Hypothesis: Poly-(R)-3-hydroxybutyrate is a major factor in intraocular pressure

Hypothesis: Poly-(R)-3-hydroxybutyrate is a major factor in intraocular pressure
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  See discussions, stats, and author profiles for this publication at: Hypothesis: Poly-(R)-3-hydroxybutyrate is amajor factor in intraocular pressure  Article   in  Medical Hypotheses · May 2009 DOI: 10.1016/j.mehy.2008.10.036 · Source: PubMed CITATIONS 7 READS 139 5 authors , including: Some of the authors of this publication are also working on these related projects: Origins and Evolution of Cellular Life   View projectpathomechanism of neurological disorders such as Parkinson diseases   View projectVic NorrisUniversité de Rouen 180   PUBLICATIONS   2,852   CITATIONS   SEE PROFILE Rosetta N. ReuschMichigan State University 76   PUBLICATIONS   2,318   CITATIONS   SEE PROFILE All content following this page was uploaded by Rosetta N. Reusch on 27 July 2014. The user has requested enhancement of the downloaded file.  Hypothesis: Poly-(R)-3-hydroxybutyrate is a major factor in intraocular pressure Vic Norris a, * , Helene Bresson-Dumont b , Etienne Gardea c , Rosetta N. Reusch d , Denis Gruber c a Department of Biology, University of Rouen, Mont Saint Aignan 76821, France b Clinique Sourdille, 8, rue Camille-Flammarion, 44000 Nantes, France c Service d’ophtalmologie, CHU Charles Nicolle, Rouen 76000, France d Department of Microbiology and Molecular Genetics, Biomedical Physical Sciences Bldg. Michigan State University, East Lansing, MI 48823, USA a r t i c l e i n f o  Article history: Received 12 October 2008Accepted 15 October 2008 s u m m a r y Short chain poly-(R)-3-hydroxybutyrate (cPHB) is a ubiquitous molecule that readily adheres to others,thatiscovalentlyaddedtoproteinsandthatforms,withpolyphosphate,ionchannels.HighlevelsofcPHBhave been implicated in atherosclerosis and in diabetes. Here, we propose a hypothesis in which cPHBadheres to filaments in the extracellular matrix and this raises intraocular pressure. In a corollary, wepropose that a positive feedback relationship between cPHB adherence to filaments, raised IOP and fila-ment stretching constitutes a switch.   2009 Elsevier Ltd. All rights reserved. Introduction Poly-(R)-3-hydroxybutyrate (PHB) is a linear head-to-tailhomopolymer of (R)-(3-hydroxybutyric acid) (Fig. 1). Althoughsometimes known as a storage material of high molecular massin certain bacteria, a short chain formof PHB witha lowmolecularmass (<150) appears to be ubiquitous in prokaryotes and eukary-otes. Short chain PHB is always found complexed to other macro-molecules and has been termed complexed PHB or cPHB. cPHB isamphiphilic and can form hydrophobic bonds via the methylgroups and hydrogen bonds (or coordinate bonds to cations) viaits ester carbonyl oxygens. It can bind to hydrophobic regions of proteins and make them more hydrophilic or the reverse. In addi-tion, the extremeflexibilityof cPHBallows it to change orientationor bonding pattern when the environment changes [1,2]. cPHB is amajor constituent of ion channels and pumps where it forms apartnership with polyphosphate [3–5]. cPHB is also known to dis-solve in membranes where it can allow passage of ions, and per-haps fluids, without selective ion channels [6]. Less well-knownis that cPHB has been found in a wide variety of tissues includingthe eye [7], that cPHB addition to proteins is a major class of  post-translational modification [8,9], and that cPHB is implicatedinseveraldiseases,includingdiabetes[7].Itisthereforeinterestingto explore the possible involvement of cPHB in other, possibly re-lated, diseases. One of these diseases is glaucoma.A major risk factor for most glaucomas and focus of modellingand treatment is increased intraocular pressure, IOP. Intraocularpressure is a function of production of liquid aqueous humor bythe ciliary body of the eye and its drainage through the trabecularmeshwork. The pathology of intraocular hypertension is mostlybased on drainage problems through the trabecular meshworkand Schlemm’s canal [10]. In most cases of primary open angleglaucoma, POAG, the aqueous humor outflow from the anterioreye chamber is impeded which raises IOP and causes ganglion celldeath in the neural retina (for references see [11,12]).As a ubiquitous molecule with several modes of action that isalready implicated in several diseases, cPHB could play a role inmany different processes associated with glaucoma including flowthrough the meshwork, regulation of the cytoskeleton, mechano-transduction, modelling of the extracellular matrix and instabilityin blood flow. Here, we propose a hypothesis and a corollary thatspell out the roles that cPHB could play and we argue that thehypothesis is plausible by reporting a preliminary result. If thishypothesis can be validated, novel and relatively cheap therapiesbased on lowering cPHB levels could be envisaged.  The hypothesis Accumulation of PHB in the trabecular meshwork traps debris,triggers aggregation of fibers and hence reduces the size of thepores and the flow of the aqueous humor through the meshwork.This reduction in efflux results in higher ocular pressures. The ini-tial accumulation of PHB in the meshwork of glaucoma patients isdueeithertohigherlevelsofcirculatingPHBintheaqueoushumoror to particular characteristics of the meshwork that cause it tohaveagreateraffinityforPHBortoboth.Acorollarytothehypoth-esis is that a positive feedback relationship or switch exists suchthat binding of PHB to the meshwork causes a higher ocular pres-sure that stretches the filamentous constituents of the meshworkand thereby reduces their charge density and causes key confor-mational changes; thisloweredchargedensityandconformationalchange lead to more PHB binding to the meshwork. 0306-9877/$ - see front matter   2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.mehy.2008.10.036 *  Corresponding author. Tel.: +33 235528383. E-mail address: (V. Norris).Medical Hypotheses 73 (2009) 398–401 Contents lists available at ScienceDirect Medical Hypotheses journal homepage:  Predictions cPHB levels in the meshwork of patients should be higher than those inthe rest of the population An effective absence of cPHB in the meshwork of such patientswould disprove the hypothesis and, before proposing it, we deter-mined cPHB levels in five patients using the crotonic acid assay(see [7] and Fig. 2). The ratios of cPHB to total protein were 270, 420, 420, 510 and 970 l g/g. These are three times higher on aver-agethanthehighcPHBlevelsfoundintheeyesofdiabeticrats(seebelow). High cPHB levels should be found in diseases known to affect glaucoma Diabetics have been consideredto be three times more likely todevelop primary open angle glaucoma [13] but this is an area of controversy [14,15]. Insulin resistance indices are associated withIOP (  p  <0.05) and IOP is higher in subjects with metabolic syn-drome [16]. cPHB is a metabolic intermediate (Fig. 2) that consti- tutes 70% of the ketone bodies produced during ketoacidosis inuncontrolled type 1 diabetes. Not surprisingly, the concentrationof cPHBintheplasmaof diabeticratswasfoundtobesixfoldhigh-erthanthatofhealthycontrolratswhilstlevelsofcPHBintheeyesofdiabeticratsarethreetimeshigher(around20 l g/gwetweight)thanthoseof normalrats(around7 l g/gwet weight)(roughly130and 45 l g/g, respectively, when a 55% protein and 70% water con-tent are taken into account) [7].Arteriosclerosis is associated with glaucoma and risk factors forarteriosclerosis are also risk factors for an increase in IOP (but forarterial stiffness see [17] and for other explanations see [10]). Hemodynamically significant carotid artery stenosis, for example,is associated with glaucoma [18]. In a recent study of 16,000 peo-ple, mean IOP was significantly higher in the high-risk coronaryheart disease group than in the low-risk group based [19]. Lipidandcarbohydratemetabolismiscentraltothedevelopmentofdia-betesandalsotothesynthesisofcPHBandcholesterol.Sharingthecommon intermediate, acetoacetyl-CoA, causes both cPHB andcholesterol synthesis to be regulated by changes in intracellularconcentrations of acetyl-CoA (Fig. 2). Acetyl-CoA concentrationsareinturnsensitivetoglucoselevels. Consequently, long-termrisein ambient glucose may be expected to result in increased concen-trations of both cPHB and cholesterol. It is again significant thatplasmacPHBconcentrationscorrelatestronglywithatherogenicli-pid profiles, that cPHB is present in the very low density and lowdensity lipoproteins (but it is virtually absent from the high den-sity lipoprotein), and that cPHB is present in plaques where itmay nucleate atheroma formation [20,21]. High cPHB levels should characterize groups at risk of glaucoma Higher age, thinner corneal thickness, and myopia are also riskfactors for primary open angle glaucoma. People with a family his-tory of glaucoma have about a 6% chance of developing glaucoma.Those of African descent are more likely to suffer from glaucoma,Asians are prone to develop angle-closure glaucoma, and Inuithave a 20–40 times higher risk than Caucasians of developingprimary angle closure glaucoma (see for example [13,22]). Thesegroups are predicted to have higher levels of cPHB. Predictions from the corollary of a switch: lower cPHB levels intrabulectomy patients The positive feedback between binding of cPHB to the mesh-work and filament stretching depends on ion interactions with fil-aments that either are – or can become – charged. This is the caseof the extracellularmatrices of the meshwork beams, juxtacanalic-ular region and Schlemm’s canal inner wall which contain fibrillarand non-fibrillar collagens, elastin-containing microfibrils, matri-cellular and structural organizing proteins, glycosaminoglycansand proteoglycans. In particular, the glycosaminoglycans containmany carboxyl and sulfate groups, are negatively charged at phys-iological pH and have long been implicated in reducing outflow asshown, for example, by injection of enzymes that degrade them(for references see [23]) whilst the corneoscleral meshwork con- Fig. 2.  Pathways for PHB production (from [7] with permission). Fig. 1.  The (R)-(3-hydroxybutyric acid) subunit of poly-(R)-3-hydroxybutyrate(PHB). V. Norris et al./Medical Hypotheses 73 (2009) 398–401  399  tainsalargeproportionofelastin,afilamentthatmaybindcalciumto become highly charged [24]. Operations to restore flow and re-duceIOPmayreducethestretchingofthemeshwork.Significantly,elastincan be stretched1.5  . A less extendedmeshwork wouldbeexpected to have a higher density of charge on its constituent fila-ments and, provided that the cPHB switch is reversible, this is pre-dicted to lead to a decrease in cPHB bound to the meshwork.Hence, following a trabeculectomy, the meshwork of patientsshould reveal a significant decrease in cPHB levels. Although thisprediction has to verified, it should be noted that there is an abun-dance of mechanisms whereby the dynamics of the extracellularmatrix may ensure IOP homeostasis in response to pressure andmechanical stretching of the meshwork; these mechanisms in-clude the turnover of the matrix, splicing of alternative mRNAsandcytoskeletal reorganizations (for referencessee [23]). Mechan-ically induced changes affecting the extracellular matrix and asso-ciated with open angle glaucoma have indeed been identified (forreferences see [25]). For example, mechanical stimulation of lam-ina cribrosa cells (which lie in a region of the optic nerve headundergoing stretching in POAG) resulted in differential expressionof genes encoding several components and modulators of theextracellular matrix including elastin, collagen IV, VI, VIII, IX, XIV,fibulin 1, thrombospondin 1, and VEGF [25]. Discussion There are several ways in which cPHB as a ubiquitous moleculemight act synergistically to raise IOP. First, cPHB is a ‘sticky’ mole-culethatassociatesreadilywithothers,that asshownhereispres-ent within the eye [7] and that could directly obstruct aqueousflow. Second, post-translational modification of proteins by cova-lent addition of cPHB could, for example, modify their propertiesand hence modify the behaviour of trabecular meshwork cells[8]. Third, insertion of cPHB and its partner, polyphosphate, intomembranes creates voltage-operated calcium channels into theplasma membrane [26] and hence their insertion into the plasmamembrane of meshwork cells could seriously perturb calcium sig-nalling. Fourth, the capacity of cPHB to allow ions to traverse themembrane  without   passing through selective ion channels [6]would also constitute a major perturbation (and indeed could leadtoaroleforcPHBinothereyediseaseswhereionchannelsarepar-ticularly important such as diabetic retinopathy [27,28]).One of the common problems in the regulation of complex sys-temssuchasaqueousflowintheeyeishowtoobtain–andhowtoavoid – coherent, coordinated behaviour. In such coordination,mechanotransduction,cPHB,andioninteractionsonchargedintra-cellular and extracellular filaments could play a central role. Ioninteractions with filaments can take more than one form. Forexample,calciumandothercounterionscondenseontoanddiffusealong linear polymers such as actin filaments or glycosaminogly-cans at a critical value of the charge density of the polymer [29–31]; these counterions also  decondense  from charged filamentswhen the charge density is lowered as by stretching. Anotherexample of ion interaction with filaments is that of calcium bind-ing to specific glycine sites in elastin to change their charge andconformation, possibly triggering biomineralization [24]. In both examples, stretching of the filaments should affect their interac-tions with ions which cPHB could modify: cPHB interaction withextracellular elastin and other filaments could stabilise certainconformations and charge densities whilst cPHB interaction withactin and other intracellular filaments (or with associated regula-tory proteins), via perhaps cPHB addition to residues that are nor-mally phosphorylated, could interfere with feedback cycles of protein phosphorylation and filament assembly [32]. Finally,cPHB/polyphosphate ion channels located within focal adhesionscould mechanically couple ion changes inside and outside cellsvia the direct diffusion of ions along intracellular and extracellularfilaments. This could underpin two way signalling (for referencessee [11]) and some of this signalling should be expected to havea basis in mechanical relationships with the extracellular matrix[25,33].A considerable pharmaceutical armory already exists for treat-ing glaucoma. Prostaglandin analogs increase uveoscleral or tra-becular outflow, topical beta-adrenergic receptor antagonistsdecreaseaqueoushumorproduction(asdoalpha2-adrenergicago-nists which also increase uveoscleral outflow), carbonic anhydraseinhibitors decrease secretion of aqueous humor, and miotic agentsor parasympathomimetics tighten the trabecular meshwork to in-crease outflow. Should cPHB indeed be a major factor in raisingIOP, drugs designed to lower cPHB levels should be added to thislist. Moreover, once an enzyme capable of hydrolyzing PHB specif-ically has been identified and purified, this enzyme might be in- jected into the eye to clear PHB and thereby restore aqueous flow. 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