Sangly P. Srinivas

Temporal progression and spatial repeatability of tear breakup

Purpose. This study used image analysis to compare the temporal progression and spatial reoccurrence of the area of tear film breakup (AB) in dry eye and normal subjects. Methods. Tear breakup was induced in 10 control and 10 dry eye subjects during the Staring Tear Breakup Dynamics (S-TBUD) test, which involves keeping one eye open for as long as possible, termed the maximum blink interval (MBI). Video imaging of tear film fluorescence measured the onset and progression of the AB. AB location and area were mapped. The progression of ABs from the first trial, the rate of tear breakup or dry area growth rate (DAGR), and the overlap of ABs in three successive trials 5 minutes apart were computed by custom MATLAB programs. Results. The final AB before the blink was significantly greater (average, 30.7% ± 12.5% vs. 16.1% ± 9.2%) and the MBI was significantly less (average, 19.5 ± 9.0 seconds vs. 56.5 ± 38.9 seconds) among dry eye subjects compared with controls (p < 0.05, Mann-Whitney U test). The DAGR was four times greater among dry eye subjects, who also showed significantly more tear breakup in the central cornea than controls (p < 0.0001, Mann-Whitney U test). When the final image from three successive trials was overlapped, tear breakup occurred more often in the same location in three trials than would be expected by the overlap of independent points. Conclusions. Structural influences such as the “black line” or corneal lid defects appeared to influence the recurrence of breakup in the same region. The S-TBUD quantitative image analysis technique demonstrates that the tear film of subjects with dry eye continues to rapidly destabilize after an initial first break; thus, a low TBUT was combined with a high DAGR. The central corneal region of subjects with dry eye appeared especially susceptible to increased tear breakup when compared with controls.

Dynamic Regulation of Barrier Integrity of the Corneal Endothelium

The corneal endothelium maintains stromal deturgescence, which is a prerequisite for corneal transparency. The principal challenge to stromal deturgescence is the swelling pressure associated with the hydrophilic glycosaminoglycans in the stroma. This negative pressure induces fluid leak into the stroma from the anterior chamber, but the rate of leak is restrained by the tight junctions of the endothelium. This role of the endothelium represents its barrier function. In healthy cornea, the fluid leak is counterbalanced by an active fluid pump mechanism associated with the endothelium itself. Although this pump-leak hypothesis was postulated several decades ago, the mechanisms underlying regulation of the balance between the pump and leak functions remain largely unknown. In the last couple of decades, the ion transport systems that support the fluid pump activity have been discovered. In contrast, despite significant evidence for corneal edema secondary to endothelial barrier dysfunction, the molecular aspects underlying its regulation are relatively unknown. Recent findings in our laboratory, however, indicate that barrier integrity (i.e., structural and functional integrity of the tight junctions) of the endothelium is sensitive to remodeling of its peri-junctional actomyosin ring, which is located at the apical junctional complex. This review provides a focused perspective on dynamic regulation of the barrier integrity of endothelium vis-à-vis plasticity of the peri-junctional actomyosin ring and its association with cell signaling downstream of small GTPases of the Rho family. Based on findings to date, it appears that development of specific pharmacological strategies to treat corneal edema in response to inflammatory stress would be possible in the near future.

Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6)

In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin‐luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing‐type chamber. We developed a LL pulse protocol to determine the rate of ATP release (RATP) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure RATP as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 μl of A6 cells released ATP at a rate of 66 ± 8 fmol min−1. A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H2O)−1 elevated RATP rapidly to a peak value of 1.89 ± 0.11 pmol min−1 (RATPpeak) followed by a plateau phase reaching 0.51 ± 0.07 pmol min−1 (RATPplat). Both RATPpeak and RATPplat values increased with the degree of dilution. The magnitude of RATPplat remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 ± 0.08 pmol min−1 was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H2O)−1. This RATP value, induced in the absence of cell swelling, is comparable to RATPplat. Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose‐response curves for peak [Ca2+]i increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with RATPpeak. The link between the ATP release and the fast [Ca2+]i transient was also demonstrated by the depression of both phenomena by Cl− removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin‐sensitive rise of [Ca2+]i during the hyposmotic shock.

Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration

Monthly intraocular injections are widely used to deliver protein-based drugs that cannot cross the blood-retina barrier for the treatment of leading blinding diseases such as age-related macular degeneration (AMD). This invasive treatment carries significant risks, including bleeding, pain, infection, and retinal detachment. Further, current therapies are associated with a rate of retinal fibrosis and geographic atrophy significantly higher than that which occurs in the described natural history of AMD. A novel therapeutic strategy which improves outcomes in a less invasive manner, reduces risk, and provides long-term inhibition of angiogenesis and fibrosis is a felt medical need. Here we show that a single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses CNV in primate and murine AMD models. Moreover, this treatment suppressed subretinal fibrosis, which is currently not addressed by clinical therapies. Murine vision, as tested by OptoMotry, significantly improved with nearly 40% restoration of visual loss induced by CNV. We found no evidence of ocular or systemic toxicity from nanoparticle treatment. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy.

Barrier Dysfunction of the Corneal Endothelium in Response to TNF-α: Role of p38 MAP Kinase

PURPOSE TNF-alpha is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF-alpha-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells. METHODS Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cell-substrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-alpha was visualized by immunofluorescence. The expression of TNF-alpha receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-alpha was determined by Western blot analysis. RESULTS Exposure to TNF-alpha induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-alpha also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine. CONCLUSIONS TNF-alpha breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-alpha-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.

Cell signaling in regulation of the barrier integrity of the corneal endothelium.

The barrier integrity of the corneal endothelium, which is conferred by its tight and adherens junctions, is critical for the maintenance of deturgescence of the corneal stroma. Although characteristically leaky, the barrier integrity restricts fluid leakage into the stroma such that the rate of leak does not exceed the rate of the endothelial active fluid transport directed toward the aqueous humor. At a molecular level, the barrier integrity is influenced by the actin cytoskeleton and microtubules, which are coupled to tight and adherens junctions via a variety of linker proteins. Since the cytoskeleton is affected by Rho family small GTPases and p38 MAP kinase, among others, many pathophysiological stimuli induce plasticity to the cytoskeleton and thereby elicit dynamic regulation of the barrier integrity. This review presents an overview of the impact of several bioactive factors on the barrier integrity of the corneal endothelium through altered actin cytoskeleton and/or disassembly of microtubules. The main focus is on the effect of TNF-α (tumor necrosis factor-α) which is a pro-inflammatory molecule found in the intraocular milieu during allograft rejection and anterior uveitis. This cytokine elicits acute activation of p38 MAP kinase, induces disassembly of microtubules, disrupts the peri-junctional actomyosin ring, and concomitantly breaks down the barrier integrity. These effects of TNF-α could be inhibited by stabilizing the microtubules, co-treating with a selective p38 MAP kinase inhibitor, and elevating intracellular cAMP via A2B receptors or direct exposure to forskolin. Overall, the corneal edema following a potential breakdown of the endothelial barrier integrity can be rescued pharmacologically by inhibiting specific cell-signaling mechanisms.

Microtubule Stabilization Opposes the (TNF-α)-Induced Loss in the Barrier Integrity of Corneal Endothelium

Microtubule disassembly breaks down the barrier integrity in a number of epithelial and endothelial monolayers. This study has investigated effects of TNF-alpha, which is implicated in corneal allograft rejection, on microtubules and barrier integrity in cultured bovine corneal endothelial cells. Exposure to TNF-alpha led to disassembly of the microtubules, and also caused disruption of the perijunctional actomyosin ring (PAMR). As a measure of barrier integrity, trans-endothelial electrical resistance (TER) was determined based on electrical cell-substrate impedance sensing in realtime. Exposure to TNF-alpha caused a slow decline in TER for > 20 h, and a similar exposure to cells grown on porous culture inserts led to a significant increase in permeability to FITC dextran. These changes, indicating a loss of barrier integrity, were also reflected by dislocation of ZO-1 at the cell border and disassembly of cadherins. These effects of TNF-alpha were inhibited upon stabilization of microtubules by pre-treatment with paclitaxel or epothilone B. Microtubule stabilization may be a useful strategy to overcome (TNF-alpha)-induced loss of the barrier integrity of corneal endothelium during inflammation associated with transplant rejection and uveitis.

Mucoadhesive Chitosan–Dextran Sulfate Nanoparticles for Sustained Drug Delivery to the Ocular Surface

PURPOSE To characterize nanoparticles produced by self-assembly of oppositely charged polymers, cationic chitosan (CS), and anionic dextran sulfate (DS), for drug delivery to the ocular surface. The goal is to overcome the short residence time of topical drugs through their sustained release from mucoadhesive nanoparticles. METHODS Chitosan-dextran sulfate nanoparticles (CDNs) were produced by mixing CS and DS; polyethylene glycol-400 was used as a surface stabilizing agent. Fourier transform infrared spectroscopy (FTIR) spectra of CS, DS, and CDNs were determined in the wavenumber range of 4,000-700 cm(-1) to assess the ionic interactions in the formation of CDNs. The physicochemical properties, entrapment efficacy, and dissolution profile of CDNs were investigated using Rhodamine B (RhB) and Nile Red (NR) as drug analogs. The mucoadhesiveness of the CDNs was assessed by imaging the retention of the fluorescein isothiocyanate-labeled CDNs on the cornea ex vivo, which was subjected to shear stress by a steady stream of saline solution. RESULTS CDNs were obtained by the polyelectrolyte complexation technique. The FTIR spectra of CDNs showed spectral shifts in the amine and sulfate regions, confirming an involvement of electrostatic interactions between cationic CS and anionic DS. The CDNs were spherical in shape and segregated. They possessed a particle size of ~400 nm with a polydispersity index of 0.3 and exhibited a zeta potential of ~40 mV. A high entrapment efficacy of up to 80% was observed with both RhB and NR. In the dissolution experiments, NR was released from CDNs within 60 min, but RhB was not released. This indicates that the release of drugs could depend on their molecular interactions with the particle. Exposure of CDNs to lysozyme, which is found in tears, had no effect on the mean particle size or the surface charge. Instillation of NR, RhB, and FITC in the presence of saline irrigation resulted in their rapid disappearance (<5 min) from the corneal surface. In contrast, fluorescent CDNs showed retention on the cornea even after 60 min. CONCLUSIONS Cationic and biocompatible mucoadhesive CDNs have been developed for sustained drug delivery to the ocular surface. The CDNs were stable to lysozyme and showed prolonged adherence to the corneal surface.

The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells.

PURPOSE Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility. METHODS BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses. RESULTS Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP. CONCLUSIONS Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.

Measurement of rapid changes in cell volume by forward light scattering.

Light scattering is an empirical technique employed to measure rapid changes in cell volume. This study describes a new configuration for the method of light scattering and its corroboration by measurements of cell height (as a measure of cell volume). Corneal endothelial cells cultured on glass cover-slips were mounted in a perfusion chamber on the stage of an inverted microscope. A beam of light was focused on the cells from above the stage at an angle of 40° to the plane of the stage. The scattered light intensity (SLI), captured by the objective and referred to as forward light scatter (FLS), increased and decreased in response to hyposmotic and hyperosmotic shocks, respectively. The rapid increase and decrease in SLI corresponded to cell swelling and shrinkage, respectively. Subsequently, SLI decreased and increased as expected for a regulatory volume decrease (RVD) and increase (RVI), respectively. These data are in agreement with measurements of cell height, demonstrating that the method of light scatter in FLS mode is useful for monitoring rapid changes in cell volume of cultured cells. Changes in SLI caused by gramicidin were consistent with cell volume changes induced by equilibration of NaCl and KCl concentrations across the cell membrane. Similarly, an additional decrease in SLI was recorded during RVD upon increasing K+ conductance by valinomycin. Decreasing K+ conductance of the cell membrane with Ba2+ changed the time course of SLI consistent with the effect of the K+ channel blocker on RVD. Bumetanide and dihydro-ouabain inhibited increases in SLI during RVI. In conclusion, FLS is a valid method for qualitative analysis of cell volume changes with a high time resolution.