Inflamed Microvessels Research Articles

A Stochastic Process Algebra Approach to Simulation of Autoreactive Lymphocyte Recruitment

This article presents a stochastic model of lymphocyte recruitment in inflamed brain microvessels. Recent studies about the inflammatory process of the brain that leads to multiple sclerosis have revealed that lymphocyte extravasation is a sequence of dynamical states, mediated by partially overlapped interactions of different adhesion molecules and activation factors. This study’s model of lymphocyte recruitment is based on process algebras for mobile systems. The biochemical system is modelled as a set of concurrent processes of the biochemical stochastic π-calculus. Processes are driven by suitable probability distributions that quantitatively describe the rates and the times at which reactions to simulations occur. The results of the model reproduce, within the estimated experimental errors, the functional behavior of the data obtained from laboratory measurements.

Acquired microvascular dysfunction in inflammatory bowel disease: loss of nitric oxide-mediated vasodilation

Background & Aims: Inflammatory bowel disease (IBD; i.e., Crohn’s disease, ulcerative colitis) is characterized by refractory inflammatory ulceration and damage to the intestine. Mechanisms underlying impaired healing are not defined. Because microvascular dysfunction resulting in diminished vasodilatory capacity and tissue hypoperfusion is associated with impaired wound healing, we hypothesized that microvascular dysfunction may also occur in chronic IBD. Methods: Intact submucosal arterioles from control, involved, and uninvolved IBD specimens were assessed using in vitro videomicroscopy to assess endothelium-dependent vasodilation in response to acetylcholine (Ach) and fluorescence microscopy to detect oxyradicals. Results: Normal microvessels dilated in a dose-dependent and endothelium-dependent manner to Ach (maximum, 82% ± 2%; n = 34). Inhibition of nitric oxide synthase with N G -nitro- l-arginine methyl ester ( l-NAME) reduced maximal dilation to 54% ± 6% ( P < 0.05, n = 7), and further reduction was observed after inhibiting cyclooxygenase (indomethacin; 23% ± 10%, n = 6). Chronically inflamed IBD microvessels showed significantly reduced Ach-induced vasodilation (maximum, 15% ± 2%; n = 33), with no effect of l-NAME. Indomethacin eliminated the remaining Ach-induced vasodilation, resulting in frank vasoconstriction (−54% ± 9%, n = 6). Uninvolved IBD gut vessels and non-IBD inflammatory controls responded in a fashion similar to normal vessels. IBD-involved microvessels generated significantly higher levels of reactive oxygen species compared with control and uninvolved IBD vessels ( P < 0.01). Conclusions: Human intestinal microvessels from chronically inflamed IBD show microvascular endothelial dysfunction, characterized by loss of NO-dependent dilation that may contribute to reduced perfusion, poor wound healing, and maintenance of chronic inflammation.

Combined anticoagulant and antiselectin treatments prevent lethal intravascular coagulation

AbstractWidespread microvascular injury followed by vessel obstruction may lead to disseminated intravascular coagulation (DIC). We describe a murine model wherein leukocytes interacting with inflamed microvessels in vivo are activated by antibodies. Treatment of tumor necrosis factor α (TNF-α)–primed mice with anti–Ly-6G antibodies reproduced many of the features of septic or traumatic shock including microvessel obstruction and coagulation, severe vasculitis, respiratory difficulties, and vascular leakage. Mice lacking either E-selectin or P-selectin were protected from this reaction as were animals treated with a combination of either selectin-blocking antibodies and heparin or a selectin antagonist plus heparin. Combined blockade of leukocyte/platelet adhesion and coagulation may provide convincing protection in DIC.

Mechanisms of heterogeneous endothelial cytoplasmic calcium increases in venular microvessels.

Localized inflammatory leaky sites form at regions of the microvessel wall with the largest increase in endothelial cell cytoplasmic calcium concentration, [Ca(2+)](i). We investigated the mechanisms that modulate localized increases in [Ca(2+)](i) in individual endothelial cells of microvessels after exposure to ATP. [Ca(2+)](i) was measured by using digital fluorescence microscopy and fura-2 in the endothelial cells forming the walls of individually perfused frog mesenteric microvessels. The spread of [Ca(2+)](i) from a localized mechanical stimulus was also measured. The peak [Ca(2+)](i) after ATP showed marked heterogeneity, ranging from 227 to 1469 nM from resting values of 69 +/- 5 nM. After depolarization with high-potassium solutions, the endothelial cells with the largest peak increase in [Ca(2+)](i) had the largest fractional reduction. Localized increases in [Ca(2+)](i) due to mechanical stimulus did not spread. The key mechanism regulating the heterogeneity in initial peak increase in [Ca(2+)](i) is a calcium-dependent process regulated by the calcium influx itself. One such mechanism, the calcium-dependent opening of additional potassium channels leading to membrane hyperpolarization and increased driving force for calcium entry through passive conductance pathways, accounts for a significant amount of the heterogeneity of [Ca(2+)](i) in our experiments. Further investigations of both localized calcium influx and membrane potentials in the endothelial cells of intact microvessels in both frog and mammals using the imaging methods developed for these investigations are needed to understand the formation of localized leaky sites in inflamed microvessels.

Mechanisms of Heterogeneous Endothelial Cytoplasmic Calcium Increases in Venular Microvessels

Objective: Localized inflammatory leaky sites form at regions of the microvessel wall with the largest increase in endothelial cell cytoplasmic calcium concentration, [Ca2+]i. We investigated the mechanisms that modulate localized increases in [Ca2+]i in individual endothelial cells of microvessels after exposure to ATP. Methods: [Ca2+]i was measured by using digital fluorescence microscopy and fura-2 in the endothelial cells forming the walls of individually perfused frog mesenteric microvessels. The spread of [Ca2+]i from a localized mechanical stimulus was also measured. Results: The peak [Ca2+]i after ATP showed marked heterogeneity, ranging from 227 to 1469 nM from resting values of 69 ± 5 nM. After depolarization with high-potassium solutions, the endothelial cells with the largest peak increase in [Ca2+]i had the largest fractional reduction. Localized increases in [Ca2+]i due to mechanical stimulus did not spread. Conclusion: The key mechanism regulating the heterogeneity in initial peak increase in [Ca2+]i is a calcium-dependent process regulated by the calcium influx itself. One such mechanism, the calcium-dependent opening of additional potassium channels leading to membrane hyperpolarization and increased driving force for calcium entry through passive conductance pathways, accounts for a significant amount of the heterogeneity of [Ca2+]i in our experiments. Further investigations of both localized calcium influx and membrane potentials in the endothelial cells of intact microvessels in both frog and mammals using the imaging methods developed for these investigations are needed to understand the formation of localized leaky sites in inflamed microvessels.

Production of Soluble Integrin α2β1 Heterodimer Complex Functionally Active in Vitro and in Vivo

Integrin α2β1, which is a membrane protein consisting of noncovalently bound α2 and β1 chains, mediates cell binding to collagen and plays a role in platelet functions. DNAs encoding the chimeric proteins in which the extracellular domains of each α2 and β1 chain was fused to hinge and Fc regions of human IgG1γ chain were cotransfected into CHO cells. Soluble integrin α2β1 (sα2β1) in which α2 and β1 chains were covalently bound by disulfide bonds was recovered from the culture supernatant. sα2β1 maintained functional characteristics of cell surface α2β1 as indicated by cation-dependent binding to collagen and conformational changes induced by cations or ligand. Intravenously administered sα2β1 in rats colocalized with collagen in inflamed microvessels. Moreover, sα2β1-conjugated liposome administered intravenously reduced bleeding time of the thrombocytopenic mice. These results indicated that sα2β1 has pharmaceutical utilities as an agent for detecting injured vessels and a component of platelet substitute.

Differential requirements for core2 glucosaminyltransferase for endothelial L-selectin ligand function in vivo.

L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 beta1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF-alpha-treated cremaster muscles and in Peyer's patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2(-/-)) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2(-/-) mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer's patch HEV was not significantly different between core2(-/-) and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2(-/-) mice, but significant rolling in controls. In Peyer's patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2(-/-) mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2(-/-) and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer's patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT-I-deficient mice.

Alpha 4 integrin-dependent leukocyte recruitment does not require VCAM-1 in a chronic model of inflammation.

Rats immunized with Mycobacterium butyricum in Freund's adjuvant develop a chronic vasculitis, with large increases in leukocyte rolling and adhesion in mesenteric postcapillary venules that are significantly inhibited with an alpha 4 integrin Ab. Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that alpha 4 integrin-dependent leukocyte rolling and adhesion was inhibited with a beta 1 integrin, but not a beta 7 integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for alpha 4 beta 1 integrin in the vasculature. However, alpha 4 beta 1 integrin-dependent interactions were not reduced by monoclonal or polyclonal VCAM-1 Abs or a VCAM-1 antisense oligonucleotide despite increased VCAM-1 expression in the mesenteric vasculature. To ensure that the VCAM-1 Abs were functional and used at saturating concentrations, blood from Ab-treated rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Sufficient alpha 4 integrin or VCAM-1 Ab was present to inhibit leukocyte interactions with rat VCAM-1 by 95-100%. Under in vitro flow conditions, only mononuclear leukocytes were recruited from blood of control rats onto purified VCAM-1. However, neutrophils were also recruited onto VCAM-1 from whole blood of adjuvant-immunized animals via alpha 4 integrin. Another ligand for alpha 4 beta 1 integrin is the connecting segment-1 (CS-1) region of fibronectin. An Ab to the CS-1 portion of fibronectin, which did not reduce rolling and adhesion in adjuvant arthritis animals, completely inhibited leukocyte adhesion to CS-1 under static conditions. These findings provide the first evidence that alpha 4 beta 1 integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.

Inhibition of gastric secretion relieves post-prandial urgency, diarrhea, and cramps in a variety of GI disorders

While several endothelial cell adhesion molecules (CAM) have been implicated as mediators of the leukocyte-endothelial cell adhesion observed in inflamed microvessels, P-selectin appears to be the major CAM that accounts for the very rapid (minutes) leukocyte rolling observed in these vessels. P-selectin can be rapidly mobilized from a preformed storage pool (WeibelPalade bodies) within endothelial cells by different stimuli, including histamine, thrombin, hydrogen peroxide, and leukotrienes. The objective of this study was to determine whether bradykinin, a well-known participant in inflammatory reactions can elicit P-selectin expression in the gastrointestinal microvasculature. The dual radiolabelled antibody technique was used to quantify endothelial cell P-selectin expression in rat metentery, small intestine, large intestine, pancreas and stomach. Intraperitoneal administration of bradykinin (25-200 lag) induced a dose-dependent up-regulation of P-selectin in most of the gastrointestinal organs. The increased P-selectin in these vascular beds occured within 15 min after bradykinin administration; no increases were noted 3 hrs after bradykinin. In order to determine whether the bradykinin B 2 receptor mediates the increased P-selectin expression elicited by bradykinin, some animals were pretreated with a Bz-receptor antagonist (HOE). In all gastrointestinal organs, HOE completely prevented the bradykinin-induced increase in P-selectin expression. These findings indicate that bradykinin can mobilize the preformed pool of P-selectin in vascular endothelial ceils and that bradykinin-mediated P-selectin involves the engagement of B~-receptors. Supported by P01 DK43785

The role of shear forces in ischemia/reperfusion-induced neutrophil rolling and adhesion.

Intravital microscopy was used to examine the role of reduced shear forces on neutrophil-endothelium interactions in single 25-40 microm diameter vessels in the feline postischemic mesenteric microvasculature. Neutrophil rolling, neutrophil adhesion, and microvascular permeability alterations were determined in vessels exposed to ischemia/reperfusion in untreated animals and in cats that received feline plasma directly into the superior mesenteric artery to increase intestinal blood flow and venular shear rates. Ischemia, followed by reperfusion, caused a profound increase in venular shear forces during the initial hyperemic response, which then decreased to below control values by 10 min of reperfusion and to less than 50% of control by 60 min of reperfusion. Associated with the decrease in blood flow was a profound increase in neutrophil rolling and neutrophil adhesion and an increase in microvascular permeability. Also, leukocyte rolling velocity decreased dramatically to 10-20% of control values during the first 10 min of reperfusion. Infusion of autologous plasma into the intestinal vasculature to maintain the shear forces at control levels during reperfusion did not affect the flux of rolling neutrophils. The rolling velocity of the neutrophils was not increased despite dramatically improved shear rates. Improved shear rates did reduce the number of adherent cells, resulting in less vascular dysfunction. The reduction in shear forces through inflamed microvessels does not contribute to the increased leukocyte rolling flux but is an essential, permissive component for neutrophil adhesion and subsequent vascular dysfunction in postischemic microvasculature.

Variable restriction of albumin diffusion across inflamed cerebral microvessels of the anaesthetized rat.

1. The possibility of restricted diffusion of macromolecules in single cerebral venular capillaries that have become leaky due to inflammation was investigated by comparing the permeabilities to Lucifer Yellow (457 Da; PLY) and rhodamine-labelled albumin (69 kDa; PRh-A). 2. The dyes were trapped between two micro-occlusion probes and the permeabilities were measured from the rates of decrease in dye fluorescence at low intraluminal hydrostatic pressure. 3. Removal of one probe had little effect on PLY but did reduce PRh-A, consistent with the influence of convection on diffusion through 22 nm wide transendothelial slits 1 micron deep. 4. Direct comparisons were made over time between PLY and PRh-A in six vessels while hydrostatic pressure effects were controlled. In all vessels PRh-A:PLY varied from being similar to the ratio of the free diffusion coefficients to virtually zero even though PLY remained high. The question of the source of this variable restriction to albumin is discussed in terms of the secretion and sloughing of glycosaminoglycans and the possible role of transient formation of transendothelial gaps.

Effects of Human Neutrophil Elastase (HNE) on Neutrophil Function In Vitro and in Inflamed Microvessels

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil-neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

Leukocyte-induced vascular protein leakage in cat mesentery

The overall objective of this study was to determine whether leukocyte adherence and/or emigration is a prerequisite for the increased vascular protein leakage associated with acute inflammation. An in vivo preparation was used to monitor intestinal vascular protein leakage as well as polymorphonuclear leukocyte (PMN) adhesion and emigration in feline mesenteric microvessels exposed to platelet-activating factor (PAF) and leukotriene B4 (LTB4). Local intra-arterial infusion of PAF (4 ng/min) produced a fourfold increase in vascular protein leakage. A 50-fold higher concentration of LTB4 had no effect on vascular protein efflux. LTB4, however, did potentiate the PAF-induced vascular protein leakage. Both inflammatory mediators caused leukocytes to adhere to endothelial cells in postcapillary venules; however, leukocyte emigration was observed only in the presence of PAF. PAF-induced leukocyte adhesion and emigration and the increased vascular protein leakage were inhibited by a monoclonal antibody (MoAb IB4) directed against the common beta-subunit of the adhesive glycoprotein complex CD11/CD18. MoAb IB4 also prevented LTB4-induced leukocyte adhesion. Both PAF and LTB4 caused degranulation of cat PMNs in vitro, yet superoxide production was stimulated by PAF only. The data derived from these in vivo and in vitro studies indicate that leukocyte adhesion per se does not necessarily lead to increased vascular protein leakage and leukocyte emigration. Adhesion-dependent PMN functions such as emigration and superoxide production may play an important role in producing the alterations in vascular integrity observed in inflamed microvessels.