Homocysteinylated albumin promotes increased monocyte-endothelial cell adhesion and up-regulation of MCP1, Hsp60 and ADAM17

Rationale

The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties.

Objective

Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels.

Methods and Results

Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2.

Conclusions

Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.     

Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease

Background and Aims

Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro.

Methodology and Principal Findings

The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF). Nitric oxide (NO) donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS) increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups.

Conclusion/Significance

Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially explained by a reduced eNOS expression. In addition, our data show that the disease primes endothelial cells in vivo, which keep the acquired phenotype in culture.     

Endothelium Trans Differentiated from Wharton's Jelly Mesenchymal Cells Promote Tissue Regeneration: Potential Role of Soluble Pro-Angiogenic Factors

Background

Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton''s jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton''s jelly (hWMSCs) can accelerate tissue repair in vivo.

Methods

Mesenchymal stem cells were isolated from human Wharton''s jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO).

Results

hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures.

Conclusion

These results demonstrate that mesenchymal stem cells isolated from Wharton''s jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.     

Diminished Neurogenic Femoral Artery Vasoconstrictor Response in a Zucker Obese Rat Model: Differential Regulation of NOS and COX Derivatives

Objective

Peripheral arterial disease is one of the macrovascular complications of type 2 diabetes mellitus. This study addresses femoral artery regulation in a prediabetic model of obese Zucker rats (OZR) by examining cross-talk between endothelial and neural factors.

Methods and Results

Arterial preparations from lean (LZR) and OZR were subjected to electrical field stimulation (EFS) on basal tone. Nitric oxide synthase (NOS) and cyclooxygenase (COX) isoform expression patterns were determined by immunohistochemical labelling and Western blotting. Results indicate significantly reduced noradrenergic contractions in preparations from OZR compared with those of LZR. Functional inhibition of endothelial NOS (eNOS) indicated a predominant role of this isoform in LZR and its modified activity in OZR. Neural (nNOS) and inducible NOS (iNOS) were activated and their expression was higher in femoral arteries from OZR. Neurotransmission modulated by large-conductance Ca²⁺-activated (BK_Ca) or voltage-dependent (K_V) K⁺ channels did not seem compromised in the obese animals. Endothelial COX-1 and COX-2 were expressed in LZR and an additional adventitial location of COX-2 was also observed in OZR, explaining the higher COX-2 protein levels detected in this group. Prostanoids derived from both isoforms helped maintain vasoconstriction in LZR while in OZR only COX-2 was active. Superoxide anion inhibition reduced contractions in endothelium-intact arteries from OZR.

Conclusions

Endothelial dysfunction led to reduced neurogenic vasoconstriction in femoral arteries from OZR. In a setting of obesity, NO-dependent nNOS and iNOS dilation activity could be an alternative mechanism to offset COX-2- and reactive oxygen species-mediated vasoconstriction, along with impaired endothelial NO relaxation.     

Nitric oxide synthases in infants and children with pulmonary hypertension and congenital heart disease

Rationale

Nitric oxide is an important regulator of vascular tone in the pulmonary circulation. Surgical correction of congenital heart disease limits pulmonary hypertension to a brief period.

Objectives

The study has measured expression of endothelial (eNOS), inducible (iNOS), and neuronal nitric oxide synthase (nNOS) in the lungs from biopsies of infants with pulmonary hypertension secondary to cardiac abnormalities (n = 26), compared to a control group who did not have pulmonary or cardiac disease (n = 8).

Methods

eNOS, iNOS and nNOS were identified by immunohistochemistry and quantified in specific cell types.

Measurements and main results

Significant increases of eNOS and iNOS staining were found in pulmonary vascular endothelial cells of patients with congenital heart disease compared to control infants. These changes were confined to endothelial cells and not present in other cell types. Patients who strongly expressed eNOS also had strong expression of iNOS.

Conclusion

Upregulation of eNOS and iNOS occurs at an early stage of pulmonary hypertension, and may be a compensatory mechanism limiting the rise in pulmonary artery pressure.     

The Timing of the Shrew: Continuous Melatonin Treatment Maintains Youthful Rhythmic Activity in Aging Crocidura russula

Background

Laboratory conditions nullify the extrinsic factors that determine the wild expected lifespan and release the intrinsic or potential lifespan. Thus, wild animals reared in a laboratory often show an increased lifespan, and consequently an increased senescence phase. Senescence is associated with a broad suite of physiological changes, including a decreased responsiveness of the circadian system. The time-keeping hormone melatonin, an important chemical player in this system, is suspected to have an anti-aging role. The Greater White-toothed shrew Crocidura russula is an ideal study model to address questions related to aging and associated changes in biological functions: its lifespan is short and is substantially increased in captivity; daily and seasonal rhythms, while very marked the first year of life, are dramatically altered during the senescence process which starts during the second year. Here we report on an investigation of the effects of melatonin administration on locomotor activity of aging shrews.

Methodology/Principal Findings

1) The diel fluctuations of melatonin levels in young, adult and aging shrews were quantified in the pineal gland and plasma. In both, a marked diel rhythm (low diurnal concentration; high nocturnal concentration) was present in young animals but then decreased in adults, and, as a result of a loss in the nocturnal production, was absent in old animals. 2) Daily locomotor activity rhythm was monitored in pre-senescent animals that had received either a subcutaneous melatonin implant, an empty implant or no implant at all. In non-implanted and sham-implanted shrews, the rhythm was well marked in adults. A marked degradation in both period and amplitude, however, started after the age of 14–16 months. This pattern was considerably delayed in melatonin-implanted shrews who maintained the daily rhythm for significantly longer.

Conclusions

This is the first long term study (>500 days observation of the same individuals) that investigates the effects of continuous melatonin delivery. As such, it sheds new light on the putative anti-aging role of melatonin by demonstrating that continuous melatonin administration delays the onset of senescence. In addition, the shrew appears to be a promising mammalian model for elucidating the precise relationships between melatonin and aging.     

HIV-1 RNA Dysregulates the Natural TLR Response to Subclinical Endotoxemia in Kenyan Female Sex-Workers

Background

Subclinical endotoxemia has been reported in HIV-1 infected persons and may drive systemic immune activation and pathogenesis. Proinflammatory responsiveness to endotoxin (LPS) is mediated by Toll-like receptor 4 (TLR4). We therefore examined the association between plasma LPS levels, HIV RNA, and TLR4 expression and cytokine responses in the blood of HIV infected and uninfected participants in a cohort of female sex-workers in Kenya.

Methodology/Principal Findings

Ex vivo plasma and peripheral blood mononuclear cells (PBMC) were assessed for LPS and TLR mRNA, respectively. The effects of HIV single stranded RNA, a TLR8 ligand, on TLR4 and LPS signaling were further assessed in short term PBMC culture. Both HIV uninfected and infected subjects frequently had low detectable LPS levels in their plasmas. Significantly increased LPS levels were associated with chronic HIV-1 infection, both treated and untreated, but not with other acute or semi-chronic conditions reported. In HIV-uninfected subjects, TLR4 mRNA expression levels correlated inversely with plasma LPS levels, suggesting chronic endotoxin ‘tolerance’ in vivo. A similar effect of reduced TLR4 mRNA was seen in short term PBMC culture after stimulation with LPS. Interestingly, the apparent in vivo tolerance effect was diminished in subjects with HIV infection. Additionally, pre-stimulation of PBMC with LPS lead to proinflammatory (TNF-α) tolerance to subsequent LPS stimulation; however, pre-treatment of PBMC with HIV single-stranded RNA40, could enhance TLR4-mediated LPS responsiveness in vitro.

Conclusions/Significance

Thus, dysregulation of endotoxin tolerance by HIV-1 RNA may exacerbate HIV chronic immune activation and pathogenesis.     

Sphingosine-1-phosphate promotes the differentiation of adipose-derived stem cells into endothelial nitric oxide synthase (eNOS) expressing endothelial-like cells

Background

Adipose tissue provides a readily available source of autologous stem cells. Adipose-derived stem cells (ASCs) have been proposed as a source for endothelial cell substitutes for lining the luminal surface of tissue engineered bypass grafts. Endothelial nitric oxide synthase (eNOS) is a key protein in endothelial cell function. Currently, endothelial differentiation from ASCs is limited by poor eNOS expression. The goal of this study was to investigate the role of three molecules, sphingosine-1-phosphate (S1P), bradykinin, and prostaglandin-E1 (PGE1) in ASC endothelial differentiation. Endothelial differentiation markers (CD31, vWF and eNOS) were used to evaluate the level of ASCs differentiation capability.

Results

ASCs demonstrated differentiation capability toward to adipose, osteocyte and endothelial like cell phenotypes. Bradykinin, S1P and PGE were used to promote differentiation of ASCs to an endothelial phenotype. Real-time PCR showed that all three molecules induced significantly greater expression of endothelial differentiation markers CD31, vWF and eNOS than untreated cells. Among the three molecules, S1P showed the highest up-regulation on endothelial differentiation markers. Immunostaining confirmed presence of more eNOS in cells treated with S1P than the other groups. Cell growth measurements by MTT assay, cell counting and EdU DNA incorporation suggest that S1P promotes cell growth during ASCs endothelial differentiation. The S1P1 receptor was expressed in ASC-differentiated endothelial cells and S1P induced up-regulation of PI3K.

Conclusions

S1P up-regulates endothelial cell markers including eNOS in ASCs differentiated to endothelial like cells. This up-regulation appears to be mediated by the up-regulation of PI3K via S1P1 receptor. ASCs treated with S1P offer promising use as endothelial cell substitutes for tissue engineered vascular grafts and vascular networks.     

Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO

Background

Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.

Methods

Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.

Results

Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.

Conclusion

Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.     

Deletion of Aldose Reductase from Mice Inhibits Diabetes-Induced Retinal Capillary Degeneration and Superoxide Generation

Purpose

Pharmacologic inhibition of aldose reductase (AR) previously has been studied with respect to diabetic retinopathy with mixed results. Since drugs can have off-target effects, we studied the effects of AR deletion on the development and molecular abnormalities that contribute to diabetic retinopathy. Since recent data suggests an important role for leukocytes in the development of the retinopathy, we determined also if AR in leukocytes contributes to leukocyte-mediated death of retinal endothelial cells in diabetes.

Methods

Wild-type (WT; C57BL/6J) and AR deficient (AR^−/−) mice were made diabetic with streptozotocin. Mice were sacrificed at 2 and 10 months of diabetes to evaluate retinal vascular histopathology, to quantify retinal superoxide production and biochemical and physiological abnormalities in the retina, and to assess the number of retinal endothelial cells killed by blood leukocytes in a co-culture system.

Results

Diabetes in WT mice developed the expected degeneration of retinal capillaries, and increased generation of superoxide by the retina. Leukocytes from diabetic WT mice also killed more retinal endothelial cells than did leukocytes from nondiabetic animals (p<0.0001). Deletion of AR largely (P<0.05) inhibited the diabetes-induced degeneration of retinal capillaries, as well as the increase in superoxide production by retina. AR-deficiency significantly inhibited the diabetes-induced increase in expression of inducible nitric oxide synthase (iNOS) in retina, but had no significant effect on expression of intercellular adhesion molecule-1 (ICAM-1), phosphorylated p38 MAPK, or killing of retinal endothelial cells by leukocytes.

Conclusions

AR contributes to the degeneration of retinal capillaries in diabetic mice. Deletion of the enzyme inhibits the diabetes-induced increase in expression of iNOS and of superoxide production, but does not correct a variety of other pro-inflammatory abnormalities associated with the development of diabetic retinopathy.     

The critical role of lipopolysaccharide in the upregulation of aquaporin 4 in glial cells treated with Shiga toxin

Background

In 2011, there was an outbreak of Shiga toxin-producing Escherichia coli (STEC) infections in Japan. Approximately 62 % of patients with hemolytic-uremic syndrome also showed symptoms of encephalopathy. To determine the mechanisms of onset for encephalopathy during STEC infections, we conducted an in vitro study with glial cell lines and primary glial cells.

Results

Shiga toxin 2 (Stx-2) in combination with lipopolysaccharide (LPS), or LPS alone activates nuclear factor-κB (NF-κB) signaling in glial cells. Similarly, Stx-2 in combination with LPS, or LPS alone increases expression levels of aquaporin 4 (AQP4) in glial cells. It is possible that overexpression of AQP4 results in a rapid and increased influx of osmotic water across the plasma membrane into cells, thereby inducing cell swelling and cerebral edema.

Conclusions

We have showed that a combination of Stx-2 and LPS induced apoptosis of glial cells recently. Glial cells are indispensable for cerebral homeostasis; therefore, their dysfunction and death impairs cerebral homeostasis and results in encephalopathy. We postulate that the onset of encephalopathy in STEC infections occurs when Stx-2 attacks vascular endothelial cells of the blood–brain barrier, inducing their death. Stx-2 and LPS then attack the exposed glial cells that are no longer in contact with the endothelial cells. AQP4 is overexpressed in glial cells, resulting in their swelling and adversely affecting cerebral homeostasis. Once cerebral homeostasis is affected in such a way, encephalopathy is the likely result in STEC patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0184-5) contains supplementary material, which is available to authorized users.     

E-Selectin Mediated Adhesion and Migration of Endothelial Colony Forming Cells Is Enhanced by SDF-1α/CXCR4

Objective

Endothelial-colony forming cells (ECFCs) can be readily expanded from human umbilical cord blood and can facilitate repair of endothelial injury. E-selectin and SDF-1α are produced following endothelial injury and can regulate endothelial progenitor homing. Mechanisms of vascular repair specific to the mode of injury have not been well described in homogenous cell populations such as ECFCs and are needed for development of more effective vascular repair strategies.

Methods and Results

Lipopolysaccharide (LPS)-induced endotoxic injury to mature human umbilical vein endothelial cells (HUVEC) was compared with hypoxic and radiation injury. E-selectin expression in HUVEC cells is markedly increased (208-fold) following LPS-induced injury and facilitates increased ECFC adhesion and migration function in vitro. SDF-1α expression remains unchanged in LPS-treated HUVEC cells but increases more than 2 fold in fibroblasts undergoing similar endotoxic injury. SDF-1α induces expression of E-selectin ligands on ECFCs and facilitates greater E-selectin-mediated adhesion and migration of ECFCs in a CXCR4-dependent manner. Induction of E-selectin expression in HUVECs following hypoxic or radiation injury is negligible, however, while SDF-1α is increased markedly following hypoxia, highlighting injury-specific synergism between mediators of vascular repair.

Conclusion

E-selectin mediates adhesion and migration of ECFCs following endotoxic endothelial injury. SDF-1α augments E-selectin mediated ECFC adhesion and migration in a CXCR4-dependent manner.     

2-Hydroxy Arachidonic Acid: A New Non-Steroidal Anti-Inflammatory Drug

Background

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a family of COX1 and COX2 inhibitors used to reduce the synthesis of pro-inflammatory mediators. In addition, inflammation often leads to a harmful generation of nitric oxide. Efforts are being done in discovering safer NSAIDs molecules capable of inhibiting the synthesis of pro-inflammatory lipid mediators and nitric oxide to reduce the side effects associated with long term therapies.

Methodology/Principal Findings

The analogue of arachidonic acid (AA), 2-hydroxy-arachidonic acid (2OAA), was designed to inhibit the activities of COX1 and COX2 and it was predicted to have similar binding energies as AA for the catalytic sites of COX1 and COX2. The interaction of AA and 2OAA with COX1 and COX2 was investigated calculating the free energy of binding and the Fukui function. Toxicity was determined in mouse microglial BV-2 cells. COX1 and COX2 (PGH2 production) activities were measured in vitro. COX1 and COX2 expression in human macrophage-like U937 cells were carried out by Western blot, immunocytochemistry and RT-PCR analysis. NO production (Griess method) and iNOS (Western blot) were determined in mouse microglial BV-2 cells. The comparative efficacy of 2OAA, ibuprofen and cortisone in lowering TNF-α serum levels was determined in C57BL6/J mice challenged with LPS. We show that the presence of the –OH group reduces the likelihood of 2OAA being subjected to H* abstraction in COX, without altering significantly the free energy of binding. The 2OAA inhibited COX1 and COX2 activities and the expression of COX2 in human U937 derived macrophages challenged with LPS. In addition, 2OAA inhibited iNOS expression and the production of NO in BV-2 microglial cells. Finally, oral administration of 2OAA decreased the plasma TNF-α levels in vivo.

Conclusion/Significance

These findings demonstrate the potential of 2OAA as a NSAID.     

Paracrine Effects of Bone Marrow–Derived Endothelial Progenitor Cells: Cyclooxygenase-2/Prostacyclin Pathway in Pulmonary Arterial Hypertension

Background

Endothelial dysfunction is the pathophysiological characteristic of pulmonary arterial hypertension (PAH). Some paracrine factors secreted by bone marrow–derived endothelial progenitor cells (BMEPCs) have the potential to strengthen endothelial integrity and function. This study investigated whether BMEPCs have the therapeutic potential to improve monocrotaline (MCT)-induced PAH via producing vasoprotective substances in a paracrine fashion.

Methods and Results

Bone marrow-derived mononuclear cells were cultured for 7 days to yield BMEPCs. 24 hours or 3 weeks after exposure to BMEPCs in vitro or in vivo, the vascular reactivity, cyclooxygenase-2 (COX-2) expression, prostacyclin (PGI₂) and cAMP release in isolated pulmonary arteries were examined respectively. Treatment with BMEPCs could improve the relaxation of pulmonary arteries in MCT-induced PAH and BMEPCs were grafted into the pulmonary bed. The COX-2/prostacyclin synthase (PGIS) and its progenies PGI₂/cAMP were found to be significantly increased in BMEPCs treated pulmonary arteries, and this action was reversed by a selective COX-2 inhibitor, NS398. Moreover, the same effect was also observed in conditioned medium obtained from BMEPCs culture.

Conclusions

Implantation of BMEPCs effectively ameliorates MCT-induced PAH. Factors secreted in a paracrine fashion from BMEPCs promote vasoprotection by increasing the release of PGI₂ and level of cAMP.     

Mechanical ventilation with high tidal volumes attenuates myocardial dysfunction by decreasing cardiac edema in a rat model of LPS-induced peritonitis

Background

Injurious mechanical ventilation (MV) may augment organ injury remote from the lungs. During sepsis, myocardial dysfunction is common and increased endothelial activation and permeability can cause myocardial edema, which may, among other factors, hamper myocardial function. We investigated the effects of MV with injuriously high tidal volumes on the myocardium in an animal model of sepsis.

Methods

Normal rats and intraperitoneal (i.p.) lipopolysaccharide (LPS)-treated rats were ventilated with low (6 ml/kg) and high (19 ml/kg) tidal volumes (Vt) under general anesthesia. Non-ventilated animals served as controls. Mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO) and pulmonary plateau pressure (P_plat) were measured. Ex vivo myocardial function was measured in isolated Langendorff-perfused hearts. Cardiac expression of endothelial vascular cell adhesion molecule (VCAM)-1 and edema were measured to evaluate endothelial inflammation and leakage.

Results

MAP decreased after LPS-treatment and Vt-dependently, both independent of each other and with interaction. MV Vt-dependently increased CVP and Pplat and decreased CO. LPS-induced peritonitis decreased myocardial function ex vivo but MV attenuated systolic dysfunction Vt-dependently. Cardiac endothelial VCAM-1 expression was increased by LPS treatment independent of MV. Cardiac edema was lowered Vt-dependently by MV, particularly after LPS, and correlated inversely with systolic myocardial function parameters ex vivo.

Conclusion

MV attenuated LPS-induced systolic myocardial dysfunction in a Vt-dependent manner. This was associated with a reduction in cardiac edema following a lower transmural coronary venous outflow pressure during LPS-induced coronary inflammation.     

Modulating the Adhesion of Haematopoietic Stem Cells with Chemokines to Enhance Their Recruitment to the Ischaemically Injured Murine Kidney

Introduction

Renal disease affects over 500 million people worldwide and is set to increase as treatment options are predominately supportive. Evidence suggests that exogenous haematopoietic stem cells (HSCs) can be of benefit but due to the rarity and poor homing of these cells, benefits are either minor or transitory. Mechanisms governing HSC recruitment to injured renal microcirculation are poorly understood; therefore this study determined (i) the adhesion molecules responsible for HSC recruitment to the injured kidney, (ii) if cytokine HSC pre-treatment can enhance their homing and (iii) the molecular mechanisms accountable for any enhancement.

Methods

Adherent and free-flowing HSCs were determined in an intravital murine model of renal ischaemia-reperfusion injury. Some HSCs and animals were pre-treated prior to HSC infusion with function blocking antibodies, hyaluronidase or cytokines. Changes in surface expression and clustering of HSC adhesion molecules were determined using flow cytometry and confocal microscopy. HSC adhesion to endothelial counter-ligands (VCAM-1, hyaluronan) was determined using static adhesion assays in vitro.

Results

CD49d, CD44, VCAM-1 and hyaluronan governed HSC adhesion to the IR-injured kidney. Both KC and SDF-1α pre-treatment strategies significantly increased HSC adhesion within injured kidney, whilst SDF-1α also increased numbers continuing to circulate. SDF-1α and KC did not increase CD49d or CD44 expression but increased HSC adhesion to VCAM-1 and hyaluronan respectively. SDF-1α increased CD49d surface clustering, as well as HSC deformability.

Conclusion

Increasing HSC adhesive capacity for its endothelial counter-ligands, potentially through surface clustering, may explain their enhanced renal retention in vivo. Furthermore, increasing HSC deformability through SDF-1α treatment could explain the prolonged systemic circulation; the HSC can therefore continue to survey the damaged tissue instead of becoming entrapped within non-injured sites. Therefore manipulating these mechanisms of HSC recruitment outlined may improve the clinical outcome of cellular therapies for kidney disease.     

Enterocyte Shedding and Epithelial Lining Repair Following Ischemia of the Human Small Intestine Attenuate Inflammation

Background

Recently, we observed that small-intestinal ischemia and reperfusion was found to entail a rapid loss of apoptotic and necrotic cells. This study was conducted to investigate whether the observed shedding of ischemically damaged epithelial cells affects IR induced inflammation in the human small gut.

Methods and Findings

Using a newly developed IR model of the human small intestine, the inflammatory response was studied on cellular, protein and mRNA level. Thirty patients were consecutively included. Part of the jejunum was subjected to 30 minutes of ischemia and variable reperfusion periods (mean reperfusion time 120 (±11) minutes). Ethical approval and informed consent were obtained. Increased plasma intestinal fatty acid binding protein (I-FABP) levels indicated loss in epithelial cell integrity in response to ischemia and reperfusion (p<0.001 vs healthy). HIF-1α gene expression doubled (p = 0.02) and C3 gene expression increased 4-fold (p = 0.01) over the course of IR. Gut barrier failure, assessed as LPS concentration in small bowel venous effluent blood, was not observed (p = 0.18). Additionally, mRNA expression of HO-1, IL-6, IL-8 did not alter. No increased expression of endothelial adhesion molecules, TNFα release, increased numbers of inflammatory cells (p = 0.71) or complement activation, assessed as activated C3 (p = 0.14), were detected in the reperfused tissue.

Conclusions

In the human small intestine, thirty minutes of ischemia followed by up to 4 hours of reperfusion, does not seem to lead to an explicit inflammatory response. This may be explained by a unique mechanism of shedding of damaged enterocytes, reported for the first time by our group.     

Pro-inflammatory endothelial cell dysfunction is associated with intersectin-1s down-regulation

Background

The response of lung microvascular endothelial cells (ECs) to lipopolysaccharide (LPS) is central to the pathogenesis of lung injury. It is dual in nature, with one facet that is pro-inflammatory and another that is cyto-protective. In previous work, overexpression of the anti-apoptotic Bcl-X_L rescued ECs from apoptosis triggered by siRNA knockdown of intersectin-1s (ITSN-1s), a pro-survival protein crucial for ECs function. Here we further characterized the cyto-protective EC response to LPS and pro-inflammatory dysfunction.

Methods and Results

Electron microscopy (EM) analyses of LPS-exposed ECs revealed an activated/dysfunctional phenotype, while a biotin assay for caveolae internalization followed by biochemical quantification indicated that LPS causes a 40% inhibition in biotin uptake compared to controls. Quantitative PCR and Western blotting were used to evaluate the mRNA and protein expression, respectively, for several regulatory proteins of intrinsic apoptosis, including ITSN-1s. The decrease in ITSN-1s mRNA and protein expression were countered by Bcl-X_L and survivin upregulation, as well as Bim downregulation, events thought to protect ECs from impending apoptosis. Absence of apoptosis was confirmed by TUNEL and lack of cytochrome c (cyt c) efflux from mitochondria. Moreover, LPS exposure caused induction and activation of inducible nitric oxide synthase (iNOS) and a mitochondrial variant (mtNOS), as well as augmented mitochondrial NO production as measured by an oxidation oxyhemoglobin (oxyHb) assay applied on mitochondrial-enriched fractions prepared from LPS-exposed ECs. Interestingly, expression of myc-ITSN-1s rescued caveolae endocytosis and reversed induction of iNOS expression.

Conclusion

Our results suggest that ITSN-1s deficiency is relevant for the pro-inflammatory ECs dysfunction induced by LPS.     

Effects of blood products on inflammatory response in endothelial cells in vitro

Background

Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products.

Methods

The inflammatory response from pre-activated (endotoxin-stimulated) and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC), platelet concentrates (PC) and fresh frozen plasma (FFP) was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated.

Results

Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product.

Conclusion

Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.