Autophagy in Vascular Disease

Abstract

Autophagy, or “self eating,” refers to a regulated cellular process for the lysosomal-dependent turnover of organelles and proteins. During starvation or nutrient deficiency, autophagy promotes survival through the replenishment of metabolic precursors derived from the degradation of endogenous cellular components. Autophagy represents a general homeostatic and inducible adaptive response to environmental stress, including endoplasmic reticulum stress, hypoxia, oxidative stress, and exposure to pharmaceuticals and xenobiotics. Whereas elevated autophagy can be observed in dying cells, the functional relationships between autophagy and programmed cell death pathways remain incompletely understood. Preclinical studies have identified autophagy as a process that can be activated during vascular disorders, including ischemia–reperfusion injury of the heart and other organs, cardiomyopathy, myocardial injury, and atherosclerosis. The functional significance of autophagy in human cardiovascular disease pathogenesis remains incompletely understood, and potentially involves both adaptive and maladaptive outcomes, depending on model system. Although relatively few studies have been performed in the lung, our recent studies also implicate a role for autophagy in chronic lung disease. Manipulation of the signaling pathways that regulate autophagy could potentially provide a novel therapeutic strategy in the prevention or treatment of human disease.

Macroautophagy (autophagy) is a regulated cellular pathway for the turnover of cytoplasmic organelles and proteins through a lysosome-dependent degradation process (1–5). During autophagy, newly formed vesicles with a double-membrane structure, termed autophagosomes or autophagic vacuoles (AVs), surround and sequester cytosolic targets. The captured material may include insoluble protein aggregates resulting from subcellular damage, organelles, or pathogenic particles (e.g., viruses, bacteria). Maturing autophagosomes subsequently fuse with lysosomes to form single, membrane-bound autolysosomes, where the engulfed components are digested by lysosomal hydrolases. This degradation process regenerates metabolic precursor molecules (e.g., amino acids, fatty acids) that can be reused for anabolic pathways and ATP synthesis (1–5). By cellular recycling, autophagy provides an endogenous mechanism for prolonging survival during starvation (2). Autophagy also serves a vital function by facilitating the turnover of damaged or dysfunctional organelles, such as mitochondria, peroxisomes, and endoplasmic reticulum (ER), thereby maintaining a healthy population of these organelles (6–8). The occurrence of autophagy under basal conditions, as well as its induction by various stimuli (e.g., xenobiotics, cytokines, infections, ER stress, and oxidative stress) suggests essential roles for this process in cellular homeostasis and adaptation to adverse environments (8–11). Additional types of autophagy, including chaperone-mediated autophagy and microautophagy, have been described elsewhere (12).

To date, autophagy has been implicated in a number of fundamental biological processes, including aging, immunity, development, tumorigenesis, and cell death and differentiation (2, 13–17). The regulation and functional significance of autophagy in human diseases, however, are currently not well known. Recent preclinical and cellular studies predict revolutionary translational applications for autophagy, including potential therapeutic strategies based on the manipulation of this process (17, 18). Because many studies have been conducted in animal models of disease, very little is currently known about the physiological function of autophagy in the clinical progression of human disease. Autophagy potentially represents a compensatory mechanism to maintain homeostasis during the early stages of disease progression (17–19). The occurrence of autophagy during late stages of disease progression may represent either a continued adaptive response to cellular stress or an insufficient defense against the pathogenic process. On the other hand, autophagy can conceivably exert a significant contributory role in disease pathogenesis if, in fact, its dysregulation or excessive activation directly affects cell death pathways (16, 20–22). This latter pathway is often referred to as caspase-independent or autophagic cell death (type II programmed cell death). Indeed, many recent studies in cardiac injury or disease models are suggestive of both protective and deleterious roles of autophagy, depending on the model system (23, 24). Recent progress has implicated autophagy in a broad spectrum of human diseases, including cancer (11, 25), neurodegenerative disorders (26), inflammatory bowel disease (27–30), and chronic lung disease (31). Many of these studies are united by similar speculative arguments that ultimately leave the functional significance of autophagy in these disease contexts an open-ended question. This review focuses on the involvement of autophagy in diseases of the heart and cardiovascular system. Some of the results of these studies have been previously reported in the form of abstracts (32–34).

AUTOPHAGIC PATHWAY

The molecular signaling pathways that regulate autophagy have been partially elucidated (Figure 1). Autophagy falls under negative regulation by a signaling pathway involving class I phosphatidylinositol 3-kinase (PI3K), and mammalian target of rapamycin (mTOR). Through this pathway, the antibiotic, rapamycin, acts as a potent inducer of autophagy by inhibiting mTOR. A series of genes critical for the regulation of autophagy (Atg) were initially characterized in yeast, followed by the identification of their mammalian homologs, each with distinct roles in the induction or progression of the autophagic pathway (5, 12). The Bcl-2–interacting protein, beclin 1 (Atg6), which is monoallelically deleted in many human cancers, functions as a haploinsufficient tumor-suppressor gene, and acts as major regulator of autophagy in mammalian cells (35, 36). Beclin 1 forms a macromolecular complex with class III PI3K (Vps34), p150 (Vps15), and Atg14, which assimilate with the autophagosome. The ultraviolet radiation resistance–associated tumor-suppressor gene protein (termed “UVRAG”) also binds this complex in competition with Atg14, and associates to the late endosome (36). Increased production of phosphatidylinositol-3-phosphate by Vps34 regulates the formation of nascent autophagosomes (vesicle nucleation) through the recruitment of cytosolic factors (36). The elongation of the newly formed autophagosome also requires the action of two ubiquitin-like conjugation systems. In the first of these pathways, Atg5 is conjugated to Atg12 by Atg7 (E1-like) and Atg10 (E2-like) enzymes (37). The resulting Atg5–Atg12 forms a ternary complex with Atg16L (38). In the second pathway, the microtubule-associated protein-1 light chain 3 (LC3; Atg8) is conjugated to the cellular lipid phosphatidyl-ethanolamine (PE) (38, 39). Atg4 cleaves the pro form of LC3B to generate the lipid conjugation site at the C-terminal glycine residue of LC3. Conjugation of PE with LC3 occurs from the sequential action of Atg7 (E1-like) and Atg3 (E2-like) activities. In mammals, the conversion of LC3 from LC3-I (free form) to LC3-II (PE-conjugated form) is regarded as a critical step in autophagosome formation (40). The appearance of punctate staining in green fluorescence protein (GFP)-LC3–expressing cells and tissues is regarded as an indicator of autophagosome formation. The recruitment of LC3-II to the autophagosome is mediated by the Atg5–Atg12–Atg16L complex, which also facilitates LC3 conjugation (41). The Atg5–Atg12–Atg16L complex participates in elongation of the phagophore, and subsequently dissociates from the autophagosome upon maturation. During autophagosome–liposome fusion, LC3 is degraded by lysosomal hydrolases within the autophagosome, or delipidated at the membrane surface by Atg4B. The GTPase, Rab7, traffics to the autophagosome membrane and, together with the lysosomal-associated membrane proteins, LAMP-1 and LAMP-2, facilitates the fusion of the autophagosome with the lysosome (42, 43). In the final stages of the autophagic pathway, encapsulated cargo is degraded to component parts by lysosomal proteases and released.

Figure 1.

Autophagic pathway. Autophagic activation proceeds through a series of steps that include: (1) vessel nucleation; (2) vessel elongation and autophagosome formation, with assimilation of material to be degraded; (3) autophagosome–lysosome fusion; and (4) autophagolysosomal digestion of encapsulated cargo. Beclin 1, in complex with class III phosphotidylinositol 3-kinase (PI3K) and Atg14, acts as a major positive regulator of autophagy. The rapamycin-sensitive mammalian target of rapamycin (mTOR)/class I PI3K pathway acts as a major negative regulator of autophagy. Autophagosome formation requires two ubiquitin-like conjugation systems: the Atg8 (microtubule-associated protein-1 light chain 3 [LC3]) conjugation system, and the Atg5–Atg12 conjugation system. Autophagosome–lysosome fusion requires Rab7 and the lysosomal proteins, LAMP1 and LAMP2.

AUTOPHAGY AND APOPTOSIS CROSS-TALK

The terms “autophagic cell death” or type II programmed cell death have been used to refer to cell death that occurs in association with apparent increases in AV formation, and independently of caspases. This definition has previously been used to define a cell death pathway distinct from apoptosis (type I programmed cell death). Apoptosis is characterized by cell surface blebbing, chromatin condensation, DNA fragmentation, and activation of caspases. The relationships between the signaling pathways that regulate autophagy and those which regulate apoptosis remain incompletely understood (20–22). Proteins involved in the regulation of autophagy interact with apoptosis-regulatory pathways. For example, beclin 1 forms a complex with Bcl-2 family members, including Bcl-2 and Bcl-X_L (44, 45). Binding of Bcl-2 family proteins to beclin 1 inhibits autophagy by preventing the association of beclin 1 with the class III PI3K complex. The autophagic protein, Atg5, may affect extrinsic apoptosis pathways through interactions with the Fas-associated death domain protein (46). Atg5 also promotes apoptosis through a calpain-dependent truncation product, which binds to and inhibits Bcl-X_L (47). The BH3-only protein, BNIP3, can act as an activator of autophagy in cardiomyocytes during ischemia–reperfusion injury (48). A newly described BH3-only binding protein, ApoL1, when overexpressed, promotes cell death in an Atg5- and Atg7-dependent fashion (49). Several well known regulators of apoptosis pathways, such as p53, have been shown to also regulate autophagy (20). For example, p53 targets the expression of damage-regulated autophagic modulator, which stimulates autophagy (50).

The morphological distinctions between autophagy and apoptosis remain incompletely delineated, as the two processes are not always mutually exclusive and can sometimes occur simultaneously in the same cell type (20–22). Increased autophagic activity is thought to primarily represent an adaptive process. In this regard, the occurrence of autophagy in dying cells may represent an unsuccessful attempt at cell preservation. The studies of Boya and colleagues (51) demonstrated that increased AV formation does not necessarily represent increased autophagic activity or flux, as AV numbers can also increase as the result of impaired autophagosome–lysosome fusion. Furthermore, cells displaying elevated AV formation resulting from impaired fusion were not necessarily committed to death, and such death that occurred under these conditions could be inhibited by caspase inhibitors or inhibitors of mitochondrial permeability transition pore opening, indicative of an apoptotic pathway. Suppression of autophagy by beclin 1 or LC3 knockdown, or by chemical inhibitors, such as 3-methyladenine (3-MA), was shown to promote apoptosis and caspase-3 activation in starved HeLa cells. These studies generally support a role for autophagy as a means for prolonging cell survival, particularly during starvation (51).

On the other hand, because autophagy functions primarily as a degradative process, deregulated autophagy, leading to excessive degradation of vital cellular components, may conceivably be a causative factor or protagonist of cell death (20–22). Recent studies indicate that autophagy may effectuate an alternative pathway to cell death in apoptosis-deficient cells. For example, in Bax^−/−Bak^−/− murine embryonic fibroblasts, which cannot activate intrinsic apoptosis, treatment with chemotherapeutic agents results in nonapoptotic cell death accompanied by excessive AV formation (52). Furthermore, this caspase-independent cell death was blocked by chemical inhibitors of autophagy or by genetic knockdown of autophagic proteins (i.e., beclin 1, Atg5) (52). Autophagy was found to occur as a response to chemical agents associated with induction of the ER stress response. Induction of autophagy with such agents promoted cell death in apoptosis-compromised Bax^−/−Bak^−/− fibroblasts, but inhibited cell death in normal fibroblasts (53). Inhibition of caspase activation using chemicals, such as Z-Val-Ala-Asp (OMe)-fluoromethylketone (Z-VAD-FMK), has been reported to promote cell death in several cell types, including L929 fibrosarcoma cells and U937 monocytic cells, associated with increased autophagy (54–57). The cell death induced by Z-VAD-FMK was associated with increased reactive oxygen species production (55–57), occurring as a result of the increased turnover of antioxidant enzymes (e.g., catalase) by autophagic degradation (56). Genetic knockdown of autophagic proteins (e.g., beclin 1 or Atg7) inhibited caspase-independent (Z-VAD-FMK–induced) cell death (55, 57). Yu and colleagues (56, 57) suggested that autophagy and caspase-independent cell death could be activated by specific inhibition of caspase-8 in L929 cells, although further studies suggested that inhibition of calpain activity was also required. In direct contrast to the aforementioned studies, the studies of Wu and colleagues (58) proposed that autophagy serves as a cell survival mechanism in Z-VAD-FMK–induced L929 cell death. In these studies, activators of autophagy, such as rapamycin, protected against Z-VAD-FMK–induced cell death, whereas inhibitors, such as chloroquine, promoted Z-VAD-FMK–induced cell death (58). The reasons for these apparent discrepancies between studies in L929 cells remain unclear.

The functional consequences of autophagy may also vary in a cell type– or inducer-specific fashion. Activation of autophagy by chemical inducers of the ER stress response promoted cell survival in transformed cell lines, but promoted cell death in the corresponding untransformed lines (59). Inhibition of autophagy by Atg5 knockdown induced fibroblast apoptosis in response to extrinsic apoptosis activators (i.e., Fas and TNF-α), but inhibited apoptosis in response to apoptosis-inducing agents, such as menadione and ultraviolet radiation (60). Finally, several recent studies have implicated autophagy as a protagonist of apoptosis in stress models. For example, knockdown of LC3B or beclin 1 inhibited the initiation of extrinsic apoptosis pathways and caspase-8,-9 activation after acute cigarette smoke exposure in vitro (31, 61). Additional studies are needed to define the dynamic equilibrium between autophagy, apoptosis, and cell death in disease pathogenesis (Figure 2) (22, 23).

Figure 2.

Potential roles of autophagy in disorders of the cardiovascular system. Autophagy, as an endogenous, inducible response to cellular stress, may have both adaptive and maladaptive consequences, depending on the experimental context. The beneficial roles of autophagy have been associated with the homeostatic turnover of damaged cellular organelles and protein, thus promoting the recycling of vital metabolic building blocks, and the regeneration of energy equivalents. In this regard, autophagy is generally regarded as a survival mechanism. On the other hand, excessive autophagy may be associated with aberrant degradation of intracellular constituents, and potentially can lead to type II (autophagic) programmed cell death. The relationships between autophagy and apoptosis (type I programmed cell death) remain incompletely understood. Both impaired and excessive autophagy have been proposed to lead to apoptosis.

AUTOPHAGY AND STARVATION

Autophagic activation is widely recognized as a cellular response to starvation. The transgenic mice overexpressing GFP-LC3 (62), upon experiencing short-term starvation (3 d), displayed accumulation of AV, as determined by LC3-GFP punctate staining, and increased expression of autophagic proteins (i.e., LC3B-II and cathepsin D) in cardiomyocytes. These changes were accompanied by loss of energy charge (ATP depletion). Inhibition of autophagy in starved mice using bafilomycin-A1 accelerated the loss of ATP, and resulted in increased left-ventricular dilation and loss of cardiac function (63). Mice in which Atg5 was genetically deleted (Atg5^−/−), which display impaired autophagy in response to starvation, were more susceptible to cardiac dysfunction after starvation (64). Recent studies indicate activation of mTOR signaling in the heart during hypercholesterolemic states, and imply impaired autophagy in states of excessive nutrients (65).

AUTOPHAGY IN ISCHEMIA–REPERFUSION INJURY

Ischemia–reperfusion (I/R) refers to the arrest and restitution of blood flow that may occur during cardiac arrest and myocardial infarction, shock, organ transplantation, respiratory failure, or by mechanical intervention. Interruptions in blood flow (ischemia) can lead to reduced Po₂ in tissues (hypoxia). Prolonged states of ischemia can result in loss of energy charge (ATP depletion) and necrotic cell death. The reoxygenation of tissue after ischemic/hypoxic episodes results in the increased production of reactive oxygen species at the time of reperfusion, which promotes tissue injury. In addition, I/R may cause injury through the recruitment of proinflammatory leukocytes.

A number of studies have reported increased AV formation or altered autophagic activity during I/R injury, or chronic ischemia (66). Although many studies have been performed in cardiac I/R models, other models, such as renal, hepatic, and cerebral I/R, have been studied as well. There is no consensus as to whether autophagy represents a protective or deleterious phenomenon during I/R, and the functional significance of autophagy has been proposed to vary between the ischemic and reperfusion phases, with the duration of ischemia, or with the vascular bed (66). Autophagy has also been proposed to contribute to the cardioprotective mechanisms during ischemic preconditioning (67).

Myocardial I/R

Early experiments using hypoxia/reoxygenation of isolated rat or rabbit hearts were suggestive of activation of a lysosomal–autophagic pathway, as evidenced by redistribution of cathepsin D activity and aberrant lysosomal proliferation (68). Consistent with this finding, a number of recent studies have reported increased AV formation and increased indicators of autophagy in myocardial ischemic or I/R injury. Chronic ischemia induced by coronary stenosis in pigs caused increased AV formation, and increased expression of autophagic markers (e.g., LC3, beclin 1, and cathepsins B and D) in the myocardium, with maximal response after repeated cycles of ischemia (69). In the isolated rat heart model, experimental I/R caused increased autophagy, with evidence of mitochondrial degeneration and encapsulated mitochondria (mitophagy) (48). Increased AV formation was also observed in mouse myocardium during ischemia, and further increased during reperfusion, as evidenced by increased punctate staining in the myocardium of GFP-LC3 mice. In this study, Matsui and colleagues (70) reported different modes of autophagic regulation during the ischemia and reperfusion phase of mouse I/R injury. Induction of autophagy occurred during the ischemic phase, and required activation of 5′-AMP–activated protein kinase (AMPK), and inhibition of the mTOR pathway. Further increases in AV formation during reperfusion required primarily beclin 1, and occurred despite down-regulation of AMPK and up-regulation of mTOR in this phase. Beclin 1^+/− mice displayed reduced autophagy during I/R, and were resistant to I/R-induced cardiac injury (70). The authors concluded that the functional role of autophagy represents a protective response during ischemia, and a potentially deleterious response during reperfusion (70). In cultured cardiomyocytes, glucose deprivation, an ischemia mimetic, activated autophagy through up-regulation of AMPK and down-regulation of mTOR (70). Autophagy was essential for cardiomyocyte survival in this model. Contrary results were obtained in H9c2 cardiac myoblasts, whereby inhibition of autophagy with 3-MA protected against cell death by glucose deprivation (71). In cultured HL-1 cardiac myoblasts, simulated I/R increased steady-state autophagosome levels, as evidenced by electron microscopy (EM) and GFP-LC3 punctate staining. This accumulation of AV was determined to result from impaired autophagic flux associated with decreased lysosomal turnover of AVs after simulated I/R. Autophagic flux was impaired during the ischemic/hypoxic phase, and recovered partially during the reoxygenation phase (72). Overexpression of beclin 1 in this model protected against cardiomyocyte apoptotic cell death, whereas small interfering (siRNA)-directed knockdown of beclin 1 promoted apoptosis (72). The results in isolated cardiomycytes, taken together, imply protective, but sometimes variable, roles for autophagy with simulated I/R in vitro. In contrast, a deleterious role for autophagy was implicated in cardiac I/R in vivo.

Renal I/R

Renal I/R has been shown to induce autophagy in several studies. In uninephrectomized Wistar rats subjected to renal I/R (1 h ischemia, 4 h reperfusion), autophagic protein expression was increased in the tubular epithelium in conjunction with increased oxidative stress and enhanced apoptosis. Short-term I/R preconditioning prevented the autophagic, apoptotic, and pro-oxidant effects of sustained renal I/R (73). During renal I/R injury, increased autophagosome formation occurred in tubular epithelial cells, as assayed in the GFP-LC3 mice and by morphological evidence of mitophagy by EM analysis. Simultaneous overexpression of Bcl-2 in the Bcl-2/GFP-LC3 mice resulted in a reduction of I/R-induced autophagosome formation in the tubular epithelia, as well as reduction in tubular damage (74). In renal proximal tubular epithelial cells (H2-K), hypoxia induced increases in LC3-positive autophagosomes and LAMP-2–positive lysosomes. Hypoxia-inducible autophagosome formation was further increased with protease inhibitors, suggesting that hypoxia increased autophagic flux in kidney tubular epithelial cells (75).

Hepatic I/R

Cold/warm I/R injury, resulting from hepatic orthotopic liver transplantation in rats, was associated with increased autophagy in hepatocytes. Administration of chemical inhibitors of autophagy (i.e., wortmannin, protease inhibitors) reduced hepatocyte injury during liver transplantation and improved transplantation outcome (76). This study implied that autophagy potentially impairs the success of graft survival. In contrast, in mice subjected to hepatic I/R, impaired hepatic autophagy was associated with mitochondrial dysfunction and hepatocyte cell death. Augmentation of autophagy by starvation or infection with adenovirus (i.e., beclin 1, LC3) preserved hepatocyte function and prevented hepatocyte cell death after reoxygenation (77).

Cerebral I/R

Hypoxic ischemia in mice subjected to carotid artery occlusion resulted in neuronal cell injury associated with increased AV formation and partial activation of the apoptotic program (78). Induction of ischemia by permanent middle cerebral artery occlusion in mice resulted in increased AV formation and expression of autophagic markers (i.e., LC3, cathepsin D). Inhibition of autophagy with chemical inhibitors (i.e., 3-MA, bafilomycin A1) reduced neuronal injury during focal ischemia (79). Recent studies using the cerebral artery occlusion model demonstrate that lentiviral siRNA–directed knockdown of beclin 1 improved outcome of cerebral ischemic injury, with evidence for increased populations of progenitor cells and reduced neural cell apoptosis (80). The results so far indicate detrimental roles of autophagy with respect to cerebral ischemic injury.

AUTOPHAGY IN HEART DISEASE: CARDIOMYOPATHY/HEART FAILURE

Genetic deficiency in the autophagic protein, LAMP-2, results in cardiomyopathy (Danon disease) associated with increased AV accumulation in the cardiomyocytes (81). LAMP-2 knockout mice display impaired autophagic flux and AV accumulation in the heart and other tissues (82). Cardiac defects evident in these mice include abnormal cardiomyocyte morphology and reduced contractility (82). These studies indicate that dysfunctional autophagy can lead to cardiac disease (81, 82). Morphological or biochemical signs of autophagy were reported in several studies of human cardiac tissue taken from patients with heart failure. In myocardium from patients with dilated cardiomyopathy, degenerated cardiomyocytes displayed extensive AV formation and evidence of mitophagy by EM analysis (83), as well as evidence for accumulation of ubiquitin–protein aggregates in autophagic (monodansylcadaverine-positive) cardiomyocytes (84). In similar studies using cardiac tissue from patients with heart failure with ischemic cardiomyopathy or dilated cardiomyopathy, a fraction of nonapoptotic cardiomyocytes were positive for autophagy (85).

Increased hemodynamic stress in the heart leads to compensatory remodeling processes involving abnormal increases in cardiomyocyte size. In experimental cardiac hypertrophy induced by supravalvular aortic constriction, AV formation was significantly reduced relative to sham-operated mice. The authors concluded that hypertrophic hearts had impaired autophagic function (86). Impairment of autophagy, as in conditional knockout mice genetically deficient in Atg5, resulted in cardiac hypertrophy, left-ventricular dilatation and contractile dysfunction, accompanied by increased levels of ubiquitination (87). Furthermore, Atg5 knockout mice displayed no cardiac abnormality during early development, but developed cardiac dysfunction and left-ventricular dilatation 1 week after treatment with pressure overload (87). The authors concluded that up-regulation of autophagy in failing hearts can serve as an adaptive response for protecting cells from hemodynamic stress (87).

In adult mice, pressure overload induced by aortic banding induced heart failure and greatly increased cardiac autophagy (88). Load-induced autophagic activity, which was highly localized to the basal septum, peaked at 48 hours and remained significantly elevated for at least 3 weeks. Beclin 1^+/− mice displayed reduced autophagy during pressure overload, and were resistant to pressure-induced cardiac remodeling. Conversely, beclin 1 overexpression increased autophagic activity and promoted pathologic remodeling (88). In patients with isolated valvular aortic stenosis, autophagy was found in a higher proportion of cells than apoptosis, and correlated with left-ventricular systolic dysfunction (89). These studies, taken together, suggest that autophagy, which is strongly induced by pressure overload as a response to increased intracellular protein aggregation, plays a contributory or maladaptive role in pressure-induced cardiac dysfunction and disease pathogenesis. Furthermore, autophagy was proposed to play a crucial role in the transition from hypertrophy to cardiac failure (88–90).

AUTOPHAGY IN ATHEROSCLEROSIS

Atherosclerosis is an inflammatory disease of the vasculature characterized by plaque formation. Although relatively few direct studies have been done, autophagy has recently been implicated as a protective mechanism during the development of atherosclerosis (91, 92). Increased autophagy can be detected in autophagic plaques, and may stabilize plaques by preventing smooth muscle cell apoptosis (91, 92). Autophagy can be induced in cultured vascular cells by a number of proatherogenic stimuli, (e.g., oxidized low-density lipoprotein, oxidized lipids and lipid peroxidation end-products, proinflammatory states, ER-stress agents, hypoxia) and, in this regard, may represent a mechanism for the processing of oxidatively modified proteins (91, 92). Verheye and colleagues (93) observed increased caspase-independent cell death that occurs with increased autophagy in macrophages of atherosclerotic plaques. In this context, autophagy could be regarded as a potential clearance mechanism for macrophages in atherosclerotic plaques. Thus, autophagy-dependent cell death of plaque macrophages may represent an adaptive rather than maladaptive process in this disease.

AUTOPHAGY IN PULMONARY DISEASE

In contrast to the heart, relatively few studies have examined the regulation and functional role of autophagy in the lung and pulmonary vascular system. We have recently described increased autophagy in clinical specimens of the lung from patients with chronic obstructive pulmonary disease (COPD) relative to normal tissue, as evidenced by morphological and biochemical markers (31). This evidence included EM evaluation of lung tissue morphology, as well as increased expression and activation of autophagic regulator proteins (i.e., LC3B, beclin 1, Atg5, Atg7). Similar evidence of increased autophagy was observed in the lungs of mice that were subjected to chronic inhalation of cigarette smoke (31). Western blot analysis of autophagic protein activation (e.g., LC3B) in human lung tissue samples from a panel of clinical pulmonary conditions revealed the most pronounced activation in COPD, whereas little detectable activation in other types of lung disease (i.e., idiopathic pulmonary fibrosis, cystic fibrosis, sarcoidosis, and systemic sclerosis) (31).

In our recent studies, we have also observed increased indices of autophagy in clinical lung tissue samples of patients with pulmonary arterial hypertension (PAH) (32). The activation of autophagic markers (i.e., LC3B, beclin1, Atg7), as determined by Western analysis, and AV formation, as determined by EM analysis, were elevated in PAH lung tissue relative to normal controls. Furthermore, activation of autophagic markers (i.e., LC3B, beclin1, Atg7), and AV formation in lung tissue were also found to be elevated in animal models of PAH, including chronic hypoxia exposure in mice, and monocrotaline exposure in rats (33, 34). Increased LC3B protein activation, as well as AV formation, was also observed as a function of hypoxia in cultured human vascular endothelial and smooth muscle cells. Specific siRNA-dependent knockdown of LC3B enhanced hypoxic cell proliferation in vitro (32). Although the functional significance of autophagy in PAH remains unclear, these initial observations are suggestive of an adaptive mechanism during disease pathogenesis.

CONCLUSIONS

The role of autophagy in cardiac and cardiovascular disorders has been extensively studied in vitro and in in vivo animal models (66, 92), whereas only few studies to date, including our own studies in COPD (31) or PAH (32–34), have examined this process in pulmonary or pulmonary vascular systems. Additional preclinical and cellular data suggest a possible relevance of autophagy in other lung diseases, such as lung cancer, acute lung injury, and acute respiratory distress syndrome. Please see additional article by Ryter and Choi appearing in this symposium for more detailed discussion of these topics (94).

The multiplicity of studies exploring both protective and deleterious roles for this process suggests a fundamental homeostatic mechanism that, upon deregulation, can lead to dysfunction (18). Studies attempting to correlate autophagy with human vascular disease pathogenesis in human clinical samples are limited to date, and include observations of autophagic cells in human heart failure (83–85). Current progress in genetics suggests that impaired expression of autophagy-related genes in subpopulations, due to gene polymorphisms, may render susceptibility to inflammatory diseases of the bowel (27–30). Genetic polymorphism in autophagy genes remains a largely unexplored area with respect to cardiopulmonary diseases. Although the basic molecular machinery of autophagy has now been well characterized in lower organisms and mammals, much remains to be learned of the functional significance of this process in pathophysiological states (1–5, 12). The relationships between autophagy and cell death regulation remain incompletely understood, and further examination of these relationships may reveal mechanisms of disease pathogenesis (16, 20–23). Manipulation of autophagy or its component regulatory proteins may represent a novel therapeutic strategy in the prevention or treatment of human disease (11, 17, 18). Further progress in this domain will depend on the development of novel activators or inhibitors of autophagy that can be applied to clinical practice, in addition to those available for experimental research.