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1.
Matrix Biol Plus ; 22: 100149, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38831847

ABSTRACT

Although the mechanism for activation of latent TGFß1 and TGFß3 is understood to involve the binding of the TGFß propeptide (LAP) to both an integrin and an insoluble substrate, the activation of latent TGFß2 has been unclear because the TGFß2 LAP does not have the classical integrin binding sequence found in the other two TGFß isoform LAPs. To assess the potential requirement for covalent linkage with a matrix or cell surface protein for the activation of latent TGFß2, we generated mice in which the TGFß2 Cys residue predicted to be involved in binding was mutated to Ser (Tgfb2C24S). We reasoned that, if covalent interaction with a second molecule is required for latent TGFß2 activation, mutant mice should display a Tgfb2 null (Tgfb2-/-)-like phenotype. Tgfb2C24S mice closely phenocopy Tgfb2-/- mice with death in utero between E18 and P1 and with congenital heart and kidney defects similar to those described for Tgfb2-/- mice. The mutant latent TGFß2 is secreted at levels similar to WT, yet TGFß signaling monitored as nuclear pSmad2 is suppressed. We conclude that, like latent TGFß1, latent TGFß2 activation requires binding to an immobilized matrix or plasma membrane molecule.

2.
JACC Basic Transl Sci ; 9(2): 185-199, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38510715

ABSTRACT

The severity of aortic stenosis (AS) is associated with acquired von Willebrand syndrome (AVWS) and gastrointestinal bleeding, leading to anemia (Heyde's syndrome). We investigated how anemia is linked with AS and AVWS using the LA100 mouse model and patients with AS. Induction of anemia in LA100 mice increased transforming growth factor (TGF)-ß1 activation, AVWS, and AS progression. Patients age >75 years with severe AS had higher plasma TGF-ß1 levels and more severe anemia than AS patients age <75 years, and there was a correlation between TGF-ß1 and anemia. These data are compatible with the hypothesis that the blood loss anemia of Heyde's syndrome contributes to AS progression via WSS-induced activation of platelet TGF-ß1 and additional gastrointestinal bleeding via WSS-induced AVWS.

3.
Matrix Biol ; 121: 41-55, 2023 08.
Article in English | MEDLINE | ID: mdl-37217119

ABSTRACT

To assess the contribution of individual TGF-ß isoforms to aortopathy in Marfan syndrome (MFS), we quantified the survival and phenotypes of mice with a combined fibrillin1 (the gene defective in MFS) hypomorphic mutation and a TGF-ß1, 2, or 3 heterozygous null mutation. The loss of TGF-ß2, and only TGF-ß2, resulted in 80% of the double mutant animals dying earlier, by postnatal day 20, than MFS only mice. Death was not from thoracic aortic rupture, as observed in MFS mice, but was associated with hyperplastic aortic valve leaflets, aortic regurgitation, enlarged aortic root, increased heart weight, and impaired lung alveolar septation. Thus, there appears to be a relationship between loss of fibrillin1 and TGF-ß2 in the postnatal development of the heart, aorta and lungs.


Subject(s)
Haploinsufficiency , Marfan Syndrome , Animals , Mice , Aorta , Fibrillin-1/genetics , Marfan Syndrome/genetics , Phenotype , Transforming Growth Factor beta2/genetics
4.
bioRxiv ; 2023 Jun 24.
Article in English | MEDLINE | ID: mdl-38586035

ABSTRACT

Collagen VI-related disorders (COL6-RDs) are a group of rare muscular dystrophies caused by pathogenic variants in collagen VI genes (COL6A1, COL6A2, and COL6A3). Collagen type VI is a heterotrimeric, microfibrillar component of the muscle extracellular matrix (ECM), predominantly secreted by resident fibroadipogenic precursor cells in skeletal muscle. The absence or mislocalizatoion of collagen VI in the ECM underlies the non-cell autonomous dysfunction and dystrophic changes in skeletal muscle with an as of yet elusive direct mechanistic link between the ECM and myofiber dysfunction. Here, we conduct a comprehensive natural history and outcome study in a novel mouse model of COL6-RDs (Col6a2-/- mice) using standardized (Treat-NMD) functional, histological, and physiologic parameter. Notably, we identify a conspicuous dysregulation of the TGFß pathway early in the disease process and propose that the collagen VI deficient matrix is not capable of regulating the dynamic TGFß bioavailability at baseline and also in response to muscle injury. Thus, we propose a new mechanism for pathogenesis of the disease that links the ECM regulation of TGFß with downstream skeletal muscle abnormalities, paving the way for developing and validating therapeutics that target this pathway.

5.
Hum Mol Genet ; 31(19): 3281-3289, 2022 09 29.
Article in English | MEDLINE | ID: mdl-35567544

ABSTRACT

A disproportionate tall stature is the most evident manifestation in Marfan syndrome (MFS), a multisystem condition caused by mutations in the extracellular protein and TGFß modulator, fibrillin-1. Unlike cardiovascular manifestations, there has been little effort devoted to unravel the molecular mechanism responsible for long bone overgrowth in MFS. By combining the Cre-LoxP recombination system with metatarsal bone cultures, here we identify the outer layer of the perichondrium as the tissue responsible for long bone overgrowth in MFS mice. Analyses of differentially expressed genes in the fibrillin-1-deficient perichondrium predicted that loss of TGFß signaling may influence chondrogenesis in the neighboring epiphyseal growth plate (GP). Immunohistochemistry revealed that fibrillin-1 deficiency in the outer perichondrium is associated with decreased accumulation of latent TGFß-binding proteins (LTBPs)-3 and -4, and reduced levels of phosphorylated (activated) Smad2. Consistent with these findings, mutant metatarsal bones grown in vitro were longer and released less TGFß than the wild-type counterparts. Moreover, addition of recombinant TGFß1 normalized linear growth of mutant metatarsal bones. We conclude that longitudinal bone overgrowth in MFS is accounted for by diminished sequestration of LTBP-3 and LTBP-4 into the fibrillin-1-deficient matrix of the outer perichondrium, which results in less TGFß signaling locally and improper GP differentiation distally.


Subject(s)
Marfan Syndrome , Animals , Fibrillin-1/genetics , Fibrillin-2 , Fibrillins , Latent TGF-beta Binding Proteins/genetics , Latent TGF-beta Binding Proteins/metabolism , Marfan Syndrome/genetics , Mice , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism
6.
Bone ; 143: 115762, 2021 02.
Article in English | MEDLINE | ID: mdl-33212319

ABSTRACT

Parathyroid hormone (PTH) is necessary for the regulation of calcium homeostasis and PTH (1-34) was the first approved osteoanabolic therapy for osteoporosis. It is well established that intermittent PTH increases bone formation and that bone remodeling and several cytokines and chemokines play an essential role in this process. Earlier, we had established that the chemokine, monocyte chemoattractant protein-1 (MCP-1/CCL2), was the most highly stimulated gene in rat bone after intermittent PTH injections. Nevertheless, MCP-1 function in bone appears to be complicated. To identify the primary cells expressing MCP-1 in response to PTH, we performed in situ hybridization of rat bone sections after hPTH (1-34) injections and showed that bone-lining osteoblasts are the primary cells that express MCP-1 after PTH treatment. We previously demonstrated MCP-1's importance by showing that PTH's anabolic effects are abolished in MCP-1 null mice, further implicating a role for the chemokine in this process. To establish whether rhMCP-1 peptide treatment could rescue the anabolic effect of PTH in MCP-1 null mice, we treated 4-month-old wild-type (WT) mice with hPTH (1-34) and MCP-1-/- mice with rhMCP-1 and/or hPTH (1-34) for 6 weeks. Micro-computed tomography (µCT) analysis of trabecular and cortical bone showed that MCP-1 injections for 6 weeks rescued the PTH anabolic effect in MCP-1-/- mice. In fact, the combination of rhMCP-1 and hPTH (1-34) has a synergistic anabolic effect compared with monotherapies. Mechanistically, PTH-enhanced transforming growth factor-ß (TGF-ß) signaling is abolished in the absence of MCP-1, while MCP-1 peptide treatment restores TGF-ß signaling in the bone marrow. Here, we have shown that PTH regulates the transcription of the chemokine MCP-1 in osteoblasts and determined how MCP-1 affects bone cell function in PTH's anabolic actions. Taken together, our current work indicates that intermittent PTH stimulates osteoblastic secretion of MCP-1, which leads to increased TGF-ß signaling, implicating it in PTH's anabolic actions.


Subject(s)
Anabolic Agents , Transforming Growth Factor beta , Anabolic Agents/pharmacology , Animals , Bone and Bones , Chemokine CCL2 , Mice , Osteoblasts , Parathyroid Hormone/pharmacology , Rats , X-Ray Microtomography
7.
Sci Rep ; 10(1): 13477, 2020 08 10.
Article in English | MEDLINE | ID: mdl-32778777

ABSTRACT

Osteoarthritis (OA) affects nearly 10% of the population of the United States and other industrialized countries and, at present, short of surgical joint replacement, there is no therapy available that can reverse the progression of the disease. Adenosine, acting at its A2A receptor (A2AR), is a critical autocrine factor for maintenance of cartilage homeostasis and here we report that injection of liposomal suspensions of either adenosine or a selective A2AR agonist, CGS21680, significantly reduced OA cartilage damage in a murine model of obesity-induced OA. The same treatment also improved swelling and preserved cartilage in the affected knees in a rat model of established post-traumatic OA (PTOA). Differential expression analysis of mRNA from chondrocytes harvested from knees of rats with PTOA treated with liposomal A2AR agonist revealed downregulation of genes associated with matrix degradation and upregulation of genes associated with cell proliferation as compared to liposomes alone. Studies in vitro and in affected joints demonstrated that A2AR ligation increased the nuclear P-SMAD2/3/P-SMAD1/5/8 ratio, a change associated with repression of terminal chondrocyte differentiation. These results strongly suggest that targeting the A2AR is an effective approach to treat OA.


Subject(s)
Adenosine/pharmacology , Cartilage/drug effects , Osteoarthritis/drug therapy , Adenosine/administration & dosage , Adenosine/analogs & derivatives , Adenosine/metabolism , Animals , Cartilage/metabolism , Cartilage, Articular/drug effects , Cartilage, Articular/metabolism , Cell Differentiation , Chondrocytes/metabolism , Disease Models, Animal , Injections, Intra-Articular/methods , Liposomes/administration & dosage , Liposomes/metabolism , Liposomes/pharmacology , Male , Mice , Mice, Inbred C57BL , Osteoarthritis/metabolism , Phenethylamines/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction
8.
Am J Hum Genet ; 102(4): 706-712, 2018 04 05.
Article in English | MEDLINE | ID: mdl-29625025

ABSTRACT

The major diseases affecting the thoracic aorta are aneurysms and acute dissections, and pathogenic variants in 11 genes are confirmed to lead to heritable thoracic aortic disease. However, many families in which multiple members have thoracic aortic disease do not have alterations in the known aortopathy genes. Genes highly expressed in the aorta were assessed for rare variants in exome sequencing data from such families, and compound rare heterozygous variants (p.Pro45Argfs∗25 and p.Glu750∗) in LTBP3 were identified in affected members of one family. A homozygous variant (p.Asn678_Gly681delinsThrCys) that introduces an additional cysteine into an epidermal growth factor (EGF)-like domain in the corresponding protein, latent TGF-ß binding protein (LTBP-3), was identified in a second family. Individuals with compound heterozygous or homozygous variants in these families have aneurysms and dissections of the thoracic aorta, as well as aneurysms of the abdominal aorta and other arteries, along with dental abnormalities and short stature. Heterozygous carriers of the p.Asn678_Gly681delinsThrCys variant have later onset of thoracic aortic disease, as well as dental abnormalities. In these families, LTBP3 variants segregated with thoracic aortic disease with a combined LOD score of 3.9. Additionally, heterozygous rare LTBP3 variants were found in individuals with early onset of acute aortic dissections, and some of these variants disrupted LTBP-3 levels or EGF-like domains. When compared to wild-type mice, Ltbp3-/- mice have enlarged aortic roots and ascending aortas. In summary, homozygous LTBP3 pathogenic variants predispose individuals to thoracic aortic aneurysms and dissections, along with the previously described skeletal and dental abnormalities.


Subject(s)
Aortic Aneurysm, Thoracic/genetics , Aortic Dissection/genetics , Genetic Predisposition to Disease , Latent TGF-beta Binding Proteins/genetics , Mutation/genetics , Adult , Aged, 80 and over , Animals , Blood Pressure/genetics , Female , Homozygote , Humans , Male , Mice , Middle Aged , Pedigree
9.
Matrix Biol ; 71-72: 90-99, 2018 10.
Article in English | MEDLINE | ID: mdl-29217273

ABSTRACT

The latent transforming growth factor (TGF) ß binding proteins (LTBP) are crucial mediators of TGFß function, as they control growth factor secretion, matrix deposition, presentation and activation. Deficiencies in specific LTBP isoforms yield discrete phenotypes representing defects in bone, lung and cardiovascular development mediated by loss of TGFß signaling. Additional phenotypes represent loss of unique TGFß-independent features of LTBP effects on elastogenesis and microfibril assembly. Thus, the LTBPs act as sensors for the regulation of both growth factor activity and matrix function.


Subject(s)
Latent TGF-beta Binding Proteins/deficiency , Latent TGF-beta Binding Proteins/metabolism , Transforming Growth Factor beta/metabolism , Animals , Bone Diseases/metabolism , Cardiovascular Diseases/metabolism , Extracellular Matrix/metabolism , Humans , Lung Diseases/metabolism , Signal Transduction
10.
Eur J Oral Sci ; 125(1): 8-17, 2017 02.
Article in English | MEDLINE | ID: mdl-28084688

ABSTRACT

Latent-transforming growth factor beta-binding protein 3 (LTBP-3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor-ß (TGF-ß). To investigate the role of LTBP-3 in tooth formation we performed micro-computed tomography (micro-CT), histology, and scanning electron microscopy analyses of adult Ltbp3-/- mice. The Ltbp3-/- mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation-stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous-like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP-3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF-ß signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3-/- mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.


Subject(s)
Amelogenesis/genetics , Dental Enamel/abnormalities , Latent TGF-beta Binding Proteins/genetics , Ameloblasts/metabolism , Amelogenesis Imperfecta/genetics , Animals , Dental Enamel/ultrastructure , Genotype , Male , Mice , Mice, Mutant Strains , Microscopy, Electron, Scanning , Mutation , Osteochondrodysplasias/genetics , Phenotype , Tooth Calcification/genetics , Transforming Growth Factor beta/genetics , X-Ray Microtomography
11.
Biochem Biophys Res Commun ; 482(4): 1387-1392, 2017 Jan 22.
Article in English | MEDLINE | ID: mdl-27956181

ABSTRACT

The four latent transforming growth factor-ß (TGF-ß) binding proteins LTBP1-4 are extracellular matrix-associated proteins playing a critical role in the activation of TGF-ß. The LTBP1 gene forms two major transcript variants (i.e. Ltbp1S and Ltbp1L) that are derived from different promoters. We have previously shown the importance of LTBP1 in vivo by using three different Ltbp1 null mice that were either deleted for exons 1 and 2 (Ltbp1L knockout), exon 5 (Ltbp1ΔEx5), or exon 8 (Ltbp1ΔEx8). While the Ltbp1L knockout and the Ltbp1ΔEx8 are perinatal lethal and die of cardiovascular abnormalities, the Ltbp1ΔEx5 is viable because it expresses a short form of Ltbp1L that lacks 55 amino acids (Δ55 variant of Ltbp1) formed by splicing out exon 5, while lacking the Ltbp1S variant. Since only the Ltbp1ΔEx5 mouse is viable, we have used this model to address aspects of puberty, fertility, age-dependent reproduction, and ovary function. We report for the first time a function of LTBP1 in female reproduction. The Ltbp1ΔEx5 females showed impaired fertility associated with delayed sexual maturity (p = 0.0074) and ovarian cyst formation in females older than 40 weeks (p = 0.0204).


Subject(s)
Infertility, Female/genetics , Latent TGF-beta Binding Proteins/genetics , Ovarian Cysts/metabolism , Alternative Splicing , Animals , Carrier Proteins/metabolism , Cells, Cultured , Estrogens/blood , Exons , Extracellular Matrix/metabolism , Female , Fertility , Fertilization in Vitro , Genotype , Latent TGF-beta Binding Proteins/deficiency , Male , Mice , Mice, Knockout , Polymerase Chain Reaction , Progesterone/blood
12.
Article in English | MEDLINE | ID: mdl-27252363

ABSTRACT

The bioavailability of members of the transforming growth factor ß (TGF-ß) family is controlled by a number of mechanisms. Bona fide TGF-ß is sequestered into the matrix in a latent state and must be activated before it can bind to its receptors. Here, we review the molecules and mechanisms that regulate the bioavailability of TGF-ß and compare these mechanisms with those used to regulate other TGF-ß family members. We also assess the physiological significance of various latent TGF-ß activators, as well as other extracellular modulators of TGF-ß family signaling, by examining the available in vivo data from knockout mouse models and other biological systems.


Subject(s)
Biological Availability , Latent TGF-beta Binding Proteins/metabolism , Signal Transduction/physiology , Transforming Growth Factor beta/physiology , Animals , Carrier Proteins/metabolism , Extracellular Matrix/metabolism , Fibrillins/metabolism , Glycosylation , Humans , Mice , Mice, Knockout , Phylogeny , Protein Domains , Protein Multimerization
13.
J Endocrinol ; 228(3): R73-83, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26698564

ABSTRACT

Prolactinomas are the most frequently observed pituitary adenomas and most of them respond well to conventional treatment with dopamine agonists (DAs). However, a subset of prolactinomas fails to respond to such therapies and is considered as DA-resistant prolactinomas (DARPs). New therapeutic approaches are necessary for these tumors. Transforming growth factor ß1 (TGFß1) is a known inhibitor of lactotroph cell proliferation and prolactin secretion, and it partly mediates dopamine inhibitory action. TGFß1 is secreted to the extracellular matrix as an inactive latent complex, and its bioavailability is tightly regulated by different components of the TGFß1 system including latent binding proteins, local activators (thrombospondin-1, matrix metalloproteases, integrins, among others), and TGFß receptors. Pituitary TGFß1 activity and the expression of different components of the TGFß1 system are regulated by dopamine and estradiol. Prolactinomas (animal models and humans) present reduced TGFß1 activity as well as reduced expression of several components of the TGFß1 system. Therefore, restoration of TGFß1 inhibitory activity represents a novel therapeutic approach to bypass dopamine action in DARPs. The aim of this review is to summarize the large literature supporting TGFß1 important role as a local modulator of pituitary lactotroph function and to provide recent evidence of the restoration of TGFß1 activity as an effective treatment in experimental prolactinomas.


Subject(s)
Drug Resistance, Neoplasm , Pituitary Gland/metabolism , Pituitary Neoplasms/drug therapy , Prolactinoma/drug therapy , Transforming Growth Factor beta1/drug effects , Transforming Growth Factor beta1/physiology , Animals , Cell Proliferation , Dopamine/physiology , Dopamine Agonists/therapeutic use , Estradiol/physiology , Humans , Lactotrophs/physiology , Pituitary Neoplasms/physiopathology , Prolactin/antagonists & inhibitors , Prolactin/metabolism , Prolactinoma/physiopathology
14.
Proc Natl Acad Sci U S A ; 112(45): 14012-7, 2015 Nov 10.
Article in English | MEDLINE | ID: mdl-26494287

ABSTRACT

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, caused by mutations of the microfibrillar protein fibrillin-1, that predisposes affected individuals to aortic aneurysm and rupture and is associated with increased TGFß signaling. TGFß is secreted from cells as a latent complex consisting of TGFß, the TGFß propeptide, and a molecule of latent TGFß binding protein (LTBP). Improper extracellular localization of the latent complex can alter active TGFß levels, and has been hypothesized as an explanation for enhanced TGFß signaling observed in MFS. We previously reported the absence of LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFß signaling in MFS might be due to defective interaction of latent TGFß complexes containing LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we genetically suppressed Ltbp3 expression in a mouse model of progressively severe MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved survival, essentially no aneurysms, reduced disruption and fragmentation of medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their aortas. These data suggest that, in MFS, improper localization of latent TGFß complexes composed of LTBP-3 and TGFß contributes to aortic disease progression.


Subject(s)
Aortic Aneurysm, Thoracic/metabolism , Latent TGF-beta Binding Proteins/metabolism , Marfan Syndrome/complications , Marfan Syndrome/genetics , Multiprotein Complexes/metabolism , Transforming Growth Factor beta/metabolism , Analysis of Variance , Animals , Aortic Aneurysm, Thoracic/etiology , DNA, Complementary/biosynthesis , Fibrillin-1 , Fibrillins , Immunohistochemistry , Latent TGF-beta Binding Proteins/deficiency , Mice , Microfilament Proteins/genetics , Muscle, Smooth, Vascular/cytology , Real-Time Polymerase Chain Reaction
15.
Article in English | MEDLINE | ID: mdl-26379781

ABSTRACT

BACKGROUND: Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-ß, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-ß is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-ß must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-ß by cleavage between R(58) and L(59) residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R(58) (R(58) LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R(58) cleavage edges and functions as a footprint of PLK-dependent TGF-ß activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L(59) (L(59) LAP-DPs). We demonstrated that the L(59) LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis. RESULTS: We established a specific sandwich ELISA to quantify L(59) LAP-DPs as low as 2 pM and measured L(59) LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L(59) LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-ß receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L(59) LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) α1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L(59) LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with α-smooth muscle actin (αSMA) expression in liver tissues. At this time, αSMA-positive cells as well as R(58) LAP-DPs were seen in their liver tissues. CONCLUSIONS: L(59) LAP-DPs reflect PLK-dependent TGF-ß activation and the increase in αSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.

16.
Matrix Biol ; 47: 44-53, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25960419

ABSTRACT

The LTBPs (or latent transforming growth factor ß binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFß by covalently binding the TGFß propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFß precursor in the trans-golgi network but LAP and TGFß remain strongly bound through non-covalent interactions. LAP, TGFß, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFß latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFß activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFß regulation, TGFß-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.


Subject(s)
Latent TGF-beta Binding Proteins/physiology , Animals , Extracellular Matrix/physiology , Fibrillins , Humans , Microfilament Proteins/physiology , Protein Isoforms/physiology , Signal Transduction , Transforming Growth Factor beta/physiology
17.
J Immunol Methods ; 421: 27-35, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25769417

ABSTRACT

Much of our understanding of gut-microbial interactions has come from mouse models. Intestinal immunity is complex and a combination of host genetics and environmental factors play a significant role in regulating intestinal immunity. Due to this complexity, no mouse model to date gives a complete and accurate representation of human intestinal diseases, such as inflammatory bowel diseases. However, intestinal tissue from patients undergoing bowel resection reflects a condition of severe disease that has failed treatment; hence a more dynamic perspective of varying inflammatory states in IBD could be obtained through the analyses of pinch biopsy material. Here we describe our protocol for analyzing mucosal pinch biopsies collected predominantly during colonoscopies. We have optimized flow cytometry panels to analyze up to 8 cytokines produced by CD4+ and CD8+ cells, as well as for characterizing nuclear proteins and transcription factors such as Ki67 and Foxp3. Furthermore, we have optimized approaches to analyze the production of cytokines, including TGF-beta from direct ex vivo cultures of pinch biopsies and LPMCs isolated from biopsies. These approaches are part of our workflow to try and understand the role of the gut microbiota in complex and dynamic human intestinal diseases.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Colitis, Ulcerative/immunology , Crohn Disease/immunology , Cytokines/metabolism , Biopsy , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Colon/cytology , Colon/immunology , Colonoscopy , Forkhead Transcription Factors/metabolism , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Ki-67 Antigen/metabolism , Microbiota/immunology , Transforming Growth Factor beta/metabolism
18.
Matrix Biol ; 43: 61-70, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25805620

ABSTRACT

Latent transforming growth factor-ß binding protein-1 (LTBP-1) is an extracellular protein that is structurally similar to fibrillin and has an important role in controlling transforming growth factor-ß (TGF-ß) signaling by storing the cytokine in the extracellular matrix and by being involved in the conversion of the latent growth factor to its active form. LTBP-1 is found as both short (LTBP-1S) and long (LTBP-1L) forms, which are derived through the use of separate promoters. There is controversy regarding the importance of LTBP-1L, as Ltbp1L knockout mice showed multiple cardiovascular defects but the complete null mice did not. Here, we describe a third line of Ltbp1 knockout mice generated utilizing a conditional knockout strategy that ablated expression of both L and S forms of LTBP-1. These mice show severe developmental cardiovascular abnormalities and die perinatally; thus these animals display a phenotype similar to previously reported Ltbp1L knockout mice. We reinvestigated the other "complete" knockout line and found that these mice express a splice variant of LTBP-1L and, therefore, are not complete Ltbp1 knockouts. Our results clarify the phenotypes of Ltbp1 null mice and re-emphasize the importance of LTBP-1 in vivo.


Subject(s)
Genes, Lethal , Heart Defects, Congenital/genetics , Heart Defects, Congenital/veterinary , Latent TGF-beta Binding Proteins/genetics , Protein Isoforms/metabolism , Alternative Splicing , Animals , Animals, Newborn , Cells, Cultured , Exons , Fibroblasts/metabolism , Fibroblasts/pathology , Heart Defects, Congenital/pathology , Latent TGF-beta Binding Proteins/metabolism , Mice , Mice, Knockout , Protein Isoforms/genetics , Signal Transduction , Transforming Growth Factor beta/metabolism
19.
J Orthop Res ; 33(6): 832-9, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25641114

ABSTRACT

Tendon disorders are common and lead to significant disability, pain, healthcare cost, and lost productivity. A wide range of injury mechanisms exist leading to tendinopathy or tendon rupture. Tears can occur in healthy tendons that are acutely overloaded (e.g., during a high speed or high impact event) or lacerated (e.g., a knife injury). Tendinitis or tendinosis can occur in tendons exposed to overuse conditions (e.g., an elite swimmer's training regimen) or intrinsic tissue degeneration (e.g., age-related degeneration). The healing potential of a torn or pathologic tendon varies depending on anatomic location (e.g., Achilles vs. rotator cuff) and local environment (e.g., intrasynovial vs. extrasynovial). Although healing occurs to varying degrees, in general healing of repaired tendons follows the typical wound healing course, including an early inflammatory phase, followed by proliferative and remodeling phases. Numerous treatment approaches have been attempted to improve tendon healing, including growth factor- and cell-based therapies and rehabilitation protocols. This review will describe the current state of knowledge of injury and repair of the three most common tendinopathies--flexor tendon lacerations, Achilles tendon rupture, and rotator cuff disorders--with a particular focus on the use of animal models for understanding tendon healing.


Subject(s)
Tendon Injuries/etiology , Tendon Injuries/therapy , Wound Healing , Animals , Humans , Models, Animal , Tendon Injuries/epidemiology
20.
Hum Mol Genet ; 24(11): 3038-49, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-25669657

ABSTRACT

Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.


Subject(s)
Amelogenesis Imperfecta/genetics , Latent TGF-beta Binding Proteins/genetics , Osteochondrodysplasias/genetics , Adolescent , Amelogenesis Imperfecta/diagnostic imaging , Animals , Base Sequence , Child , Consanguinity , DNA Mutational Analysis , Female , Frameshift Mutation , Genetic Association Studies , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation, Missense , Osteochondrodysplasias/diagnostic imaging , Pedigree , Radiography , Sequence Deletion
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