Integrated actions of transforming growth factor-beta(1) and connective tissue growth factor in renal fibrosis

Matrix accumulation in the renal tubulointerstitium is predictive of a progressive decline in renal function. Transforming growth factor-beta(1) (TGF-beta(1)) and, more recently, connective tissue growth factor (CTGF) are recognized to play key roles in mediating the fibrogenic response, independently of the primary renal insult. Further definition of the independent and interrelated effects of CTGF and TGF-beta(1) is critical for the development of effective antifibrotic strategies. CTGF (20 ng/ml) induced fibronectin and collagen IV secretion in primary cultures of human proximal tubule cells (PTC) and cortical fibroblasts (CF) compared with control values (P < 0.005 in all cases). This effect was inhibited by neutralizing antibodies to either TGF-beta or to the TGF-beta type II receptor (TbetaRII). TGF-beta(1) induced a greater increase in fibronectin and collagen IV secretion in both PTC (P < 0.01) and CF (P < 0.01) compared with that observed with CTGF alone. The combination of TGF-beta(1) and CTGF was additive in their effects on both PTC and CF fibronectin and collagen IV secretion. TGF-beta(1) (2 ng/ml) stimulated CTGF mRNA expression within 30 min, which was sustained for up to 24 h, with a consequent increase in CTGF protein (P < 0.05), whereas CTGF had no effect on TGF-beta(1) mRNA or protein expression. TGF-beta(1) (2 ng/ml) induced phosphorylated (p)Smad-2 within 15 min, which was sustained for up to 24 h. CTGF had a delayed effect on increasing pSmad-2 expression, which was evident at 24 h. In conclusion, this study has demonstrated the key dependence of the fibrogenic actions of CTGF on TGF-beta. It has further uniquely demonstrated that CTGF requires TGF-beta, signaling through the TbetaRII in both PTCs and CFs, to exert its fibrogenic response in this in vitro model.

Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.

Either interleukin-12 or interferon-gamma can correct the dendritic cell defect induced by transforming growth factor beta(1) in patients with myeloma

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44(+)) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up-regulate the expression of the B7 co-stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor beta(1) (TGFbeta(1)) and interleukin (IL)-10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti-TGFbeta(1). As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up-regulation was reduced in both stage I and stage III. It was demonstrated that both IL-12 and interferon-gamma neutralized the failure to stimulate CD80 up-regulation by huCD40LT in vitro. IL-12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123-) suggesting that there would be a predominantly T-helper cell type response. The addition of IL-12 or interferon-gamma to future immunotherapy trials involving these patients should be considered.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.

Carvedilol blocks beta(2)- more than beta(1)-adrenoceptors in human heart

Objective: To understand the basis of the effectiveness of carvedilol in heart failure by determining its specific properties at human heart and beta(2)-adrenoceptors. Methods: The positive inotropic effects of noradrenaline (in the presence of the beta(2)-selective antagonist ICI118551) and adrenaline (in the presence of the beta(1)-selective antagonist CGP20712), mediated through beta(1)- and beta(2)-adrenoceptors, respectively, were investigated in atrial and ventricular trabeculae. The patch-clamp technique was used to investigate effects of noradrenaline and adrenaline on L-type Ca2+ current in human atrial myocytes. Results: Carvedilol was a 13-fold more potent competitive antagonist of the effects of adrenaline at 1 2-adrenoceptors (-logK(B) = 10.13 +/- 0.08) than of noradrenaline at beta(1)-adrenoceptors (-logK(B) = 9.02 +/- 0.07) in human right atrium. Chronic carvedilol treatment of patients with non-terminal heart failure reduced the inotropic sensitivity of atrial trabeculae to noradrenaline and adrenaline 5.6-fold and 91.2-fold, respectively, compared to beta(1)-blocker-treated patients, consistent with persistent preferential blockade of beta(2)-adrenoceptors. In terminal heart failure carvedilol treatment reduced 1.8-fold and 25.1-fold the sensitivity of right ventricular trabeculae to noradrenaline and adrenaline, respectively, but metoprolol treatment did not reduce the sensitivity to the catecholamines. Increases of current (I-Ca,I-L) produced by noradrenaline and adrenaline were not different in atrial myocytes obtained from non-terminal heart failure patients treated with metoprolol or carvedilol, consistent with dissociation of both beta-blockers from the receptors. Conclusions: Carvedilol blocks human cardiac beta(2)-adrenoceptors more than beta(1)-adrenoceptors, thereby conceivably contributing to the beneficial effects in heart failure. The persistent blockade of beta-adrenoceptors is attributed to accumulation of carvedilol in cardiac tissue. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Overexpression of transforming growth factor-beta is associated with increased hyaluronan content and impairment of repair in Marfan syndrome aortic aneurysm

Background-Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGF beta 1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34). Methods and Results-MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGF beta 1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGF beta 1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects = 5, versus control 8%, n = 3, P < 0.05; AF: 28%, n = 5 versus 7%, n = 5, P < 0.05; SF: 29%, n = 3 versus 4%, n = 3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and beta-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGF beta 1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or beta-3 integrin compared with controls. Conclusions-There was overexpression of TGF-beta in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.

High glucose-mediated effects on endothelial cell proliferation occur via p38 MAP kinase

The mitogen-activated protein ( MAP) kinases contribute to altered cell growth and function in a variety of disease states. However, their role in the endothelial complications of diabetes mellitus remains unclear. Human endothelial cells were exposed for 72 h to 5 mM ( control) or 25 mM ( high) glucose or 5 mM glucose plus 20 mM mannitol ( osmotic control). The roles of p38 and p42/44 MAP kinases in the high glucose-induced growth effects were determined by assessment of phosphorylated MAP kinases and their downstream activators by Western blot and by pharmacological inhibition of these MAP kinases. Results were expressed as a percentage ( means +/- SE) of control. High glucose increased the activity of total and phosphorylated p38 MAP kinase ( P < 0.001) and p42/44 MAP kinase ( P < 0.001). Coexposure of p38 MAP kinase blocker with high glucose reversed the antiproliferative but not the hypertrophic effects associated with high-glucose conditions. Transforming growth factor (TGF)-beta1 increased the levels of phosphorylated p38 MAP kinase, and p38 MAP kinase blockade reversed the antiproliferative effects of this cytokine. The high glucose-induced increase in phosphorylated p38 MAP kinase was reversed in the presence of TGF-beta1 neutralizing antibody. Although hyperosmolarity also induced antiproliferation (P < 0.0001) and cell hypertrophy (P < 0.05), there was no change in p38 activity, and therefore inhibition of p38 MAP kinase had no influence on these growth responses. Blockade of p42/44 MAP kinase had no effect on the changes in endothelial cell growth induced by either high glucose or hyperosmolarity. High glucose increased p42/44 and p38 MAP kinase activity in human endothelial cells, but only p38 MAP kinase mediated the antiproliferative growth response through the effects of autocrine TGF-beta1. High glucose-induced endothelial cell hypertrophy was independent of activation of the MAP kinases studied. In addition, these effects were independent of any increase in osmolarity associated with high-glucose exposure.

Short-term peaks in glucose promote renal fibrogenesis independently of total glucose exposure

Postprandial hyperglycemia is implicated as a risk factor predisposing to vascular complications. This study was designed to assess recurrent short-term increases in glucose on markers of renal fibrogenesis. Human renal cortical fibroblasts were exposed to fluctuating short-term (2 h) increases to 15 mM D-glucose, three times a day over 72 h, on a background of 5 mM D-glucose. To determine whether observed changes were due to fluctuating osmolality, identical experiments were undertaken with cells exposed to L-glucose. Parallel experiments were performed in cells exposed to 5 mM D-glucose and constant exposure to either 15 or 7.5 mM D-glucose. Fluctuating D-glucose increased extracellular matrix, as measured by proline incorporation ( P < 0.05), collagen IV ( P < 0.005), and fibronectin production ( P < 0.001), in association with increased tissue inhibitor of matrix metalloproteinase (MMP) ( P < 0.05). Sustained exposure to 15 mM D-glucose increased fibronectin ( P < 0.001), in association with increased MMP-2 ( P = 0.01) and MMP-9 activity ( P < 0.05), suggestive of a protective effect on collagen matrix accumulation. Transforming growth factor-beta(1) (TGF-beta(1)) mRNA was increased after short-term (90 min) exposure to 15 mM glucose (P < 0.05) and after 24-h exposure to 7.5 mM ? ( P < 0.05). Normalization of TGF-beta(1) secretion occurred within 48 h of constant exposure to an elevated glucose. Fluctuating L-glucose also induced TGF-beta(1) mRNA and a profibrotic profile, however, to a lesser extent than observed with exposure to fluctuating D-glucose. The results suggest that exposure to fluctuating glucose concentrations increases renal interstitial fibrosis compared with stable elevations in D-glucose. The effects are, in part, due to the inherent osmotic changes.

Regulatory T-cells infiltrate periodontal disease tissues

CD4(+) CD25(+) regulatory T ( Tr) cells are critical in regulating the immune response and thereby play an important role in the defense against infection and control of autoimmune diseases. Our previous studies demonstrated the involvement of autoimmune responses in periodontitis. The aim of this study was to identify CD4(+) CD25(+) Tr cells in periodontitis tissues and compare them with those in gingivitis tissues. Immunohistological analysis of CD4, CD25, and CTLA- 4 and the gene expression analysis of FOXP3, TGF- beta 1, and IL-10 on gingival biopsies revealed the presence of CD4(+) CD25(+) Tr cells in all tissues. In periodontitis, the percentage of CD4(+) CD25(+) Tr cells increased with increasing proportions of B-cells relative to T- cells. FOXP3, a characteristic marker for CD4(+) CD25(+) Tr cells, TGF- beta 1 and IL-10 were expressed more highly in periodontitis compared with gingivitis. These findings suggest that CD4(+) CD25(+) Tr cells and possibly other regulatory T- cell populations do exist and may play regulatory roles in periodontal diseases.

Cardiac chymase: pathophysiological role and therapeutic potential of chymase inhibitors

On release from cardiac mast cells, alpha-chymase converts angiotensin I (Ang I) to Ang II. In addition to Ang II formation, alpha-chymase is capable of activating TGF-beta 1 and IL-1 beta, forming endothelins consisting of 31 amino acids, degrading endothelin-1, altering lipid metabolism, and degrading the extracellular matrix. Under physiological conditions the role of chymase in the mast cells of the heart is uncertain. In pathological situations, chymase may be secreted and have important effects on the heart. Thus, in animal models of cardiomyopathy, pressure overload, and myocardial infarction, there are increases in both chymase mRNA levels and chymase activity in the heart. In human diseased heart homogenates, alterations in chymase activity have also been reported. These findings have raised the possibility that inhibition of chymase may have a role in the therapy of cardiac disease. The selective chymase inhibitors developed to date include TY-51076, SUN-C8257, BCEAB, NK320, and TEI-E548. These have yet to be tested in humans, but promising results have been obtained in animal models of myocardial infarction, cardiomyopathy, and tachycardia-induced heart failure. It seems likely that orally active inhibitors of chymase could have a place in the treatment of cardiac diseases where injury-induced mast cell degranulation contributes to the pathology.

The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts

Objectives:The aim of this study was to assess the biological rationale for the use of platelet-rich plasma (PRP) by evaluating the effect of different concentrations of PRP on osteoblasts (OB) and fibroblasts (FB) function in vitro. Materials and methods:PRP was obtained from volunteer donors using standard protocols. Primary human cultures of oral FBs and OBs were exposed to both activated and non-activated plasma as well as various concentrations of PRP (2.5 x, 3.5 x and max (4.2-5.5 x)). Cell proliferation was evaluated after 24 and 72 h using an MTT proliferation assay. Production of osteocalcin (OCN), osteoprotegerin (OPG) and transforming growth factor beta 1 (TGF-beta 1) was evaluated in OB after 24 and 72 h. Statistical analysis was performed using one-way ANOVA. Results:PRP-stimulated cell proliferation in both OBs and FBs. The effect of different PRP concentrations on cell proliferation was most notable at 72 h. The maximum effect was achieved with a concentration of 2.5 x, with higher concentrations resulting in a reduction of cell proliferation. Upregulation of OCN levels and downregulation of OPG levels were noted with increasing PRP concentrations at both 24 and 72 h. TGF-beta 1 levels were stimulated by increasing concentrations of PRP, with the increased levels being maintained at 72 h. Conclusions:PRP preparations exert a dose-specific effect on oral FBs and OBs. Optimal results were observed at a platelet concentration of 2.5 x, which was approximately half of the maximal concentrate that could be obtained. Increased concentrations resulted in a reduction in proliferation and a suboptimal effect on OB function. Hence, different PRP concentrations may have an impact on the results that can be obtained in vivo.

The use of heparan sulfate to augment fracture repair in a rat fracture model

Fracture healing is a complex process regulated by numerous growth and adhesive factors expressed at specific stages during healing. The naturally occurring, cell surface-expressed sugar, heparan sulfate (HS), is known to bind to and potentiate the effects of many classes of growth factors, and as such, may be a potential candidate therapy for enhancing bone repair. This study investigated the local application of bone-derived HS in the repair of rat femoral fractures. After 2 weeks, there was a significant increase in the callus size of rats administered with 5 mu g HS compared to the control and 50 mu g HS groups, presumably due to increased trabecular bone volume rather than increased cartilage production. In addition, 5 mu g HS increased the expression of ALP, Runx2, FGF-1, IGF-II, TGF-beta 1, and VEGF. It is hypothesized that these increases resulted from changes in HS-mediated receptor/ligand interactions that increase local growth factor production to augment bone formation. The findings of this study demonstrate the anabolic potential of HS in bone repair by recruiting and enhancing the production of endogenous growth factors at the site of injury. (c) 2006 Orthopaedic Research Society.

CSF-1 as a regulator of macrophage activation and immune responses

Macrophage activation is a key determinant of susceptibility and pathology in a variety of inflammatory diseases. The extent of macrophage activation is tightly regulated by a number of pro-inflammatory cytokines (e.g. IFN-gamma, IL-2, GM-CSF, IL-3) and anti-inflammatory cytokines (e.g. IL-4, IL-10, TGF-beta). Macrophage colony-stimulating factor (CSF-1/M-CSF) is a key differentiation, growth and survival factor for monocytes/macrophages and osteoclasts. The role of this factor in regulating macrophage activation is often overlooked. This review will summarize our current understanding of the effects of CSF-1 on the activation state of mature macrophages and its role in regulating immune responses.