Gene expression in TNF treated rat aortic rings cultured in collagen or fibrin gels.
ABSTRACT: Angiogenesis in cultures of rat aorta begins with neovessels sprouting from the aortic explant within the first three days of culture. We used microarrys to examine the effects of TNF-alpha on gene expression in both fibrin and collagen gels during the first 48 hours or culture. Rat aortic rings were cultured in either collagen or fibrin maticies. Half of the cultures from each matrix group were treated with 10ng/ml recombinant rat TNF-alpha, and half were left untreated. These cultures were used to prepare total RNA
Project description:Angiogenesis in cultures of rat aorta begins with neovessels sprouting from the aortic explant within the first three days of culture. We used microarrys to examine the effects of TNF-alpha on gene expression in both fibrin and collagen gels during the first 48 hours or culture. Overall design: Rat aortic rings were cultured in either collagen or fibrin maticies. Half of the cultures from each matrix group were treated with 10ng/ml recombinant rat TNF-alpha, and half were left untreated. These cultures were used to prepare total RNA
Project description:Angiogenesis in collagen gel cultures of rat aorta begins with neovessels sprouting from the aortic explant within the first three days of culture. We used microarrays to detail the pattern of gene expression underlying initial 24 hours of growth, prior to the sprouting of visible neovessles, and identified distinct classes of up-regulated genes during this process. Either freshly harvested aortic rings, representing day 0, or collagen gel cultures of rat aorta were grown in serum free medium and used to prepare total RNA.
Project description:The goal of this study was to define the role of tumor necrosis factor-? (TNF?) in the cascade of gene activation that regulates aortic angiogenesis in response to injury.Angiogenesis was studied by culturing rat or mouse aortic rings in collagen gels. Gene expression was evaluated by quantitative reverse transcription-polymerase chain reaction, microarray analysis, immunocytochemistry, and ELISA. TNF? gene disruption and recombinant TNF? or blocking antibodies against vascular endothelial growth factor (VEGF) or TNF receptors were used to investigate TNF?-mediated angiogenic mechanisms. Resident aortic macrophages were depleted with liposomal clodronate. Angiogenesis was preceded by overexpression of TNF? and TNF?-inducible genes. Studies with isolated cells showed that macrophages were the main source of TNF?. Angiogenesis, VEGF production, and macrophage outgrowth were impaired by TNF? gene disruption and promoted by exogenous TNF?. Antibody-mediated inhibition of TNF receptor 1 significantly inhibited angiogenesis. The proangiogenic effect of TNF? was suppressed by blocking VEGF or by ablating aortic macrophages. Exogenous TNF?, however, maintained a limited proangiogenic capacity in the absence of macrophages and macrophage-mediated VEGF production.Overexpression of TNF? is required for optimal VEGF production and angiogenesis in response to injury. This TNF?/VEGF-mediated angiogenic pathway requires macrophages. The residual capacity of TNF? to stimulate angiogenesis in macrophage-depleted aortic cultures implies the existence of a VEGF-independent alternate pathway of TNF?-induced angiogenesis.
Project description:Angiogenesis in collagen gel cultures of rat aorta begins with neovessels sprouting from the aortic explant within the first three days of culture. We used microarrays to detail the pattern of gene expression underlying initial 24 hours of growth, prior to the sprouting of visible neovessles, and identified distinct classes of up-regulated genes during this process. Overall design: Either freshly harvested aortic rings, representing day 0, or collagen gel cultures of rat aorta were grown in serum free medium and used to prepare total RNA.
Project description:Engineered three-dimensional biomaterials are known to affect the regenerative capacity of stem cells. The extent to which these materials can modify cellular activities is still poorly understood, particularly for adipose-derived stem cells (ASCs). This study evaluates PEGylated fibrin (P-fibrin) gels as an ASC-carrying scaffold for encouraging local angiogenesis by comparing with two commonly used hydrogels (i.e., collagen and fibrin) in the tissue-engineering field. Human ASCs in P-fibrin were compared to cultures in collagen and fibrin under basic growth media without any additional soluble factors. ASCs proliferated similarly in all gel scaffolds but showed significantly elongated morphologies in the P-fibrin gels relative to other gels. P-fibrin elicited higher von Willebrand factor expression in ASCs than either collagen or fibrin while cells in collagen expressed more smooth muscle alpha actin than in other gels. VEGF was secreted more at 7 days in fibrin and P-fibrin than in collagen and several other angiogenic and immunomodulatory cytokines were similarly enhanced. Fibrin-based matrices appear to activate angiogenic signaling in ASCs while P-fibrin matrices are uniquely able to also drive a vessel-like ASC phenotype. Collectively, these results suggest that P-fibrin promotes the angiogenic potential of ASC-based therapeutic applications.
Project description:Completely biological tissue replacements can be fabricated by entrapping cells in a molded fibrin gel. Over time, the fibrin is degraded and replaced with cell-produced extracellular matrix. However, the relationship between fibrin degradation and matrix deposition has not been elucidated. We developed techniques to quantify fibrin degradation products (FDP) and examine plasmin activity in the conditioned medium from fibrin-based constructs. Fibrin-based tissue constructs fabricated with vascular smooth muscle cells (vSMC) were cultured for 5 weeks in the presence of varied concentrations of the fibrinolysis inhibitor -aminocaproic acid and cellularity, and deposited collagen and elastin were measured weekly. These data revealed that increasing concentrations of -aminocaproic acid led to delayed and diminished FDP production, lower vSMC proliferation, and decreased collagen and elastin deposition. FDP were shown to have a direct biological effect on vSMC cultures and vSMC within the fibrin-based constructs. Supplementing construct cultures with 250 or 500?g/mL FDP led to 30% higher collagen deposition than the untreated controls. FDP concentrations as high as 250?g/mL were estimated to exist within the constructs, indicating that FDP generation during remodeling of the fibrin-based constructs exerted direct biological activity. These results help explain many of the positive outcomes reported with fibrin-based tissue constructs in the literature, as well as demonstrate the importance of regulating plasmin activity during their fabrication.
Project description:Collagen- and fibrin-based gels are extensively used to study cell behaviour. However, 2D-3D, collagen-fibrin, and in vivo-in vitro comparisons of gene expression, cell shape and mechanotransduction have not been reported. Here we compared chick tendon fibroblasts (CTFs) at three stages of embryonic development with CTFs cultured in collagen- or fibrin-based tissue engineered constructs (TECs).
Project description:Abdominal aortic aneurysms (AAAs) involve chronic overexpression of proteases in the aortic wall that result in disruption of elastic fibers and consequent loss of vessel elasticity. Nearly 75% of AAAs contain flow-obstructing, fibrin-rich intraluminal thrombi (ILT), which act as a) a bioinert shield, protecting the underlying AAA wall from high hemodynamic stresses, and b) a reservoir of inflammatory cells and proteases that cause matrix breakdown. For these reasons, restoring flow through the aorta lumen and facilitating transmural diffusion of therapeutics from circulation to the AAA wall must be achieved by slow thrombolysis of the ILT to render it porous without rapid breakdown. Intravenously dosed tissue plasminogen activator (tPA) has been shown to rapidly lyse ILTs in acute stroke and myocardial infarctions. For future use in opening up AAA segments, in this study, we investigated the ability of tPA released from poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) to slowly lyse fibrin clots without inducing proteolytic injury and matrix synthesis-inhibitory effects on cultured rat aneurysmal smooth muscle cells (EaRASMCs). Fibrin clot lysis time was greatly extended over that in presence of exogenous tPA. Surface functionalization of NPs with a cationic amphiphile allowed them to bind to anionic fibrin clot, release tPA at a slower rate and to lyse the clot as a front proceeding outwards in unlike the more rapid and homogenous lysis that occurred due to anionic PLGA NPs. Elastic matrix content was decreased in EaRASMC cultures exposed to byproducts of clot lysis with exogenous tPA, but not tPA-NPs, and was likely due to increased proteolytic activity (MMPs, plasmin) in EaRASMC cultures exposed to exogenous tPA-lysed clots. Our results suggest that gradual ILT lysis via slow release of tPA from NPs will be likely beneficial over exogenous tPA delivery in preserving elastic matrix content and attenuating matrilysis in the adjoining AAA wall, in vivo, while rendering the ILT porous to facilitate transmural delivery of endoluminally delivered AAA therapeutics.
Project description:Fibrin deposition within joints is a prominent feature of arthritis, but the precise contribution of fibrin(ogen) to inflammatory events that cause debilitating joint damage remains unknown. To determine the importance of fibrin(ogen) in arthritis, gene-targeted mice either deficient in fibrinogen (Fib-) or expressing mutant forms of fibrinogen, lacking the leukocyte receptor integrin alphaMbeta2 binding motif (Fibgamma390-396A) or the alphaIIbbeta3 platelet integrin-binding motif (FibgammaDelta5), were challenged with collagen-induced arthritis (CIA). Fib- mice exhibited fewer affected joints and reduced disease severity relative to controls. Similarly, diminished arthritis was observed in Fibgamma390-396A mice, which retain full clotting function. In contrast, arthritis in FibgammaDelta5 mice was indistinguishable from that of controls. Fibrin(ogen) was not essential for leukocyte trafficking to joints, but appeared to be involved in leukocyte activation events. Fib- and Fibgamma390-396A mice with CIA displayed reduced local expression of TNF-alpha, IL-1beta, and IL-6, which suggests that alphaMbeta2-mediated leukocyte engagement of fibrin is mechanistically upstream of the production of proinflammatory mediators. Supporting this hypothesis, arthritic disease driven by exuberant TNF-alpha expression was not impeded by fibrinogen deficiency. Thus, fibrin(ogen) is an important, but context-dependent, determinant of arthritis, and one mechanism linking fibrin(ogen) to joint disease is coupled to alphaMbeta2-mediated inflammatory processes.