Chengxin Zhou

Corneal Stromal Cells use both High- and Low-Contractility Migration Mechanisms in 3-D Collagen Matrices

Corneal keratocyte migration can impact both corneal clarity and refractive outcome following injury or refractive surgery. In this study, we investigated how culture conditions, ECM properties, and Rho kinase activity regulate the mechanics of keratocyte migration, using a nested collagen matrix model. Time-lapse imaging demonstrated that both serum and PDGF stimulate keratocyte migration into the outer matrix. Although the velocity of cell migration was similar, cells in serum were bipolar and induced significant matrix deformation during migration, whereas PDGF induced extension of branching dendritic processes with smaller, more localized force generation. These differences in cell-induced matrix reorganization were verified with a global matrix contraction assay and confocal reflection imaging, using both bovine and rat tail collagen. When constructs were detached from the substrate to lower the effective stiffness, migration was significantly reduced in serum; but was unchanged in PDGF. These differences in migration mechanics were mediated, in part, by Rho kinase. Overall, corneal keratocytes can effectively migrate through collagen matrices using varying degrees of cellular force generation. Low-contractility migration may facilitate keratocyte repopulation of the stroma following surgery or injury, without altering the structural and mechanical properties that are critical to maintaining corneal transparency.

Techniques for assessing 3-D cell–matrix mechanical interactions in vitro and in vivo

Cellular interactions with extracellular matrices (ECM) through the application of mechanical forces mediate numerous biological processes including developmental morphogenesis, wound healing and cancer metastasis. They also play a key role in the cellular repopulation and/or remodeling of engineered tissues and organs. While 2-D studies can provide important insights into many aspects of cellular mechanobiology, cells reside within 3-D ECMs in vivo, and matrix structure and dimensionality have been shown to impact cell morphology, protein organization and mechanical behavior. Global measurements of cell-induced compaction of 3-D collagen matrices can provide important insights into the regulation of overall cell contractility by various cytokines and signaling pathways. However, to understand how the mechanics of cell spreading, migration, contraction and matrix remodeling are regulated at the molecular level, these processes must also be studied in individual cells. Here we review the evolution and application of techniques for imaging and assessing local cell-matrix mechanical interactions in 3-D culture models, tissue explants and living animals.

The Role of Titanium Surface Microtopography on Adhesion, Proliferation, Transformation, and Matrix Deposition of Corneal Cells

PURPOSE Titanium (Ti) is an excellent implantable biomaterial that can be further enhanced by surface topography optimization. Despite numerous data from orthopedics and dentistry, the effect of Ti surface topography on ocular cells is still poorly understood. In light of the recent adaptation of Ti in the Boston Keratoprosthesis artificial cornea, we attempted to perform an extended evaluation of the effect of Ti surface topography on corneal cell adhesion, proliferation, cytotoxicity, transformation, and matrix deposition. METHODS Different surface topographies were generated on medical grade Ti-6Al-4V-ELI (extra-low interstitial), with linearly increased roughness (polished to grit blasted). Biological response was evaluated in vitro using human corneal limbal epithelial (HCLE) cells, stromal fibroblasts (HCF), and endothelial cells (HCEnC). RESULTS None of the Ti surface topographies caused cytotoxicity to any of the three corneal cell types. However, rough Ti surface inhibited HCLE and HCF cell adhesion and proliferation, while HCEnC proliferation was unaffected. Long-term experiments with HCF revealed that rough Ti surface with R(a) (the arithmetic average of the profile height from the mean line) ≥ 1.15 μm suppressed HCF focal adhesion kinase phosphorylation, changed fibroblast morphology, and caused less aligned and reduced deposition of collagen matrix as compared to smooth Ti (R(a) ≤ 0.08 μm). In the presence of transforming growth factor β1 (TGFβ1) stimulation, rough Ti inhibited alpha-smooth muscle actin (α-SMA) expression and collagen deposition, leading to decreased myofibroblast transformation and disorganization of the collagen fibrils as compared to smooth Ti. CONCLUSIONS This study suggests that Ti surface topography regulates corneal cell behavior in a tissue-dependent manner that varies across the corneal strata. Contrary to the accepted paradigm, smooth surface topography can enhance cell adhesion and proliferation and increase matrix deposition by corneal cells.

MMP regulation of corneal keratocyte motility and mechanics in 3-D collagen matrices.

Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.

Sustained Subconjunctival Delivery of Infliximab Protects the Cornea and Retina Following Alkali Burn to the Eye

Purpose Tumor necrosis factor (TNF)-α is upregulated in eyes following corneal alkali injury and contributes to corneal and also retinal damage. Prompt TNF-α inhibition by systemic infliximab ameliorates retinal damage and improves corneal wound healing. However, systemic administration of TNF-α inhibitors carries risk of significant complications, whereas topical eye-drop delivery is hindered by poor ocular bioavailability and the need for patient adherence. This study investigates the efficacy of subconjunctival delivery of TNF-α antibodies using a polymer-based drug delivery system (DDS). Methods The drug delivery system was prepared using porous polydimethylsiloxane/polyvinyl alcohol composite fabrication and loaded with 85 μg of infliximab. Six Dutch-belted pigmented rabbits received ocular alkali burn with NaOH. Immediately after the burn, subconjunctival implantation of anti-TNF-α DDS was performed in three rabbits while another three received sham DDS (without antibody). Rabbits were followed with photography for 3 months. Results After 3 months, the device was found to be well tolerated by the host and the eyes exhibited less corneal damage as compared to eyes implanted with a sham DDS without drug. The low dose treatment suppressed CD45 and TNF-α expression in the burned cornea and inhibited retinal ganglion cell apoptosis and optic nerve degeneration, as compared to the sham DDS treated eyes. Immunolocalization revealed drug penetration in the conjunctiva, cornea, iris, and choroid, with residual infliximab in the DDS 3 months after implantation. Conclusions This reduced-risk biologic DDS improves corneal wound healing and provides retinal neuroprotection, and may be applicable not only to alkali burns but also to other inflammatory surgical procedures such as penetrating keratoplasty and keratoprosthesis implantation.

Chemical burns of the eye: The role of retinal injury and new therapeutic possibilities

Purpose: To propose a new treatment paradigm for chemical burns to the eye - in the acute and chronic phases. Methods: Recent laboratory and clinical data on the biology and treatment of chemical burns are analyzed. Results: Corneal blindness from chemical burns can now be successfully treated with a keratoprosthesis, on immediate and intermediate bases. Long term outcomes, however, are hampered by early retinal damage causing glaucoma. New data suggest that rapid diffusion of inflammatory cytokines posteriorly (TNF-&agr;, etc) can severely damage the ganglion cells. Prompt anti-TNF-&agr; treatment is markedly neuroprotective. Long term profound reduction of the intraocular pressure is also vital. Conclusion: A new regimen, in addition to standard treatment, for severe chemical burns is proposed. This involves tumor necrosis factor alpha (TNF-&agr;) inhibition promptly after the accident (primarily for retinal neuroprotection), prophylactic maximal lowering of the intraocular pressure (starting immediately), and keratoprosthesis implantation in a later quiet state.

Neuroglial Remodeling After Various Ocular Injuries is Regulated by Microglia

Reactive microglia and infiltrating peripheral monocytes have been implicated in many neurodegenerative diseases of the retina and central nervous system (CNS). However, their specific contribution in retinal degeneration remains unclear. We recently showed that peripheral monocytes that infiltrate the retina after ocular injury in mice become permanently engrafted into the tissue, establishing a pro-inflammatory phenotype that promotes neurodegeneration. Here, we show in mice that microglia regulate the process of neuroglia remodeling during ocular injury, and their depletion results in marked upregulation of inflammatory markers, such as Il17f, Tnfsf11, Ccl4, Il1a, Ccr2, Il4, Il5, and Csf2 in the retina, abnormal engraftment of peripheral CCR2+ CX3CR1+ monocytes into the retina and is associated with increased retinal ganglion cell (RGC) loss, retinal nerve fiber layer thinning, and RPE65+ cell migration onto the retinal surface. Furthermore, we show that other types of ocular injuries, such as penetrating corneal trauma and ocular hypertension, also cause similar changes. However, optic nerve crush injury mediated RGC loss evokes neither peripheral monocyte response in the retina, nor RPE65+ cell migration, although peripheral CX3CR1+ and CCR2+ monocytes infiltrate the optic nerve injury site and remain present for months. Our study suggests that microglia are key regulators of peripheral monocyte infiltration and RPE migration and their depletion results in abnormal neuroglia remodeling that exacerbates neuroretinal tissue damage. This mechanism of retinal damage through neuroglia remodeling may be clinically important for the treatment of patients with ocular injuries, including surgical traumas.Reactive microglia and infiltrating peripheral monocytes have been implicated in many neurodegenerative diseases of the retina and central nervous system (CNS). However, their specific contribution in retinal degeneration remains unclear. We recently showed that peripheral monocytes that infiltrate the retina after ocular injury become permanently engrafted into the tissue, establishing a pro-inflammatory phenotype that promotes neurodegeneration. Here, we show that microglia regulate the process of neuroglia remodeling during ocular injury, and their depletion results in abnormal engraftment of peripheral CCR2+ CX3CR1+ monocytes into the retina and is associated with increased retinal ganglion cell (RGC) loss, retinal nerve fiber layer thinning, and RPE65+ cell migration onto the retinal surface. Furthermore, we show that other types of ocular injuries, such as penetrating corneal trauma and ocular hypertension, also cause similar changes. However, optic nerve crush injury mediated RGC loss evokes neither peripheral monocyte response in the retina, nor RPE65+ cell migration, although peripheral CX3CR1+ and CCR2+ monocytes infiltrate the optic nerve injury site and remain present for months. Our study suggests that microglia are key regulators of peripheral monocyte infiltration and RPE migration and their depletion results in abnormal neuroglia remodeling that exacerbates neuroretinal tissue damage. This mechanism of retinal damage though neuroglia remodeling may be clinically important for the treatment of patients with ocular injuries, including surgical traumas. Significance statement Microglial cells are key regulator for peripheral monocytes engraftment into the retina following injury to the eye.

Permanent neuroglial remodeling of the retina following infiltration of CSF1R-inhibition resistant peripheral monocytes.

Previous studies have demonstrated that ocular injury can lead to prompt infiltration of bone marrow-derived peripheral monocytes into the retina. However, the ability of these cells to integrate into the tissue and become microglia has not been studied. Here we show that such peripheral monocytes not only infiltrate into the retina after ocular injury, but that they engraft permanently, migrate to the three distinct microglia strata, and adopt a microglia-like morphology. However, contrary to the original microglia, after injury the engrafted peripheral monocytes are resistant to depletion by colony stimulating factor 1 receptor (CSF1R) inhibitor and remain pro-inflammatory, expressing high levels of major histocompatibility complex II (MHC-II) for the long-term. In the absence of ocular injury, on the other hand, the peripheral monocytes that repopulate the retina after CSF1R inhibition remain sensitive to CSF1R inhibition and can be re-depleted. The observed permanent neuroglia remodeling after injury constitutes a major potential immunological change that may contribute to progressive retinal degeneration. These findings may be relevant also to other degenerative conditions of the retina and central nervous system. Significance statement: Ocular injury causes permanent neuroglia remodeling that promotes neuroinflammation.

The Role of Microglia and Peripheral Monocytes in Retinal Damage after Corneal Chemical Injury

Eyes that have experienced alkali burn to the surface are excessively susceptible to subsequent severe glaucoma and retinal ganglion cell loss, despite maximal efforts to prevent or slow down the disease. Recently, we have shown, in mice and rabbits, that such retinal damage is neither mediated by the alkali itself reaching the retina nor by intraocular pressure elevation. Rather, it is caused by the up-regulation of tumor necrosis factor-α (TNF-α), which rapidly diffuses posteriorly, causing retinal ganglion cell apoptosis and CD45+ cell activation. Herein, we investigated the involvement of peripheral blood monocytes and microglia in retinal damage. Using CX3CR1+/EGFP::CCR2+/RFP reporter mice and bone marrow chimeras, we show that peripheral CX3CR1+CD45hiCD11b+MHC-II+ monocytes infiltrate into the retina from the optic nerve at 24 hours after the burn and release further TNF-α. A secondary source of peripheral monocyte response originates from a rare population of patrolling myeloid CCR2+ cells of the retina that differentiate into CX3CR1+ macrophages within hours after the injury. As a result, CX3CR1+CD45loCD11b+ microglia become reactive at 7 days, causing further TNF-α release. Prompt TNF-α inhibition after corneal burn suppresses monocyte infiltration and microglia activation, and protects the retina. This study may prove relevant to other injuries of the central nervous system.