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In JoVE (1)
Other Publications (18)
- Biochemical and Biophysical Research Communications
- British Journal of Pharmacology
- Biochemical and Biophysical Research Communications
- Clinical and Experimental Pharmacology & Physiology
- Tissue Engineering
- Redox Report : Communications in Free Radical Research
- Inflammation & Allergy Drug Targets
- Blood
- Arteriosclerosis, Thrombosis, and Vascular Biology
- Circulation Research
- The Journal of Biological Chemistry
- Circulation Research
- The Journal of Experimental Medicine
- Nature Reviews. Cardiology
- Current Pharmaceutical Design
- Clinical and Experimental Pharmacology & Physiology
- Arteriosclerosis, Thrombosis, and Vascular Biology
- Proteome Science
Articles by Kevin J. Woollard in JoVE
JoVE Immunology and Infection
Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure
Vascular Pharmacology Laboratory, Baker IDI Heart and Diabetes Institute, Monash University
This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.
Other articles by Kevin J. Woollard on PubMed
Effects of Oral Vitamin C on Monocyte: Endothelial Cell Adhesion in Healthy Subjects
Monocyte recruitment and retention in the vasculature is influenced by oxidative stress and is involved in cardiovascular disease (CVD). Individuals with low plasma ascorbate are at elevated risk of CVD. It is unknown whether vitamin C supplementation affects monocyte adhesion to endothelial cells (ECs) in healthy non-smokers. In a randomised double-blind crossover study the effect of vitamin C supplementation (six weeks, 250 mg/day) was determined in subjects with normal (HIC) and below average (LOC) plasma vitamin C concentration at baseline (mean=67 microM, n=20, mean=32 microM, n=20, respectively). LOC subjects showed 30% greater monocyte adhesion to ECs. This was significantly reduced by 37% (P<0.02) following vitamin C supplementation to levels of HIC monocyte adhesion. No differences in plasma malondialdehyde concentrations were observed between groups or after supplementation. In conclusion, vitamin C supplementation normalises monocyte adhesion in subjects with low plasma vitamin C (LOC). This process may be related to a direct effect on monocytes, independent of lipid peroxidation.
The Anti-inflammatory Actions of Methotrexate Are Critically Dependent Upon the Production of Reactive Oxygen Species
1 The mechanism of action by which methotrexate (MTX) exerts its anti-inflammatory and immunosuppressive effects remains unclear. The aim of this study is to investigate the hypothesis that MTX exerts these effects via the production of reactive oxygen species (ROS). 2 Addition of MTX (100 nM-10 micro M) to U937 monocytes induced a time and dose dependent increase in cytosolic peroxide [peroxide](cyt) from 6-16 h. MTX also caused corresponding monocyte growth arrest, which was inhibited (P<0.05) by pre-treatment with N-acetylcysteine (NAC; 10 mM) or glutathione (GSH; 10 mM). In contrast, MTX induction of [peroxide](cyt) in Jurkat T cells was more rapid (4 h; P<0.05), but was associated with significant apoptosis at 16 h at all doses tested (P<0.05) and was significantly inhibited by NAC or GSH (P<0.05). 3 MTX treatment of monocytes (10 nM-10 micro M) for 16 h significantly reduced total GSH levels (P<0.05) independently of dose (P>0.05). However, in T-cells, GSH levels were significantly elevated following 30 nM MTX treatment (P<0.05) but reduced by doses exceeding 1 micro M compared to controls (P<0.05). 4 MTX treatment significantly reduced monocyte adhesion to 5 h and 24 h LPS (1 micro g ml(-1)) activated human umbilical vein endothelial cells (HUVEC; P<0.05) but not to resting HUVEC. Pre-treatment with GSH prevented MTX-induced reduction in adhesion. 5 In conclusion, ROS generation by MTX is important for cytostasis in monocytes and cytotoxicity T-cells. Furthermore, MTX caused a reduction in monocyte adhesion to endothelial cells, where the mechanism of MTX action requires the production of ROS. Therefore its clinical efficacy can be attributed to multiple targets.
Vitamin C Supplementation in Normal Subjects Reduces Constitutive ICAM-1 Expression
Regulation of monocyte adhesion molecule gene expression is via redox sensitive transcription factors. We have investigated whether dietary antioxidant supplementation with vitamin C (250 mg/day) can modulate monocyte ICAM-1 expression in healthy male subjects with low plasma vitamin C at baseline. In a randomised, double-blind, crossover study, monocyte ICAM-1 mRNA was analysed using quantitative reverse transcriptase PCR. Protein was determined by flow cytometry (monocytes) and ELISA (plasma). Monocyte numbers were unaltered by supplementation. Subjects with low plasma vitamin C (<50 microM) prior to supplementation expressed higher levels of monocyte ICAM-1mRNA, and showed a significant (50%) reduction in ICAM-1mRNA expression after 6 weeks of 250 mg/day vitamin C supplementation (p<0.05). This was paralleled by a reduction in sICAM-1 (p<0.05). For the first time, these results show that dietary vitamin C can modulate monocyte ICAM-1 gene expression in vivo, where regulation of gene expression represents a novel mechanism for benefit from dietary antioxidants.
Soluble Bio-markers in Vascular Disease: Much More Than Gauges of Disease?
1. In recent years demonstration of a direct association between slightly elevated serum levels of soluble proteins including the acute phase response proteins, selectins and intercellular adhesion molecules and the risk of developing vascular disease have been widely reported. These studies may provide the clinician with an insight into disease diagnosis, prognosis and disease activity. 2. The simplest interpretation of this data is that soluble proteins are just sensitive markers of inflammation. However, they may in fact be modulating inflammation directly through interaction with circulating cells. 3. Recent work has shown that these soluble proteins do indeed remain active and can bind to functional ligands expressed by circulating leucocytes. The current review focuses on the soluble proteins C-reactive protein and soluble P-selectin and describes previous studies characterizing their interaction with immune cells to modulate the pathogenesis of vascular disease. 4. The current review focuses on the soluble proteins C-reactive protein and soluble P-selectin and describes previous studies characterizing their interaction with immune cells to modulate the pathogenesis of vascular disease.
Gravity Spun Polycaprolactone Fibers for Applications in Vascular Tissue Engineering: Proliferation and Function of Human Vascular Endothelial Cells
Poly(epsilon-caprolactone) (PCL) fibers produced by wet spinning from solutions in acetone under lowshear (gravity-flow) conditions resulted in fiber strength of 8 MPa and stiffness of 0.08 Gpa. Cold drawing to an extension of 500% resulted in an increase in fiber strength to 43 MPa and stiffness to 0.3 GPa. The growth rate of human umbilical vein endothelial cells (HUVECs) (seeded at a density of 5 x 10(4) cells/mL) on as-spun fibers was consistently lower than that measured on tissue culture plastic (TCP) beyond day 2. Cell proliferation was similar on gelatin-coated fibers and TCP over 7 days and higher by a factor of 1.9 on 500% cold-drawn PCL fibers relative to TCP up to 4 days. Cell growth on PCL fibers exceeded that on Dacron monofilament by at least a factor of 3.7 at 9 days. Scanning electron microscopy revealed formation of a cell layer on samples of cold-drawn and gelatin-coated fibers after 24 hours in culture. Similar levels of ICAM-1 expression by HUVECs attached to PCL fibers and TCP were measured using RT-PCR and flow cytometry, indicative of low levels of immune activation. Retention of a specific function of HUVECs attached to PCL fibers was demonstrated by measuring their immune response to lipopolysaccharide. Levels of ICAM-1 expression increased by approximately 11% in cells attached to PCL fibers and TCP. The high fiber compliance, favorable endothelial cell proliferation rates, and retention of an important immune response of attached HUVECS support the use of gravity spun PCL fibers for three-dimensional scaffold production in vascular tissue engineering.
Alpha-tocopherol Supplementation Does Not Affect Monocyte Endothelial Adhesion or C-reactive Protein Levels but Reduces Soluble Vascular Adhesion Molecule-1 in the Plasma of Healthy Subjects
Vascular monocyte retention in the subintima is pivotal to the development of cardiovascular disease and is facilitated by up-regulation of adhesion molecules on monocytes/endothelial cells during oxidative stress. Epidemiological studies have shown that cardiovascular disease risk is inversely proportional to plasma levels of the dietary micronutrients, vitamin C and vitamin E (alpha-tocopherol). We have tested the hypothesis that alpha-tocopherol supplementation may alter endothelial/monocyte function and interaction in subjects with normal ascorbate levels (> 50 microM), as ascorbate has been shown to regenerate tocopherol from its oxidised tocopheroxyl radical form in vitro. Healthy male subjects received alpha-tocopherol supplements (400 IU RRR-alpha-tocopherol/day for 6 weeks) in a placebo-controlled, double-blind intervention study. There were no significant differences in monocyte CD11b expression, monocyte adhesion to endothelial cells, plasma C-reactive protein or sICAM-1 concentrations post-supplementation. There was no evidence for nuclear translocation of NF-kappaB in isolated resting monocytes, nor any effect of alpha-tocopherol supplementation. However, post-supplementation, sVCAM-1 levels were decreased in all subjects and sE-selectin levels were increased in the vitamin C-replete group only; a weak positive correlation was observed between sE-selectin and alpha-tocopherol concentration. In conclusion, alpha-tocopherol supplementation had little effect on cardiovascular disease risk factors in healthy subjects and the effects of tocopherol were not consistently affected by plasma vitamin C concentration.
Therapeutic Targeting of P-selectin in Atherosclerosis
P-selectin is an inflammatory adhesion molecule expressed on activated platelets and endothelial cells. The role of inflammatory cells and adhesion molecules in the development and progression of vascular diseases has been well studied in the past two decades and it is now recognised that many of the cellular and molecular events that underlie atherosclerotic vascular disease are inflammatory in nature. The critical role of P-selectin in both leukocyte recruitment and vascular disease progression has been confirmed in knockout animal models, where P-selectin knockout mice crossed with apoE deficient mice exhibit significantly reduced atherosclerosis and leukocyte recruitment in the plaque. Being the primary adhesion molecule in initiating cell activation and cell adhesion to platelets and endothelial cells, P-selectin is therefore an attractive therapeutic target in vascular disease. However the basic tenet of targeting P-selectin may be complicated by the presence of a soluble form of P-selectin (sP-selectin). sP-selectin, lacking the cytosolic/transmembrane domain, has been identified circulating in plasma and is thought to either be derived from the secretion of an alternatively spliced protein that lacks the transmembrane domain and/or from proteolytic cleavage of the membrane form, thus reflecting the activated state of both platelets and/or endothelial cells. This review will discuss the role of P-selectin in inflammatory disease particularly in atherosclerosis and will highlight current in vitro and in vivo discoveries.
Conversion of Platelets from a Proaggregatory to a Proinflammatory Adhesive Phenotype: Role of PAF in Spatially Regulating Neutrophil Adhesion and Spreading
The ability of platelets to provide a highly reactive surface for the recruitment of other platelets and leukocytes to sites of vascular injury is critical for hemostasis, atherothrombosis, and a variety of inflammatory diseases. The mechanisms coordinating platelet-platelet and platelet-leukocyte interactions have been well defined and, in general, it is assumed that increased platelet activation correlates with enhanced reactivity toward other platelets and neutrophils. In the current study, we demonstrate a differential role for platelets in supporting platelet and neutrophil adhesive interactions under flow. We demonstrate that the conversion of spread platelets to microvesiculated procoagulant (annexin A5-positive [annexin A5+ve]) forms reduces platelet-platelet adhesion and leads to a paradoxical increase in neutrophil-platelet interaction. This enhancement in neutrophil adhesion and spreading is partially mediated by the proinflammatory lipid, platelet-activating factor (PAF). PAF production, unlike other neutrophil chemokines (IL-8, GRO-alpha, NAP-2, IL-1beta) is specifically and markedly up-regulated in annexin A5+ve cells. Physiologically, this spatially controlled production of PAF plays an important role in localizing neutrophils on the surface of thrombi. These studies define for the first time a specific proinflammatory function for annexin A5+ve platelets. Moreover, they demonstrate an important role for platelet-derived PAF in spatially regulating neutrophil adhesion under flow.
High-density Lipoprotein Reduces the Human Monocyte Inflammatory Response
Whereas the anti-inflammatory effects of high-density lipoprotein (HDL) on endothelial cells are well described, such effects on monocytes are less studied.
Pathophysiological Levels of Soluble P-selectin Mediate Adhesion of Leukocytes to the Endothelium Through Mac-1 Activation
Plasma soluble P-selectin (sP-selectin) levels are increased in pathologies associated with atherosclerosis, including peripheral arterial occlusive disease (PAOD). However, the role of sP-selectin in regulating leukocyte-endothelial adhesion is unclear. The aim of this study was to assess the ability of exogenous and endogenous sP-selectin to induce leukocyte responses that promote their adhesion to various forms of endothelium. In flow chamber assays, sP-selectin dose-dependently increased neutrophil adhesion to resting human iliac artery endothelial cells. Similarly, sP-selectin induced neutrophil adhesion to the endothelial surface of murine aortae and human radial venous segments in ex vivo flow chamber experiments. Using intravital microscopy to examine postcapillary venules in the mouse cremaster muscle, in vivo administration of sP-selectin was also found to significantly increase leukocyte rolling and adhesion in unstimulated postcapillary venules. Using a Mac-1-specific antibody and P-selectin knockout mouse, it was demonstrated that this finding was dependent on a contribution of Mac-1 to leukocyte rolling and endothelial P-selectin expression. This was confirmed in an ex vivo perfusion model using viable mouse aorta and human radial vessels. In contrast, with tumor necrosis factor-alpha-activated endothelial cells and intact endothelium, where neutrophil adhesion was already elevated, sP-selectin failed to further increase adhesion. Plasma samples from PAOD patients containing pathologically elevated concentrations of sP-selectin also increased neutrophil adhesion to the endothelium in a sP-selectin-dependent manner, as demonstrated by immunodepletion of sP-selectin. These studies demonstrate that raised plasma sP-selectin may influence the early progression of vascular disease by promoting leukocyte adhesion to the endothelium in PAOD, through Mac-1-mediated rolling and dependent on endothelial P-selectin expression.
Erythrocyte Hemolysis and Hemoglobin Oxidation Promote Ferric Chloride-induced Vascular Injury
The release of redox-active iron and heme into the blood-stream is toxic to the vasculature, contributing to the development of vascular diseases. How iron induces endothelial injury remains ill defined. To investigate this, we developed a novel ex vivo perfusion chamber that enables direct analysis of the effects of FeCl(3) on the vasculature. We demonstrate that FeCl(3) treatment of isolated mouse aorta, perfused with whole blood, was associated with endothelial denudation, collagen exposure, and occlusive thrombus formation. Strikingly exposing vessels to FeCl(3) alone, in the absence of perfused blood, was associated with only minor vascular injury. Whole blood fractionation studies revealed that FeCl(3)-induced vascular injury was red blood cell (erythrocyte)-dependent, requiring erythrocyte hemolysis and hemoglobin oxidation for endothelial denudation. Overall these studies define a unique mechanism of Fe(3+)-induced vascular injury that has implications for the understanding of FeCl(3)-dependent models of thrombosis and vascular dysfunction associated with severe intravascular hemolysis.
Dissociation of Pentameric to Monomeric C-reactive Protein on Activated Platelets Localizes Inflammation to Atherosclerotic Plaques
C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentamer (pentameric CRP) in plasma. The in vivo existence of monomeric (m)CRP has been postulated, but its function and source are not clear. We show that mCRP is deposited in human aortic and carotid atherosclerotic plaques but not in healthy vessels. pCRP is found neither in healthy nor in diseased vessels. As source of mCRP, we identify a mechanism of dissociation of pCRP to mCRP. We report that activated platelets, which play a central role in cardiovascular events, mediate this dissociation via lysophosphatidylcholine, which is present on activated but not resting platelets. Furthermore, the dissociation of pCRP to mCRP can also be mediated by apoptotic monocytic THP-1 and Jurkat T cells. The functional consequence is the unmasking of proinflammatory effects of CRP as demonstrated in experimental settings that are pathophysiologically relevant for atherogenesis: compared to pCRP, mCRP induces enhanced monocyte chemotaxis; monocyte activation, as determined by conformational change of integrin Mac-1; generation of reactive oxygen species; and monocyte adhesion under static and physiological flow conditions. In conclusion, we demonstrate mCRP generation via pCRP dissociation on activated platelets and H(2)O(2)-treated apoptotic THP-1 and Jurkat T cells, thereby identifying a mechanism of localized unmasking of the proinflammatory properties of CRP. This novel mechanism provides a potential link between the established cardiovascular risk marker, circulating pCRP, and localized platelet-mediated inflammatory and proatherogenic effects.
Langerhans Cell (LC) Proliferation Mediates Neonatal Development, Homeostasis, and Inflammation-associated Expansion of the Epidermal LC Network
Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin(+) cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10-20-fold. After the first week of life, we observed low-level proliferation of langerin(+) cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.
Monocytes in Atherosclerosis: Subsets and Functions
Chronic inflammation drives atherosclerosis, the leading cause of cardiovascular disease. Over the past two decades, data have emerged showing that immune cells are involved in the pathogenesis of atherosclerotic plaques. The accumulation and continued recruitment of leukocytes are associated with the development of 'vulnerable' plaques. These plaques are prone to rupture, leading to thrombosis, myocardial infarction or stroke, all of which are frequent causes of death. Plaque macrophages account for the majority of leukocytes in plaques, and are believed to differentiate from monocytes recruited from circulating blood. However, monocytes represent a heterogenous circulating population of cells. Experiments are needed to address whether monocyte recruitment to plaques and effector functions, such as the formation of foam cells, the production of nitric oxide and reactive oxygen species, and proteolysis are critical for the development and rupture of plaques, and thus for the pathophysiology of atherosclerosis, as well as elucidate the precise mechanisms involved.
P-selectin Antagonism in Inflammatory Disease
Inflammation plays a fundamental role in many chronic diseases, including atherosclerosis associated cardiovascular disease. Adhesion of immune cells plays a critical role in the inflammatory response and indeed the pathophysiology of inflammatory related diseases. P-selectin is an inflammatory adhesion molecule, enabling the recruitment of leukocytes to the endothelium and to activated platelets involved with the growing thrombus. P-selectin is critical in the progression of atherosclerosis as evidenced by knockout animal models where P-selectin knockout mice crossed with apoE deficient mice exhibit significantly reduced atherosclerosis and leukocyte recruitment in the plaque. A soluble form of P-selectin also exists, which may have pro-atherogenic and pro-thrombotic effects. Thus targeting of P-selectin remains a strong clinical candidate for developing novel therapeutic strategies in inflammatory diseases. This review will discuss the role of P-selectin and describe the function of P-selectin antagonists as clinical targets.
High-density Lipoprotein: a Potent Inhibitor of Inflammation
1. Inflammation is an important process, driving the progression of atherosclerosis. Stemming inflammation may be a mechanism to inhibit the progression of this disease. 2. High-density lipoprotein (HDL), a particle inversely related to cardiovascular disease, has been described as having a number of anti-inflammatory functions. It has been shown that HDL inhibits endothelial inflammation in both in vitro and in vivo models, reducing the expression of key cell adhesion molecules. In addition, HDL has been shown to have an effect on the coagulation pathway by inhibiting platelet activation and reducing thrombus formation. Furthermore, by reducing the activation of leucocytes, HDL can inhibit leucocyte recruitment to the endothelium. 3. High-density lipoprotein infusion studies conducted in patients with inflammatory diseases have shown that acute treatment with HDL can effectively inhibit inflammation in vivo. Thus, HDL has been proven to be a potent inhibitor of inflammation, acting on a number of pathways, and this may suggest that HDL could be applied as an anti-inflammatory molecule for a number of diseases. 4. The present review highlights these important studies and reviews data on the anti-inflammatory effects of HDL from in vitro and in vivo studies, in both humans and animal models of atherosclerosis and inflammatory-related diseases.
Neutrophil Activation is Attenuated by High-density Lipoprotein and Apolipoprotein A-I in in Vitro and in Vivo Models of Inflammation
Neutrophils play a key role in the immune response but can undesirably exacerbate inflammation. High-density lipoproteins (HDL) are antiinflammatory particles, exerting beneficial cardiovascular influences. We determined whether HDL exerts antiinflammatory effects on neutrophils and explored the mechanisms by which these occur.
A Proteomic Analysis of C-reactive Protein Stimulated THP-1 Monocytes
C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP.
