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1.  Determination of erythrocyte sodium sensitivity in man 
Pflugers Archiv  2013;465(10):1459-1466.
Sodium buffer capacity of vascular endothelium depends on an endothelial glycocalyx rich in negatively charged heparan sulfate. It has been shown recently that after the mechanical interaction of blood with heparan sulfate-depleted endothelium, erythrocytes also lose this glycocalyx constituent. This observation led to the conclusion that the vascular sodium buffer capacity of an individual could be derived from a blood sample. A test system (salt blood test (SBT)) was developed based upon the sodium-dependent erythrocyte zeta potential. Erythrocyte sedimentation velocity was measured in isosmotic, biopolymer-supplemented electrolyte solutions of different sodium concentrations. Erythrocyte sodium sensitivity (ESS), inversely related to erythrocyte sodium buffer capacity, was expressed as the ratio of the erythrocyte sedimentation velocities of 150 mM over 125 mM Na+ solutions (ESS = Na+150/Na+125). In 61 healthy individuals (mean age, 23 ± 0.5 years), ESS ranged between 2 and 8. The mean value was 4.3 ± 0.19. The frequency distribution shows two peaks, one at about 3 and another one at about 5. To test whether ESS reflects changes of the endothelial glycocalyx, a cultured endothelial monolayer was exposed for 3 hours to a rhythmically moving blood layer (drag force experiment). When applying this procedure, we found that ESS was reduced by about 21 % when the endothelium was pretreated for 4 days with the glycocalyx protective agent WS 1442. In conclusion, the SBT could possibly serve as an in vitro test system for the evaluation of erythrocyte/vascular salt sensitivity allowing follow-up measurements in the prevention and treatment of vascular dysfunctions.
PMCID: PMC3778990  PMID: 23686295
Erythrocyte glycocalyx; Heparan sulfate; Endothelial glycocalyx; Red blood cell sedimentation velocity; Vascular sodium sensitivity
2.  A physiological concept unmasking vascular salt sensitivity in man 
Pflugers Archiv  2012;464(3):287-293.
About one third of the population worldwide is supposed to be salt sensitive which is a major cause for arterial hypertension later in life. For preventive actions it is thus desirable to identify salt-sensitive individuals before the appearance of clinical symptoms. Recent observations suggest that the vascular endothelium consists of two salt-sensitive barriers in series, the glycocalyx that buffers sodium and the endothelial cell membrane that contains sodium channels. Glycocalyx sodium buffer capacity and sodium channel activity are conversely related to each other. For proof of concept, a so-called salt provocation test (SPT) was developed that should unmask vascular salt sensitivity in humans at virtually any age. Nineteen healthy subjects, ranging from 25 to 63 years of age, underwent two series of 1-h blood pressure measurements after acute ingestion of a salt cocktail with or without addition of a sodium channel blocker effective in vascular endothelium. Differential analysis of the changes in diastolic blood pressure (net ∆DP) identified 12 individuals (63 %) as being salt resistant (net ∆DP = −0.05 ± 0.62 mmHg) and seven individuals (37 %) as being salt sensitive (net ∆DP = +6.98 ± 0.75 mmHg). Vascular salt sensitivity was not related to the age of the study participants. It is concluded that the SPT could be useful for identifying vascular salt sensitivity in humans already early in life.
PMCID: PMC3423571  PMID: 22744228
Epithelial sodium channel (ENaC); Amiloride; Hypertension; Endothelial glycocalyx; Salt provocation test
3.  Salt Sensitivity and Hypertension: A Paradigm Shift from Kidney Malfunction to Vascular Endothelial Dysfunction 
Hypertension is a complex trait determined by both genetic and environmental factors and is a major public health problem due to its high prevalence and concomitant increase in the risk for cardiovascular disease. With the recent large increase of dietary salt intake in most developed countries, the prevalence of hypertension increases tremendously which is about 30% of the world population. There is substantial evidence that suggests some people can effectively excrete high dietary salt intake without an increase in arterial BP, and another people cannot excrete effectively without an increase in arterial BP. Salt sensitivity of BP refers to the BP responses for changes in dietary salt intake to produce meaningful BP increases or decreases. The underlying mechanisms that promote salt sensitivity are complex and range from genetic to environmental influences. The phenotype of salt sensitivity is therefore heterogeneous with multiple mechanisms that potentially link high salt intake to increases in blood pressure. Moreover, excess salt intake has functional and pathological effects on the vasculature that are independent of blood pressure. Epidemiologic data demonstrate the role of high dietary salt intake in mediating cardiovascular and renal morbidity and mortality. Almost five decades ago, Guyton and Coleman proposed that whenever arterial pressure is elevated, pressure natriuresis enhances the excretion of sodium and water until blood volume is reduced sufficiently to return arterial pressure to control values. According to this hypothesis, hypertension can develop only when something impairs the excretory ability of sodium in the kidney. However, recent studies suggest that nonosmotic salt accumulation in the skin interstitium and the endothelial dysfunction which might be caused by the deterioration of vascular endothelial glycocalyx layer (EGL) and the epithelial sodium channel on the endothelial luminal surface (EnNaC) also play an important role in nonosmotic storage of salt. These new concepts emphasize that sodium homeostasis and salt sensitivity seem to be related not only to the kidney malfunction but also to the endothelial dysfunction. Further investigations will be needed to assess the extent to which changes in the sodium buffering capacity of the skin interstitium and develop the treatment strategy for modulating the endothelial dysfunction.
PMCID: PMC4520886  PMID: 26240595
Salt; Sensitivity; Hypertension; Kidney malfunction; Endothelial dysfunction
4.  Sodium selective erythrocyte glycocalyx and salt sensitivity in man 
Pflugers Archiv  2014;467(6):1319-1325.
Negatively charged surfaces of erythrocytes (RBC) reflect properties of the endothelial glycocalyx. Plasma electrolytes counteract these charges and thus control the repulsive forces between RBC and endothelium. Although Na+ is supposed to exert a rather high affinity to the RBC surface, a direct comparison between Na+ and K+ in counteracting the RBC surface has been never made. Therefore, we measured Na+/K+ selectivity of the RBC surface in 20 healthy volunteers applying the previously published salt blood test (SBT). It turned out that the Na+/K+ selectivity ratio of the RBC glycocalyx is on average 6.1 ± 0.39 (ranging from 3 to 9 in different individuals). Considering standard plasma Na+ and K+ concentrations, binding probability of Na+/K+ at the RBC surface is about 180:1. The SBT reveals that plasma K+ counteracts only about 7 % of the negative charges in the RBC glycocalyx. As an in vivo proof of principle, a volunteer’s blood was continuously tested over 6 months while applying a glycocalyx protective polyphenol-rich natural compound (hawthorn extract). It turned out that RBC Na+ sensitivity (the inverse of Na+ buffer capacity) decreased significantly by about 25 % while Na+/K+ selectivity of the RBC glycocalyx declined only slightly by about 8 %. Taken together, (i) plasma Na+ selectively buffers the negative charges of the RBC glycocalyx, (ii) the contribution of K+ in counteracting these negative surface charges is small, and (iii) natural polyphenols applied in vivo increase RBC surface negativity. In conclusion, low plasma Na+ is supposed to favor frictionless RBC-slipping through blood vessels.
PMCID: PMC4435639  PMID: 25027385
Glycocalyx; Sodium sensitivity; Salt blood test; Surface charge; Hawthorn; Polyphenol
5.  Increase in Vascular Injury of Sodium Overloaded Mice May be Related to Vascular Angiotensin Modulation 
PLoS ONE  2015;10(6):e0128141.
This study aimed to analyzing the effect of chronic sodium overload upon carotid and femoral injury, and its relation to vascular angiotensin modulation. Male C57Bl6 mice were divided in: control (cont), receiving 1% NaCl solution for 2 weeks (salt-2) or 12 weeks (salt-12). Two-weeks before the end of the study, a 2mm catheter was implanted around the left femoral and carotid arteries to induce injury. Blood pressure (BP) and heart rate (HR) were measured at the end of the study by tail plethysmography. Arteries were collected and prepared for histological analysis to determine arterial thickening and perivascular collagen deposition. Angiotensin II and Ang(1-7) were quantified in fresh arteries using the HPLC method. There were no differences in body weight, BP and HR. Intima/media ratio had a similar increase in both injured arteries of cont and salt-2 mice, but a more pronounced increase was observed in salt-12 mice (31.1±6%). On the other hand, sodium overload modified perivascular collagen deposition, increasing thick fibers (cont: 0.5%; salt-2: 3.4%; salt-12: 0.6%) and decreasing thin fibers (cont: 7.4%; salt-2: 0.5%; salt-12: 6.8%) in non-injured arteries. Injured arteries presented similar collagen fiber distribution. Angiotensin quantification showed increased Ang(1-7) in salt treated mice (salt-2: +72%; salt-12: +45%) with a concomitant decrease in Ang II (salt-2: -54%; salt-12: -60%). Vascular injury increased significantly Ang(1-7) in salt-12 mice (+80%), maintaining Ang II reduction similar to that of a non-injured artery. The lack of changes in BP and HR suggests that the structural changes observed may be due to non-hemodynamic mechanisms such as local renin-angiotensin system. Collagen evaluation suggests that sodium overload induces time-related changes in vascular remodeling. The increase of artery injury with concomitant increase in Ang(1-7) in 12-week treated mice shows a direct association between the duration of salt treatment and the magnitude of vascular injury.
PMCID: PMC4451144  PMID: 26030299
6.  Influence of dietary salts on the cardiovascular effects of low-dose combination of ramipril and felodipine in spontaneously hypertensive rats 
British Journal of Pharmacology  1998;123(2):195-204.
In spontaneously hypertensive rat (SHR) we examined over a 4-week period the influence of control low sodium diet, common salt-enriched diet (sodium chloride 6% of the dry weight of the chow) and a novel mineral salt-enriched diet (potassium-, magnesium-, and l-lysine-enriched mineral salt added at a 75% higher level of 10.5% to produce the same sodium chloride concentration of 6%) on the cardiovascular effects produced by a low-dose combination of an angiotensin converting enzyme inhibitor ramipril (0.25 mg kg−1 day−1 in the food) and a calcium channel blocker felodipine (0.4 mg kg−1 day−1 subcutaneously via an osmotic minipump).Common salt, but not the mineral salt, accelerated the development of hypertension and induced left ventricular and renal hypertrophy in SHR. Neither common salt nor mineral salt significantly affected heart rate.The combination of ramipril and felodipine decreased systolic blood pressure and prevented the development of left ventricular hypertrophy effectively during the common salt diet without any significant effect on the heart rate. The cardiovascular effects of the drug combination were improved by the low sodium diet or by replacement of high common salt in the diet by mineral salt.Responses of endothelium-intact mesenteric arterial rings in vitro were examined at the end of the four-week study. The combination of ramipril and felodipine markedly improved the endothelium-dependent vascular relaxation responses to acetylcholine and enhanced the endothelium-independent vascular relaxation responses to sodium nitroprusside in SHR on control and common salt diets. Replacement of common salt in the diet by mineral salt improved the endothelium-dependent vascular relaxation responses to acetylcholine. The drug combination attenuated the α-adrenoceptor-mediated vascular contractile responses to noradrenaline during the common salt diet.Ramipril and felodipine in combination increased plasma renin activity by 1.9–3.2 fold without affecting serum aldosterone levels.Our findings suggest that the cardiovascular effect of the low-dose combination of ramipril and felodipine was maintained during high salt intake. However, salt restriction or replacement of common salt in the diet by the potassium- and magnesium-enriched mineral salt improved the cardiovascular effects of the drug combination. In the face of a high intake of sodium, a part of the beneficial cardiovascular effects of the drug combination is apparently mediated by improved endothelium-dependent and endothelium-independent vascular relaxation responses and attenuated α-adrenoceptor-mediated vascular contractile responses.
PMCID: PMC1565153  PMID: 9489606
Ramipril; felodipine; spontaneously hypertensive rat; left ventricular hypertrophy; arterial smooth muscle; sodium; potassium; magnesium
7.  Developing a national salt reduction strategy for Mongolia 
The increase in prevalence of risk factors such as hypertension has contributed to an incremental rise in non-communicable diseases (NCDs) in Mongolia over recent decades, such that they now account for 80% of all deaths in the country. Salt reduction is one of the most cost-effective interventions to reduce the burden of NCDs.
In 2011, the Ministry of Health (MOH) instigated the development of a national salt reduction strategy for Mongolia. As part of a 2-week national consultation and training program on salt reduction, it established an inter-sectoral working party and organized a series of bilateral meetings and visits to factories. Actions arising included a baseline survey of population salt consumption patterns and the implementation of a series of pilot salt reduction initiatives.
The results of the baseline assessment revealed that average daily intake of salt, based on 24 hour urine samples from a representative national sample (n=1,027), was 11.06±5.99 g in 2011, more than double the World Health Organization (WHO) five grams recommendation. Moreover, while most participants knew that salt was bad for health, few were taking efforts to reduce intake, and many were consuming highly salty meals and tea; salt in tea alone was estimated to contribute 30% of daily salt intake. A pilot Pinch Salt intervention to reduce salt consumption of factory workers was undertaken in Ulaanbaatar (UB) city between 2012 and 2013, and was associated with a reduction of 2.8 g of salt intake. Ongoing food industry initiatives have led to significant reductions in salt levels in bread, and companies producing processed meat have indicated a willingness to reduce salt. Relevant stakeholders have also supported the campaign by participating in annual World Salt Awareness Week events. The activities to date have demonstrated the potential for action and there is now a need scale these up to a national level to ensure that Mongolia is in a strong position to achieve a 30% reduction in population salt intake by 2025. The main goal of the Mongolian national salt reduction strategy is to create a social, economic and legal environment that supports salt reduction, including by influencing food supply, increasing partnerships between government and relevant stakeholders, and creating an enabling environment to support improved consumer choices. The strategy will be implemented from 2015 to 2025, with an interim review of progress in 2020.
Given that Mongolia has one of the highest rates of stroke in the world, which is strongly associated with population-wide blood pressure (BP) levels, the addition of a population-based stroke surveillance program would provide a reliable direct assessment of the impact of these salt reduction initiatives on the health of the Mongolian people. The results from this research would likely be widely generalizable to other populations experiencing similar lifestyle transitional changes.
PMCID: PMC4451311  PMID: 26090334
Sodium; salt intake; 24-hour urine; economic region; population; Mongolia
8.  Fenofibrate lowers blood pressure in salt-sensitive but not salt-resistant hypertension 
Journal of hypertension  2013;31(4):820-829.
Peroxisome proliferator-activated receptor α agonists reduce blood pressure in rodents, but clinical trials provide conflicting data regarding their effects in humans. We tested the hypothesis that the effect of fenofibrate on blood pressure depends on salt sensitivity.
Thirty-one hypertensive volunteers (17 salt-resistant, 14 salt-sensitive) completed a randomized, crossover, double-blind protocol with three dietary phases: low salt diet (10 mmol/day) followed by two consecutive high salt diets (200 mmol/day), each for 6 days. During high salt, volunteers were randomized to fenofibrate 160 mg/day or placebo. Hemodynamic and metabolic parameters were measured on the last morning of each treatment arm.
Fenofibrate reduced triglycerides similarly in salt-sensitive and salt-resistant volunteers. Fenofibrate did not affect blood pressure in salt-resistant volunteers. In salt-sensitive volunteers, fenofibrate significantly decreased diastolic (P =0.02 versus placebo) and mean arterial (P = 0.04 versus placebo) blood pressure during high salt. In all volunteers, the decrease in systolic pressure during fenofibrate correlated inversely with the salt sensitivity of mean arterial pressure as a continuous variable. Fenofibrate significantly decreased heart rate, plasma renin activity, and renal vascular resistance during high salt in salt-sensitive volunteers, but not salt-resistant volunteers. Fenofibrate did not affect sodium excretion or weight gain during high salt. The effect of salt intake and fenofibrate on plasma and urine epoxyeicosatrienoic acid concentrations differed in salt-resistant and salt-sensitive volunteers.
Fenofibrate reduces blood pressure, heart rate and renal vasoconstriction in salt-sensitive volunteers, but not in salt-resistant volunteers. These findings have implications for the treatment of hyperlipidemia in hypertensive individuals.
PMCID: PMC3800119  PMID: 23385647
blood pressure; fenofibrate; hypertension; peroxisome proliferator-activated receptor α; salt
9.  Lack of Association between Self-reported Saltiness of Eating and Actual Salt Intake 
Reducing salt intake is known to be an important factor for lowering blood pressure and preventing cardiovascular disease. Estimating amount of salt intake is a necessary step towards salt intake reduction. Self-reported saltiness of diet is a method most easily used to measure a patient's salt intake. The purpose of this study was to examine the value of self-reported saltiness of diet in measuring salt intake.
We used data from 681 participants who visited a health center at a university hospital between August 2003 and November 2005. A self-administered questionnaire was used to collect information on self-reported saltiness of diet, other dietary habits and lifestyle factors. Salt intake was estimated on the basis of 24-hour dietary recall with a computer-aided nutritional analysis program (CAN-Pro 2.0, Korean Nutrition Society).
There was no statistically significant difference between the mean salt intake of the self-reported salty diet group (13.7 ± 4.8 g/d) and the self-reported unsalty diet group (13.3 ± 4.4 g/d). If we assume calculated salt intake as the gold standard, the sensitivity and specificity of self-reported saltiness were 39.5% and 63.6%, respectively. Salt intake was increased with higher calorie intake, frequency of eating breakfast (≥5 times/wk) and being satiated with usual diet in men, but it was increased only with higher calorie intake in women. Regardless of actual salt intake, the group satiated with a usual diet tended to be in the group of self-reported salty diet.
Self-reported saltiness of diet was not associated with actual salt intake. Further studies will be needed on the simpler and more objective tools to estimate salt intake.
PMCID: PMC3383500  PMID: 22745893
Saltiness; Salt Intake; Self Report; Diet Records
10.  Endothelin-1-induced contraction in isolated aortae from normotensive and DOCA-salt hypertensive rats: effect of magnesium. 
British Journal of Pharmacology  1996;119(7):1367-1374.
1. The contractile responses to endothelin-1 and the effect on these of various magnesium concentrations, were studied in isolated aortic rings from normotensive Sprague-Dawley rats and deoxycorticosterone acetate-salt (DOCA-salt) hypertensive rats. 2. Contractions induced by endothelin-1 were smaller in endothelium-denuded aortae from DOCA-salt hypertensive rats than in those from normotensive rats. The absence of calcium in the medium attenuated endothelin-1-induced contractions of aortae from both normotensive and DOCA-salt rats, but the contraction was greater in aortae from DOCA-salt hypertensive rats. Ryanodine (which inhibits the release of intracellular calcium) inhibited endothelin-1-induced contractions in aortae from DOCA-salt hypertensive rats to a greater extent than in aortae from normotensive rats. 3. A high extracellular magnesium concentration (4.8 mM) attenuated endothelin-1-induced contractions in tissues from DOCA-salt hypertensive rats but not in tissues from normotensive rats. In the absence of calcium, a high concentration of magnesium attenuated endothelin-1-induced contraction in aortae from both normotensive and hypertensive rats. In the presence of ryanodine, a high concentration of magnesium did not modify the contraction in preparations from either strain. 4. Absence of magnesium attenuated endothelin-1-induced contractions in aortae from both normotensive and DOCA-salt hypertensive rats. In the absence of calcium, removal of magnesium totally inhibited endothelin-1-induced contraction in tissues from normotensive rats but had no effect in those from hypertensive rats. In the presence of ryanodine, the lack of magnesium inhibited endothelin-1-induced contractions in aortae from DOCA-salt hypertensive rats but increased the sensitivity to endothelin-1 of aortae from normotensive rats. 5. The presence of endothelium did not modify the effect of high magnesium on endothelin-1-induced contractions in aortae from normotensive and DOCA-salt hypertensive rats. Conversely, the attenuating effect of magnesium removal on endothelin-1-induced contractions did not occur when endothelium was present. 6. In conclusion, endothelin-1-induced contraction was blunted in aortae from DOCA-salt hypertensive rats. The blunted response was related to altered calcium utilization during contraction. Changes in extracellular magnesium concentration differentially alter endothelin-1-induced contraction in aortae from normotensive and hypertensive rats, possibly by interfering with calcium utilization during contraction. Magnesium may be required for the contractile response to endothelin-1 and increasing magnesium may limit the vascular effects of endothelin-1 in blood vessels from DOCA-salt hypertensive rats.
PMCID: PMC1915813  PMID: 8968545
11.  Modulation of Endothelial Glycocalyx Structure under Inflammatory Conditions 
Mediators of Inflammation  2014;2014:694312.
The glycocalyx of the endothelium is an intravascular compartment that creates a barrier between circulating blood and the vessel wall. The glycocalyx is suggested to play an important role in numerous physiological processes including the regulation of vascular permeability, the prevention of the margination of blood cells to the vessel wall, and the transmission of shear stress. Various theoretical models and experimental approaches provide data about changes to the structure and functions of the glycocalyx under various types of inflammatory conditions. These alterations are suggested to promote inflammatory processes in vessels and contribute to the pathogenesis of number of diseases. In this review we summarize current knowledge about the modulation of the glycocalyx under inflammatory conditions and the consequences for the course of inflammation in vessels. The structure and functions of endothelial glycocalyx are briefly discussed in the context of methodological approaches regarding the determination of endothelial glycocalyx and the uncertainty and challenges involved in glycocalyx structure determination. In addition, the modulation of glycocalyx structure under inflammatory conditions and the possible consequences for pathogenesis of selected diseases and medical conditions (in particular, diabetes, atherosclerosis, ischemia/reperfusion, and sepsis) are summarized. Finally, therapeutic strategies to ameliorate glycocalyx dysfunction suggested by various authors are discussed.
PMCID: PMC3997148  PMID: 24803742
12.  Influence of salty food preference on daily salt intake in primary care 
A salt preference questionnaire may be a convenient and cost-effective method for predicting salt intake; however, the influence of salt preference on daily salt intake is unclear. This study aimed at revealing the effectiveness of the salt preference question in determining the daily salt intake in primary care outpatients.
Patients and methods
This cross-sectional study included 1,075 outpatients (men, n=436, 40.6%) at six primary care institutions in Japan. Primary outcomes included a salty food preference assessed by using one question and a daily salt intake, assessed using early morning second urine samples. Multivariate analyses determined the relationships between the salt intake and the two salt preference levels.
The mean age was 67.6±14.6 years, and 594 (55.3%) preferred salty foods. The daily salt intake was 12.3±4.0 g per day and 11.4±3.7 g per day in the salt preference and nonsalt preference groups, respectively (P<0.001). A salt intake <10 g per day was consumed by 169 (28.5%) and 181 (37.6%) patients (P=0.001), respectively, and <6 g salt per day was consumed by 28 (4.7%) and 26 (5.4%) patients (P=0.606), respectively. The patients who preferred salty foods consumed a significantly larger amount of salt per day than those who did not prefer salty foods (β coefficient, 0.621; 95% confidence interval [CI], 0.146–1.095). There was no difference in the number of patients who consumed <10 g salt per day (adjusted odds ratio [ad-OR], 1.29; 95% CI, 0.99–1.69) or <6 g salt per day (ad-OR, 1.39; 0.90–1.69) between the groups.
Preference for salty foods was positively associated with daily salt intake. However, daily salt intake was not always appropriate, even in the patients who did not prefer salty foods. Behavioral interventions for salt restriction after an assessment of daily salt intake are necessary for primary care patients, regardless of their preference for salty foods.
PMCID: PMC3998866  PMID: 24790467
salt preference; daily salt intake; outpatients; primary care; salt restriction; spot urine method
It will be seen from the above that we have studied the conditions associated with the deposit of calcareous salts: (I) in connection with normal and pathological ossification, and (2) in pathological calcification as exhibited in (a) atheroma of the vessels; (b) calcification of caseating tubercular lesions; (c) calcification of inflammatory new growth, and (d) degenerating tumors; and we have induced experimentally deposits of calcareous salts in the lower animals: (a) within celloidin capsules containing fats and soaps; (b) in the kidney, and (c) in connection with fat necrosis. I. We have found that bone formation and pathological calcareous infiltration are wholly distinct processes. In the former there is no evidence of associated fatty change, and the cells associated with the process of deposition of calcium are functionally active. In the latter there is an antecedent fatty change in the affected areas, and the cells involved present constant evidences of degeneration. The view that would seem to account best for the changes observed in the latter case is that with lowered vitality the cells are unable to utilize the products brought to them by the blood, or which they continue to absorb, so that the normal series of decompositions associated with their metabolism fails to take place and hence they interact among themselves in the cytoplasm with the result that insoluble compounds replace soluble ones. II. Besides the fact that calcification is always preceded by fatty change within the cells, another fact should be emphasized. namely: that combination of the fats present with calcium salts to form calcium soaps tends to occur. The stages immediately preceding these are difficult to follow with anything approaching certainty, perhaps because the earlier stages vary under different conditions. In fat necrosis, for instance, the cells affected are normally storehouses for neutral fats, and as long as they remain healthy neutral fats alone are present in them. When they are subjected to the action of the pancreatic juice with its fat-splitting ferment the cells are killed and coincidently the neutral fats are decomposed, fatty acids being deposited. The fatty acids now slowly combine with the calcium salts. In degenerating lipomata the process would seem to be similar. But in other cases the cells are not obviously fat-containing in the normal state; nevertheless prior to calcification they undergo so-called fatty degeneration, which is really a form of cell degeneration accompanied by fat infiltration. As regards the source of the cell fats in general we may safely accept: 1. That fats are transported in the blood as diffusible soaps. 2. That taken up by the cells these soaps may either— (a) Be reconverted into neutral fats and become stored in the cytoplasm as such, or (b) undergo assimilation proper, becoming part and parcel of the cell substance, in which case they are not recognizable by the ordinary microchemical tests. 3. If these two possibilities be accepted it follows that the appearance of fats and soaps in the degenerating cell may be due to either— (a) Absorption or infiltration of soaps from the surrounding medium, the degenerating cell retaining the power of splitting off the fat but being unable to utilize this in metabolism. (b) Cytoplasmic disintegration with dissociation of the soap-albumen combination or, more broadly, liberation of the fats from their combination with the cytoplasm. The appearances seen in the cells of atheromatous areas indicate that the first of these does occur. III. In areas undergoing calcareous infiltration we have demonstrated. the presence of soaps, and this often in such quantities that they can be isolated and estimated by gross chemical methods. By microchemical methods also we have been able to show that after removing all the neutral fats and fatty acids by petroleum ether there remains behind a substance giving with Sudan III the reaction we associate with the presence of soap. And experimentally we have produced these soaps within the organism, more particularly by placing capsules containing fats and fatty acids within the tissues and after several days finding that the capsules contain calcium soaps and possess a calcium content far in excess of that of the normal blood and lymph. IV. While these are the facts, certain of the details of this reaction demand elucidation. The existence of sodium and it may be potassium soaps in the degenerated cells is comprehensible if we accept that these are present in the circulating lymph and simply undergoing absorption. But even then, as these are diffusible substances how is it to be explained that they become stored up in these particular areas? We have found that, as a matter of fact, in regions which give the reaction for soaps, but which give no reaction for calcium (which therefore presumably contain at most amounts of the insoluble calcium soap too small to need consideration, the ordinary solvents for potassium and sodium soaps do not forthwith remove the stainable material; they are relatively insoluble. The reason for this insolubility is suggested by the observations made in the test tube, that soap solutions mixed with solutions of white of egg or blood serum form a precipitate of combined soap and albumen, which likewise is insoluble in water and alcohol. The indications are therefore that in cells undergoing degeneration, with degeneration of the cytoplasm, certain albuminous molecules unite with the soaps present to form relatively insoluble soap-albuminate. V. With regard to calcium soaps, these are also present and in certain stages appear to be the dominating form in the affected tissues. Two questions suggest themselves, viz.: what is the source of calcium, and what is the process by which they become formed? As to the source, the amount present in well-marked calcification is far in excess of the normal calcium contents of the affected tissue. If in the kidneys of experimental calcification three hundred times as much calcium may be present as in the normal kidney (von Kossa), the calcium must be conveyed to the part by the blood and lymph, and that this is so is demonstrated, as we have pointed out, by the distribution of the infiltration in solid organs, that like ovarian fibroids have undergone necrosis, in which the earliest deposits are superficial. As to the process, there are three possibilities: 1. That sodium and potassium soaps and soap albuminates are first formed and that interaction occurs between them and the diffused calcium salts from the lymph, the less soluble-calcium replacing the sodium and potassium. 2. That under certain conditions the calcium salts act directly on the neutral fats present in the degenerating cells. 3. That the neutral fats are first broken down into fatty acids and that these react with the calcium salts to form the soaps. We are assured that the first process occurs and that because in the boundary zone of areas of calcification we can detect soapy particles devoid of calcium, identical in position and arrangement with the particles more deeply placed which give the calcium reactions. But this is not the only reaction. In case of fat necrosis we see clearly that the third process is in evidence. And we are far from being convinced that the second does not also obtain. We have been impressed by the large accumulation of neutral fats in the cells in cases of early atheroma and the absence at any stage of the process of recognizable fatty acid. While soaps, it is true, are compounds of fatty acids with alkalies, it is recognized in ordinary domestic life that they can be formed by the direct action of strong lye upon ordinary fats, and this even in the cold. It is quite possible therefore that there occurs a similar direct process in the organism. The point is worth noting, however, that this does not occur in healthy cells the seat of fatty infiltration. We therefore leave this an open question, only laying down that, as indicated by the hyalin albuminous matrix left when calcium salts are dissolved out of an area of calcification, there must exist a calcium soap- or fat-albuminate similar to the potassium and sodium soap-albuminates already mentioned—such an albuminate as we can form with calcium soaps in the test tube. VI. In old areas of calcification soaps are largely if not entirely wanting, although these are to be detected at the periphery, when the process is still advancing. The reactions given by these older areas are almost entirely those of calcium phosphate, though some calcium carbonate is at times to be made out. This seems surely to indicate that the final stage in calcification is an interaction between the calcium soap-albuminates and substances containing phosphoric and carbonic acids. Such substances, it is needless to say, are present in considerable amounts in the lymph and blood. We must conclude that the acid sodium phosphates of the lymph act on the calcium soap, the highly insoluble calcium phosphates being formed (plus the albuminous moiety of the original compound) and diffusible sodium soap being liberated, while similarly alkaline carbonates form calcium carbonate and liberate sodium and potassium soaps. Calcium phosphate and calcium carbonate thus become the insoluble earthy salts of old crystalline areas of calcification. VII. As already stated very little soap is to be found in these old areas. It is possibly worth suggestion that the soaps liberated in this last reaction, as they diffuse out, again react with diffusible calcium salts and form calcium soaps which once more react with the alkaline salts to produce the phosphates and carbonates; that, in short, they have a katalytic action. Certain it is that old calcareous areas are extraordinarily dense, and have a coarse crystalline structure, wholly at variance with the finely granular appearance of the more recent areas, and these large crystalline masses, it would seem, can only be formed by successive deposition of new material and eventual fusion, as the interspaces become filled in between the original masses.
PMCID: PMC2124594  PMID: 19867016
Journal of hypertension  2010;28(5):1020-1026.
Several mechanisms have been proposed for salt-sensitive hypertension with most focusing on impaired renal sodium handling. We tested the hypothesis that abnormalities in peripheral vascular responsiveness to angiotensin-II might also exist in salt-sensitive hypertension because of the interplay of the renin-angiotensin system and dietary sodium.
Blood pressure response to angiotensin-II infusion was studied in 295 hypertensives and 165 normotensives after 7 days of high (200 mEq/day) and low (10 mEq/day) dietary sodium.
Normotensives demonstrated higher blood pressure response to angiotensin-II on high-salt than low-salt diet whereas hypertensives had similar responses on both diets, i.e., the high-salt response was not enhanced compared to low-salt. Additionally, hypertensives had a significantly greater high-salt blood pressure response to norepinephrine than to angiotensin-II. There was no correlation between the high-salt hormone levels and the difference in blood pressure response to angiotensin-II between the two diets. When stratified by blood pressure response to dietary salt restriction, individuals with salt sensitivity of blood pressure demonstrated abnormal high-salt blood pressure responsiveness to angiotensin-II. To assess if this represented increased tissue renin-angiotensin system activity in the vasculature, blood pressure responses to angiotensin were compared before and after captopril in 20 hypertensives on a high-salt diet. Subjects with the greatest blood pressure lowering effect to captopril had similar high and low-salt blood pressure responses to angiotensin-II at baseline and a significant increase in the high-salt response after captopril.
Hypertensives have an abnormal vascular response to angiotensin-II infusion on a high-salt diet. Dysregulated tissue renin-angiotensin system activity may play a role in this abnormal response. These findings raise an intriguing novel possibility for the pathophysiologic mechanism of salt-sensitive hypertension.
PMCID: PMC2945810  PMID: 20216091
hypertension; salt sensitivity; salt-sensitive; sodium; angiotensin II response; tissue renin-angiotensin system
15.  Sodium renders endothelial cells sticky for red blood cells 
Negative charges in the glycocalyx of red blood cells (RBC) and vascular endothelial cells (EC) facilitate frictionless blood flow through blood vessels. Na+ selectively shields these charges controlling surface electronegativity. The question was addressed whether the ambient Na+ concentration controls RBC-EC interaction. Using atomic force microscopy (AFM) adhesion forces between RBC and endothelial glycocalyx were quantified. A single RBC, mounted on an AFM cantilever, was brought in physical contact with the endothelial surface and then pulled off. Adhesion forces were quantified (i) after enzymatic removal of negative charges in the glycocalyx, (ii) under different ambient Na+ and (iii) after applying the intracellular aldosterone receptor antagonist spironolactone. Removal of negative surface charges increases RBC-EC interaction forces. A stepwise increase of ambient Na+ from 133 to 140 mM does not affect them. However, beyond 140 mM Na+ adhesion forces increase sharply (10% increase of adhesion force per 1 mM increase of Na+). Spironolactone prevents this response. It is concluded that negative charges reduce adhesion between RBC and EC. Ambient Na+ concentration determines the availability of free negative charges. Na+ concentrations in the low physiological range (below 140 mM) allow sufficient amounts of vacant negative charges so that adhesion of RBC to the endothelial surface is small. In contrast, Na+ in the high physiological range (beyond 140 mM) saturates the remaining negative surface charges thus increasing adhesion. Aldosterone receptor blockade by spironolactone prevents Na+ induced RBC adhesion to the endothelial glycocalyx. Extrapolation of in vitro experiments to in vivo conditions leads to the hypothesis that high sodium intake is likely to increase the incidence of thrombotic events.
PMCID: PMC4485165  PMID: 26175691
endothelial glycocalyx; spironolactone; aldosterone; thrombosis; atomic force microscopy
16.  Spatio-temporal development of the endothelial glycocalyx layer and its mechanical property in vitro 
The endothelial glycocalyx is a thin layer of polysaccharide matrix on the luminal surface of endothelial cells (ECs), which contains sulphated proteoglycans and glycoproteins. It is a mechanotransducer and functions as an amplifier of the shear stress on ECs. It controls the vessel permeability and mediates the blood–endothelium interaction. This study investigates the spatial distribution and temporal development of the glycocalyx on cultured ECs, and evaluates mechanical properties of the glycocalyx using atomic force microscopy (AFM) nano-indentation. The glycocalyx on human umbilical vein endothelial cells (HUVECs) is observed under a confocal microscope. Manipulation of the glycocalyx is achieved using heparanase or neuraminidase. The Young's modulus of the cell membrane is calculated from the force–distance curve during AFM indentation. Results show that the glycocalyx appears predominantly on the edge of cells in the early days in culture, e.g. up to day 5 after seeding. On day 7, the glycocalyx is also seen in the apical area of the cell membrane. The thickness of the glycocalyx is approximately 300 nm–1 μm. AFM indentation reveals the Young's modulus of the cell membrane decreases from day 3 (2.93 ± 1.16 kPa) to day 14 (0.35 ± 0.15 kPa) and remains unchanged to day 21 (0.33 ± 0.19 kPa). Significant difference in the Young's modulus is also seen between the apical (1.54 ± 0.58 kPa) and the edge (0.69 ± 0.55 kPa) of cells at day 7. By contrast, neuraminidase-treated cells (i.e. without the glycocalyx) have similar values between day 3 (3.18 ± 0.88 kPa), day 14 (2.12 ± 0.78 kPa) and day 21 (2.15 ± 0.48 kPa). The endothelial glycocalyx in vitro shows temporal development in the early days in culture. It covers predominantly the edge of cells initially and appears on the apical membrane of cells as time progresses. The Young's modulus of the glycocalyx is deduced from Young's moduli of cell membranes with and without the glycocalyx layer. Our results show the glycocalyx on cultured HUVECs has a Young's modulus of approximately 0.39 kPa.
PMCID: PMC3405740  PMID: 22417911
vascular endothelium; the glycocalyx; confocal microscopy; atomic force microscopy nano-indentation; Young's modulus
17.  Two barriers for sodium in vascular endothelium? 
Annals of Medicine  2012;44(Suppl 1):S143-S148.
Vascular endothelium plays a key role in blood pressure regulation. Recently, it has been shown that a 5% increase of plasma sodium concentration (sodium excess) stiffens endothelial cells by about 25%, leading to cellular dysfunction. Surface measurements demonstrated that the endothelial glycocalyx (eGC), an anionic biopolymer, deteriorates when sodium is elevated. In view of these results, a two-barrier model for sodium exiting the circulation across the endothelium is suggested. The first sodium barrier is the eGC which selectively buffers sodium ions with its negatively charged prote-oglycans.The second sodium barrier is the endothelial plasma membrane which contains sodium channels. Sodium excess, in the presence of aldosterone, leads to eGC break-down and, in parallel, to an up-regulation of plasma membrane sodium channels. The following hypothesis is postulated: Sodium excess increases vascular sodium permeability. Under such con-ditions (e.g. high-sodium diet), day-by-day ingested sodium, instead of being readily buffered by the eGC and then rapidly excreted by the kidneys, is distributed in the whole body before being finally excreted. Gradually, the sodium overload damages the organism.
PMCID: PMC3470790  PMID: 22471931
Aldosterone; atomic force microscopy; endothelial dysfunction; endothelial glycocalyx; epithelial sodium channel; hypertension; mechanical stiffness; salt intake; spironolactone; stiff endothelial cell syndrome
18.  Lifestyle modifications to prevent and control hypertension. 5. Recommendations on dietary salt. Canadian Hypertension Society, Canadian Coalition for High Blood Pressure Prevention and Control, Laboratory Centre for Disease Control at Health Canada, Heart and Stroke Foundation of Canada 
OBJECTIVE: To provide updated, evidence-based recommendations concerning the effects of dietary salt intake on the prevention and control of hypertension in adults (except pregnant women). The guidelines are intended for use in clinical practice and public education campaigns. OPTIONS: Restriction of dietary salt intake may be an alternative to antihypertensive medications or may supplement such medications. Other options include other nonpharmacologic treatments for hypertension and no treatment. OUTCOMES: The health outcomes considered were changes in blood pressure and in morbidity and mortality rates. Because of insufficient evidence, no economic outcomes were considered. EVIDENCE: A MEDLINE search was conducted for the period 1966-1996 using the terms hypertension, blood pressure, vascular resistance, sodium chloride, sodium, diet, sodium or sodium chloride dietary, sodium restricted/reducing diet, clinical trials, controlled clinical trial, randomized controlled trial and random allocation. Both trials and review articles were obtained, and other relevant evidence was obtained from the reference lists of the articles identified, from the personal files of the authors and through contacts with experts. The articles were reviewed, classified according to study design and graded according to level of evidence. In addition, a systematic review of all published randomized controlled trials relating to dietary salt intake and hypertension was conducted. VALUES: A high value was placed on the avoidance of cardiovascular morbidity and premature death caused by untreated hypertension. BENEFITS, HARMS AND COSTS: For normotensive people, a marked change in sodium intake is required to achieve a modest reduction in blood pressure (there is a decrease of 1 mm Hg in systolic blood pressure for every 100 mmol decrease in daily sodium intake). For hypertensive patients, the effects of dietary salt restriction are most pronounced if age is greater than 44 years. A decrease of 6.3 mm Hg in systolic blood pressure and 2.2 mm Hg in diastolic blood pressure per 100 mmol decrease in daily sodium intake was observed in people of this age group. For hypertensive patients 44 years of age and younger, the decreases were 2.4 mm Hg for systolic blood pressure and negligible for diastolic blood pressure. A diet in which salt is moderately restricted appears not to be associated with health risks. RECOMMENDATIONS: (1) Restriction of salt intake for the normotensive population is not recommended at present, because of insufficient evidence demonstrating that this would lead to a reduced incidence of hypertension. (2) To avoid excessive intake of salt, people should be counselled to choose foods low in salt (e.g., fresh fruits and vegetables), to avoid foods high in salt (e.g., pre-prepared foods), to refrain from adding salt at the table and minimize the amount of salt used in cooking, and to increase awareness of the salt content of food choices in restaurants. (3) For hypertensive patients, particularly those over the age of 44 years, it is recommended that the intake of dietary sodium be moderately restricted, to a target range of 90-130 mmol per day (which corresponds to 3-7 g of salt per day). (4) The salt consumption of hypertensive patients should be determined by interview. VALIDATION: These recommendations were reviewed by all of the sponsoring organizations and by participants in a satellite symposium of the fourth International Conference on Preventive Cardiology. They have not been clinically tested. SPONSORS: The Canadian Hypertension Society, the Canadian Coalition for High Blood Pressure Prevention and Control, the Laboratory Centre for Disease Control at Health Canada, and the Heart and Stroke Foundation of Canada.
PMCID: PMC1230337  PMID: 10333851
19.  Effect of sulodexide on endothelial glycocalyx and vascular permeability in patients with type 2 diabetes mellitus 
Diabetologia  2010;53(12):2646-2655.
Endothelial glycocalyx perturbation contributes to increased vascular permeability. In the present study we set out to evaluate whether: (1) glycocalyx is perturbed in individuals with type 2 diabetes mellitus, and (2) oral glycocalyx precursor treatment improves glycocalyx properties.
Male participants with type 2 diabetes (n = 10) and controls (n = 10) were evaluated before and after 2 months of sulodexide administration (200 mg/day). The glycocalyx dimension was estimated in two different vascular beds using sidestream dark field imaging and combined fluorescein/indocyanine green angiography for sublingual and retinal vessels, respectively. Transcapillary escape rate of albumin (TERalb) and hyaluronan catabolism were assessed as measures of vascular permeability.
Both sublingual dimensions (0.64 [0.57–0.75] μm vs 0.78 [0.71–0.85] μm, p < 0.05, medians [interquartile range]) and retinal glycocalyx dimensions (5.38 [4.88–6.59] μm vs 8.89 [4.74–11.84] μm, p < 0.05) were reduced in the type 2 diabetes group compared with the controls whereas TERalb was increased (5.6 ± 2.3% vs 3.7 ± 1.7% in the controls, p < 0.05). In line with these findings, markers of hyaluronan catabolism were increased with diabetes (hyaluronan 137 ± 29 vs 81 ± 8 ng/ml and hyaluronidase 78 ± 4 vs 67 ± 2 U/ml, both p < 0.05). Sulodexide increased both the sublingual and retinal glycocalyx dimensions in participants with diabetes (to 0.93 [0.83–0.99] μm and to 5.88 [5.33–6.26] μm, respectively, p < 0.05). In line, a trend towards TERalb normalisation (to 4.0 ± 2.3%) and decreases in plasma hyaluronidase (to 72 ± 2 U/ml, p < 0.05) were observed in the diabetes group.
Type 2 diabetes is associated with glycocalyx perturbation and increased vascular permeability, which are partially restored following sulodexide administration. Further studies are warranted to determine whether long-term treatment with sulodexide has a beneficial effect on cardiovascular risk.
Trial registration NTR780/ ISRCTN82695186
An unrestricted Novartis Foundation for Cardiovascular Excellence grant (2006) to M. Nieuwdorp/E. S. G. Stroes, Dutch Heart Foundation (grant number 2005T037)
PMCID: PMC2974920  PMID: 20865240
Diabetes mellitus type 2; Endothelial glycocalyx; Hyaluronan; Sulodexide; Vascular permeability
20.  Chronic angiotensin II infusion causes differential responses in regional sympathetic nerve activity in rats 
Hypertension  2010;55(3):644-651.
Angiotensin II (AngII) – induced hypertension in experimental animals has been proposed to be due in part to activation of the sympathetic nervous system. This sympathetic activation appears to be accentuated in animals consuming a high salt diet (AngII-salt hypertension). However, accurate quantification of sympathetic activity is difficult and controversy remains. A particularly important question is: What are the critical vascular beds targeted by increased sympathetic nerve activity (SNA) in AngII-salt hypertension? To address this issue, mean arterial pressure (MAP) and renal (RSNA) or lumbar SNA (LSNA) were continuously recorded during a 5 day control period, 11 days of AngII (150 ng/kg/min, sc) and a 5 day recovery period in conscious rats on a high salt (2% NaCl) diet. Whereas MAP reached a new steady-state level of 30-35 mmHg above control levels by the end of the AngII period, RSNA decreased by 40% during the first 7 days of AngII and then returned towards control levels by day 10 of AngII. In contrast, LSNA remained at control levels throughout the AngII period. In another experiment we measured hindlimb norepinephrine (NE) spillover in conscious rats on normal (0.4%) or high (2.0%) salt diets before and during 14 days of AngII administration. AngII had no significant affect on hindlimb NE spillover in either group. We conclude that chronic AngII modulates renal and lumbar SNA differentially in rats consuming a high salt diet and that AngII-salt hypertension in the rat is not caused by increased SNA to the renal or hindlimb vascular beds.
PMCID: PMC2856065  PMID: 20100996
hypertension; renal nerve activity; lumbar nerve activity; norepinephrine spillover; sympathetic
21.  High dietary salt does not significantly affect plasma 25-hydroxyvitamin D concentrations of Sprague Dawley rats 
BMC Research Notes  2010;3:332.
The Dahl salt-sensitive rat, but not the Dahl salt-resistant rat, develops hypertension and hypovitaminosis D when fed a high salt diet. Since the salt-sensitive rat and salt-resistant rat were bred from the Sprague Dawley rat, the aim of this research was to test the hypothesis that salt-resistant and Sprague Dawley rats would be similar in their vitamin D endocrine system response to high salt intake.
Sprague Dawley, salt-sensitive, and salt-resistant rats were fed high (80 g/kg, 8%) or low (3 g/kg, 3%) salt diets for three weeks. The blood pressure of Sprague Dawley rats increased from baseline to week 3 during both high and low salt intake and the mean blood pressure at week 3 of high salt intake was higher than that at week 3 of low salt intake (P < 0.05). Mean plasma 25-hydroxyvitamin D concentrations (marker of vitamin D status) of Sprague Dawley, salt-sensitive, and salt-resistant rats were similar at week 3 of low salt intake. Mean plasma 25-hydroxyvitamin D concentrations of Sprague Dawley and salt-resistant rats were unaffected by high salt intake, whereas the mean plasma 25-hydroxyvitamin D concentration of salt-sensitive rats at week 3 of high salt intake was only 20% of that at week 3 of low salt intake.
These data indicate that the effect of high salt intake on the vitamin D endocrine system of Sprague Dawley rats at week 3 was similar to that of salt-resistant rats. The salt-sensitive rat, thus, appears to be a more appropriate model than the Sprague Dawley rat for assessing possible effects of salt-sensitivity on vitamin D status of humans.
PMCID: PMC3018403  PMID: 21143930
22.  Potassium Inhibits Dietary Salt-Induced Transforming Growth Factor-β Production 
Hypertension  2009;54(5):1159-1163.
Human and animal studies demonstrate an untoward effect of excess dietary NaCl (salt) intake on cardiovascular function and life span. The endothelium in particular augments the production of transforming growth factor (TGF)-β, a fibrogenic growth factor, in response to excess dietary salt intake. This study explored the initiating mechanism that regulates salt-induced endothelial cell production of TGF-β. Male Sprague-Dawley rats were given diets containing different amounts of NaCl and potassium for 4 days. A bioassay for TGF-β demonstrated increased (35.2%) amounts of active TGF-β in the medium of aortic ring segments from rats on the high-salt diet compared with rats maintained on a 0.3% NaCl diet. Inhibition of the large-conductance, calcium-activated potassium channel inhibited dietary salt-induced vascular production of TGF-β but did not affect production of TGF-β by ring segments from rats on the low-salt diet. Immunohistochemical and Western analyses demonstrated the α subunit of the calcium-activated potassium channel in endothelial cells. Increasing medium [K+] inhibited production of dietary salt-induced vascular production levels of total and active TGF-β but did not alter TGF-β production by aortic rings from rats on the 0.3% NaCl diet. Increasing dietary potassium content decreased urinary active TGF-β in animals receiving the high-salt diet but did not change urinary active TGF-β in animals receiving the low-salt diet. The findings demonstrated an interesting interaction between the dietary intake of potassium and excess NaCl and further showed the fundamental role of the endothelial calcium-activated potassium channel in the vascular response to excess salt intake.
PMCID: PMC2766016  PMID: 19738156
dietary sodium chloride; potassium channel; endothelium; aorta; iberiotoxin; physiology
23.  Whole-body recruitment of glycocalyx volume during intravenous adenosine infusion 
Physiological Reports  2013;1(5):e00102.
Adenosine-mediated recruitment of microvascular volume in heart and muscle has been suggested to include, in addition to vasodilation of resistance vessels, an increased accessibility of the endothelial glycocalyx for flowing plasma as a result of an impairment of its barrier properties. The aim of the current study was to investigate the effect of systemic intravenous administration of adenosine on the glycocalyx-dependent exclusion of circulating blood at a whole-body level. In anesthetized goats (N = 6), systemic blood-excluded glycocalyx volume was measured by comparing the intravascular distribution volume of the suggested glycocalyx accessible tracer dextrans with a molecular weight of 40 kDa (Dex-40) to that of circulating plasma, derived from the dilution of labeled red blood cells and large vessel hematocrit. Systemic glycocalyx volume was determined at baseline and during intravenous infusion of adenosine (157 ± 11.6 μg/kg min−1). Blood-inaccessible glycocalyx volume decreased from 458.1 ± 95.5 to 18.1 ± 62.2 mL (P < 0.01) during adenosine administration. While circulating plasma volume did not change significantly (617.1 ± 48.5 vs. 759.2 ± 47.9 mL, NS), the decrease in blood-excluded glycocalyx volume was associated with a decrease in Dex-40 distribution volume (from 1075.2 ± 71.0 to 777.3 ± 60.0 mL, P < 0.01). Intravenous administration of adenosine is associated with a robust impairment of whole-body glycocalyx barrier properties, reflected by a greatly reduced exclusion of circulating blood compared to small dextrans. The observed decrease in Dex-40 distribution volume suggests that the reduction in glycocalyx volume coincides with a reduction in tracer-accessible vascular volume.
PMCID: PMC3841038  PMID: 24303174
Adenosine; glycocalyx; indicator dilution; systemic
24.  Effect of chronic ETA-selective endothelin receptor antagonism on blood pressure in experimental and genetic hypertension in rats 
British Journal of Pharmacology  1997;121(5):935-940.
Chronic treatment with a combined ETA/ETB endothelin receptor antagonist has been shown to reduce blood pressure in experimental rat models of hypertension in which endothelin-1 gene overexpression occurs in the walls of blood vessels, particularly small, resistance-sized arteries, but not in those genetic or experimental models of hypertension in which there is no overexpression of vascular endothelin-1. Failure of some experimental models of hypertension to respond to treatment with the combined ETA/ETB endothelin antagonist may be due in part to blockade of vasorelaxant endothelial ETB receptors which could in theory reduce the efficacy of endothelin antagonism.In this study the orally active ETA-selective endothelin antagonists A-127722.5 and LU 135252 were used in chronic experiments on deoxycorticosterone acetate (DOCA)-salt hypertensive rats (which overexpress vascular endothelin-1 and respond with blood pressure lowering to combined ETA/ETB endothelin receptor antagonism), on spontaneously hypertensive rats (SHR) (which do not overexpress vascular endothelin-1 and do not respond with blood pressure lowering to the combined ETA/ETB receptor antagonist), and in 1-kidney 1 clip Goldblatt (1-K 1C) hypertensive rats (which present mild overexpression of vascular endothelin-1 but do not respond with blood pressure lowering to the combined ETA/ETB receptor antagonist). Additionally, it has been suggested that interruption of the renin-angiotensin system may sensitize responses to endothelin antagonism. Accordingly, SHR were treated with an angiotensin converting enzyme inhibitor, cilazapril, in addition to the ETA receptor antagonist.Blood pressure of DOCA-salt hypertensive rats was lowered by a mean of 24 and of 27 mmHg (P<0.01) by A-127722.5 after 4 weeks of treatment, when given orally at two different doses (10 and 30 mg kg−1 day−1), and by 18 mmHg by LU 135252 50 mg kg−1 day−1.SHR treated with A-127722.5 for 8 weeks starting at 12 weeks of age exhibited the same progressive rise in blood pressure as untreated SHR. Addition of cilazapril resulted in similar reduction of blood pressure in A-127722.5-treated and untreated SHR.Treatment of 1-K 1C hypertensive rats with the dose of LU 135252 which lowered blood pressure in DOCA-salt hypertensive rats did not cause any reduction in blood pressure relative to untreated rats.These results demonstrate that treatment with either dose of the selective ETA receptor antagonists A-127722.5 or LU 135252 caused reductions in blood pressure similar to those obtained for a combined ETA/ETB endothelin antagonist. Blood pressure was lowered only in hypertensive rats known to overexpress vascular endothelin-1 (DOCA-salt hypertensive rats) but not in those which do not (SHR) or only have mild vascular overexpression of endothelin-1 gene (1-K 1C hypertensive rats). Reduction in activity of the renin-angiotensin system in SHR did not sensitize blood pressure to potential hypotensive effects of an ETA-selective receptor antagonist.
PMCID: PMC1564777  PMID: 9222550
Spontaneously hypertensive rats (SHR); DOCA-salt hypertensive rats; one-kidney one clip Goldblatt hypertensive rats; angiotensin converting enzyme inhibition; renin-angiotensin system; blood vessels; endothelin-1 gene expression; A-127722.5; LU 135252
25.  Eplerenone prevents salt-induced vascular stiffness in Zucker diabetic fatty rats: a preliminary report 
Aldosterone levels are elevated in a rat model of type 2 diabetes mellitus, the Zucker Diabetic fatty rat (ZDF). Moreover blood pressure in ZDF rats is salt-sensitive. The aim of this study was to examine the effect of the aldosterone antagonist eplerenone on structural and mechanical properties of resistance arteries of ZDF-rats on normal and high-salt diet.
After the development of diabetes, ZDF animals were fed either a normal salt diet (0.28%) or a high-salt diet (5.5%) starting at an age of 15 weeks. ZDF rats on high-salt diet were randomly assigned to eplerenone (100 mg/kg per day, in food) (ZDF+S+E), hydralazine (25 mg/kg per day) (ZDF+S+H), or no treatment (ZDF+S). Rats on normal salt-diet were assigned to eplerenone (ZDF+E) or no treatment (ZDF). Normoglycemic Zucker lean rats were also divided into two groups receiving normal (ZL) or high-salt diet (ZL+S) serving as controls. Systolic blood pressure was measured by tail cuff method. The experiment was terminated at an age of 25 weeks. Mesenteric resistance arteries were studied on a pressurized myograph. Specifically, vascular hypertrophy (media-to-lumen ratio) and vascular stiffness (strain and stress) were analyzed. After pressurized fixation histological analysis of collagen and elastin content was performed.
Blood pressure was significantly higher in salt-loaded ZDF compared to ZDF. Eplerenone and hydralazine prevented this rise similarily, however, significance niveau was missed. Media-to-lumen ratio of mesenteric resistance arteries was significantly increased in ZDF+S when compared to ZDF and ZL. Both, eplerenone and hydralazine prevented salt-induced vascular hypertrophy. The strain curve of arteries of salt-loaded ZDF rats was significantly lower when compared to ZL and when compared to ZDF+S+E, but was not different compared to ZDF+S+H. Eplerenone, but not hydralazine shifted the strain-stress curve to the right indicating a vascular wall composition with less resistant components. This indicates increased vascular stiffness in salt-loaded ZDF rats, which could be prevented by eplerenone but not by hydralazine. Collagen content was increased in ZL and ZDF rats on high-salt diet. Eplerenone and hydralazine prevented the increase of collagen content. There was no difference in elastin content.
Eplerenone and hydralazine prevented increased media-to-lumen ratio in salt-loaded ZDF-rats, indicating a regression of vascular hypertrophy, which is likely mediated by the blood pressure lowering-effect. Eplerenone has additionally the potential to prevent increased vascular stiffness in salt-loaded ZDF-rats. This suggests an effect of the specific aldosterone antagonist on adverse vascular wall remodelling.
PMCID: PMC3217853  PMID: 22008236

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