The rs2018736 fibulin-5 polymorphism as a determinant for pelvic organ prolapse: a case-control study.

OBJECTIVE: Fibulin-5 is a connective tissue component and may play a role in pelvic organ prolapse (POP) pathogenesis. This study aimed to verify the association of the rs2018736 polymorphism of the fibulin-5 gene with POP in postmenopausal Brazilian women, and to determine the risk factors for POP. METHOD: This observational, cross-sectional, case-control study assessed postmenopausal women with advanced POP (stages III and IV) and control women (stages 0 and I) by examination and peripheral blood sample collection. DNA sequences were analyzed by real-time reverse-transcriptase polymerase chain reaction. A logistic regression model was used with p < 0.05 for significance. RESULTS: A total of 565 participants were evaluated (325 POP and 240 control). The homozygous C allele of rs2018736 (CC) was protective against POP (odds ratio [OR] 0.49, 95% confidence interval [CI] 0.26-0.91). Age (OR 1.09, 95% CI 1.05-1.13), number of pregnancies (OR 1.14, 95% CI 1.01-1.28), vaginal delivery (OR 5.32, 95% CI 2.58-11.01), forceps delivery (OR 3.34, 95% CI 1.72-6.47), weight of newborn (OR 1.0007, 95% CI 1.0002-1.0011), family history of POP (OR 2.35, 95% CI 1.24-4.44), hypertension (OR 1.74, 95% CI 1.01-3.00) and diabetes (OR 2.19, 95% CI 1.07-4.48)] were independent predictors for POP; cesarean (OR 0.02, 95% CI 0.005-0.09) was protective. CONCLUSION: The rs2018736-CC genotype of the fibulin-5 gene has a protective role against POP.

Extracellular SOD modulates canonical TNFα signaling and α5ß1 integrin transactivation in vascular smooth muscle cells.

TNFα activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). The extracellular superoxide anion (O2•-) produced is essential for the pro-inflammatory effects of the cytokine but the specific contributions of O2•- to signal transduction remain obscure. Extracellular superoxide dismutase (ecSOD, SOD3 gene) is a secreted protein that binds to cell surface heparin sulfate proteoglycans or to Fibulin-5 (Fib-5, FBLN5 gene), an extracellular matrix protein that also associates with elastin and integrins. ecSOD converts O2•- to hydrogen peroxide (H2O2) which prevents NO• inactivation, limits generation of hydroxyl radical (OH•), and creates high local concentrations of H2O2. We hypothesized that ecSOD modifies TNFα signaling in VSMCs. Knockdown of ecSOD (siSOD3) suppressed downstream TNFα signals including MAPK (JNK and ERK phosphorylation) and NF-κB activation (luciferase reporter and IκB phosphorylation), interleukin-6 (IL-6) secretion, iNOS and VCAM expression, and proliferation (Sulforhodamine B assay, PCNA western blot). These effects were associated with significant reductions in the expression of both Type1 and 2 TNFα receptors. Reduced Fib-5 expression (siFBLN5) similarly impaired NF-κB activation by TNFα, but potentiated FAK phosphorylation at Y925. siSOD3 also increased both resting and TNFα-induced phosphorylation of FAK and of glycogen synthase kinase-3ß (GSK3ß), a downstream target of integrin linked kinase (ILK). These effects were dependent upon α5ß1 integrins and siSOD3 increased resting sulfenylation (oxidation) of both integrin subunits, while preventing TNFα-induced increases in sulfenylation. To determine how ecSOD modified TNFα-induced inflammation in intact blood vessels, mesenteric arteries from VSMC-specific ecSOD knockout (KO) mice were exposed to TNFα (10 ng/ml) in culture for 48 h. Relaxation to acetylcholine and sodium nitroprusside was impaired in WT but not ecSOD KO vessels. Thus, ecSOD association with Fib-5 supports pro-inflammatory TNFα signaling while tonically inhibiting α5ß1 integrin activation.

Genetic variants and haplotypes in fibulin-5 (FBLN5) are associated with pseudoexfoliation glaucoma but not with pseudoexfoliation syndrome.

Pseudoexfoliation (PEX) is a multifactorial age-related disease involving deposition of extracellular proteinaceous aggregates on anterior ocular tissues. The present study aims to identify functional variants in fibulin-5 (FBLN5) as risk factors for the development of PEX. Thirteen tag single-nucleotide polymorphisms (SNPs) in FBLN5 were genotyped using TaqMan SNP genotyping technology to identify association between SNPs of FBLN5 and PEX in an Indian cohort comprising 200 control and 273 PEX patients (169 PEXS and 104 PEXG). Functional analysis of risk variants was done through luciferase reporter assays and electrophoretic mobility shift assay (EMSA) using human lens epithelial cells. Genetic association and risk haplotype analysis showed a significant association of rs17732466:G>A (NC_000014.9:g.91913280G>A) and rs72705342:C>T (NC_000014.9:g.91890855C>T) within FBLN5 as risk factors with the advanced severe stage of the disease, pseudoexfoliation glaucoma (PEXG). Reporter assays showed allele-specific regulatory effect of rs72705342:C>T on gene expression, wherein, construct containing the risk allele showed a significant decrease in the reporter activity compared with the one with protective allele. EMSA further validated higher binding affinity of the risk variant to nuclear protein. In silico analysis predicted binding sites for two transcription factors, GR-α and TFII-I with risk allele at rs72705342:C>T, which were lost in the presence of protective allele. The EMSA showed probable binding of both these proteins to rs72705342. In conclusion, the present study identified the novel association of two genetic variants in FBLN5 with PEXG but not with PEXS, distinguishing between the early and the later forms of PEX. Further, rs72705342:C>T was found to be a functional variant.

Quantitative analysis of circulating levels of vimentin, clusterin and fibulin-5 in patients with pseudoexfoliation syndrome and glaucoma.

Pseudoexfoliation (PEX) is a multifactorial age-related disease involving deposition of extracellular fibrillar material on anterior ocular tissues. If left untreated, the early stage of deposition termed as pseudoexfoliation syndrome (PEXS) may lead to the advanced stage of pseudoexfoliation glaucoma (PEXG) characterised by increased intraocular pressure, damage to the optic nerve and subsequent irreversible blindness. The etiology of PEX is complex and identification of novel factors associated with the disease is needed. This study aimed to identify the involvement of vimentin in pseudoexfoliation pathology and assess the levels of vimentin, clusterin and fibulin-5 in the circulating fluids of PEX patients compared to controls. Eighty-seven participants (35 controls, 35 PEXS and 17 PEXG) were enrolled for this case-control study. The expression of vimentin in lens capsules of patients and age-sex matched controls was assayed by qRT-PCR, western blotting and immunohistochemistry. Aqueous humor (AH) vimentin levels and plasma levels of vimentin, clusterin and fibulin-5 were assayed through ELISA. Increased vimentin was observed in the lens capsule of patients compared to controls at mRNA and protein levels. Compared to control (11.5 ± 1.4 ng/ml [±SEM]), the AH vimentin concentrations were significantly higher (ANOVA p = 0.01) in PEXS (16.4 ± 1.8 ng/ml) and PEXG (20.1 ± 2.5 ng/ml). Compared to controls (372.2 ± 15.1 ng/ml), plasma vimentin levels were significantly higher (ANOVA, p < 0.001) in PEXS (449.9 ± 15.7 ng/ml) and PEXG (535.5 ± 25.0 ng/ml). The plasma and aqueous humor levels of vimentin showed a positive correlation of 0.31. The plasma levels of clusterin were 298.9 ± 19.0 µg/ml, 367.8 ± 25.6 µg/ml and 272.9 ± 16.8 µg/ml in controls, PEXS and PEXG, respectively and were significantly higher in PEXS (p = 0.03) compared to control. Plasma fibulin-5 levels were 149 ± 32.2 pg/ml, 187.6 ± 32.3 pg/ml and 203.8 ± 27.3 pg/ml in controls, PEXS and PEXG, respectively and there was no significant difference in its levels between the groups (ANOVA p = 0.49). In conclusion, vimentin is upregulated in PEX affected eyes. Increased vimentin levels in plasma and AH differentiate PEXS and PEXG from controls.

Decreased Expression of EC-SOD and Fibulin-5 in Alveolar Walls of Lungs From COPD Patients

Introduction: The aim of this study is to analyze the expression of the main oxidant scavenger superoxide dismutase (EC-SOD), its main binding protein Fibulin-5 and several oxidative and nitrosative-derived products in the lung of COPD patients and controls.Materials and methods: Lung tissue samples from 19 COPD patients and 20 control subjects were analyzed. The architecture of elastic fibres was assessed by light and electron microscope histochemical techniques, and levels of EC-SOD and fibulin-5 were analyzed by immunohistochemistry and RT-PCR. The impact of oxidative stress on the extracellular matrix was estimated by immunolocalization of 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and 3-nitrotyrosine (3-NYT) adducts.Results: Alveolar walls of COPD patients exhibited abnormal accumulations of collapsing elastic fibres, showing a pierced pattern in the amorphous component. The semiquantitative analysis revealed that COPD patients have a significantly reduced expression of both EC-SOD and fibulin-5 (0.59±0.64 and 0.62±0.61, respectively) in alveolar, bronchiolar and arteriolar walls compared to control subjects (1.39±0.63 and 1.55±0.52, respectively, p<0.05). No significant changes in mRNA levels of these proteins were observed between groups. Among the oxidation markers, malondialdehyde was the best in distinguishing COPD patients.Conclusions: COPD patients show a reduced expression of EC-SOD and fibulin-5 in the lung interstitium. Paralleling the reduction of EC-SOD levels, the decrease of fibulin-5 expression in COPD lungs supports the hypothesis of an impaired pulmonary antioxidant response in COPD patients. (AU)

YY1 affects the levels and function of fibulin-5 in ox-LDL-treated vascular smooth muscle cells.

Fibulin-5 is reportedly involved in the pathological process of atherosclerosis (AS) where low expression has been frequently observed in ruptured atherosclerotic plaques. The aim of the present study was to determine the effects of fibulin-5 on the responses of vascular smooth muscle cells (VSMC) to oxidized low-density lipoprotein (ox-LDL). The expression of fibulin-5 was studied in human aortic-VSMCs (HA-VSMCs) treated with ox-LDL. Fibulin-5 was first overexpressed by the transfection of Ov-Fibulin-5 plasmids in HA-VSMCs challenged with ox-LDL to investigate its influence on cell proliferation, migration and invasion using Cell Counting Kit-8, wound healing and Transwell assays. Yin Yang-1 (YY1) was bioinformatically predicted to bind to the promoter sites of fibulin-5, which was subsequently confirmed by dual-luciferase reporter gene assay. Fibulin-5 overexpression was able to suppress cell proliferation, invasion and migration, which was effectively reversed by YY1 silencing by the transfection of siRNA-Fibulin-5 plasmids which could induced fibulin-5 silencing. YY1 binding sites in the promoter region of fibulin-5 were identified and confirmed in vitro by chromatin immunoprecipitation assay and dual-luciferase reporter gene assay. The present results suggested that as a modulator of fibulin-5, YY1 alleviated ox-LDL-induced proliferation, invasion, migration and phenotypic transition from differentiated contractile phenotype to dedifferentiated phenotype in VSMCs. However, the mechanism underlying the YY1-mediated regulation of fibulin-5 expression needs to be confirmed further in vivo. Nevertheless, targeting fibulin-5 and YY1 could be further developed for AS therapy.

Elastic Fibre Proteins in Elastogenesis and Wound Healing.

As essential components of our connective tissues, elastic fibres give tissues such as major blood vessels, skin and the lungs their elasticity. Their formation is complex and co-ordinately regulated by multiple factors. In this review, we describe key players in elastogenesis: fibrillin-1, tropoelastin, latent TGFß binding protein-4, and fibulin-4 and -5. We summarise their roles in elastogenesis, discuss the effect of their mutations on relevant diseases, and describe their interactions involved in forming the elastic fibre network. Moreover, we look into their roles in wound repair for a better understanding of their potential application in tissue regeneration.

Decreased Expression of EC-SOD and Fibulin-5 in Alveolar Walls of Lungs From COPD Patients.

INTRODUCTION: The aim of this study is to analyze the expression of the main oxidant scavenger superoxide dismutase (EC-SOD), its main binding protein Fibulin-5 and several oxidative and nitrosative-derived products in the lung of COPD patients and controls. MATERIALS AND METHODS: Lung tissue samples from 19 COPD patients and 20 control subjects were analyzed. The architecture of elastic fibres was assessed by light and electron microscope histochemical techniques, and levels of EC-SOD and fibulin-5 were analyzed by immunohistochemistry and RT-PCR. The impact of oxidative stress on the extracellular matrix was estimated by immunolocalization of 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and 3-nitrotyrosine (3-NYT) adducts. RESULTS: Alveolar walls of COPD patients exhibited abnormal accumulations of collapsing elastic fibres, showing a pierced pattern in the amorphous component. The semiquantitative analysis revealed that COPD patients have a significantly reduced expression of both EC-SOD and fibulin-5 (0.59±0.64 and 0.62±0.61, respectively) in alveolar, bronchiolar and arteriolar walls compared to control subjects (1.39±0.63 and 1.55±0.52, respectively, p<0.05). No significant changes in mRNA levels of these proteins were observed between groups. Among the oxidation markers, malondialdehyde was the best in distinguishing COPD patients. CONCLUSIONS: COPD patients show a reduced expression of EC-SOD and fibulin-5 in the lung interstitium. Paralleling the reduction of EC-SOD levels, the decrease of fibulin-5 expression in COPD lungs supports the hypothesis of an impaired pulmonary antioxidant response in COPD patients.

[Corrigendum] Nogo­B promotes the epithelial­mesenchymal transition in HeLa cervical cancer cells via Fibulin­5.

Following the publication of the above article, an interested reader drew to the authors' attention that the 'NB­4' and 'NB­2' panels for the invasion and migration assays shown in Fig. 3B and C on p. 113 appeared to contain overlapping data, such that the data may have been derived from the same original source, even though the panels were intending to have shown results obtained under different experimental conditions. The authors have re­examined their raw data and realized that these data were inadvertently mixed up when Fig. 3B and C were assembled. A corrected version of Fig. 3, showing the data as they should have appeared for the 'NB­4' and 'NB­2' invasion and migration assay experiments in Fig. 3B and C, is shown on the next page. The authors sincerely apologize for the errors that were introduced into Fig. 3 of the published article, and thank the Editor of Oncology Reports for allowing them the opportunity to publish a Corrigendum. All the authors agree to the publication of the authors, and they apologize to the readership for any inconvenience caused. [Oncology Reports 29: 109­116, 2013; DOI: 10.3892/or.2012.2069].

Mouse Knockout Models for Pelvic Organ Prolapse: a Systematic Review.

INTRODUCTION AND HYPOTHESIS: Mouse knockout (KO) models of pelvic organ prolapse (POP) have contributed mechanistic evidence for the role of connective tissue defects, specifically impaired elastic matrix remodeling. Our objective was to summarize what mouse KO models for POP are available and what have we learned from these mouse models about the pathophysiological mechanisms of POP development. METHODS: We conducted a systematic review and reported narrative findings according to PRISMA guidelines. Two independent reviewers searched PubMed, Scopus and Embase for relevant manuscripts and conference abstracts for the time frame of January 1, 2000, to March 31, 2021. Conference abstracts were limited to the past 5 years. RESULTS: The search strategy resulted in 294 total titles. We ultimately included 25 articles and an additional 11 conference abstracts. Five KO models have been studied: Loxl1, Fbln5, Fbln3, Hoxa11 and Upii-sv40t. Loxl1 and Fbln5 KO models have provided the most reliable and predictable POP phenotype. Loxl1 KO mice develop POP primarily from failure to heal after giving birth, whereas Fbln5 KO mice develop POP with aging. These mouse KO models have been used for a wide variety of investigations including genetic pathways involved in development of POP, biomechanical properties of the pelvic floor, elastic fiber deposition, POP therapies and the pathophysiology associated with mesh complications. CONCLUSIONS: Mouse KO models have proved to be a valuable tool in the study of specific genes and their role in the development and progression of POP. They may be useful to study POP treatments and POP complications.

MiR-552-3p facilitated cell proliferation, migration and invasion by sponging Fibulin 5 in non-small cell lung cancer via activation of ERK/GSK3ß/ß-catenin signaling pathway.

Apart from the fact that miR-552-3p is known to promote cell progression among various cancers, its function on non-small cell lung cancer (NSCLC) is unknown which therefore emerges as the purpose of this research. TargetScan, Starbase, miRWalk, miRDB and the Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) were utilized to analyze the target genes of miR-552-3p. NSCLC cells were transfected with miR-552-3p mimic, miR-552-3p inhibitor, Fibulin 5 (FBLN5) overexpression plasmid, and small interfering FBLN5 (siFBLN5) and treated with extracellular regulated protein kinases (ERK) pathway inhibitor PD98059. MiR-552-3p, FBLN5, p-ERK, ERK, p-glycogen synthase kinase 3ß (GSK3ß) and ß-catenin levels were detected through quantitative reverse transcription-polymerase chain reaction and western blot. The binding sites between miR-552-3p and FBLN5 were predicted by TargetScan, which was tested through dual luciferase reporter analysis. Cell viability, migration and invasion were determined by cell counting kit-8 (CCK-8) assay, wound healing assay and transwell assay, respectively. MiR-552-3p expression was upregulated in NSCLC and FBLN5 functioned as its target. MiR-552-3p mimic promoted proliferation, migration, invasion, p-ERK, p-GSK3ß and ß-catenin expressions in NSCLC cells while miR-552-3p inhibitor did the opposite. Overexpressed FBLN5 suppressed proliferation, migration, invasion, p-ERK, p-GSK3ß and ß-catenin expressions in NSCLC cells whereas siFBLN5 exerted the effects opposite to overexpressed FBLN5. PD98059 enhanced the effect of overexpressed FBLN5 on NSCLC cell migration and invasion while reversing the effect of siFBLN5. MiR-552-3p facilitated cell proliferation, migration and invasion in NSCLC through sponging FBLN5 via activation of ERK/GSK3ß/ß-catenin pathway.

PAD2-mediated citrullination of Fibulin-5 promotes elastogenesis.

The formation of elastic fibers is active only in the perinatal period. How elastogenesis is developmentally regulated is not fully understood. Citrullination is a unique form of post-translational modification catalyzed by peptidylarginine deiminases (PADs), including PAD1-4. Its physiological role is largely unknown. By using an unbiased proteomic approach of lung tissues, we discovered that FBLN5 and LTBP4, two key elastogenic proteins, were temporally modified in mouse and human lungs. We further demonstrated that PAD2 citrullinated FBLN5 preferentially in young lungs compared to adult lungs. Genetic ablation of PAD2 resulted in attenuated elastogenesis in vitro and age-dependent emphysema in vivo. Mechanistically, citrullination protected FBLN5 from proteolysis and subsequent inactivation of its elastogenic activity. Furthermore, citrullinated but not native FBLN5 partially rescued in vitro elastogenesis in the absence of PAD activity. Our data uncover a novel function of citrullination, namely promoting elastogenesis, and provide additional insights to how elastogenesis is regulated.

Effect of fibulin-5 on aldosterone-induced apoptosis in human ascending aortic smooth muscle cells.

The aim of the current study was to investigate the effect of aldosterone on apoptosis in human aortic smooth muscle cells (HA-VSMC) and to determine the role of fibulin-5 in the aldosterone-induced apoptosis of HA-VSMC cells. Through the construction of a fibulin-5 eukaryotic overexpression vector and a short hairpin RNA interference plasmid, fibulin-5 was overexpressed and silenced, respectively. The role of fibulin-5 in the aldosterone-induced apoptosis of HA-VSMC was subsequently determined. The overexpression of fibulin-5 inhibited the apoptosis of cells, particularly at low concentrations of aldosterone; a smaller effect on apoptosis was induced by high concentrations of aldosterone. fibulin-5 knockdown promoted the apoptosis of cells induced by high concentrations of aldosterone but had a smaller effect on the apoptosis of cells induced by low concentrations of aldosterone. Therefore, the results of the current study indicate that fibulin-5 inhibits the aldosterone-induced apoptosis of HA-VSMC cells and that this effect may be altered by changing the aldosterone concentration.

A Non-Invasive Determination of LOXL1 and Fibulin-5 Levels in the Vaginal Secretions of Women with and Without Pelvic Organ Prolapse.

OBJECTIVES: The aim of this study was to determine if lysyl oxidase-like 1 (LOXL1) and Fibulin-5 (Fib-5), two crucial proteins in the elastin metabolism pathway, are detectable in the vaginal secretions of women with and without pelvic organ prolapse (POP). We then sought to quantify levels of these proteins in relation to prolapse. METHODS: Vaginal secretions were obtained from 48 subjects (13 (27.1%) without and 35 (72.9%) with POP-Q Stage 2-4 prolapse). Eleven (22.9%) subjects were premenopausal and 37 (77.1%) were postmenopausal. Presence of LOXL-1 and Fibulin-5 within specimens were first identified via western blotting. Enzyme-Linked Immunosorbent Assays specific for LOXL1 and Fibulin-5 were conducted to quantify total protein secretion. RESULTS: LOXL1 was detected in 45/48 (93.8%) and Fibulin-5 was seen in 24/48 (50%) of subjects. LOXL1 values were lower in women without prolapse (13.3 ng/100 mg median, 24.4 IQR) vs. those with prolapse (26.4 ng/100 mg, 102.2 IQR). On multivariate analysis controlling for age, women with prolapse had a 544% (p=0.0042 higher LOXL1 protein level compared to those without. There was no significant differences in LOXL1 or Fibulin-5 protein detection with relation to menopausal status in bivariate analysis. CONCLUSIONS: This is the first published report of non-invasively measuring urogenital LOXL1 and Fibulin-5. In vaginal secretions, LOXL1 protein is higher in subjects with POP than those without.

Transcriptome Analysis of Hypoxic Lymphatic Endothelial Cells Indicates Their Potential to Contribute to Extracellular Matrix Rearrangement.

Lymphedema (LE) affects millions of people worldwide. It is a chronic progressive disease with massive development of fibrosclerosis when untreated. There is no pharmacological treatment of lymphedema. The disease is associated with swelling of the interstitium of the affected organ, mostly arm or leg, impressive development of adipose tissue, fibrosis and sclerosis with accumulation of huge amounts of collagen, and Papillomatosis cutis. Malnutrition and reduced oxygenation of the affected tissues is a hallmark of lymphedema. Here, we investigated if the hypoxia of lymphatic endothelial cells (LECs) might contribute to fibrosis. We applied RNASeq and qPCR to study the concordant changes of the exome of three human foreskin-derived LEC isolates after 4 days of hypoxia (1% O2) vs. normoxia (21% O2). Of the approximately 16,000 genes expressed in LECs, 162 (1%) were up- or down-regulated by hypoxia. Of these, 21 genes have important functions in the production or modification of the extracellular matrix (ECM). In addition to the down-regulation of elastin, we found up-regulation of druggable enzymes and regulators such as the long non-coding RNA H19, inter-alpha-trypsin inhibitor heavy chain family member 5 (ITIH5), lysyl-oxidase (LOX), prolyl 4-hydroxylase subunit alpha 1 (P4HA1), procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2), and others that are discussed in the paper. Initial lymphatics do not produce a continuous basement membrane; however, our study shows that hypoxic LECs have an unexpectedly high ability to alter the ECM.

Therapeutic effect of Jianpi Liqi Fang combined with transcatheter arterial chemoembolization in patients with hepatocellular carcinoma and spleen deficiency syndrome.

OBJECTIVE: To investigate the therapeutic effect of the Jianpi Liqi Fang ( ，JPLQF) combined with transcatheter arterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC) and spleen deficiency syndrome (SDS) and identify a potential indicator of efficacy. METHODS: Ninety-nine patients with HCC who were diagnosed with SDS, non-spleen deficiency syndrome (NSDS), or no syndrome (NS) were treated with JPLQF combined with TACE for three periods. Therapeutic efficacy was compared among the groups. Plasma proteins were screened using label-free discovery analysis and verified via enzyme-linked immunosorbent assay (ELISA). Furthermore, receiver operating characteristic (ROC) curves were analyzed to evaluate therapeutic indicators. RESULTS: After treatment, the Karnofsky Performance Status was significantly improved in the SDS group and significantly better than that in the NS group. The Traditional Chinese Medicine (TCM) syndrome scores were lower in the SDS group after treatment and lower than those in the NSDS group. However, alanine aminotransferase, carbohydrate antigen 19-9, alpha-fetoprotein, and carcinoembryonic antigen levels and white blood cell and platelet counts did not differ among the groups. Serum aspartate aminotransferase levels in the SDS group were significantly lower after treatment than before treatment, and total bilirubin levels were significantly lower in the SDS group than in the NSDS group. Label-free analysis identified 24 differentially expressed proteins (DEPs) between the SDS and NS groups, including 17 and 7 upregulated and downregulated proteins, respectively. Fibulin-5 (FBLN5) displayed the largest difference in expression between the groups. ELISA confirmed that FBLN5 levels were significantly lower in the NSDS and NS groups than in the SDS group. Following treatment with JPLQF and TACE, FBLN5 expression was upregulated only in the SDS group. Furthermore, ROC curve analysis indicated that FBLN5 may serve as a potential indicator of the efficacy of JPLQF combined with TACE in patients with HCC and SDS. CONCLUSION: JPLQF combined with TACE improved quality of life, clinical TCM symptoms, and liver function in patients with HCC and SDS. FBLN5 expression was significantly altered by treatment with JPLQF and TACE in patients with HCC and SDS.

Upregulation of PTK7 and ß-catenin after vaginal mechanical dilatation: an examination of fibulin-5 knockout mice.

INTRODUCTION AND HYPOTHESIS: Pelvic organ prolapse (POP) in women is associated with deficiency of elastic fibers, and fibulin-5 is known to be a critical protein in the synthesis of elastin. The purpose of this study is to investigate the related pathway for the synthesis of elastin via fibulin-5 using fibulin-5 knockout mice. METHODS: Fibulin-5 knockout mice were generated using the CRISPR/Cas9 system, and vaginal dilatation was used to mimic vaginal delivery. We divided the mice into three groups: Fbln5+/+ mice immediately after dilatation (Fbln5+/+ day0), Fbln5+/+ mice 3 days after dilatation (Fbln5+/+ day3) and Fbln5-/- mice 3 days after dilatation (Fbln5-/- day3). Proteins related to elastogenesis in the vaginal wall were measured by liquid chromatography mass spectrometry (LC-MS/MS) analysis, and differences in the expression of these proteins between the Fbln5-/- mice and the Fbln5+/+ mice were analyzed using western blotting. RESULTS: In the LC-MS/MS analysis, protein tyrosine kinase 7 (PTK7) was not detected in the Fbln5-/- day3 group, although the expression increased by > 1.5 times between the Fbln5+/+ day0 and day3 groups. PTK7 and ß-catenin are known to act in the Wnt/ß-catenin pathway, and both were upregulated after dilatation in the Fbln5+/+ mice, though not in the Fbln5-/- mice. CONCLUSION: Our findings suggest that these proteins are involved in elastogenesis via fibulin-5, and the impairment of these proteins might be the underlying cause of POP manifestation.

Decreased Expression of EC-SOD and Fibulin-5 in Alveolar Walls of Lungs From COPD Patients.

INTRODUCTION: The aim of this study is to analyze the expression of the main oxidant scavenger superoxide dismutase (EC-SOD), its main binding protein Fibulin-5 and several oxidative and nitrosative-derived products in the lung of COPD patients and controls. MATERIALS AND METHODS: Lung tissue samples from 19 COPD patients and 20 control subjects were analyzed. The architecture of elastic fibres was assessed by light and electron microscope histochemical techniques, and levels of EC-SOD and fibulin-5 were analyzed by immunohistochemistry and RT-PCR. The impact of oxidative stress on the extracellular matrix was estimated by immunolocalization of 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and 3-nitrotyrosine (3-NYT) adducts. RESULTS: Alveolar walls of COPD patients exhibited abnormal accumulations of collapsing elastic fibres, showing a pierced pattern in the amorphous component. The semiquantitative analysis revealed that COPD patients have a significantly reduced expression of both EC-SOD and fibulin-5 (0.59±0.64 and 0.62±0.61, respectively) in alveolar, bronchiolar and arteriolar walls compared to control subjects (1.39±0.63 and 1.55±0.52, respectively, p<0.05). No significant changes in mRNA levels of these proteins were observed between groups. Among the oxidation markers, malondialdehyde was the best in distinguishing COPD patients. CONCLUSIONS: COPD patients show a reduced expression of EC-SOD and fibulin-5 in the lung interstitium. Paralleling the reduction of EC-SOD levels, the decrease of fibulin-5 expression in COPD lungs supports the hypothesis of an impaired pulmonary antioxidant response in COPD patients.

New insight into clinical heterogeneity and inheritance diversity of FBLN5-related cutis laxa.

BACKGROUND: FBLN5-related cutis laxa (CL) is a rare disorder that involves elastic fiber-enriched tissues and is characterized by lax skin and variable systemic involvement such as pulmonary emphysema, arterial involvement, inguinal hernias, hollow viscus diverticula and pyloric stenosis. This type of CL follows mostly autosomal recessive (AR) and less commonly autosomal dominant patterns of inheritance. RESULTS: In this study, we detected a novel homozygous missense variant in exon 6 of FBLN5 gene (c.G544C, p.A182P) by using whole exome sequencing in a consanguineous Iranian family with two affected members. Our twin patients showed some of the clinical manifestation of FBLN5-related CL but they did not present pulmonary complications, gastrointestinal and genitourinary abnormalities. The notable thing about this monozygotic twin sisters is that only one of them showed ventricular septal defect, suggesting that this type of CL has intrafamilial variability. Co-segregation analysis showed the patients' parents and relatives were heterozygous for detected variation suggesting AR form of the CL. In silico prediction tools showed that this mutation is pathogenic and 3D modeling of the normal and mutant protein revealed relative structural alteration of fibulin-5 suggesting that the A182P can contribute to the CL phenotype via the combined effect of lack of protein function and partly misfolding-associated toxicity. CONCLUSION: We underlined the probable roles and functions of the involved domain of fibulin-5 and proposed some possible mechanisms involved in AR form of FBLN5-related CL. However, further functional studies and subsequent clinical and molecular investigations are needed to confirm our findings.

Dysregulated assembly of elastic fibers in fibulin-5 knockout mice results in a tendon-specific increase in elastic modulus.

Elastic fiber assembly is coordinated in part by fibulin-5, a matricellular protein. When fibulin-5 is not available to guide elastogenesis, elastin forms into disconnected globules instead of the dense elastic fiber core found in healthy tissues. Despite the growing evidence for a significant role of elastic fibers in tendon mechanics and the clinical relevance to cutis laxa, a human disease which can be caused by a mutation in the gene encoding fibulin-5, it is unknown how malformed elastic fibers affect tendon function. Therefore, this study investigated the effects of dysregulated elastic fiber assembly in tendons from fibulin-5 knockout mice in comparison to wild-type controls. Due to evidence for a more prominent role of elastic fibers in tendons with higher functional demands, both the energy-storing Achilles tendon and the more positional tibialis anterior tendon were evaluated. The linear modulus of knockout Achilles tendons was increased compared to controls, yet there was no discernible change in mechanical properties of the tibialis anterior tendon across genotypes. Transmission electron microscopy confirmed the presence of malformed elastic fibers in knockout tendons while no other changes to tendon composition or structure were found. The mechanism behind the increase in linear modulus in fibulin-5 knockout Achilles tendons may be greater collagen engagement due to decreased regulation of strain-induced structural reorganization. These findings support the theory of a significant, functionally distinct role of elastic fibers in tendon mechanics.