A lightbulb moment for vasoregulation

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  • Quite by chance, Professor Dan Berkowitz, a clinician and scientist at Johns Hopkins University School of Medicine, has reignited the discussion of the phenomenon of photorelaxation. Prof Berkowitz and colleagues have described a new mechanism for this activity, with the potential for clinical therapeutic use in hypertension and other diseases.

    Professor Berkowitz describes the initial rediscovery of the phenomenon of photorelaxation, whereby the muscular walls of blood vessels relax in response to light stimulation, as “a serendipitous finding”. He tells how a postdoctoral fellow in his lab noticed the phenomenon following a change to motion controlled lighting in the building they occupy. “Dr Sikka noticed that every time he walked into the laboratory [and activated the lights], the blood vessels in his organ bath experiments relaxed,” he said. “After some further light-based experiments, we were convinced that light-induced relaxation occurs in blood vessels and we were excited to explore the phenomenon and its underlying mechanism.”

    It wasn’t all plain sailing though, a search of the literature uncovered earlier work by the Nobel prize-winning vascular biologist Robert F. Furchgott. The team were initially disappointed when they discovered that the phenomenon had been described over 40 years earlier, but a closer look revealed significant gaps with the interpretation at the time. “The mechanisms thought to be responsible for this phenomenon were improbable and poorly understood,” said Prof Berkowitz. This sparked renewed interest from the team in getting to the bottom of this unusual activity.

    Heading to the light
    There are a limited number of ways that biological systems can detect and respond to light stimuli, the most commonly known of these being those related to sight. Opsins, or light receptors, are a group of evolutionarily ancient receptors which include visual opsins such as rhodopsin. These are present in the mammalian retina and are responsible for our ability to see. In contrast, non-visual opsins such as melanopsin are important in synchronising an organism to changes in light periods, the circadian rhythm. Prof Berkowitz hypothesised that it was these non-visual opsins such as melanopsin, which were responsible for the vasorelaxation they had witnessed in their laboratory setups. Devising a series of experiments to test this hypothesis, they set about bringing to light a hitherto misunderstood feature of vascular biology.

    The first step was to discover and characterise which opsin receptors were present in the blood vessels which they studied. This was achieved by analysing messenger RNA from vascular cells via the polymerase chain reaction (PCR) method. They found both melanopsin (Opn4) and panopsin (Opn3) in a variety of blood vessels. “Our finding that functional Opn4 receptors are present in the vasculature is consistent with nonvisual functions of Opn4,” said Prof Berkowitz, going on to point out that, “finding photoreceptors in blood vessels is not entirely surprising, because Opn4 is known to regulate retinal blood vessel development.”

    Putting the puzzle together
    So, the necessary photoreceptors are present, but are they responsible for the observed vasorelaxation and if so, what is the mechanism by which they act? The next piece of the puzzle lay in removing or blocking these receptors and running the experiments again. Using both specially bred mice which lacked the gene for Opn4 and chemical inhibitors of the receptors, the team showed that this abolished the photorelaxation effect observed in their experiments.

    The way that vascular tone is measured experimentally is usually in a specialised apparatus called a myograph. Here two fine wires are inserted into the lumen of the blood vessel (the inside of the tube) and clamped to sensitive strain gauges. Slight tension is carefully applied to provide a baseline measurement from which relaxation or constriction can be accurately determined. The technique requires skill and patience to perform, particularly when working with the fine vessels of mouse models.

    Using this technique, Prof Berkowitz and his team were able to show an intensity-dependent response to light stimulus in wild-type mice as opposed to the knockout mice which lacked the Opn4 receptor. The same experiments were then repeated with normal mice, but this time with chemical inhibitors of the Opn4 receptor. These showed the same effect as with the knockout mice. This clear evidence of Opn4 mediated photorelaxation of blood vessels was described by one commentator as, “A brilliant study… [which]provides an intriguing molecular explanation for a phenomenon that has puzzled vascular biologists for more than half a century.”

    He noticed that every time he walked into the laboratory, the blood vessels in his experiments relaxed.

    Human pulmonary artery smooth muscles cells in cell culture stained with antibodies to show the cytoskeleton, (the protein actin) in green and nucleus in blue. The red dots are the result of the interaction of two proteins in the cell, opsin 3 and the protein that regulates it, G-protein receptor kinase 2. The red dots light up only when the proteins of interest are in close proximity to one another suggesting that they are interacting.

    Colour is key
    The team’s success didn’t stop there. Having identified the receptor, further confirmation could be obtained by adjusting the wavelength of the light stimulus. Opn4 is known to be activated by short wavelength light (the blue end of the spectrum), and the team confirmed that light in this range produced the maximal response.

    By first using light sources that just produced red, green or blue light, then later a monochromator which could adjust the wavelength in thirty-nanometer increments, they were able to narrow down the range of activity to 400-500nm wavelength that constitutes what we perceive as blue light.

    The careful and progressive experimental design also took account of uncovering more of the mechanism by which the signal is transduced. They ruled out an endothelium (the cells lining the lumen) mediated pathway by removing the endothelium from the vessels before light stimulation. They further ruled out classical signalling pathways such as nitric oxide production using drugs which blocked production as well as chemical scavengers to mop up any remaining stores.

    This list of ‘unsuccessful’ experiments was steadily leading them towards hyperpolarisation (a change in the voltage across a cell membrane due to movement of ions) of the vascular smooth muscle cells as an explanation for the effect. This was duly confirmed by electrophysiological recordings of smooth muscle cells in the preparation. This mechanism is related to that found in the ‘simple’ receptors of invertebrates, though the physiological mechanism by which this hyperpolarisation comes about in the case of mammalian blood vessels, is complex and not easy to ascertain.

    One interesting branch of the investigation looked at the rapidity with which the response is desensitised. This is clearly mediated by the G-protein receptor kinase GRK2, which phosphorylates the receptor (the addition of a phosphoryl group to a molecule, critical for regulating many biological processes) so that it cannot reactivate the signalling pathway. This fits with the nature of the Opn4 receptor and the team showed that the desensitisation was just as quick to recover, requiring a mere thirty minutes of darkness to return to normal response.

    A brilliant study… [which]provides an intriguing molecular explanation for a phenomenon that has puzzled vascular biologists for more than half a century.

    A bright future
    Prof Berkowitz and his colleagues have gone on to show that this effect is more widespread than previously thought, with evidence from the pulmonary arteries (which carry blood from the heart to the lungs) of rats, cows and pigs. They have even begun to show the possibility of using this improved understanding of the action of Opn4 therapeutically.

    By inhibiting GRK2 and casting blue light to the periphery of the pulmonary vasculature in rats which exhibit pulmonary hypertension (high blood pressure in the pulmonary circulation which has widespread health effects), they were able to induce sustained vasorelaxation. This raises the potential of ‘phototherapy’ where GRK inhibitors and control of light exposure are used together to adjust vasoreactivity. Light stimulation, or other targeting of these opsin receptors, may provide another option for treatment in cases where current therapies are ineffective or undesirable.

    Dr Berkowitz in his Hopkins lab surrounded by equipment for studying blood vessel function.

    What started as an energy saving measure (changing laboratory lighting controls), through being picked up by a keen-eyed researcher and developed by Prof Berkowitz and colleagues, has shed light on an area of vascular biology that has remained in the dark for nearly half a century. The implications of this research for the treatment of hypertension in the future could be significant, but here and now it is showing that with attention to detail, good experimental design, and just a little luck, there are still amazing discoveries to be made.

    What do these findings represent for the field of cardiovascular research and the treatment of hypertension?
    This work suggests that classic sensory GPCR’s (such as light receptors) present in extrasensory sights may represent novel targets for safe and effective light based therapies in which altered vasoreactivity is a significant component. Indeed, this system thus represents an “endogenous optogenetic system which can be recruited for vascular endogenous”.

    References

    • Barreto Ortiz S, Hori D, Nomura Y, Yun X, Jiang H, Yong H, Chen J, Paek S, Pandey D, Sikka G, Bhatta A, Gillard A, Steppan J, Hyung Kim J, Adachi H, Barodka V.M, Romer L, An S.S, Shimoda L.A, Santhanam L, Berkowitz D.E. (2018).‘Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition’. American Journal of Physiology, Vol. 314 (1).
    • Vanhoutte P.M. (2014). ’PDE and sGC hand in hand to see the light’. Proceedings of the National Academy of Sciences, Vol.111(50), pp.17704-17705;
    • Sikka G.G,Hussmann P, Pandey D, Cao S, Hori D, Taek Park J, Steppan J, Kim J.H, Barodka V, Myers A.C, Santhanam L, Nyhan D, Halushka M.K, Koehler R.C, Snyder S.H, Shimoda L.A, Berkowitz D.E. (2014). ‘Melanopsin mediates light-dependent relaxation in blood vessels’. Proceedings of the National Academy of Sciences, Vol.111 (50), pp.17977-17982.
  • Research Objectives
    The regulation of the dilation and constriction of blood is important as it regulates blood flow, a process that is abnormal in vascular disease. Berkowitz and his team have identified a new mechanism that regulates blood vessels; a type of light sensitive receptor that causes blood vessels to dilate when illuminated. They have thus identified a light-activated molecular switch that can be used to treat diseases such as Raynaud’s phenomenon in which constriction of the blood vessels is abnormal and causes pain and injury to the fingers.

    Funding
    NIH R01-HL124213

    Collaborators

    • Gutam Sikka, MD
    • Sebastian Barreto Ortiz, PhD
    • Lakshmi Santhanam, PhD
    • Deepesh Pandey, PhD
    • Larissa Shimoda, PhD
    • Steven An, PhD

    Bio
    Dr Berkowitz is a Clinician and Scientist at Johns Hopkins University School of Medicine, and Professor of Anesthesiology and Critical Care Medicine (ACCM) and Biomedical Engineering. He is currently the Vice Chairman for ACCM Research. He directs an integrated vascular biology laboratory funded by the NIH, American Heart Association, and NASA. His laboratory studied vascular biology and pathobiology of atherosclerosis, ageing, and radiation.

    Contact
    Dr Dan E. Berkowitz, MD
    The John Hopkins Hospital – John Hopkins Medicine
    1800 Orleans Street
    Zayed Tower 6208
    Baltimore, MD, 21287
    USA

    E: dberkowi@jhmi.edu
    T: +1 443 831 7254
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  • A lightbulb moment for vasoregulation

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