Browsing by Subject "Matrix Metalloproteinases"
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Item The Functional Roles of Rho-Kinase and Matrix Metalloproteinases in Regulating Corneal Stromal Cell Mechanics in 3-D Collagen Matrices(2013-11-26) Zhou, Chengxin; Grinnell, Frederick; Petroll, W. Matthew; Luby-Phelps, Katherine; Tang, Liping; Alexandrakis, GeorgiosThe main focus of my research has been on understanding the biomechanical and biochemical mechanisms of cell-extracellular matrix (ECM) interactions during corneal wound healing, which may allow the development of new therapeutic strategies to promote corneal regeneration. Previous studies have established that the Rho GTPases play a central role in regulating the cytoskeletal changes associated with cell mechanical activity. A novel force monitoring system was successfully developed to investigate the role of Rho in corneal cell force generation in 3-D collagen matrices. Maximum tractional force generated by 9 million corneal fibroblasts in serum culture was around 265 Dynes. Inhibition of Rho kinase by Y-27632 induced a 69% force reduction. These results demonstrated that Rho/Rho kinase play a key role in mediating contractile force generation of corneal stromal fibroblasts in serum culture. I also investigated the functions of Rho GTPase signaling in corneal stromal fibroblast migration and cell-ECM interactions using a 3-D nested matrix construct. The experimental results showed that both the amount and the speed of corneal fibroblast migration and local collagen matrix reorganization were significantly inhibited by Y-27632. Following the inhibition, cells extended thinner dendritic processes into the outer matrix, and generated tractional forces at their leading edge. However, cells were unable to generate contractile forces needed to retract their tail and pull the cell body forward through the collagen matrix. I also studied the role of Matrix metalloproteinases (MMPs) in corneal cell mechanics, since these have been recognized as an influential component in extracellular matrix turnover and corneal repair. I first assessed the expression and collagenolytic activities of MMPs by primary corneal keratocyte in response to different signaling factors. I then studied the functions of MMPs in regulating keratocyte migration, cell-induced matrix contraction, and cell protrusive activity in 3-D collagen matrices. This study suggested that, in serum free PDGF culture, although collagenolysis was limited to a pericellular scale, primary corneal keratocytes utilized MMPs to facilitate cell migration, ECM contraction, cell spreading in 3-D collagen matrices. Thus MMPs may play a key role in facilitating cell-collagen matrix interactions by corneal keratocytes, without producing widespread disruption of corneal ECM structure.Item Gene Expression Changes in Response to Severe Burn Injury in Rat Achilles Tendon(2017-01-17) Buller, Dustin; Mitchell, Wes; Manchanda, Kshitij; Song, Juquan; Hernandez, PaulaIMPORTANCE: Severe burn injury, occurring at a global rate of 5/100,000 per year, can result in serious sequelae such as infection, shock, and direct organ damage. It has been shown that severe burn also results in both systemic inflammation and distant inflammatory effects, specifically in skeletal muscle and bone. However, we are unaware of any work that investigates similar effects in the tendon, which connects muscle and bone. OBJECTIVE: To determine whether, after distant severe burn injury, changes occur in gene expression, protein synthesis, and biomechanical properties in rat Achilles tendon. METHODS: Rats were subjected to thoracolumbar full-thickness severe burn injury under anesthesia before harvesting the Achilles tendon at time points of 6h, 1d, 3d, 7d, and 14d. Unburned rats were used for control. RNA expression of collagen I (Col1), collagen III (Col3), MMP9, MMP13, IL-1β, IL-6, TNF-α, and tenomodulin was measured using qPCR. Quantified Western blots were performed to assess relative protein content for Col1, Col3, and MMP9. Finally, biomechanical testing was performed on a separate group of rats to assess biomechanical changes at 14d versus control (n = 8). RESULTS: The MMP9 gene was upregulated on the order of 20-fold at 14d (p = 0.0016) and 10-fold at 3d (p = 0.0255) and MMP13 showed a trend toward 12-fold increase at 3d (p = 0.079). A significant increase in expression of IL-1β and a trend toward significance IL-6 were also observed at 3d (p < 0.01, and p = 0.059, respectively). Differences in expression of Col1, Col3, TNF-α, and tenomodulin were not significant compared to control. Preliminary data from Western blots showed 5-fold decrease of collagen 1 at 7d (p < 0.01) and 2-fold increase of collagen 3 at 14d (p = 0.016). Significant changes were not found in MMP9. Preliminary biomechanical data shows a trend toward a 4-fold decrease in stiffness in the burn group. All p values are by t-test. CONCLUSIONS: These results newly confirm the existence of inflammation in tendon distant from the burn site after severe burn injury. Specifically, these changes could indicate initial matrix remodeling as carried out by the MMPs beginning at 3d, followed by collagen deposition with a decreased Col1:Col3 ratio, resulting in decreased tissue stiffness. These findings are consistent with the repair processes known to occur in other tissues after inflammation. Additionally, IL-6 and IL-1β may have a more significant role in post-burn acute-phase inflammation than other acute-phase reactants like TNF-α in the Achilles tendon.Item Matrix metalloproteinases: from biology to therapeutic strategies in cardiovascular disease(2000-02-10) Benjamin, Ivor J.