Accumulation Of Plasma Proteins Research Articles

#363 Altered filtration of middle-sized molecules is related to cardiovascular disease-related proteins

Abstract Background and Aims Cardiovascular (CV) complications, driven by complex vascular-metabolic interactions, are the leading cause of frailty in chronic kidney disease (CKD) complications. Advancing knowledge on cardio-renal interactions is a key to preventive and treatment strategies. Still, the cost-effective tools and biomarkers for early detection of altered glomerular filtration that subsequently leads to accelerated cardiovascular aging and, thus, higher CV mortality are lacking. Our objective was to investigate how molecules of varying masses, specifically cystatin C and creatinine, are filtered in kidney in relation to variations of plasma protein levels that might accelerate vascular changes, ultimately leading to early vascular aging and chronic kidney disease (CKD). Method From the Malmo Diet Cancer cohort, 4426 out of the 5061 participants had cardiovascular and renal biomarkers measured (mean age of 57 ± 6 years). These biomarkers included factors related to tissue growth and repair (EGFR), matrix metalloproteinases (MMP-12), proinflammatory cytokines (TNF-R1, TNF-R2, CXCL13, CXCL5), markers of vascular repair (SCF), homeostatic cytokines (CCL19), anti-apoptotic proteins (REG-4), adipokines (FABP4, LEP), as well as other cardiovascular proteins (NT-pro-BNP, MB) and indicators of kidney function. These 13 proteins were analyzed using multiplex proximity extension assay (Olink Cardiovascular II and Oncology panels), and have molecular masses either like creatinine (0.113 kDa) or cystatin C (13.3 kDa). These proteins were previously shown to be associated with measured glomerular filtration rate (mGFR) by Christensson A et al. (Proteomics Clin App, 2018). Kidney function was estimated by using cystatin C (CAPA, CKD-EPIcys) and creatinine (Lund-Malmo revised, CKD-EPIcr) equations. The filtration ratio was presented as CAPA/LMrev and CKD-EPIcys/CKD-EPIcr. The subgroups of this ratio were <0.70. Results Mean kidney function was as follows: 65 (±15)/ 69 (±16)/ 78 (±13) and 76 (±14) mL/min/1.73 m2 (CAPA/ CKD-EPIcys/ LMrev and CKD-EPIcr, respectively). The CAPA/LMrev filtration ratio was 0.84 (±0.18), whereas CKD-EPIcys/cr was 0.91 (±0.19). The filtration ratio negatively correlated with TNF-R1, TNF-R2, SCF, MMP-12, FABP4, LEP, NT-pro-BNP, CCL19, CXCL5, CXCL13 and REG-4; positively to EGFR and MB (all p < 0.001). In the whole cohort, participants with filtration ratio below 0.7 had significantly higher levels of TNF-R1, TNF-R2, MMP12, FABP4, LEP, NT-pro-BNP, CCL19, REG-4, and CXCL13, but similar levels of EGFR. CXCL5 and SCF levels were higher at <0.70 compared to 0.84-0.99 and >1.0 (Fig. 1). Interestingly, in women, MB was highest at <0.70; SCF level was higher at <0.70 compared to 0.84-0.99 and >1.00; EGFR level was highest at 0.70-0.84 compared to 0.84-0.99. In men, higher FABP4, LEP, CCL19, and MB were observed at <0.70 compared to 0.84-0.99 and >1.00. Conclusion Altered glomerular filtration for middle-sized molecules is related to the accumulation of plasma proteins that have similar molecular mass to creatinine and cystatin C and have sex-specific patterns. The proteins studied have been previously shown to contribute to vascular aging, cardiac damage and remodeling, and chronic kidney disease, and thus enlighten complex mechanisms behind cardio-renal interactions.

Proteomics Analysis of R-Ras Deficiency in Oxygen Induced Retinopathy.

Small GTPase R-Ras regulates vascular permeability in angiogenesis. In the eye, abnormal angiogenesis and hyperpermeability are the leading causes of vision loss in several ischemic retinal diseases such as proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP). Oxygen-induced retinopathy (OIR) is the most widely used experimental model for these ischemic retinopathies. To shed more light on how the R-Ras regulates vascular permeability in pathological angiogenesis, we performed a comprehensive (>2900 proteins) characterization of OIR in R-Ras knockout (KO) and wild-type (WT) mice by sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. OIR and age-matched normoxic control retinas were collected at P13, P17, and P42 from R-Ras KO and WT mice and were subjected to SWATH-MS and data analysis. The most significant difference between the R-Ras KO and WT retinas was an accumulation of plasma proteins. The pathological vascular hyperpermeability during OIR in the R-Ras KO retina took place very early, P13. This led to simultaneous hypoxic cell injury/death (ferroptosis), glycolytic metabolism as well compensatory mechanisms to counter the pathological leakage from angiogenic blood vessels in the OIR retina of R-Ras deficient mice.

Pulmonary injury and oxidative stress in rats induced by inhaled sulfur mustard is ameliorated by anti-tumor necrosis factor-α antibody

Sulfur mustard (SM) is a bifunctional alkylating agent that causes severe injury to the respiratory tract. This is accompanied by an accumulation of macrophages in the lung and the release of the proinflammatory cytokine, tumor necrosis factor (TNF)α. In these studies, we analyzed the effects of blocking TNFα on lung injury, inflammation and oxidative stress induced by inhaled SM. Rats were treated with SM vapor (0.4 mg/kg) or air control by intratracheal inhalation. This was followed 15–30 min later by anti-TNFα antibody (15mg/kg, i.v.) or PBS control. Animals were euthanized 3 days later. Anti-TNFα antibody was found to blunt SM-induced peribronchial edema, perivascular inflammation and alveolar plasma protein and inflammatory cell accumulation in the lung; this was associated with reduced expression of PCNA in histologic sections and decreases in BAL levels of fibrinogen. SM-induced increases in inflammatory proteins including soluble receptor for glycation end products, its ligand, high mobility group box-1, and matrix metalloproteinase-9 were also reduced by anti-TNFα antibody administration, along with increases in numbers of lung macrophages expressing TNFα, cyclooxygenase-2 and inducible nitric oxide synthase. This was correlated with reduced oxidative stress as measured by expression of heme oxygenase-1 and Ym-1. Together, these data suggest that inhibiting TNFα may represent an efficacious approach to mitigating acute lung injury, inflammatory macrophage activation, and oxidative stress induced by inhaled sulfur mustard.

Accumulation of Plasma Proteins in Neurons, Purkinje Cells, and Bergmann Glia as a Marker of Functional Disruption of Blood-Brain Barrier in the Early Phase of Traumatic Brain Injury in the Post Mortem Examination

Accumulation of Plasma Proteins in Neurons, Purkinje Cells, and Bergmann Glia as a Marker of Functional Disruption of Blood-Brain Barrier in the Early Phase of Traumatic Brain Injury in the Post Mortem Examination

A Pilot Study on Thickened Blood Vessels in Endometrial Curettage in Dysfunctional Uterine Bleeding

Background: Dysfunctional uterine bleeding (DUB) is a common disease. About 10% of patients registered with DUB in Gynecology OPD for treatment in developing countries. There has been a change in the rhythmic cyclic menstruation that causes excessive and irregular bleeding. Lack of ovulation or oligo-ovulation leads to permanent estrogenic effects without opposing the progesterone effects. Other causes of the estrogen domain may have similar effects. The relative estrogen domain causes several changes in the uterine vascular system that causes menstrual bleeding.
 Objective: To determine the frequency and significance of thickened blood vessels in curettage of the endometrium in dysfunctional uterine bleeding.
 Materials and methods: It was a Retrospective cross-sectional pilot study conducted at Department of Gynecology and Obstetrics, Jinnah Hospital Lahore for one-year duration from January 2017 to December 2017.Hematoxylin and eosin (H&E) stained sections 30 consecutive incident cases of endometrial curettage due to dysfunctional uterine bleeding were examined by an optical microscope, with particular regard to the number of thickened blood vessels. All patients with history of endometrial curettage with dysfunctional uterine bleeding were included while curettage of uterine mucosa in patients with a specific etiology, such as endometritis, atypical hypertrophy, retained products, intrauterine devices (intrauterine device) were excluded.
 Results: Over 50% of patients were perimenopausal, i.e. 40-55 years of age. All endometrial procedures included thickened blood vessels. On average, there were about 8 blood vessels thickened by curettage of the endometrium.
 Conclusion: Estrogen-induced vascular lesions result in greater permeability, which can lead to the accumulation of various plasma proteins with increased intramural vascular thickening. These thickened veins may not contract properly, causing excessive bleeding and prolongation.

Common fibrin deposition and tissue plasminogen activator downregulation in nasal polyps with distinct inflammatory endotypes

Common fibrin deposition and tissue plasminogen activator downregulation in nasal polyps with distinct inflammatory endotypes

Endothelial Tpl2 Regulates Vascular Barrier Function Via Tight Junction Protein Claudin-5 Promoting CNS Cell Infiltration and Tumor Metastasis

Increased vascular permeability and leakage are hallmarks of several pathologies and determine disease progression and severity by facilitating inflammatory / metastatic cell infiltration. Using tissue-specific genetic ablation in endothelial cells, we have investigated in vivo the role of Tpl2, a MAP3 kinase with pleiotropic effects in inflammation and cancer. In response to pro-inflammatory stimuli, endothelial Tpl2 deletion alters Tight Junction (TJ) claudin-5 protein expression and attenuates the increase in vascular permeability, inflammatory cell infiltration and accumulation of plasma proteins. This results in significantly attenuated disease scores in Experimental Autoimmune Encephalomyelitis and significantly less tumor nodules in a haematogenic lung cancer metastasis model. Accordingly, pharmacologic inhibition of Tpl2 or siRNA-mediated Tpl2 knockdown recapitulates our findings and reduces lung metastatic tumor invasions. These results establish a novel endothelial-specific role for Tpl2 in the regulation of TJ-mediated endothelial permeability and highlight the therapeutic potential of Tpl2 blockade in chronic inflammatory and metastatic diseases.

Sequence and time course of depletion of cardiac cellular proteins and accumulation of plasma proteins in rat early ischemic myocardium.

In order to investigate the sequence and time course of fibronectin (Fn), fibrinogen (Fg), complement (C5), myoglobin (Mb), actin (HHF35), and desmin (Dm) for the diagnosis of early myocardial ischemia, the myocardial ischemia model was established in rats, the positive reaction areas of Fn, Fg and C5 and the depletion areas of Mb, HHF35 and Dm in the ischemic cardiomyocytes were studied with immunohistochemistry, image analysis technique and statistical system. The results showed that the depletion of Dm, HHF35 and Mb, and the positive staining of Fg and C5 in ischemic cardiomyocytes were found as early as 15 min after the myocardial ischemia, but the positive staining of Fn occurred till 3 h after myocardial ischemia. With the prolongation of ischemia, the areas of the depletion of Dm, HHF35, Mb and the positive staining of Fg, C5 and Fn gradually enlarged. It is suggested that all the six immunohistochemical markers are more sensitive than routine H&E staining, and that Dm, HHF35, Mb, Fg, C5 are more sensitive markers than Fn for detection of early myocardial ischemia.

A Disintegrin and Metalloproteinase 15‐Mediated Glycocalyx Disruption Contributes to Vascular Leakage During Septic Injury

Disruption of vascular endothelial barrier results in excessive accumulation of plasma proteins and fluid within the interstitial space, exacerbating tissue injury caused by infection or sepsis. There is an urgent need to explore effective strategies to restore vascular barrier integrity in patients with sepsis. Glycocalyx, an endothelial surface coating matrix composed of glycoproteins and proteinglycans attached to glycoaminoglycan chains, has been shown to be protective of vascular barrier function. Degradation of glycocalyx has been implicated in the progression of septic injury, evidenced by increased serum levels of glycocalyx degradation products, including soluble CD44 and hyaluronic acid in sepsis patients, correlating with severity of injury. Currently, the molecular mechanisms underlying glycocalyx degradation are poorly understood. Here, we investigated whether and how this process involves a disintegrin and metalloproteinase 15 (ADAM15), a transmembrane sheddase capable of cleaving the ectodomains of membrane‐bounded proteins. Based on intravital microscopic measurements of dextran (150 kD) exclusion, we comparatively assessed glycocalyx thickness in wild‐type and ADAM15 knockout mice subjected to septic insult via lipopolysaccharides (LPS) injection. Our results indicated that LPS treatment induced a remarkable reduction in glycocalyx thickness in wide type mice, a response significantly attenuated in ADAM15−/− mice, suggesting the critical role of ADAM15 in LPS‐induced glycocalyx injury. Moreover, glycocalyx in mouse pulmonary microvasculature was observed by electron microscopy. Consistently, ADAM15 deficiency prevented the loss of glycocalyx coverage on the endothelial surface in pulmonary vessels following LPS challenge. To elucidate the underlying molecular details, we performed an in vitro cleavage assay, which confirmed the ability of recombinant ADAM15 to cleave CD44, an important glycoprotein expressed in glycocalyx, in a dose‐dependent manner. Further proteomic analysis revealed that the cleavage site of CD44 fell within the membrane‐proximal region of its ectodomain. Functionally, incubating endothelial cells with CD44 ectodomain resulted in barrier dysfunction and adherens junction disorganization. In line with these in vitro findings, mice with impaired glycocalyx exhibited vascular hyperpermeability after LPS challenge, as indicated by increased Evans Blue‐bound albumin extravasation in the lungs as well as enhanced albumin leakage from mesenteric vessels. In contrast, microvessels from ADAM15−/− mice with well‐preserved glycocalyx structure displayed an attenuated permeability response. Taken together, our studies suggest a novel function of ADAM15 in mediating glycocalyx injury during septic challenge.Support or Funding InformationThis work was supported by the National Institutes of Health Grants GM097270 and HL070752.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Upper and lower airway remodelling mechanisms in asthma, allergic rhinitis and chronic rhinosinusitis: The one airway concept revisited.

Allergic rhinitis (AR), chronic rhinosinusitis (CRS) and asthma often co-exist. The one airway model proposes that disease mechanisms occurring in the upper airway may mirror lower airway events. Airway remodelling is the term used to describe tissue structural changes that occur in a disease setting and reflect the dynamic process of tissue restructuring during wound repair. Remodelling has been long identified in the lower airways in asthma and is characterized by epithelial shedding, goblet cell hyperplasia, basement membrane thickening, subepithelial fibrosis, airway smooth muscle hyperplasia and increased angiogenesis. The concept of upper airway remodelling has only recently been introduced, and data so far are limited and often conflicting, an indication that more detailed studies are needed. Whilst remodelling changes in AR are limited, CRS phenotypes demonstrate epithelial hyperplasia, increased matrix deposition and degradation along with accumulation of plasma proteins. Despite extensive research over the past years, the precise cellular and molecular mechanisms involved in airway remodelling remain incompletely defined. This review describes our current rather limited understanding of airway remodelling processes in AR, CRS and asthma and presents mechanisms both shared and distinct between the upper and lower airways. Delineation of shared and disease-specific pathogenic mechanisms of remodelling between the sinonasal system and the lung may guide the rational design of more effective therapeutic strategies targeting upper and lower airways concomitantly and improving the health of individuals with inflammatory airway diseases.

Efek Anti Inflamasi Enzim Bromelin Nanas Terhadap Osteoartritis

<p>Inflammation is a complex reaction of the body's immune system in the vascular tissue that causes the accumulation and activation of leukocytes and plasma proteins that occur during infection, poisoning or cell damage. Inflammation can be treated by using anti-inflammatory drugs such as NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) that serve as pain reliever, febrifuge, and anti-inflammatory. One of the most common chronic inflammatory diseases is osteoarthritis. Osteoarthritis is the most common form of arthritis in society,which has a major impact on public health problems. Currently there are many therapeutic modalities of both pharmacology and non-pharmacology for the management of osteoarthritis, therefore it is necessary to recommend effective treatment of osteoarthritis in Indonesia based on the latest scientific evidence. The use of NSAIDs that do not correspond to doses can cause side effects such as bleeding, gastric ulcers, worsening asthma symptoms and acute kidney failure. This is the health problem in Indonesia, especially for ordinary people who use NSAIDs with excessive doses, because they want to quickly relieve pain. Therefore, it is necessary to look for a safe anti-inflammatory drug and can reduce the pain caused to arthritis. <em>Ananascomosus</em> (pineapple) has long been used for various medical purposes. There are four different proteases in the pineapple, the two major enzymes in pineapple defined as bromelain’s stems and bromelain’s fruit. In the latest study there are various therapeutic benefits that have been proposed for bromelin, such as anti-inflammatory, reducing swelling, reducing pain, facilitating the digestive system, accelerating wound healing and acting as an anticoagulant.<strong></strong></p>

ITRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis.

Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency.

Assessing large-vessel endothelial permeability using near-infrared fluorescence imaging--brief report.

Loss of endothelial barrier function in arterial blood vessels is characteristic of vascular pathologies, including atherosclerosis. Here, we present a near-infrared fluorescence (NIRF) imaging methodology for quantifying endothelial permeability and macromolecular uptake in large arteries in the mouse and evaluate its applicability for studying mechanisms of vascular inflammation. To validate the NIRF methodology, macrovascular inflammation was induced in C57bl/6 mice by local tumor necrosis factor-α stimulation of the carotid artery or in apolipoprotein E-deficient mice by Western diet for 4 weeks. Evans blue dye, serving as plasma protein marker and fluorescent in the near-infrared spectrum, was given intravenously at different doses. Carotids and aorta were excised, and Evans blue dye fluorescence was assessed through whole vessel scan in an infrared imaging system. NIRF correlated to extraction-absorbance methodology for Evans blue dye quantification and was superior at discriminating plasma protein accumulation in tumor necrosis factor-α-stimulated carotids. NIRF allowed for focal quantification of increased arterial wall Evans blue dye uptake in (apolipoprotein E-deficient) mice. Importantly, NIRF left vessels intact for subsequent histological analysis or quantification of leukocyte subpopulations by flow cytometry. The described NIRF methodology provides a sensitive and rapid tool to locate and quantify macromolecular uptake in the wall of arterial blood vessels in vascular pathologies in mice.

Immune Dysregulation and Endothelial Dysfunction in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) corresponds to a heterogeneous group of severe clinical conditions characterized by precapillary pulmonary hypertension (PH) diagnosed when mean pulmonary artery pressure equals or exceeds 25 mm Hg at rest with normal pulmonary artery wedge pressure (≤15 mm Hg). According to the current clinical classification, PAH can be idiopathic (IPAH), heritable, drug or toxin induced, or associated with other diseases (eg, connective tissue diseases [CTDs], congenital heart diseases, HIV infection, and portal hypertension).1 PAH has a complex and multifactorial pathogenesis in which excessive migration and proliferation of pulmonary vascular cells (ie, endothelial cells [ECs] and smooth muscle cells [SMCs]) and dysregulated immune responses are critical contributors to the inappropriate pulmonary vascular remodeling. PAH is a fatal condition leading to right heart failure and death within 2 to 3 years after diagnosis if left untreated. During the last decade, therapeutic options for the treatment of this disease have improved exercise capacity, quality of life, and long-term outcomes. However, there is currently no cure available, and further insight into the disease pathophysiology is needed to advance drug development and to improve patient management. It is now widely accepted that altered immune mechanisms play a significant role in PAH by recruiting inflammatory cells, remodeling the pulmonary vasculature, and promoting autoimmune responses.2–4 Inflammation is a general term for the local accumulation of fluid, plasma proteins, and white blood cells that is initiated by physical injury, infection, or a local immune response. These phenomena represent the innate immune response. The innate immune response contributes to the activation of adaptive immunity, which is the response of antigen-specific lymphocytes to antigen, including the development of immunologic memory. The unique features of adaptive immunity, based on clonal selection of lymphocytes bearing antigen-specific receptors, provide the ability to recognize all pathogens specifically …

Restoration of dietary-fat induced blood–brain barrier dysfunction by anti-inflammatory lipid-modulating agents

BackgroundSeveral studies have identified use of non-steroidal-anti-inflammatory drugs and statins for prevention of dementia, but their efficacy in slowing progression is not well understood. Cerebrovascular disturbances are common pathological feature of Alzheimer’s disease. We previously reported chronic ingestion of saturated fatty acids (SFA) compromises blood–brain barrier (BBB) integrity resulting in cerebral extravasation of plasma proteins and inflammation. However, the SFA-induced parenchymal accumulation of plasma proteins could be prevented by co-administration of some cholesterol lowering agents. Restoration of BBB dysfunction is clinically relevant, so the purpose of this study was to explore lipid-lowering agents could reverse BBB disturbances induced by chronic ingestion of SFA’s.MethodsWild-type mice were fed an SFA diet for 12 weeks to induce BBB dysfunction, and then randomised to receive atorvastatin, pravastatin or ibuprofen in combination with the SFA-rich diet for 2 or 8 weeks. Abundance of plasma-derived immunoglobulin-G (IgG) and amyloid-β enriched apolipoprotein (apo)-B lipoproteins within brain parenchyme were quantified utilising immunofluorescence microscopy.ResultsAtorvastatin treatment for 2 and 8 weeks restored BBB integrity, indicated by a substantial reduction of IgG and apo B, particularly within the hippocampus. Pravastatin, a water-soluble statin was less effective than atorvastatin (lipid-soluble). Statin effects were independent of changes in plasma lipid homeostasis. Ibuprofen, a lipid-soluble cyclooxygenase inhibitor attenuated cerebral accumulation of IgG and apo B as effectively as atorvastatin. Our findings are consistent with the drug effects being independent of plasma lipid homeostasis.ConclusionOur findings suggest that BBB dysfunction induced by chronic ingestion of SFA is reversible with timely introduction and sustained treatment with agents that suppress inflammation.

Cutaneous polyarteritis nodosa: A report of 16 cases with clinical and histopathological analysis and a review of the published work

Sixteen cases of cutaneous polyarteritis nodosa referred to our Department from 1985 to 2003 were studied clinically and histopathologically. Laboratory data, treatments and clinical courses were also evaluated retrospectively. All cases had nodules and/or indurated erythemas on their lower extremities. All cases showed necrotizing vasculitis of small muscular arteries in the subcutaneous tissues and/or occlusion of those arteries histopathologically. Fifteen cases also had accumulation of plasma protein in vessels of the dermis and subcutaneous tissues. Laboratory data showed high activity of platelets and coagulation in some cases. Eleven cases had been effectively treated with non-steroidal anti-inflammatory drugs. Eight cases were observed for at least 5 years (the longest for approximately 19 years) and had good prognoses and no systemic involvement. Cutaneous polyarteritis nodosa seems to be a benign disease, and differs from systemic polyarteritis nodosa although their histopathological features are common. Cutaneous polyarteritis nodosa might involve local dysfunction of the circulation from the dermis to the subcutaneous area. A review of the published work shows that the cause(s) of most cases of cutaneous polyarteritis nodosa is unknown, that no controlled trials for treatment of cutaneous polyarteritis nodosa compared to polyarteritis nodosa have been reported, and that no definitively effective therapy for cutaneous polyarteritis nodosa has been established.

Transfusion-Related Acute Lung Injury (TRALI) Requires Heterotypic Interactions of Platelets with Specific Neutrophil Microdomains

Neutrophil and platelet recruitment and activation into inflamed organs play key roles in the acute inflammatory response. Leukocyte recruitment is initiated by interactions with endothelial selectins, followed by activation of integrins which interact with their counter-receptors on the inflamed endothelium. Before migrating into the surrounding tissue, neutrophils actively crawl on the inflamed endothelium displaying a characteristic polarization with distinct microdomains at the leading (LE) and trailing edges (TE). During this period, they establish frequent interactions with circulating erythrocytes through the LE, which in the context of sickle cell disease can lead to vascular occlusion (Turhan et al., P.N.A.S. 2002 and unpublished data). During high-speed multichannel fluorescence intravital microscopy (MFIM) experiments, we have noted that neutrophils (identified by CD45+ Gr-1+ F4/80- expression) crawling on the inflamed endothelium actively interact with circulating platelets (identified in vivo by CD41 expression). In conditions in which inflammation was induced by surgical trauma only, platelet interactions were relatively rare and mediated by both the TE (identified in vivo by L-selectin clustering) and LE (opposite to L-selectin clusters). By contrast, in venules inflamed by the administration of TNFα, platelet interactions with the LE of crawling neutrophils were markedly increased. We have previously reported (Blood 110: 145; Nov. 2007) that sickle erythrocyte interactions in inflamed venules occurred at the LE of neutrophils through E-selectin-induced activation of the integrin Mac-1. We analyzed the role of E-selectin and Mac-1 in platelet capture and found a marked reduction in captures at the LE in mice lacking E-selectin, whereas these were virtually absent in mice deficient in Mac-1. These findings indicate that the increase in platelet capture during inflammation is the result of Mac-1 activation by endothelial E-selectin. Transfusion-related acute lung injury (TRALI), the leading cause of transfusion-related morbidity and mortality, is an acute inflammatory process mediated by intravascular neutrophils. Using a murine model of TRALI in which Balb/c mice are infused with high doses of anti-MHC-I antibodies (anti-H2d), we find that the induction of lung injury (assessed by accumulation of plasma proteins in the broncho-alveolar lavage (BAL)) correlates with a moderate thrombocytopenia and that depletion of platelets prior to anti-H2d infusion results in complete prevention of lung injury. We thus reasoned that neutrophil-platelet interactions might be required for TRALI. To assess this possibility, we analyzed intravascular events by MFIM of inflamed cremasteric venules before and after injection of anti-H2d. Platelet captures by crawling neutrophils were increased by 2-fold after anti-H2d antibody infusion, and this increase was prevented by blocking E-selectin. Infusion of anti-H2d markedly increased (9-fold) vascular permeability (measured by FITC-dextran leakage) that could be prevented by blocking E-selectin or Mac-1, or by depleting platelets. We next assessed in vivo activation of crawling neutrophils after anti-H2d infusion by analyzing the presence of reactive oxygen species (ROS) using the fluorescent probe dihydroxyrhodamine-123. We found that ROS were rapidly induced in crawling leukocytes following anti-H2d infusion, and that this induction could be blunted by platelet depletion, indicating a requirement for heterotypic interactions during neutrophil activation. To determine whether observations made in the cremasteric circulation correlated with the pulmonary injury caused by anti- H2d infusion, we measured protein accumulation in BAL fluids of mice pretreated with antibodies against E-selectin or Mac-1. Inhibition of either receptor strongly prevented the development of lung injury. In addition, n-acetyl-cysteine, a ROS scavenging molecule, completely prevented lung injury induced by anti-H2d. These results suggest that E-selectin-induced, Mac-1-mediated platelet capture by the LE of crawling neutrophils result in ROS production, which are responsible for the vascular and organ injuries observed in this model of TRALI. The heterotypic interactions described here share mechanisms with those triggering vaso-occlusive episodes in a humanized murine model of sickle cell disease, suggesting that they play important and previously unappreciated contributions to thrombo-inflammatory complications.

Monitoring the Capillary‐Alveolar Leakage in an A.R.D.S. Model Using Broncho‐Alveolar Lavage

We developed a modified broncho-alveolar lavage (BAL) technique in order to perform repeated measurements of capillary-alveolar leakage of a macromolecule in oleic acid (OA)-induced lung injury. BAL was performed in anesthetized dogs in a closed lung sampling site, using a bronchoscope fitted with an inflatable cuff. Fluorescein-labeled Dextran (FITC-D70) was continuously infused and its concentration measured in plasma and BAL fluid. A two-compartment model (blood and alveoli) was used to calculate K(AB), the transport-rate coefficient of FITC-D70 from blood to alveoli. K(AB) was estimated every 15 minutes over three hours. K(AB), close to zero at base-line both in control (n = 3) and in OA-injured lungs (n = 7), reached a peak in permeability 30 minutes after the induction of OA injury (K(AB) = 1.43 +/- 0.31 . 10(- 3) . minutes(- 1)), followed by a slow recovery. We conclude that this technique allows the monitoring of capillary-alveolar transport of macromolecules in in vivo experimental models. This monitoring may prove useful to study the mechanisms of the exudative stage of acute lung injury and to test therapies aimed at slowing the alveolar accumulation of plasma proteins and procoagulant factors that contribute to alveolar fibrosis.

Montelukast inhibits inflammatory responses in small airways of the Guinea-pig

Increased resistance in the small airways is a major contributor of airway obstruction in asthma. The role of leukotrienes (LT) in determining inflammation and obstruction of small size bronchi is not completely understood. Here, we have examined the effect of the cysteinyl-leukotriene ( CysLT 1) receptor antagonist, montelukast, against the bronchoconstriction and inflammatory responses induced by exogenous leukotriene and by allergen challenge in small size (⩽1 mm) Guinea-pig bronchi. Montelukast potently (pA 2 8.3) inhibited the contraction induced by LTD 4 in small bronchi taken from naïve Guinea-pigs. Furthermore, montelukast reduced the contraction produced by in vitro ovalbumin (OVA) challenge in small size bronchi from sensitized Guinea-pigs. Montelukast (10 μg kg −1) also blocked plasma protein extravasation and accumulation of inflammatory cells (eosinophils) induced by OVA challenge in small intra-parenchymal bronchi of OVA sensitized animals. These findings provide additional evidence that CysLT 1 receptor antagonism reduces allergic reactions that cause contractile and inflammatory responses in Guinea-pig small airways during OVA challenge. If the anti-bronchospastic and anti-inflammatory actions of the CysLT 1 receptor antagonists observed in the small airways of Guinea-pigs occur also in man these effects may contribute to the beneficial effects of montelukast in asthmatic patients.

Tissue remodelling in upper airways: where is the link with lower airway remodelling?

Tissue remodelling reported in upper airways include epithelial hyperplasia, increased matrix deposition in the nasal or paranasal lining, matrix degradation and accumulation of plasma proteins. Genetic influences, foetal exposures and early life events may contribute to structural changes such as subepithelial fibrosis from an early age. Other structural alterations are related to duration of the disease and long-term uncontrolled inflammation. Structural changes may increase alteration of the protective functions of the upper airways namely by affecting mucociliary clearance and conditioning of inspired air. The sequences of tissue changes during wound repair of upper airway mucosa after surgery are illustrative of the complexicity of tissue modelling and remodelling and could be considered as an important source of information for a better understanding of the complex relationship between inflammatory reaction, of the subsequent tissue damages and fibroblast metabolism of upper airways.