Characterization of shear-sensitive genes in the normal rat aorta identifies Hand2 as a major flow-responsive transcription factor.

OBJECTIVE: Shear forces play a key role in the maintenance of vessel wall integrity. Current understanding regarding shear-dependent gene expression is mainly based on in vitro or in vivo observations with experimentally deranged shear, hence reflecting acute molecular events in relation to flow. Our objective was to combine computational fluid dynamic (CFD) simulations with global microarray analysis to study flow-dependent vessel wall biology in the aortic wall under physiological conditions. METHODS AND RESULTS: Male Wistar rats were used. Animal-specific wall shear stress (WSS) magnitude and vector direction were estimated using CFD based on aortic geometry and flow information acquired by magnetic resonance imaging. Two distinct flow pattern regions were identified in the normal rat aortic arch; the distal part of the lesser curvature being exposed to low WSS and a non-uniform vector direction, and a region along the greater curvature being subjected to markedly higher levels of WSS and a uniform vector direction. Microarray analysis identified numerous novel mechanosensitive genes, including Trpc4 and Fgf12, and confirmed well-known ones, e.g. Klf2 and Nrf2. Gene ontology analysis revealed an over-representation of genes involved in transcriptional regulation. The most differentially expressed gene, Hand2, is a transcription factor previously shown to be involved in extracellular matrix remodeling. HAND2 protein was endothelial specific and showed higher expression in the regions exposed to low WSS with disturbed flow. CONCLUSIONS: Microarray analysis validated the CFD-defined WSS regions in the rat aortic arch, and identified numerous novel shear-sensitive genes. Defining the functional importance of these genes in relation to atherosusceptibility may provide important insight into the understanding of vascular pathology.

Bimatoprost effects on aqueous humor dynamics in monkeys.

The effects of bimatoprost on aqueous humor dynamics were quantified in monkey eyes. Uveoscleral outflow was measured by the anterior chamber perfusion method, using FITC-dextran. Total outflow facility was determined by the two-level constant pressure method. Aqueous flow was measured with a scanning ocular fluorophotometer. Uveoscleral outflow was 0.96 +/- 0.19 muL min(-1) in vehicle-treated eyes and 1.37 +/- 0.27 muL min(-1) (n = 6; P < .05) in eyes that received bimatoprost 0.01% b.i.d. x 5 days. Bimatoprost had no effect on total outflow facility, which was 0.42 +/- 0.05 muL min(-1) at baseline and 0.42 +/- 0.04 muL min(-1) after bimatoprost treatment. Bimatoprost had no significant effect on aqueous humor flow. This study demonstrates that bimatoprost increases uveoscleral outflow but not total outflow facility or aqueous humor flow, indicating that it lowers intraocular pressure in ocular normotensive monkeys by a mechanism that exclusively involves uveoscleral outflow.

Prostanoid EP4 receptor stimulation produces ocular hypotension by a mechanism that does not appear to involve uveoscleral outflow.

PURPOSE: As part of a systematic elucidation of the pharmacology of prostaglandin's (PG) effects on intraocular pressure in the monkey, the prototypical selective prostanoid EP(4) receptor agonist (3,7-dithia PGE(1)) was examined. It was found to be highly efficacious in nonhuman primates, and its mechanism of ocular hypotensive activity was investigated. METHODS: Intraocular pressure (IOP) was measured by pneumatonometry in conscious monkeys restrained in custom-designed chairs. All other animal experiments were performed in animals sedated with ketamine or anesthetized with ketamine/diazepam and given drug or vehicle for various lengths of time. Aqueous flow was determined by fluorophotometry. Total outflow facility was measured by the two-level, constant-pressure method and by 2-minute tonography in both normotensive and hypertensive monkey eyes. Uveoscleral outflow was measured by perfusing the anterior chamber with FITC-labeled dextran for 30 minutes at a fixed IOP of approximately 15 mm Hg. Isometric responses to drugs were measured in longitudinal and radial preparations of monkey and human isolated ciliary smooth muscle specimens. RESULTS: The selective EP(4) receptor agonist 3,7-dithia PGE(1) and an isopropyl ester prodrug thereof reduced IOP in monkeys. A single dose of 3,7-dithia PGE(1) isopropyl ester, at a 0.01% or 0.1% dose, decreased IOP in the glaucomatous monkey in the range of 40% to 50%. Studies on total outflow facility by the two-level, constant-pressure perfusion method and tonography indicated that EP(4) receptor stimulation facilitated aqueous humor outflow facility. No effect on aqueous flow was apparent. In contrast to all PGs and prostamides studied to date, 3,7-dithia PGE(1) exerted no effect on uveoscleral outflow measured directly. Moreover, it did not relax longitudinal or radial preparations of isolated human or monkey ciliary muscles. CONCLUSIONS: The EP(4) receptor agonist 3,7-dithia PGE(1) is a highly efficacious IOP-lowering drug in monkeys. It has no effect on uveoscleral outflow but does increase total outflow facility, which accounts for a substantial proportion of the ocular hypotensive activity.

Uveoscleral outflow--a review.

The uveoscleral outflow route was described more than 40 years ago. Part of aqueous leaves the eye through the iris root. The ciliary muscle, and there are large species differences in the fraction of aqueous outflow that leaves the eye through this route. In non-human primates 40-50% of aqueous leaves the eye by the uveoscleral route. In human eyes most data has been collected by indirect calculations, with results suggesting a similar fraction, at least in eyes from younger individuals. An age-dependent reduction in uveoscleral flow in human eyes may explain the initial difference seen between non-human primate and human eyes. Unlike trabecular outflow, intraocular pressures within the normal range have little effect on uveoscleral outflow. This may be explained by the fact that changes in intraocular pressure have little effect on the pressure gradient for flow through the ciliary muscle, which is likely to be the rate-limiting step in uveoscleral outflow. The state of the ciliary muscle is important and contraction reduces while relaxation increases uveoscleral flow. Similar effects are achieved with cholinergic agonists and antagonists. Epinephrine increases uveoscleral flow, most likely through stimulating beta(2)-adrenergic receptors. Prostaglandin F(2alpha) and prostaglandin F(2alpha)-analogues effectively reduce intraocular pressure by increasing uveoscleral flow. This is mediated by structural changes in the extracellular matrix of the ciliary muscle, and is likely to contribute to a valuable excess route for aqueous and proteins during intraocular inflammation. Whether uveoscleral flow plays a significant role in any other eye disease is not clear. Thus, 40 years later we are able to successfully increase aqueous flow through the uveoscleral route, a valuable contribution to glaucoma treatment, but we still have only a limited understanding on its physiological role.

The prostanoid EP2 receptor agonist butaprost increases uveoscleral outflow in the cynomolgus monkey.

PURPOSE: To investigate the ocular hypotensive effect of the prostanoid EP2 receptor agonist butaprost and to establish its mechanism of action. METHODS: All experiments were performed in cynomolgus monkeys after topical application of butaprost (0.1%). The effects of butaprost on aqueous humor flow were determined by fluorophotometry. Total outflow facility was measured by the two-level, constant-pressure perfusion method, and uveoscleral outflow was determined by perfusion of FITC-labeled dextran through the anterior chamber. Effects on ocular morphology were studied after tissue fixation with transcardial perfusion by paraformaldehyde and immersion fixation of the globe, in animals subjected to long-term treatment with butaprost. Conscious ocular normotensive monkeys and monkeys with unilateral ocular hypertension were used for intraocular pressure (IOP) studies. RESULTS: Butaprost had no significant effect on aqueous humor flow or total outflow facility in ocular normotensive monkeys. Uveoscleral outflow was significantly higher in the butaprost treated eyes than in vehicle treated eyes, 1.03 +/- 0.20 vs. 0.53 +/- 0.18 microL.min(-1). After a 1-year treatment with butaprost, the morphology of the ciliary muscle was changed, showing increased spaces between ciliary muscle bundles and the apparent formation of new outflow channels. In many instances, changes were observed in the trabecular meshwork as well. Butaprost, in a single 0.1% dose, decreased IOP significantly in ocular normotensive monkeys and reduced IOP in laser-induced glaucomatous monkey eyes to the same level as that in the ocular normotensive contralateral eyes. CONCLUSIONS: The prostanoid EP2 receptor agonist butaprost appears to lower IOP by increasing uveoscleral outflow, according to both physiological and morphologic findings. Although the prostanoid EP2 receptor is structurally and functionally distinct from the FP receptor, the effects of EP2 and FP receptor stimulation on aqueous humor outflow are similar.

Angiotensin converting anzyme (ACE) activity in porcine ocular tissue: effects of diet and ACE inhibitors.

The aim of the present experiments was to determine angiotensin converting enzyme (ACE) activity in different parts of the porcine eye, and to examine whether an atherogenic diet influenced ACE activity. Female mini-pigs were fed a standard diet or a diet with high cholesterol to produce atherosclerosis. The animals were killed by an overdose of pentobarbital, and the eyes were enucleated and dissected into iris, ciliary body, retina, and choroid. Crude tissue homogenates were used for determination of ACE activity, which was done with a radioenzymatic assay. In pigs fed a normal diet, basal ACE activity was 18.1 +/- 1.6, 13.6 +/- 1.9, 4.4 +/- 0.6, and 44.7 +/- 8.5 units/mg for iris, ciliary body, retina, and choroid, respectively. The ACE activities in ocular tissues from the pigs that had been fed an atherogenic diet were not significantly different. Nor was the ACE activity in the abdominal aorta and serum significantly different between the two groups. In both groups, the ACE inhibitors captopril and enalaprilat, caused a significant inhibition of the ACE activity in the choroid and ciliary body, with enalaprilat being more potent. In the retina, ACE activity was inhibited significantly only in the group fed a normal diet, whereas ACE activity in the iris was not significantly inhibited in either group. We did not find any differences in ACE activity between pigs fed a normal diet and pigs fed an atherogenic diet, which is in disagreement with previous studies that showed an increased ACE activity in aorta from atherosclerotic mini-pigs. The reason for this discrepancy is not clear, but lower cholesterol levels are one possibility.