Blood Cell-vessel Wall Interactions Research Articles

Insights into Sickle Cell Disease through the Retinal Microvasculature: Adaptive Optics Scanning Light Ophthalmoscopy Correlates of Clinical OCT Angiography.

Clinical OCT angiography (OCTA) of the retinal microvasculature offers a quantitative correlate to systemic disease burden and treatment efficacy in sickle cell disease (SCD). The purpose of this study was to use the higher resolution of adaptive optics scanning light ophthalmoscopy (AOSLO) to elucidate OCTA features of parafoveal microvascular compromise identified in SCD patients. Case series of 11 SCD patients and 1 unaffected control. A total of 11 eyes of 11 SCD patients (mean age, 33 years; range, 23-44; 8 female, 3 male) and 1 eye of a 34-year-old unaffected control. Ten sequential 3 × 3 mm parafoveal OCTA full vascular slab scans were obtained per eye using a commercial spectral domain OCT system (Avanti RTVue-XR; Optovue). These were used to identify areas of compromised perfusion near the foveal avascular zone (FAZ), designated as regions of interest (ROIs). Immediately thereafter, AOSLO imaging was performed on these ROIs to examine the cellular details of abnormal perfusion. Each participant was imaged at a single cross-sectional time point. Additionally, 2 of the SCD patients were imaged prospectively 2 months after initial imaging to study compromised capillary segments across time and with treatment. Detection and characterization of parafoveal perfusion abnormalities identified using OCTA and resolved using AOSLO imaging. We found evidence of abnormal blood flow on OCTA and AOSLO imaging among all 11 SCD patients with diverse systemic and ocular histories. Adaptive optics scanning light ophthalmoscopy imaging revealed a spectrum of phenomena, including capillaries with intermittent blood flow, blood cell stasis, and sites of thrombus formation. Adaptive optics scanning light ophthalmoscopy imaging was able to resolve single sickled red blood cells, rouleaux formations, and blood cell-vessel wall interactions. OCT angiography and AOSLO imaging were sensitive enough to document improved retinal perfusion in an SCD patient 2 months after initiation of oral hydroxyurea therapy. Adaptive optics scanning light ophthalmoscopy imaging was able to reveal the cellular details of perfusion abnormalities detected using clinical OCTA. The synergy between these clinical and laboratory imaging modalities presents a promising avenue in the management of SCD through the development of noninvasive ocular biomarkers to prognosticate progression and measure the response to systemic treatment.

An integrated assay to probe endothelial glycocalyx-blood cell interactions under flow in mechanically and biochemically well-defined environments

Cell-cell and cell-glycocalyx interactions under flow are important for the behaviour of circulating cells in blood and lymphatic vessels. However, such interactions are not well understood due in part to a lack of tools to study them in defined environments. Here, we develop a versatile in vitro platform for the study of cell-glycocalyx interactions in well-defined physical and chemical settings under flow. Our approach is demonstrated with the interaction between hyaluronan (HA, a key component of the endothelial glycocalyx) and its cell receptor CD44. We generate HA brushes in situ within a microfluidic device, and demonstrate the tuning of their physical (thickness and softness) and chemical (density of CD44 binding sites) properties using characterisation with reflection interference contrast microscopy (RICM) and application of polymer theory. We highlight the interactions of HA brushes with CD44-displaying beads and cells under flow. Observations of CD44+ beads on a HA brush with RICM enabled the 3-dimensional trajectories to be generated, and revealed interactions in the form of stop and go phases with reduced rolling velocity and reduced distance between the bead and the HA brush, compared to uncoated beads. Combined RICM and bright-field microscopy of CD44+ AKR1 T-lymphocytes revealed complementary information about the dynamics of cell rolling and cell morphology, and highlighted the formation of tethers and slings, as they interacted with a HA brush under flow. This platform can readily incorporate more complex models of the glycocalyx, and should permit the study of how mechanical and biochemical factors are orchestrated to enable highly selective blood cell-vessel wall interactions under flow.

Endothelial Glycocalyx Structure in the Intact Carotid Artery: A Two-Photon Laser Scanning Microscopy Study

Background: The endothelial glycocalyx (EG) is the carbohydrate-rich luminal lining of endothelial cells that mediates permeability and blood cell-vessel wall interactions. To establish an atheroprotective role of the EG, adequate imaging and quantification of its properties in intact, viable, atherogenesis-prone arteries is needed. Methods: Carotid arteries of C57Bl6/J mice (n = 22) were isolated including the bifurcation, mounted in a perfusion chamber, and perfused with fluorescent lectin wheat germ agglutinin-fluorescein isothiocyanate. The EG was visualized through the vessel wall using two-photon laser scanning microscopy. An image quantification protocol was developed to assess EG thickness, which was sensitive to hyaluronidase-induced changes. Results: In the lesion-protected common carotid artery, EG thickness was found to be 2.3 ± 0.1 µm (mean ± SEM), while the surface area devoid of (wheat germ agglutinin-sensitive) EG was 8.9 ± 4.2%. Data from the external carotid artery were similar (2.5 ± 0.1 µm; 9.1 ± 5.0%). In the atherogenesis-prone internal carotid artery the EG-devoid surface area was significantly higher (27.4 ± 5.5%, p < 0.05); thickness at the remaining areas was 2.5 ± 0.1 µm. Conclusion: The EG can be adequately imaged and quantified using two-photon laser scanning microscopy in intact, viable mounted carotid arteries. Spatial EG differences could underlie atherogenesis.

The endothelial glycocalyx: composition, functions, and visualization

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

Hypoxia/Reoxygenation Induced Blood Cell Adhesion in Cerebral Venules of Sickle Cell Transgenic (β S ) Mice: The Two Faces of eNOS

Oxidative stress has been implicated in blood cell-vessel wall interactions of βSmice. Intravital fluorescence microscopy and bone marrow (BM) transplantation were used to examine the influence of different superoxide- and nitric oxide-directed interventions on leukocyte and platelet adhesion in cerebral venules of βS mice. Two h hypoxia and 4 h reoxygenation elicited intense leukocyte and platelet adhesion responses in βS mice that were attenuated by genetic deficiency of vascular gp91phox or overexpression of vascular CuZnSOD; allopurinol treatment was without effect. Treatment with L-NAME or genetic deficiency of vascular eNOS also decreased cell adhesion responses whereas treatment with DETA-NO or genetic overexpression of vascular eNOS exacerbated the cell adhesion responses; genetic deficiency of vascular iNOS was without effect. Sepiapterin, similar to PEG-SOD, effectively reduced the enhanced cell adhesion responses in βS mice. In conclusion, vascular NADPH oxidase and eNOS are major contributors to the pro-inflammatory and pro-thrombogenic phenotype assumed by the cerebral microvasculature of βS mice. These findings suggest that the inflammatory environment created by βS BM leads to NADPH oxidase activation in endothelial cells as well as the functional inactivation of vascular eNOS enzyme, and possibly includes roles for peroxynitrite formation and/or eNOS uncoupling. (HL26441, P01-HL55552)

Critical role of endothelial cell-derived nitric oxide synthase in sickle cell disease-induced microvascular dysfunction

Superoxide, which can limit nitric oxide bioavailability, has been implicated in blood cell–vessel wall interactions observed in sickle cell transgenic (β S) mice. Here we report that nonselective chemical inhibition of nitric oxide synthase isoforms dramatically reduces the enhanced leukocyte and platelet adhesion normally observed in cerebral venules of β S mice. Although genetic deficiency of vascular wall inducible nitric oxide synthase does not alter adhesion responses in β S mice, a significant attenuation is noted in β S mice with vascular wall endothelial nitric oxide synthase (eNOS) deficiency, while the adhesion responses are exacerbated when eNOS is overexpressed in microvessels. The eNOS-mediated enhancement of blood cell adhesion is reversible by pretreatment with sepiapterin (which generates the eNOS cofactor tetrahydrobiopterin) or polyethyleneglycol–superoxide dismutase, implicating a role for eNOS-dependent superoxide production. These findings suggest that an imbalance between eNOS-derived nitric oxide and superoxide, both generated by the vessel wall, is critical to the proinflammatory and prothrombogenic phenotype that is assumed by the microvasculature in sickle cell disease.

Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion.

Although the beta2-integrins have been implicated in the pathogenesis of cerebral ischemia-reperfusion (I/R) injury, the relative contributions of the alpha-subunits to the pathogenesis of ischemic stroke remains unclear. The objective of this study was to determine whether and how genetic deficiency of either lymphocyte function-associated antigen-1 (LFA-1) or macrophage-1 (Mac-1) alters the blood cell-endothelial cell interactions, tissue injury, and organ dysfunction in the mouse brain exposed to focal I/R. Middle cerebral artery occlusion was induced for 1 h (followed by either 4 or 24 h of reperfusion) in wild-type mice and in mice with null mutations for either LFA-1 or Mac-1. Neurological deficit and infarct volume were monitored for 24 h after reperfusion. Platelet- and leukocyte-vessel wall adhesive interactions were monitored in cortical venules by intravital microscopy. Mice with null mutations for LFA-1 or Mac-1 exhibited significant reductions in infarct volume. This was associated with a significant improvement in the I/R-induced neurological deficit. Leukocyte adhesion in cerebral venules did not differ between wild-type and mutant mice at 4 h after reperfusion. However, after 24 h of reperfusion, leukocyte adhesion was reduced in both LFA-1- and Mac-1-deficient mice compared with their wild-type counterparts. Platelet adhesion was also reduced at both 4 and 24 h after reperfusion in the LFA-1- and Mac-1-deficient mice. These findings indicate that both alpha-subunits of the beta2-integrins contribute to the brain injury and blood cell-vessel wall interactions that are associated with transient focal cerebral ischemia.

Cerebral microvascular responses to hypercholesterolemia: roles of NADPH oxidase and P-selectin.

Although hypercholesterolemia is widely accepted as a major risk factor for coronary artery and peripheral vascular diseases, its role in the pathogenesis of stroke is controversial. The objectives of this study were to determine how hypercholesterolemia affects the cerebral microcirculation under resting conditions and after ischemia-reperfusion (I/R). Platelet- and leukocyte-endothelial cell interactions and oxidant production (using the oxidant-sensitive fluorochrome dihydrorhodamine-123) were monitored by intravital videomicroscopy in the cerebral microvasculature of mice placed on either a normal (ND) or cholesterol-enriched diet (HCD). Platelets labeled with carboxyfluorescein diacetate succinimidyl ester (CFDASE) and leukocytes labeled with rhodamine 6G were seen to roll and firmly adhere, with a corresponding increase in oxidant production, in venules of mice on HCD, but not ND. Immunoneutralization of P-selectin attenuated the platelet- and leukocyte-endothelial cell interactions and the enhanced oxidant production associated with HCD. A GPIIb/IIIa blocking antibody did not alter the blood cell-vessel wall interactions to HCD. Mice deficient in the NADPH oxidase subunit gp91(phox) exhibited significantly blunted platelet and leukocyte recruitment responses to HCD. Focal I/R also elicited inflammatory and prothrombogenic responses in cerebral venules and these were exaggerated in mice on HCD. These results implicate an oxidant-dependent, P-selectin-mediated mechanism in the blood cell-vessel wall interactions induced by hypercholesterolemia in the brain and demonstrate that the deleterious effects of I/R on the brain are exacerbated by this cardiovascular risk factor.

Hematologic disorders and ischemic stroke

Hematologic disorders that induce a thrombotic tendency contribute to overall ischemic stroke risk and may directly cause cerebral ischemia in patients without other risk factors. These disorders include platelet dysfunction, prothrombotic coagulopathies, defective fibrinolysis, abnormal red blood cell-vessel wall interactions, and antiphospholipid antibody syndromes. Multiple mechanisms promoting ischemia may underlie specific hematologic abnormalities. Improved markers for activation of platelets, coagulation and fibrinolysis are now being used to correlate prothrombotic mechanisms with ischemic pathology and, thus, to make therapeutic approaches more precise. Nevertheless, finding the ideal balance between preventing cerebral ischemia and incurring hemorrhagic toxicity remains difficult.

10 The importance of blood cell-vessel wall interactions in tumour metastasis

Tumour cell dissemination is a complex process, depending on the ability of malignant cells to escape from the primary tumour and penetrate and flow through the bloodstream. Circulating tumour cells can adhere to the vessel wall, dissolve the basal lamina and extravasate, giving origin to metastases. Interactions between tumour cells, blood platelets and leukocytes favour tumour cell adhesion to the vessel wall, migration in extravascular spaces and growth in secondary sites. The biochemical and molecular mechanisms regulating tumour cell adhesion to the vessel wall and intercellular contacts have been studied extensively in recent years. Moreover, it has been shown that either tumour cells or blood cells release growth factors and inflammatory proteins, such as cytokines and chemokines, that may be involved in tumour cell migration and proliferation. Finally, tumour cells and cells of the surrounding tissue possess procoagulant and fibrinolytic properties that may be important in modulating the extracellular matrix around the tumour, to allow tumour cell invasion and progression. We have described the cell types (i.e. blood platelets, leukocytes, endothelial cells), the matrix components (i.e. fibronectin, thrombospondin and laminin) and the growth factors/cytokines (i.e. platelet-derived growth factor, transforming growth factor beta, tumour necrosis factor) involved in these processes. In particular, we have described cell-cell and cell-matrix interactions, cell migration and release of growth factors, cytokines, chemotactic peptides and proteolytic enzymes. This survey has also considered a few innovative approaches for the prevention and cure of cancer and metastasis that are based on these new concepts.

4 Transcellular biosynthesis of arachidonic acid metabolites: From in vitro investigations to in vivo reality

The discovery that arachidonic acid metabolism in a multicellular environment could be different from that expected from the sum of individual cell types has led to the concept of transcellular metabolism. In this process, several cells can contribute to the formation of a novel compound with potent biological action. The study of this mode of synthesis is important in the context of the current appraisal of thrombotic diseases as part of an inflammatory reaction. In this context, blood cell-vessel wall interactions present a regulated expression of adhesive molecules on either type of cell. These complex processes are initiated by signalling molecules such as cytokines that can deeply modify the phenotype of endothelial cells, which may ultimately lead to a change in the vascular tone and to atherosclerotic complications. Such reaction processes are part of the autocrine-endocrine system whereby cells can control and modify their own phenotype through the action of a local network of mediators. In this context, arachidonic acid metabolites may be an important part of unifying signal molecules that participate in these changes. The significance of transcellular biosynthesis where combined cells acquire a different metabolic potential can be viewed as an additional modification of blood cell and vessel cell phenotype in thrombotic diseases.

Synthesis of lipoxygenase and epoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries.

Coronary vascular injury promotes blood cell-vessel wall interactions that influence arachidonic acid metabolism and coronary blood flow patterns. Since lipoxygenase and cytochrome P-450 epoxygenase metabolites of arachidonic acid are synthesized by vascular and inflammatory cells and have a variety of important biological actions, we investigated the metabolism of arachidonic acid by these pathways in normal and stenosed, endothelially injured canine coronary arteries. We found and confirmed by gas chromatography/mass spectrometry that primarily 12- and 15-hydroxyeicosatetraenoic acids (HETEs) are synthesized by both coronary artery segments. Lesser amounts of 11-, 9-, 8-, and 5-HETEs are also produced. 15-Ketoeicosatetraenoic acid is also synthesized. The synthesis of 14C-HETEs is fivefold to 10-fold greater by the stenosed than the normal coronary artery. Specific radioimmunoassays indicated that the stenosed coronary artery synthesized 93 +/- 14 and 1,102 +/- 154 ng/g of tissue of 15- and 12-HETE, respectively, while the normal coronary artery produced 17 +/- 3 and 162 +/- 68 ng/g of tissue of 15- and 12-HETE, respectively. Products comigrating with 14,15-; 11,12-; 8,9-; and 5,6-epoxyeicosatrienoic acids (EETs) and the corresponding dihydroxyeicosatrienoic acids (DHETs) were detected predominantly in stenosed coronary arteries by high-pressure liquid chromatography. The structures of the EETs were confirmed by GC/MS. The EETs and prostaglandin I2 produced endothelium-independent, concentration-related relaxations of dog coronary artery rings. These data indicate that normal and stenotic coronary arteries metabolize arachidonic acid to HETEs, DHETs, and EETs along with prostaglandins; however, the synthesis of these metabolites is greater in the stenosed, endothelially injured vessel. The EETs may be synthesized during the development of cyclic flow variations and counteract the vasoconstrictor effects of thromboxane A2.

Dipyridamole — evaluation of an established antithrombotic drug in view of modern concepts of blood cell-vessel wall interactions

The effect of dipyridamole on the local antithrombotic activities of endothelium has been evaluated. Human whole blood was allowed to flow over an endothelial cell-derived extracellular matrix partially covered by human endothelial cells. Half-maximal supression of platelet aggregate formation occurred with approximately 5 μM dipyridamole. Similarly, a pronounced inhibition of thrombus formation was observed by in vivo microscopy and computer-assisted morphometric analysis, after oral treatment of non-anesthetized hamsters with dipyridamole, 5 mg/kg. This strong suppression of thrombus formation was maintained in animals on a long-term cholesterol-supplemented diet. The antithrombotic potential of dipyridamole has been clearly demonstrated, both in vitro and in vivo using these more complex approaches employing quantitative microscopy.