Identification of intra-abdominal hyaluronan in peritoneal fluid of equine patients presenting with an acute abdomen

Abstract

Objective

To determine levels of hyaluronan (HA) within peritoneal fluid from healthy horses and horses admitted for acute abdominal crisis. Additionally, to identify the cellular sources of HA within the peritoneal cavity.

Animals

Seven client owned horses and 19 horses donated to KSU-CVM.

Procedures

Abdominal fluid was collected from horses admitted for acute abdominal crisis and from healthy horses. HA levels were determined by ELISA. Equine mesothelial cells were aseptically harvested from horses immediately post-euthanasia, and digested in a 25 cm² cell culture flask containing 0.25% trypsin-EDTA. Primary cell cultures were maintained for total protein and RNA harvested from confluent monolayers. Western blot immunoassays were utilized to detect expression of mesothelial cell markers: cytokeratin 8 and 18, vimentin, calretinin, mesothelin and CD44. RT-PCR was utilized to detect genetic expression of hyaluronan synthase-2 (HAS-2) from cultured and native equine tissue.

Results

A significant difference was found between the mean abdominal HA levels from horses in acute abdominal crisis (1203.3 ± 23.13 ng/ml) compared with healthy horses (228.4 ± 83.63). Harvested cells were maintained and immunoblotting analyses confirmed expression of the mesothelial markers. Gene expression of HAS-2 from cultured mesothelial cells and fibroblasts was confirmed with RT-PCR.

Conclusion and Clinical Relevance

Results demonstrate a significant increase in peritoneal HA levels in equine patients suffering from an acute abdominal crisis. Cultured equine mesothelial cells and fibroblasts are capable of producing HA through HAS-2. Further investigation should focus on establishing the effect of exogenous HA administration on mesothelial cell function in our clinical patients.

Introduction

Intra-abdominal adhesions are important post-operative complications that occur following celiotomy in horses resulting in future episodes of abdominal pain.¹ Horses that develop post-operative adhesions generally have a poor prognosis for long term survival.² For all horses undergoing surgery for small intestinal pathology, the re-operative rate and/or euthanasia rate has been reported to be as high as 22%³. However, the risk of adhesion formation can be reduced by atraumatic tissue handling, meticulous hemostasis, minimizing bacterial contamination, limiting the introduction of foreign material, preventing tissue desiccation, timely surgical intervention and aggressive post-operative medical management of endotoxemia and ileus. Clinical conditions rarely offer themselves as ideal situations. Thus, additional prophylactic measures have been developed to combat adhesion formation, including the application of agents to the surface of the bowel at the time of surgery^4,5. These treatments may have direct effects on mesothelia cells.

The mesothelium is an extensive monolayer whose function is essential to the health of the abdominal cavity. Mesothelia maintain a virtually frictionless environment within the abdominal cavity that facilitates normal bowel motility. Additionally, mesothelial cells are capable of rapid migration after serosal injuries⁶ and modulate serosal inflammation via the production and secretion of various pro- and anti-inflammatory mediators.^6–10 Proposed avenues of mesothelial surface reconstitution include the migration of mesothelial cells, which is in part facilitated by endogenous HA, the predominant glycosaminoglycan secreted by mesothelial cells.

Preservation of peritoneal mesothelium is critical for preventing adhesion formation, and pharmacological manipulation is one method for maintaining mesothelial integrity. Two therapeutic agents utilized to decrease abdominal adhesion formation are HA¹¹, and bioabsorbable hyaluronate-carboxymethylcellulose.¹² Presumably, these agents offer a lubricated barrier to prevent the formation of fibrin on disrupted serosal surfaces; however, other mechanisms of action may be responsible for the therapeutic benefit provided by such treatments, particularly in the case of HA.

Synthesizes of HA has been documented in human mesothelia and three membrane bound HA synthase isoforms (HAS-1, HAS-2 and HAS-3) have been identified at the inner face of the plasma membrane.¹³To date, only mRNA for HAS-2 has been documented in equine origin tissue.¹⁴ Additionally, only HAS-1 and HAS-3 protein has been documented in equine cumulus cells.¹⁵ After HA is produced, HA can bind two main classes of cell surface receptors, CD44 and receptor for hyaluronan mediated motility (RHAMM). Subsequent to receptor binging, a variety of physiological events are initiated including cell migration, cell adhesion, and cell proliferation^16–18, all of which are vital to the wound healing process. It has been shown that CD44 is present in equine lymphocytes, serosa, peritoneum, omentum, and mesentery¹⁹, however, precise biological significance of this expression has yet to be demonstrated in the horse.

There are few data regarding the function of mesothelial cells in equine abdomen despite the wealth of information available for other species.^20–23 Clearly, a more thorough understanding of equine peritoneal mesothelial cell migration and the pathogenesis of equine adhesion formation at a cellular level will promote prevention or enhance treatment of post-operative abdominal adhesions. However, to our knowledge, the presence and production of HA within normal and abnormal equine abdominal fluid has not been reported. Furthermore, an established equine peritoneal mesothelial cell line is not available nor are there techniques for harvesting and maintaining primary equine mesothelial cells. The source of HA within the equine abdomen may be identified and its effect on mesothelial cell migration or its role in fibrinolysis could then be investigated in vitro. Therefore, this study was undertaken to determine whether there were differences in the level and site of HA production in peritoneal fluid from normal horses and horses presenting an abdominal crisis. The expression (mRNA) of HAS-2 was measured in primary mesothelial cells and fibroblasts that were harvested from the equine peritoneal cavity and maintained under cell culture condition. Additionally native equine mesenteric tissue and synovium from healthy horses was analyzed for the HAS-2 mRNA. Primary cell cultures (mesothelial cells and fibroblasts) were analyzed with Western blot techniques to confirm cell line identity initially determined by microscopic morphology.

B. Materials and Methods

Animals

The Institutional Animal Care and Use Committee of Kansas State University (KSU) approved all procedures involving horses. To measure the concentration of HA in the abdominal cavity, peritoneal fluid was collected from 13 horses; 6 healthy horses from the KSU teaching herd and 7 client-owned horses admitted to the veterinary teaching hospital for an acute abdominal crisis that underwent exploratory celiotomy. Mesothelial cell harvesting techniques were conducted in an additional 13 horses. All horses were donated to the veterinary teaching hospital, 11 of which had no history of abdominal disease, and the other 2 had recent previous abdominal surgery for an unrelated study 2 weeks previous.

Peritoneal Fluid Collection/Analysis

Aseptic peritoneal samples were obtained using an 18 gauge needle collected in to blood tubes (serum and EDTA) (standing) or a 12 mL syringe (at surgery). Samples were taken from a point approximately 10 cm caudal to the xyphoid process on ventral midline in the standing horse (non-surgical) or at the time of the exploratory celiotomy. Duration of abdominal discomfort, location of the intestinal lesion, and outcome of the surgery (euthanized or discharged) were recorded for the client-owned horses that underwent an exploratory laparotomy (Table 1).

Table 1.

Summary of signalment (F=female, G=gelding, QH=Quarter Horse, Arab=Arabian, App=Appalossa, Andal=Andalusian), gross appearance, abdominocentesis findings, colic duration, surgical lesion, outcome of surgery (E = euthanized, D = discharged) and peritoneal HA level for horses that presented in an acute abdominal crisis.

Signalment	Gross Appearance	TNCC (103/uL),	TP (g/dL)	Colic Duration	Lesion	Outcome	HA Level (ng/mL)
F, 2yr, QH	Clear	0.9	2.0	12 hrs	360° large colon volvulus	E	1255
F, 10yr, QH	Turbid	14.0	2.2	12 hrs	Ileal impaction	D	1135
G, 9yr, Arab	Turbid	3.0	4.3	12 hrs	Large colon displacement, Severe gas distention proximal enteritis	D	1204
G, 3yr, QH	Clear	1.1	2.5	10 hrs	Large colon displacement	D	1196
G, 6yr, QH	Serosanguinous	0.7	1.8	6 hrs	Epiploic foramen entrapment; no resection	D	1176
F, 17yr, App	Serosanguinous	4.8	3.0	6 hrs	Strangulating lipoma	D	1146
F, 16yr, Andal	Serosanguinous	3.8	4.1	24 hrs	Strangulating lipoma	E	1309

Abdominocentesis samples were collected for cytological and HA analysis. Plasma from each horse was collected via venipuncture at the same time. Abdominal fluid and plasma were stored at −80°C for later analysis. A 96-well commercially available enzyme-linked immunosorbant assay^a (ELISA) was utilized for the determination of HA levels as described previously.²¹ All samples were analyzed in triplicate. The assay was performed accounting to manufacture’s specifications with the standards provided. Using equine plasma samples, the reported intra-assay and inter-assay coefficients of variation are 4.9 and 11%, respectfully.²⁴

Equine Mesothelial Cell Harvest and Culture

Equine mesothelial cells were harvested aseptically immediately post-euthanasia. Horses were placed in dorsal recumbency and a ventral midline approach was used. Three techniques were used to harvest the cells: mesenteric sacks (n=10 horses), omental explants (n=2 horses) and a mesenteric explant (n=3 horses). The mesenteric sack technique consisted of a purse string suture pre-placed in a circular portion of mesentery between two arterial arcades of the mid-jejunum. The mesentery was sharply transected outside the suture line and the purse string suture was tightened leaving a small opening to the lumen of the sack. The sack technique was developed here in order to expose just the mesothelium to the digesting enzymes while limiting digestion contact to sub-mesothelia tissue which may contain fibroblasts. Eight mesenteric sack samples were harvested from each horse for a total of 80 samples. Omental and mesenteric explants were obtained by isolating and sharply transecting one sample (approximately 10 cm × 10 cm) from each horse. All samples were washed and immediately transported to the laboratory in phosphate buffered saline (PBS).

Mesenteric sacks were filled with warmed saline (37°C) containing 0.25% trypsin-EDTA^b, the purse string suture was tightened, and the samples were incubated at 37°C with gentle agitation. Incubation time was determined empirically and ranged from 15 to 45 min with more dissociation appreciated between 30 and 45 min. Dissociation was stopped with a 50% dilution of complete medium. The culture medium consisted of medium M199^c supplemented with 10% fetal bovine serum^d, penicillin (100 IU/mL)/streptomycin (100 mg/mL)^c, L-glutamine (2 µg/mL)^b, insulin (5 µg/mL)^c and hydrocortisone (0.4 µg/mL)^b. Dissociated cells were transferred to a cell culture flask^e (25 cm²) and incubated as described below.

Omental and mesenteric specimens were placed into cell culture flasks (25 cm²), and incubated with pre-warmed 0.9% saline containing 0.25% trypsin-EDTA (5 mL) with agitation for 15 minutes at 37°C. Tissues were removed and trypsin-EDTA was neutralized with equal parts of complete medium. The solution containing cells, trypsin-EDTA and medium was transferred to a 15 mL conical tube and centrifuged at 300 × g for 5 min. Pelleted cells were suspended in medium and seeded onto cell culture flasks.

All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Cells were grown to a confluent monolayer, changing the medium 24 hrs after the initial seeding. Medium was changed every 48 to 72 hours afterwards. Light photomicroscopy was performed on live cells using an inverted, phase-contrast microscope^f with attached camera^g and digital software^h on a daily basis. Time to confluence (monolayer of cells covering bottom of the entire tissue flask) was documented.

Subcultures

Once confluent, additional passages of cells were obtained by discarding the medium and adding 1 mL of 0.25% trypsin-EDTA to each cell culture flask for 2 minutes. The trypsin-EDTA and lifted cells were removed and placed immediately into a new cell culture flask (25cm²) along with 5 mL of culture medium. The original tissue flask was washed three times with 5 mL PBS and culture medium was replaced (5 mL). Samples were maintained subsequently at 37°C in a humidified atmosphere containing 5% CO₂.

To minimize fibroblast contamination of mesothelial cell cultures, plates were examined for areas of fibroblastic overgrowth. Fibroblasts were identified by cell morphology under light microscopy. The areas of fibroblast growth were scraped with a cell scraper and then 1 mL of 0.25% trypsin-EDTA solution was added to each cell culture flask for approximately 1 minute, fibroblast lifting was monitored continuously. Trypsin-EDTA and fibroblasts were aspirated and placed into new cell culture flasks with 5 mL of culture medium. These subcultured fibroblasts were used for further analyses. The original culture flask was washed with three cycles of 5 mL of culture medium to aid in the removal of any loose fibroblasts that remained, was fed and returned to the incubator.

RT-PCR Analysis of Hyaluronan Synthase 2

To determine relative expression of mRNA coding for HAS-2, RNA was extracted from cultured equine peritoneal mesothelial cells, cultured equine peritoneal fibroblasts, freshly isolated native equine mesentery, ovary, and synovium using a commercially available kitⁱ and stored at −8000B0;C. Tissues were disrupted, lysed and homogenized in the provided lysis buffer with the addition of β-mercaptoethanol (10 µL/1mL lysis buffer). Quality and quantity of RNA were measured by utilizing the A₂₆₀/A₂₈₀ ratio with a Nanodrop ND-1000 Spectrophotometer^j. Aliquoted samples were frozen at −80° C.

The expression of HAS-2 was analyzed utilizing PCR with real time reporting. Equine specific primers for HAS-2 and 18S were used as described previously.¹⁴ Analyses were conducted using cultured mesothelia and fibroblasts from 3 different horses. RT-PCR was performed on a Cepheid SmartCycler^l using SYBR green I and a commercial kit (SuperScript III Platinum One-Step qRT-PCR System, Invitrogen, Grand Island NY). Each reaction was performed using 100 ng RNA. The thermocycling protocol was as follows: one cycle of 48°C for 45 min and 94°C for 2 min, followed by 40 cycles of 94°C for 30 sec, 55°C for 1 min, and 68°C for 2 min. PCR products were processed using a commercially available kit^m, and gel electrophoresis confirmed that there was a single product for each primer pair. The purified PCR products were sequenced by dye terminator cycle sequencing method using GenomeLab DTCS-Quick Start Kit^k and a Beckman Coulter CEQ 8000XLⁿ genetic analysis system. Product identity was verified by comparison with the sequence reported for equine HAS-2 (NCBI Entrez accession number AY56582).

Western blot assay

Immunoblot assays were performed to identify protein expression of mesothelial cell markers from the cultured cells that exhibited morphology consistent with mesothelia (cytokeratins, calrentinin, mesothelin) and fibroblasts (vimentin). Proteins of interest included pan cytokeratin (mouse anti-human pan cytokeratin clone C-11 monoclonal antibody^c), cytokeratin 8 and 18 (mouse anti-human cytokeratin 8 and 18 monoclonal antibody^c), vimentin (mouse anti-vimentin clone V9 monoclonal antibody^c), calretinin (rabbit anti-human calretinin polyclonal antibody^o), CD44 (rat anti-human CD44S^o), and mesothelin (mouse anti-human mesothelin monoclonal antibody^c). Although these antibodies are not equine specific, cross reactivity was confirmed and controlled by using protein harvested from rat epithelial cell lines (IEC-6), equine heart, rat brain, rat heart and RIPA lysis buffer^p to ensure that antibodies identified a protein of interest at the reported mobility of the expected target.

Confluent monolayers of equine mesothelial cells and fibroblasts were harvested for protein analysis. Cells were washed several times with chilled PBS and placed in RIPA buffer for total cell protein isolation. Cell lysate protein concentrations were determined with a bicinchoninic acid assay^q. Aliquots were stored at −80°C until used for immunoblotting.

Protein (20 µg) was suspended in SDS gel loading buffer with and without 2-mercaptoethonal for reducing and non-reducing conditions, respectively. Protein was then loaded and resolved electrophoretically on a 4–12% SDS–polyacrylamide gel, for subsequent wet transfer to a nitrocellulose membrane^r. Membranes underwent blocking of non-specific binding sites with 5% non-fat dry milk in tris-buffered saline containing 1.0% Tween 20, for 1 h at RT. After washing three times for 5 min with phosphate buffered saline containing 0.1% Tween 20, blots were incubated with primary antibodies overnight at 4°C. Membranes were washed 3 times for 15 min in phosphate buffered saline containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated goat anti-mouse^s or goat anti-rabbit IgG^t for 1 h at room temperature. Antibodies were detected by enhanced chemiluminescence using SuperSignal West FEMTO Chemiluminescent Substrate^u. Pre-stained color mobility standards were resolved in with each gel and used to estimate the molecular mass associated with immunoreactive bands. Membranes were stripped^v and re-probed with a rabbit anti-actin antibody^w for 1 h at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h at room temperature. Antibodies were detected by enhanced chemiluminescence. Densitometric analysis was performed using AlphaEaseFC^x to document the immunoreactivity of each target protein. Labeling intensity of the target proteins was normalized to the density of actin in the respective lanes.

Statistical Analysis

Statistical comparisons for HA concentration within plasma and peritoneal fluid, total protein, total nucleated cell count and age among horses suffering from acute abdominal crisis and healthy horses were made utilizing t-tests of unequal variances. Results of immunoblot assays were also evaluated with paired t-tests of unequal variances to assess differences in protein expression between cell types based on morphology. Data are presented as mean ± SE. Statistical significance was set at a P ≤ 0.05.

C. Results

Peritoneal HA levels

Peritoneal samples were collected from 7 horses with an average abdominal crisis of 11.7 ± 2.3 hours. Four strangulating lesions were identified at the time of surgery: 2 strangulating lipomas, 1 epiploic entrapment, and 1 360° large colon volvulus. There were 3 non-strangulating lesions: 2 large colon displacements and 1 ileal impaction. HA levels in peritoneal fluid, as measured by ELISA, demonstrated a significant difference between the mean abdominal HA levels between horses suffering from an abdominal crisis (1203.3 ± 23.13 ng/ml) compared with healthy horses (228.4 ± 83.63 ng/mL. Plasma HA levels, however, were not different. When compared to horses without abdominal disease (1.0 ± 0.4 g/dL), peritoneal total protein levels in diseased horses (2.9 ± 0.4 g/dL) were significantly higher. A difference was not detected between the mean peritoneal total nucleated cell counts of the normal horses (2650/µL ± 1024/µL) and diseased horses (4040/µL ± 1789/µL).

Mesothelial cell harvest

Viable cells were obtained in 3 of 10 (horses) attempts utilizing the mesenteric sack technique. Confluent monolayers were obtained from 2 samples, one at day 22 (one horse with peritonitis of 6 days duration from a previous surgical procedure and from a healthy horse, Figure 1). Fibroblastic contamination of the mesothelia cells was observed following 3 separate harvests (Figure 2). Viable cells were obtained in all explant harvests with trypsin-EDTA exposure times of 15 minutes (n=3 horses). Omental explant samples yielded confluent monolayers by day 5 (n=2 horses), whereas the mesenteric explant harvest did not result in cells that reached confluence (n=3 horses).

Figure 1. Photograph of mesothelial cells.

Microphotograph of equine peritoneal mesothelial cells. A) Cells harvested from mesenteric sack sample taken from a horse with peritonitis. Confluence was obtained at 22 days.

Figure 2. Fibroblastic (left) contamination of mesothelia cells monolayers (right).

Microphotograph of equine fibroblastic (left) contamination of primary equine peritoneal mesothelial cells (right). Cells were defined based on morphology and results of protein expression markers.

Morphological features

From omental explant samples, the yield of equine peritoneal mesothelial cells was extremely high and the primary flasks were populated densely at seeding. The cells appeared as large clusters and adopted a fibroblastic, spindle-shaped morphology after initial seeding, becoming polygonal upon confluence. Confluent monolayers were obtained after 4 to 5 days of culture with the cells becoming polygonal by day 7. Two additional passages of cells were obtained by day 10 with similar times to confluence. The omental explant culture method yielded a relatively pure population of mesothelial cells represented by no endothelial cell contamination (characterized by the formation of capillary-like tubules) and minimal fibroblast contamination.

The morphological characteristics of the mesenteric sack samples varied based on the disease status of the horses. In samples taken from a horse with peritonitis of 6 days duration at the time of tissue harvest, the initial fibroblastic morphology of the mesothelial cells was similar to the equine peritoneal mesothelial cells harvested from omentum, however the time to confluence and polygonal morphology was longer at 22 days. Mesenteric cells were harvested successfully from two additional horses, however the cells were larger. Equine mesothelial cells harvested from a horse with no abdominal disease grew to confluence at 39 days. Cells harvested from a horse euthanized after an acute abdominal crisis due to incarcerated small intestine in the gastro-splenic space became senescent (characterized by a loss of proliferation and an increase in cell size followed over time with microscopy). Fibroblast contamination was morphologically evident in samples from 2 of the 3 horses when samples were collected by mesenteric sacks; however there was no evidence of endothelial contamination.

An individual mesenteric explant sample (normal horse with no history of abdominal disease) yielded a higher seeding density than the mesenteric sack samples. The initial morphology was polygonal with a gradual increase in cell size, consistent with senescence. No fibroblast or endothelial cell contamination evident, based on visual inspection of cell morphology.

RT-PCR

Primary cell lines typically harvested high quality RNA (mesothelial cells range 426.1–447.8ng/µl, fibroblasts range 278.8–293.0ng/µl, A₂₆₀/A₂₈₀ ratio range 1.95– 1.97 with 2.0 being considered pure). Gene expression of HAS-2 by cultured equine mesothelial cells and fibroblasts, were confirmed by RT-PCR with the expected product size of 486 kB (Figure 3). Native mesentery, synovium and ovary (positive controls) also confirmed expression of HAS-2. Sequencing for all RNA sources confirmed the presence of HAS-2 with a 96–100% homology for this segment compared with the published equine sequences.

Figure 3. DNA Gel of PCR products.

Target cDNA size for HAS-2 was confirmed by 1.5% TAE-agarose gel electrophoresis in the presence of ethidium bromide. Arrow indicates the size of the generated product.

Western blot assay – mesothelial cell markers

Western blot immunoassay of mesothelial cells harvested from mesenteric sacks and omental explants confirmed expression of cell markers pan-cytokeratin, cytokeratin 8 and 18, calretinin, mesothelin and vimentin. Pan-cytokeratin immunoreactivity was observed as a single, dense band with a mobility of 58kDa. There was significantly more relative protein expression of pan-cytokeratin within the cultured mesothelial cells compared with cultured fibroblasts (Figure 4a). Cytokeratin 8 and 18 antibodies revealed two dominant bands, as expected. One band indicated a molecular mass between 50 and 75 kDa and the other was between 30 and 50 kDa. There was more relative protein expression of these cytokeratins in mesothelial cells although a significant difference was not found (P=0.09). A dominant 69 kDa band was detected with mesothelin antibody. There was significantly more relative protein expression of mesothelin within the cultured mesothelial cells compared with cultured fibroblasts (Figure 4b). Vimentin antibody labeled a single band with an apparent mass of 50 kDa band (Figure 4c). Conversely, there was no difference in relative protein expression of these vimentin within the cultured fibroblasts compared with mesothelial cell. Mesothelial cells were probed with an antibody for calretinin. Consistently, a faint band was detected at 69 kDa in the mesothelial cell lane, which is the reported mobility for the premature protein. However, a dense band was detected at 29 kDa in the rat brain whole cell lane, which would represent the reported molecular mass of the mature mesothelin protein. Antibodies for CD44 detected a dense band at 90 and 70 kDa mobilities, (Figure 5) within mesothelia cell and fibroblast lanes.

Figure 4.

Summary of densitometric analysis of immunoblots for target proteins expressed by cultured equine mesothelial cells and fibroblasts, along with exemplar western blots For all immunoblots, target protein bands were normalized by actin and reported as relative densitometric analysis. A) Expression of pan cytokeratin. B) Expression of mesothelin C) Expression of vimentin. Graphs represent 3 separate harvests, and a significant difference (*) in protein expression between mesothelial cells and fibroblasts.

Figure 5.

Representative immunoblot for protein expression of CD44. Expression of a dominant 90 kDa band was present in both the mesothelial cells and fibroblasts for CD44 (indicated by the arrow) RIPA isolation buffer served as a negative control. Species reactivity had been previously confirmed in horses.¹⁹

D. Discussion

This is the first report of a comparison between peritoneal HA levels in healthy horses and horses experiencing an abdominal crisis. The observations indicate that diseased horses have significantly higher HA along with higher total protein levels in the peritoneal cavity. Our results are consistent with previous clinical findings documenting increases in total protein prior to increases in total nucleated cell counts in the peritoneal fluid from horses suffering from moderate to severe abdominal pain.^24,25 Additionally, our findings are similar to those reported for other species in both experimental and clinical studies demonstrating elevation levels of HA with acute inflammation of abdominal cavity.^20,21 Moreover, human peritoneal mesothelial cells increase HA production when exposed to inflammatory cytokines^22,23 or after in-vitro wounding (trauma) of human peritoneal mesothelial cell monolayers.¹⁸ Increases in peritoneal HA levels might therefore be expected in cases of acute inflammation, regardless of the dominant pathology.

Increases in peritoneal HA levels were not accompanied by increases in plasma HA levels, indicating that the elevated HA in the diseased peritoneal cavity was locally produced. These findings are also similar to those from humans with acute inflammation where it was determined that most, if not all, of the peritoneal HA was derived from mesothelial cells.²³ Results from previous studies suggest that an up-regulation of HA synthase expression in mesothelial cells occurred in response to peritoneal cytokines and pro-inflammatory mediators as documented in previous studies of peritonitis.²³ It was not possible to determine if the increased HA measured in our clinical cases was a response to inflammatory cytokines, although the data suggest that this may be the case. In addition, presence of pro-inflammatory mediators within the peritoneal fluid from horses with ischemic intestine has been well documented.²⁶

The HA ELISA used in this study has been used on human serum and plasma to assess the degree of liver fibrosis and cirrhosis in chronic liver disease. It has been used previously to determine plasma HA concentration levels in 120 horses.²⁷ The mean concentration of plasma HA was reported to be 89 ng/mL, which is less than the levels determined in this study. Plasma HA concentrations of 25 resting horses were reported to range from 190 to 760 ng/mL when a specific radioimmunoassay was utilized²⁸, which is greater than the healthy horses and less than the horses with abdominal disease in the present study. There appears to be a large but reproducible inter-individual variability in plasma HA concentrations that exist among healthy horses. Our study found no differences in plasma concentrations of HA from disease and healthy horses, however significance differences in HA concentrations within peritoneal fluid were observed, which suggests that HA plays a local role during abdominal crises in horses.

HA is known to be an important mediator of inflammation, as it enhances in vitro wound healing¹⁸ and is an important modulator of peritoneal mesothelial cell migration in humans.^{18, 29–31} HA is synthesized at the inner face of the plasma membrane and is extruded to the extracellular space as it is produced.¹³ Human mesothelial cells are prolific HA producers and express all 3 HA synthase isoforms.¹⁸ To determine whether HAS-2 is present in equine mesothelial cells, the development of an equine mesothelial cell harvesting technique was pursued as well as harvesting RNA from native tissue. Cultured cells yielded a PCR product of expected size and sequence when probed with HAS-2 primers. Native omentum and mesentery are primary comprised of mesothelial cells however, connective tissue and other cell types are present. It is difficult to determine the exact cell type(s) that are responsible for HA production from full thickness harvests. HAS-2 was expressed by cultured equine fibroblasts, mesenteric native tissue and cultured mesothelial cells. Since mesothelial cells are the primary lining of the peritoneal cavity, the increase in peritoneal HA detected in the peritoneal fluid from the diseased horses likely originated from mesothelial cells. Additional studies are necessary to determine whether these cells are the primary source of HA and to determine if HAS-1 and 3 are also expressed by equine mesothelial cells.

The present study describes 3 techniques for isolation and maintenance of a primary cell line of equine peritoneal mesothelial cells. Methods included mesenteric sacks and explants of omental and mesenteric tissues. The isolation of human mesothelia cells from omental specimens has been well established and is reproducible.³² Cultured human and rat mesothelial cells demonstrate a heterogeneous phenotype that has been characterized morphologically to include polygonal to elongated appearances.^33–35 Consistent with a description by Sylianou et. al.³³ mesothelial cells from the omental explant samples and the mesenteric sack samples from the horse with peritonitis appeared multipolar or even elongated during the initial stages of growth. When grown to confluence, the mesothelial cells became polygonal in appearance and morphologically distinct from the spindle-like connective tissue fibroblasts.

Of the techniques utilized, samples collected via omental explant harvests provided the highest yield and shortest time to confluence. Samples collected from the horse with peritonitis (used in another study, 2 weeks prior) grew to confluence rapidly which may be due to stimulation by the inflammatory process.²³ In general, the mesenteric mesothelial cells were larger in size (senescent) with a prolonged time to confluence when compared to omental samples. Further research is necessary to determine the usefulness of the primary equine mesothelial cell line in order to establish their contribution in inflammation, wound healing, fibrinolysis, transport and HA biosynthesis.

Characterization of primary cell lines relies on both morphology and the cells ability to produce proteins that have been documented to be expressed in the native cell. Protein expression of calretinin, pan cytokeratin, cytokeratins 8 and 18, vimentin, mesothelin and CD44 have been utilized in previous human and equine studies.^{19, 36–39} We chose a bank of antibodies to detect vimentin, pan cytokeratin, cytokeratins 8 and 18 and CD44. The outcome suggests strongly that the primary cultured cells were characterized accurately as mesothelial cells. Equine specific antibodies to these protein are not available commercially, however, the results support the conservation of these epitopes used to characterize mesothelia across species.

In summary, the results demonstrated that equine peritoneal HA levels were elevated locally in diseased states compared to healthy horses suggesting role for HA in horses with abdominal crisis. Furthermore, equine mesothelial cells were isolated from native tissue and a primary cell line can be maintained with high purity (as confirmed by protein expression) utilizing 3 harvesting techniques. Analysis of cultured equine mesothelial cells revealed that these cells express mRNA coding for HAS-2 indicating that they are capable of producing HA. Peritoneal HA may be vital to wound healing within the equine abdomen following abdominal surgery. More importantly, gaining knowledge of the interrelationship between mesothelial cells and their production of HA will be critical in the prevention and/or treatment of adhesions post-operatively.