Calcium Signal Profiles in Vascular Endothelium from Cdh5-GCaMP8 and Cx40-GCaMP2 Mice.

INTRODUCTION: Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothelium under connexin 40 promoter, have identified the unique properties of endothelial calcium signals. However, Cx40-GCaMP2 mouse is associated with a narrow dynamic range and lack of signal in the venous endothelium. Recent studies have proposed many GCaMPs (GCaMP5/6/7/8) with improved properties although their performance in endothelium-specific calcium studies is not known. METHODS: We characterized a newly developed mouse line that constitutively expresses GCaMP8 in the endothelium under the VE-cadherin (Cdh5-GCaMP8) promoter. Calcium signals through endothelial IP3 receptors and TRP vanilloid 4 (TRPV4) ion channels were recorded in mesenteric arteries (MAs) and veins from Cdh5-GCaMP8 and Cx40-GCaMP2 mice. RESULTS: Cdh5-GCaMP8 mice showed lower baseline fluorescence intensity, higher dynamic range, and higher amplitudes of individual calcium signals than Cx40-GCaMP2 mice. Importantly, Cdh5-GCaMP8 mice enabled the first recordings of discrete calcium signals in the intact venous endothelium and revealed striking differences in IP3 receptor and TRPV4 channel calcium signals between MAs and mesenteric veins. CONCLUSION: Our findings suggest that Cdh5-GCaMP8 mice represent significant improvements in dynamic range, sensitivity for low-intensity signals, and the ability to record calcium signals in venous endothelium.

Testosterone induces leucocyte migration by NADPH oxidase-driven ROS- and COX2-dependent mechanisms.

The mechanisms whereby testosterone increases cardiovascular risk are not clarified. However, oxidative stress and inflammation seem to be determinants. Herein, we sought to determine whether exogenous testosterone, at physiological levels, induces leucocyte migration, a central feature in immune and inflammatory responses and the mediating mechanisms. We hypothesized that testosterone induces leucocyte migration via NADPH oxidase (NADPHox)-driven reactive oxygen species (ROS) and cyclooxygenase (COX)-dependent mechanisms. Sixteen-week-old Wistar rats received an intraperitoneal injection (5 ml) of either testosterone (10(-7) mol/l) or saline. Rats were pre-treated with 5 ml of sodium salicylate (SS, non-selective COX inhibitor, 1.25 × 10(-3) mol/l, 1 h prior to testosterone or saline), flutamide (androgen receptor antagonist, 10(-5) mol/l), apocynin (NADPHox inhibitor, 3 × 10(-4) mol/l), N-[2-Cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS398, COX2 inhibitor, 10(-4) mol/l) or saline, 4 h before testosterone or saline administration. Leucocyte migration was assessed 24 h after testosterone administration by intravital microscopy of the mesenteric bed. Serum levels of testosterone were measured by radioimmunoassay. NADPHox activity was assessed in membrane fractions of the mesenteric bed by dihydroethidium (DHE) fluorescence and in isolated vascular smooth muscle cells (VSMC) by HPLC. NADPHox subunits and VCAM (vascular cell adhesion molecule) expression were determined by immunoblotting. Testosterone administration did not change serum levels of endogenous testosterone, but increased venular leucocyte migration to the adventia, NADPHox activity and expression (P < 0.05). These effects were blocked by flutamide. SS inhibited testosterone-induced leucocyte migration (P<0.05). Apocynin and NS398 abolished testosterone-induced leucocyte migration and NADPHox activity (P<0.05). Testosterone induces leucocyte migration via NADPHox- and COX2-dependent mechanisms and may contribute to inflammatory processes and oxidative stress in the vasculature potentially increasing cardiovascular risk.

F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries and veins.

OBJECTIVE: Vascular endothelial-cadherin- and integrin-based cell adhesions are crucial for endothelial barrier function. Formation and disassembly of these adhesions controls endothelial remodeling during vascular repair, angiogenesis, and inflammation. In vitro studies indicate that vascular cytokines control adhesion through regulation of the actin cytoskeleton, but it remains unknown whether such regulation occurs in human vessels. We aimed to investigate regulation of the actin cytoskeleton and cell adhesions within the endothelium of human arteries and veins. APPROACH AND RESULTS: We used an ex vivo protocol for immunofluorescence in human vessels, allowing detailed en face microscopy of endothelial monolayers. We compared arteries and veins of the umbilical cord and mesenteric, epigastric, and breast tissues and find that the presence of central F-actin fibers distinguishes the endothelial phenotype of adult arteries from veins. F-actin in endothelium of adult veins as well as in umbilical vasculature predominantly localizes cortically at the cell boundaries. By contrast, prominent endothelial F-actin fibers in adult arteries anchor mostly to focal adhesions containing integrin-binding proteins paxillin and focal adhesion kinase and follow the orientation of the extracellular matrix protein fibronectin. Other arterial F-actin fibers end in vascular endothelial-cadherin-based endothelial focal adherens junctions. In vitro adhesion experiments on compliant substrates demonstrate that formation of focal adhesions is strongly induced by extracellular matrix rigidity, irrespective of arterial or venous origin of endothelial cells. CONCLUSIONS: Our data show that F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries from veins. We conclude that the biomechanical properties of the vascular extracellular matrix determine this endothelial characteristic.

Shear-dependent adhesion of leukocytes and lectins to the endothelium and concurrent changes in thickness of the glycocalyx of post-capillary venules in the low-flow state.

OBJECTIVE: To elucidate shear-dependent effects of deformation of the endothelial glycocalyx on adhesion of circulating ligands in post-capillary venules, and delineate effect of MMPs. METHODS: Adhesion of WBCs and lectin-coated FLMs (0.1 µm diameter) to EC of post-capillary venules in mesentery was examined during acute reductions in shear rates (γ·, hemorrhagic hypotension). Adhesion was examined with or without superfusion with 0.5 µm doxycycline to inhibit MMPs. Thickness of the glycocalyx was measured by exclusion of fluorescent 70 kDa dextran from the EC surface. RESULTS: During superfusion with Ringers, rapid reductions in γ· resulted in a significant rise in WBC adhesion and a twofold rise in microsphere adhesion. With addition of doxycycline WBC and FLM adhesion increased twofold under high- and low-flow conditions. FLM adhesion was invariant with γ· throughout the network in the normal (high)-flow state. With reductions in γ·, thickness of the glycocalyx increased significantly, with or without doxycycline. CONCLUSIONS: The concurrent increase in WBC and FLM adhesion with increased thickness of the glycocalyx during reductions in shear suggests that glycocalyx core proteins recoil from their deformed steady-state configuration, which increases exposure of binding sites for circulating ligands.

Relative roles of doxycycline and cation chelation in endothelial glycan shedding and adhesion of leukocytes.

Leukocyte [white blood cell (WBC)] adhesion and shedding of glycans from the endothelium [endothelial cells (ECs)] in response to the chemoattractant f-Met-Leu-Phe (fMLP) has been shown to be attenuated by topical inhibition of matrix metalloproteases (MMPs) with doxycycline (Doxy). Since Doxy also chelates divalent cations, these responses were studied to elucidate the relative roles of cation chelation and MMP inhibition. WBC-EC adhesion, WBC rolling flux, and WBC rolling velocity were studied in postcapillary venules in the rat mesentery during superfusion with the cation chelator EDTA or Doxy. Shedding and accumulation of glycans on ECs, with and without fMLP, were quantified by the surface concentration of lectin (BS-1)-coated fluorescently labeled microspheres (FLMs) during constant circulating concentration. Without fMLP, low concentrations of EDTA (1-3 mM) increased FLM-EC sequestration due to disruption of the permeability barrier with prolonged exposure. In contrast, with 0.5 µM Doxy alone, FLM adhesion remained constant (i.e., no change in glycan content) on ECs, and WBC adhesion increased with prolonged superfusion. Without fMLP, EDTA did not affect firm WBC-EC adhesion but reduced WBC rolling flux in a dose-dependent manner. With fMLP, EDTA did not inhibit WBC adhesion, whereas Doxy did during the first 20 min of superfusion. Thus, the inhibition by Doxy of glycan (FLM) shedding and WBC adhesion in response to fMLP results from MMP inhibition, in contrast to cation chelation. With either Doxy or the MMP inhibitor GM-6001, WBC rolling velocity decreased by 50%, as in the case with fMLP, suggesting that MMP inhibition reduces sheddase activity, which increases the adhesiveness of rolling WBCs. These events increase the effective leukocrit on the venular wall and increase firm WBC-EC adhesion. Thus, MMP inhibitors have both a proadhesion effect by reducing sheddase activity while exerting an antiadhesion effect by inhibiting glycocalyx shedding and subsequent exposure of adhesion molecules on the EC surface.

Inhibition of VEGF or TGF-{beta} signaling activates endothelium and increases leukocyte rolling.

OBJECTIVE: Motivated by the central roles that vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta play in the assembly and maintenance of the vasculature, we examined the impact of systemic VEGF or TGF-beta signal inhibition on endothelial activation as detected by leukocyte-endothelial interactions. METHODS AND RESULTS: VEGF or TGF-beta inhibition, accomplished using adenovirus expression of soluble Flt1 (Ad-sFlt1) or soluble endoglin (Ad-sEng), resulted in a significant increase in the number of leukocytes rolling along the mesenteric venous endothelium and a significant decrease in rolling velocity in Ad-sEng mice. Neutralization of VEGF or TGF-beta resulted in endothelial surface expression of P-selectin and impaired peripheral vasodilatation. Neither inhibition of VEGF nor TGF-beta was associated with platelet or leukocyte activation, as detected by the activation markers platelet P-selectin and the active integrin alphaIIbbetaIII, or by leukocyte expression of L-selectin. Soluble vascular cell adhesion molecule (VCAM)-1 and E-selectin were increased in sEng-expressing mice, indicating higher levels of these adhesion receptors. CONCLUSIONS: VEGF or TGF-beta neutralization leads to impaired endothelium-mediated vasodilatation and elevated expression of surface adhesion molecules, resulting in increased leukocyte adhesion. These results indicate an essential role for both VEGF and TGF-beta in maintaining the endothelium in a nonactivated state and have implications for therapeutic approaches that neutralize VEGF or TGF-beta.

Electron microscopy and electron probe analysis of mitochondrial cation accumulation in smooth muscle.

The contractile responses to barium and the ultrastructure and ionic composition of mitochondria were studied in vascular smooth muscle. In normal rabbit portal anterior mesenteric vein (PAMV) and main pulmonary artery (MPA) smooth muscle mitochondria were frequently associated with the surface vesicles. The average distance between the outer mitochondrial and inner surface vesicle membrane was 4-5 nm. Ba contractures of MPA were tonic and of PAMV were phasic. Incubation of MPA and PAMV with Ba resulted in the accumulation of mitochondrial granules, followed in the MPA by massive mitochondrial swelling. Oligomycin and anoxia inhibited the appearance of mitochondrial electron-opaque granules and prevented the Ba-induced mitochondrial swelling in the MPA. Electron probe analysis of mitochondria in PAMV incubated with Ba and containing granules showed characteristic Ba signals over the mitochondria. Electron probe X-ray microanalysis also showed a highly significant (P < 0.001) correlation of P with mitochondrial Ba, in an estimated elemental ratio of approximately 3 Ba/4 P. Mitochondrial granules were still prominent after block staining of the osmium-fixed, Ba-loaded PAMV, but electron probe microanalysis showed no Ba, but only U, emissions. Tissues incubated with strontium had electron-opaque mitochondrial granules and deposits in the sarcoplasmic reticulum. X-ray microanalysis of mitochondria containing granules showed the presence of characteristic Sr and Ca emissions. The presence of Sr was similarly verified in the sarcoplasmic reticulum. These findings indicate the energy dependent uptake of divalent cations, in association with phosphate, by mitochondria in vascular smooth muscle in situ and the possibility that mitochondria may contribute to the regulation of intracellular divalent cation levels in smooth muscle.

Thick filaments in vascular smooth muscle.

Two sets of myofilaments were demonstrated after incubation of strips of rabbit portal-anterior mesenteric vein under moderate stretch in a physiological salt solution. Thick filaments had a mean diameter of 18 nm and reached a maximum length of 1.4 microm with a mean length of 0.61 microm. In transverse sections, 2.5-5 nm particles were resolved as subunits of the thick filaments. Thin filaments had an average diameter of 8.4 nm and generally conformed to the structure believed to represent actin filaments in smooth and striated muscles. In the areas of maximum concentration there were 160-328 thick filaments/microm(2) and the lowest ratio of thin to thick filaments was 12:1. Thick filaments were present in approximately equal numbers in vascular smooth muscle relaxed by theophylline, in Ca(++)-free solution, or contracted by norepinephrine. The same preparatory procedures used with vascular smooth muscle also enabled us to visualize thick filaments in guinea pig and rabbit taenia coli and vas deferens.

Cyclic stretch controls the expression of CD40 in endothelial cells by changing their transforming growth factor-beta1 response.

BACKGROUND: CD40 is a costimulatory molecule that acts as a central mediator of various immune responses, including those involved in the progression of atherosclerosis. Correspondent to its function, CD40 is present not only on many immune cells, such as antigen-presenting cells and T cells, but also on nonimmune cells, such as endothelial cells. METHODS AND RESULTS: Ex vivo analyses in mice revealed that CD40 is strongly expressed in distinct venous and capillary but not arterial endothelial cell populations. Therefore, we analyzed to what extent determinants of an arterial environment control CD40 expression in these cells. In vitro studies indicated that the presence of smooth muscle cells or exposure to cyclic stretch significantly downregulates CD40 expression in human endothelial cells. Interestingly, endothelial cells cocultured with smooth muscle cells upregulated CD40 expression in response to cyclic stretch through a transforming growth factor-beta1/activin-receptor-like kinase-1 (Alk-1)-dependent mechanism. To corroborate that this mechanism also operates in arteries in vivo, we analyzed the expression of Alk-1 and CD40 at atherosclerosis-prone sites of the mouse aorta that also appear to be exposed to increased stretch. In wild-type mice, both Alk-1 and CD40 revealed a comparably heterogeneous expression pattern along the aortic arch that matched those sites in low-density lipoprotein-receptor-deficient mice where atherosclerotic lesions develop. CONCLUSIONS: Cyclic stretch thus increases the abundance of CD40 in endothelial cells through transforming growth factor-beta1/Alk-1 signaling. This mechanism in turn may be responsible for the heterogeneous expression of CD40 at arterial bifurcations or curvatures and would support a site-specific proinflammatory response that is typical for the early phase of atherosclerosis.

Sympathetic innervation of human mesenteric artery and vein.

BACKGROUND: Innervation of blood vessels shows inter-species variability. There are few studies on the innervation of human vessels; thus, healthy mesenteric vessels were studied to identify the expression of immunomarkers and the morphology of sympathetic innervation as the basis for a study of mesenteric vessels in inflammatory bowel disease. METHODS AND RESULTS: Electron microscopy studies examined the relationships of nerves to smooth muscle cells. In veins, nerves were distributed throughout the medial smooth muscle coat, often in close apposition (50 nm) to smooth muscle cells. In arteries, nerves were located at the adventitial-medial border, few closer than 2,000 nm to smooth muscle cells, often with interposing connective tissue and Schwann cell processes. There was a significantly greater nerve density in veins than in arteries (227 vs. 41 mm(2); p = 0.03). Immunohistochemical studies revealed the presence of sympathetic and sensory-motor nerves in arteries and veins. CONCLUSIONS: It is suggested that in humans with an upright stance, the mesenteric venous system plays a particularly important role in controlling mesenteric capacitance, which is reflected by their dense innervation. It is speculated that transmitters released from perivascular nerves supplying the human mesenteric arteries may play a long-term (trophic) role in addition to short-term signalling roles.

Using intravascular autoradiography for an estimation of proliferative activity of rat mesenteric microvessel endothelial cells during the first month of postnatal development.

BACKGROUND/METHODS: We applied a novel method for studying endothelial cells (EC) by using autoradiography of cells labeled by 3H-thymidine: photo emulsion was administered into the vascular bed. In the flat transparent organ, this method allows to compare the mitotic activity of endothelium (MAE) depending on vessel diameter and animal age. RESULTS: The number of the labeled ECs in animals was found to be almost equal in all vessels at the same age. The amount of labeled vessels and the density of the labeled nuclei increase towards near-capillary vessels of a diameter < or =10 microm. With age, MAE decreases in all categories of vessels. However, in 12-day-old animals, MAE temporarily increases. It was noted that high MAE in the bed of these rats precedes or coincides with the period of accelerated weight gain of the digestive system supplied by an increase in organ blood flow. CONCLUSIONS: By using the endothelial autoradiography method that we developed, we obtained the following evidence: (1) the number of ECs in the synthetic phase of the mitotic cycle in mesenterial vessels is approximately identical in animals of the same age, and (2) this number decreases with age, except during the period of accelerated organ growth, when MAE increases.

A small molecule, orally active, alpha4beta1/alpha4beta7 dual antagonist reduces leukocyte infiltration and airway hyper-responsiveness in an experimental model of allergic asthma in Brown Norway rats.

alpha(4)beta(1) and alpha(4)beta(7) integrins are preferentially expressed on eosinophils and mononuclear leukocytes and play critical roles in their recruitment to inflammatory sites. We investigated the effects of TR14035, a small molecule, alpha(4)beta(1)/alpha(4)beta(7) dual antagonist, in a rat model of allergic asthma. Actively sensitized rats were challenged with aerosol antigen or saline on day 21, and the responses evaluated 24 and 48-h later. TR14035 (3 mg kg(-1), p.o.) was given 1-h before and 4-h after antigen or saline challenge. Airway hyper-responsiveness to intravenous 5-hydroxytryptamine was suppressed in TR14035-treated rats. Eosinophil, mononuclear cell and neutrophil counts, and eosinophil peroxidase and protein content in the bronchoalveolar lavage fluid (BALF) were decreased in TR14035-treated rats. Histological study showed a marked reduction of lung inflammatory lesions by TR14035. At 24-h postchallenge, antigen-induced lung interleukin (IL)-5 mRNA upregulation was suppressed in TR14035-treated rats. By contrast, IL-4 levels in BALF were not significantly affected by TR14035 treatment. IL-4 selectively upregulates vascular cell adhesion molecule-1 (VCAM-1), which is the main endothelial ligand of alpha(4) integrins. Intravital microscopy within the rat mesenteric microcirculation showed that 24-h exposure to 1 microg per rat of IL-4 induced a significant increase in leukocyte rolling flux, adhesion and emigration. These responses were decreased by 48, 100 and 99%, respectively in animals treated with TR14035. In conclusion, TR14035, by acting on alpha(4)beta(1) and alpha(4)beta(7) integrins, is an orally active inhibitor of airway leukocyte recruitment and hyper-responsiveness in animal models with potential interest for the treatment of asthma.

Ubiquitous elevation of matrix metalloproteinase-2 expression in the vasculature of patients with abdominal aneurysms.

BACKGROUND: Patients with abdominal aortic aneurysms (AAAs) exhibit arterial dilation and altered matrix composition throughout the vasculature. Matrix metalloproteinase-2 (MMP-2) is the dominant elastase in small AAAs, and overexpression of MMP-2 in vascular smooth muscle cells (SMCs) may be a primary etiological event in aneurysm genesis. The aim of this study was to investigate MMP-2 production in vascular tissue remote from the abdominal aorta. METHODS AND RESULTS: Inferior mesenteric vein (IMV) was harvested from patients undergoing aneurysm repair (n=21) or colectomy for diverticular disease (n=13, control). Matrix composition of the vessels was determined by stereological techniques. MMPs were extracted from tissue homogenates and quantified by gelatin zymography and ELISA. MMP-2, membrane type-1 MMP (MT1-MMP), and tissue inhibitor of metalloproteinases type 2 (TIMP-2) expression were determined by Northern analysis. SMCs were isolated from IMV, and the production and expression of MMP-2 and TIMP-2 in the SMC lines were quantified. Tissue homogenates and isolated inferior mesenteric SMCs from patients with aneurysms demonstrated significantly elevated MMP-2 levels, with no difference in TIMP-2 or MT1-MMP. These differences were a result of increased MMP-2 expression. Histological examination revealed fragmentation of elastin fibers within venous tissue obtained from patients with AAA and a significant depletion of the elastin within the media. In situ zymography localized elastolysis to medial SMCs. CONCLUSIONS: Patients with AAA have elevated MMP-2 levels in the vasculature remote from the aorta. This finding is due to increased MMP-2 expression from SMCs, a characteristic maintained in tissue culture. These data support both the systemic nature of aneurysmal disease and a primary role of MMP-2 in aneurysm formation.

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

Effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation during early sepsis treatment.

OBJECTIVES: Experimental studies on sepsis have demonstrated that ethyl pyruvate is endowed with antioxidant and anti-inflammatory properties. This study aimed to investigate the effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation in a live Escherichia coli-induced sepsis model in rats. METHODS: Male Wistar rats were administered an intravenous suspension of E. coli bacteria or were subjected to a sham procedure. Three hours after bacterial infusion, the rats were randomized into the following groups: a control group without treatment, a group treated with lactated Ringer's solution (4 mL/kg, i.v.), and a group treated with lactated Ringer's solution (4 mL/kg, i.v.) plus ethyl pyruvate (50 mg/kg). At 24 h after bacterial infusion, leukocyte-endothelial interactions were investigated using intravital microscopy, and the expression of P-selectin and intercellular adhesion molecule-1 was evaluated via immunohistochemistry. White blood cell and platelet counts were also determined at baseline and 3 h and 24 h after E. coli inoculation. RESULTS: The non-treated and lactated Ringer's solution-treated groups exhibited increases in the numbers of rolling leukocytes (∼2.5-fold increase), adherent cells (∼3.0-fold), and migrated cells (∼3.5-fold) compared with the sham group. In contrast, treatment with Ringer's ethyl pyruvate solution reduced the numbers of rolling, adherent and migrated leukocytes to the levels observed in the sham group. Additionally, the expression of P-selectin and intercellular adhesion molecule-1 was significantly increased on mesenteric microvessels in the non-treated group compared with the sham group (p<0.001). The expression of both adhesion molecules was reduced in the other groups, with ethyl pyruvate being more effective than lactated Ringer's solution. Infusion of bacteria caused significant leukopenia (3 h), followed by leukocytosis with granulocytosis (24 h). There was also an intense and progressive reduction in the number of platelets. However, no differences were observed after treatment with the different solutions. CONCLUSIONS: The presented data suggest that ethyl pyruvate efficiently reduces the inflammatory response in the mesenteric microcirculation in an experimental model of sepsis induced by live E. coli and is associated, at least in part, with down-regulation of P-selectin and intercellular adhesion molecule-1.

Comparison of the contractile and calcium-increasing properties of platelet-activating factor and endothelin-1 in the rat mesenteric artery and vein.

In the present study, the properties of endothelin-1 (ET-1) and platelet-activating factor (PAF) in inducing contraction and increased intracellular-free calcium level in rat mesenteric arteries and veins were studied. Furthermore, measurements of cytosolic ([Ca](c)) and nuclear ([Ca](n)) Ca(2+) were performed by confocal microscopy. PAF, at a concentration of 1 microM, and the selective ET(B) agonists, IRL-1620 and sarafotoxin S6C (100 nM), induced a marked constriction and increase in [Ca](i) in the mesenteric vein but not in the artery. On the other hand, endothelin-1 (1 - 100 nM) induced a significant concentration-dependent nifedipine-insensitive increase in tension and [Ca](i) in both arteries and veins. Those responses to endothelin-1 were significantly reduced by the ET(A) receptor antagonist, BQ-123 (10(-6) M), on both types of vessels, whereas the selective ET(B) receptor antagonist, BQ-788, inhibited only the venous responses. The mixed ET(A)/ET(B) receptor antagonist, SB 209670, reduced the ET-1-induced venous responses to the same level of that found in presence of BQ-123 or BQ-788. However, concomitant applications of BQ-123 and BQ-788 reduced the vasoconstriction below to that induced by ET(A) or ET(B) blockade without further affecting [Ca](i). PAF and the selective ET(B) agonists IRL-1620, induced a sustained increase of [Ca](c) and [Ca](n) solely in venous cells and ET-1 in both arterial and venous smooth muscle cells. Thus, PAF increases total intracellular calcium concentration and tension on the smooth muscle cells from venous origin only. Furthermore, ET-1-induced vasoactive as well as [Ca](i) and [Ca](n) increasing effects are mediated by distinct receptors on venous and arterial smooth muscles.

Do adhesion molecules importantly regulate leukocyte kinetics within intraacinar microvessels of the lung?

Precise assessment of blood cell kinetics in the pulmonary microcirculation is extremely difficult because pulmonary microvascular architecture contains arterioles, venules and capillaries in an exceedingly intricate and densely convoluted fashion. Conventional epiluminescence microscopy may not be suitable for investigation of blood cell kinetic in the pulmonary microcirculation, in which arterioles, venules and capillary networks are not located in the same plane. To overcome these impediments, we recently developed a real-time confocal laser luminescence microscope with a high-speed analysis component having the capacity to yield confocal-images of rapidly moving cells at a rate of 1,000 frames/sec and at sufficiently high magnification. In the current review, we will first introduce the details of our newly developed observation system constructed with a view to estimation of blood cell dynamics in the intraacinar microcirculation of the lung. Applying this novel method to isolated perfused rat lungs, we will secondly address the issue of whether or not leukocyte-endothelium interactions in the pulmonary microcirculation qualitatively differ from those serving in the systemic microcirculation. We will particularly shed light on possible roles of endothelial ICAM-1, endothelial P-selectin and leukocyte L-selectin in distorting leukocyte kinetics in the intraacinar microvessels under a variety of diseased conditions, including prolonged exposure to a hyperoxic environment inducing a significant upregulation of ICAM-1 as well as P-selectin on the pulmonary microvascular endothelium, and stimulation of leukocytes by an IL-8 analog causing downregulation of leukocyte L-selectin but inverse upregulation of CD18-related integrins.

Hypoxia causes leukocyte adherence to mesenteric venules in nonacclimatized, but not in acclimatized, rats.

Although the effects of ischemia-reperfusion have received considerable attention, few studies have directly evaluated the microcirculatory response to systemic hypoxia. The overall objective of this study was to assess the effect of environmental hypoxia on adhesive interactions of circulating leukocytes with rat mesenteric venules by using intravital microscopy. Experiments were designed to 1) characterize the adhesive interactions of circulating leukocytes to venules during acute hypoxia produced by a reduction in inspired PO(2), 2) evaluate the role of nitric oxide in these adhesive interactions, 3) determine whether the effect of hypoxia on leukocyte adhesive interactions differs between acclimatized and nonacclimatized rats, and 4) assess whether compensatory changes in nitric oxide formation contribute to this difference. The results showed that acute hypoxia promotes leukocyte-endothelial adherence in mesenteric venules of nonacclimatized rats. The mechanism of this response is consistent with depletion of nitric oxide within the microcirculation. In contrast, no leukocyte-endothelial adherence occurred during hypoxia in rats acclimatized to hypobaric hypoxia. The results are consistent with increased nitric oxide formation due to expression of inducible nitric oxide synthase during the acclimatization period. Further studies are needed to establish the cause of nitric oxide depletion during acute hypoxia as well as to define the compensatory responses that attenuate hypoxia-induced leukocyte-endothelial adherence in the microvasculature of acclimatized rats.

Leukotriene B(4) promotes reactive oxidant generation and leukocyte adherence during acute hypoxia.

Acute systemic hypoxia produces rapid leukocyte adherence in the rat mesenteric microcirculation, although the underlying mechanisms are not fully known. Hypoxia is known to increase reactive oxygen species (ROS) generation, which could result in formation of the lipid inflammatory mediator leukotriene B(4) (LTB(4)). The goal of this study was to examine the role of LTB(4) in hypoxia-induced microvascular alterations. Using intravital microscopy, we determined the effect of the LTB(4) antagonist, LTB(4)-dimethyl amide (LTB(4)-DMA), on ROS generation and leukocyte adherence in mesenteric venules during hypoxia. Exogenous LTB(4) increased ROS generation to 144 +/- 8% compared with control values and also promoted leukocyte adherence. These responses to LTB(4) were blocked by pretreating the mesentery with LTB(4)-DMA. Leukopenia did not significantly attenuate the LTB(4)-induced increase in ROS generation (142 +/- 12.1%). LTB(4)-DMA substantially, though not completely, reduced hypoxia-induced ROS generation from 66 +/- 18% to 11 +/- 4% above control values. Hypoxia-induced leukocyte adherence was significantly attenuated by LTB(4)-DMA. Our results support a role for LTB(4) in the mechanism of hypoxia-induced ROS generation and leukocyte adherence in the rat mesenteric microcirculation.

Oligotide attenuates leukocyte-endothelial cell interaction via P-selectin in the rat mesenteric vascular bed.

A novel single stranded polydeoxyribonucleotide (oligotide) was studied for its ability to modulate leukocyte-endothelial cell interaction, by means of intravital microscopy in the rat mesenteric microvasculature. Superfusion of the rat mesentery with 50 microM NG-nitro-L-arginine methyl ester (L-NAME), caused a significant, time-dependent increase in leukocyte rolling and adherence compared to control rats superfused with Krebs-Henseleit solution. However, oligotide (15 mg/kg i.v.) consistently reduced the L-NAME-induced leukocyte rolling (62 +/- 14 vs. 23 +/- 3 cells/min; P < 0.02) and adherence (11 +/- 2 vs. 4 +/- 1 cells/100 microns length of venule P < 0.01), without altering systemic blood pressure or mesenteric venular shear rate. Moreover, immunohistochemical localization of P-selectin expression on mesenteric venules was significantly increased (P < 0.01) after exposure to L-NAME, which was significantly attenuated by oligotide (P < 0.05). Similar results were also obtained by flow cytometric analysis performed on rat platelets. Stimulation of rat platelets with L-NAME significantly (P < 0.05) increased the fluorescence intensity of P-selectin, while the concomitant treatment of isolated rat platelets with L-NAME plus oligotide significantly (P < 0.005) attenuated P-selectin fluorescence intensity. Our data demonstrate that in vivo administration of oligotide can reduce leukocyte rolling and adherence in the mesenteric rat microvasculature by attenuating P-selectin expression, and confirming the key role of nitric oxide as an important regulator of leukocyte-endothelial cell interaction.