Update on the important new drug target in cardiovascular medicine - the vascular glycocalyx.

We reviewed this subject in 2009, pointing out that, to the process of atherothrombosis, glycocalyx dysfunction and damage must be added to the previous known causitive factors. Glycocalyx dysfunction is possibly the very first step in the process of atherothrombosis, being a protective layer between the endothelial cells and the blood. We emphasise the unique feature of glycocalyx mediated vasodilatation in that it is initiated purely by mechanical changes, i.e., changes in vascular wall shear stress, allowing conduit arteries to adjust diameter to demanded blood flow rate. The predeliction of atheroma to sites of low shear stress, the inhibition of the shear response by lumenal hyperglycaemia, and the fact that the response is mediated by nitric oxide (NO), an anti-atheromatous agent has led to the hypothesis that impairment of this pathway is pro-atherogenic. In the microcirculation it has been shown that the glycocalyx must be added to the factors involved in the Starling hypothesis of tissue fluid generation and exchange. As a consequence glycoalyx dysfunction in hyperglycaemia has been postulated to cause oedema and microalbinuria. We suggested that perhaps the arterial glycocalyx will become the most important for future early prevention of people at risk of cardiovascular disease. The advances in this subject since 2009 are the subject of the present review. What has struck us when searching the literature is that research into the glycocalyx has increased very much and now comes from many disciplines; e.g., diabetes, hypertension, bioengineering, physiology, critical care, cardiology, shock. This update is by no means exhaustive, but hopes, again, to bring to the attention of the pharmaceutical industry, the need for grants in the appropriate experimental models.

Dilatation in the femoral vascular bed does not cause retrograde relaxation of the iliac artery in the anaesthetized pig.

AIM: We tested the hypothesis that dilatation of a feeding artery may be elicited by transmission of a signal through the tissue of the arterial wall from a vasodilated peripheral vascular bed. METHODS: In eight pentobarbital anaesthetized pigs, acetylcholine (ACh, an endothelium-dependent vasodilator) was injected intra-arterially above (upstream) and below (downstream) a test segment of the left iliac artery, the diameter of which was measured continuously by sonomicrometry. RESULTS: Under control conditions, ACh injections upstream and downstream of the test segment caused dilatation. Downstream injection dilated the peripheral arterioles, resulting in increased blood flow and proximal dilatation. This is a shear stress, nitric oxide (NO)-dependent response. The experiment was then repeated after applying a stenosis to prevent the increased flow caused by downstream injection of ACh; the stenosis was placed either above the site of diameter measurement to allow retrograde conduction, or below that site to prevent distally injected ACh reaching the measurement site. Under these conditions, downstream injection of ACh had a minimal effect on the shear stress of the test segment with no increase in test segment diameter. This was not due to endothelial damage or dysfunction as injection of ACh upstream still caused a large increase in test segment diameter. CONCLUSIONS: Our results indicate that dilatation of the feeding artery of a vasodilated bed is caused by increased shear stress within the feeding artery and not via a signal transmitted through the arterial wall from below.

Hypothesis: arterial glycocalyx dysfunction is the first step in the atherothrombotic process.

We present evidence that the 0.5 microm thick gel layer, lining the inner wall of healthy blood vessels, the glycocalyx, is the first line of defence against atherothrombotic disease. All blood vessel linings are coated with this gel, a highly negatively charged structure, rich in anionic sites mostly represented by the sialic acid moieties of glycoproteins and the sulphate and carboxyl groups of heparan-sulphate proteoglycans. Blood flow in arteries is associated with a shear stress at the glycocalyx, which signals the underlying endothelial cells to release nitric oxide (NO), an anti-atherogenic factor. Sites of low shear stress in the arterial tree are more susceptible to atheroma due to lack of NO generation through this mechanism, whereas exercise, by increasing blood flow and shear stress, is protective. We postulate that risk factors for atherothrombosis act by impairing glycocalyx function. That luminal hyperglycaemia causes glycocalyx dysfunction has already been shown; we postulate this to be the first step in the atherothrombotic process in patients with diabetes mellitus and metabolic syndrome (insulin resistance). There is also evidence of glycocalyx defects from exposure to oxidized low-density lipoprotein. We postulate that other risk factors will have a similar action on the glycocalyx as the initiating factor in the disease process, e.g. smoking, hyperlipidaemias and hyperhomocystenaemia. These predictions can now be tested in a large animal model of shear-stress-mediated arterial dilatation.

The antiplatelet drug target in atherosclerotic diseases.

The aim of this review is (1) to give a rationale for anti-platelet therapy based on mechanisms of platelet rich arterial thrombosis, (2) to point out the pitfalls involved in monitoring therapy with platelet function tests and (3) to outline the potential clinical applications of such therapy based on the various modes of action of anti-platelet drugs. The primary event in arterial thrombosis is platelet-mediated, either due to increased shear or exposed collagen, followed by fibrin-rich thrombosis. Anti-platelet therapy needs to be monitored but most platelet function tests, now in use, do not reflect in vivo function; the anticoagulant used for blood samples removes extra-cellular calcium ions, platelets are often separated before the test, or very high doses of agonist are used: all of these can give misleading results. We review means whereby platelet function can be monitored in whole blood samples anticoagulated with the pure thrombin inhibitor, hirudin. We review the available methods of modifying platelet activity and are particularly interested in agents that do not cause bleeding. Present therapy causes bleeding by interference with COX1, the P2Y(12) receptor or the platelet fibrinogen receptor complex, all of which can be associated with bleeding complications. In contrast, serotonin does not influence formation of haemostatic layers although it is implicated in shear-induced aggregation and thrombus propagation by positive feedback from the large amount of intraplatelet serotonin. We suggest that further investigation of selective serotonin 5HT(2) antagonism would allow effective management of intravascular thrombosis without bleeding complications. This would be safer both as prophylaxis and would also allow cardioprotection of vascular patients undergoing surgical operations.

Differential inhibition by hyperglycaemia of shear stress- but not acetylcholine-mediated dilatation in the iliac artery of the anaesthetized pig.

Clinical hyperglycaemia affects vascular endothelial function, but the effect on shear stress-induced arterial dilatation has not yet been established. We hypothesized that hyperglycaemia would inhibit this response via impaired glycocalyx mechanotransduction. Experiments were carried out in the anaesthetized pig in which pressure, blood flow and diameter of the left iliac artery were measured at two sites: proximal (d1) and distal (d2). Infusion of glucose, sufficient to raise blood glucose to 16-30 mm along the whole length of the artery, attenuated the shear stress-dependent dilatation in both sections of the artery with preservation of the responses to acetylcholine. The distal site was then isolated using snares and the lumen exposed to blood containing 25-35 mm glucose for 20 min. In the control situation, after exposure of both sections to normoglycaemia (5.7 mm glucose), both sections of artery showed increases in diameter in response to shear stress and acetylcholine. Hyperglycaemia attenuated the shear stress-dependent dilatation in the distal section only (P < 0.25), but not the response to acetylcholine. It is concluded from these results that the hyperglycaemia-impaired dilatation is consistent with loss of mechanotransducing properties of the endothelial glycocalyx by hyperglycaemia. These findings offer a possible explanation for the increased incidence of vascular disease in diabetic patients.

Chronic catecholamine depletion switches myocardium from carbohydrate to lipid utilisation.

PURPOSE: Chronic cardiac transplantation denervation (i.e., global sympathetic denervation with myocardial catecholamine depletion, plus parasympathetic denervation) is known to inhibit myocardial oxidation of glucose. It is not known whether this is due to increased utilization of lactate, lipid or ketone bodies. The purpose of the present study was to test the hypothesis that the extraction and contribution of blood-borne fatty acids (FA) to overall oxidative energy conversion is increased. METHODS: In anaesthetised dogs (control n = 6, cardiac denervated n = 6), we investigated fatty acid (FA) utilization. The studies were made at least four weeks after surgical cardiac denervation. Measurements were made of total FAs and with a radio-labelled tracer (U-14C palmitate). RESULTS: The contribution of FA utilisation to overall substrate oxidation rose from 31% (control) to 48% (cardiac denervated). The increase in the ratio (%) of CO2 production from palmitate oxidation to total CO2 production increased from 4.0 +/- 1.8 (control) to 10.6 +/- 5.8 (denervated, p = 0.04). The time from uptake of FA to release of CO2 product was unaltered. CONCLUSION: We conclude that the contribution of FA oxidation to overall energy conversion is increased in chronically denervated hearts, which is postulated to result from a decline in the active form of pyruvate dehydrogenase. This would appear to be a result of chronic catecholamine depletion.

Thrombosis in one coronary artery causes generalized coronary vasoconstriction in a dog model of unstable angina.

We investigated the effect of thrombosis in one coronary artery upon the vascular resistance of another coronary artery. In previous investigations, using an animal model of unstable angina, we have observed increased resistance downstream from thrombus within a left circumflex coronary artery (LCx) stenosis and vasoconstriction of collateral vessels from the left anterior descending artery (LAD) supplying the distal LCx vascular bed. In the present paper, we induced thrombosis within a stenosis of the LCx of 16 beagle dogs, and observed the changes in blood flow to the myocardium supplied by the LAD using the radioactive microsphere technique. This blood flow decreased with thrombosis (P = 0.005) in these animals, whereas it did not do so in three time-control experiments. The pressures across the coronary vascular bed, i.e. arterial pressure to coronary venous pressure (coronary sinus catheter), did not change. Thus the vascular resistance of the LAD bed increased significantly from 147 +/- ll.5 mmHg/ml/sec/g of tissue to 172 +/- 13.4 mmHg/ml/sec/g of tissue (P = 0.02). As the LAD territory is not perfused with blood from the artery containing thrombus, we conclude that the effect observed is caused either by release of vasoconstrictors from the thrombus into the general circulation, or by activation of a neural reflex vasoconstriction. The study suggests that unstable angina involving thrombosis in one coronary artery is a global coronary vascular disease.

Measurement of coronary collateral flow and resistance in the presence of an open critical stenosis, and the response to intra-arterial thrombosis.

OBJECTIVE: (1) Can one measure coronary collateral flow around an open critical stenosis? (2) Does intracoronary platelet thrombosis affect native coronary collateral vessels? METHODS: We measured regional myocardial blood flow by the radioactive microsphere technique in seven anaesthetised dogs with an ultrasonic flowmeter on the circumflex branch of the left coronary artery (LCx). Measurements were made (a) in a control period, (b) after induction of a tight stenosis on the LCx, and (c) after additional arterial damage at the stenosis to induce intraluminal thrombosis. Collateral flow was calculated from LCx tissue flow(in ml/min/g tissue) minus LCx flowmeter flow which is in ml/min. Therefore, it was necessary to use scaling by reference back to the control measurements and conversion to ml/min/g tissue equivalent. RESULTS: LCx stenosis induced collateral flow from the other coronary arteries into the LCx area of supply, which decreased (mean+/-S.E.) from 0.23+/-0.03 to 0.15+/-0.05 ml/min/g tissue with thrombosis. Collateral resistance correspondingly increased with thrombosis from 187.6+/-18. 2 to 1069+/-544 mmHg/ml/min/g (P<0.02). CONCLUSION: Coronary collateral flow around an open stenosis can be measured by reference back to control conditions. The coronary collaterals vasoconstrict in the presence of thrombosis even though they are in the stream of blood coming from normal coronary arteries.

Downstream resistance effects of intracoronary thrombosis in the stenosed canine coronary artery.

OBJECTIVE: The presence is well established in unstable angina of intracoronary thrombosis in a stenosed epicardial coronary artery. The effects of the thrombus formation on the distal microcirculation are however still unclear. METHODS: We adapted the Folts canine model of left circumflex coronary arterial stenosis and intracoronary thrombosis by the insertion of a pressure catheter distal to the stenosis and by the use of 15 microns radioactive microspheres for measurement of regional myocardial blood flow. This permitted measurement during circumflex artery occlusion of collateral flow, downstream vascular resistance and collateral resistance. RESULTS: Distal circumflex resistance, obtained by dividing the distal circumflex coronary pressure gradient by the collateral flow, significantly increased with thrombosis (94.47 +/- 35.72 to 120.06 +/- 34.47; p = 0.0018) mmHg/ml/min/g. Changes in collateral flow and resistance in the presence of thrombosis, during maximum ischaemic vasodilatation, were inconsistent. CONCLUSION: Thrombosis causes increased vascular resistance in the microcirculation distal to the site of injury. This may be of clinical relevance in unstable angina, characterised by episodes of thrombus growth and embolization, in which ischaemic episodes may be worsened by generalised downstream vascular changes.

Chronic cardiac denervation affects the speed of coronary vascular regulation.

OBJECTIVE: We tested the hypothesis that the rate of adaptation of coronary metabolic vasodilatation and autoregulation is modulated by the cardiac nerves. METHODS: Anaesthetised dogs (seven innervated (control) and seven with denervated hearts) were subjected to controlled pressure perfusion of the left main coronary artery. Heart rate was controlled by pacing. RESULTS: The steady state autoregulation curves and metabolic regulation curves were similar in the two groups. A sudden increase or decrease in heart rate was associated with a faster response (22% shorter half-times) in the innervated than the denervated dogs (P < 0.001). A sudden increase or decrease in coronary arterial perfusion pressure was associated with a slower response (24% longer half-times) in the innervated than the denervated hearts (P < 0.005). CONCLUSIONS: We conclude that the speed of response to metabolic and perfusion pressure changes is partly mediated by cardio-cardiac reflexes. Reflex coronary vasodilatation appears to reinforce the metabolic vasodilatation of a heart rate increase and oppose the vasoconstriction in response to increased perfusion pressure.

Depletion of endogenous dopamine stores and shift in beta-adrenoceptor subtypes in cardiac tissue following five weeks of chronic denervation.

Surgical ablation of extrinsic cardiac nerve fibers results in a chronically denervated state of the left ventricle of the heart. The present study was performed to elucidate the effect of a period of 5 weeks of chronic denervation on cardiac catecholamine levels in general and dopamine in particular. Moreover, the possible effect on cardiac beta-adrenoceptor subtypes was investigated. Experiments were performed on adult dogs. In addition to adrenaline and noradrenaline the tissue levels of dopamine were found to be severely depressed. A significant shift from beta1- to beta2-adrenoceptor subtype was observed, while the total beta-adrenoceptor density remained unaffected. The present findings indicate that catecholamine synthesis in chronically denervated hearts is impaired upstream of dopamine and that a shift in beta-adrenoceptor subtype occurs already within a relatively short period of five weeks of denervation, and suggest that the lack of endogenous catecholamines influence the relative expression levels of the two subtypes of beta-adrenoceptors present in cardiac tissue.

Metabolic alterations in the chronically denervated dog heart.

OBJECTIVE: Previous studies have shown that chronic cardiac denervation impairs myocardial glucose oxidation. To investigate this further we tested whether the tissue content of glucose transporters, activity of glycolytic enzymes or metabolic capacity of pyruvate dehydrogenase were altered. Moreover, we investigated whether the decline in glucose utilization was associated with an upregulation of proteins and enzymes involved in fatty acid handling. Chronic cardiac denervation results also in decreased left ventricular efficiency. We explored whether alterations in mitochondrial properties could be held responsible for this phenomenon. METHODS: Twelve adult dogs were included in the study. In 6 of them chronic cardiac denervation was accomplished by surgical ablation of the extrinsic nerve fibers. The other 6 dogs were sham-operated. Biopsies were obtained from the left ventricle after 4-5 weeks of denervation. The content or enzymatic activity of proteins involved in fatty acid and glucose handling was assessed. Features of glutamate oxidation were measured in freshly isolated mitochondria. RESULTS: The content or activity of a set of fatty acid handling proteins did not change during chronic cardiac denervation. In contrast GLUT1 content significantly increased in the chronically denervated left ventricle, while the active form of pyruvate dehydrogenase declined (p < 0.05). Glutamate oxidation characteristics in freshly isolated mitochondria were not affected by chronic denervation. CONCLUSION: The impairment of glucose oxidation in the chronically denervated myocardium is most likely caused by a decline of pyruvate dehydrogenase in its active form. It is unlikely that the decrease in work efficiency is caused by alterations in mitochondrial properties.

Beneficial effect of the calcium-sensitizing drug EMD 57033 in a canine model of dilated heart failure.

1. Pacing-induced heart failure was studied in eight dogs. Heart failure was induced by right ventricular pacing at 250-260 beats/min for 6 weeks. Evidence of heart failure was determined clinically and by measurement of left ventricular (LV) dimensions by transoesophageal echocardiography. 2. Haemodynamic measurements of LV pressure, maximum rate of rise of LV pressure (LVdP/dtmax), cardiac output, mean arterial pressure, heart rate, pulmonary artery and pulmonary wedge pressures were made during infusion of solvent (control) and the calcium sensitizer EMD 57033 (0.6 mg min-1 kg-1). 3. The degree of heart failure varied from mild to severe in different individuals, but in each case EMD 57033 exerted a positive inotropic effect on LV haemodynamics and dimension. 4. The positive inotropic effect of the calcium sensitizer was manifest by increased peak LVdP/dt with a subsequent increase in cardiac output at the same mean arterial pressure. 5. This study clearly demonstrates that there is the potential for improvement of contractility of the failing myocardium of the intact mammal by an agent with a mechanism of action which does not involve an increase in intracellular calcium.

Tachycardia-induced failure alters contractile properties of canine ventricular myocytes.

OBJECTIVE: Rapid cardiac pacing has been used as a model for experimentally-induced cardiomyopathy. However, its relevance to human heart failure is not clear at present because little is known about changes in size and function of ventricular myocytes. We have therefore studied the responses to graded increases in frequency and calcium in canine ventricular myocytes from failing hearts. The aim of our study was to evaluate the resemblance between canine pacing-induced and human end-stage heart failure. METHODS: Myocytes were isolated from the left ventricular wall of dogs that were in heart failure after 6 weeks of pacing at 250 beats/min. Cell shortening was measured by edge detection. RESULTS: Clinical signs of failure included dyspnea, ascites, and heart dilatation; the hemodynamic parameters were: LVdP/dtmax 1613 +/- 149 vs. 4713 +/- 304 mmHg/s in 6 control dogs; LVEDP 17.2 +/- 4.4 vs 5.6 +/- 1.1 mmHg; LV volume 60.5 +/- 6.2 vs. 30-35 ml. Myocytes from failing hearts were longer and thinner than those from controls (from factor: 0.40 +/- 0.01 vs. 0.47 +/- 0.01, P < 0.001, > 30 cells/heart). With 6 mM Ca2+ and at 0.5 Hz, contraction amplitude was significantly attenuated in myocytes from failing hearts: 6.6 +/- 0.9% cell shortening vs. 10.0 +/- 0.8% in controls (P < 0.05). This deficit was exacerbated at higher stimulation rates. Time-to-peak contraction and time-to-50% relaxation were not altered. There was no difference in sensitivity to thapsigargin. CONCLUSION: As with cells from human failing hearts, contraction amplitude showed rate-dependent depression in this animal model, whereas features like slowing of contraction and relaxation and reduced sensitivity to thapsigargin, were not reproduced.

Failure of thrombin inhibition to prevent intracoronary thrombosis in the dog.

1. Recurrent occlusion after thrombolysis may be caused by thrombin receptor-mediated platelet thrombosis occurring in a residual stenosis. To test the relative importance of the platelet thrombin receptor under conditions of high shear and endothelial damage (the Folts model of intracoronary thrombosis) we used the specific thrombin inhibitor recombinant hirudin. 2. A critical coronary artery stenosis overlying an area of crushed endothelium was used in a repeated measures study of eight open-chest anaesthetized dogs. In the control period, recurrent thrombosis occurred at an average rate (+/- SD) of 4.4 +/- 1.4 ml/min2. Infusion of recombinant hirudin at 1.6 mg h-1 kg-1 abolished recurrent thrombosis in three dogs, but the thrombosis rate averaged 4.7 +/- 2.9 ml/min2 in the remaining five animals. 3. Haematological measurements demonstrated the activity of recombinant hirudin: thrombin time rose from 13 +/- 3 s to > 165 s universally (P < 0.01), partial thromboplastin time rose from 14 +/- 2 s to 29 +/- 10 s (P < 0.01). Bleeding time rose from 2.3 +/- 0.8 min to 4.7 +/- 1.8 min (P < 0.05). 4. It is concluded that specific thrombin inhibition, despite affecting coagulation, is relatively ineffective in preventing intracoronary thrombosis under conditions of high shear.

Natriuresis caused by blood volume expansion in dogs is not mediated by the renal nerves.

The influence of renal denervation on the renal response to rapid blood volume expansion was investigated by measurement of urine volume, sodium and osmolar excretion rates, sodium p-aminohippurate and free water clearances, and systemic haemodynamic and plasma hormone (atrial natriuretic peptide, arginine vasopressin and plasma renin activity (PRA)) changes in ten control and ten renal-denervated chloralose-anaesthetized mongrel dogs. Following renal denervation, blood volume expansion caused a similar increase in sodium excretion to that in the control group. Renal denervation resulted in systemic vasodilatation and decreased PRA. During fluid loading the right atrial pressure and pulmonary capillary wedge pressure were significantly higher in the control group. In the control group, volume expansion caused a large fall in PRA, whereas in the renal-denervated dogs, PRA remained suppressed. We conclude, from the fact that natriuresis is preserved after renal denervation, that this response is not mediated by a reflex with efferent limb in the renal nerves.

The pressure-flow relation in the canine coronary artery: combined effects of critical stenosis and intracoronary thrombosis.

OBJECTIVE: To characterise the effect of coronary intra-arterial thrombosis upon the downstream vascular bed. BACKGROUND: The vascular response downstream from a coronary intra-arterial thrombus has not previously been characterised. We postulated that downstream vasoconstriction might result from the presence of endothelial damage with consequent growth of platelet-rich thrombus. METHODS: We measured the pressure gradient and flow across, and the pressure/flow ratio distal to, a canine left circumflex artery stenosis with and without endothelial damage causing intracoronary thrombosis. We also observed the effects of transient complete conclusions. RESULTS: At occlusion, the pressure gradient was maximal; relief of occlusion caused a rapid increase flow and distal pressure with a rapid decrease in stenosis pressure gradient and resistance. Subsequently there was a period of stable stenosis resistance with pressure gradient and flow declining; distal pressure therefore increased at this time. Finally in the thrombus group only, stenosis resistance increased again towards re-occlusion. During occlusion, distal pressure averaged 49 +/- 18 mmHg in the presence of thrombus vs. 22 +/- 4 mmHg in its absence (P < 0.001). Following release of occlusion, the flow increased faster than distal pressure, so that the ratio (distal pressure/flow) fell rapidly. Subsequently, distal pressure continued to increase after flow had reached a peak and begun to decline, suggesting vasoconstriction. In the presence of thrombus, the distal pressure/flow ratio was higher than in the absence of thrombus, both at maximal vasodilation (P < 0.005) and at maximum vasoconstriction (P < 0.025). CONCLUSIONS: During cyclic flow variations the stenosis resistance changes are exactly as expected from thrombus growth and embolisation. The distal pressure/flow ratio showed a time-dependent increase which appeared greater when conditions favoured intracoronary thrombosis.