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Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl\(^{−/−}\)) and platelet-specific Mgl-deficient (platMgl\(^{−/−}\)) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl\(_3\)-induced injury was markedly reduced in Mgl\(^{−/−}\) mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl\(^{−/−}\) mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl\(^{−/−}\) mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis.
T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy.
Ultra-high field cardiac MRI in large animals and humans for translational cardiovascular research
(2023)
A key step in translational cardiovascular research is the use of large animal models to better understand normal and abnormal physiology, to test drugs or interventions, or to perform studies which would be considered unethical in human subjects. Ultrahigh field magnetic resonance imaging (UHF-MRI) at 7 T field strength is becoming increasingly available for imaging of the heart and, when compared to clinically established field strengths, promises better image quality and image information content, more precise functional analysis, potentially new image contrasts, and as all in-vivo imaging techniques, a reduction of the number of animals per study because of the possibility to scan every animal repeatedly. We present here a solution to the dual use problem of whole-body UHF-MRI systems, which are typically installed in clinical environments, to both UHF-MRI in large animals and humans. Moreover, we provide evidence that in such a research infrastructure UHF-MRI, and ideally combined with a standard small-bore UHF-MRI system, can contribute to a variety of spatial scales in translational cardiovascular research: from cardiac organoids, Zebra fish and rodent hearts to large animal models such as pigs and humans. We present pilot data from serial CINE, late gadolinium enhancement, and susceptibility weighted UHF-MRI in a myocardial infarction model over eight weeks. In 14 pigs which were delivered from a breeding facility in a national SARS-CoV-2 hotspot, we found no infection in the incoming pigs. Human scanning using CINE and phase contrast flow measurements provided good image quality of the left and right ventricle. Agreement of functional analysis between CINE and phase contrast MRI was excellent. MRI in arrested hearts or excised vascular tissue for MRI-based histologic imaging, structural imaging of myofiber and vascular smooth muscle cell architecture using high-resolution diffusion tensor imaging, and UHF-MRI for monitoring free radicals as a surrogate for MRI of reactive oxygen species in studies of oxidative stress are demonstrated. We conclude that UHF-MRI has the potential to become an important precision imaging modality in translational cardiovascular research.
B cell maturation and immunoglobulin (Ig) repertoire selection are governed by expression of a functional B cell receptor (BCR). Naïve B cells co-express their BCR as IgM and IgD isotype. However, the role of the additionally expressed IgD on naïve B cells is not known. Here we assessed the impact of IgD on naïve B cell maturation and Ig repertoire selection in 8 individuals from 3 different families with heterozygous loss-of-function or loss-of expression mutations in IGHD. Although naïve B cells from these individuals expressed IgM on their surface, the IGHD variant in heterozygous state entailed a chimeric situation by allelic exclusion with almost half of the naïve B cell population lacking surface IgD expression. Flow cytometric analyses revealed a distinct phenotype of IgD-negative naïve B cells with decreased expression of CD19, CD20 and CD21 as well as lower BAFF-R and integrin-β7 expression. IgD-negative B cells were less responsive in vitro after engaging the IgM-BCR, TLR7/9 or CD40 pathway. Additionally, a selective disadvantage of IgD-negative B cells within the T2 transitional and mature naïve B cell compartment as well as reduced frequencies of IgMlo/- B cells within the mature naïve B cell compartment lacking IgD were evident. RNA-Ig-seq of bulk sorted B cell populations showed an altered selection of distinct VH segments in the IgD-negative mature naïve B cell population. We conclude that IgD expression on human naïve B cells is redundant for generation of naïve B cells in general, but further shapes the naive B cell compartment starting from T2 transitional B cells. Our observations suggest an unexpected role of IgD expression to be critical for selection of distinct Ig VH segments into the pre-immune Ig repertoire and for the survival of IgMlo/- naïve B cells known to be enriched in poly-/autoreactive B cell clones.
Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.
In tumor therapy anti-angiogenic approaches have the potential to increase the efficacy of a wide variety of subsequently or co-administered agents, possibly by improving or normalizing the defective tumor vasculature. Successful implementation of the concept of vascular normalization under anti-angiogenic therapy, however, mandates a detailed understanding of key characteristics and a respective scoring metric that defines an improved vasculature and thus a successful attempt. Here, we show that beyond commonly used parameters such as vessel patency and maturation, anti-angiogenic approaches largely benefit if the complex vascular network with its vessel interconnections is both qualitatively and quantitatively assessed. To gain such deeper insight the organization of vascular networks, we introduce a multi-parametric evaluation of high-resolution angiographic images based on light-sheet fluorescence microscopy images of tumors. We first could pinpoint key correlations between vessel length, straightness and diameter to describe the regular, functional and organized structure observed under physiological conditions. We found that vascular networks from experimental tumors diverted from those in healthy organs, demonstrating the dysfunctionality of the tumor vasculature not only on the level of the individual vessel but also in terms of inadequate organization into larger structures. These parameters proofed effective in scoring the degree of disorganization in different tumor entities, and more importantly in grading a potential reversal under treatment with therapeutic agents. The presented vascular network analysis will support vascular normalization assessment and future optimization of anti-angiogenic therapy.
Introduction
Pro-thrombotic events are one of the prevalent causes of intensive care unit (ICU) admissions among COVID-19 patients, although the signaling events in the stimulated platelets are still unclear.
Methods
We conducted a comparative analysis of platelet transcriptome data from healthy donors, ICU, and non-ICU COVID-19 patients to elucidate these mechanisms. To surpass previous analyses, we constructed models of involved networks and control cascades by integrating a global human signaling network with transcriptome data. We investigated the control of platelet hyperactivation and the specific proteins involved.
Results
Our study revealed that control of the platelet network in ICU patients is significantly higher than in non-ICU patients. Non-ICU patients require control over fewer proteins for managing platelet hyperactivity compared to ICU patients. Identification of indispensable proteins highlighted key subnetworks, that are targetable for system control in COVID-19-related platelet hyperactivity. We scrutinized FDA-approved drugs targeting indispensable proteins and identified fostamatinib as a potent candidate for preventing thrombosis in COVID-19 patients.
Discussion
Our findings shed light on how SARS-CoV-2 efficiently affects host platelets by targeting indispensable and critical proteins involved in the control of platelet activity. We evaluated several drugs for specific control of platelet hyperactivity in ICU patients suffering from platelet hyperactivation. The focus of our approach is repurposing existing drugs for optimal control over the signaling network responsible for platelet hyperactivity in COVID-19 patients. Our study offers specific pharmacological recommendations, with drug prioritization tailored to the distinct network states observed in each patient condition. Interactive networks and detailed results can be accessed at https://fostamatinib.bioinfo-wuerz.eu/.
Cyclophilin a is not acetylated at lysine-82 and lysine-125 in resting and stimulated platelets
(2022)
Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed—neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets.
Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s\(^{−1}\)). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A\(_2\) (TxA\(_2\)), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.
Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2\(^{wt}\)) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP\(^{wt}\)) and its phosphorylation-deficient mutant RKIP\(^{S153A}\), known inhibitors of the ERK1/2 signaling cascade. RKIP\(^{wt}\) and RKIP\(^{S153A}\) attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.
Candida auris is a globally emerging fungal pathogen responsible for causing nosocomial outbreaks in healthcare associated settings. It is known to cause infection in all age groups and exhibits multi-drug resistance with high potential for horizontal transmission. Because of this reason combined with limited therapeutic choices available, C. auris infection has been acknowledged as a potential risk for causing a future pandemic, and thus seeking a promising strategy for its treatment is imperative. Here, we combined evolutionary information with reverse vaccinology approach to identify novel epitopes for vaccine design that could elicit CD4+ T-cell responses against C. auris. To this end, we extensively scanned the family of proteins encoded by C. auris genome. In addition, a pathogen may acquire substitutions in epitopes over a period of time which could cause its escape from the immune response thus rendering the vaccine ineffective. To lower this possibility in our design, we eliminated all rapidly evolving genes of C. auris with positive selection. We further employed highly conserved regions of multiple C. auris strains and identified two immunogenic and antigenic T-cell epitopes that could generate the most effective immune response against C. auris. The antigenicity scores of our predicted vaccine candidates were calculated as 0.85 and 1.88 where 0.5 is the threshold for prediction of fungal antigenic sequences. Based on our results, we conclude that our vaccine candidates have the potential to be successfully employed for the treatment of C. auris infection. However, in vivo experiments are imperative to further demonstrate the efficacy of our design.
The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a ‘molecular scissor’ that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.
During ischemic stroke, infarct growth before recanalization diminishes functional outcome. Hence, adjunct treatment options to protect the ischemic penumbra before recanalization are eagerly awaited. In experimental stroke targeting two different pathways conferred protection from penumbral tissue loss: (1) enhancement of hypoxic tolerance of neurons by deletion of the calcium channel subunit Orai2 and (2) blocking of detrimental lymphocyte–platelet responses. However, until now, no preclinical stroke study has assessed the potential of combining neuroprotective with anti-thrombo-inflammatory interventions to augment therapeutic effects. We induced focal cerebral ischemia in Orai2-deficient (Orai2\(^{-/-}\)) mice by middle cerebral artery occlusion (MCAO). Animals were treated with anti-glycoprotein Ib alpha (GPIbα) Fab fragments (p0p/B Fab) blocking GPIbα–von Willebrand factor (vWF) interactions. Rat immunoglobulin G (IgG) Fab was used as the control treatment. The extent of infarct growth before recanalization was assessed at 4 h after MCAO. Moreover, infarct volumes were determined 6 h after recanalization (occlusion time: 4 h). Orai2 deficiency significantly halted cerebral infarct progression under occlusion. Inhibition of platelet GPIbα further reduced primary infarct growth in Orai2\(^{-/-}\) mice. During ischemia–reperfusion, upon recanalization, mice were likewise protected. All in all, we show that neuroprotection in Orai2\(^{-/-}\) mice can be augmented by targeting thrombo-inflammation. This supports the clinical development of combined neuroprotective/anti-platelet strategies in hyper-acute stroke.
Targeting of a conserved epitope in mouse and human GPVI differently affects receptor function
(2022)
Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6\(^{tg/tg\)). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.
LIM and SH3 protein 1 was originally identified as a structural cytoskeletal protein with scaffolding function. However, recent data suggest additional roles in cell signaling and gene expression, especially in tumor cells. These novel functions are primarily regulated by the site-specific phosphorylation of LASP1. This review will focus on specific phosphorylation-dependent interaction between LASP1 and cellular proteins that orchestrate primary tumor progression and metastasis. More specifically, we will describe the role of LASP1 in chemokine receptor, and PI3K/AKT signaling. We outline the nuclear role for LASP1 in terms of epigenetics and transcriptional regulation and modulation of oncogenic mRNA translation. Finally, newly identified roles for the cytoskeletal function of LASP1 next to its known canonical F-actin binding properties are included.
Background
Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low.
Objectives
To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA‐binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre–B‐cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc).
Methods
To avoid off‐target effects, we generated iPSCs carrying the reverse tetracycline‐responsive transactivator M2 (rtTA‐M2) in the Rosa26 locus and expressed the factors from Tet‐inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation.
Results
Overexpression of GATA1 and Pbx1 increased MK output 2‐ to 2.5‐fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro–generated platelets were functional in spreading on fibrinogen or collagen‐related peptide.
Conclusion
We demonstrate that the use of rtTA‐M2 transgenic iPSCs transduced with Tet‐inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.
Simultaneous measurements of 3D wall shear stress and pulse wave velocity in the murine aortic arch
(2021)
Purpose
Wall shear stress (WSS) and pulse wave velocity (PWV) are important parameters to characterize blood flow in the vessel wall. Their quantification with flow-sensitive phase-contrast (PC) cardiovascular magnetic resonance (CMR), however, is time-consuming. Furthermore, the measurement of WSS requires high spatial resolution, whereas high temporal resolution is necessary for PWV measurements. For these reasons, PWV and WSS are challenging to measure in one CMR session, making it difficult to directly compare these parameters. By using a retrospective approach with a flexible reconstruction framework, we here aimed to simultaneously assess both PWV and WSS in the murine aortic arch from the same 4D flow measurement.
Methods
Flow was measured in the aortic arch of 18-week-old wildtype (n = 5) and ApoE\(^{−/−}\) mice (n = 5) with a self-navigated radial 4D-PC-CMR sequence. Retrospective data analysis was used to reconstruct the same dataset either at low spatial and high temporal resolution (PWV analysis) or high spatial and low temporal resolution (WSS analysis). To assess WSS, the aortic lumen was labeled by semi-automatically segmenting the reconstruction with high spatial resolution. WSS was determined from the spatial velocity gradients at the lumen surface. For calculation of the PWV, segmentation data was interpolated along the temporal dimension. Subsequently, PWV was quantified from the through-plane flow data using the multiple-points transit-time method. Reconstructions with varying frame rates and spatial resolutions were performed to investigate the influence of spatiotemporal resolution on the PWV and WSS quantification.
Results
4D flow measurements were conducted in an acquisition time of only 35 min. Increased peak flow and peak WSS values and lower errors in PWV estimation were observed in the reconstructions with high temporal resolution. Aortic PWV was significantly increased in ApoE\(^{−/−}\) mice compared to the control group (1.7 ± 0.2 versus 2.6 ± 0.2 m/s, p < 0.001). Mean WSS magnitude values averaged over the aortic arch were (1.17 ± 0.07) N/m\(^2\) in wildtype mice and (1.27 ± 0.10) N/m\(^2\) in ApoE\(^{−/−}\) mice.
Conclusion
The post processing algorithm using the flexible reconstruction framework developed in this study permitted quantification of global PWV and 3D-WSS in a single acquisition. The possibility to assess both parameters in only 35 min will markedly improve the analyses and information content of in vivo measurements.
In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.
Effects of cocoa genotypes on coat color, platelets and coagulation parameters in French Bulldogs
(2021)
A nonsense variant in HPS3, c.2420G>A or p.Trp807*, was recently discovered as the cause for a brown coat color termed cocoa in French Bulldogs. Here, we studied the genotype–phenotype correlation regarding coat color in HPS3 mutant dogs that carried various combinations of mutant alleles at other coat color genes. Different combinations of HPS3, MLPH and TYRP1 genotypes resulted in subtly different shades of brown coat colors. As HPS3 variants in humans cause the Hermansky–Pudlak syndrome type 3, which in addition to oculocutaneous albinism is characterized by a storage pool deficiency leading to bleeding tendency, we also investigated the phenotypic consequences of the HPS3 variant in French Bulldogs on hematological parameters. HPS3 mutant dogs had a significantly lowered platelet dense granules abundance. However, no increased bleeding tendencies in daily routine were reported by dog owners. We therefore conclude that in dogs, the phenotypic effect of the HPS3 variant is largely restricted to pigmentation. While an effect on platelet morphology is evident, we did not obtain any indications for major health problems associated with the cocoa coat color in French Bulldogs. Further studies will be necessary to definitely rule out very subtle effects on visual acuity or a clinically relevant bleeding disorder.
Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, successful recanalization of occluded vessels is the primary therapeutic aim, but even if it is achieved, not all patients benefit. Although blockade of platelet aggregation did not prevent infarct progression, cerebral thrombosis as cause of secondary infarct growth has remained a matter of debate. As cerebral thrombi are frequently observed after experimental stroke, a thrombus-induced impairment of the brain microcirculation is considered to contribute to tissue damage. Here, we combine the model of transient middle cerebral artery occlusion (tMCAO) with light sheet fluorescence microscopy and immunohistochemistry of brain slices to investigate the kinetics of thrombus formation and infarct progression. Our data reveal that tissue damage already peaks after 8 h of reperfusion following 60 min MCAO, while cerebral thrombi are only observed at later time points. Thus, cerebral thrombosis is not causative for secondary infarct growth during ischemic stroke.
Background
Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII.
Objective
The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model.
Methods
A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models.
Results
These hF12\(^{KI}\) mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12\(^{KI}\) mice in an arterial thrombosis model without affecting bleeding times.
Conclusion
These data establish the newly generated hF12\(^{KI}\) mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.
Background
In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood.
Methods
To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1\(^{−/−}\) mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion.
Results
We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion.
Conclusions
Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization.
Growth, ageing and atherosclerotic plaque development alter the biomechanical forces acting on the vessel wall. However, monitoring the detailed local changes in wall shear stress (WSS) at distinct sites of the murine aortic arch over time has been challenging. Here, we studied the temporal and spatial changes in flow, WSS, oscillatory shear index (OSI) and elastic properties of healthy wildtype (WT, n = 5) and atherosclerotic apolipoprotein E-deficient (Apoe\(^{−/−}\), n = 6) mice during ageing and atherosclerosis using high-resolution 4D flow magnetic resonance imaging (MRI). Spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated, allowing the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and local correlations between WSS, pulse wave velocity (PWV), plaque and vessel wall characteristics. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe\(^{−/−}\) mice, and we identified the circumferential WSS as potential marker of plaque size and composition in advanced atherosclerosis and the radial strain as a potential marker for vascular elasticity. Two-dimensional (2D) projection maps of WSS and OSI, including statistical analysis provide a powerful tool to monitor local aortic hemodynamics during ageing and atherosclerosis. The correlation of spatially resolved hemodynamics and plaque characteristics could significantly improve our understanding of the impact of hemodynamics on atherosclerosis, which may be key to understand plaque progression towards vulnerability.
Platelet activation and thrombus formation have been implicated to be detrimental for intraportal pancreatic islet transplants. The platelet‐specific collagen receptor glycoprotein VI (GPVI) plays a key role in thrombosis through cellular activation and the subsequent release of secondary mediators. In aggregometry and in a microfluidic dynamic assay system modeling flow in the portal vein, pancreatic islets promoted platelet aggregation and triggered thrombus formation, respectively. While platelet GPVI deficiency did not affect the initiation of these events, it was found to destabilize platelet aggregates and thrombi in this process. Interestingly, while no major difference was detected in early thrombus formation after intraportal islet transplantation, genetic GPVI deficiency or acute anti‐GPVI treatment led to an inferior graft survival and function in both syngeneic mouse islet transplantation and xenogeneic human islet transplantation models. These results demonstrate that platelet GPVI signaling is indispensable in stable thrombus formation induced by pancreatic islets. GPVI deficiency resulted in thrombus destabilization and inferior islet engraftment indicating that thrombus formation is necessary for a successful intraportal islet transplantation in which platelets are active modulators.
Atherosclerosis is an inflammatory disease of large and medium-sized arteries, characterized by the growth of atherosclerotic lesions (plaques). These plaques often develop at inner curvatures of arteries, branchpoints, and bifurcations, where the endothelial wall shear stress is low and oscillatory. In conjunction with other processes such as lipid deposition, biomechanical factors lead to local vascular inflammation and plaque growth. There is also evidence that low and oscillatory shear stress contribute to arterial remodeling, entailing a loss in arterial elasticity and, therefore, an increased pulse-wave velocity. Although altered shear stress profiles, elasticity and inflammation are closely intertwined and critical for plaque growth, preclinical and clinical investigations for atherosclerosis mostly focus on the investigation of one of these parameters only due to the experimental limitations. However, cardiovascular magnetic resonance imaging (MRI) has been demonstrated to be a potent tool which can be used to provide insights into a large range of biological parameters in one experimental session. It enables the evaluation of the dynamic process of atherosclerotic lesion formation without the need for harmful radiation. Flow-sensitive MRI provides the assessment of hemodynamic parameters such as wall shear stress and pulse wave velocity which may replace invasive and radiation-based techniques for imaging of the vascular
function and the characterization of early plaque development. In combination with inflammation imaging, the analyses and correlations of these parameters could not only significantly advance basic preclinical investigations of atherosclerotic lesion formation and progression, but also the diagnostic clinical evaluation for early identification of high-risk plaques, which are prone to rupture. In this review, we summarize the key applications of magnetic resonance imaging for the evaluation of plaque characteristics through flow sensitive and morphological measurements. The simultaneous measurements of functional and structural parameters will further preclinical research on atherosclerosis and has the potential to fundamentally improve the detection of inflammation and vulnerable plaques in patients.
Background
Coronavirus disease 2019 (COVID-19) associated coagulopathy (CAC) leads to thromboembolic events in a high number of critically ill COVID-19 patients. However, specific diagnostic or therapeutic algorithms for CAC have not been established. In the current study, we analyzed coagulation abnormalities with point-of-care testing (POCT) and their relation to hemostatic complications in patients suffering from COVID-19 induced Acute Respiratory Distress Syndrome (ARDS). Our hypothesis was that specific diagnostic patterns can be identified in patients with COVID-19 induced ARDS at risk of thromboembolic complications utilizing POCT.
Methods
This is a single-center, retrospective observational study. Longitudinal data from 247 rotational thromboelastometries (Rotem®) and 165 impedance aggregometries (Multiplate®) were analysed in 18 patients consecutively admitted to the ICU with a COVID-19 induced ARDS between March 12th to June 30th, 2020.
Results
Median age was 61 years (IQR: 51–69). Median PaO2/FiO2 on admission was 122 mmHg (IQR: 87–189), indicating moderate to severe ARDS. Any form of hemostatic complication occurred in 78 % of the patients with deep vein/arm thrombosis in 39 %, pulmonary embolism in 22 %, and major bleeding in 17 %. In Rotem® elevated A10 and maximum clot firmness (MCF) indicated higher clot strength. The delta between EXTEM A10 minus FIBTEM A10 (ΔA10) > 30 mm, depicting the sole platelet-part of clot firmness, was associated with a higher risk of thromboembolic events (OD: 3.7; 95 %CI 1.3–10.3; p = 0.02). Multiplate® aggregometry showed hypoactive platelet function. There was no correlation between single Rotem® and Multiplate® parameters at intensive care unit (ICU) admission and thromboembolic or bleeding complications.
Conclusions
Rotem® and Multiplate® results indicate hypercoagulability and hypoactive platelet dysfunction in COVID-19 induced ARDS but were all in all poorly related to hemostatic complications..
Atherosclerotic lesions are populated by cells of the innate and adaptive immune system, including CD8\(^+\) T cells. The CD8\(^+\) T cell infiltrate has recently been characterized in mouse and human atherosclerosis and revealed activated, cytotoxic, and possibly dysfunctional and exhausted cell phenotypes. In mouse models of atherosclerosis, antibody-mediated depletion of CD8\(^+\) T cells ameliorates atherosclerosis. CD8\(^+\) T cells control monopoiesis and macrophage accumulation in early atherosclerosis. In addition, CD8\(^+\) T cells exert cytotoxic functions in atherosclerotic plaques and contribute to macrophage cell death and necrotic core formation. CD8\(^+\) T cell activation may be antigen-specific, and epitopes of atherosclerosis-relevant antigens may be targets of CD8\(^+\) T cells and their cytotoxic activity. CD8\(^+\) T cell functions are tightly controlled by costimulatory and coinhibitory immune checkpoints. Subsets of regulatory CD25\(^+\)CD8\(^+\) T cells with immunosuppressive functions can inhibit atherosclerosis. Importantly, local cytotoxic CD8\(^+\) T cell responses may trigger endothelial damage and plaque erosion in acute coronary syndromes. Understanding the complex role of CD8\(^+\) T cells in atherosclerosis may pave the way for defining novel treatment approaches in atherosclerosis. In this review article, we discuss these aspects, highlighting the emerging and critical role of CD8\(^+\) T cells in atherosclerosis.
Comparison of the central human and mouse platelet signaling cascade by systems biological analysis
(2020)
Background
Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC).
Results
The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.
Conclusions
Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
The CXCL12-CXCR4 axis plays a vital role in many steps of breast cancer metastasis, but the molecular mechanisms have not been fully elucidated. We previously reported that activation of CXCR4 by CXCL12 promotes the nuclear localization of LASP1 (LIM and SH3 protein 1). The nuclear LASP1 then interacts with Snail1 in triple-negative breast cancer (TNBC) cell lines. In this study, we report that the nuclear accumulation and retention of Snail1 was dependent on an increase in nuclear LASP1 levels driven by active CXCR4. The CXCR4-LASP1 axis may directly regulate the stabilization of nuclear Snail1, by upregulating nuclear levels of pS473-Akt, pS9-GSK-3β, A20, and LSD1. Furthermore, the activation of CXCR4 induced association of LASP1 with Snail1, A20, GSK-3β, and LSD1 endogenously. Thus, nuclear LASP1 may also regulate protein-protein interactions that facilitate the stability of Snail1. Genetic ablation of LASP1 resulted in the mislocalization of nuclear Snail1, loss of the ability of TNBC cells to invade Matrigel and a dysregulated expression of both epithelial and mesenchymal markers, including an increased expression of ALDH1A1, a marker for epithelial breast cancer stem-like cells. Our findings reveal a novel role for the CXCR4-LASP1 axis in facilitating the stability of nuclear localized Snail1.
Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR‐ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR‐ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1‐mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell‐mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.
The serine/threonine protein kinase AKT1 is a downstream target of the chemokine receptor 4 (CXCR4), and both proteins play a central role in the modulation of diverse cellular processes, including proliferation and cell survival. While in chronic myeloid leukemia (CML) the CXCR4 is downregulated, thereby promoting the mobilization of progenitor cells into blood, the receptor is highly expressed in breast cancer cells, favoring the migratory capacity of these cells. Recently, the LIM and SH3 domain protein 1 (LASP1) has been described as a novel CXCR4 binding partner and as a promoter of the PI3K/AKT pathway. In this study, we uncovered a direct binding of LASP1, phosphorylated at S146, to both CXCR4 and AKT1, as shown by immunoprecipitation assays, pull-down experiments, and immunohistochemistry data. In contrast, phosphorylation of LASP1 at Y171 abrogated these interactions, suggesting that both LASP1 phospho-forms interact. Finally, findings demonstrating different phosphorylation patterns of LASP1 in breast cancer and chronic myeloid leukemia may have implications for CXCR4 function and tyrosine kinase inhibitor treatment.
Objective
Bridging the gap between experimental stroke and patients by ischemic blood probing during the hyperacute stage of vascular occlusion is crucial to assess the role of inflammation in human stroke and for the development of adjunct treatments beyond recanalization.
Methods
We prospectively observed 151 consecutive ischemic stroke patients with embolic large vessel occlusion of the anterior circulation who underwent mechanical thrombectomy. In all these patients, we attempted microcatheter aspiration of 3 different arterial blood samples: (1) within the core of the occluded vascular compartment and controlled by (2) carotid and (3) femoral samples obtained under physiological flow conditions. Subsequent laboratory analyses comprised leukocyte counting and differentiation, platelet counting, and the quantification of 13 proinflammatory human chemokines/cytokines.
Results
Forty patients meeting all clinical, imaging, interventional, and laboratory inclusion criteria could be analyzed, showing that the total number of leukocytes significantly increased under the occlusion condition. This increase was predominantly driven by neutrophils. Significant increases were also apparent for lymphocytes and monocytes, accompanied by locally elevated plasma levels of the T‐cell chemoattractant CXCL‐11. Finally, we found evidence that short‐term clinical outcome (National Institute of Health Stroke Scale at 72 hours) was negatively associated with neutrophil accumulation.
Interpretation
We provide the first direct human evidence that neutrophils, lymphocytes, and monocytes, accompanied by specific chemokine upregulation, accumulate in the ischemic vasculature during hyperacute stroke and may affect outcome. These findings strongly support experimental evidence that immune cells contribute to acute ischemic brain damage and indicate that ischemic inflammation initiates already during vascular occlusion. Ann Neurol 2020;87:466–479
Endothelial–platelet interactions in influenza‐induced pneumonia: A potential therapeutic target
(2020)
Every year, influenza viruses spread around the world, infecting the respiratory systems of countless humans and animals, causing illness and even death. Severe influenza infection is associated with pulmonary epithelial damage and endothelial dysfunction leading to acute lung injury (ALI). There is evidence that an aggressive cytokine storm and cell damage in lung capillaries as well as endothelial/platelet interactions contribute to vascular leakage, pro‐thrombotic milieu and infiltration of immune effector cells. To date, treatments for ALI caused by influenza are limited to antiviral drugs, active ventilation or further symptomatic treatments. In this review, we summarize the mechanisms of influenza‐mediated pathogenesis, permissive animal models and histopathological changes of lung tissue in both mice and men and compare it with histological and electron microscopic data from our own group. We highlight the molecular and cellular interactions between pulmonary endothelium and platelets in homeostasis and influenza‐induced pathogenesis. Finally, we discuss novel therapeutic targets on platelets/endothelial interaction to reduce or resolve ALI.
Defects of platelet intracellular signaling can result in severe platelet dysfunction. Several mutations in each of the linked genes FERMT3 and RASGRP2 on chromosome 11 causing a Glanzmann‐like bleeding phenotype have been identified so far. We report on novel variants in two unrelated pediatric patients with severe bleeding diathesis—one with leukocyte adhesion deficiency type III due to a homozygous frameshift in FERMT3 and the other with homozygous variants in both, FERMT3 and RASGRP2 . We focus on the challenging genetic and functional variant assessment and aim to accentuate the risk of obtaining misleading results due to the phenomenon of genetic linkage.
The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane‐associated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASP‐1, an actin‐binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP‐1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP‐1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton‐bound protein. We identify CD2AP as a novel LASP‐1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin‐angiotensin‐aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP‐1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.
Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{−/−}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.
Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed “reperfusion injury.” Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury.
Protein ubiquitination impacts virtually every biochemical pathway in eukaryotic cells. The fate of a ubiquitinated protein is largely dictated by the type of ubiquitin modification with which it is decorated, including a large variety of polymeric chains. As a result, there have been intense efforts over the last two decades to dissect the molecular details underlying the synthesis of ubiquitin chains by ubiquitin-conjugating (E2) enzymes and ubiquitin ligases (E3s). In this review, we highlight these advances. We discuss the evidence in support of the alternative models of transferring one ubiquitin at a time to a growing substrate-linked chain (sequential addition model) versus transferring a pre-assembled ubiquitin chain (en bloc model) to a substrate. Against this backdrop, we outline emerging principles of chain assembly: multisite interactions, distinct mechanisms of chain initiation and elongation, optimal positioning of ubiquitin molecules that are ultimately conjugated to each other, and substrate-assisted catalysis. Understanding the enzymatic logic of ubiquitin chain assembly has important biomedical implications, as the misregulation of many E2s and E3s and associated perturbations in ubiquitin chain formation contribute to human disease. The resurgent interest in bifunctional small molecules targeting pathogenic proteins to specific E3s for polyubiquitination and subsequent degradation provides an additional incentive to define the mechanisms responsible for efficient and specific chain synthesis and harness them for therapeutic benefit.
The receptor EMMPRIN is involved in the development and progression of cardiovascular diseases and in the pathogenesis of myocardial infarction. There are several binding partners of EMMPRIN mediating the effects of EMMPRIN in cardiovascular diseases. EMMPRIN interaction with most binding partners leads to disease progression by mediating cytokine or chemokine release, the activation of platelets and monocytes, as well as the formation of monocyte-platelet aggregates (MPAs). EMMPRIN is also involved in atherosclerosis by mediating the infiltration of pro-inflammatory cells. There is also evidence that EMMPRIN controls energy metabolism of cells and that EMMPRIN binding partners modulate intracellular glycosylation and trafficking of EMMPRIN towards the cell membrane. In this review, we systematically discuss these multifaceted roles of EMMPRIN and its interaction partners, such as Cyclophilins, in cardiovascular disease.
Norrin is a secreted signaling molecule activating the Wnt/β-catenin pathway. Since Norrin protects retinal neurons from experimental acute injury, we were interested to learn if Norrin attenuates chronic damage of retinal ganglion cells (RGC) and their axons in a mouse model of glaucoma. Transgenic mice overexpressing Norrin in the retina (Pax6-Norrin) were generated and crossed with DBA/2J mice with hereditary glaucoma and optic nerve axonal degeneration. One-year old DBA/2J/Pax6-Norrin animals had significantly more surviving optic nerve axons than their DBA/2J littermates. The protective effect correlated with an increase in insulin-like growth factor (IGF)-1 mRNA and an enhanced Akt phosphorylation in DBA/2J/Pax6-Norrin mice. Both mouse strains developed an increase in intraocular pressure during the second half of the first year and marked degenerative changes in chamber angle, ciliary body and iris structure. The degenerations were slightly attenuated in the chamber angle of DBA/2J/Pax6-Norrin mice, which showed a β-catenin increase in the trabecular meshwork. We conclude that high levels of Norrin and the subsequent constitutive activation of Wnt/β-catenin signaling in RGC protect from glaucomatous axonal damage via IGF-1 causing increased activity of PI3K-Akt signaling. Our results identify components of a protective signaling network preventing degeneration of optic nerve axons in glaucoma.
Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.
In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.
Background:
Ischemic stroke causes a strong inflammatory response that includes T cells, monocytes/macrophages, and neutrophils. Interaction of these immune cells with platelets and endothelial cells facilitates microvascular dysfunction and leads to secondary infarct growth. We recently showed that blocking of platelet glycoprotein (GP) receptor Ib improves stroke outcome without increasing the risk of intracerebral hemorrhage. Until now, it has been unclear whether GPIb only mediates thrombus formation or also contributes to the pathophysiology of local inflammation.
Methods:
Focal cerebral ischemia was induced in C57BL/6 mice by a 60-min transient middle cerebral artery occlusion (tMCAO). Animals were treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab). Rat immunoglobulin G (IgG) Fab was used as control treatment. Stroke outcome, including infarct size and functional deficits as well as the local inflammatory response, was assessed on day 1 after tMCAO.
Results:
Blocking of GPIb reduced stroke size and improved functional outcome on day 1 after tMCAO without increasing the risk of intracerebral hemorrhage. As expected, disruption of GPIb-mediated pathways in platelets significantly reduced thrombus burden in the cerebral microvasculature. In addition, inhibition of GPIb limited the local inflammatory response in the ischemic brain as indicated by lower numbers of infiltrating T cells and macrophages and lower expression levels of inflammatory cytokines compared with rat IgG Fab-treated controls.
Conclusion:
In acute ischemic stroke, thrombus formation and inflammation are closely intertwined (“thrombo-inflammation”). Blocking of platelet GPIb can ameliorate thrombo-inflammation.
Atherosclerosis is the main underlying cause for cardiovascular events such as myocardial infarction and stroke and its development might be influenced by immune cells. Dendritic cells (DCs) bridge innate and adaptive immune responses by presenting antigens to T cells and releasing a variety of cytokines. Several subsets of DCs can be discriminated that engage specific transcriptional pathways for their development. Basic leucine zipper transcription factor ATF-like 3 (Batf3) is required for the development of classical CD8α\(^{+}\) and CD103\(^{+}\) DCs. By crossing mice deficient in Batf3 with atherosclerosis-prone low density lipoprotein receptor (Ldlr\(^{−/-}\))-deficient mice we here aimed to further address the contribution of Batf3-dependent CD8α\(^{+}\) and CD103\(^{+}\) antigen-presenting cells to atherosclerosis. We demonstrate that deficiency in Batf3 entailed mild effects on the immune response in the spleen but did not alter atherosclerotic lesion formation in the aorta or aortic root, nor affected plaque phenotype in low density lipoprotein receptor-deficient mice fed a high fat diet. We thus provide evidence that Batf3-dependent antigen-presenting cells do not have a prominent role in atherosclerosis.
Limited comprehension of aneurysm pathology has led to inconclusive results from clinical trials. miRNAs are key regulators of post-translational gene modification and are useful tools in elucidating key features of aneurysm pathogenesis in distinct entities of abdominal and popliteal aneurysms. Here, surgically harvested specimens from 19 abdominal aortic aneurysm (AAA) and 8 popliteal artery aneurysm (PAA) patients were analyzed for miRNA expression and histologically classified regarding extracellular matrix (ECM) remodeling and inflammation. DIANA-based computational target prediction and pathway enrichment analysis verified our results, as well as previous ones. miRNA-362, -19b-1, -194, -769, -21 and -550 were significantly down-regulated in AAA samples depending on degree of inflammation. Similar or inverse regulation was found for miR-769, 19b-1 and miR-550, -21, whereas miR-194 and -362 were unaltered in PAA. In situ hybridization verified higher expression of miR-550 and -21 in PAA compared to AAA and computational analysis for target genes and pathway enrichment affirmed signal transduction, cell-cell-interaction and cell degradation pathways, in line with previous results. Despite the vague role of miRNAs for potential diagnostic and treatment purposes, the number of candidates from tissue signature studies is increasing. Tissue morphology influences subsequent research, yet comparison of distinct entities of aneurysm disease can unravel core pathways.
Background
Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation.
Objective
To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke. Methods: 5-HT transporter knockout mice (5Htt\(^{-/-}\)) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke.
Results
In 5Htt\(^{-/-}\) platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca\(^{2+}\) entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt\(^{-/-}\) platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt\(^{-/-}\) mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt\(^{-/-}\) mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice.
Conclusion
Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization.
Lung cancer is currently the leading cause of cancer related mortality due to late diagnosis and limited treatment intervention. Non-coding RNAs are not translated into proteins and have emerged as fundamental regulators of gene expression. Recent studies reported that microRNAs and long non-coding RNAs are involved in lung cancer development and progression. Moreover, they appear as new promising non-invasive biomarkers for early lung cancer diagnosis. Here, we highlight their potential as biomarker in lung cancer and present how bioinformatics can contribute to the development of non-invasive diagnostic tools. For this, we discuss several bioinformatics algorithms and software tools for a comprehensive understanding and functional characterization of microRNAs and long non-coding RNAs.
The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines.
By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines.
In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown.
Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression.
The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies.
The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit+/CXCR-4+ cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases.
Atherosclerotic lesions that critically narrow the artery can necessitate an angioplasty and stent implantation. Long-term therapeutic effects, however, are limited by excessive arterial remodeling. We here employed a miniaturized nitinol-stent coated with star-shaped polyethylenglycole (star-PEG), and evaluated its bio-functionalization with RGD and CXCL1 for improving in-stent stenosis after implantation into carotid arteries of mice. Nitinol foils or stents (bare metal) were coated with star-PEG, and bio-functionalized with RGD, or RGD/CXCL1. Cell adhesion to star-PEG-coated nitinol foils was unaltered or reduced, whereas bio-functionalization with RGD but foremost RGD/CXCL1 increased adhesion of early angiogenic outgrowth cells (EOCs) and endothelial cells but not smooth muscle cells when compared with bare metal foils. Stimulation of cells with RGD/CXCL1 furthermore increased the proliferation of EOCs. In vivo, bio-functionalization with RGD/CXCL1 significantly reduced neointima formation and thrombus formation, and increased re-endothelialization in apoE\(^{-/-}\) carotid arteries compared with bare-metal nitinol stents, star-PEG-coated stents, and stents bio-functionalized with RGD only. Bio-functionalization of star-PEG-coated nitinol-stents with RGD/CXCL1 reduced in-stent neointima formation. By supporting the adhesion and proliferation of endothelial progenitor cells, RGD/CXCL1 coating of stents may help to accelerate endothelial repair after stent implantation, and thus may harbor the potential to limit the complication of in-stent restenosis in clinical approaches.