Tissue Plasminogen Activator Promotes Postischemic Neutrophil Recruitment via Its Proteolytic and Nonproteolytic Properties
Bernd UhlGabriele ZuchtriegelDaniel Puhr‐WesterheideMarc PraetnerMarkus RehbergMatthias P. FabritiusMaximilian HessenauerMartin HolzerAndrej KhandogaRobert FürstStefan ZahlerFritz KrombachChristoph A. Reichel
48
Citation
35
Reference
10
Related Paper
Citation Trend
Abstract:
Neutrophil infiltration of the postischemic tissue considerably contributes to organ dysfunction on ischemia/reperfusion injury. Beyond its established role in fibrinolysis, tissue-type plasminogen activator (tPA) has recently been implicated in nonfibrinolytic processes. The role of this serine protease in the recruitment process of neutrophils remains largely obscure.Using in vivo microscopy on the postischemic cremaster muscle, neutrophil recruitment and microvascular leakage, but not fibrinogen deposition at the vessel wall, were significantly diminished in tPA(-/-) mice. Using cell transfer techniques, leukocyte and nonleukocyte tPA were found to mediate ischemia/reperfusion-elicited neutrophil responses. Intrascrotal but not intra-arterial application of recombinant tPA induced a dose-dependent increase in the recruitment of neutrophils, which was significantly higher compared with stimulation with a tPA mutant lacking catalytic activity. Whereas tPA-dependent transmigration of neutrophils was selectively reduced on the inhibition of plasmin or gelatinases, neutrophil intravascular adherence was significantly diminished on the blockade of mast cell activation or lipid mediator synthesis. Moreover, stimulation with tPA caused a significant elevation in the leakage of fluorescein isothiocyanate dextran to the perivascular tissue, which was completely abolished on neutrophil depletion. In vitro, tPA-elicited macromolecular leakage of endothelial cell layers was abrogated on the inhibition of its proteolytic activity.Endogenously released tPA promotes neutrophil transmigration to reperfused tissue via proteolytic activation of plasmin and gelatinases. As a consequence, tPA on transmigrating neutrophils disrupts endothelial junctions allowing circulating tPA to extravasate to the perivascular tissue, which, in turn, amplifies neutrophil recruitment through the activation of mast cells and release of lipid mediators.Keywords:
Cremaster muscle
Intravital microscopy
Neutrophil Extracellular Traps
Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation. The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of calcium signaling in the arteriolar endothelium.
Cremaster muscle
Intravital microscopy
Cite
Citations (11)
Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation. The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of calcium signaling in the arteriolar endothelium.
Cremaster muscle
Intravital microscopy
Cite
Citations (46)
Inflammation is essential in the protection of the organism and wound repair, but in cases of chronic inflammation can also cause microvasculature deterioration. Thus, inflammation monitorization studies are important to test potential therapeutics. The intravital microscopy (IVM) technique monitors leukocyte trafficking in vivo, being a commonly used procedure to report systemic conditions. Although the cremaster muscle, an established protocol for IVM, may affect the hemodynamics because of its surgical preparation, only male animals are used, and longitudinal studies over time are not feasible. Thinking how this impacts future studies, our aim is to understand if the IVM technique can be successfully performed using the ear lobe instead of the cremaster muscle. Elevated IL-1β plasmatic concentrations confirmed the systemic inflammation developed in a diabetic animal model, while the elevated number of adherent and rolling leukocytes in the ear lobe allowed for the same conclusion. Thus, this study demonstrates that albeit its thickness, the ear lobe protocol for IVM is efficient, non-invasive, more reliable, cost-effective and timesaving.
Cremaster muscle
Intravital microscopy
Lobe
Ear lobe
Cite
Citations (0)
Leukocyte rolling and adhesion are generally observed in venules but rarely observed in arterioles. With the use of intravital microscopy, we found that a 4-h treatment with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) dose dependently induced leukocyte rolling and adhesion in arterioles of the mouse cremaster muscle. The rolling response lasted more than 24 h and was completely inhibited by treatment with the sulfated polysaccharide fucoidin. Moreover, we found that costimulation with IL-1beta and TNF-alpha for 4 h synergistically increased arteriolar leukocyte rolling, i.e., threshold doses of IL-1beta and TNF-alpha together caused a more than 10-fold increase of rolling in arterioles compared with the sum of the individual responses. This rolling interaction was abolished by treatment with a monoclonal antibody directed against P-selectin (RB40.34), but it apparently was unaffected by a monoclonal antibody against L-selectin (MEL-14). Taken together, our functional data show that IL-1beta and TNF-alpha separately induce and synergistically increase P-selectin-dependent leukocyte rolling and firm adhesion in mouse cremaster arterioles.
Cremaster muscle
Intravital microscopy
Alpha (finance)
P-selectin
Cite
Citations (65)
Neutrophil Extracellular Traps
Intravital microscopy
Cite
Citations (12)
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.
Cremaster muscle
Intravital microscopy
CXCL1
Cite
Citations (8)
Cremaster muscle
Intravital microscopy
Cite
Citations (21)
Cremaster muscle
Intravital microscopy
Ischemic Preconditioning
Cite
Citations (12)
Inspection of the microcirculation in living tissues has been accomplished using various models. The rat cremaster muscle has been used for this purpose for more than 20 years. In our study, for the first time, the pedicle of the cremaster muscle has been catheterised indirectly in order to perfuse and drain the muscle and to obtain blood samples. The assessment of the effects of a variety of perfusants on the microcirculation after reperfusion injury can be carried out by using this model. © 1999 Wiley-Liss, Inc. MICROSURGERY 19:389–391 1999
Cremaster muscle
Intravital microscopy
Cite
Citations (0)
The role of leukocytes in the decreased perfusion following ischemia in skeletal muscle was examined in the microcirculation of the rat cremaster muscle. The isolated muscle was viewed with an intravital microscope. Diameters of A1 and A2 arterioles and collecting venules were determined hourly. The number of leukocytes rolling along the venular walls was determined from a videotape. Nonischemic (control) rats (n = 10) were observed for 6 hours. The ischemic group (n = 10) was observed for one hour, the iliac and femoral arteries and veins were then clamped for 4 hours, released, and the muscle was observed for another two hours. No change in arteriole or venule diameters occurred in the control group. The diameters of the arterioles in the ischemic group decreased significantly during reperfusion but, the venule diameters did not. There was a significant reduction during reperfusion but, perfused capillaries following ischemia compared to control. There was a small but not significant increase in the number of rolling leukocytes in the ischemic group. The extent of leukocyte rolling in postcapillary venules was found to not correlate with the decrease in capillary perfusion that occurs after ischemia and reperfusion. However, the decrease in capillary flow was associated with reduced arteriole diameters.
Cremaster muscle
Venule
Arteriole
Intravital microscopy
Cite
Citations (20)