Gene expression profiling of Elastin (ELN) knock-down in human fibroblast MRC5
Ontology highlight
ABSTRACT: The ELN gene encodes tropoelastin which is used to generate elastic fibers that insure proper tissue elasticity. Decreased amounts of elastic fibers and/or accumulation of bioactive products of their cleavage, named elastokines, are thought to contribute to aging. Cellular senescence, characterized by a stable proliferation arrest and by the senescence-associated secretory phenotype (SASP), increases with aging. Here, we identified that decrease in ELN either by siRNA in normal human fibroblasts results in premature senescence. Surprisingly this effect is independent of elastic fiber degradation or elastokines production, but it relies on the rapid increase in HMOX1 after ELN downregulation. These results unveil a role for ELN in protecting cells from cellular senescence through a non-canonical mechanism.
Project description:Elastic fibers provide reversible elasticity to the large arteries and are assembled during development when hemodynamic forces are increasing. Mutations in elastic fiber genes are associated with cardiovascular disease. Mice lacking expression of the elastic fiber genes elastin (Eln-/-), fibulin-4 (Efemp2-/-), or lysyl oxidase (Lox-/-) die at birth with severe cardiovascular malformations. All three genetic knockout models have elastic fiber defects, aortic wall thickening, and arterial tortuosity. However, Eln-/- mice develop arterial stenoses, while Efemp2-/- and Lox-/- mice develop ascending aortic aneurysms. We performed comparative gene array analyses of these three genetic models for two vascular locations and developmental stages to determine differentially expressed genes and pathways that may explain the common and divergent phenotypes. We first examined arterial morphology and wall structure in newborn mice to confirm that the lack of elastin, fibulin-4, or lysyl oxidase expression provided the expected phenotypes. We then compared gene expression levels for each genetic model by three-way ANOVA for genotype, vascular location, and developmental stage. We found three genes upregulated by genotype in all three models, Col8a1, Igfbp2, and Thbs1, indicative of a common response to severe elastic fiber defects in developing mouse aorta. Genes that are differentially regulated by vascular location or developmental stage in all three models suggest mechanisms for location or stage specific disease pathology. Comparison of signaling pathways enriched in all three models shows upregulation of integrins and matrix proteins involved in early wound healing, but not of mature matrix molecules such as elastic fiber proteins or fibrillar collagens.
Project description:Elastin wild type Eln+/+ and Eln+/- mouse aorta and aortic valve tissue. In the study, we demonstrated differential gene expression in juvenile elastin deficient mouse valve tissue. In the study, we hybridized RNA from Elastin wild type (Eln+/+) aorta tissue, elastin wild type (Eln+/+) aortic valve tissue, elastin (Eln+/-) aorta tissue and elastin (Eln+/-) aortic valve tissue to Affymetrix GeneChip Mouse Genome 430 2.0 Array
Project description:Pseudoxanthoma elasticum (PXE) is characterized by ectopic calcification, however, despite the widely spread effect of pro/anti-calcifying systemic factors associated with the genetic metabolic condition, it not known why elastic fibers (EF) in the same patient are mainly fragmented or highly mineralized in clinically unaffected (CUS) and affected (CAS) skin, respectively. Cellular morphology and secretome were investigated in vitro in CUS and CAS fibroblasts. Data reveal that in CAS fibroblasts focal adhesions and stress fibers are differently distributed and organized thus affecting cell-matrix interactions (i.e., collagen gel retraction). These changes are known to influence the extracellular matrix (ECM) and the signals provided to cells. Differentially expressed secreted proteins (e.g., altered balance of MMPs/TIMP, of pro-/anti-calcifying proteoglycans, of elastic-fibers associated glycoproteins) may alter the stability of EF and the ECM milieu, creating local microenvironments guiding the level of matrix remodeling at an extent that may lead to degradation (in CUS) or to degradation and calcification (in CAS) of the elastic component, further highlighting the active role of fibroblasts and of ECM in pathologic mineral deposition.
Project description:Elastin wild type Eln+/+ and Eln+/- mouse aorta and aortic valve tissue. In the study, we demonstrated differential gene expression in juvenile elastin deficient mouse valve tissue.
Project description:Generally, cancer tissue is palpated as a hard mass. On the other hand, it is not clear the nature of elasticity in cancer tissue. The aim in this study is to evaluate clinical utility of measuring elastic module in colorectal cancer tissue. Using a tactile sensor, we measured the elastic module of 106 surgically resected colorectal cancer tissues. The data of the elastic module were compared with the clinicopathological findings including stromal features represented by azan and α-SMA positive area ratio in tumor area. Finally cDNA microarray profile of the tumor with high elastic module was compared with that with low elastic module Higher elastic module in tumor was associated with pathological T-, N- and M-Stage (p < 0.001, p = 0.001 and p = 0.011, respectively). Patients with high elastic module showed shorter disease free survival than patients with low elastic module. The elastic module showed strongly positive correlation with azan positive area ratio (r = 0.908) and α-SMA positive area ratio (r = 0.921). Finally, the cDNA microarray data of the tumor with high elastic module revealed distinct gene expression profile from that with low elastic module. Assessment of elasticity of colorectal cancer tissue can be available for more accurate clinical stage or prognosis estimation. Distinct phenotypical feature of the high elastic module tumor and their strong association with stromal feature seemed to be suggesting the existence of biological mechanism involved in this phenomenon, which may contribute to the future therapy. We selected four samples from the highest and the lowest EM with RIN > 6.0 measured with a 2100 Bioanalyzer (Aligent Tochnologies, Santa Clara, CA, USA) and with stage II (pTNM pathologic classification). We excluded one sample in each group because of high GAPDH.
Project description:To gain new insights into molecular changes in skeletal muscle aging and disease with a special focus on differential alternative splicing and senescence, we performed RNA-seq on tibialis muscles from 15 months old (adult, a) and 27 months old (old, o) rats. Whole (w) muscle samples were prepared for both age groups (w/a and w/o). Additionally, for both age groups, muscle fibers were isolated. For adult rats, this resulted in one group (f/a). For old rats, fibers were subjected to Laser capture microdissection guided by expression of aging markers Tmem158 and Cdkn1a, giving rise to two groups, aging marker-positive fibers (f/o+) and aging marker-negative fibers (f/o-). The entire experiment comprises 5 groups (w/a, w/o, f/a, f/o+, f/o-), with 5 animals per group, ie. 25 samples total.
Project description:Elastic fibers are required for expansion and recoil during contraction and relaxation of vessels and heart valves. The aortic valve (AoV) extracellular matrix is generated and maintained by valvular interstitial cells (VICs), a heterogeneous population of cells derived from a mixture of developmental precursors such as endocardium, neural crest and second heart field. Although most studies have focused on VICs that exhibit a fibroblastic phenotype, significant subpopulations present with neuronal, glial, smooth muscle and melanocytic phenotypes. Relatively little is known about which sub population of VICs regulate and produce elastic fibers, though elastin (Eln) abnormalities result in congenital AoV defects and Eln degradation initiates AoV diseases. The present study establishes the timing ofElnexpression in the murine AoV. We performed RT-qPCR and found thatElnpeaks at late embryogenesis (embryonic day 17.5) and early postnatal (P) stages and decreases to low levels in adulthood. Using spatial transcriptomics in postnatal day 3 AoV, we segregated AoV cells from other cardiac cells and demonstrated thatElnexpression correlates with that of genes known to regulate elastogenesis, including the common smooth muscle cell marker - Acta2. By combining RNAscopein situhybridization with immunofluorescence, we confirmed that VICs that expressElnco-express alpha smooth muscle actin. Additionally, some of theEln/alpha smooth muscle actin expressing cells also express melanocytic markers. As previously reported in adult mice, we show a relationship between AoV pigment and elastic fiber patterning during early postnatal stages and further show that melanocytes may play a critical role in elastogenesis. Our results indicate that in the murine AoVElnis produced during early postnatal stages by cells that co-express phenotypic markers of various cell types, including smooth muscle actin and melanocytic specific genes.
Project description:Generally, cancer tissue is palpated as a hard mass. On the other hand, it is not clear the nature of elasticity in cancer tissue. The aim in this study is to evaluate clinical utility of measuring elastic module in colorectal cancer tissue. Using a tactile sensor, we measured the elastic module of 106 surgically resected colorectal cancer tissues. The data of the elastic module were compared with the clinicopathological findings including stromal features represented by azan and α-SMA positive area ratio in tumor area. Finally cDNA microarray profile of the tumor with high elastic module was compared with that with low elastic module Higher elastic module in tumor was associated with pathological T-, N- and M-Stage (p < 0.001, p = 0.001 and p = 0.011, respectively). Patients with high elastic module showed shorter disease free survival than patients with low elastic module. The elastic module showed strongly positive correlation with azan positive area ratio (r = 0.908) and α-SMA positive area ratio (r = 0.921). Finally, the cDNA microarray data of the tumor with high elastic module revealed distinct gene expression profile from that with low elastic module. Assessment of elasticity of colorectal cancer tissue can be available for more accurate clinical stage or prognosis estimation. Distinct phenotypical feature of the high elastic module tumor and their strong association with stromal feature seemed to be suggesting the existence of biological mechanism involved in this phenomenon, which may contribute to the future therapy.
Project description:Skeletal muscle is a key tissue in human aging, which affects different muscle fiber types unequally. We developed a highly sensitive single muscle fiber proteomics workflow to study human aging and show that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations. Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Aging mitochondria decrease expression of the redox enzyme monoamine oxidase A. Slow fibers upregulate a subset of actin and myosin chaperones, whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging. We conclude that single muscle fiber analysis by proteomics can elucidate pathophysiology in a sub-type specific manner.