topical treatment on full-thickness cutaneous wounds in rats. METHODS: The wounds were harvested on days 3, 7, 15, and 20 post-surgery. Wound contraction rate, quantitative immunohistochemistry [macrophages, myofibroblasts, capillaries, collagens (col) I and III, transforming growth factor ß3ß (TGFß3)], and tensile strength were assessed. RESULTS: Treated wounds were smaller, contracted earlier and had increased angiogenesis, fewer CD68+ and M2 macrophages on days 7 and 15, but higher on day 20. Myofibroblasts appeared on days 3 to 7 in untreated wounds and on days 7 to 15 in treated wounds. TGFß3 levels were higher in the treated wounds, less dense collagen fibers, lower col I/III ratios and a higher tensile strength. CONCLUSION: These results demonstrate the important anti-inflammatory role of treatment and the associated modulation of macrophages, myofibroblasts, and TGFß3 levels. Collagen fibers in the treated wounds were more organized and less dense, similar to unwounded skin, which likely contributed to the higher tensile strength.

Healing effect of andiroba-based emulsion in cutaneous wound healing via modulation of inflammation and transforming growth factor beta 3

Abstract Purpose: To evaluate the effects and mechanisms of andiroba-based emulsion (ABE) topical treatment on full-thickness cutaneous wounds in rats. Methods: The wounds were harvested on days 3, 7, 15, and 20 post-surgery. Wound contraction rate, quantitative immunohistochemistry [macrophages, myofibroblasts, capillaries, collagens (col) I and III, transforming growth factor β3β (TGFβ3)], and tensile strength were assessed. Results: Treated wounds were smaller, contracted earlier and had increased angiogenesis, fewer CD68+ and M2 macrophages on days 7 and 15, but higher on day 20. Myofibroblasts appeared on days 3 to 7 in untreated wounds and on days 7 to 15 in treated wounds. TGFβ3 levels were higher in the treated wounds, less dense collagen fibers, lower col I/III ratios and a higher tensile strength. Conclusion: These results demonstrate the important anti-inflammatory role of treatment and the associated modulation of macrophages, myofibroblasts, and TGFβ3 levels. Collagen fibers in the treated wounds were more organized and less dense, similar to unwounded skin, which likely contributed to the higher tensile strength.

Expression of inflammatory and fibrogenetic markers in acne hypertrophic scar formation: focusing on role of TGF-ß and IGF-1R.

Acne vulgaris is a universal skin disease and it may leave a scar when the original skin lesion disappears. These scars can cause cosmetic problems and psychological burden, leading to poor quality of life of patients. Acne scars are classified into atrophic scars and hypertrophic scars. As most of the acne scars are atrophic, many studies have been conducted focusing on the treatment of atrophic lesions. This study was conducted to investigate the underlying pathogenesis of acne hypertrophic scars by identifying roles of fibrogenetic and inflammatory markers. Skin biopsy samples were obtained from hypertrophic scars of face and back and from adjacent normal tissues as control group. Some samples from back were immature hypertrophic scars and the other samples were in mature stages. Immunohistochemistry staining and quantitative PCR were performed for fibrogenetic and inflammatory markers. Both in mature and immature hypertrophic scars, vimentin and α-SMA were increased. Production of TGF-ß3 protein as well as transcription of TGF-ß3 was also significantly elevated. In contrast, expression of TGF-ß1 showed no increase. Instead, expression levels of SMAD2 and SMAD4 were increased. Elevations of CD45RO, TNF-α and IL-4 and reduction of IL-10 were observed. In immature hypertrophic scars, IGF-1R and insulin-degrading enzyme expression were increased. Increased apoptosis was observed in immature stages of hypertrophic scars but not in mature stages. Elevations of TGF-ß3, SMAD2 and SMAD4 in hypertrophic scars and increase of IGF-1R in immature stages may give some clues for acne hypertrophic scar formation.

Transforming growth factor-ß (TGF-ß) signaling in healthy human fetal skin: a descriptive study.

BACKGROUND: TGF-ß plays an important role in growth and development but is also involved in scarring and fibrosis. Differences for this growth factor are known between scarless fetal wound healing and adult wound healing. Nonetheless, most of the data in this area are from animal studies or in vitro studies and, thus, information about the human situation is incomplete and scarce. OBJECTIVE: The aim of this study was to compare the canonical TGF-ß signaling in unwounded human fetal and adult skin. METHODS: Q-PCR, immunohistochemistry, Western Blot and Luminex assays were used to determine gene expression, protein levels and protein localization of components of this pathway in healthy skin. RESULTS: All components of the canonical TGF-ß pathway were present in unwounded fetal skin. Compared to adult skin, fetal skin had differential concentrations of the TGF-ß isoforms, had high levels of phosphorylated receptor-Smads, especially in the epidermis, and had low expression of several fibrosis-associated target genes. Further, the results indicated that the processes of receptor endocytosis might also differ between fetal and adult skin. CONCLUSION: This descriptive study showed that there are differences in gene expression, protein concentrations and protein localization for most components of the canonical TGF-ß pathway between fetal and adult skin. The findings of this study can be a starting point for further research into the role of TGF-ß signaling in scarless healing.

[Tetrachlorodibenzo-p-dioxin-induced cleft palate because of partial loss of cell polarity to interfere with apoptosis during early developmental stage].

OBJECTIVE: In this study, folic acid (FA) was tested for antiteratogenic effects on Tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate in fetal mice. METHODS: In the present study, pregnant mice were dosed with TCDD 24 µg/kg and with or without FA 5 mg/kg body weight on gestation day 10. Control group mice received sesame oil 50 ml/kg body weight on gestation day(GD)10. The mice were sacrificed on GD12.5, GD13.5, GD14.5, GD15.5 and GD16.5. From each pregnant mouse on GD16.5, embryos were obtained to examine under a dissecting microscope, and routine histology was performed for detection and classification of palatal clefts. The fetuses were prepared for histologic examination, scanning electron microscope and TdT-mediated X-dUTP nick and labeling (TUNEL). On GD12.5, GD13.5, GD14.5 and GD15.5. Meanwhile, real-time (RT)-PCR was employed to detect the mRNA expression levels about arylhydrocarbon receptor (AHR) and transforming growth factor (TGF)ß3 in this animal model. RESULTS: Total frequencies of clefts were 70.2% in TCDD group(group B) and 66.3% in TCDD+FA group(group C) in relation to control fetuses(group A). Filopodia disappeared completely at the medial edge epithelia surface on GD15.5 (group A), GD12.5 (group B) and GD14.5 (group C). the RT-PCR results showed that TGF -ß3 expression was down-regulated on GD13.5 and GD14.5 compared to the control. CONCLUSIONS: It is found that folic acid has no protects agaist 2.3.7.8-TCDD-indued cleft palate in the experiment. Meanwhile, TCDD repressed the TGF-ß3 expression during the palatal development. Anormal apoptosis was induced by 2, 3, 7, 8-TCDD at the medial edge epithelia (MEE) during the early development stage.

Molecular events on tooth socket healing in diabetic rabbits.

Healing of extraction sockets involves complex cellular events such as repair and regeneration of tissue. These events are precisely controlled and regulated by specific signalling molecules such as transforming growth factor beta (TGF-ß), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), and insulin-like growth factor (IGF), which are well-conserved proteins involved in the initial response to injury and repair in soft and hard tissues. We studied 48 rabbits, which were divided into 3 groups of 16 each: the control group, the untreated diabetic group, and the insulin-treated diabetic group. The lower incisor of each rabbit was extracted and, after 2, 10, 20, and 30 days of healing, the expression of TGFß-3, VEGF, IGF-1R, and BMP-4 in the sockets was measured immunohistochemically. Rabbits with untreated diabetes expressed less TGFß-3 than the other groups throughout the healing periods, whereas IGF-1R expression was higher than that in the other groups. This increase in IGF-1R expression was responsible for increasing the healing time in rabbits in the untreated group. The healing of bone in diabetic rabbits that were not treated with insulin was prolonged because of a delay in the onset of cell proliferation and osteoblast differentiation, and the insulin treatment had a direct effect on the expression of TGFß-3 and IGF-1R, which accelerated healing of the socket.

Repeated intrauterine exposures to inflammatory stimuli attenuated transforming growth factor-ß signaling in the ovine fetal lung.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is one of the most common complications after preterm birth and is associated with intrauterine exposure to bacteria. Transforming growth factor-ß (TGFß) is implicated in the development of BPD. OBJECTIVES: We hypothesized that different and/or multiple bacterial signals could elicit divergent TGFß signaling responses in the developing lung. METHODS: Time-mated pregnant Merino ewes received an intra-amniotic injection of lipopolysaccharide (LPS) and/or Ureaplasma parvum serovar 3 (UP) at 117 days' and/or 121/122 days' gestational age (GA). Controls received an equivalent injection of saline and or media. Lambs were euthanized at 124 days' GA (term = 150 days' GA). TGFß1, TGFß2, TGFß3, TGFß receptor (R)1 and TGFßR2 protein levels, Smad2 phosphorylation and elastin deposition were evaluated in lung tissue. RESULTS: Total TGFß1 and TGFß2 decreased by 24 and 51% after combined UP+LPS exposure, whereas total TGFß1 increased by 31% after 7 days' LPS exposure but not after double exposures. Alveolar expression of TGFßR2 decreased 75% after UP, but remained unaltered after double exposures. Decreased focal elastin deposition after single LPS exposure was prevented by double exposures. CONCLUSIONS: TGFß signaling components and elastin responded differently to intrauterine LPS and UP exposure. Multiple bacterial exposures attenuated TGFß signaling and normalized elastin deposition.

Downregulated gene expression of TGF-ßs in diabetic oral wound healing.

BACKGROUND: Healing of tooth extraction sockets in poorly controlled diabetic patients is often delayed and accompanied by severe infection. The exact cellular and molecular mechanisms underlying the pathogenesis of this complication are still not fully understood. OBJECTIVES: The purpose of this study was to investigate molecular changes associated with delayed oral wound healing in diabetes. MATERIALS AND METHODS: Six to eight weeks old male type 2 diabetes and age matched control inbred mice were used and maxillary molar tooth extractions were performed. At 4 and 7 days after tooth extraction, the edentulous mucosa of the mice were harvested, and analyzed for histology and gene expression of key wound healing factors. RESULTS: In the diabetic model, histological analysis showed that epithelial tissue migration for wound closure was delayed after tooth extraction compared to the control. Quantitative real-time PCR revealed that expression of the TGF-ß1, TGF-ß2, TGF-ß3, TGFßRII and TGFßRIII genes was significantly downregulated in the diabetic model at 4 and 7 days after tooth extraction. CONCLUSION: These results suggest that delayed wound healing of oral mucosa in diabetes may be associated with decreased expression levels of these regulatory genes which play important roles in controlling epithelial wound closure.

Time-coordinated prevalence of extracellular HGF, FGF2 and TGF-ß3 in crush-injured skeletal muscle.

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon-guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early-differentiated satellite cells up-regulate a secreted neural chemorepellent Sema3A upon in vivo muscle-crush injury. The study also revealed that Sema3A expression is up-regulated in primary satellite-cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)-ß2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time-course of extracellular HGF, FGF2 and TGF-ß3 concentrations after crush-injury of Gastrocnemius muscle in the rat lower hind-limb, using a combination of a non-homogenization/non-spin extraction of extracellular wound fluids and enhanced chemiluminescence-Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2-8 days post-crush, whereas active TGF-ß3 increases after 12 days, providing a better understanding of the time-coordinated levels of HGF, FGF2 and TGF-ß3 that drive regulation of Sema3A expression during regenerative intramuscular moto-neuritogenesis.

Evidence for transforming growth factor-beta 3 gene polymorphism in nonsyndromiccleft lip and palate patients from indian sub-continent

Objectives: Orofacial clefts are major human birth defects with complex etiology. Previous studies have proposed Transforming growth factor - beta 3 (TGF-Beta3) gene as a key player in contributing to non-syndromic cleft lip and palate, however none of the studies have yet included Indian population. Hence this study was designed to detectTGF-Beta3 gene polymorphism in nonsyndromic cleft lip and palate patients from Indian population which is genetically distinct from previously studied populations. Study Design: Peripheral blood samples of forty non-syndromic cleft lip and palate patients and forty unaffected individuals were collected for a case - control study design. Ethical clearance from the institutional review board and informed consent from all subjects was obtained. DNA extracted from the cases and controls was amplified using polymerase chain reaction (PCR) with TGF-Beta3 specific primers. The obtained fragments were sequenced and TGF-Beta3 gene polymorphisms were assessed based on the number of CA repeats. Results: Chi -square test was used to compare the case and control groups. Results showed a significant difference in the number of CA repeats between the case and the control groups (p=0.01).Conclusion: This study confirms the crucial role of TGF-Beta3 in the fusion of palatal shelves during development and further, provides novel evidence of TGF-Beta3 gene polymorphism in the etiology of nonsyndromic cleft lip and palate in Indian subpopulation (AU)

Biochemical changes during goiter induction by methylmercaptoimidazol and inhibition by delta-iodolactone in rat.

BACKGROUND: We have demonstrated that the administration of delta-iodolactone (i.e., 5-iodo-delta lactone) of arachidonic acid (IL-delta), a mediator in thyroid autoregulation, prevents goiter induction by methylmercaptoimidazol (MMI) in rats. Other studies have shown that transforming growth factor beta-1 (TGF-beta1) mimics some of the actions of excess iodide, but its participation in autoregulation is disputed. The present studies were performed to test the hypotheses that IL-delta decreases thyroid growth by inhibition of cell proliferation and/or by stimulation of apoptosis due to oxidative stress, that TGF-beta is stimulated by an excess of iodide and by IL-delta, and that c-Myc and c-Fos expression are upregulated during goiter induction and downregulated during goiter inhibition. METHODS: Rats were treated with MMI alone or together with iodide or IL-delta. Thyroid weight, cell number, cell proliferation, apoptosis, and oxidative stress were determined. Proliferating cell nuclear antigen (PCNA), TGF-beta1, TGF-beta3, c-Myc, and c-Fos were measured by Western blot. RESULTS: MMI caused a progressive increase in thyroid weight accompanied by an increase in cell number, asymmetry of the ploidy histograms, and PCNA, c-Fos, and c-Myc expression. In addition, an early increase of apoptosis was observed. Peroxides as well as glutathione peroxidase and catalase activities were also increased in goitrous animals. The inhibitory action of IL-delta on goiter formation was accompanied by the inhibition of cell proliferation evidenced by a significant decrease in cell number, PCNA expression, and asymmetry of the ploidy histograms. A transient stimulation of apoptosis after 7 days of treatment was also observed. MMI administration stimulated TGF-beta1 but not TGF-beta3 synthesis. IL-delta alone caused a slight increase of TGF-beta3 but not TGF-beta1, whereas potassium iodide (KI) stimulated both isoforms and MMI reversed KI effect on TGF-beta1 expression but not on TGF-beta3. CONCLUSIONS: The goiter inhibitory action of IL-delta is due to the inhibition of cell proliferation and the transient stimulation of apoptosis. This latter action does not involve oxidative stress. TGF-beta1 does not play a role in the autoregulatory pathway mediated by IL-delta. Iodide stimulates TGF-beta3 without the need of being organified. These results suggest that there may be more than one pathway involved in the autoregulatory mechanism.

Developmental changes in cellular and extracellular structural macromolecules in the secondary palate and in the nasal cavity of the mouse.

The aim of this study was to analyse the hitherto largely unknown expression patterns of some specific cellular and extracellular molecules during palate and nasal cavity development. We showed that epithelia of the developing palate and the vomerine epithelium express similar sets of structural proteins. With the exception of keratin 15, which becomes barely detectable in the elevated palatal shelves, nearly all of these proteins become upregulated at the presumptive areas of fusion and in the adhering epithelia of the palate and nasal septum. In vivo and in vitro analyses indicated that reduction in the amount of keratin 15 protein is independent of Tgfbeta-Alk5 signalling. Foxa1 expression also highlighted the regionalization of the palatal and nasal epithelia. Owing to the lack of reliable markers of the palatal periderm, the fate of peridermal cells has been controversial. We identified LewisX/stage-specific embryonic antigen-1 as a specific peridermal marker, and showed that numerous peridermal cells remain trapped in the medial epithelial seam (MES). The fate of these cells is probably apoptosis together with the rest of the MES cells, as we provided strong evidence for this event. Heparan sulphate, chondroitin-6-sulphate, and versican displayed dynamically changing distribution patterns. The hitherto-unknown innervation pattern of the developing palate was revealed. These findings may be of value for unravelling the pathogenesis of palatal clefting.

Expression of decorin in esophageal cancer in relation to the expression of three isoforms of transforming growth factor-beta (TGF-beta1, -beta2, and -beta3) and matrix metalloproteinase-2 activity.

To determine the role of the reactive stroma in cancer progression, we investigated decorin (DCN) and transforming growth factor-beta (TGF-beta expression, and matrix metalloproteinase-2 (MMP-2) activity in the tumorous esophagus. We found statistically insignificantly decreased levels of DCN expression in the pathological tissues. No obvious alterations in TGF-beta expression were noticed. The highly significant increase in MMP-2 activity in cancers did not result in elevated levels of TGF-beta dimers. Therefore, the system of TGF-beta liberation from its complex with DCN by activated MMP-2 does not seem to contribute to esophageal cancerogenesis, although this hypothesis should be reevaluated with a larger study group.

Inhibition of proliferation and transforming growth factor beta3 protein expression by peroxisome proliferators-activated receptor gamma ligands in human uterine leiomyoma cells.

BACKGROUND: Rosiglitazone is known as the most potent and specific peroxisome proliferators-activated receptor gamma (PPAR-gamma) ligand. It has potentially far-reaching effects on pathophysiological processes, from cancer to atherosclerosis and diabetes. However, it is not clear whether rosiglitazone affects the protein expression of transforming growth factor beta3 (TGF-beta3) and the cell proliferation in human uterine leiomyoma cells in vitro. METHODS: Human uterine leiomyoma tissues were dissected and cultured. Cells were divided into 5 groups: one control group and other four groups with different concentrations of rosiglitazone (10(-7), 10(-8), 10(-9) and 10(-10) mol/L). Cells were cultured for 72 hours in serum-free Dulbecco's modified Eagle's medium. MTT reduction assay was used to detect the cell proliferation. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of PPAR-gamma and TGF-beta3. Immunofluorescence staining was used to detect the expressions of PPAR-gamma and TGF-beta3 proteins. RESULTS: MTT reduction assay indicated that the treatment with rosiglitazone (from 10(-7) to 10(-9) mol/L) resulted in an inhibition of the cell growths after 72 hours (P < 0.01). RT-PCR analysis revealed that 10(-7) mol/L rosiglitazone significantly affected the gene expression at 72-hour: PPAR-gamma mRNA expression was up-regulated and TGF-beta3 mRNA was down-regulated and rosiglitazone at the concentration of 10(-7) mol/L affected these most effectively (P < 0.01). Immunofluorescence staining demonstrated that treatment with 10(-7) mol/L rosiglitazone resulted in the significant changes of PPAR-gamma and TGF-beta3 protein expressions compared with the other treatment groups and the control group at 72-hour (P < 0.01). All the effects of rosiglitazone on uterine leiomyoma cells were dose- and time-dependent in vitro. CONCLUSIONS: The present study demonstrates that the PPAR-gamma activator, rosiglitazone, inhibits the cell proliferation partly through the regulations of PPAR-gamma and TGF-beta3 expressions. The cross-talk between the signal pathways of PPAR-gamma and TGF-beta3 may be involved in the process.

The expression of transforming growth factor beta in pregnant rat myometrium is hormone and stretch dependent.

From a quiescent state in early pregnancy to a highly contractile state in labor, the myometrium displays tremendous growth and remodeling. We hypothesize that the transforming growth factor beta (TGFbeta) system is involved in the differentiation of pregnant myometrium throughout gestation and labor. Furthermore, we propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial TGFbetas. The expression of TGFbeta1-3 mRNAs and proteins was examined by real-time PCR, Western immunoblot, and localized with immunohistochemistry in the rat uterus throughout pregnancy and labor. Tgfbeta1-3 genes were expressed differentially in pregnant myometrium. Tgfbeta2 gene was not affected by pregnancy, whereas the Tgfbeta1 gene showed a threefold increase during the second half of gestation. In contrast, we observed a dramatic bimodal change in Tgfbeta3 gene expression throughout pregnancy. Tgfbeta3 mRNA levels first transiently increased at mid-gestation (11-fold on day 14) and later at term (45-fold at labor, day 23). Protein expression levels paralleled the changes in mRNA. Treatment of pregnant rats with the progesterone (P4) receptor antagonist RU486 induced premature labor on day 19 and increased Tgfbeta3 mRNA, whereas artificial maintenance of elevated P4 levels at late gestation (days 20-23) caused a significant decrease in the expression of Tgfbeta3 gene. In addition, Tgfbeta3 was up-regulated specifically in the gravid horn of unilaterally pregnant rats subjected to a passive biological stretch imposed by the growing fetuses, but not in the empty horn. Collectively, these data indicate that the TGFbeta family contributes in the regulation of myometrial activation at term integrating mechanical and endocrine signals for successful labor contraction.