Randal O. Dull

Heparan sulfate proteoglycan is a mechanosensor on endothelial cells.

Abstract— The objective of this study was to test whether a glycosaminoglycan component of the surface glycocalyx layer is a fluid shear stress sensor on endothelial cells (ECs). Because enhanced nitric oxide (NO) production in response to fluid shear stress is a characteristic and physiologically important response of ECs, we evaluated NOx (NO2− and NO3−) production in response to fluid shear stress after enzymatic removal of heparan sulfate, the dominant glycosaminoglycan of the EC glycocalyx, from cultured ECs. The significant NOx production induced by steady shear stress (20 dyne/cm2) was inhibited completely by pretreatment with 15 mU/mL heparinase III (E.C.4.2.2.8) for 2 hours. Oscillatory shear stress (10±15 dyne/cm2) induced an even greater NOx production than steady shear stress that was completely inhibited by pretreatment with heparinase III. Addition of bradykinin (BK) induced significant NOx production that was not inhibited by heparinase pretreatment, demonstrating that the cells were still able to produce abundant NO after heparinase treatment. Fluorescent imaging with a heparan sulfate antibody revealed that heparinase III treatments removed a substantial fraction of the heparan sulfate bound to the surfaces of ECs. In summary, these experiments demonstrate that a heparan sulfate component of the EC glycocalyx participates in mechanosensing that mediates NO production in response to shear stress. The full text of this article is available online at http://www.circresaha.org.

The effect of varying albumin concentration and hydrostatic pressure on hydraulic conductivity and albumin permeability of cultured endothelial monolayers

An in vitro model of the endothelial transport barrier was developed using bovine aortic endothelial cell monolayers cultured on a porous polycarbonate substrate. Hydraulic conductivity (Lp) was measured by a bubble tracking technique at varying pressure differentials and albumin concentrations. The effective albumin permeability (Pc) was determined by measuring the flux of fluorescent-labeled albumin across monolayers at varying hydrostatic pressures. Lp determined at pressure differentials between 5.0 and 10 cm H2O demonstrated a strong dependence on albumin concentration, decreasing approximately 10-fold from 21.3 x 10(-7) +/- 3.18 x 10(-7) cm/sec/cm H2O (mean +/- SEM) at 0.0 g/dl to 2.35 x 10(-7) +/- 0.20 x 10(-7) cm/sec/cm H2O at 1.0 g/dl albumin. Increasing the albumin concentration from 1.0 to 4.0 g/dl reduced Lp by an additional 16% to 1.97 x 10(-7) +/- 0.17 x 10(-7) cm/sec/cm H2O. Furthermore, Lp was moderately dependent on the pressure differential, increasing by about a factor of two with a doubling of the pressure differential. The effective permeability (Pc) was also dependent on the pressure differential. At an albumin concentration of 4.0 g/dl, Pc increased from 1.37 x 10(-6) +/- 0.26 x 10(-6) cm/sec at 0.0 cm H2O to 5.06 x 10(-6) +/- 1.92 x 10(-6) cm/sec at 10 cm H2O. Analysis of Pc and Jv data, however, demonstrates that water and albumin do not share a common pathway in crossing the endothelial monolayer. These data suggest the existence of a large pore pathway for albumin. Thus, the in vitro system has many of the transport characteristics of intact vessels in vivo and should be useful for physiological studies of the endothelial transport barrier.

Mechanisms of flow-mediated signal transduction in endothelial cells: kinetics of ATP surface concentrations.

Intracellular free calcium ([Ca2+]i) was measured in single cells of a confluent endothelial monolayer subjected to defined flow. Flow medium containing adenosine triphosphate (ATP) was used to study the influence of flow forces upon agonist-response coupling as mediated via the P2y-purinoceptor. [Ca2+]i responses were highly sensitive to the fluid motion at the cell surface; consecutive small increases of flow stimulated large [Ca2+]i transients with the levels returning to baseline at the new flow rate within 250 s. The characteristics of [Ca2+]i transients were also influenced by decreasing flow. Since potent ectonucleotidases at the endothelial cell surface rapidly degrade ATP, we postulated that a combination of flow and degradative enzymes regulates the mass transport of ATP in the boundary layer. The hypothesis predicts that step increases of flow exceed the capacity of the ectonucleotidases and allow ATP to reach the receptor. Experiments were conducted to compare ATP and ADP beta S, a nonhydrolyzable ATP analog that resists degradation by surface ectonucleotidases, and calculations of ATP mass transport to the cell surface were compared to estimates of surface clearance rates. Calculations of mass transport coefficients for ATP in the boundary layer demonstrated that changes of flow which elicited a prominent [Ca2+]i response represented 26-73% changes in the mass transport of ATP from the bulk fluid. When steadystate mass transport coefficients for ATP under various flow conditions were compared with the estimated rate constant for surface degradation of ATP, ratios close to unity were obtained. These results suggest that both boundary layer mass transport and ATP clearance rates can be rate-limiting for flow-mediated activation of the P2y-receptor. The experiments provide evidence for differential signal transduction responses in the endothelium driven by diffusion gradients (derived from both the blood and the vessel wall), which are likely to vary widely in the complex flow fields encountered in vivo.

A Murine Model of Inflammatory Bladder Disease: Cathelicidin Peptide Induced Bladder Inflammation and Treatment With Sulfated Polysaccharides

PURPOSEnStudies show that LL-37 is a naturally occurring urinary defensin peptide that is up-regulated during urinary tract infections. Although normal urinary LL-37 levels are antimicrobial, we propose that increased LL-37 may trigger bladder inflammation. We further suggest that anti-inflammatory sulfated polysaccharides known as semi-synthetic glycosaminoglycan ether compounds can treat/prevent LL-37 mediated bladder inflammation.nnnMATERIALS AND METHODSnC57BL/6 mice were catheterized/instilled with LL-37 (320 μM, 150 μl) for 45 minutes. Animals were sacrificed at 12 and 24 hours, and tissues were examined using hematoxylin and eosin. Separate experiments were performed for myeloperoxidase to quantify inflammation. GM-1111 semi-synthetic glycosaminoglycan ether treatments involved instillation of 10 mg/ml for 45 minutes directly before or after LL-37. Tissues were harvested at 24 hours. To compare semi-synthetic glycosaminoglycan ether efficacy, experiments were performed using 10 mg/ml heparin. Finally, tissue localization of semi-synthetic glycosaminoglycan ether was examined using a fluorescent GM-1111-Alexa Fluor® 633 conjugate.nnnRESULTSnProfound bladder inflammation developed after LL-37. Greater tissue inflammation occurred after 24 hours compared to that at 12 hours. Myeloperoxidase assays revealed a 21 and 61-fold increase at 12 and 24 hours, respectively. Semi-synthetic glycosaminoglycan ether treatment after LL-37 showed mild attenuation of inflammation with myeloperoxidase 2.5-fold below that of untreated bladders. Semi-synthetic glycosaminoglycan ether treatment before LL-37 demonstrated almost complete attenuation of inflammation. Myeloperoxidase results mirrored those in controls. In heparin treated bladders minimal attenuation of inflammation occurred. Finally, instillation of GM-1111-Alexa Fluor 633 revealed urothelial coating, significant tissue penetration and binding to endovasculature.nnnCONCLUSIONSnWe developed what is to our knowledge a new model of inflammatory bladder disease by challenge with the naturally occurring urinary peptide LL-37. We also noted that a new class of anti-inflammatory sulfated polysaccharides prevents and mitigates bladder inflammation.

How does the arterial endothelium sense flow? Hemodynamic forces and signal transduction.

The focal nature of atherosclerotic lesions is associated with patterns of altered blood flow in the major arteries (1–3), although the precise nature of the flow in such regions is unclear. At the interface between flowing blood and the arterial wall, a confluent monolayer of endothelial cells operates as a signal-transduction system for hemodynamic forces associated with flow. Investigations of the influence of pressure, stretch and shear stress upon endothelial biology have therefore been conducted with a view to linking the precise flow profiles and the vessel wall pathophysiology. It is now clear that early atherogenesis develops in the presence of an intact endothelial monolayer (4–6, 10) consistent with the pivotal role that the endothelium may play in this disease process. The mechanisms by which physical forces influence endothelial biology, however, have yet to be fully defined.

The use of an endothelium-targeted cationic copolymer to enhance the barrier function of lung capillary endothelial monolayers.

Acute changes in lung capillary permeability continue to complicate procedures such as cardiopulmonary bypass, solid organ transplant, and major vascular surgery and precipitate the more severe disease state Adult Respiratory Distress Syndrome (ARDS). To date there is no treatment targeted directly to the lung microvasculature. We hypothesized that biomimetic polymers could be used to enhance passive barrier function by reducing the porosity of the endothelial glycocalyx and attenuate mechanotransduction by restricting the motion of the glycoproteins implicated in signal transduction. To this end, cationic copolymers containing methacrylamidopropyl trimethylammonium chloride (P-TMA Cl) have been developed as an infusible therapy to target the lung capillary glycocalyx in order to mechanically enhance the capillary barrier and turn off pressure-induced mechanotransduction. Copolymers were tested for functional efficacy by measuring both albumin permeability (P(DA)) and hydraulic conductivity (L(p)) across cultured endothelial monolayers. P-TMA Cl significantly decreased P(DA) in normal and inflamed cells and attenuated pressure-induced increases in L(p). Decreases in L(p) across endothelial monolayers in the presence of P-TMA Cl is evidence of a dampening of mechanotransduction-induced barrier dysfunction. We show the potential for biomimetic polymers targeted to lung endothelium as a viable therapy to enhance endothelial barrier function thereby attenuating a major component of vascular inflammation.

Study of the therapeutic benefit of cationic copolymer administration to vascular endothelium under mechanical stress.

Pulmonary edema and the associated increases in vascular permeability continue to represent a significant clinical problem in the intensive care setting, with no current treatment modality other than supportive care and mechanical ventilation. Therapeutic compound(s) capable of attenuating changes in vascular barrier function would represent a significant advance in critical care medicine. We have previously reported the development of HPMA-based copolymers, targeted to endothelial glycocalyx that are able to enhance barrier function. In this work, we report the refinement of copolymer design and extend our physiological studies to demonstrate that the polymers: 1) reduce both shear stress and pressure-mediated increase in hydraulic conductivity, 2) reduce nitric oxide production in response to elevated hydrostatic pressure and, 3) reduce the capillary filtration coefficient (K(fc)) in an isolated perfused mouse lung model. These copolymers represent an important tool for use in mechanotransduction research and a novel strategy for developing clinically useful copolymers for the treatment of vascular permeability.

ACE Phenotyping as a Guide Toward Personalized Therapy With ACE Inhibitors

Background: Angiotensin-converting enzyme (ACE) inhibitors (ACEI) are widely used in the management of cardiovascular diseases but with significant interindividual variability in the patient’s response. Objectives: To investigate whether interindividual variability in the response to ACE inhibitors is explained by the “ACE phenotype”—for example, variability in plasma ACE concentration, activity, and conformation and/or the degree of ACE inhibition in each individual. Methods: The ACE phenotype was determined in plasma of 14 patients with hypertension treated chronically for 4 weeks with 40 mg enalapril (E) or 20 mg E + 16 mg candesartan (EC) and in 20 patients with hypertension treated acutely with a single dose (20 mg) of E with or without pretreatment with hydrochlorothiazide. The ACE phenotyping included (1) plasma ACE concentration; (2) ACE activity (with 2 substrates: Hip-His-Leu and Z-Phe-His-Leu and calculation of their ratio); (3) detection of ACE inhibitors in patient’s blood (indicator of patient compliance) and the degree of ACE inhibition (ie, adherence); and (4) ACE conformation. Results: Enalapril reduced systolic and diastolic blood pressure in most patients; however, 20% of patients were considered nonresponders. Chronic treatment results in 40% increase in serum ACE concentrations, with the exception of 1 patient. There was a trend toward better response to ACEI among patients who had a higher plasma ACE concentration. Conclusion: Due to the fact that “20% of patients do not respond to ACEI by blood pressure drop,” the initial blood ACE level could not be a predictor of blood pressure reduction in an individual patient. However, ACE phenotyping provides important information about conformational and kinetic changes in ACE of individual patients, and this could be a reason for resistance to ACE inhibitors in some nonresponders.

Hydraulic Conductivity and Shear Dependent Albumin Permeability of Cultured Endothelial Cell Monolayers

Endothelial cells regulate transmural volume flux and permeability of the vascular wall to macromolecules. Alteration of wall permeability may be an early event in atherogenesis. In order to study transport properties of the endothelial monolayer in isolation from the underlying structures of the arterial wall, we developed an in vitro model employing bovine aortic endothelial cells grown on polycarbonate filters. Hydraulic conductivity (Lp) was measured under static conditions (zero wall shear stress) by a bubble tracking technique in both the presence and absence of albumin in the culture media. Endothelial permeability to flourescently labelled albumin (Pe) was measured under static conditions and during exposure to steady wall shear stress of 1 or 10 dynes/cm2 in a parallel plate flow chamber with 1% albumin in the culture media on both sides of the monolayer.

Contents Vol. 53, 2016

s Mechanisms of Vasodilatation 12th International Symposium Rochester, Minn., USA, November 6–9, 2016 Guest Editors: M.V. Miller (Rochester, Minn.); R.C. Webb (Augusta, Ga.); M.P. Vanhoutte (Hong Kong)