Neuronal and astrocytic interactions modulate brain endothelial properties during metabolic stresses of in vitro cerebral ischemia.

Neurovascular and gliovascular interactions significantly affect endothelial phenotype. Physiologically, brain endothelium attains several of its properties by its intimate association with neurons and astrocytes. However, during cerebrovascular pathologies such as cerebral ischemia, the uncoupling of neurovascular and gliovascular units can result in several phenotypical changes in brain endothelium. The role of neurovascular and gliovascular uncoupling in modulating brain endothelial properties during cerebral ischemia is not clear. Specifically, the roles of metabolic stresses involved in cerebral ischemia, including aglycemia, hypoxia and combined aglycemia and hypoxia (oxygen glucose deprivation and re-oxygenation, OGDR) in modulating neurovascular and gliovascular interactions are not known. The complex intimate interactions in neurovascular and gliovascular units are highly difficult to recapitulate in vitro. However, in the present study, we used a 3D co-culture model of brain endothelium with neurons and astrocytes in vitro reflecting an intimate neurovascular and gliovascular interactions in vivo. While the cellular signaling interactions in neurovascular and gliovascular units in vivo are much more complex than the 3D co-culture models in vitro, we were still able to observe several important phenotypical changes in brain endothelial properties by metabolically stressed neurons and astrocytes including changes in barrier, lymphocyte adhesive properties, endothelial cell adhesion molecule expression and in vitro angiogenic potential.

Inflammation induces neuro-lymphatic protein expression in multiple sclerosis brain neurovasculature.

BACKGROUND: Multiple sclerosis (MS) is associated with ectopic lymphoid follicle formation. Podoplanin+ (lymphatic marker) T helper17 (Th17) cells and B cell aggregates have been implicated in the formation of tertiary lymphoid organs (TLOs) in MS and experimental autoimmune encephalitis (EAE). Since podoplanin expressed by Th17 cells in MS brains is also expressed by lymphatic endothelium, we investigated whether the pathophysiology of MS involves inductions of lymphatic proteins in the inflamed neurovasculature. METHODS: We assessed the protein levels of lymphatic vessel endothelial hyaluronan receptor and podoplanin, which are specific to the lymphatic system and prospero-homeobox protein-1, angiopoietin-2, vascular endothelial growth factor-D, vascular endothelial growth factor receptor-3, which are expressed by both lymphatic endothelium and neurons. Levels of these proteins were measured in postmortem brains and sera from MS patients, in the myelin proteolipid protein (PLP)-induced EAE and Theiler's murine encephalomyelitis virus (TMEV) induced demyelinating disease (TMEV-IDD) mouse models and in cell culture models of inflamed neurovasculature. RESULTS AND CONCLUSIONS: Intense staining for LYVE-1 was found in neurons of a subset of MS patients using immunohistochemical approaches. The lymphatic protein, podoplanin, was highly expressed in perivascular inflammatory lesions indicating signaling cross-talks between inflamed brain vasculature and lymphatic proteins in MS. The profiles of these proteins in MS patient sera discriminated between relapsing remitting MS from secondary progressive MS and normal patients. The in vivo findings were confirmed in the in vitro cell culture models of neuroinflammation.

Metabolic modulation of cytokine-induced brain endothelial adhesion molecule expression.

OBJECTIVE: Cytokines contribute to cerebro-vascular inflammatory and immune responses by inducing ECAMs' expression. Ischemic insults can be separated into aglycemic and hypoxic components. However, whether aglycemia, hypoxia or OGD plays a major role in dysregulating BBB or promotes immune cell infiltration via ECAMs' expression is not clear. We investigated how expression of ICAM-1, VCAM-1, MAdCAM-1, PECAM-1, E- and P-selectin in response to TNF-α, IL-1ß and IFN-γ was altered by aglycemia (A), hypoxia (H) or combined oxygen glucose deprivation (OGD). METHODS: A cell surface enzyme linked immunoabsorbent assay (cell surface ELISA) was used to analyze ECAM expression. RESULTS: We observed that ICAM-1 and PECAM-1 expressions were insensitive to hypoxia, aglycemia or OGD. Conversely, VCAM-1 and E-selectin were increased by hypoxia, but not by aglycemia. MAdCAM-1 and P-selectin were induced by hypoxia, and decreased by aglycemia. Patterns of cytokine-regulated ECAMs' expression were also modified by metabolic conditions. CONCLUSIONS: Our results indicate that patterns of inflammation-associated ECAMs represent cumulative influences from metabolic stressors, as well as cytokine activation. The expression of ECAMs following tissue injury reflects mechanistic interactions between metabolic disturbances, and alterations in tissue cytokines. Normalization of tissue metabolism, as well as cytokine profiles, may provide important targets for therapeutic treatment of inflammation.

PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration.

The normal function of poly (ADP-ribose) polymerase-1 (PARP-1) is the routine repair of DNA damage by adding poly (ADP ribose) polymers in response to a variety of cellular stresses. Recently, it has become widely appreciated that PARP-1 also participates in diverse physiological and pathological functions from cell survival to several forms of cell death and has been implicated in gene transcription, immune responses, inflammation, learning, memory, synaptic functions, angiogenesis and aging. In the CNS, PARP inhibition attenuates injury in pathologies like cerebral ischemia, trauma and excitotoxicity demonstrating a central role of PARP-1 in these pathologies. PARP-1 is also a preferred substrate for several 'suicidal' proteases and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes and matrix metalloproteinases (MMPs)) on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights. These PARP-1 signature fragments are recognized biomarkers for specific patterns of protease activity in unique cell death programs. This review focuses on specific suicidal proteases active towards PARP-1 to generate signature PARP-1 fragments that can identify key proteases and particular forms of cell death involved in pathophysiology. The roles played by some of the PARP-1 fragments and their associated binding partners in the control of different forms of cell death are also discussed.

Differential PARP cleavage: an indication of heterogeneous forms of cell death and involvement of multiple proteases in the infarct of focal cerebral ischemia in rat.

AIM: Poly (ADP-ribose) polymerase (PARP) is a nuclear repair enzyme whose role is widely depicted in various physiological and pathological processes. In the present study, we wanted to check the status of PARP and the role of various cell death proteases involved in apoptotic and non-apoptotic forms of cell death during transient focal cerebral ischemia in rat model. The activation of these proteases can result in the production of PARP fragments which can be treated as specific signature fragments to the particular protease involved in the pathology and hence the type of cell death. RESULTS: In the ischemic samples, we observed activation of calpain, cathepsin-b, caspase-3, and granzyme-b which were known to act on and cleave PARP to produce specific signature fragments by Western blot and immunohistochemical analysis. Cresyl violet staining showed the presence of apoptotic and necrotic cell deaths. Further we observed interaction of AIF and gra-b with PARP in double immunofluorescence and co-immunoprecipitation experiments. CONCLUSION: Activation of calpains, cathepsin-b, caspase-3, and granzyme-b correlated with either apoptotic or necrotic cell deaths in cresyl violet staining. The appearance of PARP signature fragments gives a clear idea on the involvement of particular protease in the pathology. Appearance of signature fragments like 89- and 50-kDa indicates the involvement of apoptotic and necrotic cell death in the pathology. Further interaction of AIF and gra-b with PARP also indicates the involvement of non-apoptotic modes of cell death during the pathology of focal cerebral ischemia.

Granzyme-b mediated cell death in the spinal cord-injured rat model.

Spinal cord injury initiates a complex series of inflammatory and immune responses including the influx of monocytes, macrophages, T-cells, NK cells and so on, into the injured area. In the present study, we found a significant increase in the levels of granzyme-b (gra-b) from the first day after the transection until the third day, with decrease in intensity thereafter. The chemokine IP-10/CXCL10 was also found to be elevated along with gra-b correlating with the infiltration of CD-8(+) cytotoxic T lymphocytes (CTLs) into the injured spinal cord. We observed an increase in the levels of the 64 kDa poly ADP ribose polymerase fragment, known to be a signature fragment produced by gra-b. Localization of gra-b in TUNEL positive neurons indicates that gra-b might play a crucial role in neuronal death and contributes to the pathophysiology of spinal cord injury.

Activation of calpain, cathepsin-b and caspase-3 during transient focal cerebral ischemia in rat model.

Calpains, cathepsins and caspases play crucial role in mediating cell death. In the present study we observed a cascade of events involving the three proteases during middle cerebral artery occlusion (MCAo) in Wistar rats. The rats were MCA occluded and reperfused at various time points. We observed a maximal increase in the levels of calpains during 1h and 12 h after reperfusion than permanently occluded rats. Further, these levels were reduced by 1st and 3rd day of reperfusion. Similarly the cathepsin-b levels were significantly increased during 1h and 12 h, of reperfusion, followed by activation of caspase-3 which reached maximal levels by 1st and 3rd day of reperfusion. The sequential activation of calpains, cathepsin-b and cleaved caspase-3 is evident by the Western blot analysis which was further confirmed by the cleavage of substrates like PSD-95 and spectrin. The differences in the regional distribution and elevation of these proteases at different reperfusion time periods indicates that differential mode of cell death occur in the brain during cerebral ischemia in rat model.

Proteolytic breakdown of cytoskeleton induces neurodegeneration during pathology of murine cerebral malaria.

Fatal murine cerebral malaria is known to induce cellular degeneration by altering cellular morphology and integrity of cell. The morphology and integrity of the cell mainly depends on the cytoskeletal network of the cell. Increased proteolysis of cytoskeletal proteins accompanied by aggravated suicidal proteases activation leads to cellular degeneration. In the present study, we investigated the roles of apoptotic and necrotic cell death proteases, caspase-3, calpain-1 and cathepsin-b in the proteolysis of neuronal cytoskeletal proteins in mouse model of fatal cerebral malaria. We found increased levels of calpain-1, cathepsin-b and caspase-3, with extensive cross talks between these suicidal proteases. Increased levels of these proteases correlated with the enhanced proteolysis of several cytoskeletal proteins including neuronal cytoskeleton proteolytic signature fragments. Further, we also observed that increased levels of these proteases correlated with the appearance of neuronal death that exhibited apo-necrotic continuum. Our results confirm that activation of multiple suicidal proteases, their cross talks and breakdown of the cytoskeletal proteins increase neuronal degeneration and lead to exacerbation of cerebral malaria pathology.

Role of cytotoxic protease granzyme-b in neuronal degeneration during human stroke.

Infiltration of leukocytes into post-ischemic cerebrum is a well-described phenomenon in stroke injury. Because CD-8(+) T-lymphocytes secrete cytotoxic proteases, including granzyme-b (Gra-b) that exacerbates post-ischemic brain damage, we investigated roles of Gra-b in human stroke. To study the role of Gra-b in stroke, ischemic and non-ischemic tissues (from post-mortem stroke patients) were analyzed using immunoblotting, co-immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin-V immunostaining, and in vitro neuron survival assays. Activated CG-SH cells and supernatants were used to model leukocyte-dependent injury. Non-ischemic brain tissues were used as non-pathological controls. Non-activated CG-SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra-b and interferon-gamma inducible protein-10 (IP-10/CXCL10) than non-ischemic controls. In stroke, poly (ADP-ribose) polymerase-1 and heat shock protein-70 were cleaved to canonical proteolytic "signature" fragments by Gra-b. Gra-b was also found to bind to Bid and caspase-3. Gra-b also co-localized with Annexin-V(+) /TUNEL(+) in degenerating neurons. Importantly, Gra-b inhibition protected both normal and ischemia-reperfused neurons against in vitro neurotoxicity mediated by activated CG-SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra-b levels in the post-stroke brain can induce contact-dependent and independent post-ischemic neuronal death to aggravate stroke injury.

Emerging roles of lymphatics in inflammatory bowel disease.

The mobilization and recruitment of blood and lymphatic vasculatures are widely described in inflammatory bowel diseases (IBDs). Although angiogenesis contributes to intense gut inflammation, it remains unclear whether and when lymphangiogenesis amplifies or protects in IBD. The prolonged maintenance of lymphatic (over blood vessels) in inflammation indicates that lymphatic-blood vessel interactions may regulate IBD pathogenesis and restitution. Although lymphatic expansion helps to restore fluid balance and clear cytokines and immune cells, lymphatic failure results in accumulation of these factors and exacerbates IBD. Lymphatic obstruction and remodeling may impair lymphatic pumping, leading to repeated rounds of lymphangiogenesis. Early descriptions of Crohn's disease and ulcerative colitis describe colon lymphatic congestion, remodeling, expansion, and many other features that are recapitulated in experimental IBD and also by intestinal lymphatic obstruction, supporting lymphangitis as a cause and consequence of IBD. Growth factors, cytokines, gut flora, Toll receptors, and leukocytes all regulate inflammation and gut lymphatic remodeling in IBD. This review summarizes the importance of lymphatics and lymphangiogenesis in IBD etiology that may be useful in diagnosis and therapy of gut inflammation.

Discovery of transcriptional programs in cerebral ischemia by in silico promoter analysis.

In stroke, gene transcription plays a central role in processes such as neuroinflammation and neuroregeneration. To predict new transcriptional regulatory mechanisms in cerebral ischemia, we applied a computational approach combining two kinds of information: the results of a microarray analysis in a mouse model of stroke and in silico detection of transcription factor (TF) binding sites in promoter regions of the genes on the array. By using a discriminative logistic regression model, we identified binding sites significantly associated with the up-regulation of genes. Out of 356 TF binding sites defined in TRANSFAC, we could link 32 to gene up-regulation in cerebral ischemia. These sites bind both TFs with an established and a so far unknown role in cerebral ischemia. To evaluate the results further we investigated whether two TFs, CCAAT/enhancer binding protein beta (C/EBP beta) and vitamin D receptor (VDR), are activated as predicted. Immunohistochemistry demonstrated that C/EBP beta and VDR translocated to the nucleus in cerebral ischemia. Chromatin immunoprecipitation revealed increased binding of C/EBP beta to the promoter of its target gene saa3. In addition, we found evidence for the up-regulation of VDR in brain samples from human stroke patients. These results confirm the activation of C/EBP beta and VDR in cerebral ischemia. Thus, our in silico analysis may provide additional information on transcriptional regulation in stroke and suggests several novel transcriptional programs for further exploration.

Multiple apoptogenic proteins are involved in the nuclear translocation of Apoptosis Inducing Factor during transient focal cerebral ischemia in rat.

Apoptosis Inducing Factor is a mitochondrial protein which upon translocation to nucleus causes large scale DNA fragmentation. The stimulus for the cytosolic release and nuclear translocation for this protein still remains to be understood. The role of calpains, cathepsin-b, Poly ADP (ribose) Polymerase and granzyme-b in the nuclear translocation of AIF has been investigated in the pathology of cerebral ischemia. Calpains, cathepsin-b and PARP-1 which were mostly confined to cytosol, lysosomes and nucleus respectively were found to be elevated in the mitochondrial fraction interacting with AIF in the western blot analysis and double immunofluorescence analysis. Western blot and immunohistochemical analysis revealed elevated levels of granzyme-b secreted by cytotoxic T lymphocytes and natural killer cells in the infarct of ischemic mouse brain. Co-immunoprecipitation revealed and western blot analysis the interaction and break down of Heat Shock Protein-70 an endogenous inhibitor of AIF into signature fragments by granzyme-b facilitating the nuclear translocation of AIF. Break down of HSP-70 correlated with the nuclear translocation of AIF observed in western and immunohistochemical analysis. These results indicate that multiple proteases were involved in the nuclear translocation of AIF during the pathology of cerebral ischemia.