Biology subjects

Neeves, K. B.

Publications and source records attributed to Neeves, K. B..

7 recordsLinked to original sources

bioRxiv2025

Female fibroblast activation is estrogen-mediated in sex-specific 3D-bioprinted pulmonary artery adventitia models

Pulmonary arterial hypertension (PAH) impacts male and female patients in different ways. Female patients exhibit a greater susceptibility to disease (4:1 female-to-male ratio) but live longer after diagnosis than male patients. This complex sexual dimorphism is known as the estrogen paradox. Prior studies suggest that estrogen signaling may be pathologic in the pulmonary vasculature and protective in the heart, yet the mechanisms underlying these sex-differences in PAH remain unclear. PAH is a form of a pulmonary vascular disease that results in scarring of the small blood vessels, leading to impaired blood flow and increased blood pressure. Over time, this increase in blood pressure causes damage to the heart. Many previous studies of PAH relied on male cells or cells of undisclosed origin for in vitro modeling. Here we present a dynamic, 3D-bioprinted model that incorporates cells and circulating sex hormones from female patients to specifically study how female patients respond to changes in microenvironmental stiffness and sex hormone signaling. Poly(ethylene glycol)-alpha methacrylate (PEGMA)-based hydrogels containing female human pulmonary artery adventitia fibroblasts (hPAAFs) from idiopathic PAH (IPAH) or control donors were 3D bioprinted to mimic pulmonary artery adventitia. These biomaterials were initially soft, like healthy blood vessels, and then stiffened using light to mimic vessel scarring in PAH. These 3D-bioprinted models showed that stiffening the microenvironment around female IPAH hPAAFs led to hPAAF activation. On both the protein and gene-expression levels, cellular activation markers significantly increased in stiffened samples and were highest in IPAH patient-derived cells. Treatment with a selective estrogen receptor modulator reduced expression hPAAF activation markers, demonstrating that hPAAF activation is a one pathologic response mediated by estrogen signaling in the vasculature, validating that drugs currently in clinical trials could be evaluated in sex-specific 3D-bioprinted pulmonary artery adventitia models.

bioengineering↗

bioRxiv2024

Harnessing micrometer-scale tPA beads for high plasmin flux and accelerated fibrinolysis

Rapid restoration of blood flow is critical in treating acute ischemic stroke. Current fibrinolytic therapies using tissue plasminogen activator (tPA) are limited by low recanalization rates and risks of off-target bleeding. Here, we present a strategy using tPA immobilized on micrometer-scale beads to enhance local plasmin generation. We synthesized tPA-functionalized beads of varying sizes (0.1 m and 1.0 m) and evaluated their efficacy. In vitro assays demonstrated that 1.0 m tPA-beads generated higher plasmin generation compared to free tPA and 0.1 m beads, overcoming antiplasmin inhibition and promoting a self-propagating wave of fibrinolysis. In a murine model of acute ischemic stroke, intravenous administration of 1.0 m tPA-beads at doses nearly two orders of magnitude lower than the standard free tPA dose led to rapid and near-complete thrombus removal within minutes. This approach addresses kinetic and transport limitations of current therapies and may reduce the risk of hemorrhagic complications. TeaserMicrometer-scale tPA beads improve stroke treatment by enhancing local clot-dissolving action.

bioengineering↗

bioRxiv2023

Magnetically Powered Microwheel Thrombolysis of Occlusive Thrombi in Zebrafish

Tissue plasminogen activator (tPA) is the only FDA approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for [~]25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels ({micro}wheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Providing a living microfluidic analog, transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here we show that tPA-functionalized {micro}wheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and {micro}wheels, as minimal to no recanalization is achieved with tPA alone, {micro}wheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-{micro}wheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-{micro}wheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered {micro}wheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

bioengineering↗

bioRxiv2022

Magnetically powered chitosan milliwheels for rapid translation, barrier function rescue, and delivery of therapeutic proteins to the inflamed gut epithelium

Inflammatory bowel disease (IBD) is mediated by an overexpression of tumor necrosis factor- (TNF) by mononuclear cells in the intestinal mucosa. Intravenous delivery of neutralizing anti-TNF antibodies can cause systemic immunosuppression and up to one-third of people are non-responsive to treatment. Oral delivery of anti-TNF could reduce adverse effects; however, it is hampered by antibody degradation in the harsh gut environment during transit and poor bioavailability. To overcome these shortcomings, we demonstrate magnetically powered hydrogel particles that roll along mucosal surfaces, provide protection from degradation, and sustain local release of anti-TNF. Iron oxide nanoparticles are embedded into a crosslinked chitosan hydrogel and sieved to produce 100-200 m particles called milliwheels (m-wheels). Once loaded with anti-TNF, these m-wheels release 10% to 80% of their payload over one week at a rate that depends on crosslinking density and pH. A rotating magnetic field induces a torque on the m-wheels that results in rolling velocities greater than 500 m/s on glass and mucus-secreting cells. The permeability of TNF challenged gut epithelial cell monolayers was rescued in the presence of anti-TNF carrying m-wheels which both neutralized the TNF and created an impermeable patch over leaky cell junctions. With the ability to translate over mucosal surfaces at high speed, provide sustained release directly to the inflamed epithelium, and provide barrier rescue, m-wheels demonstrate a potential strategy to deliver therapeutic proteins for the treatment of IBD.

bioengineering↗

bioRxiv2021

Quantifying Central Metabolic Fluxes in Human Platelets Using Metabolic Flux Analysis

Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary 13C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies.

systems biology↗

bioRxiv2021

Breaking the fibrinolytic speed limit with microwheel co-delivery of tissue plasminogen activator and plasminogen

Fibrinolysis is the enzymatic degradation of fibrin, the biopolymer that gives blood clots their mechanical integrity. To reestablish blood flow in vessels occluded by clots, tissue plasminogen activator (tPA) can be used; however, its efficacy is limited by transport to and into a clot and by the depletion of its substrate, plasminogen. To overcome these rate limitations, we design a platform to co-deliver tPA and plasminogen based on microwheels (wheels), wheel-like assemblies of superparamagnetic colloidal beads that roll along surfaces at high speeds and carry therapeutic payloads in applied magnetic fields. By experimentally measuring fibrinolysis of plasma clots at varying concentrations of tPA and plasminogen, the biochemical speed limit was first determined. These data, in conjunction with measurements of wheel translation, activity of immobilized tPA on beads, and plasminogen release kinetics from magnetic mesoporous silica nanoparticles (mMSN), were used in a mathematical model to identify the optimal tPA:plasminogen ratio and guide the coupling of plasminogen-loaded mMSN to tPA functionalized superparamagnetic beads. Once coupled, particle-bead assemblies form into a co-delivery vehicle that rolls to plasma clot interfaces and lyses them at rates comparable to the biochemical speed limit. With the addition of mechanical action provided by rotating wheels to penetrate clots, this barrier was exceeded by rates 40-fold higher lysis by 50 nM tPA. This co-delivery of an immobilized enzyme and its substrate via a microbot capable of mechanical work has the potential to target and rapidly lyse clots that are inaccessible by mechanical thrombectomy devices or recalcitrant to systemic tPA delivery.

bioengineering↗

bioRxiv2018

A mathematical model of flow-mediated coagulation identifies factor V as a modifier of thrombin generation in hemophilia A

Hemophilia A is a bleeding disorder categorized as severe, mild, and moderate deficiencies in factor VIII (FVIII). Within these categories the variance in bleeding severity is significant and the origins unknown. The number of parameters that could modify bleeding are so numerous that experimental approaches are not feasible for considering all possible combinations. Consequently, we turn to a mathematical model of coagulation under flow to act as a screening tool to identify parameters that are most likely to enhance thrombin generation. We performed global sensitivity analysis on 110,000 simulations that varied coagulation factor levels by 50-150% of their normal values in humans while holding FVIII levels at 1%. These simulations identified low factor V (FV) levels as the strongest candidate, with additional enhancement when combined with high prothrombin levels. This prediction was confirmed in two experimental models: Partial FV inhibition boosted fibrin deposition in flow assays performed at 100 1/s on collagen-tissue factor surfaces using whole blood from individuals with mild and moderate FVIII deficiencies. Low FV (~50%) or partial FV inhibition also augmented thrombin generation in FVIII-inhibited or FVIII-deficient plasma in calibrated automated thrombography. These effects were amplified by high prothrombin levels in both experimental models. Our mathematical model suggests a mechanism in which FV and FVIII compete to bind to factor Xa to initiate thrombin generation in low FV, FVIII-deficient blood. This unexpected result was made possible by a mechanistic mathematical model, providing an example of the potential of such models in making predictions in complex biological networks.

systems biology↗