Vickery Trinkaus-Randall

Boston University | BU · Departments of Biochemistry ane Cell Biology and of Ophthalmology

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound ed...

The corneal epithelium is an avascular structure that has a unique wound healing mechanism, which allows for rapid wound closure without compromising vision. This wound healing mechanism is attenuated in diabetic patients, resulting in poor clinical outcomes and recurrent non-healing erosion. We investigated changes in cellular calcium signaling ac...

Maintaining the clarity of the cornea is essential for vision, and is achieved through an exquisite array of collagen fibrils and proteoglycans in the corneal stroma. Alterations in the identity and modifications of the glycosaminoglycans (GAGs) are seen both throughout the normal wound healing process and in pathological conditions resulting in co...

The cornea is exposed daily to a number of mechanical stresses including shear stress from tear film and blinking. Over time, these stressors can lead to changes in the extracellular matrix that alter corneal stiffness, cell-substrate structures, and the integrity of cell-cell junctions. We hypothesized that changes in tissue stiffness of the corne...

Epithelial wound healing is essential to repair the corneal barrier function after injury and requires coordinated epithelial sheet movement over the wounded region. The presence and role of pannexin1 on multilayered epithelial sheet migration was examined in unwounded and wounded corneal epithelium from C57BL/6J (B6) control and diet-induced obese...

Chemical indicators are used to study calcium signaling events in the context of live cell imaging. Fluo-3 AM, Fluo-4 AM, and Cal-520 AM are three commonly used fluorescent indicators derived from the calcium chelator BAPTA. Here we describe sample protocols that detail how these indicators are used in in vitro and ex vivo experiments to analyze th...

The cornea is avascular, which makes it an excellent model to study matrix protein expression and tissue stiffness. The corneal epithelium adheres to the basement zone and the underlying stroma is composed of keratocytes and an extensive matrix of collagen and proteoglycans. Our goal was to examine changes in corneas of 8- and 15-week mice and comp...

Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. We developed a method to characterize the injury-induced sustained Ca²⁺ mobilizations that travel between c...

Association of P2X7 and pannexin1 protein in epithelial cells. HCLE cells were cultured until confluent, and cross-linking was performed with formaldehyde in situ, as previously described [12]. Each crosslinked experimental sample (labeled “CL”) and its corresponding control were heated at two different temperature settings: 65°C (to maintain cross...

Sustained Ca2+ oscillations detected after scratch-wounding. Confluent cells were preincubated with 5 μM of Fluo3-AM for 30 minutes. Cells were scratch-wounded and imaged for 2 hours in an environmental chamber mounted on a Zeiss 880 confocal microscope (10x). Images were taken every 3 seconds, with the movie at 25 fps. Scale bar = 60 μm. (AVI)

Sustained Ca2+ oscillations induced by UTP. Confluent HCLE cells were preincubated with 5μM of Fluo3-AM for 30 minutes. Cells were stimulated with 25 μM UTP and imaged for 45 minutes in an environmental chamber mounted on a Zeiss 880 confocal microscope (20x). Images were taken every 3 seconds, with the movie at 25 fps. Scale Bar = 50 μm. (AVI)

Ca2+ mobilizations in organ culture. Mouse corneas were preincubated with 15 μM Fluo3-AM for 30 minutes and CellMask Deep Red Plasma membrane stain at recommended concentration for 5 minutes. Cells were scratch-wounded and imaged for at least 15 mins in an environmental chamber mounted on a Zeiss 880 confocal microscope with AIRYSCAN Fast Module (2...

Representative kymograph of cells at least 2 cell rows away from the wound edge. Compared to the kymographs made from cells at the wound edge (LE), the Ca2+ response showed less intensity. Brackets on the left and each horizontal line represent activity of a single cell (n = 7). (TIF)

Sustained Ca2+ oscillations induced by BzATP stimulation. Confluent HCLE cells were preincubated with 5 μM of Fluo3-AM for 30 minutes. Cells were stimulated with 25 μM BzATP and imaged for 45 minutes in an environmental chamber mounted on a Zeiss 880 confocal microscope (20x). Images were taken every 3 seconds, with the movie at 25 fps. Scale Bar =...

Pannexin scrambled peptide does not inhibit rate of wound closure. Confluent cells were treated with 100 μM Scrambled Panx control peptide for an hour before being preincubated with 5 μM Fluo3-AM for 30 minutes. Cells were scratch-wounded and imaged for 16 hours in an environmental chamber mounted on a Zeiss 880 confocal microscope (20x). Images we...

Ca2+ mobilizations and cell shape. Confluent HCLE cells were preincubated with 5 μM Fluo3-AM for 30 minutes and CellMask Deep Red Plasma membrane stain at recommended concentration for 5 minutes. Cells were scratch-wounded and imaged for 45 minutes in an environmental chamber mounted on a Zeiss 880 confocal microscope (40x oil). Images were taken e...

10Panx significantly attenuates wound closure rate. Confluent HCLE cells were treated with 100 μM 10Panx inhibitory peptide for an hour before being preincubated with 5 μM Fluo3-AM for 30 minutes. Cells were scratch-wounded and imaged for 16 hours in an environmental chamber mounted on a Zeiss 880 confocal microscope (20x). Images were taken every...

The cornea is an excellent model tissue to study how cells adapt to periods of hypoxia as it is naturally exposed to diurnal fluxes in oxygen. It is avascular, transparent, and highly innervated. In certain pathologies, such as diabetes, limbal stem cell deficiency, or trauma, the cornea may be exposed to hypoxia for variable lengths of time. Due t...

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood....

The cornea is the outermost part of the eye, and as a result, can be damaged by debris and other irritants. While the cornea can repair itself from minor injuries, improper wound repair can further complicate the issue. Type II Diabetes is known to hinder the body's wound healing ability, with complications in epithelial wound repair being detected...

Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. We developed a method to characterize the injury-induced sustained Ca2+ mobilizations that travel between c...

The cornea is a transparent avascular tissue that transmits light to the retina and covers the lens and iris. According to the World Health Organization, blindness due to injury and disease in the cornea is the fourth leading cause of preventable blindness. Previous research demonstrates that epithelial cells release nucleotides that activate purin...

Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, w...

Pulmonary neuroendocrine cells (PNECs) are the only innervated airway epithelial cells. To what extent neural innervation regulates PNEC secretion and function is unknown. Here, we discover that neurotrophin 4 (NT4) plays an essential role in mucus overproduction after early life allergen exposure by orchestrating PNEC innervation and secretion of...

Nucleotide release and purinergic signaling make up the earliest response to corneal injury and are vital for proper wound healing. In this study, we review the importance of nucleotide release in the injury response and focus on the contribution of 2 receptors that mediate purinergic signaling, P2Y2 and P2X7. These receptors mediate the early resp...

The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic...

Vascular disease and its associated complications are the number one cause of death in the Western world. Both extracellular matrix stiffening and dysfunctional endothelial cells contribute to vascular disease. We examined endothelial cell calcium signaling in response to VEGF as a function of extracellular matrix stiffness. We developed a new anal...

The process of wound healing involves a complex network of signaling pathways working to promote rapid migration and wound closure. Secreted nucleotides and purinergic receptors have been shown to play a major role in the calcium wave and subsequent calcium‐dependent signaling that is essential for proper healing. While the P2X7 purinergic receptor...

Corneal disease and injury remain a major cause of blindness according to the World Health Organization. Certain conditions such as diabetes, corneal infection, and corneal ulceration may cause the cornea to be exposed to a hypoxic state for extended periods of time. Over 95% of the new cases of diabetes are Type II. In these patients, a simple wou...

This review highlights recent findings describing how purines modulate 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 m...

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 we...

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...

The process of wound healing must be tightly regulated to achieve successful restoration of injured tissue. Previously, we demonstrated that when injury to corneal epithelium occurs, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca(2+) wave from the origin of the wound to neighboring cells. In the present s...

Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleo...

Purpose: We have previously shown that TGF-β3 (T3) stimulates extracellular matrix (ECM) assembly while maintaining antifibrotic characteristics in a model using human corneal fibroblasts (HCFs). This model, however, requires non-physiological levels of serum. In the current study, we tested whether T3 could stimulate human corneal keratocytes (HC...

Epithelial wounds usually heal relatively quickly, but repair may be impaired by environmental stressors, such as hypoxic or diabetic states, rendering patients vulnerable to a number of corneal pathologies. Though this response appears simple, at first, years of research have uncovered the complicated biochemical pathways coordinating the wound he...

Vascular endothelial growth factor (VEGF) drives both endothelial cell maintenance and normal/pathological angiogenesis. Recent research has suggested that mechanical signaling due to the stiffness of the cell contact surface may also have profound effects on endothelial cell behavior. To study VEGF signaling as a function of substrate stiffness, e...

Previously, our lab demonstrated that ATP release upon injury induces Ca2+ mobilization in corneal epithelial cells via purinergic signaling. This mobilization was shown to be critical for the promotion of corneal wound repair. Most recently, we demonstrated that glutamatergic and purinergic receptors modulate communication between corneal epitheli...

Purinergic signaling is an important early event in corneal wound healing. The P2X7 receptor has been shown to play a role in epithelial cell migration after injury in scratch wounds after knockdown with siRNA. We have shown that a truncated variant of P2X7, known for its role in cell death, alters receptor function to promote proliferation after w...

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. I...

Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2) receptors resulting in mobilization of a Ca(2+) wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of...

Neuronal wound media induces pERK. HCLEs were cultured to confluence and stimulated with control media or neuronal wound media for 5 or 15 min. Lysates were probed with an antibody directed against pERK and reprobed with an antibody directed against ERK. pERK was normalized to ERK using ImageJ. Images are representative of 3 independent experiments...

Previously, the authors demonstrated that the lack of the P2X(7) receptor impairs epithelial wound healing and stromal collagen organization in the cornea. The goal here is to characterize specific effects of the P2X(7) receptor on components of the corneal stroma extracellular matrix. Unwounded corneas from P2X(7) knockout mice (P2X(7) (-/-)) and...

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...

Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X7 receptors leading to cation fluxes and cytotoxicity. The c...

Corneal integrity is critical for vision. Corneal wounds frequently heal with scarring that impairs vision. Recently, human umbilical cord mesenchymal stem cells (cord stem cells) have been investigated for tissue engineering and therapy due to their availability and differentiation potential. In this study, we used cord stem cells in a 3-dimension...

1837 AL amyloidosis results from the production and aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) by a clonal bone marrow plasma cell dyscrasia. Currently, there are no genetically reproducible animal models of AL amyloidosis. We engineered transgenic mice to express an amyloidogenic human λ6 LC. We used the cytomegalovirus (...

The release of nucleotides after injury activates purinergic receptors, leading to phosphorylation of site-specific residues on epidermal growth factor receptor (EGFR). To elucidate the differences between the injury-induced response and that induced by exogenous EGF, we examined recruitment of docking proteins, internalization of EGFR, and migrati...

Primary amyloidosis (AL) results from overproduction of unstable monoclonal immunoglobulin light chains (LCs) and the deposition of insoluble fibrils in tissues, leading to fatal organ disease. Glycosaminoglycans (GAGs) are associated with AL fibrils and have been successfully targeted in the treatment of other forms of amyloidosis. We investigated...

Injury to epithelial cells results in the release of ATP and stimulation of purinergic receptors and is thought to alter cell migration and wound repair. Medium from the injured cells triggers Ca(2+) mobilization and phosphorylation of ERK, both of which are inhibited if the medium is pretreated with apyrase. To understand the wound repair mechanis...

Aberrant constitutive expression of NF-κB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERα)-negative breast cancers versus ERα-positive ones, due in part to repression of RelB synthesis by ERα s...

Our goal was to develop a 3-D multi-cellular construct using primary human corneal fibroblasts cultured on a disorganized collagen substrate in a scaffold-free environment and to use it to determine the regulation of proteoglycans over an extended period of time (11 weeks). Electron micrographs revealed multi-layered constructs with cells present i...

The purpose of this study was to demonstrate the presence of vinculin and alpha2 integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured...

Previously, the authors demonstrated that BzATP, a P2X(7) receptor agonist, enhanced corneal epithelial migration in vitro. The goal here was to characterize the role of the P2X(7) receptor in the repair of in vivo corneal epithelial debridement wounds and in the structural organization of the corneal stroma. Epithelial debridement was performed on...

To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of vitamin C. The cells were allowed to synthesize a fib...

The early events that occur rapidly after injury trigger signal cascades that are essential for proper wound closure of corneal epithelial cells. We hypothesize that injury releases ATP, which stimulates purinergic receptors and elicits the phosphorylation of epidermal growth factor receptor (EGFR) tyrosine residues and subsequent cell migration by...

Amyloidosis is a disease of protein misfolding that ultimately impairs organ function. Previously, we demonstrated that amyloidogenic light chains (kappa1, lambda6, and lambda3 subtypes), internalized by cardiac fibroblasts, enhanced sulfation of secreted glycosaminoglycans. In this study, we investigated the internalization and cellular traffickin...

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 long-term cell migration are essential for wound closure. We have demonstrated previously that injury resulted in...

Extracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here,...

Amyloidoses are a group of disorders characterized by abnormal folding of proteins that impair organ function. We investigated the cellular response of primary cardiac fibroblasts to amyloidogenic light chains and determined the corresponding change in proteoglycan expression and localization. The cellular response to 11 urinary immunoglobulin ligh...

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 phosphoryl...

Wound healing is a complex process that involves cell communication, migration, proliferation, and changes in gene expression. One of the first events after injury is the rapid release of Ca(2+) that propagates as a wave to neighboring cells (Klepeis et al. [2001]: J. Cell. Sci. 114:4185-4195). Our goal was to examine the signaling events induced b...

This chapter reviews that intercellular calcium waves in astrocytes represent a phenomenon whereby a wave of increases in free cytosolic calcium concentration ([Ca2+]i) spreads from an initially stimulated cell across an astrocytic syncytium. Three key factors merged to trigger the discovery of calcium waves in the astrocyte syncytium. The first wa...

This chapter reviews that billions of neurons, interconnected to a large network, perform numerous cognitive and regulatory tasks. Most work on the modeling of brain functions is based on the modelling of neuronal networks. The vast majority of cells in the brain, however, are nonneuronal cells or glial cells; about 90% of all brain cells are glial...

Epidermal growth factor (EGF) stimulates integrin beta4 expression and synthesis in corneal epithelium through ligand binding to the EGF receptor, receptor dimerization and activation of the intracellular domain. We hypothesized that inhibition of EGF receptor messenger RNA (mRNA) would block integrin beta4 expression, which is induced by EGF. We a...

We developed a method to purify decorin core protein from tissue with the goal of preserving its native structure and biological function. Currently, most procedures rely on the use of denaturing reagents potentially altering the biological activity. Decorin was purified from corneal stromas without the use of detergents or chaotropic reagents. Pro...