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BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7 x 10(-8), HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4 x 10(-8), HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4 x 10(-8), HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific association. The 17q21.31 locus was also associated with ovarian cancer risk in 8,211 BRCA2 carriers (P = 2 x 10(-4)). These loci may lead to an improved understanding of the etiology of breast and ovarian tumors in BRCA1 carriers. Based on the joint distribution of the known BRCA1 breast cancer risk-modifying loci, we estimated that the breast cancer lifetime risks for the 5% of BRCA1 carriers at lowest risk are 28%-50% compared to 81%-100% for the 5% at highest risk. Similarly, based on the known ovarian cancer risk-modifying loci, the 5% of BRCA1 carriers at lowest risk have an estimated lifetime risk of developing ovarian cancer of 28% or lower, whereas the 5% at highest risk will have a risk of 63% or higher. Such differences in risk may have important implications for risk prediction and clinical management for BRCA1 carriers.
Family studies suggest a genetic component to the etiology of chronic kidney disease (CKD) and end stage renal disease (ESRD). Previously, we identified 16 loci for eGFR in genome-wide association studies, but the associations of these single nucleotide polymorphisms (SNPs) for incident CKD or ESRD are unknown. We thus investigated the association of these loci with incident CKD in 26,308 individuals of European ancestry free of CKD at baseline drawn from eight population-based cohorts followed for a median of 7.2 years (including 2,122 incident CKD cases defined as eGFR < 60ml/min/1.73m(2) at follow-up) and with ESRD in four case-control studies in subjects of European ancestry (3,775 cases, 4,577 controls). SNPs at 11 of the 16 loci (UMOD, PRKAG2, ANXA9, DAB2, SHROOM3, DACH1, STC1, SLC34A1, ALMS1/NAT8, UBE2Q2, and GCKR) were associated with incident CKD; p-values ranged from p = 4.1e-9 in UMOD to p = 0.03 in GCKR. After adjusting for baseline eGFR, six of these loci remained significantly associated with incident CKD (UMOD, PRKAG2, ANXA9, DAB2, DACH1, and STC1). SNPs in UMOD (OR = 0.92, p = 0.04) and GCKR (OR = 0.93, p = 0.03) were nominally associated with ESRD. In summary, the majority of eGFR-related loci are either associated or show a strong trend towards association with incident CKD, but have modest associations with ESRD in individuals of European descent. Additional work is required to characterize the association of genetic determinants of CKD and ESRD at different stages of disease progression.
Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6β1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.
Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca2\(^{+}\)-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term "subthreshold IKK activation". This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33. We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo. Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure.
The interaction of CLEC-2 on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signalling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signalling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the centre of the platelet to form a single structure. Fluorescence life-time imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilised Podoplanin using direct stochastic optical reconstruction microscopy (dSTORM). These findings provide mechanistic insight by which CLEC-2 signalling promotes adhesion to Podoplanin and regulation of Podoplanin signalling thereby contributing to lymphatic vasculature development.
Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets and thus is essential for hemostasis and thrombosis. Integrin activation requires the transmission of a signal from the small cytoplasmic tails of the α or β subunit to the large extracellular domains resulting in conformational changes of the extracellular domains to enable ligand binding. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic tails. Previous studies suggested Hic-5 as a novel regulator of integrin αIIbβ3 activation and platelet aggregation in mice. To assess this in more detail, we generated Hic-5-null mice and analyzed activation and aggregation of their platelets in vitro and in vivo. Surprisingly, lack of Hic-5 had no detectable effect on platelet integrin activation and function in vitro and in vivo under all tested conditions. These results indicate that Hic-5 is dispensable for integrin αIIbβ3 activation and consequently for arterial thrombosis and hemostasis in mice.
The brain-derived neurotrophic factor BDNF plays a critical role in neuronal development and the induction of L-LTP at glutamatergic synapses in several brain regions. However, the cellular and molecular mechanisms underlying these BDNF effects have not been firmly established. Using in vitro cultures of cortical neurons from knockout mice for Pld1 and Rsk2, BDNF was observed to induce a rapid RSK2-dependent activation of PLD and to stimulate BDNF ERK1/2-CREB and mTor-S6K signalling pathways, but these effects were greatly reduced in Pld1\(^{-/-}\) neurons. Furthermore, phospho-CREB did not accumulate in the nucleus, whereas overexpression of PLD1 amplified the BDNF-dependent nuclear recruitment of phospho-ERK1/2 and phospho-CREB. This BDNF retrograde signalling was prevented in cells silenced for the scaffolding protein PEA15, a protein which complexes with PLD1, ERK1/2, and RSK2 after BDNF treatment. Finally PLD1, ERK1/2, and RSK2 partially colocalized on endosomal structures, suggesting that these proteins are part of the molecular module responsible for BDNF signalling in cortical neurons.
Objective: The assessment of response to lithium maintenance treatment in bipolar disorder (BD) is complicated by variable length of treatment, unpredictable clinical course, and often inconsistent compliance. Prospective and retrospective methods of assessment of lithium response have been proposed in the literature. In this study we report the key phenotypic measures of the "Retrospective Criteria of Long-Term Treatment Response in Research Subjects with Bipolar Disorder" scale currently used in the Consortium on Lithium Genetics (ConLiGen) study.
Materials and Methods: Twenty-nine ConLiGen sites took part in a two-stage case-vignette rating procedure to examine inter-rater agreement [Kappa (\(\kappa\))] and reliability [intra-class correlation coefficient (ICC)] of lithium response. Annotated first-round vignettes and rating guidelines were circulated to expert research clinicians for training purposes between the two stages. Further, we analyzed the distributional properties of the treatment response scores available for 1,308 patients using mixture modeling.
Results: Substantial and moderate agreement was shown across sites in the first and second sets of vignettes (\(\kappa\) = 0.66 and \(\kappa\) = 0.54, respectively), without significant improvement from training. However, definition of response using the A score as a quantitative trait and selecting cases with B criteria of 4 or less showed an improvement between the two stages (\(ICC_1 = 0.71\) and \(ICC_2 = 0.75\), respectively). Mixture modeling of score distribution indicated three subpopulations (full responders, partial responders, non responders).
Conclusions: We identified two definitions of lithium response, one dichotomous and the other continuous, with moderate to substantial inter-rater agreement and reliability. Accurate phenotypic measurement of lithium response is crucial for the ongoing ConLiGen pharmacogenomic study.
Agricultural Policies Exacerbate Honeybee Pollination Service Supply-Demand Mismatches Across Europe
(2014)
Declines in insect pollinators across Europe have raised concerns about the supply of pollination services to agriculture. Simultaneously, EU agricultural and biofuel policies have encouraged substantial growth in the cultivated area of insect pollinated crops across the continent. Using data from 41 European countries, this study demonstrates that the recommended number of honeybees required to provide crop pollination across Europe has risen 4.9 times as fast as honeybee stocks between 2005 and 2010. Consequently, honeybee stocks were insufficient to supply >90% of demands in 22 countries studied. These findings raise concerns about the capacity of many countries to cope with major losses of wild pollinators and highlight numerous critical gaps in current understanding of pollination service supplies and demands, pointing to a pressing need for further research into this issue.
Maternal cigarette smoking and its effect on neonatal lymphocyte subpopulations and replication
(2013)
Background
Significant immunomodulatory effects have been described as result of cigarette smoking in adults and pregnant women. However, the effect of cigarette smoking during pregnancy on the lymphocyte subpopulations in newborns has been discussed, controversially.
Methods
In a prospective birth cohort, we analyzed the peripheral lymphocyte subpopulations of smoking (SM) and non-smoking mothers (NSM) and their newborns and the replicative history of neonatal, mostly naive CD4 + CD45RA + T cells by measurements of T-cell-receptor-excision-circles (TRECs), relative telomere lengths (RTL) and the serum cytokine concentrations.
Results
SM had higher lymphocyte counts than NSM. Comparing SM and NSM and SM newborns with NSM newborns, no significant differences in proportions of lymphocyte subpopulations were seen. Regardless of their smoking habits, mothers had significantly lower naive T cells and higher memory and effector T cells than newborns. NSM had significantly lower percentages of CD4 + CD25++ T cells compared to their newborns, which was not significant in SM. There were no differences regarding cytokine concentrations in newborns of SM and NSM. However, NSM had significantly higher Interleukin-7 concentrations than their newborns. Regardless of smoking habits of mothers, newborns had significantly longer telomeres and higher TRECs than their mothers. Newborns of SM had significantly longer telomeres than newborns of NSM.
Conclusions
Apart from higher lymphocyte counts in SM, our results did not reveal differences between lymphocyte subpopulations of SM and NSM and their newborns, respectively. Our finding of significantly longer RTL in newborns of SM may reflect potential harm on lymphocytes, such as cytogenetic damage induced by smoking.
Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1’s mode of action differs between the two species.
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.
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.
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.
Background: Faster rates of cognitive decline are likely to result in earlier onset of cognitive impairment and dementia. d-galactose, a derivative of lactose, is used in animal studies to induce neurodegeneration. Milk is the primary source of lactose in the human diet, and its effects on cognitive decline have not been fully evaluated. Objective: Assess the association of milk intake with change in cognitive function over 20 years. Methods: A total of 13,751 participants of the Atherosclerosis Risk in Communities (ARIC) cohort completed a food frequency questionnaire and three neurocognitive evaluations from 1990 through 2013. Two single nucleotide polymorphisms (SNPs) were used to determine lactase persistence (LCT-13910 C/T for Whites and LCT-14010 G/C for Blacks). Mixed-effects models were used to study the association of milk intake with cognitive change. Multiple imputations by chained equations were used to account for attrition. Results: Milk intake greater than 1 glass/day was associated with greater decline in the global z-score over a 20-year period. The difference in decline was 0.10 (95% CI: 0.16, 0.03) z-scores, or an additional 10% decline, relative to the group reporting “almost never” consuming milk. Conclusions: Replication of these results is warranted in diverse populations with greater milk intake and higher variability of lactase persistence genotype.
Background: The COVID-19 pandemic has led to a flood of — often contradictory — evidence. HCWs had to develop strategies to locate information that supported their work. We investigated the information-seeking of different HCW groups in Germany. Methods: In December 2020, we conducted online surveys on COVID-19 information sources, strategies, assigned trustworthiness, and barriers — and in February 2021, on COVID-19 vaccination information sources. Results were analyzed descriptively; group comparisons were performed using χ\(^2\)-tests. Results: For general COVID-19-related medical information (413 participants), non-physicians most often selected official websites (57%), TV (57%), and e-mail/newsletters (46%) as preferred information sources — physicians chose official websites (63%), e-mail/newsletters (56%), and professional journals (55%). Non-physician HCWs used Facebook/YouTube more frequently. The main barriers were insufficient time and access issues. Non-physicians chose abstracts (66%), videos (45%), and webinars (40%) as preferred information strategy; physicians: overviews with algorithms (66%), abstracts (62%), webinars (48%). Information seeking on COVID-19 vaccination (2700 participants) was quite similar, however, with newspapers being more often used by non-physicians (63%) vs. physician HCWs (70%). Conclusion: Non-physician HCWs more often consulted public information sources. Employers/institutions should ensure the supply of professional, targeted COVID-19 information for different HCW groups.
Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1\(^{-/-}\) and Pld2\(^{-/-}\) mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes.
Rare variants in at least 10 genes, including BRCA1, BRCA2, PALB2, ATM, and CHEK2, are associated with increased risk of breast cancer; however, these variants, in combination with common variants identified through genome-wide association studies, explain only a fraction of the familial aggregation of the disease. To identify further susceptibility genes, we performed a two-stage whole-exome sequencing study. In the discovery stage, samples from 1528 breast cancer cases enriched for breast cancer susceptibility and 3733 geographically matched unaffected controls were sequenced. Using five different filtering and gene prioritization strategies, 198 genes were selected for further validation. These genes, and a panel of 32 known or suspected breast cancer susceptibility genes, were assessed in a validation set of 6211 cases and 6019 controls for their association with risk of breast cancer overall, and by estrogen receptor (ER) disease subtypes, using gene burden tests applied to loss-of-function and rare missense variants. Twenty genes showed nominal evidence of association (p-value < 0.05) with either overall or subtype-specific breast cancer. Our study had the statistical power to detect susceptibility genes with effect sizes similar to ATM, CHEK2, and PALB2, however, it was underpowered to identify genes in which susceptibility variants are rarer or confer smaller effect sizes. Larger sample sizes would be required in order to identify such genes.
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.
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.
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.
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.