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Changes in our society such as the increasing cost of retirement and age redistribution toward a larger elderly population will require humans to remain highly functional until an advanced age. As a consequence, chronic illnesses that are primarily responsible for reducing functionality and life expectancy will require improved prevention and therapeutic strategies. In a global way, cardiovascular disease and cancer represent the most challenging disorders to maintaining the functional integrity of our fellow humans. A new theory has been derived from recent progress in our understanding of atherosclerosis as a key mechanism for cardiovascular disease and of cancer. Instructively, this theory provides a bridge at the stem cell level, linking most chronic disorders. Antioxid. Redox Signal. 11, 401–406.
Atherosclerosis has been described as an inflammatory process triggered by risk factors that have been carefully characterized by studies like the Framingham Heart Study. Hence, cigarette smoking, diabetes mellitus, elevated low-density lipoprotein cholesterol, reduced high-density lipoprotein cholesterol, hypertension, or excessive inflammation all can contribute to injuring the arterial wall. However, a couple of years ago, we showed for the first time that atherosclerosis is not simply the result of direct injury to the arterial wall, but instead the consequence of such injury in a context in which the intrinsic repair process for the arterial wall is no longer fully functional (6, 7). Our laboratory (11) and other groups of investigators (5) have shown that arterial repair is triggered by arterial injury, which, in turn, provokes a local inflammatory reaction (7) (Fig. 1). This inflammatory reaction, if sufficiently pronounced, can lead to a generalized signaling process that helps recruit progenitor cells and, in particular, endothelial progenitor cells that originate from the bone marrow, the spleen, and other adult stem cell reservoirs and circulate with other blood cells to specific areas of arteries that are prone to develop lesions of atherosclerosis (5, 17). Endothelial progenitor cells (EPCs) are capable of forming a bond with the extracellular matrix and cells of the damaged arterial wall, and of integrating within the surface layer of the arterial wall (Fig. 2). There, they may contribute to regenerating a functional endothelium through cell growth, differentiation, and many other activities that include paracrine functions (3). We have shown that mice that are exposed to proatherosclerotic factors, such as a high lipid content of the blood, lose over time their capacity to mount a functional repair activity (11, 17) (Fig. 3). We have also shown that the loss of functional arterial repair in sick mice is associated with the exhaustion or the disappearance of a specific marrow population that can otherwise differentiate, within appropriate conditions, into mature and functional endothelial cells (26).
By using gene-expression analysis, we have reconstituted the natural history of atherosclerosis by analyzing changes in gene expression that are associated with successive stages of the atherosclerotic process (11) (Fig. 3). With an apolipoprotein E (ApoE) knockout model on a high-fat diet in which mice rapidly develop lesions rather typical of human atherosclerosis (total cholesterol usually exceeds 1,000mg/dl), we have characterized the molecular signature for various stages of the progression of atherosclerosis. Through the identification of genes whose expression is pathognomonic for each stage of the disease, we reconstituted the molecular puzzle of atherosclerosis progression (11) (Fig. 3). Early on, genes that characterize the arterial wall have to do with altered metabolism. At that stage, lesions of atherosclerosis are undetectable (before 6 weeks of age). Next, lesions (fatty streaks) become barely detectable (up to 12 weeks); genes that characterize this stage through their expression all have to do with inflammation. Later on, and as lesions become more organized and involve thicker atheroma (12-plus weeks), the genes whose expression is specific to this stage have to do with tissue remodeling.
We also were able to define a molecular signature that corresponds to efficient repair of the arterial wall (11). Genes that constitute this pool often have to do with cellular functions that are important for stem cells. We used this “repair signature” to survey arterial walls at various stages of the atherosclerosis disease process for the presence of successful arterial repair. Although this signature was detectable early in the disease process (before the presence of detectable atherosclerotic lesions), the molecular signature for efficient repair became blurred and no longer significantly detectable as early lesions of atherosclerosis started to appear (11). At later stages of the atherosclerotic process, no signs of active and efficient arterial repair could be detected. These studies contributed to the conclusion that atherosclerosis is not simply the response of the arterial wall to an injury, but instead represents the altered response of the arterial wall in which injury continues to apply, once an effective repair response can no longer be recruited to the arterial wall (7) (Fig. 4).
Today, the molecular mechanism(s) responsible for the loss in ability to produce functional endothelial progenitor cells capable of maintaining an effective repair response are not fully identified. Early evidence suggests that such mechanisms may involve changes in chromatin conformation (through epigenetic alterations) for precursor cells within the bone marrow and other reservoirs for progenitor cells (4). It is also highly likely that micro-RNAs (miRNAs) are implicated in the obsolescence of bone-marrow–derived endothelial progenitor cells.
These studies are supported by a body of research performed in human conditions that result from atherosclerosis, such as coronary artery disease, myocardial infarction, and stroke. It has been shown that the level of circulating endothelial progenitor cells is lower in the blood of patients who experience risk factors for coronary artery disease (9) or advanced arterial lesions (12, 16, 22). Furthermore, Werner and colleagues (22) showed that patients with low levels of circulating EPCs are at high risk for coronary events. Moreover, they showed that the level of EPCs that circulate in the blood is inversely proportional to the mortality of patients with coronary artery disease (22). Such associations support the concept that maintaining a functional repair system that involves competent EPCs is critical for a life sheltered from coronary artery disease and, for patients at risk, from deadly events. The replacement of dysfunctional EPCs in patients with atherosclerosis and advanced coronary artery disease remains elusive. Even in animal models, the specific progenitor cells capable of repairing arteries have not been fully identified (26). What is known is that after a lifetime of atherosclerosis, it is highly likely that these cells cannot simply be extracted from the bone marrow of patients or animal models (17). It is tempting to speculate that with adequate treatment of marrow progenitor cells in vitro before injection into patients, functional repair capacity may be restored (2, 20).
Instructively, it appears that for most cancers that affect humans, a specific triggering factor can be identified. Cigarette smoking for lung cancer, asbestos for mesothelioma, Helicobacter pylori for gastric cancer, Epstein–Barr virus for Burkitt lymphoma, and ultraviolet rays (UV) of sunlight for melanoma represent well-known examples. These injuries, when applied over the long term to a specific target organ, can promote the development of a cancer illness in susceptible patients. Some cancers have not yet been associated with a specific noxious stimulus, but it is increasingly clear that an association exists between such injuries and the disease process. Recent progress in cancer research indicates that the biology of stem cells may be implicated in tumor formation (18). Metastases often limit the life expectancy of patients who have cancer. The development of metastases was believed to be at a late stage in cancer progression, but new evidence suggests that cancer cells metastasize early on and lodge in homing tissues early with the onset of cancer (15). In their new location, mutations and selection lead to oncogene activation and disruption of suppressor genes, allowing the heightened growth of metastases.
It is highly likely that risk factors that contribute to the development of organ-specific cancers trigger a chronic repair process that is, for a while, responsible for the maintenance of affected tissues in a rather functional state (Fig. 5). It is possible that the maintenance of a noxious stimulus for a long period for a patient with a predisposition to a given type of cancer will eventually lead to transformation of the tissue-specific repair stem cell(s), thus leading to tumor formation. It is also likely that aging of the immune system leads to reduced surveillance capacity for the elimination of cells that have undergone malignant transformation. Hence, disruption of repair mechanisms can lead to two very different types of chronic illnesses: (a) exhaustion of repair cells for atherosclerosis with its thromboembolic complications of acute myocardial infarction, sudden cardiac death, symptomatic coronary artery disease, stroke, and other symptomatic cerebral vascular disease; and (b) transformation of repair cells for cancer illnesses. A deficient repair process is probably sustaining most chronic illnesses based on available information at this point (8).
Kaposi sarcoma is triggered by the human herpesvirus 8 (HHV8). The likelihood for Kaposi sarcoma to develop in HHV8-infected patients is particularly high in patients infected with the human immunodeficiency virus (patients with AIDS). A key gene for the development of Kaposi sarcoma is coded for by the HHV8 genome, vGPCR. vGPCR, when expressed on the surface of endothelial cells, is required for their transformation in Kaposi sarcoma cells and for the generation of Kaposi sarcoma in nude mice (13). We have discovered that the small GTP-binding protein Rac1, when constitutively activated and expressed in cells that also express smooth muscle cell α-actin, can induce Kaposi sarcoma lesions that are identical to lesions found in human patients (unpublished observation). Among the various targets of Rac1, which functions as a biologic timer for specific cellular activities, the most relevant is NADPH-oxidase and, in particular, the isoforms that bind Rac1 for activity (14).
We have shown that Rac1 is an important regulator of mitogenic activity induced by oncogenic Ras in transformed fibroblasts. The mitogenic and transforming effect of activated Rac1 requires activation of NADPH-oxidase and production of superoxide and derived reactive oxygen species (14). The fact that activated Rac1, when expressed in cells that also express smooth muscle actin, can induce Kaposi sarcoma came as a surprise and was not anticipated. The mouse Kaposi sarcoma (KS) induced by constitutively activated Rac1 (Rac CA) is indistinguishable from the human KS. Interestingly, mouse KS lesions occurred primarily in homozygous animals, mainly in males (as human KS), and are located in hair-less regions such as the tail, nose, and ears of mice overexpressing Rac CA. Spindle cells and immature vascular structures are typical of human KS in Rac CA lesions. We demonstrated that the activation of NADPH-oxidase and production of reactive oxygen species are necessary for Rac-induced mitogenesis and oncogenic transformation. By suppressing reactive oxygen species, resulting from the activation of NADPH-oxidase by Rac1, we were able to prevent mitogenic response to activated Rac1, transformation of cells, and formation of KS in mice expressing Rac CA (unpublished observation).
While we were attempting to identify genes that could contribute to susceptibility to atherosclerosis and cardiovascular complications in a framework that was unrelated to work described in prior sections of this review, three genes that belong to the Rac1 pathways were found to be critical for definition of risk for CAD. In a genome-wide scan study called GENECARD for early-onset CAD, we reported on the association of segments of the genome with the development of premature CAD and cardiac events such as myocardial infarction. A region strongly associated with such events was located on chromosome 3q13. On combing the 3q13 region (21), we identified single-nucleotide polymorphisms associated with CAD within genes that belong to the Rac1 pathway. Genes coding for the proteins kalirin, CDGAP, and MYLK are all implicated in the susceptibility for premature CAD. The protein kalirin functions to accelerate the exchange of the GTP nucleotide bound to Rac1 and other Rho-family proteins. The protein CDGAP accelerates the hydrolysis of the GTP nucleotide bound to Rac1. Finally, the protein MYLK is a kinase that functions downstream from Rac1. Hence, in a nonbiased way, three proteins and their single-nucleotide polymorphisms (SNPs) that belong to the Rac1 pathway were implicated in susceptibility for developing premature coronary artery disease. It remains to be defined whether SNPs that are associated with susceptibility for CAD directly affect protein level or structure in a way that would modify the activity of the pathway. Because Rac1 contributes to pathways that involve oncogenes such as Ras, as well as critical pathways involved in stem cell biology, such as the canonic Wnt pathway (23), it is tempting to speculate that polymorphisms of proteins of the Rac1 pathway may affect repair processes and the repair stem cells that are important for prevention of both CAD and cancers.
The connection between cancer and atherosclerosis through Rac1 is puzzling, but highly interesting. It could be that the connection has to do with chronic injury and tissue repair, in which Rac1 is likely to play a pivotal role. Chronic repair reactions ongoing in arterial vessels or in areas of the skin (for Kaposi sarcoma) may constitute the defective processes that involve Rac1. In the case of atherosclerosis, the disease process may occur once the repair process becomes insufficient to maintain the homeostasis of arteries (due to progenitor cell exhaustion). Polymorphisms in genes that code for proteins of the Rac1 pathway may increase the susceptibility for a deficient repair process or exhaustion of progenitor cells capable of arterial repair or both. In the case of cancer, the constitutive activation of Rac1 may lead to deterioration of the cells involved in repairing damaged skin, which eventually leads to the oncogenic differentiation (KS). In both cases, production of reactive oxygen species through activation of NADPH-oxidase may represent an important contributor. Our studies illustrate the importance of furthering our understanding of cellular repair activities involving stem cells and other progenitor cells to unveil new mechanisms for chronic illnesses that affect cohorts of humans. Such studies will create new opportunities for therapeutic interventions to predict, prevent, and reverse chronic disease processes, opportunities that may represent the greatest advance in medicine for the 21st century.
I thank Carmen Alsina, Irene Hung, Magaly Robitaille, and Christine Morris for their editorial contributions to this review; I thank all the students, research and clinical fellows, young faculty, and staff who have worked in my laboratory or worked as my colleagues, without whom this work would not have been possible; I thank Jay and Jeanie Schottenstein and friends at The Ohio State University for this wonderful award; I thank my wife Emily and sons Alex, Zach, and Dylan for their immeasurable support; and finally, I thank the American Heart Association, NIH, and NHLBI for funding our work.