The genetic and functional relevance of the High Temperature Requirement A1 protein (HtrA1) and of a HtrA1-single nucleotide polymorphism for Alzheimer’s Disease

Morbus Alzheimer ist ein multifaktorielles Syndrom, dem genetisch heterogene Erkrankungen zugrunde liegen. Als Ursachen für die sporadisch auftretenden Fällen wird ein komplexes Vererbungsmuster angenommen. Neben dem bestätigten Risikofaktor Apolipoprotein E Gen, wurde eine Region auf Chromosom 10q26 identifiziert, die das Erkrankungsalter von Morbus Alzheimer modifiziert. Das HtrA1 Gen liegt in diesem chromosomalen Abschnitt. Kürzlich wurde gezeigt, dass es sich bei dem funktionellen Single Nucleotide Polymorphism (SNP) rs11200638 in der HtrA1 Promotorregion um einen genetischen Risikofaktor für altersbedingte Makuladegeneration, eine Erkrankung, die pathophysiologische Gemeinsamkeiten mit Morbus Alzheimer aufweist, handelt. Ziel dieser Arbeit war es, die Assoziation zwischen dem SNP rs11200638 und dem Erkrankungsalter von Alzheimer Patienten sowie die funktionelle Bedeutung von HtrA1 für Morbus Alzheimer zu untersuchen. Die Analyse des SNP rs11200638 an 455 Alzheimer Patienten zeigte eine deutliche Modifikation des Erkrankungsbeginns. Zudem sind der Effekt des HtrA1 SNP rs11200638 und der etablierte Effekt des APOE Genotyps auf das Erkrankungsalter additiv und unabhängig voneinander. Die Ergebnisse der genetischen Untersuchungen wurden durch funktionelle Daten gestützt. Im Gehirn von Alzheimer Patienten mit HtrA1 A-Allel ist die Anzahl neuritischer Plaques und neurofibrillärer Tangles signifikant höher als bei HtrA1-GG Trägern; auch dieser Einfluss des SNP rs11200638 ist unabhängig vom APOE-Status. Zudem zeigt sich in humanem Autopsiegewebe von Alzheimer Patienten eine signifikant erhöhte HtrA1-RNA-Expression im Vergleich zu altersgleichen Kontrollen. Zellkulturexperimente lassen vermuten, dass die HtrA1-Expression über ein Spaltprodukt des Amyloid Precursor Proteins reguliert werden kann. Unsere Ergebnisse sowie bisherige Daten, die zeigen, dass HtrA1 Aß abbauen kann und mit Alzheimerschen Plaques kolokalisiert, unterstützen die Hypothese, dass HtrA1 eine wichtige Rolle in der Pathogenese des Morbus Alzheimer spielt. Möglicherweise ist HtrA1 eine weitere Aß-abbauende Protease, die sich als potentielles Ziel einer therapeutischen Intervention bei Morbus Alzheimer eignen könnte.The genetics of Alzheimer’s disease (AD) is complex. While rare mutations in only a few genes cause early onset of the disease, the risk for late onset and thus for the great majority of the population seems to be modulated by genetic variations and environmental factors. To date, the only well established susceptibility factor for late onset AD is the ε4 allele of Apolipoprotein E. In the present work it was shown that a single nucleotide polymorphism (rs11200638) in the promoter region of the gene encoding the protein quality control protease HtrA1 is associated with a remarkable modification of age-at-onset. Moreover, HtrA1 is a predictive indicator for age-at-onset, which is independent of Apolipoprotein E. The results of the genetic study are supported by an analysis of AD pathology revealing a striking increase in neuritic plaques and neurofibrillary tangles in carriers of the HtrA1-A allele compared to non-carriers. In addition, neuropathologically confirmed AD brains exhibit a significant increase of HtrA1-mRNA levels compared to age-matched controls. The mechanism of HtrA1 regulation might be controlled by the amyloid precursor protein intracellular domain (AICD). These and previous data demonstrating that HtrA1 degrades Aß and is colocalized with neuritic plaques suggest that HtrA1 plays a role in AD pathogenesis providing additional support for the notion that Aß degrading proteases might represent promising drug targets for therapeutic intervention

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

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

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 alpha IIb) 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

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

Platelet-tumor-aggregate formation <i>in vivo</i>.

<p><b>a)</b> GFP-transfected B16-D5 melanoma cells were injected intravenously into wildtype mice. After 1 hour, lung tissue was obtained for immunofluorescence analysis. Photomicrographs show mouse lung tissue stained with antibodies directed against platelet GPIIb (CD41, red) and B16-D5 (GFP, green); nuclei were stained with DAPI (blue). Bars, 40μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x objective. <b>b-d)</b> Arrest of DCF-tagged B16-D5 melanoma cells was visualized in the pulmonary vasculature by intravital confocal videofluorescence microscopy (IVM) in GPIIb^+/+ and GPIIb^-/- littermate mice immediately and after 1 hour. <b>b)</b> Number of metastatic events was quantified. Results are given as percentage of firmly adherent B16-D5 in GPIIb^-/- mice compared to its WT littermate (n = 5–6 experiments per group; *<i>P</i><0.01 acute; **<i>P</i><0.001 after 1 hour). <b>c)</b> Size of B16-aggregates was quantified. Results are given as percentage of B16-aggregate size in GPIIb^-/- mice compared to its WT littermate (n = 5–6; *<i>P</i><0.05 after 1 hour). <b>d)</b> Photomicrographs show representative IVM images obtained in GPIIb^+/+ and GPIIb^-/-. In GPIIb^+/+ mice, arrest of large multicellular aggregates is frequently observed (left). Arrest of DCF-tagged B16-D5 is visualized in precapillary vessels (right). Bars, 20μm. <b>d e-f)</b> GPIIb^+/+ (WT) or GPIIb^-/- platelets were injected into GPIIb^-/- mice just prior to administration of DCF-labeled B16-D5. IVM was performed immediately and after 1 hour. <b>e)</b> Number of metastatic events was quantified. Results are given as percentage of firmly adherent B16-D5 in GPIIb^-/- littermates receiving GPIIb^-/- platelets (n = 4; *<i>P</i><0.05). <b>f)</b> Size of B16-aggregates was quantified. Results are given as percentage of B16-aggregate size in GPIIb^-/- littermates receiving GPIIb^-/- platelets (n = 3; P = n.s.).</p

FKN positive structures in an advanced atherosclerotic lesion.

<p>Image of an advanced atherosclerotic plaque (VI) isolated by CEA from the human carotid artery. Insets magnify FKN positive structures within the plaque (upper row). Bottom row shows consecutive stainings for smooth muscle cells (SMA), endothelium and neovessels (Factor VIII), and macrophages (CD68). Upper scale bar: 200 µm, lower scale bar: 40 µm. Asterisk indicates the luminal side of the vessel.</p