TRIM28 prevents autoinflammatory T cell development in vivo.

TRIM28 is a component of heterochromatin complexes whose function in the immune system is unknown. By studying mice with conditional T cell-specific deletion of TRIM28 (CKO mice), we found that TRIM28 was phosphorylated after stimulation via the T cell antigen receptor (TCR) and was involved in the global regulation of CD4(+) T cells. The CKO mice had a spontaneous autoimmune phenotype that was due in part to early lymphopenia associated with a defect in the production of interleukin 2 (IL-2) as well as incomplete cell-cycle progression of their T cells. In addition, CKO T cells showed derepression of the cytokine TGF-ß3, which resulted in an altered cytokine balance; this caused the accumulation of autoreactive cells of the T(H)17 subset of helper T cells and of Foxp3(+) T cells. Notably, CKO Foxp3(+) T cells were unable to prevent the autoimmune phenotype in vivo. Our results show critical roles for TRIM28 in both T cell activation and T cell tolerance.

The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes.

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.

Egr2 and Egr3 in regulatory T cells cooperatively control systemic autoimmunity through Ltbp3-mediated TGF-ß3 production.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by autoantibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic autoimmunity in mice and humans. We have previously identified a subpopulation of CD4+ regulatory T cells, CD4+CD25-LAG3+ cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-ß3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-ß3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously developed an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4+CD25-LAG3+ cells from Egr2/3DKO mice completely lost the capacity to produce TGF-ß3, the excessive germinal center reaction in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4+CD25-LAG3+ cells or treatment with a TGF-ß3-expressing vector. Intriguingly, latent TGF-ß binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-ß3 production from CD4+CD25-LAG3+ cells. Because Egr2 and Egr3 did not demonstrate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-ß3 production by CD4+CD25-LAG3+ cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE.

Effect of TGFß1, TGFß3 and keratinocyte conditioned media on functional characteristics of dermal fibroblasts derived from reparative (Balb/c) and regenerative (Foxn1 deficient; nude) mouse models.

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin's pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor ß family (TGFß) expression showed significantly higher levels of Tgfß3 transcript between nude and Balb/c mice but no differences were detected for Tgfß1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFß3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFß3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.

Effect of tamoxifen on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs.

End-to-end anastomosis in the treatment for bile duct injury during laparoscopic cholecystectomy has been associated with stricture formation. The aim of this study was to experimentally investigate the effect of oral tamoxifen (tmx) treatment on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs. Twenty-six pigs were divided into three groups [sham (n = 8), control (n = 9) and tmx (n = 9)]. The common bile ducts were transected and anastomosed in the control and tmx groups. Tmx (40 mg/day) was administered orally to the tmx group, and the animals were euthanized after 60 days. Fibrosis was analysed by Masson's trichrome staining. Picrosirius red was used to quantify the total collagen content and collagen type I/III ratio. mRNA expression of transforming growth factor (TGF)-ß1, -ß2 and -ß3 was quantified using real-time polymerase chain reaction (qRT-PCR). The control and study groups exhibited higher fibrosis than the sham group, and the study group showed lower fibrosis than the control group (P = 0.011). The control and tmx groups had higher total collagen content than the sham group (P = 0.003). The collagen type I/III ratio was higher in the control group than in the sham and tmx groups (P = 0.015). There were no significant differences in the mRNA expression of TGF-ß1, -ß2 and -ß3 among the groups (P > 0.05). Tmx decreased fibrosis and prevented the change in collagen type I/III ratio caused by the procedure.

Transforming growth factor-ß3 regulates cell junction restructuring via MAPK-mediated mRNA destabilization and Smad-dependent protein degradation of junctional adhesion molecule B (JAM-B).

Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the blood-testis barrier (BTB) as well as between Sertoli and germ cells at the apical ectoplasmic specializations (ES) in the testis. The expression of JAM-B is tightly regulated to modulate the passage of spermatocytes across the BTB as well as the release of mature spermatozoa from the seminiferous epithelium. Transforming growth factor beta (TGF-ß) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the effects of TGF-ß3 on the expression of JAM-B as well as the underlying mechanisms on how TGF-ß3 regulates JAM-B expression to facilitate the disassembly of the BTB and apical ES. Our results revealed that TGF-ß3 suppresses JAM-B at post-transcriptional and post-translational levels. Inhibitor, siRNA knockdown and co-immunoprecipitation have shown that TGF-ß3 induces JAM-B protein degradation via ubiquitin-proteasome pathway. Immunofluorescence staining further confirmed that blockage of ubiquitin-proteasome pathway could abrogate TGF-ß3-induced loss of JAM-B at the cell-cell interface. siRNA knockdown and immunofluorescence staining also demonstrated that activation of Smad signaling is required for TGF-ß3-induced JAM-B protein degradation. In addition, TGF-ß3 reduces JAM-B mRNA levels, at least in part, via post-transcriptional regulation. mRNA stability assay has confirmed that TGF-ß3 promotes the degradation of JAM-B transcript and TGF-ß3-mediated mRNA destabilization requires the activation of ERK1/2 and p54 JNK signal cascades. Taken together, TGF-ß3 significantly downregulates JAM-B expression via post-transcriptional and post-translational modulation and results in the disruption of BTB and apical ES.

Topical photodynamic therapy following excisional wounding of human skin increases production of transforming growth factor-ß3 and matrix metalloproteinases 1 and 9, with associated improvement in dermal matrix organization.

BACKGROUND: Animal studies report photodynamic therapy (PDT) to improve healing of excisional wounds; the mechanism is uncertain and equivalent human studies are lacking. OBJECTIVES: To explore the impact of methyl aminolaevulinate (MAL)-PDT on clinical and microscopic parameters of human cutaneous excisional wound healing, examining potential modulation through production of transforming growth factor (TGF)-ß isoforms. METHODS: In 27 healthy older men (60-77 years), a 4-mm punch biopsy wound was created in skin of the upper inner arm and treated with MAL-PDT three times over 5 days. An identical control wound to the contralateral arm was untreated and both wounds left to heal by secondary intention. Wounds were re-excised during the inflammatory phase (7 days, n = 10), matrix remodelling (3 weeks, n = 8) and cosmetic outcome/dermal structure (9 months, n = 9). Production of TGF-ß1, TGF-ß3 and matrix metalloproteinases (MMPs) was assessed by immunohistochemistry alongside microscopic measurement of wound size/area and clinical assessment of wound appearance. RESULTS: MAL-PDT delayed re-epithelialization at 7 days, associated with increased inflammation. However, 3 weeks postwounding, treated wounds were smaller with higher production of MMP-1 (P = 0·01), MMP-9 (P = 0·04) and TGF-ß3 (P = 0·03). TGF-ß1 was lower than control at 7 days and higher at 3 weeks (both P = 0·03). At 9 months, MAL-PDT-treated wounds showed greater, more ordered deposition of collagen I, collagen III and elastin (all P < 0·05). CONCLUSIONS: MAL-PDT increases MMP-1, MMP-9 and TGF-ß3 production during matrix remodelling, ultimately producing scars with improved dermal matrix architecture.

The perivascular niche governs an autoregulatory network to support breast cancer metastasis.

Unravelling the autoregulatory network that induces and maintains cancer stem cell state may provide novel effective therapies against breast cancer metastasis. The perivascular niche develops elements that initiate the autoregulatory machine to induce and maintain cancer stem cells, but not EMT, among newly arrived tumour cells. Inhibition of one or more primary key elements that trigger this circuit may result in the prevention or cure of breast cancer metastasis.

Nuclear export factor 3 is involved in regulating the expression of TGF-ß3 in an mRNA export activity-independent manner in mouse Sertoli cells.

The NXF (nuclear export factor) family members are implicated in the transport of mRNA from the nucleus to the cytoplasm. Recently, some members of the NXF family have been reported to play divergent functional roles, such as post-transcriptional regulation, translational control, regulation of mRNA stability and trafficking. However, little is known about the roles of NXF3 in spermatogenesis. In the present study, we found that mouse NXF3, specifically expressed in principal cells in segment II of the caput epididymis, as well as Sertoli cells in the mouse testis, was required to mediate TGF-ß (transforming growth factor ß)-induced down-regulation of Tgfb3/TGF-ß3 mRNA expression and protein secretion in Sertoli cells. In addition, NXF3 was also involved in TGF-ß-induced transcriptional regulation of other genes associated with Sertoli cell maturation and the restructuring of the Sertoli cell BTB (blood-testis barrier), such as Gata1 (GATA-binding protein 1), Wt1 (Wilms's tumour homologue 1), Cldn11 (claudin11) and Cdkn1a (cyclin-dependent kinase inhibitor 1A or p21(Cip1)). The transcriptional regulation of NXF3 was mediated through physical interaction with STRAP (serine/threonine kinase receptor-associated protein), where NXF3 inhibited the complex formation among Smad7, STRAP and activated type I TGF-ß receptor. Taken together, our data provide mechanistic insights into the roles of NXF3 in TGF-ß-mediated expression of Tgfb3 and other genes. NXF3 may be implicated in Sertoli cell maturation and the extensive restructuring of the Sertoli cell BTB.

Analysis of expression of TNF-alpha and TGF-beta3 in intrinsic ureteropelvic junction obstruction.

OBJECTIVES: Ureteropelvic junction (UPJ) obstruction is of critical importance to understand the histopathology of UPJ obstruction in terms of therapy planning and follow-up. For this purpose, our study was conducted with TNF-α and TGF-ß markers to investigate possible underlying problems in intrinsic UPJ obstruction. METHODS: Of the patients who had undergone surgery in our clinic, 36 UPJ segments of patients who had undergone dismembered pyeloplasty surgery due to UPJ obstruction and 14 UPJ segments of the patients who had undergone nephrectomy were collected to form 2 groups. All histological sections were examined by applying immunohistochemical transforming growth factor beta 3 (TGF-ß3) and tumour necrosis factor alpha (TNF-α) monoclonal antibody dyes. RESULTS: The mean staining values for TNF-α in mucosal tissue and mucosa were 0.53±0.84 and 0.58±0.84, respectively in the obstruction group, whereas the values observed in the control group were 0.86±0.36 and 0.93±0.47, respectively. While the mean staining values in the obstruction group in mucosal tissue and mucosa for TGF-ß3 were 1.75±0.73 and 2.17±0.77, respectively, the values established in the control group were 1.14±0.66 and 1.43±0.93, respectively. The difference between the obstruction and control groups were statistically significant for both values (p<0.05). CONCLUSION: Only a limited number of studies have been carried out on this particular issue. Data from the present study indicate that TGF-ß3 and TNF-α may play a role in the histopathogenesis of UPJ obstruction (Tab. 1, Fig. 1, Ref. 18).

A synthetic gene increases TGFß3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule.

Human transforming growth factor-ß3 (TGFß3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFß3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFß3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFß3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFß3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFß3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFß3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFß3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFß3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFß3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFß3.

An improved strategy of TGFß3 expression in Escherichia coli: Exploiting folding modulators for a switch from misfolded to folded form.

Transforming growth factor beta 3 (TGFß3) exhibits a complex native structure featuring the presence of multiple disulfide bonds forming the active dimer. Consequently, its heterologous expression in microbial system invariably leads to inclusion body (IB) formation. In this study, we observed an interesting phenomenon of switching a significant fraction of misfolded TGFß3 to folded form by modulating the cellular protein folding machinery. We carried out co-expression experiments with chaperones and demonstrated the requirement of a coordinated action of DnaK-DnaJ-GrpE and GroESL, to achieve the native soluble conformation of TGFß3, during over-expression in E. coli. The novelty of this study lies in the fact that orchestration of a group of chaperones to work in concert for efficient folding and assembly of TGFß3-like cytokines has not been widely explored. Additionally, we have also demonstrated that presence of osmolytes (sorbitol or trehalose) in the growth media have an appreciable impact on the solubility of TGFß3. We have further shown a synergism between the effects of molecular chaperone and osmolytes on the solubility of TGFß3. We have confirmed the functionality of soluble TGFß3 by performing binding interactions with its cognate receptor TßRII. Our study delineates the fact that an effective combination of chaperones or optimum concentration of compatible osmolyte, can efficiently abrogate competing aggregation pathways and help attain the native conformation of a cysteine rich cytokine in a facile manner.

Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients.

BACKGROUND: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). PATIENTS AND METHODS: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFbeta1, TGFbeta2, TGFbeta3 in oral SCC. RESULTS: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFbeta isoforms, we found that Smad2 mRNA and TGFbeta1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFbeta1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. CONCLUSION: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFbeta signaling should be better clarified in the future.

Therapeutic Effects of Focused Ultrasound on Expression of Notch1, C-Fos and Transforming Growth Factor-ß3 in Vulvar Skin of SD Rats with Vulvar Lichen Simplex Chronicus.

The purpose of this study was to investigate the therapeutic effects of focused ultrasound on the expression of notch1, c-fos and transforming growth factor-ß3 (TGF-ß3) in genital skin of SD rats with vulvar lichen simplex chronicus (LSC). Fifty-six female SD rats with LSC were randomly divided into therapy and sham groups. The therapy group was exposed to focused ultrasound. The sham group received the same therapy with an instrument that had no power output. Four wk after a singly focused ultrasound therapy, histologic analyses revealed that recovered SD rats accounted for 75% of SD rats in the therapy group and 10.7% in the sham group. Total collagen fiber density in the superficial layer of dermis in the therapy group was significantly lower than that in the sham group. Notch1 and c-fos protein expression in the therapy group was significantly lower than that in the sham group, with the opposite effect present for TGF-ß3. Focused ultrasound therapy may inhibit superficial collagen fibrosis in the dermis by affecting expression of notch1, c-fos and TGF-ß3 in vulvar skin tissue and consequently reduce the recurrence rate of LSC.

Expression of integrin alphavbeta6 and TGF-beta in scarless vs scar-forming wound healing.

Oral mucosal wounds heal with reduced scar formation compared with skin. The epithelial integrin alphavbeta6 is induced during wound healing, and it can activate fibrogenic transforming growth factor beta1 (TGF-beta1) and anti-fibrogenic TGF-beta3 that play key roles in scar formation. In this study, expression of beta6 integrin and members of the TGF-beta pathway were studied in experimental wounds of human gingiva and both gingiva and skin of red Duroc pigs using real-time PCR, gene microarrays, and immunostaining. Similar to human wounds, the expression of beta6 integrin was induced in the pig wounds 7 days after wounding and remained upregulated >49 days. The alphavbeta6 integrin was colocalized with both TGF-beta isoforms in the wound epithelium. Significantly higher expression levels of beta6 integrin and TGF-beta1 were observed in the pig gingival wounds compared with skin. Early gingival wounds also expressed higher levels of TGF-beta3 compared with skin. The spatio-temporal colocalization of alphavbeta6 integrin with TGF-beta1 and TGF-beta3 in the wound epithelium suggests that alphavbeta6 integrin may activate both isoforms during wound healing. Prolonged expression of alphavbeta6 integrin along with TGF-beta3 in the gingival wound epithelium may be important in protection of gingiva from scar formation.

Infarct size is increased in female post-MI rats treated with rapamycin.

Rapamycin represents a recognized drug-based therapeutic approach to treat cardiovascular disease. However, at least in the female heart, rapamycin may suppress the recruitment of putative signalling events conferring cardioprotection. The present study tested the hypothesis that rapamycin-sensitive signalling events contributed to the cardioprotective phenotype of the female rat heart after an ischemic insult. Rapamycin (1.5 mg/kg) was administered to adult female Sprague-Dawley rats 24 h after complete coronary artery ligation and continued for 6 days. Rapamycin abrogated p70S6K phosphorylation in the left ventricle of sham rats and the noninfarcted left ventricle (NILV) of 1-week postmyocardial-infarcted (MI) rats. Scar weight (MI 0.028 +/- 0.006, MI+rapamycin 0.064 +/- 0.004 g) and surface area (MI 0.37 +/- 0.08, MI+rapamycin 0.74 +/- 0.03 cm2) were significantly larger in rapamycin-treated post-MI rats. In the NILV of post-MI female rats, rapamycin inhibited the upregulation of eNOS. Furthermore, the increased expression of collagen and TGF-beta3 mRNAs in the NILV were attenuated in rapamycin-treated post-MI rats, whereas scar healing was unaffected. The present study has demonstrated that rapamycin-sensitive signalling events were implicated in scar formation and reactive fibrosis. Rapamycin-mediated suppression of eNOS and TGF-beta3 mRNA in post-MI female rats may have directly contributed to the larger infarct and attenuation of the reactive fibrotic response, respectively.

Collagen I promotes epithelial-to-mesenchymal transition in lung cancer cells via transforming growth factor-beta signaling.

Epithelial-to-mesenchymal transition (EMT) is a fundamental biological process whereby epithelial cells lose their polarity and undergo a transition to a mesenchymal phenotype. When cancer cells invade adjacent tissues, they use a mechanism akin to EMT, and understanding the molecular mechanisms that drive this transition will facilitate studies into new targets for prevention of metastasis. Extracellular stimuli, such as growth factors, and their cytosolic effectors cooperate to promote EMT. In highly fibrotic cancers like lung cancer, it is thought that extracellular matrix molecules, including collagen, can initiate signals that promote EMT. Here, we present data showing that collagen I induces EMT in non-small cell lung cancer cell lines, which is prevented by blocking transforming growth factor (TGF)-beta3 signaling. In addition, we show that collagen I-induced EMT is prevented by inhibitors of phosphoinositide 3-kinase and extracellular signal-related kinase signaling, which promotes transcription of TGF-beta3 mRNA in these cells. Thus, our data are consistent with the hypothesis that collagen I induces EMT in lung cancer cells by activating autocrine TGF-beta3 signaling. Epidermal growth factor also seems to initiate EMT via a TGF-dependent mechanism.

Site-specific production of TGF-beta in oral mucosal and cutaneous wounds.

Wound healing in the oral mucosa is clinically distinguished by rapid healing and lack of scar formation compared with dermal wounds. Mechanisms of favorable mucosal healing are yet to be elucidated. Utilizing a murine model of equivalent-size mucosal and skin wounds, we verified the rapid reepithelializaton and reduction in scarring of oral wounds reported in humans. Collagen fibrillar structure in oral wounds rapidly approached the size of normal collagen fibrils, while the collagen ultrastructure in skin remained immature through the later phases of healing. To determine whether the transforming growth factor-beta (TGF-beta) contributes to the lack of scar formation in oral mucosa, we compared the expression and production in oral and skin wounds. The RNase protection assay demonstrated significantly lower levels of TGF-beta1 expression in oral wounds compared with dermal wounds, and no changes were observed in the expression levels of TGF-beta2 or TGF-beta 3. ELISA analysis confirmed that oral wounds contained lower levels of TGF-beta1 levels compared with dermal wounds, along with a significant increase in the ratio of TGF-beta 3 to -beta1. These findings showed reduced scarring in oral wounds at the ultrastructural level, and provide evidence that site-specific differences in TGF-beta production contributes to the superior healing of oral wounds.

Glycated adducts induce mesothelial cell transdifferentiation: role of glucose and icodextrin dialysis solutions.

BACKGROUND: In peritoneal dialysis (PD), the peritoneum is exposed to intermediate Amadori adducts (AmAs) and advanced (AGE) glycated products of proteins. The aim of this study was to test the capacity of AmAs created in different PD solutions (PDSs) to elicit a fibroblast-like transdifferentiation of human peritoneal mesothelial cells (HPMCs) in culture. METHODS: HPMCs were incubated for 12 hours with AmA obtained by human serum albumin (HSA) incubated for 6 days with commercial 3.86% glucose (Glu), 1.36% Glu and 7.5% icodextrin (Ico) PDS. Mesenchymal (vimentin), epithelial (cadherin) and myofibroblastic (Type I collagen and alpha smooth muscle cell actin [ASMA]) markers were evaluated (RT-PCR, immunostaining and Western blot), as well as TGF-b3 synthesis (ELISA and Western blot). RESULTS: Ico-PDS was less active than 3.86% and 1.36% Glu-PDS in glycating albumin (p<0.001). AmA-HSA-Glu 3.86% and 1.36% induced a significantly higher increase in vimentin and Type I collagen mRNA expression than AmA-HSA-Ico (p<0.0001). By contrast, AmA-HSA-Glu 3.86% and 1.36% induced a reduction in cadherin mRNA expression which was significantly different from AmA- HSA-Ico (p<0.0001). RT-PCR data were confirmed by immunostaining and Western blot analysis. AmA-HSA-Glu 3.86% and 1.36% induced a significantly higher increase in ASMA mRNA expression than AmA-HSA-Ico (p<0.0001). AmA-HSA-Glu 3.86% and 1.36% stimulated ASMA and TGF-b3 synthesis which were significantly higher than AmA-HSA-Ico (p<0.001 and p<0.01, respectively). CONCLUSIONS: Our data suggest that Glu-PDS, but not Ico-PDS, can turn on the fibroblastic-like transdifferentiation in HPMCs, and this mechanism may result in peritoneal sclerosis.

Placental overexpression of transforming growth factor-beta3 in the HELLP syndrome.

OBJECTIVE: To evaluate the placental expression of transforming growth factor-beta3 (TGF-beta3) in patients with HELLP syndrome and pre-eclampsia compared to controls, and its correlation to Doppler velocimetry analysis of the utero-placental blood flow. STUDY DESIGN: Real-time PCR analysis was performed, after cesarean section, in placental samples from 10 women affected by HELLP syndrome, 10 women with pre-eclampsia and 10 controls. Pulsatility indices on Doppler waveform analysis from uterine and umbilical arteries were measured. RESULTS: The mean TGF-beta3 expression was significantly higher in patients with HELLP syndrome compared with the control group (p < 0.001), and no difference was observed in the pre-eclampsia group. TGF-beta3 expression correlated positively with umbilical PI (p < 0.001). CONCLUSIONS: TGF-beta3 may play a key role as regulator of a variety of cellular events occurring during HELLP syndrome, high local expression of this growth factor may be responsible for remodeling of the placental structure, which results in the dysfunction of maternal-fetal circulation.