Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.     

Atherogenic region and diet diminish glycocalyx dimension and increase intima-to-media ratios at murine carotid artery bifurcation

It was hypothesized that endothelial glycocalyx perturbation contributes to increased vulnerability of the arterial wall exposed to atherogenic risk factors. Glycocalyx and intima-to-media ratios (IMR) were studied at a low- and a high-risk region within the murine carotid artery (common region) and internal carotid branch (sinus region) in control C57BL/6J (C57BL6) and age-matched C57BL/6J/apoE*3-Leiden (apoE*3; on an atherogenic diet) mice. Electron micrographs revealed significantly thinner glycocalyces [73 (SD 36) vs. 399 (SD 174) nm, P < 0.05] and greater IMR [0.096 (SD 0.045) vs. 0.044 (SD 0.023), P < 0.05] at the sinus region of C57BL6 mice than in the common region. Thinner glycocalyces [100 (SD 27) vs. 399 (SD 174) nm, P < 0.05] and greater IMR [0.071 (SD 0.024) vs. 0.044 (SD 0.023), P < 0.05] were also observed in the common region of age-matched apoE*3 mice on an atherogenic diet for 6 wk vs. C57BL6 mice on a normal diet. Greater IMR were due to greater intima layers, without significant changes in media layer dimension. In addition, atherogenic diet resulted in increased endothelial cell thickness at the sinus region [0.85 (SD 0.49) vs. 0.53 (SD 0.28) microm, P < 0.05] but not at the common region [0.66 (SD 0.37) vs. 0.62 (SD 0.32) microm]. It is concluded that both regional and diet-induced increases in atherogenic risk are associated with smaller glycocalyx dimensions and greater IMR and that vascular sites with diminished glycocalyx are more vulnerable to proinflammatory and atherosclerotic sequelae.     

In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin EC(50) = 3.3 x 10(-6) mol/l, peak 21Delta% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine EC(50) = 1.3 x 10(-6) mol/l, peak 27Delta% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16Delta% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-L-arginine [1Delta% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16Delta% (SD 3) vs. 10Delta% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats.     

Shear stress-induced redistribution of the glycocalyx on endothelial cells in vitro

The glycocalyx is the inner most layer of the endothelium that is in direct contact with the circulating blood. Shear stress affects its synthesis and reorganization. This study focuses on changes in the spatial distribution of the glycocalyx caused by shear stimulation and its recovery following the removal of the shear stress. Sialic acid components of the glycocalyx on human umbilical vain endothelial cells are observed using confocal microscopy. The percentage area of the cell membrane covered by the glycocalyx, as well as the average fluorescence intensity ratio between the apical and edge areas of the cell is used to assess the spatial distribution of the glycocalyx on the cell membrane. Our results show that following 24 h shear stimulation, the glycocalyx relocates near the edge of endothelial cells (i.e., cell–cell junction regions). Following the removal of the shear stress, the glycocalyx redistributes and gradually appears in the apical region of the cell membrane. This redistribution is faster in the early hours ( $<$ 4 h) after shear stimulation than that in the later stage (e.g., between 8 and 24 h). We further investigate the recovery of the glycocalyx after its enzyme degradation under either static or shear flow conditions. Our results show that following 24 h recovery under shear flow, the glycocalyx reappears predominantly near the edge of endothelial cells. Static and shear flow conditions result in notable changes in the spatial recovery of the glycocalyx, but the difference is not statistically significant. We hypothesize that newly synthesized glycocalyx is not structurally well developed. Its weak interaction with flow results in less than significant redistribution, contrary to what has been observed for a well-developed glycocalyx layer.     

Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release

Endothelium-derived nitric oxide (NO) is synthesized in response to chemical and physical stimuli. Here, we investigated a possible role of the endothelial cell glycocalyx as a biomechanical sensor that triggers endothelial NO production by transmitting flow-related shear forces to the endothelial membrane. Isolated canine femoral arteries were perfused with a Krebs-Henseleit solution at a wide range of perfusion rates with and without pretreatment with hyaluronidase to degrade hyaluronic acid glycosaminoglycans within the glycocalyx layer. NO production rate was evaluated as the product of nitrite concentration in the perfusate and steady-state perfusion rate. The slope that correlates the linear relation between perfusion rate and NO production rate was taken as a measure for flow-induced NO production. Hyaluronidase treatment significantly decreased flow-induced NO production to 19 +/- 9% of control (mean +/- SD; P < 0.0001 vs. control; n = 11), whereas it did not affect acetylcholine-induced NO production (88 +/- 17% of pretreatment level, P = not significant; n = 10). We conclude that hyaluronic acid glycosaminoglycans within the glycocalyx play a pivotal role in detecting and amplifying the shear force of flowing blood that triggers endothelium-derived NO production in isolated canine femoral arteries.     

Fibrin D-dimer impairs the accumulation and anticoagulant properties of heparan sulphate and stimulates secretion of plasminogen activator inhibitor-1 by rabbit coronary endothelial cells

Fibrin split product D-dimer (DD) is most probably involved in the development of vascular disorders. At 1.5 microM concentration DD inhibited the incorporation of D-[1-(3)H]glucosamine hydrochloride and [2-(14)C]acetate x Na into pericellular heparan sulphate (HS) of rabbit coronary endothelial cells without affecting other groups of glycosaminoglycans (GAGs). At the same time, DD reduced HS ability to bind antithrombin (AT) and suppressed NO production. The effect of DD on pericellular GAGs was similar to that of N(omega)-methyl-L-arginine, the competitive inhibitor of endothelial NO synthase (eNOS). L-Ascorbic acid, eNOS activator, increased the level of endogenous NO in the DD-treated cells, and restored HS accumulation and antithrombin binding. It is suggested that DD influence on endothelial HS may be mediated by NO production. Another effect of DD, namely, stimulation of plasminogen activator inhibitor-1 (PAI-1) secretion did not depend on the NO level. The decreased HS content, reduced anticoagulant properties of HS, and increased PAI-1 secretion disorganized the endothelial matrix, and promoted fibrin formation and vascular damage. This points to DD as an important factor in the development of vascular disorders.     

Sex differences in left ventricular function and beta-receptor responsiveness following prolonged strenuous exercise.

Sex differences in neuroendocrine and metabolic responses to prolonged strenuous exercise (PSE) have been well documented. The aim of this investigation was to examine sex differences in left ventricular function and cardiac beta-receptor responsiveness following a single bout of PSE. Nine male and eight female triathletes were examined during three separate sessions: before, immediately after, and 24 h following a half-ironman triathlon using dobutamine stress echocardiography. Steady-state graded infusions of dobutamine were used to assess beta-receptor responsiveness. Slopes calculated from linear regressions between dobutamine doses and changes in heart rate and contractility for each participant were used as an index of beta-receptor responsiveness. Despite no change in preload, fractional area change decreased from baseline after the race in both men and women, with a greater decrease in men [men: 54.1% (SD 2.1) to 50.7% (SD 3.4) vs. women: 55.4% (SD 2.7) to 53.3% (SD 2.5); P < 0.05]. The amount of dobutamine necessary to increase heart rate by 25 beats/min [men: 29.6 microg x kg(-1) x min(-1) (SD 6.6) to 42.7 microg x kg(-1) x min(-1) (SD 12.9) vs. women: 23.5 microg x kg(-1) x min(-1) (SD 4.0) to 30.0 microg x kg(-1) x min(-1) (SD 7.8); P < 0.05] and contractility by 10 mmHg/cm2 [men: 20.9 microg x kg(-1) x min(-1) (SD 5.1) to 37.0 microg x kg(-1) x min(-1) (SD 11.5) vs. women: 22.6 microg x kg(-1) x min(-1) (SD 6.4) to 30.7 microg x kg(-1) x min(-1) (SD 7.2); P < 0.05] was greater in both men and women postrace. However, the amount of dobutamine required to induce these changes was greater in men, reflecting larger beta-receptor alterations in male triathletes following PSE relative to women. These data suggest that following an acute bout of PSE, male triathletes demonstrate an attenuated chronotropic and inotropic response to beta-adrenergic stimulation compared with female triathletes.     

Inflammation- and ischemia-induced shedding of venular glycocalyx

Alterations in the composition of the glycocalyx of venular endothelium in postcapillary venules (rat mesentery) were explored in models of inflammation and ischemia-reperfusion injury. Lectins were covalently linked to fluorescently labeled microspheres (0.1-microm diameter) or directly labeled with FITC. Adhesion of lectins specific for glucose and galactose residues of glycosaminoglycans (GAGs) and other components of the endothelial glycocalyx decreased dramatically after superfusion of the mesentery with the chemoattractant N-formylmethionyl-leucyl-phenylalanine and during reperfusion after 60-min ischemia. These reductions were significantly attenuated by superfusion with pertussis toxin (PTX), suggesting that shedding of glycocalyx was mediated by G proteins. Adhesion of microspheres linked with antibody for syndecan-1, a major proteoglycan to which GAGs are bound, revealed increased labeling as GAGs were lost and permitted greater numbers of spheres to adhere to the protein core, which was not shed. Induction of ischemia by occluding proximal microvessels for 60 min resulted in a 40% increase in galactosaminoglycans and a 15% increase in glucosaminoglycans on the endothelium, which was not inhibited by PTX. Reperfusion of vessels led to a rapid loss of GAGs that was inhibited by pretreatment with PTX, with 40% of galactosaminoglycans and 25% of glucosaminoglycans accumulated being removed by G protein-mediated shedding and the remainder freely convected away by fluid shear. We conclude that the composition of the glycocalyx results from a balance of the rate of biosynthesis of GAGs by the endothelial cell and their shedding, which may be mediated by intracellular and/or membrane-bound proteases or lyases released or activated by G protein signaling.     

Endothelial glycocalyx as a critical signalling platform integrating the extracellular haemodynamic forces and chemical signalling

The glycocalyx covers the human mammalian cells and plays important roles in stroke, inflammation and atherosclerosis. It has also been shown to be involved in endothelial mechanotransduction of shear stress. Shear stress induces the remodelling of the major component of the glycocalyx including glypican‐1, a cell membrane heparan sulphate proteoglycan. Other factors, such as sphingosine‐1‐phosphate (S1P), protect the glycocalyx against syndecan‐1 ectodomain shedding and induce the synthesis of heparan sulphate. In this study, we reviewed the role of shear stress and S1P in glycocalyx remodelling and revealed that the glycocalyx is a critical signalling platform, integrating the extracellular haemodynamic forces and chemical signalling, such as S1P, for determining the fate of endothelial cells and vascular diseases. This review integrated our current understanding of the structure and function of the glycocalyx and provided new insight into the role of the glycocalyx that might be helpful for investigating the underlying biological mechanisms in certain human diseases, such as atherosclerosis.     

Effect of the gonadal status and the gender on glycosaminoglycans profile in the lower urinary tract of dogs

Glycosaminoglycans (GAGs) form a functional component of connective tissues that affect the structural and functional integrity of the lower urinary tract (LUT). The specific GAGs of physiological relevance are both nonsulfated (hyaluronan) and sulfated GAGs (chondroitin sulphate [CS], dermatan sulphate [DS], keratan sulphate [KS], and heparan sulphate [HS]). As GAG composition in the LUT is hormonally regulated, we postulated that gonadectomy-induced endocrine imbalance alters the profile of GAGs in the canine LUT. Four regions of the LUT (body and neck of the bladder as well as the proximal and distal urethra) from 20 clinically healthy dogs (5 intact males, 5 intact anoestrus females, 4 castrated males, and 6 spayed females) were collected, wax-embedded and sectioned. Alcian blue staining at critical electrolyte concentrations was performed on the sections to determine total GAGs, hyaluronan, total sulfated GAGs, combined components of CS and DS, as well as KS and HS. The amount of staining was evaluated in 3 tissue layers, i.e., epithelium, subepithelial stroma and muscle within a region. Overall, hyaluronan (67.1%) was the predominant GAG in the LUT. Among sulfated GAGs, a combined component of KS and HS was found to be 61.8% and 38.2% for CS and DS. Gonadal status significantly affected GAG profiles in the LUT (P < 0.01). All GAG components were lower (P < 0.05) in body of the bladder of gonadectomized dogs. Total sulfated GAGs and a combined component of KS and HS were lower (P < 0.05) in all 4 regions of gonadectomized dogs. Except for a combined component of CS and DS, decreases in all GAGs were found more consistently in the muscle compared to other tissue layers. Differences between genders became obvious only when considered along with the effect of gonadal status. In gonadectomized dogs, changes in GAG components in the LUT were more consistent in females compared to males; this may partly explain different levels of risk in the development of urinary incontinence between genders. Quantitative differences in GAG profiles found between intact and gonadectomized dogs indicate a potential role of gonadectomy-induced endocrine imbalance in modifying GAG composition in the canine LUT. Profound alteration in the pattern of GAGs in gonadectomized dogs may compromise structural and functional integrity of the LUT and is possibly involved in the underlying mechanism of urinary incontinence post neutering.     

Partial recovery of the endothelial glycocalyx upon rosuvastatin therapy in patients with heterozygous familial hypercholesterolemia

The endothelial glycocalyx has been shown to serve as a protective barrier between the flowing blood and the vessel wall in experimental models. The aim of this study was to evaluate whether hypercholesterolemia is associated with glycocalyx perturbation in humans, and if so, whether statin treatment can restore this. We measured systemic glycocalyx volume (V(G)) in 13 patients with heterozygous familial hypercholesterolemia (FH) after cessation of lipid-lowering therapy for a minimum of 4 weeks and 8 weeks after initiating rosuvastatin therapy. Normocholesterolemic subjects were used as controls. V(G) was estimated by subtracting the intravascular distribution volume of a glycocalyx permeable tracer (dextran 40) from that of a glycocalyx impermeable tracer (labeled erythrocytes). V(G) in untreated FH patients [LDL 225 +/- 57 mg/dl (mean +/- SD)] was significantly reduced compared with controls (LDL 93 +/- 24 mg/dl) (V(G) 0.8 +/- 0.3 vs. 1.7 +/- 0.6, respectively, P < 0.001). After normalization of LDL levels (95 +/- 33 mg/dl) upon 8 weeks of statin treatment, V(G) recovered only partially (V(G) 1.1 +/- 0.4 L, P = 0.04). The endothelial glycocalyx is profoundly reduced in FH patients, which may contribute to increased atherogenic vulnerability. This perturbation is partially restored upon short-term statin therapy.     

Effects of different types of fluid shear stress on endothelial cell proliferation and survival

We attempted to clarify the effect of different types of shear stress on endothelial cell (EC) proliferation and survival. Bovine aortic ECs were subjected to either steady laminar, 1 Hz pulsatile, or 1 Hz to and fro shear at 14 dyne/cm(2). % of BrdU positive EC was 14.3 +/- 1.6% in steady, 21.5 +/- 3.2% in pulsatile, and 11.4 +/- 2.4% in to and fro after 4 h, respectively (P < 0.05). Pulsatile shear compared with static control. Rapamycin reduced BrdU incorporation in all shear regimens (P < 0.001). However, it was still higher in EC exposed to pulsatile shear than the other regimens (P < 0.005). PD98059 completely abolished the increased BrdU incorporation in all shear regimens, including pulsatile shear. Pulsatile shear had significantly elevated ERK1/2 phosphorylation at 5 min compared with steady (P < 0.05) and to and fro shear (P < 0.01) while there was no significant difference in pp70(S6k) phosphorylation between any shear regimen. The ratio of apoptotic cells in serum deprived EC in the presence of steady laminar, pulsatile and to and fro shear for 4 h were 2.7 +/- 0.78%, 2.7 +/- 0.42%, and 2.9 +/- 0.62%, respectively while after the addition of serum for 4 h, it was 4.3 +/- 0.73%. All shear regimens phosphorylated AKT in a time-dependent manner with no significant difference between regimens. Our results demonstrate that different types of shear stress regimens have different effects on EC and may account for the variable response of EC to hemodynamics in the circulation.     

Temporal and spatial differences in glycosaminoglycan synthesis by fetal lung fibroblasts

In studies of the ontogeny of fibroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [3H]glucosamine and Na2 35SO4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts (t1/2 = 12 h) than from that of epithelial cells (t1/2 = 48 h). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation.     

Permeability of human venous endothelial cell monolayers perfused in microcarrier cultures: effects of flow rate, thrombin, and cytochalasin D.

We have applied a multiple isotope dilution technique to examine junctional permeability of human umbilical vein endothelial cells (HUVEC) in vitro. Primary cultures were grown to confluence on porous Cytodex-3 microcarrier beads, packed into 0.3 ml columns (3 x 10(6) cells) and perfused at varying flow rates (0.3-1.2 ml/min) with HEPES-buffered Tyrodes solution containing unlabeled cyanocobalamin, insulin, and albumin. Columns were challenged periodically with mixtures of radioactive tracers of different molecular size. Permeability to 22Na+, [57Co]cyanocobalamin (1.3 kD), [125I]insulin (6 kD) or [125I]albumin (66 kD) was assessed relative to [131I]IgG (160 kD, impermeant reference tracer) by comparing column elution profiles. Although the single passage extraction of [125I]albumin by beads alone approximated 40%, the presence of confluent HUVEC rendered these beads effectively impermeable to albumin. High junctional extractions were measured for cyanocobalamin (0.79 +/- 0.02, n = 28) and insulin (0.51 +/- 0.05, n = 14) in cultures perfused at 0.3-0.4 ml/min, and tracer extraction decreased as perfusion rates increased. Permeability coefficients for cyanocobalamin (9.66 x 10(-5) cm/s) and insulin (4.18 x 10(-5) cm/s) increased significantly during perfusion with thrombin (10 U/ml) or cytochalasin D (1 microgram/ml), whereas permeability to albumin (0.39 x 10(-5) cm/s) remained unchanged. Morphological studies, using the glycocalyx stain ruthenium red, revealed that thrombin or cytochalasin D increased the penetration of the stain into junctions between endothelial cells.     

Change in properties of the glycocalyx affects the shear rate and stress distribution on endothelial cells

The endothelial glycocalyx mediates interactions between the blood flow and the endothelium. This study aims to evaluate, quantitatively, effects of structural change of the glycocalyx on stress distribution and shear rate on endothelial cells. In the study, the endothelial glycocalyx is modeled as a surface layer of fiber matrix and when exposed to laminar shear flow, the matrix deforms. Fluid velocity and stress distribution inside the matrix and on cell membranes are studied based on a binary mixture theory. Parameters, such as the height and porosity of the matrix and the drag coefficient between fluid and matrix fibrils, are based on available data and estimation from experiments. Simple theoretical solutions are achieved for fluid velocity and stress distribution in the surface matrix. Degradation of the matrix, e.g., by enzyme digestion, is represented by reductions in the volume fraction of fibrils, height, and drag coefficient. From a force balance, total stress on endothelial surface remains constant regardless of structural alteration of the glycocalyx. However, the stress that is transmitted to endothelial cells by direct "pulling" of fiber branches of the glycocalyx is reduced significantly. Fluid shear rate at the cell membrane, on the other hand, increases. The study gives quantitative insight into the effect of the structural change of the glycocalyx on the shear rate and pulling stress on the endothelium. Results can be used to interpret experiments on effects of the glycocalyx in shear induced endothelial responses.     

Myocardial perfusion reserve in adults with cyanotic congenital heart disease

In patients with cyanotic congenital heart disease (CCHD), a right-to-left shunt results in systemic hypoxemia. Systemic hypoxemia incites a compensatory erythrocytosis, which increases whole blood viscosity. We considered that these changes might adversely influence myocardial perfusion in CCHD patients. Basal and hyperemic (intravenous dipyridamole) perfusion measurements were obtained with [13N]ammonia positron emission tomographic imaging in left (LV) and right (RV) ventricular and septal myocardium in 14 adults with CCHD [age: 34.1 yr (SD 6.5)]; hematocrit: 62.2% (SD 4.8)] and 10 healthy controls [age: 34.1 yr (SD 6.5)]. In patients, basal perfusion measurements were higher in LV [0.77 (SD 0.24) vs. 0.55 ml x min(-1) x g(-1) (SD 0.09), P < 0.02], septum [0.71 (SD 0.16) vs. 0.49 ml x min(-1) x g(-1) (SD 0.09), P < 0.001], and RV [0.77 (SD 0.30) vs. 0.38 ml x min(-1) x g(-1) (SD 0.09), P < 0.001]. However, basal measurements normalized for the rate-pressure product were similar to those of controls. Calculated oxygen delivery relative to rate-pressure product was higher in the patients [2.2 (SD 0.8) vs. 1.6 (SD 0.4) x 10(-5) ml O2 x min(-1) x g tissue(-1) x (beats x mmHg)(-1) in the LV, P < 0.05, and 2.0 (SD 0.7) vs. 1.4 (SD 0.3) x 10(-5) ml O2 x min(-1) x g tissue(-1) x (beats x mmHg)(-1) in the septum, P < 0.01]. Hyperemic perfusion measurements in CCHD patients did not differ from controls [LV, 1.67 (SD 0.60) vs. 1.95 ml x min(-1) x g(-1) (SD 0.46); septum, 1.44 (SD 0.56) vs. 1.98 ml x min(-1) x g(-1) (SD 0.69); RV, 1.56 (SD 0.56) vs. 1.65 ml x min(-1) x g(-1) (SD 0.64), P = not significant], and coronary vascular resistances were comparable [LV, 55 (SD 25) vs. 48 mmHg x ml(-1) x g x min (SD 16); septum, 67 (SD 35) vs. 50 mmHg x ml(-1) x g x min (SD 21); RV, 59 (SD 26) vs. 61 mmHg x ml(-1) x g x min (SD 27), P = not significant]. These findings suggest that adult CCHD patients have remodeling of the coronary circulation to compensate for the rheologic changes attending chronic hypoxemia.     

Angiogenesis and its hormonal control in the corpus luteum of the pregnant rat

Beginning on Day 8 of pregnancy (Day 1 = sperm in vaginal smear), rats were injected i.p. with [3H] thymidine (TDR), killed 3 h later, and corpora lutea (CL) were dissected and saved for determining radioactivity in the acid-insoluble fraction or for autoradiography to determine labeling index (LI) of luteal and endothelial cells. An approximate doubling in DNA content in CL occurred between Days 13 and 14, with a high level maintained through Day 23. This was reflected in an abrupt increase in [3H] TDR incorporation on Day 13, with the peak reached on Day 14 and a subsequent decline to baseline values on Day 18. Autoradiography revealed that the LI of luteal endothelial cells rose from 2.1% on Day 12 to 10.0% on Day 14, and the LI of luteal cells correspondingly increased from 0.3% to 2.3%. Hypophysectomy (H) on Day 12 resulted, by Day 14, in no change in serum progesterone (P4) and TDR incorporation and LI of endothelial cells. However, after H and hysterectomy (HS) on Day 12, by Day 14, animals had low values for LI of endothelial and luteal cells, [3H] TDR incorporation and serum P4. After H + HS at Day 12, animals injected daily with estradiol cyclopentylpropionate (200 micrograms/day) on Days 12-14 had serum P4, [3H] TDR incorporation and LI of endothelial cells comparable to intact controls but not to luteal cells. However, similar treatment with testosterone cypionate (200 micrograms/day) or P4 (10 mg/day) did not maintain [3H] TDR incorporation or LI of either cell type, although serum P4 and estradiol levels were restored to normal values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.     

Oxygen release from arterioles with normal flow and no-flow conditions.

The rate of oxygen release from arterioles ( approximately 55 microm diameter) was measured in the hamster window chamber model during flow and no-flow conditions. Flow was stopped by microvascular transcutaneous occlusion using a glass pipette held by a manipulator. The reduction of the intra-arteriolar oxygen tension (Po2) was measured by the phosphorescence quenching of preinfused Pd-porphyrin, 100 microm downstream from the occlusion. Oxygen release from arterioles was found to be 53% greater during flow than no-flow conditions (2.6 vs. 1.7 x 10(-5) ml O2.cm(-2).s(-1), P < 0.05). Acute hemodilution with dextran 70 was used to reduce vessel oxygen content, significantly increase wall shear stress (14%, P < 0.05), reduce Hct to 28.4% (SD 1.0) [vs. 48.8% (SD 1.8) at baseline], lower oxygen supply by the arterioles (10%, P < 0.05), and increase oxygen release from the arterioles (39%, P < 0.05). Hemodilution also increased microcirculation oxygen extraction (33% greater than nonhemodilution, P < 0.05) and oxygen consumption by the vessel wall, as shown by an increase in vessel wall oxygen gradient [difference in Po2 between the blood and the tissue side of the arteriolar wall, nonhemodiluted 16.2 Torr (SD 1.0) vs. hemodiluted 18.3 Torr (SD 1.4), P < 0.05]. Oxygen released by the arterioles during flow vs. nonflow was increased significantly after hemodilution (3.6 vs. 1.8 x 10(-5) ml O2.cm(-2).s(-1), P < 0.05). The oxygen cost induced by wall shear stress, suggested by our findings, may be >15% of the total oxygen delivery to tissues by arterioles during flow in this preparation.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.