Platelet GPIIb supports initial pulmonary retention but inhibits subsequent proliferation of melanoma cells during hematogenic metastasis.

Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin αIIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb-/-) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb-/- mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb-/- mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb-/- mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases.

Critical role of platelet glycoprotein ibα in arterial remodeling.

OBJECTIVE: Arteriogenesis is strongly dependent on the recruitment of leukocytes, especially monocytes, into the perivascular space of growing collateral vessels. On the basis of previous findings that platelets are central players in inflammatory processes and mediate the recruitment of leukocytes, the aim of this study was to assess the role of platelets in a model of arterial remodeling. APPROACH AND RESULTS: C57Bl6 wild-type mice, IL4-R/Iba mice lacking the extracellular domain of the glycoprotein Ibα (GPIbα) receptor, and mice treated with antibodies to block GPIbα or deplete circulating platelets were studied in peripheral arteriogenesis. Using a novel model of intravital 2-photon and epifluorescence imaging, we visualized and quantified the interaction of platelets with leukocytes and the vascular endothelium in vivo. We found that transient platelet adhesion to the endothelium of collateral vessels was a major event during arteriogenesis and depended on GPIbα. Furthermore, leukocyte recruitment was obviously affected in animals with defective platelet GPIbα function. In IL4-R/Iba mice, transient and firm leukocyte adhesion to the endothelium of collateral vessels, as well as leukocyte accumulation in the perivascular space, were significantly reduced. Furthermore, we detected platelet-leukocyte aggregates within the circulation, which were significantly reduced in IL4-R/Iba animals. Finally, platelet depletion and loss of GPIbα function resulted in poor reperfusion recovery as determined by laser Doppler imaging. CONCLUSIONS: Thus, GPIbα-mediated interactions between platelets and endothelial cells, as well as leukocytes, support innate immune cell recruitment and promote arteriogenesis-establishing platelets as critical players in this process.

Capillary and arteriolar pericytes attract innate leukocytes exiting through venules and 'instruct' them with pattern-recognition and motility programs.

Coordinated navigation within tissues is essential for cells of the innate immune system to reach the sites of inflammatory processes, but the signals involved are incompletely understood. Here we demonstrate that NG2(+) pericytes controlled the pattern and efficacy of the interstitial migration of leukocytes in vivo. In response to inflammatory mediators, pericytes upregulated expression of the adhesion molecule ICAM-1 and released the chemoattractant MIF. Arteriolar and capillary pericytes attracted and interacted with myeloid leukocytes after extravasating from postcapillary venules, 'instructing' them with pattern-recognition and motility programs. Inhibition of MIF neutralized the migratory cues provided to myeloid leukocytes by NG2(+) pericytes. Hence, our results identify a previously unknown role for NG2(+) pericytes as an active component of innate immune responses, which supports the immunosurveillance and effector function of extravasated neutrophils and macrophages.

A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis.

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.

Fractalkine is expressed in early and advanced atherosclerotic lesions and supports monocyte recruitment via CX3CR1.

Fractalkine (CX3CL1, FKN) is expressed in the inflamed vascular wall and absence of FKN reduces atherogenesis. Whether FKN is expressed throughout all stages of atherosclerotic disease and whether it directly contributes to monocyte recruitment to atherosclerotic lesions is not known. We collected human atherosclerotic plaque material and blood samples from patients with carotid artery disease undergoing endarterectomy. Plaques were analyzed by immunohistochemistry and qPCR. We found that FKN is expressed at all stages of atherosclerotic lesion formation, and that the number of FKN-expressing cells positively correlates with the number of CX3CR1-positive cells in human carotid artery plaques. In the circulation, soluble FKN levels are significantly elevated in the presence of high-grade (sub-occlusive) stenosis. To determine the role of the FKN-CX3CR1 axis for monocyte adhesion in vivo we then performed intravital videofluorescence microscopy of the carotid artery in ApoE(-/-) mice. Notably, FKN-CX3CR1 interactions are critical for recruitment of circulating monocytes to the injured atherosclerotic vascular wall. Thus, this chemokine dyad could represent an attractive target for anti-atherosclerotic strategies.

Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates.

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.

Modulation of gene expression and cytoskeletal dynamics by the amyloid precursor protein intracellular domain (AICD).

Amyloidogenic processing of the amyloid precursor protein (APP) results in the generation of beta-amyloid, the main constituent of Alzheimer plaques, and the APP intracellular domain (AICD). Recently, it has been demonstrated that AICD has transactivation potential; however, the targets of AICD-dependent gene regulation and hence the physiological role of AICD remain largely unknown. We analyzed transcriptome changes during AICD-dependent gene regulation by using a human neural cell culture system inducible for expression of AICD, its coactivator FE65, or the combination of both. Induction of AICD was associated with increased expression of genes with known function in the organization and dynamics of the actin cytoskeleton, including alpha2-Actin and Transgelin (SM22). AICD target genes were also found to be differentially regulated in the frontal cortex of Alzheimer's disease patients compared with controls as well as in AICD/FE65 transiently transfected murine cortical neurons. Confocal image analysis of neural cells and cortical neurons expressing both AICD and FE65 confirmed pronounced changes in the organization of the actin cytoskeleton, including the destabilization of actin fibers and clumping of actin at the sites of cellular outgrowth. Our data point to a role of AICD in developmental and injury-related cytoskeletal dynamics in the nervous system.