Investigation of the transforming growth factor-beta 1 signalling pathway as a possible link between hyperphosphataemia and renal fibrosis in feline chronic kidney disease.

Chronic kidney disease (CKD) is common in geriatric cats, and is characterised in the majority of cases by tubulointerstitial inflammation and fibrosis. Hyperphosphataemia is a frequent complication of CKD and is independently associated with severity of renal fibrosis and disease progression. Transforming growth factor-beta 1 (TGF-ß1) signalling is thought to be a convergent pathway which mediates the progression of renal fibrosis in CKD. The aims of this study were to explore the interaction between increased extracellular phosphate and the TGF-ß1 signalling pathway by investigating: (a) the effect of a commercially available, phosphate-restricted, diet on urinary TGF-ß1 excretion in cats with CKD; and (b) the role of increased extracellular phosphate in regulating proliferation, apoptosis, and expression of genes related to TGF-ß1 signalling and extracellular matrix (ECM) production in feline proximal tubular epithelial cells (FPTEC) and cortical fibroblasts from cats with azotaemic CKD (CKD-FCF). The dietary intervention study revealed no effect of dietary phosphate restriction on urinary active TGF-ß1 excretion after 4-8 weeks (P=0.98), despite significantly decreasing serum phosphate (P<0.001). There was no effect of increased growth media phosphate concentration (from 0.95mM to 2mM and 3.5mM) on proliferation (P=0.99) and apoptotic activity in FPTEC (P=0.22), or expression of genes related to ECM production and the TGF-ß1 signalling pathway in FPTEC and CKD-FCF (P>0.05). These findings suggest the beneficial effects of dietary phosphate restriction on progression of feline CKD may not occur through modulation of renal TGF-ß1 production, and do not support a direct pro-fibrotic effect of increased extracellular phosphate on feline renal cells.

Characterisation of Crandell-Rees Feline Kidney (CRFK) cells as mesenchymal in phenotype.

The Crandell-Rees Feline Kidney Cell (CRFK) is an immortalised cell line derived from the feline kidney that is utilised for the growth of certain vaccinal viruses. Confusion exists as to whether CRFK are epithelial or mesenchymal in phenotype. The aim of this study was to characterise CRFK cells via immunofluorescence, enzyme cytochemistry, western blotting, RT-qPCR for S100A4 and comparison to primary feline proximal tubular epithelial cells (FPTEC) and feline cortical fibroblasts (FCF). CRFK cells were of fusiform morphology and appeared similar to FCF. CRFK expressed the mesenchymal intermediate filament (IF) protein vimentin together with two cell adhesion molecules associated with feline fibroblasts (CD29 and CD44), and lacked expression of the epithelial IF cytokeratin, myogenic IF desmin and endothelial marker von Willebrand factor (vWF). In addition, CRFK did not demonstrate brush border enzyme activity typical of FPTEC. S100A4 gene expression, implicated in both neoplastic transformation and epithelial to mesenchymal transition, was highly upregulated in CRFK in comparison to the primary feline renal cells. CRFK appear phenotypically similar to fibroblasts, rather than tubular epithelial cells, and may have undergone neoplastic transformation or epithelial-to-mesenchymal transition after extensive passaging. This finding may have potential implications for future research utilising this cell line.

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor ß1.

BACKGROUND: Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-ß1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-ß1. RESULTS: Cortical fibroblast cultures were successfully established from the kidney tissue of cats with normal kidney function (FCF) and cats with chronic kidney disease (CKD-FCF). Both cell types expressed the mesenchymal markers vimentin, CD44 and CD29, and were negative for the epithelial marker cytokeratin, mesangial cell marker desmin and endothelial cell marker vWF. Only CKD-FCF expressed VCAM-1, a cell marker associated with inflammation. Incubation with TGF-ß1 (0-10 ng/ml) induced a concentration dependent change in cell morphology, and upregulation of myofibroblast marker gene α-SMA expression alongside collagen 1α1, fibronectin, TGF-ß1 and CTGF mRNA. These changes were blocked by the TGF-ß1 receptor 1 antagonist SB431542 (5 µM). CONCLUSIONS: FCF and CKD-FCF can be cultured via a simple method and represent a model for the investigation of the progression of fibrosis in feline CKD. The findings of this study suggest TGF-ß1 may be involved in fibroblast-myofibroblast transition in feline CKD, as in other species.

Urinary active transforming growth factor ß in feline chronic kidney disease.

The cytokine transforming growth factor beta 1 (TGF-ß1) has been widely implicated in the development and progression of renal fibrosis in chronic kidney disease (CKD) in humans and in experimental models. The aims of this study were to assess the association between urinary active TGF-ß1 and (a) development of CKD in a cross-sectional study, (b) deterioration of renal function over 1 year in a longitudinal study, and (c) renal histopathological parameters in cats. A human active TGF-ß1 ELISA was validated for use in feline urine. Cross-sectional analysis revealed no significant difference in urinary active TGF-ß1:creatinine ratio (aTGF-ß1:UCr) between groups with differing renal function. Longitudinally, non-azotaemic cats that developed CKD demonstrated a significant (P = 0.028) increase in aTGF-ß1:UCr approximately 6 months before the development of azotaemia, which remained elevated (P = 0.046) at diagnosis (approximately 12 months prior, 8.4 pg/mg; approximately 6 months prior, 22.2 pg/mg; at CKD diagnosis, 24.6 pg/mg). In the histopathology study, aTGF-ß1:UCr was significantly higher in cats with moderate (P = 0.02) and diffuse (P = 0.005) renal fibrosis than in cats without fibrosis. Cats with moderate renal inflammation had significantly higher urinary active aTGF-ß1 concentrations than cats with mild (P = 0.035) or no inflammatory change (P = 0.004). The parameter aTGF-ß1:UCr was independently associated with Log urine protein:creatinine ratio in a multivariable analysis of clinicopathological parameters and interstitial fibrosis score in a multivariable analysis of histopathological features. These results suggest that urinary aTGF-ß1 reflects the severity of renal pathology. Increases in urinary aTGF-ß1 followed longitudinally in individual cats may indicate the development of CKD.

Lipopolysaccharide and toll-like receptor 4 in dogs with congenital portosystemic shunts.

Surgical attenuation of a congenital portosystemic shunt (CPSS) results in increased portal vein perfusion, liver growth and clinical improvement. Portal lipopolysaccharide (LPS) is implicated in liver regeneration via toll-like receptor (TLR) 4 mediated cytokine activation. The aim of this study was to investigate factors associated with LPS in dogs with CPSS. Plasma LPS concentrations were measured in the peripheral and portal blood using a limulus amoebocyte lysate (LAL) assay. LPS concentration was significantly greater in the portal blood compared to peripheral blood in dogs with CPSS (P = 0.046) and control dogs (P = 0.002). LPS concentrations in the peripheral (P = 0.012) and portal (P = 0.005) blood of dogs with CPSS were significantly greater than those of control dogs. The relative mRNA expression of cytokines and TLRs was measured in liver biopsies from dogs with CPSS using quantitative PCR. TLR4 expression significantly increased following partial CPSS attenuation (P = 0.020). TLR4 expression was significantly greater in dogs that tolerated complete CPSS attenuation (P = 0.011) and those with good portal blood flow on pre-attenuation (P = 0.004) and post-attenuation (P = 0.015) portovenography. Serum interleukin (IL)-6 concentration was measured using a canine specific ELISA and significantly increased 24 h following CPSS attenuation (P < 0.001). Portal LPS was increased in dogs with CPSS, consistent with decreased hepatic clearance. TLR4 mRNA expression was significantly associated with portal blood flow and increased following surgery. These findings support the concept that portal LPS delivery is important in the hepatic response to surgical attenuation. Serum IL-6 significantly increased following surgery, consistent with LPS stimulation via TLR4, although this increase might be non-specific.

Markers of angiogenesis associated with surgical attenuation of congenital portosystemic shunts in dogs.

BACKGROUND: Dogs with congenital portosystemic shunts (CPSS) have hypoplasia of the intrahepatic portal veins. Surgical CPSS attenuation results in the development of the intrahepatic portal vasculature, the precise mechanism for which is unknown, although new vessel formation by angiogenesis is suspected. HYPOTHESIS: That the degree of portal vascular development and the increase in portal vascularization after CPSS attenuation is significantly associated with hepatic vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) gene expression and serum VEGF concentration. ANIMALS: Client-owned dogs with CPSS undergoing surgical treatment. Forty-nine dogs were included in the gene expression data and 35 in the serum VEGF data. MATERIALS AND METHODS: Dogs surgically treated by partial or complete CPSS attenuation were prospectively recruited. Relative gene expression of VEGF and VEGFR2 was measured in liver biopsy samples taken at initial and follow-up surgery using quantitative polymerase chain reaction. Serum VEGF concentration was measured before and after CPSS attenuation using a canine specific ELISA. Statistical significance was set at the 5% level (P ≤ .05). RESULTS: There was a significant increase in the mRNA expression of VEGFR2 after partial attenuation (P = .006). Dogs that could tolerate complete attenuation had significantly greater VEGFR2 mRNA expression than those that only tolerated partial attenuation (P = .037). Serum VEGF concentration was significantly increased at 24 (P < .001) and 48 (P = .003) hours after attenuation. CONCLUSIONS AND CLINICAL IMPORTANCE: These findings suggest that intrahepatic angiogenesis is likely to occur after the surgical attenuation of CPSS in dogs, and contributes to the development of the intrahepatic vasculature postoperatively.

Vascular endothelial growth factor (VEGF) and VEGF receptor expression in biopsy samples of liver from dogs with congenital portosystemic shunts.

Surgical attenuation of a congenital portosystemic shunt (CPSS) results in increased liver mass, development of intrahepatic portal vasculature and improved liver function. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The aim of this study was to investigate the role of VEGF and its receptor in the hepatic response to CPSS surgery. The study included 99 dogs with CPSS treated with either partial or complete suture attenuation. Forty-four dogs with partial attenuation underwent a second surgery for complete attenuation. The expression of VEGF and VEGF receptor 2 (VEGFR2) in biopsy samples of liver was assessed by immunohistochemistry with rabbit anti-human VEGF polyclonal antibody and mouse anti-human VEGFR2 monoclonal antibody. Expression of these molecules was graded. The proportion of samples expressing VEGF was significantly greater in samples from dogs with CPSS compared with control samples (P=0.04) and the proportion of samples expressing VEGFR2 was significantly greater in control samples compared with samples from dogs with CPSS (P=0.04). VEGF labelling grade decreased significantly (P=0.038) and VEGFR2 increased significantly (P=0.046) between first and second surgery. The decrease in VEGF may reflect transient expression, preferential expression of other factors, reperfusion of existing vessels and/or increased angiogenesis before surgery in the form of arterialization and subsequent reduction due to improved portal blood flow. Partial suture attenuation was associated with a degree of 'normalization' of VEGF and VEGFR2 expression when compared with the control samples. Further investigation is needed to provide more information on the hepatic response to CPSS surgery.

Triacylglycerol-rich lipoproteins derived from healthy donors fed different olive oils modulate cytokine secretion and cyclooxygenase-2 expression in macrophages: the potential role of oleanolic acid.

PURPOSE: Current evidence suggests that consumption of virgin olive oil (VOO) helps to protect against the development of atherosclerosis and that minor components such as oleanolic acid contribute to this effect. In this study, the effects of triacylglycerol-rich lipoproteins (TRLs) derived from olive oil on inflammatory processes in macrophages and how they are modulated by oleanolic acid was investigated. METHODS: TRLs isolated from healthy volunteers 2 and 4 h after a test meal containing VOO, pomace olive oil (POO) (the second pressing of olive oil, enriched in minor components) or POO enriched with oleanolic acid (OPOO) were incubated with macrophages derived from the human monocyte cell line, THP-1. RESULTS: All types of TRLs caused a decrease of about 50% in the secretion of monocyte chemoattractant protein-1 (MCP-1) by the cells. Interleukin (IL)-6 secretion was also significantly decreased by 2 and 4 h VOO TRLs and by 4 h OPOO TRLs. In contrast, increased IL-1ß secretion was observed with all 2 h TRL types, and increased tumour necrosis factor-α (TNF-α) production with 2 h VOO and POO, but not OPOO, TRLs. TRLs isolated after 4 h, however, had no significant effects on TNF-α secretion and increased IL-1ß secretion only when they were derived from VOO. Cyclooxygenase-2 (COX-2) mRNA expression was strongly down-regulated by all types of TRLs, but protein expression was significantly depressed only by 4 h OPOO TRLs. CONCLUSION: These findings demonstrate that TRLs derived from olive oil influence inflammatory processes in macrophages and suggest that oleanolic acid may have beneficial effects.

Influence of chylomicron remnants on human monocyte activation in vitro.

BACKGROUND AND AIMS: Atherosclerosis is known to be an inflammatory disease and there is increasing evidence that chylomicron remnants (CMR), the lipoproteins which carry dietary fats in the blood, cause macrophage foam cell formation and inflammation. In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is increased, and clearance of CMR from blood may be delayed, however, whether CMR contribute directly to monocyte activation and subsequent egress into the arterial wall has not been established. Here, the contribution of CMR to activation of monocyte pro-inflammatory pathways was assessed using an in vitro model. METHODS AND RESULTS: Primary human monocytes and CMR-like particles (CRLP) were used to measure several endpoints of monocyte activation. Treatment with CRLP caused rapid and prolonged generation of reactive oxygen species by monocytes. The pro-inflammatory chemokines MCP-1 and IL-8 were secreted in nanogram quantities by the cells in the absence of CRLP. IL-8 secretion was transiently increased after CRLP treatment, and CRLP maintained secretion in the presence of pharmacological inhibitors of IL-8 production. In contrast, exposure to CRLP significantly reduced MCP-1 secretion. Chemotaxis towards MCP-1 was increased in monocytes pre-exposed to CRLP and was reversed by addition of exogenous MCP-1. CONCLUSION: Our findings indicate that CRLP activate human monocytes and augment their migration in vitro by reducing cellular MCP-1 expression. Our data support the current hypothesis that CMR contribute to the inflammatory milieu of the arterial wall in early atherosclerosis, and suggest that this may reflect direct interaction with circulating blood monocytes.

The expression of angiogenic growth factors and their receptors in ovarian follicles throughout the estrous cycle in the ewe.

Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n=5) or before (n=5), during (n=4), and after (n=4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by in situ hybridization using digoxigenin-11-UTP-labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles >or=2mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5mm) and medium (2.5 to 4.0mm) follicles, but large follicles (>4.0mm) expressed higher mRNA and protein levels (all P<0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size.

C-type natriuretic peptide regulation of guanosine-3',5'-cyclic monophosphate production in human endothelial cells.

In vascular smooth muscle cells, relaxant actions of guanosine--3',5'-cyclic monophosphate (cGMP) are well recognized, but there is increasing evidence that cGMP also plays regulatory roles in vascular endothelium. However, the autacoid and endocrine mechanisms controlling cGMP production in endothelium are not well understood. The objective of these studies was to examine the mechanisms of cGMP accumulation in human umbilical vein endothelial cells (HUVEC) in response to natriuretic peptides. Expression in HUVEC of natriuretic peptide receptors, particulate guanylyl cyclases (GC)-A and GC-B, was confirmed by RT-PCR and Western blot analysis. In the presence of the phosphodiesterase inhibitor IBMX 500 microM, 3 h incubation of HUVEC with B-type natriuretic peptide (BNP) (preferential GC-A agonist) or C-type natriuretic peptide (CNP) (preferential GC-B agonist) stimulated concentration-dependent increases in cGMP production. At 10 and 100 nM, we observed two to three-fold greater potency of CNP compared to BNP. In the absence of IBMX, CNP-stimulated cGMP accumulation was significantly less than cGMP accumulation in response to sodium nitroprusside 1 mM. This greater sensitivity of GC-B-derived cGMP to phosphodiesterases suggests compartmentalization of two pools of cGMP from particulate and soluble guanylyl cyclases. Although CNP 100 nM and 1 microM was observed to increase nitrite + nitrate (stable metabolites of NO) production in HUVEC two-fold above basal level, the soluble guanylyl cyclase inhibitor ODQ 10 microM did not significantly modify CNP-stimulated cGMP accumulation suggesting that endothelial actions of CNP may be NO-independent. In conclusion, these studies indicate functional signaling by natriuretic peptides in endothelial cells, supporting possible roles of these mediators in regulating endothelial cell function.

The effect of estradiol on COX-2, EP2, and EP4 mRNA expression and the extracellular matrix in the cervix of the hypogonadotrophic, ovariectomized ewe.

There is a degree of cervical relaxation in the ewe at estrus that is regulated by changes in prostaglandin synthesis, prostaglandin receptor expression, and changes in the cervical extracellular matrix. It is likely that these are regulated by changes in periovulatory hormones, particularly estradiol. This study determined the effect of estradiol benzoate on the mRNA expression of cyclooxygenase-2 (COX-2) and the prostaglandin E receptors EP(2) and EP(4), the concentration of cervical hyaluronan, and the proportion of smooth muscle and collagen in the cervix of the hypogonadotrophic ovariectomized ewe (Ovis aries). Ovariectomized hypogonadotrophic ewes were given 100 microg estradiol benzoate, and their cervices were collected 0, 24, and 48 h thereafter to determine the expression of cervical COX-2, EP(2), and EP(4) mRNA by in situ hybridization, the concentration of hyaluronan by ELISA, and the proportion of smooth muscle and collagen by Masson's trichrome staining. Estradiol benzoate increased the mRNA expression of COX-2 and EP(4) within 24h after treatment (P<0.05), whereas EP(2) mRNA, hyaluronan, and the ratio of smooth muscle to collagen did not change within 48 h after treatment. The COX-2, EP(2), and EP(4) mRNA expression were greatest in the smooth muscle layers (P<0.05) and least in the luminal epithelium (P<0.05). In conclusion, we inferred that estradiol regulates cervical COX-2 and EP(4) mRNA expression and may regulate cervical relaxation via the synthesis of prostaglandin E(2) and activation of the PGE(2) receptors EP(2) and EP(4).

Regulation of platelet activating factor-induced equine platelet activation by intracellular kinases.

Lipopolysaccharide (LPS) can activate equine platelets directly or indirectly, via leukocyte-derived platelet activating factor (PAF). Thromboxane (Tx) production by LPS-stimulated equine platelets requires p38 MAPK and this kinase has been suggested as a therapeutic target in endotoxaemia. The present study has utilised selective inhibitors to investigate the role of p38 MAPK and two other kinases, phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC), in regulating PAF-induced Tx production, aggregation and 5-HT release in equine platelets, and the modification of these responses by LPS. LPS enhanced PAF-induced 5-HT release, an effect that was reduced by the p38 MAPK inhibitor, SB203580 (60 +/- 8% reduction; n = 6). SB203580 did not affect responses to PAF alone; whereas inhibition of PKC reduced PAF-induced 5-HT release, Tx production and aggregation (maximal inhibition by the PKCdelta inhibitor, rottlerin: 69 +/- 13%, 63 +/- 14% and 97 +/- 1%, respectively; n = 6). Wortmannin and LY249002, which inhibit PI3K, also caused significant inhibition of PAF-induced aggregation (maximal inhibition 78 +/- 3% and 88 +/- 2%, respectively; n = 6). These data suggest that inhibition of platelet p38 MAPK may be of benefit in equine endotoxaemia by counteracting some of the effects of LPS. However, detrimental effects of platelet activation mediated by PAF and not enhanced by LPS are unlikely to be markedly affected.

Introduction to the Biochemical Society Focused Meeting on Diet and Cardiovascular Health: chylomicron remnants and their emerging roles in vascular dysfunction in atherosclerosis.

Although it has been known for many years that dietary lipids influence the development of atherosclerosis, in the past this has been attributed to their effects on blood cholesterol levels. Recent work, however, has shown that CMRs (chylomicron remnants), the lipoproteins which carry dietary lipids in the blood, potentially have a direct role in initiating atherogenesis by influencing vascular function. The Diet and Cardiovascular Health: Chylomicron Remnants and Their Emerging Roles in Vascular Dysfunction in Atherosclerosis Meeting focused attention on studies which have shown that CMRs influence vascular function via interactions with cells of the artery wall, including endothelial cells and macrophages, and also highlighted the part played by CMRs in the development of premature atherosclerosis in conditions such as the metabolic syndrome, which are an increasing cause of heart disease in developed countries.

Chylomicron remnants: mediators of endothelial dysfunction?

Vascular disease is initiated by activation of the endothelium characterized by the predominance of pro-inflammatory and pro-coagulant changes in endothelial cells (ECs) referred to collectively as 'endothelial dysfunction'. There is increasing evidence that lipoproteins of dietary origin modulate EC function and the use of artificial chylomicron remnant-like particles (CRLPs) in vitro is now beginning to shed light on the molecular mechanisms through which these particles influence cell behaviour. CRLPs enriched in n-6 PUFAs (polyunsaturated fatty acids) influence the production of vasoactive mediators by ECs in a pro-inflammatory manner. Thus CRLPs reduce the synthesis and release of nitric oxide and alter the balance of release of vasodilator versus vasoconstrictor eicosanoids. These changes are accompanied by induction of cyclo-oxygenase-2 expression and activity as well as increased expression of adhesion molecules and the antioxidant defence enzyme haem oxygenase-1. CRLPs also activate a number of intracellular signalling pathways, including NF-kappaB (nuclear factor kappaB) and MAPKs (mitogen-activated protein kinases), which may be involved in mediating their effects on gene expression. The effects of CRLPs on EC behaviour can also be modulated by the type of fat/oxidation status of the particles. These findings support the hypothesis that lipoproteins of dietary origin directly regulate molecular events in the vascular wall.

Dietary fats induce human monocyte activation in vitro.

In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is amplified, and migration of monocytes into the walls of the aorta and large arteries is increased, due partly to de novo expression or activation of monocyte adhesion molecules. Although there is increasing evidence that CMRs (chylomicron remnants) are strongly atherogenic, the outcomes of interactions between blood monocytes and circulating CMRs are not known. Here, we have studied the effects of CRLPs (CMR-like particles) on THP-1 human monocyte oxidative burst. The particles induced a significant increase in reactive oxygen species within 1 h, which persisted for 24 h. We suggest that monocyte-CMR interactions may be important in early atherosclerosis when many activated monocytes are found in susceptible areas of the artery wall.

Cartilage oligomeric matrix protein and hyaluronan levels in synovial fluid from horses with osteoarthritis of the tarsometatarsal joint compared to a control population.

REASONS FOR PERFORMING STUDY: Quantification of cartilage oligomeric matrix protein (COMP) levels within synovial fluid from the tarsometatarsal joint has not previously been reported and an effective synovial fluid marker would allow monitoring of disease progression and treatment. OBJECTIVES: To quantify levels of COMP and hyaluronan (HA) in synovial fluid from the tarsometatarsal joint, identify differences in levels from horses with osteoarthritis (OA) of the tarsometatarsal joint compared to a control population and to correlate levels with radiographic changes in horses with OA. METHODS: Synovial fluid was collected from the tarsometatarsal joint of 25 horses without hindlimb lameness (controls) and 25 lame horses, subjected to analgesia of the joint. COMP concentrations were measured using a homologous inhibition ELISA. Immunoblots of synovial fluid from 3 lame horses and 3 controls were performed to identify fragmentation of COMP. Hyaluronan (HA) concentration in synovial fluid was determined using a competition ELISA. Radiographs of the lame horses with OA were scored and correlated with levels of COMP and HA. RESULTS: Concentrations of COMP in OA of the tarsometatarsal joint were significantly lower than in the control samples. An additional fragment band of COMP (approximately 30 kDa) was identified on the immunoblots of the horses with OA and this fragment was not identified in controls. No significant difference was identified in the HA or HA:COMP ratio between lame and control horses. There was no correlation between levels of synovial fluid COMP and HA, and radiographic changes. CONCLUSIONS AND POTENTIAL RELEVANCE: Lowered levels of COMP in synovial fluid of tarsometatarsal joints correlates with the presence of osteoarthritis. However, a single value cannot be used to stage the disease process. Levels of HA may not be a useful marker for this disease. Decreased, rather than increased COMP levels, may reflect significant loss of cartilage in established osteoarthritis. A specific assay for the COMP fragment generated with osteoarthritis may allow the earlier detection of clinical cases.

Direct interaction of dietary lipids carried in chylomicron remnants with cells of the artery wall: implications for atherosclerosis development.

The development of atherosclerotic lesions in the artery wall is a complex process involving the endothelium, lipid engorged macrophages (foam cells) and smooth muscle cells. In recent years it has become clear that chylomicron remnants, the lipoproteins which carry lipids of dietary origin in the blood, are strongly atherogenic, and there is increasing evidence to indicate that this is due to direct interaction of the remnant particles with cells of the artery wall. Chylomicron remnants have been demonstrated to inhibit endothelium dependent vasorelaxation and to activate signal transduction pathways associated with inflammation in cultured endothelial cells. They have also been shown to be taken up by smooth muscle cells and macrophages, and to cause the extensive lipid accumulation associated with foam cell formation, as well as influencing the expression of key genes regulating macrophage lipid uptake and metabolism. Furthermore, oxidative modification of the remnant particles is not required for many of these effects. Chylomicron remnants, therefore, have multiple direct effects on three major cell types of the arterial wall which are likely to promote the development of atherosclerotic lesions. These effects may be modulated by various lipids carried by the particles, including the type of fat (saturated or unsaturated or oxidised fat), micronutrients such as vitamins and carotenoids which have antioxidant properties, and orally administered lipophilic drugs. Delayed clearance of chylomicron remnants from the blood occurs in a number of dyslipidemias associated with premature atherosclerosis development, and the potentially atherogenic effects of the particles would clearly be enhanced in these circumstances. Thus, elucidation of the mechanisms involved will aid in the identification of new drug targets which may be particularly useful for these conditions.

Chylomicron-remnant-like particles modify production of vasoactive mediators by endothelial cells.

Endothelial-cell dysfunction is a critical initiating event in the pathogenesis of atherosclerosis. Although there is evidence to suggest that chylomicron remnants (CMRs), lipoproteins derived from the diet, influence endothelial-cell function to generate a pro-atherogenic phenotype, the mechanisms involved remain undefined. We have examined the effects of CMR-like particles (CMR-LPs) on human endothelial-cell function, focusing on the cyclo-oxygenase (COX) and nitric oxide synthase (NOS) pathways. CMR-LPs strongly enhanced the expression of the inducible cyclo-oxygenase COX-2 and increased prostacyclin synthesis in a biphasic manner. Studies with the COX-2-selective inhibitor NS-398 confirmed the COX-2 dependency of the later increase in prostanoid production. Pre-incubation with CMR-LPs reduced basal and thrombin-stimulated cGMP generation, whereas expression of endothelial NOS was not modified by remnant treatment.