Research

In the treatment of cardiovascular disease in the clinic today, one of the most successful approaches has been the development of drugs that elicit their therapeutic effects through actions on membrane receptors. Drugs targeting receptors belonging to the sympathetic nervous system and the renin angiotensin-aldosterone system are widely used.

A new regulatory system is now emergingthat regulates most of the cardiovascular tissues – the extracellular nucleotide system. The extracellular nucleotides (ATP, ADP, UTP and UDP) can be released from sympathetic nerves, platelets, erythrocytes, endothelial, cardiac and inflammatory cells in response to physiological stimuli. They act on P2 receptors located on the surface of the same cell types including vascular smooth muscle cells (VSMC). The P2 receptor family constitute one of the largest receptor groups known and consists of 15 subtypes. Molecular cloning has revealed seven subtypes of the P2X-receptor family and eight P2Y receptor subtypes. This unanticipated diversity of both ionotropic P2X and metabotropic P2Y receptors provides a possibility to develop selective agents for specific therapeutic modalities in the treatment of cardiovascular disease.

As will be illustrated below, there are great opportunities to understand and develop therapies for cardiovascular disease by targeting P2 receptors. The platelet ADP-antagonist clopidogrel (Plavix), already have an established role in the prevention of myocardial infarction. However, at least two more P2-receptors remain to be blocked on the platelets. In the heart at least three P2-receptors could be blocked as a treatment for heart failure, arrhythmias and protection against ischemia. Or agonists could be used as inotropic agents. In the endothelium several receptors could be targeted to release t-PA and improve endothelial function. The proliferative effects of extracellular nucleotides on VSMC could be antagonised as a treatment for restenosis after PCI, chronic transplant rejection and diabetes microangiopathy. Contractile VSMC receptors could be targets for antihypertensive therapies and for pulmonary hypertension. High expression of P2 receptors on monocytes and other inflammatory cells in the atherosclerotic plaque are other interesting targets to treat atherosclerosis.

Our strategy has been to examine the physiological effects in cardiovascular tissue, to carefully establish the expression patterns for the 15 subtypes in human cardiovascular tissue, to establish the role of the extracellular nucleotide system in cardiovascular disease processes and to develop new chemical compounds that may serve as drug candidates.

The red blood cell – a regulator of blood flow

The matching of oxygen supply with demand requires a mechanism that increases blood flow in response to decreased tissue oxygen levels. Red blood cells (RBC) contain millimolar amounts of ATP and possess the membrane-bound glycolytic enzymes necessary for its production. Work by especially Ellsworth and Sprague has suggested that ATP is released in response to reductionsin oxygen tension and pH, and that vessels dilate in response to low O2 levels only when blood vessels are perfused with RBCs. In vivo studies in man have demonstrated that ATP is released in working skeletal muscle circulation depending on the number of unoccupied hemoglobin O2 binding sites. The released ATP then binds to P2Y receptors on the endothelium and stimulates vasodilatation by the release of nitric oxide (NO), prostaglandins and as we have shown endothelium-derived hyperpolarizing factor (EDHF). Thus, the RBC functions as an O2 sensor, contributing to the regulation of blood flow and O2 delivery, by releasing ATP depending on the oxygenation state of hemoglobin. This new paradigm, in which the RBC is not considered a “passive bag” that transports oxygen, but instead a regulator of its own destination is currently becoming accepted in the research community. A new dimension was added when we described that the ADP receptor P2Y13 is a negative feedback pathway that turns off ATP release when extracellular ATP/ADP levels increases. This is similar to physiologically important signalling systems such as noradrenaline release from sympathetic nerves that is inhibited by presynaptic a2 receptors.

Our findings were based on two methodological developments. First, we developed the first method for mRNA quantification in RBC. Secondly, we developed a microdialysis based in vitro (and in vivo) assay for measurement of extracellular ATP in blood.

Endothelium – blood flow regulation

Since many years we have been a leading group in the understanding of the important endothelial effects of extracellular nucleotides. We were first to describe that nucleotides mediate their dilatory effects not only via NO and prostaglandins, but also via EDHF, and that the dilatory responses are altered in congestive heart failure.

As an interventional cardiologist, specialised in PCI, I am interested in the postischemic increase in flow that follows upon opening of an occluded vessel. In a pig model using a new P2Y1 (ADP) antagonist we have been able to show that the peak of reactive hyperemia is mediated to 40% by ADP released and subsequently dilating the coronary vessel bed via P2 receptors on endothelium (60). This fits previous evidence that adenosine is responsible for the late reactive hyperemia phase. We have formulated a purine hypytheses to explain reactive hyperemia: ATP is released from myocardium and RBC, mediates the very early phase via P2Y2 receptors, is degraded to ADP that binds to P2Y1 receptors and mediates the peak, followed by degradation to adenosine mediating the late phase. The role of ATP will be tested using the first developed P2Y2 receptor antagonists (Prof Jacobson, NIH). Possibilities for measuring ATP release in the coronary sinus in patients during PCI are currently investigated.

We have shown that ATP and UTP release t-PA in man. Using P2-antagonists we have data indicating that t-PA release during myocardial ischemia is in part ATP/ADP mediated.

Platelets

ADP is together with thromboxane the most important positive feedback mechanisms by platelets, important for the full activation and aggregation that is necessary for thrombus formation. The clinical importance has been demonstrated by the P2Y12 receptor antagonist clopidogrel (Plavix) that is more efficient than aspirin in preventing myocardial infarctions and together with aspirin it is widely used in the treatment of acute coronary syndromes.

We have developed the first mRNA quantification method for any gene in platelets (49), in which we could demonstrate the presence of three P2-receptor subtypes. The methodology was then used for the first clinical studies of gene expression in platelets (55). Systemic lupus (SLE) patients that has an increased morbidity and mortality in ischemic heart disease was found to have a decreased expression P2Y12 receptor while P2Y1 and P2X1 receptors where unchanged. This could have implications for the choice platelet inhibitor for SLE patients. Furthermore, the abundantly expressed complement inhibitor Clusterin was markedly downregulated, and this loss of protection against complement activation may be detrimental and contribute to atherosclerosis. Furthermore, we have been able to show that the mRNA in circulating platelets is translationally active and synthesize the major part of circulating PAI. We found that patients with myocardial infarction has aspirin resistance that may depend on increased plasma ADP-levels.

Heart

In a rat model of congestive heart failure we found a prominent upregulation at the mRNA level of P2X1 and P2Y2 receptors. Now we have made a comprehensive study of P2 receptor expression in human heart (right and left ventricles and atria). This revealed at least three new inotropic receptors activated with ATP, UTP and UDP, respectively. The expression findings were confirmed by functional and pharmacological studies on mouse cardiomyocytes. The inotropic effect is in the same magnitude as for beta-1 receptors activated by adrenaline. We have done the first studies demonstrating that not only ATP but also UTP is released during myocardial infarction in man and in a pig model were samples are taken from a catheter in the coronary sinus.

Heart protection with hypothermia

We have recently developed a method of rapid hypothermia (colling) immediately before balloon angioplasty in patients with acute myocardial infarction. The method results in a 40% decrease in infarct size in pigs. A clinical safety/feasibility study will be started soon.

Vascular Smooth muscle cells

We have done pioneering work demonstrating that extracellular nucleotides are growth factors for VSMC by stimulating several P2Y receptors via intracellular pathways including Gq-proteins, protein kinase C and tyrosine phosphorylation, leading to increased immediate early gene expression, cell number, DNA and protein synthesis.Furthermore, we have shown that expression of the P2 receptors are important in VSMC phenotype regulation and vascular adaptation. Mitogenic P2Y receptors are upregulated and contractile P2X receptors are downregulated as the SMC shift from a contractile to a synthetic phenotype.The specific contractile receptor pattern in human coronary arteries and bypass vessels (saphenous vein and internal mammary artery) revealed differences in receptor expression that may be important for drug development. The powerful contractile effects of UDP acting on P2Y6 receptors have been revealed in vein grafts and may contribute to explain the lower patency of vein grafts. Recently, based on our mRNA quantifications we revealed that the P2Y12 ADP receptor that previously was believed to have a very restricted expression to platelets, in fact is a potent contractile receptor in human arteries. The vasospasm was not blocked by clopidogrel (that does not reach the systemic circulation).

Single Nucleotide Polymorphisms (SNP)

We have recently found an overrepresentation of a SNP in the P2Y11 receptor in patients with myocardial infarction (n=3600). This is interesting because we have demonstrated that the P2Y11 receptor is present on inflammatory cells. Furthermore, the SNP is common (20% of controls) and could be important as a prognostic determinant. Subgroup analysis strongly support a genetic mechanism with a clear genetic dosage effect in patients with family history or homozygots. By examining risk factors in 6000 individual, we found that the probable mechanism of action is a proinflammatory effect, because CRP was elevated in carriers of this Thr-87 polymorphism, while blood pressure, cholesterol, BMI were unaffected. Thus, we have found a new genetic link between inflammation and myocardial infarction!

Pharmacology

We test new chemical compounds for their actions on P2 receptors with the hope to find agonistic or antagonistic effect that could aid the design of future therapeutics.

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