Hans-Anton Lehr

Protection From Oxidized LDL–Induced Leukocyte Adhesion to Microvascular and Macrovascular Endothelium In Vivo by Vitamin C but Not by Vitamin E

BACKGROUNDnThe ability of oxidized LDL (oxLDL) to stimulate leukocyte-endothelium interaction is considered to be an important aspect of its proatherogenic action. Using intravital fluorescence microscopy in the dorsal skinfold chamber model in hamsters, we have previously shown that systemic administration of oxLDL stimulates leukocyte adhesion to microvascular endothelium through a mechanism that involves the generation and action of reactive oxygen species (ROS).nnnMETHODS AND RESULTSnThrough the combined use of scanning electron microscopy and intravital microscopy in the same animal model, we demonstrate that oxLDL-induced leukocyte adhesion is not confined to the microcirculation but can also be observed on aortic endothelium. OxLDL-induced leukocyte adhesion to both microvascular and macrovascular endothelium was almost entirely prevented by pretreatment of the hamsters with dietary or intravenous vitamin C, which has the capacity to scavenge and neutralize ROS (arterioles: 20.5 +/- 16.4 cells/mm2 [diet] and 16.3 +/- 23.8 cells/mm2 [IV] versus 74.2 +/- 47.5 cells/mm2 [control, P < .01]; aorta: 1.0 +/- 0.4 cells/mm2 [diet] and 1.1 +/- 0.5 cells/mm2 [IV] versus 14.7 +/- 6.0 cells/mm2 [control, P < .01], 15 minutes after oxLDL, n = 7 animals per group). Vitamin C pretreatment also completely prevented oxLDL-induced leukocyte-platelet aggregate formation in the blood-stream but did not affect leukocyte rolling along the microvascular endothelium. No inhibitory effect on any of the studied parameters was observed as a result of pretreatment of the animals with the lipid-soluble antioxidants vitamin E and probucol.nnnCONCLUSIONSnThe protective effects of vitamin C on oxLDL-induced leukocyte adhesion and aggregate formation were seen at vitamin C plasma levels that can easily be reached in humans by diet or supplementation, suggesting that this could be one of the mechanisms by which vitamin C contributes to the well-documented protraction of atherogenesis as observed in large epidemiological surveys.

Cigarette smoke elicits leukocyte adhesion to endothelium in hamsters: Inhibition by CuZn-SOD

Although cigarette smoking has been identified as a major risk factor for cardiovascular diseases, the underlying pathomechanism is largely unknown. Using a dorsal skinfold chamber model in Syrian golden hamsters for intravital microscopy on striated muscle microcirculation, we investigated whether cigarette smoke (CS) affects the adhesion of circulating leukocytes to the endothelium, a constant feature of early atherogenesis and a hallmark of ischemia-reperfusion injury. Awake hamsters were exposed for 5 min to the mainstream smoke of one cigarette (2R1 research cigarette), inducing nicotine, cotinine, and carboxyhemoglobin plasma levels comparable to levels found in human smokers. In control animals (n = 7), CS exposure elicited the rolling and subsequent adhesion of fluorescently stained leukocytes to the endothelium of arterioles and postcapillary venules. Leukocyte/endothelium interaction was preceded by an early rise in xanthine oxidase activity and intravascular hemolysis. Leukocyte adhesion and xanthine oxidase (XO) activation were significantly attenuated in hamsters pretreated with superoxide dismutase (5 mg/kg, 10 min prior to CS, n = 7), suggesting a key role of superoxide in this event. These in vivo results suggest a novel pathomechanism of CS-induced cardiovascular pathology.

CD4+CD25+ regulatory T cells suppress contact hypersensitivity reactions by blocking influx of effector T cells into inflamed tissue

CD4+CD25+ regulatory T cells (Treg) exert suppressive functions on effector T cells in vitro and in vivo. However, the exact cellular events that mediate this inhibitory action remain largely unclear. To elucidate these events, we used intravital microscopy in a model of contact hypersensitivity (CHS) and visualized the leukocyte‐endothelium interaction at the site of antigen challenge in awake C57BL/6 mice. Injection of Treg i.v. into sensitized mice at the time of local hapten challenge significantly inhibited rolling and adhesion of endogenous leukocytes to the endothelium. A similar inhibition of leukocyte recruitment could be recorded after injection of Treg‐derived tissue culture supernatant. Thus, these data indicate that soluble factors may account for the suppressive effects. Accordingly we found that IL‐10, but not TGF‐β, was produced by Treg upon stimulation and that addition of anti‐IL‐10 antibodies abrogated the suppressive effects of Treg and tissue culture supernatant in CHS reactions. Moreover, CD4+CD25+ T cells isolated from IL‐10–/– mice were not able to suppress the immune response induced by hapten treatment in C57BL/6 mice. In conclusion, our data suggest that cytokine‐dependent rather than cell‐cell contact‐dependent mechanisms play a pivotal role in the suppression of CHS reactions by Treg in vivo.

Oxidized LDL-induced leukocyte/endothelium interaction in vivo involves the receptor for platelet-activating factor.

Leukocyte adhesion and subendothelial emigration, constant hallmarks of early atherogenesis, have been ascribed to the action of oxidized low-density lipoprotein (oxLDL). Using intravital fluorescence microscopy in the skinfold-chamber model in hamsters, we have previously shown that systemic administration of oxLDL stimulates leukocyte adhesion in vivo through a mechanism that depends on the generation and/or action of both leukotrienes and superoxide radicals. On the basis of the fact that oxygen radical-catalyzed peroxidation of phospholipids results in the generation of fragments with short sn2 residues, which besides authentic platelet-activating factor (PAF), activate the receptor for PAF on leukocytes and thereby induce leukocyte adhesion, we asked whether pretreatment of hamsters with a specific PAF receptor antagonist (WEB2170; 1 mg/kg of body weight IV, 10 minutes before oxLDL) attenuates leukocyte adhesion after injection of oxLDL (4 mg/kg of body weight IV, oxidized by 7.5 mumol/L Cu2+ for 18 hours at 37 degrees C). We demonstrate herein that in contrast to untreated control animals in which oxLDL elicited rolling and adhesion of circulating leukocytes to the endothelium of venules and arterioles, oxLDL-induced leukocyte adhesion was significantly attenuated in WEB2170-pretreated animals. These changes cannot be ascribed to alterations of microhemodynamic parameters and, hence, wall shear conditions. This finding indicates that oxLDL-induced leukocyte/endothelium interaction involves the PAF receptor, which may function both as a receptor for authentic PAF or for PAF-like lipids that are generated in a free radical-catalyzed peroxidation of phospholipids.

Superoxide-dependent stimulation of leukocyte adhesion by oxidatively modified LDL in vivo.

Low density lipoprotein modified by oxidation (Ox-LDL) causes adhesion of leukocytes to the endothelium, a feature common in early atherogenesis. Because leukocyte adhesion under various pathophysiological conditions involves superoxide generation, we explored the possibility that superoxide is likewise involved in leukocyte adhesion in response to Ox-LDL. For our studies, we used the dorsal skin fold chamber model for intravital microscopic observation of leukocyte-endothelium interactions in hamsters. We show here that injection of human LDL (4 mg/kg LDL cholesterol oxidatively modified by incubation in 7.5 microM Cu2+ for 18 hours at 37 degrees C) elicited in control hamsters (n = 7) the rolling and adhesion of circulating leukocytes along the endothelium of arterioles and postcapillary venules. This adhesion was significantly attenuated when hamsters were pretreated with bovine copper-zinc-superoxide dismutase (CuZn-SOD, 0.25 mg/kg, n = 7) or heparin (2,000 IU/kg, n = 7). The CuZn-SOD infusion and the heparin-induced release of extracellular SOD from endothelial cell surfaces to plasma resulted in nearly equal plasma SOD activities. Further inhibition of Ox-LDL-induced leukocyte adhesion could not be achieved by increasing the dose of CuZn-SOD to 5 mg/kg (n = 6). Pretreatment of the hamsters with inactivated CuZn-SOD showed no effect. These results indicate that Ox-LDL stimulates leukocyte adhesion through a superoxide-dependent step, and they indicate a possible mechanism by which antioxidants might inhibit the onset of experimental and clinical atherosclerosis.

Reduction of postischemic leukocyte-endothelium interaction by adenosine via A2 receptor

SummaryThe adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated.The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg−1 min−1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion.Adenosine and CGS 21,689 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p<0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction.These findings indicate that adenosine reduces postischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.

Role of oxygen radicals in the microcirculatory manifestations of postischemic injury

Reperfusion after transient tissue ischemia constitutes an irrevocable need to preserve tissue viability. However, release of prolonged ischemia will either result in failure of the microcirculation to reperfusion (no-reflow) and thus the prolongation of hypoxia, or in restoration of blood flow resulting in reoxygenation of the inflicted tissue. While ischemia damages the tissue primarily through hypoxia-induced depletion of energy stores, reoxygenation paradoxically contributes to tissue damage through the formation of oxygen radicals, the release of chemoattractant mediators (TNF, IL-1, LTB4), and the activation of circulating polymorphonuclear leukocytes (PMNs). Through the action of chemoattractant mediators and the upregulation of leukocytic (CD11/CD18) and endothelial adhesion receptors (ICAM, GMP-140), activated PMNs adhere to the endothelium, release further chemoattractants and oxygen radicals and undertain a vicious circle, which will ultimately result in further tissue damage. Both theno-reflow phenomenon and the events initiated by reflow — termed herein as thereflow-paradox — contribute to the failure of the nutritive microvascular perfusion and loss of tissue viability following ischemia and reperfusion.

Decreased plasma ubiquinone-10 concentration in patients with mevalonate kinase deficiency

ABSTRACT: Patients with mevalonate kinase deficiency suffer from psychomotor retardation, ataxia with progredient cerebellar atrophy, and myopathy. The pathophysiology of the disease remains unclear. The mevalonate kinase product, cholesterol, is within the normal range in patient plasma and fibroblasts. In search of the pathophysiology of this disorder, another mevalonate kinase product, ubiquinone-10, was studied. The concentrations of ubiquinone-10 in patient plasma (n = 6) and ubiquinol-10 in patient LDL (n = 2) and the synthesis of ubiquinone-10 in patient fibroblasts (n = 4) were determined. After oxidative modification of LDL by copper in vitro, the concentrations of α-tocopherol and polyunsaturated fatty acids in LDL and the relative electrophoretic mobility of LDL were measured to determine the antioxidant capacity of LDL samples of two affected siblings. The ubiquinone-10 concentrations in plasma samples (median = 508 μg/L, range = 488–642 μg/L) versus controls (median = 613 μg/L, range = 564–809 μg/L; p < 0.005) were decreased. In LDL samples of two affected siblings, the concentration of ubiquinol-10 and the resistance to oxidation in vitro were found decreased during intercurrent patient crisis condition. In patient fibroblasts (median = 533 dpm/mg protein, range = 399–1 047 dpm/mg protein) versus controls (median = 40 731 dpm/mg protein, range = 12 774–54 739 dpm/mg protein), the synthesis of ubiquinone was found to be decreased. We conclude that mevalonate kinase deficiency leads to a decreased synthesis of ubiquinone-10 and that ubiquinone-10 deficiency is responsible for the clinical progression of this disease characterized by increased lipid peroxidation, cerebellar atrophy, cataract development, and myopathy with increased creatine kinase activity.

Improvement of skin flap perfusion by subdermal injection of recombinant human basic fibroblast growth factor.

The effect of subcutaneously injected recombinant human basic fibroblast growth factor (bFGF) was studied in an arterial skin flap model on the ear of the hairless mouse. Fifty-three male, hairless mice were randomly assigned to 4 groups and pretreated in two different time intervals with different doses of human bFGF. Microvascular perfusion of the skin flaps was determined over a 5-day period by means of intravital microscopy after intravenous injection of the fluorescence marker fluorescein isothiocyanate—dextran (M, 150,000). Human bFGF (2,700 ng) injected 6 days before flap creation could not improve perfusion of the flap (n = 10) when compared with controls. However, when applied 18 days before flap creation (n = 13), the same dose resulted in a significant reduction of nonperfused tissue at day 5 after flap creation (12.3% vs 26.8%, p < 0.01). Eighteen-day pretreatment with 1,200 ng (n = 10) and 480 ng (n = 10) had no significant effect on skin flap perfusion. We conclude, therefore, that successful pretreatment with bFGF for prevention of skin flap necrosis is time and dose dependent.

Impact of Ischemia on Tissue Oxygenation and Wound Healing: Intravital Microscopic Studies on the Hairless Mouse Ear Model

To determine the effect of ischemia on tissue oxygenation and the healing of experimental wounds, chronic ischemia was induced in the ears of hairless mice by ligating 2 of the 3 main nutritional arteries. Tissue ischemia was verified by measurement of transcutaneous pO2 (tcpO2) prior to and on days 3, 6, 9, 12 and 15 after vessel ligation. TcpO2 values decreased from 24 to 6 mm Hg at day 2 after ligation, and slowly recovered to 12 mm Hg at day 12 after vessel ligation. In animals treated with the vasoactive drug buflomedil (3 mg/kg BW i.v., startling 2 days after vessel ligation) tcpO2 values were significantly higher on days 6 and 9 when compared to saline-treated control animals. In order to find out whether the enhanced tissue oxygenation resulted in enhancement of healing of wounds, we created circular wounds (diameter = 2.5 mm, depth 0.1 mm) on ischemic ears 2 days after vessel ligation. The wound surface area was measured by means of intravital fluorescence microscopy and digital planimetry at 3-day intervals until the time of wound closure. These experiments were performed on buflomedil-treated and control animals receiving equivalent amounts of saline. The reduction of wound surface area was accelerated and wound closure time was reduced from 15 days in control animals to 12 days in buflomedil-treated animals (p < 0.01). Functional capillary density as well as the microhemodynamic parameters microvessel diameter and red blood cell velocity were not different between buflomedil-treated and control animals.