Signalling In Space And Time: Intracellular Cyclic AMP Dynamics In Human Vascular Smooth Muscle

Blood vessels constantly change their diameter to match blood flow to tissue needs for oxygen. These adjustments are made by the contraction and relaxation of muscle cells within blood vessel walls. This makes understanding the mechanisms that control muscle contractility important for understanding normal blood flow around the body and how this changes during exercise, with age or in diseases like diabetes or high blood pressure. When a tissue becomes starved of oxygen and needs more blood it sends 'relaxation' signals to the arterial muscle cells. These signals are relayed from the cell surface to the cell interior by a small diffusible messenger molecule called cyclic AMP which functions to distribute the message to multiple sites within the cell to induce relaxation. A fundamental question is how a highly diffusive messenger that can move freely in the cell manages to deliver information to the correct intracellular 'address'?

One way to solve the problem would be if cyclic AMP moved about within vascular muscle cells in complex 'waves' that co-ordinated the correct arrival of the relaxation signal at different cellular targets. These patterns can be generated by enzymes called phosphodiesterases (PDEs) that degrade cyclic AMP and restrict its free movement in the cell. Barriers of PDEs, like flood defences, could channel cyclic AMP towards its intended destination ensuring the message reaches the correct intracellular targets in the correct order. Arterial cells possess many different types of PDE enzyme that should allow them to generate these complex cyclic AMP dispersal patterns, but little is known about this in vascular smooth cells. This is a major gap in our understanding of blood vessel physiology and of particular interest to the pharmaceutical industry since genetic differences in the activity of PDEs (and also the enzymes that produce cyclic AMP) are linked to susceptibility to high blood pressure and stroke. Drugs that target PDEs could be useful in a number of diseases, but their usage is currently restricted due to serious side-effects because of our limited knowledge about how these enzymes work in normal cells.

In this project we will use state-of-the-art molecular sensors anchored at specific points within human arterial cells to track the real-time flow of cyclic AMP around the cell. Differences in the timing of activation of these sensors will allow us to determine where the cyclic AMP 'wave' is at any one time within the cell. We can also use drugs that selectively inhibit different types of PDE to tell us which of these enzymes is important in channelling the cyclic AMP signal. We believe that different cell-surface signals from different hormones and neurotransmitters generate distinct patterns of cyclic AMP dispersal and that the maintenance of these patterns is crucial to normal blood vessel relaxation. We will carry out experiments in human cells from two different arteries, the coronary artery and the pulmonary artery. These arteries carry out very different physiological roles: the coronary artery feeds the heart muscle with oxygenated blood, while the pulmonary artery carries deoxygenated blood from the heart to the lungs to pick up more oxygen. It is important that we identify any potential differences in how PDEs work between different arteries as this will direct future research aimed at identifying drugs that can dilate one artery while leaving other unaffected, thus reducing the side effects of therapies aimed at modulating blood flow in the body.

The overall outcome of this project will be to: 1) identify the molecular mechanisms that ensure that our arteries dilate to optimise the flow of blood and oxygen around the body; 2) explain how genetic variation in cyclic AMP signalling protein activity can result in differences in blood flow and blood pressure, and 3) ultimately help in the development of future therapies that target the cyclic AMP signalling axis.

Most hormones and neurotransmitters that relax vascular smooth muscle cells (VSMCs) to widen blood vessels act at surface receptors that increase intracellular 3'-5'-cyclic adenosine monophosphate (cAMP). cAMP is a diffusible messenger that relays the signal to downstream effectors which in turn modulate multiple proteins to relax VSMCs. Different vasodilators modulate distinct protein populations at different time points, leading to variation in the speed, degree and longevity of relaxation which underlies normal vascular reactivity. In VSMCs, we know little about how cAMP manages to transfer signals to only intended targets and in the correct sequence.
Fidelity of cAMP signal transfer can be achieved by clustering target proteins in defined subcellular locations and by using phosphodiesterases (PDEs) to restrict cAMP diffusion and funnel it to these targets. We will use cytosolic FRET-based biosensors to measure real-time cAMP changes in human VSMCs from two different arterial beds in response to different vasodilators. Selective pharmacological and genetic PDE knock-down will allow us to identify PDE isoforms that regulate cAMP dispersal in response to different vasodilators. To determine cAMP's spatio-temporal dispersal pattern we will use FRET-based biosensors anchored to different subcellular locations. Localized probes combined with the high temporal resolution of our photometry system will allow us to measure differences in the time taken for cAMP to reach different parts of the cell. To locate the 'address' to which the cAMP is being directed we will measure activity of the cAMP effector, protein kinase A (PKA) using the FRET-based reporter, AKAR4. We will anchor AKAR4 to different subcellular locations to determine the spatial-temporal activation of different PKA pools in response to vasodilators. We will combine this with mass-spectrometry-based proteomics to quantitatively evaluate the phosphorylation of proteins involved in PKA-induced vasorelaxation.

The economic and societal impact of our project stems from defining the molecular mechanisms that underpin vascular smooth muscle contractility. This has direct implications for understanding the control of blood flow and pressure.

A. Public understanding of science and societal issues: Who might benefit?
Public
How might they benefit?
Up to 30% of all deaths in the UK in 2019 were due to heart and circulatory disease and the UK spends £9 billion every year treating the disease itself and combating its development through education about risk factors such as smoking, obesity and physical inactivity [1]. Risk factors are more common in deprived areas of the UK. Liverpool has the 7th highest level of average deprivation in England (out of 326 local authorities), and people living in Liverpool are twice as likely to die prematurely from cardiovascular disease compared to those living in the least deprived region [1]. We will engage our local communities through our extensive outreach programme to inform on the importance of blood pressure control. This includes Christmas Lectures for secondary schools; 'Meet the Scientists' events at the World Museum, Liverpool , a Faculty-run programme of free Saturday drop-in events that engages over 6000 visitors annually (conceived and developed by Co-I, RBJ); family science fairs at Ness Gardens, Wirral; and the Scouse Science Alliance Blog.

B. Exploitation of scientific knowledge: Who might benefit?
Pharmaceutical Sector
How might they benefit?
Our work will identify the specific phosphodiesterases (PDEs) involved in regulating vasodilator responses in coronary and pulmonary vascular beds. This will be of direct immediate interest to industry. Close to 100 different PDE variants exist but few PDE inhibitors are used clinically due to lack of efficacy and/or debilitating side effects, reflecting the poorly understood roles these enzymes play in shaping physiological cAMP gradients. Our study will fill this knowledge gap for human vascular smooth muscle. Pharmaceutical interest is reflected in the large number of clinical trials registered for PDE inhibitors (ClinicalTrials.gov). Companies sponsoring these trials include Pfizer (PDE2 inhibitors), Gilead Sciences/AstraZeneca (PDE3 inhibitors); GlaxoSmithKline, Takeda; Astellas Pharma, Celgene, Pfizer (PDE4 inhibitors). Trials are investigating novel PDE inhibitors for the treatment of diseases for which PDE inhibitors are already approved (erectile dysfunction, chronic obstructive pulmonary disease and pulmonary hypertension), and the use of PDE inhibitors for additional diseases: asthma, vasospasm/Raynaud's, peripheral vascular disease, small vessel stroke. Our first objective will be to release our results in high-profile open access journals and through dissemination at international conferences. We will also undertake important follow-on studies to assess differential effects of existing PDE inhibitors on relaxation of intact normal human coronary and pulmonary arteries obtained through colleagues at the Liverpool Heart and Chest Hospital. We will work closely with UoL's Research and Partnerships Development team who work with organisations across all sectors to identify and develop partnerships/ collaborations, and guide staff through IP and the management of knowledge exchange projects.

C. Enhancing quality of life and health: Who might benefit?
Clinicians
How might they benefit?
Our study will generate the first detailed comparison of the functional role of PDEs in different arteries. Ultimately, understanding if different populations of PDEs control relaxation in different vascular beds opens up the possibility of generating compounds that target one PDE group to relax one artery while leaving the other unaffected. This ability to induce local as opposed to systemic effects would have significant therapeutic potential.

[1] Heart & Circulatory Disease Statistics 2019, BHF & IAHR, Univ of Birmingham