Fritz Krombach

Defective smooth muscle regulation in cGMP kinase I‐deficient mice

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP‐dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross‐activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP‐dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI‐deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.

Optimized dispersion of nanoparticles for biological in vitro and in vivo studies.

BackgroundThe aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies.ResultsTiO2 (rutile) dispersions were prepared in distilled water, PBS, or RPMI 1640 cell culture medium. Different ultrasound energies, various dispersion stabilizers (human, bovine, and mouse serum albumin, Tween 80, and mouse serum), various concentrations of stabilizers, and different sequences of preparation steps were applied. The size distribution of dispersed nanoparticles was analyzed by dynamic light scattering and zeta potential was measured using phase analysis light scattering. Nanoparticle size was also verified by transmission electron microscopy. A specific ultrasound energy of 4.2 × 105 kJ/m3 was sufficient to disaggregate TiO2 (rutile) nanoparticles, whereas higher energy input did not further improve size reduction. The optimal sequence was first to sonicate the nanoparticles in water, then to add dispersion stabilizers, and finally to add buffered salt solution to the dispersion. The formation of coarse TiO2 (rutile) agglomerates in PBS or RPMI was prevented by addition of 1.5 mg/ml of human, bovine or mouse serum albumin, or mouse serum. The required concentration of albumin to stabilize the nanoparticle dispersion depended on the concentration of the nanoparticles in the dispersion. TiO2 (rutile) particle dispersions at a concentration lower than 0.2 mg/ml could be stabilized by the addition of 1.5 mg/ml albumin. TiO2 (rutile) particle dispersions prepared by this method were stable for up to at least 1 week. This method was suitable for preparing dispersions without coarse agglomerates (average diameter < 290 nm) from nanosized TiO2 (rutile), ZnO, Ag, SiOx, SWNT, MWNT, and diesel SRM2975 particulate matter.ConclusionThe optimized dispersion method presented here appears to be effective and practicable for preparing dispersions of nanoparticles in physiological solutions without creating coarse agglomerates.

Histones from Dying Renal Cells Aggravate Kidney Injury via TLR2 and TLR4

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

Synchronized renal tubular cell death involves ferroptosis

Significance Cell death by regulated necrosis causes tremendous tissue damage in a wide variety of diseases, including myocardial infarction, stroke, sepsis, and ischemia–reperfusion injury upon solid organ transplantation. Here, we demonstrate that an iron-dependent form of regulated necrosis, referred to as ferroptosis, mediates regulated necrosis and synchronized death of functional units in diverse organs upon ischemia and other stimuli, thereby triggering a detrimental immune response. We developed a novel third-generation inhibitor of ferroptosis that is the first compound in this class that is stable in plasma and liver microsomes and that demonstrates high efficacy when supplied alone or in combination therapy. Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia–reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the first-in-class compound ferrostatin-1 (Fer-1). Even in conditions with extraordinarily severe IRI, 16-86 exerts strong protection to an extent which has not previously allowed survival in any murine setting. In addition, 16-86 further potentiates the strong protective effect on IRI mediated by combination therapy with necrostatins and compounds that inhibit mitochondrial permeability transition. Renal tubules thus represent a tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. Finally, ferroptosis mediates postischemic and toxic renal necrosis, which may be therapeutically targeted by ferrostatins and by combination therapy.

Ultrafine Particles Exert Prothrombotic but Not Inflammatory Effects on the Hepatic Microcirculation in Healthy Mice In Vivo

Background—Air pollution episodes are strongly associated with increased cardiovascular morbidity and mortality. The effect of ultrafine particles (UFPs), when translocated after inhalation, on the microcirculation of extrapulmonary organs remains unclear. Methods and Results—In C57BL/6 mice, either carbon black UFPs (1×107 and 5×107) or vehicle was infused intra-arterially. Two hours after infusion, platelet- and leukocyte-endothelial cell interactions, sinusoidal perfusion, endothelial fibrin(ogen) deposition, and phagocytic activity of Kupffer cells were analyzed by intravital video fluorescence microscopy in the liver microvasculature. Expression of fibrin(ogen), von Willebrand factor (vWF), and P-selectin on hepatic endothelium was determined by immunostaining. Apoptotic cells were quantified in TUNEL-stained tissue sections. Application of UFPs caused significantly enhanced platelet accumulation on endothelium of postsinusoidal venules and sinusoids in healthy mice. UFP-induced platelet adhesion was not preceded by platelet rolling but was strongly associated with fibrin deposition and an increase in vWF expression on the endothelial surface. In contrast, inflammatory parameters such as the number of rolling/adherent leukocytes, P-selectin expression/translocation, and the number of apoptotic cells were not elevated 2 hours after UFP exposure. In addition, UFPs did not affect sinusoidal perfusion and Kupffer cell function. Conclusions—UFPs induce platelet accumulation in the hepatic microvasculature of healthy mice that is associated with prothrombotic changes on the endothelial surface of hepatic microvessels. Accumulation of particles in the liver exerts a strong procoagulatory impact but does not trigger an inflammatory reaction and does not induce microvascular/hepatocellular tissue injury.

von Willebrand factor promotes leukocyte extravasation

von Willebrand factor (VWF) is an important player in hemostasis but has also been suggested to promote inflammatory processes. Gene ablation of VWF causes a simultaneous defect in P-selectin expression making it difficult to identify VWF-specific functions. Therefore, we analyzed whether blocking antibodies against VWF would be able to interfere with neutrophil extravasation. We found that these antibodies inhibited neutrophil recruitment into thioglycollate-inflamed peritoneum and KC-stimulated cremaster by approximately 50%. Whereas platelet-VWF was not involved, the contribution of VWF to granulocyte recruitment was strictly dependent on the presence of platelets and the accessibility of their VWF-receptor glycoprotein Ib. Surprisingly, platelet P-selectin was largely dispensable for leukocyte extravasation, in agreement with our observation that anti-VWF antibodies did not affect leukocyte rolling and adhesion. Searching for possible effects downstream of leukocyte capture, we found that anti-VWF antibodies significantly inhibited thioglycollate-induced vascular permeability. The increase of permeability was independent of circulating granulocytes, showing that it was not a side effect of neutrophil diapedesis. Collectively, our results demonstrate that VWF-associated platelets strongly support neutrophil extravasation at a step downstream of leukocyte docking to the vessel wall. This step could be related to leukocyte diapedesis facilitated by destabilization of the endothelial barrier.

The role of JAM-A and PECAM-1 in modulating leukocyte infiltration in inflamed and ischemic tissues

Innate and adaptive immunological responses are accompanied by leukocyte adhesion to the blood‐vessel wall and their subsequent infiltration into the underlying tissues. In the majority of the cases, leukocytes cross the endothelium by squeezing through the border of apposed endothelial cells, a process that is known as diapedesis. Many data suggest that proteins at endothelial junctions establish homophilic interactions with identical proteins, which are present on leukocytes. These interactions might then direct the passage of leukocytes through the endothelial border. In this review, we focus on two endothelial junctional proteins [junctional adhesion molecule‐A (JAM‐A) and PECAM], which play an important role in leukocyte diapedesis. In vivo data with blocking antibodies or inactivation of JAM‐A and PECAM genes indicate that the role of these two proteins depends on the stimulus and the experimental model used.

Single-walled carbon nanotubes activate platelets and accelerate thrombus formation in the microcirculation

OBJECTIVES Although ambient nanoparticles have been shown to exert prothrombotic effects, manufactured nanoparticles are in this aspect less well investigated. Thus, the aim of this study was to characterize the effects of diesel, titanium dioxide rutile, and single-walled carbon nanotube nanoparticles on (i) platelet activation in vitro and (ii) on macro- and microcirculatory thrombus formation in vivo. METHODS Platelet P-selectin expression was measured by flow cytometry after incubation of whole blood with diesel (0.1mg/mL), titanium dioxide (0.1mg/mL) or single-walled nanotubes (0.001-0.1mg/mL). Platelet-granulocyte complexes were analyzed in whole blood and platelet aggregometry was performed with platelet-rich plasma. Upon systemic administration of nanoparticles (1mg/kg) to anesthetized mice, ferric chloride-induced thrombus formation was measured in small mesenteric arteries using in vivo microscopy. In separate experiments, diesel (1mg/kg), titanium dioxide (1mg/kg), or single-walled nanotubes (0.01-1mg/kg) were injected into anesthetized mice and light/dye-induced thrombus formation was investigated in the cremasteric microcirculation. RESULTS Diesel and titanium dioxide nanoparticles did not activate platelets or exert prothrombotic effects. In contrast, single-walled nanotubes significantly increased platelet P-selectin expression, the number of platelet-granulocyte complexes, and platelet aggregability in vitro, and reduced the occlusion time in mesenteric arteries as well as in cremasteric arterioles. CONCLUSION Our study shows that single-walled carbon nanotubes, but not diesel or titanium dioxide nanoparticles, induce platelet activation in vitro and exert prothrombotic effects in the microcirculation in vivo.

CD4+ T cells contribute to postischemic liver injury in mice by interacting with sinusoidal endothelium and platelets

The mechanisms by which T cells contribute to the hepatic inflammation during antigen‐independent ischemia/reperfusion (I/R) are not fully understood. We analyzed the recruitment of T cells in the postischemic hepatic microcirculation in vivo and tested the hypothesis that T cells interact with platelets and activate sinusoidal endothelial cells, resulting in microvascular dysfunction followed by tissue injury. Using intravital videofluorescence microscopy, we show in mice that warm hepatic I/R (90/30‐140 min) induces accumulation and transendothelial migration of CD4+, but not CD8+ T cells in sinusoids during early reperfusion. Simultaneous visualization of fluorescence‐labeled CD4+ T cells and platelets showed that approximately 30% of all accumulated CD4+ T cells were colocalized with platelets, suggesting an interaction between both cell types. Although interactions of CD4+/CD40L−/− T cells with CD40L−/− platelets in wild‐type mice were slightly reduced, they were almost absent if CD4+ T cells and platelets were from CD62P−/− mice. CD4 deficiency as well as CD40‐CD40L and CD28‐B7 disruption attenuated postischemic platelet adherence in the same manner as platelet inactivation with a glycoprotein IIb/IIIa antagonist and reduced neutrophil transmigration, sinusoidal perfusion failure, and transaminase activities. Treatment with an MHC class II antibody, however, did not affect I/R injury. In conclusion, we describe the type, kinetic, and microvascular localization of T cell recruitment in the postischemic liver. CD4+ T cells interact with platelets in postischemic sinusoids, and this interaction is mediated by platelet CD62P. CD4+ T cells activate endothelium, increase I/R‐induced platelet adherence and neutrophil migration via CD40‐CD40L and CD28‐B7‐dependent pathways, and aggravate microvascular/hepatocellular injury. (HEPATOLOGY 2006;43:306–315.)

Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue

Leukocyte infiltration of reerfused tissue is a key event in the pathogenesis of ischemia‐reperfusion. However, the role of chemokine receptors Ccr1, Ccr2, and Ccr5 for each single step of the postischemic recruitment process of leukocytes has not yet been characterized. Leukocyte rolling, firm adherence, transendothelial, and extravascular migration were analyzed in the cremaster muscle of anaesthetized C57BL/6 mice using near‐infrared reflected light oblique transillumination microscopy. Prior to 30 min of ischemia as well as at 5, 30, 60, 90, and 120 min after onset of reperfusion, migration parameters were determined in wild‐type, Ccr1−/−, Ccr2−/−, and Ccr5−/− mice. Sham‐operated wild‐type mice without ischemia were used as controls. No differences were detected in numbers of rolling leukocytes among groups. In contrast, the number of firmly adherent leukocytes was increased significantly in wild‐type mice as compared with sham‐operated mice throughout the entire reperfusion phase. Already after 5 min of reperfusion, this increase was reduced significantly in Ccr1−/− and Ccr5−/− mice, whereas only in Ccr2−/− mice, was adherence attenuated significantly at 120 min after onset of reperfusion. Furthermore, after 120 min of reperfusion, the number of transmigrated leukocytes (>80% Ly‐6G+ neutrophils) was elevated in wild‐type mice as compared with sham‐operated animals. This elevation was significantly lower in Ccr1−/−, Ccr2−/−, and Ccr5−/− mice. Leukocyte extravascular migration distances were comparable among groups. In conclusion, these in vivo data demonstrate that Ccr1, Ccr2, and Ccr5 mediate the postischemic recruitment of neutrophils through effects on intravascular adherence and subsequent transmigration.