Heme oxygenase-1 is a modulator of inflammation and vaso-occlusion in transgenic sickle mice (original) (raw)

These data highlight the critical importance of HO-1 in ameliorating vascular inflammation and vaso-occlusion in murine models of SCD. Nath and colleagues demonstrated increased HO-1 in the kidney vasculature of a patient with SCD as well as increased HO-1 in circulating ECs (39). Jison et al. (40) showed that sickle blood mononuclear cells have elevated HO-1 and biliverdin reductase mRNA even during steady state. Thus, chronic hemolysis and oxidative stress in SCD result in upregulation of HO-1 in patients as well as in transgenic sickle mice. It is likely that during acute hemolytic crises the patient with SCD increases HO-1 even further to deal with the increased heme burden and oxidative stress. It is this upregulation that may facilitate the resolution of vaso-occlusion. This adaptation allows the degradation of toxic heme and the production of CO, an antiinflammatory vasodilator; biliverdin and bilirubin, both antioxidants; and ferritin, an iron chelator.

Since we do not completely understand the pathobiology of human SCD, it is impossible to know how well the sickle mouse models mimic human disease. Probably none of the models displays all of the attributes of human SCD. They can best be described as tools to understand human SCD (41). For our studies, the sickle mouse models mimic human SCD in several important ways: they have rbc sickling in response to hypoxia; hemolysis; organ pathology with rbc congestion and infarction as in the human disease; excessive ROS production; a vigorous and chronic inflammatory response with activated endothelium; and impaired blood flow, especially in response to hypoxia/reoxygenation (2, 4, 7, 37, 42–45). Most of the studies described in this paper focused on the less severe murine model of SCD, the S+S-Antilles mouse (46), and fewer on the more severe murine model, BERK mice (47). However, BERK mice, which are more anemic (hematocrit ~22%) than S+S-Antilles mice, have similar levels of HO-1 expression, EC activation (2, 37), and organ pathology with rbc congestion and infarction but respond to hypoxia/reoxygenation with even more vaso-occlusion. Manci and colleagues (48) reported that BERK mice were similar to humans with SCD in having erythrocytic sickling, vascular ectasia, intravascular hemolysis, exuberant hematopoiesis, cardiomegaly, glomerulosclerosis, visceral congestion, hemorrhages, multiorgan infarcts, pyknotic neurons, and progressive siderosis. However, the BERK mice differed from humans with SCD in having splenomegaly; splenic hematopoiesis; more severe hepatic infarcts; less severe pulmonary manifestations; no significant vascular intimal hyperplasia; and only a trend toward vascular medial hypertrophy. Thus, these notable differences warrant careful consideration when parallels to human SCD are drawn (48). Our studies suggest that HO-1 upregulation may be important in inhibiting vascular inflammation in these murine transgenic models of human SCD. It is uncertain whether HO-1 plays a similar role in human SCD patients. Linkage of additional HO-1 expression in these SCD mouse models to other endpoints such as improvements in hemolysis and anemia or organ pathology and lifespan, conditions more directly relevant to the human disease, would strengthen these findings as being potentially relevant to human SCD.

Based on our current understanding of the interrelationships of hemolysis, oxidative stress, EC activation, blood cell adhesion, and vaso-occlusion, we have proposed a model that delineates how oxidative stress and inflammation contribute to the pathophysiology of vaso-occlusion in SCD (2, 3). We hypothesize that Hb, heme, and iron derived from hemolysis of sickle rbcs promote excessive ROS production, leading to EC activation and adhesion molecule expression on the vessel wall, which in turn promote the adhesion of sickle rbcs and leukocytes to endothelium, leading to vaso-occlusion. This paper demonstrates that the same pathophysiology induces adaptive cytoprotective proteins such as HO-1 in the endothelium that can ameliorate and prevent vaso-occlusion and the accompanying vascular inflammation commonly seen in SCD patients. We propose that it is the balance between these pro-oxidative and antioxidative forces that determines whether or not a vessel becomes occluded. In steady state these forces are likely balanced. However, a pro-oxidative insult such as hypoxia or an infection can easily tip the delicate balance in favor of hemolysis, oxidative stress, inflammation, and vaso-occlusion, causing the vessel wall to adapt by producing more cytoprotective proteins such as HO-1, which put the vasculature back in a balanced steady state. Induction of HO-1 arrives too late in the pathology of crises, and perhaps those patients that do not have significant clinical manifestations of the disease are those that have a greater homeostatic HO-1 response.

HO-1 degrades heme, which not only removes a major catalytic source of ROS and oxidative stress but also at the same time produces CO and biliverdin, which have their own antiinflammatory effects. CO gas is produced by HO-1–mediated opening of the heme ring. CO is a colorless, odorless gas that has traditionally been considered a dangerous poison. This toxicity is in part due to its high affinity for Hb (245 times greater than that of O2), which alters O2 transport and delivery. CO also can interact with other heme proteins as discussed below. At low concentrations CO can be therapeutic. CO mimics many of the protective effects of HO-1, as well as some of the functions of NO (22, 24). Like NO, CO activates the heme protein guanylate cyclase and inhibits platelet activation and aggregation (22, 24). CO participates in the regulation of vascular tone in hepatic sinusoidal cells, suggesting that NO and CO share control of these relaxation processes (49). Exogenous inhaled CO, at approximately 250 ppm, and in some studies as low as 10 ppm (50), reduces inflammatory responses in several models of oxidant injury in ways similar to those of HO-1 overexpression (22). Also, CO liberated by CO-releasing molecules significantly suppresses the inflammatory response elicited by LPS in cultured macrophages (51). Like NO (52), CO interacts with signal transduction pathways, inhibits proinflammatory genes, and augments antiinflammatory cytokines (22, 24, 53, 54). Specifically, it selectively activates p38 MAPK signaling pathways in a guanylate cyclase–independent manner (22, 53). CO also inhibits proliferation of VSMCs and has antiapoptotic effects on cells (22, 24).

As noted above, CO seems to play a role similar to that of NO. NO has taken an important role in the pathogenesis and therapy of SCD (34, 55, 56). SCD patients have a reduction in basal and stimulated NO production and bioavailability. The deficiency of NO is in part due to elevated plasma Hb and excessive ROS production in both SCD patients and transgenic sickle mice (34). Therapeutic CO may be more effective, because the efficacy of NO is hampered by its ability to form reactive nitrogen species. Under oxidative conditions, NO reacts with ROS, resulting in the formation of highly reactive ONOO– (peroxynitrite) (57). Peroxynitrite does not prevent or ameliorate disease like NO does but, in contrast, exacerbates oxidative inflammatory stress (58, 59). Unlike NO, CO does not contain any unpaired free electrons and is, therefore, relatively inert.

Studies over 30 years ago by Beutler (60) suggested that CO binding to hemoglobin S shifts the oxygen dissociation curve to the left and can actually inhibit hemoglobin S deoxygenation, hemoglobin S polymerization, and rbc hemolysis. Less hemolysis means less plasma Hb, less oxidative stress, less vascular inflammation, enhanced bioavailability of NO, and ultimately less vaso-occlusion. NO and peroxynitrite can induce HO-1 activity and thus form a feedback loop, where CO takes over NO functions under conditions of oxidative stress (21). In our studies, inhaled CO was able to inhibit vaso-occlusion in the skin and NF-κB activation in the lungs, suggesting a potential therapeutic benefit at low doses in SCD patients.

Another product of heme metabolism by HO-1, as noted above, is biliverdin. In mammals, biliverdin is rapidly converted by biliverdin reductase to bilirubin. Both biliverdin and bilirubin are antioxidants with probable physiological relevance in plasma and the extravascular space (61). Exogenous bilirubin has been demonstrated to be cytoprotective in models of ischemia/reperfusion injury (22, 24). At micromolar concentrations, both biliverdin and bilirubin efficiently scavenge peroxyradicals and thereby inhibit lipid peroxidation (62, 63). Biliverdin and bilirubin also may counteract intracellular nitrosative stress reactions (64, 65). In our studies, biliverdin injected i.p. was able to inhibit vascular stasis in the skin and NF-κB activation in the lungs similarly to inhaled CO, suggesting that biliverdin also may have therapeutic benefits in SCD. Biliverdin reductase might also be involved in transcriptional regulation of HO-1 (66).

Some of HO-1’s protective effects in sickle mice were likely due to ferritin induction. In our initial studies, the induction of HO-1 was accompanied by the induction of apoferritin (18). Apoferritin, made up of heavy and light chains, protects cells by its capacity to bind 4,500 iron molecules and through its heavy chain ferroxidase activity (23, 67, 68). Ferritin can store nonreactive Fe3+ in the core of the ferritin complex. This handling of catalytic Fe2+ is essential for ferritin’s ability to protect cells against oxidative stress (13, 69, 70) by interrupting Fenton chemistry, exemplified in the Haber-Weiss reaction for hydroxyl radical generation. Animal models that overexpress the H-ferritin chain withstand ischemia/reperfusion injury and oxidative stress without the concomitant increase in HO-1 (71). In vivo models that increased HO-1 by hemin or Hb injections all increased ferritin levels (36, 72, 73). Dissecting the intimate relationship between HO-1, Fe release from heme, and ferritin has depended on the use of HO-1 inhibitors and iron chelators. Additional studies in sickle mice are needed to dissect the relative contributions of HO-1 and ferritin to inhibition of oxidative stress, inflammation, and vaso-occlusion in SCD. We conjecture that ferritin may be an important cytoprotectant in iron-burdened SCD animals and patients.

Other defenses against Hb and heme released into the vasculature include plasma haptoglobin and hemopexin. However, these defenses are likely overwhelmed in SCD, as evidenced by the low haptoglobin concentrations seen in our sickle mice and low plasma hemopexin levels in SCD patients (74–77). Moreover, the heme content and the overexpression of HO-1 in organs of sickle mice suggest that this is indeed the case.

Markers of hemolysis are associated with a clinical subphenotype of pulmonary hypertension, leg ulceration, priapism, and risk of death in SCD patients (78, 79). Hemolysis may drive vasculopathy, but, by inducing HO-1 expression, hemolysis may ultimately limit vaso-occlusion. Perhaps HO-1 induction by hemolysis may explain the protection against atherosclerosis in SCD patients. Ironically, we used hemin, a “bad actor” in SCD, to therapeutically increase HO-1 and inhibit vascular inflammation and vaso-occlusion. Hematin therapy could be an approach in SCD patients. However, before HO-1 induction, hematin could potentially be pro-oxidative and also contribute to iron overload. Future therapeutic studies in our laboratory will focus on pharmacological therapies such as statins to increase HO-1 expression in SCD. Two recent papers demonstrated that some of the vasoprotective effects of statins, drugs used to decrease cholesterol and vascular inflammation in atherosclerosis, may be due to their ability to induce HO-1 (80, 81). Alternatively, direct administration of CO or biliverdin might be a therapeutic modality by which to treat or prevent sickle crises. In addition, functional polymorphisms in the HO-1 gene promoter region that control the ability to upregulate HO-1 (82, 83) could potentially explain some of the phenotypic differences in severity seen in SCD patients. These findings suggest that HO-1 and its products are promising new avenues of therapy in SCD.