Porcine osteochondrosis: Deficiencies in transforming growth factor-β and insulin-like growth factor-I

Osteochondrosis and dyschondroplasia are common multifocal disturbances of endochondral ossification in many species of domestic animals, and are characterized by the retention of avascular cartilage. These cartilage disorders are characterized by a failure of chondrocyte differentiation, matrix mineralisation and its replacement by bone. Rabbit polyclonal antibodies to transforming growth factor-beta (TGF-) and to insulin-like growth factor-I (IGF-I) were used to detect the two growth factors in normal and osteo-chondrotic porcine epiphyses. In the normal pig epiphyses IGF-I and TGF- were present in the chondrocytes of the epiphyseal hyaline cartilage and IGF-I was readily localised to the hypertrophic chondrocytes in the growth cartilage adjacent to the epiphyseal ossification centre. Both growth factors were found to be deficient in chondrocytes at sites of osteochondrosis. Both these growth factors are thought to be involved in the cascade of events associated with chondrocyte function during endochondral ossification. Deficiencies in TGF- and IGF-I demonstrated in porcine osteochondrosis and previously shown in avian dyschondroplasia suggest further similarities in the pathogenesis of these conditions.     

Cell division autoantigen 1 enhances signaling and the profibrotic effects of transforming growth factor-β in diabetic nephropathy

Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-β (TGF-β) signaling in a number of cellular systems; here we found that its levels were elevated in the kidneys of two animal models of diabetic renal disease. The localization of CDA1 to tubular cells and podocytes in human kidney sections was similar to that seen in the rodent models. CDA1 small interfering RNA knockdown markedly attenuated, whereas its overexpression increased TGF-β signaling, modulating the expression of TGF-β, TGF-β receptors, connective tissue growth factor, collagen types I, III, IV, and fibronectin genes in HK-2 cells. CDA1 and TGF-β together were synergistic in stimulating TGF-β signaling and target gene expression. CDA1 knockdown effectively blocked TGF-β-stimulated expression of collagen genes. This was due to its ability to modulate the TGF-β type I, but not the type II, receptor, leading to increased phosphorylation of Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinase. Furthermore, the Smad3 inhibitor, SIS3, markedly attenuated the activities of CDA1 in stimulating TGF-β signaling as well as gene expression of collagens I, III, and IV. Thus, our in vitro and in vivo findings show that CDA1 has a critical role in TGF-β signaling in the kidney.     

Enhanced gene expression of transforming growth factor-α and c-met in rat urinary bladder cancer

To investigate the roles of growth factors in bladder cancer, changes in the expression of messenger RNAs (mRNAs) for several growth factors and their receptors were examined during rat bladder carcinogenesis induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Northern blot analysis showed that the contents of mRNAs for transforming growth factor- (TGF-) and c-met/hepatocyte growth factor (HGF) receptor increased with BBN treatment. Epidermal growth factor (EGF) receptor mRNA was hardly affected by the treatment; while mRNA for fibroblast growth factor (FGF) receptor 1 and transforming growth factor- (TGF-) type II receptor decreased with BBN treatment. A rat bladder tumor cell line, NBT-II, expressed both TGF- and c-met mRNAs, and HGF showed apparent scattering and growth-stimulating effects on the cells. These results indicate the possibility that TGF- produced by a bladder cancer, in addition to urinary EGF, plays a role in the development of bladder cancer, and that enhanced cell motility due to activation of the c-met/HGF receptor participates in the invasion and metastasis of the cancer cells.     

Transforming growth factor-β induces vascular endothelial growth factor-C expression leading to lymphangiogenesis in rat unilateral ureteral obstruction

Inflammation is recognized as an important contributor to lymphangiogenesis; however, in tubulointerstitial lesions in human chronic kidney diseases, this process is better correlated with the presence of myofibroblasts rather than macrophages. As little is known about the interaction between lymphangiogenesis and renal fibrosis, we utilized the rat unilateral ureteral obstruction model to analyze inflammation, fibrosis, lymphangiogenesis, and growth factor expression. Additionally, we determined the relationship between vascular endothelial growth factor-C (VEGF-C), an inducer of lymphangiogenesis, and the profibrotic factor, transforming growth factor-β1 (TGF-β1). The expression of both TGF-β1 and VEGF-C was detected in tubular epithelial and mononuclear cells, and gradually increased, peaking 14 days after ureteral obstruction. The kinetics and localization of VEGF-C were similar to those of TGF-β1, and the expression of these growth factors and lymphangiogenesis were linked with the progression of fibrosis. VEGF-C expression was upregulated by TGF-β1 in cultured proximal tubular epithelial cells, collecting duct cells, and macrophages. Both in vitro and in vivo, the induction of VEGF-C along with the overall appearance of lymphatics in vivo was specifically suppressed by the TGF-β type I receptor inhibitor LY364947. Thus, TGF-β1 induces VEGF-C expression, which leads to lymphangiogenesis.     

Connective tissue growth factor mediates the profibrotic effects of transforming growth factor-β produced by tubular epithelial cells in response to high glucose

Background It was reported that connective tissue growth factor (CTGF) was expressed in the tubular epithelial cells of the diabetic kidney. CTGF has, among other factors, been implicated in mediating the downstream, profibrotic effects of transforming growth factor-β (TGF-β), though is precise role in interstitial fibrogenesis in the diabetic kidney has not yet been clarified.Methods We employed a coculture system involving cultured murine proximal tubular epithelial cells (mProx24) and renal fibroblasts (TFB), as a model of the subepithelial mesenchyme in the kidney in order to examine the profibrotic effects of CTGF derived from mProx24 cells in response to high glucose (30 mM).Results We showed that glucose stimulated CTGF expression in cultured mProx24 in both a dose- and a time-dependent manner, and that this effect was mediated by increased levels of TGF-β. We also found that high glucose significantly stimulated TFB cells to produce profibrotic molecules, such as type I collagen, the EIIIA isoform of fibronectin, and plasminogen activator inhibitor-1. The induction of these molecules was both direct and indirect, the latter induction being mediated by mProx24 cell-derived CTGF, which, in turn, was induced by TGF-β that was produced by the mProx24 cells.Conclusions CTGF plays an important role in mediating renal interstitial fibrogenesis in response to high glucose and, as such, is a reasonable target for anti-fibrotic therapy.     

Transforming growth factor-β pathway is activated in cholecystolithiasis

Background The etiopathogenesis of cholecystolithiasis is not well defined. Primary dysmotility of the organ, due to fibrosis of the gallbladder wall or muscular dysfunction, is suggested as a crucial factor. Transforming growth factor-beta (TGF-) and connective tissue growth factor (CTGF) are involved in several fibrotic disorders and play a critical role in fibrogenesis, thereby changing the physiological function of the organs. In the present study we analyzed the role of TGF- and its downstream target CTGF in patients with cholecystolithiasis.Methods Gallbladders were obtained from 16 individuals undergoing surgery for symptomatic cholecystolithiasis. Normal human gallbladder tissue samples from five individuals without any history of gallbladder disease were obtained through an organ donor transplantation program. Northern blot analysis, in situ hybridization, and immunohistochemistry were used to analyze the expression of TGF-1 and CTGF in the gallbladder tissue samples.Results By northern blot analysis there was an enhanced TGF-1 mRNA expression (eightfold increase; P<0.04) in the cholecystolithiasis tissue samples in comparison with normal controls. There was also a concomitant increase in CTGF (41-fold increase; P<0.01). By in situ hybridization and immunohistochemistry, CTGF mRNA was localized mainly in the mucosa layer, while intensive staining of the smooth muscle cells with TGF-1 and CTGF was observed. In addition, TGF-1 immunoreactivity was also localized in the fibroblasts and inflammatory cells. TGF-1 m-RNA levels showed a significant relationship with the degree of fibrosis in the tissue samples (P<0.04, r=0.5).Conclusion Our data indicate that TGF- and CTGF are involved in ultrastructural tissue changes in patients with cholecystolithiasis. Activation of the TGF- pathway, predominantly in the remaining mucosa and submucosal layer, indicates that extracellular matrix (ECM) synthesis with subsequent gallbladder wall fibrosis is an important step in gallbladder dysfunction in this disorder.     

Endothelial cell transforming growth factor-β receptor activation causes tacrolimus-induced renal arteriolar hyalinosis

Arteriolar hyalinosis is a common histological finding in renal transplant recipients treated with the calcineurin inhibitor tacrolimus; however, the pathophysiologic mechanisms remain unknown. In addition to increasing transforming growth factor (TGF)-β levels, tacrolimus inhibits calcineurin by binding to FK506-binding protein 12 (FKBP12). FKBP12 alone also inhibits TGF-β receptor activation. Here we tested whether tacrolimus binding to FKBP12 removes an inhibition of the TGF-β receptor, allowing ligand binding, ultimately leading to receptor activation and arteriolar hyalinosis. We found that specific deletion of FKBP12 from endothelial cells was sufficient to activate endothelial TGF-β receptors and induce renal arteriolar hyalinosis in these knockout mice, similar to that induced by tacrolimus. Tacrolimus-treated and knockout mice exhibited significantly increased levels of aortic TGF-β receptor activation as evidenced by SMAD2/3 phosphorylation, along with increased collagen and fibronectin expression compared to controls. Treatment of isolated mouse aortas with tacrolimus increased TGF-β receptor activation and collagen and fibronectin expression. These effects were independent of calcineurin, absent in endothelial denuded aortic rings, and could be prevented by the small molecule TGF-β receptor inhibitor SB-505124. Thus, endothelial cell TGF-β receptor activation is sufficient to cause vascular remodeling and renal arteriolar hyalinosis.     

Urinary transforming growth factor-β1 in children with obstructive uropathy

Aim: Obstructive uropathies (OU) in childhood constitute one of the major causes of chronic renal insufficiency. Transforming growth factor‐β1 (TGF‐β1) is considered to be the major fibrogenic growth factor. The aim of the present study was to investigate urinary TGF‐β1 levels in children with obstructive and non‐obstructive uropathies (NOU). Methods: This study involved 19 children with OU, 11 children with non‐obstructive hydronephrosis and 21 healthy children. Urinary TGF‐β1, proteinuria, microalbuminuria and urinary α1‐microglobulin were measured, and renal function was assesed. The results were statistically analyzed. Results: Mean urinary TGF‐β1 concentrations in patients with OU were significantly higher than those with NOU (4.14 ± 0.67 creatinine vs 1.80 ± 0.24 pg/mmol creatinine, P < 0.05) and healthy controls (1.66 ± 0.28 pg/mmol creatinine, P < 0.05). Positive correlations of urinary TGF‐β1 concentrations with proteinuria (r = 0.87, P < 0.0001) and urinary α1‐microglobulin (r = 0.82, P = 0.0002) were found in patients with OU. Conclusion: Children with OU have higher urinary TGF‐β1 than children with NOU. Urinary TGF‐β1 may be a useful non‐invasive tool for the differential diagnosis between OU and NOU in children. A positive correlation of TGF‐β1 with markers of renal tissue damage in patients with OU was found.     

FK506 inhibits the mice glomerular mesangial cells proliferation by affecting the transforming growth factor-β and Smads signal pathways

TGF-β1 plays an important role in the pathogenesis of chronic renal diseases. Although the specific mechanism is unknown, a major factor is the potent fibrogenic activity of TGF-β1 in the chronic progression of renal diseases. TGF-β1 closely correlates with renal fibrosis in cooperation with several fibrosis-promoting molecules. Recently it has been studied that, Smad proteins as intracellular mediators of TGF-β signaling pathways provide important insights into the mechanisms determining the specificity of TGF-β action in various renal cells. Some studies have proved that immunosuppressants can affect TGF-β expression, but the mechanisms are unclear. In this study, we investigated the effect of FK506 on mesangial cells via TGF-β and Smads signal pathways. Our results shows that FK506 effectively blocked the TGF-β/Smad signaling pathway by downregulation of TGF-β receptor, and played an important role in TGF-β1-induced Smad2 expression in mice mesangial cells. FK506 can inhibit the TGF-β1-stimulated cell proliferation via Smad-related pathways. And reduced the Smad2 protein and mRNA expression. Altogether, this study provided a theoretical proof for the protective and treating effect of FK506 on kidneys.     

Plasma transforming growth factor-β1 levels in patients with erectile dysfunction

Aim: To evaluate the plasma TGF-β1 level in erectile dysfunction (ED) patients of various causes. Methods: Sixty-two patients with ED and 26 potent men were subjected to the study. Based on multidisciplinary work-ups, including medical history, physical examinations, blood tests with lipid profile and hormones, penile duplex Doppler ultrasonogram and neurophysiological tests, causes for ED were classified as psychogenic (n=15), neurogenic (n=16) and vasculogenic (n=31). The plasma TGF-β1 level was measured by the ELISA method. Results: The plasma TGF-β1 level was significantly increased in the ED group (6.7 ± 4.9 ng/mL), compared to the control (4.0±2.1 ng/mL) (P <0.01). In the ED groups, there was a significant increase in the vasculogenic group (9.0 ± 5.5 ng/mL), compared to the psychogenic (3.8 ± 1.8 ng/mL) and neurogenic groups (4.8 ± 3.2 ng/mL) (P<0.01). Of the vascular risk factors, both the smoking (7.5 ± 4.7 ng/mL) and dyslipidemia groups (7.4 ± 4.4 ng/mL) showed significantly increased     

Transforming growth factor-β and prostate cancer

Summary This review focuses on the possible role of transforming growth factor- isoforms 1–3 (TGF) in prostate cancer. TGF1 appears to inhibit the cellular proliferation of normal prostate cells. Surprisingly, TGF1 is overexpressed in prostate cancer. To help explain this apparent paradox, it has been revealed that with tumor progression, prostate cancer cells acquire reduced sensitivity to the growth-inhibitory effects of TGF1. Aberrations of the TGF1 signaling pathway at the prereceptor, receptor, or postreceptor level may lead to prostate cancer cell resistance to TGF1 growth inhibition. Indirectly, elevated levels of TGF1 may induce host effects that may be beneficial to prostate tumor growth by suppressing the immune system, promoting angiogenesis and extracellular matrix formation, and enhancing metastatic potential. Consequently, TGF1 appears to be important in prostate carcinogenesis and tumorigenicity. TGF2 and TGF3 are only briefly presented as very little is known about their role in prostate cancer.     

Transforming growth factor-β in calcium alginate beads for the treatment of articular cartilage defects in the rabbit

Purpose: Articular cartilage has only limited capability for intrinsic repair. The use of growth factors has been suggested to improve the repair of cartilage after injury. Reliable delivery systems for these agents are needed. In this study we tested calcium alginate for the delivery of TGF-β in the treatment of osteochondral defects in the rabbit knee. Type of Study: Randomized trial animal study and basic science study. Methods: In vitro, to establish the kinetics of TGF-β release from the alginate, ¹²⁵I- labeled TGF-β was suspended in 1.2% sodium alginate at concentrations of 1 μg/mL and 10 μg/mL. Beads were formed from 50 μL aliquots and placed into standard culture medium by immersion in calcium chloride solution and incubated at 37° C. A gamma counter was used to measure the amount of TGF-β that was released into the medium at various time points. In vivo, osteochondral defects were created in the trochlear grooves of 32 New Zealand White rabbits. Defects were treated with plain alginate or with alginate containing TGF-β at 20 ng/mL or 2,000 ng/mL. Untreated defects served as a control. Animals were killed after 6 and 12 weeks. Knee joints were evaluated grossly with a 12-point grading scale. Histologic sections of the repair tissue were stained with Safranin O and evaluated using a 24-point grading scale by 2 independent blinded observers. Mean scores and standard deviations were calculated. P values were determined using the Student t test. Results: The TGF-β was released at a surprisingly slow but steady rate. Release rates extrapolated from the gamma counter measurements were 0.25% per hour and 0.33% per hour, for the 1 μg/mL and 10 μg/mL beads, respectively. Gross analysis scores at 6 and 12 weeks resulted in higher scores for both TGF-β groups without reaching statistical significance. The lower TGF-β concentration reached the highest scores, whereas the higher concentration (2,000 ng/mL) resulted in increased osteophyte formation. Histologic analysis at 6 weeks resulted in average scores ranging from 14.5 for empty defects and 18.1 for alginate-treated defects, to 20.0 and 20.3 for the 2,000 ng/mL and 20 ng/mL TGF-β groups, respectively (P <.05). At 12 weeks, histologic scores ranged from 14.9 for empty and 14.5 for alginate to 20.1 and 20.5 for the 2,000 ng/mL and 20 ng/mL TGF-β groups, respectively (P <.05). These results indicate a significant improvement of the quality of the repair tissue at 6 and 12 weeks with TFG-β treatment, especially at the lower concentration. Conclusions: The use of alginate allows the controlled delivery of TGF-β selectively to the site of injury, potentially avoiding systemic side effects. Furthermore, treatment with TGF-β appears to improve the repair of articular cartilage defects. Longer-term studies are needed to assess whether the benefits of the TGF-β treatment can be sustained.Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 18, No 8 (October), 2002: pp 892–900     

Ontogeny of expression of transforming growth factor-β and its receptors and their possible relationship with scarless or scar-forming healing in human fetal and postnatal skins

Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming growth factor-β (TGF-β) isoforms and their receptors in wound healing process, their roles in scarless wound repair in early gestation,their functions in human fetal skin development and structural and functional maintenance are still not well understood. In this study, we explore the expression and distribution characteristics of three TGF-β isoforms, their receptors, i. e.     

Transforming growth factor-β1 in the urine of young children with urinary tract infection

Urinary tract infection (UTI) is a frequent cause of morbidity during the first years of life and may lead to renal insufficiency. Transforming growth factor-1 (TGF-) is both immunoregulatory and an important mediator of interstitial fibrosis. TGF- was detected in the urine of 52% of 48 children aged 1–24 months with a first episode of UTI (94% due to Escherichia coli) and no obstructive nephropathy compared with 0 of 20 healthy young children (P<0.001). TGF- was detected in the urine only during the early stage (<1 day) after initiation of treatment. It was detected more frequently (P=0.06) and in significantly higher concentrations (P=0.046) in children with a normal ^99m Tc-dimercaptosuccinic acid scan compared with those with abnormal scans performed 3–14 days after the diagnosis of UTI, suggesting a regulatory role in fibrogenesis and outcome of pyelonephritis in childhood.     

Local effects of transforming growth factor-β1 on rat calvaria: Changes depending on the dose and the injection site

Transforming growth factor-β₁ (TGF-β₁) has opposite effects on osteoblastic cells in vitro, namely an inhibitory or stimulatory effect on cell differentiation. Because these effects are dependent on TGF-β₁ concentration or culture condition, we investigated whether the in vivo effects of TGF-β₁ on bone formation in infant rat calvaria were affected by the dose or the injection site. Human platelet-derived TGF-β₁ was injected subcutaneously onto the periosteal surface of parietal bone of 4-week-old rats at doses of 5 or 20 ng/100 μl per animal for 14 days, and the local effect on bone formation was examined by bone histomorphometry. TGF-β₁ treatment for 7 days decreased the mineral apposition rate, bone formation rate, and elongated mineralization lag time at the injection site. This change became more prominent when treatment continued for 14 days. These changes were restricted to the TGF-β₁-exposed area. Multiple subcutaneous injections of a relatively high dose (200 ng/100 μl per animal) of TGF-β₁ induced woven bone formation, in addition to marked inhibition of bone formation rate and prolongation of mineralization lag time. On the other hand, direct exposure of TGF-β₁ in the subperiosteal layer induced woven bone with periosteal cell proliferation even at a single injection of a low dose (5 or 50 ng/25 μl) of TGF-β₁. In conclusion, the in vivo effects of TGF-β₁ on bone formation varied depending on its concentration and injection site. Also, subcutaneous injection of relatively low doses of TGF-β₁ inhibited local lamellar bone formation. Received: Feb. 2, 1998 / Accepted: Aug. 25, 1998