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Interview: Dr. Shawn J. Green, PhD

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Dr. Shawn J. Green has been awarded numerous patents and published over 100 articles in such journals as Science, Nature Medicine, and Nature. He served as an associate editor for the American Society of Microbiology Infection Immunity and a reviewer for several biomedical journals. He held academic appointments at Catholic, Howard, and Johns Hopkins. He also served as a board member for Virginia BIO. While serving at Walter Reed Army Institute of Research, he received the Meritorious Service Medal for his work on host response to intracellular pathogens and cytokine regulation of nitric oxide synthesis.

Shawn has started a number of life science initiatives, including Berkeley Test, Labbook BSML, and Maxcyte. He also served as a founding director of discovery research at EntreMed to commercialize the work of Dr. Judah Folkman, who founded the field of angiogenesis research, which has led to the discovery of a number of therapies based on inhibiting or stimulating neovascularization.

Dr. Green received his PhD on characterizing a hyaluronate receptor and MBA in finance & strategy from Georgetown University. Presently, he serves as an advisor to a healthcare investment bank and various start-ups.

Q. Nitrates are one of our most popular research topics. You've actually been in the thick of the research. How did you get started in this area?

Being in the thick of the research is probably stretching it. However, I did have the good fortune to contribute in a small way to how cells make and regulate nitrate in response to an infection through a newly discovered enzymatic pathway. In the late 80s, nitrate was recognized as a downstream byproduct of nitric oxide, a short-lived gas. Nitric oxide is produced by a variety of different cells, and affects a spectrum of biological processes, from the dilation of blood vessels to combating parasites.

Since that time, I’ve actually been standing on the sidelines, watching the transformation of nitrate, which was previously viewed as an incorrectly-characterized carcinogen and toxin, to an inert byproduct of nitric oxide synthesis, to a vital micronutrient that is critical to endothelial function. This once underappreciated salt found in arugula, spinach, bok choy, celery, plus other leafy greens and beets, is emerging as the principle heart health bioactive associated with plant-based diets.

My introduction to nitrate and how the body makes and regulates it was through the good fortune of meeting Drs. John Hibbs at the University of Utah and Michael Marletta at the University of Michigan. If the Karolinska Institute were to have given out more than three Nobel prizes, Moncada, Hibbs, and Marletta would have certainly rounded out the top five laureates in 1998, for discovering nitric oxide as a signaling molecule in our body, especially since their contribution significantly added to our understanding of the nitric oxide pathway.

In 1989, my first assignment as a medical service corps captain at the Walter Reed Army Institute of Research was to understand how infected cells, in particular, the macrophage – a sentinel immune cell found in organs and around blood vessels – is often able to rid itself of parasites and intracellular bacteria that break into the very cell that is suppose to provide the first line of protection.

At this time, Hibbs and Marletta, independently, were piecing together a decade’s worth of work on how the macrophage can be activated to kill cancer cells. Hibbs recognized that L-arginine’s conversion to L-citrulline was a required step, with a nitrogen group from L-arginine being stripped away as a key part of this newfound biochemical process. At the same time, Michael found that immune-activated macrophages produced nitrate.

With those pieces of information in mind, both Hibbs and Marletta recognized that nitrogen oxide derived from L-arginine must be the missing link. The soon-to-be Nobel laureates, Ignarro and Furchgott, were independently reporting that nitric oxide was produced by smooth muscle cells to relax endothelial cells, which tied nicely into Murad’s discovery that nitric oxide generated from nitroglycerin causes vasodilation. Becoming aware of each other’s work through their respective publications helped the pieces come together regarding nitric oxide production in our body and its importance in a number of physiological outcomes.

Both John Hibbs and Michael Marletta surmised that this L-arginine-dependent, nitric oxide generating biochemical pathway likely reached beyond the killing of tumor cells to control the growth of infections. John discovered that an analogue of the semi-essential amino acid L-arginine inhibited the macrophage’s ability to kill tumors by blocking L-arginine from being used by the cell’s nitric oxide synthase, hence, blocking nitric oxide and the downstream stable byproduct nitrate.

In a collaborative effort, we demonstrated that this analogue, which blocks non-specific tumor killing, was the same for killing parasites that resided within cells, hence, demonstrating for the first time that the nitric oxide pathway is turned on by the infectious agent. We also identified the cytokines (chemical mediators from other immune cells) that regulated nitric oxide synthase in infected cells. The byproducts, nitrate and nitrite, became surrogates for this process. Michael Marletta was also characterizing the enzyme(s) that uses L-arginine to make nitric oxide, and in doing so makes an antibody to the nitric oxide synthase. With this antibody, we were able to zero in on the cell types involved in making nitric oxide in relationship to the infectious process and mediators involved in regulating it.

Collectively, we identified how the pathway was turned on and off in different cells by various cytokines. This became important because nitric oxide is both a regulatory molecule and toxin; the type of nitric oxide synthase, location, quantity, and duration of nitric oxide synthesis dictated whether tumors and parasite were killed or blood vessels dilated, therefore, fine-tuning the regulation of nitric oxide synthesis remains an ongoing research pursuit.

Q. So when exactly did nitrates start being pegged as potentially important and beneficial?

Since nitric oxide is short-lived, the relatively stable byproducts – nitrate and nitrite – were found to be useful surrogates of the nitric oxide pathway. However, in the mid 90s, nitrate emerged as more than a mere byproduct of the nitric oxide synthase pathways, but as a precursor for nitric oxide, independent of the nitric oxide synthase pathway. It became ever so apparent to me when I was asked to write a News & View review article for Nature Medicine, Nitric Oxide in Mucosal Immunity, on a paper by Callum Duncan and Nigel Benjamin at the University of Aberdeen Medical School. They first suggested a nitric oxide synthase-independent source of nitric oxide via acidification of nitrite in the stomach and subsequently showed the reduction of nitrate to nitrite by symbiotic bacteria on the posterior surface of the tongue.

At the same time, Jon Lundberg and Eddie Witzberg at the Karolinska Institute were publishing the first demonstration of nitric oxide synthase: independent nitric oxide production from inorganic nitrate and nitrite in humans. These seminal observations, in my estimation, set the stage that dietary nitrate is beneficial. And here we are, nearly decade later, with a growing list of clinical studies that provide a compelling case that dietary nitrate, especially, when delivered through leafy greens and beets, may explain the basis of the heart-healthy effects of the Mediterranean diet and other plant-based diets, such as the DASH diet.

Q. You went from being a bench researcher to working at a healthcare investment bank. How did that happen?

I’ve always enjoyed the collaborative discovery process while working at the bench and I look forward to the day I go back. Many of the basic science research questions that I was involved with often had a commercial trajectory to it: will this project reach beyond the publications and have an impact in the drug discovery process or healthcare space? I had the opportunity to work on advancing drug candidates in oncology, develop a medical device for cell-based targeting of therapeutics, and create an informatics platform to access biomedical data more meaningfully for scientists, which led me to work with various startups and serve as an advisor to a healthcare investment bank.

Q. Saliva and nitrates is a topic that you know a ton about – can you give us a few key takeaways?

As you know, increasing nitrate-rich plant food intake boosts circulating nitrite and nitric oxide-mediated outcomes, i.e., improved endothelial function. One of the oddities of nitrate handling is that such a large proportion is concentrated in the salivary glands. Estimates suggest that nearly 1/3 of nitrate load is secreted in saliva, which peaks within two hours after eating a nitrate-rich arugula salad or drinking a nitrate-rich beet juice. This peak in salivary nitrite correlates with plasma nitrite and functional outcomes, such as blood pressure lowering in hypertensive people.

It is becoming generally accepted that nitrate-rich plant based diets are a principal source for nitric oxide production in the body. And this alternative or dietary nitrate pathway to nitric oxide synthase may become more relevant, especially as we grow older and sedentary, which corresponds with a loss of L-arginine-dependent endothelial nitric oxide synthase expression and activity. What’s even most striking about the alternative or dietary nitrate-nitrite-nitric oxide pathway is:

First, dietary sources of inorganic nitrate derived plant-based foods are chemically reduced by both enzymatic and non-enzymatic means, ie, XOR, acidosis, etc., and requires recycling via the entero-salivary loop from the gut the to salivary gland and back to the gut,

Second, elevated levels of nitrite in blood and saliva align with functional outcomes, i.e., blood pressure reduction.

Third, bioconversion of nitrate to nitrite in saliva is obligatory for functional outcomes: if saliva secretion and nitrite is interrupted, the blood pressure lowering effect is blunted.

Webb and colleagues at William Harvey in the UK in 2008 elegantly reinforced the obligatory role of saliva in humans. Here, ingestion of beet juice containing excessively high nitrate by healthy volunteers markedly reduced blood pressure up to 8 mmHg. When saliva was disrupted, either through spitting or interrupting the conversion of dietary nitrate to nitrite in the mouth, the reduction of blood pressure was abated; by blocking the saliva from recirculating, it prevented a rise in plasma levels, and blocked a decrease in blood pressure, confirming that this was attributable to the conversion of nitrate to nitrite in the mouth.

There was a lag period of approximately one to two hours after ingestion, with a peak drop in blood pressure occurring after 2.5 to three hours. This time course of reduction in blood pressure correlated with the appearance and peak levels of nitrite in the circulation and saliva; again, an effect that was absent in individuals within whom the entero-salivary circuit was disrupted by avoidance of swallowing. These observations, together with the fact that nitrite, and not nitrate, concentration correlated with the decreases in blood pressure implicate nitrite reduced from nitrate as the functional mediator, NO precursor & recycled byproduct of beetroot juice-induced effects on blood pressure.

By interrupting saliva levels in volunteers that already had elevated saliva levels, Kapil and coworkers in 2013 (William Harvey, UK) showed a rise in blood pressure. This was striking because it aligned with a precipitous drop in both salivary and blood nitrite.

Liu’s report in 2013 assessed the acute vascular effects of an easily-achieved intake of nitrate, derived from a single meal containing nitrate-rich spinach, resulted in an eightfold increase in saliva levels, with a corresponding decrease in pulse pressure, systolic, and higher large artery elasticity index. Other correlates of saliva levels that are reflective of blood pressure changes continue to be published; i.e., Sobko in 2010 showing that Japanese traditional diets abundant in leafy greens elevated both plasma and saliva levels, with a corresponding blood pressure decrease.

Q. Can you explain why dietary nitrates may be more desirable than nitrites?

It is well-appreciated that dietary inorganic nitrate ingested via greens and beets is both safe and effective at lowering blood pressure and increase athletic performance by decreasing oxygen demand. Since nitrate derived from vegetables is converted to nitrite, one may think that it may be more effective to short-circuit the pathway and consider a dietary supplement consisting of nitrite as a more attractive strategy. However, only about 8% of ingested nitrate is converted to nitrite, hence, the plasma nitrite is relatively low and the half-life of nitrite is approximately 15 to 30 minutes, in comparison to nitrate, which has a half-life of five to seven hours. With that said, it may be prudent to eat natural vegetable sources of nitrate, such as whole vegetables and vegetable juice, thereby, allowing the body to naturally ‘dose the body’ through this circuitous pathway involving the bioconversion via saliva.

Q. How about non-exercise benefits of nitrates?

The biggest cause of death worldwide is cardiovascular. However, dietary lifestyle modification can actually delay cardiovascular damage, if not reverse it. Daily inorganic nitrate consumption through leafy greens and beets may be as effective as an anti-hypertension drug intervention in terms of reducing blood pressure.

Ahluwalia and coworkers at Queen Mary University of London just reported in that patients with high blood pressure, who drank nitrate-rich beetroot juice daily, experienced a decrease in blood pressure of about eight mmHg, which for many patients brought their blood pressure levels back into the ‘normal’ range. The typical reduction in blood pressure through a single anti-hypertensive drug is nine mmHg. Therefore, these findings suggest a role for dietary nitrate as an effective, easy and affordable treatment in managing blood pressure with similar results to drug treatment. And keep in mind that every two mmHg increase in blood pressure increases the likelihood of death from heart disease by 7% and stroke by 10%.

Aside from a reduction in blood pressure, study participants experienced a 20% increase in blood vessel dilation capacity and around a 10% reduction in arterial stiffness. These changes in blood vessel function have been shown, by other studies, to be associated with substantial reductions in heart disease. There were no adverse side effects from the daily dietary nitrate.

Q. Sifting through the twenty or so patents that you hold, I see a lot concerning angiogenesis. We had previously interviewed a cancer-angiogenesis researcher – what applications of angiogenesis inhibitors did you research?

Ironically, the regulation of nitric oxide is in many ways similar to angiogenesis. Both are tightly controlled. And in both cases, too much is bad and too little is equally problematic.

In the case of inducible nitric oxide synthase, mediators referred to as cytokines are released from neighboring immune cells that both turn on the synthase to combat infection and quickly dampen it with resolution of the infection and before causing extensive tissue inflammation and damage. In like fashion, a homeostatic balance between positive and negative endogenous angiogenesis regulators governs vascular growth, where neovascularization is normally suppressed. Unfortunately when angiogenic growth factor production is upregulated, or when endogenous inhibitors are downregulated, angiogenesis is accelerated to supply tumors and other vascular-dependent disease.

With that said, I was most intrigued with endogenous antiangiogenic candidates produced by the body to keep the pro-angiogenic factors in check. While serving as scientific director of EntreMed, we collaborated closely with Judah Folkman at Children’s Hospital Boston at Harvard in exploring the merit of a number of endogenous factors, including metastatin, endostatin, angiostatin, and 2-methoxyestradiol (Panzem), which is an intriguing estrogen breakdown product that exhibits potent anti-proliferative activity.

Epidemiologic studies have suggested that increased consumption of certain whole foods containing natural sources of antiangiogenic molecules, including glucosinolate-rich brassica vegetables, resveratrol-containing grapes, nitrate-rich leafy greens, and other bioactives such as lycopene and cryptoxanthin, can improve health.

Prevention of angiogenic-based diseases by ensuring the balance of anti-angiogencs may be accomplished through lifestyle dietary choices.

Q. We have quite a few scientists who read the Study Deep Dives, and some are interested in pursuing business. Any tips for them?

Scientists interested in bringing their discoveries to the marketplace should consider reading two books. The first is Rework by the founders of 37signals, Jason Fried & Davis Heinemeier Hansson, and the second is The Art of Start by Guy Kawasaki. My tip is to read these books with your invention, product, or service platform in mind. I sense it will be self-evident as to what the next step will be. And afterwards give a shout to brainstorm … I am always interested in learning more about the next disruptive business.

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