Candesartan prevents arteriopathy progression in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy model.

Cerebral small vessel disease (CSVD) causes dementia and gait disturbance due to arteriopathy. Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a hereditary form of CSVD caused by loss of high-temperature requirement A1 (HTRA1) serine protease activity. In CARASIL, arteriopathy causes intimal thickening, smooth muscle cell (SMC) degeneration, elastic lamina splitting, and vasodilation. The molecular mechanisms were proposed to involve the accumulation of matrisome proteins as substrates or abnormalities in transforming growth factor ß (TGF-ß) signaling. Here, we show that HTRA1-/- mice exhibited features of CARASIL-associated arteriopathy: intimal thickening, abnormal elastic lamina, and vasodilation. In addition, the mice exhibited reduced distensibility of the cerebral arteries and blood flow in the cerebral cortex. In the thickened intima, matrisome proteins, including the hub protein fibronectin (FN) and latent TGF-ß binding protein 4 (LTBP-4), which are substrates of HTRA1, accumulated. Candesartan treatment alleviated matrisome protein accumulation and normalized the vascular distensibility and cerebral blood flow. Furthermore, candesartan reduced the mRNA expression of Fn1, Ltbp-4, and Adamtsl2, which are involved in forming the extracellular matrix network. Our results indicate that these accumulated matrisome proteins may be potential therapeutic targets for arteriopathy in CARASIL.

Modulation of TGF­ß activity by latent TGF­ß­binding protein 1 in human osteoarthritis fibroblast­like synoviocytes.

Osteoarthritis (OA) is a common degenerative joint disease; however, its underlying pathogenesis remains to be elucidated. Previous studies have demonstrated that the transforming growth factor­ß (TGF­ß) signaling pathway has a role in the initiation and development of OA. Additionally, latent TGF­ß­binding protein­1 (LTBP­1) modulates the activity of the TGF­ß­mothers against decapentaplegic (Smad) signaling pathway in numerous diseases, including malignant glioma. The present study demonstrated that expression of LTBP­1 is increased in OA synovial tissues compared with normal synovial tissues. The effect of TGF­ß was identified to be mediated by phosphorylated(p)­(Smad)2/3, which may activate activin­like kinase (ALK)5 receptor, and by p­Smad1/5/8, which may induce ALK1, thereby stimulating expression of matrix metalloproteinase­(MMP)­13 in OA fibroblast­like synoviocytes (FLS). Compared with normal FLS, OA FLS demonstrated an increased p­Smad1/5/8:p­Smad2 ratio, which led to elevated MMP­13 expression and aggravation of OA. Furthermore, knockdown of the LTBP­1 gene by siRNA transfection in OA FLS reduced p­Smad1/5/8 expression without affecting TGF­ß mRNA levels, although p­Smad2 expression increased. It was also demonstrated that OA FLS exhibited increased proliferation compared with normal FLS in vitro. Furthermore, siRNA­mediated downregulation of LTBP­1 reduced proliferation of OA FLS. In conclusion, the present study demonstrated that an alteration in the p­Smad1/5/8:p­Smad2 ratio as well as association between p­Smad1/5/8 and MMP­13 expression in human OA FLS, may contribute to the development of OA. The results of the present study suggested that LTBP­1 is a modulator of the TGF­ß signaling pathway in human OA FLS, which may aid in elucidating the mechanism underlying the pathology of OA.

Increased expression of latent TGF-ß-binding protein 4 affects the fibrotic process in scleroderma by TGF-ß/SMAD signaling.

Scleroderma is a fibrosis-related disorder characterized by cutaneous and internal organ fibrosis, and excessive collagen deposition in extracellular matrix (ECM) is a major cause of fibrosis. Transforming growth factor-ß (TGF-ß)/SMAD signaling has a central role in the pathogenesis of fibrosis by inducing abnormal collagen accumulation in ECM, and latent TGF-ß-binding protein 4 (LTBP-4) affects the secretion of latent TGF-ß to ECM. A previous study indicated that bleomycin (BLM) treatment increased LTBP-4 expression in lung fibroblasts of Thy-1 knockout mice with lung fibrosis, and LTBP-4 further promoted TGF-ß bioavailability as well as SMAD3 phosphorylation. However, the expression and function of LTBP-4 in human scleroderma remain unclear. We aimed to investigate the potential role of LTBP-4 in scleroderma through clinical, in vivo and in vitro studies. LTBP-4 and TGF-ß expressions were significantly upregulated in systemic scleroderma (SSc) patients' plasma compared with normal controls (LTBP-4, 1,215±100.2 vs 542.8±41.7 ng/ml, P<0.0001; TGF-ß, 1.5±0.2 vs 0.7±0.1 ng/ml, P=0.0031), while no significant difference was found between localized scleroderma (LSc) and normal controls. The plasma concentrations of LTBP-4 and TGF-ß were even higher in SSc patients with lung fibrosis (LTBP-4, 1462± 137.3 vs 892.8±113.4 ng/ml, P=0.0037; TGF-ß, 2.0±0.4 vs 0.9±0.2 ng/ml, P=0.0212) and esophagus involvement (1390±134.4 vs 940.7±127.0 ng/ml, P=0.0269; TGF-ß, 1.9±0.3 vs 0.9±0.2 ng/ml, P=0.0426). The area under receiver operating characteristics (ROC) curve of LTBP-4 was 0.86. Immunohistochemistry measurement also demonstrated a higher LTBP-4 expression in sclerotic skin tissue of LSc and SSc compared with normal controls. More positive fibroblasts were also found in BLM-induced scleroderma mouse model than the saline-treated group. In in vitro studies, knockdown of LTBP-4 in SSc skin fibroblasts prominently reduced downstream COL1A1, COL1A2, and COL3A1 mRNA level by 84%, 82%, and 43%, respectively, and other fibrosis-related genes' expression were also decreased. Furthermore, extracellular TGF-ß level and the SMAD2/3 phosphorylation were inhibited through LTBP-4 knockdown treatment, suggesting that the knockdown of LTBP-4 reduced the collagen expression through TGF-ß/SMAD signaling pathway. Taken together, these data suggest that LTBP-4 affects fibrotic process in scleroderma, and the high expression of LTBP-4 in SSc plasma may serve as a clinical biomarker in diagnosing this disease. In addition, this study also lays the theoretical foundation for targeting LTBP-4 as treatment of scleroderma.

LTBP2 is a prognostic marker in head and neck squamous cell carcinoma.

Latent transforming growth factor (TGF)-beta binding protein 2 (LTBP2) belongs to the fibrillin/LTBP extracellular matrix glycoprotein superfamily. It plays vital roles in tumorigenesis through regulating TGFß activity, elastogenesis and maintenance of the extracellular matrix (ECM) structure. In this study, we determined the expression levels of LTBP2 mRNA and protein in head and neck squamous cell carcinoma (HNSCC) tissues and adjacent normal tissues by quantitative reverse transcription PCR (qRT-PCR) and tissue microarray immunohistochemistry analysis (TMA-IHC) respectively. LTBP2 protein levels in cancer tissues were correlated with HNSCC patients' clinical characteristics and overall survival. Both LTBP2 mRNA and protein levels were significantly higher in HNSCC tissues than in adjacent normal tissues. High LTBP2 protein level was associated with lymph node metastasis and higher pTNM stages. High LTBP2 protein level is an independent prognostic marker in HNSCC. Our data suggest that LTBP2 acts as an oncogene in HNSCC development and progression. Detection of LTBP2 expression could be a useful prognosis marker and targeting LTBP2 may represent a novel strategy for cancer treatment through regulating activities of TGFß.

Latent transforming growth factor-ß binding proteins (LTBP-1 and LTBP-2) and gingiva keratinization.

OBJECTIVE: Transforming growth factor-beta (TGF-ß) proteins are involved in epithelial keratinization. The major function of latent TGF-ß binding proteins (LTBPs) is modulating TGF-ß activity. However, whether LTBP-1 and LTBP-2 play roles in gingiva keratinization remains unclear. MATERIALS AND METHODS: Human keratinized gingiva and non-keratinized alveolar mucosa were processed for LTBP-1, LTBP-2, cytokeratin-1 (K1), cytokeratin-4 (K4), and TGF-ß immunohistochemical (IHC) staining. Porcine heterotopically transplanted connective tissues and newly grown epithelia were harvested for IHC staining. The expression levels of LTBP-1 and LTBP-2 were compared between differentiated and undifferentiated human normal oral keratinocytes (hNOK). The expression of LTBP-1 and LTBP-2 was knocked down in a cell line (OEC-M1) to evaluate the effects on the expression of K1, K4, and involucrin (INV). RESULTS: In human and porcine specimens, LTBP-2 expression patterns distinguished keratinized and non-keratinized oral epithelia. Western blotting results showed that K1, LTBP-1, and INV proteins were upregulated in differentiated hNOK. In OEC-M1 cells, LTBP-2 knockdown resulted in upregulated the expression of K1 and INV and downregulated the expression of K4. LTBP-1 knockdown resulted in opposite effects. CONCLUSION: The expression patterns of LTBP-2 differ in keratinized gingiva and non-keratinized mucosa. LTBP-1 and LTBP-2 are involved in the keratinization of oral epithelium; however, the underlying mechanism remains to be elucidated.

Expressional alterations of fibrillin-1 during wound healing of human dental pulp.

INTRODUCTION: The degradation of fibrillins, the major constituents of microfibrils, is known to facilitate the release of active transforming growth factor-ß (TGF-ß), a signaling molecule contributing to mineralized tissue barrier formation in exposed dental pulps. To examine the involvement of fibrillins in the barrier formation, we examined the temporospatial expression of (1) genes and proteins of fibrillins and (2) factors possibly associated with fibrillin degradation and cytodifferentiation in exposed human pulps. Human pulp slice cultures were also examined for the role of fibrillins in mineralization. METHODS: Clinically healthy pulps were mechanically exposed and capped with mineral trioxide aggregate. After 7 to 42 days, the teeth were processed for immunohistochemical and cytochemical staining of fibrillin-1, fibrillin-2, latent TGF-ß-binding protein (LTBP)-1, matrix metalloproteinase-3 (MMP-3), alkaline phosphatase (ALP), and in situ hybridization of fibrillin-1. Pulp tissue slices cultured with ß-glycerophosphate were analyzed for fibrillin-1, fibrillin-2, and ALP with the immunohistochemical/cytochemical staining and quantitative reverse-transcriptase polymerase chain reaction. RESULTS: Fibrillin-1-immunoreactivity was seen until 7 days but turned into undetectable since 14 days in the pulpal area just beneath the exposure site. MMP-3-immunoreaction was transiently detected at 14 days. At 42 days when the mineralized barrier was evident, fibrillin-1-immunoreactivity and fibrillin-1 expression remained down-regulated. Fibrillin-2, LTBP-1, and ALP were constantly detected in the fibrillin-1-undetectable area. Pulp slices cultured with ß-glycerophosphate showed mineralization with up-regulation of ALP and down-regulation of fibrillin-1. CONCLUSIONS: Degradation and down-regulation of fibrillin-1 expression took place during the mineralized tissue barrier formation in exposed pulps in vivo and ß-glycerophosphate-induced pulpal mineralization in vitro.

The distribution of fibrillin-2 and LTBP-2, and their co-localisation with fibrillin-1 in adult bovine tail disc.

We investigated the distribution of fibrillin-2 and LTBP-2 (latent TGF-ß binding protein-2) in the intervertebral disc of the adult bovine tail. The association of fibrillin-2 and of LTBP-2 with fibrillin-1 was examined by dual immunofluorescence staining. Both fibrillin-2 and LTBP-2 were found extensively distributed in all regions of the disc with the organisation of the network varying significantly region to region. In the outer annulus fibrosus (OAF) both fibrillin-2 and LTBP-2 co-localised with fibrillin-1 forming fibres running parallel to the collagen fibres of the lamellae with the microfibrillar network staining densely in between the adjacent lamellae and also at the boundaries of the collagen bundle compartments. In the inner annulus fibrosus (IAF) and nucleus pulposus (NP), co-localised fibrillin-1,2 and LTBP-2 formed a chondron-like structure around the cell. By contrast, the inter-territorial matrix of the IAF and NP contained a dense network of fibrillin-2 but only sparse/filamentous fibres of fibrillin-1 and LTBP-2. Dual immunostaining revealed that in this region, fibrillin-2 was highly colocalised with elastin. The LTBP-2 network co-localised well with that of fibrillin-1 in all regions and indeed is reported to bind strongly to fibrillin-1. However, interestingly LTBP-2 but not fibrillin-1 or fibrillin-2 was removed by hyaluronidase but not collagenase pre-digestion. Our results suggest that fibrillin-2 and LTBP-2 could play an important role in disc function.

Comparative immunolocalization of the elastin fiber-associated proteins fibrillin-1, LTBP-2, and MAGP-1 with components of the collagenous and proteoglycan matrix of the fetal human intervertebral disc.

STUDY DESIGN: A comparative immunolocalization study of elastin-associated proteins and established intervertebral disc (IVD) extracellular matrix (ECM) components. OBJECTIVE: To localize for the first time, elastic fiber­associated proteins with structural fibrillar components in the annulus fibrosus (AF) of the fetal IVD. SUMMARY OF BACKGROUND DATA: Elastin has been identified histochemically in adult bovine, human, and immature rat IVDs, and in fetal human IVDs using electron microscopy; however, no immunolocalization studies have been undertaken for associated components in human fetal IVDs. METHODS: En-bloc fixation of thoracolumbar spinal segments in formalin and Histochoice followed by standard histochemical processing, paraffin embedding, microtome sectioning, and identification of IVD ECM components using a range of specific mono- and polyclonal antibodies and bright-field and laser scanning confocal microscopy. RESULTS: The elastic fiber-associated proteins fibrillin-1, LTBP-2, and MAGP-1 were prominently immunolocalized in the outer lamellar layers of the AF of the human fetal IVD. Dual localization of selected components by confocal microscopy demonstrated that versican and LTBP-2 were colocalized with fibrillin-1 microfibrils in the AF lamellae with a similar distribution to the elastin fibers. LTBP-2 was also associated with pericellular perlecan in the outer AF. These interconnections between elastin-associated proteins resulted in an elastic network, which connected the AF cells with the adjacent cartilaginous vertebral bodies. CONCLUSION: Specific immunolocalization of fibrillin-1, MAGP-1, and versican with elastin in the outer AF of the fetal human IVD has been demonstrated. We deduce from the established distributions of the elastin-associated proteins and their known interactivities with matrix components that these stabilize and aid in the integration of the elastic fibers in the annular lamellae and may be responsible for the generation of tensional forces in the outer AF, which direct the assembly of this tissue.

Localization of latent transforming growth factor-ß binding protein-1 in human coronary atherosclerotic plaques.

BACKGROUND: Transforming growth factor-ß (TGFß) and its receptors have been detected by immunohistochemistry in the normal vessel wall and in atherosclerotic lesions of human coronary arteries. However, TGFß is normally secreted as an inactive complex associated with a latent TGFß-binding protein (LTBP). Therefore, detection of TGFß antigen only in the arterial wall does not imply the activated form of the growth factor. METHODS AND RESULTS: In situ hybridization and immunohistochemistry demonstrated LTBP1 mRNA and protein expression throughout the media and intima of early coronary artery lesions, with the highest levels of protein at the luminal surface. In advanced lesions, LTBP1 mRNA and protein were detected mainly in regions of high cell density, such as the fibrous cap. CONCLUSIONS: Assays of the TGFß signalling pathway will be required to determine the activity associated with TGFß antigen in the vessel wall.

Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway.

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta1 mRNA level. The increase in stored TGF-beta1 was associated with enhanced LTBP-1 protein and mRNA levels. These dysregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta1 and LTBP-1 staining and between their respective mRNA expressions. In parallel, decorin was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta.