Assessment of Commercial Off-the-Shelf Tissue Adhesives for Sealing Military-Relevant Corneal Perforation Injuries.

INTRODUCTION: Open-globe ocular injuries have increased in frequency in recent combat operations due to increased use of explosive weaponry. Unfortunately, open-globe injuries have one of the worst visual outcomes for the injured warfighter, often resulting in permanent loss of vision. To improve visual recovery, injuries need to be stabilized quickly following trauma, in order to restore intraocular pressure and create a watertight seal. Here, we assess four off-the-shelf (OTS), commercially available tissue adhesives for their ability to seal military-relevant corneal perforation injuries (CPIs). MATERIALS AND METHODS: Adhesives were assessed using an anterior segment inflation platform and a previously developed high-speed benchtop corneal puncture model, to create injuries in porcine eyes. After injury, adhesives were applied and injury stabilization was assessed by measuring outflow rate, ocular compliance, and burst pressure, followed by histological analysis. RESULTS: Tegaderm dressings and Dermabond skin adhesive most successfully sealed injuries in preliminary testing. Across a range of injury sizes and shapes, Tegaderm performed well in smaller injury sizes, less than 2 mm in diameter, but inadequately sealed large or complex injuries. Dermabond created a watertight seal capable of maintaining ocular tissue at physiological intraocular pressure for almost all injury shapes and sizes. However, application of the adhesive was inconsistent. Histologically, after removal of the Dermabond skin adhesive, the corneal epithelium was removed and oftentimes the epithelium surface penetrated into the wound and was adhered to inner stromal tissue. CONCLUSIONS: Dermabond can stabilize a wide range of CPIs; however, application is variable, which may adversely impact the corneal tissue. Without addressing these limitations, no OTS adhesive tested herein can be directly translated to CPIs. This highlights the need for development of a biomaterial product to stabilize these injuries without causing ocular damage upon removal, thus improving the poor vision prognosis for the injured warfighter.

Characterization of an anterior segment organ culture model for open globe injuries.

Open-globe injuries have poor visual outcomes and have increased in frequency. The current standard of care is inadequate, and a therapeutic is needed to stabilize the injury until an ophthalmic specialist is reached. Unfortunately, current models or test platforms for open-globe injuries are insufficient. Here, we develop and characterize an open-globe injury model using an anterior segment organ-culture platform that allows therapeutic assessment for up to 72 h post-injury. Anterior segments maintained in organ culture were kept at physiological intraocular pressure throughout, and puncture injuries were created using a novel pneumatic-powered system. This system can create high-speed, military-relevant injuries up to 4.5 mm in diameter through the cornea. From intraocular pressure readings, we confirmed a loss of pressure across the 72 h after open-globe injury. Proof-of-concept studies with a Dermabond tissue adhesive were performed to show how this model system could track therapeutic performance for 72 h. Overall, the organ-culture platform was found to be a suitable next step towards modeling open-globe injuries and assessing wound closure over the critical 72 h post-injury. With improved models such as this, novel biomaterial therapeutics development can be accelerated, improving care, and, thus, improving the prognosis for the patients.

Amniotic membrane allografts maintain key biological properties post SCCO2 and lyophilization processing.

Amniotic membrane (AM) has been shown to enhance corneal wound healing due to the abundance of growth factors, cytokines, and extracellular matrix (ECM) proteins inherent to the tissue. As such, AM has garnered widespread clinical utility as a biological dressing for a number of ophthalmic and soft tissue applications. The preparation, sterilization, and storage procedures used to manufacture AM grafts are extremely important for the conservation of inherent biological components within the membrane. Current processing techniques use harsh chemicals and sterilization agents that can compromise the fundamental wound healing properties of AM. Furthermore, commercially available cryopreserved AM products require specific storage conditions (e.g., ultra-low freezers) thereby limiting their clinical availability in austere environments. Supercritical carbon dioxide (SCCO2) technology allows for the sterilization of biological tissues without the resulting degradation of integral ECM proteins and other factors often seen with current tissue sterilization processes. With this study we demonstrate that lyophilized AM, sterilized using SCCO2, maintains similar biochemical properties and biocompatibility as that of commercially available AM products requiring specialized cold storage conditions.

Development and Characterization of a Benchtop Corneal Puncture Injury Model.

During recent military operations, eye-related injuries have risen in frequency due to increased use of explosive weaponry which often result in corneal puncture injuries. These have one of the poorest visual outcomes for wounded soldiers, often resulting in blindness due to the large variations in injury shape, size, and severity. As a result, improved therapeutics are needed which can stabilize the injury site and promote wound healing. Unfortunately, current corneal puncture injury models are not capable of producing irregularly shaped, large, high-speed injuries as seen on the battlefield, making relevant therapeutic development challenging. Here, we present a benchtop corneal puncture injury model for use with enucleated eyes that utilizes a high-speed solenoid device suitable for creating military-relevant injuries. We first established system baselines and ocular performance metrics, standardizing the different aspects of the benchtop model to ensure consistent results and properly account for tissue variability. The benchtop model was evaluated with corneal puncture injury objects up to 4.2 mm in diameter which generated intraocular pressure levels exceeding 1500 mmHg. Overall, the created benchtop model provides an initial platform for better characterizing corneal puncture injuries as seen in a military relevant clinical setting and a realistic approach for assessing potential therapeutics.

A Mouse Model of Repetitive Blast Traumatic Brain Injury Reveals Post-Trauma Seizures and Increased Neuronal Excitability.

Repetitive blast traumatic brain injury (TBI) affects numerous soldiers on the battlefield. Mild TBI has been shown to have long-lasting effects with repeated injury. We have investigated effects on neuronal excitability after repetitive, mild TBI in a mouse model of blast-induced brain injury. We exposed mice to mild blast trauma of an average peak overpressure of 14.6 psi, repeated across three consecutive days. While a single exposure did not reveal trauma as indicated by the glial fibrillary acidic protein indicator, three repetitive blasts did show significant increases. As well, mice had an increased indicator of inflammation (Iba-1) and increased tau, tau phosphorylation, and altered cytokine levels in the spleen. Video-electroencephalographic monitoring 48 h after the final blast exposure demonstrated seizures in 50% (12/24) of the mice, most of which were non-convulsive seizures. Long-term monitoring revealed that spontaneous seizures developed in at least 46% (6/13) of the mice. Patch clamp recording of dentate gyrus hippocampus neurons 48 h post-blast TBI demonstrated a shortened latency to the first spike and hyperpolarization of action potential threshold. We also found that evoked excitatory postsynaptic current amplitudes were significantly increased. These findings indicate that mild, repetitive blast exposures cause increases in neuronal excitability and seizures and eventual epilepsy development in some animals. The non-convulsive nature of the seizures suggests that subclinical seizures may occur in individuals experiencing even mild blast events, if repeated.

PEGylated Platelet-Free Blood Plasma-Based Hydrogels for Full-Thickness Wound Regeneration.

Objective: To develop a cost-effective and clinically usable therapy to treat full-thickness skin injuries. We accomplished this by preparing a viscoelastic hydrogel using polyethylene glycol (PEG)-modified platelet-free plasma (PEGylated PFP) combined with human adipose-derived stem cells (ASCs). Approach: PEGylated PFP hydrogels were prepared by polymerizing the liquid mixture of PEG and PFP±ASCs and gelled either by adding calcium chloride (CaCl2) or thrombin. Rheological and in vitro studies were performed to assess viscoelasticity and the ability of hydrogels to direct ASCs toward a vasculogenic phenotype, respectively. Finally, a pilot study evaluated the efficacy of hydrogels±ASCs using an athymic rat full-thickness skin wound model. Results: Hydrogels prepared within the range of 11 to 27 mM for CaCl2 or 5 to 12.5 U/mL for thrombin exhibited a storage modulus of ∼62 to 87 Pa and ∼47 to 92 Pa, respectively. The PEGylated PFP hydrogels directed ASCs to form network-like structures resembling vasculature, with a fourfold increase in perivascular specific genes that were confirmed by immunofluorescent staining. Hydrogels combined with ASCs exhibited an increase in blood vessel density when applied to excisional rat wounds compared with those treated with hydrogels (110.3 vs. 95.6 BV/mm2; p < 0.05). Furthermore, ASCs were identified in the perivascular region associated with newly forming blood vessels. Innovation: This study demonstrates that PFP modified with PEG along with ASCs can be used to prepare cost-effective stable hydrogels, at the bed-side, to treat extensive skin wounds. Conclusion: These results indicate that PEGylated plasma-based hydrogels combined with ASCs may be a potential regenerative therapy for full-thickness skin wounds.

Treatment of corneal chemical alkali burns with a crosslinked thiolated hyaluronic acid film.

PURPOSE: The study objective was to test the utilization of a crosslinked, thiolated hyaluronic acid (CMHA-S) film for treating corneal chemical burns. METHODS: Burns 5.5mm in diameter were created on 10 anesthetized, male New Zealand white rabbits by placing a 1N NaOH soaked circular filter paper onto the cornea for 30s. Wounds were immediately rinsed with balanced salt solution (BSS). CMHA-S films were placed in the left inferior fornix of five injured and five uninjured animals. Five animals received no treatment. At 0h, 48h, 96h, and on day 14 post chemical burn creation, eyes were evaluated by white light imaging, fluorescein staining, and optical coherence tomography (OCT). Corneal histology was performed using H&E and Masson's Trichrome stains. RESULTS: Image analysis indicated biocompatible CMHA-S treatment resulted in significant decreases in the areas of corneal opacity at 48h, 96h, and on day 14 postoperatively. A significant increase in re-epithelialization was seen 14days post injury. CMHA-S treated corneas showed significantly less edema than untreated burns. No pathological differences were observed in corneal histological samples as a result of CMHA-S treatment. CONCLUSIONS: CMHA-S films facilitate re-epithelialization and decrease the area of corneal opacity in our corneal alkali burn rabbit model.

Fibrin-based stem cell containing scaffold improves the dynamics of burn wound healing.

For severe burn injuries, successful medical intervention is accomplished by rapidly and safely providing physical barriers that can cover damaged skin tissues, thereby preventing critical danger of extensive bleeding and infection. Despite availability of a large assortment of wound coverage options, the etiology of wound healing is rather complex leading to significant defects in skin repair. The use of cell-mediated treatment approaches in combination with bioengineered wound coverage constructs may provide the missing tool to improve wound healing outcomes. In this study, we have used an engineered 3D PEGylated fibrin (P-fibrin) gel as a scaffold for adipose derived stem cells (ASCs) delivery into the burn injury model. We were able to confirm the presence of ASCs in the wound site two weeks after the initial injury. Delivery of ASCs-containing gels was associated with improved vascularization of the injured area at early time points accompanied by an increased abundance of mannose receptor expressing cells. Moreover, the application of P-fibrin biomaterial exhibited positive effects on early mononuclear cell recruitment and granulation tissue formation without negatively affecting wound closure kinetics or extent of connective tissue deposition. Collectively, our data support the feasibility of using P-fibrin gels in wound dressing applications requiring controlled delivery of viable cells.

Magnetic Nanoparticles as a Potential Vehicle for Corneal Endothelium Repair.

The corneal endothelium is paramount to the health and function of the cornea as damage to this cell layer can lead to corneal edema, opacification, and ultimately vision loss. Transplantation of the corneal endothelium is associated with numerous limitations, including graft rejection, thus an alternative therapeutic treatment is needed to restore endothelial layer integrity. We hypothesize that a nanotechnology-based approach using superparamagnetic iron oxide nanoparticles (SPIONPs) can ultimately be used to guide corneal endothelial cells (CECs) to injured areas via an external magnetic force without changing their morphology or viability. In this feasibility study we examined the effects of SPIONPs on the morphology and viability of bovine CECs in the presence of a magnetic force. The CECs were exposed to increasing SPIONP concentrations and the viability and cytoskeletal structure assessed over 3 days via metabolic analysis and rhodamine phalloidin staining. Significant differences (p < .05) in the metabolic activity of the CECs (100 × 10(6) SPIONP/cell) occurred in the presence of magnetic force versus those with no magnetic force. No differences were observed in the cytoskeleton of CECs in the presence or absence of magnetic force for all SPIONP concentrations. These SPIONPs will next be evaluated with human CECs for future applications.

Effects of hyaluronic acid conjugation on anti-TNF-α inhibition of inflammation in burns.

Biomaterials capable of neutralizing specific cytokines could form the basis for treating a broad range of conditions characterized by intense, local inflammation. Severe burns, spanning partial- to full-thickness of the dermis, can result in complications due to acute inflammation that contributes to burn progression, and early mediation may be a key factor in rescuing thermally injured tissue from secondary necrosis to improve healing outcomes. In this work, we examined the effects on burn progression and influence on the inflammatory microenvironment of topical application of anti-tumor necrosis factor-α (anti-TNF-α) alone, mixed with hyaluronic acid (HA) or conjugated to HA. We found that non-conjugated anti-TNF-α decreased macrophage infiltration to a greater extent than that conjugated to HA; however, there was little effect on the degree of progression or IL-1ß levels. A simple transport model is proposed to analyze the results, which predicts qualitative and quantitative differences between untreated burn sites and those treated with the conjugates. Our results indicate that conjugation of anti-TNF-α to high molecular weight HA provides sustained, local modulation of the post-injury inflammatory responses compared to direct administration of non-conjugated antibodies.

Enhanced wound vascularization using a dsASCs seeded FPEG scaffold.

The bioengineering of autologous vascular networks is of great importance in wound healing. Adipose-derived stem cells (ASCs) are of interest due to their ability to differentiate toward various cell types, including vascular. We hypothesized that adult human ASCs embedded in a three-dimensional PEG-fibrin (FPEG) gel have the ability to modulate vascularization of a healing wound. Initial in vitro characterization of ASCs isolated from discarded burn skin samples (dsASCs) and embedded in FPEG gels indicated they could express such pericyte/smooth muscle cell markers as α-smooth muscle actin, platelet-derived growth factor receptor-ß, NG2 proteoglycan, and angiopoietin-1, suggesting that these cells could potentially be involved in a supportive cell role (i.e., pericyte/mural cell) for blood vessels. Using a rat skin excision model, wounds treated with dsASCs-FPEG gels showed earlier collagen deposition and wound remodeling compared to vehicle FPEG treated wounds. Furthermore, the dsASCs-seeded gels increased the number of vessels in the wound per square millimeter by day 16 (~66.7 vs. ~36.9/mm(2)) in these same studies. dsASCs may support this increase in vascularization through their trophic contribution of vascular endothelial growth factor, as determined by in vitro analysis of mRNA and the protein levels. Immunohistochemistry showed that dsASCs were localized to the surrounding regions of large blood-perfused vessels. Human dsASCs may play a supportive role in the formation of vascular structures in the healing wound through direct mechanisms as well as indirect trophic effects. The merging of autologous grafts or bioengineered composites with the host's vasculature is critical, and the use of autologous dsASCs in these procedures may prove to be therapeutic.

Bilayer hydrogel with autologous stem cells derived from debrided human burn skin for improved skin regeneration.

The objective of this study was to demonstrate that stem cells isolated from discarded skin obtained after debridement can be used with collagen and fibrin-based scaffolds to develop a tissue-engineered vascularized dermal equivalent. Discarded tissue samples were collected from severely burned patients undergoing wound debridement. Stem cells were isolated from the adipose tissue layer and their growth and immunophenotype characterized. To develop a skin equivalent, debrided skin adipose stem cells (dsASCs) were added to a collagen-polyethylene glycol (PEG) fibrin-based bilayer hydrogel and analyzed in vitro. The effect of the bilayered hydrogels on wound healing was demonstrated using an excision wound model in athymic rats. The dsASCs isolated from all samples were CD90, CD105, and stromal cell surface protein-1 positive, similar to adipose stem cells isolated from normal human lipoaspirates. Within the bilayer hydrogels, dsASCs proliferated and differentiated, maintained a spindle-shaped morphology in collagen, and developed a tubular microvascular network in the PEGylated fibrin. Rat excision wounds treated with bilayer hydrogels showed less wound contraction and exhibited better dermal matrix deposition and epithelial margin progression than controls. Stem cells can be isolated from the adipose layer of burned skin obtained during debridement. When dsASCs are incorporated within collagen-PEGylated fibrin bilayer hydrogels, they develop stromal and vascular phenotypes through matrix-directed differentiation without use of growth factors. Preliminary in vivo studies indicate that dsASC-bilayer hydrogels contribute significantly to wound healing and provide support for their use as a vascularized dermal substitute for skin regeneration to treat large surface area burns.

Role of the retinal vascular endothelial cell in ocular disease.

Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell.

Development of a vascularized skin construct using adipose-derived stem cells from debrided burned skin.

Large body surface area burns pose significant therapeutic challenges. Clinically, the extent and depth of burn injury may mandate the use of allograft for temporary wound coverage while autografts are serially harvested from the same donor areas. The paucity of donor sites in patients with burns involving large surface areas highlights the need for better skin substitutes that can achieve early and complete coverage and retain normal skin durability with minimal donor requirements. We have isolated autologous stem cells from the adipose layer of surgically debrided burned skin (dsASCs), using a point-of-care stem cell isolation device. These cells, in a collagen-polyethylene glycol fibrin-based bilayer hydrogel, differentiate into an epithelial layer, a vascularized dermal layer, and a hypodermal layer. All-trans-retinoic acid and fenofibrate were used to differentiate dsASCs into epithelial-like cells. Immunocytochemical analysis showed a matrix- and time-dependent change in the expression of stromal, vascular, and epithelial cell markers. These results indicate that stem cells isolated from debrided skin can be used as a single autologous cell source to develop a vascularized skin construct without culture expansion or addition of exogenous growth factors. This technique may provide an alternative approach for cutaneous coverage after extensive burn injuries.

Constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

Engineering a bilayered hydrogel to control ASC differentiation.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM). Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

Episcleral venous pressure responses to topical nitroprusside and N-Nitro-L-arginine methyl ester.

PURPOSE: To determine the episcleral venous pressure (EVP) responses to nitroprusside (NP) and L-NAME. METHODS: In anesthetized rabbits (n = 36), arterial pressure and IOP were measured by direct cannulation, and carotid blood flow and heart rate were measured with an ultrasound flowmeter and cardiotachometer. EVP was measured in two groups with a servonull system. Group 1 (n = 13) was given NP (50 microL, 10 mg/mL). Group 2 (n = 10) was given L-NAME (100 microL, 10 mg/mL) followed by NP (50 microL, 10 mg/mL). In group 3 (n = 13), fluorophotometric aqueous flow was measured before and after NP (100 microL, 10 mg/mL). RESULTS: Systemic parameters were unaffected by treatment in all groups. In group 1, NP increased EVP from 9.1 +/- 0.6 to 11.6 +/- 0.8 mm Hg (P < 0.01) and IOP from 18.7 +/- 1.4 to 23.9 +/- 1.6 mm Hg (P < 0.01). In group 2, L-NAME lowered EVP from 11.5 +/- 1.2 to 8.8 +/- 1.0 mm Hg (P < 0.01) and subsequent NP increased EVP to 13.9 +/- 1.7 mm Hg (P < 0.01 versus L-NAME and baseline). L-NAME decreased IOP from 20.8 +/- 1.7 to 16.7 +/- 1.8 mm Hg (P < 0.01), and then it increased to 20.7 +/- 1.3 mm Hg after NP (P < 0.01 versus L-NAME and P > 0.05 versus baseline). In group 3, NP increased IOP from 16.6 +/- 0.7 to 20.0 +/- 0.9 mm Hg (P < 0.01) but did not alter aqueous flow (2.65 +/- 0.3 vs. 3.0 +/- 0.3 microL/min, P > 0.05). CONCLUSIONS: Because a topical NO donor raises EVP and a topical NO synthase inhibitor lowers EVP, the authors conclude that EVP is modulated by NO.

Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells.

PURPOSE: EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. METHODS: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 microL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). RESULTS: Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2+/-VEGF compared to IgG control+/-VEGF (P<0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. CONCLUSIONS: These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.

Topical proparacaine and episcleral venous pressure in the rabbit.

PURPOSE: To determine the effect of proparacaine-induced topical anesthesia on episcleral venous pressure (EVP). METHODS: In anesthetized rabbits (n = 11), EVP was measured with a servonull micropressure system, with glass pipettes with 2- to 3-microm tips used to cannulate episcleral veins. Additional measurements included arterial, intraocular, and orbital venous pressures obtained by direct cannulation, to assess the ocular pressure gradients, and carotid blood flow and heart rate, to verify preparation stability. The protocol entailed 5 to 10 minutes of stable baseline recording followed by topical application of proparacaine (0.5%, 10 microL) with continued measurements for another 5 to 15 minutes. RESULTS: Baseline EVP without topical anesthesia was 12.3 +/- 1.1 mm Hg. EVP decreased significantly to 8.7 +/- 0.9 mm Hg within minutes after application of proparacaine. A small decrease also occurred in intraocular pressure. All other measured variables were unchanged. CONCLUSIONS: These results suggest that the episcleral circulation is under tonic neural control and that either an upstream resistance site is under tonic vasodilatory control or a downstream site is under vasoconstrictor control.

Proteomic profiling of human retinal and choroidal endothelial cells reveals molecular heterogeneity related to tissue of origin.

PURPOSE: The ocular vascular endothelium plays a key role in the development of several leading retinal causes of blindness in Western nations. Choroidal endothelial cells are integral to the subretinal neovascular lesions that characterize the exudative form of late age-related macular degeneration (AMD), and retinal endothelial cells participate in the initiation of diabetic retinopathy and posterior uveitis. Vascular endothelial cells at different sites exhibit considerable molecular diversity. This diversity has implications for understanding the pathogenesis of tissue-specific diseases and for the development of targeted therapies to treat these conditions. Previous work from our group has identified significant differences in the gene transcript profiles of human retinal and choroidal endothelial cells. Because the proteome ultimately determines the behavior of any given cell, however, it is critical to determine whether molecular differences exist at the level of protein expression. METHODS: Retinal and choroidal endothelial cells were separately isolated from five sets of human eyes by enzymatic digestion with type II collagenase followed by anti-CD31 antibody-conjugated magnetic bead separation. Cells were washed to remove serum peptides in the culture medium, and lysed by sonication in buffer containing 2% sodium dodecyl sulfate. Protein was then precipitated with acetone. Retinal and choroidal endothelial samples from each donor were labeled with Cy3 and Cy5, respectively, mixed with a Cy2-labeled pooled protein sample to facilitate spot matching across gels, and separated by two-dimensional difference gel electrophoresis (2D-DIGE). Following a global normalization, differentially abundant protein spots that were visible in at least four of five donor gels were detected by the significance analysis of microarrays method, with false discovery rate set at 5%. Corresponding spots were excised from additional DIGE-labeled or Coomassie-stained 2D electrophoretic gels. Protein identification was performed by liquid chromatography and tandem mass spectrometry. RESULTS: Of 123 protein spots detected by 2D-DIGE that qualified for statistical analysis, we found 31 spots that demonstrated a significant difference in abundance between retinal endothelial samples versus choroidal endothelial samples. For 17 proteins, over 50% of the spectral counts could be matched to a single protein in the digested spot. Eleven proteins were more abundant in retinal endothelial cells (i.e., inorganic pyrophosphatase, protein disulfide isomerase A3, calreticulin, peroxiredoxin-4, protein disulfide isomerase, serpin B9, F-actin capping protein subunit beta, coactosin-like protein, vimentin, cathepsin B, and a high molecular weight form of annexin A3). Six proteins were more abundant in choroidal endothelial cells (i.e., glutathione peroxidase 1, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), heat-shock protein beta-1, superoxide dismutase (Cu-Zn), nucleoside diphosphate kinase A, and a low molecular weight form of annexin 3). CONCLUSIONS: Our data indicate that the proteomes of retinal and choroidal vascular endothelial cells are different. Several differentially expressed proteins are implicated in the regulation of angiogenesis; these include cathepsin B and UCH-L1, proteins with transcripts that were also differently expressed according to microarray. Our observations further suggest that angiogenesis within the retina, a component of severe diabetic retinopathy and posterior uveitis, may be controlled by different mechanisms to those regulating choroidal neovascularization, as occur in exudative AMD. Future studies to establish the role of these angiogenic proteins in disease may suggest potential new targets for tissue-specific therapies.