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/bloodABSTRACT
Latent TGF-ß-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.
Subject(s)
Cilia/genetics , Latent TGF-beta Binding Proteins/genetics , Microfibrils/genetics , Animals , Cell Line , Ectopia Lentis/genetics , Ectopia Lentis/pathology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fibrillin-1 , Fibrillins , Gene Knockout Techniques , Gene Targeting , Genotype , Glaucoma/genetics , Humans , Latent TGF-beta Binding Proteins/metabolism , Mice , Mice, Knockout , Microfilament Proteins/metabolism , Mutation , Phenotype , Protein BindingABSTRACT
Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-ß binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-ß because TGF-ß-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-ß-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.
Subject(s)
Extracellular Matrix Proteins/metabolism , Latent TGF-beta Binding Proteins/physiology , Recombinant Proteins/metabolism , Animals , HEK293 Cells , Humans , Latent TGF-beta Binding Proteins/metabolism , Mice , Mice, Knockout , Protein Binding , RNA InterferenceABSTRACT
PURPOSE: We aimed to predict the neurological prognosis of cardiac arrest (CA) patients using quantitative imaging biomarkers extracted from brain computed tomography images. METHODS: We retrospectively enrolled 86 CA patients (good prognosis, 32; poor prognosis, 54) who were treated at three hospitals between 2017 and 2019. We then extracted 1131 quantitative imaging biomarkers from whole-brain and local volumes of interest in the computed tomography images of the patients. The data were split into training and test sets containing 60 and 26 samples, respectively, and the training set was used to select representative quantitative imaging biomarkers for classification. In univariate analysis, the classification was evaluated using the p-value of the Brunner-Munzel test and area under the receiver operating characteristic curve (AUC) for the test set. In multivariate analysis, machine learning models reflecting nonlinear and complex relations were trained, and they were evaluated using the AUC on the test set. RESULTS: The best performance provided p = 0.009 (<0.01) and an AUC of 0.775 (95% confidence interval, 0.590-0.960) for the univariate analysis and an AUCof0.813 (95% confidence interval, 0.640-0.985) for the multivariate analysis. Overall, the gray level with the maximum gradient in the histogram of the three-dimensionally low-pass-filtered image was an important feature for prediction across the analyses. CONCLUSIONS: Quantitative imaging biomarkers can be used in neurological prognosis prediction for CA patients. Relevant biomarkers may contribute to protocolized computed tomography image acquisition to ensure proper decision support in acute care.
ABSTRACT
Sterol regulatory element-binding protein 2 (SREBP-2) transcription factor has been identified as a key protein in cholesterol metabolism through the transactivation of the LDL receptor and cholesterol biosynthesis genes. Here, we generated mice lacking microRNA (miR)-33, encoded by an intron of the Srebp2, and showed that miR-33 repressed the expression of ATP-binding cassette transporter A1 (ABCA1) protein, a key regulator of HDL synthesis by mediating cholesterol efflux from cells to apolipoprotein A (apoA)-I. In fact, peritoneal macrophages derived from miR-33-deficient mice showed a marked increase in ABCA1 levels and higher apoA-I-dependent cholesterol efflux than those from WT mice. ABCA1 protein levels in liver were also higher in miR-33-deficient mice than in WT mice. Moreover, miR-33-deficient mice had significantly higher serum HDL cholesterol levels than WT mice. These data establish a critical role for miR-33 in the regulation of ABCA1 expression and HDL biogenesis in vivo.
Subject(s)
Cholesterol, HDL/metabolism , Introns , MicroRNAs/metabolism , Sterol Regulatory Element Binding Protein 2/genetics , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Base Sequence , Cattle , Cell Line , Chickens , Cholesterol, HDL/genetics , Female , Humans , Macrophages/metabolism , Mice , MicroRNAs/genetics , Molecular Sequence Data , Sequence Alignment , Sterol Regulatory Element Binding Protein 2/metabolismABSTRACT
Fibrillin microfibrils are extracellular matrix structures with essential functions in the development and the organization of tissues including blood vessels, bone, limbs and the eye. Fibrillin-1 and fibrillin-2 form the core of fibrillin microfibrils, to which multiple proteins associate to form a highly organized structure. Defining the components of this structure and their interactions is crucial to understand the pathobiology of microfibrillopathies associated with mutations in fibrillins and in microfibril-associated molecules. In this study, we have analyzed both in vitro and in vivo the role of fibrillin microfibrils in the matrix deposition of latent TGF-ß binding protein 1 (LTBP-1), -3 and -4; the three LTBPs that form a complex with TGF-ß. In Fbn1(-/-) ascending aortas and lungs, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. In addition, in cultures of Fbn1(-/-) smooth muscle cells or lung fibroblasts, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. Fibrillin-2 is not involved in the deposition of LTBP-1 in Fbn1(-/-) extracellular matrix as cells deficient for both fibrillin-1 and fibrillin-2 still incorporate LTBP-1 in their matrix. However, blocking the formation of the fibronectin network in Fbn1(-/-) cells abrogates the deposition of LTBP-1. Together, these data indicate that LTBP-3 and LTBP-4 association with the matrix depends on fibrillin-1 microfibrils, whereas LTBP-1 association depends on a fibronectin network.
Subject(s)
Fibronectins/metabolism , Gene Expression Regulation/physiology , Latent TGF-beta Binding Proteins/metabolism , Microfilament Proteins/metabolism , Animals , DNA, Complementary/genetics , DNA, Complementary/metabolism , Fibrillin-1 , Fibrillin-2 , Fibrillins , Fibroblasts/metabolism , Fibronectins/genetics , Latent TGF-beta Binding Proteins/genetics , Lung/cytology , Mice , Mice, Knockout , Microfilament Proteins/genetics , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , RNA/genetics , RNA/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat-containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5-deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171-175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168-171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.
Subject(s)
Elastic Tissue/metabolism , Extracellular Matrix Proteins/physiology , Microfibrils/metabolism , Tropoelastin/metabolism , Aging/metabolism , Amino Acid Oxidoreductases/metabolism , Animals , Cell Line , Culture Media, Serum-Free/pharmacology , Elastic Tissue/ultrastructure , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Humans , Mice , Microfibrils/ultrastructure , Regeneration/physiology , Skin/metabolism , Skin/ultrastructureABSTRACT
Great arteries, as well as lungs and skin, contain elastic fibers as important components to maintain their physiological functions. Although recent studies have revealed that a glycoprotein fibulin-4 (FBLN4) is indispensable for the assembly of mature elastic fibers, it remains to be elucidated how FBLN4 takes part in elastogenesis. Here, we report a dose-dependent requirement for FBLN4 in the development of the elastic fibers in arteries, and a specific role of FBLN4 in recruiting the elastin-cross-linking enzyme, lysyl oxidase (LOX). Reduced expression of Fbln4, which was achieved with a smooth muscle-specific Cre-mediated gene deletion, caused arterial stiffness. Electron-microscopic examination revealed disorganized thick elastic laminae with aberrant deposition of elastin. Aneurysmal dilation of the ascending aorta was found when the Fbln4 expression level was reduced to an even lower level, whereas systemic Fbln4 null mice died perinatally from rupture of the diaphragm. We also found a specific interaction between FBLN4 and the propeptide of LOX, which efficiently promotes assembly of LOX onto tropoelastin. These data suggest a mechanism of elastogenesis, in which a sufficient amount of FBLN4 is essential for tethering LOX to tropoelastin to facilitate cross-linking.
Subject(s)
Arteries/metabolism , Elastin/metabolism , Extracellular Matrix Proteins/metabolism , Protein-Lysine 6-Oxidase/metabolism , Animals , Arteries/ultrastructure , Extracellular Matrix Proteins/genetics , Gene Deletion , Immunohistochemistry , Mice , Mice, Knockout , Microscopy, Electron , Polymerase Chain Reaction , Protein BindingABSTRACT
A 78-year-old woman who sustained traumatic liver injury with hemorrhagic shock was hospitalized. She was admitted to the ICU after blood transfusion and emergent angiography. AKI was observed on the following day. Blood transfusion was continued because initial assessment was prerenal AKI due to hypovolemia. Despite transfusion of blood products and administration of diuretics, aggravated renal dysfunction, and low urine output continued, resulting in respiratory failure due to pulmonary edema. Renal venous congestion was suspected as the primary cause of AKI, since IVC compression from a hematoma with IVC injury was observed on CT imaging captured on admission, and renal Doppler ultrasonography demonstrated an intermittent biphasic pattern of renal venous flow. It was finally concluded that renal venous congestion resulted from IVC compression, since urine output increased remarkably after RRT without additional diuretics, and follow-up CT and renal Doppler ultrasonography revealed improvements in IVC compression and renal venous flow pattern, respectively. Renal venous congestion has been often reported to be associated with acute decompensated heart failure and, to our knowledge, this is the first report to describe trauma-induced renal venous congestion. Trauma patients are at risk for renal venous congestion due to massive blood transfusion after recovery from hemorrhagic shock; therefore, if they develop AKI that cannot be explained by other etiologies, physicians should consider the possibility of trauma-induced renal venous congestion and perform renal Doppler ultrasonography.
Subject(s)
Hyperemia/diagnosis , Kidney Diseases/diagnosis , Liver/injuries , Shock, Hemorrhagic/complications , Wounds and Injuries/complications , Aged , Female , Humans , Hyperemia/diagnostic imaging , Hyperemia/etiology , Kidney Diseases/diagnostic imaging , Kidney Diseases/etiology , UltrasonographyABSTRACT
BACKGROUND: The lack of precise information on the epidemiology of peripheral intravascular catheter (PIVC)-related phlebitis and complications in critically ill patients results in the absence of appropriate preventive measures. Therefore, we aimed to describe the epidemiology of the use of PIVCs and the incidence/occurrence of phlebitis and complications in the intensive care unit (ICU). METHODS: This prospective multicenter cohort study was conducted in 23 ICUs in Japan. All consecutive patients aged ≥ 18 years admitted to the ICU were enrolled. PIVCs inserted prior to ICU admission and those newly inserted after ICU admission were included in the analysis. Characteristics of the ICU, patients, and PIVCs were recorded. The primary and secondary outcomes were the occurrence and incidence rate of PIVC-related phlebitis and complications (catheter-related blood stream infection [CRBSI] and catheter failure) during the ICU stay. RESULTS: We included 2741 patients and 7118 PIVCs, of which 48.2% were inserted in the ICU. PIVC-related phlebitis occurred in 7.5% (95% confidence interval [CI] 6.9-8.2%) of catheters (3.3 cases / 100 catheter-days) and 12.9% (95% CI 11.7-14.2%) of patients (6.3 cases / 100 catheter-days). Most PIVCs were removed immediately after diagnosis of phlebitis (71.9%). Grade 1 was the most common phlebitis (72.6%), while grade 4 was the least common (1.5%). The incidence rate of CRBSI was 0.8% (95% CI 0.4-1.2%). In cases of catheter failure, the proportion and incidence rate per 100 intravenous catheter-days of catheter failure were 21% (95% CI 20.0-21.9%) and 9.1 (95% CI 8.7-10.0), respectively. CONCLUSION: PIVC-related phlebitis and complications were common in critically ill patients. The results suggest the importance of preventing PIVC-related complications, even in critically ill patients. TRIAL REGISTRATION: UMIN-CTR, the Japanese clinical trial registry (registration number: UMIN000028019 , July 1, 2017).
ABSTRACT
Fibulin-4 is a matricellular protein required for extracellular matrix (ECM) assembly. Mice deficient in fibulin-4 (Fbln4-/- ) have disrupted collagen and elastin fibers and die shortly after birth from aortic and diaphragmatic rupture. The function of fibulin-4 in ECM assembly, however, remains elusive. Here, we show that fibulin-4 is required for the activity of lysyl oxidase (LOX), a copper-containing enzyme that catalyzes the covalent cross-linking of elastin and collagen. LOX produced by Fbln4-/- cells had lower activity than LOX produced by wild-type cells due to the absence of lysine tyrosyl quinone (LTQ), a unique cofactor required for LOX activity. Our studies showed that fibulin-4 is required for copper ion transfer from the copper transporter ATP7A to LOX in the trans-Golgi network (TGN), which is a necessary step for LTQ formation. These results uncover a pivotal role for fibulin-4 in the activation of LOX and, hence, in ECM assembly.
Subject(s)
Elastin , Protein-Lysine 6-Oxidase , Animals , Collagen/metabolism , Copper , Elastin/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Mice , Protein-Lysine 6-Oxidase/genetics , Protein-Lysine 6-Oxidase/metabolismABSTRACT
We treated two patients with severe respiratory failure due to coronavirus disease 2019 (COVID-19). Case 1 was a 73-year-old woman, and Case 2 was a 65-year-old-man. Neither of them had a history of autoimmune disease. Chest computed tomography scans before the antiviral therapy showed bilateral multiple patchy ground-glass opacities (GGO) consistent with COVID-19 pneumonia. The GGO regressed over the course of the antiviral treatment; however, new non-segmental patchy consolidations emerged, which resembled those of interstitial lung disease (ILD), specifically collagen vascular disease-associated ILD. We tested the patients' sera for autoantibodies and discovered that both patients had high anti-SSA/Ro antibody titers. In Case 1, the patient recovered with antiviral therapy alone. However, in Case 2, the patient did not improve with antiviral therapy alone but responded well to corticosteroid therapy (methylprednisolone) and made a full recovery. The relationship between some immunological responses and COVID-19 pneumonia exacerbation has been discussed previously; our discovery of the elevation of anti-SSA/Ro antibodies suggests a contribution from autoimmunity functions of the immune system. Although it is unclear whether the elevation of anti-SSA/Ro antibodies was a cause or an outcome of aggravated COVID-19 pneumonia, we hypothesize that both patients developed aggravated the COVID-19 pneumonia due to an autoimmune response. In COVID-19 lung injury, there may be a presence of autoimmunity factors in addition to the known effects of cytokine storms. In patients with COVID-19, a high level of anti-SSA/Ro52 antibodies may be a surrogate marker of pneumonia severity and poor prognosis.
Subject(s)
Antibodies, Antinuclear/immunology , Coronavirus Infections/immunology , Lung Diseases, Interstitial/immunology , Pneumonia, Viral/immunology , Respiratory Insufficiency/immunology , Aged , Amides/therapeutic use , Antiviral Agents/therapeutic use , Benzamidines , Betacoronavirus , COVID-19 , Coronavirus Infections/complications , Coronavirus Infections/diagnostic imaging , Coronavirus Infections/drug therapy , Female , Glucocorticoids/therapeutic use , Guanidines/therapeutic use , Humans , Hydroxychloroquine/therapeutic use , Lung Diseases, Interstitial/diagnostic imaging , Lung Diseases, Interstitial/drug therapy , Lung Diseases, Interstitial/etiology , Male , Methylprednisolone/therapeutic use , Pandemics , Pneumonia, Viral/complications , Pneumonia, Viral/diagnostic imaging , Pneumonia, Viral/drug therapy , Pregnenediones/therapeutic use , Pyrazines/therapeutic use , Recovery of Function , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/immunology , Respiratory Insufficiency/etiology , SARS-CoV-2 , Severity of Illness Index , Tomography, X-Ray ComputedABSTRACT
Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4-/- mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4-/- embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4-/- mice do not develop intact elastic fibers but contain irregular elastin aggregates. Electron microscopy revealed that the elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that elastin cross-links in fibulin-4-/- tissues were largely diminished. However, expression of tropoelastin or lysyl oxidase mRNA was unaffected in fibulin-4-/- mice. In addition, fibulin-4 strongly interacts with tropoelastin and colocalizes with elastic fibers in culture. These results demonstrate that fibulin-4 plays an irreplaceable role in elastogenesis.
Subject(s)
Elastic Tissue/physiology , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Fetal Death/genetics , Animals , Aorta/abnormalities , Aorta/embryology , Cells, Cultured , Desmosine/metabolism , Elastic Tissue/abnormalities , Elastin/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Silencing , Humans , Lung/abnormalities , Lung/embryology , Lung/pathology , Mice , Mice, Mutant Strains , Protein-Lysine 6-Oxidase/metabolism , Tropoelastin/metabolismABSTRACT
AIM: In order to elucidate the factors for willingness to perform CPR, we evaluated the responses of college students to questionnaires before and after basic life support (BLS) training. METHODS: Before and after participating in a small group BLS course, 259 students completed questionnaires. A logistic regression model was used to elucidate independent factors for their willingness to attempt resuscitation. RESULTS: Factors associated with willingness to perform BLS for strangers were "anxiety for a bad outcome" (odds ratio (OR) 0.08) and "having knowledge of automated external defibrillator (AED)" (OR 4.5) before training. The proportion of students showing willingness to perform BLS increased from 13% to 77% after the training even when the collapsed person is a stranger. After training, "anxiety for being sued because of a bad outcome" (OR 0.3), and "anxiety for infection" (OR 3.8) were significant factors. Those who preferred to perform BLS without ventilation increased from 40% to 79% (p<0.0001). CONCLUSION: The proportion of students showing willingness to perform BLS increased after the training. Significant association between "anxiety for infection" and willingness to perform BLS might indicate that those who wish to perform BLS developed their awareness of risk of infection more than the counterparts. For future guidelines for resuscitation and the instruction consensus, the reluctance of bystanders to perform CPR due to the hesitation about mouth-to-mouth ventilation should be reconsidered with other recent reports indicating the advantage of compression-only CPR.
Subject(s)
Attitude , Cardiopulmonary Resuscitation/education , Students/psychology , Educational Measurement , Female , Heart Arrest/therapy , Humans , Male , Retrospective Studies , Surveys and Questionnaires , Young AdultABSTRACT
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/metabolismABSTRACT
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/physiologyABSTRACT
The contributions of TGF-ß signaling to cancer are complex but involve the inflammatory microenvironment as well as cancer cells themselves. In mice encoding a TGF-ß mutant that precludes its binding to the latent TGF-ß binding protein (Tgfb1(-/C33S)), we observed multiorgan inflammation and an elevated incidence of various types of gastrointestinal solid tumors due to impaired conversion of latent to active TGF-ß1. By genetically eliminating activators of latent TGF-ß1, we further lowered the amount of TGF-ß, which enhanced tumor frequency and multiorgan inflammation. This model system was used to further investigate the relative contribution of TGF-ß1 to lymphocyte-mediated inflammation in gastrointestinal tumorigenesis. Toward this end, we generated Tgfb1(-/C33S);Rag2(-/-) mice that lacked adaptive immune function, which eliminated tumor production. Analysis of tissue from Tgfb1(-/C33S) mice indicated decreased levels of P-Smad3 compared with wild-type animals, whereas tissue from Tgfb1(-/C33S);Rag2(-/-) mice had normal P-Smad3 levels. Inhibiting the inflammatory response normalized levels of interleukin (IL)-1ß and IL-6 and reduced tumor cell proliferation. In addition, Tgfb1(-/C33S);Rag2(-/-) mice exhibited reduced paracrine signaling in the epithelia, mediated by hepatocyte growth factor produced by gastric stroma. Together, our results indicate that many of the responses of the gastric tissue associated with decreased TGF-ß1 may be directly or indirectly affected by inflammatory processes, which accompany loss of TGF-ß1, rather than a direct effect of loss of the cytokine.
Subject(s)
Cytokines/metabolism , Stomach Neoplasms/metabolism , Transforming Growth Factor beta1/physiology , Animals , Carcinogenesis/metabolism , Gastric Mucosa/immunology , Gastric Mucosa/metabolism , Gastritis/metabolism , Gastritis/pathology , Hepatocyte Growth Factor/metabolism , Inflammation Mediators/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Paracrine Communication , Proto-Oncogene Proteins c-met/metabolism , Signal Transduction , Stomach Neoplasms/immunologyABSTRACT
TGF-ß and its signaling pathways are important mediators in the suppression of cancers of the gastrointestinal tract. TGF-ß is released from cells in a latent complex consisting of TGF-ß, the TGF-ß propeptide [latency associated protein (LAP)], and a latent TGF-ß binding protein (LTBP). We previously generated mice in which the LTBP-binding cysteine residues in LAP TGF-ß1 were mutated to serine precluding covalent interactions with LTBP. These Tgfb1(C33S/C33S) mice develop multiorgan inflammation and tumors consistent with reduced TGF-ß1 activity. To test whether further reduction in active TGF-ß levels would yield additional tumors and a phenotype more similar to Tgfb1(-/-) mice, we generated mice that express TGF-ß1(C33S) and are deficient in either integrin ß8 or TSP-1, known activators of latent TGF-ß1. In addition, we generated mice that have one mutant allele and one null allele at the Tgfb1 locus, reasoning that these mice should synthesize half the total amount of TGF-ß1 as Tgfb1(C33S/C33S) mice, and the amount of active TGF-ß1 would be correspondingly decreased compared with Tgfb1(C33S/C33S) mice. These compound-mutant mice displayed more severe inflammation and higher tumor numbers than the parental Tgfb1(C33S/C33S) animals. The level of active TGF-ß1 in compound mutant mice seemed to be decreased compared with Tgfb1(C33S/C33S) mice as determined from analyses of surrogate markers of active TGF-ß, such as P-Smad2, C-Myc, KI-67, and markers of cell-cycle traverse. We conclude that these mutant mice provide a useful system for modulating TGF-ß levels in a manner that determines tumor number and inflammation within the gastrointestinal tract.
Subject(s)
Gastrointestinal Neoplasms/metabolism , Gastrointestinal Neoplasms/pathology , Mice, Mutant Strains , Transforming Growth Factor beta1/metabolism , Animals , Immunoblotting , Immunohistochemistry , Mice , Real-Time Polymerase Chain ReactionABSTRACT
The cytokine transforming growth factor-beta (TGF-ß) has multiple effects in both physiological and pathological conditions. TGF-ß is secreted as part of a tripartite complex from which it must be released in order to bind to its receptor. Sequestration of latent TGF-ß in the extracellular matrix (ECM) is crucial for proper mobilization of the latent cytokine and its activation. However, contrary to expectation, loss-of-function mutations in genes encoding certain matrix proteins that bind TGF-ß yield elevated, rather than decreased, TGF-ß levels, posing a 'TGF-ß paradox.' In this review, we discuss recent findings concerning the relationship of TGF-ß, ECM molecules, and latent TGF-ß activation and propose a model to resolve the 'TGF-ß paradox.'
Subject(s)
Extracellular Matrix/metabolism , Transforming Growth Factor beta/metabolism , Animals , HumansABSTRACT
BACKGROUND: Cholesterol efflux from cells to apolipoprotein A-I (apoA-I) acceptors via the ATP-binding cassette transporters ABCA1 and ABCG1 is thought to be central in the antiatherogenic mechanism. MicroRNA (miR)-33 is known to target ABCA1 and ABCG1 in vivo. METHODS AND RESULTS: We assessed the impact of the genetic loss of miR-33 in a mouse model of atherosclerosis. MiR-33 and apoE double-knockout mice (miR-33(-/-)Apoe(-/-)) showed an increase in circulating HDL-C levels with enhanced cholesterol efflux capacity compared with miR-33(+/+)Apoe(-/-) mice. Peritoneal macrophages from miR-33(-/-)Apoe(-/-) mice showed enhanced cholesterol efflux to apoA-I and HDL-C compared with miR-33(+/+)Apoe(-/-) macrophages. Consistent with these results, miR-33(-/-)Apoe(-/-) mice showed reductions in plaque size and lipid content. To elucidate the roles of miR-33 in blood cells, bone marrow transplantation was performed in these mice. Mice transplanted with miR-33(-/-)Apoe(-/-) bone marrow showed a significant reduction in lipid content in atherosclerotic plaque compared with mice transplanted with miR-33(+/+)Apoe(-/-) bone marrow, without an elevation of HDL-C. Some of the validated targets of miR-33 such as RIP140 (NRIP1) and CROT were upregulated in miR-33(-/-)Apoe(-/-) mice compared with miR-33(+/+)Apoe(-/-) mice, whereas CPT1a and AMPKα were not. CONCLUSIONS: These data demonstrate that miR-33 deficiency serves to raise HDL-C, increase cholesterol efflux from macrophages via ABCA1 and ABCG1, and prevent the progression of atherosclerosis. Many genes are altered in miR-33-deficient mice, and detailed experiments are required to establish miR-33 targeting therapy in humans.