Mechanisms of triple-negative breast cancer extravasation: Impact of the physical environment and endothelial glycocalyx.

Cancer metastasis is the leading cause of death for those afflicted with cancer. In cancer metastasis, the cancer cells break off from the primary tumor, penetrate nearby blood vessels, and attach and extravasate out of the vessels to form secondary tumors at distant organs. This makes extravasation a critical step of the metastatic cascade. Herein, with a focus on triple-negative breast cancer, the role that the prospective secondary tumor microenvironment's mechanical properties play in circulating tumor cells' extravasation is reviewed. Specifically, the effects of the physically regulated vascular endothelial glycocalyx barrier element, vascular flow factors, and subendothelial extracellular matrix mechanical properties on cancer cell extravasation are examined. The ultimate goal of this review is to clarify the physical mechanisms that drive triple-negative breast cancer extravasation, as these mechanisms may be potential new targets for anti-metastasis therapy.

Endothelial Dysfunction in Atherosclerotic Cardiovascular Diseases and Beyond: From Mechanism to Pharmacotherapies.

The endothelium, a cellular monolayer lining the blood vessel wall, plays a critical role in maintaining multiorgan health and homeostasis. Endothelial functions in health include dynamic maintenance of vascular tone, angiogenesis, hemostasis, and the provision of an antioxidant, anti-inflammatory, and antithrombotic interface. Dysfunction of the vascular endothelium presents with impaired endothelium-dependent vasodilation, heightened oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Recent studies have implicated altered endothelial cell metabolism and endothelial-to-mesenchymal transition as new features of endothelial dysfunction. Endothelial dysfunction is regarded as a hallmark of many diverse human panvascular diseases, including atherosclerosis, hypertension, and diabetes. Endothelial dysfunction has also been implicated in severe coronavirus disease 2019. Many clinically used pharmacotherapies, ranging from traditional lipid-lowering drugs, antihypertensive drugs, and antidiabetic drugs to proprotein convertase subtilisin/kexin type 9 inhibitors and interleukin 1ß monoclonal antibodies, counter endothelial dysfunction as part of their clinical benefits. The regulation of endothelial dysfunction by noncoding RNAs has provided novel insights into these newly described regulators of endothelial dysfunction, thus yielding potential new therapeutic approaches. Altogether, a better understanding of the versatile (dys)functions of endothelial cells will not only deepen our comprehension of human diseases but also accelerate effective therapeutic drug discovery. In this review, we provide a timely overview of the multiple layers of endothelial function, describe the consequences and mechanisms of endothelial dysfunction, and identify pathways to effective targeted therapies. SIGNIFICANCE STATEMENT: The endothelium was initially considered to be a semipermeable biomechanical barrier and gatekeeper of vascular health. In recent decades, a deepened understanding of the biological functions of the endothelium has led to its recognition as a ubiquitous tissue regulating vascular tone, cell behavior, innate immunity, cell-cell interactions, and cell metabolism in the vessel wall. Endothelial dysfunction is the hallmark of cardiovascular, metabolic, and emerging infectious diseases. Pharmacotherapies targeting endothelial dysfunction have potential for treatment of cardiovascular and many other diseases.

Atherosclerosis and endothelial mechanotransduction: current knowledge and models for future research.

Endothelium health is essential to the regulation of physiological vascular functions. Because of the critical capability of endothelial cells (ECs) to sense and transduce chemical and mechanical signals in the local vascular environment, their dysfunction is associated with a vast variety of vascular diseases and injuries, especially atherosclerosis and subsequent cardiovascular diseases. This review describes the mechanotransduction events that are mediated through ECs, the EC subcellular components involved, and the pathways reported to be potentially involved. Up-to-date research efforts involving in vivo animal models and in vitro biomimetic models are also discussed, including their advantages and drawbacks, with recommendations on future modeling approaches to aid the development of novel therapies targeting atherosclerosis and related cardiovascular diseases.

Diosmin and its glycocalyx restorative and anti-inflammatory effects on injured blood vessels.

The endothelium, a crucial homeostatic organ, regulates vascular permeability and tone. Under physiological conditions, endothelial stimulation induces vasodilator endothelial nitric oxide (eNO) release and prevents adhesion molecule accessibility and leukocyte adhesion and migration into vessel walls. Endothelium dysfunction is a principal event in cardiovascular disorders, including atherosclerosis. Minimal attention is given to an important endothelial cell structure, the endothelial glycocalyx (GCX), a negatively charged heterogeneous polysaccharide that serves as a protective covering for endothelial cells and enables endothelial cells to transduce mechanical stimuli into various biological and chemical activities. Endothelial GCX shedding thus plays a role in endothelial dysfunction, for example by increasing vascular permeability and decreasing vessel tone. Consequently, there is increasing interest in developing therapies that focus on GCX repair to limit downstream endothelium dysfunction and prevent further downstream cardiovascular events. Here, we present diosmin (3',5,7-trihydroxy-4'-methoxyflavone-7-rhamnoglucoside), a flavone glycoside of diosmetin, which downregulates adhesive molecule expression, decreases inflammation and capillary permeability, and upregulates eNO expression. Due to these pleiotropic effects of diosmin on the vasculature, a possible unidentified mechanism of action is through GCX restoration. We hypothesize that diosmin positively affects GCX integrity along with GCX-related endothelial functions. Our hypothesis was tested in a partial ligation left carotid artery (LCA) mouse model, where the right carotid artery was the control for each mouse. Diosmin (50 mg/kg) was administered daily for 7 days, 72 h after ligation. Within the ligated mice LCAs, diosmin treatment elevated the activated eNO synthase level, inhibited inflammatory cell uptake, decreased vessel wall thickness, increased vessel diameter, and increased GCX coverage of the vessel wall. ELISA showed a decrease in hyaluronan concentration in plasma samples of diosmin-treated mice, signifying reduced GCX shedding. In summary, diosmin supported endothelial GCX integrity, to which we attribute diosmin's preservation of endothelial function as indicated by attenuated expression of inflammatory factors and restored vascular tone.

Imaging the effect of the circadian light-dark cycle on the glymphatic system in awake rats.

The glymphatic system functions in the removal of potentially harmful metabolites and proteins from the brain. Dynamic, contrast-enhanced MRI was used in fully awake rats to follow the redistribution of intraventricular contrast agent entrained to the light-dark cycle and its hypothetical relationship to the sleep-waking cycle, blood flow, and brain temperature in specific brain areas. Brain areas involved in circadian timing and sleep-wake rhythms showed the lowest redistribution of contrast agent during the light phase or time of inactivity and sleep in rats. Global brain redistribution of contrast agent was heterogeneous. The redistribution was highest along the dorsal cerebrum and lowest in the midbrain/pons and along the ventral surface of the brain. This heterogeneous redistribution of contrast agent paralleled the gradients and regional variations in brain temperatures reported in the literature for awake animals. Three-dimensional quantitative ultrashort time-to-echo contrast-enhanced imaging was used to reconstruct small, medium, and large arteries and veins in the rat brain and revealed areas of lowest redistribution overlapped with this macrovasculature. This study raises new questions and theoretical considerations of the impact of the light-dark cycle, brain temperature, and blood flow on the function of the glymphatic system.

Flow-regulated endothelial glycocalyx determines metastatic cancer cell activity.

Cancer metastasis and secondary tumor initiation largely depend on circulating tumor cell (CTC) and vascular endothelial cell (EC) interactions by incompletely understood mechanisms. Endothelial glycocalyx (GCX) dysfunction may play a significant role in this process. GCX structure depends on vascular flow patterns, which are irregular in tumor environments. This work presents evidence that disturbed flow (DF) induces GCX degradation, leading to CTC homing to the endothelium, a first step in secondary tumor formation. A 2-fold greater attachment of CTCs to human ECs was found to occur under DF conditions, compared to uniform flow (UF) conditions. These results corresponded to an approximately 50% decrease in wheat germ agglutinin (WGA)-labeled components of the GCX under DF conditions, vs UF conditions, with undifferentiated levels of CTC-recruiting E-selectin under DF vs UF conditions. Confirming the role of the GCX, neuraminidase induced the degradation of WGA-labeled GCX under UF cell culture conditions or in Balb/C mice and led to an over 2-fold increase in CTC attachment to ECs or Balb/C mouse lungs, respectively, compared to untreated conditions. These experiments confirm that flow-induced GCX degradation can enable metastatic CTC arrest. This work, therefore, provides new insight into pathways of secondary tumor formation.

Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression, localization, and function via glycocalyx degradation.

BACKGROUND: Endothelial-dependent atherosclerosis develops in a non-random pattern in regions of vessel bending and bifurcations, where blood flow exhibits disturbed flow (DF) patterns. In contrast, uniform flow (UF), normal endothelium, and healthy vessel walls co-exist within straight vessels. In clarifying how flow protectively or atherogenically regulates endothelial cell behavior, involvement of the endothelial surface glycocalyx has been suggested due to reduced expression in regions of atherosclerosis development. Here, we hypothesized that pro-atherosclerotic endothelial dysfunction occurs as a result of DF-induced reduction in glycocalyx expression and subsequently impairs endothelial sensitivity to flow. Specifically, we propose that glycocalyx degradation can induce pro-atherosclerotic endothelial dysfunction through decreased caveolin-1 and endothelial nitric oxide synthase expression and localization. METHODS: We studied endothelial cells in atherosclerotic-prone DF and atherosclerotic-resistant UF conditions in parallel plate flow culture and in C57Bl/6 mice. The effects of flow conditioning on endothelial cell behavior were quantified using immunocytochemistry. The glycocalyx was fluorescently labeled for wheat germ agglutinin, which serves as a general glycocalyx label, and heparan sulfate, a major glycocalyx component. Additionally, mechanosensitivity was assessed by immunocytochemical fluorescence expression and function of caveolin-1, the protein that forms the mechanosignaling caveolar invaginations on the endothelial surface, total endothelial-type nitric oxide synthase (eNOS), which synthesizes nitric oxide, and serine 1177 phosphorylated eNOS (eNOS-pS1177), which is the active form of eNOS. Caveolin function and eNOS expression and activation were correlated to glycocalyx expression. Heparinase III enzyme was used to degrade a major glycocalyx component, HS, to identify the role of the glycocalyx in caveoin-1 and eNOS-pS1177 regulation. RESULTS: Results confirmed that DF reduces caveolin-1 expression and abolishes most of its subcellular localization preferences, when compared to the effect of UF. DF down-regulates caveolin-1 mechanosignaling, as indicated by its reduced colocalization with serine 1177 phosphorylated endothelial-type nitric oxide synthase (eNOS-pS1177), a vasoregulatory signaling molecule whose activity is regulated by its residence in caveolae. As expected, DF inhibited glycocalyx expression compared to UF. In the absence of heparan sulfate, a major glycocalyx component, UF-conditioned endothelial cells exhibited near DF-like caveolin-1 expression, localization, and colocalization with eNOS-pS1177. CONCLUSIONS: This is the first demonstration of a flow-defined role of the glycocalyx in caveolae expression and function related to vasculoprotective endothelial mechanosensitivity that defends against atherosclerosis. The results suggest that a glycocalyx-based therapeutic targeted to areas of atherosclerosis development could prevent disease initiation and progression.

Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1, PI3K, FAK, and p38.

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5 h to examine shear stress-induced induction of COX-2/PGI2 Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5ß1-integrin, upregulation of COX-2, and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of α5ß1-integrin, upregulation of COX-2 gene and protein expression, and release of PGI2 in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1-/- mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI2 levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI2 release compared with wild-type animals.NEW & NOTEWORTHY In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I2 (PGI2) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI2 mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1).

Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target.

PURPOSE OF REVIEW: The cell surface-attached extracellular glycocalyx (GCX) layer is a major contributor to endothelial cell (EC) function and EC-dependent vascular health and is a first line of defense against vascular diseases including atherosclerosis. Here, we highlight our findings regarding three GCX-dependent EC functions, which are altered when GCX is shed and in atherosclerosis. We discuss why the GCX is a viable option for the prevention and treatment of atherosclerosis. RECENT FINDINGS: GCX regulated EC activities such as barrier and filtration function, active cell-to-cell communication, and vascular tone mediation contribute to function of the entire vascular wall. Atheroprone vessel regions, including bifurcation sites, exhibit breakdown in GCX. This GCX degradation allows increased lipid flux and thereby promotes lipid deposition in the vessel walls, a hallmark of atherosclerosis. GCX degradation also alters EC-to-EC communication while increasing EC-to-inflammatory cell interactions that enable inflammatory cells to migrate into the vessel wall. Inflammatory macrophages and foam cells, to be specific, appear in early stages of atherosclerosis. Furthermore, GCX degradation deregulates vascular tone, by causing ECs to reduce their expression of endothelial nitric oxide synthase (eNOS) which produces the vasodilator, nitric oxide. Loss of vasodilation supports vasoconstriction, which promotes the progression of atherosclerosis. Common medicinal atherosclerosis therapies include lipid lowering and anti-platelet therapies. None of these treatments specifically target the endothelial GCX, although the GCX is at the front-line in atherosclerosis combat. This review demonstrates the viability of targeting the GCX therapeutically, to support proper EC functionality and prevent and/or treat atherosclerosis.

Unraveling neurovascular mysteries: the role of endothelial glycocalyx dysfunction in Alzheimer's disease pathogenesis.

While cardiovascular disease, cancer, and human immunodeficiency virus (HIV) mortality rates have decreased over the past 20 years, Alzheimer's Disease (AD) deaths have risen by 145% since 2010. Despite significant research efforts, effective AD treatments remain elusive due to a poorly defined etiology and difficulty in targeting events that occur too downstream of disease onset. In hopes of elucidating alternative treatment pathways, now, AD is commonly being more broadly defined not only as a neurological disorder but also as a progression of a variety of cerebrovascular pathologies highlighted by the breakdown of the blood-brain barrier. The endothelial glycocalyx (GCX), which is an essential regulator of vascular physiology, plays a crucial role in the function of the neurovascular system, acting as an essential vascular mechanotransducer to facilitate ultimate blood-brain homeostasis. Shedding of the cerebrovascular GCX could be an early indication of neurovascular dysfunction and may subsequently progress neurodegenerative diseases like AD. Recent advances in in vitro modeling, gene/protein silencing, and imaging techniques offer new avenues of scrutinizing the GCX's effects on AD-related neurovascular pathology. Initial studies indicate GCX degradation in AD and other neurodegenerative diseases and have begun to demonstrate a possible link to GCX loss and cerebrovascular dysfunction. This review will scrutinize the GCX's contribution to known vascular etiologies of AD and propose future work aimed at continuing to uncover the relationship between GCX dysfunction and eventual AD-associated neurological deterioration.

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses.

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer.

Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

Specificity in the participation of connexin proteins in flow-induced endothelial gap junction communication.

Endothelial cell (EC) dysfunction and atherosclerotic plaque formation coincide with human circulatory regions where blood flow is altered (disturbed). In areas of undisturbed uniform blood flow, including the majority of the vasculature, the vessel wall is relatively atherosclerotic lesion-resistant with normal endothelium. The molecular mechanisms of blood flow regulation of EC function and atherogenesis are unclear. We hypothesize that EC dysfunction potentiating atherosclerosis is related to disturbed flow (DF)-induced EC gap junctional intercellular communication (GJIC) changes via the gap junction connexin (Cx) 37, 40, and 43 proteins, which are involved in EC proliferation and vasoactivity that are known to be altered in atherosclerosis. We investigated human EC GJIC using an in vitro model of the hemodynamic features found in atherosclerotic-prone DF regions in vivo. Using dye transfer assays, Cx-specific mimetic peptide inhibitors, proliferation assays, and immunocytochemistry, we correlated functional GJIC via gap junction channels formed by hemichannels composed of the two most abundant endothelial Cx-Cx40 and Cx43-to EC proliferation and expression of vasoactive endothelial-type nitric oxide synthase (eNOS). We found that, in uniform flow conditions, substantial GJIC was conducted through gap junctions containing Cx40 hemichannels and correlated to a nonproliferative EC phenotype and membrane localization of eNOS, similar to physiological conditions. In DF, GJIC was largely attained through Cx43 hemichannel-containing gap junctions, EC phenotype was proliferative (attributed to loss of contact inhibition), and intracellular eNOS was more abundant than membrane eNOS, typical of atherosclerotic sites in vivo. This is the first in vitro study to demonstrate local hemodynamically defined Cx protein specificity in human EC GJIC with a potential role in endothelial dysfunction characteristic of early atherosclerosis.

Fluid shear stress induces the clustering of heparan sulfate via mobility of glypican-1 in lipid rafts.

The endothelial glycocalyx plays important roles in mechanotransduction. We recently investigated the distribution and interaction of glycocalyx components on statically cultured endothelial cells. In the present study, we further explored the unknown organization of the glycocalyx during early exposure (first 30 min) to shear stress and tested the hypothesis that proteoglycans with glycosaminoglycans, which are localized in different lipid microdomains, respond distinctly to shear stress. During the initial 30 min of exposure to shear stress, the very early responses of the glycocalyx and membrane rafts were detected using confocal microscopy. We observed that heparan sulfate (HS) and glypican-1 clustered in the cell junctions. In contrast, chondroitin sulfate (CS), bound albumin, and syndecan-1 did not move. The caveolae marker caveolin-1 did not move, indicating that caveolae are anchored sufficiently to resist shear stress during the 30 min of exposure. Shear stress induced significant changes in the distribution of ganglioside GM1 (a marker for membrane rafts labeled with cholera toxin B subunit). These data suggest that fluid shear stress induced the cell junctional clustering of lipid rafts with their anchored glypican-1 and associated HS. In contrast, the mobility of CS, transmembrane bound syndecan-1, and caveolae were constrained during exposure to shear stress. This study illuminates the role of changes in glycocalyx organization that underlie mechanisms of mechanotransduction.

Endothelial glycocalyx sensitivity to chemical and mechanical sub-endothelial substrate properties.

Glycocalyx (GCX) is a carbohydrate-rich structure that coats the surface of endothelial cells (ECs) and lines the blood vessel lumen. Mechanical perturbations in the vascular environment, such as blood vessel stiffness, can be transduced and sent to ECs through mechanosensors such as GCX. Adverse stiffness alters GCX-mediated mechanotransduction and leads to EC dysfunction and eventually atherosclerotic cardiovascular diseases. To understand GCX-regulated mechanotransduction events, an in vitro model emulating in vivo vessel conditions is needed. To this end, we investigated the impact of matrix chemical and mechanical properties on GCX expression via fabricating a tunable non-swelling matrix based on the collagen-derived polypeptide, gelatin. To study the effect of matrix composition, we conducted a comparative analysis of GCX expression using different concentrations (60-25,000 µg/mL) of gelatin and gelatin methacrylate (GelMA) in comparison to fibronectin (60 µg/mL), a standard coating material for GCX-related studies. Using immunocytochemistry analysis, we showed for the first time that different substrate compositions and concentrations altered the overall GCX expression on human umbilical vein ECs (HUVECs). Subsequently, GelMA hydrogels were fabricated with stiffnesses of 2.5 and 5 kPa, representing healthy vessel tissues, and 10 kPa, corresponding to diseased vessel tissues. Immunocytochemistry analysis showed that on hydrogels with different levels of stiffness, the GCX expression in HUVECs remained unchanged, while its major polysaccharide components exhibited dysregulation in distinct patterns. For example, there was a significant decrease in heparan sulfate expression on pathological substrates (10 kPa), while sialic acid expression increased with increased matrix stiffness. This study suggests the specific mechanisms through which GCX may influence ECs in modulating barrier function, immune cell adhesion, and mechanotransduction function under distinct chemical and mechanical conditions of both healthy and diseased substrates.

Imaging the endothelial glycocalyx in vitro by rapid freezing/freeze substitution transmission electron microscopy.

OBJECTIVE: Recent publications questioned the validity of endothelial cell (EC) culture studies of glycocalyx (GCX) function because of findings that GCX in vitro may be substantially thinner than GCX in vivo. The assessment of thickness differences is complicated by GCX collapse during dehydration for traditional electron microscopy. We measured in vitro GCX thickness using rapid freezing/freeze substitution (RF/FS) transmission electron microscopy (TEM), taking advantage of the high spatial resolution provided by TEM and the capability to stably preserve the GCX in its hydrated configuration by RF/FS. METHODS AND RESULTS: Bovine aortic EC (BAEC) and rat fat pad EC were subjected to conventional or RF/FS-TEM. Conventionally preserved BAEC GCX was ≈0.040 µm in thickness. RF/FS-TEM revealed impressively thick BAEC GCX of ≈11 µm and rat fat pad EC GCX of ≈5 µm. RF/FS-TEM also discerned GCX structure and thickness variations due to heparinase III enzyme treatment and extracellular protein removal, respectively. Immunoconfocal studies confirmed that the in vitro GCX is several micrometers thick and is composed of extensive and well-integrated heparan sulfate, hyaluronic acid, and protein layers. CONCLUSIONS: New observations by RF/FS-TEM reveal substantial GCX layers on cultured EC, supporting their continued use for fundamental studies of GCX and its function in the vasculature.

Developing a transwell millifluidic device for studying blood-brain barrier endothelium.

Blood-brain barrier (BBB) endothelial cell (EC) function depends on flow conditions and on supportive cells, like pericytes and astrocytes, which have been shown to be both beneficial and detrimental for brain EC function. Most studies investigating BBB EC function lack physiological relevance, using sub-physiological shear stress magnitudes and/or omitting pericytes and astrocytes. In this study, we developed a millifluidic device compatible with standard transwell inserts to investigate BBB function. In contrast to standard polydimethylsiloxane (PDMS) microfluidic devices, this model allows for easy, reproducible shear stress exposure without common limitations of PDMS devices such as inadequate nutrient diffusion and air bubble formation. In no-flow conditions, we first used the device to examine the impact of primary human pericytes and astrocytes on human brain microvascular EC (HBMEC) barrier integrity. Astrocytes, pericytes, and a 1-to-1 ratio of both cell types increased HBMEC barrier integrity via reduced 3 and 40 kDa fluorescent dextran permeability and increased claudin-5 expression. There were differing levels of low 3 kDa permeability in HBMEC-pericyte, HBMEC-astrocyte, and HBMEC-astrocyte-pericyte co-cultures, while levels of low 40 kDa permeability were consistent across co-cultures. The 3 kDa findings suggest that pericytes provide more barrier support to the BBB model compared to astrocytes, although both supportive cell types are permeability reducers. Incorporation of 24-hour 12 dynes per cm2 flow significantly reduced dextran permeability in HBMEC monolayers, but not in the tri-culture model. These results indicate that tri-culture may exert more pronounced impact on overall BBB permeability than flow exposure. In both cases, monolayer and tri-culture, flow exposure interestingly reduced HBMEC expression of both claudin-5 and occludin. ZO-1 expression, and localization at cell-cell junctions increased in the tri-culture but exhibited no apparent change in the HBMEC monolayer. Under flow conditions, we also observed HBMEC alignment in the tri-culture but not in HBMEC monolayers, indicating supportive cells and flow are both essential to observe brain EC alignment in vitro. Collectively, these results support the necessity of physiologically relevant, multicellular BBB models when investigating BBB EC function. Consideration of the roles of shear stress and supportive cells within the BBB is critical for elucidating the physiology of the neurovascular unit.

Endothelial Glycocalyx-Mediated Intercellular Interactions: Mechanisms and Implications for Atherosclerosis and Cancer Metastasis.

PURPOSE: The endothelial glycocalyx (GCX) plays a critical role in the health of the vascular system. Degradation of the GCX has been implicated in the onset of diseases like atherosclerosis and cancer because it disrupts endothelial cell (EC) function that is meant to protect from atherosclerosis and cancer. Examples of such EC function include interendothelial cell communication via gap junctions and receptor-mediated interactions between endothelial and tumor cells. This review focuses on GCX-dependent regulation of these intercellular interactions in healthy and diseased states. The ultimate goal is to build new knowledge that can be applied to developing GCX regeneration strategies that can control intercellular interaction in order to combat the progression of diseases such as atherosclerosis and cancer. METHODS: In vitro and in vivo studies were conducted to determine the baseline expression of GCX in physiologically relevant conditions. Chemical and mechanical GCX degradation approaches were employed to degrade the GCX. The impact of intact versus degraded GCX on intercellular interactions was assessed using cytochemistry, histochemistry, a Lucifer yellow dye transfer assay, and confocal, intravital, and scanning electron microscopy techniques. RESULTS: Relevant to atherosclerosis, we found that GCX stability determines the expression and functionality of Cx43 in gap junction-mediated EC-to-EC communication. Relevant to cancer metastasis, we found that destabilizing the GCX through either disturbed flow-induced or enzyme induced GCX degradation results in increased E-selectin receptor-mediated EC-tumor cell interactions. CONCLUSION: Our findings lay a foundation for future endothelial GCX-targeted therapy, to control intercellular interactions and limit the progression of atherosclerosis and cancer.

Quantitative Imaging of Blood-Brain Barrier Permeability Following Repetitive Mild Head Impacts.

This was an exploratory study designed to evaluate the feasibility of a recently established imaging modality, quantitative ultrashort time-to-echo contrast enhanced (QUTE-CE), to follow the early pathology and vulnerability of the blood brain barrier in response to single and repetitive mild head impacts. A closed-head, momentum exchange model was used to produce three consecutive mild head impacts aimed at the forebrain separated by 24 h each. Animals were measured at baseline and within 1 h of impact. Anatomical images were collected to assess the extent of structural damage. QUTE-CE biomarkers for BBB permeability were calculated on 420,000 voxels in the brain and were registered to a bilateral 3D brain atlas providing site-specific information on 118 anatomical regions. Blood brain barrier permeability was confirmed by extravasation of labeled dextran. All head impacts occurred in the absence of any structural brain damage. A single mild head impact had measurable effects on blood brain barrier permeability and was more significant after the second and third impacts. Affected regions included the prefrontal ctx, basal ganglia, hippocampus, amygdala, and brainstem. Our findings support the concerns raised by the healthcare community regarding mild head injuries in participants in organized contact sports and military personnel in basic training and combat.

Mild repetitive head impacts alter perivascular flow in the midbrain dopaminergic system in awake rats.

Head injury is a known risk factor for Parkinson's disease. Disruption in the perivascular clearance of metabolic waste and unwanted proteins is thought to be a contributing factor to disease progression. We hypothesized that repetitive mild head impacts, without evidence of structural brain damage, would increase microgliosis and AQP4 expression and depolarization and alter perivascular flow in the midbrain dopaminergic system. Adult male rats were subjected to sham, or two mild head impacts separated by 48 h. Three weeks later, fully awake rats were imaged using dynamic, contrast-enhanced MRI to follow the distribution of intraventricular gadobenate dimeglumine contrast agent. Images were registered to and analysed using a 3D MRI rat atlas providing site-specific data on 171 different brain areas. Following imaging, rats were tested for cognitive function using the Barnes maze assay. Histological analyses of tyrosine hydroxylase, microglia activation and AQP4 expression and polarization were performed on a parallel cohort of head impacted rats at 20 days post insult to coordinate with the time of imaging. There was no change in the global flux of contrast agent between sham and head impacted rats. The midbrain dopaminergic system showed a significant decrease in the influx of contrast agent as compared to sham controls together with a significant increase in microgliosis, AQP4 expression and depolarization. There were no deficits in cognitive function. The histology showed a significant level of neuroinflammation in the midbrain dopaminergic system 3 weeks post mild repetitive head impact but no loss in tyrosine hydroxylase. MRI revealed no structural brain damage emphasizing the potential serious consequences of mild head impacts on sustained brain neuroinflammation in this area critical to the pathophysiology of Parkinson's.