Heme Oxygenases in Cardiovascular Health and Disease

Abstract

Heme oxygenases are composed of two isozymes, Hmox1 and Hmox2, that catalyze the degradation of heme to carbon monoxide (CO), ferrous iron, and biliverdin, the latter of which is subsequently converted to bilirubin. While initially considered to be waste products, CO and biliverdin/bilirubin have been shown over the last 20 years to modulate key cellular processes, such as inflammation, cell proliferation, and apoptosis, as well as antioxidant defense. This shift in paradigm has led to the importance of heme oxygenases and their products in cell physiology now being well accepted. The identification of the two human cases thus far of heme oxygenase deficiency and the generation of mice deficient in Hmox1 or Hmox2 have reiterated a role for these enzymes in both normal cell function and disease pathogenesis, especially in the context of cardiovascular disease. This review covers the current knowledge on the function of both Hmox1 and Hmox2 at both a cellular and tissue level in the cardiovascular system. Initially, the roles of heme oxygenases in vascular health and the regulation of processes central to vascular diseases are outlined, followed by an evaluation of the role(s) of Hmox1 and Hmox2 in various diseases such as atherosclerosis, intimal hyperplasia, myocardial infarction, and angiogenesis. Finally, the therapeutic potential of heme oxygenases and their products are examined in a cardiovascular disease context, with a focus on how the knowledge we have gained on these enzymes may be capitalized in future clinical studies.

FIGURE 1.

Schematic of the heme oxygenase reaction pathway. Heme oxygenases degrade heme b to sequentially yield carbon monoxide (CO), ferrous iron (Fe²⁺), and biliverdin IXα with the reaction requiring 3 mol of molecular oxygen and 7 electrons. In mammals, bilirubin IXα is subsequently reduced to bilirubin IXα by an NADPH-dependent biliverdin reductase.

FIGURE 2.

Signaling pathways and transcription factors modulating HMOX1 expression via different response elements (RE). Various extra- and intracellular stimuli activate the mitogen-activated protein kinases (MAPK), protein kinase C (PKC), 5'-AMP-activated protein kinase (AMPK), and phosphoinositide 3-kinase/RAC-alpha serine/threonine-protein kinase (PI3K/Akt) signaling pathways, leading to Hmox1 activation through the transcription factor Nrf2. Oxidative stress, including reactive nitrogen species (168, 373), also induce HMOX1 expression at least in part via Nrf2. Other transcription factors, including activator protein-1 (AP-1), signal transducer and activator of transcription 3 (STAT3), Yin Yang 1 (YY1), and hypoxia inducible factor-1α (HIF-1α) induce HMOX1 expression through various stimuli such as oxidative stress, hypoxia, heme, and interkeukin-6 (IL-6). Bach1 is a repressor of HMOX1 expression.

FIGURE 3.

Cross-section of a hematoxylin and eosin-stained section of the left carotid artery from a Sprague-Dawley rat. The artery is lined by a single layer of endothelial cells (also referred to as the tunica intima) sitting on top of the internal elastic lamina. Following this is the tunica media composed of VSMC and elastic tissue. The final layer is the tunica adventitia that is comprised mainly of fibroblasts and connective tissue and that is supported by the external elastic lamina.

FIGURE 4.

Hmox1 expression impacts on cardiomyocytes, vascular cells, and stem cells in a cell-type specific, in part opposing manner. In EC, increased Hmox1 expression promotes cell proliferation and decreases apoptosis inflammation and oxidative injury. Conversely, Hmox1 increases apoptosis and decreases the proliferation of VSMC. Hmox1 expression has been reported to increase apoptosis of macrophages and decrease inflammation. In various stem cells, Hmox1 expression has been associated with normal differentiation and protection against oxidative injury, while in cardiomyocytes, Hmox1 expression decreases apoptosis and oxidative stress.

FIGURE 5.

Schematic showing the mechanisms by which CO and bilirubin affect VSMC proliferation. CO has been reported to 1) activate soluble guanylate cyclase (sGC) leading to increased cGMP; 2) decrease cytokine-mediated apoptosis; 3) decrease NADPH oxidase 1 (Nox1) activity; 4) decrease T-type Ca²⁺ channel activity; 5) increase cell cycle progression through the p38 MAPK pathway; and 6) increase caveolin expression. Bilirubin has been reported to decrease cell cycle progression through hypophosphorylation of retinoblastoma protein (Rb) and by increasing intracellular Ca²⁺, leading to a decrease in the transcription factor Yin Yang 1 (YY1) and altered transcription of genes important for VSMC cell cycle control.

FIGURE 6.

Potential pathways involved in hypertension that are modulated by Hmox1 and its products. Increased Hmox1 activity and CO both decrease the activity of nucleus tractus solitarii. CO activates MAPK, leading to inhibition of Jun amino-terminal kinases (JNK) 1/2 with subsequent mitigation of inflammation. CO can also activate sGC, leading to an increase in cGMP. This effect along with activation of high-conductance calcium-activated K⁺ channels (K-ca) leads to vasodilation. Additionally, CO inhibits cyclooxygenase-1 (COX) 1/2 and cytochrome P-450 (CYP-4) with subsequent reduction in thromboxane (TXA₂) and 20-hydroxyeicosatetraenoic acid (20-HETE), respectively, both of which are vasoconstrictors and have sodium retention properties. Angiotensin II (ANG II)-mediated increase in superoxide generation with ensuing increased sodium reabsorption and renal vascular resistance is also known to be attenuated by HMOX1 induction and generation of CO and bilirubin. The activity of the Na⁺-K⁺-Cl⁻ cotransporter (NKCC) in the thick ascending loop of Henle is also directly modulated by Hmox1 activity, leading to a decrease in Na⁺ absorption.

FIGURE 7.

Interaction of stromal derived factor-1 (SDF-1), Hmox1, and endothelial nitric oxide synthase (eNOS) in response to vascular injury. Vascular injury, e.g., wounding or ischemia (110), results in the release of SDF-1 and subsequent induction of the phosphatidylinositol-4,5-bisphosphate 3-kinase/RAC-alpha serine/threonine-protein kinase/eNOS (PI3K/Akt/eNOS) pathway in bone marrow stromal cells. NO and Hmox1 (309, 598, 601) are critical for the mobilization of endothelial progenitor cells (EPC) from the bone marrow to the circulation. In circulating EPC, Hmox1 and eNOS release CO and NO, respectively, which induce phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and its redistribution to the leading edge of the cells (282). This promotes migration and vascular repair. In addition, there is also evidence suggesting that Hmox1 regulates SDF-1 expression (309, 601).

FIGURE 8.

Potential modulatory roles of Hmox1 in atherogenesis. Hmox1 expression in macrophages, foam cells, EC, and VSMC has been associated with decreases in EC injury; EC dysfunction; expression of the adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1); release of the inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1), IL-1β, TNF-α, and IL-6; and decrease in oxidative damage. In addition, increased Hmox1 expression has been reported to increase the stability of atherosclerotic lesions.

FIGURE 9.

Probucol (PB) inhibits injury-induced intimal hyperplasia, and this is prevented by Yin Yang 1 (YY-1) knockdown. Carotid arteries obtained from Zucker rats fed normal food without (Ctrl) or with 1% PB for 14 days before and 14 days after balloon injury and receiving YY1 siRNA or YY-1 scrambled RNA (scRNA) immediately before injury. Injury was assessed by intima-to-media- ratio. [From Beck et al. (28).]

FIGURE 10.

Promoter polymorphisms in HMOX1 and their clinical significance. Two single nucleotide polymorphisms (SNP) have been identified in the HMOX1 promoter, G(-1135)A, and T(-413)A. Their clinical significance remains inconclusive, as a result of limited and inconsistent data. The more extensively studied association of the dinucleotide length polymorphism (GT)_n with various cardiovascular diseases has been reviewed in two recent meta-analyses. The results suggest that this polymorphism may have clinical implications in some populations. Superscripts refer to the following references: ^a403, ^b402, ^c323, ^d432, and ^e103.

FIGURE 11.

Multipotent mesenchymal stem cells (MSC) derived from Hmox1^−/− mice have lower angiogenic potential than MSC from wild-type (Hmox1^+/+) animals. An equal number of cells were placed on growth factor-reduced Matrigel and cultured in endothelial basal medium-2 for 9 days. The figure shows representative images of 3 separate experiments. Bar = 100 μm. [From Zarjou et al. (631).]

FIGURE 12.

Potential therapeutic modulation of Hmox1 and its products. Upstream of its enzymatic activity, Hmox1 may be modulated via transcription factors and gene therapy to increase or decrease expression of Hmox1 at the mRNA and protein level employing microRNA (miRNA), short interfering RNA (siRNA), and/or short hairpin RNA (shRNA). Pharmacological inhibitors or inducers may be used to modulate Hmox1 enzymatic activity. CO (via inhalation or via CORMs), biliverdin, and bilirubin may also be used therapeutically.