The rapid growth rate in feedlot cattle is likely to promote joint abnormalities like osteochondrosis dissecans (OCD) and osteoarthritis (OA) with subsequent lameness. We have identified a small leucine-rich repeat proteoglycan (SLRP) biomarker at cleavage site 262GLGHNQIRM (BGN262) arising from the fragmentation of biglycan (BGN) in subchondral bone associated with OA. With a validated custom-made ELISA, BGN262 has been quantified in serum from cattle. The concentration of BGN262 in serum from cattle raised in the conventional and all-natural production systems increased at harvest compared to the starting period. The limitation of the study is the small sample size. However, the promising results encourage a further evaluation of BGN262 and its potential as a biomarker for subchondral bone pathology in cattle.

Objective: This study aimed to test a novel treatment combination (TC) (equivalent to sildenafil, mepivacaine, and glucose) with disease-modifying properties compared to Celestone (R) bifas (R) (CB) in a randomized triple-blinded phase III clinical study in horses with mild osteoarthritis (OA). Joint biomarkers (reflecting the articular cartilage and subchondral bone remodelling) and clinical lameness were used as readouts to evaluate the treatment efficacy. Methods: Twenty horses with OA-associated lameness in the carpal joint were included in the study and received either TC (n = 10) or CB (n = 10) drug intra-articularly-twice in the middle carpal joint with an interval of 2 weeks (visit 1 & 2). Clinical lameness was assessed both objectively (Lameness locator) and subjectively (visually). Synovial fluid and serum were sampled for quantification of the extracellular matrix (ECM) neo-epitope joint biomarkers represented by biglycan (BGN262) and cartilage oligomeric matrix protein (COMP156). Another two weeks later clinical lameness was recorded, and serum was collected for biomarkers analysis. The overall health status was compared pre and post-intervention by interviewing the trainer. Results: Post-intervention, SF BGN262 levels significantly declined in TC (P = 0.002) and COMP156 levels significantly increased in CB (P = 0.002). The flexion test scores improved in the TC compared to CB (P =0.033) and also had an improved trotting gait quality (P =0.044). No adverse events were reported. Conclusion: This is the first clinical study presenting companion diagnostics assisting in identifying OA phenotype and evaluating the efficacy and safety of a novel disease-modifying osteoarthritic drug.

Objective:We aimed to delineate a novel soluble Biglycan Neo-epitope-BGN262 in saliva from young reference and osteoarthritic horses in conjunction with the influence of short-term training exercise, riding surface hardness, circadian rhythm, and feeding on its soluble levels. Design:A custom-made inhibition ELISA was used for the quantification of BGN262 in saliva. Cohort 1: A cross-sectional study comprising reference (N ​= ​19) and OA horses (N ​= ​9) with radiographically classified subchondral bone sclerosis. Receiver operating characteristic curve analysis was performed to evaluate the robustness of BGN262. Cohorts 2 (N ​= ​5) & 3 (N ​= ​7): Longitudinal studies of sampling during a short-term training exercise (sand-fibre) and a cross-over design of short-training exercise on 2 different riding arenas (sand and sand-fibre), respectively. Capillary western immunoassay was used to determine the BGN262 molecular size in a selection of saliva samples collected from cohort 1. Results:Cohort 1: Salivary BGN262 levels were significantly higher in the OA group. The Receiver operating characteristic curve analysis showed an area under the curve of 0.8304 [0.6386 to 1.022], indicating a good separation from the reference group. Cohorts 2 & 3: Salivary BGN262 levels significantly changed during the exercise on sand and sand-fibre arena, with a trend towards higher levels for sand-fibre. The size of the BGN262 fragment determined by Capillary western assay was 18 ​kDa. Conclusions:The data presented show saliva BGN262 levels as a novel biomarker in evaluating the influence of exercise, and interaction with riding arenas alongside assessing osteoarthritis severity.

Nerve Growth Factor (NGF) is a signalling molecule for pain and inflammation. NGF is increased in synovial fluid from osteoarthritic humans and animals, compared to healthy controls. Monoclonal antibody therapy directed against NGF has been approved to treat pain in osteoarthritic dogs but despite many years of trialling, therapy has not been approved for human use. One reason for this is that adverse reactions with rapidly progressing osteoarthritis has occurred in some individuals. More detailed knowledge of NGF expression in joints is needed. In this study, capillary-based Simple Western was used to analyse NGF in cultured equine chondrocytes. Chondrocytes were collected post mortem from three macroscopically healthy intercarpal joints and three intercarpal joints with mild osteoarthritic changes. The chondrocytes were expanded to passage one and seeded in chondrogenic medium to maintain the phenotype. On day four, cells were either stimulated with LPS or kept untreated in medium. All cells were harvested on day five. Wes analysis of lysates did not show mature NGF but two proforms, 40 and 45 kDa, were identified. Results were confirmed with western blot. The same proforms were expressed in chondrocytes from healthy and osteoarthritic joints. Acute inflammation induced by LPS stimulation did not change the forms of expressed NGF. Capillary Simple Western offers a sensitive and sample-sparing alternative to traditional western blot. However, confirmation of peaks is imperative in order to avoid misinterpretation of findings. In addition, in this case the method did not offer the possibility of quantification advertised by the manufacturers.

Objective: Native biglycan (BGN), which can undergo proteolytic cleavage in pathological conditions, is well known to be involved in bone formation and mineralization. This study aimed to delineate the specific cleavage fragment, a neo-epitope for BGN (BGN(262)), in synovial fluid (SF) from young racehorses in training, osteoarthritic (OA) joints with subchondral bone sclerosis (SCBS), and chip fracture joints.Design: A custom-made inhibition ELISA was developed to quantify BGN(262) in SF. Cohort 1: A longitudinal study comprising 10 racehorses undergoing long-term training. Cohort 2: A cross-sectional study comprising joints from horses (N = 69) with different stages of OA and radiographically classified SCBS. Cohort 3: A cross-sectional study comprising horses (N = 9) with chip fractures. Receiver operating characteristic (ROC) curve analysis was performed (healthy joints vs chip joints) to evaluate BGN(262) robustness.Results: Cohort 1: SF BGN(262) levels from racehorses showed a statistical increase during the first 6 months of the training period. Cohort 2: BGN(262) levels were significantly higher in the SF from severe SCBS joints. Cohort 3: SF BGN(262) levels in chip fracture joints showed a significant increase compared to normal joints. The ROC analysis showed an AUC of 0.957 (95% C.I 0.868-1.046), indicating good separation between the groups.Conclusions: The data presented show that BGN(262) levels increase in SF in correlation with the initiation of training, severity of SCBS, and presence of chip fractures. This suggests that BGN262 is a potential predictor and a novel biomarker for early changes in subchondral bone (SCB), aiming to prevent catastrophic injuries in racehorses. (c) 2022 The Author(s). Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

Significance Statement Hypertension and elevated levels of LDL cholesterol (LDLc) are vital risk factors for cardiovascular disease (CVD) in patients with CKD. Although statins indisputably reduce plasma cholesterol levels, not all patients with renal disease benefit from them, making CVD the leading cause of CKD-related mortality. CKD induces hyperelongation of hepatic heparan sulfate (HS) chains from HS proteoglycans (HSPG), thereby increasing the HSPG–proprotein convertase subtilisin kexin type-9 (PCSK9) interaction. These changes associate with elevated LDLc levels, without affecting LDL-receptor expression. This study, using rat models, discloses a novel mechanism behind hypercholesterolemia in CKD with prospects for future investigation of the HSPG-PCSK9 interaction and development of novel heparin-related glycomimetics targeting interruption of HSPG-PCSK9 binding. Background Dyslipidemia is an important risk factor in CKD. The liver clears triglyceride-rich lipoproteins (TRL) via LDL receptor (LDLR), LDLR-related protein-1 (LRP-1), and heparan sulfate proteoglycans (HSPGs), mostly syndecan-1. HSPGs also facilitate LDLR degradation by proprotein convertase subtilisin/kexin type 9 (PCSK9). Progressive renal failure affects the structure and activity of hepatic lipoprotein receptors, PCSK9, and plasma cholesterol. Methods Uninephrectomy- and aging-induced CKD in normotensive Wistar rats and hypertensive Munich-Wistar-Frömter (MWF) rats. Results Compared with 22-week-old sex- and strain-matched rats, 48-week-old uninephrectomized Wistar-CKD and MWF-CKD rats showed proteinuria, increased plasma creatinine, and hypercholesterolemia (all P<0.05), which were most apparent in hypertensive MWF-CKD rats. Hepatic PCSK9 expression increased in both CKD groups (P<0.05), with unusual sinusoidal localization, which was not seen in 22-week-old rats. Heparan sulfate (HS) disaccharide analysis, staining with anti-HS mAbs, and mRNA expression of HS polymerase exostosin-1 (Ext-1), revealed elongated HS chains in both CKD groups. Solid-phase competition assays showed that the PCSK9 interaction with heparin-albumin (HS-proteoglycan analogue) was critically dependent on polysaccharide chain length. VLDL binding to HS from CKD livers was reduced (P<0.05). Proteinuria and plasma creatinine strongly associated with plasma cholesterol, PCSK9, and HS changes. Conclusions Progressive CKD induces hepatic HS elongation, leading to increased interaction with PCSK9. This might reduce hepatic lipoprotein uptake and thereby induce dyslipidemia in CKD. Therefore, PCSK9/HS may be a novel target to control dyslipidemia.

The regenerative medicine field has been revolutionized by the direct conversion of one cell type to another by ectopic expression of lineage-specific transcription factors. The direct reprogramming of fibroblasts to induced cardiac myocytes (iCMs) by core cardiac transcription factors (Gata4, Mef2c, Tbx5) both in vitro and in vivo has paved the way in cardiac regeneration and repair. Several independent research groups have successfully reported the direct reprogramming of fibroblasts in injured myocardium to cardiac myocytes employing a variety of approaches that rely on transcription factors, small molecules, and micro RNAs (miRNAs). Recently, this technology has been considered for local repair of the pacemaker and the cardiac conduction system. To address this, we will first discuss the direct reprograming advancements in the setting of working myocardium regeneration, and then elaborate on how this technology can be applied to repair the cardiac pacemaker and the conduction system.

BackgroundChronic renal transplant dysfunction is characterized by loss of renal function and tissue remodeling, including chronic inflammation and lymph vessel formation. Proteoglycans are known for their chemokine presenting capacity. We hypothesize that interruption of the lymphatic chemokine-proteoglycan interaction interferes with the lymphatic outflow of leukocytes from the renal graft and might decrease the anti-graft allo-immune response.MethodsIn a rat renal chronic transplant dysfunction model (female Dark-Agouti to male Wistar Furth), chemokines were profiled by qRT-PCR in microdissected tubulo-interstitial tissue. Disruption of lymphatic chemokine-proteoglycan interaction was studied by (non-anticoagulant) heparin-derived polysaccharides in vitro and in renal allografts. The renal allograft function was assessed by rise in plasma creatinine and urea.ResultsWithin newly-formed lymph vessels of transplanted kidneys, numerous CD45(+) leukocytes were found, mainly MHCII+, ED-1(-), IDO-, HIS14(-), CD103(-) antigen presenting cells, most likely representing a subset of dendritic cells. Treatment of transplanted rats with regular heparin and two different (non-)anticoagulant heparin derivatives revealed worsening of kidney function only in the glycol-split heparin treated group despite a two-fold reduction of tubulo-interstitial leukocytes (p<0.02). Quantitative digital image analysis however revealed increased numbers of intra-lymphatic antigen-presenting cells only in the glycol-split heparin group (p<0.01). The number of intra-lymphatic leukocytes significantly correlates with plasma creatinine and urea, and inversely with creatinine clearance.ConclusionsTreatment of transplanted rats with glycol-split heparin significantly increases the number of intra-lymphatic antigen presenting cells, by increased renal diffusion of lymphatic chemokines, thereby increasing the activation and recruitment of antigen presenting cells towards the lymph vessel. This effect is unwanted in the transplantation setting, but might be advantageous in e.g., dendritic cell vaccination.

Our study was to assess and evaluate the effects of anti-tubercular drugs on hematological, renal and hepatic function tests in Warangal district population, Telangana state, India. This observational prospective study was carried out in the department of respiratory medicine, Government Tuberculosis (TB) Hospital, Hanamkonda, Telangana, India. Seventy tuberculosis patients were randomly selected with normal liver and kidney function tests. Renal and liver function tests were done using spectrophotometric methods using the biosystem and flame photometer. Results obtained were collected and analyzed with the statistical package of social sciences (SPSS Version 17). Descriptive and comparative analysis was conducted. The 0.05 level of significance was used as a cut-off for statistical significance. After 4 weeks of treatment, the study revealed that the characteristic renal function tests; mean plasma concentration of creatinine (mg/dL) increased from 0.77±0.18 to 2.99±1.64 and uric acid levels (mg/dL) increased from 3.05±0.86 to 4.59±0.93 showing a significant difference (p=<0.0001). The characteristic liver function tests increased significantly (p=<0.0001) with mean plasma concentration of aspartate aminotransferase (AST) (IU/L) increased from 22.47± 15.24 to 62.44±13.52 and alanine aminotransferase (ALP) (IU/L) increased from 27.05±15.28 to 64.28±15.56. Hematological Parameters also decreased significantly (p=<0.0001) with Hemoglobin (gm %) decreasing from 11.8±2.4 to 9.43±1.9 and RBC (m/cmm) decreasing from 3.8±0.9 to 3.23±0.79 in patients taking anti tubercular drugs. In this study, serious adverse reactions to anti-tuberculous drugs were common and resulted in changes in hematological, liver and renal function, increased hospitalization costs as well as prolongation of therapy. Drug-induced Hematological, renal and hepatic toxicities indicate adjustment of dosage and optimization of drug regimen along with proactive assessment of organ function in appropriate manner with frequent laboratory testing.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan involved in regenerative growth and cellular adhesion. We hypothesized that the induction of tubular syndecan-1 is a repair response to incipient renal damage in apparently stable, uncomplicated renal transplant recipients. We quantified tubular syndecan-1 in unselected renal protocol biopsies taken 1 yr after transplantation. Spearman rank correlation analysis revealed an inverse correlation between tubular syndecan-1 expression and creatinine clearance at the time of biopsy (r = -0.483, P < 0.03). In a larger panel of protocol and indication biopsies from renal transplant recipients, tubular syndecan-1 correlated with tubular proliferation marker Ki67 (r = 0.518, P < 0.0001). In a rat renal transplantation model, 2 mo after transplantation, mRNA expression of syndecan-1 and its major sheddase, A disintegrin and metalloproteinase-17, were upregulated (both P < 0.03). Since shed syndecan-1 might end up in the circulation, in a stable cross-sectional human renal transplant population (n = 510), we measured plasma syndecan-1. By multivariate regression analysis, we showed robust independent associations of plasma syndecan-1 with renal (plasma creatinine and plasma urea) and endothelial function parameters (plasma VEGF-A, all P < 0.01). By various approaches, we were not able to localize syndecan-1 in vessel wall or endothelial cells, which makes shedding of syndecan-1 from the endothelial glycocalyx unlikely. Our data suggest that early damage in transplanted kidneys induces repair mechanisms within the graft, namely, tubular syndecan-1 expression for tubular regeneration and VEGF production for endothelial repair. Elevated plasma syndecan-1 levels in renal transplantation patients might be interpreted as repair/survival factor related to loss of tubular and endothelial function in transplanted kidneys.

Syndecan-1 is a transmembrane heparan sulfate (HS) proteoglycan present on hepatocytes and involved in uptake of triglyceride-rich lipoproteins via its HS polysaccharide side chains. We hypothesized that altered hepatic syndecan-1 metabolism could be involved in dyslipidemia related to renal transplantation. In a rat renal transplantation model elevated plasma triglycerides were associated with fivefold increased expression of hepatic syndecan-1 mRNA (p < 0.01), but not protein. Expression of syndecan-1 sheddases (ADAM17, MMP9) and heparanase was significantly up-regulated after renal transplantation (all p < 0.05). Profiling of HS side chains revealed loss of hepatic HS upon renal transplantation accompanied by significant decreased functional capacity for VLDL binding (p = 0.02). In a human renal transplantation cohort (n = 510), plasma levels of shed syndecan-1 were measured. Multivariate analysis showed plasma syndecan-1 to be independently associated with triglycerides (p < 0.0001) and inversely with HDL cholesterol (p < 0.0001). Last, we show a physical association of syndecan-1 to HDL from renal transplant recipients (RTRs), but not to HDL from healthy controls. Our data suggest that after renal transplantation loss of hepatic HS together with increased syndecan-1 shedding hampers lipoprotein binding and uptake by the liver contributing to dyslipidemia. Our data open perspectives toward improvement of lipid profiles by targeted inhibition of syndecan-1 catabolism in renal transplantation.

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

The pathogenesis of chronic transplant dysfunction (CTD) is multifactorial and is histologically characterized by interstitial fibrosis and tubular atrophy, transplant vasculopathy and focal segmental glomerulosclerosis, and is also associated by an inflammatory component. To date, no effective therapies are available to treat renal CTD. Heparan sulfate proteoglycans are crucial players in tissue homeostasis via their heparan sulfate polysaccharide side chains. We hypothesized that heparan sulfate proteoglycans could be promising therapeutic targets to limit CTD, by their involvement in cell recruitment and complement cascades. To test this hypothesis, in a rat model for renal CTD, by daily subcutaneous injections, we intervened with vehicle, unfractionated heparin, and the non-anticoagulant heparinoids (N-acetyl heparin and periodate-oxidized, borohydrate-reduced (RO-) heparin) for 9 weeks. Both these non-anticoagulant heparinoids have been described to exert similar anti-inflammatory properties as heparin. On the one hand, RO-heparin treatment ameliorated the cortical tubulo-interstitial accumulation of CD45+ inflammatory cells compared to the vehicle treated group (week 9: 0.8% surface area in RO-heparin group versus 1.8% surface area in vehicle group; p<0.02). On the other hand, properdin deposition was increased ̃2-fold in RO-heparin compared to vehicle (p<0.05), without affecting factor H, C3 and Membrane Attack Complex deposition. A tendency for lower proteinuria and glomerulosclerosis was present in the RO-heparin group, but not in the other heparin(oid) groups. Heparin(oid) treatment did not affect body weight, graft survival, blood pressure, and renal function. These data demonstrate that specifically modified heparinoids can reduce inflammation in experimental CTD, and suggest that carefully selected non-anticoagulant heparan sulfate glycomimetics could have protective effects as adjunct therapy for CTD.

Syndecan-1, a heparan sulfate proteoglycan, has an important role in wound healing by binding several growth factors and cytokines. As these processes are also crucial in damage and repair after renal transplantation, we examined syndecan-1 expression in human control kidney tissue, renal allograft protocol biopsies, renal allograft biopsies taken at indication, and non-transplant interstitial fibrosis. Syndecan-1 expression was increased in tubular epithelial cells in renal allograft biopsies compared with control. Increased epithelial syndecan-1 in allografts correlated with low proteinuria and serum creatinine, less interstitial inflammation, less tubular atrophy, and prolonged allograft survival. Knockdown of syndecan-1 in human tubular epithelial cells in vitro reduced cell proliferation. Selective binding of growth factors suggests that syndecan-1 may promote epithelial restoration. Bilateral renal ischemia/reperfusion in syndecan-1-deficient mice resulted in increased initial renal failure and tubular injury compared with wild-type mice. Macrophage and myofibroblast numbers, tubular damage, and plasma urea levels were increased, and tubular proliferation reduced in the kidneys of syndecan-1 deficient compared with wild-type mice 14 days following injury. Hence syndecan-1 promotes tubular survival and repair in murine ischemia/reperfusion injury and correlates with functional improvement in human renal allograft transplantation. Kidney International (2012) 81, 651-661; doi:10.1038/ki.2011.425; published online 11 January 2012