interleukin-8 and Ischemia

Injection of endothelial progenitor cells (EPC) expanded ex vivo has been shown to increase neovascularization in preclinical models of ischemia and in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. Given the potential usefulness of EPC as a cell therapy product, their thorough characterization is of major importance. This review describes the two cell populations currently called EPC and the means to find differential phenotypic markers. We have shown that BMP2/4 are specific markers of late EPC and play a key role in EPC commitment and outgrowth during neovascularization. Several authors have attempted to expand EPC ex vivo in order to obtain a homogeneous cell therapy product. One possible mean of expanding EPC ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation increases angiogenic properties of EPC through activation of SDF-1, angiopoietin and IL-8 pathways. This review summarizes the characterization of EPC and different methods of ex vivo expansion.

Topics: Adult; Angiopoietin-2; Animals; Biomarkers; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Cell Differentiation; Cell Lineage; Cells, Cultured; Chemokine CXCL12; Endothelial Cells; Humans; Interleukin-8; Ischemia; Mice; Multipotent Stem Cells; Neovascularization, Physiologic; Peptide Fragments; Receptor, PAR-1; Receptors, CXCR; Thrombin; Tissue Culture Techniques

Topics: Animals; Hepatitis; Humans; Interleukin-8; Ischemia; Liver; Neutrophils; Reperfusion Injury

Cardiac patch implantation helps maximize the paracrine function of grafted cells and serves as a reservoir of soluble proangiogenic factors required for the neovascularization of infarcted hearts. We have previously fabricated a cardiac patch, EF-HAM, composed of a human amniotic membrane (HAM) coated with aligned PLGA electrospun fibers (EF). In this study, we aimed to evaluate the biocompatibility and angiogenic effects of EF-HAM scaffolds with varying fiber thicknesses on the paracrine behavior of skeletal muscle cells (SkM). Conditioned media (CM) obtained from SkM-seeded HAM and EF-HAM scaffolds were subjected to multiplex analysis of angiogenic factors and tested on HUVECs for endothelial cell viability, migration, and tube formation analyses. All three different groups of EF-HAM scaffolds demonstrated excellent biocompatibility with SkM. CM derived from SkM-seeded EF-HAM 7 min scaffolds contained significantly elevated levels of proangiogenic factors, including angiopoietin-1, IL-8, and VEGF-C compared to plain CM, which was obtained from SkM cultured on the plain surface. CM obtained from all SkM-seeded EF-HAM scaffolds significantly increased the viability of HUVECs compared to plain CM after five days of culture. However, only EF-HAM 7 min CM induced a higher migration capacity in HUVECs and formed a longer and more elaborate capillary-like network on Matrigel compared with plain CM. Surface roughness and wettability of EF-HAM 7 min scaffolds might have influenced the proportion of skeletal myoblasts and fibroblasts growing on the scaffolds and subsequently potentiated the angiogenic paracrine function of SkM. This study demonstrated the angioinductive properties of EF-HAM composite scaffold and its potential applications in the repair and regeneration of ischemic tissues.

Topics: Amnion; Angiopoietin-1; Biocompatible Materials; Cell Movement; Cell Survival; Culture Media, Conditioned; Fibroblasts; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Ischemia; Muscle Cells; Muscle, Skeletal; Neovascularization, Physiologic; Polylactic Acid-Polyglycolic Acid Copolymer; Regeneration; Tissue Engineering; Tissue Scaffolds; Vascular Endothelial Growth Factor A

Diabetic retinopathy (DR) is characterized by microvascular changes including ischemia. Degradation and metabolic changes of various retinal cells occur during ischemia. Ischemic region containing more cells will lead to greater metabolic impairment. We analyzed the non-perfusion region (NPR) by integrating histologic mapping with ultra-widefield fluorescein angiography (UWF FA) images. We also investigated the correlations of the weighted ischemic index (ISI) considering the regional distribution of retinal cells with cytokines, macular edema (ME), and neovascularization (NV). In this study, 32 patients with treatment-naïve DR and 21 age-matched control participants were included. The difference between the non-weighted and weighted ISI of NPR with leakage was greatest at the posterior region. The weighted ISI of NPR with leakage was correlated with MCP-1, IL-8, IL-6, PlGF, and VEGF-A levels, while the non-weighted ISI of NPR with leakage was correlated with IL-8 and IL-6 levels. The presence of baseline ME or NV in patients with DR was associated with the weighted ISI, with a stronger association when cones and rods were weighted. The weighted ISI reflecting both metabolic activity and cell distribution demonstrated a better correlation with clinical features and was more valuable in NPR with leakage than non-weighted ISI, which previous studies conventionally used.

Topics: Diabetes Mellitus; Diabetic Retinopathy; Fluorescein Angiography; Humans; Interleukin-6; Interleukin-8; Ischemia; Macular Edema; Retinal Vessels; Tomography, Optical Coherence; Vascular Endothelial Growth Factor A; Visual Acuity

Patients with Severe Limb Ischemia (SLI) have a high risk of amputation and mortality. Here, we investigated a panel of serum biomarkers with the aim of identifying biomarkers for major events and mechanisms that contribute to disease progression in established SLI. A panel of biomarkers including GROα, HGF, SCF, SCGFβ, SDF1α, TRAIL, IL-6, IL-8, FGFβ, GCSF, GMCSF, IP10, MCP1, PDGFbb, RANTES, TNFα, VEGF, sICAM, sVCAM, TM, and E-selectin was measured in serum samples from a subset (n = 108) of the JUVENTAS cohort. The primary outcome was major events, defined as major amputation or death. The inflammatory biomarkers IL-6, IL-8, GROα and IP-10 were significantly elevated in patients who reached a major endpoint. Results were validated in a secondary cohort (n = 146). Cox regression showed that adjusted hazard ratios were 1.40 (95% CI: 1.15-1.70, p = 0.0007) and 1.48 (95% CI 1.16-1.87, p = 0.001) for IL-6 and IP-10 in a fully adjusted model containing both biomarkers. A prediction model using IL-6 and IP-10 showed predictive accuracy with an AUC of ~ 78% in both discovery and validation cohorts, which is higher than previously published models. We conclude that inflammatory biomarkers predict major events in patients with SLI and allow the creation of biomarker-based risk-prediction models.

Topics: Aged; Amputation, Surgical; Biomarkers; Chemokine CXCL1; Chemokine CXCL10; Chemokines; Cytokines; Extremities; Female; Humans; Inflammation; Interleukin-6; Interleukin-8; Ischemia; Male; Middle Aged; Peripheral Arterial Disease; Predictive Value of Tests; Proportional Hazards Models; Survival Analysis

Acute exercise-induced inflammation is implicated in mediating the beneficial adaptations to regular exercise. Evidence suggests that reduced oxygen and/or blood flow to contracting muscle alters cytokine appearance. However, the acute inflammatory responses to hypoxic/ischemic exercise have been documented with inconsistent results and may not accurately reflect the ischemia produced during exercise in patients with ischemic cardiovascular diseases. Therefore, we determined the extent to which local inflammation is involved in the response to ischemic exercise. Fourteen healthy males performed unilateral isometric forearm contractions for 30 min with and without experimental ischemia. Blood was drawn at baseline, 5 and 10 min into exercise, at the end of exercise, and 30, 60, and 120 min after exercise. Oxygen saturation levels, as measured by near-infrared spectroscopy, were reduced by 10% and 41% during nonischemic and ischemic exercise, respectively. Nonischemic exercise did not affect cytokine values. Ischemia enhanced concentrations of basic fibroblast growth factor, interleukin (IL)-6, IL-10, tumor necrosis factor-alpha, and vascular endothelial growth factor during exercise, but IL-8 was not influenced by ischemic exercise. In conclusion, the present study demonstrates that ischemic, small-muscle endurance exercise elicits local inflammatory cytokine production compared with nonischemic exercise.

Topics: Adult; Cardiovascular Diseases; Cytokines; Exercise; Fibroblast Growth Factors; Forearm; Humans; Inflammation; Interleukin-10; Interleukin-6; Interleukin-8; Ischemia; Isometric Contraction; Male; Muscle, Skeletal; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factors; Young Adult

To develop cell therapies for ischemic diseases, endothelial progenitor cells (EPCs) have been expected to play a pivotal role in vascular regeneration. It is desirable to use a molecular marker that is related to the function of the cells. Here, a quantitative polymerase chain reaction array revealed that early EPCs derived from CD133(+) cells exhibited significant expression of MMP-9. Some populations of early EPCs expressed MMP-9 on the cell surface and others did not. We also attempted to separate the proangiogenic fraction from early EPCs derived from CD133(+) cells using a functional cell surface marker, and we then analyzed the MMP-9(+) and MMP-9(-) cell fractions. The MMP-9(+) cells not only revealed higher invasion ability but also produced a high amount of IL-8. Moreover, the stimulative effect of MMP-9(+) cells on angiogenesis in vitro and in vivo was prohibited by anti-IL-8 antibody. These data indicate that MMP-9 is one of the useful cell surface markers for the separation of angiogenic cells. Our treatment of early EPCs with hyaluronidase caused not only a downregulation of cell-surface MMP-9 but also a decrease in invasion ability, indicating that membrane-bound MMP-9, which is one of the useful markers for early EPCs, plays an important role in angiogenesis. Stem Cells 2016;34:1251-1262.

Topics: AC133 Antigen; Animals; Biomarkers; Cell Fractionation; Cell Membrane; Cell Separation; Endothelial Progenitor Cells; Flow Cytometry; Gene Expression Profiling; Hindlimb; Human Umbilical Vein Endothelial Cells; Humans; Hyaluronoglucosaminidase; Interleukin-8; Ischemia; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Neovascularization, Physiologic; RNA, Messenger

Wnt/β-catenin signaling has an important role in the angiogenic activity of endothelial cells (ECs). Bach1 is a transcription factor and is expressed in ECs, but whether Bach1 regulates angiogenesis is unknown.. This study evaluated the role of Bach1 in angiogenesis and Wnt/β-catenin signaling.. Hind-limb ischemia was surgically induced in Bach1(-/-) mice and their wild-type littermates and in C57BL/6J mice treated with adenoviruses coding for Bach1 or GFP. Lack of Bach1 expression was associated with significant increases in perfusion and vascular density and in the expression of proangiogenic cytokines in the ischemic hindlimb of mice, with enhancement of the angiogenic activity of ECs (eg, tube formation, migration, and proliferation). Bach1 overexpression impaired angiogenesis in mice with hind-limb ischemia and inhibited Wnt3a-stimulated angiogenic response and the expression of Wnt/β-catenin target genes, such as interleukin-8 and vascular endothelial growth factor, in human umbilical vein ECs. Interleukin-8 and vascular endothelial growth factor were responsible for the antiangiogenic response of Bach1. Immunoprecipitation and GST pull-down assessments indicated that Bach1 binds directly to TCF4 and reduces the interaction of β-catenin with TCF4. Bach1 overexpression reduces the interaction between p300/CBP and β-catenin, as well as β-catenin acetylation, and chromatin immunoprecipitation experiments confirmed that Bach1 occupies the TCF4-binding site of the interleukin-8 promoter and recruits histone deacetylase 1 to the interleukin-8 promoter in human umbilical vein ECs.. Bach1 suppresses angiogenesis after ischemic injury and impairs Wnt/β-catenin signaling by disrupting the interaction between β-catenin and TCF4 and by recruiting histone deacetylase 1 to the promoter of TCF4-targeted genes.

Topics: Acetylation; Animals; Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; beta Catenin; Binding Sites; Cell Movement; Cell Proliferation; Disease Models, Animal; Down-Regulation; Endothelial Cells; Fanconi Anemia Complementation Group Proteins; Female; HEK293 Cells; Hindlimb; Histone Deacetylase 1; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Ischemia; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Muscle, Skeletal; Neovascularization, Physiologic; p300-CBP Transcription Factors; Promoter Regions, Genetic; Protein Binding; RNA Interference; Transcription Factor 4; Transcription Factors; Transfection; Vascular Endothelial Growth Factor A; Wnt Signaling Pathway; Wnt3A Protein

The beneficial effects of mesenchymal stem cells (MSCs) are mediated partly by the paracrine production of cytoprotective and trophic factors. Vascular endothelial growth factor (VEGF) is released from MSCs as a paracrine trophic factor and contributes to the therapeutic effects of the stem cell by regulating angiogenesis and promoting revascularization in injured tissues. Interleukin-8 (IL-8), an inflammatory chemokine with potent proangiogenic properties, is upregulated in the ischemic brain and has been shown to promote homing of bone marrow-derived cells to injured sites. However, the effect of IL-8 on MSCs paracrine function remains unknown. We found that IL-8 induced VEGF production and phosphorylation of Akt and ERK. Both effects could be blocked by inhibitors (LY294002, PD098059) or siRNA-mediated silencing of Akt and ERK in human bone marrow MSCs (hBM-MSCs). IL-8-induced VEGF production in hBM-MSCs significantly increased tube formation on Matrigel compared with basal secreted VEGF. In a rat stroke model, administration of IL-8-treated hBM-MSCs decreased the infarction volume and increased angiogenesis in the ischemic boundary zone compared with hBM-MSC treatment alone. In conclusion, IL-8 stimulates VEGF production in hBM-MSCs in part via the PI3K/Akt and MAPK/ERK signal transduction pathways and that administration of IL-8-treated hBM-MSCs increases angiogenesis after stroke. This approach may be used to optimize MSC-based therapies for numerous diseases including stroke, myocardial ischemia, and spinal cord injury.

Topics: Animals; Bone Marrow Cells; Brain; Cells, Cultured; Chromones; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Humans; Interleukin-8; Ischemia; Mesenchymal Stem Cells; Mice; Morpholines; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Vascular Endothelial Growth Factor A

Endothelial activation contributes to the development of vascular inflammation and subsequent vascular diseases, particularly atherosclerosis. AGGF1, a new member of angiogenic factors with a FHA and a G-patch domain, has been shown critical for the regulation of vascular differentiation and angiogenesis. In this study, we found that various inflammatory cytokines strongly induced the expression of AGGF1 in endothelial cells (ECs) and identified AGGF1 as a novel anti-inflammatory factor both in vivo and in vitro. Overexpression of AGGF1 significantly repressed the expression of pro-inflammatory molecules such as E-Selectin, ICAM-1, and IL-8 and the adhesion of monocytes onto ECs activated by TNF-α. Conversely, the knockdown of AGGF1 resulted in the increased expressions of these pro-inflammatory molecules and the enhanced monocyte-EC interaction. We further demonstrated that AGGF1 potently attenuated TNF-α triggered NF-κB pathway, as indicated by the decreased promoter activity, nuclear distribution and phosphorylation of NF-κB p65 subunit as well as the increased protein level of IκBα. This inhibitory effect of AGGF1 was further proved through blocking the phosphorylation of ERK induced by TNF-α. Finally, we showed that the FHA domain of AGGF1 was required for its anti-inflammatory effect. Thus, our findings for the first time demonstrate that AGGF1 suppresses endothelial activation responses to TNF-α by antagonizing the ERK/NF-κB pathway, which makes AGGF1 a promising therapeutic candidate for the prevention and treatment of inflammatory diseases.

Topics: Angiogenic Proteins; Animals; Cell Adhesion; Cell Line; E-Selectin; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Ischemia; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Monocytes; NF-kappa B; Phosphorylation; Promoter Regions, Genetic; RNA Interference; RNA, Small Interfering; Signal Transduction; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Harnessing outgrowth endothelial cells (OECs) for vasoreparative therapy and tissue engineering requires efficient ex vivo expansion. How such expansion impacts on OEC function is largely unknown. In this study, we show that OECs become permanently cell-cycle arrested after ex vivo expansion, which is associated with enlarged cell size, β-galactosidase activity, DNA damage, tumor suppressor pathway activation, and significant transcriptome changes. These senescence hallmarks were coupled with low telomerase activity and telomere shortening, indicating replicative senescence. OEC senescence limited their regenerative potential by impairing vasoreparative properties in vitro and in vivo. Integrated transcriptome-proteome analysis identified inflammatory signaling pathways as major mechanistic components of the OEC senescence program. In particular, IL8 was an important facilitator of this senescence; depletion of IL8 in OECs significantly extended ex vivo lifespan, delayed replicative senescence, and enhanced function. While the ability to expand OEC numbers prior to autologous or allogeneic therapy remains a useful property, their replicative senescence and associated impairment of vasorepair needs to be considered. This study also suggests that modulation of the senescence-associated secretory phenotype could be used to optimize OEC therapy.

Topics: Adult; Animals; Cell- and Tissue-Based Therapy; Cellular Senescence; Disease Models, Animal; Endothelial Cells; Eye; Fetal Blood; Gene Knockdown Techniques; Humans; Interleukin-8; Ischemia; Mice; Mice, Inbred C57BL; Regeneration; RNA, Small Interfering; Signal Transduction; Young Adult

The aim of this prospective study was to examine whether serum neurotensin, interleukin (IL)-6, and IL-8 are early predictor of bowel ischaemia especially in clinically equivocal cases. To this end, 56 patients were assigned to the following groups according to their disease: bowel ischaemia (group 1: n = 14), small bowel obstruction (group 2: n = 12), acute inflammation (group 3: n = 6), perforation (group 4: n = 8), and colorectal adenocarcinoma (group 5: n = 16). Fifteen healthy controls were assigned to group 6. Blood samples were obtained at enrollment, all measurements were done blindly, and all patients underwent surgery. Pretreatment doubtful diagnosis comprised of ileus, mild abdominal pain, and indeterminate imaging. Blood urea nitrogen, lactic acidosis, diagnostic workup, and IL-6 were predictors of diagnosis in univariate analysis. In multivariate analysis, IL-6 (P < 0.001) and diagnostic workup (P < 0.01) were independent predictors of the definite diagnosis. Neurotensin and IL-8 did not differentiate among groups. Considering clinically doubtful cases, IL-6 perfectly differentiates mesenteric ischaemia (of infarction/embolic/occlusive aetiology) from the rest of the indeterminate pathologies. The optimum cut-off point for IL-6 was 27.66 pg/mL. The value of serum IL-6 (27.66 pg/mL) had sensitivity = 1 and specificity = 1. In conclusion, plasma IL-6 measurement on admission might be an additional diagnostic tool that can predict bowel ischaemia in doubtful clinical situations.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers; Case-Control Studies; Colorectal Neoplasms; Diagnosis, Differential; Female; Humans; Interleukin-6; Interleukin-8; Intestinal Obstruction; Intestine, Small; Ischemia; Male; Middle Aged; Neurotensin; Prospective Studies

Mesenchymal stem cells (MSCs) accelerate regeneration of ischemic or injured tissues by stimulation of angiogenesis through a paracrine mechanism. Tumor necrosis factor-α (TNF-α)-activated MSCs secrete pro-angiogenic cytokines, including IL-6 and IL-8. In the present study, using an ischemic hindlimb animal model, we explored the role of IL-6 and IL-8 in the paracrine stimulation of angiogenesis and tissue regeneration by TNF-α-activated MSCs. Intramuscular injection of conditioned medium derived from TNF-α-treated MSCs (TNF-α CM) into the ischemic hindlimb resulted in attenuated severe limb loss and stimulated blood perfusion and angiogenesis in the ischemic limb. Immunodepletion of IL-6 and IL-8 resulted in attenuated TNF-α CM-stimulated tissue repair, blood perfusion, and angiogenesis. In addition, TNF-α CM induced migration of human cord blood-derived endothelial progenitor cells (EPCs) through IL-6- and IL-8-dependent mechanisms in vitro. Intramuscular injection of TNF-α CM into the ischemic limb led to augmented homing of tail vein-injected EPCs into the ischemic limb in vivo and immunodepletion of IL-6 or IL-8 from TNF-α CM attenuated TNF-α CM-stimulated homing of EPCs. In addition, intramuscular injection of recombinant IL-6 and IL-8 proteins resulted in increased homing of intravenously transplanted EPCs into the ischemic limb and improved blood perfusion in vivo. These results suggest that TNF-α CM stimulates angiogenesis and tissue repair through an increase in homing of EPCs through paracrine mechanisms involving IL-6 and IL-8.

Topics: Adipocytes; Animals; Blotting, Western; Cell Movement; Cell Proliferation; Cells, Cultured; Culture Media, Conditioned; Fluorescent Antibody Technique; Hindlimb; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Interleukin-8; Ischemia; Mesenchymal Stem Cells; Mice; Mice, Nude; Necrosis; Neovascularization, Physiologic; Stem Cells; Tumor Necrosis Factor-alpha; Wound Healing

Peripheral arterial diseases, the major complication of diabetes, can result in lower limb amputation. Since endothelial progenitor cells (EPCs) are involved in neovascularization, the aim of this study was to examine whether EPCs isolated from Wharton's jelly (WJ-EPCs) of the umbilical cord, a rich source of mesenchymal stem cells, could reduce ischemia-induced hind limb injury in diabetic mice. We evaluated the effects of WJ-EPC transplantation on hind limb injury caused by femoral artery ligation in mice with streptozotocin (STZ)-induced diabetes. We found that the ischemic hind limb in mice with STZ-induced diabetes showed decreased blood flow and capillary density and increased cell apoptosis and that these effects were significantly inhibited by an injection of WJ-EPCs. In addition, hypoxia-inducible factor-1α (HIF-1α) and interleukin-8 (IL-8) were highly expressed in transplanted WJ-EPCs in the ischemic skeletal tissues and were present at high levels in hypoxia-treated cultured WJ-EPCs. Moreover, incubation of the NOR skeletal muscle cell line under hypoxic conditions in conditioned medium from EPCs cultured for 16 h under hypoxic conditions resulted in decreased expression of pro-apoptotic proteins and increased expression of anti-apoptotic proteins. The inhibition of HIF-1α or IL-8 expression by EPCs using HIF-1α siRNA or IL-8 siRNA, respectively, prevented this change in expression of apoptotic-related proteins. Wharton's jelly in the umbilical cord is a valuable source of EPCs, and transplantation of these EPCs represents an innovative therapeutic strategy for treating diabetic ischemic tissues. The HIF-1α/IL-8 signaling pathway plays a critical role in the protective effects of EPCs in the ischemic hind limb of diabetic mice.

Topics: Animals; Apoptosis; Cell Hypoxia; Cell Movement; Cells, Cultured; Diabetes Mellitus, Experimental; Femoral Artery; Hindlimb; Human Umbilical Vein Endothelial Cells; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; Ischemia; Male; Mice; Mice, Inbred ICR; Muscle, Skeletal; Neovascularization, Physiologic; Peripheral Arterial Disease; Regional Blood Flow; Signal Transduction; Stem Cell Transplantation; Stem Cells; Transcriptional Activation; Wharton Jelly

Cells from a human chondrocyte cell line were studied in 1% oxygen and/or a lower glucose concentration (5.5 mM), compared to the routine culture conditions of normoxia and high glucose. HIF-1α, IL-1β, IL-6, IL-8, COX-2, TNFα, LIF, MMP-3, MMP-13, and reactive oxygen species (ROS) were evaluated, respectively. Effects of hypoxia inducing expression of HIF-1α were statistically significant at 72 h (p<0.05). Increased production of ROS by hypoxia was also observed with passage of time (p<0.05). The effects of hypoxia on HIF-1α and IL-1β were potentiated by 5.5 mM glucose, especially after 48 h (p<0.05). IL-8 production was significantly induced in 1% O(2), with 5.5 mM glucose (p<0.01). IL-8 mRNA expression and production in response to IL-1β were potentiated by hypoxia/ischemia (p<0.05, p<0.01, respectively). Up-regulation of IL-1β, ROS, and IL-8 by hypoxia/ischemia in human chondrocytes may occur in correlation with HIF-1α. IL-8 response to IL-1β may be potentiated synergically by hypoxia/ischemia, as an effector of hypoxia/ischemia. The results may suggest aggressive biology of the ordinary cartilage hypoxia/ischemia in the context of arthro-degeneration.

Topics: Cartilage, Articular; Cell Line; Chondrocytes; Glucose; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-1beta; Interleukin-8; Ischemia; Reactive Oxygen Species; Up-Regulation

To clarify the mechanism by which chronic bladder ischemia causes bladder functional changes, and to investigate the involvement of oxidative stress and pro-inflammatory cytokines, and the effects of the phytotherapeutic drug, Eviprostat, on these biochemical marker levels and bladder function.. Male Sprague-Dawley rats aged 15 weeks were divided into three groups. Arterial injury was experimentally induced by balloon endothelial injury of the iliac arteries, and a 2% cholesterol diet was given for 8 weeks. Rats in the arterial-injury group were given daily oral vehicle or Eviprostat, whereas sham-operated animals on a regular diet (0.09% cholesterol) were given vehicle for the last 2 weeks. Eight weeks after surgery, the levels of bladder pro-inflammatory cytokines, as well as bladder and urinary oxidative-stress markers, were determined. Cystometrograms were carried out without anesthesia or restraint.. Bladder and urinary oxidative-stress markers, and bladder pro-inflammatory cytokine levels were significantly increased in the arterial-injury group, and Eviprostat markedly suppressed these increase. The cystometrograms showed that arterial injury decreased the intermicturition interval without affecting the micturition pressure. This decrease was reversed by Eviprostat treatment.. Oxidative stress and pro-inflammatory cytokines might be involved in the development of overactive bladder by atherosclerosis-induced chronic bladder ischemia. Eviprostat might provide an attractive treatment option for individuals with bladder dysfunction due to chronic bladder ischemia because of its anti-oxidant and anti-inflammatory properties.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Atherosclerosis; Deoxyguanosine; Drug Combinations; Ethamsylate; Interleukin-8; Ischemia; Male; Malondialdehyde; Models, Animal; Oxidative Stress; Plant Extracts; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Urinary Bladder; Urinary Bladder, Overactive; Urination; Urodynamics

Peripheral blood is an easily accessed source for stem cell production; however, the number of cells produced is relatively low. We hypothesized that ischemic preconditioning may serve as a safe method to increase the number of CD34+ cells that can be harvested and cultured in a short period. This study was conducted to test this hypothesis by examining the safety and efficacy of brief, transient ischemia of the lower limbs to augment the number of cells that can be produced from blood of healthy volunteers. Following induction of ischemia, blood samples were withdrawn at baseline, 30 min, 12 h and 24 h. The number of progenitor cells was determined by flow cytometry after the harvested cells were cultured for 5 days. We also analyzed the blood samples to determine IL-8 and VEGF concentrations. No serious adverse events were observed. The total number of cells increased from 0.46 ± 0.1 × 10(6) cells/ml in the pretreatment blood samples to 0.7 ± 0.1 × 10(6) cells/ml in blood taken 12 h after the conclusion of transient ischemia, p = 0.0029. The number of CD34+ cells increased from 4.23 ± 0.8 × 10(4) cells/ml in the pretreatment samples to 7.17 ± 1.34 × 10(4) cells/ml in blood taken 12 h after ischemia, p = 0.0001. The harvested stem cells maintained their ability to construct tubular structures. The augmentation in the number of CD34+ cells was positively correlated with the increase of IL-8, but not with VEGF concentrations. Ischemic preconditioning is a safe and effective technique to increase the availability of stem cells for therapeutic purposes.

Topics: Antigens, CD34; Biomarkers; Extremities; Humans; Interleukin-8; Ischemia; Ischemic Preconditioning; Neovascularization, Physiologic; Nitric Oxide; Stem Cells; Vascular Endothelial Growth Factor A

Angiogenesis is regulated by the local balance between angiogenic stimulators and inhibitors and is maintained by muscle-derived angiogenic factors in ischemic tissues.. Our objectives were to investigate the effect of cold shock domain protein A (CSDA) as an endogenous angiogenesis inhibitor and to develop a novel strategy of therapeutic angiogenesis by blocking CSDA expression.. In human skeletal muscle cells, CSDA was upregulated during hypoxia when cells were damaged and apoptosis was induced. CSDA expression could repress the activity of hypoxia inducible factor-1α and nuclear factor κB, because CSDA can competitively bind the hypoxia response element and the nuclear factor κB-binding element. As a result, vascular endothelial growth factor-A, interleukin-6, and interleukin-8 secretions from skeletal muscle cells were decreased. Further, CSDA depletion increased the secretion level of these angiogenic factors. In a hindlimb ischemia model, transfer of short-hairpin RNA targeting CSDA ameliorated ischemia without direct transfer of angiogenic factors. In this ischemic tissue, vascular endothelial growth factor-A, interleukin-6, and CXCL2 protein levels were increased.. CSDA appears to play a critical role as an endogenous angiogenesis inhibitor in skeletal muscle, and RNA interference targeting of CSDA is a promising gene therapy strategy for treating peripheral arterial disease.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Blotting, Western; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; Chemokine CXCL2; Electrophoretic Mobility Shift Assay; Fluorescent Antibody Technique; Heat-Shock Proteins; Hindlimb; Humans; Immunoenzyme Techniques; Interleukin-6; Interleukin-8; Ischemia; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Necrosis; Real-Time Polymerase Chain Reaction; Vascular Endothelial Growth Factor A

Today, there is no continuous monitoring of the bronchial epithelial lining fluid. This study used microdialysis as a method of continuous monitoring of early lung cytokine response secondary to intestinal ischemia-reperfusion in pigs. The authors aimed to examine bronchial microdialysis for continuous monitoring of IL-1β, TNF-α, IL-8, and fluorescein isothiocyanate Dextran 4,000 Da (FD-4). The superior mesenteric artery was cross-clamped for 120 min followed by 240 min of reperfusion (ischemia group, n = 8). Four sham-operated pigs served as controls. The pigs were anesthetized and normoventilated (peak inspiratory pressure, <20 cm H2O; positive end-expiratory pressure, 7 cm H2O). Samples from bronchial and luminal intestinal and arterial microdialysis catheters (flow-rate of 1 μL/min) were collected during reperfusion in 60-min fractions. Samples were analyzed for TNF-α, IL-1β, IL-8, and FD-4. Data are presented as median (interquartile range). A lung biopsy was collected at the end of the experiment. During reperfusion, there was an increase in bronchial concentrations of both IL-8 (3.70 [1.47-8.93] ng/mL per h vs. controls, 0.61 [0.47-0.91] ng/mL per h; P < 0.001) and IL-1β (0.32 [0.05-0.56] ng/mL per h vs. controls, 0.07 [0.04-0.10] ng/mL per h; P = 0.008). In the intestinal lumen, IL-8 was increased in the ischemia group (6.33 [3.13-9.23] ng/mL per h vs. controls, 0.89 [0.21-1.86] ng/mL per h; P < 0.001). The FD-4 did not differ between groups. Pulmonary vascular resistance and pulmonary shunt increased versus controls. During reperfusion, PaO2/FiO2 ratio decreased in the ischemia group. Histology was normal in both groups. Bronchial microdialysis detects altered levels of cytokines in the epithelial lining fluid and can be used for continuous monitoring of the immediate local lung cytokine response secondary to intestinal ischemia-reperfusion.

Topics: Acute Lung Injury; Animals; Body Fluids; Bronchi; Constriction; Cytokines; Epithelial Cells; Hemodynamics; Interleukin-1beta; Interleukin-8; Intestines; Ischemia; Mesenteric Artery, Superior; Microdialysis; Reperfusion Injury; Sus scrofa; Swine; Time Factors; Tumor Necrosis Factor-alpha

To determine the intracellular mechanisms mediating the angiogenic effects of integrin alpha v beta 5 overexpression in circulating angiogenic cells (CACs).. Integrin alpha v beta 5 is expressed on angiogenic endothelial cells, and integrin alpha v beta 5 activation was shown to improve the reparative functions of endothelial progenitors within the cardiovascular system. CACs were transiently transfected with the full-length cDNA of human integrin beta 5 (CAC-ITGB5) or control-vector (CAC-vector). Integrin beta 5 overexpression was confirmed using flow cytometry, Western blot, and PCR analysis; it enhanced the angiogenic capacities of CACs in vitro (spheroid and Matrigel angiogenesis assay) and stimulated new vessel formation in vivo (murine hind limb ischemia model). Overexpression of ITGB5 resulted in integrin alpha v beta 5 phosphorylation and activation of Src kinase and signal transducer and activator of transcription (STAT) 3. Furthermore, elevated mRNA and protein expression of the CXC chemokine CXCL8 and the CC chemokine CCL2 was detected in CAC-ITGB5, and conditioned medium from CAC-ITGB5 enhanced the sprouting of coincubated human endothelial cells in a STAT3-, CXCL8-, and CCL2-dependent manner.. Src kinase-mediated activation of STAT3 and subsequent angiogenic gene expression mediate the effects of integrin alpha v beta 5 and may be exploited to enhance the paracrine activities of CACs.

Topics: Animals; Blotting, Western; Cell Adhesion; Cell Movement; Cells, Cultured; Chemokine CCL2; Culture Media, Conditioned; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Flow Cytometry; Hindlimb; Humans; Integrin beta Chains; Interleukin-8; Ischemia; Mice; Mice, Nude; Muscle, Skeletal; Neovascularization, Physiologic; Paracrine Communication; Phosphorylation; Receptors, Vitronectin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; src-Family Kinases; STAT3 Transcription Factor; Time Factors; Transfection; Up-Regulation

Notch signaling regulates vascular development. However, the implication of the Notch ligand Delta-like 4 (Dll4) in postischemic angiogenesis remains unclear.. We investigated the role of Dll4/Notch signaling in reparative angiogenesis using a mouse model of ischemia.. We found Dll4 weakly expressed in microvascular endothelial cells of normoperfused muscles. Conversely, Dll4 is upregulated following ischemia and localized at the forefront of sprouting capillaries. We analyzed the effect of inhibiting endogenous Dll4 by intramuscular injection of an adenovirus encoding the soluble form of Dll4 extracellular domain (Ad-sDll4). Dll4 inhibition caused the formation of a disorganized, low-perfused capillary network in ischemic muscles. This structural abnormality was associated to delayed blood flow recovery and muscle hypoxia and degeneration. Analysis of microvasculature at early stages of repair revealed that Dll4 inhibition enhances capillary sprouting in a chaotic fashion and causes excessive leukocyte infiltration of ischemic muscles. Furthermore, Dll4 inhibition potentiated the elevation of the leukocyte chemoattractant CXCL1 (chemokine [C-X-C motif] ligand 1) following ischemia, without altering peripheral blood levels of stromal cell-derived factor-1 and monocyte chemoattractant protein-1. In cultured human monocytes, Dll4 induces the transcription of Notch target gene Hes-1 and inhibits the basal and tumor necrosis factor-alpha-stimulated production of interleukin-8, the human functional homolog of murine CXCL1. The inhibitory effect of Dll4 on interleukin-8 was abolished by DAPT, a Notch inhibitor, or by coculturing activated human monocytes with Ad-sDll4-infected endothelial cells.. Dll4/Notch interaction is essential for proper reparative angiogenesis. Moreover, Dll4/Notch signaling regulates sprouting angiogenesis and coordinates the interaction between inflammation and angiogenesis under ischemic conditions.

Topics: Adaptor Proteins, Signal Transducing; Animals; Calcium-Binding Proteins; Cells, Cultured; Chemokine CXCL1; Chemotaxis, Leukocyte; Coculture Techniques; Disease Models, Animal; Endothelial Cells; Hindlimb; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Ischemia; Laser-Doppler Flowmetry; Leukocytes; Male; Mice; Muscle, Skeletal; Neovascularization, Physiologic; Receptors, Notch; Regeneration; Regional Blood Flow; Signal Transduction; Time Factors; Transfection; Ultrasonography

We evaluated the healing potential of human fetal aorta-derived CD133(+) progenitor cells and their conditioned medium (CD133(+) CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2x10(4) CD133(+) or CD133(-) cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133(+) cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133(-) cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133(+) cells accelerated wound closure as compared with CD133(-) or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133(+) cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133(+) CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by co-administering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133(+) CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133(+) CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133(+) cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.

Topics: AC133 Antigen; Animals; Antigens, CD; Aorta; Cell Differentiation; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Diabetes Mellitus, Experimental; Diabetic Foot; Fetal Stem Cells; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Ischemia; Lower Extremity; Male; Membrane Proteins; Mice; Neovascularization, Physiologic; Paracrine Communication; Peptides; Signal Transduction; Stem Cell Transplantation; Time Factors; Vascular Endothelial Growth Factor A; Wnt Proteins; Wound Healing

We sought to evaluate the association between ischemic times, cytokines-interleukin (IL)-6, IL-1b, tumor necrosis factor-alpha, sIL-2r, IL-8, and IL-10-and alterations in gaseous exchange.. This prospective study of 42 orthotopic liver transplantation (OLT) recipients examined ischemic times and respiratory variables measured as alterations in intrapulmonary shunt and in the Po(2)/Fio(2) ratio. Centrifuged blood samples were frozen at -80 degrees C for storage. The Inmulite-One system (Euro/Dpc, Gwynedd, UK) was used to determine the concentration of cytokines. For statistical analysis, we used the Pearson correlation coefficient.. The average cold ischemic time was 478 minutes (range, 35-929) and warm ischemic time was 69.58 minutes (range, 20-180). The warm ischemic time affected the degree of shunt at the end of the operation (P < .027) and the levels of IL-10 (P < .018) and IL-6 (P < .000). The final degree of shunting and IL-10 (P < .044) showed a correlation. The cold ischemic time affected IL-1 (P < .046) and IL-8 levels (P < .023). The reperfusion syndrome was correlated with the final levels of IL-10 (P < .064) and of IL-8 (P < .066).. Warm and cold ischemic times affect the final cytokine levels and the degree of intrapulmonary shunt.

Topics: Cytokines; Humans; Inflammation; Interleukin-1; Interleukin-10; Interleukin-6; Interleukin-8; Ischemia; Liver Circulation; Liver Transplantation; Oxygen; Oxygen Consumption; Partial Pressure; Portal Vein; Reperfusion; Reperfusion Injury

Circulating endothelial progenitor cells (EPCs) counts were determined in patients with sickle cell disease (SCD) to elucidate their role in SCD-related ischemia-induced angiogenesis and reendothelialization.. Circulating EPC counts (KDR(+)/CD34(+)/Cd45(dim) cells) and their relation to serum levels of EPC mobilizing growth factors erythropoietin, vascular endothelial growth factor, and interleukin-8 were investigated in SCD patients during asymptomatic state (n=66) and painful crisis (n=36) and compared to healthy controls (n=13).. EPC counts were comparable between controls (0; range, 0-1.1 cells/mL) and patients (0; range, 0-0 cells/mL) in asymptomatic state, but were significantly higher during painful crisis (41.7; range, 0-186 cells/mL; p<0.05). Also in a paired analysis of 12 patients who were included both during asymptomatic state and painful crisis, EPC counts increased significantly during painful crisis (from 0 [range, 0-0] to 26 [range, 0-149 cell/mL; p<0.05). EPC counts were not related to any of the measured growth factors.. The higher EPC counts during painful crisis might indicate a role for EPC mobilization in reendothelialization. As a relationship of EPCs with the established mobilizing growth factors, measured in this study was not observed, the mechanism of EPC mobilization in SCD remains to be elucidated.

Topics: Adult; Anemia, Sickle Cell; Endothelial Cells; Erythropoietin; Female; Humans; Interleukin-8; Ischemia; Leukocyte Count; Male; Neovascularization, Pathologic; Pain; Stem Cells; Vascular Endothelial Growth Factor A

Recruitment of various stem and progenitor cells is crucial for the regeneration of an injured organ. Levels of uric acid, one of the prototypical "alarm signals," surge after ischemia-reperfusion injury. Exogenous uric acid rapidly mobilizes endothelial progenitor cells and hematopoietic stem cells and protects the kidney from ischemia. The relatively fast responses to uric acid suggest that preformed second messengers may be released from a storage pool. Here, it is reported that monosodium urate (MSU) results in exocytosis of Weibel-Palade bodies in vitro and in vivo, leading to the release of IL-8, von Willebrand factor, and angiopoietin 2 in the culture medium or circulation. Confocal and immunoelectron microscopy confirmed depletion of von Willebrand factor in MSU-treated aortic endothelial cells. Angiopoietin 2 alone induced exocytosis of Weibel-Palade bodies, mobilized hematopoietic stem cells and depleted splenic endothelial progenitor cells, partially reproducing the actions of MSU. In addition, pretreatment with angiopoietin 2 protected the kidneys from an ischemic insult, suggesting that the previously reported renoprotection conferred by MSU likely results from exocytosis of Weibel-Palade bodies. Furthermore, experiments with toll-like receptor 4 (TLR-4)-and TLR-2-deficient mice demonstrated that uric acid-induced exocytosis of Weibel-Palade bodies is mediated by TLR-4 and that uric acid-induced release of IL-8 requires both TLR-2 and TLR-4. In summary, these results suggest that exocytosis of Weibel-Palade bodies links postischemic repair with inflammation and mobilization of stem cells.

Topics: Angiopoietin-2; Cells, Cultured; Exocytosis; Hematopoietic Stem Cells; Humans; Interleukin-8; Ischemia; Reperfusion Injury; Stem Cells; Toll-Like Receptor 2; Toll-Like Receptor 4; Uric Acid; von Willebrand Factor; Weibel-Palade Bodies

These studies were undertaken to evaluate human microvascular endothelial cell (MEC) synthesis of interleukin-8 (IL-8), a potent neutrophil chemoattractant, under in vitro conditions of ischemia and reperfusion. IL-8 and other related CXC chemokines are believed to mediate tissue injury in a variety of pathologic conditions in humans. MEC grown on microcarrier beads were exposed to 3 or 6 h of in vitro ischemia followed by 2 h of reperfusion. Conditioned medium, MEC protein, and total RNA extracts were assayed for IL-8 using an ELISA. During ischemia alone, MEC increased intracellular, but not extracellular levels of IL-8 secretion. In contrast, reperfusion markedly stimulated both intracellular and extracellular IL-8 secretion. Neither 3 h of ischemia alone or followed by reperfusion altered steady-state levels of IL-8 mRNA when compared to pre-ischemic levels. In contrast, after 6 h of ischemia alone and ischemia followed by reperfusion, IL-8 mRNA was increased eight- and sixfold, respectively, when compared to pre-ischemic levels. These studies demonstrate an inverse relationship between the rate of IL-8 protein secretion and the steady-state levels of IL-8 mRNA during ischemia and reperfusion. During ischemia and reperfusion both the increase in cell-associated IL-8 protein and the release of IL-8 into the medium is dependent on de novo protein synthesis rather than the intracellular accumulation of IL-8. These experiments indicate that post-ischemic modulation of IL-8 release and synthesis following ischemia reperfusion will require strategies directed towards inhibition of IL-8 transcription and in depth knowledge of the mechanisms regulating IL-8 secretion.

Topics: Cell Survival; Cells, Cultured; Endothelial Cells; Humans; Interleukin-8; Ischemia; Microcirculation; Reperfusion; RNA, Messenger

Because hypoxia increases extracellular adenosine levels and stimulates angiogenesis, we evaluated the relative roles of reduced oxygen concentrations and adenosine receptor activation in the production of angiogenic factors. In vitro, we analyzed the effects of hypoxia and adenosine on the secretion of angiogenic factors from human microvascular endothelial cells (HMEC-1). To study the effects of hypoxia alone, we scavenged adenosine from the hypoxic medium with adenosine deaminase, and we used the stable adenosine analog 5'-N-ethylcarboxamidoadenosine (NECA) to study the effects of stimulation of adenosine receptors. In the absence of adenosine, hypoxia stimulated vascular endothelial growth factor (VEGF) but not interleukin-8 (IL-8) secretion from HMEC-1. In contrast, NECA stimulated both VEGF and IL-8 secretion. VEGF secretion was increased 1.9 +/- 0.04-fold with NECA (10 microM) and 1.7 +/- 0.1-fold with hypoxia (5% O(2)) but 3.8 +/- 0.1-fold when these two stimuli were combined. Thus, adenosine receptors act in a cooperative fashion with hypoxia to stimulate VEGF and induce IL-8 secretion not stimulated by hypoxia alone. In vivo, antagonism of adenosine receptors with caffeine abrogated VEGF up-regulation induced by local injection of NECA into the mouse hind limb and produced a 46% reduction of neovascularization in a mouse ischemic hind limb model. Our study suggests that adenosine actions are not redundant but rather are complementary to the direct effects of hypoxia. Stimulation of adenosine receptors not only contributes to the overall effect of hypoxia but also has additional actions in the regulation of angiogenic factors. Thus, adenosine receptors represent a potential therapeutic target for regulation of neovascularization.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cells, Cultured; Gene Expression Regulation; Hindlimb; Humans; Hypoxia; Interleukin-8; Ischemia; Male; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Receptors, Purinergic P1; RNA, Messenger; Vascular Endothelial Growth Factor A

E-selectin plays critical roles in tethering leukocytes to endothelial cells (ECs). We studied the role of E-selectin in endothelial progenitor cell (EPC) homing and vasculogenesis. After ischemia, the expression of E-selectin on ECs peaked 6 to 12 hours and returned to baseline at 24 hours, whereas the level of soluble E-selectin (sE-selectin) in serum increased over 24 hours and remained high at day 7. Mouse bone marrow-derived EPCs expressed not only E-selectin but also its ligand. Homing of circulating EPCs to ischemic limb was significantly impaired in E-selectin knock-out mice, as well as wild-type mice pretreated with blocking antibody against E-selectin, which was rescued by local sE-selectin injection. Mechanism for this is that sE-selectin stimulated not only ECs to express ICAM-1, but also EPCs to secrete interleukin-8 (IL-8), leading to enhanced migration and incorporation to ECs capillary formation. In therapeutic aspect, local treatment with sE-selectin enhanced efficacy of EPC transplantation for vasculogenesis and salvage of ischemic limb. Conversely, when E-selectin was knocked down by E-selectin small interfering RNA, blood flow recovery after EPC transplantation was significantly impaired. But this impaired vasculogenesis was rescued by sE-selectin. In conclusion, these data demonstrate E-selectin is a pivotal molecule for EPCs' homing to ischemic limb and vasculogenesis.

Topics: Animals; Cell Movement; E-Selectin; Endothelial Cells; Hindlimb; Interleukin-8; Ischemia; Mice; Mice, Knockout; Muscle, Skeletal; Neovascularization, Pathologic; RNA, Small Interfering; Stem Cell Transplantation; Stem Cells; Time Factors; Transplantation, Homologous

The aim of this study was to examine whether chronic infections and genetic factors of the host play roles in the pathophysiology of acute noncardioembolic ischemic stroke. Blood samples from 59 subjects with ischemic stroke and 52 control patients were investigated by nested PCR for the presence of C. pneumoniae DNA, HCMV DNA and enterovirus RNA, by ELISA for the levels of antibodies to C. pneumoniae, HCMV, HSV, HHV-6, EBV and the inflammatory chemokine IL-8, and by PCR for promoter polymorphism of the IL-8 and CD14 host genes. Associations of stroke with the HCMV IgG and HSV-1 IgA antibody levels were observed. No association of stroke was detected with the presence of C. pneumoniae, HCMV or enterovirus nucleic acids in the peripheral blood, C. pneumoniae IgM, IgG and IgA, the HSV IgG, the EBV IgG, or HHV-6 IgG antibody levels, the pathogen burden, the IL-8 or CD14 promoter polymorphisms, or with the serum levels of IL-8 in the overall study population. These results are consistent with the hypothesis that certain pathogens are involved in the development of ischemic stroke.

Topics: Adult; Aged; Antibodies, Viral; Chlamydophila Infections; Chlamydophila pneumoniae; Chronic Disease; Cytomegalovirus; Cytomegalovirus Infections; DNA, Bacterial; DNA, Viral; Enterovirus; Enterovirus Infections; Enzyme-Linked Immunosorbent Assay; Genetic Predisposition to Disease; Herpesviridae; Herpesviridae Infections; Herpesvirus 1, Human; Humans; Interleukin-8; Ischemia; Lipopolysaccharide Receptors; Middle Aged; Polymerase Chain Reaction; Polymorphism, Genetic; Promoter Regions, Genetic; Risk Factors; RNA, Viral; Stroke

In recent years it has become increasingly clear that a cross-talk between the inflammatory response and blood coagulation exists, although many of the underlying mechanisms remain unclear. In the present study we investigated the potential anti-inflammatory properties of two different anticoagulant compounds, i.e. active-site inactivated FVIIa (FVIIai) and fondaparinux sodium, a selective FXa inhibitor, administered as pretreatment in a model of intestinal I/R in rats.. Endothelial barrier permeability was assessed using the vascular leakage of radiolabelled human serum albumin, tissue neutrophil sequestration was quantitated by myeloperoxidase (MPO) activity, and plasma levels of macrophage inflammatory protein (MIP)-2 were examined using an enzyme-linked-immuno-sorbent assay after 40 min of intestinal ischaemia and 6 h of reperfusion in the rat (n = 34). Pretreatment with FVIIai or fondaparinux sodium was administered 90 min before initiation of ischaemia.. Endothelial-barrier permeability in all examined organs, myeloperoxidase activity in the lungs, and ileum and MIP-2 levels in plasma increased after intestinal I/R. Pretreatment with FVIIai decreased the endothelial barrier permeability and MPO activity in the ileum, and a tendency towards decreased permeability was also observed in the lungs. Fondaparinux did not affect the endothelial barrier permeability or MPO activity. Both FVIIai and fondaparinux decreased the MIP-2 levels in plasma after intestinal I/R.. Inhibition of the TF-FVIIa complex by FVIIai can attenuate inflammatory responses in connection with intestinal I/R-injury and could represent a potentially important therapeutic strategy for the prevention of organ dysfunction. Potential anti-inflammatory properties of fondaparinux and other inhibitors of FXa are not excluded and need further investigation.

Topics: Animals; Anticoagulants; Blood Cell Count; Cell Membrane Permeability; Chemokine CXCL2; Chemokines, CXC; Endothelium; Factor VIIa; Factor Xa Inhibitors; Fondaparinux; Hemostasis; Intercellular Signaling Peptides and Proteins; Interleukin-8; Intestines; Ischemia; Male; Neutrophil Infiltration; Peroxidase; Polysaccharides; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Reperfusion Injury

Oxidative stress seems to contribute to cardiopulmonary bypass (CPB)-related postoperative complications. Pediatric patients are particularly prone to these complications. With this in mind, we measured oxidative stress markers in blood plasma of 20 children undergoing elective heart surgery before, during, and up to 48 h after cessation of CPB, along with inflammatory parameters and full analysis of iron status. Ascorbate levels were decreased by approximately 50% (P < 0.001) at the time of aorta cross-clamp removal (or pump switch-off in 4 patients with partial CPB), and associated with corresponding increases in dehydroascorbate (P < 0.001, r = -0.80) and malondialdehyde (P < 0.01, r = -0.59). In contrast to the immediate oxidative response, peak levels of IL-6 and IL-8 were not observed until 3-12 h after CPB cessation. The early loss of ascorbate correlated with duration of CPB (P < 0.002, r = 0.72), plasma hemoglobin after cross-clamp removal (P < 0.001, r = 0.70), and IL-6 and IL-8 levels at 24 and 48 h after CPB (P < 0.01), but not with postoperative lactate levels, strongly suggesting that hemolysis, and not inflammation or ischemia, was the main cause of early oxidative stress. The correlation of ventilation time with early changes in ascorbate (P < 0.02, r = 0.55), plasma hemoglobin (P < 0.01, r = 0.60), and malondialdehyde (P < 0.02, r = 0.54) suggests that hemolysis-induced oxidative stress may be an underlying cause of CPB-associated pulmonary dysfunction. Optimization of surgical procedures or therapeutic intervention that minimize hemolysis (e.g., off-pump surgery) or the resultant oxidative stress (e.g., antioxidant treatment) should be considered as possible strategies to lower the rate of postoperative complications in pediatric CPB.

Topics: Ascorbic Acid; C-Reactive Protein; Cardiac Surgical Procedures; Cardiopulmonary Bypass; Child; Child, Preschool; Dehydroascorbic Acid; Heart Defects, Congenital; Hemolysis; Humans; Infant; Interleukin-6; Interleukin-8; Iron; Ischemia; Malondialdehyde; Neutrophils; Oxidative Stress; Pneumonia; Postoperative Complications; Prospective Studies

Two types of cells are cultured from the human peripheral blood, early endothelial progenitor cells (EPCs) and outgrowth endothelial cells (OECs), as previously reported. Here, we further characterize these cells, especially with respect to their different origins and functions both in vitro and in vivo. We also investigated whether the combination of these different cell types shows synergism during neovascularization.. Early EPCs were heterogeneously made up of both CD14+ monocyte-derived cells, which secrete cytokines, and CD14(-)-derived cells, which contain high levels of (CD34+)KDR+ cells. OECs were cultured almost exclusively from CD14- cells, not CD14+ cells, and were distinct from mature endothelial cells in terms of proliferation potential, KDR+ expression level, and telomerase activity. A portion of cells from CD14- cells and early EPCs produced rapidly proliferating, capillary-forming cells in both the Matrigel plug and the ischemic hind limb similar to OECs. Early EPCs and OECs expressed receptors for vascular endothelial growth factor and interleukin-8, cytokines secreted by early EPCs. There was a differential increase in matrix metalloproteinases (MMPs): MMP-9 in early EPCs and MMP-2 in OECs. In vitro, the angiogenic capability of the 2 cell types was augmented by mutual interaction through cytokines and MMPs. Injection of a mixture of the 2 cells resulted in superior neovascularization in vivo to any single-cell-type transplantation.. Distinct origins of the different types of EPCs exist that have different functions in neovascularization. Mixed transplantation of these cells results in synergistic neovascularization through cytokines and MMPs.

Topics: Animals; Autocrine Communication; Blood Cells; Cell Lineage; Cells, Cultured; Cellular Senescence; Cytokines; Endothelial Cells; Female; Hindlimb; Humans; Interleukin-8; Ischemia; Matrix Metalloproteinases; Mice; Mice, Nude; Neovascularization, Physiologic; Paracrine Communication; Stem Cell Transplantation; Stem Cells; Vascular Endothelial Growth Factor A

Antithrombin (AT) reveals its antiinflammatory activity by promoting endothelial release of prostacyclin (PGI(2)) in vivo. Since neuroinflammation is critically involved in the development of ischemia/reperfusion (I/R)-induced spinal cord injury (SCI), it is possible that AT reduces the I/R-induced SCI by attenuating the inflammatory responses. We examined this possibility using rat model of I/R-induced SCI in the present study. AT significantly reduced the mortality and motor disturbances by inhibiting reduction of the number of motor neurons in animals subjected to SCI. Microinfarctions of the spinal cord seen after reperfusion were markedly reduced by AT. AT significantly enhanced the I/R-induced increases in spinal cord tissue levels of 6-keto-PGFIalpha, a stable metabolite of PGI2. AT significantly inhibited the I/R-induced increases in spinal cord tissue levels of TNF-alpha, rat interleukin-8 and myeloperoxidase. In contrast,Trp(49) -modified AT did not show any protective effects. Pretreatment with indomethacin significantly reversed the protective effects of AT. An inactive derivative of factor Xa, which selectively inhibits thrombin generation, has been shown to fail to reduce SCI. Taken together, these observations strongly suggested that AT might reduce I/R-induced SCI mainly by the antiinflammatory effect through promotion of endothelial production of PGI(2). These findings also suggested that AT might be a potential neuroprotective agent.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Antithrombins; Coloring Agents; Disease Models, Animal; Epoprostenol; Factor Xa; Humans; Inflammation; Interleukin-8; Ischemia; Male; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Spinal Cord; Spinal Cord Injuries; Tetrazolium Salts; Time Factors; Tryptophan; Tumor Necrosis Factor-alpha

Endothelial progenitor cells (EPC) in one study group is not the same as EPC in other investigators, suggesting that EPC is not a single type of cell population. In this study, we tried to demonstrate the heterogeneity of EPC.. We cultured total mononuclear cells from human peripheral blood to get two types of EPC sequentially from the same donors. We called them early EPC and late EPC. Early EPC with spindle shape showed peak growth at 2 to 3 weeks and died at 4 weeks, whereas late EPC with cobblestone shape appeared late at 2 to 3 weeks, showed exponential growth at 4 to 8 weeks, and lived up to 12 weeks. Late EPC was different from early EPC in the expression of VE-cadherin, Flt-1, KDR, and CD45. Late EPC produced more nitric oxide, incorporated more readily into human umbilical vein endothelial cells monolayer, and formed capillary tube better than early EPC. Early EPC secreted angiogenic cytokines (vascular endothelial growth factor, interleukin 8) more so than late EPC during culture in vitro. Both types of EPC showed comparable in vivo vasculogenic capacity.. We found two types of EPC from a source of adult peripheral blood that might have different roles in neovasculogenesis based on the identified differences.

Topics: Animals; Cell Division; Cell Line; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Endothelium, Vascular; Female; Femoral Artery; Gene Expression Profiling; Gene Expression Regulation, Developmental; Hindlimb; Humans; Interleukin-8; Ischemia; Mice; Mice, Nude; Neovascularization, Physiologic; NIH 3T3 Cells; Stem Cells; Umbilical Veins; Vascular Endothelial Growth Factor A

It has been suggested that inflammatory mediators such as cytokines released during intestinal ischemia and reperfusion increase permeability in the lungs. Cytokines exist at concentrations several hundred times higher at the site of inflammation than in the blood. When absorbed, the locally produced cytokines may affect multiple remote organs. We thus investigated whether the isolation of the intestine in a bag during ischemia and reperfusion can reduce subsequent lung injury.. Rats were divided into three groups: group 1, simple laparotomy (sham); group 2, intestinal ischemia and reperfusion (I/R); and group 3, intestinal ischemia and reperfusion with an intestinal bag (IB). Lung permeability was assessed using the Evans Blue leakage method. Cytokines (interleukin-1beta, tumor necrosis factor alpha, interleukin-8) in the plasma and ascites were measured by enzyme-linked immunosorbent assay.. The increase in lung permeability of I/R significantly decreased in IB (1.73 +/- 0.48 vs 1.05 +/- 0.22, P < 0.01). The plasma cytokine concentrations were also lower in IB than in I/R. In addition, the cytokine levels in the intestinal bag fluid were extremely high.. The isolation of the intestine during ischemia and reperfusion was found to reduce the degree of subsequent lung injury, possibly due to the reduced absorption of locally produced cytokines via the parietal peritoneum.

Topics: Analysis of Variance; Animals; Biomarkers; Capillary Permeability; Cytokines; Disease Models, Animal; Interleukin-1; Interleukin-8; Intestines; Ischemia; Lung Diseases; Male; Probability; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Sensitivity and Specificity; Tumor Necrosis Factor-alpha

Cellular damage and inflammation after ischemia contribute to sustained acute renal failure (ARF).. To quantify cellular damage and inflammation in postischemic ARF and identify markers of renal functional outcome, urine specimens from 40 renal allograft recipients, including 30 cadaveric (9 "sustained ARF" and 21 "recovery" subjects) and 10 living donor allografts ("LD"), were analyzed for actin, gamma-glutamyl transpeptidase (GGTP), lactate dehydrogenase (LDH), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) during the first posttransplant week.. On day 0, urinary actin, GGTP, IL-6, and IL-8 were elevated in recipients destined to have sustained ARF compared with those destined to recover. Median values per gram of urine creatinine in the sustained ARF, recovery, and LD groups were 263.9, 0.0, and 0.0 microg for actin; 5000.0, 892.9, and 5555.6 U for GGTP; 193.1, 27.2, and 10.5 ng for IL-6; and 382.0, 17.8, and 18.5 ng for IL-8, respectively. In contrast, urinary LDH and TNF-alpha increased in recipients with recovering function compared with those who had sustained ARF. The corresponding median values were 36.7 and 16.3 U (recovery versus sustained ARF) for LDH, and 18.4 and 7.6 ng (LD versus sustained ARF) for TNF-alpha. Computational analyses using the Receiver Operating Characteristic Curve found that elevated urinary actin, IL-6, and IL-8 on day 0 were strong predictors of sustained ARF, where the calculated areas under the curve were 0.75, 0.91, and 0.82, respectively.. Increased urinary actin, IL-6, and IL-8 may be useful markers for the prediction of sustained ARF after ischemia.

Topics: Actins; Acute Kidney Injury; Adult; Biomarkers; Creatinine; Female; gamma-Glutamyltransferase; Humans; Interleukin-6; Interleukin-8; Ischemia; Kidney; Kidney Function Tests; Kidney Transplantation; L-Lactate Dehydrogenase; Male; Middle Aged; ROC Curve; Transplantation, Homologous; Tumor Necrosis Factor-alpha

Cytokines have been shown to play an important role in promoting inflammation in the setting of ischemia-reperfusion injury. However, their role in human lung transplantation has not been systematically explored. This study was undertaken to examine the kinetics of cytokine release in 18 consecutive human lung transplantation procedures and to examine the relationships between their levels and donor factors, length of ischemic time, and allograft function. TNF-alpha, IFN-gamma, IL-10, IL-12, and IL-18 were found at higher levels during the ischemic time, whereas IL-8 predominantly increased after reperfusion. IL-8 levels after 2 h of reperfusion correlated with lung function assessed by the Pa(O2 )/FI(O(2)) ratio, the mean airway pressure, and the APACHE score during the first 24 postoperative hours. The length of ICU stay also correlated with IL-8 levels after 2 h of reperfusion. Longer ischemic time was associated with significantly higher levels of IL-18 before reperfusion, and older donors had significantly lower levels of IL-10 after reperfusion. We have demonstrated the importance of IL-8 in predicting early graft function after human lung transplantation. In addition, we showed that donor age and ischemic time may influence release of specific cytokines during ischemia-reperfusion.

Topics: Adult; Age Factors; Aged; APACHE; Female; Graft Survival; Humans; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-18; Interleukin-8; Ischemia; Length of Stay; Lung; Lung Transplantation; Male; Middle Aged; Predictive Value of Tests; Reperfusion Injury; Respiratory Function Tests; Time Factors; Tissue Donors; Tumor Necrosis Factor-alpha

A previous study demonstrated that continuous enteric luminal perfusion of fetal bovine serum (FBS) protects the small intestine from total ischemia/reperfusion injury (IRI) and increases the intestinal mass. In this study, we further investigated the changes in plasma interleukin-8 (IL-8) level caused by total ischemia/reperfusion of the small intestine and the effect of FBS on plasma IL-8 levels. A 3-h total ischemia was induced in a 15-cm segment of terminal ileum and then reperfusion was instituted. Luminal perfusion of FBS was conducted via an osmotic minipump connected to the stomach through a fine polyethylene tube, starting 3 days prior to total ischemia. The rats were killed after 10 and 30 min and 1 and 3 h of total ischemia, and 1, 6, and 12 h or 1, 2, and 3 days after initiation of reperfusion. Plasma IL-8 was measured by enzyme-linked immunosorbent assay. The results were compared among the FBS-treated and untreated groups. The plasma IL-8 level was elevated from 1 h of total ischemia to 6 h after initiation of reperfusion ( P< 0.05) with a peak of 641.5 +/- 36.9 pg/ml in the untreated group and 471.6 +/- 42.2 pg/ml in the treated group. Luminal perfusion of FBS significantly suppressed plasma IL-8 levels after 1 h of total ischemia and 1 h after initiation of reperfusion ( P< 0.05). The results suggest that FBS might play a role in the treatment of total IRI of the small intestine.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Interleukin-8; Intestine, Small; Ischemia; Male; Rats; Rats, Inbred Lew; Reperfusion Injury; Serum Albumin, Bovine

The interrelation of adhesion between leukocyte and endothelium was studied by several irritation factors.. Leukocyte adhesion was observed by impulse electricity irritation, ischemia/reperfusion, endotoxin and IL-8 in venular of rat mesentery.. The results showed these irritation factors resulted in a significant increase in the number of leukocytes adhesion along the venular endothelium of rat mesentery. IL-8 leaded to the most increase of leukocytes adhesion. Especially treated by IL-8 for 30 minutes. The number of leukocytes adhesion of the others was approximately identical.. The study suggests that impulse electricity irritation, ischemia/reperfusion, endotoxin and IL-8 are able to induce leukocytes and endothelium adhesion, and IL-8 of them has the most effect.

Topics: Animals; Cell Adhesion; Electric Stimulation; Endotoxins; Interleukin-8; Ischemia; Leukocytes; Microvessels; Rats; Rats, Wistar; Reperfusion Injury

Despite increasing evidence supporting the involvement of neutrophils in ischemic and postischemic damages, the mechanisms underlying the early recruitment of these cells are not completely understood. In this report, the effects of conditioned media from hypoxic endothelial cells on neutrophil chemotaxis were investigated by biochemical and morphological studies. We showed that conditioned media collected from several endothelial cell origins submitted to hypoxia as well as ischemic rat liver perfusion liquids have a chemotactic activity for neutrophils. The role of various chemoattractant molecules like HETEs, platelet-activating factor, and cytokines such as interleukin-8 and interleukin-1 was examined in the same model. Chemotactic peptide contribution was ruled out as boiled conditioned media still trigger chemotaxis. However, cell treatment with cyclooxygenase inhibitors, neutralization of PGF(2alpha) biological activity with polyclonal antibodies, and the neutrophil preincubation with a specific PGF(2alpha) antagonist, all dramatically inhibited neutrophil chemotaxis. A strong chemoattractant effect of pure exogenous PGF(2alpha) or of a synthetic analog was also observed. The major effect of PGF(2alpha) on neutrophil chemotaxis was confirmed ex vivo in a rat liver perfusion ischemic model. These results suggest that PGF(2alpha), a prostanoid abundantly released by the endothelium of hypoxic or ischemic tissues, is a chemoattractant molecule that might be involved in the early recruitment of neutrophils in ischemic organs.

Topics: Animals; Cells, Cultured; Chemotactic Factors; Culture Media, Conditioned; Dinoprost; Endothelium, Vascular; Female; Humans; Hydroxyeicosatetraenoic Acids; Hypoxia; Interleukin-1; Interleukin-8; Ischemia; Liver Circulation; Neutrophil Infiltration; Prostaglandin Antagonists; Prostaglandins F; Rats; Rats, Wistar

Hepatic ischemia/reperfusion (I/R) results in tumor necrosis factor (TNF) release. Kupffer cells (KC) are one source of this TNF. This study investigates the effects of hepatic I/R combined with lipopolysaccharide (LPS) on the lung and liver injury that follow hepatic I/R and on hepatic release of TNF, epithelial neutrophil activating protein (ENA-78), and macrophage inflammatory protein-2 (MIP-2). The effects of these experimental conditions on TNF production by primary rat KC in vitro were also investigated. Rats were subjected to hepatic I/R alone, hepatic I/R + LPS, sham laparotomy alone, or sham laparotomy + LPS and pulmonary MPO, pulmonary microvascular permeability, hepatic neutrophil influx, hepatic injury, and hepatic TNF, ENA-78, and MIP-2 production were measured. These experiments demonstrated that hepatic I/R in conjunction with LPS results in a more severe lung and liver injury and increased hepatic TNF, ENA-78, and MIP-2 release. The effects of these experimental conditions on rat KC TNF production demonstrated that hepatic I/R + LPS results in a more significant release of TNF as compared to LPS alone or I/R alone. Hepatic I/R plus LPS results in a more severe lung and liver injury and is likely secondary to a more significant and prolonged release of TNF by KC. This may provide a mechanism for development of multiple organ system failure in some patients undergoing hepatic resection, hepatic transplantation, complex vascular operations, or in the setting of hypovolemic shock. Portal endotoxemia related to mesenteric venous congestion or other systemic insults may have a significant impact on post-operative complications and recovery in the setting of a local or global hepatic I/R injury.

Topics: Animals; Cells, Cultured; Chemokine CXCL2; Chemokine CXCL5; Chemokines; Chemokines, CXC; Interleukin-8; Ischemia; Kupffer Cells; Lipopolysaccharides; Liver; Lung; Lung Injury; Male; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha

Hepatic ischemia/reperfusion (I/R) results in a neutrophil-dependent lung and liver injury. The process of neutrophil recruitment and activation in this injury is at least partially dependent on the presence of the ELR+ CXC chemokines. Other investigations have shown that ELR- CXC chemokines can block ELR+ CXC chemokine neutrophil recruitment and activation in vitro. To begin to investigate the role of the balance between these 2 types of molecules in vivo in neutrophil recruitment and activation following hepatic I/R, we used our rat model of lobar hepatic I/R and pretreated animals with pharmacologic doses of gamma-interferon (gamma-IFN). gamma-IFN is known to upregulate some of the ELR- CXC chemokines, including gamma-IFN-inducible protein (IP-10) and monokine-induced by gamma-IFN (MIG), as well as down-regulate ELR+ CXC chemokine production. Following hepatic I/R or sham laparotomy, hepatic and pulmonary levels of the ELR- chemokines, IP-10 and MIG, and the ELR+ chemokines, rat cytokine-induced neutrophil chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), and epithelial neutrophil activating protein (ENA-78) were determined by ELISA, and lung and liver injury were assessed. In response to gamma-IFN, hepatic and pulmonary levels of the ELR- chemokines were increased and the levels of the ELR+ chemokines were decreased. Immunohistochemical staining confirmed the hepatocyte as the source of these molecules, as well as the changes in chemokine levels in response to gamma-IFN. There was an associated significant decrease in liver and lung injury, although there was no significant decrease in neutrophil influx in either tissue. This suggests that the alteration in the balance of ELR+ to ELR- CXC chemokines results in a decrease in tissue injury through a mechanism other than through an alteration in tissue neutrophil levels.

Topics: Amino Acid Sequence; Animals; Chemokine CXCL10; Chemokine CXCL5; Chemokines, CXC; Interleukin-8; Ischemia; Liver; Liver Circulation; Lung; Male; Neutrophils; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The objectives of this study were to 1) determine the time-course of T-lymphocyte adhesion to monolayers of human umbilical vein endothelial cell (HUVEC) that were exposed to 60 min of anoxia followed by 24 h of reoxygenation, and 2) define the mechanisms responsible for the hyperadhesivity of postanoxic HUVEC to human T-lymphocytes.. Human peripheral blood mononuclear leukocytes were isolated from heparinized peripheral blood. T-lymphocytes were obtained by negative selection using a MACS column. HUVEC monolayers were exposed to anoxia/reoxygenation (A/R), and then reacted with 51Cr -labeled T-lymphocytes in adhesion assays.. A/R leads to an increased adhesion of T-lymphocytes to HUVEC monolayers, with peak responses occurring at 8 h after reoxygenation. This adhesion response was largely attributed to the CD4+ T-cell subset. The hyperadhesivity of A/R-exposed HUVEC was inhibited by monoclonal antibodies directed against either LFA-1, VLA-4, ICAM-1, or VCAM-1, indicating a contribution of these adhesion molecules and their ligands. Moreover, T-cell hyperadhesivity was attenuated by anti- IL-8. consistent with a role for this chemokine in the adhesion response. Protein synthesis inhibitors (actinomycin D and cycloheximide) as well as chemical inhibitors of (and binding ds-oligonucleotides to) NFkappaB and AP-1 significantly attenuated the A/R-induced T-lymphocyte adhesion responses. The kinetics of VCAM-1 on post-anoxic HUVEC correlated with the T-lymphocyte adhesion response.. A/R elicits a T-lymphocyte-endothelial cell adhesion response that involves transcription-dependent surface expression of VCAM-1.

Topics: Antibodies, Monoclonal; Benzamides; Cell Adhesion; Cell Adhesion Molecules; Cell Hypoxia; Cells, Cultured; Cysteine Endopeptidases; Endothelium, Vascular; Humans; Interleukin-8; Ischemia; Kinetics; Leupeptins; Multienzyme Complexes; NF-kappa B; Oxygen; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Reperfusion Injury; T-Lymphocyte Subsets; Thionucleotides; Time Factors; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Cell Adhesion Molecule-1

21-aminosteroids (lazaroids) have demonstrated the protective effect against cerebral ischemic injury through the inhibition of lipid peroxidation. We examined whether lazaroids affected the production of proinflammatory and antiinflammatory cytokines in ischemic spinal cord injury model.. Anesthetized New Zealand white rabbits underwent a 20-minute infrarenal aortic cross-clamping (AXC) with pretreatment of either an intravenous 3 mg/kg lazaroid U74389G (group L; n = 10) or the same volume saline (group P; n = 10). Sham operation group (group S; n = 6) underwent only exposure of the aorta. Plasma concentrations of interleukin (IL)-8, -1beta, -1 receptor antagonist (IL-1ra) and tumor necrosis factor (TNF)-alpha were measured at four time points. Functional assessment with Tarlov score at 24 and 48 hours after pretreatment, pathologic assessment of the spinal cord, and measurements of cytokine levels in the spinal cord were performed.. The maximum elevation of plasma IL-8 and -1ra levels occurred at 1 hour after declamping in four measurement points. Plasma IL-8 and -1ra levels in group L were significantly lower than those in group P (*p < 0.05). Plasma TNFalpha peaked at 5 minutes after declamping, but decreased afterwards. Plasma TNFalpha levels were not different among three groups. Spinal IL-8 levels in group L (0.98 +/- 0.34 ng/g tissue) were lower than those in group P (7.26 +/- 2.26 ng/g tissue)(*p < 0.05). Spinal IL-1ra and TNFalpha were not significantly different. Tarlov score and pathologic assessment were better in group L.. Lazaroid U-74389G reduced the production of systemic IL-8 and -1ra and spinal IL-8 when AXC caused spinal cord injury. These results indicate that lazaroids may attenuate ischemic endothelial cell injury or activation of leukocytes.

Topics: Animals; Interleukin 1 Receptor Antagonist Protein; Interleukin-8; Ischemia; Neuroprotective Agents; Pregnatrienes; Rabbits; Receptors, Interleukin-1; Sialoglycoproteins; Spinal Cord Injuries

A growing amount of data supports the role of inflammation in the pathophysiology of atherosclerotic diseases but the cellular source of cytokines has not been clearly identified. Cytokines could be produced by inflammatory cells, activated endothelial and smooth muscle cells, and by the tissue exposed to recurrent ischemia. Accordingly, we evaluated whether hypoperfusion induces gene expression of interleukin (IL)-1beta and IL-6 in the skeletal muscle of patients with peripheral arterial disease and critical limb ischemia.. Skeletal muscle biopsies were obtained, during a femoral-distal bypass, from normoperfused (control) and hypoperfused skeletal muscles in 8 patients. Gene expression was assessed by semiquantitative reverse transcriptase-polymerase chain reaction, using glyceraldehyde-phosphate-deydrogenase mRNA levels as a normalization factor.. In the hypoperfused biopsies, the level of IL-1beta gene expression was significantly higher in all but 2 patients (mean upregulation > 8.8 fold, p = 0.043), and the level of IL-6 gene expression was significantly higher in all but 1 patient (mean upregulation > 23.7 fold, p = 0.031).. We report that IL-1beta and IL-6 gene expression is markedly upregulated in hypoperfused skeletal muscle of patients with critical lower limb ischemia. To our knowledge this is the first report of a local activation of the inflammatory cascade at the level of hypoperfused skeletal muscle. This activation, which could worsen symptoms and tissue viability and be involved in the pathophysiology of reperfusion injury, might be considered as a therapeutic target. It remains to be investigated whether our results may also apply to coronary artery disease.

Topics: Aged; Aged, 80 and over; Arterial Occlusive Diseases; Female; Gene Expression; Humans; Interleukin-1; Interleukin-8; Ischemia; Leg; Male; Middle Aged; Muscle, Skeletal

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Dogs; Immunosuppressive Agents; Interleukin-8; Ischemia; Liver; Neutrophils; Pyrazoles; Pyridines; Reperfusion; RNA, Messenger; Transcription, Genetic

To study the mechanisms responsible for ischemia-reperfusion lung injury, we developed an anesthetized rabbit model in which the effects of lung deflation, lung inflation, alveolar gas composition, hypothermia, and neutrophils on reperfusion pulmonary edema could be studied. Rabbits were anesthetized and ventilated, and the left pulmonary hilum was clamped for either 2 or 4 h. Next, the left lung was reperfused and ventilated with 100% oxygen. As indexes of lung injury, we measured arterial oxygenation, extravascular lung water, and the influx of a vascular protein (131I-labeled albumin) into the extravascular space of the lungs. The principal results were that 1) all rabbits with the deflation of the lung during ischemia for 4 h died of fulminant pulmonary edema within 1 h of reperfusion; 2) inflation of the ischemic lung with either 100% oxygen, air, or 100% nitrogen prevented the reperfusion lung injury; 3) hypothermia at 6-8 degreesC also prevented the reperfusion lung injury; 4) although circulating neutrophils declined during reperfusion lung injury, there was no increase in interleukin-8 levels in the plasma or the pulmonary edema fluid, and, furthermore, neutrophil depletion did not prevent the reperfusion injury; and 5) ultrastructural studies demonstrated injury to both the lung endothelium and the alveolar epithelium after reperfusion in deflated lungs, whereas the inflated lungs had no detectable injury. In summary, ischemia-reperfusion injury to the rabbit lung can be prevented by either hypothermia or lung inflation with either air, oxygen, or nitrogen.

Topics: Animals; Body Fluids; Cell Count; Hypothermia, Induced; Interleukin-8; Ischemia; Lung; Neutrophils; Osmolar Concentration; Oxygen; Pulmonary Alveoli; Pulmonary Circulation; Pulmonary Edema; Rabbits; Reperfusion Injury; Vinblastine

To study plasma concentrations of interleukin 6 (IL-6) and interleukin 8 (IL-8) in patients with splanchnic hypoxia, as documented by gastric intramucosal measurements (pH-i), during major abdominal surgery and the relationship between IL-6 and IL-8 concentrations and postoperative complications as well as clinical outcome.. A prospective study.. Twelve patients scheduled for major abdominal surgery with no evidence of coexisting infectious disease.. Six out of seven samples from patients with postoperative complications showed intraoperative pH-i levels lower than 7.32 and IL-6 levels higher than 300 pg/ml. Seven out of nine samples from patients without complications showed pH-i levels higher than 7.32 and IL-6 levels lower than 300 pg/ml. The difference in the pattern of distribution was statistically significant (p < 0.01). Only two out of seven samples of patients with postoperative complications showed intraoperative pH-i levels lower than 7.32 and IL-8 levels higher than 60 pg/ml. It was not possible to identify a clear distribution pattern of data points for IL-6 and IL-8 during the postoperative period.. Intraoperative splanchnic ischemia, as documented by gastric intramucosal pH-i, is directly correlated to the increase of IL-6 plasma levels and to the incidence of postoperative complications, while IL-8 levels showed no correlation with surgical complications.

Topics: Adult; Aged; Cell Hypoxia; Gastric Mucosa; Humans; Hydrogen-Ion Concentration; Incidence; Interleukin-6; Interleukin-8; Intraoperative Complications; Ischemia; Laparotomy; Linear Models; Middle Aged; Postoperative Complications; Predictive Value of Tests; Prospective Studies; Splanchnic Circulation; Treatment Outcome

Topics: Animals; Antibodies, Monoclonal; Chemokines, CXC; Chemotactic Factors; Growth Substances; Intercellular Signaling Peptides and Proteins; Interleukin-8; Intestine, Small; Ischemia; Rats; Rats, Inbred Lew; Reperfusion Injury; Tumor Necrosis Factor-alpha

As a mediator of neurogenic inflammation and pain, we hypothesized that levels of the neuropeptide Substance P (SP) would be elevated in patients with sickle cell disease (SCD) with vaso-occlusive pain crisis. SP is a known stimulator of tumor necrosis factor-alpha (TNF-alpha) release and a promoter of interleukin-8 (IL-8), which are reported to be increased in SCD. These cytokines enhance adhesion of leukocytes to endothelium and may play a role in vaso-occlusive events. Serum levels of IL-8, TNFalpha, and SP were studied in three groups of children aged 2 to 18 years: 30 well children with SCD, 21 with SCD in pain crisis, and 20 healthy age-matched controls. Serum levels of SP were elevated in all SCD patients and were highest in patients in pain crisis. The percentage of sera with detectable levels of IL-8 (>5.0 pmol/L) was increased in SCD patients as compared with the control group. IL-8 levels were similar for well SCD patients and those with pain. TNFalpha levels were not significantly different among the three groups. In three children with SCD, SP was measured at baseline and again during pain crisis. In each case, serum levels during pain crisis were higher than they were when the patient was well. We conclude that levels of SP are high in patients with SCD and increase during pain crisis. These results imply that SP plays a prominent role in the pain and inflammation of SCD and may be a measurable laboratory marker of vaso-occlusive crisis. We speculate that neurokinin receptor antagonists may have a therapeutic potential in the treatment of crisis pain.

Topics: Acute Disease; Adolescent; Anemia, Sickle Cell; Child; Child, Preschool; Female; Humans; Interleukin-8; Ischemia; Male; Pain; Substance P; Tumor Necrosis Factor-alpha

This study investigates the protective mechanism of prostaglandin E1 (PGE1) against hepatic ischemia-reperfusion injury in vivo. It has been demonstrated that activated leukocytes contribute to ischemia-reperfusion injury, and that administration of the monoclonal antibody (mAb) for adhesion molecules reduces the injury by inhibiting leukocyte-endothelial cell adhesion. We therefore attempted to find out whether PGE1 has an effect on the inhibition of neutrophil adherence to endothelial cells after reperfusion.. We administered anti-intercellular adhesion molecule 1 (ICAM-1) mAb, antiserum against rat polymorphonuclear leukocytes, or PGE1 to a rat model of left lobar ischemia for 60 min followed by reperfusion. Leukocyte adherence was observed by intravital fluorescence microscopy. The effect of PGE1 on the expression of adhesion molecules was analyzed by immunohistochemistry and flow cytometry.. Ischemia-reperfusion caused endothelial dysfunction and hepatocellular injury with leukostasis in postsinusoidal venules. Anti-ICAM-1 mAb administration or leukopenia ameliorated both the hepatocellular injury and endothelial dysfunction. Although PGE1 administration did not affect the serum interleukin-8 level, it significantly decreased hepatic injury and leukostasis in the reperfused liver. Immunohistochemical findings showed that PGE1 decreased ICAM-1 expression on endothelial cells, but did not affect lymphocyte function-associated antigen 1, and membrane attack complex 1 on neutrophils in flow cytometric analysis.. We conclude that PGE1 protects the liver against ischemia-reperfusion injury by reducing leukocyte-endothelial cell adhesion via down-modulation of ICAM-1 expression on the endothelium.

Topics: Alprostadil; Animals; Antibodies, Monoclonal; Aspartate Aminotransferases; Cell Adhesion; Endothelium, Vascular; Flow Cytometry; Intercellular Adhesion Molecule-1; Interleukin-8; Ischemia; Liver; Lymphocyte Function-Associated Antigen-1; Macrophage-1 Antigen; Male; Microscopy, Fluorescence; Neutrophils; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The liver is highly susceptible to a number of pathological insults, including ischemia/reperfusion injury. We have previously employed an animal model of hepatic ischemia/reperfusion injury, and have shown that this injury induces the production and release of hepatic-derived tumor necrosis factor alpha (TNF-alpha), which mediates, in part, local liver injury following hepatic reperfusion. In the present study, we have extended these previous observations to assess whether an interrelationship exists between TNF-alpha and the neutrophil chemoattractant/activating factor, epithelial neutrophil activating protein, that may account for some of the pathology of neutrophil-mediated ischemia/reperfusion-induced liver injury. We observed that hepatic ischemia/reperfusion injury leads to: (1) a coincident increase in hepatic neutrophil sequestration, elevated serum alanine aminotransferase (ALT) levels, and hepatic production of epithelial neutrophil activating protein; (2) passive immunization with neutralizing antibodies to TNF-alpha resulted in significant suppression of hepatic-derived epithelial neutrophil activating protein; and (3) neutralization of epithelial neutrophil activating protein by passive immunization significantly attenuated neutrophil sequestration in the liver and serum ALT levels. These findings support the notion that local expression of hepatic epithelial neutrophil activating protein produced in response to TNF-alpha is an important mediator of the local neutrophil-dependent hepatic injury associated with hepatic ischemia/reperfusion.

Topics: Alanine Transaminase; Animals; Chemokine CXCL5; Chemokines, CXC; Cytokines; Interleukin-8; Ischemia; Liver; Male; Neutrophil Activation; Neutrophils; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha

Topics: Animals; Interleukin-8; Intestinal Mucosa; Intestine, Small; Ischemia; Male; Mesenteric Arteries; Rats; Rats, Inbred Lew; Reperfusion Injury; Temperature; Time Factors; Tumor Necrosis Factor-alpha

Topics: Animals; Interleukin-8; Intestine, Small; Ischemia; Male; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Time Factors

Topics: Animals; Cytokines; Hemostasis, Surgical; Humans; Interleukin-6; Interleukin-8; Ischemia; Liver; Liver Diseases; Tumor Necrosis Factor-alpha

Topics: Animals; Chemokines, CXC; Chemotactic Factors; Gadolinium; Gene Expression; Growth Substances; Intercellular Signaling Peptides and Proteins; Interleukin-8; Ischemia; Kupffer Cells; Liver; Male; Rats; Rats, Wistar; Reperfusion Injury

Polymorphonuclear leukocytes (PMN) contribute to post-ischemic injury in many organs and in a variety of clinical situations. PMN accumulate in both lungs during unilateral lung ischemia in sheep, but the mechanism has not been defined. In this study, we tested the hypothesis that PMN accumulation is a response to chemotactic signals generated during lung ischemia. Chemotactic activity was measured in a modified Boyden chamber using normal sheep PMN as the responding cells. Increased chemotactic activity was observed in both plasma and lung lymph in a time-dependent manner after ischemia. These data indicate that a chemotactic substance immunoreactive to interleukin-8 antibody is formed as a result of unilateral lung ischemia in sheep in vivo and is a possible mediator of PMN inflammation in this model.

Topics: Acute-Phase Reaction; Animals; Chemotaxis, Leukocyte; Immune Sera; Interleukin-8; Ischemia; Leukocyte Count; Lung; Lymph; Neutrophils; Sheep

Re-establishing blood flow to ischaemic tissues causes greater injury than that induced during the ischaemic period. This type of tissue injury, reperfusion injury, is involved in frostbite, multiple organ failure after hypovolaemia and in myocardial infarction. Depletion of neutrophils alleviates reperfusion injury, implying a causal role of neutrophil infiltration. Among members of the recently discovered family of chemotactic cytokines (chemokines), interleukin-8 (IL-8) is a major neutrophil chemotactic and activating factor produced by various types of human cells. We investigated its pathophysiological role in a rabbit model of a lung reperfusion injury. Reperfusion of ischaemic lung caused neutrophil infiltration and destruction of pulmonary structure, as well as local production of IL-8. Furthermore, the administration of a neutralizing monoclonal antibody against IL-8 prevented neutrophil infiltration and tissue injury, proving a causal role of locally produced IL-8 in this model.

Topics: Animals; Antibodies, Monoclonal; Bronchoalveolar Lavage Fluid; Chemotaxis, Leukocyte; Female; Guinea Pigs; Interleukin-8; Ischemia; Lung; Lung Diseases; Neutrophils; Rabbits; Reperfusion Injury