BACKGROUND
laminitis is a painful and debilitating disease of the equine foot.
the specific molecular factors initiating laminitis are unknown, but it is often associated with insulin resistance and obesity and can be precipitated by diseases such as colic and diarrhea
<cit> .
the acute phase of the disease is characterized by the onset of severe lameness and disintegration of the laminae, which connect the hoof wall to the underlying dermis and third phalanx
<cit> .
in many cases, the pathology progresses until the weight of the horse causes dorsopalmar rotation of the third phalanx, detaching it from the hoof wall.
this condition is usually not reversible and euthanasia is often the only humane option.
in many instances, horses that survive an episode of acute laminitis are crippled for life.
this stage of the disease is termed chronic laminitis or “founder”.
acute laminitis transitions to the chronic form of the disease in an estimated 75% of cases
<cit> , leaving the majority of afflicted horses permanently lame.
despite intense efforts to understand the root cause of acute laminitis, much less attention has been focused on the pathophysiology of chronic laminitis.
a handful of studies have investigated changes in laminar morphology, metabolism, and gene expression in foundered horses
<cit> , although several recent studies have focused on the efficacy of different management strategies to ameliorate pain and improve quality of life
<cit> .
little is known, therefore, about how chronic foot pain and inflammation affect the horse on a global level.
a recent study quantified behavioral changes indicative of pain in horses with chronic laminitis; these changes were associated with alterations in morphology and gene expression of the lateral digital nerve and dorsal root ganglia that are consistent with neuropathic pain, suggesting that some of the pain associated with chronic laminitis results from peripheral nerve damage outside of the foot
<cit> .
in addition, wagner et al.
found that horses with chronic laminitis show excessive dermal inflammatory responses to a panel of allergens
<cit> , suggesting an inappropriate activation of the systemic immune response.
to our knowledge, however, these studies are the only published reports describing basic scientific investigations of chronic laminitis.
the dearth of information regarding how chronic laminar inflammation affects the horse as a whole is a major obstacle to understanding why certain horses experience recurrent bouts of laminitis and how these bouts can be prevented.
as a first step towards characterizing global alterations in the physiology of horses with chronic laminitis, we used dige followed by lc-ms/ms to detect differentially expressed plasma proteins in foundered horses.
plasma was chosen as the sample matrix because the blood is a primary means of transport for numerous effector hormones and cytokines of the immune and endocrine systems, which have been suggested to be altered in chronic laminitis
<cit> .
RESULTS
population characteristics
the control  and laminitis  groups were balanced for age and gender.
none of the horses in the con group exhibited signs of lameness at the time of sample collection.
three of the four horses in the lmn group were persistently lame; the fourth experienced intermittent lameness but was sound on the day of sample collection.
two of the permanently lame horses were being treated with a low level of phenylbutazone , a non-steroidal anti-inflammatory drug commonly used in veterinary practice.
horses in both groups were in good body condition and did not exhibit any evidence of injury or illness other than laminitis.
white blood cell counts obtained on the same day that plasma was collected for dige were within normal parameters and did not differ between groups .
detection of differentially expressed spots
in order to view global changes in the plasma of horses with chronic laminitis, we performed dige on plasma depleted of albumin and igg.
the decyder software detected ~ <dig> individual spots,  <dig> of which were differentially expressed in the lmn group as compared to controls .
manual inspection of the spot histograms in the dia module revealed that  <dig> of the  <dig> spots were most likely artefacts based on the size and shape of the 3d intensity profile.
ten spots  were chosen for identification using lc-ms/ms .
table
<dig> describes the relative fold change in intensity and associated p value for each of the  <dig> spots.
we found no evidence to suggest that the horse in the lmn group that was not lame at the time of sampling differed from the other horses in the group or was a possible outlier.
in addition, no differences were noted between the two horses treated with phenylbutazone and those that were unmedicated.
protein identification
twenty one proteins were identified from the lc-ms/ms data generated from the  <dig> digested spots .
human keratin and trypsin derivatives were assumed to be contaminants  and not included in the final list of differentially expressed proteins.
likewise, although the majority of plasma albumin was depleted during sample preparation, it was identified in one of the differentially expressed spots but was not included in table
<dig>  proteins identified from spots upregulated in lmn horses included angiotensinogen, alpha-2-macroglobulin, and apolipoprotein a-iv; these are presented at the top of table
<dig>  antithrombin and complement factor c <dig> precursor were identified in both up- and down-regulated spots.
the remainder of the differentially expressed proteins were from spots downregulated in the lmn group.
predicted molecular weight of each protein, the number of peptides identified from each protein, and the percentage of the total sequence covered by the identified peptides are also shown.
annotation and ontology
the horse genome, though sequenced, is only partially annotated.
therefore, the human orthologs were used to identify gene function and overrepresented gene ontologies among differentially expressed proteins .
five of the proteins identified by ms were found in multiple spots and one protein showed differential expression of two of its subunits .
therefore, of the  <dig> proteins, only  <dig> were unique.
all  <dig> proteins belonged to the extracellular compartment or were secretory molecules stored within platelet alpha granules.
molecular functions of the proteins were predominated by serine endopeptidase inhibitory activity.
overrepresented biological process ontologies included response to wounding, defense response, coagulation, and inflammatory response.
eleven of the  <dig> proteins were identified as being involved in the inter-related coagulation, kininogen, and complement cascades.
the remaining proteins included alpha-2-hs glycoprotein  and fetuin b, both molecular chaperones and participants in the acute phase response
<cit> , the immunoglobulin heavy chains alpha and mu , and apolipoprotein a-iv , which is an anti-inflammatory molecule
<cit> .
immunoglobulin concentrations
the differences in iga and igm concentrations suggested by dige were not confirmed by elisa.
iga concentrations were similar between con and lmn groups , as were igm concentrations .
coagulation panel
because the majority of differentially expressed proteins were involved in the coagulation cascade, we assessed the physiological relevance of our dige results by testing coagulation parameters in seven lmn horses: another three horses with chronic laminitis were used in addition to the four horses used for the dige experiment.
five of the seven horses, including three of the four used for the dige study, had prolonged prothrombin times , indicating that the lmn group was statistically different from the reference population .
it should be noted that none of the animals in the lmn group had a history of coagulopathy.
fibrinogen concentration derived from prothrombin time was elevated in six of seven animals, including all four horses used for the dige experiment, although this finding was not corroborated by the more accurate clauss method of fibrinogen quantification.
fibrin d-dimer was elevated in only one animal.
normal clinical reference ranges are as follows: pt: prothrombin time,  <dig>  –  <dig>  s. ptt: partial thromboplastin time,  <dig>  –  <dig>  sec.
at: antithrombin activity, > <dig> % nhp.
fib pt: fibrinogen , 207– <dig> mg/dl.
fib clauss: fibrinogen , 96– <dig> mg/dl.
d dimer: d dimer of fibrin,  <dig> –  <dig> ng/ml.
asterisks indicate deviation from normal reference range.
apoa-iv concentration
immunoblotting for equine apoa-iv using an anti-human antibody was successful, with a single band detected at ~ <dig> kda.
no other bands were detected on the blot
<cit> .
densitometry showed a significant  elevation of apoa-iv in horses with chronic laminitis .
discussion
the data presented herein describe, for the first time, plasma proteome changes in chronic equine laminitis.
our results suggest that a number of proteins involved in immune regulation are differentially expressed in horses with chronic equine laminitis.
in particular, the anti-inflammatory protein apoa-iv was elevated approximately two-fold in foundered horses.
although no major deficits in the coagulation cascade were seen in the present study, possible alterations in the complement and kininogen cascades have yet to be explored.
our results suggest that chronic inflammation of the laminae of the hoof might be associated with system-wide changes in immune function and that this hypothesis warrants further investigation.
these conclusions are discussed in detail below.
technological considerations
dige has been used successfully to identify contributing factors and biomarkers of a number of human diseases, including multiple sclerosis
<cit> , acute and chronic liver disease
<cit> , and various cancers
<cit> .
to our knowledge, however, dige has only been applied to horses in two previous studies; these authors used dige with and without depletion of abundant serum proteins to investigate equine recurrent uveitis
<cit> , a chronic inflammatory disorder of the eye with many similarities to human autoimmune uveitis.
as in our study, antibodies directed against human proteins removed a large portion of abundant equine serum proteins, including albumin and igg
<cit> .
zipplies et al.
identified kininogen, apolipoprotein a-iv, igm, and alpha-2-hs glycoprotein as differentially expressed in eru
<cit> ; the overlap between their results and ours may be because eru and chronic laminitis are both chronic inflammatory diseases and similar pathways are likely involved in disease pathogenesis.
it must be noted, however, that the direction of change seen in apoa-iv and kininogen in the present study was not consistent with that seen by zipplies et al., although the reason for this remains unknown.
of the  <dig> differentially expressed proteins identified in this study,  <dig> have already been associated with other chronic diseases in rats
<cit> , humans
<cit> , and horses
<cit>  using dige.
to our knowledge, coagulation factor x and iga have not been associated with either acute or chronic diseases using dige based proteomics.
the high level of redundancy in differentially expressed proteins among various disease states could result from the known bias of dige towards detecting relatively high abundance proteins.
alternatively, there could be a core group of proteins that are differentially expressed during chronic inflammation.
analogous to the “acute phase reactants” seen during acute trauma, infection, or stress, these “chronic phase reactants” might serve a similar purpose in modulation of the immune response.
further standardization of experimental proteomics methods and meta-analysis of a larger number of studies is necessary to confirm or refute this hypothesis.
coagulation and complement
in our study, the majority  of differentially expressed proteins belonged to the inter-related coagulation, complement, and kininogen cascades.
the coagulation cascade in lmn horses was characterized by the decreased expression of clotting factors such as factor x and fibrinogen and the increased expression of the inhibitor antithrombin, all of which would inhibit clotting.
this was balanced, however, by an increase in alpha-2-macroglobulin and a decrease in protein s; the effect of these two changes would be to promote clotting through the inhibition of the anti-coagulant protein c. changes in abundance of liver-associated clotting proteins are dependent upon the rates of both production and consumption, so the dige results provided limited information about the ability of the blood to clot.
we therefore assessed the functional significance of our results using a coagulation panel, which showed a slightly prolonged prothrombin  time.
it should be noted that, even though pt time was elevated above normal levels in  <dig> horses, the clinical relevance of this finding is yet to corroborated by other approaches.
findings from the coagulation panel were sufficient to rule out the presence of major coagulopathies such as disseminated intravascular coagulation and antithrombin deficiency, but unfortunately do not provide insight as to the mechanism underlying the changes in clotting proteins seen with dige.
one possible explanation for the alteration in liver-associated proteins is the over-expression of interleukin- <dig>  by white blood cells of horses with chronic laminitis .
il- <dig> has the potential to both positively and negatively regulate hepatocyte expression of a number of acute phase proteins
<cit> ; specifically, it has been shown to induce expression of angiotensinogen and alpha-2-macroglobulin
<cit> , both of which were upregulated in our study.
further investigation of this possibility is currently underway in our laboratory.
as two of the horses in the lmn group were being treated with the nsaid phenylbutazone at the time of sample collection, this had the potential to influence our results.
although, to the best of our knowledge, phenylbutazone use has not been proven to directly alter the expression of any of the diffferentially expressed proteins in the current study, we cannot rule out this possibility.
in addition, nsaids are known to affect the coagulation cascade through their regulation of prostaglandins; however, we saw no obvious differences in any of the clotting parameters tested in the two horses receiving treatment  as compared with the rest of the group.
indeed, previous reports suggest that phenylbutazone at the dose used in the current study had no effect on thrombus formation, fibrinogen concentration, or factor xa activity in rats
<cit> .
apoa-iv
our results showed a significant upregulation of apolipoprotein a-iv in horses with chronic laminitis as compared to controls.
apoa-iv is an essential component of chylomicrons and is produced by the small intestine, particularly in response to the ingestion of triglycerides
<cit> .
in this way, it functions as a satiety factor
<cit> , but it is also known to have anti-oxidant
<cit>  and anti-inflammatory
<cit>  properties.
vowinkel and colleagues
<cit>  showed that apoa-iv ameliorates experimentally-induced colitis in mice by attenuating the upregulation of p-selectin and decreasing microvascular adhesion of platelets and leukocytes.
studies in horses and dogs have shown extensive polymorphism of apoa-iv
<cit> ; in humans, certain alleles have been associated with elevated blood glucose and triglyceride levels as well as body mass index
<cit> .
the tissue sources and physiological functions of apoa-iv have yet to be explored in horses.
evidence exists, however, that serum apoa-iv levels are influenced by the gastrointestinal microflora in mice
<cit> , which, like horses, are hindgut fermenters.
preliminary data from our laboratory suggest that the composition of fecal microflora is altered in horses with chronic laminitis as compared with controls, thus providing a possible explanation for the elevation of apoa-iv seen in the present study.
the underlying mechanism is currently unknown, although the composition of the hindgut microflora play an important role in the development of acute laminitis
<cit>  and could possibly contribute to exacerbation of symptoms in chronic laminitis as well.
future studies will focus on these questions and their relevance to the problem of equine laminitis.
CONCLUSIONS
our results suggest that chronic equine laminitis is associated with specific changes in the plasma proteome, which might be reflective of alterations in the immune system that are not confined to the foot.
specifically, the altered abundance of complement proteins and acute phase reactants suggests a chronic activation of the innate immune response.
our results provide a base upon which to build future studies with the ultimate goal of attenuating inflammation and reducing the pain associated with this devastating disease.

 laminitis is a painful and debilitating disease of the equine foot. the specific molecular factors initiating laminitis are unknown, but it is often associated with insulin resistance and obesity and can be precipitated by diseases such as colic and diarrhea. the acute phase of the disease is characterized by the onset of severe lameness and disintegration of the laminae, which connect the hoof wall to the underlying dermis and third phalanx. in many cases, the pathology progresses until the weight of the horse causes dorsopalmar rotation of the third phalanx, detaching it from the hoof wall. this condition is usually not reversible and euthanasia is often the only humane option. in many instances, horses that survive an episode of acute laminitis are crippled for life. this stage of the disease is termed chronic laminitis or “founder”. acute laminitis transitions to the chronic form of the disease in an estimated 75% of cases, leaving the majority of afflicted horses permanently lame. despite intense efforts to understand the root cause of acute laminitis, much less attention has been focused on the pathophysiology of chronic laminitis. a handful of studies have investigated changes in laminar morphology, metabolism, and gene expression in foundered horses, although several recent studies have focused on the efficacy of different management strategies to ameliorate pain and improve quality of life. little is known, therefore, about how chronic foot pain and inflammation affect the horse on a global level. a recent study quantified behavioral changes indicative of pain in horses with chronic laminitis; these changes were associated with alterations in morphology and gene expression of the lateral digital nerve and dorsal root ganglia that are consistent with neuropathic pain, suggesting that some of the pain associated with chronic laminitis results from peripheral nerve damage outside of the foot. in addition, wagner et al. found that horses with chronic laminitis show excessive dermal inflammatory responses to a panel of allergens, suggesting an inappropriate activation of the systemic immune response. to our knowledge, however, these studies are the only published reports describing basic scientific investigations of chronic laminitis. the dearth of information regarding how chronic laminar inflammation affects the horse as a whole is a major obstacle to understanding why certain horses experience recurrent bouts of laminitis and how these bouts can be prevented. as a first step towards characterizing global alterations in the physiology of horses with chronic laminitis, we used dige followed by lc-ms/ms to detect differentially expressed plasma proteins in foundered horses. plasma was chosen as the sample matrix because the blood is a primary means of transport for numerous effector hormones and cytokines of the immune and endocrine systems, which have been suggested to be altered in chronic laminitis.  population characteristics the control and laminitis groups were balanced for age and gender. none of the horses in the con group exhibited signs of lameness at the time of sample collection. three of the four horses in the lmn group were persistently lame; the fourth experienced intermittent lameness but was sound on the day of sample collection. two of the permanently lame horses were being treated with a low level of phenylbutazone, a non-steroidal anti-inflammatory drug commonly used in veterinary practice. horses in both groups were in good body condition and did not exhibit any evidence of injury or illness other than laminitis. white blood cell counts obtained on the same day that plasma was collected for dige were within normal parameters and did not differ between groups. detection of differentially expressed spots in order to view global changes in the plasma of horses with chronic laminitis, we performed dige on plasma depleted of albumin and igg. the decyder software detected ~ individual spots, of which were differentially expressed in the lmn group as compared to controls. manual inspection of the spot histograms in the dia module revealed that of the spots were most likely artefacts based on the size and shape of the 3d intensity profile. ten spots were chosen for identification using lc-ms/ms. table describes the relative fold change in intensity and associated p value for each of the spots. we found no evidence to suggest that the horse in the lmn group that was not lame at the time of sampling differed from the other horses in the group or was a possible outlier. in addition, no differences were noted between the two horses treated with phenylbutazone and those that were unmedicated. protein identification twenty one proteins were identified from the lc-ms/ms data generated from the digested spots. human keratin and trypsin derivatives were assumed to be contaminants and not included in the final list of differentially expressed proteins. likewise, although the majority of plasma albumin was depleted during sample preparation, it was identified in one of the differentially expressed spots but was not included in table proteins identified from spots upregulated in lmn horses included angiotensinogen, alpha-2-macroglobulin, and apolipoprotein a-iv; these are presented at the top of table antithrombin and complement factor c precursor were identified in both up- and down-regulated spots. the remainder of the differentially expressed proteins were from spots downregulated in the lmn group. predicted molecular weight of each protein, the number of peptides identified from each protein, and the percentage of the total sequence covered by the identified peptides are also shown. annotation and ontology the horse genome, though sequenced, is only partially annotated. therefore, the human orthologs were used to identify gene function and overrepresented gene ontologies among differentially expressed proteins. five of the proteins identified by ms were found in multiple spots and one protein showed differential expression of two of its subunits. therefore, of the proteins, only were unique. all proteins belonged to the extracellular compartment or were secretory molecules stored within platelet alpha granules. molecular functions of the proteins were predominated by serine endopeptidase inhibitory activity. overrepresented biological process ontologies included response to wounding, defense response, coagulation, and inflammatory response. eleven of the proteins were identified as being involved in the inter-related coagulation, kininogen, and complement cascades. the remaining proteins included alpha-2-hs glycoprotein and fetuin b, both molecular chaperones and participants in the acute phase response, the immunoglobulin heavy chains alpha and mu, and apolipoprotein a-iv, which is an anti-inflammatory molecule. immunoglobulin concentrations the differences in iga and igm concentrations suggested by dige were not confirmed by elisa. iga concentrations were similar between con and lmn groups, as were igm concentrations. coagulation panel because the majority of differentially expressed proteins were involved in the coagulation cascade, we assessed the physiological relevance of our dige results by testing coagulation parameters in seven lmn horses: another three horses with chronic laminitis were used in addition to the four horses used for the dige experiment. five of the seven horses, including three of the four used for the dige study, had prolonged prothrombin times, indicating that the lmn group was statistically different from the reference population. it should be noted that none of the animals in the lmn group had a history of coagulopathy. fibrinogen concentration derived from prothrombin time was elevated in six of seven animals, including all four horses used for the dige experiment, although this finding was not corroborated by the more accurate clauss method of fibrinogen quantification. fibrin d-dimer was elevated in only one animal. normal clinical reference ranges are as follows: pt: prothrombin time, – s. ptt: partial thromboplastin time, – sec. at: antithrombin activity, > % nhp. fib pt: fibrinogen, 207– mg/dl. fib clauss: fibrinogen, 96– mg/dl. d dimer: d dimer of fibrin, – ng/ml. asterisks indicate deviation from normal reference range. apoa-iv concentration immunoblotting for equine apoa-iv using an anti-human antibody was successful, with a single band detected at ~ kda. no other bands were detected on the blot. densitometry showed a significant elevation of apoa-iv in horses with chronic laminitis. discussion the data presented herein describe, for the first time, plasma proteome changes in chronic equine laminitis. our results suggest that a number of proteins involved in immune regulation are differentially expressed in horses with chronic equine laminitis. in particular, the anti-inflammatory protein apoa-iv was elevated approximately two-fold in foundered horses. although no major deficits in the coagulation cascade were seen in the present study, possible alterations in the complement and kininogen cascades have yet to be explored. our results suggest that chronic inflammation of the laminae of the hoof might be associated with system-wide changes in immune function and that this hypothesis warrants further investigation. these conclusions are discussed in detail below. technological considerations dige has been used successfully to identify contributing factors and biomarkers of a number of human diseases, including multiple sclerosis, acute and chronic liver disease, and various cancers. to our knowledge, however, dige has only been applied to horses in two previous studies; these authors used dige with and without depletion of abundant serum proteins to investigate equine recurrent uveitis, a chronic inflammatory disorder of the eye with many similarities to human autoimmune uveitis. as in our study, antibodies directed against human proteins removed a large portion of abundant equine serum proteins, including albumin and igg. zipplies et al. identified kininogen, apolipoprotein a-iv, igm, and alpha-2-hs glycoprotein as differentially expressed in eru; the overlap between their results and ours may be because eru and chronic laminitis are both chronic inflammatory diseases and similar pathways are likely involved in disease pathogenesis. it must be noted, however, that the direction of change seen in apoa-iv and kininogen in the present study was not consistent with that seen by zipplies et al., although the reason for this remains unknown. of the differentially expressed proteins identified in this study, have already been associated with other chronic diseases in rats, humans, and horses using dige. to our knowledge, coagulation factor x and iga have not been associated with either acute or chronic diseases using dige based proteomics. the high level of redundancy in differentially expressed proteins among various disease states could result from the known bias of dige towards detecting relatively high abundance proteins. alternatively, there could be a core group of proteins that are differentially expressed during chronic inflammation. analogous to the “acute phase reactants” seen during acute trauma, infection, or stress, these “chronic phase reactants” might serve a similar purpose in modulation of the immune response. further standardization of experimental proteomics methods and meta-analysis of a larger number of studies is necessary to confirm or refute this hypothesis. coagulation and complement in our study, the majority of differentially expressed proteins belonged to the inter-related coagulation, complement, and kininogen cascades. the coagulation cascade in lmn horses was characterized by the decreased expression of clotting factors such as factor x and fibrinogen and the increased expression of the inhibitor antithrombin, all of which would inhibit clotting. this was balanced, however, by an increase in alpha-2-macroglobulin and a decrease in protein s; the effect of these two changes would be to promote clotting through the inhibition of the anti-coagulant protein c. changes in abundance of liver-associated clotting proteins are dependent upon the rates of both production and consumption, so the dige results provided limited information about the ability of the blood to clot. we therefore assessed the functional significance of our results using a coagulation panel, which showed a slightly prolonged prothrombin time. it should be noted that, even though pt time was elevated above normal levels in horses, the clinical relevance of this finding is yet to corroborated by other approaches. findings from the coagulation panel were sufficient to rule out the presence of major coagulopathies such as disseminated intravascular coagulation and antithrombin deficiency, but unfortunately do not provide insight as to the mechanism underlying the changes in clotting proteins seen with dige. one possible explanation for the alteration in liver-associated proteins is the over-expression of interleukin- by white blood cells of horses with chronic laminitis. il- has the potential to both positively and negatively regulate hepatocyte expression of a number of acute phase proteins; specifically, it has been shown to induce expression of angiotensinogen and alpha-2-macroglobulin, both of which were upregulated in our study. further investigation of this possibility is currently underway in our laboratory. as two of the horses in the lmn group were being treated with the nsaid phenylbutazone at the time of sample collection, this had the potential to influence our results. although, to the best of our knowledge, phenylbutazone use has not been proven to directly alter the expression of any of the diffferentially expressed proteins in the current study, we cannot rule out this possibility. in addition, nsaids are known to affect the coagulation cascade through their regulation of prostaglandins; however, we saw no obvious differences in any of the clotting parameters tested in the two horses receiving treatment as compared with the rest of the group. indeed, previous reports suggest that phenylbutazone at the dose used in the current study had no effect on thrombus formation, fibrinogen concentration, or factor xa activity in rats. apoa-iv our results showed a significant upregulation of apolipoprotein a-iv in horses with chronic laminitis as compared to controls. apoa-iv is an essential component of chylomicrons and is produced by the small intestine, particularly in response to the ingestion of triglycerides. in this way, it functions as a satiety factor, but it is also known to have anti-oxidant and anti-inflammatory properties. vowinkel and colleagues showed that apoa-iv ameliorates experimentally-induced colitis in mice by attenuating the upregulation of p-selectin and decreasing microvascular adhesion of platelets and leukocytes. studies in horses and dogs have shown extensive polymorphism of apoa-iv; in humans, certain alleles have been associated with elevated blood glucose and triglyceride levels as well as body mass index. the tissue sources and physiological functions of apoa-iv have yet to be explored in horses. evidence exists, however, that serum apoa-iv levels are influenced by the gastrointestinal microflora in mice, which, like horses, are hindgut fermenters. preliminary data from our laboratory suggest that the composition of fecal microflora is altered in horses with chronic laminitis as compared with controls, thus providing a possible explanation for the elevation of apoa-iv seen in the present study. the underlying mechanism is currently unknown, although the composition of the hindgut microflora play an important role in the development of acute laminitis and could possibly contribute to exacerbation of symptoms in chronic laminitis as well. future studies will focus on these questions and their relevance to the problem of equine laminitis.  our results suggest that chronic equine laminitis is associated with specific changes in the plasma proteome, which might be reflective of alterations in the immune system that are not confined to the foot. specifically, the altered abundance of complement proteins and acute phase reactants suggests a chronic activation of the innate immune response. our results provide a base upon which to build future studies with the ultimate goal of attenuating inflammation and reducing the pain associated with this devastating disease. 
we identified differentially expressed proteins; the majority of these were involved in the interrelated coagulation, clotting, and kininogen cascades. our results support the hypothesis that localized laminar inflammation may be linked to systemic alterations in immune regulation, particularly in the gastrointestinal system. in acute laminitis, the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall, leaving the horse unable to bear weight on the affected limbs. gastrointestinal inflammation has been implicated in the development of acute laminitis but has not previously been associated with chronic laminitis. we investigated this broad hypothesis by using dige to assess global differences in the plasma proteome between horses with chronic laminitis and controls. upregulation of the intestinal apolipoprotein apoa-iv in horses with chronic laminitis was confirmed by western blot. horses that survive the acute phase transition into a chronic form of laminitis, which is often termed “founder”. equine laminitis is a devastating disease that causes severe pain in afflicted horses and places a major economic burden on the horse industry.
 equine laminitis is a devastating disease that causes severe pain in afflicted horses and places a major economic burden on the horse industry. in acute laminitis, the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall, leaving the horse unable to bear weight on the affected limbs. horses that survive the acute phase transition into a chronic form of laminitis, which is often termed “founder”. some evidence suggests that chronic laminar inflammation might be associated with alterations in the endocrine and immune systems. we investigated this broad hypothesis by using dige to assess global differences in the plasma proteome between horses with chronic laminitis and controls.  we identified differentially expressed proteins; the majority of these were involved in the interrelated coagulation, clotting, and kininogen cascades. clinical testing of functional coagulation parameters in foundered horses revealed a slight delay in prothrombin clotting time, although most other indices were within normal ranges. upregulation of the intestinal apolipoprotein apoa-iv in horses with chronic laminitis was confirmed by western blot.  our results support the hypothesis that localized laminar inflammation may be linked to systemic alterations in immune regulation, particularly in the gastrointestinal system. gastrointestinal inflammation has been implicated in the development of acute laminitis but has not previously been associated with chronic laminitis. 