“Our largest environmental exposure is what we eat, and that is all perceived through the filter of our gut microbiome,” Stanley L. Hazen, MD, PhD, chair of the department of cellular and molecular medicine, section head of preventive cardiology and rehabilitation and director of the Center for Microbiome and Human Health at Cleveland Clinic, said in the presentation. “The gut microbiome is an active participant in many facets of cardiovascular disease and thrombosis.”

The initial discovery and structural identification of gut microbe-derived metabolites that are associated with CVD risk occurred nearly a decade ago with untargeted metabolomics, according to the presentation. Data from healthy patients were reviewed for the development of CVD over a period of time. Patients’ serum levels were analyzed for the chemical signatures that predicted future CVD risk, and of the metabolites that predicted risks, a third of them are linked to gut microbes, Hazen said.

A study published in Nature in 2011 found that three compounds linked to phosphatidylcholine metabolism, also termed lecithin, suggested a common pathway: choline, betaine and trimethylamine N-oxide (TMAO).

Diet and intestinal microbes are mechanically linked to atherosclerotic heart disease. A diet rich in phosphatidylcholine, a Western diet, also feeds the gut microbes. The microbes generate trimethylamine (TMA) as a waste product of dietary lecithin. After the TMA leaves the gut, it goes into the liver where it is converted to TMAO. In animal studies, TMAO accelerated heart disease development.

The clinical relevance of this was validated in a study published in Nature in 2011, which found that choline, betaine and TMAO dose-dependently track CV events. Beyond association, the study proved causation because a diet rich in choline led to TMAO generation and accelerated atherosclerosis, Hazen said.

“The relationship between plasma TMAO levels and incident CVD and mortality risks in subjects is a steeper curve than what you see with LDL cholesterol, triglycerides or C-reactive protein, for example,” Hazen said.

In a study published in Cell in 2015, a small molecule inhibitor was developed against the microbial enzyme responsible for generating TMA and TMAO, and then shown to block diet-induced atherosclerosis in animal models.

“Studies using microbial transplantation have shown the remarkable finding that susceptibility to atherosclerosis or thrombosis potential are transmittable, much in the same way an infectious disease can cause cholera or tuberculosis,” Hazen said.

The gut microbial TMAO pathway has a fundamental effect on tissue sterol metabolism. It does not change circulating cholesterol levels, but the pathway has been shown to modify tissue cholesterol ester content. Human clinical data suggest a link between TMAO and the risk for HF, MI, stroke, chronic kidney disease and adverse ventricular remodeling, Hazen said.

In patients from the Framingham study who had normal renal function at baseline and developed chronic kidney disease over 10 years, choline and TMAO were among the top molecules identified that predicted future risk for development of chronic kidney disease.

A meta-analysis published online in 2017 featuring over 26,000 patients also found that TMAO levels tracked future risk for adverse CV events, with a twofold increase in patients with higher TMAO levels. Those with lower TMAO levels had lower CV risk.

“In terms of strength of the association, that makes it almost equivalent to a CAD risk equivalent — or the diagnosis of diabetes,” Hazen said.

When studies were stratified by chronic kidney disease status, TMAO predicted future event risk in both subjects with and without kidney disease.

TMAO impacts multiple cardiac, vascular and renal targets. Dietary nutrients like carnitine and choline increase TMAO levels. Cholesterol and sterol metabolism are changed at the liver and intestine. Chronic high TMAO levels have also been shown to lead to fibrosis in the heart and kidney, according to the presentation.

In a more recent study that was published in Cell in 2016, over 4,000 subjects were examined and found to have higher plasma TMAO levels predict incident thrombotic event risk, including MI, stroke or death, and then through mechanistic studies, to show that TMO interacts with human and mouse platelets and fosters a prothrombotic phenotype in vivo. “There seems to be a sensor in the platelet that is recognizing TMAO and making it have a hair trigger more prone to clot with a lower degree of activation,” Hazen said.

Clinical studies in humans have found that titrating TMAO levels change platelet function. In a study published in Circulation in 2017, regardless of whether subjects ate a vegan/vegetarian or omnivore diet, when they were given choline supplements, their TMAO levels went up and their platelet function changed. At baseline, vegans had low TMAO levels and low levels of aggregation, but after 1 month of choline supplements, TMAO plasma levels increased 10-fold and platelet aggregation was greater. No further increases were seen in prolonged choline supplement use. Healthy volunteers who were omnivores also had low TMAO levels at baseline, which increased 15-fold at 1 month. Platelet aggregation also increased, with no further increase in TMAO or change in platelet function with longer duration of choline supplementation.

“This is the first time that anyone has shown that just what you eat changes your platelet function,” Hazen said. “This is all within the normal range. They’re not spontaneously getting clots, but how fast the clot forms is changing. So for example, if they were to nick themselves when they shaved, it could be impacted by what their TMAO level is. And analogously, if they had underlying atherosclerosis, it might affect their susceptibility for experiencing a heart attack.”

The same study published in Circulation in 2017 also analyzed the interactions between low-dose aspirin, TMAO levels and platelet function. Subjects who were omnivores took a low-dose aspirin once a day. At baseline, the extent of platelet aggregation was reduced as expected from the aspirin, but as choline supplements were added, by the first and second months, TMAO levels and platelet aggregation increased. The beneficial effect of low-dose aspirin in reducing platelet aggregation was totally lost when TMAO levels became elevated. Interestingly, low-dose aspirin was also seen to reduce TMAO levels by 40%.

“At any level of TMAO, being on aspirin had an antiplatelet effect, but the other thing that’s very striking is that the antiplatelet effect with aspirin seen at baseline was completely lost as TMAO levels increased,” Hazen said. “This raises the intriguing idea that some of so-called ‘aspirin resistance’ may be due to the changes in the level of metabolites that alter platelet function like an elevated TMAO.”

There are various ways to control TMAO levels, including diet. In a study published in Gut in 2015, patients who were vegetarian or vegan had lower urinary TMAO production rates. In another large observational diet study, it was observed that greater compliance to a Mediterranean diet was associated with lower TMAO levels.

Genetic studies that suppress the host enzyme in the liver, FMO3, that serves as the major enzyme source of TMAO generation from gut microbe-generated TMA, have shown targeting FMO3 can lower TMAO level, but at the risk of generating a higher TMA level. Though there was a reduction in atherosclerosis and favorable changes in tissue cholesterol, the animals tested in these studies smelled like rotting fish, as TMA is what causes rotting fish to smell. Subjects with a rare genetic disorder called Fish Malodor Syndrome have deficiency in FMO3, high levels of TMA, low TMAO and smell like rotting fish. Whether they are protected from atherosclerosis or not has not been determined.  

“From a scientific standpoint, studies genetically suppressing FMO3 levels and reducing atherosclerosis validate the concept of blocking the metaorganismal TMAO pathway,” Hazen said. “In more than one study, it has been observed in the LDL receptor knockout background to block atherosclerosis.”

An analog of choline, 3,3-dimethyl-1-butanol (DMB), was shown to be effective in inhibiting microbial TMA production and diet-induced atherosclerosis in vivo, according to a study published in Cell in 2015.

The highest levels of DMB were found in grapeseed oil, cold-pressed extra virgin olive oil, some red wines and Guinness lager stout, according to the presentation.

“What these results suggest is that beyond atherosclerosis, but linked to cholesterol metabolism in tissue and inflammation in the artery wall, that microbial metabolites — in this case, TMAO — impact these processes,” Hazen said. “We can measure TMAO and use it is as a predictor of risk in cardiovascular disease, but more importantly, by targeting this pathway, it may show promise as a potential therapeutic in the future for the treatment of atherosclerosis.” – by Darlene Dobkowski

Reference:

Hazen SL. Gut Microbes as a Participant and Therapeutic Target in Cardiometabolic Diseases.

Disclosure: Hazen reports he is a consultant/independent contractor for Esperion and Procter & Gamble, receives grant/research support from AstraZeneca, Pfizer, Procter & Gamble, Roche Diagnostics and Takeda and has the right to receive royalties from Cleveland Heart Lab, Frantz Biomarkers and Procter & Gamble.