Anti-transforming Growth Factor-beta Research Articles

Elevation of CSF albumin in old sheep: relations to CSF turnover and albumin extraction at blood‐CSF barrier

Albumin is the most abundant protein in both CSF and plasma, and albumin quotient is often used to assess the functions of brain barriers especially that of the blood-CSF barrier [i.e. the choroid plexus (CP) which also secretes CSF]. In this study, we took albumin as a model molecule to investigate ageing-related alterations in the CSF-CP system in sheep. We found significant ageing-related increases in the weight of lateral CP [122.4 +/- 14.0 mg in the young, 198.6 +/- 35.4 mg in the middle aged, 286.1 +/- 25.1 mg in the old (p < 0.05)], in the CSF albumin as well as the albumin quotient. Albumin protein spots in old CSF displayed wider on 2D western immunoblotting images, and had higher densities on images of 2D large gels stained with Pro-Q Emerald 488 compared to the young samples, suggesting ageing-related post-translational modification in the albumin. CSF secretion was reduced with age: 0.148 +/- 0.013 mL/min/g in the young, 0.092 +/- 0.02 mL/min/g in the middle aged, 0.070 +/- 0.013 mL/min/g in the old (p < 0.05). The (125)I-BSA extraction was not different among the sheep groups, nor was altered by temperature reduction, monensin, nocodazole, anti-transforming growth factor beta receptor II antibody, as well as unlabelled albumins. In conclusion, elevation of albumin in old CSF is associated with reduced CSF secretion by the CP, which size increases with age. (125)I-BSA extract, reflecting the extracellular space rather than the active albumin uptake in the CP, is not different between ages. These early changes in health ageing may result in the accumulation and modifications of CSF proteins leading to neurotoxicity.

Application of T cell-based transcriptomics to identify three candidate biomarkers for monitoring anti-TGFβR therapy

The development of targeted drugs would greatly benefit from the simultaneous identification of biomarkers to determine the aspects of bioactivity, drug safety and efficacy, particularly when affecting receptor-signaling pathways. However, the establishment of appropriate systems to monitor drug-induced events requires an accessible surrogate tissue for functional read out. Therefore we present a universal platform based upon T cell-based gene expression profiling for the identification of biomarkers using the antitransforming growth factor beta receptor inhibitor LY2109761 as an example. Our initial screen revealed 12 candidate genes specifically regulated in T cells by the inhibitor. In subsequent in-vitro and in-vivo analyses, the combined monitoring of independent gene regulation of three genes was established in peripheral blood mononuclear cells as novel pharmacodynamic candidate biomarkers for antitransforming growth factor beta receptor based therapies. Overall, the proposed concept of biomarker identification can be easily adapted towards other drug candidates for whom gene regulation can be established in cellular components of peripheral blood.

Regulation of microsomal prostaglandin E2synthase-1 and 5-lipoxygenase-activating protein/5-lipoxygenase by 4-hydroxynonenal in human osteoarthritic chondrocytes

IntroductionThis study aimed to investigate whether hydroxynonenal (HNE) depletion is responsible for the switch from cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) to 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX).MethodsFor COX-2 and mPGES-1 studies, human osteoarthritic chondrocytes were stimulated at different incubation times (up to 24 hours) with a single or repetitive addition of 10 μM HNE to the cultures at 2-hour intervals, up to 14 hours. For 5-LOX and FLAP studies, cells were treated with a single addition of 10 μM HNE for 24 hours, 48 hours, and 72 hours in the presence or absence of naproxen (a nonspecific COX-2 inhibitor) or antibody anti-transforming growth factor-beta 1 (TGF-β1). The protein levels of COX-2, mPGES-1 and early growth response factor-1 (Egr-1) transcription factor were evaluated by western blot, and those of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and TGF-β1 were determined with commercial kits. The levels of mPGES-1, FLAP and 5-LOX mRNA were measured by real-time RT-PCR. Transient transfection was performed to determine promoter activities of mPGES-1 and 5-LOX.ResultsSingle addition of 10 μM HNE to cultured chondrocytes induced PGE2 release as well as COX-2 and mPGES-1 expression at the protein and mRNA levels, with a plateau reached respectively at 8 and 16 hours of incubation, followed by a subsequent decline. However, repeated treatments with HNE prevented the decline of COX-2 and mPGES-1 expression that occurred with a single aldehyde addition. HNE induced mPGES-1 promoter activity, possibly through transcription factor Egr-1 activation. After 48 hours, when COX-2 expression decreased, the LTB4 level rose through 5-LOX and FLAP upregulation. The addition of naproxen to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that HNE significantly induced TGF-β1 production. The addition of anti-TGF-β1 antibody reduced HNE-induced 5-LOX and FLAP expression by 40%, indicating the partial involvement of a TGF-β1-dependent mechanism.ConclusionsOur data demonstrate that the shunt to the FLAP and 5-LOX pathway in HNE-induced human osteoarthritic chondrocytes is attributed to COX-2 and mPGES-1 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation.

Hepatic Stellate Cell-Specific Gene Silencing Induced by an Artificial MicroRNA for Antifibrosis In Vitro

We previously reported that the anti-transforming growth factor-beta1 (TGF-beta1) ribozymes directed by T7 and CMV promoters could reverse the character of activated hepatic stellate cells (HSCs) in vitro and improve fibrotic pathology in vivo. However, nonspecific elimination of the effects of TGF-beta1 without selectivity might have unfavorable consequences, such as overwhelming inflammation, tissue necrosis, etc. To establish an activated-HSC-specific gene silencing method and validate its feasibility for antifibrosis in vitro. An artificial intronic microRNA (miRNA) expression system was established, containing three parts: (1) a 1,074-bp SM-alpha actin promoter SMP8, which is a kind of RNA polymerase II promoter and has no activity in normal liver-derived cells but is switched on during the activation of HSCs, (2) intron1 modified by inserting an artificial pre-miRNA sequence against TGF-beta1, and (3) report gene enhanced green fluorescent proteins (EGFP). The feasibility of this system for artificial microRNA expression was validated through microRNA detection by real-time polymerase chain reaction (PCR). Alteration of biological characteristics of HSCs with the anti-TGF-beta1 miRNAs was preliminarily evaluated by measuring the expression levels of TGF-beta1 and its downstream molecules, including collagen I, matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP-1), etc. The microRNA expression system could successfully produce mature anti-TGF-beta1 miRNAs in an activated-HSC-specific manner. The microRNA-induced inhibition rate of TGF-beta1 reached 70% and above. Accompanied by TGF-beta1 suppression, its downstream targets such as collagen I, MMP2, TIMP-1, etc. were also significantly downregulated in vitro. Activated-HSC-cell-specific gene silencing could be induced well by the artificial intronic microRNA expression system to realize antifibrosis in vitro.

Synergistic enhancement of CD8+ T cell-mediated tumor vaccine efficacy by an anti-transforming growth factor-beta monoclonal antibody.

Transforming growth factor-beta (TGF-beta) is an immunosuppressive cytokine, having direct suppressive activity against conventional CD4(+) and CD8(+)T cells and natural killer cells, thereby inhibiting tumor immunosurveillance. Here, we investigated possible synergy between anti-TGF-beta (1D11) and a peptide vaccine on induction of antitumor immunity, and the mechanisms accounting for synergistic efficacy. The effect of combination treatment with a peptide vaccine and anti-TGF-beta was examined in a subcutaneous TC1 tumor model, as well as the mechanisms of protection induced by this treatment. Anti-TGF-beta significantly and synergistically improved vaccine efficacy as measured by reduction in primary tumor growth, although anti-TGF-beta alone had no impact. The number of tumor antigen-specific CTL with high functional avidity as measured by IFN-gamma production and lytic activity was significantly increased in vaccinated mice by TGF-beta neutralization. Although TGF-beta is known to play a critical role in CD4(+)Foxp3(+) Treg cells, Treg depletion/suppression by an anti-CD25 monoclonal antibody (PC61) before tumor challenge did not enhance vaccine efficacy, and adding anti-TGF-beta did not affect Treg numbers in lymph nodes or tumors or their function. Also, TGF-beta neutralization had no effect on interleukin-17-producing T cells, which are induced by TGF-beta and interleukin-6. Absence of type II NKT cells, which induce myeloid cells to produce TGF-beta, was not sufficient to eliminate all sources of suppressive TGF-beta. Finally, the synergistic protection induced by anti-TGF-beta vaccine augmentation was mediated by CD8(+) T cells since anti-CD8 treatment completely abrogated the effect. These results suggest that TGF-beta blockade may be useful for enhancing cancer vaccine efficacy.

Comparison of Craniofacial Phenotype in Craniosynostotic Rabbits Treated with Anti–Tgf-β2 at Suturectomy Site

Overexpression of transforming growth factor-beta 2 has been associated with craniosynostosis and resynostosis following surgery. We examined the effects of localized transforming growth factor-beta 2 inhibition on craniofacial phenotype in rabbits with craniosynostosis. Twenty-five New Zealand white rabbits with bilateral coronal craniosynostosis were divided into three treatment groups: (1) suturectomy control (n=8); (2) suturectomy with nonspecific, control immunoglobulin G antibody (n=6); and (3) suturectomy with anti-transforming growth factor-beta 2 antibody (n=11). At 10 days of age, a coronal suturectomy was performed on all rabbits. The sites in groups 2 and 3 were immediately filled with a slow-resorbing collagen gel mixed with either immunoglobulin G or anti-transforming growth factor-beta 2 antibody. Computed tomography scans of each rabbit were acquired at ages 10, 25, and 84 days. Craniofacial landmarks were collected from three-dimensional computed tomography reconstructions, and growth and form were compared among the three groups. Rabbits treated with anti-transforming growth factor-beta 2 antibody differed in form at 84 days of age compared with suturectomy control rabbits, specifically in the snout and posterior neurocranium. Growth in some areas of the skull was greater in rabbits from the anti-transforming growth factor-beta 2 group than in suturectomy control rabbits, but not significantly greater than in IgG control rabbits. We find support for the hypothesis that transforming growth factor-beta 2 inhibition alters adult form, but these changes do not appear to be localized to the suturectomy region. Slight differences in form and growth between the two control groups suggest that the presence of the collagen vehicle itself may affect skull growth.

Effect of γδ T cell supernatant on human skin fibroblast proliferation and collagen production – Possible role of transforming growth factor‐β and basic fibroblast growth factor

Perivascular mononuclear cell infiltration, vascular injury and diffuse interstitial fibrosis has been shown in systemic sclerosis (SSc). Objective To study a role of cellular immune mechanism inducing fibroblast proliferation and collagen synthesis. The interaction of gammadelta T-cell receptor bearing lymphocyte supernatant to human fibroblast proliferation and collagen synthesis was studied. The fibroblasts added with gammadelta T cell supernatant showed increased proliferation compared with those without it and that added with alphabeta supernatant. The endothelial cells added with gammadelta T cell supernatants, however, showed significantly decreased proliferation compared with those without it and that added with alphabeta T cell supernatant. The fibroblasts added with endothelial cell supernatant showed increased proliferation compared with those without it. A doubling of collagen synthesis was observed when human skin fibroblasts were maintained in all of the supernatant. The collagen synthesis was inhibited by antitransforming growth factor-beta antibody and antibasic fibroblast growth factor antibody. gammadelta T-cell-receptor bearing lymphocyte has proliferative and collagen synthesis activity to human skin fibroblasts, but suppressive to endothelial cells.

Phase I/II study of GC1008: A human anti-transforming growth factor-beta (TGFβ) monoclonal antibody (MAb) in patients with advanced malignant melanoma (MM) or renal cell carcinoma (RCC)

9028 Background: TGFβ is a pleiotropic cytokine that normally helps maintain tissue homeostasis. However, in advanced cancers, TGFβ is often overexpressed and associated with higher pathologic grade, increased frequency of metastases, and poorer survival. In such cancers, TGFβ signaling is often altered, and TGFβ becomes a tumor promoter by stimulating tumor cell proliferation/migration/invasion and angiogenesis, by activating stromal cells, and by suppressing anti-tumor immunity. GC1008 is a human IgG4 MAb capable of neutralizing TGFβ isoforms 1, 2, & 3. We report the results of an ongoing, first in cancer, multi-center trial examining the safety and effectiveness of GC1008. Methods: Cohorts of patients (Pts) with advanced MM or RCC who had failed ≥ 1 prior therapy (Tx) were treated with GC1008 at 1 of 6 dose levels (0.1, 0.3, 1, 3, 10 or 15mg/kg) in a 3+3 design. If no DLTs occurred within 28 days of the first dose, 3 additional doses were administered 2wks apart. Tumor response was determined by RECIST...

Small Molecular Inhibitor of Transforming Growth Factor-β Protects Against Development of Radiation-Induced Lung Injury

To determine whether an anti-transforming growth factor-beta (TGF-beta) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g/kg or 0.15 g/kg) beginning 7 days before RT. The rats were divided into eight groups: group 1, control chow; group 2, SM16, 0.07 g/kg; group 3, SM16, 0.15 g/kg; group 4, RT plus control chow; group 5, RT plus SM16, 0.07 g/kg; group 6, RT plus SM16, 0.15 g/kg; group 7, RT plus 3 weeks of SM16 0.07 g/kg followed by control chow; and group 8, RT plus 3 weeks of SM16 0.15 g/kg followed by control chow. The breathing frequencies, presence of inflammation/fibrosis, activation of macrophages, and expression/activation of TGF-beta were assessed. The breathing frequencies in the RT plus SM16 0.15 g/kg were significantly lower than the RT plus control chow from Weeks 10-22 (p <0.05). The breathing frequencies in the RT plus SM16 0.07 g/kg group were significantly lower only at Weeks 10, 14, and 20. At 26 weeks after RT, the RT plus SM16 0.15 g/kg group experienced a significant decrease in lung fibrosis (p = 0.016), inflammatory response (p = 0.006), and TGF-beta1 activity (p = 0.011). No significant reduction was found in these measures of lung injury in the group that received SM16 0.7 g/kg nor for the short-course (3 weeks) SM16 at either dose level. SM16 at a dose of 0.15 g/kg reduced functional lung damage, morphologic changes, inflammatory response, and activation of TGF-beta at 26 weeks after RT. The data suggest a dose response and also suggest the superiority of long-term vs. short-term dosing.

Anti‐Transforming Growth Factor Beta as a Treatment for Laryngotracheal Stenosis in a Canine Model

Laryngotracheal stenosis (LTS) represents a significant treatment dilemma faced by otolaryngologists. Recent topical use of Mitomycin C as an adjunctive treatment has proved helpful but does not completely prevent stenosis. Current literature suggests that transforming growth factor beta (TGFB) plays a significant role in the development of subglottic stenosis. We modified an existing canine model to test antitransforming growth factor beta (anti-TGFB) as a possible treatment for laryngotracheal stenosis. Pilot study in a modified canine model. Eight mixed-breed dogs underwent cautery injury to the subglottic region creating subsequent laryngotracheal stenosis. Four dogs were treated with saline injection into the injury site and four dogs were treated with a combination of intravenous (5 mg/kg on day 0 and 5 of the experiment) and local injection of anti-TGFB (50 micrograms). In the canine model of induced subglottic airway injury, a combination of intralesional and intravenous anti-TGFB results in a reduction in tracheal stenosis (P < .05) and an increase in survival time (P < .03) when compared to the saline control subjects. Anti-TGFB appears to be a useful adjunct in the treatment of LTS. Further study regarding the optimal dosing, route of administration, and timing of delivery is needed to understand the role of anti-TGFB in the treatment for laryngotracheal stenosis.

Sensory neuropeptides and epithelial cell restitution: the relevance of SP- and CGRP-stimulated mast cells

Calcitonin-gene-related peptide (CGRP) and substance P (SP) are neurotransmitters of extrinsic primary afferent neurons located within the dorsal root ganglia. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on intestinal mucosa was presumed. The mucosal protection partly depends on a CGRP-mediated modulation of mucosal blood flow. Limited data are available regarding CGRP- or SP-mediated effects on epithelial cell restitution. Having shown earlier that SP-stimulated fibroblasts but not CGRP-stimulated fibroblasts induce epithelial cell migration in vitro, the aim of this study was to explore whether mast cells mediate effects of SP and CGRP on epithelial cell restitution in vitro. Mast cells (C57) were exposed to SP [10(-12)-10(-6 M)] and CGRP [10(-12)-10(-7 M)]. After a 24-h incubation period, the cell supernatants (conditioned media, CDM) were taken from mast cell cultures and directly applied to rat intestinal epithelial cell lines-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h prior to the experiments. Epithelial cell migration was assessed by counting cells across the wound edge and epithelial cell proliferation was measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide test. CGRP significantly induced epithelial cell migration and proliferation via mast cells when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralizing anti-transforming growth factor-beta (TGF-beta) had been added to the cell cultures. SP had no effects on epithelial cells following stimulation of mast cells. CGRP modulates epithelial cell restitution in vitro mediated by mast cells. The CGRP- and mast-cell-induced epithelial cell migration is TGF-beta dependent. This observation underlines an important role for extrinsic primary afferent neurons in mucosal defence and repair and in keeping the mucosal homeostasis. This knowledge leads to a better understanding of the interaction of the enteric nervous system and wound healing and may, in the future, lead to new therapeutic approaches to inflammatory diseases of the intestine.

Histological, immunohistochemical and biological data in assessing interstitial fibrosis in patients with chronic glomerulonephritis

The aim of this study was to determine the relationship between histological, immunohistochemical (IHC) and biological data in the assessment of interstitial fibrosis in patients with glomerular diseases. A group of 41 patients with primary and secondary glomerulonephritis was studied. In order to quantify the histological changes and to assess the extent of active-inflammatory and chronic-sclerotic/fibrotic interstitial lesions, we adapted a scoring system, initially used for lupus nephritis, and ANCA-associated vasculitis. IHC labeling procedures with monoclonal antibodies anti-smooth muscle actin (SMA), anti-vimentin and anti-transforming growth factor beta (TGFbeta) were assessed using a semi-quantitative score, correlated with the histological and biological data. Our results showed that interstitial labeling of SMA correlated with scores for sclerotic/fibrotic lesions (chronicity index) and with active-inflammatory lesions (interstitial infiltrate, activity index). Interstitial vimentin correlated with the score for interstitial infiltrate. Both interstitial vimentin and TGFbeta immunopositivity correlated with sclerotic/fibrotic lesions (interstitial fibrosis, tubular atrophies, vascular hyalinosis/fibrosis, chronicity index), and negatively with glomerular filtration rate. An important correlation was found between the interstitial labeling of the two IHC markers of myofibroblasts (SMA and vimentin). We conclude that IHC studies related to clinico-biological and histological data can have an important role in the evaluation of the glomerular diseases, but the classical histological investigation assessed through quantification has still not lost its importance.

Substance P induces intestinal wound healing via fibroblasts—evidence for a TGF-β-dependent effect

Substance P (SP) and calcitonin gene-related peptide (CGRP) are neurotransmitters of the afferent sensory nervous system. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on the intestinal mucosa was presumed. In part, mucosal protection depends on a SP-mediated and CGRP-mediated modulation of mucosal blood flow after injury. We thought to explore whether there is a fibroblast-mediated effect of SP and CGRP on epithelial cell restitution in vitro. Rat kidney fibroblast (NRK-49F) cell lines were exposed to CGRP or SP in various concentrations. After incubation, the cell culture supernatants were taken from the fibroblast cultures and were directly applied to IEC-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h before the experiments. Epithelial cell migration was assessed by counting cells across the wound edge. Epithelial cell proliferation was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) test. SP significantly induced epithelial cell migration and inhibited epithelial cell proliferation via stimulation of fibroblasts when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralising anti-transforming growth factor-beta (TGF-beta) was added to the cell cultures. CGRP had no effect on epithelial cells via stimulation of fibroblasts. Neither CGRP nor SP had any effect on epithelial cell migration or proliferation when directly applied to epithelial cells. SP modulates epithelial cell restitution in vitro mediated by fibroblasts. The epithelial cell migration depends on a TGF-beta release from SP-stimulated fibroblasts. This observation underlines an important role for the sensory nervous system in mucosal defence and repair and in keeping mucosal homeostasis. Modulation of SP may be potentially useful for the treatment of various intestinal disorders characterised by injury and ineffective repair of the intestinal mucosa.

Advanced glycation end products decrease mesangial cell MMP-7: A role in matrix accumulation in diabetic nephropathy?

Increased extracellular matrix material is a pathological hallmark of diabetic nephropathy. In addition to collagens, a variety of non-collagenous glycoproteins such as fibronectin also accumulate in the kidney of diabetics. The effect of diabetes on degradative pathways, in particular those involving non-collagenous proteins, are relatively unexplored. In this study, we determined the expression of the major matrix metalloproteinase (MMP) responsible for degrading the non-collagenous matrix glycoprotein fibronectin. Furthermore, the modulation of these MMPs by advanced glycation end products (AGE), a key factor in the diabetic milieu, was explored. Exposure of mesangial cells to AGEs led to a significant reduction in MMP-7, but not MMP-3 or -10. MMP-7 expression was normalized by both aminoguanidine, an inhibitor of glycation product formation, or by a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody. In streptozotocin-induced diabetic rats, the diminution in MMP-7 expression and excessive fibronectin accumulation were attenuated by aminoguanidine. Humans with type 2 diabetes and nephropathy displayed similar alterations in MMP-7 to their rodent counterparts. Our findings suggest that diminished expression of the glycoprotein-degrading enzyme, MMP-7, may play a role in fibronectin accumulation in the diabetic kidney in response to AGEs and/or TGF-beta.

Effect of the Ocular Microenvironment in Regulating Corneal Dendritic Cell Maturation

To determine whether the ocular anterior segment (aqueous humor and cornea) actively inhibits dendritic cell (DC) maturation. Dendritic cells were injected into syngeneic corneas or conjunctivae, and their surface major histocompatibility complex class II expression in response to the local milieu was assessed using confocal microscopy. Immature DCs were cocultured with corneal supernatant or with aqueous humor to evaluate their regulation of DC phenotypic and functional maturity. In contrast to conjunctivally injected DCs, DCs injected into the cornea resisted up-regulation in expression of surface major histocompatibility complex class II. Corneal supernatant-treated and aqueous humor-treated DCs retained their immaturity, as reflected by high antigen uptake but low costimulatory molecule (CD80 and CD86) expression and poor T-cell stimulation. Anti-transforming growth factor beta(2) treatment of aqueous humor and of corneal supernatant led to complete and partial blockade of their inhibition of DC maturation, respectively. However, alpha-melanocyte-stimulating hormone and calcitonin gene-related peptide had no demonstrable effect on DC maturation. Cornea and aqueous humor, principally through transforming growth factor beta(2,) promote generation of phenotypically and functionally immature DCs. Clinical Relevance Our results indicate that relative immune quiescence in the cornea and in the anterior segment is actively maintained in part by the inhibitory effect of transforming growth factor beta(2) on resident DCs and by their suppression of T-cell-mediated immune and inflammatory responses.

Specific down‐regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes

Diabetic nephropathy (DN) remains a major complication in both type 1 and type 2 diabetes. Systemic administration of antitransforming growth factor-beta (TGF-beta) antibody has shown some promise in mouse models of DN. However, chronic blockade of the multifunctional TGB-beta could be problematic. Several downstream effects of TGF-beta are mediated by connective tissue growth factor (CTGF), which is up-regulated in several renal cells and secreted in the urine in the diabetic state. Using murine models of DN (type 1 and type 2) and a CTGF antisense oligonucleotide (ASO) of novel chimeric chemistry, we evaluated the specific role of this target in DN. In the type 1 model of DN, C57BL6 mice were made diabetic using streptozotocin injections and hyperglycemic animals were treated with CTGF ASOs (20 mg/kg/2 qw) for 4 months. ASO, but not mismatch control oligonucleotide, -treated animals showed significant reduction in target CTGF expression in the kidney with a concomitant decrease in proteinuria and albuminuria. Treatment with the CTGF ASO for 8 wk reduced serum creatinine and attenuated urinary albuminuria and proteinuria in diabetic db/db mice, a model of type 2 DN. The ASO also reduced expression of genes involved in matrix expansion such as fibronectin and collagen (I and IV) and an inhibitor of matrix degradation, PAI-1, in the renal cortex, contributing to significant reversal of mesangial expansion in both models of DN. Pathway analyses demonstrated that diabetes-induced phosphorylation of p38 MAPK and its downstream target CREB was also inhibited by the ASO. Our results strongly suggest that blocking CTGF using a chimeric ASO holds substantial promise for the treatment of DN.

Prostate‐specific antigen stimulates osteoprotegerin production and inhibits receptor activator of nuclear factor‐KappaB ligand expression by human osteoblasts

Prostate cancer cells produce a large amount of prostate-specific antigen (PSA), which is widely used as a marker for this cancer. Even though it is widely used in the diagnosis of prostate cancer, many aspects of the pathophysiologic role of PSA in bone metastasis remain obscure. The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the activation of osteoclasts, while osteoprotegerin (OPG) neutralizes the action of RANKL. Various substances that act on bone have been shown to modulate the production of RANKL and OPG by osteoblasts. In this study, we investigated the effect of PSA on the expression of OPG and RANKL mRNA and on protein production in human osteoblast-like cells. After addition of PSA and culture for 72 h, OPG mRNA expression and protein secretion by MG-63 and SaOS-2 cells showed a concentration-dependent increase. When osteoblasts were incubated with PSA (100 ng/ml), OPG mRNA expression and protein secretion increased with the passage of time. alpha1 -antichymotrypsin (ACT), which inactivates the serine protease activity of PSA, inhibited the increase of OPG mRNA expression and protein production in response to PSA, and this effect of PSA was also inhibited by anti-transforming growth factor-beta antibody. Based on our findings, PSA acts on human osteoblast-like cells via its own serine protease activity and promotes osteoblast differentiation. In addition, PSA stimulates OPG production and inhibits RANKL expression of osteoblasts, and inhibits bone resorption by osteoclasts, suggesting that it contributes to the characteristic osteoblastic features of bone metastases of prostate cancer.

Effects of angiotensin II subtype 1 receptor blockade by losartan on tubulointerstitial lesions caused by hyperoxaluria.

Hyperoxaluria is a recognized cause of tubulointerstitial lesions and this circumstance could contribute to cause chronic renal disease. The renin-angiotensin system has a critical role in the development of interstitial fibrosis, mostly by angiotensin II type 1 receptor stimulation of pro-fibrotic mechanisms. We evaluated whether angiotensin II type 1 receptor blockade prevents oxalate renal lesions. We divided 2-month-old male Sprague-Dawley rats into 4 groups, namely group 1-control, group 2-hyperoxaluria, group 3-hyperoxaluria plus losartan and group 4-losartan. For 4 weeks groups 2 and 3 received 1% ethylene glycol (precursor for oxalates) in drinking water. Losartan (40 mg./kg. body weight) was administered in groups 3 and 4 daily. At the end of the study renal lesions were evaluated using anti-alpha-smooth muscle actin, anti-collagen type III, anti-monocytes/macrophages and anti-transforming growth factor-beta1 antibodies. To evaluate oxidative stress in renal tissue total glutathione and thiobarbituric acid reactive substances in kidney homogenates were determined. Regarding renal functional parameters, creatinine clearance and urinary albumin excretion were also studied. Despite similar urinary oxalate levels compared with group 2 group 3 rats showed fewer tubulointerstitial lesions, consisting of significant lower scores for tubular atrophy, unspecific inflammatory cell infiltrate, ED1 mouse anti-rat monoclonal antibody (Serotec, Ltd., Oxford, United Kingdom) (monocytes/macrophages), crystal deposits, interstitial fibrosis, alpha-smooth muscle actin, collagen type III and tubulointerstitial transforming growth factor-beta1. Moreover, urinary albumin excretion and creatinine clearance were significantly improved in group 3 (p <0.01). Higher total glutathione and lower thiobarbituric acid reactive substances were also observed in this group (p <0.01). Thiobarbituric acid reactive substances were the most important and significant independent variable correlating with interstitial fibrosis (t ratio 4.867, p <0.04). We believe that the renal-angiotensin system interaction by losartan produces a beneficial effect against renal lesions caused by hyperoxaluria through a number of actions, including a reduction in crystal formation in the tubular fluid, inflammatory reaction control and interaction with oxidative stress. These factors lead concurrently to preserve tubular epithelial cell and renal interstitium integrity. In addition, these results suggest that the principal mechanism of action should be mediated by angiotensin II type 1 receptors.

Role of transforming growth factor-β1 (TGF-β1) in endotoxin-induced hepatic failure after extensive hepatectomy in rats

Postoperative infections after hepatectomy sometimes lead to fatal hepatic failure, but the mechanism of the hepatic failure is unclear. Wistar rats underwent 90% hepatectomy, and were then divided into three groups: (i) the SAL group, injected with normal saline; (ii) the LPS group, injected with lipopolysaccharide (LPS) every day for 1 week; and (iii) the LPS plus TGF-Ab (LPS+TGF-Ab) group, injected with LPS with anti-transforming growth factor-beta1 (TGF-beta1) antibody. We investigated survival rates, TGF-beta1 expression in the liver, liver regeneration by proliferating cell nuclear antigen labeling index, hepatocyte apoptosis by single stranded DNA labeling index, and perisinusoidal fibrosis using Masson's trichrome staining. The LPS group (30.4%) had a significantly lower survival rate than the SAL group (84%) and tended to be lower than the LPS+TGF-Ab group (49.4%). Liver regeneration in the LPS group was significantly lower than in the other groups. In the LPS group, hepatocyte apoptosis and perisinusoidal fibrosis was significantly more remarkable, and TGF-beta1 expression was significantly higher than in the SAL group. TGF-beta1 enhanced by LPS plays an important role in the mechanism of hepatic failure by infections after hepatectomy, especially in inhibition of liver regeneration, and induction of hepatocyte apoptosis and perisinusoidal fibrosis.

Gene Expression of Early Hypertrophic Scar Tissue Screened by Means of cDNA Microarrays

Hypertrophic scar is an excessive healing response that often follows thermal injury. The most outstanding morphologic change is the overdeposition of collagen, which is caused by imbalance between synthesis and metabolism of collagen. Previous studies also found that transforming growth factor-beta was the key factor controlling scar formation. However, neither anti-transforming growth factor-beta nor other methods could completely control scar formation and contraction. This fact suggests a multifactorial cause. Fortunately, cDNA microarray throws light on the general alteration at the gene level, and thus could allow us to find some new clues for understanding scar formation and contraction. In this article, we report the results obtained from the scanning of gene expression of hypertrophic scar by means of cDNA microarray. Five cases of early human postburn hypertrophic scars were selected. Total tissue RNA was extracted from each hypertrophic scar sample and the corresponding uninjured region skin tissue; mRNA was further purified by Oligotex and then was reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray containing 4,096 genes on a type of chemical material-coated glass slide. After high-stringent washing, the hybridized slides were scanned for fluorescent signal detection. Then, the expression and distribution of cytoskeletal genes such as alpha-smooth muscle actin (alpha-SMA) gene; fibroblast tropomyosin TM30(pl) gene; vimentin gene; profilin gene; and BM40 gene of hypertrophic scar at 3, 6, 9, and 12 months age were further quantitatively studied by in situ hybridization or immunohistochemistry. Our data indicated that there were 94 genes overexpressed and 3 genes down-regulated in early postburn hypertrophic scar. These altered genes were related to proto-oncogenes, apoptosis, immune regulatory genes, cytoskeletal elements, metabolism, and so forth. We also found that the detected cytoskeletal gene expression was much more intense at all time points than the control group. Consistent with clinical observation, cytoskeletal genes reached a peak at an early stage and gradually decreased. Our study implied that multiple genes are involved in scar formation and contraction. Interferon is an autosecreted cytokine that might be responsible for self-control of overgrowth of cells in wounds. The early period of hypertrophic scar formation might be a good time for preventing overgrowth and contraction of hypertrophic scar by gene therapy.