Procoagulant platelets promote immune evasion in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive tumor entity, in which immune checkpoint (IC) molecules are primarily synthesized in the tumor environment. Here, we report that procoagulant platelets bear large amounts of such immunomodulatory factors and that the presence of these cellular blood components in TNBC relates to pro-tumorigenic immune cell activity and impaired survival. Mechanistically, tumor-released nucleic acids attract platelets into the aberrant tumor microvasculature where they undergo procoagulant activation, thus delivering specific stimulatory and inhibitory IC molecules. This concomitantly promotes pro-tumorigenic myeloid leukocyte responses and compromises anti-tumorigenic lymphocyte activity, ultimately supporting tumor growth. Interference with platelet-leukocyte interactions prevented immune cell misguidance and suppressed tumor progression, nearly as effective as systemic IC inhibition. Hence, our data uncover a self-sustaining mechanism of TNBC in utilizing platelets to misdirect immune cell responses. Targeting this irregular multicellular interplay might represent a novel immunotherapeutic strategy in TNBC without side effects of systemic IC inhibition.

Vitronectin promotes immunothrombotic dysregulation in the venular microvasculature.

Microvascular immunothrombotic dysregulation is a critical process in the pathogenesis of severe systemic inflammatory diseases. The mechanisms controlling immunothrombosis in inflamed microvessels, however, remain poorly understood. Here, we report that under systemic inflammatory conditions the matricellular glycoproteinvitronectin (VN) establishes an intravascular scaffold, supporting interactions of aggregating platelets with immune cells and the venular endothelium. Blockade of the VN receptor glycoprotein (GP)IIb/IIIa interfered with this multicellular interplay and effectively prevented microvascular clot formation. In line with these experimental data, particularly VN was found to be enriched in the pulmonary microvasculature of patients with non-infectious (pancreatitis-associated) or infectious (coronavirus disease 2019 (COVID-19)-associated) severe systemic inflammatory responses. Targeting the VN-GPIIb/IIIa axis hence appears as a promising, already feasible strategy to counteract microvascular immunothrombotic dysregulation in systemic inflammatory pathologies.

B- and T-cell acute lymphoblastic leukemias evade chemotherapy at distinct sites in the bone marrow.

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support escape from treatment. Using three-dimensional fluorescence imaging of ten primary ALL xenografts we identified sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detected B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon shortterm chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment.

Uncoupled biological and chronological aging of neutrophils in cancer promotes tumor progression.

BACKGROUND: Beyond their fundamental role in homeostasis and host defense, neutrophilic granulocytes (neutrophils) are increasingly recognized to contribute to the pathogenesis of malignant tumors. Recently, aging of mature neutrophils in the systemic circulation has been identified to be critical for these immune cells to properly unfold their homeostatic and anti-infectious functional properties. The role of neutrophil aging in cancer remains largely obscure. METHODS: Employing advanced in vivo microscopy techniques in different animal models of cancer as well as utilizing pulse-labeling and cell transfer approaches, various ex vivo/in vitro assays, and human data, we sought to define the functional relevance of neutrophil aging in cancer. RESULTS: Here, we show that signals released during early tumor growth accelerate biological aging of circulating neutrophils, hence uncoupling biological from chronological aging of these immune cells. This facilitates the accumulation of highly reactive neutrophils in malignant lesions and endows them with potent protumorigenic functions, thus promoting tumor progression. Counteracting uncoupled biological aging of circulating neutrophils by blocking the chemokine receptor CXCR2 effectively suppressed tumor growth. CONCLUSIONS: Our data uncover a self-sustaining mechanism of malignant neoplasms in fostering protumorigenic phenotypic and functional changes in circulating neutrophils. Interference with this aberrant process might therefore provide a novel, already pharmacologically targetable strategy for cancer immunotherapy.

Vitronectin stabilizes intravascular adhesion of neutrophils by coordinating ß2 integrin clustering.

The recruitment of neutrophils from the microvasculature to the site of injury or infection represents a key event in the inflammatory response. Vitronectin (VN) is a multifunctional macromolecule abundantly present in blood and extracellular matrix. The role of this glycoprotein in the extravasation process of circulating neutrophils remains elusive. Employing advanced in vivo/ex vivo imaging techniques in different mouse models as well as in vitro methods, we uncovered a previously unrecognized function of VN in the transition of dynamic to static intravascular interactions of neutrophils with microvascular endothelial cells. These distinct properties of VN require the heteromerization of this glycoprotein with plasminogen activator inhibitor-1 (PAI- 1) on the activated venular endothelium and subsequent interactions of this protein complex with the scavenger receptor low-density lipoprotein receptor-related protein-1 on intravascularly adhering neutrophils. This induces p38 mitogen-activated protein kinases-dependent intracellular signaling events which, in turn, regulates the proper clustering of the b2 integrin lymphocyte function associated antigen-1 on the surface of these immune cells. As a consequence of this molecular interplay, neutrophils become able to stabilize their adhesion to the microvascular endothelium and, subsequently, to extravasate to the perivascular tissue. Hence, endothelial-bound VN-PAI-1 heteromers stabilize intravascular adhesion of neutrophils by coordinating b2 integrin clustering on the surface of these immune cells, thereby effectively controlling neutrophil trafficking to inflamed tissue. Targeting this protein complex might be beneficial for the prevention and treatment of inflammatory pathologies.

Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90.

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms - a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition - apart from its radiosensitizing potential - obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC.

Neutrophils promote venular thrombosis by shaping the rheological environment for platelet aggregation.

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIbα-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies.

Artery-Associated Sympathetic Innervation Drives Rhythmic Vascular Inflammation of Arteries and Veins.

BACKGROUND: The incidence of acute cardiovascular complications is highly time-of-day dependent. However, the mechanisms driving rhythmicity of ischemic vascular events are unknown. Although enhanced numbers of leukocytes have been linked to an increased risk of cardiovascular complications, the role that rhythmic leukocyte adhesion plays in different vascular beds has not been studied. METHODS: We evaluated leukocyte recruitment in vivo by using real-time multichannel fluorescence intravital microscopy of a tumor necrosis factor-α-induced acute inflammation model in both murine arterial and venous macrovasculature and microvasculature. These approaches were complemented with genetic, surgical, and pharmacological ablation of sympathetic nerves or adrenergic receptors to assess their relevance for rhythmic leukocyte adhesion. In addition, we genetically targeted the key circadian clock gene Bmal1 (also known as Arntl) in a lineage-specific manner to dissect the importance of oscillations in leukocytes and components of the vessel wall in this process. RESULTS: In vivo quantitative imaging analyses of acute inflammation revealed a 24-hour rhythm in leukocyte recruitment to arteries and veins of the mouse macrovasculature and microvasculature. Unexpectedly, although in arteries leukocyte adhesion was highest in the morning, it peaked at night in veins. This phase shift was governed by a rhythmic microenvironment and a vessel type-specific oscillatory pattern in the expression of promigratory molecules. Differences in cell adhesion molecules and leukocyte adhesion were ablated when disrupting sympathetic nerves, demonstrating their critical role in this process and the importance of ß2-adrenergic receptor signaling. Loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. Rhythmicity in the intravascular reactivity of adherent leukocytes resulted in increased interactions with platelets in the morning in arteries and in veins at night with a higher predisposition to acute thrombosis at different times as a consequence. CONCLUSIONS: Together, our findings point to an important and previously unrecognized role of artery-associated sympathetic innervation in governing rhythmicity in vascular inflammation in both arteries and veins and its potential implications in the occurrence of time-of-day-dependent vessel type-specific thrombotic events.

Narciclasine exerts anti-inflammatory actions by blocking leukocyte-endothelial cell interactions and down-regulation of the endothelial TNF receptor 1.

The alkaloid narciclasine has been characterized extensively as an anticancer compound. Accumulating evidence suggests that narciclasine has anti-inflammatory potential; however, the underlying mechanism remains poorly understood. We hypothesized that narciclasine affects the activation of endothelial cells (ECs), a hallmark of inflammatory processes, which is a prerequisite for leukocyte-EC interaction. Thus, we aimed to investigate narciclasine's action on this process in vivo and to analyze the underlying mechanisms in vitro. In a murine peritonitis model, narciclasine reduced leukocyte infiltration, proinflammatory cytokine expression, and inflammation-associated abdominal pain. Moreover, narciclasine decreased rolling and blocked adhesion and transmigration of leukocytes in vivo. In cultured ECs, narciclasine inhibited the expression of cell adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin and blocked crucial steps of the NF-κB activation cascade: NF-κB promotor activity, p65 nuclear translocation, inhibitor of κB α (IκBα) phosphorylation and degradation, and IκBα kinase ß and TGF-ß-activated kinase 1 phosphorylation. Interestingly, these effects were based on the narciclasine-triggered loss of TNF receptor 1 (TNFR1). Our study highlights narciclasine as an interesting anti-inflammatory compound that effectively inhibits the interaction of leukocytes with ECs by blocking endothelial activation processes. Most importantly, we showed that the observed inhibitory action of narciclasine on TNF-triggered signaling pathways is based on the loss of TNFR1.-Stark, A., Schwenk, R., Wack, G., Zuchtriegel, G., Hatemler, M. G., Bräutigam, J., Schmidtko, A., Reichel, C. A., Bischoff, I., Fürst, R. Narciclasine exerts anti-inflammatory actions by blocking leukocyte-endothelial cell interactions and down-regulation of the endothelial TNF receptor 1.

Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells.

The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as in situ cancer vaccination by exposing tumor antigens and providing adjuvants for anti-tumor immune priming. In this regard, the mode of tumor cell death and the repertoire of released damage-associated molecular patterns (DAMPs) are crucial. However, optimal dosing and fractionation of radiotherapy remain controversial. Here, we examined the initial steps of anti-tumor immune priming by different radiation regimens (20 Gy, 4 × 2 Gy, 2 Gy, 0 Gy) with cell lines of triple-negative breast cancer in vitro and in vivo. Previously, we have shown that especially high single doses (20 Gy) induce a delayed type of primary necrosis with characteristics of mitotic catastrophe and plasma membrane disintegration. Now, we provide evidence that protein DAMPs released by these dying cells stimulate sequential recruitment of neutrophils and monocytes in vivo. Key players in this regard appear to be endothelial cells revealing a distinct state of activation upon exposure to supernatants of irradiated tumor cells as characterized by high surface expression of adhesion molecules and production of a discrete cytokine/chemokine pattern. Furthermore, irradiated tumor cell-derived protein DAMPs enforced differentiation and maturation of dendritic cells as hallmarked by upregulation of co-stimulatory molecules and improved T cell-priming. Consistently, a recurring pattern was observed: The strongest effects were detected with 20 Gy-irradiated cells. Obviously, the initial steps of radiotherapy-induced anti-tumor immune priming are preferentially triggered by high single doses - at least in models of triple-negative breast cancer.

Vitronectin promotes the vascularization of porous polyethylene biomaterials.

Rapid implant vascularization is a prerequisite for successful biomaterial engraftment. Vitronectin (VN) is a matricellular glycoprotein well known for its capability to interact with growth factors, proteases, and protease inhibitors/receptors. Since such proteins are highly relevant for angiogenic processes, we hypothesized that VN contributes to the tissue integration of biomaterials. Employing different in vivo and ex vivo microscopy techniques, engraftment of porous polyethylene (PPE) implants was analyzed in the dorsal skinfold chamber model in wild-type (WT) and VN-/- mice. Upon PPE implantation, vascularization of this biomaterial was severely compromised in animals lacking this matricellular protein. Proteome profiling revealed that VN deficiency does not cause major changes in angiogenic protein composition in the implants suggesting that VN promotes PPE vascularization via mechanisms modulating the activity of angiogenic factors rather than by directly enriching them in the implant. Consequently, surface coating with recombinant VN (embedded in Matrigel®) accelerated implant vascularization in WT mice by enhancing the maturation of a vascular network. Thus, VN contributes to the engraftment of PPE implants by promoting the vascularization of this biomaterial. Surface coating with VN might provide a promising strategy to improve the vascularization of PPE implants without affecting the host's integrity. STATEMENT OF SIGNIFICANCE: Porous polyethylene (PPE) is a biomaterial frequently used in reconstructive surgery. The proper vascularization of PPE implants is a fundamental prerequisite for its successful engraftment in host tissue. Although the overall biocompatibility of PPE is good, there are less favorable application sites for its use in tissue reconstruction mostly characterized by low blood supply. Employing advanced in vivo microscopy methods and proteomic analyses in genetically engineered mice, we here describe a previously unrecognized function of vitronectin (VN) that enables this abundantly present glycoprotein to particularly promote the vascularization of PPE biomaterial. These properties of VN specifically facilitate the formation of a dense vessel network within the implant which relies on modulating the activity of angiogenic mediators rather than on the enrichment of these factors in the implant. Consequently, surface coating with this matricellular protein effectively accelerated and intensified implant vascularization which might be beneficial for its implementation at unfavorable sites for implantation without affecting the host's integrity.

Plasminogen Activator Inhibitor-1 Promotes Neutrophil Infiltration and Tissue Injury on Ischemia-Reperfusion.

OBJECTIVE: Ischemia-reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure. APPROACH AND RESULTS: Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of ß2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor-related protein-1 and mitogen-activated protein kinase-dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1 effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis. CONCLUSIONS: Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury.

The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes.

Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5ß1, αIIbß3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of ß3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5ß1 with the RGD, but essential to re-enforce the binding of α5ß1/αIIbß3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvß3 integrin levels decrease below a critical level.

Aged neutrophils contribute to the first line of defense in the acute inflammatory response.

Under steady-state conditions, aged neutrophils are removed from the circulation in bone marrow, liver, and spleen, thereby maintaining myeloid cell homeostasis. The fate of these aged immune cells under inflammatory conditions, however, remains largely obscure. Here, we demonstrate that in the acute inflammatory response during endotoxemia, aged neutrophils cease returning to the bone marrow and instead rapidly migrate to the site of inflammation. Having arrived in inflamed tissue, aged neutrophils were found to exhibit a higher phagocytic activity as compared with the subsequently recruited nonaged neutrophils. This distinct behavior of aged neutrophils under inflammatory conditions is dependent on specific age-related changes in their molecular repertoire that enable these "experienced" immune cells to instantly translate inflammatory signals into immune responses. In particular, aged neutrophils engage Toll-like receptor-4- and p38 MAPK-dependent pathways to induce conformational changes in ß2 integrins that allow these phagocytes to effectively accomplish their mission in the front line of the inflammatory response. Hence, ageing in the circulation might represent a critical process for neutrophils that enables these immune cells to properly unfold their functional properties for host defense.

A novel HSP90 inhibitor with reduced hepatotoxicity synergizes with radiotherapy to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer.

The chaperone heat shock protein 90 (HSP90) crucially supports the maturation, folding, and stability of a variety of client proteins which are of pivotal importance for the survival and proliferation of cancer cells. Consequently, targeting of HSP90 has emerged as an attractive strategy of anti-cancer therapy, and it appears to be particularly effective in the context of molecular sensitization towards radiotherapy as has been proven in preclinical models of different cancer entities. However, so far the clinical translation has largely been hampered by suboptimal pharmacological properties and serious hepatotoxicity of first- and second-generation HSP90 inhibitors. Here, we report on NW457, a novel radicicol-derived member of the pochoxime family with reduced hepatotoxicity, how it inhibits the DNA damage response and how it synergizes with ionizing irradiation to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer in vitro and in vivo.

Platelets Guide Leukocytes to Their Sites of Extravasation.

Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells "find" their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation.

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis.

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-α-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-α/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure.

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications.

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

Spatiotemporal expression dynamics of selectins govern the sequential extravasation of neutrophils and monocytes in the acute inflammatory response.

OBJECTIVE: Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure. APPROACH AND RESULTS: Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue. CONCLUSIONS: Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases.

Effects of acute systemic administration of TiO2, ZnO, SiO2, and Ag nanoparticles on hemodynamics, hemostasis and leukocyte recruitment.

It has been suggested that engineered nanomaterials (ENM), once arrived in the circulation, may affect the cardiovascular system. The aim of this in vivo study was to screen major cardiovascular effects of acute systemic administration of a panel of five nanomaterials, TiO2 anatase (NM-101), TiO2 rutile (NM-104), ZnO (NM-110), SiO2 (NM-200) and Ag (NM-300). Mice were anesthetized and the ENM were injected at a dose of 1 mg/kg via a catheter placed in the left femoral artery. Hemodynamic parameters were determined by invasive measurement of blood pressure and non-invasive measurement of heart rate. Ten minutes after injection of the ENM, the formation of light/dye-induced thrombi was assessed in the cremasteric microcirculation by intravital microscopy. In addition, the numbers of rolling, firmly adherent and transmigrated leukocytes were recorded in postcapillary cremasteric venules over a time period of 120 min after injection of ENM by intravital microscopy. The systemic administration of a single dose of the ENM tested did not dramatically alter hemodynamic parameters or affect early steps of leukocyte recruitment. However, the presence of circulating TiO2 anatase, but not of TiO2 rutile, SiO2, ZnO or Ag nanoparticles, significantly accelerated thrombus formation in the murine microcirculation. Moreover, TiO2 anatase but not TiO2 rutile nanoparticles increased murine platelet aggregation in vitro. Taken together, only one of the five systemically administered ENM, TiO2 anatase, affected hemostasis, whereas none of the ENM tested in this screening study dramatically modulated hemodynamic parameters or early steps of leukocyte recruitment.