Latent Transforming Growth Factor Binding Research Articles

Abstract P2107: Latent Transforming Growth Factor Binding Protein - 2 Deficiency Improves Cardiac Function Post-Myocardial Infarction

Cardiac fibrosis and myocardial stiffening are key factors contributing to deteriorating cardiac function during heart failure, especially after myocardial infarction (MI). In the myocardium, cardiac fibroblasts (CFs) are the main cell type driving excess extracellular matrix (ECM) protein deposition, leading to pathological remodelling of the heart. Latent Transforming Growth Factor Binding Protein 2 (LTBP2) is one of the ECM proteins released as part of the fibrotic response and is significantly upregulated in all types of heart failure. Although LTBP2 belongs to a family of proteins that regulate the release of Transforming Growth Factor - β; (TGF-β;) in the ECM, it doesn’t interact with TGF-β;. Nonetheless, LTBP2 is shown to play a role in the progression of fibrosis following heart failure by contributing to CFs activation. Therefore, we hypothesized that in the absence of LTBP2, the heart would have improved cardiac function following MI. We used LTBP2 knockout (KO) C57BL/6 mice and modelled MI by ligating the left anterior descending artery. Echocardiography was performed to analyze the cardiac function in KO and wild-type (WT) infarcted hearts at 7 and 28 days post-MI. KO mice showed significantly better ejection fractions (KO = 24.1±4.9% vs. WT = 9.2±1.4%, p-value < 0.01) and fractional shortening 28 days post-MI compared to WT mice. In conclusion, our findings show that LTBP2 contributes to the deteriorating cardiac function following MI suggesting that targeting LTBP2 may have therapeutic potential. As LTBP2 is known to be a part of the fibrotic response, future studies will investigate the fibrotic profile of WT and KO infarcted hearts using histology and CFs-specific RNA sequencing to highlight the underlying differences leading to the observed cardiac function.

Three-generation family with novel contiguous gene deletion on chromosome 2p22 associated with thoracic aortic aneurysm syndrome.

Latent transforming growth factor binding proteins (LTBP) are a family of extracellular matrix glycoproteins that play an important role in the regulation of transforming growth factor beta (TGF-ß) activation. Dysregulation of the TGF-ß pathway has been implicated in the pathogenesis of inherited disorders predisposing to thoracic aortic aneurysms syndromes (TAAS) including Marfan syndrome (MFS; FBN1) and Loeys-Dietz syndrome (LDS; TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, SMAD3). While these syndromes have distinct clinical criteria, they share clinical features including aortic root dilation and musculoskeletal findings. LTBP1 is a component of the TGF-ß pathway that binds to fibrillin-1 in the extracellular matrix rendering TGF-ß inactive. We describe a three-generation family case series with a heterozygous ∼5.1 Mb novel contiguous gene deletion of chromosome 2p22.3-p22.2 involving 11 genes, including LTBP1. The deletion has been identified in the proband, father and grandfather, who all have a phenotype consistent with a TAAS. Findings include thoracic aortic dilation, ptosis, malar hypoplasia, high arched palate, retrognathia, pes planus, hindfoot deformity, obstructive sleep apnea, and low truncal tone during childhood with joint laxity that progressed to reduced joint mobility over time. While the three affected individuals did not meet criteria for either MFS or LDS, they shared features of both. Although the deletion includes 11 genes, given the relationship between LTBP1, TGF-ß, and fibrillin-1, LTBP1 stands out as one of the possible candidate genes for the clinical syndrome observed in this family. More studies are necessary to evaluate the potential role of LTBP1 in the pathophysiology of TAAS.

Latent transforming growth factor binding protein 4 regulates transforming growth factor beta receptor stability.

Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFβ) signaling. Despite elevated extracellular TGFβ activity, downstream signaling molecules of the TGFβ pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFβ receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFβ1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFβ receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFβ signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFβ receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFβ receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.

The identification of three novel biomarkers of major adverse kidney events.

To describe the prognostic value of three novel biomarkers for acute adverse kidney events compared with routine biological markers. We used high-end MS to quantify biomarkers predictive of acute kidney injury (AKI) and major adverse kidney events (MAKE) in 100 adult patients after open heart surgery (n = 100). Early postoperatively measured LG3 (a C-terminal fragment of perlecan), LTBP2 (latent transforming growth factor binding protein-2), Cathepsin L as well as two other renal biomarkers (NGAL, Cystatin C) had greater predictive value for AKI (n = 23) and MAKE (n = 24) compared with creatinine, urea and urine output. LG3, LTBP2 and Cathepsin L deserve further exploration as biomarkers for the early identification of patients at risk of MAKE.

Novel biomarkers of acute kidney injury: time for implementation?

Novel biomarkers of acute kidney injury: time for implementation?

The composition of exfoliation material and the cells involved in its production.

The hallmark of exfoliation syndrome is the pathologic production and stable accumulation of an abnormal, fibrillar, extracellular material (XFM). Although the exact biochemical composition of the pathologic matrix product is still not known, immunohistochemical and biochemical approaches have shown XFM to represent a highly glycosylated, cross-linked, and enzymatically resistant glycoprotein/proteoglycan complex, composed of a protein core surrounded by glycoconjugates. The protein core includes basement membrane components, such as laminin, nidogen, and fibronectin, and components of the elastic fiber system, such as fibrillin-1, elastin, and latent transforming growth factor binding proteins, as well as enzymatically active components, such as metalloproteinases, the extracellular chaperone clusterin, and the cross-linking enzyme lysyl oxidase-like 1 (LOXL1). Ultrastructural evidence suggests that XFM is multifocally produced by intraocular cells, such as ciliary epithelial cells, preequatorial epithelial cells of the lens, trabecular and corneal endothelial cells, all cell types of the iris, as well as by extraocular cells, such as fibrocytes, vascular cells, and muscle cells. All cells involved in the exfoliation syndrome process disclosed common ultrastructural signs of active fibrillogenesis and metabolic activation, such as increased vesicular transport to the cell surface, XFM formation within infoldings of cellular surfaces, and a prominent rough endoplasmic reticulum. Finally, cells involved in the production of XFM displayed a gene expression pattern characterized by the upregulation of elastic components, the transient upregulation of LOXL1, and the dysregulated expression of cytoprotective gene products, matrix metalloproteinases, and their inhibitors, possibly leading to the accumulation and stable deposition of XFM.

Transcriptional profiling of the human fibrillin/LTBP gene family, key regulators of mesenchymal cell functions

The fibrillins and latent transforming growth factor binding proteins (LTBPs) form a superfamily of extracellular matrix (ECM) proteins characterized by the presence of a unique domain, the 8-cysteine transforming growth factor beta (TGFβ) binding domain. These proteins are involved in the structure of the extracellular matrix and controlling the bioavailability of TGFβ family members. Genes encoding these proteins show differential expression in mesenchymal cell types which synthesize the extracellular matrix. We have investigated the promoter regions of the seven gene family members using the FANTOM5 CAGE database for human. While the protein and nucleotide sequences show considerable sequence similarity, the promoter regions were quite diverse. Most genes had a single predominant transcription start site region but LTBP1 and LTBP4 had two regions initiating different transcripts. Most of the family members were expressed in a range of mesenchymal and other cell types, often associated with use of alternative promoters or transcription start sites within a promoter in different cell types. FBN3 was the lowest expressed gene, and was found only in embryonic and fetal tissues. The different promoters for one gene were more similar to each other in expression than to promoters of the other family members. Notably expression of all 22 LTBP2 promoters was tightly correlated and quite distinct from all other family members. We located candidate enhancer regions likely to be involved in expression of the genes. Each gene was associated with a unique subset of transcription factors across multiple promoters although several motifs including MAZ, SP1, GTF2I and KLF4 showed overrepresentation across the gene family. FBN1 and FBN2, which had similar expression patterns, were regulated by different transcription factors. This study highlights the role of alternative transcription start sites in regulating the tissue specificity of closely related genes and suggests that this important class of extracellular matrix proteins is subject to subtle regulatory variations that explain the differential roles of members of this gene family.

Latent Transforming growth factor Binding Protein 2 (LTBP2) is related to phenotypic changes suggestive for HFPEF in stable outpatients with coronary artery disease and preserved ejection fraction

Purpose: The Latent Transforming growth factor Binding Protein 2 (LTBP2) was recently discovered in an unbiased proteomics search for novel markers in patients with acute dyspnea. The highest levels of LTBP2 are found in lung tissue and plasma levels of LTBP2 are a powerful predictor of all-cause mortality in patients presenting with acute dyspnea. We evaluated the relationship between plasma levels of LTBP2 and phenotypic changes suggestive for heart failure with preserved ejection fraction (HFPEF) including parameters of neurohormonal activation, Left Ventricular (LV) remodeling and filling pressures as well as exercise capacity. Methods: We studied 136 stable patients with CAD and LVEF≥50% (mean age 68±8 years, 76% men, LVEF 63±8%, 37% in NYHA class II/III). All patients underwent resting echocardiography for evaluation of LVEF and volumes, TDI derived longitudinal systolic and diastolic velocities function and E/E', as well as a six minute walking distance test (6minWD), maximal bicycle spiroergometry (peakVO2 and VE/VCO2 slope, n=104) and blood sampling. A proteomic approach for antibody-free targeted protein quantification based on high-end mass spectrometry was used to measure LTBP2 levels. Results: Levels of LTBP2 were higher in NYHA II/III as compared to NYHA I patients (p<0.05) and were correlated with NT-proBNP levels (r=0.49, p<0.01). ROC analysis showed a comparable modest AUC of 0.61 (p<0.05) for NT-proBNP and 0.60 (p<0.05) for LTBP2 to discern NYHA I versus NYHA II/III patients. LTBP2 levels were correlated with LV volumes (r=-0.38, p<0.01 for LVEDV, r=-0.33, p<0.01 for LVESV) as well as with LV longitudinal systolic velocities (r=-0.35, p<0.01), LV early diastolic velocities (r=-0.31,p<0.01) and E/E' (r=0.28,p<0.01). In linear regression analysis, including age and creatinine, LVESV and E/E' were independent predictors for LTBP2 levels (all p<0.05). Higher levels of LTBP2 were associated with lower 6minWD (r=-0.43, p<0.001), lower peakVO2 (r=-0.38, p<0.001) and higher VE/VCO2 slope (r=0.36, p<0.001). In multiple linear regression analysis including age, creatinine, LVESV and E/E', LTBP2 levels appeared to be an independent predictor for both 6minWD and peakVO2 (p<0.01). Conclusion: In stable patients with CAD and preserved LV function, higher levels of LTBP2 are related to phenotypic changes suggestive for HFPEF including higher neurohormonal activation, adverse LV remodeling with loss of longitudinal function, higher LV filling pressures and a lower exercise capacity. Therefore, the role of LTBP2 in the development, diagnosis and outcome of HFPEF deserves further study.

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.

Latent transforming growth factor binding protein 4 (LTBP4) is downregulated in mouse and human DCIS and mammary carcinomas

BackgroundTransforming growth factor beta (TGF-ß) is able to inhibit the proliferation of epithelial cells and is involved in the carcinogenesis of mammary tumors. Three latent transforming growth factor-ß binding proteins (LTBPs) are known to modulate TGF-ß functions.MethodsThe current study analyses the expression profiles of LTBP4, its isoforms LTBP1 and LTBP3, and TGF-ß1, TGF-ß2, TGF-ß3, and SMAD2, SMAD3 and SMAD4 in human and murine (WAP-TNP8) DCIS compared to invasive mammary tumors. Additionally mammary malignant (MCF7, Hs578T, MDA-MB361) and non malignant cell lines (Hs578BsT) were analysed. Microarray, q-PCR, immunoblot, immunohistochemistry and immunofluorescence were used.ResultsIn comparison to non-malignant tissues (n = 5), LTBP4 was downregulated in all human and mouse DCIS (n = 9) and invasive mammary adenocarcinomas (n = 5) that were investigated. We also found decreased expression of bone morphogenic protein 4 (BMP4) and increased expression of its inhibitor gremlin (GREM1). Treatment of the mammary tumor cell line (Hs578T) with recombinant TGF-ß1 rescued BMP4 and GREM1 expression.ConclusionWe conclude that the lack of LTBP4-mediated targeting in malignant mammary tumor tissues may lead to a possible modification of TGF-ß1 and BMP bioavailability and function.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-011-0023-y) contains supplementary material, which is available to authorized users.

Latent transforming growth factor binding protein 4 (LTBP‐4) is downregulated in human mammary adenocarcinomas in vitro and in vivo

Transforming growth factor beta (TGF-ss) is able to inhibit proliferation of epithelial cells and is involved in the carcinogenesis of human mammary tumours. Three latent transforming growth factor-beta binding proteins (LTBP-1, -3 and -4) are involved in TGF-beta function. The aim of the study was to analyze the expression profiles of TGF-beta 1 and 2 and LTBP-4 in human mammary carcinoma cell lines as well as in human mammary tumours. Expression analysis was performed at the transcription and protein level under in vivo and in vitro conditions. LTBP-4 expression was quantitatively analysed in human carcinomas of the mammary gland and in healthy mammary tissues of the same patients. Downregulation of LTBP-4 in all investigated human mammary tumours compared to normal tissues could be demonstrated. Results also revealed that protein levels of TGF-beta 1 are downregulated and of TGF-beta 2 are upregulated in human mammary carcinoma cell lines compared to primary (normal) human mammary epithelial cells. LTBP-4 reduction in neoplasms leads to a possible decrease of TGF-beta 1 extracellular deposition with reduced TGF-beta 1 bioavailability. TGF-beta 2 was upregulated, which indicates a possible compensatory mechanism. This study demonstrated a possible functional role of LTBP-4 for TGF-beta bioavailability with respect to carcinogenesis of human mammary tumours in vivo and in vitro.

Marfan's syndrome

1978 www.thelancet.com Vol 366 December 3, 2005 Marfan’s syndrome is a multisystem connective tissue disorder of autosomal dominant inheritance. Approximately one in every 5000 individuals is affected, though this figure is probably an underestimate. The condition shows no predilection for any particular race or geographical background. It exhibits complete penetrance but variable expression and age dependency. About a quarter of affected individuals have no family history of the disease—ie, they are new mutations. Marfan’s syndrome demonstrates pleiotropy with diverse manifestations in different organ systems. Clinical diagnosis depends on a combination of major and minor signs defined in the revised 1996 Ghent criteria. The hallmark features are noted in the cardiovascular, ocular, and skeletal systems (table). The diagnosis of Marfan’s syndrome can be challenging because of phenotypic variability even between affected individuals of the same family, low specificity of many of the clinical signs, and multiple Marfan-like microfibrillar disorders with overlapping features. The gene for Marfan’s syndrome was localised to chromosome 15q21 in 1990 and cloned in 1991. FBN1 is a huge gene, spanning 110 kb of genomic DNA and comprising 65 exons. It encodes the microfibrillar protein fibrillin 1. Mutational analysis is difficult and impractical at times, in part because of the size of the gene, the heterogeneity of mutations discovered, and the lack of mutational hot spots. Moreover, there are currently no molecular techniques that offer a highly sensitive mutation detection rate of FBN1. Fibrillin 1 is a major component of extracellular microfibrils. It consists of cysteine-rich repeats interspersed by latent transforming growth factor binding protein-like and epidermal growth factor-like motifs. The microfibrils are thought to confer important biomechanical properties in connecting, anchoring, and maintaining tissues and organs. Many mutations associated with fibrillin 1 appear to confer a dominant negative mechanism of pathogenesis, but some associated with fibrillin-1 haploinsufficiency have also been described. These mutations can lead to defects in the synthesis, secretion, modification, folding, stabilisation, incorporation, and assembly of the FBN1 protein. Severe neonatal Marfan’s syndrome is associated with a cluster of mutations in the middle portion of FBN1, but there are no other established correlations between genotype and phenotype. Currently, our understanding of fibrillin biochemistry and microfibrillar assembly is limited. Experiments in mice could provide insight into fibrillin pathogenesis and microfibrillar organisation, leading to a better understanding of the pleiotropic manifestations of Marfan’s syndrome and perhaps development of a cure. Lancet 2005; 366: 1978–81

Differential Gene Expression in Pseudoexfoliation Syndrome

To identify and characterize genes differentially expressed in anterior segment tissues of eyes with pseudoexfoliation (PEX) syndrome and glaucoma. Anterior segment tissues (iris, ciliary processes, lens epithelium) were obtained from eight surgically enucleated eyes with PEX-associated open-angle or closed-angle glaucomas and eight age-matched glaucomatous control eyes without PEX. cDNA libraries were generated from three PEX and three control specimens, and their gene expression patterns were compared by means of cDNA subtraction. Differentially expressed clones from the subtracted cDNA libraries were sequenced, and their differential expression was verified by means of RT-PCR, virtual Northern blot analysis, and in situ hybridization with specific RNA probes. Subtraction of cDNA libraries identified 27 candidate genes for differential expression in PEX tissues, of which 23 genes were confirmed by virtual Northern blot, RT-PCR, and in situ hybridization. One set of genes consistently upregulated in anterior segment tissues from different patients with PEX comprised latent transforming growth factor binding proteins (LTBP-1 and -2), which are structural components of elastic microfibrils, the cross-linking enzyme transglutaminase-2 (TGase-2), tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), A-kinase anchor protein-2 (AKAP-2), apolipoprotein D, and the adenosine receptor-A3 (AdoR-A3). Genes reproducibly downregulated in PEX tissues included TIMP-1, clusterin, microsomal glutathione-S-transferase-1 (mGST-1), and serum amyloid A1. Further transcripts, such as elastase, GST-T1, integrin beta4, and dehydrocholesterol reductase, did not show a consistent differential expression pattern in tissues obtained from different patients. Although fibrillin-1 was not isolated from subtracted cDNA libraries, upregulated expression of this elastic microfibrillar component was also demonstrated by RT-PCR and in situ hybridization. Differentially expressed genes with a high level of reproducibility in different tissues and different patients with PEX syndrome are mainly related to extracellular matrix metabolism and cellular stress. The underlying pathophysiology of PEX syndrome appears to be associated with an excessive production of elastic microfibril components, enzymatic cross-linking processes, a proteolytic imbalance between matrix metalloproteinases and their inhibitors, and increased cellular and oxidative stress supporting the notion of PEX syndrome as a stress-induced elastic microfibrillopathy.

Molecular cloning of the mouse Ltbp-1 gene reveals tissue specific expression of alternatively spliced forms

Latent transforming growth factor binding proteins (Ltbp-1, -2, -3 and -4) and fibrillins (Fbn-1 and -2) are structurally related cysteine-rich extracellular matrix proteins that localize to the 10 nm microfibrils. Ltbp-1 is thought to promote the secretion and proper folding of the small latent transforming growth factor β (TGF-β) complex (TGF-β plus its propeptide) and is implicated in sequestering it in the extracellular matrix. Here we report the isolation of the mouse Ltbp-1 complementary DNA (cDNA) and gene. The longer form of the Ltbp-1 cDNA encodes a predicted 1713 amino acid protein containing 18 epidermal growth factor-like repeats, four 8-cysteine domains and several motifs that suggest interactions with α IVβ 1 and α 9β 1 integrins. Northern blotting analyses indicate that long and short Ltbp-1 transcripts are widely expressed in adult mouse tissues and most abundantly expressed in heart. Ltbp-1 is a single copy gene that maps to chromosome 17, band E (1–3) and encompasses more than 212 kb. The Ltbp-1 gene contains 34 exons and shows a similar organization to the LTBP-2 gene, suggesting that these genes originated from a common ancestral gene.