E A Mickler

Indiana University-Purdue University Indianapolis, Indianapolis, IN, United States

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Publications (22)95.25 Total impact

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    ABSTRACT: The epithelial complement inhibitory proteins (CIPs) cluster of differentiation 46 and 55 (CD46 and CD55) regulate circulating immune complex-mediated complement activation in idiopathic pulmonary fibrosis (IPF). Our previous studies demonstrated that IL-17A mediates epithelial injury via transforming growth factor β1 (TGF-β1) and down-regulates CIPs. In the current study, we examined the mechanistic role of TGF-β1 in complement activation-mediated airway epithelial injury in IPF pathogenesis. We observed lower epithelial CIP expression in IPF lungs compared to normal lungs, associated with elevated levels of complement component 3a and 5a (C3a and C5a), locally and systemically. In normal primary human small airway epithelial cells (SAECs) treated with TGF-β1 (10 ng/ml), C3a, or C5a (100 nM), we observed loss of CIPs and increased poly(ADP-ribose) polymerase (PARP) activation [also observed with RNA interference (RNAi) of CD46/CD55]. TGF-β1-mediated loss of CIPs and Snail induction [SNAI1; a transcriptional repressor of E-cadherin (E-CAD)] was blocked by inhibiting mitogen-activated protein kinase (p38MAPK; SB203580) and RNAi silencing of SNAI1. C3a- and C5a-mediated loss of CIPs was also blocked by p38MAPK inhibition. While C3a upregulated TGFb transcripts, both C3a and C5a down-regulated SMAD7 (negative regulator of TGF-β), and whereas TGF-β1 induced C3a/C5a receptor (C3aR/C5aR) expression, pharmacologic C3aR/C5aR inhibition protected against C3a-/C5a-mediated loss of CIPs. Taken together, our results suggest that epithelial injury in IPF can be collectively amplified as a result of TGF-β1-induced loss of CIPs leading to complement activation that down-regulates CIPs and induces TGF-β1 expression.-Gu, H., Mickler, E. A., Cummings, O. W, Sandusky, G. E., Weber, D. J., Gracon, A., Woodruff, T., Wilkes, D. S., Vittal, R. Crosstalk between TGF-β1 and complement activation augments epithelial injury in pulmonary fibrosis.
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 06/2014;
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    ABSTRACT: Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17-regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1-related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1-related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17-mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.
    The Journal of Immunology 09/2013; · 5.52 Impact Factor
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    ABSTRACT: Mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2, MK2), a serine/threonine kinase downstream of p38MAPK, has been implicated in inflammation and fibrosis. Compared to pathologically normal lung, there are significantly higher levels of activated MK2 in idiopathic pulmonary fibrosis (IPF) patient lung biopsies. Expression is localized to fibroblasts and epithelial cells. In the murine bleomycin model of pulmonary fibrosis, we observed robust activated MK2 expression at day 7 (pre-fibrotic stage) and day 14 (post-fibrotic stage). To determine the effect of MK2 inhibition in the post-inflammatory/pre-fibrotic and post-fibrotic stages, C57BL/6 mice received intra-tracheal bleomycin instillation (0.025U; day 0), followed by phosphate buffered saline (PBS) or MK2 inhibitor (MK2i; MMI-0100; 37.5 μg/kg), administered via either local (nebulized) or systemic (intra-peritoneal) routes. MK2i or PBS was dosed daily for 14 days subsequent to bleomycin injury, beginning at either day 7 or day 14. Regardless of mode of administration or stage of intervention, MK2i significantly abrogated collagen deposition, myofibroblast differentiation and activated MK2 expression. MK2i also decreased circulating TNF-α and IL-6 levels and modulated local mRNA expression of pro-fibrotic cytokine il-1β, matrix-related genes-col1a2, col3a1, lox, and TGF-beta family members including smad3, serpine1 (pai1) and smad6/7. In vitro, MK2i dose-dependently attenuated total MK2, myofibroblast differentiation, secretion of collagen type I, fibronectin, and FAK activation, while activated MK2 was attenuated at higher doses. Peptide mediated inhibition of MK2 impacts both inflammatory and fibrotic responses and thus may be a promising therapeutic target for IPF.
    American Journal of Respiratory Cell and Molecular Biology 03/2013; · 4.15 Impact Factor
  • American Journal of Respiratory and Critical Care Medicine 02/2013; 187(4):454-7. · 11.04 Impact Factor
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    ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models. Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses. Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local Il-17a transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (Tgf-β, Il-1β, Pdgfb), matrix (Acta2, Col1a2, Col3a1, Lox, Itgb1/6, Itga2/3) and members of the TGF-β superfamily (Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb). Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- β-related signaling pathways.
    PLoS ONE 01/2013; 8(10):e76451. · 3.53 Impact Factor
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    ABSTRACT: Obliterative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death post lung transplantation. Type V collagen [col(V)] overexpression, and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) alpha 1 chains [(α1 (V)] in normal airway epithelial cells in vitro, and detected α1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-β mRNA expression and SMAD3 activation, while downregulating SMAD7 expression in vitro. Pharmacologic inhibition of TGF-βRI tyrosine kinase, p38MAPK, or FAK prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-β mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-β leading to induction of col(V) and associated epithelial repair, - thus providing one possible link between autoimmunity and OB post lung-transplantation.
    AJP Lung Cellular and Molecular Physiology 12/2012; · 3.52 Impact Factor
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    ABSTRACT: Obliterative bronchiolitis (OB) is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining OB immunopathogenesis. In the current study, lungs from C57BL/10 H-2(b) mice that are MHC compatible, but minor histocompatability antigen incompatible, were transplanted into C57BL/6 mice. Histological features and cytokine profiles of OB were assessed. Moderate rejection (grade A3) developed by day 14, with evidence of OB at that time point. At 21 days, OB was present in 55% of grafts and moderate to severe rejection (grade A3-A4) was present in all mice. At 28 days, OB was present in 44% of mice and severe rejection (grade A4) was present in all. IL-17A, but not IL-17F, splenic mRNA transcripts and serum protein levels were increased only in mice that developed OB, whereas IL-10 transcripts and protein were increased only in non-OB mice. Neutralizing IL-17 prevented OB, down regulated acute rejection, and upregulated systemic IL-10. Collectively, these data show that transplantation of minor histoincompatible lungs from C57BL/10 mice into C57BL/6 mice results in a highly reproducible preclinical model of OB. In addition, these data indicate that neutralizing IL-17A or augmenting IL-10 could be therapeutic interventions to prevent OB.
    American Journal of Transplantation 05/2011; 11(5):911-22. · 6.19 Impact Factor
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    ABSTRACT: Primary graft dysfunction (PGD) is a major complication following lung transplantation. We reported that anti-type V collagen (col(V)) T cell immunity was strongly associated with PGD. However, the role of preformed anti-col(V) Abs and their potential target in PGD are unknown. Col(V) immune serum, purified IgG or B cells from col(V) immune rats were transferred to WKY rat lung isograft recipients followed by assessments of lung pathology, cytokines, and PaO(2)/FiO(2), an index of lung dysfunction in PGD. Immune serum, purified IgG, and B cells all induced pathology consistent with PGD within 4 days posttransfer; up-regulated IFN-gamma, TNF-alpha, and IL-1beta locally; and induced significant reductions in PaO(2)/FiO(2). Depleting anti-col(V) Abs before transfer demonstrated that IgG2c was a major subtype mediating injury. Confocal microscopy revealed strong apical col(V) expression on lung epithelial, but not endothelial cells; which was consistent with the ability of col(V) immune serum to induce complement-dependent cytotoxicity only in the epithelial cells. Examination of plasma from patients with or without PGD revealed that higher levels of preformed anti-col(V) Abs were strongly associated with PGD development. This study demonstrates a major role for anti-col(V) humoral immunity in PGD, and identifies the airway epithelium as a target in PGD.
    The Journal of Immunology 11/2008; 181(8):5738-47. · 5.52 Impact Factor
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    ABSTRACT: Immunity to type V collagen [col(V)] contributes to lung transplant rejection. Matrix metalloproteases (MMPs), which are induced by transplant-related ischemia-reperfusion injury (IRI), could expose col(V) and regulate local IRI-induced inflammation. To test the hypothesis that MMPs induce col(V) exposure and inflammation, Wistar-Kyoto rats were treated with the MMP inhibitor, COL-3, before inducing lung IRI without transplantation, and in parallel studies, Wistar-Kyoto lung donor and recipients were treated with COL-3 pre- and postisograft lung transplantation. Ischemia-reperfusion injury induced growth-related oncogene/CINC-1-dependent neutrophil influx, and up-regulated tumor necrosis factor-alpha. MMP2 and MMP9, induced at 4 and 24 hr after IRI, respectively, were associated with detection of antigenic col(V) in bronchoalveolar lavage and lung interstitium because of MMP-mediated matrix degradation. MMP-inhibitor treatment significantly reduced polymorphonuclear leukocytes, growth-related oncogene/CINC-1, and tumor necrosis factor-alpha; abrogated MMP-9 expression; and resulted in lower levels of antigenic col(V) in bronchoalveolar lavage. In the lung transplant model, inhibiting MMPs in the donor before lung harvest and in the recipient after lung transplantation resulted in improved oxygenation and diminished polymorphonuclear leukocyte influx into the isograft. MMP inhibition may be a potential therapy to prevent release of antigenic col(V) and ameliorate IRI in the transplant recipient.
    Transplantation 03/2008; 85(3):417-26. · 3.78 Impact Factor
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    ABSTRACT: Parenchymal disease in the allograft lung is associated with interstitial remodeling believed to be mediated by matrix metalloproteinases (MMPs). Recent studies suggest high levels of MMP-9 are associated with bronchiolitis obliterans syndrome (BOS) in lung transplant recipients. Since BOS occurs late in the posttransplant period and may be preceded by episodes of acute rejection or infection, which are associated with interstitial remodeling, we examined MMP profiles in allograft bronchoalveolar lavage (BAL) fluid in the early posttransplant period (preceding BOS). Gelatin zymography, protein array analysis and specific ELISA on BAL fluids from transplanted lungs indicated that MMP-8, MMP-9 and TIMP-1 were strongly expressed in allograft BAL fluid from stable patients, or those with infection or rejection compared to BAL fluid from normal volunteers. Elevated expression of MMP-8, MMP-9 and TIMP-1 occurred early, and was sustained for the 3.2 years covered in this study. Elevations of MMP-8, MMP-9 and TIMP-1 in the first 2 years posttransplant appear to be associated with lung transplantation itself, and not infection or rejection. These data suggest that ongoing and clinically silent MMP activity could perpetuate progressive disease in the allograft lung.
    American Journal of Transplantation 08/2007; 7(7):1856-61. · 6.19 Impact Factor
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    ABSTRACT: Upregulation of matrix metalloproteinases (MMPs) has been associated with chronic lung allograft rejection known as bronchiolitis obliterans syndrome. It has been suggested that MMP inhibition could prevent the rejection response. However, the effect of MMP inhibition on lung allograft rejection has not been reported. Utilizing a rat model of lung transplantation, tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were overexpressed by gene therapy in F344 rat lung allografts prior to transplantation into WKY recipient rats. Separately, WKY rats that received F344 lung allografts were treated systemically with COL-3, a global MMP inhibitor. TIMP-1 and TIMP-2 had differential effects on delayed type hypersensitivity (DTH) responses to donor antigens and type V collagen, an autoantigen involved in the rejection response. Neither TIMP-1 or TIMP-2 affected the onset of rejection pathology. COL-3 suppressed DTH responses to donor antigens and type V collagen, abrogated local production of tumor necrosis factor-alpha, and interleukin-1beta. Although it did not prevent rejection pathology, COL-3 (30 mg/kg) induced intragraft B cell hyperplasia suggestive of posttransplant proliferative disorder (PTLD). These data identify a complex role for MMPs and TIMPs in the immunopathogenesis of lung allograft rejection, and indicate their effects are not limited to matrix remodeling.
    Transplantation 04/2007; 83(6):799-808. · 3.78 Impact Factor
  • Matrix Biology. 01/2006; 25.
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    ABSTRACT: To determine if intraarticular (IA) injection of hyaluronan (HA) into the canine knee after anterior cruciate ligament transection (ACLT) alters the progression of osteoarthritis (OA) and the perception of pain in this model. OA was induced in 30 adult dogs of mixed breed by ACLT. The dogs were divided into 3 groups and given 5 weekly IA injections into the unstable knee during Weeks 1-5 and 13-17. The prophylactic treatment group received HA in the first series and saline during the second series. The therapeutic group received saline in the first series and HA in the second series. The control group received saline during both injection series. The progression of joint damage of OA was evaluated by arthroscopy 12 weeks after ACLT and by gross examination 32 weeks after ACLT. Histologic and biochemical changes of OA were evaluated. Loading of the unstable limb during gait was determined by force-plate analysis before surgery, after each series of injections, and the week before euthanasia. Arthroscopic examination 12 weeks after ACLT revealed OA changes in the cruciate-deficient knees. Chondropathy scores ranged from 0 to 8 (possible range 0-65). The mean chondropathy score was 2.5 +/- 1.3 (mean +/- SD) for the controls, 2.5 +/- 2.5 for the therapeutic group, and 2.1 +/- 1.3 for the prophylactic group. At the termination of the experiment 32 weeks after ACLT, the gross chondropathy scores were 14.0 +/- 5.2 for controls, 17.6 +/- 6.8 for the therapeutic group, and 13.3 +/- 5.0 for the prophylactic group. There were no significant differences among the means of the gross scores, the histologic scores, or biochemical composition of articular cartilage. The peak vertical ground reaction force (VGRF) generated by the unstable limb was reduced by ~60% after ACLT, and slowly increased to ~75% of the baseline value over the 32 weeks after ACLT. HA injection had no effect on the VGRF or on the pathologic changes of OA. Intraarticular HA injection did not alter the progression of OA in the cruciate-deficient canine knee or alter the loading of the unstable limb.
    The Journal of Rheumatology 03/2005; 32(2):325-34. · 3.26 Impact Factor
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    ABSTRACT: To examine the relationship between the severity of cartilage damage and the severity of meniscus damage after transection of the anterior cruciate ligament (ACLT) in adult dogs. Data were obtained from 40 dogs which underwent ACLT and from three additional sham-operated dogs that were subjected to arthrotomy but not ligament transection. Joint pathology was analysed 12, 24 or 32 weeks after surgery. The severity of damage to the articular cartilage on the femoral condyle and tibial plateau was graded with a scoring system based on that of the Sociètè Française d'Arthroscopie and meniscus damage was graded on a 0-4 scale. No damage to the meniscus or articular cartilage was observed 12 weeks after surgery in the dogs subjected only to arthrotomy. In contrast, tears of the medial meniscus were observed in two of 10 (20%) dogs examined 12 weeks after ACLT. The incidence of severe tears increased to 86% and 84% after 24 weeks and 32 weeks, respectively. Damage to the lateral meniscus was mild, with only 7.5% of all dogs with a cruciate-deficient knee having a bucket handle or complete tear. Most of the unstable knees exhibited ulceration of the articular cartilage of the femoral condyles and tibial plateaus 12 weeks (mean chondropathy score+/-standard deviation 11.9+/-8.5, N=10), 24 weeks (7.9+/-5.0, N=7), and 32 weeks (7.1+/-5.5, N=23) after ACLT. The mean chondropathy scores for the tibial plateaus were similar to those for the femoral condyles. No correlation was apparent between the severity of cartilage damage and of meniscus damage for either joint surface. Damage to the medial meniscus is a consistent feature of the pathology which develops in the canine knee after ACLT, but the severity of cartilage damage is not correlated with the severity of meniscal damage.
    Osteoarthritis and Cartilage 05/2002; 10(4):321-6. · 4.26 Impact Factor
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    G N Smith, E A Mickler, S L Myers, K D Brandt
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    ABSTRACT: To determine how the quantity and molecular weight of synovial fluid hyaluronan (HA) within the synovial fluid (SF) of osteoarthritis (OA) joints is affected by intraarticular injection of HA. Dogs in which OA was induced by transection of the anterior cruciate ligament received 5 weekly injections of HA (1.5 x 10(6) Da) in saline (10 mg/0.67 ml) or an equal volume of saline into the operated knee, beginning the day after surgery. Immediately before each injection, SF was aspirated and the volume of SF and the concentration of HA was measured (uronic acid), and the molecular weight of the HA in each sample was estimated by electrophoresis in agarose. The volume of SF in the unstable knee increased after surgery, and the molecular weight decreased from approximately 2.5 x 10(6) Da to approximately 2 x 10(6) Da. Injection of HA did not affect the volume of SF or average molecular weight of HA in samples obtained immediately before each injection or at the end of the experiment, 12 weeks after surgery. The SF HA concentration fell from a baseline value of 2.3 +/- 0.1 mg/ml to 1.1 +/- 0.2 mg/ml the day after surgery and remained low throughout the course of injections. The HA concentration 12 weeks after surgery in the HA injected knees was approximately 40% lower than the preoperative value, although it increased slightly relative to saline injected knees (1.4 +/- 0.3 vs 1.1 +/- 0.01 mg/ml, respectively; p = 0.04). Intraarticular injection of HA did not alter the volume of SF or molecular weight of HA in SF of OA canine knees, nor did it restore the HA concentration to that of normal canine SF.
    The Journal of Rheumatology 07/2001; 28(6):1341-6. · 3.26 Impact Factor
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    G N Smith, E A Mickler, K A Hasty, K D Brandt
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    ABSTRACT: To investigate the inhibition of matrix metalloproteinase 1 (MMP-1), MMP-8, and MMP-13 by doxycycline, and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases. Recombinant human MMP-1 (collagenase 1), MMP-8 (collagenase 2), and MMP-13 (collagenase 3), truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain, and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen, a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition. The activity of MMP-13 and MMP-8 against type II collagen was inhibited by 50-60% by 30 microM doxycycline, while that of MMP-1 was inhibited only 18% by 50 microM doxycycline. In contrast, in experiments with the peptolide substrate, neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 microM. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 microM doxycycline, while MMP-1 was slightly inhibited (14%) by 90 microM doxycycline. For MMP-8, inhibition was reversible upon dilution and was independent of the order in which the reagents were added. Kinetic analysis of the inhibition constant (K(i)) of MMP-8 (K(i) = 36 microM) and truncated MMP-8 (K(i) = 77 microM) indicated that inhibition was noncompetitive. Significant inhibition of MMP-13 and MMP-8 activity against collagen occurred in vitro at concentrations that were near the concentrations achieved in serum after oral dosing. Studies with truncated enzymes and 2 substrates suggest that doxycycline disrupts the conformation of the hemopexin-like domain of MMP-13 and the catalytic domain of MMP-8.
    Arthritis & Rheumatology 07/1999; 42(6):1140-6. · 7.48 Impact Factor
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    ABSTRACT: To determine if diacerhein protects against the early stages of joint damage in a canine model of osteoarthritis (OA). OA was induced in 20 adult mongrel dogs by transection of the anterior cruciate ligament of the left knee. Beginning the day after surgery, dogs in the active treatment group were dosed twice a day with capsules of diacerhein, providing a total daily dose of 40 mg/kg, for 32 weeks. Dogs in the control group received placebo capsules on the same schedule. Pathology in the unstable knee was assessed arthroscopically 16 weeks after surgery and by direct observation when the dogs were killed 32 weeks after surgery. The severity of gross joint pathology was recorded, and samples of the medial femoral condyle cartilage and the synovial tissue adjacent to the central portion of the medial meniscus were collected for histologic evaluation. Water content and uronic acid concentration of the articular cartilage from the femoral condyle were determined, and collagenolytic activity in extracts of cartilage pooled from the medial and lateral tibial plateaus was assayed against 14C-labeled collagen fibers. Diacerhein treatment slowed the progression of OA, as measured by grading of gross changes in the unstable knee at arthroscopy 16 weeks after cruciate ligament transection (P = 0.04) and at the time the animals were killed, 32 weeks after surgery (P = 0.05). However, 32 weeks after ACL transection, the mean proteoglycan concentration and water content of the OA cartilage and the level of collagenolytic activity in extracts of the cartilage were not significantly different in the diacerhein treatment group than in the placebo treatment group. Diacerhein treatment significantly reduced the severity of morphologic changes of OA compared with placebo. These findings support the view that diacerhein may be a disease-modifying drug for OA.
    Arthritis & Rheumatology 04/1999; 42(3):545-54. · 7.48 Impact Factor
  • G N Smith, S L Myers, K D Brandt, E A Mickler
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    ABSTRACT: To determine if intraarticular injections of hyaluronan (HA) protect against the early stages of joint damage in a canine model of osteoarthritis (OA). OA was induced in adult mongrel dogs by transection of the anterior cruciate ligament of the left knee. One group of dogs (n=7) was treated with 5 weekly injections of HA (MW 1,500,000) into the operated knee beginning 1 day after ligament transection. The control group (n=6) was injected with saline on the same schedule. Twelve weeks after surgery, all dogs were killed, the severity of pathologic changes of OA was graded, and composition of the cartilage and extent of aggregation of proteoglycans (PGs) synthesized in vitro by cartilage slices were determined. All dogs showed gross morphologic changes typical of OA in the unstable knee. The severity of joint pathology in HA-treated dogs was comparable with that in the saline-injected controls. In OA cartilage from the saline-treated group, the mean uronic acid concentration was 30-60% greater than that in the contralateral knee. In sharp contrast, the uronic acid concentration in OA cartilage from the HA-treated dogs was 10-30% lower than that in cartilage from the contralateral knee (P=0.02 and P=0.03, respectively, for samples from the medial and lateral femoral condyle). The extent of aggregation of PG synthesized in vitro by cartilage from HA-injected animals was similar to that synthesized by cartilage from the saline-injected dogs. In this canine model of OA, the series of intraarticular injections of HA did not alter development of osteophytosis or fibrillation. However, the PG concentration of cartilage in the OA knee was significantly reduced by this treatment, suggesting that HA therapy might adversely affect the biomechanical properties of the cartilage.
    Arthritis & Rheumatology 07/1998; 41(6):976-85. · 7.48 Impact Factor
  • G N Smith, K D Brandt, E A Mickler, K A Hasty
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    ABSTRACT: To examine, as part of an evaluation of the role of matrix metalloproteinase (MMP) inhibition in the amelioration of cartilage damage by doxycycline, the effect of pH on the inhibition of activity and reduction in stability of recombinant human neutrophil collagenase (rhMMP-8) by doxycycline in vitro. After activation with trypsin, rhMMP-8 was assayed using a peptolide substrate and a colorimetric assay. The rate of hydrolysis in the presence and absence of 30 microM doxycycline was measured over a pH range of 6.5-7.9. The molecular weight changes that accompanied activation of the proenzyme by acetylphenylmercuric acetate (APMA) in the presence and absence of doxycycline at pH 6.9 and 7.5 were studied by Western blotting. At pH values above 7.1, doxycycline inhibited the activity of the enzyme. At pH values below 7.1, no inhibition was observed. When doxycycline was present during activation with APMA at pH 7.5, significant amounts of small (< 30 kDa) fragments were generated. In contrast, when doxycycline was present during activation with APMA at pH 6.9, no small fragments were detected. The ability of doxycycline to inhibit matrix rhMMP-8 activity or to promote its degradation is lost at pH values lower than 7. Although relatively high pH values may exist in adult articular in some pathological situations, at lower pH the effect of doxycycline on proenzyme levels in the extracellular matrix may be due to an effect on the regulation of synthesis of the proenzyme, rather than to direct inhibition of the active enzyme or reduction in the level of enzyme by proteolysis.
    The Journal of Rheumatology 09/1997; 24(9):1769-73. · 3.26 Impact Factor
  • Gerald N. Smith, Marjorie Albrecht, Elizabeth A. Mickler
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    ABSTRACT: The ability of misoprostol to reverse the deleterious changes induced in cartilage by sodium salicylate was tested using osteoarthritic canine and chondrocytes. Adult mongrel dogs were subjected to anterior cruciate ligament transection and dosed with either misoprostol, salicylate, or misoprostol plus salicylate. No significant differences were noted among the three groups in either gross and histological changes or general biochemical changes. This approach was abandoned since the levels of misoprostol attained by oral dosing were much lower than those required for domonstration of misoprostol effects in vitro. Next, chondrocytes were isolated from the osteoarthritic and contralateral knee joint cartilage of dogs 12 weeks after anterior cruciate ligament transection and cultured in alginate beads. The cultures were incubated with (3)H-proline and both the genetic types of collagen synthesized and the net synthesis of (3)H-hydroxyproline were determined. No drug or combination of drugs affected the genetic types of collagen synthesized. Total protein and collagen synthesis by chondrocytes was reduced in the presence of salicylate and increased in the presence of misoprostol. When osteoarthritic chondrocytes were incubated with both agents, the salicylate effect was reversed by misoprostol. Collagen synthesis by the chondrocytes from the contralateral knee was also suppressed by salicylate, but addition of misoprostol failed to restore synthesis. In summary, misoprostol, even at very high doses, has limited chondroprotective activity in canine cartilage, as judged from collagen synthetic activity.
    American journal of therapeutics 02/1996; 3(1):17-20. · 1.29 Impact Factor

Publication Stats

368 Citations
95.25 Total Impact Points


  • 1991–2013
    • Indiana University-Purdue University Indianapolis
      • • Department of Medicine
      • • Center for Immunobiology
      • • Division of Rheumatology
      Indianapolis, IN, United States
  • 2011
    • Xi'an Jiaotong University
      • Department of General Surgery
      Xi’an, Shaanxi Sheng, China