Age and aerobic training status effects on plasma and skeletal muscle tPA and PAI-1

Introduction

Reductions in fibrinolytic potential occur with both aging and physical inactivity and are associated with an increased cardiovascular disease risk. Plasmin, the enzyme responsible for the enzymatic degradation of fibrin clots, is activated by tissue plasminogen activator (tPA), while plasminogen activator inhibitor-1 (PAI-1) inhibits its activation. Currently, fibrinolysis research focuses almost exclusively on changes within the plasma. However, tPA and PAI-1 are expressed by human skeletal muscle (SM). Currently, no studies have focused on changes in SM fibrinolytic activity with regard to aging and aerobic fitness.

Purpose

The purpose of this study was to cross-sectionally evaluate effects of age and aerobic fitness on tPA and PAI-1 expressions and activity in SM.

Methods

Twenty-six male subjects were categorized into the following groups: (1) young aerobically trained (n = 8); (2) older aerobically trained (n = 6); (3) young aerobically untrained (n = 7); and (4) older aerobically untrained (n = 5). Muscle biopsies were obtained from each subject. SM tPA activity was assessed using gel zymography and SM tPA and PAI-1 expressions were assessed using RT-PCR.

Results

Trained subjects had higher SM tPA activity compared to untrained (25.3 ± 2.4 × 10³ vs. 21.5 ± 5.6 × 10³ pixels, respectively; p = 0.03) with no effect observed for age. VO_{2 max} and SM tPA activity were also significantly correlated (r = 0.42; p < 0.04). SM tPA expression was higher in older participants, but no effect of fitness level was observed. No differences were observed for PAI-1 expression in SM.

Conclusions

Higher levels of aerobic fitness are associated with increased fibrinolytic activity in SM.     

Reduced monocyte adhesion to aortae of diabetic plasminogen activator inhibitor-1 knockout mice

Objective and design

To determine the requirement of plasminogen activator inhibitor-1-knockout (PAI-1) for monocyte adhesion in animals and cells under diabetic conditions.

Methods and subjects

Monocyte adhesion assay, enzyme-linked immunosorbent assay, and Western blotting were used in analyzing samples from PAI-1-knockout (PAI-1-KO) mice or cultured human umbilical vein endothelial cells (HUVEC).

Treatments

Diabetes in PAI-1-KO and wild-type mice was induced by intraperitoneal injection of streptozotocin (STZ). HUVEC was transfected with short interference RNA (siRNA) against PAI-1, tumor necrosis factor-α (TNFα), or toll-like receptor (TLR4), and then was treated with glycated low-density lipoproteins (glyLDL).

Results

The adhesion of monocytes to aortic intima was reduced in PAI-1-KO mice, which was associated with decreased levels of TNFα and monocyte chemotactic protein-1 (MCP-1) in plasma and cardiovascular tissue, and increased abundances of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in cardiovascular tissue compared to wild-type mice. Significant reductions in monocyte adhesion, inflammatory, and fibrinolytic regulators were detected in cardiovascular tissue or plasma in diabetic PAI-1-KO mice compared to wild-type diabetic mice. Transfection of PAI-1, TNFα or TLR4 siRNA to HUVEC inhibited glyLDL-induced monocyte adhesion to EC. PAI-1 siRNA inhibited the abundances of TLR4 and TNFα in EC.

Conclusion

The findings suggest that PAI-1 is required for diabetes-induced monocyte adhesion via interactions with uPA/uPAR, and it also regulates TLR4 and TNFα expression in vascular EC. Inhibition of PAI-1 potentially reduces vascular inflammation under diabetic condition.

     

Spinal motoneuron synaptic plasticity after axotomy in the absence of inducible nitric oxide synthase

Background

Astrocytes play a major role in preserving and restoring structural and physiological integrity following injury to the nervous system. After peripheral axotomy, reactive gliosis propagates within adjacent spinal segments, influenced by the local synthesis of nitric oxide (NO). The present work investigated the importance of inducible nitric oxide synthase (iNOS) activity in acute and late glial responses after injury and in major histocompatibility complex class I (MHC I) expression and synaptic plasticity of inputs to lesioned alpha motoneurons.

Methods

In vivo analyses were carried out using C57BL/6J-iNOS knockout (iNOS^-/-) and C57BL/6J mice. Glial response after axotomy, glial MHC I expression, and the effects of axotomy on synaptic contacts were measured using immunohistochemistry and transmission electron microscopy. For this purpose, 2-month-old animals were sacrificed and fixed one or two weeks after unilateral sciatic nerve transection, and spinal cord sections were incubated with antibodies against classical MHC I, GFAP (glial fibrillary acidic protein - an astroglial marker), Iba-1 (an ionized calcium binding adaptor protein and a microglial marker) or synaptophysin (a presynaptic terminal marker). Western blotting analysis of MHC I and nNOS expression one week after lesion were also performed. The data were analyzed using a two-tailed Student's t test for parametric data or a two-tailed Mann-Whitney U test for nonparametric data.

Results

A statistical difference was shown with respect to astrogliosis between strains at the different time points studied. Also, MHC I expression by iNOS^-/- microglial cells did not increase at one or two weeks after unilateral axotomy. There was a difference in synaptophysin expression reflecting synaptic elimination, in which iNOS^-/- mice displayed a decreased number of the inputs to alpha motoneurons, in comparison to that of C57BL/6J.

Conclusion

The findings herein indicate that iNOS isoform activity influences MHC I expression by microglial cells one and two weeks after axotomy. This finding was associated with differences in astrogliosis, number of presynaptic terminals and synaptic covering of alpha motoneurons after lesioning in the mutant mice.     

Alterations in coagulatory and fibrinolytic systems following an ultra-marathon

Purpose

The aim of this study was to examine coagulatory and fibrinolytic responses to the Western States Endurance Run (WSER, June 23 to 24, 2012). The WSER is a 161-km (100 mile) trail foot race through the Sierra Nevada Mountains that involves 6,030 m of climb and 7,001 m of descent.

Methods

We examined 12 men and 4 women [mean (95 % CI), age 44.6 years (38.7–50.6)] who completed the race (24.64 h; range 16.89–29.46). Blood samples were collected the morning before the race, immediately post-race, and 1 (D1) and 2 (D2) days post-race (corresponding to 51–54 h and 75–78 h from the start of the race, respectively). Hypercoagulable state was characterized by prothrombin fragment 1+2 (PTF 1+2) and thrombin–antithrombin complex (TAT). Fibrinolytic state was assessed by plasminogen activator inhibitor antigen (PAI-1 Ag), tissue plasminogen activator antigen (tPA Ag), and d-Dimer. Muscle damage was assessed by serum creatine kinase (CK) and myoglobin concentrations.

Results

Significant (P ≤ 0.05) increases were observed immediately post-race for thrombin generation markers, PTF 1+2 (3.9-fold) and TAT (2.4-fold); markers of fibrinolysis, tPA Ag (4.0-fold), PAI-1 Ag (4.5-fold), and d-Dimer (2.2-fold); and muscle damage markers, CK (154-fold) and myoglobin (114-fold). Most markers continued to be elevated at D1, as seen by PTF 1+2, TAT (1.5- and 1.3-fold increase at D1), and d-Dimer (2.5- and 2.1-fold increase at D1 and D2, respectively). Additionally, PTF 1+2:tPA and TAT:tPA ratios, which assessed balance between coagulation and fibrinolysis, were slightly, but significantly increased at D1 (69 and 36 %) and D2 (19 and 31 %). CK and myoglobin also remained elevated at D1 (54- and 7-fold) and D2 (25- and 2-fold) time points.

Conclusion

The WSER produced extensive muscle damage and activated the coagulation and fibrinolytic systems. Since we observed a slight imbalance response between the two systems, a limited potential for thrombotic episodes is apparent in these highly trained athletes.     

A lack of association between hyperserotonemia and the increased frequency of serum anti-myelin basic protein auto-antibodies in autistic children

Background

The purpose of this study was to investigate whether localized peripheral inflammation, such as osteoarthritis, contributes to neuroinflammation and neurodegenerative disease in vivo.

Methods

We employed the inducible Col1-IL1β^XAT mouse model of osteoarthritis, in which induction of osteoarthritis in the knees and temporomandibular joints resulted in astrocyte and microglial activation in the brain, accompanied by upregulation of inflammation-related gene expression. The biological significance of the link between peripheral and brain inflammation was explored in the APP/PS1 mouse model of Alzheimer's disease (AD) whereby osteoarthritis resulted in neuroinflammation as well as exacerbation and acceleration of AD pathology.

Results

Induction of osteoarthritis exacerbated and accelerated the development of neuroinflammation, as assessed by glial cell activation and quantification of inflammation-related mRNAs, as well as Aβ pathology, assessed by the number and size of amyloid plaques, in the APP/PS1; Col1-IL1β^XAT compound transgenic mouse.

Conclusion

This work supports a model by which peripheral inflammation triggers the development of neuroinflammation and subsequently the induction of AD pathology. Better understanding of the link between peripheral localized inflammation, whether in the form of osteoarthritis, atherosclerosis or other conditions, and brain inflammation, may prove critical to our understanding of the pathophysiology of disorders such as Alzheimer's, Parkinson's and other neurodegenerative diseases.     

Vespa tropica venom suppresses lipopolysaccharide-mediated secretion of pro-inflammatory cyto-chemokines by abrogating nuclear factor-κ B activation in microglia

Objective and design

The present study was aimed to evaluate the anti-inflammatory potentials of Vespa tropica (VT) venom and its isolated peptides. Effects of whole venom and its two peptides (Vt1512 and Vt1386) on lipopolysaccharide (LPS) challenged BV-2 murine microglial cells was evaluated.

Materials

Mouse microglial cell line, BV-2 and crude venom extract as well as purified peptides from VT along with LPS from Salmonella enterica were used for the studies.

Treatment

BV-2 cells were treated with 500 ng/ml of LPS and different doses of crude wasp venom as well as purified peptides.

Methods

We used immunoblotting, cytokine bead arrays and fluorescence activated cell sorter (FACS) to evaluate the levels of various proteins, cytokines and reactive oxygen species (ROS).

Results

Our studies suggest that treatment with whole venom significantly reduces oxidative stress and LPS-stimulated activation of microglia. Also, purified peptides from crude venom exhibited potential anti-inflammatory properties. Further, whole venom was found to be targeting Akt and p38 MAPK pathways, leading to suppressed NF-κB phosphorylation in LPS challenged BV-2 cells.

Conclusions

VT venom possesses anti-inflammatory properties and can be further explored for their therapeutic potential in treating various inflammatory conditions of the central nervous system (CNS).     

Effects of black cohosh on the plasminogen activator system in vascular smooth muscle cells

Objective

The rhizome of the Cimicifuga racemosa plant (commonly known as black cohosh) has been used for menopausal complaints. Studies regarding the cardiovascular effects of black cohosh are lacking. We investigated the effect of black cohosh on the plasminogen activator system in cultured vascular smooth muscle cells (VSMCs).

Methods

VSMCs were isolated from rat aortae. Expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) proteins were evaluated by Western blot analysis and enzyme-linked immunosorbent assay, respectively. The activities of PAI-1 and t-PA in the conditioned media were assessed by fibrin overlay zymography. A 40% 2-propanol extract of black cohosh was used.

Results

Black cohosh extract (BcEx) stimulated the protein expression of PAI-1, but it did not affect that of t-PA. Vitamin E, a potent antioxidant, inhibited the BcEx-induced increase in PAI-1 expression, while ICI 182,780, an estrogen receptor antagonist, had no effect. Fibrin overlay zymography revealed that BcEx increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA by inducing a binding to PAI-1.

Conclusions

BcEx induces PAI-1 protein expression in the VSMCs likely via an oxidant mechanism. It also stimulates the enzyme activity of PAI-1 and reduces that of free t-PA. These findings suggest that black cohosh might exert a negative influence on fibrinolysis.     

Immunoregulatory effects of AFP domains on monocyte-derived dendritic cell function

Background

Neutrophil migration to an inflamed site constitutes the first line of the innate immune response against invading microorganisms. Given the crucial role of endogenous lectins in neutrophil mobilization and activation, lectins from exogenous sources have often been considered as putative modulators of leukocyte function. Lectins purified from snake venom have been described as galactoside ligands that induce erythrocyte agglutination and platelet aggregation. This study evaluated human neutrophil migration and activation by C-type lectin BJcuL purified from Bothrops jararacussu venom.

Results

Utilizing fluorescence microscopy, we observed that biotinylated-BJcuL was evenly distributed on the neutrophil surface, selectively inhibited by D-galactose. Lectin was able to induce modification in the neutrophil morphology in a spherical shape for a polarized observed by optical microscopy and exposure to BJcuL in a Boyden chamber assay resulted in cell migration. After 30 minutes of incubation with BJcuL we found enhanced neutrophil functions, such as respiratory burst, zymozan phagocytosis and an increase in lissosomal volume. In addition, BJcuL delays late apoptosis neutrophils.

Conclusion

These results demonstrate that BJcuL can be implicated in a wide variety of immunological functions including first-line defense against pathogens, cell trafficking and induction of the innate immune response since lectin was capable of inducing potent neutrophil activation.     

Donepezil suppresses intracellular Ca<Superscript>2+</Superscript> mobilization through the PI3K pathway in rodent microglia

Background

Microglia are resident innate immune cells which release many factors including proinflammatory cytokines or nitric oxide (NO) when they are activated in response to immunological stimuli. Pathophysiology of Alzheimer’s disease (AD) is related to the inflammatory responses mediated by microglia. Intracellular Ca²⁺ signaling is important for microglial functions such as release of NO and cytokines. In addition, alteration of intracellular Ca²⁺ signaling underlies the pathophysiology of AD, while it remains unclear how donepezil, an acetylcholinesterase inhibitor, affects intracellular Ca²⁺ mobilization in microglial cells.

Methods

We examined whether pretreatment with donepezil affects the intracellular Ca²⁺ mobilization using fura-2 imaging and tested the effects of donepezil on phagocytic activity by phagocytosis assay in rodent microglial cells.

Results

In this study, we observed that pretreatment with donepezil suppressed the TNFα-induced sustained intracellular Ca²⁺ elevation in both rat HAPI and mouse primary microglial cells. On the other hand, pretreatment with donepezil did not suppress the mRNA expression of both TNFR1 and TNFR2 in rodent microglia we used. Pretreatment with acetylcholine but not donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the nicotinic α7 receptors. In addition, sigma 1 receptors were not involved in the donepezil-induced suppression of the TNFα-mediated intracellular Ca²⁺ elevation. Pretreatment with donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the PI3K pathway in rodent microglial cells. Using DAF-2 imaging, we also found that pretreatment with donepezil suppressed the production of NO induced by TNFα treatment and the PI3K pathway could be important for the donepezil-induced suppression of NO production in rodent microglial cells. Finally, phagocytosis assay showed that pretreatment with donepezil promoted phagocytic activity of rodent microglial cells through the PI3K but not MAPK/ERK pathway.

Conclusions

These suggest that donepezil could directly modulate the microglial function through the PI3K pathway in the rodent brain, which might be important to understand the effect of donepezil in the brain.

     

The anti-inflammatory action of Bothrops jararaca snake antithrombin on acute inflammation induced by carrageenan in mice

Objective and design

Antithrombin is known as the most important natural coagulation inhibitor and has been shown to have anti-inflammatory properties. The present study aimed to investigate the effects of Bothrops jararaca antithrombin on acute inflammation induced by carrageenan in mice.

Methods

We evaluated the anti-inflammatory activity of antithrombin on models of paw edema formation, cell migration and leukocyte–endothelium interaction in mice (Swiss; n = 5). Acute inflammation was induced by the administration of carrageenan (15 mg kg^?1).

Results

Treatment with B. jararaca antithrombin (1 mg kg^?1) 1 h before or after carrageenan administration significantly inhibited paw edema formation, reduced cell influx to the peritoneal cavity due to reduction in the migration of polymorphonuclear cells, and attenuated leukocyte rolling in the microcirculation of the cremaster muscle.The effects of antithrombin on vascular and cellular events of inflammation were completely abolished by treatment with the cyclo-oxygenase inhibitor indomethacin (4 mg kg^?1), suggesting the involvement of prostacyclin in the mechanism of inflammation inhibition by B. jararaca antithrombin.

Conclusion

This work showed for the first time the anti-inflammatory properties of B. jararaca antithrombin on vascular and cellular events of inflammation. These findings suggest that antithrombin is effective in preventing paw edema formation, cell migration and leukocyte rolling induced by carrageenan in mice.     

LRP1 modulates the microglial immune response via regulation of JNK and NF-κB signaling pathways

Background

Neuroinflammation is characterized by microglial activation and the increased levels of cytokines and chemokines in the central nervous system (CNS). Recent evidence has implicated both beneficial and toxic roles of microglia when over-activated upon nerve injury or in neurodegenerative diseases, including Alzheimer’s disease (AD). The low-density lipoprotein receptor-related protein 1 (LRP1) is a major receptor for apolipoprotein E (apoE) and amyloid-β (Aβ), which play critical roles in AD pathogenesis. LRP1 regulates inflammatory responses in peripheral tissues by modulating the release of inflammatory cytokines and phagocytosis. However, the roles of LRP1 in brain innate immunity and neuroinflammation remain unclear.

Methods

In this study, we determined whether LRP1 modulates microglial activation by knocking down Lrp1 in mouse primary microglia. LRP1-related functions in microglia were also assessed in the presence of LRP1 antagonist, the receptor-associated protein (RAP). The effects on the production of inflammatory cytokines were measured by quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Potential involvement of specific signaling pathways in LRP1-regulated functions including mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) were assessed using specific inhibitors.

Results

We found that knocking down of Lrp1 in mouse primary microglia led to the activation of both c-Jun N-terminal kinase (JNK) and NF-κB pathways with corresponding enhanced sensitivity to lipopolysaccharide (LPS) in the production of pro-inflammatory cytokines. Similar effects were observed when microglia were treated with LRP1 antagonist RAP. In addition, treatment with pro-inflammatory stimuli suppressed Lrp1 expression in microglia. Interestingly, NF-κB inhibitor not only suppressed the production of cytokines induced by the knockdown of Lrp1 but also restored the down-regulated expression of Lrp1 by LPS.

Conclusions

Our study uncovers that LRP1 suppresses microglial activation by modulating JNK and NF-κB signaling pathways. Given that dysregulation of LRP1 has been associated with AD pathogenesis, our work reveals a critical regulatory mechanism of microglial activation by LRP1 that could be associated with other AD-related pathways thus further nominating LRP1 as a potential disease-modifying target for the treatment of AD.

     

Evaluation of in vivo labelled dendritic cell migration in cancer patients

Background

Dendritic Cell (DC) vaccination is a very promising therapeutic strategy in cancer patients. The immunizing ability of DC is critically influenced by their migration activity to lymphatic tissues, where they have the task of priming naïve T-cells. In the present study in vivo DC migration was investigated within the context of a clinical trial of antitumor vaccination. In particular, we compared the migration activity of mature Dendritic Cells (mDC) with that of immature Dendritic Cells (iDC) and also assessed intradermal versus subcutaneous administration.

Methods

DC were labelled with ^99mTc-HMPAO or ¹¹¹In-Oxine, and the presence of labelled DC in regional lymph nodes was evaluated at pre-set times up to a maximum of 72 h after inoculation. Determinations were carried out in 8 patients (7 melanoma and 1 renal cell carcinoma).

Results

It was verified that intradermal administration resulted in about a threefold higher migration to lymph nodes than subcutaneous administration, while mDC showed, on average, a six-to eightfold higher migration than iDC. The first DC were detected in lymph nodes 20–60 min after inoculation and the maximum concentration was reached after 48–72 h.

Conclusions

These data obtained in vivo provide preliminary basic information on DC with respect to their antitumor immunization activity. Further research is needed to optimize the therapeutic potential of vaccination with DC.     

MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult

Background

Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD) through over-activation of microglia, which consequently causes the excessive production of proinflammatory and neurotoxic factors, and impacts surrounding neurons and eventually induces neurodegeneration. Hence, prevention of microglial over-activation has been shown to be a prime target for the development of therapeutic agents for inflammation-mediated neurodegenerative diseases.

Methods

For in vitro studies, mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanism by which FLZ, a squamosamide derivative, mediates anti-inflammatory and neuroprotective effects in both lipopolysaccharide-(LPS)- and 1-methyl-4-phenylpyridinium-(MPP⁺)-mediated models of PD. For in vivo studies, a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-(MPTP-) induced PD mouse model was used.

Results

FLZ showed potent efficacy in protecting dopaminergic (DA) neurons against LPS-induced neurotoxicity, as shown in rat and mouse primary mesencephalic neuronal-glial cultures by DA uptake and tyrosine hydroxylase (TH) immunohistochemical results. The neuroprotective effect of FLZ was attributed to a reduction in LPS-induced microglial production of proinflammatory factors such as superoxide, tumor necrosis factor-α (TNF-α), nitric oxide (NO) and prostaglandin E₂ (PGE₂). Mechanistic studies revealed that the anti-inflammatory properties of FLZ were mediated through inhibition of NADPH oxidase (PHOX), the key microglial superoxide-producing enzyme. A critical role for PHOX in FLZ-elicited neuroprotection was further supported by the findings that 1) FLZ's protective effect was reduced in cultures from PHOX^-/- mice, and 2) FLZ inhibited LPS-induced translocation of the cytosolic subunit of p47^PHOX to the membrane and thus inhibited the activation of PHOX. The neuroprotective effect of FLZ demonstrated in primary neuronal-glial cultures was further substantiated by an in vivo study, which showed that FLZ significantly protected against MPTP-induced DA neuronal loss, microglial activation and behavioral changes.

Conclusion

Taken together, our results clearly demonstrate that FLZ is effective in protecting against LPS- and MPTP-induced neurotoxicity, and the mechanism of this protection appears to be due, at least in part, to inhibition of PHOX activity and to prevention of microglial activation.