extfieldsofstudy: Chemistry Medicine

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Systems approach to the study of stretch and arrhythmias in right ventricular failure induced in rats by monocrotaline.

We demonstrate the synergistic bene ﬁ ts of using multiple technologies to investigate complex multi- scale biological responses. The combination of reductionist and integrative methodologies can reveal novel insights into mechanisms of action by tracking changes of in vivo phenomena to alterations in protein activity (or vice versa ). We have applied this approach to electrical and mechanical remodelling in right ventricular failure caused by monocrotaline-induced pulmonary artery hypertension in rats. We show arrhythmogenic T-wave alternans in the ECG of conscious heart failure animals. Optical mapping of isolated hearts revealed discordant action potential duration (APD) alternans. Potential causes of the arrhythmic substrate; structural remodelling and/or steep APD restitution and dispersion were observed, with speci ﬁ c remodelling of the Right Ventricular Out ﬂ ow Tract. At the myocyte level, [Ca 2 þ ]i transient alternans were observed together with decreased activity, gene and protein expression of the sarcoplasmic reticulum Ca 2 þ -ATPase (SERCA). Computer simulations of the electrical and struc- tural remodelling suggest both contribute to a less stable substrate. Echocardiography was used to estimate increased wall stress in failure, in vivo . Stretch of intact and skinned single myocytes revealed no effect on the Frank-Starling mechanism in failing myocytes. In isolated hearts acute stretch-induced arrhythmias occurred in all preparations. Signi ﬁ cant shortening of the early APD was seen in control but not failing hearts. These observations may be linked to changes in the gene expression of candidate mechanosensitive ion channels (MSCs) TREK-1 and TRPC1/6. Computer simulations incorporating MSCs and changes in ion channels with failure, based on altered gene expression, largely reproduced experimental observations.

Reduction and integration
While Systems Biology has been interpreted by some as confined to the study of genes and proteins, there is no necessity for such a narrow definition (Kohl et al., 2010;Kuster et al., 2011). In addition the overall goals of Systems Biology seem similar to those long held by Integrative Physiologists (Greenhaff and Hargreaves, 2011). The approach described here is in keeping with ideas presented in the above articles. It stems from the self-evident conviction that combining technologies to allow a reductionist (organism to gene) and/or integrative approach (gene to organism) to the study of a complex problem, such as heart failure, will reveal more information about 'the system' than study at a single level.

The right ventricle and pulmonary artery hypertension
Pulmonary artery hypertension (PAH) is a progressive disease caused by vasoconstriction and/or cell proliferation in the pulmonary vasculature. Despite new agents that specifically target the vasculature (Rhodes et al., 2009), median survival time is 5e6 years (Naeije, 2010;Galie et al., 2009). Serious consequences of increased pulmonary artery pressure are right heart hypertrophy, dilatation, arrhythmias and failure. Right ventricular (RV) failure is the major cause of death in sufferers of PAH (Haddad et al., 2008;Bogaard et al., 2009), however the RV is not currently a target for therapy, other than by the supportive use of diuretics and digoxin (Galie et al., 2009). There is an acknowledged need for further study of the failing RV (Haddad et al., 2008;Bogaard et al., 2009;Voelkel et al., 2006).
Electrical remodelling is seen in patients with PAH, for example increased QT interval and time from T-wave peak to end (Hlaing et al., 2005) and the association between QT prolongation and increased mortality (Rich et al., 2013). The RV is anatomically and functionally distinct from the left ventricle (LV) and works under different mechanical conditions. The thin wall of the RV makes it susceptible to distension by sudden increases in venous return (Bristow et al., 1998;Mebazaa et al., 2004;Voelkel et al., 2006). Mechanical stimuli, such as increased stress, due to elevated afterload and strain due to diastolic chamber dilation are predicted to be important factors in the progression to RV failure in PAH (Bristow et al., 1998;Mebazaa et al., 2004). In support of this idea, chronic pharmacological preload reduction in mice was shown to prevent the development of exercise-induced arrhythmogenic right ventricular cardiomyopathy (Fabritz et al., 2011).

MCT model
The monocrotaline (MCT) model of PAH in rats is well established. MCT is a pyrrolizidine alkaloid from the plant Crotalaria spectabilis. A single injection of MCT (60e80 mg/kg) causes injury to the vascular endothelium of the lung, hypertrophy of the pulmonary vasculature and pulmonary hypertension (Kay et al., 1967). Compensated RV hypertrophy occurs after 2e3 weeks and right heart failure after 3e5 weeks Campian et al., 2006;Bogaard et al., 2010;de Man et al., 2012). We have reported the model has a pro-arrhythmic substrate (Benoist et al., 2011(Benoist et al., , 2012 and it shares some electrical remodelling characteristics with human patients (see (Benoist et al., 2011(Benoist et al., , 2012Hlaing et al., 2005;Rich et al., 2013). There is evidence for a chronic increase in wall stress (Stones et al., 2013).

Alternans and arrhythmias
Alternans are known to be precursors of serious arrhythmias (Cutler and Rosenbaum, 2009). They manifest as beat to beat variations in amplitude of the T-wave in the ECG (Qu et al., 2010), in action potential duration (APD) and/or [Ca 2þ ]i transient amplitude (Laurita and Rosenbaum, 2008;Cutler and Rosenbaum, 2009;Weiss et al., 2011). They may be the consequence of steep APD restitution and/or dysfunction in Ca 2þ handling (Pruvot et al., 2004). In tissue, alternans can be concordant, when adjacent regions alternate in phase or be discordant where regions are out of phase. The dispersion of repolarisation created by discordant alternans makes them more likely to descend into serious arrhythmias.

Mechanical stimulation and arrhythmias in the heart
Mechanical stimuli, such as stress and strain, have important effects on the normal and diseased heart. Acute increases in cardiac chamber volume stretch the myocardium and increase its contractility (the Frank-Starling law of the heart) and the intrinsic beating frequency of the cardiac pacemaker (the Bainbridge effect). Chronic increases in chamber volume or wall stress, caused e.g. by hypertension, trigger compensatory hypertrophy but ultimately Fig. 1. A. T-wave alternans from the ECG of an unrestrained, conscious, FAIL animal. The T-wave shows a long (L), short (S) pattern. B Discordant alternans in an isolated heart from a FAIL animal revealed by optical mapping. Differences between beat 1 and 2 in zones a and b are out of phase for both AP amplitude and AP duration. C. Increased APD and dispersion of APD in the RV of a FAIL animal compared to a CON animal. D Difference in APD 80 between RVOT and mid-RV at 5 Hz. In CON hearts APD in the RVOT region was shorter than the mid-RV but longer in MCT treated hearts (***P < 0.001 N ¼ 11 CON and 9 MCT hearts), thus APD remodelling was greater in the RVOT than the mid-RV. E APD restitution was significantly steeper in FAIL than CON hearts or hearts from animals with stable hypertrophy (HYP). A, B, C and E modified from (Benoist et al., 2012). lead to heart failure. Acute stretch can also trigger arrhythmias in both ventricles (Franz et al., 1992) and atria (Bode et al., 2001) possibly via the activation of mechanosensitive ion channels (MSCs) see articles in (Kohl and Ravens, 2003;Kohl et al., 2005) and (White, 2006).
There is some evidence to suggest that stretch-activated arrhythmias are more prominent in diseased hearts, e.g. in atrial fibrillation (Bode et al., 2001), heart failure (Wang et al., 1994) and compensated hypertensive hypertrophy (Salmon et al., 1997). This may be due to changes in MSC activity and/or a pro-arrhythmic state in disease, caused by changes such as altered dispersion of repolarisation (Antzelevitch, 2005). However, given the important role stress and strain play in many cardiac dysfunctions, surprisingly few studies have investigated their acute roles in the modulation of mechanical and electrical activity in diseased tissue.

Mechanosensitive ion channels
MSCs are activated by increased tension in the lipid membrane and/or the cytoskeleton (Hamill & McBride, Jr., 1996;Hamill and Martinac, 2001). In the heart there is evidence for 2 major types of MSC (excluding volume-regulated channels); K þ -selective MSCs (MSC K ) and non-specific cationic MSCs (MSC NS ) which conduct Na þ and Ca 2þ (Kohl and Ravens, 2003;Kohl et al., 2005;White, 2006). MSC K appears to include TREK-1 (Xian et al., 2006;Kelly et al., 2006) a member of the 2 poreedomain K þ channel family (Patel and Honore, 2005). It has been reported that the levels of TREK-1 protein in the heart can be increased in systemic hypertension (Cheng et al., 2006) and by acute stretch (Zhao et al., 2007).
Although MSC NS is highly implicated in the effects of acute stretch (Kohl and Ravens, 2003;Kohl et al., 2005;White, 2006) their identity is uncertain. Research has centred upon Transient Receptor Potential (TRP) channels, since (Maroto et al., 2005) identified TRPC-1 as a vertebrate MSC NS (Barritt and Rychkov, 2005). Although this interpretation has been questioned with respect to channels expressed in cell lines (Gottlieb et al., 2008), the initial observation has sparked research in native tissue which has provided evidence for TRPC function in the heart (Ju et al., 2007) and as MSCs, in both skeletal (Yeung et al., 2005) and cardiac muscle (TRPC-6) (Dyachenko et al., 2009). TRPC-1 is increased in cardiac hypertrophy e.g. (Ohba et al., 2007) and TRPC-3 by stretch (Dalrymple et al., 2007). The potential involvement of multiple TRPC channels may be linked to channel heteromeric sub-unit structure and common function within a given cell (Beech, 2005;Dietrich et al., 2006).
MSCs are thought to modulate action potential shape and generate stretch-activated arrhythmias both by the generation of ion currents and in the case of MSC NS , the modulation of intracellular Ca 2þ , but there has been relatively few studies of MSCs in diseased tissue (Kohl and Ravens, 2003;Kohl et al., 2005). The spontaneously hypertensive rat is more susceptible to stretchactivated arrhythmias (Evans et al., 1995;Kim et al., 2012) via mechanisms associated with MSC NS (Salmon et al., 1997) and these animals are reported to have a greater density of such currents (Kamkin et al., 2000).
We are interested in the electrical and mechanical remodelling that occurs in heart failure. This article will describe our investigation of two aspects of remodelling in the MCT model; the occurrence of alternans, a pre-cursor of serious arrhythmias and the acute effect of mechanical stimulation. Our aim is to Fig. 2. A Fibre angle assessed by DT-MRI in a CON and FAIL heart. B RV fibre angle plotted against distance from mid-wall, the change in fibre angle is slower and more varied in the FAIL heart which may indicate increased structural heterogeneity. C Snapshots showing simulated ventricular tachycardia (VT) in CON and FAIL models. Red is excited tissue, blue resting: waves are rotating anticlockwise. D VT was initiated in various ventricular locations (192 simulations in total) and stability (time to breakup, e.g. into fibrillation) measured. Stability of VT decreased in FAIL compared to CON. Restoring CON geometry (GEO) but maintaining FAIL electrophysiology (EP) recovered stability, whereas CON EP with FAIL GEO decreased stability further. *P < 0.05. demonstrate the utility and feasibility of a systems approach to understand mechanisms associated with a complex problem such as heart failure induced by PAH.

Monocrotaline model of PAH
Wistar rats (200 g) were either given a single injection of saline (CON) or a single injection of 60 mg/kg MCT to induce PAH and RV failure within 3e4 weeks (FAIL). Some animals were given a lower dose of 30 mg/kg MCT to induce a non-failing hypertropic phenotype (HYP) (Benoist et al., 2011(Benoist et al., , 2012Stones et al., 2013). When FAIL animals showed signs of heart failure (e.g. weight loss, lethargy) they were humanely killed, HYP and CON animals were killed on equivalent days. All experiments were performed with local ethical and UK Home Office approval.
Methods used in this study have been previously described, unless otherwise stated; optical mapping of electrical activity, myocyte Ca 2þ transients and contraction, protein measurement by Western blotting and mRNA by real-time PCR in (Benoist et al., 2011(Benoist et al., , 2012Stones et al., 2013;Stones et al., 2009); force-pCa in permeabilised single myocytes (Cazorla et al., 2005); forcesarcomere length (SL) relationships in intact single myocytes (Calaghan and White, 2004). All experiments were performed at 37 C unless otherwise stated.

Echocardiography
Echocardiography was performed on rats anaesthetised with 5% isofluorane (mixed with O 2 ) and maintained at 1.5% during examination. Images were acquired with a GE Healthcare Vivid7 with a 10S probe at a frequency of 11.5 MHz. A non-invasive, in vivo estimate of RV wall stress was made: (s) ¼ Pr/2h where P ¼ transwall pressure difference; r ¼ radius of curvature and h ¼ wall thickness. RV systolic pressure was estimated from pulmonary artery acceleration time (PAAT) using pulse wave Doppler (Jones et al., 2002). Radius of curvature of the RV was measured in the short axis in B mode and RV wall thickness in M mode.

Whole heart stretch and arrhythmia
Isolated hearts were Langendorff perfused at 37 C with a bicarbonate-based physiological saline solution. The RV monophasic action potential duration (MAPD) was measured in hearts stimulated at 5 Hz, before and after an increase in RV volume by inflation of an indwelling fluid filled balloon. The balloon was connected to a pressure transducer allowing RV force to be measured. Stretch was achieved by inflating each RV to the individual volume that gave maximum active force development.

Ventricular structure
Ventricular structural remodelling was quantified using diffusion tensor magnetic resonance imaging (DT-MRI) as previously described (Benson et al., 2011). Briefly, fixed hearts were immersed in the perfluoropolyether Fomblin, then high-resolution (200 mm isotropic) imaging of fibre structure was performed using an NMR spectrometer with a 9.4 T magnet. Diffusion of protons was measured throughout the tissue in a set of 12 optimized directions using a three-dimensional diffusion-weighted spin-echo sequence with reduced encoding at 20 C: repetition time: 500 ms; echo time: 15 ms; diffusion gradients with 2 ms duration and 7 ms separation; b ¼ 1000 s/mm 2 . Diffusion tensors, and the eigenvectors of these tensors, were calculated from the diffusion measurements, alternans for a RV myocyte isolated from a FAIL animal stimulated at 9 Hz. B SERCA activity estimated as K SERCA was significantly reduced in RV myocytes from FAIL animals. K SERCA was calculated as the difference between the rate constant of decay of the electrically stimulated Ca 2þ -transient at 5 Hz (e, representing Ca 2þ removal by SERCA and NaeCa exchange) and caffeine stimulated Ca 2þ transient (c, representing Ca 2þ removal by NaeCa exchange). C Levels of SERCA protein, estimated by Western blot, were significantly lower in HYP and FAIL RV myocardium than CON (data expressed as % of the mean density of CON samples, N ¼ 6 in each group) D. In the HYP group SERCA density was significantly correlated with the RV weight:body weight ratio R 2 ¼ 0.7, P < 0.05). E Levels of mRNA for SERCA, estimated by real-time RT-PCR, were significantly lower in FAIL myocardium than HYP or CON (data expressed relative to a calibrator sample normalised to the housekeeper gene 18S, N ¼ CON 12, HYP 7, FAIL 14). A and B modified from (Benoist et al., 2012). *P < 0.05; **P < 0.01; ***P < 0.001. before fibre structure was quantified, principally as the fibre inclination or helix angle, from these vectors using in-house software.

Computer simulations
For structural and electrical remodelling interactions, levels of expression of mRNA for individual ion channels and action potential profiles obtained from LV and RV of CON and FAIL rats (Benoist et al., 2011) were used to scale ion channel conductances in a single rat ventricular myocyte model (Pandit et al., 2001). These were then incorporated into heterogeneous and anisotropic 3D ventricle models (3 CON and 3 FAIL) with anatomy reconstructed from DT-MRI at 200 mm resolution (Benson et al., 2011).
To simulate our in vitro stretch experiments the adapted (Pandit et al., 2001) model was further modified to reproduce our in vitromeasured MAPDs at 25%, 50% and 90% repolarisation with no stretch (Fig. 9), by reducing I to , I ss and I Ks maximal conductances by 70% and increasing their activation time constants five-fold.
MSC currents were incorporated as in (Healy and McCulloch, 2005). Currents were carried through TRPC1/6 (IMSC NS ) and TREK-1 (IMSC K ), based on (Xian et al., 2006) with parameters adjusted to reproduce outcomes measured by us in vitro. IMSC NS had a linear currentevoltage relationship given by: 10% stretch, l is the magnitude of the applied stretch (20% in this study) and EMSC NS ¼ À10 mV is the current reversal potential. IMSC K is modelled as an outwardly-rectifying current: with gMSC K ¼ 2500 nS. Note that our formulations result in IMSC K being significantly larger than IMSC NS at positive potentials, with the absolute magnitudes of the two currents being similar at resting potentials, as predicted experimentally by (Xian et al., 2006). To simulate MSCs in FAIL, we reduced gMSC NS by 11% and gMSC K by 87% based on mRNA changes (Fig. 10) and TRPC 1:TRPC6 abundance which was scaled at 8:1 based on the cycle threshold in real time RT-PCR experiments being 3 higher in TRPC6 (3 2 ¼ 8 fold reduced initial copy number). Action potentials were recorded after a train of 10 stimuli were applied at 5 Hz with a 5 nA stimulus current of 1 ms duration. For simulations with stretch, we applied 20% stretch immediately prior to the final stimulus. The level of stretch was an estimation of the change in length reported to occur from slack length (SL 1.8e1.9 mm) to the peak of the length-tension relationship (SL 2.2e2.3 mm).

Alternans: in vivo to whole heart
In vivo recordings of ECG by telemetry from conscious, unrestrained FAIL animals revealed T-wave alternans in a sub-set of Fig. 4. A There was a significant decrease in the level of mRNA for PLB In FAIL compared to HYP and CON myocardium, estimated by real-time RT-PCR, (data expressed relative to a calibrator sample and normalised to the housekeeper gene 18S (N ¼ 12 CON, 7 HYP, 14 FAIL hearts). B There was a significant decrease in the level of total PLB protein in FAIL compared to CON and HYP myocardium hearts estimated by Western blot, PLB density normalised to GAPDH (N ¼ 6 in each group). C Levels of protein for phosphorylated Serine 16 (c'AMP_dependent site) and D for phosphorylated Threonine 17 (Ca 2þ -calmodulin dependent site) were significantly reduced in FAIL compared to CON. E levels of phosphorylated Serine 16 and F of phosphorylated Theonine 17 were expressed as a % of total PLB. There was a statistically significant increase in phosphorylated Threonine 17: Total PLB for FAIL compared to CON in contrast the level of phosphorylated Serine 16: Total PLB did not change. *P < 0.05; **P < 0.01; ***P < 0.001. animals (Fig. 1A). T-wave alternans are thought to reflect underlying alternating patterns of ventricular repolarisation. Optical mapping of electrical activity from isolated whole hearts revealed discordant APD alternans, (alternans with regions out of phase, Fig. 1B). APD alternans are more likely to arise when there is increased dispersion of repolarisation (Fig. 1C, D) and/or steeper APD restitution (Fig.1E): both conditions were found in MCTtreated hearts.
It was observed that while the APD in the Right Ventricular Outflow Tract (RVOT) region was typically shorter than the APD measured midway between RV apex and base (mid-RV) in CON rats (in 11/11 hearts) it was significantly longer in MCT hearts (in 8/9 hearts) (Fig. 1C, D). This is interesting because it suggests remodelling is greater in the RVOT, a region where many arrhythmic syndromes and the majority of idiopathic ventricular arrhythmias are found. A longer APD coupled with the late activation of the RVOT is also likely to increase the dispersion of repolarisation.
Structural remodelling can affect electrical conduction and be pro-arrhythmic in its own right (Engelman et al., 2010). DT-MRI was used to measure the rotation of myocardial fibre angles across the RV wall ( Fig. 2A). In 3D DT-MRI reconstructions, there were no changes to LV structure with FAIL, but mean RV wall thickness was increased from 1.6 to 2.2 mm and the rate of transmural fibre rotation decreased from 148 to 86 /mm. The absolute change in angle was not different between CON and FAIL hearts, but the thicker RV wall meant that the rate of change of angle was less in FAIL hearts, decreasing from 73 ± 7.8 /mm to 37 ± 3.6 /mm. In addition the correlation between fibre angle and position in the RV wall was much weaker in FAIL hearts (R 2 ¼ 0.72 ± 0.05 for CON and 0.46 ± 0.07 for FAIL) suggesting a greater heterogeneity of fibre orientation (Fig. 2B, and see (Benoist et al., 2012). Ventricular dimensions and fibre angle rotation were combined with a simulation of electrical activity based on relative mRNA gene expression, the APD 90 increased in FAIL from 36 to 51 ms in LV cells and from 31 to 78 ms in RV cells. Following programmed stimulation in the RV wall, we found an increased propensity for transition to ventricular fibrillation in FAIL, with the structural and functional changes playing a synergistic role in this increase (Fig. 2C, D) suggesting that the transition in FAIL is dependent on both structural and functional remodelling.

Alternans: single cell to gene expression
There is good evidence that electrical alternans are associated with Ca 2þ -handling anomalies. The varying Ca 2þ transient amplitude is thought to generate varying inward NaeCa exchange current which in turn modulates APD. When stimulation frequency was elevated above 5 Hz, Ca 2þ transient alternans were seen in myocytes from FAIL but not CON hearts (Fig. 3A). Ca 2þ transient alternans have been linked to decreased function of Ca 2þ cycling by the sarcoplasmic reticulum Ca 2þ uptake pump (SERCA). In FAIL myocytes there was decreased SERCA activity, assessed by measuring K SERCA , the rate constant of Ca 2þ transient decay in the presence and absence of SR Ca 2þ accumulation (Fig. 3B). It should Fig. 5. Systolic RV wall stress calculated using echocardiography. A Radius of curvature (solid line) was found by fitting a circle at the widest part of the junction between the RV free wall and septum during systole measured in B-mode. B RV wall thickness (white bars) and internal diameter (arrows) measured during systole in M-mode: hypertrophy and dilatation were evident in FAIL rats. C Pulmonary artery acceleration time (PAAT) measured as the time from onset to peak flow rate using pulsed-wave Doppler. PAAT was reduced in FAIL rats. D Mean values for RV systolic pressure, RV radius of curvature and RV wall thickness and calculated wall stress (see Methods) in 7 CON and 5 FAIL hearts. There was a significant increase in all parameters in FAIL compared to CON animals. **P < 0.01; ***P < 0.001. be noted however that estimates of K SERCA based in part on the decay of electrically stimulated Ca 2þ transients will be influenced by changes in action potential repolarisation time and it is well established that the FAIL action potential is longer than the CON action potential. Consistent with decreased K SERCA was decreased SERCA protein, measured by Western Blot (Fig. 3C) and mRNA levels of the gene expressing SERCA (Fig. 3E) measured by real-time RT-PCR. Interestingly in the HYP group, levels of SERCA protein were negatively correlated with RV hypertrophy (Fig. 3D). SERCA activity is decreased by Phospholamban (PLB): this inhibition is relieved by phosphorylation of PLB. We observed a decrease in both the mRNA (Fig. 4A) and protein (Fig. 4B) for PLB in FAIL hearts. Levels of PLB phosphorylation at the Serine 16 (PKAdependent site) and Threonine-17 (Ca-Calmodulin dependent site) were reduced (Fig. 4C, D). The decrease in phosphorylated Serine 16 was not different to decrease in total PLB (Fig. 4E). The decrease in phosphorylated Threonine 17 was less than the decrease in total PLB (Fig. 4F). Some models of heart failure show decreased levels of SERCA in the presence of maintained PLB and explain decreased SERCA function in terms of altered SERCA:PLB. We did not find such a relationship in the MCT model and conclude the decreased levels of SERCA are the main reason for its decreased function.
The above section of the article is focussed on the demonstration of a systems approach to study alternans and we have not Fig. 6. A Cross-sectional area (XSA) of RV myocytes from FAIL hearts was significantly larger than CON myocytes. There were no significant differences between CON and FAIL myocytes in: B resting sarcomere length (SL). C Absolute active force, normalised to myocyte XSA. D increase in SL in response to stretch E. increase in resting tension per unit stretch. F increase in active tension per unit stretch (n ¼ 17 CON and 25 FAIL myocytes). *P < 0.05. Fig. 7. A Maximal active tension in response to alterations in pCa for triton skinned RV myocytes from CON and FAIL hearts. Experiments were performed at 25 C. Data was collected at SL 2.0 and 2.3 mm and fitted to a Hill equation. Increased SL caused a left shift in the curves. B Change in the pCa50 (index of length-dependent increase in myofilament Ca 2þ -sensitivity) in response to an increase in SL from 2.0 to 2.3 mm for RV and LV myocytes from CON and FAIL hearts. There was a significant difference in DpCa50 between RV and LV in CON myocytes but no differences between CON and FAIL (CON RV n ¼ 12; CON LV n ¼ 12; FAIL RV n ¼ 18; FAIL LV n ¼ 14 from N ¼ CON 3, FAIL 4 hearts). *P < 0.05.
given a full account of arrhythmogenic mechanisms in the MCT model. One important property not dealt with is conduction velocity. This is slowed in FAIL hearts, relative to CON hearts, as stimulation increases (Benoist et al., 2012). Thus, conduction velocity restitution is steeper in FAIL hearts and this could contribute to re-entry type arrhythmias. Table 1 gives whole animal and organ characteristics of the CON and FAIL animals used in the investigation of mechanical modulation. In agreements with previous studies there was an increase in heart weight:body weight and lung weight:body weight in FAIL animals compared to CON. The increase in HW:BW was principally due to RV hypertrophy, indexed as a significant increase in RV:LV weight.

Mechanical stimulation in MCT hearts
Echocardiography from anaesthetised animals was performed in B-mode to calculate the radius of curvature of the RV (Fig. 5A), Mmode to calculate RV wall thickness (Fig. 5B) and Doppler to calculate PAAT (Fig. 5C). Mean data (Fig. 5D) shows greater wall stress in FAIL hearts and thus evidence of chronic increased mechanical stimuli. We previously estimated a 60% increase in wall stress in FAIL hearts (Stones, 2013) based on values from different hearts measured after different experimental procedures. The values presented here were calculated for each heart individually with parameters collected simultaneously. The increase in wall stress is about twice the previous estimate.

Length-dependent changes in tension in single myocytes
To measure the response to acute increase in strain, single RV myocytes were attached to flexible carbon fibres and stretched. Changes in resting and active force were normalised to cell crosssectional area (XSA). Consistent with RV hypertrophy, the XSA of FAIL myocytes was significantly increased (Fig. 6A). There was a trend for resting SL to be shorter in FAIL myocytes but this was not statistically significant (Fig. 6B). Active force normalised to XSA was not altered (Fig. 6C). Myocytes were stretched and resting SL increased by approximately 0.11 mm (Fig. 6). The increase in tension normalised to SL increase was calculated for resting tension (Fig. 6E) and active tension (Fig. 6F) to give an index of length dependent changes. There were no significant differences between CON and FAIL myocytes. These experiments indicated little effect activity of the heart paced at 5 Hz. Inflation caused a brief disruption to the rhythm of the heart. B Interbeat interval before and immediately after balloon inflation, data show mean ± SD. There was a significant increase in the SD of the beat to beat interval in both CON and FAIL hearts, indicating a stretch-induced decrease in rhythmicity. The increase in SD was significantly greater in CON than FAIL hearts. (N ¼ 6 CON, 8 FAIL hearts). ***P < 0.001.  9. A representative monophasic action potential (MAP) traces from a CON and FAIL heart, % amplitude is shown to facilitate comparison of repolarisation. B MAP duration (MAPD) at 25, 50 and 90% repolarisation before (unmarked) and after (S) an increase in RV volume to that giving the maximal active tension in CON and FAIL hearts. Stretch caused a significant decrease in MAPD 25 in CON hearts (N ¼ 6 CON, 8 FAIL hearts). *P < 0.05. on the length-tension relationship of FAIL myocytes. However only SLs at the lower end of the length-tension curve were tested due to technical difficulties in maintaining cell attachment of intact myocytes to carbon fibres at longer SL.
To investigate length-tension relationships over a wider range of SL, permeabilised myocytes were glued to force transducers and force-pCa relationships measured at SL of 2.0 and 2.3 mm (Fig. 7). The relationships were fitted with a Hill equation. Mean data for RV myocytes are shown in (Fig. 7A). There was no significant difference in the maximally activated force of CON and FAIL myocytes nor the slope of the relationships from either the LV or RV. In both CON and FAIL myocytes an increase in SL caused a left-shift in the curve, indicative of a length-dependent increase in myofilament sensitivity to Ca 2þ . The pCa at half maximal activation (pCa 50 ) is an index of myofilament Ca 2þ sensitivity and the change in this value (DpCa 50 ) upon increased SL, an index of the length-dependant change. The DpCa 50 was significantly greater in CON RV than LV but there was no difference between CON and FAIL (Fig. 7B). There are reports of myofilament Ca 2þ sensitivity being increased, decreased and unchanged in heart failure. The effect of heart failure on the Frank-Starling mechanism is also subject of debate with studies finding the mechanism unaltered or depressed (von Lewinski et al., 2009). We have found no evidence of a change in myofilament Ca 2þ sensitivity or in its response to length changes in FAIL myocytes in this model.

Stretch activated arrhythmias in CON and FAIL hearts
Langendorff-perfused isolated whole hearts were stimulated at a frequency of 5 Hz and MAPD was monitored in response to an acute stretch to the volume that gave maximum active force development in each heart. The FAIL hearts were larger than CON hearts and this volume was greater (CON 78.3 ± 7.0 ml, N ¼ 6; FAIL 101.3 ± 8.3 ml, N ¼ 8, P < 0.05). Stretch-induced disruption of rhythm was seen in both groups immediately following stretch (Fig. 8A). Rhythmicity was assessed as the standard deviation (SD) of the beat to beat interval prior and immediately following the stretch. The stretch-induced disruption to rhythm (increase in SD of the beat to beat interval) was smaller in the FAIL hearts (Fig. 8B).
As previously reported, MAPD was longer in the RV of FAIL animals (Fig. 9A). When stable rhythm was re-established following a stretch, MAPD at 25% repolarisation was reduced in CON hearts but not FAIL hearts. There was no effect on MAPD at 50% or 90% repolarisation in either group (Fig. 9B).
These observations were somewhat unexpected given that the MCT model has a pro-arrhythmic substrate and acute stretch is an acknowledged arrhythmic stimulus. It therefore seemed likely these 2 factors would operate synergistically when FAIL hearts were stretched. An additional factor may be that our stretch-stimulus was not sufficient to generate sustained tachycardias or fibrillation and in this situation the index of rhythmicity is linked to refractoriness. It is known that the refractory period of FAIL hearts is longer than CON hearts (Benoist et al., 2011), probably because of the longer APD. The longer the refractory period the lower the maximal rate of excitation and the lower the possible range of beat to beat intervals.
The observation that in CON, APD 25 was shortened by stretch could be explained in terms of the activation of MSC NS and MSC K . At the elevated membrane potentials of the early AP both these channels, if active, would be predicted to generate outward repolarising current that would speed repolarisation. However, during late repolarisation the membrane potential would fall below the predicted equilibrium potential of MSC NS , generating an inward current that would oppose MSC K which would in turn decay as the membrane potential approached the equilibrium potential for K þ . The net effect could be no change in MAPD 90 , as observed.
Data from whole hearts suggested that the electrical response to acute stretch was not enhanced and possibly decreased in FAIL hearts. If electrical changes were dependent upon MSC, this might indicate reduced levels of MSCs in MCT hearts. To test this possibility we measured the expression of mRNA for the genes encoding channels thought to be responsible for MSC NS (TRPC 1 and 6) and MSC K (TREK-1) using real time RT-PCR. In addition we measured expression of the NaeH exchanger (NHE). Though not electrogenic, NHE has been implicated in the generation of stretch-activated current via alterations in [Na þ ]i. In the RV of FAIL hearts there was a decrease in the mRNA levels for TRPC-1 and TREK-1 but an increase in TRPC6 compared to both RV CON and LV FAIL. NHE mRNA levels were unchanged (Fig. 10).
Computer simulation of the activation of MSC NS and MSC K were performed (Fig. 11A). Using the parameters described in the Methods, based on published data for MSC NS and MSC K together with our mRNA-based remodelling data for FAIL, it was possible to reproduce stretch effects that closely mimic our in vitro observations. Stretch shortened the CON rat APD with a greater effect (early in repolarisation) than in FAIL hearts (Fig. 11B). An interesting observation from the simulation is that in FAIL reduction in MSC K with relatively maintained levels of MSC NS generates a net depolarising effect compared to CON. In vitro measurement or computer simulation of either current in isolation would not have identified this interaction. Fig. 10. Real-time RT-PCR measurement of mRNA for proteins thought to represent non-selective cationic mechanosensitive ion channels (TRPC1 and TRPC6), a potassium selective mechanosensitive ion channel (TREK-1) and a mechanosensitive ion exchanger (NHE). Expression is given for myocardium from RV and LV of CON and FAIL hearts. Data expressed relative to a calibrator sample and normalised to the housekeeper gene 18S. In the FAIL RV there was a statistically significant decrease in TRPC 1 and TREK-1 but an increase in TRPC6 compared to CON RV (N ¼ 10 CON and 12 FAIL hearts). **P < 0.01; ***P < 0.001.
Thus the decreased responsiveness of FAIL hearts to stretch may be linked to the decreased expression of MSCs and computer simulations suggest this is a feasible possibility. Decreased expression of MSCs in PAH rats may be a response to the chronic pressure and volume overload that occurs, in order to prevent excessive activation of MSCs.

Conclusion
The technologies we have used are not novel per se but in several instances their combination is for example; the use of DT-MRI, whole heart electrophysiology and real time RT-PCR to create a structural and electrical simulation of the PAH rat heart. The use of multiple methodologies at different levels has enabled us to demonstrate mechanisms associated with the generation of alternans from in vivo function to protein expression and function and to make links that could not have been made by studying a single level. In practice the experimental process was not linear, the observation of Ca 2þ transient alternans in single myocytes and steep APD restitution in isolated hearts led to further studies, both reductionist and integrative in nature. In our investigation of mechanical stimulation, using the 3 techniques; MAP recording, mRNA measurement and computer simulation give a better understanding of the phenomena (and provide synergistic evidence for a mechanism) than would have been available from a single technique. Incorporation of in vivo wall stress data and MSC expression into our simulation is a future possibility. What is apparent from our studies is that it is highly unlikely to find an individual scientist with the skills and time to perform all the techniques discussed in this study, therefore collaboration is both welcome and necessary, 'systems biology' is a team sport.

Editors' note
Please see also related communications in this issue by Ravelli et al. (2014) and Rouillard et al. (2014).


s2fieldsofstudy: Biology Chemistry

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Adsorption of the Inflammatory Mediator High-Mobility Group Box 1 by Polymers with Different Charge and Porosity

High-mobility group box 1 protein (HMGB1) is a conserved protein with a variety of biological functions inside as well as outside the cell. When released by activated immune cells, it acts as a proinflammatory cytokine. Its delayed release has sparked the interest in HMGB1 as a potential therapeutic target. Here, we studied the adsorption of HMGB1 to anionic methacrylate-based polymers as well as to neutral polystyrene-divinylbenzene copolymers. Both groups of adsorbents exhibited efficient binding of recombinant HMGB1 and of HMGB1 derived from lipopolysaccharide-stimulated peripheral blood mononuclear cells. The adsorption characteristics depended on particle size, porosity, accessibility of the pores, and charge of the polymers. In addition to these physicochemical parameters of the adsorbents, modifications of the molecule itself (e.g., acetylation, phosphorylation, and oxidation), interaction with other plasma proteins or anticoagulants (e.g., heparin), or association with extracellular microvesicles may influence the binding of HMGB1 to adsorbents and lead to preferential depletion of HMGB1 subsets with different biological activity.

Introduction
High-mobility group box 1 protein (HMGB1) is a ubiquitous nonhistone DNA binding protein with distinct intra-and extracellular functions. It is crucial for nuclear architecture and has been implicated in DNA replication, repair, and transcription. It acts as a sentinel for nucleic acid-mediated immune responses [1,2] and as a pathogenic inflammatory mediator during sterile and infectious injury [3][4][5][6].
While the secretion of HMGB1 is regulated by phosphorylation and acetylation, its extracellular biological activity and interaction with different receptors depend on the redox state of three conserved cysteine residues at positions 23, 45, and 106. With these residues in a reduced form, HMGB1 induces chemotaxis. With a disulfide bridge between C23 and C45 and a free sulfhydryl at position 106, HMGB1 interacts with toll-like receptor 4 (TLR-4) to stimulate cytokine production, while it loses its biological activity in its completely oxidized form [25,26].
The depletion of HMGB1 by extracorporeal therapies, such as hemofiltration with porous membranes [27] or hemoperfusion with adsorption columns has been reported [28,29]. As HMGB1 possesses two DNA binding domains that interact with negatively charged groups, we tested different anionic polymers for their ability and capacity to bind HMGB1, compared their adsorption efficiency to neutral polystyrene divinylbenzene-based polymers, and correlated the binding characteristics to the physicochemical properties of the polymers. We show here that porosity, size distribution, hydrophobicity, and effective charge density as well as the distribution and accessibility of functional groups on the adsorbent surface are critical determinants of the adsorption characteristics. This implies that a given polymer may preferentially bind subsets of molecules with different posttranslational or oxidative modifications and with different biological activity.

Plasma.
Venous blood was drawn into tubes containing 3.8% trisodium citrate (Vacuette, Greiner Bio-One, Kremsmuenster, Austria) from healthy adult volunteers after written informed consent. Plasma was obtained by centrifugation of the whole blood at 2000 ×g for 10 min at room temperature.

Adsorbents.
Negatively charged and neutral adsorbents of different particle sizes and porosities were tested in this study. PSDVB-16 and PSDVB-30 (trade names CG161 and CG300; both from Rohm & Haas/Dow Chemical), as well as PVP-PSDVB (trade name Cytosorb; Cytosorbents Corporation), are hydrophobic neutral resins that are under evaluation or already in clinical application as selective cytokine adsorbents in extracorporeal blood purification. DALI (Fresenius Medical Care, Bad Homburg, Germany) and ReliSorb (Resindion S.r.l.) are both methacrylate-based polymers functionalized with polyacrylate. DALI is clinically applied for whole blood lipid apheresis. As a third negatively charged polymer, we used the cellulose-based adsorbent Cellufine sulfate (Chisso Corporation), while the neutral Cellufine GCL-2000 served as negative control. Prior to the adsorption studies, adsorbents were extensively washed with pyrogen-free 0.9% NaCl and stored at 4 ∘ C in saline solution until further use.

Adsorbent Characterization.
Particle morphology was analyzed by scanning electron microscopy (SEM) using a TM-1000 Tabletop Microscope (Hitachi, Tokyo, Japan). Samples were washed with 100 vol% ethanol and dried for 12 h at 100 ∘ C in a heating cabinet. Adsorbents were subsequently sputter-coated with gold (Q150R ES, Quorum Technologies). Cellulose-based adsorbents were incubated overnight with 2.5 vol% glutaraldehyde, rinsed with dH 2 O, and dehydrated with increasing concentrations of ethanol (30 to 100 vol%) before sputter coating.
To determine the specific surface and the pore size distribution, nitrogen adsorption and desorption isotherms were recorded at −196 ∘ C and at relative pressures / 0 between 0.001 and 1.0 using an ASAP 2010 V2.00 C physisorption analyzer (Micrometrics Instrument Corp., Norcross, USA). The Brunauer-Emmett-Teller (BET) equation [30] was used to calculate the specific surface area ( BET ). The micropore volume (pore size < 2 nm) was calculated with the Horvath-Kawazoe (H-K) method [31], whereas the mesopore and macropore volume (2-50 and 50-300 nm, resp.) was obtained via the Barrett-Joyner-Halenda method (BJH) [32]. Assuming cylindrical pore geometry, the average pore diameter was calculated as = 4 / BET (with = maximum adsorbed nitrogen volume). Values for the average particle diameter and the charge density of the negatively charged adsorbents were provided by the manufacturers.

Adsorption of Recombinant HMGB1.
Adsorption of HMGB1 to the different polymers was studied in batch experiments using adsorbent-to-plasma ratios of 1, 5, and 10 vol%. Plasma was spiked with recombinant human HMGB1 to a target concentration of 200 ng/mL and incubated with the adsorbents at 37 ∘ C with gentle shaking. Spiked plasma without adsorbent served as a control. Samples were taken after 15 and 60 min and centrifuged immediately at 4600 ×g for 5 min to remove the adsorbents. Supernatants were collected, aliquoted, and stored at −80 ∘ C until further analysis. All experiments were conducted in triplicate. HMGB1 was quantified by enzyme-linked immunosorbent assay (ELISA, Shino-Test Corporation, Kanagawa, Japan) according to instructions of the manufacturer.

Adsorption of HMGB1 Derived from Stimulation of Peripheral Blood Mononuclear Cells.
Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood anticoagulated with heparin (5 IU/mL final concentration) by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare, Uppsala, Sweden) as described [33]. Cells were suspended in medium M199 supplemented with 10 vol% human plasma, 0.02 M HEPES, and 100 M PS. Aliquots of 1 × 10 6 cells per mL of medium were stimulated with LPS (0.01-1000 ng/mL) for 16 h in HydroCell Surface 24well plates (1 mL/well) in humidified atmosphere (5 vol% CO 2 , 37 ∘ C). After stimulation, the cells were pelleted by  centrifugation, the supernatant (conditioned medium) was harvested, and the concentration of released HMGB1 was quantified by ELISA. Conditioned medium was incubated for 60 min with Cellufine sulfate or PSDVB-16, respectively, at an adsorbent-to-plasma ratio of 5 vol%. Adsorbents were removed by centrifugation and HMGB1 remaining in the supernatant was quantified by ELISA.

Physicochemical Characterization of the Adsorbents.
The characteristics of the adsorbents used in this study are summarized in Table 1. The neutral polymers PSDVB-16 and PSDVB-30 are mesoporous polystyrene-divinylbenzene copolymers with average pore sizes of 15 and 30 nm, respectively. The smaller mean pore size of PSDVB-16 is reflected by its higher specific surface area in comparison to PSDVB-30. PVP-PSDVB, which is based on polystyrene-divinylbenzene coated with polyvinylpyrrolidone, exhibited the lowest pore diameter of all hydrophobic resins tested.
The two anionic methacrylate-based adsorbents DALI and ReliSorb differ not only with respect to their surface morphology [34], but also with respect to charge density, which is 530 and 300 equivalents of COOH per mL of dry adsorbent for DALI and ReliSorb, respectively. Cellufine sulfate, the third anionic polymer used in this study, has an approximately tenfold higher charge density.
Adsorption isotherms were obtained by measuring the amount of N 2 adsorbed across a wide range of relative pressures at a constant temperature. The isotherms resembled type IV according to the classification by Brunauer et al. [35], typically occurring on porous adsorbents with pores in the range of 1.5-100 nm. At lower pressures, the slope of the isotherms is given by micropores, while at higher pressures the slope reflects an increased uptake of adsorbate as pores become filled, with the inflection point typically occurring near the completion of the first monolayer ( Figure 1). The neutral polystyrene-divinylbenzene-based adsorbents showed a high BET resulting from the presence of micro-, meso-, and macropores.
The morphology of the adsorbent particles was characterized by scanning electron microscopy (SEM; Figure 2). DALI and ReliSorb, the two methacrylate-based polymers, exhibited a comparably structured inner surface but showed clear differences with respect to their outer surface, which appeared open and porous on scanning electron micrographs for ReliSorb, while it had a closed and smooth appearance for DALI. The polystyrene-divinylbenzene-based resins all had a smooth outer surface but differed with respect to their porosity, which was highest for PSDVB-30 in accordance with nitrogen adsorption measurements.

Adsorption of Recombinant HMGB1
. HMGB1 adsorption was studied both with recombinant human HMGB1 and with HMGB1 derived from stimulated peripheral blood mononuclear cells. Pathological blood levels of HMGB1 have been reported to range between 10 and 150 ng/mL [36]. Therefore, we used a target concentration of 200 ng/mL of recombinant HMGB1 in the adsorption experiments, which are summarized in Figure 3. HMGB1 was efficiently adsorbed by both neutral and anionic polymers. Binding  of HMGB1 to neutral PSDVB beads occurs via hydrophobic interactions between the amphipathic adsorbate and the hydrophobic matrix, as confirmed by the negligible HMGB1 adsorption by neutral hydrophilic cellulose beads. Regarding the polystyrene-divinylbenzene-based polymers, HMGB1 adsorption was higher for PSDVB-30 as compared to PSDVB-16, most likely due to a better accessibility of the pores due to the higher average pore diameter. PVP-PSDVB bound significantly less HMGB1 than the uncoated PSDVB polymers under identical experimental conditions despite an equivalent specific surface area. This lower efficacy can be attributed to a smaller average pore diameter as well as to diminished accessibility of the inner surface due to the coating of the polymer with polyvinylpyrrolidone. Adsorption of albumin, the most abundant plasma protein, was evaluated for the 5 vol% batch experiment. Plasma albumin levels were reduced by 1.4% to 3% for the negatively charged hydrophilic polymers and by 8% to 10% for neutral hydrophobic resins ( Table 2).
The adsorption of cytokines of higher molecular mass, in particular tumor necrosis factor alpha (TNF-; 51 kDa), to PVP-PSDVB polymers is considerably reduced due to a lack of accessibility of their inner surface [37,38]. The same may hold true for HMGB1 (30 kDa), and as discussed further below, its association with a wide range of plasma proteins as well as with microvesicles may additionally influence its binding characteristics. PSDVB-based resins are clinically applied in extracorporeal liver support to remove albuminbound metabolites such as unconjugated bilirubin [39][40][41]. Table 2: Albumin adsorption from plasma after 60 min of incubation at an adsorbent-to-plasma ratio of 5 vol%; = 3.

Adsorbent
Albumin Noteworthy, blocking of HMGB1 activity has been shown to improve hepatocyte regeneration after ischemia/reperfusion injury [2], suggesting that its removal by extracorporeal liver support systems may provide a benefit for liver regeneration. As a DNA-associated protein, we reasoned that HMGB1 would bind to anionic surfaces via electrostatic interactions. Confirming this assumption, all anionic polymers showed efficient binding of HMGB1, with the highest efficiency for Cellufine sulfate, which has a high negative charge density and a small particle diameter, resulting in a large outer surface. Application of Cellufine sulfate as adsorbent for HMGB1 in rat hemoperfusion models resulted in decreased HMGB1 serum levels and improved survival in rats with ischemia-reperfusion injury [28,29].  The two methacrylate-based adsorbents bound similar amounts of HMGB1 as Cellufine sulfate after 60 min (17.5 ± 3.0 versus 16.3 ± 2.8 versus 16.8 ± 2.9 g for Cellufine sulfate, DALI, and ReliSorb, resp.). The delayed binding of HMGB1 to DALI and ReliSorb as indicated by the higher remaining concentrations after 15 min may be due to differences in particle size, with the most favourable surface-to-volume ratio for Cellufine sulfate. This indicates rapid binding of HMGB1 to the adsorbent surface, while diffusion and adsorption to functional groups inside the particles occur gradually over time. DALI is clinically applied to remove low-density lipoproteins in patients with familial hypercholesterolemia, and the adsorption of HMBG1 may provide an additional benefit in the setting of atherosclerosis, which has been shown to trigger the release of HMGB1 from macrophages [42].

Adsorption of HMGB1 Derived from Stimulated Peripheral
Blood Mononuclear Cells. Stimulation of PBMCs with LPS resulted in a time-dependent release of HMGB1 reaching a peak after 16 h. PBMCs treated with increasing LPS concentrations (0.01-1000 ng/mL) for 16 h released HMGB1 in a concentration dependent manner (Figure 4(a)). PBMCderived HMGB1 was efficiently removed by both adsorbents tested (243 versus 113 ng/mL adsorbent for Cellufine sulfate and PSDVB-16, respectively, for an initial concentration of 13.7 ng/mL and an adsorbent-to-medium ratio of 5 vol%, Figure 4(b)).
Next to the physicochemical parameters of the adsorbent polymers, modifications of HMGB1 are likely to influence its binding to different polymers. Acetylation, phosphorylation, and oxidation are critical for the diverse biological activities of HMGB1. In an inflammatory environment, the production of reactive oxygen species induces HMGB1 oxidation, leading to a loss of its biological activity and restricting its proinflammatory role in a temporal and spatial manner. Since acetylation, phosphorylation, and oxidation induce changes in charge and conformation of HMGB1, they might influence its binding to different polymers. It is, thus, conceivable that adsorbents preferentially deplete certain subsets of HMGB1 with different biological activity.
Finally, HMGB1 is associated with microvesicles from both activated and apoptotic cells [43]. It may be embedded into microvesicles or interact with negatively charged phosphatidylserine residues on the microvesicle surface. In any case, this association influences the adsorption of HMGB1 to porous polymers due to increased size of the adsorbate, and different subsets of HMGB1 may preferentially be associated with microvesicles, again leading to selective depletion of HMGB1 variants.

Conclusions
Neutral and anionic polymers were tested for their capability to remove recombinant as well as PBMC-derived HMGB1 from human plasma. HMGB1 was efficiently adsorbed by negatively charged beads in a time-dependent manner due to electrostatic forces. It also bound to neutral porous polystyrene-based particles via hydrophobic interactions. Unmodified hydrophilic cellulose adsorbed only negligible amounts of the cytokine, despite its large pores and high surface-to-volume ratio, while Cellulose sulfate bound HMGB1 with high efficiency. Posttranslational modification and/or oxidation of HMGB1 are critical for the regulation of its biological activity. Since these modifications influence the charge as well as the conformation of HMGB1, subsets with different biological activity may show preferential interaction with adsorbent polymers or with plasma proteins deposited on these polymers, which might open a way to selectively deplete HMGB1 subsets with different biological activity.


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Conjugates of Small Molecule Drugs with Antibodies and Other Proteins

Conjugates of small molecule drugs with antibodies (ADCs) and with other proteins (protein-drug conjugates, PDC) are used as a new class of targeted therapeutics combining the specificity of monoclonal antibodies (mAbs) and other proteins with potent cytotoxic activity of small molecule drugs for the treatment of cancer and other diseases. A(P)DCs have three major components, antibody (targeting protein), linker and payload, the cytotoxic drug. Recently, advances in identifying targets, selecting highly specific mAbs of preferred isotypes, optimizing linker technology and improving chemical methods for conjugation have led to the approval of two ADCs by Food and Drug Administration (FDA) and more than 30 ADCs in advanced clinical development. However, the complex and heterogeneous nature of A(P)DCs often cause poor solubility, instability, aggregation and eventually unwanted toxicity. This article reviews the main components of A(P)DCs, and discusses the choices for drugs, linkers and conjugation methods currently used. Future work will need to focus on developments and strategies for overcoming such major problems associated with the A(P)DCs.

Introduction
An ideal anti-tumor drug kills tumor cells efficiently but leaves normal cells intact. With the emergence of antibody-drug conjugates (ADCs) we may be a step closer to this goal. Therapeutic antibodies have exquisite specificity toward antigens that they are designed to recognize. Conjugated with potent cytotoxic drugs, which are otherwise too toxic to use alone, ADCs promise to deliver these cytotoxic drugs directly into tumor cells and release the drug inside cells. Approval of Brentuximab vedotin (ADCETRIS ® ) by US FDA in 2011 has ushered many other similar ADCs into the development for cancer therapy [1,2]. Brentuximab vedotin [3] was approved to treat Hodgkin's lymphoma (HL) after failure of autologous stem cell transplantation, and systemic anaplastic large cell lymphoma (ALCL) after failure of multi-agent treatment. In February 2013, US FDA approved trastuzumab emtansine (Kadcyla™) for patients with HER2-positive, late-stage (metastatic) breast cancer [4]. It has been reported that currently more than 30 ADCs are in clinical studies [5].
This article reviews the main components of ADCs and other protein-drug conjugates (PDCs), and discusses choices for drugs and conjugations by using ADCs currently under late clinical studies as examples. Several recent reviews provide details in efficacy and adverse effects of these ADCs [1,2,6-10].

Antibodies and Targets
Therapeutic antibodies eliminate target cells through two mechanisms in general. One is through triggering signaling from the receptor, which may lead to apoptosis or disrupting signal transduction essential for growth, such as bevacizumab [11]. This mechanism is usually determined by the inherent nature of the antibody's target. Agonistic antibodies, such as PRO95780, targeting the death receptors are examples of this mechanism [12]. The other mechanism is through the effector function of antibodies, namely antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Immunoglobulin isotypes IgG1 and IgG3, are capable of mediating these effector functions [13]. However, in many cases, these cell-killing mechanisms are not sufficient to eliminate tumor cells. In addition, some antibodies with high specificity to cancer antigens may lack therapeutic effect by themselves. Conjugating these antibodies to potent cytotoxic drugs is an excellent way to empower these antibodies.
An appropriate ADC target should have elevated levels on tumor cells and limited expressions in normal cells. For example, Her2 has been shown to overexpress in significant portion of metastatic breast cancers, and with very restricted distribution in normal cells [14,15]. Identifying such cancer differential targets is one of the most important steps in developing ADCs. Higher density of the target molecules could facilitate introduction of more cytotoxic drugs into cells. In addition to selective expression in cancer cells, target molecules should undergo internalization mostly through the early endosome-lysosome pathway [16]. As the targeting moiety of the ADCs, other than characteristics a typical therapeutic antibody should have (e.g., high specificity and high affinity), the antibody preferentially triggers the internalization upon its binding to the cell surface receptor, making ADCs available to intracellular processing [17]. The internalization of antibody/receptor complex may be mediated through the direct engagement of the receptor or clustering of multiple receptors [18,19]. Many receptors, such as G protein coupled receptors (GPCRs), have adapted a mechanism to desensitize to repeated or prolonged exposure by recycling rapidly back to cytoplasmic membrane through early endosomes [20], which makes them less ideal as ADC targets. However, an ADC targeting the GPCR family member endothelin receptor type B (ETBR), Anti-ETBR-MC-vc-PAB-MMAE, has been advanced to clinical development [21], which suggests that unexplored GPCRs with effective internalization could be potential targets for the ADCs [22].
A selected list of ADCs in late stage clinical development and their components along with the details of targets, cytotoxic drugs, therapeutic area, and status is given in Table 1. GPNMB, glycoprotein nonmetastatic B; PSMA, prostate-specific membrane antigen; * Lorvotuzumab phase 2 studied has been discontinued.
Although the vast majority of reported ADCs employ humanized or fully human antibodies as the guiding molecules, other proteins could perform this function. For instance, soluble forms of the HIV-1 primary receptor CD4 (sCD4) are potentially promising molecules which could target cytotoxic drugs to and kill HIV-1-infected cells expressing the viral envelope glycoproteins (Envs) on cell surface [23]. Two recent studies showed that decreasing the molecular size of sCD4, which typically is composed of either four (D1-4) or two (D1D2) extracellular CD4 domains, to a single domain (D1) and engineering D1 through mutagenesis lead to largely reduced nonspecificity (e.g., interactions with the major histocompatibility complex class II and presumably other targets) of D1 while preserving D1 binding and cross-reactivity with the Envs [24,25]. Therefore, D1 could be superior to D1-4 and D1D2 as a component of the targeting molecules. Interestingly, a class of antibodies targeting the HIV-1 coreceptor-binding site on the Envs (designated as CD4i antibodies) synergize with sCD4 in binding and neutralization because their epitopes are better exposed or formed in the presence of sCD4 [26][27][28]. It has been proposed that sCD4-CD4i antibody fusion proteins could be better than sCD4 or the antibody alone as targeting molecules of not only ADCs or PDCs but also bispecific antibodies and chimeric antigen receptors (CARs) to guide naturally occurring killer cells for HIV-1 eradication [23].

Cytotoxic Drugs and Mechanisms of Action
It is critical to maintain the antibody pharmocokinetics, antigen binding ability and thermostability of ADCs. There are limited numbers of drug molecules that can be attached to antibody. Too many drug molecules lead to short serum half life of ADCs and increased hydrophobicity [29,30]. The current consensus is three to four molecules of drug per antibody [1,2]. In the early days of protein-drug conjugates, toxins such as ricin were used [31]. The chemotherapy drugs doxorubicin [32,33], methotrexate [34], vinca alkaloids [35], etc., have also been conjugated to antibodies. These drugs were found not potent enough to kill cancer cells, especially in solid tumors where only suboptimal amount of drug are delivered and accumulated. More potent cytotoxic drugs were later used for preparing ADCs. Although these potent cell killing drugs are too toxic to use as free drug, they have been proven to be safe and potent when conjugated with tumor specific antibodies. The drugs used in ADCs currently may be classified into two families: drugs that damage DNA and drugs that disrupt microtubule polymerization [36].
Duocarmycin and calicheamicin are two examples of DNA damaging agents. Duocarmycin was first isolated from culture of Streptomyces. It binds to the minor groove of DNA and alkylates the nucleobase adenine, which leads to DNA double strand breaks and apoptosis. It has IC50 of ~10 pM against cancer cells [37]. Synthetic analogs of duocarmycin are available in modified and pro-drug forms for stability requirement before it reaches DNA. MDX-1203, for example, a CD70 targeting ADC conjugated with duocarmycin pro-drug which is under development for treating Non-Hodgkin's lymphoma and renal cell carcinoma [38].
Calicheamicin was first isolated in 1980s from Micromonospora echinospora that live in caliche clay. It belongs to the family of enediyne antibiotics. Calicheamicins bind with DNA in the minor groove, where they then undergo a reaction analogous to the Bergman cyclization, generating a diradical species. The diradical intermediates remove hydrogen atoms from the deoxyribose backbone of DNA which results in strand breaks [39]. Calicheamicin is one of the most potent antitumor agents known. It impacts both quiescent and proliferating cells. N-acetyl-γ calicheamicin is the cytotoxic compound in both Gemtuzumab ozogamicin and Inotuzumab ozogamincin [17]. Gemtuzumab ozogamicin was the first ADC approved by US FDA for acute myeloid leukemia and later withdrawn for safety concerns [40]. Inotuzumab ozogamincin is currently in phase 3 clinical trial.
Auristatins and maytansinoids are the two most selective microtubule disrupting drugs for ADCs. Both are vinca-domain binders of β-tubulin and prevent polymerization of tubulins into microtubules and disrupt mitotic spindles [36,41]. Therefore, their cytotoxicity is mainly on dividing cells. Monomethyl auristatin E (MMAE), the payload in Brentuximab vedotin, is a synthetic dolastatin 10 derivative. Dolastatin 10 is a lipophilic pentapeptide isolated from the mollusk Dolabella auricularia [42]. As an antitumor agent, it is more potent than vinblastine and taxol. Total synthesis of dolastatin 10 was reported in 1989 [43]. Dolastatins may be sensitive to the efflux pump by the multi-drug resistant p-glycoprotein [36]. Monomethyl auristatin F (MMAF) has a charged C-terminal phenylalanine residue instead of a carboxyl residue [44]. In comparison with MMAE, MMAF has attenuated membrane translocation capacity, less potent (higher maximal tolerated dose), and much higher aqueous solubility. ADCs comprising MMAE include the FDA approved Brentuximab vedotin [3], and other ADCs that are in late stage clinical development such as Glembatumumab vedotin [45], Pinatuzuumab vedotin [46] and PSMA-ADC [47]. ADCs targeting CD30 [44] and CD22 [48] have the MMAF as a payload component which exhibited different properties and activities as compared to ADCs anti-CD30-MMAE and CD22-DM1 respectively.
Maytainsine is a type of ansamycin antibiotics originally isolated from Ethiopian shrub Maytenus serrate, which inhibits tubulin polymerization and binding at a rhizoxin binding site. Trastuzumab emtansine (T-DM1, Kadcyla ® ) targeting the human epidermal growth factor receptor (HER2) which was approved by FDA has the DM1 (derivative of maytansine) as potent cytotoxic component of the ADC [4].

Linkers and Conjugation Chemistry
The potent cytotoxic drugs are covalently attached to antibodies through linkers, and the linker stability is critical to the efficacy of ADCs. Stability of ADCs before they reach target cells is the key to minimize off-target killing and maximize tumor exposure to drugs. Upon internalization of ADCs into cells, linkers should be labile enough to ensure rapid release of drugs into cytoplasm where they bind to tubulins or DNA molecules. Design of linker/spacer is intricately related to how ADC will exert its cytotoxicity intracellularly. The choice of linker determines the release mechanism of drugs that is often limited by chemistry and biology of cancer cells.
Heterobifunctional linkers are commonly used in ADCs [49], because they allow attachment of drugs through disulfide bond and amide bond. A typical example of linker, such as 4-(-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) has N-hydroxysuccinimide (NHS) ester, which reacts with primary amines to form amide bond, and maleimide group that reacts with sulfhydryl group to form stable thioether bond. Linkers such as N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) and N-succinimidyl 3-(2-pyridyldithiol)butyrate (SPDB) allow amide bond on one end and disulfide bond on the other end, which may be cleaved in reductive environment of some cancer cells. Besides, since maytansinoids and MMAE are synthetic analogs of their natural product counterparts, sulfhydryl or amine groups can be added during synthesis, making them adaptable to various conjugation chemistry methods. Therefore, the main conjugation sites on antibodies are cysteines (both native and mutated cysteines) and lysines. Although studies on conjugations through carbohydrates [50,51] and selenocysteines [52] have been reported, they have yet to reach clinical tests.
Cysteine and lysine are two most common naturally occurring amino acids which are used to attach the drug through the linker to the antibody. IgGs have four pairs of interchain disulfide bonds, two between the heavy chains in the hinge region and one on each Fab between CH1 and CL domains ( Figure 1A). Intrachain disulfide bonds are to be left intact because they are critical to maintain the basic IgG domain structure essential for antigen recognition, stability and effector functions. Only partial reduction of the interchain disulfide bonds at the hinges gives eight potential conjugation sites. During the conjugation step, molar ratio of free drug and antibody may be adjusted such that approximately four molecules of drugs are attached to each antibody [4,53]. Brentuximab vedotin is conjugated to maleimide-VC-PAB-MMAE on its cysteines [53] (Figure 1A). In the case of Brentuximab vedotin, addition of VC dipeptide provides a proteolytic site for the lysosomal protease cathepsin B to release the drug [54]. Proteolytic activity is abundant in late endosome/lysosomes, such design facilitates rapid release of drug for efficient cell killing. Residues in blue sticks represent the more than eighty naturally occurring lysines.
The advantage of conjugation through cysteines is the relatively mild condition for reduction and conjugation. The procedure poses minimal risk to the stability of IgGs. The conjugation step is often efficient. There is an established method to monitor the intermediate product and final product ADCs [55]. However, the method gives a mixture of ADCs with 0 to 8 drugs per antibody. To overcome the heterogeneity, site specific conjugation through engineered cysteines, such as THIOMAB [56] has been explored to provide a more homogenous final ADCs with defined drug antibody ratio of 2, better stability and larger therapeutic window. It has been reported that site of conjugation also impacted on ADC stability. Site specific cysteines on heavy, light and Fc regions with various solvent accessibilities have different overall stability and therapeutic index [57].
For conjugation through lysines, the primary amine on lysine side chain provides a convenient reactive group for conjugation chemistry. Overall, in an average IgG molecule, there are more than eighty lysine residues and almost a half of them having their side chains exposed to the surface of folded IgG ( Figure 1B). More importantly, lysine is not a common residue in the CDR regions which minimizes possible interference of antigen recognition properties while lysine based conjugation of ADCs are used. However, the availability of large number of potential lysine conjugations sites compared to the small number of antibody to antibody ratio (DAR) could lead to a highly heterogeneous mixture. For example, the ADC huN901-DM1 involves about 20 different lysine residues in both light and heavy chains that are found in areas of structural flexibility and with large solvent accessibility [58]. In the recently approved trastuzumab emtansine (T-DM1), the lysine residues on the antibody trastuzumab are connected to a cytotoxic maytansine derivative DM1 through a stable thioether linker N-succinimidyl 4-(Nmaleimidomethyl) cyclohexane-1-carboxylate (SMCC) ( Figure 1B). While the N-hydroxysuccinimide ester forms a stable amide bond with the primary amine of any of the available lysine residues of the antibody at pH 7-9, the maleimide moiety undergoes Michael addition reaction with sulfhydryl groups at pH 6.5-7.5 forming stable thioether bonds which are used to attach the drug to the antibody. The conjugation process leads to the formation of a mixture of ADCs with DARs ranging from 0-9, with an average value of 3.5 [59]. The DM1 binds directly to microtubules and inhibits their assembly thereby causing cell death [60]. Although there is no releasing trigger moiety in the T-DM1, following the internalization by the target cancer cells via antigen-mediated endocytosis it was delivered to lysosomes by the vesicular trafficking, and then degraded into the lysine derivatives, lysine-N ε -SMCC-DM1 [61], which likely bind to the tubulin and cause cytotoxicity by the inhibition of microtubule polymerization.
Lysine conjugation are also used in other ADCs including inotuzumab ozogamincin that is currently in Phase 3 clinical testing for non-Hodgkin lymphoma (NHL) in combination with rituximab [62]. The ADC, inotuzumab ozogamincin has anti-CD22 antibody with 78 lysine residues available for conjugating to cytotoxic drug N-acetyl-γ-calicheamicin via acetyl butyrate hydrazine. The linker drug molecule preferentially attaches to lysines on the heavy chain. The same lysine conjugation as well as drug and linker moieties is used in the other ADC, gemtuzumab ozogamicin (Mylotarg) targeting CD33 which was approved in 2000 by FDA but voluntarily withdrawn in 2010. Both inotuzumab ozogamincin and gemtuzumab ozogamicin use a hydrazone linker connecting through the cytotoxic drug N-acetyl-γ-calicheamicin to the antibody, in which the hydrazone is first cleaved in the acidic intracellular environment followed by the reduction of disulfide bridge in the calicheamicin [63]. Overall, in the case of conjugation through lysine residue, the cytotoxic drug attachment to the antibody typically occurs at a relatively very few ε-amino groups of any of the ~80 lysine residues present in the antibody, because the preferential surface accessibility for the lysine side chains to chemical modification is limited. ADCs with the DAR value of more than certain value, for example more than five, are usually hydrophobic and can lead to solubility problem. Currently, the widely used chemical method for lysine conjugation involves the formation of a stable amide bond using activated ester of the drug molecules, and several methods for the preparation of ADCs through lysine conjugation were described [64].
Therefore, both conjugation methods described above are useful and resulting in several ADCs, two of which were approved by the FDA and others are in late clinical development. However, the complexity of ADCs often leads to heterogeneous distribution of drug molecules conjugated to antibodies and unfavorable physicochemical properties including increased aggregation with higher number of DARs, poor aqueous solubility and low stability in circulation.

Conclusions
The two recently FDA approved ADCs and growing number of ADCs in the pipeline present promising opportunities in treating various cancers and other diseases. The main optimization parameters in the development of successful ADCs include selection of targets, specificity of mAbs, drug potency, linker stability, and conjugation methods (inter-chain or engineered cysteines, lysines, and Fc glycans). Receptors or cell surface proteins that are overexpressed on tumor cells as compared to normal tissue are good candidate targets. In addition to the usual requirement of therapeutic mAbs, the mAbs used in ADCs must have sufficient aggregation-resistance to withstand the hydrophobicity brought on by adding the drug molecules, and maintain stability and affinity for targets upon conjugation. The selection of antibody isotype, the ability for internalization and tumor penetration are also important. The cytotoxic drugs used in ADCs should be rapidly released in active forms in the intracellular environment. The number of drug molecules in ADCs depend on the method of conjugation, desired potency and linkers [65]. The linkers used in ADCs are a key component, which influences the stability of ADCs and release of drug molecules [66]. Therefore, the choice of linkers used in ADCs profoundly affects the thermal stability, half life, toxicity and the overall efficacy of the ADCs [67]. In order to render better control on the drug/linker conjugation sites on antibodies and the desired DAR, better linker and conjugation methods are needed, as well as rational approaches for improving physicochemical properties and therapeutic index of ADCs.


Differential Effects of Dry Eye Disorders on Metabolomic Profile by 1H Nuclear Magnetic Resonance Spectroscopy

We used 1H NMR spectroscopy to analyze the metabolomic profile of reflex tears from patients with dry eye disorders (DEDs). 90 subjects were divided into 2 groups: (1) patients with DEDs (DEDG; n = 55) and (2) healthy subjects (CG; n = 35). Additionally, the DEDG was subdivided into 2 subgroups based on DED severity: mild-to-moderate and moderate (n = 22 and n = 33, resp.). Personal interviews and systematized ophthalmologic examinations were carried out. Reflex tears (20–30 μL) were collected by gently rubbing in the inferior meniscus of both eyelids with a microglass pipette and stored at −80°C until analysis. NMR spectra were acquired using a standard one-dimensional pulse sequence with water suppression. Data were processed and transferred to MATLAB for further chemometric analysis. Main differences in tear composition between DEDG and CG were found in cholesterol, N-acetylglucosamine, glutamate, creatine, amino-n-butyrate, choline, acetylcholine, arginine, phosphoethanolamine, glucose, and phenylalanine levels. This metabolic fingerprint helped also to discriminate between the three additional subgroups of DEDG. Our results suggest that tear metabolic differences between DEDG and CG identified by NMR could be useful in understanding ocular surface pathogenesis and improving biotherapy.

Introduction
The ocular surface (OS) is a functional unit consisting of the eyelids, lacrimal glands, cornea, conjunctiva, and tear film. Main roles of the OS are to preserve the anatomical and physiological properties of its components, to maintain the optic conditions of the eye surface dioptric, and to protect the eyes against injury by exogenous and endogenous agents [1][2][3]. A healthy tear film is fundamental for supporting OS integrity and refractive functions [1].
Dry eye disorders (DEDs) are complex pathological conditions involving the OS [2,3]. Approximately 20-50% of the patients who visit an ophthalmology clinic complain of dryness, making DEDs one of the most frequent ocular morbidities. In fact, DEDs are a growing public health problem worldwide [3]. DEDs usually affect the elderly and postmenopausal women [4,5]. However, the prevalence of DEDs may be higher than suspected, because some patients may not report their eye problems to ophthalmologists and thus remain undiagnosed. The main symptoms of DEDs include dryness, itchiness, burning, stinging, grittiness, foreign body sensation, tearing, tired eyes, redness, and blurred vision [2][3][4]. Among the recognized risk factors for DEDs are aging, being female, smoking, the use of topical or systemic medications, contact lenses wearing, laser excimer refractive surgery, weather conditions, environmental pollutants, air conditioning, hormone disorders, immune system diseases, and the use of video display terminals [6]. There are two major clinical forms of DEDs: the deficient aqueous tear production type (due to lacrimal gland dysfunction) and the increased evaporative loss type (due to meibomian gland disorder), but combinations of the two forms are usually seen in clinical practice [2,3,7,8]. DEDs are also classified according to severity, ranging from mild to moderate to severe forms. Significant reduction in body water content associated with aging may play a pivotal role in DEDs. Meibomian gland dropout, a reduction in the number of goblet cells, and laxity of the eyelids may also be contributing factors [7,8].
According to a report from the 2007 International Dry Eye Workshop [2], the main pathogenic mechanisms of DEDs are tear film instability and hyperosmolarity. Oxidative stress, inflammation, and apoptosis must also be considered in this process. All conditions lead to a chain of events that induce a series of OS clinical manifestations that taken together are referred to as DEDs.
Recent reports have proposed certain molecules and genes, as well as various clinical parameters, as presumptive DED biomarkers [7,8], including oxidant and antioxidant activities, apoptotic mediators, antibodies, cytokines and chemokines, and hormone tear levels. however, lack of specificity of these biomarkers justifies the need for a further research on DED pathogenesis.
The relevance of the metabolome-the complex array of small-molecule metabolites and metabolic by-products, including carbohydrates, peptides, and lipids, present in cells, tissues, organs, and body fluids as a result of the expression and activity of genes and proteins [9][10][11][12][13][14] to both disease and health-has long been recognized. The study of the metabolome, metabolomics, may help to establish a link between the components of the metabolome and corresponding cellular responses [15,16]. During the past 30 years, metabolomics has been used in clinical and animal studies of several diseases, including ocular pathologies. Young and Wallace reviewed the metabolic consequences of ocular diseases and explained why the multiplexed analysis inherent to metabolomics can be expected to provide data that are uniquely useful for the assessment of ocular diseases [17].
Metabolomics is defined as "the quantitative measurement of the metabolic response of living systems to pathophysiological stimuli or genetic modification" [18,19] and is based on analytical platforms such as proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) and mass spectrometry (MS), particularly, gas chromatography (GC) and liquid chromatography-(LC-) MS [18,19]. The metabolites and their concentrations provided a dataset on which multivariate statistical analysis was performed.
The NMR technique is based on the measurements of the magnetic properties of certain atomic nuclei, for example, 1 H, 31 P, and 13 C, in the metabolites. Each chemical group (CH, CH 2 , CH 3 , etc.) of each metabolite will have a unique chemical shift (in ppm). The metabolite peak will have an integral value that is directly proportional to the metabolite concentration in the sample. In MS experiments compounds are ionized to form positively or negatively charged molecules which are separated and detected according to their mass-tocharge ratio.
1 H NMR spectroscopy analysis of biofluids provides information on both the structure and the composition of low-molecular-mass metabolites in biological fluids and is a rapid and low-cost technique for exploring pathological metabolic processes. The major advantages of NMR spectroscopy include its unbiased metabolite detection, quantitative nature, and high reproducibility [18]. Unfortunately, this technique is associated with a low sensitivity compared to the other analytical methods such as GC-MS or LC-MS. The MS analysis requires more labor-intense (and destructive) tissue preparation. Detectable compounds are limited to those that can be derivatized, which can be time consuming, costly, and carrying a risk of metabolite loss. NMR-based metabolomics could be appropriate as a cost effective solution for highthroughput analysis [20].
Biotechnological advances, such as the application of NMR spectroscopy to tissue and biofluid samples, have permitted the generation of data on low-molecular-weight metabolites in samples from individuals [9][10][11]. For example, Holmes et al. analyzed the aqueous humor and vitreous body of pig eyes by means of 31 P NMR [15]. Midelfart et al. analyzed extracts of rabbit corneas and lenses by NMR spectroscopy and observed that some metabolites suggested the existence of a powerful antioxidant environment in the ocular tissues [21][22][23]. Our group reported previous experience on the metabolomics study by NMR of the aqueous humor in a rat model of hyaluronic acid-induced ocular hypertension [24]. Compounds identified in these glaucomatous samples correlated well with data obtained in similar glaucoma models by means of conventional techniques. Moreover, Mayordomo-Febrer et al. published a detailed work on the characterization of human tear metabolome using LC-MS/MS [25]. Although NMR study of tear composition remains incomplete, some works using this biofluid [23,24,[26][27][28] have demonstrated that metabolomics can be useful for monitoring eye diseases such as DEDs.
Proteomic assays have recently been reported in tear samples of dry eye patients by 2D electrophoresis (2DE) and differential gel electrophoresis (DIGE). Presumptive biomarkers of DEDs included proline rich 4 protein (LPRR4) which appeared downregulated in all types of DEDs [29]. Other authors have suggested the higher expression of apoptotic and inflammation proteins in tears from diabetic dry eye patients as performed by two-dimensional nanoliquid chromatography coupled with tandem mass spectrometry-(MS-) based proteomics [30].
In the present study, our goal was to improve knowledge on human tear composition by using 1 H NMR spectroscopybased metabolite profiling, followed by multivariate statistical analysis, in order to explore metabolite imbalances between DEDG. In addition, we investigated the possibility to build up a metabolic discriminating model that helps us to identify the three established severity ranges subgroups of DED, with the ultimate goal of improving the management of these patients.

Material and Methods
All participants firmed the informed consent and all procedures of this prospective study were subjected to the Declaration of Helsinki for the protection of human subjects in medical research. The study was approved by the Institutional Review Board of the University and Polytechnic Hospital La Fe (Valencia, Spain) (Ref. 2013/0417).

Patients and Groups.
A total of 90 subjects of both sexes, aged 25-80 years, were enrolled during ophthalmologic appointments at the study center (University and Polytechnic Hospital La Fe, Valencia, Spain) between February 2013 and September 2013 according to the main inclusion and exclusion criteria listed in Table 1.
Suitable subjects were assigned to one of the following groups: (1) patients diagnosed with DEDs (DEDG; = 55) and (2) healthy subjects as a control group (CG; = 35). From the DEDG, three subgroups were formed on the basis of clinical OS data and OSDI questionnaire: patients diagnosed with mild-to-moderate ( = 22) and moderate ( = 33) DEDs. Participants pertaining to the severe forms were excluded of the present study.

Clinical Assessment and Tear Sampling.
Each subject was interviewed about their personal and familial background and their personal characteristics and lifestyle as well as disease data (symptoms of dry eyes and subjective sensations, duration, and treatments). We used the Ocular Surface Disease Index (OSDI) questionnaire as part of the interview. This clinically validated questionnaire was designed to evaluate DEDs in terms of ocular symptoms, visual function, and environmental factors. Each of the 12 questions has to be responded to with a rating from (0 to 4), and a final score (0-100) is then calculated, with higher scores indicating moresevere DEDs [24].
Each subject was given a systematized ophthalmologic examination consisting of the following tests: measurement of best corrected visual acuity in each eye, Schirmer's test, slitlamp examination of the eye adnexa and anterior segment, and measurement of tear break-up time (BUT) with fluorescein.
Reflex tear samples were obtained by gently rubbing the inferior meniscus of both eyelids with a micro-Pasteur pipette, without damaging the OS tissues, as previously described [25]. Tears from both eyes (total volume 20-30 L) were deposited in one labeled cryotube per subject and stored at −80 ∘ C until metabolomics analysis. Patients for whom the volume of the collected tear sample was less than 10 L were rejected; because of this, it was difficult to get a significant number of severe DEDs participants. A total of 20 L of the mixture from each subject was then transferred to a 1 mm high-quality NMR tube. All 1 H NMR spectra were acquired using a standard onedimensional pulse sequence with water suppression on a Bruker Avance 600 spectrometer operating at 600.13 MHz with a 1 mm 1 H/ 13 C/ 15 N TXI probe. A total of 256 free induction decays were collected into 64 k data points with a spectral width of 14 ppm and a recycle delay of 1 s. The water signal was saturated by weak irradiation during the recycle delay. Before Fourier transformation, the free induction decay was multiplied by a 0.3 Hz exponential line broadening. Spectral chemical shift referencing on the TSP signal at 0 ppm was performed for all spectra. Spectral regions between 0.5 and 4.4 ppm and between 5.5 and 9.5 ppm were binned in segments of 0.01 ppm width (6 Hz) for multivariate analysis. Binned data were normalized to total aliphatic spectral area. We used available spectral databases and two-dimensional NMR experiments to facilitate structural identification of relevant metabolites. Spectra were processed using MestRenova 6.2 software (Mestrelab Research S.L., Santiago de Compostela, Spain) and transferred to MATLAB 7.6 (The MathWorks Inc., Natick, MA, USA) for additional processing and further analysis. Signals belonging to selected metabolites were integrated and quantified using semiautomated in-house MATLAB peak-fitting routines based on Levenberg-Marquardt optimization procedures. Resonances were assigned according to the previous literature, the Human Metabolome Database (http://www.hmdb.ca/), and characteristic cross-peak from previously mentioned 2D spectra to unequivocal assignation of the resonances.

Statistical Analyses. Chemometrics statistical analyses
were performed using in-house MATLAB scripts and PLS Toolbox 6.7 (Eigenvector Research Inc., Wenatchee, WA, USA). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were applied to NMR spectra data matrix. PLS-DA is a classification technique that combines the properties of partial leastsquares (PLS) regression with the discrimination power of discriminant analysis (DA) [20]. The main advantage of PLS-DA models is that the main sources of variability in the data are modeled by the so-called latent variables and consequently in their associated scores and loadings, allowing the visualization and understanding of different patterns and relations in the data. The PLS-DA model was tested using a leave-one-out cross-validation (CV) algorithm.
All data are expressed as mean ± standard deviation (SD). Finally, one-way analysis of variance was used for the determination of statistical significance between group means of the corresponding integrals. A difference was considered significant when < 0.05.

Results
Median ages of the subjects were 52 ± 18 years old (DEDG) and 36 ± 11 years old (CG). The percentages of men and women in the two groups (DEDG and CG) were 75% and 58%, 25% and 42%, respectively.  Data from the 1 H NMR spectra of the tear samples from all the study subjects permitted a visual comparison between the average spectra of tears from CG subjects and the DEDG patients. Average spectra for the two groups are shown in Figure 1 with some of the most intense metabolite peaks labeled.
The metabolic profiles of the groups were compared by means of principal component analysis, which is an unsupervised test for homogeneity of the set of samples (detecting the existence of possible outliers). As part of this process we determined the presence of 9 samples (3 from the CG and 6 from the DEDG) that were excluded from further analysis. Outliers are defined as those samples that are situated outside the 95% confidence interval of the Hotelling T-squared distribution in the score scatter plot.  Because the samples did not cluster spontaneously, we performed a PLS-DA that maximized the separation between the groups (Figure 2). The PLS-DA score plot clearly indicated an incipient separation with minimum overlap between DEDG and the CG samples, confirming the existence of significant differences in metabolic profile between the two groups.
Quantification of the most contributing regions in the PLS-DA model allowed us to determine the major metabolites differences in the tears of the two study groups (Table 2).
Additionally, based on previous metabolites differences found between DEDG and CG, we proceeded with a deeper analysis in order to build up a model that helps us to   discriminate between the three DED subgroups. Firstly, we constructed a PLS-DA model using the two extreme groups (severe-DED and mild-DED). After that, the intermediate group (moderate DEDs) was projected in the same discriminating space. We obtained a multivariate space where the three subgroups presented a clear differential grouping. The mild-to-moderate and the moderate DED samples were distributed as shown in Figure 3.
In Table 3 relative values of the most contributing regions in the PLS-DA discriminating model for each subgroup sample are reflected.

Discussion
The anatomy and function of the OS must be preserved by appropriate tear secretion and availability [1][2][3][4]. The tear film is formed by various layers that are secreted, in turns, by several ocular glands and tissues. DEDs are complex pathological conditions involving the OS and are among the most frequent ocular morbidities worldwide [1][2][3][4][5][6][7][8]. This research was conducted to shed light on the metabolite composition of the human tears in relation to DED manifestations and severity.  Mean age of the DED patients was 52 ± 18 years, and women made up 75% of the DEDG, in agreement with previous reports [1][2][3][4][5], confirming that age and sex are relevant factors in DED initiation and progression, as previously reported [5,6]. Statistically significant differences in the Schirmer and BUT scores were observed between the two main groups, as previously reported [1][2][3][4][5][6][7][8]. We subdivided the DEDG patients according to the severity of clinical DED manifestations (mild, moderate, or severe). The results of the Schirmer and BUT tests showed different degrees of alteration that were closely related to the intensity of clinical signs and symptoms.
1 H NMR spectroscopy allowed us to identify and quantify sets of metabolites in human tears with little sample preparation and quite small sample volumes. In addition, the metabolite profile could be acquired relatively rapidly (5 min with a short routine), with sensitivity sufficient to evaluate even subtle differences. By using the combination of NMR metabolomics and chemometric approaches, we were able to obtain and include in the analysis a large amount of data, which contribute to obtaining robust models that Zhou and Beuerman, 2012 [33] can be expected to provide useful information for further ophthalmic research [9][10][11][12][13][14][15][16]. The 1 H NMR metabolic profiles of the DEDG and the CG were clearly different from each other. The major differences in tear composition, between CG and the DEDG samples, were in N-acetylglucosamine, glutamate, creatine, amino-n-butyrate, choline, acetylcholine, arginine, phosphoethanolamine, cholesterol/lipids, glucose, and phenylalanine levels. Many of these metabolites have been cited in previous works which were carried out in human tears samples, as substrate (or by-products) of the ocular surface metabolism. In Table 4 each metabolite and its bibliographical reference are summarized.
The meibomian glands secretions comprise the lipid layer of the tear film which is essential for preventing rapid evaporation of the tears [30]. The results presented herein demonstrated lower lipid levels in tears from the DEDG as compared to the controls.
The increased levels of essential amino acids such as arginine and phenylalanine in the DED reflect the proinflammatory response to this type of ocular surface alteration [31]. However it is difficult to clearly discuss the presence and function of these amino acids, due to the variability of them found in individuals with or without alteration of the ocular surface. Furthermore, oxidative stress occurring in the cellular compartments of the ocular surface, as described in subjects with DEDs, could generate increased levels of glucose and creatine in the tear film, in a similar manner as it has been described in our work [32].
There is a strong regulatory action of parasympathetic autonomous system stimulation on the secretion of lacrimal and salivary glands. Important changes in the stimulus/secretion process associated with parasympathetic stimulation have been previously described by Bacman et al. [34]. In fact, higher levels of neurotransmitters choline and acetylcholine in tears of our DEDs patients constituted an interesting finding but elucidation of their role in dry eyes is far from complete.
In an attempt to obtain additional data on OS alterations and tear composition in patients with DEDs, we discriminated the metabolome of DED tears in three subgroups of progression (mild, moderate, and severe). Our data showed that the metabolomic profiles of each one of these DEDs subgroups differed significantly. In this scenario we suggest for the first time that DED differences in severity have a significant effect in tear metabolic content, suggesting that the tear metabolomic profile may be utilized as biomarkers and surrogated endpoints of DEDs.
In summary, metabolomics is a feasible tool for measuring the metabolite profile of human tears. The results presented herein strongly suggest that further metabolomic analyses of OS pathologies should be conducted. The identification of specific metabolites associated with particular situations (oxidative stress, inflammation, and apoptosis) will enhance the usefulness of this technique in a better understanding of the pathogenic mechanisms of eye disorders and design of new therapeutic strategies [33,35]. Finally, the combination of all this metabolomic information in a discriminating model gives us the opportunity of developing a fast, reliable, and cheap methodology for severity classification of DED patients using a small sample of tears.