| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
|
|
|||||||
|
|||||||||
Electronic Letters to:
|
|
Electronic letters published:
![[Read eLetter]](/icons/misc/sectionDOWN.gif){kind=icon}
Hydrostatic Pressure Is Not a Surrogate for IOP in Glaucoma
Author Response: Hydrostatic Pressure Is Not a Surrogate for IOP in Glaucoma
|
|
|||
|
C. Ross Ethier
Send letter to journal:
ethier{at}mie.utoronto.ca C. Ross Ethier
|
We read with interest the article by Knepper et al.1 on "Hypophosphorylation of Aqueous Humor sCD44 and Primary Open-Angle Glaucoma." While the findings regarding altered sCD44 in glaucomatous patients are intriguing, we have concerns about the claim made in the article that "increased pressure influenced the HA [hyaluronan] polymer and . . . binding sites." To draw this conclusion, the investigators examined the effects of pressure on the binding of soluble CD44 (sCD44) to hyaluronan (HA) and on the conformation of hyaluronan as visualized by rotary shadowing. They carried out experiments at either atmospheric pressure or at a pressure 40 mm Hg above atmospheric, conditions that they refer to as "0 mm Hg" and "40 mm Hg." It should be recognized that the pressure applied in these experiments was a hydrostatic pressure applied to molecules in a rigid container, which is a fundamentally different situation from the case of a pressure difference occurring across a deformable substrate. In the case of the eye the relevant pressure difference is the intraocular pressure (IOP), i.e., the difference between pressure inside the eye and atmospheric pressure. In the eye, IOP (or more correctly, the difference between IOP and episcleral venous pressure or between IOP and retrolaminar cerebrospinal fluid pressure) can result in significant mechanical strains on the tissues and macromolecules of the eye (in particular, the trabecular meshwork and the optic nerve), while the same value of IOP acting in a rigid container would have little effect. A simple estimate helps put this distinction into perspective. We know from experimental observations that a pressure difference between IOP and episcleral venous pressure of 15-50 mm Hg in live monkey and enucleated human eyes causes the trabecular meshwork to stretch very significantly.2 On the other hand, by using published values of the compressibility of water, proteins and polysaccharides,3,4 we can estimate that a 50 mm Hg increase in the hydrostatic pressure applied to these molecules in a rigid container will lead to deformations of these molecules less than 0.005%. In general, hydrostatic pressures applied to molecules in rigid containers result in miniscule deformations unless the pressures are many, many atmospheres. We are aware of no studies in the literature that show that the miniscule deformations expected in the study of Knepper et al. can give rise to significant configurational changes in macromolecules, as is claimed by the authors. The extraordinary nature of the claim made in this article can be appreciated in another way. We can estimate the free energy change of the hyaluronan due to a hydrostatic pressure increase of 40 mm Hg. Using compressibility values for polysaccharides,3,4 allowing that the density of hyaluronan is approximately 0.65 g/ml,5,6 and using a HA molecular weight of 1x106, we can calculate that the work done in compressing the hyaluronan as the hydrostatic pressure increases by 40 mm Hg is roughly 1 calorie/mole. This must equal the change in free energy of the HA molecule. On the other hand, typical binding energies such as would be associated with binding CD44 to HA are on the order of kilocalories/mole, i.e., thousands of times larger. Hence, it is again difficult to understand how this very modest hydrostatic pressure change would have any effect on binding of CD44 to hyaluronic acid. (Note also that the calculation done here is very conservative, since we used a molecular weight of 1x106 for the entire HA molecule whereas only a small part of the HA molecule is actually involved with binding the CD44). While we find the images and data presented to be interesting, such a remarkable claim about the effects of small changes in hydrostatic pressure on physicochemical properties of macromolecules calls for extraordinarily convincing experimental support. We were not convinced by the data for several reasons. First, it is important to appreciate that the pressure experienced by an object (including the samples used in this study) is the sum of atmospheric pressure (nominally 760 mm Hg) and any additional pressure imposed by the investigators. It is not indicated in the article what the atmospheric pressure was on those days that the measurements were made. As the magnitude of the atmospheric pressure can change daily by 10-20 mm Hg (and by as much as 50 mm Hg or more during storms), it is unclear what the total pressure was in the experiments that were being investigated. Were each of the replicate experiments done on days of identical atmospheric pressure? Also of concern was the lack of reported statistical variability for the data presented in Figure 6 or for the images presented in Figure 7. Repeatability is an important issue in light of the discussions about atmospheric pressure changes above. It is also worth pointing out that what might appear to be a fairly large difference in pressures (0 mm Hg vs. 40 mm Hg) is actually a difference of only 5% in total pressure (nominally 760 mm Hg vs. 800 mm Hg). This is rather modest compared to the pressure variations associated with traveling between locations having different elevations. For example, in Denver (Denver International Airport elevation 5,431 ft [1,655 m]), the ambient pressure is approximately 620 mm Hg, while in Sedom, Israel (elevation 1275 ft [390 m] below sea level) the ambient pressure is about 795 mm Hg. Is it reasonable to expect that molecular binding processes vary significantly depending on the elevation of the city that one lives in? Perhaps more tellingly, if absolute pressure rather than IOP mattered in glaucoma, then one might expect Denver to be glaucoma-free, which it is not. Can physiological systems sense and respond to pressures? Absolutely, but they invariably do so by transducing the pressure-induced stretching of a deformable substrate. An example is the carotid baroreceptor that responds to stretching of the carotid artery wall by changes in systemic blood pressure. The details of the pressurizing system used by Knepper et al. were only briefly described, but no such deformable substrate seems to have been included. Unfortunately, changing total pressure can change other experimental variables (e.g., evaporation rate in the rotary shadowing preparations and dissolved gas concentration in samples) that could have an effect on the results. Hence, it is possible that some of the observed differences between the "0 mm Hg" and "40 mm Hg" states are secondary effects. But it seems most unlikely that total pressure can explain the effects reported in Figures 6 and 7 of this manuscript. Finally, we raise one other aspect regarding the reported toxicity of sCD44 that we found puzzling. We note that the concentrations of sCD44 found to be toxic to TM cells in this study, and a previous study by this group,1,7 were 450-fold smaller than the concentration that human vascular endothelial cells are normally exposed to from blood plasma.7 It would have been useful to have used vascular endothelial cells as a control to substantiate this toxicity observation. C. Ross Ethier1 1Institute of Biomaterials and Biomedical Engineering, University of
Toronto, Ontario, Canada References 1. Knepper PA, Miller AM, Choi J, et al. Hypophosphorylation of aqueous humor sCD44 and primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 2005;46:2829-2837. |
|||
|
|
|||
|
Paul A. Knepper
Send letter to journal:
pknepper{at}northwestern.edu Paul A. Knepper
|
We appreciate the interest of Ethier and Johnson as shown in their letter to the editor regarding our recent article "Hypophosphorylation of aqueous humor sCD44 and primary open-angle glaucoma"1 and particularly their concerns about the possible effect of pressure on the conformation of hyaluronic acid (HA) and the biological consequences. HA is an extracellular and cell surface associated glycosaminoglycan that is traditionally regarded as inert biological goo,2 such as is found in extracellular matrices. Recent evidence suggests that HA functions as a micro-environmental clue that co-regulates a variety of fundamental cell processes.3 CD44 is a transmembrane receptor and binds HA. In order to explore the cell toxicity of sCD44, which is the ectodomain of CD44, we used two in vitro tests to examine the effect of pressure on HA polymer interactions under non-equilibrium conditions. In the first test, we used HA affinity columns, as described in the article,1 to determine the binding affinities of sCD44 to HA. Of particular note, graded amounts of sCD44 were loaded into columns, sealed at 0 or 40 mm Hg for 16 hours, mixed, and eluted from the immobilized HA using 0.5 M NaCl PBS and 0.2 M glycine at a pressure head of 0 or 40 mm Hg. In the second test, we used a rotary shadowing technique4 to visualize HA. Again, of particular note are the conditions in which the conformation of HA was tested. HA was dissolved in 0.5 M ammonium acetate, briefly subjected to 0 or 40 mm Hg of air pressure, and the conformation of HA was examined by electron microscopy. The control for the HA at 0 pressure was 0.2 M glycine, which disrupted the HA intertwining. We attribute the pressure-induced effects on HA conformation to be a manifestation of non-equilibrium thermodynamic conditions. The influence of pressure on sCD44 binding to HA and subsequent elution may involve pressure per se or a differential pressure effect; that is, HA was subjected to a brief, 10-15 minute, pressure head that was required to elute the bound sCD44. Similarly, the influence of brief air pressure on HA conformation for the rotary shadowing may also involve pressure per se or a differential pressure effect. In either case, the results of our studies suggest a pressure-dependent response on HA conformation. A feature of HA which is not well appreciated is that HA has clusters of contiguous CH groups, forming hydrophobic patches.5 Within HA polymers in solution, the hydrophobic patches disturb the water structure. To return to the lowest possible free energy state, the hydrophobic groups within HA polymers associate with one another. A decrease in hydrophobic surface area causes a decrease in order and thus an increase in entropy. The exact amount of bond energy is unknown, although it is likely that the hydrophobic patches utilize van der Waals attractive forces, which are quite weak, as well as secondary valencies.6 HA tertiary structures in solution reversibly change, which controls HA biological activity.6 As noted by Scott,7 HA structure in solution is transiently disrupted by mechanical stress, such as stirring or moving a synovial joint, and HA structure would revert on releasing the stress. Consequently, ambient atmospheric pressure decidedly influences an individual traveling by air from Chicago, Illinois to Denver, Colorado and may affect HA structure, but the pressure effect on the individual or on HA would be limited in time, as predicted by the second law of thermodynamics. That is, the pressure would change the free energy, and water would be forced into the hydrophobic patches. As a result, the passenger would be slightly dehydrated, a well-known consequence of high altitude,8 and the eye pressure would not change. In short, an individual acclimatizes, and HA conformation adjusts to the ambient atmospheric pressure. The second issue raised was the possible effect of sCD44 in plasma on vascular endothelial cells. Indeed, the plasma sCD44 concentration is 450 times greater than aqueous humor concentration; however, the cytotoxic effects of sCD44 on trabecular meshwork cells and retinal ganglion cells are blocked by exogenous HA.1 Moreover, smooth muscle cells and human cortical neurons are not affected by sCD44.9 Therefore, vascular endothelia may not be affected by sCD44 because vascular endothelia are insensitive to sCD44 or have a sufficient pericellular concentration of HA to block sCD44 toxicity. Although the notion that a shed protein such as sCD44 acts as a pro-death protein to susceptible cells may seem unique, recent evidence indicates that the converse occurs. The ectodomain of Klotho protein is shed and is detected in the blood and cerebrospinal fluid in humans.10 Klotho protein functions as an anti-aging protein by apparently suppressing tyrosine phosphorylation of IGF1 receptors and prevents oxidative stress.11 Absence of Klotho gene in mice leads to multiple age-related disorders. Paul A. Knepper1,2 1Department of Ophthalmology and Visual Science, University of Illinois
at Chicago, Chicago, Illinois References 1. Knepper PA, Miller AM, Choi J, et al. Hypophosphorylation of aqueous humor sCD44 and primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 2005;46:2829-2837. |
|||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
eLetters: