Experimental allergic encephalomyelitis: role of fibrin deposition in immunopathogenesis of inflammation in rats.

The immunopathogenesis of experimental allergic encephalomyelitis (EAE) is reviewed with special focus on the role of central nervous system fibrin deposition in the inflammatory cascade characterizing this autoimmune disease. Among rats sensitized to whole spinal cord or myelin basic protein of either guinea pig or bovine origin, there is a striking degree of concordance of perivascular fibrin deposits and occurrence of clinical paralytic signs. Neither paralytic signs nor fibrin deposition are temporally related to development of perivascular cellular infiltrates. Rats sensitized to neuroantigen and treated with ancrod, a polypeptide derived from the venom of Agkistrodon rhodostoma, develop profound hypofibrinogenemia, have a marked inhibition of fibrin deposition, and often exhibit no paralytic signs whatsoever. In contrast, cellular infiltrates are not demonstrably influenced by ancrod treatment. Activation of the clotting cascade at loci of developing immune injury of nervous tissue appears to result from and lead to increasing neurovascular permeability and accumulation of edema fluid. Distention of the extracellular space in central and peripheral nervous system tissues by edema fluid appears to be directly responsible for clinical abnormalities characterizing EAE in rats. Cellular infiltrates, on the other hand, appear to be an independent immune response to neuroantigenic sensitization.     

Tissue plasminogen activator-mediated fibrinolysis protects against axonal degeneration and demyelination after sciatic nerve injury

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.     

Fibrin mechanisms and functions in nervous system pathology

In brain physiology, cerebrovascular interactions regulate both, vascular functions, such as blood vessel branching and endothelial cell homeostasis, as well as neuronal functions, such as local synaptic activity and adult neurogenesis. In brain pathology, including stroke, HIV encephalitis, Alzheimer Disease, multiple sclerosis, bacterial meningitis, and glioblastomas, rupture of the vasculature allows the entry of blood proteins into the brain with subsequent edema formation and neuronal damage. Fibrin is a blood-derived protein that is not produced by cells of the nervous system, but accumulates only after disease associated with vasculature rupture. This review presents evidence from human disease and animal models that highlight the role of fibrin in nervous system pathology. Our review presents novel experimental data that extend the role of fibrin, from that of a blood-clotting protein in cerebrovascular pathologies, to a component of the perivascular extracellular matrix that regulates inflammatory and regenerative cellular responses in neurodegenerative diseases.     

The nonspecific nature of fibrin thrombi in ischemic bowel disease.

Twenty cases of ischemic bowel disease were analysed to determine the frequency and significance of fibrin thrombi in this condition. Fibrin thrombi were present in all 10 patients with occlusive ischemic bowel disease and in 7 of the 10 patients with nonocclusive ischemic bowel disease. In addition, fibrin thrombi were noted in a wide variety of specific and nonspecific inflammatory bowel diseases and in acute appendicitis. We conclude that fibrin thrombi are a nonspecific feature of tissue necrosis and that their mere presence in the bowel should not be regarded as an expression of disseminated intravascular coagulation.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

Obesity and bone metabolism

Both bone and adipose tissue change their size, shape and distribution during the whole human being's life. Many factors, including genetic factors, hormones and activity of nervous system are responsible for these changes. It is generally accepted that obesity has a protective effect on bone tissue. On the other hand some authors present an opposite results--the lack of beneficial effect of obesity on development of osteoporosis fractures. The aim of this article was to present and discuss the relations between adipose tissue and bone metabolism.     

Peripheral pathogenesis of prion diseases

Prions are infectious pathogens that cause a group of neurodegenerative diseases characterized by spongiform degeneration of the central nervous system. Prions appear to lack any informational nucleic acid. The most notable prion diseases include bovine spongiform encephalopathy, scrapie in sheep and Creutzfeldt-Jakob disease of humans. Transmission is thought to be achieved through conversion of a normal host protein into a pathological isoform. Although the main pathological changes during the course of the disease occur in the brain, the infectious agent accumulates early in lymphoid tissue. The subsequent development of clinical disease depends on the presence of an intact immune system including mature B-cells. In this article we review the state of knowledge on the routes of neuroinvasion used by the infectious agent in order to gain access to the central nervous system upon entry into extracerebral sites.     

Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis

This study deals with the effect of fibrin on the transformation of Glu-plasminogen to Glu-plasmin during fibrinolysis. It focuses particularly on changes in fibrin effector function caused by plasmin-catalysed fibrin degradation. Conversion of 125I-labelled Glu-plasminogen to Glu-plasmin was catalysed by urokinase or tissue plasminogen activator, in the presence of different preparations of progressively degraded fibrin. Plasmin catalysis of Glu-plasminogen and the fibrin (derivative) effector was inhibited by aprotinin. The presence of intact fibrin enhanced the rate of Glu-plasmin formation catalysed by tissue plasminogen activator, but not by urokinase. The presence of initially plasmin-cleaved fibrin, however, increased the rates of Glu-plasmin formation with both activators, as compared to those found with intact fibrin. The rate enhancements induced by initial plasmin degradation of the fibrin effector were associated with an increase in its affinity to both Glu-plasminogen and tissue plasminogen activator, suggesting causal relationships. The weak binding of urokinase was unaffected by fibrin degradation, indicating that effector function was solely exerted on the Glu-plasminogen moiety of urokinase-activated systems. Further degradation of fibrin decreased the stimulating effect on Glu-plasmin formation. This decrease occurred at an earlier stage of degradation with tissue plasminogen activator than with urokinase, indicating that greater integrity of the fibrin effector is necessary for its optimal interaction with the tissue plasminogen activator than with Glu-plasminogen. Concentrations of tranexamic acid that saturate low-affinity lysine-binding sites nearly completely dissociated the binding of Glu-plasminogen to degraded fibrin, but not to intact fibrin. In analogy with the binding of lysine analogues to these sites, the conformation of Glu-plasminogen may be altered by binding to degraded fibrin, thus giving rise to the increased activation rate.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung’s Disease

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.     

Antioxidants in peripheral nerve

Oxidative stress and antioxidants have been related in a wide variety of ways with nervous tissue. This review attempts to gather the most relevant information related to a) the antioxidant status in non pathologic nervous tissue; b) the hypothesis and evidence for oxidative stress (considered as the disequilibrium between prooxidants and antioxidants in the cell) as the responsible mechanism of diverse neurological diseases; and c) the correlation between antioxidant alterations and neural function, in different experimental neuropathies. Decreased antioxidant availability has been observed in different neurological disorders in the central nervous system, for example, Parkinson's disease, Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis, cerebral ischaemia, etc. Moreover, the experimental manipulation of the antioxidant defense has led in some cases to interesting experimental models in which electrophysiological alterations are associated with the metabolic modifications induced. In view of the electrophysiological and biochemical effects of some protein kinase C inhibitors on different neural experimental models, special attention is dedicated to the role of this kinase in peripheral nervous tissue. The nervous tissue, central as well as peripheral, has two main special features that are certainly related to its antioxidant metabolism: the lipid-enriched membrane and myelin sheaths, and cellular excitability. The former explains the importance of the glutathione (GSH)-conjugating activity towards 4-hydroxy-nonenal, a biologically active product of lipid peroxidation, present in nervous tissue and in charge of its inactivation. The impairment of the latter by oxidative damage or experimental manipulation of antioxidant metabolism is discussed. Work on different experimental neuropathies from author's laboratory has been primarily used to provide information about the involvement of free radical damage and antioxidants in peripheral nerve metabolic and functional impairment.     

Lack of Prion Infectivity in Fixed Heart Tissue from Patients with Creutzfeldt-Jakob Disease or Amyloid Heart Disease

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP^Sc) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP^Sc in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP^Sc. Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.     

Connective tissue growth factor binds to fibronectin through the type I repeat modules and enhances the affinity of fibronectin to fibrin

Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role.     

Suspected lysosomal storage disease in kangaroos

A probable neurovisceral lysosomal storage disease is reported, for the first time, in immature red and grey kangaroos (Macropus rufus and M. giganteus). Foamy, pale eosinophilic, periodic acid-Schiff positive, intracytoplasmic material was stored in the liver, lymphoid tissue, kidney, adrenal gland, stomach, blood vessels and central nervous system. Extensive Wallerian-type degeneration was present in the central nervous system. Electron microscopic study of one animal revealed electron dense, cytoplasmic lamellar bodies in neurons and foamy visceral cells. The disease differs from other reported storage diseases in the distribution and nature of the lesions.     

Role of the clotting system in the pathogenesis of neuroimmunologic disease

Experimental allergic encephalomyelitis (EAE) is a prototypic neuroautoimmune disease involving sensitization to central nervous system myelin basic protein (MBP). Our studies of the clotting system and ensuing fibrinolysis implicate coagulation and cleavage of fibrin within or on the luminal surface of the cerebrovasculature as events initiating the inflammation characterizing EAE. Among recipient rats injected with MPB-primed, cultured-activated lymph node cells, opening of the blood-brain barrier (BBB) and deposition of perivascular fibrin within the spinal cord occur in parallel 1 day before onset of clinical signs of EAE. Daily treatment of recipient rats with trans-4-(aminomethyl)cyclohexanecarboxylic acid, a synthetic product that specifically inhibits plasminogen activator derived from endothelial cells, results in marked reduction of increased permeability of the BBB and suppression of clinical signs of EAE. We postulate that the critical event precipitating EAE is binding of circulating MBP-reactive immune effector cells to MBP immunodeterminants on the surface of cerebrovascular endothelial cells. Coagulation and ensuing fibrinolysis occur at sites of binding of effector cells to cerebrovascular endothelium. Release of biologically active peptides cleaved from fibrin open the BBB, thereby setting the stage for the cascade of inflammatory events culminating in clinical manifestations of EAE.     

Neurite extension and in vitro myelination within three-dimensional modified fibrin matrices

The deposition of fibrin clots in vivo occurs after injury in the peripheral nervous system and their removal correlates with nerve regeneration. Fibrin clots provide a provisional matrix for invading cells, induce wound healing, and become proteolytically removed by regenerating tissue. Here, neurite extension and in vitro myelination were studied within three-dimensional fibrin matrices that were covalently modified with the sixth Ig-like domain of cell adhesion molecules L1 containing N-terminal transglutaminase substrate sequences (TG-L1Ig6) for covalent incorporation into fibrin matrices. TG-L1Ig6 is a specific receptor for alphavbeta3-integrin involved in neurite extension of PC12 cells and dorsal root ganglion neurons (DRGs). Neurite extension of PC12 cells depended on interactions between cell surface alphavbeta3 and RGD-sites provided by TG-L1Ig6. In addition, matrix properties such as fibrin crosslink density and matrix degradation by serine proteases were crucial. No involvement of matrix metalloproteinases was found. DRG neurite extension in native fibrin matrices was retarded as compared to neurite extension within L1Ig6-modified and laminin-1-containing matrices. Moreover, myelinated structures were almost exclusively found in TG-L1Ig6-modified and laminin-1-containing matrices. These results indicate that potential use of three-dimensional matrices in a biomaterials-based healing device to induce and/or help in vivo nerve regeneration requires specific structural and biological signals.     

Inhibition of the process of Glu-plasminogen activation by tissue activator with fibrin, DDE-complex, and D-dimer using alpha-2-antiplasmin

The alpha-2-antiplasmin influence on the Glu-plasminogen activation by tissue activator both on fibrin and fibrin(ogen) fragments was investigated. The kinetics of activation was studied and velocity of this process in the absence and presence of the inhibitor was calculated. It was established that alpha-2-antiplasmin decreased the velocity of Glu-plasminogen activation on desAABBfibrin, DDE-complex and DD-dimer and did no influence upon proenzyme activation on fibrinogen fragment--Ho1-DSK. In the presence of fibrin plasminogen activation linear related to the amount added tissue activator in limit concentration from 5 before 50 units/ml. It was shown that alpha-2-antiplasmin reduced the activation velocity with used concentration of tissue activator. Fibrin hydrolysis by plasmin, forming on its surface during the plasminogen activation by tissue activator, was also inhibited with alpha-2-antiplasmin. The obtained results are explained by the influence of the inhibitor on formation of the triple complex between plasminogen, tissue activator and fibrin, and competition of the alpha-2-antiplasmin for lysine-binding sites of tissue activator kringle 2 or for binding sites of the activator on fibrin.     

Growth factors, feeding regulation and the nervous system

A variety of growth factors and their receptors are present in the nervous system. Growth factors can modulate specific nervous system functions others than those related to growth, development, and tissue repair. The presence of growth factors in the brain and cerebrospinal fluid is the result of local synthesis (by neuronal, glial, vascular, and mononuclear phagocyte components), and uptake from the peripheral blood through the blood-brain barrier (in specific cases) and circumventricular organs. This paper focuses on the effects of a heterogeneous group of growth factors (acidic and basic fibroblast growth factors, insulin-like growth factors, epidermal growth factor, platelet-derived growth factor, interleukin-1 and others) on the central nervous system (CNS), in particular, on feeding regulation. Recent evidence supporting participation of growth factors in the regulation of feeding by a direct action at the level of the CNS is reviewed. Various growth factors have the ability to suppress short- and long-term food intake (FI), whereas others affect only short-term FI, or do not affect FI. Acute and chronic pathological processes stimulate the synthesis and release of growth factors in various cellular systems, and monitoring of growth factors by the CNS could be part of the regulatory signals that induce FI suppression frequently accompanying acute and chronic disease. Thus, it is proposed that a system regulating FI through growth factor-dependent mechanisms may be operative during specific physiological or pathological conditions.