Liposomal Nanocarriers Designed for Sub-Endothelial Matrix Targeting under Vascular Flow Conditions

Vascular interventions result in the disruption of the tunica intima and the exposure of sub-endothelial matrix proteins. Nanoparticles designed to bind to these exposed matrices could provide targeted drug delivery systems aimed at inhibiting dysfunctional vascular remodeling and improving intervention outcomes. Here, we present the progress in the development of targeted liposomal nanocarriers designed for preferential collagen IV binding under simulated static vascular flow conditions. PEGylated liposomes (PLPs), previously established as effective delivery systems in vascular cells types, served as non-targeting controls. Collagen-targeting liposomes (CT-PLPs) were formed by conjugating established collagen-binding peptides to modified lipid heads via click chemistry (CTL), and inserting them at varying mol% either at the time of PLP assembly or via micellar transfer. All groups included fluorescently labeled lipid species for imaging and quantification. Liposomes were exposed to collagen IV matrices statically or via hemodynamic flow, and binding was measured via fluorometric analyses. CT-PLPs formed with 5 mol% CTL at the time of assembly demonstrated the highest binding affinity to collagen IV under static conditions, while maintaining a nanoparticle characterization profile of ~50 nm size and a homogeneity polydispersity index (PDI) of ~0.2 favorable for clinical translation. When liposomes were exposed to collagen matrices within a pressurized flow system, empirically defined CT-PLPs demonstrated significant binding at shear stresses mimetic of physiological through pathological conditions in both the venous and arterial architectures. Furthermore, when human saphenous vein explants were perfused with liposomes within a closed bioreactor system, CT-PLPs demonstrated significant ex vivo binding to diseased vascular tissue. Ongoing studies aim to further develop CT-PLPs for controlled targeting in a rodent model of vascular injury. The CT-PLP nanocarriers established here show promise as the framework for a spatially controlled delivery platform for future application in targeted vascular therapeutics.

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R93W mutation in Orai1 causes impaired calcium influx in platelets

Blood ◽

10.1182/blood-2008-08-174516 ◽

2009 ◽

Vol 113 (3) ◽

pp. 675-678 ◽

Cited By ~ 89

Author(s):

Wolfgang Bergmeier ◽

Masatsugu Oh-hora ◽

Christie-Ann McCarl ◽

R. Claire Roden ◽

Paul F. Bray ◽

...

Keyword(s):

Calcium Influx ◽

Ex Vivo ◽

Cellular Responses ◽

Calcium Sensor ◽

Flow Conditions ◽

Calcium Store ◽

Inactive Form ◽

Store Operated Calcium Entry ◽

Static Conditions

Abstract The intracellular Ca2+ concentration of many nonexcitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1R93W). Platelets expressing Orai1R93W were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca2+]i. Orai1R93W platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1R93W platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1R93W platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets' procoagulant response rather than for other Ca2+-dependent cellular responses.

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Neutrophil Adhesion to Fibrinogen and Fibrin Under Flow Conditions Is Diminished by Activation and L-Selectin Shedding

Blood ◽

10.1182/blood.v89.6.2131 ◽

1997 ◽

Vol 89 (6) ◽

pp. 2131-2138 ◽

Cited By ~ 27

Author(s):

P.H.M. Kuijper ◽

H.I. Gallardo Torres ◽

J.A.M. van der Linden ◽

J.-W.J. Lammers ◽

J.J. Sixma ◽

...

Keyword(s):

Polymorphonuclear Leukocytes ◽

Random Distribution ◽

Neutrophil Adhesion ◽

Shear Stresses ◽

Flow Conditions ◽

Neuraminidase Treatment ◽

Cluster Index ◽

Custom Made ◽

Static Conditions ◽

Β2 Integrins

Abstract The adhesion of neutrophils (polymorphonuclear leukocytes [PMNs]) to immobilized fibrinogen/fibrin is mediated by β2-integrins. However, the influence of physiologic flow conditions on neutrophil adhesion to these surfaces is poorly defined. In this report, the effect of flow and neutrophil activation on adhesion to immobilized fibrinogen and fibrin was examined. For the evaluation of (the distribution of ) neutrophil adhesion, real-time video-assisted microscopy and custom-made software were used. Under flow conditions, adherent neutrophils appeared to support the subsequent margination of other neutrophils, thereby enhancing the adherence of these cells to fibrin. Consequently, neutrophils adhered in clusters, especially at higher shear stresses (eg, cluster index 1.4 at shear 80 mPa). Preactivation of PMNs with fMLP (10−7 mol/L) or 4β-phorbol, 12-myristate, 13-acetate (PMA; 100 ng/mL) resulted in approximately 50% inhibition of adhesion to fibrin and a more random distribution (cluster index <0.5). L-selectin antibodies or neuraminidase treatment of PMNs also inhibited adhesion and clustering, indicating a role for L-selectin. Under static conditions, no clustering appeared and PMN activation with fMLP or PMA caused threefold and sevenfold increased adhesion, respectively. Under these conditions, anti–L-selectin antibodies or neuraminidase did not affect adhesion. These results indicate that, under flow conditions, adherent neutrophils support adhesion of flowing neutrophils by L-selectin–mediated cell-cell interactions. Preactivated neutrophils, with lowered L-selectin expression, are less susceptible for this interaction. By this mechanism, adhered leukocytes can modulate the recruitment of leukocytes to the vessel wall at sites of inflammation.

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Platelet and Fibrin Deposition at the Damaged Vessel Wall: Cooperative Substrates for Neutrophil Adhesion Under Flow Conditions

Blood ◽

10.1182/blood.v89.1.166 ◽

1997 ◽

Vol 89 (1) ◽

pp. 166-175 ◽

Cited By ~ 79

Author(s):

P.H.M. Kuijper ◽

H.I. Gallardo Torres ◽

J.-W.J. Lammers ◽

J.J. Sixma ◽

L. Koenderman ◽

...

Keyword(s):

Vessel Wall ◽

Human Neutrophils ◽

Shear Stresses ◽

Dependent Manner ◽

Fibrin Deposition ◽

Flow Conditions ◽

Shear Rates ◽

Fibrin Network ◽

Static Conditions ◽

Blocking Antibodies

Abstract At sites of vessel wall damage, the primary hemostatic reaction involves platelet and fibrin deposition. At these sites, circulating leukocytes marginate and become activated. Adhered platelets can support leukocyte localization; however, the role of fibrin in this respect is not known. We studied the adhesion of human neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial extracellular matrix (ECM)-bound fibrin and platelets under flow conditions. ECM alone did not show PMN adhesion. ECM-coated cover slips were perfused with plasma to form a surface-bound fibrin network, and/or with whole blood to allow platelet adhesion. Unstimulated PMNs adhered to fibrin at moderate shear stress (20 to 200 mPa). ECM-bound platelets induced rolling adhesion and allowed more PMNs to adhere at higher shear (320 mPa). ECM coated with both platelets and fibrin induced more static and shear-resistant PMN adhesion. PMN adhesion to fibrin alone but not to platelet/fibrin surfaces was inhibited by soluble fibrinogen. Adhesion to fibrin alone was inhibited by CD11b and CD18 blocking antibodies. Furthermore, fibrin formed under flow conditions showed up to threefold higher PMN adhesion compared with fibrin formed under static conditions, due to structural differences. These results indicate that circulating PMNs adhere to fibrin in an integrin-dependent manner at moderate shear stresses. However, at higher shear rates (<200 mPa), additional mechanisms (ie, activated platelets) are necessary for an interaction of PMNs with a fibrin network.

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Platelet and Fibrin Deposition at the Damaged Vessel Wall: Cooperative Substrates for Neutrophil Adhesion Under Flow Conditions

Blood ◽

10.1182/blood.v89.1.166.166_166_175 ◽

1997 ◽

Vol 89 (1) ◽

pp. 166-175 ◽

Cited By ~ 3

Author(s):

P.H.M. Kuijper ◽

H.I. Gallardo Torres ◽

J.-W.J. Lammers ◽

J.J. Sixma ◽

L. Koenderman ◽

...

Keyword(s):

Vessel Wall ◽

Human Neutrophils ◽

Shear Stresses ◽

Dependent Manner ◽

Fibrin Deposition ◽

Flow Conditions ◽

Shear Rates ◽

Fibrin Network ◽

Static Conditions ◽

Blocking Antibodies

At sites of vessel wall damage, the primary hemostatic reaction involves platelet and fibrin deposition. At these sites, circulating leukocytes marginate and become activated. Adhered platelets can support leukocyte localization; however, the role of fibrin in this respect is not known. We studied the adhesion of human neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial extracellular matrix (ECM)-bound fibrin and platelets under flow conditions. ECM alone did not show PMN adhesion. ECM-coated cover slips were perfused with plasma to form a surface-bound fibrin network, and/or with whole blood to allow platelet adhesion. Unstimulated PMNs adhered to fibrin at moderate shear stress (20 to 200 mPa). ECM-bound platelets induced rolling adhesion and allowed more PMNs to adhere at higher shear (320 mPa). ECM coated with both platelets and fibrin induced more static and shear-resistant PMN adhesion. PMN adhesion to fibrin alone but not to platelet/fibrin surfaces was inhibited by soluble fibrinogen. Adhesion to fibrin alone was inhibited by CD11b and CD18 blocking antibodies. Furthermore, fibrin formed under flow conditions showed up to threefold higher PMN adhesion compared with fibrin formed under static conditions, due to structural differences. These results indicate that circulating PMNs adhere to fibrin in an integrin-dependent manner at moderate shear stresses. However, at higher shear rates (<200 mPa), additional mechanisms (ie, activated platelets) are necessary for an interaction of PMNs with a fibrin network.

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R93W Mutation in Orai1 Causes Impaired Calcium Influx in Platelets.

Blood ◽

10.1182/blood.v112.11.1838.1838 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1838-1838

Author(s):

Wolfgang Bergmeier ◽

Masatsugu Oh-hora ◽

Christie-Ann McCarl ◽

R. Claire Roden ◽

Paul F. Bray ◽

...

Keyword(s):

Calcium Influx ◽

Ex Vivo ◽

Cellular Responses ◽

Flow Conditions ◽

Excitable Cells ◽

Calcium Store ◽

Inactive Form ◽

Store Operated Calcium Entry ◽

Static Conditions

Abstract The intracellular Ca2+ concentration of non-excitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmatic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1R93W). Platelets expressing Orai1R93W were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca2+]i. Orai1R93W platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1R93W platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1R93W platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets pro-coagulant response rather than for other Ca2+-dependent cellular responses.

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Platelet and Shear Rate Promote Tumor Cell Adhesion to Human Endothelial Extracellular Matrix - Absence of a Role for Platelet Cyclooxygenase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646619 ◽

1989 ◽

Vol 61 (03) ◽

pp. 485-489 ◽

Cited By ~ 5

Author(s):

Eva Bastida ◽

Lourdes Almirall ◽

Antonio Ordinas

Keyword(s):

Cell Adhesion ◽

Shear Rate ◽

Tumor Cell ◽

Umbilical Vein ◽

Blood Platelets ◽

Flow Conditions ◽

Tumor Cell Adhesion ◽

Rate Dependent ◽

Static Conditions

SummaryBlood platelets are thought to be involved in certain aspects of malignant dissemination. To study the role of platelets in tumor cell adherence to vascular endothelium we performed studies under static and flow conditions, measuring tumor cell adhesion in the absence or presence of platelets. We used highly metastatic human adenocarcinoma cells of the lung, cultured human umbilical vein endothelial cells (ECs) and extracellular matrices (ECM) prepared from confluent EC monolayers. Our results indicated that under static conditions platelets do not significantly increase tumor cell adhesion to either intact ECs or to exposed ECM. Conversely, the studies performed under flow conditions using the flat chamber perfusion system indicated that the presence of 2 × 105 pl/μl in the perfusate significantly increased the number of tumor cells adhered to ECM, and that this effect was shear rate dependent. The maximal values of tumor cell adhesion were obtained, in presence of platelets, at a shear rate of 1,300 sec-1. Furthermore, our results with ASA-treated platelets suggest that the role of platelets in enhancing tumor cell adhesion to ECM is independent of the activation of the platelet cyclooxygenase pathway.

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Point-of-Care Diagnostic Assays and Novel Preclinical Technologies for Hemostasis and Thrombosis

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0041-1723798 ◽

2021 ◽

Vol 47 (02) ◽

pp. 120-128

Author(s):

Christina Caruso ◽

Wilbur A. Lam

Keyword(s):

Point Of Care ◽

Healing Process ◽

Coagulation Factors ◽

Wound Healing Process ◽

Shear Stresses ◽

Flow Conditions ◽

Novel Technologies ◽

Clinical Consequences ◽

Complex Wound ◽

Reproducible Analysis

AbstractHemostasis is a complex wound-healing process involving numerous mechanical and biochemical mechanisms and influenced by many factors including platelets, coagulation factors, and endothelial components. Slight alterations in these mechanisms can lead to either prothrombotic or bleeding consequences, and such hemostatic imbalances can lead to significant clinical consequences with resultant morbidity and mortality. An ideal hemostasis assay would not only address all the unique processes involved in clot formation and resolution but also take place under flow conditions to account for endothelial involvement. Global assays do exist; however, these assays are not flow based. Flow-based assays have been limited secondary to their large blood volume requirements and low throughput, limiting potential clinical applications. Microfluidic-based assays address the aforementioned limitations of both global and flow-based assays by utilizing standardized devices that require low blood volumes, offer reproducible analysis, and have functionality under a range of shear stresses and flow conditions. While still largely confined to the preclinical space, here we aim to discuss these novel technologies and potential clinical implications, particularly in comparison to the current, commercially available point-of-care assays.

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Numerical and Experimental Investigations on Vocal Fold Approximation in Healthy and Simulated Unilateral Vocal Fold Paralysis

Applied Sciences ◽

10.3390/app11041817 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1817

Author(s):

Zheng Li ◽

Azure Wilson ◽

Lea Sayce ◽

Amit Avhad ◽

Bernard Rousseau ◽

...

Keyword(s):

Computational Model ◽

Vocal Fold ◽

High Speed ◽

Computational Models ◽

Ex Vivo ◽

Vocal Fold Paralysis ◽

Future Application ◽

Experimental Investigations ◽

Type I ◽

Mri Scans

We have developed a novel surgical/computational model for the investigation of unilat-eral vocal fold paralysis (UVFP) which will be used to inform future in silico approaches to improve surgical outcomes in type I thyroplasty. Healthy phonation (HP) was achieved using cricothyroid suture approximation on both sides of the larynx to generate symmetrical vocal fold closure. Following high-speed videoendoscopy (HSV) capture, sutures on the right side of the larynx were removed, partially releasing tension unilaterally and generating asymmetric vocal fold closure characteristic of UVFP (sUVFP condition). HSV revealed symmetric vibration in HP, while in sUVFP the sutured side demonstrated a higher frequency (10–11%). For the computational model, ex vivo magnetic resonance imaging (MRI) scans were captured at three configurations: non-approximated (NA), HP, and sUVFP. A finite-element method (FEM) model was built, in which cartilage displacements from the MRI images were used to prescribe the adduction, and the vocal fold deformation was simulated before the eigenmode calculation. The results showed that the frequency comparison between the two sides was consistent with observations from HSV. This alignment between the surgical and computational models supports the future application of these methods for the investigation of treatment for UVFP.

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Growth Factor Therapy for Parkinson’s Disease: Alternative Delivery Systems

Journal of Parkinson s Disease ◽

10.3233/jpd-212662 ◽

2021 ◽

pp. 1-7

Author(s):

Sarah Jarrin ◽

Abrar Hakami ◽

Ben Newland ◽

Eilís Dowd

Keyword(s):

Parkinson’S Disease ◽

Gene Therapy ◽

Parkinson's Disease ◽

Growth Factors ◽

Growth Factor ◽

Ex Vivo ◽

Brain Regions ◽

Delivery Systems ◽

Alternative Delivery

Despite decades of research and billions in global investment, there remains no preventative or curative treatment for any neurodegenerative condition, including Parkinson’s disease (PD). Arguably, the most promising approach for neuroprotection and neurorestoration in PD is using growth factors which can promote the growth and survival of degenerating neurons. However, although neurotrophin therapy may seem like the ideal approach for neurodegenerative disease, the use of growth factors as drugs presents major challenges because of their protein structure which creates serious hurdles related to accessing the brain and specific targeting of affected brain regions. To address these challenges, several different delivery systems have been developed, and two major approaches—direct infusion of the growth factor protein into the target brain region and in vivo gene therapy—have progressed to clinical trials in patients with PD. In addition to these clinically evaluated approaches, a range of other delivery methods are in various degrees of development, each with their own unique potential. This review will give a short overview of some of these alternative delivery systems, with a focus on ex vivo gene therapy and biomaterial-aided protein and gene delivery, and will provide some perspectives on their potential for clinical development and translation.

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Assessment of Electrospun Pellethane-Based Scaffolds for Vascular Tissue Engineering

Materials ◽

10.3390/ma14133678 ◽

2021 ◽

Vol 14 (13) ◽

pp. 3678

Author(s):

Vera Chernonosova ◽

Alexandr Gostev ◽

Ivan Murashov ◽

Boris Chelobanov ◽

Andrey Karpenko ◽

...

Keyword(s):

Vascular Tissue ◽

Ex Vivo ◽

Vascular Grafts ◽

Wistar Rat ◽

Graft Occlusion ◽

Suitable Candidate ◽

Large Animals ◽

Cell Adhesion And Proliferation

We examined the physicochemical properties and the biocompatibility and hemocompatibility of electrospun 3D matrices produced using polyurethane Pellethane 2363-80A (Pel-80A) blends Pel-80A with gelatin or/and bivalirudin. Two layers of vascular grafts of 1.8 mm in diameter were manufactured and studied for hemocompatibility ex vivo and functioning in the infrarenal position of Wistar rat abdominal aorta in vivo (n = 18). Expanded polytetrafluoroethylene (ePTFE) vascular grafts of similar diameter were implanted as a control (n = 18). Scaffolds produced from Pel-80A with Gel showed high stiffness with a long proportional limit and limited influence of wetting on mechanical characteristics. The electrospun matrices with gelatin have moderate capacity to support cell adhesion and proliferation (~30–47%), whereas vascular grafts with bivalirudin in the inner layer have good hemocompatibility ex vivo. The introduction of bivalirudin into grafts inhibited platelet adhesion and does not lead to a change hemolysis and D-dimers concentration. Study in vivo indicates the advantages of Pel-80A grafts over ePTFE in terms of graft occlusion, calcification level, and blood velocity after 6 months of implantation. The thickness of neointima in Pel-80A–based grafts stabilizes after three months (41.84 ± 20.21 µm) and does not increase until six months, demonstrating potential for long-term functioning without stenosis and as a suitable candidate for subsequent preclinical studies in large animals.

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