Redox Regulation of Ischemic Angiogenesis

Abstract

Antioxidants are expected to improve cardiovascular disease (CVD) by eliminating oxidative stress, but clinical trials have not shown promising results in chronic CVD. Animal studies revealed that reactive oxygen species (ROS) exacerbate acute CVDs in which high levels of ROS are observed. However, ROS are also necessary for angiogenesis after ischemia, because ROS not only damage cells but also stimulate cell signaling required for angiogenesis. ROS affect signaling by protein modifications, especially on cysteine amino acid thiols. Although there are several cysteine modifications, S-glutathionylation (GSH adducts; -SSG), a reversible cysteine modification by glutathione (GSH), plays an important role in angiogenic signal transduction by ROS. Glutaredoxin-1 (Glrx) is an enzyme that specifically removes GSH adducts in vivo. Overexpression of Glrx inhibits, while deletion of Glrx improves revascularization after mouse hind limb ischemia. These studies indicate that increased levels of GSH adducts in ischemic muscle is beneficial in promoting angiogenesis. The underlying mechanism can be explained by multiple targets of S-gluathionylation which mediate angiogenic effects in ischemia. Increments in the master angiogenic transcriptional factor, HIF-1α reduction of the anti-angiogenic factor sFlt1, activation of endoplasmic reticulum Ca²⁺ pump, SERCA, and inhibition of phosphatases may occur as a consequence of enhanced S-glutathionylation in ischemic tissue. In summary, inducing S-glutathionylation by inhibiting Glrx may be a therapeutic strategy to improve ischemic angiogenesis in CVD.

Introduction

Reactive oxygen species (ROS) are generated mainly as result of cellular respiration, and the level of ROS is maintained at low levels by antioxidant molecules and enzymes¹. It’s well known that ROS are increased in cardiovascular diseases (CVD), such as myocardial infarction, stroke, heart failure, atherosclerosis and peripheral arterial disease². Increased ROS are considered a consequence of CVD particularly as they are produced by inflammatory leukocytes, and in addition may exacerbate disease because of deleterious cellular effects caused by “oxidative stress”³. Therefore, eliminating ROS would seem to be a promising strategy for treatment of CVD, particularly based on animal studies. However, the results of clinical trials have been far below this expectation⁴. ROS are now recognized not only as cytotoxic molecules but also as cell signaling mediators⁵. Little is known about effects of ROS related to signaling in CVD. However, animal studies showed that ROS contribute to improvement of CVD, including angiogenesis following ischemia^{6, 7}. In this review, we summarize the results of antioxidants in clinical trials and mouse CVD models, the mechanism by which ROS regulate cell signaling, and discuss how ROS influence angiogenesis. We especially focus on ROS induced signaling by S-glutathionylation or GSH protein adducts, which are a relatively stable oxidative post-translational protein modification.

Effects of antioxidants on human and mouse CVD

Levels of ROS are increased in almost every CVD. For example, ischemia reperfusion associated with myocardial infarction or stroke causes an ROS burst in mitochondria⁸. Angiotensin II, and cytokines which are up-regulated in heart failure increase ROS from NADPH oxidase⁹. Macrophage-derived foam cells release ROS in atherosclerotic plaques¹⁰. In addition, increments in ROS exacerbate CVD. For example, mouse studies showed that deletion of antioxidant enzymes including superoxide dismutase (SODs)^{11, 12} and glutamate-cysteine ligase modifier subunit (Gclm)¹³ exacerbates ischemic reperfusion injury. SOD1 or SOD2 knock out mice also experience worsened stroke^{14, 15}, and mice with deficient SOD2¹⁶ or Gclm¹⁷ have impaired cardiac function in heart failure. Furthermore, genetic polymorphisms in antioxidant enzymes, such as SOD2¹⁸ and Gclm^{17, 19, 20}, are also a risk factor for CVD. Therefore, one would expect that treating CVD with antioxidants might be a good strategy. Actually, overexpression of antioxidant enzymes such as SODs or glutathione peroxidase (Gpx) as well as treatment with the free radical scavengers, edaravone or tempol, improves animal models of myocardial infarction^{21, 22}, ischemic reperfusion^23–27 and stroke^{28, 29} (Table 1). In addition, clinical studies showed that edaravone, which has been clinically used for patients in Japan, is effective in myocardial infarction³⁰ and stroke³¹. In contrast to acute CVD, the effect of antioxidant therapeutics in chronic CVD has been limited. In mice, the antioxidant effects of probucol improved heart failure³², but clinical trials of antioxidant results in heart failure have been controversial^{33, 34} (Table 1). Several large clinical trials of antioxidants in the prevention of CVD were negative. These studies revealed that antioxidant vitamins C, E, and β-carotene, alone or in combination did not reduce risk of CVD⁴. It is not clear why these studies ended with disappointing results. One possibility is that antioxidants have not proven universally beneficial in CVD in animal models. For example, in hind limb ischemia models, overexpression of catalase⁷ or treatment with tempol⁶ impaired blood flow recovery. In spite of many studies that showed beneficial effects of antioxidants on myocardial infarction, the antioxidant N-acetyl cysteine decreased collateral growth after myocardial infarction³⁵. In addition, the cardio-protective effects of ischemic pre-conditioning are eliminated by antioxidants³⁶. Taken together, antioxidant therapeutics may be effective in acute CVD like myocardial infarction and stroke which are associated with high levels of ROS, but not in chronic CVDs. High levels of ROS may damage lipids and DNA by oxidation, result in cell death or dysfunction, and exacerbate CVD (Figure 1A). On the other hand, physiological levels of ROS can stimulate signaling by modifying protein function, but their role in CVD remains controversial. At least ROS are likely important for angiogenic responses after ischemia because not only do antioxidants impair angiogenesis, but also overexpression of Nox4³⁷, which produces hydrogen peroxide, improves angiogenesis after hind limb ischemia (Figure 1B). Thus, ROS-induced signaling, which is mediated by oxidative protein modifications, is worth considering in angiogenesis.

Table 1.

(A) Effect of Antioxidants or Antioxidant Enzymes in Animal Models of CVD, (B) Clinical Trials of Antioxidants in CVD

	CVD	Exacerbate	Improve
A
	MI	NAC (collateral growth)35	Gpx overexpression,21 edaravone22
	Ischemic reperfusion		SOD1, 23 224 and Gpx25 overexpression, edaravone,26 tempol27
	Stroke		SOD1 overexpression,28 edaravone29
	HF		Probucol32
	Hindlimb ischemia	Catalase overexpression,7 tempol6
	Ischemic preconditioning	SOD, N-2-mercaptopropionyl glycine36
	CVD	Ineffective	Improve
B
	MI		Edaravone30
	Stroke		Edaravone31
	HF	Vitamin E33	NAC (renal failure)34
	Prevention of CVD	Vitamins C and E, β-carotine4

CVD, cardiovascular disease; HF, heart failure; MI, myocardial infarction; NAC, N-acetyl cysteine.

Figure 1.

Effects of reactive oxygen species (ROS) in cardiovascular disease (CVD). (A) Effects on molecules in CVD. Oxidation of lipids and DNA results in cell death or dysfunction and might exacerbate CVD. On the other hand, oxidative protein modification can change protein function and its effects on CVD remain unclear. (B) Effects of ROS and antioxidants on ischemic CVD. Effects of ROS on cells depend on their levels. High levels of ROS induce cell death and increase necrosis; therefore, antioxidants can decrease infarct area in myocardial infarction or stroke. However, intermediate levels of ROS can induce angiogenic signaling. Therefore, antioxidants may impair angiogenesis after ischemia.

Oxidative post-translational modification of cysteine residues

Protein oxidation results from various oxidations of amino acid residues. For example, all kinds of amino acid residues can be carbonylated and this modification is well known as a marker for protein oxidation³⁸. However, compared with other amino acid oxidative modifications, many studies have shown abundant cysteine modifications by ROS. Cysteine has oxidant-sensitive thiols (-SH groups) which can be modified all kinds of ROS³⁹. Approximately 10 percent of protein thiols exist as thiolate anions (-S^-) because of local electrostatic forces, making them much more reactive than the uncharged thiol. These thiols in particular are easily oxidized by ROS to form sulfenic acid (Cys-SOH) (Figure 2 A). Because of instability of sulfenic acid, sulfinic acid (Cys-SO₂H) (Figure 2 B) is formed by additional oxidation. Further oxidation induces sulfonic acid (Cys-SO₃H) (Figure 2 C). Sulfinic acid and sulfonic acid are biologically irreversible oxidative modifications, but sulfenic acid is a reducible modification or can react to form other protein modifications. Intra-protein (Figure 2 D) or inter-protein (Figure 2 E) disulfide bonds may form between sulfenic acid groups. Disulfide bond formation also occurs between sulfenic acid and the most abundant cytosolic small molecule thiol, glutathione (GSH)(Figure 2 F), producing S-glutathionylation. Glutathione, a tri-peptide which is composed of glutamine, cysteine and glycine, can be oxidized to form the homodimer, GSSG⁴⁰. When levels of GSSG are increased under oxidant stress conditions, it can form S-glutathione adducts by thiol exchange (Figure 2 H). Nitric oxide (·NO), a free radical, mediates its bioactivity largely by modifying protein thiols (-SNO, S-nitrosylation)⁴¹ (Figure 2 G). S-nitrosylation is chemically unstable and may further react with GSH to form S-glutathionylation (Figure 2 I). GSH may be oxidized by ·NO to form S-nitrosoglutathione (GSNO), which is relatively stable compared with ·NO and also stimulates S-nitrosylation of proteins (Figure 2 G) as well S-glutathionylation (Figure 2 H)⁴². All these modifications are reported to affect protein function or signal transduction. For example, intra-protein disulfide bond formation is an important part of thioredoxin function⁴³, and inter-protein disulfide bond formation regulates ASK1 activation⁴⁴. These protein modifications are site-specific reactions, because the reaction between two intra- or inter-protein cysteine thiols completely depends on protein structure. Sulfenic, sulfinic, and sulfonic acids, S-nitrosylation and S-glutathionylation also depend on protein structure as it pertains to reactivity of cysteine residues. For example, only 1 of 14 cysteine thiols on the protein surface of SERCA exists as a thiolate anion at physiological pH, and therefore modification of its thiol represents more than 90% of the glutathione adducts as demonstrated by mutating the one cysteine to serine⁴⁵. In addition, modification of only specific cysteine residues in specific protein results in functional changes, and it is likely that protein structure has evolved to increase the reactivity of thiols that can influence the function of a particular protein in an advantageous manner. Sulfinic and sulfonic acids being irreversible modifications cause protein dysfunction and degradation, whereas reversible modifications (S-sulfenylation, S-nitrosylation and S-glutathionylation) are considered to play an important role to induce ROS-related signaling. Sulfinic, sulfonic, and glutathione modifications add a negative charge to the cysteine residue, so can introduce an additional affect on protein function. S-nitrosylation (-SNO) and sulfenic acid (-SOH) react with intracellular GSH to form the much more stable S-glutathionylation (-SSG)⁴⁶. S-glutathionylation was greatly increased in ischemic muscles for at least several days after hind limb ischemia ⁴⁷, but whether other modifications occur physiologically under the same conditions is unknown. Therefore, particularly as pertains to more chronic influences of ROS, S-glutathionylation might play an important role in angiogenesis after ischemia.

Figure 2.

[A–I] Oxidative cysteine modification. Cysteine residues in proteins can be modified by oxidation and the negative charge may change protein function. GHS, glutathione; GSNO, S-nitrosoglutathione; S-GSH, S-glutathionylation; SH, protein thiol of cysteine; S-S, disulfide band; SOH, sulfenic acid; SO₂H, sulfinic acid; SO₃H, sulfonic acid; SNO, S-nitrosylation.

Glutaredoxin and its role in ischemic revascularization

Removal of GSH protein adducts is catalyzed by a small cytosolic enzyme, glutaredoxin-1 (Glrx). Glrx, also called thioltransferase, specifically catalyzes the removal of GSH adducts from S-glutathionylated proteins using one of its own thiols on cysteine 22 or 25⁴⁸. After GSH is removed from a protein, Glrx requires GSH to be returned to an active reduced form again. The GSH which is oxidized to GSSG in the process, needs to be reduced by glutathione reductase which requires NADPH (Figure 3A). Thus, Glrx activity depends on GSH, glutathione reductase, and NADPH levels in cells. Glrx generates dynamic redox signaling by removing GSH protein adducts and thus may control pathophysiological phenotypes. Up-regulation of Glrx inhibits VEGF-induced endothelial cell migration⁴⁹ and activates NF-κB signaling⁴⁷. Deletion of endogenous Glrx in mice attenuates cardiovascular hypertrophy induced by angiotensin II in association with lower ROS production in the aorta⁵⁰. Therefore, the role of Glrx in controlling GSH adducts goes beyond antioxidant effects. Glrx expression is increased in atherosclerotic human coronary artery⁵¹ and diabetic rat retina⁵². Glrx itself is induced by NF-kB activation⁵³ and is therefore up-regulated in association with inflammation. However, the pathophysiological role of Glrx in vivo still needs to be explored.

Figure 3.

Glrx and its effect on hind limb ischemia. (A) Removal of S-glutathionylation by Glrx and its recycle by GSH. (B) Laser Doppler measurement of blood flow after hind limb ischemia in Glrx TG mice. (C) Analysis of blood flow after hind limb ischemia in Glrx KO mice. *P<0.05.

Just as anti-angiogenic effects are shown by Glrx overexpression in vitro⁴⁹, Glrx overexpressing transgenic (TG) mice showed poor blood flow recovery in hindlimb ischemia compared to control wild type mice⁴⁷ (Figure 3B). The impaired blood flow was associated with poor motor function and lower capillary density in muscle reflecting poorer angiogenesis. In contrast, deletion of Glrx in knockout (KO) mice improves revascularization after hindlimb ischemia⁵⁴ (Figure 3C). Therefore, these data indicate that endogenous Glrx has an anti-angiogenic role. In other words, oxidant-induced S-glutathionylation (GSH adducts) on protein(s) in ischemic muscles facilitate angiogenic responses and promote ischemic revascularization. There is a report indicating that Glrx overexpression enhances neovascularization in a mouse myocardial infarction model⁵⁵. However, they evaluated vascularization only by α-smooth muscle actin staining in hearts, and the results are not sufficient to conclude Glrx enhances neovascularization. Also, they showed anti-apoptotic effects in cardiomyocytes which most likely explain the better cardiac function observed in Glrx TG mice.

Role of S-glutathionylation in angiogenesis

Gain and loss of function of Glrx in vivo showed that S-glutathionylation stimulates angiogenic signaling after ischemia (Figure 4). Vascular endothelial growth factor A (VEGFa), the central angiogenic growth factor, plays an important role in angiogenesis and ischemic revascularization. S-glutathionylation regulates VEGFa production and signaling through effects on multiple proteins. First, S-glutathionylation of Cys520 of HIF1α stabilizes the protein and increases VEGFa expression⁵⁴. Second, S-glutathionylation of the NF-κB components p65⁴⁷, p50⁵⁶, and Inhibitor of kappa B kinase (IKK) β⁵⁷ inhibits NF-κB activation. Soluble Flt (sFlt)-1, which binds to VEGFa as a decoy receptor and inhibits VEGFa signaling, is regulated by NF-κB and is anti-angiogenic. Decreased S-glutathionylation of p65 caused by Glrx overexpression results in hyper-activation of NF-κB and increased sFlt1⁴⁷, indicating that S-glutathionylation has a role in suppressing anti-angiogenic sFlt1 production. Therefore, S-glutathionylation promotes VEGFa expression and activation of its signaling receptor (VEGFR2) by decreasing this inhibitory factor.

Figure 4.

Mechanism and target proteins of S-glutathionylation mediating angiogenesis pathway. (Upper) Ischemic cells produce VEGF or sFlt1. (Lower) Endothelial cells responding to the growth factor. NF-κB, nuclear factor κ B; VEGF, vascular endothelial growth factor.

In addition, downstream of VEGF receptor activation, S-glutathionylation regulates VEGFa-dependent angiogenic responses in endothelial cells. VEGFa-dependent Ca²⁺ influx is critical for angiogenic responses in endothelial cells. The sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) 2b is activated by S-glutathionylation of Cys674⁴⁵. GSH adducts on Cys674 are required for VEGF-induced increased SERCA activity (ER Ca²⁺ uptake), increased cytosolic Ca²⁺ influx, and endothelial cell migration⁴⁹. In addition, in vivo S-glutathionylation of SERCA2b C674 is essential for normal angiogenesis after hind limb ischemia in mice⁵⁸. These studies indicate that S-glutathionylation of SERCA C674 critically regulates endothelial angiogenic responses.

As another example, phosphatases, such as protein tyrosine phosphatase-1B (PTP1B) and protein phosphatase-A2 (PP2A), normally suppress VEGFa signaling and are inhibited by oxidative protein modifications⁵⁹. GSH adducts on Cys215 of PTP1B inhibit the phosphatase activity⁶⁰. In endothelial cells, PTP1B inhibits tyrosine phosphorylation of the VEGF receptor, VEGFR2⁶¹, and deletion of PTP1B in mice increases angiogenesis after myocardial infarction⁶². Therefore, the inhibition of phosphatases by GSH adducts may enhance tyrosine phosphorylation of VEGFR2, thereby enhancing activity of focal adhesion kinase, a key mediator of endothelial cell migration, and angiogenesis.

In ischemic conditions, various other proteins are S-glutathionylated. It is reported that some angiogenic proteins, such as eNOS⁶³ and sirtuin-1⁶⁴, are inhibited by S-glutathionylation, and therefore might be expected to inhibit their important role in angiogenesis. This apparent contradiction could exist for several reasons, including the possibility that the proteins are protected from S-glutathionylation during ischemia because of their subcellular localization (eg. calveoli and nucleus, respectively) or the effects are masked by other angiogenic factors. However, in vivo studies of Glrx knock out and overexpression indicate that over all enhancement of S-glutathionylation by inhibiting Glrx promotes angiogenenic responses during ischemia.

Summary

ROS are increased in CVD and considered a deleterious consequence of the diseases. However, antioxidant therapeutics in man do not prevent CVD. This might be explained by a beneficial effect of ROS on CVD, especially in ischemic angiogenesis. Although high levels of ROS cause cell damage and death, another aspect of ROS is to directly modify protein thiols and change protein function. S-glutathionylation, a relatively stable oxidative cysteine modification by GSH, increased during hind limb ischemia and apparently stimulates angogenic signaling. Studies of gain and loss of function of Glrx, which specifically removes S-glutathionylation from proteins, reveal that S-glutathionylation, on balance, stimulates angiogenesis after hind limb ischemia. S-glutathionylation increases active VEGFa by stabilizing HIF-1α and inhibiting the anti-angiogenic factor, sFlt1, as well as enhances VEGFa signaling in endothelial cells through inhibition of PTPs and activation of SERCA2. Therefore, S-glutathionylation and its regulator Glrx may be good targets for treatment to improve ischemic angiogenesis in CVD.