Targeting microbes to treat heart disease

Deep in the recesses of the gut, they wait, ready to devour whatever comes their way. The microbes that live in the human intestines are not picky; their food is our food. But what we decide to eat — whether a greasy cheeseburger or a beet salad — determines not just the microbes’ health but our own. 

Scientists have linked factors produced by the gut microbiome to diverse conditions such as Parkinson’s disease and autism; they even play a role in the effectiveness of cancer immunotherapy. Over the past two decades of his laboratory’s research, Stanley Hazen, a cardiovascular and metabolic disease physician and researcher at the Cleveland Clinic, added cardiovascular disease to that list.

When he and his team took into account genetic factors as well as lifestyle habits known to contribute to cardiovascular disease, they found that all of these factors still didn’t account for the majority of the disease risk. “There must be pathways beyond traditional risk factors that contribute to cardiovascular disease,” Hazen said. So, he and his team used mass spectrometry to profile people’s blood and asked, “are there chemical signatures in their blood that predict the future development of heart attack, stroke, or death?”

The researchers noticed that people in multiple independent cohorts who later went on to have a heart attack, stroke, or death from cardiovascular disease also had high levels of a particular molecule called trimethylamine N-oxide (TMAO) in their blood (1). When we eat foods rich in choline, such as red meat, eggs, and liver, our gut microbes metabolize choline into a molecule called trimethylamine (TMA). The liver then converts TMA into TMAO, which enters the blood circulation. 

To see if TMAO itself increased risk for cardiovascular disease, Hazen and his team fed mice a choline rich diet and saw that the mice exhibited the same atherosclerosis symptoms as the human cohort. When they treated these same mice with antibiotics to remove their gut microbiomes, the choline-induced cardiovascular symptoms never appeared.

Hazen said that this was “the eureka moment that the gut microbiome is contributing to our metabolism and is generating compounds that are impacting the host. By definition then, that means that just like we can pharmacologically block a pathway like cholesterol synthesis, maybe we can pharmacologically block a microbial pathway and lead to a beneficial effect in the host.”

Hazen and his team have now developed inhibitors that specifically target the microbial enzymes that convert choline to TMA, blocking the production of TMAO. Numerous preclinical studies have demonstrated that these inhibitors effectively treat and prevent multiple cardiovascular disorders, including ones that have no therapeutic intervention at all. These findings set the stage for new drugs for cardiovascular diseases that act by targeting the microscopic flora living inside us.

Treat TMAO like cholesterol

If eating red meat and eggs increases levels of TMAO in the blood, why not just eat less of those foods? While Hazen and his team showed that when people eliminated red meat from their diet, their blood TMAO levels decreased, that intervention may not be enough to decrease cardiac risk (2).

“While diet will have an effect, you still have to eat,” said Hazen. “A perfect analogy is cholesterol. You can go on a low cholesterol diet and lower your cholesterol, but the effect you see is modest… You can get a much bigger bang for the buck in terms of lowering cardiac risk with drugs.”

While TMAO can be metabolized from other dietary components including carnitine and lecithin, the major source of TMAO is choline. To block most of the TMAO production, Hazen and his colleagues searched for ways to inhibit the first step in the TMAO pathway: the conversion of dietary choline to TMA by the gut microbial enzymes CutC and CutD.

While diet will have an effect, you still have to eat. 
– Stanley Hazen, Cleveland Clinic

They screened multiple synthetic and natural choline derivatives and landed on a small molecule called 3,3-dimethyl-1-butanol (DMB) (3). DMB wasn’t toxic in cellular or animal studies even when the scientists administered it at very high concentrations, and it inhibited the conversion of choline to TMA, significantly decreasing the levels of TMAO in the blood. DMB did this not by killing the microbes like an antibiotic would, but by simply inhibiting CutC and CutD.

While DMB worked, it was not the most potent inhibitor. The researchers had to dose their mice with 1% DMB by volume in their drinking water to block TMA production. “1% DMB, that’s 2 proof. That’s a lot. If you convert that into capsules or how many tablespoons a day for humans, that would be too much,” Hazen explained.

To improve their next generation of inhibitors, the team designed a choline-like inhibitor such that once the microbial CutC and CutD enzymes cleaved it, the drug would covalently modify the enzyme active site, irreversibly inhibiting the conversion of choline to TMA. This new class of inhibitors, called halomethylcholines, were much better inhibitors than DMB, with fluoromethylcholine (FMC) as the strongest one among those tested (4). 

Hazen and his team are using these inhibitors as a starting point for developing microbial-targeting drugs for lowering TMAO levels in humans. But even though these drugs have not yet reached the clinic, these halomethylcholines already show promise as tools to link TMAO levels to other cardiovascular disease processes, including ones that currently have no drug treatment.

Happy accidents for AAA

As a cardiovascular disease researcher at the University of Cincinnati, Phillip Owens doesn’t typically attend conferences on nutrition. But in 2017, nutritionist Kelsey Conrad joined his lab as a PhD student. “We just happened to go to this conference, and at the conference, somebody was presenting on TMAO and how it was basically a derivative of a diet high in choline,” said Owens. “She was like, ‘That sounds like a cool project.’” 

Owens studies abdominal aortic aneurysm (AAA), which is the swelling of the aorta. Other than some back pain, AAA has no clear symptoms. Doctors typically diagnose AAA if a patient happens to get an ultrasound, CT scan, or an MRI. If the aneurysm goes undetected, it can rupture, which is typically fatal.

“There’s no pharmaceutical therapy for it at all. The only intervention is surgery,” said Owens. “If you have cardiovascular disease, atherosclerosis, heart disease, heart hypertrophy, chronic kidney disease, there’s at least something you can take in a pill form that mitigates the effects or delays the progression of the disease. But [for] AAA, there’s no therapy you can take. It’s one of those really big puzzles that we have left that we just don’t know how to treat.”

Owens and his team were excited to learn about TMAO because it seemed to fit with what they knew about the pathogenesis of AAA. TMAO can lead to the production of reactive oxygen species, which contribute to AAA (5,6). “We just made the connections and started running from there,” he said.

They first assessed TMAO levels in two independent human cohorts and found that as TMAO levels rose, so too did the size and severity of a person’s AAA (7). When they looked at mice that were fed a high choline diet, they found that AAA became more severe. But if they treated these same mice with FMC from the Hazen group, they found that it blocked AAA development. If AAA already existed, FMC halted its progression. Owens and his team also discovered that if they fed the mice TMAO in addition to FMC, the AAA progression resumed, mechanistically linking TMAO to AAA pathogenesis.

“Everything fell into place,” said Owens. “We were super excited because it’s just very rare for that to happen where everything kind of works the way that you thought it might work.”

The fortuitous coincidences didn’t stop there. To better understand exactly how TMAO contributed to AAA, Owens and his team performed RNA sequencing on human vascular smooth muscle cells cultured in vitro and on mouse aortas. They found that increased levels of TMAO in the blood associated with increased expression of protein kinase R-like endoplasmic reticulum kinase (PERK), a protein involved in the ER stress pathway. A few days after they got their PERK hit, Owens and his team piled into a car in Boston to drive up to a scientific meeting in Maine.

“We wound up getting lost in Boston, so we didn’t make it up to the Gordon conference until after the first talk was going on,” Owens said. But as they stumbled into the room, the speaker started talking about how PERK was the potential receptor for TMAO.

“We were like, ‘Oh, holy crap!’ We weren’t expecting that. We got this huge hit on PERK, and now, all of a sudden PERK is the putative receptor for TMAO!” he said. “We almost didn’t even hear the talk.”

Owens and his team linked up with the speaker, obesity and cardiovascular disease researcher Sudha Biddinger at Boston Children’s Hospital, and together they are now investigating how TMAO activates the ER stress pathway through PERK. They are searching for ways to inhibit PERK as a parallel strategy to blocking the TMAO pathway via the gut microbiome.

Owens is excited for Hazen and his team to continue developing their CutC and CutD enzyme inhibitors for use in humans. “We’ve cured aneurysm about a thousand times in mice, but we can’t seem to translate any of those inhibitors over to the human population,” but he added, now “we have a good drug that’s blocking something that is in so many different cardiovascular diseases. I’ve got a lot more hope for this one than I do for others.”

For his part, Hazen has spun his team’s work on the CutC and CutD inhibitors into a company called Zehna Therapeutics, which is part of the Cleveland Clinic. While still very interested in developing these drugs for cardiovascular diseases, the company’s first target is chronic kidney disease.

“The TMAO pathway is integrally linked to the development of renal dysfunction, and it’s a vicious feed forward cycle. As renal function declines, the ability to excrete TMAO reduces, and TMAO levels rise even more,” said Hazen. But the diseases are linked, he added. “The major cause of mortality in patients with renal dysfunction is cardiovascular.”

What excites Hazen most about developing his TMAO pathway inhibitors is that targeting specific microbial enzymes rather than human factors opens up a whole new way to treat disease.

“I am just amazed by the fact that the gut microbiome makes metabolites that impact our physiology and our disease susceptibility. It is just such a cool idea,” he said. “We’re just scratching the very beginning of this surface, and it is no doubt going to be an incredibly, rapidly advancing field in the decades to come.”

References

1. Wang, Z. et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature  472, 57-63 (2011).
2. Wang, Z. et al. Impact of chronic dietary red meat, white meat, or non-meat protein on trimethylamine N-oxide metabolism and renal excretion in healthy men and women. European Heart Journal  40, 583-594 (2019).
3. Wang, Z. et al. Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis. Cell  163, 1585-1595 (2015).
4. Roberts, A.B. et al. Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential. Nat Med  24, 1407-1417 (2018).
5. Sun, X. et al. Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome. Biochemical and Biophysical Research Communications  481, 63-70 (2016).
6. Guzik, B. et al. Mechanisms of oxidative stress in human aortic aneurysms — Association with clinical risk factors for atherosclerosis and disease severity. International Journal of Cardiology  168, 2389-2396 (2013).
7. Benson, T.W. et al. Gut Microbiota-Derived Trimethylamine N-Oxide Contributes to Abdominal Aortic Aneurysm Through Inflammatory and Apoptotic Mechanisms. Circulation  147, 1079-1096 (2023).