Human keratocytes, cultured in a stable vitamin C derivative, are capable of assembling extracellular matrix, which comprises parallel arrays of ECM fibrils. The resultant constructs, which are highly cellular, are morphologically similar to the developing mammalian stroma, where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggests a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes that govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue engineering a biomimetic stroma.

Cellular injury induces a complex series of events that involves Ca2+ signaling, cell communication, and migration. One of the first responses following mechanical injury is the propagation of a Ca2+ wave (Klepeis et al. [2001] J Cell Sci 114(Pt 23):4185-4195). The wave is generated by the extracellular release of ATP, which also induces phosphorylation of ERK (Yang et al. [2004] J Cell Biochem 91(5):938-950). ATP and other nucleotides, which bind to and activate specific purinergic receptors were used to mimic injury. Our goal was to determine which of the P2Y purinergic receptors are expressed and stimulated in corneal epithelial cells and which signaling pathways are activated leading to changes in cell migration, an event critical for wound closure. In this study, we demonstrated that the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors were present in corneal epithelial cells. A potency profile was determined by Ca2+ imaging for nucleotide agonists as follows: ATP > or = UTP > ADP > or = UDP. In contrast, negligible responses were seen for beta,gamma-meATP, a general P2X receptor agonist and adenosine, a P1 receptor agonist. Homologous desensitization of the Ca2+ response was observed for the four nucleotides. However, P2Y receptor internalization and degradation was not detected following stimulation with ATP, which is in contrast to EGFR internalization observed in response to EGF. ATP induced cell migration was comparable to that of EGF and was maximal at 1 microM. Cells exposed to ATP, UTP, ADP, and UDP demonstrated a rapid twofold increase in phosphorylation of paxillin at Y31 and Y118, however, there was no activation elicited by beta,gamma-meATP or adenosine. Additional studies demonstrated that wound closure was inhibited by reactive blue 2. These results indicate that P2Y receptors play a critical role in the injury repair process.

Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-β3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.

Corneal wound healing studies have a long history and rich literature that describes the data obtained over the past 70 years using many different species of animals and methods of injury. These studies have lead to reduced suffering and provided clues to treatments that are now helping patients live more productive lives. In spite of the progress made, further research is required since blindness and reduced quality of life due to corneal scarring still happens. The purpose of this review is to summarize what is known about different types of wound and animal models used to study corneal wound healing. The subject of corneal wound healing is broad and includes chemical and mechanical wound models. This review focuses on mechanical injury models involving debridement and keratectomy wounds to reflect the authors’ expertise.

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This review highlights recent findings that describe how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca2+ concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target may be a key issue in understanding how physiological signaling becomes pathological in ocular disease.

Systemic AL amyloidosis results from the aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) usually produced by a plasma cell clone in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as amyloid fibrils can rapidly accumulate in tissues including the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. Chemotherapy is used to eradicate the cellular source of the amyloidogenic precursor. Currently, there are no therapies that target the process of LC aggregation, fibril formation, or organ damage. We developed transgenic mice expressing an amyloidogenic 6 LC using the cytomegalovirus ( IntroductionThe systemic amyloidoses are a diverse group of protein misfolding diseases in which proteins aggregate and form fibrillar deposits in tissues. Amyloidosis can be genetic in origin (familial amyloidosis, AF) or can occur in the setting of chronic inflammation or infection (amyloidosis because of deposition of the acute phase serum amyloid A protein, AA). However the most commonly diagnosed form, amyloid light chain (AL) amyloidosis, is because of deposition of an immunoglobulin light chain (LC) usually produced by clonal plasma cells in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as LC deposits may rapidly accumulate in organs such as the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. 1 Patients with AL amyloidosis are treated with chemotherapy to eradicate the plasma cell clone in the bone marrow that is the source of the amyloidogenic protein. Unfortunately, chemotherapeutics and even newer anti-plasma cell drugs with novel mechanisms of action can cause significant toxicity in AL amyloidosis patients. Although the pathophysiology of AL amyloidosis is still not completely understood, it is hoped that patient outcomes will be improved with the development of therapies that specifically target the process of protein aggregation, fibril formation, amyloid deposition, and organ damage.Although it is clear that the overexpression of a clonal amyloidogenic LC causes AL amyloidosis, it is not clear what structural features of amyloidogenic LC are responsible for misfolding and aggregation. Furthermore, although it is well-established that glycoaminoglycans 2 and serum amyloid P component 3 can interact with LC proteins, and are found in association with amyloid fibrils, the role of these accessory molecules in fibril formation in vivo is not well understood. The role of prefibrillar LCs in organ dysfunction remains a major question in the disease pathogenesis. Data from our group have demonstrated that amyloidogenic LC can be acutely toxic to target organs, inducing oxidative stress in cells and organ culture model systems. 4,5 Amyloidogenic LCs can be internalized into cells, regulating the expression of proteoglycans and possibly mediating interactions leading to the activation of stress and other signaling pathways. 6,7 Moreover, other investigators have demonstrat...

The cornea plays a major role in the refraction of light to the retina. Therefore, the integrity and transparency of the corneal epithelium are critical to vision. Following injury, a combination of rapid signal transduction events and longterm cell migration are essential for wound closure. We have demonstrated previously that injury resulted in the release of nucleotides that induce the propagation of a Ca 2+ wave to neighboring cells. This suggests that nucleotides and their receptors are critical components of wound healing. Epidermal growth factor (EGF) and integrins also have been shown to play a role in injury. In this study, we demonstrate that pretreatment of cells with ATP and UTP inhibited the immediate wound response, while BzATP, ADP, and UDP did not affect this response. Tri-nucleotide pretreatment also reduced the EGF induced Ca 2+ response. Additionally, lower EC 50 concentrations of ATP and UTP triggered migration of cells that was enhanced further with EGF and was inhibited by the tripeptide, RGD. Results indicate that the desensitization induced by ATP and UTP was specific. While ADP and UDP cause a homologous desensitization of their own signal, they did not cause an inhibition of the wound response nor does BzATP. Neither Ca 2+ wave propagation nor cell migration occurred in response to b,g-MeATP. Together these results lead us to hypothesize that corneal epithelial wound repair is mediated by both P2Y 2 and P2Y 4 receptors.Abbreviations: BAPTA -1,2-bis(o-aminophenoxy