Sided Stimulation of Endothelial Cells Modulates Neutrophil Trafficking in an In Vitro Sepsis Model.

While the role of dysregulated polymorphonuclear leukocyte (PMN) transmigration in septic mediated tissue damage is well documented, strategies to mitigate aberrant transmigration across endothelium have yet to yield viable therapeutics. Recently, microphysiological systems (MPS) have emerged as novel in vitro mimetics that facilitate the development of human models of disease. With this advancement, aspects of endothelial physiology that are difficult to assess with other models can be directly probed. In this study, the role of endothelial cell (EC) apicobasal polarity on leukocyte trafficking response is evaluated with the µSiM-MVM (microphysiological system enabled by a silicon membrane - microvascular mimetic). Here, ECs are stimulated either apically or basally with a cytokine cocktail to model a septic-like challenge before introducing healthy donor PMNs into the device. Basally oriented stimulation generated a stronger PMN transmigratory response versus apical stimulation. Importantly, healthy PMNs are unable to migrate towards a bacterial peptide chemoattractant when ECs are apically stimulated, which mimics the attenuated PMN chemotaxis seen in sepsis. Escalating the apical inflammatory stimulus by a factor of five is necessary to elicit high PMN transmigration levels across endothelium. These results demonstrate that EC apicobasal polarity modulates PMN transmigratory behavior and provides insight into the mechanisms underlying sepsis.

Activation effects on the physical characteristics of T lymphocytes.

The deformability of leukocytes is relevant to a wide array of physiological and pathophysiological behaviors. The goal of this study is to provide a detailed, quantitative characterization of the mechanical properties of T cells and how those properties change with activation. We tested T cells and CD8+ cells isolated from peripheral blood samples of healthy donors either immediately (naïve population) or after 7 days of activation in vitro. Single-cell micropipette aspiration was used to test the mechanical properties. T cells exhibit the general characteristics of a highly viscous liquid drop with a cortical "surface" tension, T cort. The time course of each cell entry into the micropipette was measured at two different aspiration pressures to test for shear thinning behavior. The data were analyzed in the framework of an approximate mechanical model of the cell deformation to determine the cortical tension, the cell volume, the magnitude of the initial cell entry, the characteristic viscosity µ o, and the shear thinning coefficient, b. Activation generally caused increases in cellular resistance to deformation and a broadening of the distribution of cell properties. The cell volume increased substantially upon cell activation from ∼200 µm3 to ∼650 µm3. Naive and activated T cells had similar mean cortical tension (∼150 pN/µm). However, compared to naïve CD8+ cells, the cortical tension of activated CD8+ cells increased significantly to ∼250 pN/µm. Dynamic resistance of naive CD8+ T cells, as reflected in their characteristic viscosity, was ∼870 Pa and significantly increased to 1,180 Pa after in vitro activation. The magnitude of the instantaneous projection length as the cell enters the pipette (L init) was more than doubled for activated vs. naive cells. All cell types exhibited shear thinning behavior with coefficients b in the range 0.5-0.65. Increased cell size, cortical tension, and characteristic viscosity all point to increased resistance of activated T cells to passage through the microvasculature, likely contributing to cell trapping. The increased initial elastic response of cells after activation was unexpected and could point to instability in the cell that might contribute to spontaneous cell motility.

A computer vision approach for analyzing label free leukocyte trafficking dynamics on a microvascular mimetic.

High-content imaging techniques in conjunction with in vitro microphysiological systems (MPS) allow for novel explorations of physiological phenomena with a high degree of translational relevance due to the usage of human cell lines. MPS featuring ultrathin and nanoporous silicon nitride membranes (µSiM) have been utilized in the past to facilitate high magnification phase contrast microscopy recordings of leukocyte trafficking events in a living mimetic of the human vascular microenvironment. Notably, the imaging plane can be set directly at the endothelial interface in a µSiM device, resulting in a high-resolution capture of an endothelial cell (EC) and leukocyte coculture reacting to different stimulatory conditions. The abundance of data generated from recording observations at this interface can be used to elucidate disease mechanisms related to vascular barrier dysfunction, such as sepsis. The appearance of leukocytes in these recordings is dynamic, changing in character, location and time. Consequently, conventional image processing techniques are incapable of extracting the spatiotemporal profiles and bulk statistics of numerous leukocytes responding to a disease state, necessitating labor-intensive manual processing, a significant limitation of this approach. Here we describe a machine learning pipeline that uses a semantic segmentation algorithm and classification script that, in combination, is capable of automated and label-free leukocyte trafficking analysis in a coculture mimetic. The developed computational toolset has demonstrable parity with manually tabulated datasets when characterizing leukocyte spatiotemporal behavior, is computationally efficient and capable of managing large imaging datasets in a semi-automated manner.

ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

Is Size All That Matters? New Predictors of Complications and Bleeding in Renal Angiomyolipoma.

Purpose: Renal angiomyolipoma (AML) is the most common benign renal tumor. Whilst generally asymptomatic, they can cause life-threatening bleeding. Selective angioembolization (SAE) may be used to treat large symptomatic and asymptomatic AMLs. We aimed to evaluate the efficacy of SAE for symptomatic and asymptomatic renal AMLs and determine characteristics that predict spontaneous bleeding. Patients and Methods: Data were retrospectively collected from a prospectively maintained database from July 2011 to April 2022. Patients were included if AML was >4cm and they underwent subsequent SAE. Follow-up imaging was analyzed to calculate mean reduction in AML size. Clinical notes were reviewed to analyze lesion characteristics including vascularity, fat content and presence of aneurysm as well as post-procedural complications. Results: 26 patients with 30 AMLs were identified. Interval of follow-up imaging ranged from 1 to 60 months. 25 AMLs were embolized electively with 5 emergency embolizations performed for bleeding. Mean reduction in AML volume was 41% at 3 months (p=0.013) and 63% at 12 months (p=0.007). All 5 bleeding AMLs had a rich vascularity with 60% also having either aneurysms or a low fat content. Complications included post-embolic syndrome (n=9), segmental renal parenchyma devascularization (n=3), acute bleeding requiring re-embolization (n=2), nephrectomy for ongoing bleeding (n=1) and delayed bleeding managed conservatively (n=1). No deterioration in renal function was observed. Conclusion: SAE is an effective procedure for managing symptomatic and asymptomatic renal AML, with minimal significant complications. AML vascularity, fat content and aneurysms may be useful characteristics to assess future risk of bleeding in patients with renal AML.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

The Modular µSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro.

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support "on-chip" clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, an extension of the µSiM (microdevice featuring a silicon-nitride membrane) platform is introduced. The modular µSiM (m-µSiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. The utility of the m-µSiM is demonstrated by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and nonengineering, brain barriers focused laboratories. In situ and sampling-based assays of small molecule diffusion are developed and validated as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-µSiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. The ability to quickly reconfigure the m-µSiM for coculture and immune cell transmigration studies through addition of accessories and/or quick exchange of components is then demonstrated. Because the development of modified components and accessories is easily achieved, custom designs of the m-µSiM shall be accessible to any laboratory desiring a barrier-style tissue chip platform.

Molecular mechanisms underlying the heterogeneous barrier responses of two primary endothelial cell types to sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) signals to enhance or destabilize the vascular endothelial barrier depending on the receptor engaged. Here, we investigated the differential barrier effects of S1P on two influential primary endothelial cell (EC) types, human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). S1PR1 (barrier protective) and S1PR3 (barrier disruptive) surface and gene expression were quantified by flow cytometry and immunofluorescence, and RT-qPCR, respectively. Functional evaluation of EC monolayer permeability in response to S1P was quantified with transendothelial electrical resistance (TEER) and small molecule permeability. S1P significantly enhanced HUVEC barrier function, while promoting HPMEC barrier breakdown. Immunofluorescence and flow cytometry analysis showed select, S1PR3-high HPMECs, suggesting susceptibility to barrier destabilization following S1P exposure. Reevaluation of HPMEC barrier following S1P exposure under inflamed conditions demonstrated synergistic barrier disruptive effects of pro-inflammatory cytokine and S1P. The role of the Rho-ROCK signaling pathway under these conditions was confirmed through ROCK1/2 inhibition (Y-27632). Thus, the heterogeneous responses of ECs to S1P signaling are mediated through Rho-ROCK signaling, and potentially driven by differences in the surface expression of S1PR3.

Treatment of in-transit Merkel cell carcinoma by isolated limb infusion with cytotoxic drugs.

Merkel cell carcinoma of the skin is a rare but aggressive malignancy, which predominantly affects older adults with fair skin. Isolated limb infusion (ILI) using melphalan and actinomycin D was first developed as a minimally invasive treatment option to treat unresectable metastatic melanoma confined to the limb. We report on a 62-year-old male with in-transit metastases (ITMs) treated with ILI to highlight the ongoing role this treatment has when all other therapies have been exhausted. At presentation, the patient had widespread ITMs in the right leg. Positron emission tomography scan demonstrated recurrent disease in the thigh and pelvis, and it was decided to treat the patient with ILI. The patient progressed well in the immediate post-operative period. The patient was able to mobilize from Day 6 post-ILI and was discharged on Day 10. There was an immediate clinical response seen in the lesions, with necrosis developing in the larger lesions.

Epithelioid sarcoma of the penis: A penile sparing approach, and long-term implications.

A 33-year-old male presented with a one-centimetre lesion at the penoscrotal junction which was excised and revealed to be an epithelioid sarcoma (ES). A wide local excision of the lesion and subsequent neoadjuvant radiotherapy followed, with transposition of the patient's testicles laterally to protect fertility. At 3-year follow-up, the patient has no local or distant recurrence but does have a low sperm count. The patient has also had intermittent haematospermia since his treatment for which a cause has yet to be identified. This case highlights that ES of the penis can be managed successfully with surgical excision and local radiotherapy.

An observational study to determine volume changes in the functional liver remnant following portal vein embolization.

BACKGROUND AND AIM: Portal vein embolization (PVE) prior to hepatic resection reduces the risk of hepatic insufficiency in the postoperative period by redistributing blood from the embolized unhealthy liver to the healthy liver, termed the functional liver remnant (FLR). A retrospective analysis of liver volumes after embolization in a single institution was performed to identify change in volume of the FLR and determine factors affecting this change. METHODS: Between 2013 and 2015, 21 patients undergoing PVE followed by hepatic resection for varied indications (colorectal metastases, hepatocellular carcinoma, cholangiocarcinoma, etc.) were included in this study. n-butyl cyanoacrylate glue diluted with Lipiodol (35-45% strength) along with 75-100 µm of polyvinyl alcohol particles were used for embolization. Liver volumetric determination was performed before and after PVE and volume changes in the FLR were analyzed. Biochemical factors and factors affecting FLR hypertrophy were also analyzed. RESULTS: Majority of the patients (n = 18) underwent right-lobe embolization. All were performed using the ipsilateral approach. No major complications occurred with only one patient developing post-procedural ascites requiring percutaneous draining. A significant increase in the mean volume of the FLR by 63.7% ± 91.6%, P = 0.001 was noted after PVE. The FLR/total liver volume (TLV) increased significantly by 17% ± 18%. No significant demographic factors affected FLR hypertrophy and no significant biochemical changes were noted. Thirteen patients were successfully operated on after embolization. CONCLUSIONS: PVE is effective in inducing significant hypertrophy of the future FLR, prior to hepatic resection in our institution.

Optical Control of CD8+ T Cell Metabolism and Effector Functions.

Although cancer immunotherapy is effective against hematological malignancies, it is less effective against solid tumors due in part to significant metabolic challenges present in the tumor microenvironment (TME), where infiltrated CD8+ T cells face fierce competition with cancer cells for limited nutrients. Strong metabolic suppression in the TME is often associated with impaired T cell recruitment to the tumor site and hyporesponsive effector function via T cell exhaustion. Increasing evidence suggests that mitochondria play a key role in CD8+ T cell activation, effector function, and persistence in tumors. In this study, we showed that there was an increase in overall mitochondrial function, including mitochondrial mass and membrane potential, during both mouse and human CD8+ T cell activation. CD8+ T cell mitochondrial membrane potential was closely correlated with granzyme B and IFN-γ production, demonstrating the significance of mitochondria in effector T cell function. Additionally, activated CD8+ T cells that migrate on ICAM-1 and CXCL12 consumed significantly more oxygen than stationary CD8+ T cells. Inhibition of mitochondrial respiration decreased the velocity of CD8+ T cell migration, indicating the importance of mitochondrial metabolism in CD8+ T cell migration. Remote optical stimulation of CD8+ T cells that express our newly developed "OptoMito-On" successfully enhanced mitochondrial ATP production and improved overall CD8+ T cell migration and effector function. Our study provides new insight into the effect of the mitochondrial membrane potential on CD8+ T cell effector function and demonstrates the development of a novel optogenetic technique to remotely control T cell metabolism and effector function at the target tumor site with outstanding specificity and temporospatial resolution.

Changes in endothelial glycocalyx layer protective ability after inflammatory stimulus.

Leukocyte adhesion to the endothelium is an important early step in the initiation and progression of sepsis. The endothelial glycocalyx layer (EGL) has been implicated in neutrophil adhesion and barrier dysfunction, but studies in this area are few. In this report, we examine the hypothesis that damage to the structure of the EGL caused by inflammation leads to increased leukocyte adhesion and endothelial barrier dysfunction. We used human umbilical vein endothelial cells enzymatically treated to remove the EGL components hyaluronic acid (HA) and heparan sulfate (HS) as a model for EGL damage. Using atomic force microscopy, we show reductions in EGL thickness after removal of either HA or HS individually, but the largest decrease, comparable with TNF-α treatment, was observed when both HA and HS were removed. Interestingly, removal of HS or HA individually did not affect neutrophil adhesion significantly, but removal of both constituents resulted in increased neutrophil adhesion. To test EGL contributions to endothelial barrier properties, we measured transendothelial electrical resistance (TEER) and diffusion of fluorescently labeled dextran (10 kDa molecular weight) across the monolayer. Removal of EGL components decreased TEER but had an insignificant effect on dextran diffusion rates. The reduction in TEER suggests that disruption of the EGL may predispose endothelial cells to increased rates of fluid leakage. These data support the view that damage to the EGL during inflammation has significant effects on the accessibility of adhesion molecules, likely facilitates leukocyte adhesion, and may also contribute to increased rates of fluid transport into tissues.

Development of Mechanical Stability in Late-Stage Embryonic Erythroid Cells: Insights From Fluorescence Imaged Micro-Deformation Studies.

The combined use of fluorescence labeling and micro-manipulation of red blood cells has proven to be a powerful tool for understanding and characterizing fundamental mechanisms underlying the mechanical behavior of cells. Here we used this approach to study the development of the membrane-associated cytoskeleton (MAS) in primary embryonic erythroid cells. Erythropoiesis comes in two forms in the mammalian embryo, primitive and definitive, characterized by intra- and extra-vascular maturation, respectively. Primitive erythroid precursors in the murine embryo first begin to circulate at embryonic day (E) 8.25 and mature as a semi-synchronous cohort before enucleating between E12.5 and E16.5. Previously, we determined that the major components of the MAS become localized to the membrane between E10.5 and E12.5, and that this localization is associated with an increase in membrane mechanical stability over this same period. The change in mechanical stability was reflected in the creation of MAS-free regions of the membrane at the tips of the projections formed when cells were aspirated into micropipettes. The tendency to form MAS-free regions decreases as primitive erythroid cells continue to mature through E14.5, at least 2 days after all detectable cytoskeletal components are localized to the membrane, indicating continued strengthening of membrane cohesion after membrane localization of cytoskeletal components. Here we demonstrate that the formation of MAS-free regions is the result of a mechanical failure within the MAS, and not the detachment of membrane bilayer from the MAS. Once a "hole" is formed in the MAS, the skeletal network contracts laterally along the aspirated projection to form the MAS-free region. In protein 4.1-null primitive erythroid cells, the tendency to form MAS-free regions is markedly enhanced. Of note, similar MAS-free regions were observed in maturing erythroid cells from human marrow, indicating that similar processes occur in definitive erythroid cells. We conclude that localization of cytoskeletal components to the cell membrane of mammalian erythroid cells during maturation is insufficient by itself to produce a mature MAS, but that subsequent processes are additionally required to strengthen intraskeletal interactions.

Endothelial cell apicobasal polarity coordinates distinct responses to luminally versus abluminally delivered TNF-α in a microvascular mimetic.

Endothelial cells (ECs) are an active component of the immune system and interact directly with inflammatory cytokines. While ECs are known to be polarized cells, the potential role of apicobasal polarity in response to inflammatory mediators has been scarcely studied. Acute inflammation is vital in maintaining healthy tissue in response to infection; however, chronic inflammation can lead to the production of systemic inflammatory cytokines and deregulated leukocyte trafficking, even in the absence of a local infection. Elevated levels of cytokines in circulation underlie the pathogenesis of sepsis, the leading cause of intensive care death. Because ECs constitute a key barrier between circulation (luminal interface) and tissue (abluminal interface), we hypothesize that ECs respond differentially to inflammatory challenge originating in the tissue versus circulation as in local and systemic inflammation, respectively. To begin this investigation, we stimulated ECs abluminally and luminally with the inflammatory cytokine tumor necrosis factor alpha (TNF-α) to mimic a key feature of local and systemic inflammation, respectively, in a microvascular mimetic (µSiM-MVM). Polarized IL-8 secretion and polymorphonuclear neutrophil (PMN) transmigration were quantified to characterize the EC response to luminal versus abluminal TNF-α. We observed that ECs uniformly secrete IL-8 in response to abluminal TNF-α and is followed by PMN transmigration. The response to abluminal treatment was coupled with the formation of ICAM-1-rich membrane ruffles on the apical surface of ECs. In contrast, luminally stimulated ECs secreted five times more IL-8 into the luminal compartment than the abluminal compartment and sequestered PMNs on the apical EC surface. Our results identify clear differences in the response of ECs to TNF-α originating from the abluminal versus luminal side of a monolayer for the first time and may provide novel insight into future inflammatory disease intervention strategies.

Constitutive Model of Erythrocyte Membranes with Distributions of Spectrin Orientations and Lengths.

We present an analytical hyperelastic constitutive model of the red blood cell (erythrocyte) membrane based on recently improved characterizations of density and microscopic structure of its spectrin network from proteomics and cryo-electron tomography. The model includes distributions of both orientations and natural lengths of spectrin and updated copy numbers of proteins. By applying finite deformation to the spectrin network, we obtain the total free energy and stresses in terms of invariants of shear and area deformation. We generalize an expression of the initial shear modulus, which is independent of the number of molecular orientations within the network and also derive a simplified version of the model. We apply the model and its simplified version to analyze micropipette aspiration computationally and analytically and explore the effect of local cytoskeletal density change. We also explore the discrepancies among shear modulus values measured using different experimental techniques reported in the literature. We find that the model exhibits hardening behavior and can explain many of these discrepancies. Moreover, we find that the distribution of natural lengths plays a crucial role in the hardening behavior when the correct copy numbers of proteins are used. The initial shear modulus values we obtain using our current model (5.9-15.6 pN/µm) are close to the early estimates (6-9 pN/µm). This new, to our knowledge, constitutive model establishes a direct connection between the molecular structure of spectrin networks and constitutive laws and also defines a new picture of a much denser spectrin network than assumed in prior studies.

Endovascular management of recurrent hemarthrosis of the knee: a case series.

BACKGROUND: Recurrent hemarthrosis of the knee is an uncommon but potentially debilitating occurrence with multiple etiologies, including previous total knee replacement. The purpose of this study is to present data of a group of patients undergoing angiography and embolization for recurrent hemarthrosis of the knee. Patient characteristics, angiographic findings, safety and efficacy of the procedure are reported. METHODS: A retrospective single centre review of patients undergoing angiography and embolization at a tertiary referral centre in Sydney, Australia from March 2006 to April 2018 was performed. A total of 25 patients undergoing a total of 29 procedures were identified (20 female, 5 male; mean age 67), the majority of which (23/25, 92%) had a history of total knee arthroplasty. Embolization was performed in 28 of the 29 procedures (97%). The embolic agent used was either polyvinyl alcohol particles (23/28), gelatin foam (3/28), detachable microcoils (1/28) or a combination of particles and coils (1/28). RESULTS: The most commonly identified dominant vascular abnormality was periarticular synovial hypervascularity (23/25, 92%). A pseudoaneurysm was demonstrated in two patients (8%). Technical success (elimination of angiographic abnormalities) was achieved in 27 of 29 procedures (93%). There were 6 episodes of recurrence (25%) following a single embolization procedure, three of which were managed successfully with repeat embolization. There were no complications relating to skin or periarticular ischemia. CONCLUSION: Angiography and embolization is a safe and effective tool for the management of recurrent hemarthrosis of the knee following arthroplasty and should be considered first line treatment following failure of conservative management. LEVEL OF EVIDENCE: Level 4, Case Series.

Endothelial Glycocalyx Layer Properties and Its Ability to Limit Leukocyte Adhesion.

The endothelial glycocalyx layer (EGL), which consists of long proteoglycans protruding from the endothelium, acts as a regulator of inflammation by preventing leukocyte engagement with adhesion molecules on the endothelial surface. The amount of resistance to adhesive events the EGL provides is the result of two properties: EGL thickness and stiffness. To determine these, we used an atomic force microscope to indent the surfaces of cultured endothelial cells with a glass bead and evaluated two different approaches for interpreting the resulting force-indentation curves. In one, we treat the EGL as a molecular brush, and in the other, we treat it as a thin elastic layer on an elastic half-space. The latter approach proved more robust in our hands and yielded a thickness of 110 nm and a modulus of 0.025 kPa. Neither value showed significant dependence on indentation rate. The brush model indicated a larger layer thickness (∼350 nm) but tended to result in larger uncertainties in the fitted parameters. The modulus of the endothelial cell was determined to be 3.0-6.5 kPa (1.5-2.5 kPa for the brush model), with a significant increase in modulus with increasing indentation rates. For forces and leukocyte properties in the physiological range, a model of a leukocyte interacting with the endothelium predicts that the number of molecules within bonding range should decrease by an order of magnitude because of the presence of a 110-nm-thick layer and even further for a glycocalyx with larger thickness. Consistent with these predictions, neutrophil adhesion increased for endothelial cells with reduced EGL thickness because they were grown in the absence of fluid shear stress. These studies establish a framework for understanding how glycocalyx layers with different thickness and stiffness limit adhesive events under homeostatic conditions and how glycocalyx damage or removal will increase leukocyte adhesion potential during inflammation.

Microvascular Mimetics for the Study of Leukocyte-Endothelial Interactions.

INTRODUCTION: The pathophysiological increase in microvascular permeability plays a well-known role in the onset and progression of diseases like sepsis and atherosclerosis. However, how interactions between neutrophils and the endothelium alter vessel permeability is often debated. METHODS: In this study, we introduce a microfluidic, silicon-membrane enabled vascular mimetic (µSiM-MVM) for investigating the role of neutrophils in inflammation-associated microvascular permeability. In utilizing optically transparent silicon nanomembrane technology, we build on previous microvascular models by enabling in situ observations of neutrophil-endothelium interactions. To evaluate the effects of neutrophil transmigration on microvascular model permeability, we established and validated electrical (transendothelial electrical resistance and impedance) and small molecule permeability assays that allow for the in situ quantification of temporal changes in endothelium junctional integrity. RESULTS: Analysis of neutrophil-expressed ß1 integrins revealed a prominent role of neutrophil transmigration and basement membrane interactions in increased microvascular permeability. By utilizing blocking antibodies specific to the ß1 subunit, we found that the observed increase in microvascular permeability due to neutrophil transmigration is constrained when neutrophil-basement membrane interactions are blocked. Having demonstrated the value of in situ measurements of small molecule permeability, we then developed and validated a quantitative framework that can be used to interpret barrier permeability for comparisons to conventional Transwell™ values. CONCLUSIONS: Overall, our results demonstrate the potential of the µSiM-MVM in elucidating mechanisms involved in the pathogenesis of inflammatory disease, and provide evidence for a role for neutrophils in inflammation-associated endothelial barrier disruption.

When the Conventional Treatment Fails: A Rare Approach to Superior Mesenteric Arterial Embolization.

Retroperitoneal haemorrhage is a rare but potentially life-threatening event. It may occur either spontaneously or secondary to percutaneous vascular access procedures, trauma, or ruptured aortic, iliac, or mesenteric aneurysms. As a result, the clinical presentation is variable. Computed tomography and/or angiography are vital for diagnosis. Management may range from conservative treatment for stable patients to emergency laparotomy or embolization for catastrophic haemorrhage. Direct percutaneous puncture of a deep intra-abdominal pseudoaneurysm is an accepted but infrequently performed technique due to a number of diagnostic and technical challenges. We describe the successful percutaneous transabdominal angioembolization of a superior mesenteric artery rupture in a 77-year-old woman with a large retroperitoneal haematoma. This was performed after a conventional femoral transarterial approach was unsuccessful.