To characterise the clinicopathological features of amyloidosis due to EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), a newly described amyloid type.
We identified cases by ...searching the Mayo Clinic amyloid liquid chromatography and tandem mass spectrometry typing database for specimens with the universal amyloid signature proteins, abundant EFEMP1 spectra and absence of other specific amyloid precursor proteins. We also developed an immunohistochemical stain for EFEMP1 applicable to formalin-fixed tissue sections and performed electron microscopy in one case. We identified 33 specimens from 32 patients with EFEMP1 amyloid. Most patients were female (91%) with a mean age of 75 years, and most specimens (94%) were from the bowel. EFEMP1 amyloid was incidentally identified in specimens biopsied/resected for a variety of clinical indications. In bowel specimens, EFEMP1 amyloid involved blood vessels and interstitium of the lamina propria, submucosa and/or muscularis propria. Although the EFEMP1 deposits were weakly to moderately Congo red-positive with absent to weak birefringence, they were strongly positive for EFEMP1 by immunohistochemistry, had the characteristic fibrillar ultrastructure of amyloid and were readily identified by mass spectrometry.
EFEMP1 amyloid is a recently described novel amyloid type that predominantly affects the bowel of elderly females. Because EFEMP1 amyloid is only weakly Congo red-positive, it may be overlooked without a high index of suspicion. However, its characteristic microanatomical distribution is highlighted by immunohistochemistry and its identity is readily confirmed by mass spectrometry. Based on its distinctive features, we propose that EFEMP1 amyloidosis be considered a new amyloid type.
Fibulin-3 is an extracellular matrix glycoprotein that is present in elastic tissue and involved in carcinoma development. Previous studies have indicated that fibulin-3 may affect skeletal ...development, cartilage, and osteoarthritis (OA). This study aims to investigate the function of fibulin-3 on chondrocytes under tumor necrosis factor alpha (TNF-α) stimulation and in murine OA models, and explore the possible mechanism. It was found that fibulin-3 was increased in the cartilage of OA models and in the chondrogenic cells ATDC5 stimulated by TNF-α. Fibulin-3 promoted the proliferation of ATDC5 cells both in the presence and absence of TNF-α. Moreover, overexpression of fibulin-3 suppressed the chondrogenic and hypertrophic differentiation of ATDC5 cells, while knockdown of fibulin-3 caused the opposite effect. Mechanistically, fibulin-3 partially suppressed the activation of TGF-β/Smad3 signaling by inhibiting the phosphorylation of Smad3. SIS3, a Smad3 inhibitor, decreased the chondrogenesis of articular cartilages in OA models, and partially reversed the chondrogenic differentiation of ATDC5 cells caused by knockdown of fibulin-3 in the presence of TNF-α. Furthermore, co-immunoprecipitation (Co-IP) showed that fibulin-3 could only interact with TGF-β type I receptor (TβRI), although overexpression of fibulin-3 reduced the protein levels of both TβRI and TβRII. In conclusion, this study indicates that fibulin-3 modulates the chondrogenic differentiation of ATDC5 cells in inflammation partially via TGF-β/Smad3 signaling pathway.
•Fibulin-3 was existent in the cartilage and chondrocytes of osteoarthritis models.•Fibulin-3 modulated the differentiation of ATDC5 in TNF-α-induced inflammation.•Fibulin-3 partially regulated ATDC5 differentiation via TGF-β/Smad3 signaling.•Fibulin-3 possibly interacted with TβRI to repress the TGF-β/Smad3 signaling.
Breast cancer is a leading cause of cancer mortality. In particular, triple negative breast cancer (TNBC) comprise a heterogeneous group of basal-like tumors lacking estrogen receptor (ERα), ...progesterone receptor (PR) and HER2 (ErbB2). TNBC represents 15-20% of all breast cancers and occurs frequently in women under 50 years of age. Unfortunately, these patients lack targeted therapy, are typically high grade and metastatic at time of diagnosis. The mechanisms regulating metastasis remain poorly understood. We have previously shown that the kisspeptin receptor, KISS1R stimulates invasiveness of TNBC cells. In this report, we demonstrate that KISS1R signals via the secreted extracellular matrix protein, fibulin-3, to regulate TNBC invasion. We found that the fibulin-3 gene is amplified in TNBC primary tumors and that plasma fibulin-3 levels are elevated in TNBC patients compared to healthy subjects. In this study, we show that KISS1R activation increases fibulin-3 expression and secretion. We show that fibulin-3 regulates TNBC metastasis in a mouse experimental metastasis xenograft model and signals downstream of KISS1R to stimulate TNBC invasion, by activating matrix metalloproteinase 9 (MMP-9) and the MAPK pathway. These results identify fibulin-3 as a new downstream mediator of KISS1R signaling and as a potential biomarker for TNBC progression and metastasis, thus revealing KISS1R and fibulin-3 as novel drug targets in TNBC.
It is known that the actin cytoskeleton and its associated cellular interactions in the trabecular meshwork (TM) and juxtacanalicular tissues mainly contribute to the formation of resistance to ...aqueous outflow of the eye. Fibulin-3, encoded by EFEMP1 gene, has a role in extracellular matrix (ECM) modulation, and interacts with enzymatic ECM regulators, but the effects of fibulin-3 on TM cells has not been explored. Here, we report a stop codon variant (c.T1480C, p.X494Q) of EFEMP1 that co-segregates with primary open angle glaucoma (POAG) in a Chinese pedigree. In the human TM cells, overexpression of wild-type fibulin-3 reduced intracellular actin stress fibers formation and the extracellular fibronectin levels by inhibiting Rho/ROCK signaling. TGFβ1 up-regulated fibulin-3 protein levels in human TM cells by activating Rho/ROCK signaling. In rat eyes, overexpression of wild-type fibulin-3 decreased the intraocular pressure and the fibronectin expression of TM, however, overexpression of mutant fibulin-3 (c.T1480C, p.X494Q) showed opposite effects in cells and rat eyes. Taken together, the EFEMP1 variant may impair the regulatory capacity of fibulin-3 which has a role for modulating the cell contractile activity and ECM synthesis in TM cells, and in turn may maintain normal resistance of aqueous humor outflow. This study contributes to the understanding of the important role of fibulin-3 in TM pathophysiology and provides a new possible POAG therapeutic approach.
•Fibulin-3 inhibited Rho/ROCK signaling in the human trabecular meshwork (TM) cells.•TGFβ1 up-regulated fibulin-3 protein level in human TM cells via Rho/ROCK signaling.•Overexpression of fibulin-3 decreased the intraocular pressure in rat eyes.•The p.X494Q variant in EFEMP1 impaired the regulatory capacity of fibulin-3.•Fibulin-3 may maintain a normal resistance of aqueous humor outflow.
Fibulin‐3 (F3 or EFEMP1) is a disulfide‐rich, secreted glycoprotein necessary for maintaining extracellular matrix (ECM) and connective tissue integrity. Three studies have identified distinct ...autosomal recessive F3 mutations in individuals with Marfan Syndrome‐like phenotypes. Herein, we characterize how one of these mutations, c.163T>C; p.Cys55Arg (C55R), disrupts F3 secretion, quaternary structure, and function by forming unique extracellular disulfide‐linked homodimers. Dual cysteine mutants suggest that the C55R‐induced disulfide species forms because of the new availability of Cys70 on adjacent F3 monomers. Surprisingly, mutation of single cysteines located near Cys55 (i.e., Cys29, Cys42, Cys48, Cys61, Cys70, Cys159, and Cys171) also produced similar extracellular disulfide‐linked dimers, suggesting that this is not a phenomenon isolated to the C55R mutant. To assess C55R functionality, F3 knockout (KO) retinal pigmented epithelial (RPE) cells were generated, followed by reintroduction of wild‐type (WT) or C55R F3. F3 KO cells produced lower levels of the ECM remodeling enzyme, matrix metalloproteinase 2, and reduced formation of collagen VI ECM filaments, both of which were partially rescued by WT F3 overexpression. However, C55R F3 was unable to compensate for these same ECM‐related defects. Our results highlight the unique behavior of particular cysteine mutations in F3 and uncover potential routes to restore C55R F3 loss‐of‐function.
Malignant pleural mesothelioma (MPM) is difficult to diagnose. An accurate blood biomarker could prompt specialist referral or be deployed in future screening. In earlier retrospective studies, ...SOMAscan proteomics (Somalogic, Boulder, CO) and fibulin-3 seemed highly accurate, but SOMAscan has not been validated prospectively and subsequent fibulin-3 data have been contradictory.
A multicenter prospective observational study was performed in 22 centers, generating a large intention-to-diagnose cohort. Blood sampling, processing, and diagnostic assessment were standardized, including a 1-year follow-up. Plasma fibulin-3 was measured using two enzyme-linked immunosorbent assays (CloudClone used in previous studies and BosterBio, Pleasanton, CA). Serum proteomics was measured using the SOMAscan assay. Diagnostic performance (sensitivity at 95% specificity, area under the curve AUC) was benchmarked against serum mesothelin (Mesomark, Fujirebio Diagnostics, Malvern, PA). Biomarkers were correlated against primary tumor volume, inflammatory markers, and asbestos exposure.
A total of 638 patients with suspected pleural malignancy (SPM) and 110 asbestos-exposed controls (AECs) were recruited. SOMAscan reliably differentiated MPM from AECs (75% sensitivity, 88.2% specificity, validation cohort AUC 0.855) but was not useful in patients with differentiating non-MPM SPM. Fibulin-3 (by BosterBio after failed CloudClone validation) revealed 7.4% and 11.9% sensitivity at 95% specificity in MPM versus non-MPM SPM and AECs, respectively (associated AUCs 0.611 0.557–0.664, p = 0.0015) and 0.516 0.443–0.589, p = 0.671), both inferior to mesothelin. SOMAscan proteins correlated with inflammatory markers but not with asbestos exposure. Neither biomarker correlated with tumor volume.
SOMAscan may prove useful as a future screening test for MPM in asbestos-exposed persons. Neither fibulin-3 nor SOMAscan should be used for diagnosis or pathway stratification.
Age-related macular degeneration (AMD) is one of the leading causes of blindness, and choroidal neovascularization (CNV) in AMD can lead to serious visual impairment. Gene expression profiling of ...human ocular tissues have a great potential to reveal the pathophysiology of AMD. This study aimed to identify novel molecular biomarkers and gene expression signatures of AMD.
We analyzed transcriptome profiles in retinal-choroid tissues derived from donor patients with AMD in comparison with those from healthy controls using a publicly available dataset (GSE29801). We focused on the EFEMP1 gene, which was found to be differentially upregulated in AMD, especially in wet AMD eyes. Serological validation analysis was carried out to verify the expression of EFEMP1 in 39 wet AMD patients and 39 age- and gender-matched cataract controls, using an enzyme-linked immunosorbent assay (ELISA). We then investigated the role of EFEMP1 in angiogenesis through
experiments involving EFEMP1 overexpression (OE) and knockdown in human umbilical vein endothelial cells (HUVECs).
An increase in EFEMP1 expression was observed in the retinal-choroid tissues of eyes with AMD, which was more significant in wet AMD than in dry AMD. In addition, there was a significant increase in serum fibulin-3 (EFEMP1 encoded protein) concentration in patients with wet AMD compared with that in the controls. Tube formation and proliferation of EFEMP1-OE HUVECs increased significantly, whereas those of EFEMP1 knockdown HUVECs decreased significantly compared with those of the control. Additional extracellular fibulin-3 treatments did not increase tube formation and proliferation of wildtype and EFEMP1 knockdown HUVECs, indicating that the proangiogenic properties of EFEMP1 are of cell origin. We also found that vascular endothelial growth factor expression in HUVECs was upregulated by EFEMP1 overexpression and downregulated by EFEMP1 knockdown.
Our findings demonstrate EFEMP1 as a novel biomarker for CNV in AMD, providing a new target for the development of wet AMD-directed pharmaceuticals and diagnostics.
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