PubMed Health⌕ Search

Biomedical subjects

M A Watsky

Publications and source records attributed to M A Watsky.

At least 19 recordsLinked to original sources

Properties of porcine and recombinant human collagen matrices for optically clear tissue engineering applications.

Porcine and recombinant human atelocollagen I solutions were cross-linked with a water soluble carbodiimide at various stoichiometries and collagen concentrations (5-20 w/w %). The resulting hydrogels were clear and, when used as cell growth matrices, allowed cell and nerve visualization in vitro and in vivo. We have previously reported that, after six months of implantation in pigs' and rabbits' corneas, these robust hydrogels allowed regeneration of host cells and nerves to give optically clear corneas with no detected loss in thickness, indicating stable engraftment. Here, the biocompatible hydrogel formulations leading to this novel in vivo performance were characterized for amine consumption, gel hydration, thermal properties, optical clarity, refractive index, nutrient diffusion, biodegradation, tensile measurements, and average pore diameters. Gels with excellent in vitro (epithelial overgrowth, neurite penetration) and in vivo performance (clarity, touch sensitivity regeneration) had 4-11 nm pores, yet had glucose and albumin diffusive coefficients similar to mammalian corneas and allowed neurite extension through the gels.

Amines↗

Pamidronate infusion in patients with systemic sclerosis results in changes in blood mononuclear cell cytokine profiles.

A single infusion of pamidronate was given to patients with systemic sclerosis (scleroderma, SSc) to assess effects on cytokine production by peripheral blood mononuclear cells (PBMC) and lymphocyte subsets. Eighteen patients with SSc received a single intravenous dose of 60 mg of pamidronate and were followed for 6 months. Assessment of cytokine production [interferon (IFN)-gamma, interleukin (IL)-10, transforming growth factor (TGF)-beta1, tumour necrosis factor (TNF)-alpha and IL-4] by PBMC and lymphocyte subsets by flow cytometry was carried out before and after the pamidronate infusion. Unstimulated PBMC produced increased amounts of IFN-gamma and TNF-alpha and reduced levels of TGF-beta1 for up to 24 weeks after the infusion. gammadelta T cells from patients with SSc were activated in vitro and produced increased IFN-gamma. The effects of pamidronate on modulation of cytokine profiles in patients with SSc may merit future study.

Aged↗

A collagen-based scaffold for a tissue engineered human cornea: physical and physiological properties.

Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K+ channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma.

Animals↗

Phospholipid growth factors and corneal wound healing.

In many tissue types, wound healing involves cell division and migration over and into the wound area to cover and remodel the wound. LPA and other members of the phospholipid lipid growth factor (PLGF) family stimulate many of the activities involved in wound healing. In the rabbit cornea, we have found that keratocytes from wounded corneas have a volume-activated Cl- current activated by LPA and alkenyl-LPA. This current is minimally activated by cyclic PA and SPC, and is not activated by LPA in cells from uninjured corneas. Biochemical examination of PLGFs in aqueous humor and lacrimal fluid before and after wounding identified LPA, alkenyl-GP, PA, and lyso PS, with elevated PLGF activity after wounding. In recent experiments examining human corneal cell lines and cultured cells using RT-PCR, we found mRNA for EDG receptors 1-5, with an apparent increase in EDG-3, -4, and -5 following brief SDS application to cell lines, and EDG receptors 2-5 induction in late-passage human corneal epithelial cells. This work points to a significant role for PLGFs in the corneal wound-healing process.

Animals↗

Functional human corneal equivalents constructed from cell lines.

Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

Animal Testing Alternatives↗

Loss of fenamate-activated K+ current from epithelial cells during corneal wound healing.

PURPOSE: The corneal epithelium provides a barrier between the external environment and the cornea. It also serves as an ion transporting epithelium. Because of its proximity with the external environment, the corneal epithelium is frequently injured through physical or chemical insult. The purpose of this study was to determine whether corneal epithelial cell whole-cell currents change during corneal wound healing as the author of the present study has previously reported for corneal keratocytes and endothelial cells. METHODS: Rabbit corneal epithelial cells were injured by scraping, heptanol exposure, or freezing. The epithelium was allowed to heal for 12 to 74 hours. Cells were dissociated from corneas, and whole-cell currents were examined using the amphotericin-perforated-patch technique. RESULTS: Cells from the wounded corneal groups had significantly increased capacitance values, indicating increased surface area compared with that of control cells. As previously reported, the primary control whole-cell current was a fenamate-activated K+ current. An inwardly rectifying K+ current and a Cl- current were also observed. In epithelial cells from heptanol-wounded corneas, these conductances were generally unchanged. In cells from scrape- and freeze-wounded corneas, however, the fenamate-activated current was absent or significantly attenuated. CONCLUSIONS: As they do in corneal keratocytes and endothelial cells, K+ channels disappear during some models of corneal epithelial wound healing. In addition, cell capacitance, a measurement of cell surface area, increases. These results suggest that substantial K+ channel activity is not required for in vivo epithelial cell proliferation during corneal wound healing.

Animals↗

Growth factor-like phospholipids generated after corneal injury.

The present study provides evidence that growth factor-like glycerophosphate mediators of the lysophosphatidic acid (LPA) family are present in the aqueous humor and the lacrimal gland fluid of the rabbit eye. By use of a combination of HPLC, two-dimensional TLC, mass spectrometry, and the Xenopus oocyte bioassay, the LPA-like phospholipids LPA, cyclic PA, alkenyl-glycerophosphate (GP), lysophosphatidylserine, and phosphatidic acid were detected as physiological constituents of the fluids bathing the cornea. Corneal injury resulted in an increased production of some of these mediators. Alkenyl-GP, a novel member of the LPA family, has been identified in postinjury aqueous humor, establishing that it is generated endogenously. LPA and its homologues were found to be mitogenic in freshly dissociated keratocytes from uninjured corneas. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury and the increase in LPA-like activity in aqueous humor. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting cellular regeneration of the injured cornea.

Animals↗

Characterization of voltage-gated, whole-cell ionic currents from conjunctival epithelial cells.

PURPOSE: These studies were performed to characterize the voltage-gated, whole-cell ionic currents in rabbit bulbar conjunctival epithelial and goblet cells. METHODS: New Zealand White rabbits were killed, and the bulbar conjunctiva was isolated. Conjunctival cells were dissociated for patch clamp analysis of whole-cell currents. The amphotericin, perforated-patch, whole-cell technique was used. RESULTS: Conjunctival epithelial cells had a mean capacitance of 6.72 pF (SE = 0.49; n = 25). The primary currents found were an inwardly rectifying K+ current, a saturating K+ current, and an outwardly rectifying nonselective cation current. A second nonselective cation current also appeared to be present. The inward current was observed in a KCl Ringer's bath and was almost nonexistent in a NaCl bath. The current was Ba(2+)- and Cs(+)-sensitive. The second K+ current became saturated at depolarized voltages and was Ba(2+)- and quinidine-sensitive. The first outward nonselective cation current was typically less than 100 pA in amplitude and activated at voltages positive to 0 mV. Tail current experiments showed that the current was cation selective. The current was blocked by Gd3+ but not by the Cl- current blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid. The second nonselective cation current was larger and Gd(3+)-insensitive. The primary current observed in goblet cells was a large outward K+ current. CONCLUSIONS: The primary currents observed during whole-cell patch clamping of bulbar conjunctival epithelium are a Ba(2+)- and Cs(+)-sensitive, inwardly rectifying K+ current, a saturating K+ current, and two outwardly rectifying nonselective cation currents. Goblet cells contain a large outward K+ current.

Animals↗

Properties of whole-cell ionic currents in cultured human corneal epithelial cells.

PURPOSE: To identify and partially characterize the ionic currents contributing to the whole-cell conductance of cultured human corneal epithelial cells. METHODS: Epithelial cells were scraped from human donor corneas and cultured for use in patch-clamp experiments. Amphotericin B and the perforated-patch configuration were used to measure whole-cell currents in cells isolated from confluent monolayers. RESULTS: Cell monolayers exhibited cobblestone morphology and were immunopositive for corneal epithelium-specific cytokeratin. Single cells had a capacitance of 21 +/- 2 pF and expressed similar types of ionic currents regardless of passage number. In descending order of frequency of occurrence, cells exhibited a nonselective cation current active at depolarized voltages and insensitive to Ba2+ and Gd3+; an outwardly rectifying K+ current active at depolarized voltages, stimulated by flufenamic acid and inhibited by tetraethylammonium; a voltage-gated inward Na+ current; an outwardly rectifying K+ current active at hyperpolarized voltages, stimulated by flufenamic acid, blocked by Ba2+, and insensitive to diltiazem; an inwardly rectifying K+ current; and a nonselective cation current inhibited by flufenamic acid. CONCLUSIONS: Our results are consistent with those in previous studies of noncultured epithelia from rabbit and human corneas showing an outwardly rectifying K+ current active at hyperpolarized voltages and a nonselective cation current active at depolarized voltages and insensitive to Ba2+. These data suggest cultured cells may be useful in determining the physiological role of ion channels in corneal epithelia and may aid in the development of a cell-based model for the examination of the effects of wounding and toxic agents on the human cornea.

Amphotericin B↗

Dye spread through gap junctions in the corneal epithelium of the rabbit.

PURPOSE: Microelectrode dye injection of 5,6-carboxyfluorescein was used to investigate gap junctional communication in the corneal epithelium. METHODS: Dye injection started in the superficial layer and proceeded stepwise into the underlying epithelial layers until spread was observed. Intracellular [Ca2+] was manipulated by exposing the cornea to the calcium ionophore A23187 (global increase) or by increasing the [Ca2+] in the injection electrode (source cell increase). Intracellular pH was manipulated by exposing the cornea to nigericin in a low-pH KCI Ringer's (global decrease) or by lowering the pH in the injection electrode (source cell decrease). Heptanol was tested for its ability to uncouple gap junctions. Gap junctional communication was based on the layer at which spread was first observed and on the apparent dye travel distance from the point of injection. RESULTS: Control dye spread occurred, on average, in the third layer from the surface. Increased [Ca2+] in the source cell resulted in an initial spread occurring in the second layer. Globally increasing [Ca2+] with A23187 resulted in no change in the average initial spread layer. Lowering intracellular pH of the source cell did not affect the initial dye spread layer. Globally lowering intracellular pH resulted in significant gap junctional inhibition in a time-dependent manner. Dye spread distance was not significantly affected by [Ca2+] or pH manipulations. Heptanol (2.5 mM) completely inhibited dye coupling. CONCLUSION: All cell layers of the corneal epithelium contain functional gap junctions, although it appears that intercellular communication in the superficial layers does not occur under our control conditions. Intercellular communication through these junctions can be altered by various manipulations of [Ca2+] and pH.

Alcohols↗

Phorbol ester modulation of rabbit corneal endothelial permeability.

PURPOSE: Phorbol esters have been shown to have a profound influence on cellular activity in many cell types. The purpose of this study was to examine the influence of phorbol esters on the function and structure of corneal endothelial cells. METHODS: Corneas were placed under a specular microscope, and the endothelium was superfused with glutathione bicarbonate Ringer's solution (GBR); with GBR and 10 nM, 100 nM, or 1 microM 4 beta-phorbol 12-myristate 13-acetate (PMA); or with 100 nM 4-alpha-PMA. Corneal swelling curves were generated, and endothelial permeability was determined. Corneal endothelial structure was examined with a scanning electron microscope. RESULTS: Significant increases in swelling and endothelial permeability were found in corneas perfused with 100 nM PMA versus that observed in controls (swelling rate = 26 microns/hr versus 6.9 microns/hr; permeability = 6 x 10(-4) cm/min versus 3.4 x 10(-4) cm/min) and in corneas receiving 1 microM PMA versus that in controls (swelling rate = 26.3 microns/hr versus 0.12 micron/hr; permeability = 6.9 x 10(-4) cm/min versus 4.9 x 10(-4) cm/min). Application of 10 nM PMA did not significantly alter either parameter. Study with transmission electron microscope demonstrated significant morphologic changes in cells perfused with all concentrations of PMA. Corneas perfused with 100 nM 4-alpha-PMA versus 100 nM PMA had significantly lower slope and permeability values (swelling rate = 5.9 microns/hr versus 25.1 microns/hr; permeability = 3 x 10(-4) cm/min versus 6.7 x 10(-4) cm/min). CONCLUSIONS: Phorbol esters are detrimental for corneal endothelial function, creating significant corneal swelling, increases in endothelial permeability, and changes in endothelial cell structure. This effect appears to be mediated through a protein kinase C pathway.

Animals↗

Ionic channels in corneal endothelium.

Single-channel patch-clamp techniques as well as standard and perforated-patch whole cell voltage-clamp techniques have been applied to the study of ionic channels in the corneal endothelium of several species. These studies have revealed two major K+ currents. One is due to an anion- and temperature-stimulated channel that is blocked by Cs+ but not by most other K+ channel blockers, and the other is similar to the family of A-currents found in excitable cells. The A-current is transient after a depolarizing voltage step and is blocked by both 4-aminopyridine and quinidine. These two currents are probably responsible for setting the -50 to -60 mV resting voltage reported for these cells. A Ca(2+)-activated ATP-inhibited nonselective cation channel and a tetrodotoxin-blocked Na+ channel are possible Na+ inflow pathways, but, given their gating properties, it is not certain that either channel works under physiological conditions. A large-conductance anion channel has also been identified by single-channel patch-clamp techniques. Single corneal endothelial cells have input resistances of 5-10 G omega and have steady-state K+ currents that are approximately 10 pA at the resting voltage. Pairs or monolayers of cells are electrically coupled and dye coupled through gap junctions.

Animals↗

Effect of tumor necrosis factor alpha on rabbit corneal endothelial permeability.

PURPOSE: Tumor necrosis factor alpha (TNF alpha) is present in the iris and the lacrimal gland, and its concentration is increased during inflammation and after corneal wounding. Although TNF alpha has been shown to increase keratocyte and corneal epithelial interleukin production, no definitive effects of TNF alpha on corneal endothelial cells have been reported. TNF alpha has been shown to disrupt barrier function in vascular endothelial monolayers through f-actin depolymerization. A reduction in intracellular cyclic adenosine monophosphate (cAMP) concentration may play a role in this response. This study was designed to examine the role and signal transduction mechanisms of TNF alpha modulation of endothelial permeability in the cornea. In addition, it is the first examination of the effects of TNF alpha on the barrier function of a noncultured cell monolayer. METHODS: Rabbit corneal endothelial superfusions were performed under an in vitro specular microscope. Corneas were processed for permeability measurements or f-actin staining. RESULTS: TNF alpha superfused corneas had significantly higher permeabilities than controls. f-actin staining revealed that TNF alpha superfusion disrupted f-actin filaments when compared to controls. Corneas superfused with the f-actin stabilizing agent phallacidin had significantly lower permeabilities than TNF alpha superfused pairs. Permeabilities of corneas superfused with TNF alpha plus 8-bromo-cAMP (0.01 to 3 mM) were significantly lower than TNF alpha superfused pairs at all concentrations, although only significantly lower at the 0.1 mM cAMP concentration. CONCLUSIONS: TNF alpha causes an increase in corneal endothelial permeability, and this increase is mediated by disruption of f-actin filaments; cAMP appears to be involved in this response.

8-Bromo Cyclic Adenosine Monophosphate↗

Nonselective cation channel activation during wound healing in the corneal endothelium.

Rabbit corneas were injured by mechanical or thermal trauma. At several time points after wounding, corneal endothelial cells were isolated and their ion channels examined using standard and amphotericin perforated-patch whole cell patch-clamp configurations. Within 15-24 h after mechanical or thermal trauma, a nonselective cation current was observed in 79% of the cells examined that was not present in unwounded or sham-wounded corneas. By 73 h postwounding, the current was present in only 10% of the cells examined. The wound healing-induced current is outwardly rectifying, activates at depolarized voltages, shows no sign of inactivation, and is inhibited by flufenamic acid, quinidine, and acetate. In addition to this new current, it was observed that endothelial cells from freeze-wounded corneas no longer expressed the transient K+ current seen in control, sham, and mechanically wounded corneas. Corneal endothelial superfusion experiments found no significant difference in swelling rates between control and flufenamic acid-superfused wounded corneas, indicating that the wound healing-induced channel is not involved in the stromal hydration maintenance function of the corneal endothelium.

Animals↗

Lysophosphatidic acid, serum, and hyposmolarity activate Cl- currents in corneal keratocytes.

The influence of serum, lysophosphatidic acid (LPA), and hyposmotic stress on the ion channel activity of normal and cryo-injured rabbit corneal keratocytes was investigated. Whole cell currents were examined using the amphotericin perforated-patch technique. In cells from wounded corneas, fetal bovine serum activated large, holding voltage-insensitive, fast-activating, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-, flufenamic acid-, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)-blockable outward currents showing inactivation at depolarized voltages. LPA activated identical currents, also only in cells from wounded corneas. Blocker and reversal potential experiments characterized the current as a Cl- currents (Icl). Lysophosphatidylcholine (10 microM) failed to activate the current. An identical current was activated by hyposmotic stimulation in cells from control and wounded corneas. Hyposmotic stimulation also activated Icl in cells from wounded corneas that were unresponsive to LPA. We conclude that serum, LPA, and hypotonic stress activate Icl in keratocytes from wounded corneas. We also conclude that LPA is a serum factor that can activate Icl and that hyposmotic activation may work through a signaling pathway separate from that of LPA.

Animals↗

Keratocyte gap junctional communication in normal and wounded rabbit corneas and human corneas.

PURPOSE: Several studies have indicated the anatomic and biochemical presence of gap junctions in corneal keratocytes. The current study was designed to demonstrate that these gap junctions are functional in rabbit and human corneal keratocytes. This study also examined dye coupling between keratocytes migrating into the wound region of freeze-wounded rabbit corneas. METHODS: Freeze wounds were created on anesthetized rabbit corneas using a liquid nitrogen-cooled brass probe. Freeze-wounded corneas were examined at several time periods from days 0 to 5 after wounding. Nonwounded rabbit corneas also were examined. Human corneal buttons were examined immediately after removal from patients who underwent keratoplasty. Gap junctional coupling was examined by microinjecting carboxyfluorescein from microelectrodes into the basal-most keratocytes and capturing dye spread images with a cooled charge coupled device camera. RESULTS: Significant dye spread was observed between cells in the unwounded areas of corneas at wound time 0 and between cells migrating into the wound areas as early as 24 hours after wounding. In control corneas, dye spread to as many as 50 cells from the source cell. Dye spread also was seen between keratocytes in human corneas with pseudophakic bullous keratopathy and keratoconus. CONCLUSIONS: Gap junctions observed in keratocytes from normal rabbit corneas are functional. Gap junctions also are present and functional in keratocytes within unwounded and wounded regions of freeze-injured corneas. In addition, functional gap junctions are present between keratocytes in human corneas. This study confirms the long-held contention that corneal keratocytes form a large intercommunicating network within the corneal stroma.

Adult↗

Loss of keratocyte ion channels during wound healing in the rabbit cornea.

PURPOSE: Corneal keratocytes are responsible for repairing the corneal stromal matrix after injury or infection. Recent work has characterized the primary voltage-gated ion currents in keratocytes from normal, uninjured corneas. The purpose of the present study was to examine and characterize keratocyte voltage-gated ion currents from freeze-wounded rabbit corneas. METHODS: Rabbit corneas were injured using a liquid nitrogen cooled brass probe. Keratocytes were isolated from control eyes, trephined buttons of stroma encompassing the wound area, and the stromal rim surrounding the button. Ionic currents were examined using the amphotericin perforated-patch variation of the whole cell patch clamp technique. RESULTS: The delayed rectifier K+ current, described previously as the primary voltage-gated outward current in keratocytes, was found in 100% of control cells, 91% of cells isolated from the corneal rim of wounded cells, and 33% of cells isolated from the wound region. Na+ currents were also seen with a lower frequency in cells from the wound area. CONCLUSION: The majority of keratocytes migrating into a corneal freeze wound lose the voltage-gated K+ and Na+ ion channels present in cells from normal corneas. Ion channels from cells surrounding the wound site are minimally affected by the injury.

Animals↗

Ion channel involvement in the temperature-sensitive response of the rabbit corneal endothelial cell resting membrane potential.

Previous studies have shown that the resting potential (Em) of the corneal endothelium hyperpolarizes following an increase in temperature above 24 degrees C. Whole-cell studies using the perforated-patch technique were used to compare currents and Em values from isolated corneal endothelial cells at 24 and 32 degrees C. These studies revealed a small, outwardly rectifying, slowly activating, weakly voltage-dependent current with a reversal potential showing K+ selectivity (Erev = -80 mV). This current had features similar to the whole-cell current seen following addition of HCO3- to these cells. Em measurements found an average 24 mV hyperpolarization following temperature elevation in NaCl Ringer. Single channel studies found the only change in channel activity following an elevation in temperature to be an increase in the open probability (Po) of a K+ channel previously reported in this cell type to be activated by external anions. Po (-30 mV) at 24 and 32 degrees C equaled 0.003 and 0.06, respectively. Increases in Po were found at all voltages examined. This increased Po can account for the magnitude of the hyperpolarization seen in these cells following temperature elevation. Addition of HCO3- along with elevated temperature produced a synergistic effect on the increase in Po along with an increased hyperpolarization of the cell, pointing to separate mechanisms of activation from these two stimuli.

Animals↗