Physiopathology of necrobiotic xanthogranuloma with monoclonal gammopathy.

RATIONALE: Xanthomatosis associated with monoclonal gammopathy includes hyperlipidaemic xanthoma (HX), normolipidaemic xanthoma (NX) and necrobiotic xanthogranuloma (NXG). All three pathologies are characterized by skin or visceral lesions related to cholesterol accumulation, monoclonal immunoglobulin (MIg) and hypocomplementemia. The pathophysiology underlying NXG remains unknown although the involvement of MIg is suspected. OBJECTIVE: To provide further insights into the pathophysiology of NXG, we evaluated the plasma lipid phenotype, mechanisms involved in cellular cholesterol accumulation and role of MIg in an analysis of blood and plasma markers of inflammation in 16 patients with xanthomatosis [NXG (n = 8) and NX (n = 8)] associated with monoclonal IgG relative to the relevant controls. RESULTS: The lipid profile of patients with NXG was characterized by a low HDL-C phenotype and an abnormal distribution of HDL particles. Sera from patients with NXG induced cholesterol accumulation in human macrophages. This accumulation was due in part to a significant reduction in the HDL capacity to promote cholesterol efflux from macrophages, which was not found in the case of NX. The MIg of NXG and NX patients was tested positively by ELISA to recognize a large spectrum of lipoproteins. High plasma levels of pro-inflammatory cytokines (TNFα and IL-6), soluble cytokine receptors (sIL-6R, sTNFRI and sTNFRII), adhesion molecules (VCAM-1 and ICAM-1) and chemokines (MCP-1, IL-8 and MIP-1α) were observed in both patients with NXG and NX, revealing a specific xanthoma inflammatory signature which was inversely correlated with plasma levels of anti-inflammatory HDL. However, patients with NXG were distinguished by elevated levels of IL-15 and a marked increase in the rate of intermediate CD14++CD16+ monocytes. CONCLUSION: This study revealed that NXG is characterized by impaired macrophage lipid homeostasis associated with a systemic inflammatory profile that may result from the interaction of MIg and lipoproteins.

Inhibition of matrix metalloproteinases by lung TIMP-1 gene transfer or doxycycline aggravates pulmonary hypertension in rats.

Chronic hypoxic pulmonary hypertension (PH) results from persistent vasoconstriction, excess muscularization, and extracellular matrix remodeling of pulmonary arteries. The matrix metalloproteinases (MMPs) are a family of proteinases implicated in extracellular matrix turnover and hence in smooth muscle and endothelial cell migration and proliferation. Because MMP expression and activity are increased in PH, we designed the present study to investigate whether inhibition of lung MMPs in rats subjected to chronic hypoxia (CH) contributes to or protects against vascular remodeling and PH. To achieve lung MMP inhibition, rats exposed to 10% O(2) for 15 days were treated with either doxycycline (20 mg/kg per day by gavage starting 2 days before and continuing throughout the CH period) or a single dose of recombinant adenovirus (Ad) for the human tissue inhibitors of metalloproteinases-1 (hTIMP-1) gene (Ad.hTIMP-1, 10(8) plaque-forming units given intratracheally 2 days before CH initiation). Control groups either received no treatment or were treated with an adenovirus containing no gene in the expression cassette (Ad.Null). Efficacy of hTIMP-1 gene transfer was assessed both by ELISA on bronchoalveolar lavages and by hTIMP-1 immunofluorescence on lung sections. MMP inhibition in lungs was evaluated by in situ zymography and gelatinolytic activity assessment using [(3)H]gelatin. Rats treated with either doxycycline or Ad.hTIMP-1 had higher pulmonary artery pressure and right heart ventricular hypertrophy more severe than their respective controls. Worsening of PH was associated with increased muscularization and periadventitial collagen accumulation in distal arteries. In conclusion, our study provides compelling evidence that MMPs play a pivotal role in protecting against pulmonary artery remodeling.

Characterization of myogenesis from adult satellite cells cultured in vitro.

We describe several characteristics of in vitro myogenesis from adult skeletal muscle satellite cells from the rat and several amphibian species. The timing of cell proliferation and fusion into myotubes was determined, and in urodeles, myogenesis from satellite cells was clearly demonstrated for the first time. Growth factors are known to stimulate satellite cell proliferation. Acidic FGF mRNA was present in rat satellite cells during proliferation but it was not detected in myotubes. Fibronectin was synthesized in satellite cells during proliferation and expelled into the extracellular medium when the myotubes differentiated. We suggest that fibronectin plays a part in the formation of myotubes, as this process was inhibited by anti-fibronectin IgG. Adult satellite cells might differ from fetal myoblasts since they were observed to exhibit the opposite response to a phorbol ester (TPA) to that of the myoblasts. We therefore examined the possibility that the different levels of protein kinase C activity and different phorbol ester binding characteristics in the two cell types account for these opposite responses. Our results suggest that the difference is not connected with the phorbol ester receptor but might be caused by events subsequent to protein kinase C activation. Localized extracellular proteolytic activity might have a role in cell mobilization and/or fusion when satellite cells are activated. We showed that the content of plasminogen activators, chiefly urokinase, was larger in tissues from slow twitch muscles which regenerate more rapidly than fast muscles. The urokinase level rose sharply in cultures when cells fused into myotubes, and was twice as high in slow muscle cells as in fast ones. We also found that, in vitro, slow muscle satellite cells displayed greater myogenicity, but that phorbol ester inhibited their mitosis and myogenicity. We conclude that satellite cells acquire characteristics which differentiate them from myoblasts and correspond to the fast and slow muscles from which they originate.

Slow and fast rat skeletal muscles differ in their plasminogen activator activities.

Slow and fast contracting muscles differ in their innervation and electrophysiological properties as well as in their regenerating potentialities. The purpose of the present work was to investigate the expression of plasminogen activators and its possible relation to each type of muscle. Slow (Soleus) and fast (Extensor Digitorum Longus) muscles were obtained from white Wistar rats. Before sectioning the muscles, the euthanized rats were perfused with cold phosphate buffer saline to avoid interference by circulating proteases and inhibitors. Muscle extracts were pounded in an ice-cold Potter tube. Plasminogen activators (PAs) were assayed by fibrin zymography and by both liquid and solid-phase fibrin spectrophotometric assays for the detection of PAs activity. Both urokinase (uPA) and tissue-type plasminogen activator (tPA) activities corresponding to proteins of 38 kDa and 65 kDa molecular masses, were detected in the extracts. Slow muscles contained higher amounts of both activators than fast muscles, but the relative amount of uPA was higher in both types of muscles. In addition, the characteristics of each type of extracts differed somewhat: the fast muscle activity curve was typical of an accelerating process, while the slow muscle curve showed an activity probably related to already formed plasmin or to some other trypsin-like enzyme. These results suggest that the amount of plasminogen activators could be a new criterion of discrimination between slow and fast skeletal muscles.

Hormonal and metabolic changes during exercise in cirrhotic patients.

The metabolic response to exercise was compared in 10 cirrhotic patients (P) in a stable clinical condition and in 6 sedentary, age-matched, normal subjects (C) performing 32 minutes of treadmill exercise with the same constant workload corresponding to three to four times their resting oxygen uptake. Taking indirect calorimetry as reference, respiratory exchanges indicated that cirrhotic patients consumed carbohydrates almost exclusively, unlike the normal controls, who consumed lipids and glucids in about the same proportions (RQ: 0.98 +/- 0.04 v 0.87 +/- 0.04, P less than .0001). In the patients, this carbohydrate path of exercise metabolism lowered glycemia from the resting value of 5.23 +/- 0.16 mmol/L to 4.03 +/- 0.37 mmol/L (P less than .0001) and raised the plasma lactate concentration from 2.08 +/- 0.24 mmol/L at rest to 3.48 +/- 0.32 mmol/L at the eighth minute of exercise (P less than .001), thus suggesting defective liver glyconeogenesis. Fatty free acids and glycerol remained almost constant during exercise, whereas catecholamines increased. Insulin levels were high in patients at rest (67.1 +/- 14.5 U/mL v 15.1 +/- 3.5 U/mL); they declined sharply at the onset of exercise but nevertheless remained high compared to those observed in the controls (P less than .0001). Glucagon increased in exercising patients from 88.3 +/- 21.3 pg/mL to 127.4 +/- 30.6 pg/mL (NS). Esterified plasma carnitine declined in the patients from 13.0 +/- 2.2 mumol/L to 8.6 +/- 1.5 mumol/L (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular and mitochondrial toxicity of zidovudine (AZT), didanosine (ddI) and zalcitabine (ddC) on cultured human muscle cells.

Zidovudine (AZT), didanosine (ddI) and zalcitabine (ddC) are the reference antiretroviral therapy in patients with AIDS. A toxic mitochondrial myopathy can be observed in patients treated with AZT, but not with ddI and ddC. All 3 compounds can inhibit mitochondrial (mt)DNA polymerase and cause termination of synthesis of growing mtDNA strands and mtDNA depletion. The propensity to injure particular target tissues is unexplained. In our work, cultured muscle cells prepared from human muscle biopsies, were exposed to various concentrations of AZT (4-5000 micromol/l), ddI (5-1000 micromol/l) and ddC (1-1000 micromol/l) for 10 days. We evaluated cell proliferation and differentiation and measured lipid droplet accumulation, lactate production and respiratory chain enzyme activities. All 3 compounds induced a dose-related decrease of cell proliferation and differentiation. AZT seemed to be the most potent inhibitor of cell proliferation. AZT, ddI and ddC induced cytoplasmic lipid droplet accumulations, increased lactate production and decreased activities of COX (complex IV) and SDH (part of complex II). NADHR (complex I) and citrate sinthase activities were unchanged. Zalcitabine (ddC) and, to a lesser extent, ddI, were the most potent inhibitors of mitochondrial function. In conclusion, AZT, ddI and ddC all exert cytotoxic effects on human muscle cells and induce functional alterations of mitochondria possibly due to mechanisms other than the sole mtDNA depletion. Our results provide only a partial explanation of the fact that AZT, but not ddI and ddC, can induce a myopathy in HIV-infected patients. AZT myopathy might not simply result from a direct mitochondrial toxic effect of crude AZT.

Smooth muscle cell matrix metalloproteinases in idiopathic pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) results from persistent vasoconstriction, smooth muscle growth and extracellular matrix (ECM) remodelling of pulmonary arteries (PAs). Matrix metalloproteinases (MMPs) are matrix-degrading enzymes involved in ECM turnover, and in smooth muscle cell (SMC) and endothelial cell migration and proliferation. MMP expression and activity are increased in experimental PAH. Therefore, this study investigated whether similar changes occur in idiopathic PAH (IPAH; formerly known as primary pulmonary hypertension). Both in situ and in vitro studies were performed on PAs from patients undergoing lung transplantation for IPAH and from patients treated by lobectomy for localised lung cancer, who served as controls. In IPAH, MMP-tissue inhibitor of metalloproteinase (TIMP) imbalance was found in cultured PA-SMCs, with increased TIMP-1 and decreased MMP-3. MMP-2 activity was markedly elevated as a result of increases in both total MMP-2 and proportion of active MMP-2. In situ zymography and immunolocalisation showed that MMP-2 was associated with SMCs and elastic fibres, and also confirmed the MMP-3-TIMP-1 imbalance. In conclusion, the findings of this study were consistent with a role for the matrix metalloproteinase-tissue inhibitor of metalloproteinase system in pulmonary vascular remodelling in idiopathic pulmonary arterial hypertension. The matrix metalloproteinase-tissue inhibitor of metalloproteinase imbalance may lead to matrix accumulation, and increased matrix metalloproteinase-2 activity may contribute to smooth muscle cell migration and proliferation. Whether these abnormalities are potential therapeutic targets deserves further investigation.

Prevention of leucocyte elastase-induced emphysema in mice by heparin fragments.

Heparin and its derivatives inhibit human leucocyte proteinases i.e. elastase and cathepsin G, but do not inhibit porcine pancreatic elastase and Pseudomonas aeruginosa elastase. In vitro experiments, reported here, also indicate that elastin, one of the physiological substrates of human leucocyte elastase (HLE), could decrease by 30-fold the inhibitory potential of an hexadecasaccharide heparin fragment (dp 16) isolated from CY 222. Nevertheless, the inhibitory capacity of the heparin fragment still remains elevated with IC50 = 2.7 x 10(-7) M and still inhibits HLE in its free and adsorbed state to elastin. These overall data prompted us to evaluate the influence of CY 222 in HLE-induced emphysema. Emphysema was induced in mice eight weeks old, following a single instillation of 200 micrograms of HLE. CY 222 treated animals received 2.5 mg.kg-1 subcutaneously once daily, 6 days per week during 4 weeks prior to HLE instillation, and for eight weeks following HLE instillation. The heparin fragment treatment of the mice halved the mortality rate observed early following HLE instillation. After 8 weeks, surviving animals were examined for lung histological and morphometrical changes: mean linear intercept (MLI) and internal alveolar area (ISA). The CY 222 heparin fragments exerted a protective effect against HLE-induced emphysema by decreasing by 70% the MLI; these heparin fragments exerted no effect on emphysema induced by pancreatic elastase in hamsters or mice. Heparin derivatives represent a new class of physiological HLE low molecular weight inhibitors capable of preventing HLE-induced emphysema.

Dexfenfluramine-associated changes in 5-hydroxytryptamine transporter expression and development of hypoxic pulmonary hypertension in rats.

The appetite suppressant dexfenfluramine, which inhibits neuronal 5-HT uptake and elevates plasma 5-HT levels, has been associated with an increase in the relative risk of developing primary pulmonary hypertension. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PA-SMCs), an effect that depends upon activity of the 5-HT transporter (5-HTT). To investigate the relationship between dexfenfluramine and pulmonary hypertension, we examined 1) the effect of dexfenfluramine on 5-HT uptake by PA-SMCs and the mitogenic response of these cells to 5-HT, and 2) 5-HTT mRNA in lung tissue from normoxic and chronically hypoxic rats during and at discontinuation of a 4-week dexfenfluramine treatment (2 mg/kg/day). In cultured PA-SMCs, dexfenfluramine (10(-6) M) markedly reduced [3H]5-HT uptake and [3H]thymidine incorporation in response to 5-HT (10(-6) M). In lungs from rats exposed to 4-week hypoxia (10% O(2)), 5-HTT mRNA levels were higher than in normoxic rats (233.5 +/- 22.5 versus 121.8 +/- 4.8 amol/mg of RNA, P < 0.05), but were not affected by concomitant treatment with dexfenfluramine. One week after discontinuation of dexfenfluramine, 5-HTT mRNA levels increased substantially, this effect being additive with that of hypoxia (364.0 +/- 13.1 in hypoxic versus 164.2 +/- 10 amol/mg of RNA in normoxic rats). When exposure to 2 weeks of hypoxia followed discontinuation of a 4-week treatment, right ventricular hypertrophy was more severe and muscularization of distal pulmonary arteries more marked (P < 0.01) than in rats pretreated with the vehicle. These data show that, in rats, the increased 5-HTT expression that follows dexfenfluramine discontinuation promotes the development of hypoxic pulmonary hypertension.

Pharmacological and molecular characterisation of beta-adrenoceptors in adult rat diaphragm muscle.

Using pharmacological and molecular approaches to investigate beta-adrenoceptor (beta-AR) subtype expression in adult rat diaphragm, we found that adenylyl cyclase (AC) was potently stimulated by the beta2-AR-selective agonist fenoterol, weakly stimulated by the beta1-AR-selective agonist prenalterol and unaffected by the beta3-AR agonist CGP12177. AC activity in response to a submaximal isoproterenol concentration was potently inhibited by the beta2-AR-selective antagonist ICI118551, whereas the beta1-AR-selective antagonist CGP20712A was effective only in very high concentrations. (-)-[125I]-cyanopindolol ([125I]-CYP) saturation binding experiments indicated a single affinity component (dissociation constant (Kd) = 22 +/- 2 pM) for beta-AR sites (maximal beta -AR density (Bmax) = 14 +/- 2 fmol/ mg). Eadie-Hofstee analysis of [125I]-CYP displacement curves by beta1-, beta2- or beta3-AR-selective ligands allowed to characterise a homogeneous population of beta2-AR sites. Finally, reverse transcriptase-polymerase chain reaction analysis of beta-AR subtype mRNAs identified beta2-AR transcripts but no beta1- and beta3-AR mRNAs. Our results demonstrate that beta2-AR is the only beta-AR subtype expressed in the diaphragm.

Increased expression of matrix metalloproteinase-9 in nasal polyps.

To investigate the role of gelatinases in nasal polyposis, a common and disabling airway disease characterized by chronic inflammation and tissue remodelling, matrix metalloproteinase-2 (MMP-2) and MMP-9 expression was investigated in the nasal polyps (NP) of 24 patients undergoing ethmoidectomy and compared with 15 control nasal mucosal (CM) samples obtained from snorers during turbinectomy. Tissue samples were either frozen for enzymatic analysis or paraffin wax-embedded for immunohistochemistry. Zymography and quantitative image analysis showed that MMP-9 active forms were significantly increased (p<0.05) in NPs compared to CM (44 +/- 40 versus 13 +/- 19x10(3) AU/10 microg protein), while MMP-2 expression was similar in both tissues. Concomitant studies of gelatinase immunoexpression showed that MMP-9 expression was enhanced (4- to 16-fold) in surface epithelium, glands (p<0.05), and submucosal inflammatory cells (p<0.05). In addition, MMP-9 positivity was markedly increased in endothelial cells (p<0.01). In situ zymography demonstrated marked gelatinolytic activity, consistent with the immunolocalization of MMP-2 and MMP-9. These results suggest up-regulation of active MMP-9 in the glands and vessels characteristic of NPs. It is concluded that MMP-9 may play a role in the upper airway remodelling observed during nasal polyposis.

Increased expression of gelatinases and alteration of basement membrane in rat soleus muscle following femoral artery ligation.

Acute severe muscle ischaemia is characterized by significant remodelling of basement membranes of myofibres. It was hypothesized that peripheral artery insufficiency is accompanied by similar muscle extracellular matrix (ECM) changes involving matrix metalloproteinase gelatinases. Using a model of femoral artery ligation, both gelatinase activity and basement membrane component degradation were studied in hindlimb skeletal muscles. SDS-PAGE zymography of muscle homogenates showed that acute moderate ischaemia was followed by a significant transient increase in expression of 72- and 92-kDa gelatinases during 48 h; the latter probably originated from inflammatory cells. In situ zymography showed that this increase occurred chiefly at the periphery of myofibres. Immunolocalization demonstrated 72-kDa gelatinase in interspaces and at the periphery of myofibres, and suggested that this enzyme may explain the gelatinolytic activities found by in situ zymography. Type IV collagen and laminin staining showed that the gelatinase expression increase correlated with dramatic basement membrane component alterations. Our data show that even moderate ischaemia results in significant muscle basement membrane remodelling due to matrix degrading enzymes matrix metalloproteinases (MMP) gelatinases.

Gelatinase expression in pulmonary arteries during experimental pulmonary hypertension.

Structural remodelling of pulmonary vessels is an important feature of pulmonary hypertension (PH), which reflects distal artery muscularization and matrix remodelling. The matrix metalloproteinases (MMPs) are involved in extracellular matrix turnover and hence, in smooth muscle cell migration and endothelial cell migration and proliferation. Among the MMPs, gelatinases (MMP-2 and MMP-9) can degrade basement membrane components and promote cell proliferation and migration. This study evaluated gelatinases in pulmonary vessels during progressive PH in two rat models: exposure to hypoxia or monocrotaline. Zymography of tissue homogenates revealed an association of progression of hypoxic PH with a time-dependent increase in gelatinase MMP-2 activity, specific to pulmonary vessels. Increased MMP-2 activity was also found 30 days postmonocrotaline. Reverse transcription polymerase chain reaction demonstrated upregulation of MMP-2 messenger ribonucleic acid. Immunolocalization showed MMP-2 throughout the pulmonary vasculature, from the trunk to the distal vessels, with strong staining of the intima, media and adventitia. MMP-2 was found in its active form and gelatinolytic activity was correlated to PH severity. Activity localization by in situ zymography corroborated with the immunolocalization findings. In conclusion, the authors demonstrated that matrix metalloproteinase-2 activity is increased in pulmonary vessels during progression of pulmonary hypertension, probably as a result of involvement in the matrix turnover associated with vascular remodelling during pulmonary hypertension.

Modulation of activities and RNA level of the components of the plasminogen activation system during fusion of human myogenic satellite cells in vitro.

Primary cultures of human myogenic stem cells (satellite cells) mimic myogenic differentiation. During this process, the expression of the components of the plasminogen activation system underwent modulation. Activities and mRNA levels of tissue-type and urokinase-type plasminogen activator were increased in a reproducible pattern during differentiation. A modulation of the mRNA level of PAI-2 was also observed. Human satellite cells expressed a urokinase receptor and also the mRNA level of this component underwent modulation. With the exception of PAI-1 mRNA, the level of all mRNAs increased from Day 4 to Day 8, i.e., just before myoblasts fusion, and then remained high at later stages. The modulation of the plasminogen activating activity indicates that this system is directly involved in the fusion process of myogenic differentiation.

Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion.

Urokinase can form a tripartite complex binding urokinase receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1), a component of the extracellular matrix (ECM). The components of the tripartite complex are modulated throughout the in vitro myogenic differentiation process. A series of experiments aimed at elucidating the role of the urokinase tripartite complex in the fusion of human myogenic cells were performed in vitro. Myogenic cell fusion was associated with increased cell-associated urokinase-type plasminogen activator (uPA) activity, cell-associated uPAR, and uPAR occupancy. Incubation of cultures with either uPA anticatalytic antibodies, or the amino-terminal fragment of uPA (ATF), which inhibits competitively uPA binding to its receptor, or anti-PAI-1 antibodies, which inhibit uPA binding to PAI-1, resulted in a 30 to 47% decrease in fusion. Incubation of cultures with the plasmin inhibitor aprotinin did not affect fusion. Decreased fusion rates induced by interfering with uPAR/uPA/PAI-1 interactions were not associated with significant changes in mRNA levels of both the myogenic regulatory factor myogenin and its inhibitor of DNA binding, Id. Incubation of cultures with purified uPA resulted in a decrease in fusion, likely due to a competitive inhibition of PAI-1 binding of endogenous uPA. We conclude that muscle cell fusion largely depends on interactions between the members of the urokinase complex (uPAR/uPA/PAI-1), but does not require proteolytic activation of plasmin. Since the intrinsic muscle cell differentiation program appears poorly affected by the state of integrity of the urokinase complex, and since cell migration is a prerequisite for muscle cell fusion in vitro, it is likely that the urokinase system is instrumental in fusion through its connection with the cell migration process. Our results suggest that the urokinase tripartite complex may be involved in cell migration in a non conventional way, playing the role of an adhesion system bridging cell membrane to ECM.