Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix

Rationale: Although an age-related decrease in NO bioavailability contributes to vascular stiffness, the underlying molecular mechanisms remain incompletely understood. We hypothesize that NO constrains the activity of the matrix crosslinking enzyme tissue transglutaminase (TG2) via S-nitrosylation in young vessels, a process that is reversed in aging. Objective: We sought to determine whether endothelium-dependent NO regulates TG2 activity by S-nitrosylation and whether this contributes to age-related vascular stiffness. Methods and Results: We first demonstrate that NO suppresses activity and increases S-nitrosylation of TG2 in cellular models. Next, we show that nitric oxide synthase (NOS) inhibition leads to increased surface and extracellular matrix-associated TG2. We then demonstrate that endothelium-derived bioactive NO primarily mediates its effects through TG2, using TG2 ؊/؊ mice chronically treated with the NOS inhibitor L-N Gnitroarginine methyl ester (L-NAME). We confirm that TG2 activity is modulated by endothelium-derived bioactive NO in young rat aorta. In aging rat aorta, although TG2 expression remains unaltered, its activity increases and S-nitrosylation decreases. Furthermore, TG2 inhibition decreases vascular stiffness in aging rats. Finally, TG2 activity and matrix crosslinks are augmented with age in human aorta, whereas abundance remains unchanged. Conclusions: Decreased S-nitrosylation of TG2 and increased TG activity lead to enhanced matrix crosslinking and contribute to vascular stiffening in aging. TG2 appears to be the member of the transglutaminase family primarily contributing to this phenotype. Inhibition of TG2 could thus represent a therapeutic target for age-associated vascular stiffness and isolated systolic hypertension. (Circ Res. 2010;107:117-125.)Key Words: tissue transglutaminase Ⅲ S-nitrosylation Ⅲ S-nitrosation Ⅲ aging Ⅲ vascular stiffness A ging is associated with alterations in the properties of all elements of the vascular wall including endothelium, vascular smooth muscle, and matrix. 1 These changes result in increased vascular stiffness and isolated systolic hypertension. In addition, increased vascular stiffness promotes atherosclerosis at various sites in the vascular tree, such as the carotid artery. 2,3 Both dynamic changes (alterations in endothelial function and effects on vascular smooth muscle contractility), as well as structural alterations (eg, fracturing of elastin, increased collagen content, and accumulation of advanced glycation end products) have been described in aging. Vessel structure can additionally be regulated by alterations in matrix crosslinking. 1 Transglutaminases (TGs) are enzymes that catalyze a transamidation reaction, leading to the crosslinking of proteins through the formation of the stable N--(␥-glutamyl)lysine isopeptide bonds. 4,5 At least 3 of the 9 members of the TG superfamily are expressed in vascular systems. Tissue transglutaminase (TG2) in particular is ubiquitously expressed in vasculature, including in endothelial ce...

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“…ECM fraction was recovered by removing cells and nuclear material following the protocol of Soucy and Romer. 24 …”

Section: Isolation Of Cell Surface Proteins and Ecmmentioning

confidence: 99%

Decreased S -Nitrosylation of Tissue Transglutaminase Contributes to Age-Related Increases in Vascular Stiffness

Santhanam

Tuday

Webb

et al. 2010

Circulation Research

129

132

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“…Soucy and Romer discovered that capillary tube networks were formed in 24 h after seeding HUVECs at a density of 2.1 · 10 5 cells/cm 2 on FDM. 10 The present study employs a much smaller cell density (1.25 · 10 4 cells/cm 2 ) and different culture medium conditions during 5-day culture. Another report prepared an ECM by coculturing different stages of breast cancer cells and primary fibroblasts, and demonstrated that tumor ECM can sequester cell-produced tumor necrosis factor-a, which in turn activates ECs and thus significantly increase CLS lumen diameters.…”

Section: Fig 5 Cell Migration Assay On Cdms and Gelatin-coated Covementioning

confidence: 99%

“…Several studies have documented that MT1-MMP and MMP-1 are actively involved in neovascularization, wherein they cause ECM degradation by migrating ECs both in vivo and in vitro. 10,28,29 Researchers report that EC network formation is MT1-MMP dependent, which creates enough space for EC migration by degrading the surrounding matrix. 30 It is notable that the lack of CLS formation on gelatin and CHDM correlates well with the little expression of MT1-MMP on gelatin and weak expression on CHDM.…”

Section: Fig 5 Cell Migration Assay On Cdms and Gelatin-coated Covementioning

confidence: 99%

Vascular Morphogenesis of Human Umbilical Vein Endothelial Cells on Cell-Derived Macromolecular Matrix Microenvironment

Subbiah

Park

et al. 2014

Tissue Engineering Part A

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Extracellular matrix (ECM) is a highly organized network of proteins and other macromolecules that plays a critical role in cell adhesion, migration, and differentiation. In this study, we hypothesize that ECM derived from in-vitro-cultured cells possesses unique surface texture, topography, and mechanical property, and consequently carries some distinct cues for vascular morphogenesis of human umbilical vein endothelial cells (ECs). Cell-derived matrix (CDM) was obtained by culturing fibroblasts, preosteoblasts, and chondrocytes, respectively, on coverslips and then by decellularizing them using detergents and enzymes. These matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). Immunofluorescence of each CDM shows that some of the matrix components are fibronectin (FN), type I collagen, and laminin. Atomic force microscopy analysis presented that average fiber diameter ranged from 2 to 7 mm and FDM holds much larger fibers. The matrix elasticity measurements revealed that average Young's modulus of CHDM (17.7 -4.2 kPa) was much greater than that of PDM (10.5 -1.1 kPa) or FDM (5.7 -0.5 kPa). During 5-day culture, EC morphologies were dramatically changed on PDM and FDM, but those on CHDM and gelatin were rather stable, regardless of time lapse. Cell migration assay discovered quicker repopulation of the scratched areas on PDM and FDM than on gelatin and CHDM. A capillary-like structure (CLS) assembly was also notable only in the PDM and FDM, as compared with CHDM, gelatin, or FN that were very poor in CLS formation. Quantitative analysis of mean CLS branch points and branch lengths demonstrated much better angiogenic activity of ECs on PDM and FDM. Interestingly, CLS formation was closely associated with matrix remodeling by ECs and the matrix clearance on PDM with time was sharply contrasted with that on CHDM that majority of the matrix FN was reserved. It was notable that membrane type 1-matrix metalloprotease was deeply involved in the process of matrix remodeling. This study indicates that specific matrix microenvironments are very critical for vascular morphogenesis of ECs, and thus, provide a nice platform for angiogenesis study as well as vascular tissue engineering.

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“…For human fetal lung fibroblasts, versican, tenascin-C, decorin, FN, Col I, IV, and VI were the components in the CDM. 19 Analysis of the marrow stromal cell-derived ECM found that FN, LN, Col I, III, decorin, and biglycan were the major components in the CDM. 20 The advantages of the CDM appear to be due to the role of the matrix as a physical cue in stimulating chondrocyte redifferentiation.…”

Section: Figmentioning

confidence: 99%

Induction of Re-Differentiation of Passaged Rat Chondrocytes Using a Naturally Obtained Extracellular Matrix Microenvironment

Hwa

Hee

Park

et al. 2013

Tissue Engineering Part A

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Dedifferentiated human chondrocytes severely limit successful hyaline cartilage repair in clinical practice. The primary interest of this study is to evaluate the naturally obtained cell-derived matrix (CDM) as a physical microenvironment for chondrocyte re-differentiation. Once different cell types were cultured for 6 days and decellularized using detergents and enzymes, the fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM) were obtained. From scanning electron microscope observation, each CDM was found to resemble a fibrous mesh with self-assembled fibrils. Both the FDM and PDM showed a more compact matrix structure compared to the CHDM. For compositional analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis displayed numerous matrix proteins, which were quite different from each CDM in quantity and type. Specific matrix components, such as fibronectin, type I collagen (Col I), and laminin, were detected using immunofluorescent staining. In addition, the water contact angle suggests that the FDM is more hydrophilic than the PDM or CHDM. The proliferation of rat primary chondrocytes growing on CDMs was better than those growing on a plastic coverslip (control) or gelatin. Meanwhile, synthesis of glycosaminoglycan (GAG) was more effective for passaged chondrocytes (P4) cultivated on CDMs, and the difference was significant compared to cells grown on the control or on gelatin. As for the gene expression of cartilage-specific markers, CDMs exhibited good chondrocyte re-differentiation with time: the dedifferentiating marker, Col I was restrained, whereas the ratio between Col II and Col I, and between aggrecan and Col I, as an indicator of re-differentiation, was greatly improved. In addition, immunofluorescence of Col II showed a very positive signal in chondrocytes cultivated for 2 weeks on the CDMs. In an additional study, when threedimensional cell pellets made from either plate-grown or matrix-grown dedifferentiated chondrocytes (P5) were cultured for 4 weeks, the results of Safranin-O staining, immunohistochemistry of Col II, and total GAG assay suggested that matrix-grown cells were significantly better in the induction of chondrocyte re-differentiation, than those grown on the plate. This work suggests that the naturally occurring matrix, CDM, can provide a favorable surface texture for cell attachment, proliferation, and more importantly, a chondroinductive microenvironment for the re-differentiation of dedifferentiated chondrocytes.

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Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix

Cited by 85 publications

References 70 publications

Decreased S -Nitrosylation of Tissue Transglutaminase Contributes to Age-Related Increases in Vascular Stiffness

Decreased S -Nitrosylation of Tissue Transglutaminase Contributes to Age-Related Increases in Vascular Stiffness

Vascular Morphogenesis of Human Umbilical Vein Endothelial Cells on Cell-Derived Macromolecular Matrix Microenvironment

Induction of Re-Differentiation of Passaged Rat Chondrocytes Using a Naturally Obtained Extracellular Matrix Microenvironment

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