To meet demands of vascular reconstruction, there is a need for prosthetic alternatives to natural blood vessels. Here we explored a new conduit fabrication approach. Macroporous, gelatin microcarriers laden with human umbilical vein endothelial cells and aortic smooth muscle cells were dispensed into tubular agarose molds and found to adhere to form living tubular tissues. The ability of cellularized microcarriers to adhere to one another involved cellular and extracellular matrix bridging that included the formation of epithelium-like cell layers lining the lumenal and ablumenal surfaces of the constructs and the deposition of collagen and elastin fibers. The tubular tissues behaved as elastic solids, with a uniaxial mechanical response that is qualitatively similar to that of native vascular tissues and consistent with their elastin and collagen composition. Linearized measures of the mechanical response of the fabricated tubular tissues at both low and high strains was observed to increase with duration of static culture, with no significant loss of stiffness following decellularization. The findings highlight the utility of cellularized macroporous gelatin microcarriers as self-adhering building blocks for the fabrication of living tubular structures.
The extracellular matrix protein Fibulin-1 (Fbln1) has been shown to be involved in numerous processes including cardiovascular and lung development. Here we have examined the role of Fbln1 in bone formation. Alizarin red staining of skulls from Fbln1 deficient mice showed reduced mineralization of both membranous and endochondral bones. Micro CT (μCT) analysis of the calvarial bones (i.e., frontal, parietal and interparietal bones collectively) indicated that bone volume in Fbln1 nulls at neonatal stage P0 were reduced by 22% (p = 0.015). Similarly, Fbln1 null frontal bones showed a 16% (p = 0.035) decrease in bone volume, with a reduction in the interfrontal bone, and a discontinuity in the leading edge of the frontal bone. To determine whether Fbln1 played a role in osteoblast differentiation during bone formation, qPCR was used to measure the effects of Fbln1 deficiency on the expression of Osterix (Osx), a transcription factor essential for osteoblast differentiation. This analysis demonstrated that Osx mRNA was significantly reduced in Fbln1-deficient calvarial bones at developmental stages E16.5 (p = 0.049) and E17.5 (p = 0.022). Furthermore, the ability of BMP-2 to induce Osx expression was significantly diminished in Fbln1-deficient mouse embryo fibroblasts. Together, these findings indicate that Fbln1 is a new positive modulator of the formation of membranous bone and endochondral bone in the skull, acting as a positive regulator of BMP signaling.
Epidermal Growth Factor Receptor (EGFR) is a known promoter of tumor progression and is overexpressed in lung cancers. Growth factor receptors (including EGFR) are known to interact with extracellular matrix (ECM) proteins, which regulate their activation and function. Fibulin-1 (FBLN1) is a major component of the ECM in lung tissue, and its levels are known to be downregulated in non-small cell lung cancers (NSCLC). To test the possible role FBLN1 isoforms could have in regulating EGFR signaling and function in lung cancer, we performed siRNA mediated knockdown of FBLN1C and FBLN1D in NSCLC Calu-1 cells. Their loss significantly increased basal (with serum) and EGF (Epidermal Growth Factor) mediated EGFR activation without affecting net EGFR levels. Overexpression of FBLN1C and FBLN1D also inhibits EGFR activation confirming their regulatory crosstalk. Loss of FBLN1C and FBLN1D promotes EGFR-dependent cell migration, inhibited upon Erlotinib treatment. Mechanistically, both FBLN1 isoforms interact with EGFR, their association not dependent on its activation. Notably, cellderived matrix (CDM) enriched FBLN1 binds EGFR. Calu-1 cells plated on CDM derived from FBLN1C and FBLN1D knockdown cells show a significant increase in EGF mediated EGFR activation. This promotes cell adhesion and spreading with active EGFR enriched at membrane ruffles. Both adhesion and spreading on CDMs is significantly reduced by Erlotinib treatment. Together, these findings show FBLN1C/1D, as part of the ECM, can bind and regulate EGFR activation and function in NSCLC Calu-1 cells. They further highlight the role tumor ECM composition could have in influencing EGFR dependent lung cancers.
Endothelial to mesenchymal transition (EMT) that occurs during cardiac outflow tract (OFT) development is critical for formation of the semilunar valves. Fibulin-1 (Fbln1) is an extra-cellular matrix protein that is present at several sites of EMT, including the OFT (i.e., E9.5–10.5). The aim of this study was to determine the role of Fbln1 in EMT during the earliest events of OFT development. Examination of proximal OFT cushions in Fbln1 null embryos detected hypercellularity at both E9.5 (93% increase; p = 0.002) and E10.5 (43% increase; p = 0.01) as compared to wild type, suggesting that Fbln1 normally suppresses OFT endocardial cushion EMT. This was supported by studies of proximal OFT cushion explants, which showed that explants from Fbln1 null embryos displayed a 58% increase in cells migrating from the explants as compared to wild type (p = 0.005). We next evaluated the effects of Fbln1 deficiency on the expression of factors that regulate proximal OFT EMT. At E9.5, Fbln1 null proximal OFT endocardium and EMT-derived mesenchyme showed increased TGFβ2 (58% increase; p = 0.01) and increased Snail1-positive nuclei (27% increase; p = 0.0003). Histological examination of OFT cushions in Fbln1 null embryos (E9.5) also detected cells present in the cushion that were determined to be erythrocytes based on round morphology, autofluorescence, and positive staining for hemoglobin. Erythrocytes were also detected in Fbln1 null OFT cushions at E10.5. Together, the findings indicate that Fbln1 normally suppresses proximal OFT EMT preventing proximal cushion hypercellularity and blood cell accumulation.
The extracellular matrix in the tumour microenvironment can regulate cancer cell growth and progression. A pan-cancer analysis of TCGA data from 30 cancer types, identified the top 5% of matrisome genes with amplifications or deletions in their copy number, that affect their expression and cancer survival. A similar analysis of matrisome genes in individual cancers identified CTHRC1 to be significantly altered. CTHRC1, a regulator of collagen synthesis, was identified as the most prominently upregulated matrisome gene of interest across cancers. Differential gene expression analysis identified 19 genes whose expression is increased with CTHRC1. STRING analysis of these genes classified them as ‘extracellular’, involved most prominently in ECM organization and cell adhesion. KEGG analysis showed their involvement in ECM-receptor and growth factor signalling. Cytohubba analysis of these genes revealed 13 hub genes, of which MMP13, POSTN, SFRP4, ADAMTS16 and FNDC1 were significantly altered in their expression with CTHRC1 and seen to affect survival across cancers. This could in part be mediated by their overlapping roles in regulating ECM (collagen or fibronectin) expression and organisation. In breast cancer tumour samples CTHRC1 protein levels are significantly upregulated with POSTN and MMP13, further supporting the need to evaluate their crosstalk in cancers.
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