Latent-transforming Growth Factor Beta-binding Protein Research Articles

Lactate triggers KAT8-mediated LTBP1 lactylation at lysine 752 to promote skin rejuvenation by inducing collagen synthesis in fibroblasts

Decreased collagen synthesis by fibroblasts is a key aspect of skin aging. Poly-L-Lactic Acid (PLLA) is a bioabsorbable material that can release lactate continuously, stimulating endogenous collagen synthesis in the skin. Herein, this study aimed to investigate the impact of PLLA-released lactate on collagen production in fibroblasts for skin rejuvenation. Human fibroblasts were exposed to varying concentrations of PLLA in vitro, while PLLA was injected into the back skin of aged mice in vivo. Safety and efficacy of PLLA on collagen synthesis and skin rejuvenation were evaluated through Calcein-AM/PI staining, EdU proliferation assay, and analysis of collagen I and collagen III expression in fibroblasts using western blotting and immunofluorescence. To elucidate the underlying mechanisms, lactate contents in cell-free supernatant and cell lysates from PLLA-treated fibroblasts, as well as total lysine lactylation (Pan Kla) levels were measured. Additionally, we found that fibroblasts can uptake extracellular lactate released from PLLA through monocarboxylate transporter-1 (MCT1) to facilitate latent-transforming growth factor beta-binding protein 1 (LTBP1) lactylation at lysine 752 (K752) via a KAT8-dependent mechanism, then increases the protein levels of collagen I and collagen III in fibroblasts. Overall, this study highlights a valuable insight into lactylation modification of non-histone protein for skin rejuvenation.

Secreted Protein Profiling of Human Aortic Smooth Muscle Cells Identifies Vascular Disease Associations.

Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular disease, the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a contractile to a synthetic phenotype characterized by an increased proliferation, migration, production of ECM (extracellular matrix) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of cardiovascular disease, including coronary artery disease, stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies. Using human aortic SMCs from 123 multiancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted liquid chromatography-tandem mass spectrometry-based proteomic analysis of the conditioned media. We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 (latent-transforming growth factor beta-binding protein 1) in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

Abstract 15735: Antibody-Based Plasma Proteomics Profiling Reveals New Markers of Cardiorespiratory Fitness

Background: Plasma proteins can be biomarkers of cardiovascular health status but also physiologic effectors that mediate health benefits. Cardiorespiratory fitness (CRF) is an integrative measure of cardiovascular and metabolic health and independent predictor of future cardiovascular disease (CVD) and mortality risk, however limited knowledge exists regarding its molecular transducers. We sought to expand upon prior proteomic studies of CRF using an antibody-based technology (Olink TM ). Hypothesis: Olink proteomic profiling will identify new markers and potential mediators of CRF. Methods: We measured plasma proteins (N=1,472) using Olink’s platform in a pilot study within the HERITAGE Family Study (N=209 participants, mean age=34 years, 56% female, 36% Black) before and after 20 weeks of endurance exercise training (ET). We performed multivariable linear regression to measure the association between protein levels and CRF measured by CPET (VO 2 max in ml*kg -1 *min -1 ) adjusting for age and sex. Lean body mass was measured by hydrostatic weighing. Protein changes after ET were assessed using paired Student’s t-tests. Results: We identified 70 proteins significantly associated (FDR q <0.05) with VO 2 max. Among these, 14 have not previously been measured in the context of CRF, including perilipin-1, a modulator of lipid homeostasis in adipocytes (beta=-3.8, q=2.4x10 -4 ); latent-transforming growth factor beta-binding protein 3 (beta=-2.6, q=1.3x10 -3 ), a key regulator of TGF-beta activation; and hydroxysteroid 11-beta dehydrogenase 1 (beta=3.2, q=1.2x10 -3 ), which regulates cortisol metabolism. Carbonic anhydrase (CA) XIV (CA14; beta=5.4, q=1.3x10 -8 ), a member of the CA family with extracellular activity, had the strongest positive association with VO 2 max in our primary model as well as after further adjusting VO 2 max for lean body mass. Further, CA14 levels increased significantly after ET (log2 fold change: 0.14, q=0.007). Conclusions: Antibody-based plasma proteomics profiling identified new markers of CRF, including a CA isoform that increases after ET. These findings motivate further study of CA14’s mechanistic role in CA14 and expanded proteomics profiling to investigate molecular transducers of CRF.

Proteomic Biomarkers of Survival in Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes progressive lung scarring and high mortality. Reliable and accurate prognostic biomarkers are urgently needed. Objectives: To identify and validate circulating protein biomarkers of IPF survival. Methods: High-throughput proteomic data were generated using prospectively collected plasma samples from patients with IPF from the Pulmonary Fibrosis Foundation Patient Registry (discovery cohort) and the Universities of California, Davis; Chicago; and Virginia (validation cohort). Proteins associated with three-year transplant-free survival (TFS) were identified using multivariable Cox proportional hazards regression. Those associated with TFS after adjustment for false discovery in the discovery cohort were advanced for testing in the validation cohort, with proteins maintaining TFS association with consistent effect direction considered validated. After combining cohorts, functional analyses were performed, and machine learning was used to derive a proteomic signature of TFS. Measurements and Main Results: Of 2,921 proteins tested in the discovery cohort (n = 871), 231 were associated with differential TFS. Of these, 140 maintained TFS association with consistent effect direction in the validation cohort (n = 355). After cohorts were combined, the validated proteins with the strongest TFS association were latent-transforming growth factor β-binding protein 2 (hazard ratio [HR], 2.43; 95% confidence interval [CI] = 2.09-2.82), collagen α-1(XXIV) chain (HR, 2.21; 95% CI = 1.86-2.39), and keratin 19 (HR, 1.60; 95% CI = 1.47-1.74). In decision curve analysis, a proteomic signature of TFS outperformed a similarly derived clinical prediction model. Conclusions: In the largest proteomic investigation of IPF outcomes performed to date, we identified and validated 140 protein biomarkers of TFS. These results shed important light on potential drivers of IPF progression.

Height-Related Polygenic Variants Are Associated with Metabolic Syndrome Risk and Interact with Energy Intake and a Rice-Main Diet to Influence Height in KoGES

Adult height is inversely related to metabolic syndrome (MetS) risk, but its genetic impacts have not been revealed. The present study aimed to examine the hypothesis that adult height-related genetic variants interact with lifestyle to influence adult height and are associated with MetS risk in adults aged >40 in Korea during 2010-2014. Participants were divided into short stature (SS; control) and tall stature (TS; case) by the 85th percentile of adult height. The genetic variants linked to adult height were screened from a genome-wide association study in a city hospital-based cohort (n = 58,701) and confirmed in Ansan/Ansung plus rural cohorts (n = 13,783) among the Korean Genome and Epidemiology Study. Genetic variants that interacted with each other were identified using the generalized multifactor dimensionality reduction (GMDR) analysis. The interaction between the polygenic risk score (PRS) of the selected genetic variants and lifestyles was examined. Adult height was inversely associated with MetS, cardiovascular diseases, and liver function. The PRS, including zinc finger and BTB domain containing 38 (ZBTB38)_rs6762722, polyadenylate-binding protein-interacting protein-2B (PAIP2B)_rs13034890, carboxypeptidase Z (CPZ)_rs3756173, and latent-transforming growth factor beta-binding protein-1 (LTBP1)_rs4630744, was positively associated with height by 1.29 times and inversely with MetS by 0.894 times after adjusting for covariates. In expression quantitative trait loci, the gene expression of growth/differentiation factor-5 (GDF5)_rs224331, non-SMC condensin I complex subunit G (NCAPG)_rs2074974, ligand-dependent nuclear receptor corepressor like (LCORL)_rs7700107, and insulin-like growth factor-1 receptor (IGF1R)_rs2871865 was inversely linked to their risk allele in the tibial nerve and brain. The gene expression of PAIP2B_rs13034890 and a disintegrin and metalloproteinase with thrombospondin motifs-like-3 (ADAMTSL3)_rs13034890 was positively related to it. The PRS was inversely associated with MetS, hyperglycemia, HbA1c, and white blood cell counts. The wild type of GDF5_rs224331 (Ala276) lowered binding energy with rugosin A, D, and E (one of the hydrolyzable tannins) but not the mutated one (276Ser) in the in-silico analysis. The PRS interacted with energy intake and rice-main diet; PRS impact was higher in the high energy intake and the low rice-main diet. In conclusion, the PRS for adult height interacted with energy intake and diet patterns to modulate height and was linked to height and MetS by modulating their expression in the tibial nerve and brain.

Latent TGFβ-binding proteins 1 and 3 protect the larval zebrafish outflow tract from aneurysmal dilatation.

ABSTRACTAortic root aneurysm is a common cause of morbidity and mortality in Loeys-Dietz and Marfan syndromes, where perturbations in transforming growth factor beta (TGFβ) signaling play a causal or contributory role, respectively. Despite the advantages of cross-species disease modeling, animal models of aortic root aneurysm are largely restricted to genetically engineered mice. Here, we report that zebrafish devoid of the genes encoding latent-transforming growth factor beta-binding protein 1 and 3 (ltbp1 and ltbp3, respectively) develop rapid and severe aneurysm of the outflow tract (OFT), the aortic root equivalent. Similar to syndromic aneurysm tissue, the distended OFTs display evidence for paradoxical hyperactivated TGFβ signaling. RNA-sequencing revealed significant overlap between the molecular signatures of disease tissue from mutant zebrafish and a mouse model of Marfan syndrome. Moreover, chemical inhibition of TGFβ signaling in wild-type animals phenocopied mutants but chemical activation did not, demonstrating that TGFβ signaling is protective against aneurysm. Human relevance is supported by recent studies implicating genetic lesions in LTBP3 and, potentially, LTBP1 as heritable causes of aortic root aneurysm. Ultimately, our data demonstrate that zebrafish can now be leveraged to interrogate thoracic aneurysmal disease and identify novel lead compounds through small-molecule suppressor screens.This article has an associated First Person interview with the first author of the paper.

Latent TGFβ-binding proteins regulate UCP1 expression and function via TGFβ2

ObjectiveActivation of brown adipose tissue (BAT) in humans has been proposed as a new treatment approach for combating obesity and its associated diseases, as BAT participates in the regulation of energy homeostasis as well as glucose and lipid metabolism. Genetic contributors driving brown adipogenesis in humans have not been fully understood. MethodsProfiling the gene expression of progenitor cells from subcutaneous and deep neck adipose tissue, we discovered new secreted factors with potential regulatory roles in white and brown adipogenesis. Among these, members of the latent transforming growth factor beta-binding protein (LTBP) family were highly expressed in brown compared to white adipocyte progenitor cells, suggesting that these proteins are capable of promoting brown adipogenesis. To investigate this potential, we used CRISPR/Cas9 to generate LTBP-deficient human preadipocytes. ResultsWe demonstrate that LTBP2 and LTBP3 deficiency does not affect adipogenic differentiation, but diminishes UCP1 expression and function in the obtained mature adipocytes. We further show that these effects are dependent on TGFβ2 but not TGFβ1 signaling: TGFβ2 deficiency decreases adipocyte UCP1 expression, whereas TGFβ2 treatment increases it. The activity of the LTBP3–TGFβ2 axis that we delineate herein also significantly correlates with UCP1 expression in human white adipose tissue (WAT), suggesting an important role in regulating WAT browning as well. ConclusionsThese results provide evidence that LTBP3, via TGFβ2, plays an important role in promoting brown adipogenesis by modulating UCP1 expression and mitochondrial oxygen consumption.

Latent-transforming growth factor beta-binding protein-2 (LTBP-2) is required for longevity but not for development of zonular fibers

Latent-transforming growth factor beta-binding protein 2 (LTBP-2) is a major component of arterial and lung tissue and of the ciliary zonule, the system of extracellular fibers that centers and suspends the lens in the eye. LTBP-2 has been implicated previously in the development of extracellular microfibrils, although its exact role remains unclear. Here, we analyzed the three-dimensional structure of the ciliary zonule in wild type mice and used a knockout model to test the contribution of LTBP-2 to zonule structure and mechanical properties. In wild types, zonular fibers had diameters of 0.5–1.0 micrometers, with an outer layer of fibrillin-1-rich microfibrils and a core of fibrillin-2-rich microfibrils. LTBP-2 was present in both layers. The absence of LTBP-2 did not affect the number of fibers, their diameters, nor their coaxial organization. However, by two months of age, LTBP-2-depleted fibers began to rupture, and by six months, a fully penetrant ectopia lentis phenotype was present, as confirmed by in vivo imaging. To determine whether the seemingly normal fibers of young mice were compromised mechanically, we compared zonule stress/strain relationships of wild type and LTBP-2-deficient mice and developed a quasi-linear viscoelastic engineering model to analyze the resulting data. In the absence of LTBP-2, the ultimate tensile strength of the zonule was reduced by about 50%, and the viscoelastic behavior of the fibers was altered significantly. We developed a harmonic oscillator model to calculate the forces generated during saccadic eye movement. Model simulations suggested that mutant fibers are prone to failure during rapid rotation of the eyeball. Together, these data indicate that LTBP-2 is necessary for the strength and longevity of zonular fibers, but not necessarily for their formation.

Expression and clinical significance of latent-transforming growth factor beta-binding protein 2 in primary hepatocellular carcinoma.

The present study aimed to explore the expression of latent transforming growth factor β binding protein 2 (LTBP2) in patients with hepatocellular carcinoma (HCC) and their correlation to clinicopathologial features.Serum levels of LTBP2 in 60 patients with HCC, 35 patients with hepatocellular benign tumors, 60 patients with precancerous lesions of HCC, and 60 healthy volunteers were determined by enzyme-linked immunosorbent assay. The expression levels of LTBP2 at messenger RNA (mRNA) and protein levels in 60 cases of HCC and adjacent tissues were detected by quantitative real-time polymerase chain reaction and immunohisochemistry. Statistical analysis was used to analyze the relationship between LTBP2 and clinical characteristics of patients with HCC.The mRNA and protein levels of LTBP2 were significantly upregulated in HCC tissues compared to adjacent tissues. Additionally, higher serum LTBP2 level was also observed in HCC patients relative to normal controls. Further investigation demonstrated that LTBP2 expression was associated with malignant degree of tumor, tumor progression, tumor differentiation, tumor size, tumor stage and hepatitis virus infection, and has prognostic implications in HCC patients.LTBP2 might be served as a potential biomarker in diagnosis and treatment of HCC.

Abstract P3-08-19: Proteomic tracking of breast cancer metastasis progression

Abstract Background Metastases from breast cancers have not yet reached curability mainly because of the continuous evolution of cancer cells leading to treatment resistance and tumor progression. Despite the identification of the main genomic drivers of breast cancer resistance, and the development of specific targeted therapies, resistance has not been eradicated. A deeper understanding of tumor biology underlying treatment escape and tumor progression is necessary to find new targets. The aim of this study was to determine whether mass spectrometry based imaging and microproteomics are able to capture changes in protein expression and pathways occurring during metastasis progression. Methods Ten tumor samples from five progressing metastatic breast cancer patients treated at Oscar Lambret Cancer Center (France) were selected based on the availability of paired metastasis biopsy performed at two time points during the evolution of the disease. The proteomic profiles of paired tumor samples were analyzed using MALDI mass spectrometry imaging combined with microproteomics, a spatially resolved proteomic technology. Results Comparison of paired samples showed gain and loss of proteins associated with tumor progression. However, few were shared between patients. The pathways and biological processes mainly represented during tumor progression, and which were found in at least two patients, were those involved in the negative regulation of leukocyte mediated cytotoxicity, the metabolism of carboxilic acid, peptides and nucleosides, in response to stress, oxidation-reduction process, endocytosis, catabolic processes, actin regulation, and extracellular matrix organization. During metastasis evolution, few shared proteins were identified in at least 3 patients such as SRPX2, CILP1, collagen alpha-2(V) chain, Ras-related C3 botulinum toxin substrate 1, filamin-C, PDZ and LIM domain protein 2 and chloride intracellular channel protein 4. The main proteins lost during progression and found in at least 2 patients were cell surface glycoprotein MUC18, collagen alpha-2(IV) chain, polyadenylate-binding protein 2, and latent-transforming growth factor beta-binding protein 4 (LTAGP4). Results from an expanded cohort will be presented at the meeting. Conclusion MALDI mass spectrometry proteomics identified private and shared changes in proteins and biological processes associated with breast cancer metastasis progression. Citation Format: Hajjaji N, Aboulouard S, Robin YM, Bertin D, Fournier I, Bonneterre J, Salzet M. Proteomic tracking of breast cancer metastasis progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-19.

Feasibility of low field MRI and proteomics for the analysis of Tissue Engineered bone

There is a need for the replacement of bone tissue that has been lost due to trauma. On average, there are an estimated 10 million cases of fracture-related injuries in the US every year [Amini et al 2012 Crit. Rev. Biomed. Eng. 40, 363–408]. A potential solution is offered by tissue engineering which aims to create biological substitutes for natural tissues. Magnetic resonance imaging (MRI) has been proposed to assess engineering outcomes, however one challenge is the use of high field, which is costly and with regulatory effect on the cells not well understood. This study aims to illustrate the capability of a table top, low field MRI system in monitoring the development of Tissue Engineered bone, supported through proteome analysis. MR images, spin-spin (T2) relaxation, and proteomics were acquired for samples assessed at three in vitro developmental time points: week 0, week 2, and week 4. A ∼45% decrease in the T2 relaxation time was observed from weeks 0 to 4. During cellular differentiation, proteomics revealed the appearance of proteins [alkaline phosphatase (ALP), collagen I and III, fibronectin, transforming growth factor beta (TGF-β), and latent transforming growth factor beta binding protein-1 (LTBP1)] consistent with osteogenesis. The data demonstrated that low field MRI can offer a cost-effective alternative to study the development of Tissue Engineered bone, in addition to proteomics analysis of the developing tissue.

Estrogen nuclear receptors affect cell migration by altering sublocalization of AQP2 in glioma cell lines

Glioblastomas are capable of infiltrating into neighboring brain tissues. The prognosis of a male patient is worse than that of women. Here, we demonstrate the effects of estrogen on invasion of glioma cells via regulating estrogen nuclear receptors (ERα and ERβ) combined with aquaporin 2 (AQP2). In our study, we conclude that AQP2 was located mainly in the nuclei of the glioma cell lines and is capable of inhibiting cell invasion. According to the gene ontology analysis, out of 138 screened genes, three genes of ankyrin repeat and FYVE domain containing 1 (ANKFY1), lymphocyte transmembrane adaptor 1 (LAX1), and latent transforming growth factor beta-binding protein 1 (LTBP1) were found to be regulating the ERα and ERβ. The expression of ERα was found to be high, whereas the expression of both ERβ and AQP2 was low in glioma cells from patient tissues and glioblastoma cell lines. The expression levels of AQP2, ANKFY1, LAX1, and LTBP1 were upregulated by both ERα small interfering RNA (siRNA) and overexpression of ERβ. AQP2 inhibition of cell invasion was inversely influenced by LAX1siRNA. The luciferase report system indicated that AQP2 promoted the transcriptional activity of LAX1 and inhibited cell invasion. These data suggest that ERβ may function as AQP promoter in the nucleus to sustain cells' stability by promoting AQP production, while ERα acts as an antagonist of AQP2. The ratio between ERα and ERβ is likely to affect the distribution of AQP2 in the nucleus. Low level of ERβ reduces the inhibition of invasion of glioma cells influenced by high level of LAX1 expression, leading to an increase in the invasion ability of glioma cells.

Transcriptomic Signature of Right Ventricular Failure in Experimental Pulmonary Arterial Hypertension: Deep Sequencing Demonstrates Mitochondrial, Fibrotic, Inflammatory and Angiogenic Abnormalities.

Right ventricular failure (RVF) remains the leading cause of death in pulmonary arterial hypertension (PAH). We investigated the transcriptomic signature of RVF in hemodynamically well-phenotyped monocrotaline (MCT)-treated, male, Sprague-Dawley rats with severe PAH and decompensated RVF (increased right ventricular (RV) end diastolic volume (EDV), decreased cardiac output (CO), tricuspid annular plane systolic excursion (TAPSE) and ventricular-arterial decoupling). RNA sequencing revealed 2547 differentially regulated transcripts in MCT-RVF RVs. Multiple enriched gene ontology (GO) terms converged on mitochondria/metabolism, fibrosis, inflammation, and angiogenesis. The mitochondrial transcriptomic pathway is the most affected in RVF, with 413 dysregulated genes. Downregulated genes included TFAM (−0.45-fold), suggesting impaired mitochondrial biogenesis, CYP2E1 (−3.8-fold), a monooxygenase which when downregulated increases oxidative stress, dehydrogenase/reductase 7C (DHRS7C) (−2.8-fold), consistent with excessive autonomic activation, and polypeptide N-acetyl-galactose-aminyl-transferase 13 (GALNT13), a known pulmonary hypertension (PH) biomarker (−2.7-fold). The most up-regulated gene encodes Periostin (POSTN; 4.5-fold), a matricellular protein relevant to fibrosis. Other dysregulated genes relevant to fibrosis include latent-transforming growth factor beta-binding protein 2 (LTBP2), thrombospondin4 (THBS4). We also identified one dysregulated gene relevant to all disordered transcriptomic pathways, ANNEXIN A1. This anti-inflammatory, phospholipid-binding mediator, is a putative target for therapy in RVF-PAH. Comparison of expression profiles in the MCT-RV with published microarray data from the RV of pulmonary artery-banded mice and humans with bone morphogenetic protein receptor type 2 (BMPR2)-mutations PAH reveals substantial conservation of gene dysregulation, which may facilitate clinical translation of preclinical therapeutic and biomarkers studies. Transcriptomics reveals the molecular fingerprint of RVF to be heavily characterized by mitochondrial dysfunction, fibrosis and inflammation.

TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators

ABSTRACTTGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.

Latent Transforming Growth Factor-beta Binding Protein-1 as a Diagnostic Biomarker for the Detection of Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancerrelated death worldwide. The current study aims to evaluate the diagnostic role of latent transforming growth factorbeta binding protein-1 (LTBP-1) as a biomarker to distinguish hepatocellular carcinoma (HCC) from patients with liver cirrhosis. Methods: The study carried out on 90 individuals classified to healthy individuals (n=20), liver cirrhosis (n=30), and HCC (n=40). The serum level of LTBP-1 was measured by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristics (ROC) curves and area under the curve (AUC) were calculated. Results: The level of LTBP-1 was significantly higher in HCC patients than healthy and patients with cirrhosis. Furthermore, there was a significant (p<0.001) association between the level of LTBP-1 and CLIP and BCLC in HCC patients. Moreover, LTBP-1 levels were significantly (p=0.01) associated to child pugh grade in patients with cirrhosis and HCC. ROC curve analyses revealed that LTBP-1 showed a better diagnostic performance (AUC=0.970, Sensitivity: 82.50%, Specificity: 96.67%, PPV: 97.06%, NPV: 80.56%) in distinguishing HCC from cirrhosis patients, compared to AFP (AUC=0.810, Sensitivity: 62.50%, Specificity: 93.33%, PPV: 92.59%, NPV: 65.12%). Conclusion: These findings suggested that LTBP-1 may be a promising biomarker for distinguish HCC from liver cirrhosis patients.

Anatomical differences of the protein profile in the rabbit sclera during growth

We aimed to investigate the proteome changes in anatomical regions of sclera during growth and development of the rabbit. Sclera from New Zealand white rabbits of three ages (1 month, 2 months and 6 months) was dissected into three segments - anterior, equatorial, and posterior. A total of 36 samples were divided into groups by age and anatomical region. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Label-free quantification based on spectral counts was used to determine the relative protein abundance and identify proteins with statistically significant differences between age groups or anatomical regions of the sclera. Western blotting was performed to validate some of the differentially expressed proteins. A total of 840 non-redundant proteins was identified in the sclera at different ages and regions with protein and peptide false discovery rate (FDR) at ≤1.0% and ≤0.1%, respectively. Differentially expressed proteins were identified by comparing age or anatomical region. Among these, periostin showed decreasing abundance with age, while myocilin, latent-transforming growth factor beta-binding protein 2, hyaluronan, proteoglycan link protein 1 and selenbp1 showed increasing abundance with age. In mature rabbits, alcohol dehydrogenase showed region-related differences in the sclera. Periostin showed an age-related decrease while selenbp1 showed an age-related increase in abundance in the anterior region. Vitronectin and extracellular superoxide dismutase had greater expression with age in the equatorial and posterior regions, respectively. The age related differential expression of periostin and selenbp1 was confirmed by western blotting. In conclusion, the protein profile of sclera showed age- and region-related differences. The differential protein profiles provide a baseline for understanding changes in the protein expression in the young and mature rabbit that appears to show regional changes. The changes observed in the present study add to the existing knowledge about regional alterations in biomechanical properties of sclera during growth.

Glaucoma in iran and contributions of studies in iran to the understanding of the etiology of glaucoma.

Epidemiologic and genetic/molecular research on glaucoma in Iran started within the past decade. A population-based study on the epidemiology of glaucoma in Yazd, a city in central Iran, revealed that 4.4% of studied individuals were affected with glaucoma: 1.6% with high tension primary open angle glaucoma (POAG), 1.6% with normal tension POAG, and 0.4% each with primary angle closure glaucoma (PACG) and pseudoexfoliation glaucoma (PEXG), and other types of secondary glaucoma. Two notable observations were the relatively high frequency of normal tension glaucoma cases (1.6%) and the large fraction of glaucoma affected individuals (nearly 90%) who were unaware of their condition. The first and most subsequent genetic studies on glaucoma in Iran were focused on primary congenital glaucoma (PCG) showing that cytochrome P450 1B1 (CYP1B1) is the cause of PCG in the majority of Iranian patients, many different CYP1B1 mutations are present among Iranian patients but only four mutations constitute the vast majority, and the origins of most mutations in the Iranians are identical by descent (IBD) with the same mutations in other populations. Furthermore, most of the PCG patients are from the northern and northwestern provinces of Iran. A statistically significant male predominance of PCG was observed only among patients without CYP1B1 mutations. Clinical investigations on family members of PCG patients revealed that CYP1B1 mutations exhibit variable expressivity, but almost complete penetrance. A great number of individuals harboring CYP1B1 mutations become affected with juvenile onset POAG. Screening of JOAG patients showed that an approximately equal fraction of the patients harbor CYP1B1 and (myocilin) MYOC mutations; MYOC is a well-known adult onset glaucoma causing gene. Presence of CYP1B1 mutations in JOAG patients suggests that in some cases, the two conditions may share a common etiology. Further genetic analysis of Iranian PCG patients led to identification of Latent-transforming growth factor beta-binding protein 2 (LTBP2) as a causative gene for both PCG and several diseases which are often accompanied by glaucomatous presentations, such as Weill-Marchesani syndrome 3 (WMS3). The findings on LTBP2 have contributed to recognize the importance of the extracellular matrix in pathways leading to glaucoma.

Targets and candidate agents for type 2 diabetes treatment with computational bioinformatics approach.

We sought to explore the molecular mechanism of type 2 diabetes (T2D) and identify potential drug targets and candidate agents for T2D treatment. The differentially expressed genes (DEGs) were assessed between human pancreatic islets with T2D and normal islets. The dysfunctional pathways, the potential transcription factor, and microRNA targets were analyzed by bioinformatics methods. Moreover, a group of bioactive small molecules were identified based on the connectivity map database. The pathways of Eicosanoid Synthesis, TGF-beta signaling pathway, Prostaglandin Synthesis and Regulation, and Integrated Pancreatic Cancer Pathway were found to be significantly dysregulated in the progression of T2D. The genes of ZADH2 (zinc binding alcohol dehydrogenase domain containing 2), BTBD3 (BTB (POZ) domain containing 3), Cul3-based ligases, LTBP1 (latent-transforming growth factor beta binding protein 1), PDGFRA (alpha-type platelet-derived growth factor receptor), and FST (follistatin) were determined to be significant nodes regulated by potential transcription factors and microRNAs. Besides, two small molecules (sanguinarine and DL-thiorphan) were identified to be capable of reverse T2D. In the present study, a systematic understanding for the mechanism underlying T2D development was provided with biological informatics methods. The significant nodes and bioactive small molecules may be drug targets and candidate agents for T2D treatment.

Development of a new chick chorioallantoic membrane model for human pancreas adenocarcinoma

Pancreatic cancer is the fourth leading cause of cancer death in developed countries. Early‐stage pancreatic cancer is usually clinically silent, and disease only becomes apparent after the tumor invades surrounding tissues or metastasizes to distant organs. One of the urgent challenges in pancreas cancer research is to set up model systems allowing the development and testing of new therapies as well as the discovery of new diagnostic markers. The chick chorioallantoic membrane (CAM) has been successfully used to produce tumors from various cancer cell lines. Here, using BxPC3 human pancreatic cells we were able to produce within 7 days post‐grafting 30 mm3vascularized tumor on CAM. Histology analysis revealed that the cancer cells formed tumors compatible with PanIN‐3 grade human malignant adenocarcinomas. Immunohistochemistry revealed that the malignant cells were positive for carcinoembryonic antigen and cytokeratin‐7, and ‐19. Functional blood vessels were numerous between pancreatic islets. Interestingly, we were able to demonstrate that two biomarkers recently identified in our lab from human pancreas cancer tissues, latent‐transforming growth factor beta‐binding protein 2 (LTBP‐2) and transforming growth factor beta‐induced (TGFBI) were found to be also expressed in the BxPC3. The possibility to perfuse the tumor allows to expect this model to be used to detect new biomarkers as well as inject and test drugs.This work was support by the Fond Léon‐Fredericq (ULg) and FNRS.

Structure and Interdomain Interactions of a Hybrid Domain: A Disulphide-Rich Module of the Fibrillin/LTBP Superfamily of Matrix Proteins

SummaryThe fibrillins and latent transforming growth factor-β binding proteins (LTBPs) form a superfamily of structurally-related proteins consisting of calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with 8-cysteine-containing transforming growth factor β-binding protein-like (TB) and hybrid (hyb) domains. Fibrillins are the major components of the extracellular 10–12 nm diameter microfibrils, which mediate a variety of cell-matrix interactions. Here we present the crystal structure of a fibrillin-1 cbEGF9-hyb2-cbEGF10 fragment, solved to 1.8 Å resolution. The hybrid domain fold is similar, but not identical, to the TB domain fold seen in previous fibrillin-1 and LTBP-1 fragments. Pairwise interactions with neighboring cbEGF domains demonstrate extensive interfaces, with the hyb2-cbEGF10 interface dependent on Ca2+ binding. These observations provide accurate constraints for models of fibrillin organization within the 10–12 nm microfibrils and provide further molecular insights into how Ca2+ binding influences the intermolecular interactions and biomechanical properties of fibrillin-1.