Leukocyte Flux Research Articles

Effect of Roux-en-Y gastric bypass surgery on subclinical atherosclerosis, oxidative stress, and mitochondrial dynamics in leukocytes of obese patients: A one-year follow-up study

Background and Aims : Little is known about the mechanisms underlying the cardiovascular protective effect of Roux en-Y gastric bypass (RYGB) surgery. The present study aimed to investigate how RYGB influences the oxidative and mitochondrial status of leukocytes and subclinical atherosclerotic markers.Methods: This interventional study was carried out in 57 obese subjects who underwent RYGB surgery. Leukocytes were isolated at baseline and 12 months after the intervention. We evaluated oxidative stress markers by static cytometry and mitochondrial markers by western blot. Leukocyte-endothelial cell interactions - rolling flux, velocity, and adhesion - were evaluated in vitro in a parallel-plate flow chamber.Results: RYGB induced weight loss and an improvement of anthropometric clinical indicators. At the molecular level, a significant reduction of superoxide production (p<0.01) and mitochondrial membrane potential (p<0.05) was observed, while GPX1 content increased (p<0.05). In terms of mitochondria, RYGB induced upregulation of complexes (I-V) (p<0.05 and p<0.001), MIEAP (p<0.05), and Pink1 (p<0.05), and regulated the fusion-fission machinery through an increase in MFN1 (p<0.05). Likewise, a significant reduction of sICAM (p< 0.05) and sP-Selectin (p < 0.001) and of rolling flux (p< 0.05) and adhesion of leukocytes (p< 0.01) were reported at follow-up.Conclusions: Our results suggest that patients undergoing RYGB benefit from an amelioration of their prooxidant status and enhanced mitochondrial dynamics in leukocytes, which could be responsible for the reduction of subclinical markers of atherosclerosis after surgery Acknowledgements: PI19/00838, PI19/00437, FI17/00144, CD19/00180, CP19/00077 from ISCIII-ERDF (“A way to build Europe”), PROMETEO/2019/027; APOSTD/2020/145, ACIF/2020/371 from the Valencian Regional Government. Background and Aims : Little is known about the mechanisms underlying the cardiovascular protective effect of Roux en-Y gastric bypass (RYGB) surgery. The present study aimed to investigate how RYGB influences the oxidative and mitochondrial status of leukocytes and subclinical atherosclerotic markers. Methods: This interventional study was carried out in 57 obese subjects who underwent RYGB surgery. Leukocytes were isolated at baseline and 12 months after the intervention. We evaluated oxidative stress markers by static cytometry and mitochondrial markers by western blot. Leukocyte-endothelial cell interactions - rolling flux, velocity, and adhesion - were evaluated in vitro in a parallel-plate flow chamber. Results: RYGB induced weight loss and an improvement of anthropometric clinical indicators. At the molecular level, a significant reduction of superoxide production (p<0.01) and mitochondrial membrane potential (p<0.05) was observed, while GPX1 content increased (p<0.05). In terms of mitochondria, RYGB induced upregulation of complexes (I-V) (p<0.05 and p<0.001), MIEAP (p<0.05), and Pink1 (p<0.05), and regulated the fusion-fission machinery through an increase in MFN1 (p<0.05). Likewise, a significant reduction of sICAM (p< 0.05) and sP-Selectin (p < 0.001) and of rolling flux (p< 0.05) and adhesion of leukocytes (p< 0.01) were reported at follow-up. Conclusions: Our results suggest that patients undergoing RYGB benefit from an amelioration of their prooxidant status and enhanced mitochondrial dynamics in leukocytes, which could be responsible for the reduction of subclinical markers of atherosclerosis after surgery Acknowledgements: PI19/00838, PI19/00437, FI17/00144, CD19/00180, CP19/00077 from ISCIII-ERDF (“A way to build Europe”), PROMETEO/2019/027; APOSTD/2020/145, ACIF/2020/371 from the Valencian Regional Government.

Effect of Roux-en-Y Bariatric Bypass Surgery on Subclinical Atherosclerosis and Oxidative Stress Markers in Leukocytes of Obese Patients: A One-Year Follow-Up Study.

Little is known about the mechanisms underlying the cardioprotective effect of Roux en-Y gastric bypass (RYGB) surgery. Therefore, the aim of the present study was to investigate whether weight loss associated with RYGB improves the oxidative status of leukocytes and ameliorates subclinical atherosclerotic markers. This is an interventional study of 57 obese subjects who underwent RYGB surgery. We determined biochemical parameters and qualitative analysis of cholesterol, leukocyte and systemic oxidative stress markers —superoxide production, glutathione peroxidase 1 (GPX1), superoxide dismutase (SOD) activity and protein carbonylation—, soluble cellular adhesion molecules —sICAM-1 and sP-selectin—, myeloperoxidase (MPO) and leukocyte-endothelium cell interactions—rolling flux, velocity and adhesion. RYGB induced an improvement in metabolic parameters, including hsCRP and leukocyte count (p < 0.001, for both). This was associated with an amelioration in oxidative stress, since superoxide production and protein carbonylation were reduced (p < 0.05 and p < 0.01, respectively) and antioxidant systems were enhanced (GPX1; p < 0.05 and SOD; p < 0.01). In addition, a significant reduction of the following parameters was observed one year after RYGB: MPO and sICAM (p < 0.05, for both), sPselectin and pattern B of LDL particles (p < 0.001, for both), and rolling flux and adhesion of leukocytes (p < 0.05 and p < 0.01, respectively). Our results suggest that patients undergoing RYGB benefit from an amelioration of the prooxidant status of leukocytes, metabolic outcomes, and subclinical markers of atherosclerosis.

Tuning the Thromboinflammatory Response to Venous Flow Interruption by the Ectonucleotidase CD39.

Objective- Leukocyte flux contributes to thrombus formation in deep veins under pathological conditions, but mechanisms that inhibit venous thrombosis are incompletely understood. Ectonucleotide di(tri)phosphohydrolase 1 ( ENTPD1 or Cd39), an ectoenzyme that catabolizes extracellular adenine nucleotides, is embedded on the surface of endothelial cells and leukocytes. We hypothesized that under venous stasis conditions, CD39 regulates inflammation at the vein:blood interface in a murine model of deep vein thrombosis. Approach and Results- CD39-null mice developed significantly larger venous thrombi under venous stasis, with more leukocyte recruitment compared with wild-type mice. Gene expression profiling of wild-type and Cd39-null mice revealed 76 differentially expressed inflammatory genes that were significantly upregulated in Cd39-deleted mice after venous thrombosis, and validation experiments confirmed high expression of several key inflammatory mediators. P-selectin, known to have proximal involvement in venous inflammatory and thrombotic events, was upregulated in Cd39-null mice. Inferior vena caval ligation resulted in thrombosis and a corresponding increase in both P-selectin and VWF (von Willebrand Factor) levels which were strikingly higher in mice lacking the Cd39 gene. These mice also manifest an increase in circulating platelet-leukocyte heteroaggregates suggesting heterotypic crosstalk between coagulation and inflammatory systems, which is amplified in the absence of CD39. Conclusions- These data suggest that CD39 mitigates the venous thromboinflammatory response to flow interruption.

Cerium Dioxide Nanoparticle Exposure Improves Microvascular Dysfunction and Reduces Oxidative Stress in Spontaneously Hypertensive Rats.

The elevated production of reactive oxygen species (ROS) in the vascular wall is associated with cardiovascular diseases such as hypertension. This increase in oxidative stress contributes to various mechanisms of vascular dysfunction, such as decreased nitric oxide bioavailability. Therefore, anti-oxidants are being researched to decrease the high levels of ROS, which could improve the microvascular dysfunction associated with various cardiovascular diseases. From a therapeutic perspective, cerium dioxide nanoparticles (CeO2 NP) hold great anti-oxidant potential, but their in vivo activity is unclear. Due to this potential anti-oxidant action, we hypothesize that injected CeO2 NP would decrease microvascular dysfunction and oxidative stress associated with hypertension. In order to simulate a therapeutic application, spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats were intravenously injected with either saline or CeO2 NP (100 μg suspended in saline). Twenty-four hours post-exposure mesenteric arteriolar reactivity was assessed via intravital microscopy. Endothelium-dependent and –independent function was assessed via acetylcholine and sodium nitroprusside. Microvascular oxidative stress was analyzed using fluorescent staining in isolated mesenteric arterioles. Finally, systemic inflammation was examined using a multiplex analysis and venular leukocyte flux was counted. Endothelium-dependent dilation was significantly decreased in the SH rats (29.68 ± 3.28%, maximal response) and this microvascular dysfunction was significantly improved following CeO2 NP exposure (43.76 ± 4.33%, maximal response). There was also an increase in oxidative stress in the SH rats, which was abolished following CeO2 NP treatment. These results provided evidence that CeO2 NP act as an anti-oxidant in vivo. There were also changes in the inflammatory profile in the WKY and SH rats. In WKY rats, IL-10 and TNF-α were increased following CeO2 NP treatment. Finally, leukocyte flux was increased in the SH rats (34 ± 4 vs. 17 ± 3 cells/min in the normotensive controls), but this activation was decreased following exposure (15 ± 2 vs. 34 ± 4 cells/min). These results indicated that CeO2 NP may alter the inflammatory response in both SH and WKY rats. Taken together, these results provide evidence that CeO2 NP act as an anti-oxidant in vivo and may improve microvascular reactivity in a model of hypertension.

Abstract 19478: Endothelial and Cardiomyocyte -derived C-type Natriuretic Peptide Coordinate Heart Structure and Function

Background: Endothelium-derived C-type natriuretic peptide (CNP) plays a key vascular homeostatic role governing vascular tone, blood pressure, leukocyte flux, platelet reactivity and the integrity of the vessel wall. However, relatively little is known about physiological role(s) for endogenous CNP in regulating cardiac structure and function. Herein, we have utilised novel mouse strains with endothelium or cardiomyocyte -specific deletion of CNP to determine if the peptide modulates heart function under basal conditions and during cardiac stress. Methods: Blood pressure and ECG were assessed by radiotelemetry. A Langendorff heart model was used to study coronary vascular reactivity and ischemia-reperfusion (I/R) injury ex vivo. Echocardiography was performed to determine cardiac function at baseline and following pressure overload (trans-aortic constriction; 6 weeks) -induced left ventricular hypertrophy/heart failure. Results: Hearts from endothelium-specific CNP knockout (ecCNP KO) mice exhibited smaller reductions in coronary perfusion pressure (CPP) compared to wildtype (WT) littermates in response to the vasodilators bradykinin (ΔCPP: WT=31.7±2.7%, KO=21.1±2.9%, n=8, p&lt;0.05) and acetylcholine (ΔCPP: WT=36.4±4.4%, KO=18.5±3.8%, n=6, p&lt;0.05). Shear-stress induced coronary dilatation (i.e. reactive hyperaemia) was also blunted in ecCNP KO hearts (AUC: WT=2804±280 [a.u.], KO=1493±280 [a.u.], n=8, p&lt;0.05). Under basal conditions the heart rate (BPM: WT=605±5, KO 579±4, n=5, p&lt;0.001) and contractility (QA interval; WT=13.7±0.1ms, KO=14.8±0.1ms, n=5, p&lt;0.001) were significantly reduced in cardiomyocyte-specific CNP (cmCNP) KO mice compared to WT. Myocardial infarct size was larger in cmCNP KO following I/R injury ex vivo (Infarct size: WT=14.1±6.3%, KO=21.8±1.8 %, n=6, p&lt;0.05). Furthermore, cmCNP KO mice exhibited greater cardiac dysfunction following pressure-overload (e.g. fractional shortening: WT=34.4±0.9%, KO=30.5±1.4%, n=8, p&lt;0.05). Conclusion: These data suggest that CNP of endothelial and cardiomyocyte origin preserves cardiac function and morphology via the regulation of coronary vascular tone, heart rate, and myocardial contractility/hypertrophy.

Lymphatic Pump Treatment Repeatedly Enhances the Lymphatic and Immune Systems

Osteopathic practitioners utilize manual therapies called lymphatic pump techniques (LPT) to treat edema and infectious diseases. While previous studies examined the effect of a single LPT treatment on the lymphatic system, the effect of repeated applications of LPT on lymphatic output and immunity has not been investigated. Therefore, the purpose of this study was to measure the effects of repeated LPT on lymphatic flow, lymph leukocyte numbers, and inflammatory mediator concentrations in thoracic duct lymph (TDL). The thoracic ducts of five mongrel dogs were cannulated, and lymph samples were collected during pre-LPT, 4 min of LPT, and 2 hours post-LPT. A second LPT (LPT-2) was applied after a 2 hour rest period. TDL flow was measured, and TDL were analyzed for the concentration of leukocytes and inflammatory mediators. Both LPT treatments significantly increased TDL flow, leukocyte count, total leukocyte flux, and the flux of interleukin-8 (IL-8), keratinocyte-derived chemoattractant (KC), nitrite (NO2(-)), and superoxide dismutase (SOD). The concentration of IL-6 increased in lymph over time in all experimental groups; therefore, it was not LPT dependent. Clinically, it can be inferred that LPT at a rate of 1 pump per sec for a total of 4 min can be applied every 2 h, thus providing scientific rationale for the use of LPT to repeatedly enhance the lymphatic and immune system.

Multiple-Site Activation of the Cysteinyl Leukotriene Receptor 2 Is Required for Exacerbation of Ischemia/Reperfusion Injury

Transgenic overexpression of the human cysteinyl leukotriene receptor 2 (CysLT2R) in murine endothelium exacerbates vascular permeability and ischemia/reperfusion injury. Here, we explore the underlying mechanisms of CysLT2R activation-mediated inflammation and delineate the relative contributions of endogenous murine CysLT2R and the transgene-derived receptor. We created a novel mouse with only endothelial-expressed CysLT2R (endothelium-targeted overexpression mice [EC]/CysLT2R-knockout mice [KO]) by crossing EC with KO to dissect the role of endothelial CysLT2R in tissue injury. Surprisingly, we discovered that damage in EC/KO mice was not elevated (24% versus 47% EC) after ischemia/reperfusion. We examined vascular permeability and leukocyte recruitment/rolling responses in the cremaster vasculature after cysteinyl leukotriene (cysLT) stimulation. Mice possessing transgenic endothelial CysLT2R overexpression, whether EC or EC/KO, when stimulated with cysLTs, exhibited vascular hyperpermeability, declining leukocyte flux, and a transient increase in slow-rolling leukocyte fraction. Mice lacking endogenous CysLT2R (both KO [20 ± 3 cells/min] EC/KO [24 ± 3]) showed lower-rolling leukocyte flux versus wild-type (38 ± 6) and EC (35 ± 6) mice under unstimulated conditions. EC/KO mice differed from EC counterparts in that vascular hyperpermeability was not present in the absence of exogenous cysLTs. These results indicate that endothelial and nonendothelial CysLT2R niches have separate roles in mediating inflammatory responses. Endothelial receptor activation results in increased vascular permeability and leukocyte slow-rolling, facilitating leukocyte transmigration. Nonendothelial receptors, likely located on resident/circulating leukocytes, facilitate endothelial receptor activation and leukocyte transit. Activation of both receptor populations is required for injury exacerbation.

Neutrophils Usher Monocytes Into Sites of Inflammation

One of the key processes of inflammation is the transmigration of circulating leukocytes across the endothelium. Among the leukocytes, neutrophils and monocytes are large phagocytes that can respond quickly to infection or injury. On sensing danger, neutrophils and monocytes adhere to the endothelium and transmigrate to the adjacent tissue via the coordinated activities of adhesion molecules, integrins, cytokines, and chemokines.1 Once they accumulate, these myeloid cells participate in a myriad of immune inflammatory activities. The importance of this event cannot be understated, especially because the accumulation of leukocytes in tissue is a double-edged sword. On the one hand, coordinated leukocyte accumulation in injured or infected sites is required for effective pathogen elimination and tissue healing. On the other hand, uncontrolled accumulation is a defining feature of chronic diseases, such as atherosclerosis.2 Understanding leukocyte migration is essential to understanding the immune system. Article, see p 792 Neutrophils and monocytes do not accumulate all at once. In a typical acute inflammatory response, there is a well-defined sequence: neutrophils accumulate first; monocytes accumulate second. Among the monocytes, of which ≥2 subsets circulate in the mouse and human, there is yet another …

Electroacupuncture increases cerebral blood flow in a clinically relevant model of ischemic stroke

Effects of EA at clinically relevant times in evolving ischemic stroke remain essentially unexplored. The purpose of this study was to determine if electroacupuncture (EA) initiated after the onset of ischemic stroke and during reperfusion can increase CBF. Middle cerebral artery occlusion (MCAO) was performed in anesthetized, ventilated male rats via the filament method. EA treatment consisted of 5–10 min. of stimulation at 4Hz using standard needles placed in the scalp (GV20) and the paw (LI4) ipsilateral to the MCAO. EA was applied before and during ischemia and during reperfusion. CBF (mean % increase +/SD over baseline values) was measured via doppler flowmetry. Mean arterial pressure (MAP) was monitored. In an exploratory experiment, the affects of EA on the microcirculation was observed using standard cranial window and fluorescence microscopy methods. Needle placement without EA had no effect on CBF or MAP nor did EA affect MAP. EA resulted in an increase in CBF before MCAO (93% +/− 64%) (N=4), during MCAO (82 +/− 42%) (N=3) and during reperfusion (26.1 +/− 2%) (N=3). EA increased blood flow, leukocyte flux and adhesion in the cerebral microcirculation, which reversed after withdrawal of stimulation. EA treatment applied during ischemic stroke, reperfusion, or both may improve perfusion in the microcirculation and is worthy of further investigation.

Response of the Murine Urinary Bladder Microcirculation to Lipopolysaccharide from Escherichia coli and Pseudomonas aeruginosa

There is in vitro evidence demonstrating the inflammatory response to lipopolysaccharide (LPS) differs depending on the bacterial source. The objective of our study was to examine the in vivo inflammatory response to LPS from two common uropathogens, Escherichia coli (Ec) and Pseudomonas aeruginosa (Pa).LPS or saline was injected transurethrally into bladders of female C57Bl/6 mice at varying doses. The bladder was exteriorized, and the bladder microcirculation examined by intravital microscopy. Leukocyte adhesion at 4 hrs post challenge with 5 mg/kg Pa LPS was 2.9±0.6 cells/field of view and at 7 mg/kg it was 3.9±0.8 cells/field of view, a significant increase from saline control. In contrast, leukocyte adhesion 4 hours post 5 mg/kg Ec LPS challenge was 17.4±3.9 and with 7 mg/kg 19.7±2.9 cells/field of view. However, at 4.5 hours adhesion was similar with either LPS (5 mg/kg) with 18.4±3.0 and 21.2±4.0 cells/field of view, respectively. The flux of rolling leukocytes was significantly increased above baseline levels only after Ec LPS challenge. There was also a significant increase in the number of neutrophils with Ec LPS stimulation.In support of the in vitro observations, these in vivo results demonstrate that Ec LPS is more potent and induces a more rapid inflammatory response than Pa LPS.Supported by a Collaborative Health Research Projects grant from CIHR and NSERC.

Intravital Microscopy of the Murine Urinary Bladder Microcirculation

Catheter associated urinary tract infection (UTI) is one of the most common nosocomial infections. The objective of this study was to establish an in vivo mouse model of the urinary bladder microcirculation to study the inflammatory response to UTI.Female C57Bl/6 mice were challenged intravesically with 0.1, 0.5, 1, 5 or 7 mg/kg of Escherichia coli lipopolysaccharide (LPS) or saline. 3.5 hrs later, the right jugular vein was cannulated, a catheter was inserted into the bladder, the urine drained and 100 μl of warm saline injected. The bladder was gently exteriorized and the bladder microcirculation was recorded for the next hour. In some mice, leukocyte endothelial cell interactions were observed after exposure to anti‐P‐selectin or anti‐α4‐integrin Ab.LPS at a dose of 5 and 7 mg/kg resulted in a significant increase in leukocyte adhesion and rolling. Leukocyte adhesion at 4 and 4.5 hrs post saline stimulation was 0.9±1.0 and 2.7±1.0 cells/field of view, respectively, and the flux of rolling leukocytes was 21±6.4 and 47±22 cells/min. Leukocyte adhesion at the two time points after 5 mg/kg of LPS stimulation was 17±7.8 and 21±8.0 cells/field of view, respectively, and the flux of rolling leukocytes was 74±17 and 116±56 cells/min. Blockage of α4‐integrin had no effect on leukocyte rolling, whereas the use of a P‐selectin Ab significantly inhibited leukocyte rolling (54.0± 26.0 to 1.25±0.97 cells/min, p&lt;0.05).These results demonstrate that baseline conditions are maintained within the first half‐hour of microcirculatory observations. Leukocyte rolling within the bladder microcirculation is P‐selectin dependent but α4‐integrin independent. This model can be used to decipher the molecular mechanisms of leukocyte recruitment into the bladder as well as to examine in vivo responses to catheter materials and bacterial infection.

The Selective Proteasome Inhibitor Carfilzomib Is Well Tolerated in Experimental Animals with Dose Intensive Administration

Carfilzomib (CFZ) is a selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Phase I trials have demonstrated that doses of carfilzomib from 11 – 20 mg/m2 were well tolerated, inhibited blood CT-L activity by ≥80%, and resulted in objective anti-tumor responses in patients with multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL) (Blood 110:409 & 411). In order to further characterize the safety profile of CFZ, studies in mice, rats and monkeys were conducted to assess the toxicologic consequences of acute and chronic administration. In both rats and monkeys, a single dose of CFZ (equivalent to 24 mg/m2) resulted in > 80% inhibition of CT-L activity in blood and tissues with recovery occurring in most tissues with a t1/2 of 24 hr. At this dose, monkeys developed a pre-renal azotemia within 24 hr, which was characterized by increases in serum creatinine, blood urea nitrogen (BUN) levels and urine specific gravity. These changes resolved in 72 hr, were not associated with histologic changes in the kidney, and were not exacerbated by consecutive daily administrations, suggesting that CFZ may not have a direct nephrotoxic effect. A single dose in either rats or monkeys induced a transient drop in platelets within 48 hr that resolved within 7 days and neither the level of nor recovery from this thrombocytopenia were affected by consecutive daily administration. This phenomenon has also been noted with bortezomib (BTZ), and is observed clinically with both compounds, suggesting that thrombocytopenia is a hallmark of inhibition of proteasome CT-L activity. However, in contrast to BTZ, which has been reported to induce both lymphopenia and neutropenia in animals, CFZ treatment in both rats and monkeys resulted in acute increases in circulating neutrophils and monocytes which rose within 24 hr of a single dose and returned to baseline in 3 – 4 days. We were unable to detect a significant induction of cytokines or chemokines capable of mediating leukocyte fluxes. However, since the acute phase proteins fibrinogen and C-reactive protein were elevated during the same time period of leukocyte flux, cytokines may play a role in this response. In chronic studies, CFZ was administered on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle to rats and monkeys for 6 and 9 cycles, respectively. This dosing schedule results in anti-tumor activity in mouse models of MM, NHL and solid tumors and is currently being utilized in Phase 2 studies in MM and solid tumors. In these chronic studies, transient pre-renal azotemia and increases in circulating neutrophils were manifest in monkeys at 24 mg/m2 following the first dose of both the first and last cycles of treatment. However, no histologic signs of nephropathy were seen in the kidneys of rats receiving 6 cycles of treatment. In addition, chronic administration of CFZ at doses equivalent to those currently administered in the clinic did not impact on the neurobehavioral function of rats and monkeys. There was no severe degeneration in the dorsal root ganglion or in the sciatic, sural, and peroneal nerves of CFZ treated rats. The data with CFZ are in distinct contrast to the axonal swelling, demyelination and severe neural degeneration that is induced by BTZ in rodents and monkeys. In the clinic, peripheral neuropathy associated with CFZ treatment appears to be less severe and possibly less frequent than what is seen with BTZ. Overall, studies in rodents and monkeys have been largely predictive of the clinical experience to date with CFZ. Together with anti-tumor activity in human tumor xenograft models, the toxicologic data support that CFZ administered at therapeutic doses is well tolerated and prolonged treatment of patients with this agent will be feasible.

Characterization of the Human α1β1 Soluble Guanylyl Cyclase Promoter: KEY ROLE FOR NF-κB(p50) AND CCAAT-BINDING FACTORS IN REGULATING EXPRESSION OF THE NITRIC OXIDE RECEPTOR

Soluble guanylyl cyclase (sGC) is the principal receptor for NO and plays a ubiquitous role in regulating cellular function. This is exemplified in the cardiovascular system where sGC governs smooth muscle tone and growth, vascular permeability, leukocyte flux, and platelet aggregation. As a consequence, aberrant NO-sGC signaling has been linked to diseases including hypertension, atherosclerosis, and stroke. Despite these key (patho)physiological roles, little is known about the expressional regulation of sGC. To address this deficit, we have characterized the promoter activity of human α1 and β1 sGC genes in a cell type relevant to cardiovascular (patho)physiology, primary human aortic smooth muscle cells. Luciferase reporter constructs revealed that the 0.3- and 0.5-kb regions upstream of the transcription start sites were optimal for α1 and β1 sGC promoter activity, respectively. Deletion of consensus sites for c-Myb, GAGA, NFAT, NF-κB(p50), and CCAAT-binding factor(s) (CCAAT-BF) revealed that these are the principal transcription factors regulating basal sGC expression. In addition, under pro-inflammatory conditions, the effects of the strongest α1 and β1 sGC repressors were enhanced, and enzyme expression and activity were reduced; in particular, NF-κB(p50) is pivotal in regulating enzyme expression under such conditions. NO itself also elicited a cGMP-independent negative feedback effect on sGC promoter activity that is mediated, in part, via CCAAT-BF activity. In sum, these data provide a systematic characterization of the promoter activity of human sGC α1 and β1 subunits and identify key transcription factors that govern subunit expression under basal and pro-inflammatory (i.e. atherogenic) conditions and in the presence of ligand NO.

Exogenous stromal cell-derived factor-1 induces modest leukocyte recruitment in vivo

Stromal cell-derived factor-1 (SDF-1; CXCL12), a CXC chemokine, has been found to be involved in inflammation models in vivo and in cell adhesion, migration, and chemotaxis in vitro. This study aimed to determine whether exogenous SDF-1 induces leukocyte recruitment in mice. After systemic administration of SDF-1alpha, expression of the adhesion molecules P-selectin and VCAM-1 in mice was measured using a quantitative dual-radiolabeled Ab assay and leukocyte recruitment in various tissues was evaluated using intravital microscopy. The effect of local SDF-1alpha on leukocyte recruitment was also determined in cremaster muscle and compared with the effect of the cytokine TNFalpha and the CXC chemokine keratinocyte-derived chemokine (KC; CXCL1). Systemic administration of SDF-1alpha (10 microg, 4-5 h) induced upregulation of P-selectin, but not VCAM-1, in most tissues in mice. It caused modest leukocyte recruitment responses in microvasculature of cremaster muscle, intestine, and brain, i.e., an increase in flux of rolling leukocytes in cremaster muscle and intestines, leukocyte adhesion in all three tissues, and emigration in cremaster muscle. Local treatment with SDF-1alpha (1 microg, 4-5 h) reduced leukocyte rolling velocity and increased leukocyte adhesion and emigration in cremasteric venules, but the responses were much less profound than those elicited by KC or TNFalpha. SDF-1alpha-induced recruitment was dependent on endothelial P-selectin, but not P-selectin on platelets. We conclude that the exogenous SDF-1alpha enhances leukocyte-endothelial cell interactions and induces modest and endothelial P-selectin-dependent leukocyte recruitment.

Abdominal Lymphatic Pump Treatment Increases Leukocyte Count and Flux in Thoracic Duct Lymph

Previous studies suggest that rhythmic compression of the abdomen (abdominal lymphatic pump techniques, LPT) enhances immunity and resistance to infectious disease, but direct evidence of this has not been documented. In this study, the thoracic duct of eight anesthetized mongrel dogs was catheterized, so the immediate effects of LPT on lymph flow and leukocyte output could be measured. Lymph flow was measured by timed collection or ultrasonic flowmeter, and lymph was collected over ice under 1) resting (baseline) conditions, and 2) during application of LPT. The baseline leukocyte count was 4.8 +/- 1.7 x 10(6) cells/ml of lymph, and LPT significantly increased leukocytes to 11.8 +/- 3.6 x 10(6) cells/ml. Flow cytometry and differential cell staining revealed that numbers of macrophages, neutrophils, total lymphocytes, T cells and B cells were similarly increased during LPT. Furthermore, LPT significantly enhanced lymph flow from 1.13 +/- 0.44 ml/min to 4.14 +/- 1.29 ml/min. Leukocyte flux, computed from the product of lymph flow and cell count, was increased by LPT from 8.2 +/- 4.1 x 10(6) to 60 +/- 25 x 10(6) total cells/min. Similar trends were observed in macrophages, neutrophils, total lymphocytes, T cells and B cells during LPT. LPT significantly increased both thoracic duct lymph flow and leukocyte count, so lymph leukocyte flux was markedly enhanced. Increased mobilization of immune cells is likely and important mechanism responsible for the enhanced immunity and recovery from infection of patients treated with LPT.

Abdominal lymphatic pump treatment increases leukocyte count and flux in thoracic duct lymph (99.13)

Abstract Studies suggest that lymphatic pump techniques (LPT) enhance immunity and resistance to infection, but direct evidence of this has not been documented. In this study, the immediate effects of LPT on thoracic duct lymph flow and leukocyte output was measured. Lymph flow was measured and lymph was collected at baseline and during LPT. The baseline leukocyte count was 4.8 ± 1.7 x 106 cells/ml, and LPT increased leukocytes to 11.8 ± 3.6 x 106cells/ml (P&amp;lt;0.05). While macrophages, neutrophils, total lymphocytes, T cells and B cell numbers were similarly increased during LPT, their relative percentage in lymph was unaltered by LPT; however, IgA antibody forming B cells increased from 5.8% at baseline to 17% during LPT. Furthermore, LPT enhanced lymph flow 4-fold. Leukocyte flux was computed from the product of lymph flow and cell count. LPT enhanced leukocyte flux from 8.2± 4.1 x 106total cells per min to 60 ± 25 x 106cells per min. In summary, LPT significantly increased both thoracic duct lymph flow and leukocyte count, so lymphatic leukocyte flux was markedly enhanced. In addition, our data suggests that LPT can act preferentially on mucosal tissues, revealing a source of these mobilized leukocytes. Enhanced mobilization and lymphatic transport of immune cells during abdominal compression is likely an important mechanism responsible for the increased immune responses of patients treated with LPT.

Real-time imaging of mechanically injured femoral artery in mice reveals a biphasic pattern of leukocyte accumulation

Wire injury of an artery has been recognized as a standard model of vascular inflammation and atherosclerosis; however, the mechanism of leukocyte recruitment has not been studied in this model. In this study, we documented the recruitment of leukocytes to the murine femoral artery after a wire injury. A transluminal mechanical injury was generated by insertion of a wire into the femoral artery of male C57BL/6J mice. The mice were anesthetized and ventilated after tracheotomy and protected from hypothermia by a warming lamp. Body temperature and blood pH did not significantly change during the experiment. The interaction between rhodamine 6G-labeled leukocytes and the injured femoral artery was monitored using an epifluorescent microscope, and the images were evaluated using a computer-assisted image analysis program. In the absence of injury, virtually no leukocyte adhesion was observed. In contrast, the number of adherent leukocytes increased 4 and 24 h after injury and declined 72 h after injury. The rolling flux of leukocytes increased 4 h after injury and remained high up to 7 days, but it was faster 72 h after injury. We identified another peak of leukocyte adhesion 7 days after injury. Injection of anti-P-selectin antibody significantly reduced leukocyte adhesion at the early and later phases. In conclusion, we have established a novel experimental system for direct observation of leukocyte recruitment to the injured femoral artery. Our system revealed a previously undetected, unique profile of leukocyte recruitment during vascular injury.

Golgi GDP-fucose Transporter-deficient Mice Mimic Congenital Disorder of Glycosylation IIc/Leukocyte Adhesion Deficiency II

Modification of glycoproteins by the attachment of fucose residues is widely distributed in nature. The importance of fucosylation has recently been underlined by identification of the monogenetic inherited human disease "congenital disorder of glycosylation IIc," also termed "leukocyte adhesion deficiency II." Due to defective Golgi GDP-fucose transporter (SLC35C1) activity, patients show a hypofucosylation of glycoproteins and present clinically with mental and growth retardation, persistent leukocytosis, and severe infections. To investigate effects induced by the loss of fucosylated structures in different organs, we generated a mouse model for the disease by inactivating the Golgi GDP-transporter gene (Slc35c1). Lectin binding studies revealed a tremendous reduction of fucosylated glycoconjugates in tissues and isolated cells from Slc35c1(-/-) mice. Fucose treatment of cells from different organs led to partial normalization of the fucosylation state of glycoproteins, thereby indicating an alternative GDP-fucose transport mechanism. Slc35c1-deficient mice presented with severe growth retardation, elevated postnatal mortality rate, dilatation of lung alveoles, and hypocellular lymph nodes. In vitro and in vivo leukocyte adhesion and rolling assays revealed a severe impairment of P-, E-, and L-selectin ligand function. The diversity of these phenotypic aspects demonstrates the broad general impact of fucosylation in the mammalian organism.

Abdominal pump treatment increases leukocyte count and flux in thoracic duct lymph

Previous studies suggest that rhythmic compression of the abdomen (abdominal lymphatic pump, ALP) enhances immunity and resistance to infectious disease, but direct evidence of this has not been documented. Thus, the hypothesis that ALP increases leukocyte numbers in thoracic duct lymph was tested in five anesthetized, open-chest dogs. The lymph duct was catheterized, lymph flow was measured, and lymph was collected under 1) baseline conditions, 2) during ALP. ALP increased lymph flow from 0.5±0.1 to 2.7±0.3 ml/min and increased leukocytes from 1.7±0.2 × 106 to 6.2±0.4 × 106cells/ml. Relative proportions macrophages, neutrophils, total lymphocytes, T cells, and B cells did not change during ALP. Considering increases in flow and leukocyte counts, ALP enhanced leukocyte flux from 0.75±0.04 × 106to 17 ± 2 × 106cells/min. In summary, ALP significantly increased both thoracic duct lymph flow and leukocyte count, so leukocyte lymphatic flux was markedly enhanced. Enhanced mobilization and lymphatic transport of immune cells during abdominal compression is likely an important mechanism responsible for the enhanced immune responses of patients treated with ALP. (Support: NIH grant U19 AT002023)

Differences between leukocyte‐EC interactions in arterioles vs. venules may be related to ICAM‐1.

The observation that an inflammatory stimulus upregulates adhesion molecule expression in arterioles suggests an explicit role for arterioles in inflammation. We used intravital confocal microscopy to study the connection between ICAM-1 expression levels and leukocyte-EC interactions in cremaster muscle arterioles and venules of anesthetized mice (Nembutal, 74 mg/ml). To compare normal and inflammatory conditions we used control and TNF-α (500ng, intrascrotally) treated mice. We observed ICAM-1 dependent rolling in arterioles and ICAM-1 dependent adhesion in venules. Interestingly, ICAM-1 expression levels increased 2-3 fold following TNF-α treatment in both arterioles and venules, but leukocyte adhesion events were rarely found in arterioles compared to venules (0.6 vs 5.5/100μm). Note that the potential for an adhesion event was similar in both arterioles and venules since the delivered flux of leukocytes to the vessel wall was the same (10 leukocyte/40 sec). On the other hand, the number of rolling leukocytes was significantly higher at the regions of higher ICAM-1 density (r2=0.56) in arterioles but not in venules (r2=0.11). We also found that the number of firmly adhered leukocytes in venules linearly increased with ICAM-1 expression levels (r2=0.59). Although an inflammatory stimulus did not induce leukocyte adhesion in arterioles as it did in venules, elevated ICAM-1 levels were found to correlate with the increased number of rolling leukocytes in arterioles, suggesting that ICAM-1 and arterioles play an important role in inflammation. (Supported by NIH HL18208 and HL75186)