Current topics in vascular biology: An update for the 21st century

Credit for introducing the modern concept of the circulatory system to Western medicine is generally given to William Harvey, who described the continuous circulation of the blood in his epochal treatise De Motu Cordis in 1628. Over the next centuries, physicians added to their armamentarium the ability to auscultate the heart and measure blood pressure, but it was not until the latter half of the 20 ^th century that physicians and scientists developed an appreciation for the role of blood vessels themselves in the pathogenesis of the most common and debilitating cardiovascular diseases: coronary artery disease, peripheral vascular disease, myocardial infarction, and systemic arterial hypertension. It is the appreciation of this central role of the blood vessels as target organs in the major cardiovascular pathologies that led to the emergence of vascular biology as an independent field of study over the past few decades.

Major achievements in the field of vascular biology in the last century are numerous. Important mediators of vascular tone, inflammation, and proliferative capacity— most notably, nitric oxide and angiotensin II— were discovered, and pharmacologic tools to manipulate their effects were developed. An appreciation for the role of the vascular endothelium in the pathogenesis of atherosclerosis arose from the elegant studies of Russell Ross¹. The importance of inflammatory cell infiltration into atherosclerotic lesions is now understood², and therapeutic strategies to target the inflammatory response are in development.

These major achievements in the field of vascular biology have had an enormous impact on cardiovascular therapeutics over the past twenty years. New drugs— such as angiotensin-converting enzyme inhibitors, glycoprotein IIB/IIIa inhibitors, and thrombolytics— and new uses for older drugs— including aspirin and beta-blockers— have derived from fundamental discoveries by vascular biologists in the 20^th century. It is virtually impossible to find anyone familiar with the field who is willing to bet that the labors of vascular biologists in the 21^st century will be any less fruitful than those of their forebearers. With this as a prelude, the present forum provides an excellent opportunity to review some of the most interesting and timely issues and controversies in the field of vascular biology.

Oxidative stress and vascular biology. Although reactive oxygen intermediates (ROI) have well-defined physiologic roles— for example, generation of the neutrophil oxidative burst— recent data provide a compelling link between ROI generation and vascular lesion formation³. For example, physiologic concentrations of ROI (superoxide and hydrogen peroxide) can elicit growth responses in vascular smooth muscle cells⁴. It is now recognized that ROI induce activation of growth-related intracellular signaling pathways in a manner similar to that of endogenous growth factors, indicating that ROI may lie downstream of some growth factors in the course of intracellular signaling⁵. In spite of the compelling data linking ROI and atherogenesis, numerous clinical trials in humans have failed to demonstrate a compelling benefit of antioxidant therapy in patients with atherosclerosis^6-9. The discrepancy between experimental studies linking ROI generation and vascular lesion formation, on the one hand, and the lack of benefit of antioxidant strategies in clinical trials, on the other hand, has been termed the “oxidative paradox”¹⁰. Resolving this paradox by increasing our understanding of the role ROI play in vascular events is a major imperative for vascular biology in the next century.

Oxidant generation in vascular cells. Reports such as those by Berk and colleagues demonstrating that ROI may directly induce vascular smooth muscle cell proliferation⁴ led many groups to ask whether ROI are physiologic mediators of signaling events in vascular cells and, if so, what the source of these ROI are. Indeed, we now know that cells within blood vessels, including endothelial and smooth muscle cells, generate ROI^11,12. ROI are important mediators of the effects of proatherogenic factors such as platelet-derived growth factor and thrombin^13,14. In addition, thrombosis itself is likely a potent initiator of ROS generation in the vasculature¹⁵.

If ROI generation within vascular cells plays a role in signaling events associated with atherosclerotic lesion formation, then the source of these oxidative species would be a potential therapeutic target. Although there are several oxidant-generating sources within vascular cells, recent studies implicate the smooth muscle cell NAD(P)H oxidase as a mediator of oxidative events. Several components of this oxidase, which is similar to but distinct from the phagocytic NADPH oxidase, have been identified, and include p22^phox, p47^phox, and a smooth muscle-specific component called MOX1^14,16,17. In particular, p47^phox is rapidly activated in smooth muscle cells in response to stimulation with agents such as thrombin¹⁴, and regulation of this component may be an important means whereby ROI generation is controlled in smooth muscle cells, making it a potentially attractive target for intervention in vascular diseases.

Consequences of ROI generation and its link to atherogenesis. There are several downstream targets for ROI-mediated signaling within vascular cells, including the extracellular signal-regulated and stress-activated protein kinases (ERKs and SAPKs) and transcription factors such as nuclear factor-k B and the signal transducers and activators of transcription (STAT) family (reviewed in³ ). These pathways are already well-linked to proatherogenic events; for example, activation of nuclear factor-k B is a potent stimulus for inflammatory cell recruitment into vascular lesions¹⁸ and ERK activation mediates proliferative responses in smooth muscle cells¹⁹. However, it is still difficult to reconcile how short-lived ROI such as superoxide and hydrogen peroxide can mediate the long-term cellular changes associated with atherosclerosis. One possibility is that lipids, proteins, and DNA are oxidatively modified by ROS, and that these modified proteins have long-lived and deleterious consequences to the vasculature. The oxidation of low-density lipoprotein is an excellent example of such a process²⁰. In addition, 4-hydroxynonenal is an oxidatively modified fatty acid product with relatively long-lived effects that may have atherogenic properties²¹. Oxidative modification of DNA, particularly within the mitochondrial genome, may also account for some of the long-term changes in cellular function that result in progression of atherosclerosis²².

Markers of oxidative injury as indicators of atherogenesis. Similar to hypertension or hyperlipidemia, increased oxidative stress may be a risk factor for only a subset of patients with atherosclerosis. However, we presently have no good method to determine which individuals are most likely to benefit from antioxidant therapies. Imagine the likelihood of determining the efficacy of an antihypertensive medication if it was applied to the population in general, or of using cholesterol-lowering medications indiscriminately without knowledge of serum cholesterol values. There are hints that it may be possible to develop such a marker (or group of markers): the measurement of F2-isoprostanes²³ and a newly reported method for measuring mitochondrial DNA damage²² may be promising. Clinical studies of these and other oxidative markers are underway to identify patients for whom oxidative stress may be an important atherogenic risk factor. Developing strategies to target ROI as prevention or treatment of vascular disease is a critical imperative for vascular biologists in the coming years.

Angiogenesis. Angiogenesis is the process whereby new blood vessels grow from existing blood vessels. Nature has harnessed the angiogenic process to create blood vessels during development, to control reproductive events, and in response to injury, inflammation, and ischemia. In addition, angiogenesis is a major contributor to athologic conditions such as tumor growth and metastasis, diabetic microvascular disease, and proliferative retinopathies, in which cases new blood vessels either feed a pathologic process or are themselves the morbid factors. Interest in angiogenesis by vascular biologists has grown on several fronts. I will consider here the role played by angiogenesis as a contributing component in neointimal formation and atherosclerosis, but first I will examine the potential use of angiogenesis and angiogenic factors as therapies for ischemic vascular diseases.

Angiogenic strategies to treat ischemic vascular diseases. The creation of new vascular conduits to increase blood flow to ischemic tissues is an old story; coronary artery bypass surgery, after all, takes advantage of this principle. The use of angiogenic growth factors, and in particular angiogenic gene therapy, is seen as a means to the same end— a so-called “molecular bypass”. The hope of angiogenic therapies is that by inducing angiogenesis, blood flow to ischemic zones in the central or peripheral circulation can be improved in a manner that is less invasive, more beneficial, or more widely applicable than current pharmacologic, interventional, and surgical therapies.

The idea that angiogenic factors such as vascular endothelial growth factor (VEGF) could be harnessed to improve outcomes in ischemic vascular diseases was proposed by several groups, most notably the one led by Jeffrey Isner in Boston. Indeed, early studies in rabbit and canine models of ischemia demonstrated the potential of intravenously administered VEGF protein to augment collateral formation in ischemic tissues^24,25. These proof-of-principle experiments led to an explosion of interest in the role of angiogenic growth factors among vascular biologists. As the major groups in this field now have early phase human studies in progress to test the safety and efficacy of the therapeutic angiogenesis strategy, it is worth considering the status of these studies.

The Isner group has received the most notoriety for their work in this field. They have demonstrated the promise of an angiogenic gene therapy strategy by delivering the VEGF gene using plasmids, which are small, circular, relatively inert DNA molecules. Plasmids can be injected locally into tissues that are ischemic, but they are taken up unefficiently by most cells and are unprotected against cellular surveillance systems. It has generally been thought that proteins expressed by plasmids would appear transiently and in few cells. However, the Isner group demonstrated that plasmid-based angiogenic gene therapy was practical and provided long-lived effects on collateral formation in animal models²⁶.

Based on these animal studies, two Phase I trials were initiated to test VEGF plasmid therapy in humans. The first protocol examines the effects of VEGF injection into ischemic muscles of patients with critical limb ischemia²⁷, whereas the second is testing the activity of VEGF plasmid administration by direct injection via mini-thoracotomy as a treatment for patients with myocardial ischemia²⁸. The data presented so far from these Phase I studies is in the form of uncontrolled case reports, and therefore provide no conclusions with regard to the efficacy of these methods. Indeed, concerns have been raised regarding the invasiveness of the myocardial ischemia trial, insofar as it requires general anesthesia and a thoracotomy incision²⁹. Also, experimental studies have not fully addressed the potential for adverse effects— such as proliferative retinopathy, unmasking of latent tumors, and angioma formation at the sight of injection^30,31 — with these therapies. Nonetheless, initial findings from the early human studies are at least encouraging.

The results of several other ongoing, early phase human trials have now been published. The aforementioned trials testing VEGF plasmid therapy for peripheral vascular disease²⁷ and myocardial ischemia²⁸ have demonstrated promising, though preliminary results in uncontrolled studies. In addition, a study of an adenovirus expressing VEGF, administered either as an adjunct to bypass surgery or as sole therapy via thoracotomy, demonstrated the safety of these approaches in small numbers of patients³². VEGF protein has also been administered by the intracoronary route in a small non-randomized study, with no evidence of toxicity and limited improvement in some clinical endpoints³³.

Although much attention has been given to VEGF-based angiogenesis therapy, other angiogenic factors are also under consideration in human trials. Fibroblast growth factor (FGF)-1 protein has been administered via injection into the myocardium of patients undergoing bypass surgery^34,35. Although the administration of these therapies at the time of bypass limits the likelihood that clinical improvements will be observed in short-term studies, angiogenic sprouting and improvements in perfusion were reported at follow-up angiography. A similar study, in which FGF-2 was administered as sustained-release microcapsules into ischemic but non-graftable myocardium at the time of bypass, and improvements were observed in noninvasive indices of perfusion in a subset of patients³⁶. Finally, FGF-2 protein has also been administered via the intracoronary or intravenous route in a small group of unrandomized patients, again with incremental decreases in stress-inducible ischemia³⁷.

The role of angiogenesis in atherosclerotic lesion progression and neointima formation. For those who are first familiar with angiogenesis based on clinical trials for therapeutic angiogenesis, it may seem paradoxical that angiogenesis may also have a deleterious effect on vascular lesion formation. Indeed, new blood vessel formation is a long-recognized component of atherosclerotic lesions. Koestner first reported the vascularization of atherosclerotic lesions in 1876³⁸. Both across and within species, the media of blood vessels remains avascular until a critical width is achieved, beyond which point vascularization is required for medial nutrition³⁹. Increased medial blood flow in atherosclerotic lesions is due to the growth of new medial vessels rather than dilation of existing vessels⁴⁰. New vessels in atherosclerotic lesions form primarily by branching from the adventitial vasa vasorum⁴¹. The possibility that neovascularization contributes to the pathophysiology of atherosclerosis was reconsidered when Barger et al. demonstrated the presence of rich networks of vessels surrounding human atherosclerotic plaques by cinefluorography⁴².

Several mechanisms exist by which neovascularization may contribute to the clinical consequences of atherosclerosis. Neovascularization provides a source of nutrients, growth factors and vasoactive molecules to cells within the media and neointima, as evidenced by the association between neovascularization of atherosclerotic lesions and proliferation of adjacent smooth muscle cells⁴³. Intimal hemorrhage, which is associated with plaque instability, is due to rupture of the rich network of friable new capillaries surrounding lesions⁴⁴. Regulation of blood flow through plaque microvessels may contribute to the pathophysiology of vasospasm in advanced lesions⁴⁵. Remodeling of the vascular wall, a particular problem associated with the vasculopathy of aging, appears to be related in part to the process of neovascularization⁴⁶. Finally, neovascularization within human erosclerotic lesions is associated with expression of adhesion molecules, which in turn is strongly related to neointimal inflammatory cell recruitment^47,48

The preceding studies demonstrate a plausible link between angiogenesis and increased vascular lesion formation. Convincing evidence that vascular lesion formation is indeed dependent on angiogenesis has recently been provided by members of the Folkman laboratory, who demonstrated that vascular lesion formation could be inhibited by the angiogenesis inhibitors endostatin and TNP-470 in atherosclerosis-prone ApoE(–/–) mice⁴⁹. Interestingly, the mechanisms that underlie angiogenesis in vascular lesions— namely, increased VEGF production⁵⁰ — are the same mechanisms being harnessed to elicit angiogenesis therapeutically. These studies raise the possibility that an adverse consequence of therapeutic angiogenesis strategies may be the paradoxical effect of accelerating the very disease these agents are used to treat. Clearly, more work will be necessary to unravel the role angiogenesis plays in ischemic vascular disease, and the therapeutic potential of agents that modulate the angiogenic response.

New risk factors for atherogenesis. The discovery of new risk factors and the identification of appropriate primary interventions remain perhaps the biggest challenges of all in vascular biology over the next century. Oxidative stress as an emerging risk factor has been previously discussed above. We focus here on hyperhomocysteinemia, markers for chronic inflammation, and infectious agents as potential risk factors to consider in predicting the likelihood of atherosclerosis.

Hyperhomocysteinemia. Homocystinuria (severe genetic hyperhomocysteinemia) is a disease of childhood characterized by mental retardation, megaloblastic anemia, and a severe form of thrombotic vascular disease that is usually fatal in the first decades of life. The similar (though accelerated) nature of this syndrome in comparison with atherosclerosis led investigators to propose the “homocysteine theory of atherosclerosis”⁵¹, wherein less severe elevations of plasma homocysteine were postulated as a causative factor in atherogenesis. In contrast to patients with homocystinuria(in whom fasting plasma homocysteine levels may exceed 400 µmol/L), patients with hyperhomocysteinemia may have plasma homocysteine levels only at the upper limits of normal or slightly above (>15 µmol/L). Elevations of this magnitude may be due to heterozygous deficiency or hypomorphic alleles of enzymes (such as cystathionine b-synthase and methylenetetrahydrofolate reductase), vitamin deficiencies (folate and vitamins B₆ and B₁₂), and some drugs and chronic medical disorders.

Based on the known association between elevated homocysteine levels and thrombosis⁵², endothelial cell damage⁵³, and smooth muscle cell proliferation⁵⁴, it is logical to postulate a causative role for elevated homocysteine levels in atherosclerosis. Indeed, several studies have provided strong epidemiologic data in favor of such an association (for instance,^55,56), suggesting that elevation of homocysteine levels only slightly above the normal range (16 µmol/L) conferred a three-fold increased risk of coronary heart disease. However, it should be kept in mind that the data from prospective studies are not all consistent⁵⁷. Until randomized, prospective trials designed to test the effects of homocysteine-lowering agents on cardiovascular mortality are completed (several are in progress), firm conclusions regarding causality cannot be made.

Despite lingering questions regarding the putative causal link between homocysteine and atherogenesis, a place for measurement of homocysteine levels probably exists in current clinical practice, insofar as the primary treatment for mild hyperhomocysteinemia is a trial of vitamin therapy (B₆, B₁₂ and folate), even in patients who are not frankly vitamin deficient. It is therefore not unreasonable to screen patients with coronary disease (or a strong family history thereof) without other strong risk factors in an effort to uncover patients who might benefit from vitamin therapy. Of course, ongoing studies may suggest more widespread use for these vitamin supplements in all patients known to have, or who are at risk for, coronary artery disease.

Infectious agents. A link between infectious agents and atherogenesis was intermittently postulated throughout the last century⁵⁸, and the demonstration that viral infection had a causal role in atherogenesis in an animal model⁵⁹ made this link plausible. To date, a large number of infectious agents have been advanced as having a causative role in atherosclerotic lesion progression, among them herpes-family viruses, Chlamydia pneumoniae, Helicobacter pylori, and bacterial dental infections (reviewed in⁶⁰). The evidence supporting a link between infection and atherogenesis consists, in various proportions, of direct demonstration of the agent in vascular lesions, data derived from animal studies, and epidemiologic evidence based on seromarkers for infection. Some clinical evidence exists suggesting that macrolide and tetracycline therapy may be protective against myocardial infarction (perhaps by targeting C. pneumoniae)^61-64, although the results presented so far have been rather mixed. The determination of a causal role is hampered in part by the large number of infectious agents postulated to be involved. A number of clinical studies are now under way to test the infectious hypothesis by treating patients with coronary artery disease with different antibiotic regiments. The use of antimicrobial agents in all or a select group of patients with coronary disease, if proven efficacious, would be a major revolution in anti-atherosclerotic therapy analogous to the treatment of H. pylori infections in patients with gastric ulcerations.

Markers for inflammation. It is now well-accepted that atherosclerosis is a disease with a strong inflammatory component^65,66 that includes infiltration of advanced lesions with inflammatory cells². The general thought was that this inflammation was localized, intermittent, and unlikely to reveal itself systemically. Recent data suggest otherwise. Although a variety of inflammatory markers have been considered to reveal unstable atherosclerotic lesions, including soluble adhesion molecules⁶⁷, attention has focused on C-reactive protein (CRP) as a marker for risk of unstable coronary syndromes. Although, as an acute phase reactant, CRP is generally thought to be a non-specific marker of systemic inflammation, three different prospective trials (the Multiple Risk Factor Intervention Trial, the Physician’s Health Study, and the Women’s Health Study) have identified an association between CRP levels and increased risk of coronary artery disease^68-70. This risk remains after adjustment for other known risk factors for coronary disease.

Although no data exist to suggest that high CRP levels should be particularly targeted therapeutically, it is attractive to speculate that non-specific anti-inflammatory agents such as aspirin may be of special benefit in such patients. As more selective anti-inflammatory drugs (such as cyclooxygenase inhibitors) are considered as anti-atherogenic agents, the potential for an anti-inflammatory “recipe” for therapy based on circulating markers becomes a consideration.

Conclusion. Looking forward, many important issues in cardiovascular care require resolution. As our ability to probe patients noninvasively for indications of their atherogenic status improves, we can look to new therapies— antioxidants, ies of the future are certainly the ones we cannot think of yet, and that depend on the studies of a vascular biologist of the future, perhaps one in training now. Our understanding, and ability to treat, coronary artery diseases has proceeded rapidly, perhaps more rapidly than we realize. There is no reason to suspect anything other than that this pace will quicken in the years to come, to the benefit of our patients.

Acknowledgements

Research in the author’s laboratory is supported by grants HL03658 and AG15234.