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Abstract Background Synovial macrophages perform a multitude of functions that include clearance of cell debris and foreign bodies, tissue immune surveillance, and resolution of inflammation. The functional diversity of macrophages is enabled by distinct subpopulations that express unique surface markers. Proteoglycan-4 (PRG4) is an important regulator of synovial hyperplasia and fibrotic remodeling, and the involvement of macrophages in PRG4’s synovial role is yet to be defined. Our objectives were to study the PRG4’s importance to macrophage homeostatic regulation in the synovium and infiltration of pro-inflammatory macrophages in acute synovitis and investigate whether macrophages mediated synovial fibrosis in Prg4 gene-trap (Prg4GT/GT) murine knee joints. Methods Macrophage phenotyping in Prg4GT/GT and Prg4+/+ joints was performed by flow cytometry using pan-macrophage markers, e.g., CD11b, F4/80, and surface markers of M1 macrophages (CD86) and M2 macrophages (CD206). Characterizations of the various macrophage subpopulations were performed in 2- and 6-month-old animals. The expression of inflammatory markers, IL-6, and iNOS in macrophages that are CD86+ and/or CD206+ was studied. The impact of Prg4 recombination on synovial macrophage populations of 2- and 6-month-old animals and infiltration of pro-inflammatory macrophages in response to a TLR2 agonist challenge was determined. Macrophages were depleted using liposomal clodronate and synovial membrane thickness, and the expression of fibrotic markers α-SMA, PLOD2, and collagen type I (COL-I) was assessed using immunohistochemistry. Results Total macrophages in Prg4GT/GT joints were higher than Prg4+/+ joints (p) at 2 and 6 months, and the percentages of CD86+/CD206− and CD86+/CD206+ macrophages increased in Prg4GT/GT joints at 6 months (p), whereas the percentage of CD86−/CD206+ macrophages decreased (p). CD86+/CD206− and CD86+/CD206+ macrophages expressed iNOS and IL-6 compared to CD86−/CD206+ macrophages (p). Prg4 re-expression limited the accumulation of CD86+ macrophages (p) and increased CD86−/CD206+ macrophages (p) at 6 months. Prg4 recombination attenuated synovial recruitment of pro-inflammatory macrophages in 2-month-old animals (p). Clodronate-mediated macrophage depletion reduced synovial hyperplasia, α-SMA, PLOD2, and COL-I expressions in the synovium (p). Conclusions PRG4 regulates the accumulation and homeostatic balance of macrophages in the synovium. In its absence, the synovium becomes populated with M1 macrophages. Furthermore, macrophages exert an effector role in synovial fibrosis in Prg4GT/GT animals.

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome leads to diarthrodial joint arthropathy and is caused by the absence of lubricin (proteoglycan 4—PRG4), a surface-active mucinous glycoprotein responsible for lubricating articular cartilage. In this study, mice lacking the orthologous gene Prg4 served as a model that recapitulates the destructive arthrosis that involves biofouling of cartilage by serum proteins in lieu of Prg4. This study hypothesized that Prg4-deficient mice would demonstrate a quadruped gait change and decreased markers of mitochondrial dyscrasia, following intra-articular injection of both hindlimbs with recombinant human PRG4 (rhPRG4). Prg4−/− (N = 44) mice of both sexes were injected with rhPRG4 and gait alterations were studied at post-injection day 3 and 6, before joints were harvested for immunohistochemistry for caspase-3 activation. Increased stance and propulsion was shown at 3 days post-injection in male mice. There were significantly fewer caspase-3-positive chondrocytes in tibiofemoral cartilage from rhPRG4-injected mice. The mitochondrial gene Mt-tn, and myosin heavy (Myh7) and light chains (Myl2 and Myl3), known to play a cytoskeletal stabilizing role, were significantly upregulated in both sexes (RNA-Seq) following IA rhPRG4. Chondrocyte mitochondrial dyscrasias attributable to the arthrosis in CACP may be mitigated by IA rhPRG4. In a supporting in vitro crystal microbalance experiment, molecular fouling by albumin did not block the surface activity of rhPRG4.

Background Synovial tissue fibrosis is common in advanced OA with features including the presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I. Proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and is a major component of synovial fluid. PRG4 is a ligand of the CD44 receptor. Our objective was to examine the role of PRG4-CD44 interaction in regulating synovial tissue fibrosis in vitro and in vivo. Methods OA synoviocytes were treated with TGF-β ± PRG4 for 24 h and α-SMA content was determined using immunofluorescence. Rhodamine-labeled rhPRG4 was incubated with OA synoviocytes ± anti-CD44 or isotype control antibodies and cellular uptake of rhPRG4 was determined following a 30-min incubation and α-SMA expression following a 24-h incubation. HEK-TGF-β cells were treated with TGF-β ± rhPRG4 and Smad3 phosphorylation was determined using immunofluorescence and TGF-β/Smad pathway activation was determined colorimetrically. We probed for stress fibers and focal adhesions (FAs) in TGF-β-treated murine fibroblasts and fibroblast migration was quantified ± rhPRG4. Synovial expression of fibrotic markers: α-SMA, collagen type-I, and PLOD2 in Prg4 gene-trap (Prg4GT) and recombined Prg4GTR animals were studied at 2 and 9 months of age. Synovial expression of α-SMA and PLOD2 was determined in 2-month-old Prg4GT/GT&Cd44−/− and Prg4GTR/GTR&Cd44−/− animals. Results PRG4 reduced α-SMA content in OA synoviocytes (p .001). rhPRG4 was internalized by OA synoviocytes via CD44 and CD44 neutralization attenuated rhPRG4’s antifibrotic effect (p .05). rhPRG4 reduced pSmad3 signal in HEK-TGF-β cells (p .001) and TGF-β/Smad pathway activation (p .001). rhPRG4 reduced the number of stress fiber-positive myofibroblasts, FAs mean size, and cell migration in TGF-β-treated NIH3T3 fibroblasts (p .05). rhPRG4 inhibited fibroblast migration in a macrophage and fibroblast co-culture model without altering active or total TGF-β levels. Synovial tissues of 9-month-old Prg4GT/GT animals had higher α-SMA, collagen type-I, and PLOD2 (p .001) content and Prg4 re-expression reduced these markers (p .01). Prg4 re-expression also reduced α-SMA and PLOD2 staining in CD44-deficient mice. Conclusion PRG4 is an endogenous antifibrotic modulator in the joint and its effect on myofibroblast formation is partially mediated by CD44, but CD44 is not required to demonstrate an antifibrotic effect in vivo.

An amendment to this paper has been published and can be accessed via the original article.

Regulated/activated protein kinase (PRAK) plays a crucial role in modulating biological function. However, the role of PRAK in mediating cardiac dysfunction and metabolic disorders remains unclear. We examined the effects of deletion of PRAK on modulating cardiac function and insulin resistance in mice exposed to a high-fat diet (HFD). Wild-type and PRAK−/− mice at 8 weeks old were exposed to either chow food or HFD for a consecutive 16 weeks. Glucose tolerance tests and insulin tolerance tests were employed to assess insulin resistance. Echocardiography was employed to assess myocardial function. Western blot was used to determine the molecular signaling involved in phosphorylation of IRS-1, AMPKα, ERK-44/42, and irisin. Real time-PCR was used to assess the hypertrophic genes of the myocardium. Histological analysis was employed to assess the hypertrophic response, interstitial myocardial fibrosis, and apoptosis in the heart. Western blot was employed to determine cellular signaling pathway. HFD-induced metabolic stress is indicated by glucose intolerance and insulin intolerance. PRAK knockout aggravated insulin resistance, as indicated by glucose intolerance and insulin intolerance testing as compared with wild-type littermates. As compared with wild-type mice, hyperglycemia and hypercholesterolemia were manifested in PRAK-knockout mice following high-fat diet intervention. High-fat diet intervention displayed a decline in fractional shortening and ejection fraction. However, deletion of PRAK exacerbated the decline in cardiac function as compared with wild-type mice following HFD treatment. In addition, PRAK knockout mice enhanced the expression of myocardial hypertrophic genes including ANP, BNP, and βMHC in HFD treatment, which was also associated with an increase in cardiomyocyte size and interstitial fibrosis. Western blot indicated that deletion of PRAK induces decreases in phosphorylation of IRS-1, AMPKα, and ERK44/42 as compared with wild-type controls. Our finding indicates that deletion of PRAK promoted myocardial dysfunction, cardiac remodeling, and metabolic disorders in response to HFD.

Background Gout is an inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystals’ joint deposition. MSU phagocytosis by resident macrophages is a key step in gout pathogenesis. MSU phagocytosis triggers nuclear factor kappa B (NFκB) activation and production of cytokines and chemokines. Proteoglycan-4 (PRG4) is a glycoprotein produced by synovial fibroblasts and exerts an anti-inflammatory effect in the joint mediated by its interaction with cell surface receptor CD44. PRG4 also binds and antagonizes TLR2 and TLR4. The objective of this study is to evaluate the efficacy of recombinant human PRG4 (rhPRG4) in suppressing MSU-induced inflammation and mechanical allodynia in vitro and in vivo. Methods THP-1 macrophages were incubated with MSU crystals ± rhPRG4 or bovine submaxillary mucin (BSM), and crystal phagocytosis, cytokines and chemokines expression and production were determined. NFκB p65 subunit nuclear translocation, NLRP3 induction, caspase-1 activation and conversion of proIL-1β to mature IL-1β were studied. MSU phagocytosis by Prg4 +/+ and Prg4 −/− peritoneal macrophages was determined in the absence or presence of rhPRG4, BSM, anti-CD44, anti-TLR2, anti-TLR4 and isotype control antibodies. Rhodamine-labeled rhPRG4 was incubated with murine macrophages and receptor colocalization studies were performed. Lewis rats underwent intra-articular injection of MSU crystals followed by intra-articular treatment with PBS or rhPRG4. Weight bearing and SF myeloperoxidase activities were determined. Results rhPRG4 reduced MSU crystal phagocytosis at 4 h (p

Background: Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. Objective: To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. Study Design: Controlled laboratory study. Methods: Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O–fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1β (IL-1β) levels in synovial fluid and serum. Results: The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group ( P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups ( P = .23). However, no differences in microscopic damage scores were observed between the 3 groups ( P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group ( P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS ( P = .013) and rhPRG4+hyaluronan ( P = .011) groups. In serum and synovial fluid, both rhPRG4 ( P = .006; P = .017) and rhPRG4+hyaluronan groups ( P = .009; P = .03) presented decreased IL-1β levels. Conclusion: All groups exhibited significant cartilage degeneration after DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of posttraumatic osteoarthritis. Clinical Relevance: Patients with meniscal trauma are at high risk for posttraumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage after meniscal injury.

p38-Regulated/activated protein kinase (PRAK) plays a critical role in modulating cellular survival and biological function. However, the function of PRAK in the regulation of myocardial ischemic injury remains unknown. This study is aimed at determining the function of PRAK in modulating myocardial ischemia-reperfusion injury and myocardial remodeling following myocardial infarction. Hearts were isolated from adult male homozygous PRAK−/− and wild-type mice and subjected to global ischemia-reperfusion injury in Langendorff isolated heart perfusion. PRAK−/− mice mitigated postischemic ventricular functional recovery and decreased coronary effluent. Moreover, the infarct size in the perfused heart was significantly increased by deletion of PRAK. Western blot showed that deletion of PRAK decreased the phosphorylation of ERK1/2. Furthermore, the effect of deletion of PRAK on myocardial function and remodeling was also examined on infarcted mice in which the left anterior descending artery was ligated. Echocardiography indicated that PRAK−/− mice had accelerated left ventricular systolic dysfunction, which was associated with increased hypertrophy in the infarcted area. Deletion of PRAK augmented interstitial fibrosis and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive myocytes. Furthermore, immunostaining analysis shows that CD31-postive vascular density and α-smooth muscle actin capillary staining decreased significantly in PRAK−/− mice. These results indicate that deletion of PRAK enhances susceptibility to myocardial ischemia-reperfusion injury, attenuates cardiac performance and angiogenesis, and increases interstitial fibrosis and apoptosis in the infarcted hearts.

Histone deacetylases (HDACs) play a critical role in modulating cardiac function and ischemic injury. HDAC4 was found to be elevated and activated in response to injury. However, whether HDAC4 mediates cardiac function is currently unknown. In this study, we created myocyte-specific activated HDAC4 transgenic mice to examine the role of HDAC4 in mediating cardiac function during development and response to infarction. There are no differences in cardiac function and gross phenotype between wild-type and cardiomyocyte-specific HDAC4 transgenic mice at 1 mo of age. However, cardiac dysfunction and vascular growth deficiency were displayed in 6-mo-old HDAC4-transgenic mice compared with wild-type mice. Activation of HDAC4 increased heart and myocyte size, hypertrophic proteins, and interstitial fibrosis in 6-mo-old mice but not in 1-mo-old mice. To further define whether activated HDAC4 in the heart could impact myocardial function and remodeling, myocardial infarction was created in both wild-type and cardiomyocyte-specific HDAC4-transgenic mice. In myocardial infarction, the overexpression of activated HDAC4 exacerbated cardiac dysfunction and augmented cardiac remodeling and interstitial fibrosis, which was associated with the reduction of cardiokines in the heart. These results indicate the activation of HDAC4 as a crucial regulator for cardiac function in development and myocardial infarction. NEW & NOTEWORTHY We created myocyte-specific activated HDAC4-transgenic mice to examine the function of HDAC4 in mediating cardiac function. HDAC4 overexpression led to cardiac dysfunction, which was associated with increased hypertrophy and myocardial fibrosis. Furthermore, the overexpression of activated HDAC4 exacerbated cardiac dysfunction, augmented remodeling, and increased apoptosis in the infarcted heart. This is the first demonstration that transgenic overexpression of HDAC4 is crucial for modulation of cardiac function and remodeling.

Osteoarthritis (OA) is characterized by synovitis and synovial fibrosis. Synoviocytes are fibroblast-like resident cells of the synovium that are activated by transforming growth factor (TGF)-β to proliferate, migrate, and produce extracellular matrix. Synoviocytes secrete hyaluronan (HA) and proteoglycan-4 (PRG4). HA reduces synovial fibrosis in vivo, and the Prg4−/−mouse exhibits synovial hyperplasia. We investigated the antifibrotic effects of increased intracellular cAMP in TGF-β-stimulated human OA synoviocytes. TGF-β1 stimulated collagen I (COL1A1), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression, and procollagen I, α-SMA, HA, and PRG4 production, migration, and proliferation of OA synoviocytes were measured. Treatment of OA synoviocytes with forskolin (10 μM) increased intracellular cAMP levels and reduced TGF-β1-stimulated COL1A1, α-SMA, and TIMP-1 expression, with no change in PLOD2 expression. Forskolin also reduced TGF-β1-stimulated procollagen I and α-SMA content as well as synoviocyte migration and proliferation. Forskolin (10 μM) increased HA secretion and PRG4 expression and production. A cell-permeant cAMP analog reduced COL1A1 and α-SMA expression and enhanced HA and PRG4 secretion by OA synoviocytes. HA and PRG4 reduced α-SMA expression and content, and PRG4 reduced COL1A1 expression and procollagen I content in OA synoviocytes. Prg4−/−synovium exhibited increased α-SMA, COL1A1, and TIMP-1 expression compared with Prg4+/+synovium. Prg4−/−synoviocytes demonstrated strong α-SMA and collagen type I staining, whereas these were undetected in Prg4+/+synoviocytes and were reduced with PRG4 treatment. We conclude that increasing intracellular cAMP levels in synoviocytes mitigates synovial fibrosis through enhanced production of HA and PRG4, possibly representing a novel approach for treatment of OA synovial fibrosis.

Objective. Congenital deficiency of the principal boundary lubricant in cartilage (i.e., lubricin, encoded by the gene PRG4) increases joint friction and causes progressive joint failure. This study was undertaken to determine whether restoring lubricin expression in a mouse model would prevent, delay, or reverse the disease process caused by congenital deficiency. Methods. Using genetically engineered lubricin-deficient mice, we restored gene function before conception or at ages 3 weeks, 2 months, or 6 months after birth. The effect of restoring gene function (i.e., expression of lubricin) on the tibiofemoral patellar joints of mice was evaluated histologically and by ex vivo biomechanical testing. Results. Restoring gene function in mice prior to conception prevented joint disease. In 3-week-old mice, restoring gene function improved, but did not normalize, histologic features of the articular cartilage and whole-joint friction. In addition, cyclic loading of the joints produced fewer activated caspase 3-containing chondrocytes when lubricin expression was restored, as compared to that in littermate mice whose gene function was not restored (nonrestored controls). Restoration of lubricin expression in 2-month-old or 6-month-old mice had no beneficial effect on histopathologic cartilage damage, extent of whole-joint friction, or activation of caspase 3 when compared to nonrestored controls. Conclusion. When boundary lubrication is congenitally deficient and cartilage becomes damaged, the window of opportunity for restoring lubrication and slowing disease progression is limited.

Objective To evaluate recombinant human proteoglycan 4 (rhPRG4) binding to CD44 receptor and its consequence on cytokine induced synoviocyte proliferation.

Deficiency of PRG4 (lubricin), the boundary lubricant in mammalian joints, contributes to increased joint friction accompanied by superficial and upper intermediate zone chondrocyte caspase-3 activation, as shown in lubricin-null (Prg4−/−) mice. Caspase-3 activity appears to be reversible upon the restitution of Prg4 either endogenously in vivo, in a gene trap mouse, or as an applied lubricant in vitro. In this study we show that intra-articular injection of human PRG4 in vivo in Prg4−/− mice prevented caspase-3 activation in superficial zone chondrocytes and was associated with a modest decrease in whole joint friction measured ex vivo using a joint pendulum method. Non-lubricated Prg4−/− mouse cartilage shows caspase cascade activation caused by mitochondrial dysregulation, and significantly higher levels of peroxynitrite (ONOO− and −OH) and superoxide (O−2) compared to Prg4+/+ and Prg4+/− cartilage. Enzymatic activity levels of caspase 8 across Prg4 mutant mice were not significantly different, indicating no extrinsic apoptosis pathway activation. Western blots showed caspase-3 and 9 activation in Prg4−/− tissue extracts, and the appearance of nitrosylated Cys163 in the active cleft of caspase-3 which inhibits its enzymatic activity. These findings are relevant to patients at risk for arthrosis, from camptodactyl-arthropathy-coxa vara-pericarditis (CACP) syndrome and transient lubricin insufficiency due to trauma and inflammation.

We have recently shown that in vivo inhibition of histone deacetylase (HDAC) stimulates endogenous myocardial regeneration in infarcted hearts (Zhang L et al. J Pharmacol Exp Ther 341: 285–293, 2012). Furthermore, our observation demonstrates that HDAC inhibition promotes cardiogenesis, which is associated with HDAC4 reduction. However, it remains unknown as to whether specific inhibition of HDAC4 modulates cardiac stem cells (CSCs) to facilitate myocardial repair and to preserve cardiac performance. c-kit+ CSCs were isolated from adult mouse hearts and were transfected with HDAC4 siRNA to knockdown HDAC4 of c-kit+ CSCs. The transfection of HDAC4 siRNA caused a marked reduction of HDAC4 mRNA and proteins in c-kit+ CSCs. Mouse myocardial infarction (MI) was created to assess the effect of HDAC4 inhibition in c-kit+ CSCs on myocardial regeneration in vivo when cells were introduced into MI hearts. Transplantation of HDAC4 siRNA-treated c-kit+ CSCs into MI hearts improved ventricular function, attenuated ventricular remodeling, and promoted CSC-derived regeneration and neovascularization. Furthermore, Ki67 and BrdU positively proliferative myocytes increased in MI hearts receiving HDAC4 siRNA-treated c-kit+ CSCs compared with MI hearts engrafted with control siRNA-treated c-kit+ CSCs. In addition, compared with MI hearts engrafted with control adenoviral GFP-infected c-kit+ CSCs, MI hearts receiving adenoviral HDAC4-infected c-kit+ CSCs exhibited attenuated cardiac functional recovery, CSC-derived regeneration, and neovascularization, which was accompanied with adverse ventricular remodeling and decrease in Ki67 and BrdU positively proliferative myocytes. HDAC4 inhibition facilitated c-kit+ CSCs into the differentiation into cardiac lineage commitments in vitro, while HDAC4 overexpression attenuated c-kit+ CSC-derived cardiogenesis. Our results indicate that HDAC4 inhibition promotes CSC-derived cardiac regeneration and improves the restoration of cardiac function.

We have recently shown that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia and reperfusion (I/R) injury. The mechanism by which HDAC inhibition induces cardioprotection remains unknown. We sought to investigate whether the genetic disruption of gp-91, a subunit of NADPH-oxidase, would mitigate cardioprotection of HDAC inhibition. Wild-type and gp-91(-)(/-) mice were treated with a potent inhibitor of HDACs, trichostatin A (TSA, 0.1 mg/kg, i.p.). Twenty-four hours later, the perfused hearts were subjected to 30 min of ischemia and 30 min of reperfusion. HDAC inhibition in wild-type mice produced marked improvements in ventricular functional recovery and the reduction of infarct size. TSA-induced cardioprotection was eliminated with genetic deletion of gp91. Notably, Western blot and immunostaining displayed a significant increase in gp-91 in myocardium following HDAC inhibition, which resulted in a mildly subsequent increase in the production of reactive oxygen species (ROS). The pre-treatment of H9c2 cardiomyoblasts with TSA (50 nmol/l) decreased cell necrosis and increased viability in response to simulated ischemia (SI), which was abrogated by the transfection of cells with gp-91 siRNA, but not by scrambled siRNA. Furthermore, treatment of PLB-985 gp91(+/+) cells with TSA increased the resistance to SI, which also diminished with genetic disruption of gp91 in gp91(phox)-deficient PLB-985 cells. TSA treatment inhibited the increased active caspase-3 in H9c2 cardiomyoblasts and PLB-985 gp91(+/+) cells exposed to SI, which were prevented by knockdown of gp-91 by siRNA. These results suggest that a cascade consisting of gp-91 and HDAC inhibition plays an essential role in orchestrating the cardioprotective effect.

We have recently demonstrated that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia-reperfusion (I/R) injury. The mechanism by which HDAC inhibition confers myocardial protection remains unknown. The purpose of this study is to investigate whether the disruption of NF-kappaB p50 would eliminate the protective effects of HDAC inhibition. Wild-type and NF-kappaB p50-deficient mice were treated with trichostatin A (TSA; 0.1 mg/kg ip), a potent inhibitor of HDACs. Twenty-four hours later, the hearts were perfused in Langendorff model and subjected to 30 min of ischemia and 30 min of reperfusion. Inhibition of HDACs by TSA in wild-type mice produced marked improvements in left ventricular end-diastolic pressure, left ventricular rate pressure product, and the reduction of infarct size compared with non-TSA-treated group. TSA-induced cardioprotection in wild-type animals was absent with genetic deletion of NF-kappaB p50 subunit. Notably, Western blot displayed a significant increase in nuclear NF-kappaB p50 and the immunoprecipitation demonstrated a remarkable acetylation of NF-kappaB p50 at lysine residues following HDAC inhibition. EMSA exhibited a subsequent increase in NF-kappaB DNA binding activity. Luciferase assay demonstrated an activation of NF-kappaB by HDAC inhibition. The pretreatment of H9c2 cardiomyoblasts with TSA (50 nmol/l) decreased cell necrosis and increased in cell viability in simulated ischemia. The resistance of H9c2 cardiomyoblasts to simulated ischemia by HDAC inhibition was eliminated by genetic knockdown of NF-kappaB p50 with transfection of NF-kappaB p50 short interfering RNA but not scrambled short interfering RNA. These results suggest that NF-kappaB p50 acetylation and activation play a pivotal role in HDAC inhibition-induced cardioprotection.

Background Lubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. PRG4 has a homeostatic multifaceted role in the joint. PRG4 intra-articular treatment retards progression of cartilage degeneration in pre-clinical posttraumatic osteoarthritis models. The objective of this study is to evaluate the binding of recombinant human PRG4 (rhPRG4) and native human PRG4 (nhPRG4) to toll-like receptors 2 and 4 (TLR2 and TLR4) and whether this interaction underpins a PRG4 anti-inflammatory role in synovial fluid (SF) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Methods rhPRG4 and nhPRG4 binding to TLR2 and TLR4 was evaluated using a direct enzyme linked immunosorbent assay (ELISA). Association of rhPRG4 with TLR2 and TLR4 overexpressing human embryonic kidney (HEK) cells was studied by flow cytometry. Activation of TLR2 and TLR4 on HEK cells by agonists Pam3CSK4 and lipopolysaccharide (LPS) was studied in the absence or presence of nhPRG4 at 50, 100 and 150 μg/ml. Activation of TLR2 and TLR4 by OA SF and RA SF and the effect of nhPRG4 SF treatment on receptor activation was assessed. PRG4 was immunoprecipitated from pooled OA and RA SF. TLR2 and TLR4 activation by pooled OA and RA SF with or without PRG4 immunoprecipitation was compared. Results rhPRG4 and nhPRG4 exhibited concentration-dependent binding to TLR2 and TLR4. rhPRG4 associated with TLR2- and TLR4-HEK cells in a time-dependent manner. Co-incubation of nhPRG4 (50, 100 and 150 μg/ml) and Pam3CSK4 or LPS reduced TLR2 or TLR4 activation compared to Pam3CSK4 or LPS alone (p

Previously, we have reported that thalidomide can modulate the immune responses in female B6C3F1 mice. Furthermore, thalidomide immunomodulation increased primary host resistance to intravenously infected Listeria monocytogenes. The present study was intended to evaluate the mechanisms underlying the enhanced host resistance to L. monocytogenes by focusing on the neutrophils. Female B6C3F1 mice were treated intraperitoneally with thalidomide (100 mg/kg) for 15 days. Exposure to thalidomide increased the numbers of neutrophils in the spleens and livers of L. monocytogenes-infected mice when compared to the L. monocytogenes-infected control mice. Additionally, the percentage of neutrophils was also significantly increased after Thd treatment in L. monocytogenes-infected mice. Further studies using antibodies to deplete corresponding cells indicated that thalidomide-mediated increase in primary host resistance (both the moribundity and colony counts in the liver and spleen) to L. monocytogenes infection was due to its effect on neutrophils but not CD8+ T cells or NK cells. Finally, Thd exposure also increased host resistance to secondary host resistance to L. monocytogenes infection, and depletion of neutrophils abolished the protective effect. In conclusion, thalidomide enhanced host resistance to both primary and secondary L. monocytogenes infections by a neutrophil-related mechanism in female B6C3F1 mice.

Ethanol has been recognized as an immunosuppressive agent for many years. Effects of high levels of ethanol consumption on immune functions have been extensively studied, but little is known about the effects of low levels (scuh as 5% ethanol) of ethanol consumption. Herein we report that exposure of mice to 5% ethanol for 4-8 weeks decreases IL-2-augmented splenic NK cell activity, decreases the numbers of NK cells in spleen and liver, decreases the number of granulocytes (Gr-l+) in bone marrow and spleen, and decreases the percentages of B cells in liver. In contrast, the percentages of CD4+CD8+ thymocytes, CD4+CD8- splenocytes, CD4+CD8- liver nonparenchymal cells, CD3+ splenocytes, and CD3+ bone marrow cells were increased. Furthermore, exposure to 5% ethanol increases STAT5 activation in T cells and liver cells while decreases STAT5 activation in NK cells. Taken together, these findings suggest that low levels of ethanol consumption can differentially modulate immune cells in thymus, spleen, bone marrow and liver, which may be due to differential regulation of STAT5 activation by ethanol.

SummaryObjectiveStudy the impact of intra-articular interleukin-1 receptor antagonist (IL-1 ra) treatment on lubricin biosynthesis following anterior cruciate ligament transection (ACLT) in the rat and evaluate the effect of combined IL-1 ra and recombinant human lubricin (rhPRG4) treatments on chondrocyte apoptosis.MethodsACLT was performed in male Lewis rats. Treatments included IL-1 ra or vehicle (n = 36 in each group). IL-1 ra intra-articular dosing was performed on days 1, 3, 5 and 7 following ACLT using Anakinra (150 mg/ml; 40 μl). At 3 and 5 weeks, animals were sacrificed and RNA was isolated. Histological analyses included Safranin O and H&E. Lubricin synovial fluid (SF) lavage concentrations were determined at 5 weeks. ACLT animals were treated with a single injection of vehicle, IL-1 ra (75 mg/ml; 40 μl), rhPRG4 (200 μg/ml; 40 μl), or IL-1 ra + rhPRG4 (75 mg/ml + 200 μg/ml; 40 μl) (n = 6 in each group) on day 7 following ACLT and cartilage was probed for cleaved caspase-3 at 5 weeks.ResultsIL-1 ra treatment improved lubricin expression (P

To evaluate the binding of recombinant human proteoglycan 4 (rhPRG4) to CD44 receptor and its consequences on cytokine-induced synoviocyte proliferation.The binding of rhPRG4 to CD44 and competition with high molecular weight (HMW) hyaluronic acid (HA) was evaluated using a direct enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance. Sialidase A and O-glycosidase digestion of rhPRG4 was performed, and CD44 binding was evaluated using ELISA. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were stimulated with interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) for 48 hours in the presence or absence of rhPRG4 or HMW HA at 20, 40, and 80 μg/ml, and cell proliferation was measured. The contribution of CD44 was assessed by coincubation with a CD44 antibody (IM7). The antiproliferative effect of rhPRG4 was investigated following treatment of PRG4(-/-) mouse synoviocytes with IL-1β or TNFα in the presence or absence of IM7.Recombinant human PRG4 bound CD44 and interfered with the binding of HMW HA to CD44. Removal of sialic acid and O-glycosylations significantly increased CD44 binding by rhPRG4 (P 0.001). Both rhPRG4 and HMW HA at 40 and 80 μg/ml significantly suppressed IL-1β-induced proliferation of RA FLS (P 0.05). Recombinant human PRG4 at 20, 40, and 80 μg/ml significantly suppressed TNFα-induced RA FLS proliferation (P 0.05). CD44 neutralization reversed the effect of rhPRG4 on IL-1β- and TNFα-stimulated RA FLS and the effect of HMW HA on IL-1β-stimulated RA FLS. Recombinant human PRG4 inhibited cytokine-induced proliferation of PRG4(-/-) synoviocytes, which could be prevented by blocking CD44.PRG4 (lubricin) is a novel putative ligand for CD44 and may control synoviocyte overgrowth in inflammatory arthropathies via a CD44-mediated mechanism.

Background: Although the Murine Local Lymph Node Assay (LLNA) is efficient in identifying chemicals with sensitizing potential, there is increasing need for alternative end points. Cinnamaldehyde (CIN) was chosen for evaluation based on its moderate potency and extensive use in fragrance materials. Objectives: The purpose of the present studies is to incorporate some alternative end points, such as phenotypic analysis and cytokine production, into a modified LLNA/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to CIN. Methods: Several nontraditional end points, including the analysis of lymphocyte subpopulations, B7 costimulatory molecule and cytokine messenger RNA (mRNA) expression, and intracellular interferon-γ (IFN-γ) levels, were incorporated into a modified murine local lymph node (LLNA)/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to cinnamaldehyde (CIN). Results: The alternate end points used in these studies support the classification of CIN as a moderately potent sensitizer. Dermal treatment with CIN resulted in an increase in the percentage of B cells in the auricular lymph nodes (ALNs) and expression of the costimulatory molecule, B7-2, on B cells. Lymph node cells also showed increased transforming growth factor-β1, migration-inhibition factor, and mild increases in IFN-γ and interleukin-2 cytokine mRNA expression. Although the increase in IFN-γ mRNA expression did not translate into increased intracellular IFN-γ levels, the absolute number of T cells producing IFN-γ in the ALNs increased. Conversely, the MEST did not classify CIN as a contact allergen. Conclusion: The nontraditional end points used in the LLNA/IA were not as sensitive as the traditional radioisotope method used to assess cell proliferation. However, they may help identify compounds inappropriately classified as sensitizers or nonsensitizers by the LLNA and MEST.

Hair dye components such as pyrogallol and cresol have been shown previously to promote allergic reactions such as rashes, dermal inflammation, irritation and dermatitis. The objective of this study was to determine the contact sensitization potential of pyrogallol (PYR) and 5-amino-o-cresol (AOC) when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the local lymph node assay. For PYR, significant increases in the proliferation of lymph node cells were observed at concentrations of 0.5% (w/v) and higher. For AOC, borderline increases, albeit significant, in auricular lymph node cell proliferation were observed at 5% and 10%. Results from the irritancy assay suggested that PYR, but not AOC, was an irritant. To further delineate whether PYR was primarily an irritant or a contact sensitizer, the mouse ear swelling test (MEST) was conducted. A significant increase in mouse ear thickness was observed at 72 hr following challenge with 0.5% PYR in mice that had been sensitized with 5% PYR. In contrast, no effects were observed in the MEST in mice sensitized and challenged with the highest achievable concentration of AOC (10%). Additional studies examining lymph node subpopulations and CD86 (B7.2) expression by B cells further support the indication that PYR was a sensitizer in BALB/c mice. The results demonstrate that PYR is both a sensitizer and an irritant in female BALB/c mice. However, the contact sensitization potential of AOC is minimal in this strain of mouse.

Osteoarthritis is a complex disease involving the mechanical breakdown of articular cartilage in the presence of altered joint mechanics and chondrocyte death, but the connection between these factors is not well established. Lubricin, a mucinous glycoprotein encoded by the PRG4 gene, provides boundary lubrication in articular joints. Joint friction is elevated and accompanied by accelerated cartilage damage in humans and mice that have genetic deficiency of lubricin. Here, we investigated the relationship between coefficient of friction and chondrocyte death using ex vivo and in vitro measurements of friction and apoptosis. We observed increases in whole-joint friction and cellular apoptosis in lubricin knockout mice compared with wild-type mice. When we used an in vitro bovine explant cartilage-on-cartilage bearing system, we observed a direct correlation between coefficient of friction and chondrocyte apoptosis in the superficial layers of cartilage. In the bovine explant system, the addition of lubricin as a test lubricant significantly lowered the static coefficient of friction and number of apoptotic chondrocytes. These results demonstrate a direct connection between lubricin, boundary lubrication, and cell survival and suggest that supplementation of synovial fluid with lubricin may be an effective treatment to prevent cartilage deterioration in patients with genetic or acquired deficiency of lubricin.

The name of the third author was spelled incorrectly. The correct name is: Shougang Zhuang.

Objective.Symptomatic osteoarthritis (OA) is a common painful disease with limited treatment options. A rising number of patients with OA have been treated with intraarticular injections of hyaluronic acid, including the high-molecular-weight hylan G-F 20, which is injected following arthrocentesis. We investigated the effectiveness of hylan G-F 20 to lower coefficient of friction (COF) and prevent chondrocyte apoptosisin vitro.Methods.A disc-on-disc bovine cartilage bearing was used to measure the static and kinetic COF when lubricated with hylan G-F 20, human synovial fluid (HSF), and phosphate buffered saline (PBS). Following friction testing, we stained paraffin-embedded sections of these cartilage bearings for activated caspase-3, a marker of apoptosis.Results.Bearings lubricated with hylan G-F 20 had kinetic COF values that were similar to bearings lubricated with PBS, but significantly higher than those lubricated with HSF. There were no significant differences in static COF values in bearings lubricated with hylan G-F 20 as compared to PBS or HSF. However, bearings lubricated with HSF had significantly lower static COF values compared to bearings lubricated with PBS. The mean percentage of caspase-3-positive chondrocytes in the superficial and upper intermediate zones of bearings lubricated with hylan G-F 20 was significantly higher compared to that of bearings lubricated with HSF or unloaded controls, but significantly lower than in those lubricated with PBS.Conclusion.These findings indicate that joint lubrication may prevent chondrocyte apoptosis by lowering the COF. Further, removal of synovial fluid prior to hylan G-F 20 injection may be detrimental to cartilage health.

Objective To investigate whether cartilage degeneration is prevented or minimized following intraarticular injections of lubricin derived from human synoviocytes in culture, recombinant human PRG4 (rhPRG4), or human synovial fluid (SF) in a rat model of anterior cruciate ligament (ACL) injury. Methods Unilateral ACL transection (ACLT) was performed in Lewis rats (n = 45). Nine animals were left untreated. The remaining rats were given intraarticular injections (50 μl/injection) of either phosphate buffered saline (PBS) (n = 9), human synoviocyte lubricin (200 μg/ml; n = 9), rhPRG4 (200 μg/ml; n = 9), or human SF lubricin (200 μg/ml; n = 9) twice weekly beginning on day 7 after injury. Joints were harvested on day 32 after injury. Histologic analysis was performed using Safranin O–fast green staining, and articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)–modified Mankin criteria. Histologic specimens were immunoprobed for lubricin and sulfated glycosaminoglycans. A 24-hour urine collection was performed on days 17 and 29 postinjury, and urinary C-terminal telopeptide of type II collagen (CTX-II) levels were measured. Results Treatment with human synoviocyte lubricin resulted in significantly lower OARSI scores for cartilage degeneration compared with no treatment or PBS treatment (P < 0.05). Increased immunostaining for lubricin in the superficial zone chondrocytes and on the surface of cartilage was observed in lubricin-treated, but not untreated or PBS-treated, joints. On day 17, urinary CTX-II levels in human synoviocyte lubricin– and human SF lubricin–treated animals were significantly lower than those in untreated animals (P = 0.005 and P = 0.002, respectively) and in PBS-treated animals (P = 0.002 and P < 0.001, respectively). Conclusion After treatment with any of the 3 types of lubricin evaluated in this study, a reduction in cartilage damage following ACLT was evident, combined with a reduction in type II collagen degradation. Our findings indicate that intraarticular lubricin injection following an ACL injury may be beneficial in retarding the degeneration of cartilage and the development of posttraumatic OA.

Objectives Recent evidence has demonstrated the importance of histone deacetylases (HDAC) in the control of hypertrophic responses in the heart. However, it remains unknown whether inhibition of HDACs plays a role in myocardial ischemia and reperfusion (I/R) injury. We hypothesize that HDAC inhibition triggers preconditioning-like effects against I/R injury. Methods and results Isolated mouse hearts were perfused with 3 cycles of 5-minute infusion and 5-minute washout of 50 nM of trichostatin A (TSA), a potent inhibitor of HDACs to mimic early pharmacologic preconditioning. This was followed by 30 min of ischemia and 30 min of reperfusion. In addition, mice were treated with saline or TSA (0.1 mg/kg, i.p.) to investigate delayed pharmacologic preconditioning. Twenty-four hours later, the hearts were subjected to I/R. Ventricular function and infarct size were measured, and HDAC 3, 4 and 5 were assessed by Western blot and immunofluorescence. HDAC and p38 mitogen-activated protein kinase activities were determined. TSA produced marked improvements in post-ischemic ventricular function recovery and a reduction in infarct size in both early and delayed preconditioning. Cardioprotection elicited by TSA was abrogated by SB203580, an inhibitor of p38. HDAC 3, 4 and 5 proteins were detected in mouse myocardium. TSA treatments resulted in a significant inhibition of HDAC activity. HDAC inhibition caused a dramatic increase in phosphorylation of p38 and p38 activity. Notably, HDAC inhibition also resulted in remarkable acetylation of p38 at lysine residues. Conclusion These results suggest that inhibition of HDACs triggers pharmacologic preconditioning to protect the ischemic heart, which involves p38 activation.

Previously, we have reported that thalidomide can modulate the immune responses in female B6C3F1 mice. Furthermore, thalidomide immunomodulation increased primary host resistance to intravenously infected Listeria monocytogenes. The present study was intended to evaluate the mechanisms underlying the enhanced host resistance to L. monocytogenes by focusing on the neutrophils. Female B6C3F1 mice were treated intraperitoneally with thalidomide (100 mg/kg) for 15 days. Exposure to thalidomide increased the numbers of neutrophils in the spleens and livers of L. monocytogenes-infected mice when compared to the L. monocytogenes-infected control mice. Additionally, the percentage of neutrophils was also significantly increased after Thd treatment in L. monocytogenes-infected mice. Further studies using antibodies to deplete corresponding cells indicated that thalidomide-mediated increase in primary host resistance (both the moribundity and colony counts in the liver and spleen) to L. monocytogenes infection was due to its effect on neutrophils but not CD8+ T cells or NK cells. Finally, Thd exposure also increased host resistance to secondary host resistance to L. monocytogenes infection, and depletion of neutrophils abolished the protective effect. In conclusion, thalidomide enhanced host resistance to both primary and secondary L. monocytogenes infections by a neutrophil-related mechanism in female B6C3F1 mice.

Previously, we have reported that thalidomide (Thd) treatment can modulate the immune responses in female B6C3F1 mice. The present study was designed to evaluate whether or not these immunomodulatory responses were of sufficient magnitude to alter host resistances in a number of pathogen and tumor models. B6C3F1 mice were treated intraperitoneally with Thd (30–150 mg/kg) for 14 or 28 days, then inoculated with either Plasmodium yeolii, PYB6 fibrosarcoma tumor cells, B16F10 melanoma tumor cells, Listeria monocytogenes, or Streptococcus pneumoniae. Significant dose-dependent protection against B16F10 and L. monocytogenes was observed in mice that were treated with Thd. Furthermore, time course study using bacterial colony-forming units per spleen and liver as the endpoints indicated that the protective effect of Thd on host resistance to L. monocytogenes was time-dependent. In contrast, Thd treatment did not affect host resistance to P. yeolii, S. pneumoniae and PYB6 tumor. Additionally, the effect of Thd on the phagocytic function of the mononuclear phagocyte system (MPS) was evaluated following intravenous injection of 51Cr-labeled sRBCs. The overall phagocytic activity of MPS was not significantly altered by Thd treatment. In conclusion, these results demonstrate that Thd immunomodulation altered host resistance to B16F10 and L. monocytogenes; and selective modulation of Thd on the immune system may be responsible for the pathogen or tumor-specific effect of this compound.

The isoflavone genistein (4,7,4'-trihydroxyisoflavone) is a phytoestrogen found in high levels in soy products that has been associated with decreased incidences of breast and prostate cancers. The potential effects of genistein on the immune system were evaluated in adult female B6C3F1 mice. Groups of mice were exposed to vehicle or genistein by gavage for 28 d. The doses of genistein used were 2, 6 and 20 mg/kg body. Consistent with the chemopreventive effect of genistein, exposure to this compound significantly increased host resistance to B16F10 tumor as reflected by a decrease in the number of lung tumor nodules after tumor cell injection at the middle and high dose levels. Inhibition of B16F10 tumor formation was not due to a direct effect of serum genistein and/or its metabolites on the proliferation of B16F10 tumor cells. When innate and acquired immune responses were evaluated, a dose-related increase of cytotoxic T-cell activity was observed in genistein-treated mice with significant changes observed at the middle and high dose levels. Furthermore, in vitro interleukin (IL)-2-stimulated natural killer (NK) cell activity was significantly enhanced in the high genistein dose group, although the basal NK cell activity was not affected. Although no affect on the mixed lymphocyte responses and anti-CD3 antibody-mediated splenocyte proliferation was observed, exposure to genistein significantly increased basal splenocyte proliferation. Exposure to genistein did not alter the activity of the mononuclear phagocyte system and the cytotoxic/cytostatic function of thioglycollate-recruited peritoneal cells on B16F10 tumor cells. Finally, exposure to genistein did not produce biologically meaningful changes in spleen immunoglobulin (Ig)M and IgG antibody-forming cell responses. In conclusion, genistein enhanced host resistance as evaluated in the B16F10 tumor model, which may be related to the increases in the activities of cytotoxic T cells and NK cells.

We and others have demonstrated that HDAC inhibition protects the heart against myocardial injury. It is known that Akt-1 and MAP kinase play an essential role in modulation of myocardial protection and cardiac preconditioning. Our recent observations have shown that Akt-1 was activated in post-myocardial infarction following HDAC inhibition. However, it remains unknown whether MKK3 and Akt-1 are involved in HDAC inhibition-induced myocardial protection in acute myocardial ischemia and reperfusion injury. We sought to investigate whether the genetic disruption of Akt-1 and MKK3 eliminate cardioprotection elicited by HDAC inhibition and whether Akt-1 is associated with MKK3 to ultimately achieve protective effects. Adult wild type and MKK3⁻/⁻, Akt-1⁻/⁻ mice received intraperitoneal injections of trichostatin A (0.1 mg/kg), a potent inhibitor of HDACs. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after twenty four hours to elicit pharmacologic preconditioning. Left ventricular function was measured, and infarct size was determined. Acetylation and phosphorylation of MKK3 were detected and disruption of Akt-1 abolished both acetylation and phosphorylation of MKK3. HDAC inhibition produces an improvement in left ventricular functional recovery, but these effects were abrogated by disruption of either Akt-1 or MKK3. Disruption of Akt-1 or MKK3 abolished the effects of HDAC inhibition-induced reduction of infarct size. Trichostatin A treatment resulted in an increase in MKK3 phosphorylation or acetylation in myocardium. Taken together, these results indicate that stimulation of the MKK3 and Akt-1 pathway is a novel approach to HDAC inhibition -induced cardioprotection.

SummaryObjectiveTo evaluate the impact of forced joint exercise following acute knee injury on lubricin metabolism and its relationship to cartilage degeneration and to assess chondroprotection of a single-dose purified human lubricin injection in exercised injured joints.MethodsAnterior cruciate ligament transection (ACLT) was performed in rats with six experimental groups; 3-week post-ACLT, 3-week post-ACLT + exercise, 5-week post-ACLT, 5-week post-ACLT + exercise, and 5-week post-ACLT + exercise treated with intra-articular phosphate buffered saline (PBS) or lubricin. Joint exercise was achieved using a rotating cylinder at a speed of 6 rpm for 30 min daily, 5 days a week starting 1 week following surgery. Cartilage lubricin expression in injured joints was determined. Histological analyses included Safranin O/Fast Green, activated caspase-3, and lubricin mRNA in-situ hybridization. Assessment of cartilage damage was performed by osteoarthritis research society international (OARSI) modified Mankin scoring and urinary CTXII (uCTXII) levels.ResultsAt 3 weeks, lubricin expression in exercised ACLT joints was significantly (P

Lubricin, a mucinous glycoprotein secreted by synoviocytes and chondrocytes plays an important role in reducing the coefficient of friction in mammalian joints. Elevated cartilage surface friction is thought to cause chondrocyte loss; however, its quantification and methodological approaches have not been reported. We adapted a stereological method and incorporated vital cell staining to assess cellular loss in superficial and upper intermediate zones in lubricin deficient mouse cartilage. The femoral condyle cartilage of the intact knees from lubricin wild type (Prg4 +/+), heterozygote (Prg4 +/-), and knockout (Prg4 -/-) mice was imaged using fluorescein diacetate (FDA), propidium iodide (PI), and Hoechst staining, and confocal microscopy. Three dimensional reconstructions of confocal images to a depth of 14 μm were analyzed using Matlab to determine the volume fraction occupied by chondrocytes in cartilage of both medial and lateral femoral condyles. Living chondrocyte volume fraction was defined as FDA stained chondrocyte volume/total volume of superficial + upper intermediate zone. Living and dead (total) chondrocyte volume fraction was defined as FDA + PI stained chondrocyte volume/total volume of superficial + upper intermediate zone. MicroCT provided an orthogonal measure of cartilage thickness. Immunohistology for activated caspase-3 and TUNEL staining were performed to evaluate the presence of apoptotic chondrocytes in Prg4 mutant mice. Living chondrocyte volume fraction of the medial femoral condyle was significantly lower in Prg4 -/- mice compared to Prg4 +/+ (p = 0.002) and Prg4 +/- (p = 0.002) littermates. There was no significant difference in medial condyle chondrocyte volume fraction between Prg4 +/+ and Prg4 +/- mice (p = 0.82). No significant differences were observed for the chondrocyte volume fraction for the lateral condyle (p > 0.26). Cartilage thickness increased in the medial condyle for Prg4 -/- mice compared to Prg4 +/+ (p = 0.02) and Prg4 +/- (p = 0.03) littermates, and the lateral condyle for Prg4 -/- mice compared to Prg4 +/+ (p 0.05 for all comparisons). Increased Caspase-3 activation is observed in Prg4 deficient mice compared to Prg4 sufficient littermates. Absence of Prg4 induces loss of chondrocytes in the superficial and upper intermediate zone of mouse cartilage that is quantifiable by a novel image processing technique.