Heparin Interaction with the Primed Polymorphonuclear Leukocyte CD11b Induces Apoptosis and Prevents Cell Activation

Heparin is known to have anti-inflammatory effects, yet the mechanisms are not completely understood. In this study, we tested the hypothesis that heparin has a direct effect on activated polymorphonuclear leukocytes (PMNLs), changing their activation state, and can explain its anti-inflammatory effect. To test our hypothesis, we designed both in vitro and ex vivo studies to elucidate the mechanism by which heparin modulates PMNL functions and therefore the inflammatory response. We specifically tested the hypothesis that priming of PMNLs renders them more susceptible to heparin. Amplified levels of CD11b and increased rate of superoxide release manifested PMNL priming. Increase in cell priming resulted in a dose-dependent increase in heparin binding to PMNLs followed by augmented apoptosis. Blocking antibodies to CD11b inhibited heparin binding and abolished the apoptotic response. Moreover, heparin caused a significant dose-dependent decrease in the rate of superoxide release from PMNLs, which was blunted by blocking antibodies to CD11b. Altogether, this study shows that the interaction of heparin with the PMNL CD11b results in cell apoptosis and explains heparin’s anti-inflammatory effects

Elastase and Cathepsin G from Primed Leukocytes Cleave Vascular Endothelial Cadherin in Hemodialysis Patients

Aims. To test the hypothesis that primed PMNLs in blood of chronic kidney disease patients release the active form of elastase and cathepsin G causing degradation of vital proteins and promote tissue damage. Methods. RT-PCR, immunocytochemical staining, immunoblotting, and FACS analyses were used to study these enzymes in hemodialysis patients (HD) versus healthy normal controls (NC). Using PMNLs and endothelial cells cocultivation system we measure the effect of HD PMNLs on the endothelial VE-cadherin, an essential protein for maintaining endothelial integrity. Results. Levels of elastase and cathepsin G were reduced in PMNLs of HD patients, while mRNA enzymes levels were not different. Elevated levels of the active form of these enzymes were found in blood of HD patients compared to NC.HD plasma had higher levels of soluble VE-cadherin present in three molecular forms: whole 140 kDa molecule and two fragments of 100 and 40 kDa. Cocultivation studies showed that primed PMNLs cleave the endothelial cadherin, resulting in a 100 kDa fragment. Conclusions. Elastase and cathepsin G are elevated in the plasma of HD patients, originating from primed PMNLs. In these patients, chronic elevation of these enzymes contributes to cleavage of VE-cadherin and possible disruption of endothelial integrity

Unexpected Normal Colloid Osmotic Pressure in Clinical States with Low Serum Albumin

<div><p>Background</p><p>In clinical states associated with systemic oxidative stress (OS) and inflammation such as chronic kidney disease (CKD), oxidative modifications of serum albumin impair its quantification, resulting in apparent hypoalbuminemia. As the maintenance of oncotic pressure/colloid osmotic pressure (COP) is a major function of albumin, this study examined the impact of albumin oxidation on COP, both in-vivo and in-vitro.</p><p>Methods</p><p>Patients with proteinuria and patients on chronic hemodialysis (HD) with systemic inflammation and OS were enrolled. Blood samples were collected from 134 subjects: 32 healthy controls (HC), proteinuric patients with high (n = 17) and low (n = 31) systemic inflammation and from 54 patients on chronic hemodialysis (HD) with the highest levels of OS and inflammation.</p><p>Results</p><p>In-vitro oxidized albumin showed significantly higher COP values than non-oxidized albumin at identical albumin levels. In vivo, in hypoalbuminemic HD patients with the highest OS and inflammation, COP values were also higher than expected for the low albumin levels. The contribution to COP by other prevalent plasma proteins, such as fibrinogen and immunoglobulins was negligible.</p><p>We imply that the calculation of COP based on albumin levels should be revisited in face of OS and inflammation. Hence, in hypoalbuminemic proteinuric patients with systemic OS and inflammation the assumption of low COP should be verified by its measurements.</p></div

The association of COP with albumin detection index in sera.

<p>The association of COP (measured in serum) with albumin detection index was analyzed using selected subject's sera with albumin levels within a narrow range of 1.9–2.9 g/dl. HC sera with albumin levels within this range were obtained by partial albumin depletion (Alb-depl.HC).</p

Heparin Interaction with the Primed Polymorphonuclear Leukocyte CD11b Induces Apoptosis and Prevents Cell Activation.

Heparin is known to have anti-inflammatory effects, yet the mechanisms are not completely understood. In this study, we tested the hypothesis that heparin has a direct effect on activated polymorphonuclear leukocytes (PMNLs), changing their activation state, and can explain its anti-inflammatory effect. To test our hypothesis, we designed both in vitro and ex vivo studies to elucidate the mechanism by which heparin modulates PMNL functions and therefore the inflammatory response. We specifically tested the hypothesis that priming of PMNLs renders them more susceptible to heparin. Amplified levels of CD11b and increased rate of superoxide release manifested PMNL priming. Increase in cell priming resulted in a dose-dependent increase in heparin binding to PMNLs followed by augmented apoptosis. Blocking antibodies to CD11b inhibited heparin binding and abolished the apoptotic response. Moreover, heparin caused a significant dose-dependent decrease in the rate of superoxide release from PMNLs, which was blunted by blocking antibodies to CD11b. Altogether, this study shows that the interaction of heparin with the PMNL CD11b results in cell apoptosis and explains heparin's anti-inflammatory effects

COP values of purified albumin from HC and HD sera.

<p>Albumin was purified from HD and HC sera, diluted to give equal concentrations and used for COP measurements after addition (of equal volume of each sample) to a normal (HC) sample with known albumin level and index (a single HC serum was used in each experiment). Albumin quantification by BCG (<b>A</b>), COP values (calculated per 1g/dl of the purified albumin and given as % of unmodified HSA) (<b>B</b>), and the albumin detection index values (<b>C</b>) are given as mean+SD. Glycoxidized (AGE) and unmodified (U) HSA that were treated identically, served as positive and negative controls, respectively. # indicates a significant p value (p<0.05). N = 5.</p

Characteristics of the study population.

<p>Characteristics of the study population.</p

COP of in-vitro oxidized human serum albumin.

<p>Commercial HSA was glycoxidized <b>(A,C)</b> or oxidized by hypochlorite <b>(B,D)</b> <i>in-vitro</i> to produce AGE-albumin (AGE) and AOPP, respectively. COP values were correlated with albumin levels that were measured by BCG <b>(A,B)</b> or by OD₂₈₀ <b>(C,D)</b> in the oxidized (●) and control (C;□) samples.</p

Albumin levels and COP values in the study groups.

<p>Sera samples from HC, proteinuria patients with low or high degree of inflammation (normal CRP. and high CRP, respectively), HD patients and HC sera after partial albumin depletion (Alb-depl.HC) were used for determination of albumin levels by BCG and measurements of COPCOP values were correlated with albumin levels measured by BCG in sera of all subjects <b>(A).</b> The separate regression lines are given for each subjects' group and for HC sera after partial albumin depletion (Alb-depl.HC) <b>(B)</b>. The BCG-measured albumin levels were also correlated with the albumin detection index (insert in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159839#pone.0159839.g001" target="_blank">Fig 1A</a>).</p

<i>In-vivo</i> oxidized albumin– a pro-inflammatory agent in hypoalbuminemia

<div><p>Hypoalbuminemia of Hemodialysis (HD) patients is an independent cardiovascular risk factor, however, there is no mechanistic explanation between hypoalbuminemia and vascular injury. In the event of oxidative stress and inflammation to which HD patients are exposed, albumin is oxidized and undetected by common laboratory methods, rendering an apparent hypoalbuminemia. We wanted to show that these circulating modified oxidized albumin molecules cause direct vascular damage, mediating inflammation. Once these <i>in-vivo</i> albumin modifications were reduced <i>in- vitro</i>, the apparent hypoalbuminemia concomitantly with its inflammatory effects, were eliminated. Albumin modification profiles from 14 healthy controls (HC) and 14 HD patients were obtained by mass spectrometry (MS) analyses before and after reduction <i>in- vitro</i>, using redox agent 1,4 dithiothreitol (DTT). Their inflammatory effects were explored by exposing human umbilical endothelial cells (HUVEC) to all these forms of albumin. Albumin separated from hypoalbuminemic HD patients increased endothelial mRNA expression of cytokines and adhesion molecules, and augmented secretion of IL-6. This endothelial inflammatory state was almost fully reverted by exposing HUVEC to the <i>in-vitro</i> reduced HD albumin. MS profile of albumin modifications peaks was similar between HD and HC, but the intensities of the various peaks were significantly different. Abolishing the reversible oxidative modifications by DTT prevented endothelial injury and increased albumin levels. The irreversible modifications such as glycation and sulfonation show low intensities in HD albumin profiles and are nearly unobserved in HC. We showed, for the first time, a mechanistic link between hypoalbuminemia and the pro-inflammatory properties of <i>in-vivo</i> oxidized albumin, initiating vascular injury.</p></div