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Abstract

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme to carbon monoxide (CO), iron, and biliverdin. Biliverdin is subsequently metabolized to bilirubin by the enzyme biliverdin reductase. Although interest in HO-1 originally centered on its heme-degrading function, recent findings indicate that HO-1 exerts other biologically important actions. Emerging evidence suggests that HO-1 plays a critical role in growth regulation. Deletion of the HO-1 gene or inhibition of HO-1 activity results in growth retardation and impaired fetal development, whereas HO-1 overexpression increases body size. Although the mechanisms responsible for the growth promoting properties of HO-1 are not well established, HO-1 can indirectly influence growth by regulating the synthesis of growth factors and by modulating the delivery of oxygen or nutrients to specific target tissues. In addition, HO-1 exerts important effects on critical determinants of tissue size, including cell proliferation, apoptosis, and hypertrophy. However, the actions of HO-1 are highly variable and may reflect a role for HO-1 in maintaining tissue homeostasis. Considerable evidence supports a crucial role for HO-1 in blocking the growth of vascular smooth muscle cells (SMCs). This antiproliferative effect of HO-1 is mediated primarily via the release of CO, which inhibits vascular SMC growth via multiple pathways. Pharmacologic or genetic approaches targeting HO-1 or CO to the blood vessel wall may represent a promising, novel therapeutic approach in treating vascular proliferative disorders. © 2003 Wiley-Liss, Inc.

Heme oxygenase (HO) plays a central role in regulating intracellular heme levels by catalyzing the first and rate-limiting step in the degradation of heme (Tenhunen et al., 1968, 1969). HO cleaves the α-meso carbon bridge of heme, yielding equimolar quantities of carbon monoxide (CO), biliverdin, and free iron (Fig. 1). This oxidative reaction is inhibited by various metalloporphyrins, including zinc or tin protoporphyrin-IX (Yoshinaga et al., 1992). Biliverdin is subsequently metabolized to bilirubin by biliverdin reductase and free iron is promptly sequestered by ferritin. To date, three distinct isoforms of HO (HO-1, HO-2, and HO-3) have been identified (Maines et al., 1986; Maines, 1989; McCoubrey et al., 1997). These isozymes are products of different genes and vary markedly in their tissue distribution and properties. HO-2 is a 36-kDa protein, that is, for the most part, constitutively expressed and present in high levels in the brain and testes. HO-3 is a recently cloned 33-kDa protein that closely resembles HO-2 but with much lower catalytic activity. In contrast, HO-1 is an inducible 32-kDa protein that is ubiquitously distributed.

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Figure 1. The heme oxygenase (HO) catalyzed reaction. Heme is cleaved by heme oxygenase to yield equimolar amounts of iron (Fe), carbon monoxide (CO), and biliverdin. Biliverdin is subsequently metabolized to bilirubin by the enzyme biliverdin reductase. M, P, and V represent methyl, propionyl, and vinyl groups, respectively.

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Although interest in HO-1 originally centered on its ability to degrade heme, recent findings indicate that HO-1 exerts other critical biological actions. HO-1 possesses important antioxidant and anti-inflammatory functions, and its robust induction by a wide variety of inimical stimuli is believed to represent a fundamental adaptive response to cellular stress (Keyse and Tyrrell, 1989; Stocker, 1990; Willis et al., 1996; Siow et al., 1999; Immenschuh and Ramadori, 2000; Durante, 2002a; Hill-Kapturczak et al., 2002). In addition, emerging evidence suggests a crucial role for HO-1 in regulating growth. In this review, we will highlight studies from our laboratory and others that characterize the effect of HO-1 and its products in controlling growth. Particular attention will be given to studies examining the molecular mechanisms by which HO-1 regulates vascular smooth muscle cell (SMC) growth and the possible clinical application of HO-1 or CO in treating occlusive vascular disease.

HO-1 AND GROWTH REGULATION

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

The importance of HO-1 in growth control is illustrated by observations made in HO-1 deficient (HO-1−/−) mice and humans. Poss and Tonegawa (1997a) generated HO-1−/− mice by targeted deletion of the mouse HO-1 gene and found that they are significantly smaller than wild type littermates. Similarly, the first human case of HO-1 deficiency exhibited severe growth retardation, and the patient died at a young age (Yachie et al., 1999). More recently, Sabaawy et al. (2001) found that the systemic administration of a retrovirus containing the human HO-1 gene to young rats results in a marked increase in body weight and size relative to control animals. The increase in somatic growth is proportionate and is not associated with an increase in food intake. These findings clearly implicate HO-1 as a critical regulator of growth; however, the mechanisms responsible for this effect are not yet known. Interestingly, Cheriathundam et al. (1998) found a significant correlation between hepatic levels of HO-1 and growth hormone in transgenic animals, raising the possibility that HO-1 may influence the production or cellular action of growth hormones.

HO-1 also plays an important role in fetal growth. HO-1 expression is decreased in the placentas of mothers with pre-eclampsia or with intrauterine growth restricted fetuses (Ahmed and Perkins, 2000; Ahmed et al., 2000; Barber et al., 2001; Wang and Yu, 2002). In addition, inhibition of placental HO-1 activity in rats via the administration of the HO inhibitor, zinc deuteroporphyrin-IX 2,4-bis-glycol, results in a significant decrease in pup size, whereas infection with an HO-1 adenovirus increases pup size (Kreiser et al., 2002). The growth enhancing action of HO-1 may arise from its ability to enhance fetal oxygenation and substrate delivery. Induction of placental HO-1 expression augments placental blood flow, while inhibition of HO-1 activity increases resistance in the perfused placenta (Ahmed et al., 2000b; Lyall et al., 2000; McLaughlin et al., 2000). This vasodepressor function of HO-1 is likely mediated via the liberation of CO since CO relaxes blood vessels in numerous vascular beds, including human placenta (Durante and Schafer, 1998; Wang, 1998; Zhang et al., 2001; Bainbridge et al., 2002). The CO-induced vasorelaxation of placental resistance blood vessels is likely mediated via the activation of soluble guanylate cyclase, since 1-H-(1,2,4)oxadiazolo(4,3-α)quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor, attenuates this response (Bainbridge et al., 2002). In addition, HO-1 may also increase placental blood flow by stimulating the production of corticotrophin releasing hormone, which dilates placental vessels in a nitric oxide-dependent manner (Barker and Corder, 1999; Navarra et al., 2001). Furthermore, HO-1 may augment placental vascularization by inducing the expression of vascular endothelial derived growth factor (VEGF), a potent angiogenic agent (Kreiser et al., 2002). Aside from promoting feto-placental blood flow, HO-1 may also directly stimulate fetal growth by inducing the release of growth factors. In this respect, HO-1 activity is associated with marked increases in the placental expression of insulin-like growth factor binding protein-1 and its receptor (Kreiser et al., 2002).

HO-1 AND CELL PROLIFERATION

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

HO-1 has been demonstrated to stimulate the proliferation of numerous cell types. In epidermal keratinocytes, the administration of nitric oxide donors induces HO-1 expression and enhances cell growth. The proliferative effect of the nitric oxide donors is totally abolished by the HO inhibitor, tin protoporphyrin-IX, suggesting a key role for HO-1 in keratinocyte proliferation (Clark et al., 1997). Consistent with this proposal, a striking increase in HO-1 expression is observed in the hyperproliferating epidermis during cutaneous wound repair or in biopsies from psoriatic skin lesions (Hanselmann et al., 2001). Similarly, HO-1 induction stimulates cell cycle progression and proliferation in vascular endothelium (Deramaudt et al., 1998; Li Volti et al., 2002). Transduction of the HO-1 gene into microvascular endothelial cells (ECs) also promotes the formation of capillary-like tube structures when ECs are embedded within a matrigel matrix (Deramaudt et al., 1998; Malaguarnera et al., 2002). These latter findings suggest that HO-1 may play an important role in angiogenesis. The mechanism by which HO-1 is able to stimulate the growth of vascular endothelium is not known, however, the ability of HO-1 to stimulate the synthesis of VEGF from vascular cells may contribute to its proliferative action (Dulak et al., 2002).

Elevated expression of HO-1 has also been observed in various tumor cells, including human adenocarcinoma, hepatoma, glioblastoma, melanoma, and squamous carcinoma cells (Goodman et al., 1997; Doi et al., 1999; Tsuki et al., 1999; Deininger et al., 2000; Torisu-Itakura et al., 2000). Furthermore, increased HO-1 immunoreactive staining is found in benign prostatic hyperplasia and malignant prostate tissue (Maines and Abrahamsson, 1996). Interestingly, an intra-arterial injection of the HO inhibitor, zinc protoporphyrin-IX, to solid hepatoma tumors strongly suppresses their growth, suggesting a role for HO-1 in tumor growth (Doi et al., 1999). Since the growth of solid tumors is dependent on the maintenance of adequate blood flow, the ability of HO-1 to stimulate angiogenesis may contribute to tumor cell growth. Consistent with this proposal, two recent clinical studies linked HO-1 expression with angiogenesis in human gliomas and human vertical growth melanomas (Nishie et al., 1999; Torisu-Itakura et al., 2000). Additional studies examining the role of HO-1 in the development and progression of cancer are warranted.

The effect of HO-1 on cell proliferation is, however, highly variable and appears to be cell-specific. In contrast to its proliferative effect on keritinocytes, endothelium, and tumor cells, HO-1 exerts a potent antiproliferative effect in epithelial cells. The induction of HO-1 reduces angiotensin II-mediated kidney epithelial cell proliferation, and inhibition of HO activity augments renal cell growth (Aizawa et al., 2001). In addition, overexpression of HO-1 in human pulmonary or renal tubular epithelial cells results in a marked decrease in cell growth and DNA synthesis that can be reversed by the selective HO inhibitor, tin protoporphyrin-IX (Lee et al., 1996; Inguaggiato et al., 2001). Flow cytometric analysis reveals that transfection of HO-1 in both cell types increases the number of cells in the G0/G1 phase of the cell cycle and decreases the entry of cells in the S phase of the cell cycle. The pulmonary epithelial cells overexpressing HO-1 also accumulate at the G2/M phase and they fail to progress through the cell cycle when stimulated with serum. These findings suggest that HO-1 may block cell cycle progression in epithelial cells at multiple sites.

Substantial evidence indicates that HO-1 is also a negative regulator of growth in vascular SMCs. Inhibition of HO-1 activity by zinc or tin protoporphyrin-IX potentiates the mitogenic action of serum, angiotensin II, platelet-derived growth factor, endothelin, and hypoxia in vascular SMCs (Morita et al., 1997; Togane et al., 2000; Durante, 2002b; Peyton et al., 2002). In addition, the induction of HO-1 expression by hemin administration or by gene transfer blocks growth in porcine and rat aortic SMCs (Duckers et al., 2001; Liu et al., 2002a, Zhang et al., 2002). Along another line of evidence, vascular SMCs obtained from HO-1 knock out mice exhibit enhanced SMC proliferation and DNA synthesis compared to SMCs from wild type animals (Duckers et al., 2001).

The inhibition of vascular SMC proliferation by HO-1 appears to be mediated via the release of CO. The CO scavenger, hemoglobin, potentiates the proliferative response of vascular SMC to several growth factors (Togane et al., 2000; Peyton et al., 2002). In addition, we observed that the administration of physiologically relevant concentrations of CO (100–200 ppm) inhibits SMC proliferation and DNA synthesis in response to several growth factors (Liu et al., 2002a; Peyton et al., 2002). These results are consistent with earlier data by Morita et al. (1997) which show that high doses of CO (50,000 ppm) blocks the proliferation of hypoxic SMCs. CO appears to block vascular SMC growth by increasing the intracellular levels of guanosine 3′-5′-cyclic monophosphate (cGMP). CO activates both crude and purified preparations of soluble guanylate cyclase and elevates cGMP levels in vascular SMCs (Ramos et al., 1989; Brune et al., 1990; Stone and Marletta, 1994; Christodoulides et al., 1995; Morita et al., 1995). Furthermore, inhibition of soluble guanylate cyclase by ODQ blocks the rise in cGMP and the antiproliferative action of HO-1 (Duckers et al., 2001). Moreover, the protein kinase G inhibitor, R-p-bromo-guanosine-3′,5′-monophosphorothiate, restores SMC growth in the presence of HO-1, indicating a primary role for the cGMP signaling pathway in the antiproliferative action of CO.

Recent studies indicate that the HO-1/CO system interacts with various components of the cell cycle machinery. Adenovirus-mediated overexpression of HO-1 or the exogenous administration of CO arrests SMC in the G1/S transition phase of the cell cycle (Duckers et al., 2001; Peyton et al., 2002). The inhibition of cell cycle progression by CO is associated with a marked decrease in the phosphorylation of retinoblastoma, a critical event required for S-phase entry and DNA synthesis (Peyton et al., 2002). Consistent with this finding, the inhibition of hypoxia-mediated SMC growth by CO is coupled with a decrease in the levels of the transcription factor E2F-1, which is a downstream target of phosphorylated retinoblastoma (Morita et al., 1997). CO also selectively inhibits the expression of cyclin A mRNA and protein while having no effect on the expression of cyclin D1 and E (Peyton et al., 2002). In addition, inhibition of cyclin A expression by CO results in the suppression of cyclin A-associated kinase activity and cyclin-dependent kinase-2 (cdk-2) activity, independent of any changes in the level of cdk-2 protein. Because cdk2 is a key regulator of both G1 and S phase cell progression (Braun-Dellaeus et al., 1998), the ability of CO to block cdk2 activity may provide a potent mechanism by which CO inhibits SMC proliferation. Interestingly, the HO-1-derived release of CO induces the expression of the cdk inhibitor, p21, suggesting that CO may block cdk2 activity via multiple mechanisms (Duckers et al., 2001). A functional link between HO-1 and p21 in growth regulation is supported by the finding that the antiproliferative action of HO-1 is significantly reduced in vascular SMCs obtained from p21 null mice (Duckers et al., 2001). Furthermore, SMCs obtained from HO-1 knock out animals that exhibit enhanced growth have a corresponding reduction in p21 levels compared to wild type mice (Duckers et al., 2001).

In addition to directly inhibiting vascular SMC proliferation, HO-1 may also indirectly modulate cell growth by affecting the release of growth factors. Morita and Kourembanas (1995) have shown in a co-culture system that HO-1 decreases the synthesis of endothelin-1 and platelet-derived growth factor from ECs. This inhibitory action of HO-1 is mediated by CO; however, it does not involve the cGMP signaling pathway since the administration of lipophilic analogues of cGMP or the inhibition of soluble guanylate cyclase has no effect on the expression of either of these mitogens. Similarly, studies in our laboratory and others found that HO-1-derived CO inhibits platelet aggregation, thereby preventing the release of growth factors from platelet alpha granules (Wagner et al., 1997; Sato et al., 2001). In this case, the anti-aggregatory effect of CO is mediated by cGMP since it is associated with a marked increase in platelet cGMP and is prevented by soluble guanylate cyclase inhibitors (Brune and Ullrich, 1987; Wagner et al., 1997). Thus, the HO-1-catalyzed release of CO is able to function in a paracrine fashion to block the release of growth factors from vascular cells and circulating blood cells in both a cGMP-dependent and independent fashion.

More recently, a report found that CO also inhibits the proliferation of non-vascular SMCs (Song et al., 2002). Physiologically relevant concentrations of CO (50–500 ppm) arrests human airway SMCs in the G0/G1 phase of the cell cycle. This CO-induced growth arrest is associated with the up-regulation of p21 and the down-regulation of cyclin D1. Interestingly, the antiproliferative effect of CO in airway smooth muscle is not mediated via the activation of soluble guanylate cyclase, but rather through the inhibition of the ERK1/ERK2 mitogen-activated protein kinase (MAPK) pathway. These results indicate that distinct signaling pathways mediate the inhibition of growth by CO in vascular and non-vascular smooth muscle.

The ability of HO-1 to exert opposing effects on proliferation in different cell types is intriguing. One possibility is that HO-1 may function as a critical enzyme in tissue protection and/or repair. In this respect, the capacity of HO-1 to block SMC growth while stimulating EC proliferation may play a critical vasoprotective role by preserving blood flow and blood fluidity at sites of arterial injury. Similarly, the inhibition of airway SMC and pulmonary and renal epithelial cell growth by HO-1 may operate to overcome the adverse remodeling response that occurs in lung and kidney disease. Furthermore, the stimulation of keritinocyte proliferation by HO-1 may serve to accelerate cutaneous wound healing. In this respect, the capacity of HO-1 to increase the growth of certain tumor cells may represent an extreme instance of tissue repair.

HO-1 AND CELL DEATH

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

Since tissue growth is largely determined by the balance between cell proliferation and cell death, HO-1 may also influence tissue size by regulating cell viability. Indeed, considerable evidence suggests an important role for HO-1 in cytoprotection. The induction of HO-1 by chemical inducers or selective overexpression of HO-1 provides cellular protection in cultured cells and in several animal models of brain, heart, kidney, lung, and liver failure (Abraham et al., 1995; Agarwal et al., 1995; Amersi et al., 1999; Otterbein et al., 1999; Panahain et al., 1999; Clark et al., 2000; Hangiashi et al., 2000; Yet et al., 2001; Coito et al., 2002). Studies with HO-1−/− mice also support the functional significance of HO-1 in cytoprotection. HO-1−/− mice demonstrate a significant embryonic loss and die within 1 year of birth (Poss and Tonegawa, 1997a,b). Adult HO-1−/− mice exhibit normochromic, microcytic anemia, and develop a progressive chronic inflammation in the kidney and liver. Embryonic fibroblasts from HO-1 deficient mice are also highly susceptible to heme- or hydrogen peroxide-mediated cytotoxicity and exposure of HO-1 deficient mice to endotoxin results in increased hepatocellular necrosis and mortality from endotoxin shock. The first human case of HO-1 deficiency exhibited similar characteristics, including severe, persistant endothelial damage in vivo and extreme sensitivity to hemin-induced cell injury in a cell line derived from this patient (Yachie et al., 1999).

The cytoprotection afforded by HO-1 is mediated by several distinct mechanisms. Increased HO-1 activity results in the degradation of the heme moiety, a potentially toxic pro-oxidant to the bile pigments biliverdin and bilirubin. Both biliverdin and bilirubin are potent antioxidants capable of scavenging peroxy radicals, inhibiting lipid peroxidation, and blocking the production of superoxide (Stocker et al., 1987; Kwak et al., 1991; Llesuy and Tomaro, 1994; Dore et al., 1999). Furthermore, HO-1 induction is accompanied by increased ferritin synthesis, which exerts an additional antioxidant effect by sequestering free iron (Vile and Tyrell, 1993).

Aside from its antioxidant effect, HO-1 can directly affect cell viability by blocking programmed cell death or apoptosis. Soares et al. (1998) first demonstrated that the overexpression of HO-1 prevents apoptosis in ECs. This was subsequently confirmed in numerous other cell types in response to a wide spectrum of apoptotic stimuli (Ferris et al., 1999; Brouard et al., 2000; Petrache et al., 2000; Shiraishi et al., 2000; Pileggi et al., 2001). The antiapoptotic effect of HO-1 is reversed in the presence of HO-1 inhibitors or in cells overexpressing antisense HO-1 (Petrache et al., 2000). Inhibition of apoptosis following the expression of HO-1 has also been reported in animal models of inflammation, ischemia-reperfusion, hypoxia, and organ transplantation while inhibition of HO-1 activity or deletion of the HO-1 gene promotes apoptosis in these animal models (Hancock et al., 1998; Soares et al., 1998; Yet et al., 1999; Ke et al., 2002; Vulapalli et al., 2002).

Recent studies have focused on defining the molecular mechanism by which HO-1 blocks apoptosis. Several discrete mechanisms have been proposed. In embryonic fibroblasts, HO-1 may prevent apoptosis by decreasing the cellular levels of the pro-oxidant, iron (Ferris et al., 1999). HO-1-mediated protection in these cells is associated with increased cellular iron efflux through the up-regulation of an iron pump that remains to be fully characterized. In contrast, survival in cultured vascular cells is mediated via the release of CO since the cytoprotection afforded by HO-1 is reversed by the CO scavenger, hemoglobin. Moreover, the exogenous administration of CO inhibits apoptosis in these cells (Brouard et al., 2000; Liu et al., 2002b). However, a proapoptotic effect of CO in ECs has also been reported (Thom et al., 2000). The reasons for these divergent results are not known but may reflect differences in the dose and duration of CO exposure and/or the vascular source of ECs. Interestingly, CO abrogates apoptosis by activating descrete signaling pathways in vascular cells. In ECs, the antiapoptotic action of CO results from the activation of the p38 MAPK while the activation of soluble guanylate cyclase contributes to the inhibition of apoptosis in vascular SMCs (Brouard et al., 2000; Liu et al., 2002b). Although the mechanisms by which CO interferes with the apoptotic cascade have not been fully characterized, we recently found that CO inhibits the expression of the proapoptotic protein, p53, and the release of cytochrome c from the mitochondria (Liu et al., 2002b). The ability of CO to block mitochondrial cytochrome c release, a fundamental step in the apoptotic pathway, may provide a mechanism by which CO can serve as a general inhibitor of apoptosis.

While the majority of evidence supports a cytoprotective role for HO-1, this is not a universal finding. Paradoxically, studies in our laboratory found that high level HO-1 expression following adenoviral-mediated gene transfer induces SMC apoptosis both in vitro and in vivo (Tulis et al., 2001a; Liu et al., 2002c). Under these conditions, the apoptotic action of HO-1 appears to be mediated via the release of the bile pigments, biliverdin, and bilirubin, which at high concentration are known inducers of apoptosis (Silva et al., 2001; Liu et al., 2002c). Reversal of HO-1-related cytoprotection with increased expression has also been documented in fibroblasts (Suttner and Dennery, 1999). In this instance, the cytotoxicity results from the liberation of free iron, which can generate reactive oxygen species via the Fenton reaction. In addition, the induction of HO-1 in Leydig cells triggers apoptosis in neighboring premeiototic germ cells by releasing CO (Ozawa et al., 2002). These findings clearly demonstrate that HO-1 and its products can exert divergent effects on cell survival depending on dose and cell type.

HO-1 AND CELL HYPERTROPHY

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

In addition to regulating cellular hyperplasia, studies in the heart suggest that HO-1 may modulate cellular hypertrophy. Pressure overload of the right ventricle, induced experimentally by hypoxia or pulmonary artery banding, increases HO-1 expression in cardiomyocytes (Katayose et al., 1993). Interestingly, HO-1 knockout mice exposed to hypoxia exhibit significantly greater ventricular hypertrophy compared to wild type controls, suggesting that the induction of HO-1 may function in an autocrine manner to limit cardiac hypertrophy (Yet et al., 1999). In support of this proposal, the induction of HO-1 by stannous chloride attenuates left ventricular/body weight ratio in genetically hypertensive rats (Seki et al., 1999). These findings also demonstrate that HO-1 attenuates both load-dependent and -independent forms of cardiac hypertrophy. The mechanism by which HO-1 inhibits cardiac hypertrophy has not been established; however, it is unlikely that CO is responsible since the exogenous administration of this gas induces cardiac hypertrophy (Penney and Bugaisky, 1992; Leonnechen et al., 1999). Finally, the effect of HO-1 on cell hypertrophy is cell-specific. In kidney epithelial cells, stable overexpression of HO-1 results in cell hypertrophy, as reflected by a significant increase in the protein/DNA ratio (Inguaggiato et al., 2001). The hypertrophic response to HO-1 may be mediated by p21 since HO-1 markedly increases the expression of p21 in kidney epithelial cells and p21 induces hypertrophy in these cells (Terada et al., 1999; Inguaggiato et al., 2001).

HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

HO-1 plays a key role in regulating vascular disease in humans. This is underscored by the observation that a microsatellite polymorphism in the promoter region of the HO-1 gene that is linked to decreased inducibility is associated with restenosis and increased vascular inflammation after percutaneous transluminal angioplasty, susceptibility to coronary artery disease in type 2 diabetic patients and in Japanese patients with coronary risk factors, and the development of abdominal aortic aneurysms (Exner et al., 2001; Chen et al., 2002; Kaneda et al., 2002; Schillinger et al., 2002a,b). Since excessive growth of vascular SMCs is an important contributing factor to a number of vascular disease states, including restenosis following angioplasty, atherosclerosis, and hypertension (Zierler et al., 1982; Ross, 1986; Schwartz et al., 1986), increasing HO-1 expression in blood vessels may offer a promising approach in treating vascular disorders. As shown in Figure 2, the expression of HO-1 in SMCs may block the growth of vascular SMCs via multiple mechanisms. The HO-1-catalyzed release of CO may function in an autocrine manner to directly inhibit vascular SMC proliferation by arresting cells in the G0/G1 phase of the cell cycle. The luminal release of CO from SMCs may also act in a paracrine fashion to block the release of vascular mitogens from adjacent ECs and circulating platelets. Furthermore, HO-1-derived CO may contribute to the re-endothelialization of the vessel wall at sites of vascular injury by stimulating EC growth and by protecting ECs from apoptosis. This would further serve to down-regulate SMC proliferation since ECs release several antiproliferative autacoids (Van Belle et al., 1998). Finally, the HO-1-mediated formation of biliverdin and bilirubin may promote the loss of SMCs in vascular lesions by inducing SMC apoptosis.

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Figure 2. Regulation of vascular smooth muscle cell (SMC) growth by HO-1. HO-1 blocks the growth of vascular SMCs via multiple mechanisms. The HO-1-catalyzed release of CO may function in an autocrine manner to directly inhibit vascular SMC proliferation by arresting cells in the G0/G1 phase of the cell cycle. The luminal release of CO from SMCs may also act in a paracrine fashion to block the release of vascular mitogens from adjacent endothelial cells and circulating platelets. Furthermore, HO-1-derived CO may down-regulate SMC proliferation at sites of vascular injury by promoting the re-endothelialization of the vessel wall by stimulating endothelial cell (EC) growth and protecting ECs from apoptosis. Finally, the HO-1-mediated formation of biliverdin and bilirubin may promote the loss of SMCs in vascular lesions by inducing SMC apoptosis. IEL represents internal elastic lamina.

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Considerable evidence indicates that HO-1 exerts a vasoprotective role by altering the arterial remodeling response to balloon angioplasty. The pathological remodeling response of restenosis is characterized by the local loss of ECs and the formation of a SMC-rich neointima. Several animal studies have documented that the induction of HO-1 by hemin inhibits neointima formation following balloon injury (Aizawa et al., 1999; Togane et al., 2000; Tulis et al., 2001b). In contrast, inhibition of HO-1 activity by metalloporphyrins or deletion of the HO-1 gene potentiates intimal thickening following arterial injury (Togane et al., 2000; Duckers et al., 2001). More recently, localized adenovirus-mediated delivery of the HO-1 gene to rat carotid and pig femoral arteries following balloon angioplasty suppressed neointimal development (Duckers et al., 2001; Tulis et al., 2001a). The reduction in neointima formation following HO-1 gene transfer is dependent on HO-1 activity since the HO inhibitor, tin protoporphyrin-IX, completely restores neointima formation back to control levels (Tulis et al., 2001a).

Genetic or pharmacological approaches that target HO-1 have also been successfully used to treat other vascular disorders. Induction of HO-1 by chemical inducers results in the reduction of atherosclerotic lesions in LDL-receptor knockout mice and prevents transplant arteriosclerosis in mouse cardiac allografts (Hancock et al., 1998; Ishikawa et al., 2001). Similarly, adenovirus-mediated transfer of HO-1 to arteries significantly attenuates the development of atherosclerosis in apoE null mice and graft arteriosclerosis in a rat model of chronic allogeneic aorta rejection (Juan et al., 2001; Bouche et al., 2002). In addition, up-regulation of HO-1 by heme analogues or by retroviral gene delivery lowers blood pressure in spontaneously hypertensive rats (Levere et al., 1990; Johnson et al., 1995; Sabaawy et al., 2001) while the targeted overexpression of HO-1 in lungs protects against pulmonary vessel wall hypertrophy induced by hypoxia (Minamino et al., 2001).

Although animal studies strongly support the usefulness of HO-1 in ameliorating vascular disease, there are potential limitations of targeting HO-1 to the vessel wall. In particular, pharmacological induction of HO-1 suffers from a lack of specificity, while gene transfer to blood vessels using adenoviral vectors results in inflammation and only transient transgene expression (O'Brien, 2000). In this respect, the recent development of recombinant adeno-associated virus may circumvent the problems associated with the use of first or second generation adenoviruses (Flotte and Carter, 1995). This virus is non-pathogenic and elicits an attenuated host inflammatory response. Furthermore, the adeno-associated virus has tropism for many cell types and is able to integrate into the host genome, thereby permitting prolonged expression of the transgene. Adeno-associated virus-mediated delivery of the HO-1 gene to the heart has recently been demonstrated and may provide an optimum delivery vehicle for sustained expression of HO-1 and long-term vascular protection (Melo et al., 2002).

Since many of the antiproliferative actions of HO-1 are mediated via the release of CO, the exogenous administration of CO may provide an alternate strategy in preventing aberrant SMC growth. Inhalation of low doses of CO (10–500 ppm) has been shown to protect tissues against hyperoxia, transplant rejection, and ischemia-reperfusion (Otterbein et al., 1999; Fujita et al., 2001; Sato et al., 2001). Presently, comparable concentrations of inhaled CO are used diagnostically to estimate lung diffusing capacity in patients, raising the possibility that inhaled CO may be used therapeutically (Otterbein et al., 2000). Recently, Motterlini et al. (2002) have developed the use of transition metal carbonyls as CO-releasing molecules. This represents a novel approach to administering this gas and offers the potential for the localized delivery of CO to sites of vascular injury. For example, the advential or luminal areas of injured vessels can be selectively coated with CO-releasing molecules, thus bypassing possible hypoxic effects associated with the systemic administration of CO. In addition, vascular stents can be layered with CO-releasing agents to prevent in-stent restenosis. The development of new CO-releasing molecules that possess better biocompatibility and stability represents an attractive area of future research.

Finally, rather than delivering CO to the artery, an alternative strategy is to enhance the biological potency of endogenously generated CO via the use of CO-sensitizing compounds. In particular, the 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole derivative, YC-1, has been shown to markedly potentiate the activation of soluble guanylate cyclase by CO (Friebe et al., 1996). YC-1 enhances soluble guanylate cyclase activity by stabilizing the active configuration of soluble guanylate cyclase while decreasing the dissociation rate of CO from the activated enzyme (Friebe et al., 1996; Friebe and Koesling, 1998). We found that YC-1 inhibits the proliferation of cultured vascular SMCs and that topical perivascular application of YC-1 to blood vessels stimulates arterial cGMP levels and attenuates intimal hyperplasia following experimental balloon injury (Tulis et al., 2000, 2002). These results suggest that YC-1 may provide a new therapeutic approach in reducing endovascular injury through CO-mediated, cGMP-dependent processes.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

Recent studies demonstrate an emerging role for HO-1 in regulating growth. Ablation of the HO-1 gene or inhibition of HO-1 activity in animals and humans results in growth retardation and impaired fetal development. Conversely, systemic HO-1 gene delivery to animals induces a marked increase in body weight and size. Although the mechanisms responsible for the growth promoting properties of HO-1 are not completely understood, HO-1 may influence growth via several mechanisms. In particular, HO-1 may indirectly modulate growth by stimulating the release of growth factors or by increasing the delivery of oxygen and nutrients to target tissues. In addition, HO-1 may exert important direct effects on critical determinants of tissue size, including cell proliferation, apoptosis, and hypertrophy. The actions of HO-1 on these various cellular functions are remarkably diverse and may reflect a role for HO-1 in maintaining tissue homeostasis. Further studies evaluating the role of specific HO-1 products and their molecular mechanisms of action may provide additional insight into the heterogenous actions of HO-1. Finally, abundant evidence implicates HO-1 as a novel and potent inhibitor of vascular SMC growth. This antiproliferative effect is primarily mediated by the release of CO, which inhibits SMC growth via multiple pathways. Genetic approaches targeting HO-1 to the vessel wall or pharmacological interventions using CO-releasing or CO-sensitizing agents offer a promising therapeutic modality in treating occlusive vascular disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

I thank Dr. Andrew I. Schafer, Dr. David A. Tulis, and Dr. Hong Wang for helpful discussions and critical review of the manuscript.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. HO-1 AND GROWTH REGULATION
  4. HO-1 AND CELL PROLIFERATION
  5. HO-1 AND CELL DEATH
  6. HO-1 AND CELL HYPERTROPHY
  7. HO-1 AS A THERAPEUTIC TARGET IN VASCULAR DISEASE
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED
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