The Future of Vascular Biology and Medicine.

U.S. Department of Veterans Affairs Public Access Author manuscript Circulation. Author manuscript; available in PMC 2017 June 19. Published in final edited form as: Circulation. 2016 June 21; 133(25): 2603–2609. doi:10.1161/CIRCULATIONAHA.116.023513.

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The Future of Vascular Biology and Medicine Scott Kinlay, MBBS, PhD, Veterans Affairs Boston Healthcare System, MA; Brigham and Women's Hospital and Harvard Medical School, Boston, MA Thomas Michel, MD, PhD, and Brigham and Women's Hospital and Harvard Medical School, Boston, MA Jane A. Leopold, MD Brigham and Women's Hospital and Harvard Medical School, Boston, MA

Keywords


biology vessels; gastrointestinal microbiome; hypertension; pulmonary; peripheral arterial disease During the past several decades, landmark discoveries in the field of vascular biology have evolved our understanding of the biology of blood vessels and the pathobiology of local and systemic vascular disease states and have led to novel disease-modifying therapies for patients. In 1980, Furchgott and Zawadzki1 radically changed our focus in blood vessel research with the discovery of endothelium-derived nitric oxide and its effects on vascular tone. This seminal discovery redefined blood vessels as dynamic organs with autocrine, paracrine, and endocrine functions that are capable of regulating their own environment. The later recognition that many risk factors associated with vascular disease perturb nitric oxidemediated homeostatic mechanisms lent further credence to the concept of blood vessels as complex organs rather than inert tubes.


In the decades to follow, research in vascular biology predominantly targeted 3 facets of vascular function: vasomotor tone, inflammation, and the balance between thrombosis and thrombolysis. This occurred because atherosclerotic cardiovascular disease reached epidemic levels and atherothrombosis was found to feature disturbances in these functions that preceded visible pathology and clinical manifestations of the disease. Furthermore, modification of responsible causal factors reversed impaired vascular function (eg, lowering levels of low-density lipoproteins in atherosclerosis), and clinical studies began to validate the importance of preclinical vascular biology research in the treatment of hypertension,

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atherosclerosis, pulmonary vascular disease, erectile dysfunction, Raynaud phenomenon, and neointimal proliferation after mechanical vascular intervention.


More recently, advances in molecular biology and –omics technologies have facilitated in vitro and in vivo studies that revealed that blood vessels regulate their own redox milieu, metabolism, mechanical environment, and phenotype, in part, through complex interactions between cellular components of the blood vessel wall and circulating factors. These interactors include stem, progenitor, and differentiated cells; microRNAs, long noncoding RNAs, and DNA; and, hormones, proteins, and lipids. Dysregulation of these carefully orchestrated homeostatic interactions has also been implicated as the mechanism by which risk factors for cardiopulmonary vascular disease lead to vascular dysfunction, structural remodeling, and, ultimately, adverse clinical events, including myocardial infarction, stroke, critical limb ischemia, and pulmonary hypertension.

New Directions in Vascular Biology

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With all that is known, what directions will vascular biology research take over the coming years, and how will we advance the field? Although some future avenues will arise as the natural evolution of previous work, there are also some experimental and conceptual trends that we believe are evident. The majority of studies examining vascular cell functional responses are performed using passaged cells cultured on hard plastic dishes in an artificial medium, which does not mimic the biological, biochemical, or mechanical conditions present in normal or diseased blood vessels. Possible platforms to replace our current models could range from a self-contained blood vessel organ-on-a-chip system to one that is composed of 3-dimensional flexible tubular biomaterials that could be layered with vascular cells, perfused with typical blood or serum components, pressurized, and subjected to varied flow conditions. Studies performed in these systems will be complemented by in silico analyses done with supercomputing to predict structural or functional changes with resolution to the levels of single vascular cells. Second, with respect to in vivo studies, we will continue to search for better models and grow less reliant on the knockout mouse. This model system also does not take into account the notion that epigenetic or posttranslational modification of a protein may play a greater role in disease biology by altering expression, putative binding partners, downstream signaling pathways, and response to inhibitors/ medications. Add to the mix human-mouse species-related differences, and it is not surprising that many findings obtained in these models are not confirmed in human studies.2 Thus, it becomes clear that the knockout mouse model is more of a crude tool. On the basis of this conclusion, identifying accessible in vivo models that are more relevant and predictable for human vascular disease is necessary. There will also be a conceptual change in how we approach new questions in vascular biology, with a shift away from a reductionist approach (ie, 1 gene–1 protein–1 disease) to one akin to network analysis and systems biology in which interactions between genes, proteins, and metabolites are considered simultaneously. Furthermore, although the entire vascular system is one connected functional unit, vascular beds are phenotypically different and are likely to take on organ-specific properties through cross-talk with structural cells. In addition, we will begin to explore mechanisms that govern interindividual patient variability

Circulation. Author manuscript; available in PMC 2017 June 19.

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by examining key gene-environment interactions that affect the vasculature such as that between humans and commensal microbes. This latter nascent area in blood vessel research is discussed further as an example of a new frontier in vascular biology that will likely use the aforementioned principles in future studies to define how the microbiome regulates cardiopulmonary vascular disease.


The Microbiome Is Associated With Vascular Dysfunction New and emerging technologies are continuing to reveal that the microbiome plays a critical role in the maintenance of vascular health and the development of vascular disease (Figure 1). The diversity of the human microbiome between individuals, in both species and patterns of colonization, may underlie the differential phenotypic expression of vascular disease and ultimately establish a new paradigm in personalized medicine.


The microbial world has long been viewed as adverse to human health. The advent of germ theory in the 19th century implicated microbes as the source of infection and death. The development of antibiotics and the implementation of hygienic public health standards served to mitigate the effects of pathogenic microorganisms, even while the emergence of antibiotic resistance attenuated the efficacy of the powerful antimicrobial agents developed in the latter half of the 20th century. A less malignant view of the interactions between humans and microbes evolved from the discovery that vitamins essential for human health (eg, vitamin K and some B vitamins) are the products of microbial metabolism in the gut.3 Thus, the relationship between humans and microbes refects a commensal interaction in which it appears that both have coevolved to their mutual benefit. Despite this view, our awareness of critical connections between the microbial ecology of the gut and the pathobiology of the vascular wall has emerged only recently.


Studies of gnotobiotic (ie, pathogen-free) mice have provided important insights into the role of microbes in vascular biology. It has long been known that gnotobiotic mice have stunted growth, which is consistent with the role of gut bacteria in energy and vitamin metabolism. These mice also have marked abnormalities in vascular development that can be reversed with the restoration of normal gut flora, suggesting that the gut epithelium is able to transduce signals initiated by resident microbes to the microvasculature.4 In some mouse lines, gnotobiotic animals are more prone to the development of atherosclerosis than genetically identical mice with normal microbial flora.5 Interestingly, gnotobiotic mice are resistant to other systemic disorders associated with vascular dysfunction, including dietinduced obesity and diabetes mel-litus. The resistance to these diseases is lost with restoration of normal bacterial flora.6 Taken together, these early observations in gnotobiotic mice established a clear link between the gut microbiome and vascular pathobiology and did so in the absence of traditional predisposing risk factors. There are, however, difficulties in extrapolating discoveries made in gnotobiotic mice to humans, in whom the biological and biochemical diversity of the microbiome differs by orders of magnitude. In recent years, we have begun to appreciate the magnitude of the interaction between the microbiome and the human body. The Human Microbiome Project used next-generation DNA sequencing to identify thousands of distinct bacterial species that are resident in and


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on the normal human body.7 The aggregate number of bacteria far exceeds the total number of human cells and accounts for 1% to 3% of the total body mass (ie, 0.7–2.1 kg in a 70-kg person). DNA sequence analysis also revealed that microbes residing in different anatomic sites such as the gut, skin, respiratory tract, and genitourinary system are characterized by distinct enzymatic pathways that are adapted to metabolize nutrients present in the local environment.7 The greatest mass and diversity of bacterial species are found in the human colon. The gut microbiome consists of ≈1000 distinct bacterial species that coexist with the genes and gene products of their host to make up the human metagenome. Sequencing of 16S ribosomal RNA found in all bacteria identified 2 large and diverse groups that predominate in the human gut, Firmicutes and Bacteroidetes.8 Subsequent studies related changes in the levels of these bacteria to human disease and uncovered a complex interplay between human genetic variability, the effects of diet, and the composition of the gut microbiome in the development of obesity and hypertension.9,10

Mechanisms by Which the Microbiome Mediates Vascular Disease VA Author Manuscript VA Author Manuscript

To understand the mechanism(s) by which the microbiome regulates vascular function, metabolomic profiling was used to identify specific bacteria-derived molecules related to energy metabolism and vascular homeostasis. Further analysis identified trimethylamine as the gut metabolite and bacteria-derived chemical with the clearest association with cardiovascular disease.11 In clinical studies, unbiased metabolic profiling further revealed a significant increase in the levels of trimethylamine-N-oxide (TMAO) and related metabolites in plasma samples from patients with increased risk for cardiovascular disease compared with matched control subjects.12 TMAO is formed by bacterial metabolism of choline and phosphatidylcholine in the gut to yield trimethylamine, which is oxidized in the liver by the enzyme flavin monooxygenase-3 to form TMAO (Figure 2).11 Studies in human subjects, cultured cells, and animal models have converged on a comprehensive view of TMAO as a critical molecule associated with atherosclerosis, myocardial infarction, stroke, insulin resistance, and chronic kidney disease. TMAO has become the target of several therapeutic interventions, ranging from schemes to reduce dietary intake of trimethylamine precursors to manipulations of the gut microbiome to reduce trimethylamine synthesis. The revelation that atherosclerosis susceptibility could be transmitted from an atherosclerosis-prone strain of mice to another strain typically resistant to atherosclerosis simply by the transplantation of gut microbes, an effect that was closely related to TMAO levels, provided additional causal evidence to support the role of the gut microbiome in regulating atherosclerosis.13

The Microbiome and Peripheral Vascular Disease Peripheral artery disease is an often underappreciated and understudied manifestation of atherosclerosis. Peripheral arterial disease either is asymptomatic and portends an increased risk of adverse cardiovascular events or is present as symptomatic claudication with exercise or ischemic pain at rest with evidence of tissue loss. Obstructive atherosclerosis, which reduces perfusion to the limbs, defines this disorder. The ankle-brachial index, the ratio of systolic pressure at the ankle to the highest brachial pressure, is a highly sensitive and


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specific indicator of peripheral artery disease.14 There is, however, a substantial variability in the correlation between limb perfusion pressure measured by the ankle-brachial index and symptoms or function in patients with peripheral artery disease. There is also limited mechanistic understanding of the disease pathobiology at the cellular, molecular, and metabolic levels to explain the mismatch between symptoms and perfusion pressure.15 Furthermore, studies using supervised exercise programs or cilostazol, 2 mainstays in the treatment of claudication, show impressive improvements in walking function without making substantial changes in the resting ankle-brachial index.16–18 This suggests that the manifestations of peripheral artery disease are dependent not only on perfusion pressure of the conduit arteries of the legs but also on other as-yet unrecognized factors. Previous studies have shown that exercise improves endothelial function15,19–22 and were focused on identifying related mechanisms, including increased nitric oxide synthase activity,23,24 decreased expression of genes related to vascular inflammation,25 enhanced production of endothelial progenitor cells to promote endothelial repair and angiogenesis,26 and telomere stabilization to prevent endothelial cell senescence.27

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Accumulating evidence suggests that the microbiome might regulate the development of peripheral arterial disease and exercise-induced angiogenesis, which is widely thought of as the physiological mechanism by which exercise improves peripheral vascular disease symptoms. The gut microbiota has been shown to contribute to nitric oxide levels by reducing nitrate to ammonia with the generation of gaseous nitric oxide through nonenzymatic nitrite reduction.28 Exercise-induced angiogenesis has also been related to activation of AMP-activated protein kinase, hypoxia inducible factor-1a, and peroxisome proliferator–activated receptor-γ coactivator-1α, which stimulate vascular endothelial growth factor and skeletal muscle angiogenesis, potentially through interactions with muscle innervation and β-adrenergic signals.29 In fact, expression and activity of these molecules have been regulated by changes in the gut microbiome.5,30 Moreover, exercise has been linked to changes in the microbiome in experimental models and increased diversity in the gut microbiome in humans. In a murine model of diet-induced obesity, after 12 weeks of exercise, there was a shift in the gut microbiota that correlated with the exercise dose and differed from the effects observed with diet alone.31 In humans, exercise correlates with microbiome diversity with up to 22 distinct phyla identified in professional athletes compared with healthy individuals matched for age, ethnicity, and body mass index who had only 9 to 11 distinct phyla.32 Although these early studies do not provide causal or definitive evidence to link exercise to the gut microbiome and improved peripheral vascularization, they underscore the complexity needed in experimental design for future studies in this area. They may also explain why clinical trials of cell-based therapies as a method to increase angiogenesis that failed to take into account the effects of the microbiome on cell activity have been largely unsuccessful.16 Thus, exploring the effects of physical activity on the gut microbiome in the future may provide insights into new therapies for peripheral vascular disease that do not rely on improving the ankle-brachial index.


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Diet and the Microbiome


There is very little exploration of the effect of diet on vascular function in patients with peripheral artery disease; however, extrapolating from studies in patients with coronary artery disease had led to an endorsement of a Mediterranean diet for these patients. This diet is made up of foods higher in mono-unsaturated fats, with greater proportions of fruit, vegetables, and whole grains than processed foods and the selection of fatty fish over red meats.33 This dietary pattern is associated with lower rates of cardiovascular events related to atherosclerosis. The effects of diets on vascular function are also sparse. Diets rich in fruit and vegetables34 and some foods high in flavonoids35,36 improve vascular function in limited studies.


Studies of the gut microbiome in rodent models show that in response to changes in dietary intake of carbohydrates, fat, and fiber, there are changes in the gut microbiota at the phylum level. Similar studies in humans have shown the same trend, but there is significant interindividual variability. There is also regional variability in the microbiome, and the diversity and composition can refect an industrialized versus agrarian diet. Thus, it may be possible to predict disease risk vis-à-vis diets rich in phosphatidylcholine, a source of choline, or dietary carnitine, which is ultimately metabolized to TMAO.37 Investigators have exploited knowledge about these relationships to identify the choline analog 3,3-dimethyl-1butanol as an inhibitor of trimethylamine formation that can be given orally to mice. In animals fed a high-choline or high-carnitine diet, dimethyl-1-butanol decreased TMAO levels and inhibited atherosclerosis development.38 The presence of other microbes in the gut, including archaea, bacteriophages, and fungi, has also been associated with diet and, in some cases, linked to disease. Interestingly, analyses of the gut microbiome and the metabolome from individuals who follow the Mediterranean diet revealed that there was an association between dietary compliance and increased fecal short-chain fatty acid abundance that likely refected the presence of Firmicutes and Bacteroidetes bacteria and lower levels of TMAO.39 It is also interesting to speculate that there may be a relationship between the gut microbiome and bacteria resident in atherosclerotic plaques. Analysis of directional coronary atherectomy specimens obtained from patients with atherosclerotic epicardial coronary artery disease revealed the presence of a number of diverse bacterial species, including members from the phylum Firmacutes.40 Future studies will need to determine if these 2 reservoirs of microbes have distinct origins or if plaque bacteria represents a “metastasis” from the gut; if the bacteria resident in atherosclerotic plaque are merely a structural finding or have functional implications; and if interventions modify both areas equally.

The Potential Role of the Microbiome in Pulmonary Hypertension Although we are only just beginning to understand the role of the gut microbiome in systemic vascular disease, even less is known about the effects of the microbiome on pulmonary vascular structure and function, especially in pathological conditions characterized by pulmonary vascular remodeling such as pulmonary arterial hypertension. The current World Health Organization identifies 5 groups of patients on the basis of disease pathogenesis and pathophenotype41; however, there is growing recognition that subsets of


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patients in different groups share commonalities that are not immediately attributable to any one broad factor. The biological basis of these similarities is not yet known, but it is plausible that the gut microbiome, possibly in combination with the lung microbiome, may modulate genetic, epigenetic, or environmental factors that predispose to aberrant pulmonary vascular remodeling and pulmonary arterial hypertension.


From what is known about the gut microbiome–regulating response in the systemic circulation, it is certainly possible that it also affects the development of pulmonary vascular diseases. As mentioned, studies that examine the gut microbiome effects in the pulmonary compartment also need to consider the importance of the lung microbiome. This distinction achieves importance because the lining of the trachea and the bronchi contains heavily glycosylated proteins, similar to the gut, whereas the more distal airways are coated with lipid-containing surfactant. Thus, the proximal airways may support a microbiome that is similar to what is observed in the gut.42 Whether this occurs and has any pathophysiological significance for pulmonary vascular remodeling remains to be determined. Interestingly, it is also recognized that components of the microbiome respond to autocrine and paracrine signaling molecules released by human cells, including several that have been implicated in the pathobiology of pulmonary hypertension such as steroid hormones, catecholamines, and the cytokines interleukin-6 and tumor necrosis factor-α.42


Other data supporting a putative role for the gut microbiome in pulmonary vascular disease come from lung ischemia/ reperfusion studies performed in mice. In these studies, mice were administered antibiotics that localized to the intestine. Treatment with antibiotics attenuated both lung and circulating inflammatory markers and decreased alveolar damage. When alveolar macrophages were isolated from mice treated with antibiotics, they were found to generate fewer and lower levels of cytokines compared with control macrophages. Thus, this study suggested that the intestinal microbiome could play a role in regulating the inflammatory response in pulmonary disease.43 Other supporting evidence comes from studies that examined the effect of Escherichia coli, a component of the gut microbiome, on the inflammatory response in endothelial cells. These bacteria constitutively release nanosized vesicles that contain pathogen-associated molecular patterns, lipoproteins, and DNA. When endothelial cells are exposed to these vesicles, they demonstrate an acute inflammatory response by upregulating expression of the endothelial cell adhesion molecules E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1.44 The gut microbiome may also regulate vascular remodeling in pulmonary hypertension through effects on pulmonary vascular smooth muscle cells. In the systemic vasculature, there is evidence that the gut microbiome regulates smooth muscle cell proliferation and the formation of neointimal hyperplasia in response to injury after balloon angioplasty. In one study, rats treated with the antibiotic vancomycin had demonstrated changes in gut flora that were evident 4 weeks after initiation of treatment. The rats were shown to have decreased abundance of Firmicutes and an increase in the ratio of Bacteroidetes to Firmicutes. Antibiotic treatment was also found to lower serum levels of butyrate, one of the beneficial short-chain fatty acids produced by fermentation of dietary fiber. After vascular injury, neointimal hyperplasia was more severe in vancomycin-treated animals compared with


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controls; however, neointimal hyperplasia was ameliorated when antibiotic-treated animals were supplemented with butyrate. Butyrate limited neointimal formation through a mechanism that involved decreased aortic smooth muscle cell proliferation, cell cycle progression, and migration.45


Experimental evidence further links diet and the gut microbiome with pulmonary hypertension. In studies performed in apolipoprotein E knockout mice fed a Paigen diet (high fat, high choline) for 8 weeks, mice were found to have pulmonary hypertension with pulmonary vascular remodeling compared with mice fed a regular chow diet.46 Because choline is the precursor for TMAO, it is likely that the gut microbiome influenced the development of pulmonary arterial remodeling and pulmonary hypertension in this model. Furthermore, diet has been shown to improve pulmonary hemodynamics in a chronically hypoxic rat model of pulmonary hypertension. This study demonstrated that rats fed a diet containing fish oil (part of the Mediterranean diet) had lower mean pulmonary artery pressures, fibrosis, right ventricular hypertrophy, and platelet aggregation compared with rats with pulmonary hypertension fed other high-fat diets. This translated into lower mortality rates.47 Considered together, these findings indicate that it is very likely that the gut microbiome influences pulmonary vascular disease and the development of pulmonary hypertension.

Conclusions


At present, the causal pathways and molecular mechanisms whereby the gut microbiome initiates and perpetuates cardiopulmonary vascular disease remain incompletely characterized. However, early studies in the field indicate that future in vitro and in vivo studies of the blood vessel function must now contend with an additional layer of complexity in experimental design. These studies will need to examine local and remote interaction with the gut micro-biota and metabolites; for in vitro studies often done in the presence of antibiotics, this remains a challenge. Further adding to the experimental complexity, the microbiome may be altered by diet and by drugs. Thus, in addition to standardizing diets for in vivo studies, the effects of drugs (and delivery vehicle) will need to be evaluated. For example, it has been suggested that some of the beneficial effects of the antidiabetic drug metformin may be a consequence of the effect of the drug on the gut microbiome (Table).48 This underscores the possibility that other pathway inhibitors and receptor blockers commonly used as medications may also modulate the gut microbiome, thereby confounding interpretation of past studies that failed to rigorously account for drug use. A detailed characterization of drug effects on the gut microbiome could also lead to the repurposing of existing medications that may be called metabiotic therapies to refect the higher-order modulation of the microbial environment by drugs to achieve a salutary response.

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VA Author Manuscript Figure 1.


The gut microbiome. The gut microbiome is composed of up to 1000 different species of bacteria, the majority of which belong to the phyla Bacteroidetes and Firmicutes. The microbiome performs several homeostatic functions, is subject to interindividual variability, and has been implicated in disease.

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VA Author Manuscript Figure 2.


The gut microbiome and atherosclerosis. Dietary intake of foods high in phosphatidylcholine results in increased formation of trimethylamine (TMA) by the gut microbiome. TMA is then transformed into trimethylamine-N-oxide (TMAO) in the liver by flavin-containing monooxygenase-3 (FMO3). Elevated levels of TMAO have been implicated in atherosclerosis.

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Table

Categorization of Therapeutics That Target the Gut Microbiome


Therapeutic Approach

Definition and Therapeutic Target in the Gut

Prebiotic therapy

Use of foods (eg, high-fiber grains, fruits, and vegetables) or drugs that support the colonization or growth of favorable gut microorganisms

Probiotic therapy

Ingestion of live bacteria that promote a healthy gut microbiome, ranging from certain foods (eg, yogurt) to humanto-human fecal transplantations or selected bacterial strains

Antibiotic therapy

Use of microbicidal drugs to selectively target gut bacteria that cause disease

Metabiotic therapy

Design and administration of drugs that modulate the metabolic state of the gut to promote the growth of favorable microorganisms

VA Author Manuscript VA Author Manuscript Circulation. Author manuscript; available in PMC 2017 June 19.

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The specialty of pulmonary vascular medicine in China: historical development and future directions.

Breaking down the silos of medicine: the future of vascular neurosurgery.

The future of emergency medicine.

The future of general medicine.

The future of general medicine.

The future of US medicine.

The future of occupational medicine.

Shaping the future of synthetic biology.

The future of vascular surgery.

Social media and the future of medicine.

Psychological medicine and the future of psychiatry.

Phospholipases in biology and medicine.

Ultrasonics in medicine and biology.

Mouse medicine and human biology.

Genomic medicine and the future of physiotherapy.

Accelerating precision biology and medicine with computational biology and bioinformatics.

Adipose tissue-derived stromal vascular fraction in regenerative medicine: a brief review on biology and translation.

Plant Vascular Biology 2013: vascular trafficking.

Molecular biology in medicine.

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The translational biology of remyelination: past, present, and future.

The future of critical care medicine within emergency medicine.