Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), is found in plasma and platelets. PAI-1 circulates in complex with vitronectin (Vn), an interaction that stabilizes PAI-1 in its active conform. In this study, we examined the binding of platelet-derived Vn and PAI-1 to the surface of isolated platelets. Flow cytometry indicate that, like P-selectin, PAI-1, and Vn are found on the surface of thrombin-or calcium ionophore-activated platelets and platelet microparticles. The binding of PAI-1 to the activated platelet surface is Vn-dependent. Vn mediates the binding of PAI-1 to platelet surfaces through a high affinity (K d of 80 nM) binding interaction with the NH 2 terminus of vimentin, and this Vn-binding domain is expressed on the surface of activated platelets and platelet microparticles. Immunological and functional assays indicate that only ؊5% of the total PAI-1 in platelet releasates is functionally active, and it co-precipitates with Vn, and the vimentin-enriched cytoskeleton fraction of activated platelet debris. The remaining platelet PAI-1 is inactive, and does not associate with the cytoskeletal debris of activated platelets. Confocal microscopic analysis of platelet-rich plasma clots confirm the co-localization of PAI-1 with Vn and vimentin on the surface of activated platelets, and platelet microparticles. These findings suggest that platelet vimentin may regulate fibrinolysis in plasma and thrombi by binding platelet-derived Vn⅐PAI-1 complexes.

Vasopressins are evolutionarily conserved peptide hormones. Mammalian vasopressin functions systemically as an antidiuretic and regulator of blood and cardiac flow essential for adapting to terrestrial environments. Moreover, vasopressin acts centrally as a neurohormone involved in social and parental behavior and stress response. Vasopressin synthesis in several cell types, storage in intracellular vesicles, and release in response to physiological stimuli are highly regulated and mediated by three distinct G protein coupled receptors. Other receptors may bind or cross-bind vasopressin. Vasopressin is regulated spatially and temporally through transcriptional and post-transcriptional mechanisms, sex, tissue, and cell-specific receptor expression. Anomalies of vasopressin signaling have been observed in polycystic kidney disease, chronic heart failure, and neuropsychiatric conditions. Growing knowledge of the central biological roles of vasopressin has enabled pharmacological advances to treat these conditions by targeting defective systemic or central pathways utilizing specific agonists and antagonists.

Localized and reversible plasma membrane disruption is a promising technique employed for the targeted deposition of exogenous therapeutic compounds for the treatment of disease. Indeed, the plasma membrane represents a significant barrier to successful delivery, and various physical methods using light, sound, and electrical energy have been developed to generate cell membrane perforations to circumvent this issue. To restore homeostasis and preserve viability, localized cellular repair mechanisms are subsequently triggered to initiate a rapid restoration of plasma membrane integrity. Here, we summarize the known emergency membrane repair responses, detailing the salient membrane sealing proteins as well as the underlying cytoskeletal remodeling that follows the physical induction of a localized plasma membrane pore, and we present an overview of potential modulation strategies that may improve targeted drug delivery approaches.

In endothelial cells, microRNA-126 (miR-126) promotes angiogenesis, and modulating the intracellular levels of this gene could suggest a method to treat cardiovascular diseases such as ischemia. Novel ultrasound-stimulated microbubbles offer a means to deliver therapeutic payloads to target cells and sites of disease. The purpose of this study was to investigate the feasibility of gene delivery by stimulating miR-126-decorated microbubbles using gentle acoustic conditions (stable cavitation). A cationic DSTAP microbubble was formulated and characterized to carry 6 µg of a miR-126 payload per 109 microbubbles. Human umbilical vein endothelial cells (HUVECs) were treated at 20–40% duty cycle with miR-126-conjugated microbubbles in a custom ultrasound setup coupled with a passive cavitation detection system. Transfection efficiency was assessed by RT-qPCR, Western blotting, and endothelial tube formation assay, while HUVEC viability was monitored by MTT assay. With increasing duty cycle, the trend observed was an increase in intracellular miR-126 levels, up to a 2.3-fold increase, as well as a decrease in SPRED1 (by 33%) and PIK3R2 (by 46%) expression, two salient miR-126 targets. Under these ultrasound parameters, HUVECs maintained >95% viability after 96 h. The present work describes the delivery of a proangiogenic miR-126 using an ultrasound-responsive cationic microbubble with potential to stimulate therapeutic angiogenesis while minimizing endothelial damage.

Introduction: Hypertrophic cardiomyopathy is a disease characterized by the abnormal growth of cardiomyocytes (CMs), a marker of which is the early downregulation of miR-1. The delivery of miR-1 to hypertrophic CMs presents as a promising treatment strategy, however there are still limitations in the safe and efficient delivery of gene therapeutics.Ultrasound (US) and microbubbles is an emerging approach to site-specific gene delivery. Microbubbles have long been employed in clinical cardiology as a diagnostic contrast agent. Recent work has demonstrated that under specific conditions, they are capable of forming temporary pores on neighboring cell membranes, allowing the delivery of otherwise impermeable macromolecules. This presents an exciting approach to the treatment of cardiovascular disease (CVD) as a targeted and inherently image-guided therapeutic delivery platform. Objective: The goal of this study is to determine the feasibility of viable US and microbubble mediated delivery of miR-1 to hypertrophic CMs with a view towards reversing the disease. Secondly, we aim to show a correlation between bubble echoes and resulting gene delivery to introduce clinical metrics for the translation of this technique. Methods: CMs were harvested from neonatal rat pups (1-3 days old) and hypertrophied using phenylephrine (100μM). Suspension of healthy or diseased CMs, microbubbles and free miR-1 were placed in a chamber within a 37°C degassed water bath. These samples were insonicated at a mechanical index (MI) ranging from 0.063-0.25 for 2 minutes (20 cycles; 5 kHz PRF) and bubble echoes were recorded via a co-aligned transducer to be processed offline. miR-1 delivery was confirmed with RT-qPCR, cell viablity assesed with MTT, protein expression was measured by immunoblotting and cell size via microscopy. All experiments preformed are at least n=2 independent samples. Result and Conclusion: Treated healthy and diseased CMs demonstrate an upregulation in miR-1 with increasing MI, from 0.063 to 0.188. At MI=0.12, a 2.22 and 1.95-fold increase in miR-1 was shown in diseased and healthy CMs respectively ( p= 0.07, p= 0.02) while maintaining viability (86% and 96% respectively). These miR-1 levels begin to show the reversal of hypertrophy as quantified by increases in target protein expression (1.93-fold in TWF1; 1.38-fold in MEF2A; 1.36-fold in CX43) and cell size decrease (16%, p =0.035) back towards heathy levels. Echo data highlights that relative miR-1 expression positively correlates with bubble disruption ( r =0.76, p =0.04), suggesting a means by which treatment efficiency can be monitored in a clinical setting.In conclusion, we demonstrated that US and microbubbles can be used to successfully delivery miR-1 to CMs, with the potential to reverse the disease phenotype. These findings demonstrate the feasibility of US as an image-guided delivery method for molecular therapeutics in CVD.

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