cutting edge research updates

REVIEW

Cutting Edge Research UpdatesJames A. Orsini, DVM, ACVS

ABSTRACT

The most important research findings pertain to a betterunderstanding of the timing and progression of biochem-ical andcellular eventsoccurringduring thedevelopmentalstage of laminitis. Using molecular biological techniques,a critical gene was identified for the upregulation of thethrombo-spondin motif known as aggrecanase-1. Aggre-can and other large polysulfated proteoglycans are impor-tant components of the lamellar structure; therefore withthe presence of aggrecanase identified as one of the earlieroccuring substances present in laminitis, this and otherchemokines and cytokines may play an important role inlamellar tissue failure.

Keywords: Horse; Equine; Laminitis; Foot; Research

FULL CIRCLEA lot has unfolded in the field of laminitis research in recentyears. Perhaps themost significant groupof findings pertainsto the timing and sequence of events, biochemical as well ascellular, during the developmental stage (DEV) of laminitis,that is, before the horse starts showing signs of foot pain.Recently, we have come full circle in our understanding

of laminitis, primarily returning again to the disease as aninflammatory event. For decades, the disease has beenknown as laminitis, literally “inflammation of the laminae,”and for a long time that was where our rudimentary under-standing of the disease process began and ended. But thenour research led us to believe that the lamellar inflamma-tion, evident in biopsy or necropsy samples of the lamellartissues by the time the horse begins showing signs of lame-ness (LAM), was secondary to vascular compromise and/or degradation of the basement membrane by certain ma-trix metalloproteinases (MMPs), particularly MMP-2,MMP-9, and MMP-14.More recently, studies using the techniques of molecular

biologyhave shown that these vascular andenzymatic eventsare precededdby several hours in some casesdby the upre-gulation of several genes that code for inflammatory

From the Department of Clinical Studies, New Bolton Center, University of

Pennsylvania, Kennett Square, PA.

Summary of the 5th International Equine Conference on Laminitis and

Diseases of the Foot.

Reprint Requests: James A. Orsini, DVM, New Bolton Center, University of

Pennsylvania, 382 West Street Road, Kennett Square, PA 19348.

0737-0806/$ - see front matter

� 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.jevs.2010.07.008

Journal of Equine Veterinary Science � Vol 30, No 9 (2010)

chemokines and cytokines, and one very big gun: a disinte-grin and metalloproteinase with thrombospondin motifs(ADAMTS-4), also known as aggrecanase-1. Aggrecanand other large polysulfated proteoglycans are importantcomponents of the lamellar structure (see Lamellae Revis-ited, later in the text), so it seems particularly significantthat upregulation of aggrecanase in the lamellar tissues isone of the earliest events in laminitis, occurring before thedegradation of the basement membrane.1,2

TimelineFigure 1 shows a timeline of biochemical and cellular eventsthat occur during DEV, using the carbohydrate overload(CHO) model of laminitis. Note that the upregulation ofinterleukin-6 (IL-6) and ADAMTS-4 occurs several hoursbefore MMP-2 is upregulated.2,3 Additionally, the tissueinhibitor of MMP-2 (TIMP-2) is downregulated severalhours before the upregulation of MMP-2. As their namesuggests, the TIMPs control the corresponding MMPs,so it is interesting to note that the “brake” for one of thekey MMPs in the pathophysiology of laminitis is inhibitedearly in DEV. Although what exactly triggers the down-regulation of TIMP-2 still remains to be determined.

LeukocytesThe extravasation and lamellar infiltration of leukocytes isanother early event, beginning several hours before LAMin the CHO, black walnut extract (BWE), and hyperinsuli-nemia (HI) models of laminitis.2,3 However, at least in theCHO model, histologic changes in the basement mem-brane begin before leukocytic infiltration (Fig. 1), therebydownplaying an initiating role for leukocytes.2

Calprotectin (CP) is a protein that is present in all neutro-phils, activatedmonocyte/macrophages, and stressed epithe-lium. Belknap recently showed that leukocyte emigrationpreceded epithelial stress in the BWE model becauseCP-positive leukocytes were present during DEV, whereaslamellar epithelial cells were positive for CP only at LAM.However, in the CHOmodel, leukocyte emigration seemedto occur simultaneously with the onset of epithelial stress.4

Although neutrophils are of primary interest in inflam-matory events, it has been recently discovered that thereis a normal population of mononuclear cells in the lamellae,consisting of macrophages and both B and T lympho-cytes.3 The provocative, protective, and/or reparativepotential of these cells remain to be fully evaluated. Whatwe do know is that, at the onset of lameness, the majorityof lamellar leukocytes in the BWE model are neutrophils

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Figure 1. Timeline of the events occurring during thedevelopmental phase of laminitis caused bycarbohydrate overload. The starting point for eachhorizontal arrow indicates the time at which change(e.g. upregulation) was first identified on biopsy in theexperimental horses. (Reprinted with permission fromChristopher C. Pollitt). IL, interleukin; TC,transcription; TIMP, tissue inhibitor of matrixmetalloproteinase; ADAMTS-4, aggrecanase; MMP,matrix metalloproteinase; Ln, laminin; Col IV, collagentype 4; BM, basement membrane (as detected usingPAS [periodic acid-Schiff] staining).

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(and are accompanied by an increase in myeloperoxidaseactivity), whereas the majority in the CHO model aremononuclear cells, including monocytes. This differencemay be important in the increased incidence of lamellar fail-ure in the CHO model.4 Interestingly, neutrophil influxhas also been reported in laminitis induced by HI.4

Chemokines and CytokinesThere are some additional differences between the CHOand BWE models of laminitis that are worth noting. Dur-ing DEV there is a marked increase in proinflammatory cy-tokine signaling in bothmodels; however, there is a distinctdifference in the temporal patterns, with cytokine expres-sion being delayed in the CHO model.5

In the BWEmodel, some cytokines and chemokines peakearly in DEV (1.5 hours after BWE administration throughthe nasogastric tube). They continue to show increased geneexpression later in DEV (at onset of leucopenia, approxi-mately 3e4 hours after BWE administration) and also atthe onset of lameness. The cytokine that shows the mostdramatic increase is IL-6, which is the one that most consis-tently correlates with morbidity and mortality in humansepsis. Lamellar IL-6 undergoes a several 100-fold increaseduring DEV in BWE-induced laminitis and increases by>1,000-foldatLAM.Lamellar expressionof IL-1b, a central

proinflammatory cytokine commonly characterized as an“immediate early gene,” peaks (approximately 50-foldincrease) at the 1.5-hours time point and continues toincrease (approximately 10-fold increase) at LAM.5

Two chemokines associated with sepsis-related inflam-matory injury, chemokine (C-X-C motif) ligand (CXCL1)(Gro a) and CXCL8 (IL-8), peak at the 1.5-hours timepoint during DEV in the BWE model, exhibiting 120- to150-fold increases. Their expression then decreases rapidly,with CXCL1 returning to baseline at LAM.5

By comparison, the CHO model shows significant in-creases in CXCL1 and CXCL8 expression during DEV(at the onset of fever), but the upregulation of proinflam-matory cytokines is delayed until LAM, when increases ingene expression of approximately 2,200-fold for IL-6and 10-fold for IL-1b are reported.5

Interestingly, tumor necrosis factor alpha, the cytokinebest characterized to increase in human cases (and experi-mental models) of sepsis, is neither upregulated in thelamellae of any of the laminitis models5 nor is it increasedsystemically in the CHO model. Frank recently showedthat various inflammatory cytokines were increased in theblood of horses after CHO, with the exception of tumornecrosis factor alpha.6 In that particular study, systemicincreases in interleukins 1b, -6, -8, and -10 preceded clinicallaminitis, which suggests that systemic inflammation playsa role in the development of CHO-induced laminitis.6

SummaryIn summary, many of the events which we thought wouldtrigger the destruction of the lamellar interface endedupoc-curring too late to actually cause any destruction. Proteaseswhich are upregulated early, such as ADAMTS-4 or perhapsa still undiscovered protease, seem to be important targetsfor ongoing research. Also, vigorous anti-inflammatorytherapy during the DEV and early LAM stages of laminitisnowassumes importance beyond the comfort of the patient.In the light of these recent findings, it seems plausible thatanti-inflammatory therapymay actually be capableof haltingthe development of laminitis or limiting its severity.

LAMELLAE REVISITEDAnother recent revision in our understanding of the equinefoot pertains to the structure and function of the lamellae.One current perspective on hoof loading has the distalphalanx suspended within the hoof capsule through the in-terdigitation of the epidermal and dermal lamellae (Figs.2A, B). In this model, the “suspensory apparatus of the dis-tal phalanx” is thought to be the principle weight-bearingmechanism, facilitating the painless transfer of force be-tween the ground and the skeleton.7

However, recent findings on the extracellular compo-nents of the lamellae indicate a more complicated picture,

Figure 2. (A) Diagram of the forces interacting withinthe distal limb of the horse during static weight-bearing.W: weight of the horse acting through the centre of thehoof; G: ground reaction force (GRF) generated inresponse to W; g: component of the GRF acting uponthe bearing border of the hoof; c: resolved compressivecomponent of g acting in a disto-proximal directionwithin the hoof wall; T: tensile forces acting across thesuspensory apparatus of the distal phalanx (see 2B); D:action of the deep digital flexor tendon; t: tensilereaction force in the heels. (B) Suspensory apparatus ofthe distal phalanx. (Both diagrams reprinted withpermission from Simon Collins.)

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in which the lamellae apparently sustain significant com-pressive loads. Tissues that are subjected to strong com-pressive forces (e.g. articular cartilage) are richly endowedwith large polysulfated proteoglycans, whereas tissuesthat are not subjected (e.g. lungs, liver) contain little poly-sulfated proteoglycans, if any. These large molecules formmacromolecular complexes with hyaluronan. Because oftheir strong negative charge, they assume a space-fillingconfiguration, and attract and hold water within the tis-sues. Furthermore, hydrated proteoglycanehyaluronancomplexes typically are packaged in collagen and other ten-sile extracellular matrix proteins. The resultant gel-filledcompartments are poorly compressible, endowing the tis-sue with resistance to compressive forces.1

Using cDNA-specific probes, Black recently documentedthat the genes encoding aggrecan, versican, chondroitinsynthase, and hyaluronan synthase are constitutivelyexpressed in normal digital lamellae. Aggrecanase, orADAMTS-4, was also detected in the lamellar extracts,most likely representing pre-secretion enzyme andproteoglycan-bound enzyme.1 Presumably, it has a similarlyconstitutive role in the healthy hoof as the MMPs. There-fore, just as in articular cartilage, large polysulfated proteo-glycans would seem to play an important role in thestructure and function of the digital lamellae.Stretching of the lamellae during jumping and other ex-

ercise, as well as distortions resulting from positionalchanges in the coffin bone relative to the hoof wall duringnormal movement, may cause the lamellae to undergostrong compressive forces. Such loading may explain theclose parallels in the content of extracellular matrix proteo-glycans between articular cartilage and digital lamellae. Notsurprisingly, there are similarities in the main pathologiesof these tissues (osteoarthritis and laminitis, respectively).1

These parallels are actively being investigated for theirshared therapeutic potential as much as for a fuller under-standing of their shared mechanisms.

Differential DistributionOn the basis of analyses of articular cartilage, the large pro-teoglycan complexes are expected tobe composedof aback-bone of linear hyaluronan, decorated at multiple sites withaggrecan and/or versican core proteins, each with multipleglycosaminoglycan side chains of chondroitin sulfate andlesser amounts of keratan sulfate. This arrangement of a hy-aluronan backbone, aggrecan and versican core proteins,and glycosaminoglycan side chains has recently been re-ported to be present in the digital lamellae.8

Within the lamellae, there are areas of overlap around thedermal-epidermal junction, consistent with assembly ofclassical large polysulfated proteoglycan complexes thatare associated with resistance to compression. However,there are also areas of unique distribution, indicating addi-tional or specialized functions. For example, aggrecan and

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versican core proteins as well as hyaluronan are concen-trated in the extracellularmatrix of the secondary epidermallamellae, around the basal epithelial cells. Chondroitin sul-fates and keratan sulfate also show varying and unique dis-tributions throughout the lamellae, such as concentrationin the secondary dermal lamellae and diffuse staining inthe primary dermal lamellae.8 The significance of this differ-ential distribution warrants further exploration.

Lamellar Glucose MetabolismGlucose supplies the energy essential formaintaining the in-tegrity of the dermal-epidermal interface within the hoof.However, despite much speculation regarding insulin resis-tance, glucosemetabolism, and their links to laminitis, thereis little information regarding as towhere, how, andwhetherglucose is consumed by the living cells of the equine hoof.Pollitt recently conducted a series of experiments to in-

vestigate the use of glucose by the lamellar tissues in healthyhorses at rest.9Using seven adult horses, blood glucose andlactate concentrations were simultaneously measured fromthese vessels: the transverse facial artery, the jugular vein,and a digital vein at the level of the pastern in one foreleg.Antibodies directed against the glucose transport

proteins-1, -3, and -4, and the insulin receptor were usedfor immunolocalization of these proteins in biopsy samplestaken from the gluteus muscle and from the dermal-epidermal interface of all seven horses. In addition, histo-chemical methods were used to localize the enzyme lactatedehydrogenase in the dermal-epidermal interface, with thegluteus muscle serving as the positive control.The researchers reported that the horse’s hoof as com-

pared with its head consumes more glucose and producesmore lactate. Furthermore, the lamellar epidermal basalcells stained strongly for lactate dehydrogenase, indicatinga reliance on anaerobic metabolism for energy production.In other words, even at rest the lamellae in healthy horsesare net lactate producers and seem to depend on anaerobicglycolysis for their energy supply. Therefore, the dermal-epidermal interface may be quite indifferent to the oxygen(but not the glucose) status of its blood supply.Pollitt also reported that glucose transport protein-1 is

the major epidermal cell glucose transporter in the hoof. Incontrast to the gluteus muscle, the hoof dermal-epidermalinterface does not rely on insulin for its glucose uptake.9

OTHER HEADLINES

Pathophysiology

Laminitis Laboratory at Penn Develops a LaminitisDiscovery Database. The Laminitis Laboratory at the Uni-versity of Pennsylvania has established an Equine LaminitisDiscovery Database using tissue and serum samples fromhealthy and laminitic horses (both naturally-occurring and

experimentally-induced laminitis). Banked tissues includeserum, plasma, frozen isolated cells, and tissue from severalregions of the dorsal foot (skin, coronary band, and proxi-mal-, mid-, and distal-lamellar regions). Archived informa-tion includes gross images and histopathology results. Thiscollaborative tissue bank and information database relieson the involvement of local veterinarians, horse owners,and laminitis researchers to ensure that adequate age-,breed-, and sex-matched controls and sample sizes forvarious types and stages of laminitis are available for study.10

Epidermal Lamellar Basal Cells May Hold a Key toLaminitis Pathophysiology and Recovery. The epidermallamellar basal cells are at the dermal-epidermal interface,the “ground zero” of laminitis pathology. Disruption ofepidermal lamellar basal cells adhesion to the basementmembrane and adjacent cells, cell death, and disturbancesin cell proliferation are all key events. Although actively pro-liferating epithelial progenitor cells in the normal hoofmostly localize to the coronary and proximal lamellar re-gions, all lamellar regions maintain proliferative potentialand may contribute toward the “lamellar wedge” and sub-sequent vascular and bone pathology seen with chroniclaminitis. These cells may also hold a key to laminitis path-ophysiology at the cellular level and to the therapeutic and/or regenerative potential of epidermal tissue autografts.10

Equine Genome Aids Laminitis Research. On the basis ofhigh-throughput transcriptome and proteome analysis,completionof the equinegenome sequencenowallows lam-initis researchers tomove from studies limited to preselectedgene andprotein expressionprofiles to amore global viewofthedisease.Researchers atUPenn are investigatinggene andprotein expression during experimental laminitis inductionusing a CHO model (oligofructose overload) and a HImodel (prolonged euglycemic-hyperinsulinemic clamp).Researchers are using an equine whole-genome array repre-senting 21,500 genes for differential gene expression andpathway profiling studies. Differential gene expressiondoes not always translate to differential protein expression;therefore, proteomic studies in the Laminitis Laboratoryare being performed in parallel on the same samples.10

Oxidant Stress IsNot a PrimaryCause of Lamellar Injuryor Failure in CHO or BWE Laminitis. Although earlystages of oxidant stress (lipid peroxidation) were identifiedin the BWE model of laminitis, there was no evidence ofmore severe lamellar oxidant stress (protein carbonylation)in either the BWE or the CHO model. Investigation ofthe lamellar regulation of protective antioxidant enzymesshowed upregulation of both superoxide dismutase-2(SOD2 or mitochondrial SOD) as well as phospholipid hy-droperoxide glutathione peroxidase-4 in the lamellae at theonset of LAM. Therefore, although early reversible oxidant

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stress may occur in the BWE model, end-stage oxidantstress does not seem to occur in either model of laminitisand therefore would not be an ideal primary candidate forcausing lamellar injury and failure.3

Apoptosis May Be a Common EndpointWith the DifferentCauses of Laminitis. The number of cells undergoing orhaving undergone apoptosis (programmed cell death) inthe lamellar basal epithelium is significantly increasedin the CHO model of laminitis. Many factors implicatedin the pathogenesis of laminitis, including hypoxia andinflammatory cytokines, may induce cell death in epithelialcells. Apoptosis is another, andmay in fact be the, commonendpoint for the various causes of laminitis.11

Toll-like Receptor Signaling Is Upregulated in Laminitisand Metabolic Syndrome. Numerous proinflammatorysubstances are present in increased quantities in horseswith laminitis. Investigators at Louisiana State Universitydemonstrated that mediators along the toll-like receptorsignaling pathway are upregulated in the liver of horseswith laminitis and in those predisposed to laminitis becauseof metabolic syndrome.12

Human Sepsis Research May Aid LaminitisTreatment. Signaling of Janus kinase and the signal trans-ducers and activators of transcription (STATs) is being stud-ied as a therapeutic target in human sepsis research, toameliorate inflammatory injury. In horses, both STAT1and STAT3 signaling is activated in the lamellae in theBWE model, and STAT3 signaling is activated in theCHO model.3

Nuchal Crest Fat Is the Most Hormonally Responsive Fatin Horses With EMS. Unlike human beings, in whom vis-ceral fat depots are the most important sites for producingan inflammatory response, the nuchal crest fat has beenshown to be the most responsive site in horses with equinemetabolic syndrome (EMS).3 In a separate study, substan-tial weight gain (approximately 20% of starting bodyweight) resulted in decreased insulin sensitivity, althoughit caused only modest changes in cytokine mRNA expres-sion in subcutaneous adipose tissue.13

Insulin May Contribute to Laminitis Risk in SeveralWays. Themechanisms by whichHI and insulin resistancelead to laminitis may be different from the mechanisms in-volved in CHO-induced laminitis because insulin does notincrease beyond the normal reference range in the CHOmodel of laminitis. HI may contribute to laminitis risk inseveral ways, including vascular derangements and activa-tion of the insulin-like growth factor receptor in lamellarepithelial cells, causing proliferation and potentially weak-ening the lamellar bond. HI may further predispose to

laminitis by lowering the threshold at which other triggerfactors induce the condition.11

Amine Metabolites May Contribute to Pasture-associatedLaminitis. Endothelial cell dysfunction, in which vasodila-tor pathways are inhibited and vasoconstrictormediators arereleased from the endothelium,may be an important predis-posing event in pasture-induced laminitis. Generation ofamine metabolites by cell surface amine oxidases may con-tribute, as they increase the pressor response to vasoconstric-tors, such as 5-hydroxytryptamine (serotonin), and inhibitthe vasodilatory responses that are dependent onendothelium-derived nitric oxide. Increased intake of inulin(a complex carbohydrate similar tograss fructans) can lead toinsulin resistance and increased formation of amines in theGI tract of ponies. Plasma amine oxidase activity is higherin the summer months, when horses and ponies are eatinggrass (and when laminitis prevalence peaks), as comparedwith the winter months, when they are being fed hay.14

Endocrine Studies at UTenn Yield Useful Findings. Keyfindings from endocrine studies conducted at the Univer-sity of Tennessee include the following:6

� Higher insulinemic responses to hay or pasture grass werereported in horses with EMS or equine Cushing’s disease

� Exaggerated insulinemic responses to dexamethasonewere reported in horses with EMS, suggesting thataffected horses are more susceptible to stress-inducedinsulin resistance

� Insulin sensitivity decreased in healthy horses who werehospitalized for 2 weeks, indicating that stress may con-tribute to insulin resistance in hospitalized horses

� Hyperinsulinemia results from both increased insulinsecretion and reduced clearance

� Horses with EMS lost weight when fed with L-thyroxineat a rate of 48 to 72 mg/d for 6 months

Previous Exposure to Endotoxin May Predispose Horses toLaminitis After CHO. Administration of lipopolysaccha-ride (7 ng/kg/h IV for 8 hours) 24 hours before theadministration of oligofructose (5 g/kg PO) tended toincrease the clinical severity of laminitis (P¼ .063). This find-ingmayhave implications forhorses that suffer fromrepeatedCHO events when turned out on pasture for several days ina row. Interestingly, L-thyroxine (48 mg/d per os [PO])ameliorated the insulin resistance induced by the administra-tion of an endotoxin.6

Treatment and Prevention

Lidocaine CRI Is Not an Effective Anti-inflammatoryTreatment for BWE Laminitis. In the BWE modelof laminitis, constant-rate infusion (CRI) of lidocaine

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was ineffective in decreasing proinflammatory cytokineexpression by, or leukocyte infiltration of, the lamellartissue. In fact, the lidocaine CRI increased (P < .05)endothelial activation, as evidenced by increased concen-trations of E-selectin mRNA. This work suggests thatlidocaine by CRI is not anti-inflammatory and shouldnot be used as such in the treatment of laminitis.3

Cecal Buffering May Prevent Lamellar Degradation inCHO Laminitis. Commonly used treatments for lamini-tis, such as NSAIDs, heparin, and nitroglycerin patches,when administered alone are insufficient to inhibit clinicalsigns and histological changes in the CHO model. How-ever, the use of a buffer solution to regulate cecal pHmay reduce the chance of lamellar degradation andsubsequent structural failure. The buffer solution studiedcomprised 3.5 g Al(OH)3, 65.6 g Mg(OH)2, and 1.2 gdimethicone, administered intracecally once, 8 hoursafter CHO. This treatment ameliorated the lamellarexpression of MMP-2 and -9, the lamellar apoptoticindex, and an increase in putrescine concentration in thececum.15

Corticosteroids May Be Worth a Second Look in HorsesWith Intestinal Obstruction. Treatment with hydrocorti-sone before decompressionmay reduce the lamellar inflam-mation associated with intestinal obstruction. Neutrophilgelatinase-associated lipocalin (a leukocyte marker) andMMP-2 and -9 were increased in the lamellae of horsesthat underwent jejunal obstruction. Pretreatment withhydrocortisone (4 mg/kg IV, 60 minutes beforedecompression) ameliorated the increase in neutrophilgelatinase-associated lipocalineMMP-9 complex lamellarconcentrations.15

Pentoxifylline Inhibits MMP Production More EffectivelyThan Tetracyclines. Pentoxifylline (8.5 mg/kg IV q12hours, diluted in 1 L saline) effectively inhibits MMPproduction during DEV in the CHO model and duringendotoxemia. In contrast, doxycycline (10 mg/kg POq12 hours) and oxytetracycline (20 mg/kg IV q12 hours)did not inhibit MMP production sufficiently to preventthe development of Obel grade 3 lameness in the CHOmodel. However, oxytetracycline (but not doxycycline)substantially inhibited MMP-9 production in the low-dose endotoxin model.12

Phenotype Can Predict Pasture-associated Laminitis inPonies. Obesity (body condition score > 7/9), the pres-ence of a cresty neck (i.e. regional adiposity), and HI (insu-lin > 32 mU/L) predict the development of laminitisunder spring grazing conditions in ponies expressing theEMS, phenotype.13

The next report in this conference series wrap-up isClin-ical UpdatesdLaminitis. Please read on for more practicalstrategies for dealing with laminitis.

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