Ultrastructure of equine endothelial cells exposed to endotoxin and flunixin meglumine and equine neutrophils

An in vitro system of cultured equine endothelial cells was evaluated as a model for endotoxin (ET) exposure in the horse. Primary cell lines from pulmonary vessels and aortas were cultured from tissues of 6 horses. Effects of ET alone with and without serum and in combination with the cyclo-oxygenase inhibitor flunixin meglumine and isolated equine neutrophils were evaluated by transmission electron microscopy. Cells plus serum were incubated with 10, 25, 50, or 100 micrograms of ET/ml of incubation medium for 1, 3, 8, or 24 hours. Cells without serum were cultured for 1 and 3 hours. Flunixin meglumine was used at a concentration of 20 micrograms/ml. Cells also were incubated in the presence of 1,000, 5,000, or 20,000 neutrophils/ml plus ET and in the presence of a combination of ET and flunixin meglumine for 1 or 3 hours. Endotoxin alone did not cause cell damage, and the only evidence of an effect was an increased number of secondary lysosomes at incubation hour 8. At incubation hour 24, cells appeared normal. Endotoxin plus neutrophils caused cells to become round and detach from the growth substrate. Cell pathologic changes included swollen and distorted mitochondria and cytoplasmic vacuolization. Response to the ET plus neutrophil combination was variable and ranged from 5% to 50% of the cells being affected. The variability appeared to have some correlation with cell age, as well as individual preparation of neutrophils.     

Analysis of serum and lymphocyte surface IgM of healthy and immunodeficient horses with monoclonal antibodies

Nine monoclonal antibodies which reacted with equine immunoglobulin (Ig)M and not other equine Ig and serum proteins were prepared. Cells producing antibodies (C 1.9) which precipitated with IgM and bound to staphylococcal protein A were triple-cloned (C 1.9/3.2) and the antibodies further characterized. Monoclonal antibody C 1.9/3.2 reacted with an IgM determinant present on serum IgM from horses of several breeds. Studies with 125I-labeled IgM revealed the presence of this determinant on all IgM molecules. The monoclonal antibody enabled quantitation of IgM in presuckling foal and adult horse sera, using rocket electrophoresis. This procedure was used because presumably it gives a positive precipitation reaction over a wide range of antigen-antibody ratios. The C 1.9/3.2 monoclonal antibody recognized an exposed mu-chain determinant on live B lymphocytes, as determined by immunofluorescence. Also, IgM-containing cells could be identified in acetone-fixed frozen sections of lymphoid tissue. Sera from several other species carry the determinant identified by C 1.9/3.2, suggesting that the reagent may be useful for IgM studies in other species.     

Equine infectious anaemia: detection of antibodies using an immunofluorescence test

An indirect immunofluorescence test for detecting antibodies to equine infectious anaemia virus is presented. Using monolayers of equine dermal cells within a defined period after infection, discrete fluorescent spots were observed in the cytoplasm of as many as 95 per cent of the cells. These inclusions appeared as ring-like structures when high titred sera were employed but became spots when the sera were diluted. Cells showing optimal antigen fluorescence were used immediately or after storage at -70 degrees C. The fluorescence test detected lower levels of antibody than the immunodiffusion test, and results were available in less than two hours. It should be useful as a confirmatory assay with the immunodiffusion test.     

Diagnostic value of intestinal alkaline phosphatase in horse serum

Antiserum directed against equine intestinal Alkaline Phosphatase (ALP) was produced in rabbits and used to develop a sensitive and quantitative assay for the detection of intestinal ALP in equine serum. This assay was then used to measure the half-life of intravenously injected intestinal ALP and to determine if the intestinal ALP was present in normal horse sera, sera from horses presented for lesions not involving the gastrointestinal tract and sera from horses presented with lesions involving the gastrointestinal tract. The results suggest that intestinal ALP is not likely to appear in equine serum even when gastrointestinal disease is present and, therefore, appears to be of no diagnostic value.     

A comparison of equine and bovine sera as sources of lipopolysaccharide-binding protein activity in equine monocytes incubated with lipopolysaccharide

Lipopolysaccharide-binding protein (LBP) is an acute phase protein that binds the lipid A moiety of lipopolysaccharide (LPS) and transfers LPS monomers to soluble CD14 in plasma or membrane bound CD14 on mononuclear phagocytes. The result of these interactions is activation of the TLR4 receptor complex, and the synthesis and release of inflammatory mediators. Inclusion of LBP in cellular assays increases the sensitivity of cells expressing CD14 to LPS. Therefore, the objectives of this study were to (1) compare differentially treated sera from cattle and horses as sources of LBP activity using LPS-induced expression of procoagulant activity (PCA) by equine monocytes as a readout and (2) evaluate the use of commercial equine serum as a source of LBP activity using LPS concentration response and time course studies to validate the response. Monocytes were isolated from eight horses and incubated with five different serum preparations in the presence or absence of Escherichia coli LPS. The sera tested were heat-inactivated fetal bovine serum (HI-FBS), pooled commercial equine serum (CES), heat-inactivated pooled commercial equine serum (HI-CES), autologous equine serum (AES), and heat-inactivated autologous equine serum (HI-AES). In the absence of LPS, monocytes from half of the horses in the study had increased expression of PCA when incubated with HI-FBS alone; PCA was unaffected by incubation with the other sera. There was a four-fold increase in PCA when monocytes were incubated with LPS in the presence of CES, HI-CES or AES compared to LPS without serum. The combination of HI-FBS and LPS increased PCA 20-fold compared to LPS without serum. The HI-AES serum lacked significant LBP activity. Whereas maximal expression of PCA was induced by 1ng/ml of LPS in the absence of serum, inclusion of 1% CES reduced the LPS concentration required for maximal PCA to 30pg/ml. Monocytes incubated with LPS in the presence of CES had increased PCA at 3h and peaked at 6h. In conclusion, monocytes from many horses are directly stimulated by HI-FBS, suggesting that HI-FBS is not an optimal source of LBP for in vitro studies of LPS with equine monocytes. In contrast, CES and AES are effective sources of LBP activity for such studies, as they do not directly induce activation. Although the heat inactivation process did not affect the LBP activity in CES, it ablated LBP activity in AES. Consequently, investigators are advised to utilize either CES or AES in future studies, but not heat-inactivated AES.     

Surface antigens on equine sarcoid cells and normal dermal fibroblasts as assessed by xenogeneic antisera

To characterise the expression of surface antigens on equine sarcoid cells compared to normal equine fibroblasts, immune sera were produced in rabbits against transformed cells of a virus-containing sarcoid cell line (Mc-1) and normal dermal fibroblasts, respectively. The specificities of the sera were analysed by antibody-dependent cellular cytotoxicity against 51Cr-labelled target cells using human lymphocytes as effector cells. Anti-Mc-1 antiserum induced strong cytotoxicity against transformed cells of two sarcoid cell lines (Mc-1 and Bay Mc-1), whereas the cytotoxicity against transformed cells of equine testis, or against cells grown in short term or primary cultures derived either from sarcoids or normal dermis was low or absent. In contrast, anti-normal dermal fibroblasts antiserum did not induce antibody-dependent cellular cytotoxicity against Mc-1 or Bay Mc-1 but reacted strongly against both normal fibroblasts and primary cultured cells derived from sarcoids. The apparent specificity of the anti-Mc-1-induced cytotoxicity was confirmed by absorption of the anti-serum with either normal dermal fibroblasts and equine testis cells or Mc-1 cells. Immunoprecipitation of 125I-surface labelled Mc-1 cells by absorbed anti-serum and analysis by SDS-PAGE and autoradiography revealed that anti-Mc-1 antibodies reacted with at least four surface components with a wide range of molecular weights. Immunofluorescence with rabbit anti-human beta 2mu serum indicated that Mc-1 cells in contrast to equine lymphocytes and fibroblasts did not express class 1 major histocompatibility complex antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Common membrane neoantigens on bovine papilloma virus-induced fibroma cells from cattle and horses.

Cultured cells from bovine papilloma virus (BPV)-induced fibroblastic tumors and normal dermis of cattle, horses, and hamsters were examined for cell membrane or internal neoantigens, using the indirect immunofluorescence technique. Sera from cattle and horses bearing BPV-induced fibromas cross reacted with cell membranes of tumor, but not with normal dermal cells of both species. The reaction could be blocked with homologous, but not heterologous, serum of these 2 species. Immunofluorescence was not detected with sera from hamsters bearing BPV-induced sarcomas if incubated with bovine, equine, or hamster cells. Internal neoantigens were not found in any of the acetone-fixed tumor cells, using sera from the 3 species. Both tumor and normal cells were all found free of BPV antigen, using direct immunofluorescence.     

Isolation and characterization of equine microvascular endothelial cells in vitro

The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobblestone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors, Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.     

Establishing a reproducible method for the culture of primary equine corneal cells

Objective  To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation.
Procedures  Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining.
Results  All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogenous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery.
Discussion  This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.     

Effect of fetal bovine serum and heat-inactivated fetal bovine serum on microbial cell wall-induced expression of procoagulant activity by equine and canine mononuclear cells in vitro

OBJECTIVE: To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. SAMPLE POPULATION: Mononuclear cells from 18 horses and 3 dogs. PROCEDURES: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. RESULTS: Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPS-binding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosan-induced procoagulant activity of canine cells. CONCLUSION AND CLINICAL RELEVANCE: Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPS and zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.     

Evaluation of hyaluronidase activity in equine and bovine sera and equine synovial fluid samples by use of enzyme zymography

OBJECTIVE: To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes. SAMPLE POPULATION: Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses. PROCEDURE: Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis. RRESULTS: Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7 Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints.     

Serological studies on leptospirosis in domestic animals in Quebec.

During a period of 30 months, from January 1977 to June 1979, Leptospira agglutinins were detected in 355 (6%) of 5841 bovine sera, 52 (10.1%) of 511 porcine sera, one (5%) of 20 equine sera and one (12.5%) of eight canine sera. Bovine, porcine and equine sera reacted predominantly with L. pomona. Reactors to L. hardjo/sejroe, L. icterohaemorrhagiae and L. grippotyphosa were also detected in cattle. One porcine serum reacted with L. grippotyphosa and one canine serum with L. icterohaemorrhagiae. Al the sera originated from suspected cases of leptospirosis.     

Laboratory diagnosis of African horse sickness: comparison of serological techniques and evaluation of storage methods of samples for virus isolation

Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA described in this paper was group specific. It did not require calibration with a standard positive serum but did yield elevated values with negative sera that were repeatedly frozen and thawed or heat inactivated. The IFA test was sensitive but could not be used on some field sera as the control cells exhibited fluorescence, possibly due to the animal being recently vaccinated with cell culture material. Sixty-two experimental sera were compared by VN, CF, AGID, and ELISA. Forty sera, 10 positive and 30 negative, were correctly classified by the 5 serologic assays. The 22 remaining sera gave mixed reactions. The AGID had no false positive results but had false negative results for up to 20% of the samples, depending upon the comparison. The VN, CF, and ELISA were similar in their variability. The length of time that virus could be recovered from a viremic blood sample was compared in an evaluation of storage methods for virus isolation samples. Washed erythrocytes were held at 4 C, washed erythrocytes plus stabilizer were held at -70 C, and blood that was drawn into a preservative (oxalate/phenol/glycerol) was held at 4 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inability of kaolin treatment to remove nonspecific inhibitors from equine serum for the hemagglutination inhibition test against equine H7N7 influenza virus.

The hemagglutination inhibition test is used by many diagnostic and surveillance laboratories for detection of antibodies to influenza viruses. It is well known that the hemagglutination inhibition test is affected by nonspecific inhibitors present in equine serum. Several serum treatments are in use to remove these inhibitors, including treatment with kaolin. Discrepant results were observed in the authors' laboratories when using kaolin treatment before testing equine sera for antibodies against equine influenza virus (EIV) subtype-1 (H7N7). It is demonstrated here that kaolin treatment leads to false positive results when testing for antibodies against EIV subtype-1, as compared to other standard serum treatments (trypsin-periodate, receptor-destroying enzyme). Against EIV subtype-2 (H3N8), however, false positive results were not evident. Trypsinperiodate and receptor-destroying enzyme (RDE) treatments appear to be superior to kaolin for removal of nonspecific inhibitors from equine serum and should be used for serological diagnosis and surveillance of equine influenza virus.     

Rapid and specific serodiagnosis of western equine encephalitis virus infection in horses

Paired sera from 28 nonvaccinated horses with serologically confirmed western equine encephalitis (WEE) virus infections were evaluated for immunoglobulin (Ig)M and IgG directed against WEE virus, by use of enzyme immunoassay. Twenty-one of the horses developed greater than or equal to 4-fold increases or decreases in serum IgM titers in paired serum samples, confirming the diagnosis of WEE in these horses. Of the remaining 7 horses, 1 had stable IgM titers, 1 had a 2-fold increase in IgM titer between paired sera, 2 had 2-fold decreases in IgM titer, and for 3 horses adequate volumes were not available for both sera of the pair. Twenty-nine of 56 blood samples collected from these 28 horses had been collected within the first 3 days after clinical disease was recognized; all 28 horses and 48 of 53 available serum samples had IgM antibody to WEE virus. Immunoglobulin M also was detected in sera of 27 of 45 other nonvaccinated horses that had illnesses clinically compatible with WEE. Sera with IgM did not have cross-reacting IgM against eastern equine encephalitis virus. Therefore, the sensitivity, specificity, and lack of persistence of IgM was useful in the rapid diagnosis of WEE virus infections in horses.     

Agents of equine viral encephalomyelitis: correlation of serum and cerebrospinal fluid antibodies.

A survey was conducted by testing 115 paired equine serum and cerebrospinal fluid samples by hemagglutination-inhibition for antibodies to Powassan and snowshoe hare viruses, and by virus neutralization for antibodies to equine herpesvirus type 1. Twenty-five samples were from horses with spontaneous neurological disease and the remainder from horses euthanized because of various nonneurological disorders. All sera and cerebrospinal fluids were negative for antibodies to Powassan virus. Fifty-one sera (44.3%) and 15 cerebrospinal fluids (13.0%) had antibodies to snowshoe hare virus. Ninety-eight sera (85.2%) and four cerebrospinal fluids (3.5%) were positive for antibodies to equine herpesvirus type 1. Powassan virus was inoculated intracerebrally into one, and intravenously into four ponies. Neurological signs associated with a nonsuppurative encephalomyelitis occurred in three ponies. Antibodies to Powassan virus were detected in sera of all animals but in cerebrospinal fluids of only two. Powassan virus was isolated from brain and spinal cord of only the intracerebrally inoculated animal.     

Antibody isotypes in sera of equine fetuses aborted due to Leptospira interrogans serovar pomona-type kennewicki infection

Leptospira-specific antibody isotypes in sera of late term equine fetuses aborted due to Leptospira interrogans serovar pomona-type kennewicki infection were characterized and compared with those of their dams. IgM was the dominant Leptospira-Specific isotype in both fetuses and mares. However, IgGa was the isotype in highest concentration in petal sera and strong Leptospira-specific IgGa but no IgGb and little or no IgG(T) were detected. In contrast, although IgGb was quantitatively the dominant isotype in mares serum, Leptospira-specific serum IgG in aborting mares was dominated by IgG(T) but also included large amounts of IgGa and IgGb. IgGa and IgGb were quantitatively the dominant isotypes in sera of fetuses and mares, respectively. Affinity purified IgGa from fetuses did not agglutinate leptospires but serum devoid of IgGa did, suggesting that IgM is the principal agglutinating antibody. It is concluded that the equine fetus is deficient in IgGb and IgG(T) synthesis.     

Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.     

Autologous conditioned serum: the comparative cytokine profiles of two commercial methods (IRAP and IRAP II) using equine blood

Reasons for performing study: Osteoarthritis (OA) is one of the most prevalent and debilitating conditions affecting the horse. Autologous conditioned serum (ACS), commercially available as IRAP and IRAP II, is a recently developed treatment for OA in which plasma is prepared from venous blood by incubation with glass beads for 24 h. This product has been shown to increase anti‐inflammatory cytokines and growth factors in human blood. However, data for equine ACS preparations are lacking. Objectives: To characterise the protein profiles produced by commercially available ACS systems in equine blood. Methods: Blood was drawn from 5 horses into 6 groups: red top vacutainer (control), IRAP and IRAP II, with and without heparin. Samples were collected 1 or 24 h post draw and analysed for IL‐1ra, IL‐10, IGF‐1, TGF‐β, TNF‐α and IL‐1β using ELISAs. Results: Twenty‐four hour IRAP and IRAP II samples contained significantly higher levels of all cytokines relative to 1 h serum controls. At 24 h, IRAP II contained significantly higher levels of IL‐1ra and IRAP contained significantly higher levels of TNF‐α, compared to 24 h controls. In addition, TGF‐β, IL‐10 and IL‐1β in IRAP and IRAP II sera were similar to 24 h serum controls. The addition of heparin significantly reduced levels of IGF‐1, TNF‐α and TGF‐β, and significantly elevated levels of IL‐1ra. Conclusions: The cytokine profile that IRAP II produced is modestly better than IRAP. Incubation of whole blood in glass tubes stimulated cytokine synthesis, although not as efficiently as IRAP II. Potential relevance: Although high levels of IL‐1ra were found in ACS, elevation of other factors suggests these cytokines play a previously understated role in clinical improvements. Because ACS has been shown to alleviate clinical symptoms of OA, the present study suggests that factors other than IL‐1ra alone might be involved in its clinical efficacy. Species‐dependent elevations of cytokines warrant further investigation and optimisation of the systems appears to be necessary based on the differences between human and equine blood.     

Endothelial cells and angiogenesis in the horse in health and disease—A review

The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progenitor cells; equine endothelial cells and angiogenesis (heterogeneity, markers and assessment); endothelial regulatory molecules in equine angiogenesis; angiogenesis research in equine reproduction (ovary, uterus, placenta and conceptus, testis); angiogenesis research in pathological conditions (tumours, ocular pathologies, equine wound healing, musculoskeletal system and laminitis). The review also includes a table that summarizes in vitro studies on equine endothelial cells, either describing the isolation procedure or using previously isolated endothelial cells. A particular challenge of the review was that results published are fragmentary and sometimes even contradictory, raising more questions than they answer. In conclusion, angiogenesis is a major factor in several diseases frequently occurring in horses, but relatively few studies focus on angiogenesis in the horse. The challenge for the future is therefore to continue exploring new therapeutic angiogenesis strategies for horses to fill in the missing pieces of the puzzle.