Browsing by Subject "Extracellular Matrix"

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Adhesion molecules-potential targets of new anti-inflammatory therapies
(1993-11-04) Lipsky, Peter E.
Corneal Stromal Remodeling after Photorefractive Keratectomy
(2017-01-17) Su, Shan; Kivanany, Pouriska; Grose, Kyle; Petroll, W. Matthew
The cornea is an optically clear tissue that contributes 2/3 of the eye's refractive power, making it a target for vision correction procedures. A small percentage of patients who receive corneal surgery experience loss of corneal transparency (haze). Haze occurs when stromal cells in the cornea (keratocytes) become activated and transform into fibroblasts or myofibroblasts, which can generate contractile forces that may disrupt the collagen architecture. We investigated the wound healing process following photorefractive keratectomy (PRK, a clinical vision correction procedure) using 3-D imaging both in vivo and in situ. We hypothesized that following PRK in rabbits, keratocytes located within the injured stroma, where collagen is intact, would align with the collagen lamellae and will not produce fibrosis. In contrast, we expected keratocytes anterior to the wound, where collagen is disrupted, to undergo myofibroblast transformation and produce significant haze. Twelve New Zealand white rabbits were scanned one week before surgery using an in vivo confocal microscope. PRK was performed on the right eyes of the rabbits (9 diopter spherical photocorrection) with subsequent scans at 7 days, 21 days, 3 months, and 6 months. Custom software was used to build 3-D reconstructions and measure stromal thickness and haze. Some rabbits were sacrificed at each time point to obtain in situ confocal images. Thickness measurements at pre-scan, 7 days, 21 days, 3 months, and 6 months were: 335, 208, 265, 293, 313um. The decrease and subsequent increase in thickness is consistent with removal of tissue for PRK, followed by tissue regeneration. Haze measurements at the same time points were: 1797, 4453, 6906, 3212, 3433 intensity units. The increase and subsequent decrease in backscatter suggests two phases of wound healing. In situ confocal imaging showed that cells within the native stroma were aligned with collagen lamellae without prominent stress fibers at 7 and 21 days, while cells anterior to the injured stroma at 21 days aligned randomly and displayed prominent stress fibers. At 3 months, these cells aligned with correlation to the collagen and did not express stress fibers. At 6 months, the cell/collagen arrangement was similar to uninjured tissue. Our results suggest that the collagen lamellae direct fibroblast patterning during repopulation of the native stroma, without inducing fibrosis or significant haze. In contrast, cells accumulating on top of the stroma initially align randomly and produce hazy, fibrotic tissue. Remarkably, over time, cells remodel the fibrotic tissue to produce a lamellar structure that is similar to the native corneal stroma.
Decellularized Normal and Cancer Susceptible Mice Colons to Study the Contribution of the Extracellular Matrix to Cell Behavior and Colon Cancer Progression
(2020-05-01T05:00:00.000Z) Alabi, Busola Ruth; Fiolka, Reto; Shay, Jerry W.; Conacci-Sorrell, Maralice; Eberhart, Robert; Welch, Tre
Current 3D culture models to study colorectal cancer lack architectural support and signaling proteins provided by the tissue extracellular matrix (ECM) which may influence cell behavior and cancer progression. Therefore, the ability to study cancer cells in the context of a matrix that is physiologically more relevant and to understand how the ECM affects cancer progression has been understudied. To address this, I developed an ex-vivo 3D system, provided by intact wild type (WT) and colon cancer susceptible decellularized mouse colons (DMC), to support the growth of human cancer cells. DMC are free of viable cells but still contain extracellular matrix proteins including subsets of collagens. Stiffness, an important mechanical property, is also maintained in DMCs. Importantly, I observed that the DMC is permissive for cell proliferation and differentiation of a human colon cancer cell line (HT-29). Notably, the ability of cells in the WT DMC to differentiate was also greater when compared to Matrigel TM, an extracellular matrix extract from a mouse tumor cell line. Additionally, I observed using invasion assays that DMC obtained from polyps from a colon cancer susceptible mouse model facilitated increased cell migration/invasion of colorectal cancer cells and immortalized non-tumor colonic epithelial cells compared to DMC from WT mice. Finally, using mass spectrometry, I identified extracellular matrix proteins that are more abundant in DMC from a colorectal cancer mouse model compared to age and sex-matched WT mice. I propose that these abundantly expressed proteins in the tumor microenvironment are potentially involved in colorectal cancer progression.
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, Georgios
The 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.
Multi-Dimensional Imaging of Cell and Extracellular Matrix Interactions During Corneal Wound Healing
(2018-04-04) Kivanany, Pouriska Bigvand; Xu, Chet; Petroll, W. Matthew; Eberhart, Robert C.; Mootha, Venkateswara; Schmidtke, David
Corneal haze is a leading cause of blindness, and may occur after injury, surgery, or chemical burns. The cornea's thickest layer, the stroma, accounts for two-thirds of the eye's refractive power, and contains highly aligned collagen fibrils needed for transparency. Stromal cells, or keratocytes, help maintain the extracellular matrix (ECM). After injury or surgery, keratocytes within the wound undergo apoptosis, while keratocytes in the wound margin transform and activate into fibroblasts and myofibroblasts, leading to contraction, deposition of fibrotic ECM, and haze. Thus, spatial and temporal investigations of cell/ECM interactions are needed for further insight into this healing process. In this research, freeze-injury (FI), lamellar keratectomy (LK), and photorefractive keratectomy (PRK) in vivo rabbit models are used, and cell/ECM interactions are tracked using an in vivo confocal microscope as well as second harmonic generation (SHG) and multiphoton imaging. These in vivo models revealed that in the short term, intrastromal cells migrated in parallel to the collagen lamellae, while cells over the stromal wound bed transformed to myofibroblasts and were randomly arranged. Stromal haze initially increased after injury, but gradually decreased over time. Long term, collagen became more organized, more quiescent cells replaced myofibroblasts, and normal stromal thickness regenerated (after keratectomy) below the ablation surface. Punctate F-actin labeling was detected along cells that co-aligned with native and regenerated collagen, suggesting critical involvement of cell/ECM interactions in stromal remodeling and regeneration. Collagen lamellae appeared to provide a template for fibroblast patterning during both intrastromal migration and stromal regrowth. Overall, cells appear to regenerate and remodel the ECM to produce a lamellar structure similar to the native stroma. These results provide novel insights into the mechanisms of cell/ECM interactions involved in various stages of wound healing in the cornea, and may lead to potential therapies that prevent or reverse fibrosis in the clinic.