Biomedical subjects

B'Ann True Gabelt

Publications and source records attributed to B'Ann True Gabelt.

5 recordsLinked to original sources

PubMed2006

Caldesmon transgene expression disrupts focal adhesions in HTM cells and increases outflow facility in organ-cultured human and monkey anterior segments.

Cytoskeleton modulating compounds have been shown to lower intraocular pressure (IOP) and increase outflow facility. Caldesmon is one protein that is involved in the regulation of actin stress fiber formation. The effects of rat non-muscle caldesmon (Cald) gene over-expression on focal adhesions in human trabecular meshwork (HTM) cells and on outflow facility in organ-cultured human and monkey anterior segments were determined. Treatment of HTM cells with adenovirus-delivered caldesmon (AdCaldGFP) resulted in characteristic changes in the actin cytoskeleton and matrix adhesions within 24-48 hr post-transduction. Stress fibers gradually disappeared and novel actin structures were formed (see manuscript by Grosheva et al., this issue). In cells with disrupted stress fibers, vinculin-containing focal adhesions were also disrupted. In organ-cultured anterior segments, baseline outflow facility (microl min-1 mmHg-1) for all anterior segments averaged (mean+/-sem): human, 0.19+/-0.03 (n=12); monkey, 0.36+/-0.02 (n=19). In human anterior segments, transduction with 10(7) plaque forming units of AdGFPCald increased outflow facility by 43+/-21% (p<or=0.11, n=6) at 66 hr compared to baseline and corrected for the changes in outflow facility of the contralateral vehicle treated segment. Using the same time point, i.e. 2-3 days after injection, outflow facility in monkey anterior segments, transduced with 1.5x10(7) plaque forming units of AdGFPCald was increased by 35+/-18%, p<0.2, n=10 compared to baseline and corrected for the change in outflow facility in the contralateral AdGFP treated segment. Combining human (66 hr) and monkey (2-3 days) data, outflow facility was increased by 38+/-13%, p<0.02, n=16. Additional analysis of maximum responses in monkey anterior segments from 1 to 6 days after transduction showed outflow facility was increased by 66+/-18%, p<0.01, n=10. Caldesmon over-expression, which relaxes cultured HTM cells and disrupts their actin cytoskeleton and cell-matrix adhesions, also appears to increase outflow facility in organ-cultured human and monkey anterior segments. This suggests that over-expression of the caldesmon gene in the TM may be an effective approach for the gene therapy of glaucoma.

Actins↗

PubMed2006

Monkey organ-cultured anterior segments: technique and response to H-7.

In the intact primate eye in vivo, it may be difficult to determine whether an outflow facility response is due to direct effects on the trabecular meshwork or indirectly via an effect on ciliary muscle tone unless complicated surgical ciliary muscle disinsertion or isotope accumulation procedures are used. We established a monkey anterior segment organ culture system modeled after the human anterior segment organ culture system and determined its characteristics and response to H-7, an agent shown to increase trabecular outflow in vivo and in other organ culture systems. Outflow facility studies conducted using the monkey organ-cultured anterior segment system showed that: baseline values were comparable to those measured in vivo; washout occurred with time; the two-level constant pressure perfusion technique gave results comparable to the constant rate technique; species differences were found comparing baseline outflow facility and intraocular pressure in rhesus and cynomolgus monkey organ-cultured anterior segments; there was no effect of age on outflow facility in either species by one-way ANOVA; anterior segment exchange perfusion increased outflow facility if measured within 1 hr of the exchange; the magnitude of the response to H-7 was comparable to those reported in vivo and in other in vitro systems. The onset of the response to H-7 was delayed in most cases (day after H-7 exchange), compared to in vivo monkey studies (onset 6-9 min) and in vitro human and porcine organ culture systems (within 1 hr). Also the duration of the response (2-3 days from the onset) was longer than that found for in vitro human studies (1 day). Thus, the monkey organ-cultured anterior segment system can be used to determine the effects of pharmacological and biological agents on trabecular outflow.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2006

Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells.

Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell-matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP-caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 microm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility.

Actins↗

PubMed2005

Changes in aqueous humor dynamics with age and glaucoma.

Changes in aqueous humor dynamics with age and in glaucoma have been studied for several decades. More recently, techniques have been developed which confirm earlier studies showing that outflow facility decreases with age and in glaucoma and add the newer finding that uveoscleral outflow also decreases. Morphologic studies in aging and glaucoma eyes have shown an increase in accumulation of extracellular material in both the trabecular meshwork and ciliary muscle and a loss of trabecular meshwork cells, which contribute to this reduction in outflow and result in an increase in intraocular pressure. A reduction in hyaluronic acid and increases in fibronectin and thrombospondin contribute to the change in the extracellular environment. Imbalances in responses to age-related stresses such as oxidative damage to long-lived molecules, protein cross-linking and loss of elasticity could trigger excess production of factors such as transforming growth factor beta, interleukin-1 and CD44S that could stimulate pathways leading to increases in fibronectin, transformation of trabecular meshwork cells to a myoepithelial state and decrease the breakdown in extracellular matrix material, allowing excess to accumulate. Ultimately trabecular outflow and uveoscleral outflow are reduced and intraocular pressure becomes elevated, adding more stress and perpetuating the pathological condition. Future research to identify additional factors and clarify their roles in these processes could lead to alternative therapies for age and glaucoma related changes in the eye.

Aging↗

PubMed2003

Aqueous humor dynamics and trabecular meshwork and anterior ciliary muscle morphologic changes with age in rhesus monkeys.

PURPOSE: To determine in rhesus monkeys the age-dependence of uveoscleral outflow (Fu) and morphology of the trabecular meshwork (TM) and anterior ciliary muscle (CM). METHODS: Intraocular pressure (IOP) was measured by Goldmann applanation tonometry in monkeys under ketamine anesthesia. After anterior chamber cannulation under pentobarbital anesthesia, aqueous humor formation (AHF), anterior chamber volume, trabecular outflow, and Fu were determined isotopically. The CM and TM were examined by light and electron microscopy. RESULTS: IOP increased significantly with age in monkeys aged 3 to 29 years. AHF and anterior chamber volume were unchanged. Fu was decreased, and trabecular outflow increased in monkeys aged 25 to 29 years compared with the remaining monkeys. Morphologically, there was a significant increase in the thickness of the elastic fibers of the trabeculum ciliare covering the anterior tips of the CM, and an increase in extracellular material between the muscle tips. The number of TM cells decreased with age, whereas the amount of fibrillar material and sheath-derived plaques increased. This increase was less pronounced in the middle filtering portion of the cribriform region than in the anterior and posterior portions. CONCLUSIONS: The decline in Fu in very old rhesus monkeys with normal IOP parallels that seen in normotensive aging humans. This may be correlated with thickening of the elastic fiber sheath in the CM tips in addition to other morphologic changes. The TM findings are analogous to those in the aging human eye and are consistent with the age-related decrease in outflow facility reported in both humans and monkeys.

Aging↗