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Cameron, S. J.

Publications and source records attributed to Cameron, S. J..

8 recordsLinked to original sources

Fitness costs of antibiotic resistance mutations in rich media do not reliably predict those in infection-like environments

The acquisition of antibiotic resistance is typically associated with a fitness cost, reducing the competitive ability of cells in the absence of antibiotics. Our ability to predict these costs informs our ability to predict how likely it is that resistance will subside in the absence of treatment. Fitness costs are typically measured in laboratory cultures using a nutritionally rich medium for growth. Here, we tested the impact of infection-like environments on the prevalence and magnitude of fitness costs associated with antibiotic resistance mutations in the opportunistic pathogen Pseudomonas aeruginosa. We assessed two different clinically relevant infection niches, the lung and urinary tract, through the use of infection-niche mimicking media that models the chemical compositions of these niches. The fitness costs of resistance mutations in rich media did not reliably predict those in infection-like environments, on average costs were increased in the lung-mimicking environment and costly mutations were less prevalent in the urinary tract-mimicking environment. Synthetic human urine significantly enhanced the fitness of P. aeruginosa with resistance mutations in nfxB, encoding a multidrug efflux pump transcriptional repressor, and we identified oxalate as the likely component driving this effect. Our findings highlight the importance of environmental chemical composition for shaping the fitness costs of resistance in P. aeruginosa and suggest that nfxB mutants may have a selective advantage in the urinary tract, mediated by the presence of oxalate.

microbiology↗

Platelets are Protective in Early Abdominal Aortic Aneurysm Formation

BackgroundAbdominal aortic aneurysm (AAA) is a disease associated with the pathophysiologic degradation of the tunica media resulting in aortic dilatation, systemic inflammation, and dysregulated hemostasis. Beyond role its role in initiating primary hemostasis, platelets are a source of ROS, inflammatory cytokines and growth factors necessary for angiogenesis and vascular remodeling. Although platelets contribute to the progression of established aneurysms, their role in the initiation of AAA remains undefined. MethodsLow density lipoprotein receptor deficient (Ldlr-/-) mice were examined for platelet accumulation in the angiotensin II (AngII) model of AAA utilizing in vivo labeling techniques. Two platelet antagonists (clopidogrel and aspirin), a thrombin inhibitor (dabigatran) or genetic deficiencies (protease-activated receptor 4, P2Y12, Lnk) were administered to AngII-infused mice to determine the role of platelets in initiation of AAA. The effect of platelet depletion was examined in multiple mouse strains of AngII-induced AAA and two additional aneurysm models. PheWAS and meta-analysis was analyzed in humans for platelet gene SNPs associated with AAA. ResultsWe show that platelets are recruited rapidly to the aorta after the initiation of AngII infusion. Genetic deficiency of platelet receptors had no effect on abdominal aortic diameter, but augmented rupture-induced death in littermate versus placebo controls during AngII-induced AAA. Moreover, Ldlr-/- mice receiving anti-platelet inhibitors or a thrombin inhibitor also had augmented rupture-induced death. Platelet depletion preceding aneurysm formation resulted in pervasive rupture-induced death in several mouse strains and with three different mouse models of AAA. ConclusionsInhibition of platelet function is detrimental in an early expanding aortic lumen resulting in catastrophic rupture and hemodynamic failure in murine AAA models.

pathology↗

Protection of β2GPI Deficient Mice from Thrombosis Reflects a Defect in PAR3-facilitated Platelet Activation

BackgroundAntibodies to {beta}2-glycoprotein I ({beta}2GPI) cause thrombosis in antiphospholipid syndrome, however the role of {beta}2GPI itself in regulation of coagulation pathways in vivo is not well understood. MethodsWe developed {beta}2GPI-deficient mice (Apoh-/-) by deleting exon 2 and 3 of Apoh using CRISPR/Cas9 and compared the propensity of wild-type (WT) and Apoh-/-mice to develop thrombosis using rose bengal and FeCl3-induced carotid thrombosis, laser-induced cremaster arteriolar injury, and inferior vena cava (IVC) stasis models. We also compared tail bleeding times and assessed platelet activation in WT and Apoh-/- mice in the absence and presence of exogenous {beta}2GPI. ResultsCompared to WT littermates, Apoh-/- mice demonstrated a prolonged time to occlusion of the carotid artery after exposure to rose bengal or FeCl3, and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Similarly, significantly smaller thrombi were retrieved from the IVC of Apoh-/-mice 48 hours after IVC occlusion. The activated partial thromboplastin time (aPTT) and prothrombin time, as well as aPTT reagent- and tissue factor-induced thrombin generation times using plasma from Apoh-/- and WT mice revealed no differences. However, we observed significant prolongation of tail bleeding in Apoh-/- mice, and reduced P-selectin expression and binding of fibrinogen to the activated 2b{beta}3 integrin on platelets from these mice after stimulation with low thrombin concentrations; these changes were reversed by exogenous {beta}2GPI. An antibody to PAR3 blocked thrombin-induced activation of WT, but not Apoh-/- platelets, as well as the ability of {beta}2GPI to restore the activation response of Apoh-/- platelets to thrombin. {beta}2GPI deficiency did not affect platelet activation by a PAR4-activator peptide, or ADP. ConclusionsIn mice, {beta}2GPI may mediate procoagulant activity by enhancing the ability of PAR3 to present thrombin to PAR4, promoting platelet activation at low thrombin concentrations. Key PointsO_LI{beta}2GPI deficient mice are protected from experimental arterial, venous, and microvascular thrombosis. C_LIO_LI{beta}2GPI deficient mice display prolonged tail bleeding times and reduced PAR3-facilitated platelet activation by low concentrations of thrombin. C_LI

cell biology↗

Glycoprotein VI is Critical for the Detection and Progression of Abdominal Aortic Aneurysms

A common feature in patients with abdominal aortic aneurysms (AAA) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation impacts the pathogenesis of AAA. Using RNA-sequencing, we identify that the platelet-associated transcripts are significantly enriched in the ILT compared to the adjacent aneurysm wall and healthy control aortas. We found that the platelet specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of AAA patients. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in two independent AAA patient cohorts is highly predictive of a AAA diagnosis and associates more strongly with aneurysm growth rate when compared to D-dimer in humans. Finally, intervention with the anti-GPVI antibody (J) in mice with established aneurysms blunted the progression of AAA in two independent mouse models. In conclusion, we show that levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, where none currently exist. KEY POINTSO_LISoluble glycoprotein VI, which is a platelet-derived blood biomarker, predicts a diagnosis of AAA, with high sensitivity and specificity in distinguishing patients with fast from slow-growing AAA. C_LIO_LIBlockade of glycoprotein VI in mice with established aneurysms reduces AAA progression and mortality, indicating therapeutic potential. C_LI

pathology↗

Dysregulated Platelet Function and Thrombosis in Patients with Post-Acute Sequelae of COVID-19

BackgroundPost-acute sequelae of COVID-19 (PASC), also referred as Long-COVID, sometimes follows COVID-19, a disease caused by SARS-CoV-2. While SARS-CoV-2 is well-known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. AimOur objective was to evaluate the platelet function and thrombotic potential in patients following recovery from SARS-CoV-2 with clear symptoms of PASC. MethodsPASC patients and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by Light Transmission Aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions. ResultsA mild increase in platelet aggregation in PASC patients through the thromboxane receptor was observed and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in PASC patients compared to age- and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in PASC patients. Plasma from PASC patients was an extremely potent activator of washed, healthy platelets - a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals. ConclusionsPASC patients show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exists in PASC patients to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

pharmacology and toxicology↗

Sex-specific niche signaling contributes to sexual dimorphism following stem cell transplantation

Hematopoietic stem cell (HSC) transplantation (HST) is a curative treatment for many hematopoietic cancers and bone marrow (BM) disorders but is currently limited by numerous complications including a lengthy recovery period, prolonged neutropenia resulting in severe infections and bleeding, and a high incidence of graft vs. host disease (GVHD). While clinical studies have demonstrated that sex mismatch, notably male recipients with female donor cells, results in increased risk of GVHD (likely due to male recipient minor histocompatibility antigens targeted by donor female T-cells 1), increased non-relapse mortality, and decreased overall survival, the mechanisms underlying sex-determinants on hematopoiesis and post-transplant recovery are not clear. In this manuscript we have identified: 1) unique expression of hematopoietic niche factors in the BM and spleens of male and female mice, 2) altered kinetics of hematopoietic reconstitution following transplantation when male vs. female BM is used as the donor cell source, 3) a sex-specific role for the recipient niche in promoting post HST recovery, and 4) a dose-dependent role for exogenous sex hormones in maintaining hematopoietic stem and progenitor cells (HSPCs). Taken together, these data demonstrate that sex-specific cellular and molecular signaling occurs during hematopoietic regeneration. Further identifying novel sex-dependent determinants of regeneration following transplantation will not only enhance understanding of steady state versus regeneration hematopoiesis but may also reveal unique (and potentially sex-specific) therapeutic targets to accelerate hematologic recovery. Key PointsO_LIMale and female mice display altered kinetics of regeneration following HST due to unique niche factors in hematopoietic compartments. C_LIO_LIExogenous steroid sex hormones uniquely regulate the pool of hematopoietic stem and progenitor cells and may impact transplantation outcomes. C_LI

immunology↗

A Sex-Specific Switch in Platelet Receptor Signaling Following Myocardial Infarction

BACKGROUNDA Sex-specific, personalized approach to anti-platelet therapy may be important in patients with myocardial infarction (MI).\n\nOBJECTIVESOur goal was to determine whether platelets activate differently in healthy men and women compared to following MI.\n\nMETHODSBlood was obtained from healthy subjects or patients presenting acutely with ST-segment Elevation Myocardial Infarction (STEMI) and non-ST Segment Elevation Myocardial Infarction (NSTEMI). Platelet function through surface receptor activation was examined in healthy subjects, in patients with MI, and in age- and strain-matched mice before and after MI. Multivariate regression analyses revealed clinical variables associated with platelet receptor sensitivity at the time of MI.\n\nRESULTSPlatelets from healthy women are dose-dependently more active compared to men, particularly through the platelet thromboxane signaling pathway (7.8-fold increase in women vs. 3.0-fold in men, P=0.02). At the time of MI, platelet activation through surface protease-activated receptor 1 (PAR1) was less in women than men (3.5-fold vs. 8.5-fold, respectively, P=0.0001). Multivariate regression analyses revealed male sex (P=0.04) and NSTEMI (P=0.003) as independent predictors of enhanced platelet PAR1 signaling at the time of MI. Similar to humans, healthy female mice showed preferential thrombin-mediated platelet activation compared to male mice (8.7-fold vs. 4.8-fold, respectively; P<0.001). In the immediate post-MI environment, male mice showed preferential thrombin-mediated platelet activation compared to female mice (12.4-fold vs. 5.5-fold, respectively; P<0.001).\n\nCONCLUSIONSThese results outline a previously unrecognized sex-dependent platelet phenotype where inhibition of thrombin signaling in the peri-MI environment--particularly in males--may be an important consideration.\n\nCONDENSED ABSTRACTPreclinical studies evaluating anti-platelet drugs are generally conducted in platelets isolated from healthy individuals. Growing evidence suggests changes in platelet signaling properties in certain disease conditions compared to healthy platelets may alter the response to anti-platelet medications. This investigation revealed that platelets from men and women who are healthy and following MI signal differently, particularly through thromboxane and PAR1 receptors. This effect was especially noted in patients with NSTEMI compared to STEMI. These observations raise the possibility of considering a sex-specific anti-platelet regimen for males and females in atheroembolic vascular diseases such as NSTEMI.

molecular biology↗

Acetylcholine inhibits platelet activation and regulates hemostasis

Platelets are key mediators of thrombosis. Many agonists of platelet activation are known, but there are fewer identified endogenous inhibitors of platelets, such as prostacyclin and nitric oxide (NO). Acetylcholinesterase inhibitors such as donepezil can cause bleeding in patients, but the underlying mechanisms are not well understood. We hypothesized that acetylcholine is an endogenous inhibitor of platelets.\n\nWe measured the effect of acetylcholine or analogues of acetylcholine upon human platelet activation ex vivo. We characterized expression of components of the acetylcholine signaling pathway in human platelets. We tested the effect of a subunit of the acetylcholine receptor, CHRNA7, on acetylcholine signaling in platelets. Acetylcholine and analogues of acetylcholine inhibited platelet activation, as measured by P-selectin translocation and GPIIbIIIA conformational changes. Conversely, we found that antagonists of the acetylcholine receptor such as pancuronium enhance platelet activation. Furthermore, drugs inhibiting acetylcholinesterase such as donepezil also inhibit platelet activation, suggesting that platelets release acetylcholine. We found that NO mediates acetylcholine inhibition of platelets. Human platelets express members of the acetylcholine signaling pathway including CHRNA2, CHRNA7, CHRNB1, and ACHE. Platelets from mice lacking Chrna7 are hyperactive when stimulated by thrombin and resistant to inhibition by acetylcholine. Furthermore, acetylcholinesterase inhibitors prolonged bleeding in wild-type mice. Knockout mice lacking Chrna7 subunits of the acetylcholine receptor display prolonged bleeding as well.\n\nOur data suggest that acetylcholine is an endogenous inhibitor of platelet activation. The cholinergic system may be a novel target for anti-thrombotic therapies.

pharmacology and toxicology↗