Mechanisms of Isolevuglandin-protein Adduct Formation in Inflammation and Hypertension

Abstract

Inflammation has been implicated in the pathogenesis of hypertension and recent evidence suggests that isolevuglandin (IsoLG)-protein adducts play a role. Several hypertensive stimuli contribute to formation of IsoLG-protein adducts including excess dietary salt and catecholamines. The precise intracellular mechanisms by which these hypertensive stimuli lead to IsoLG-protein adduct formation are still not well understood; however, there is now evidence implicating NADPH-oxidase derived reactive oxygen species (ROS) in this process. ROS oxidize arachidonic acid leading to formation of IsoLGs, which non-covalently adduct to lysine residues and alter protein structure and function. Recent studies suggest that these altered proteins act as neo-antigens leading to an autoimmune state that results in hypertension. The goal of this mini-review is to highlight some of the hypertensive stimuli and the mechanisms contributing to IsoLG-protein adduct formation leading to inflammation and hypertension.

INTRODUCTION

Hypertension is a growing health care burden and is the leading cause of mortality due to myocardial infarction, stroke, heart failure, and chronic kidney disease. Recently, the American Heart Association (AHA) and the American College of Cardiology (ACC) developed new classification criteria that put nearly half of the American population in the hypertensive category (1). Unfortunately, the National Health and Nutritional Examination Study (NHANES) found that blood pressure is uncontrolled in approximately one-half of hypertensive patients and despite extensive research, the pathogenesis of essential hypertension remains elusive (2, 3).

In general, alterations in renal and systemic vascular function are required to sustain an elevation in blood pressure (4). In the past decade, it has become apparent that inflammation is important for the initiation and maintenance of these pathophysiologic changes and that both the innate and adaptive immune systems contribute to hypertension. Virtually all cells of the innate and adaptive immune system, including monocytes, dendritic cells (DCs), macrophages, T cells, and B cells have been implicated in the genesis of hypertension (5). Several studies have emphasized a role of CD8⁺ T cells, which are specifically activated by antigenic peptides presented in class 1 major histocompatibility complexes (MHC-I) (6). These studies combined suggest an autoimmune etiology of hypertension. However, the intra and intercellular processes required for the initiation of this autoimmune response are not characterized.

Activation of the immune system in hypertension occurs by multiple potential mechanisms (7). Sympathetic activation of immune cells occurs directly by the binding of catecholamines to adrenergic receptors expressed on immune cells (8). This has been shown to produce the elaboration of pro-inflammatory cytokines IL-1 β and IL-6 (9). Moreover, deletion of anti-oxidant enzymes in the brain enhances T-cell activation (10). Interestingly, elevated extracellular sodium concentrations have been shown to stimulate pro-inflammatory cytokine secretion by T-cells (11, 12). Sodium increases intracellular ROS by activation of NADPH oxidase (13). Finally, mechanical stretch induces the secretion of pro-inflammatory cytokines and the production of intracellular ROS from endothelial cells, leading to activation of inflammatory pathways (14). These studies suggest a central role of ROS in the initiation of inflammation and hypertension.

One consequence of ROS production is arachidonic acid peroxidation leading to formation of IsoLGs (5, 15-17). IsoLGs were first discovered in 1985 by Dr. Salomon who found that they form from non-enzymatic rearrangement of prostaglandin H2 as byproducts of the cyclooxygenase pathway (18). They are also formed as products of the isoprostane pathway by rearrangement of the prostaglandin H2-like endoperoxide intermediates (19). While the cyclooxygenase pathway yields only 2 gamma-ketoaldehydes, the isoprostane pathway yields 64 different IsoLG isomers. IsoLGs are highly labile and form covalent bonds to lysine residues leading to post-translational protein modifications (20) (Figure 1). The identification of the IsoLG-adducted peptides and the mechanism by which they are processed are unknown; however, recent studies have provided insight into their role in autoimmune mediated hypertension. In this review, we summarize findings providing insights into the mechanistic processes and the hypertensive stimuli contributing to IsoLG-protein adduct formation in hypertension.

Figure 1: IsoLG-protein adduct formation.

Isolevuglandins (IsoLGs) are formed from lipid peroxidation of arachidonic acid. They are highly labile and form covalent bonds with lysine residues of nearby proteins leading to formation of IsoLG-protein adducts.

ISOLEVUGLANDINS AND HYPERTENSION

IsoLG-protein adducts as biomarkers of hypertension:

IsoLG-protein adducts have been found to accumulate in inflammatory diseases including asthma and alcoholic liver disease (21, 22). In the past few years, we found that IsoLG-protein adducts are also increased in hypertension. Immunohistochemistry analysis using a single chain antibody that recognizes IsoLG-lysine adducts on any protein independent of the amino acid backbone (23) revealed a 2-fold increase of IsoLG-protein adducts in the heart and aorta of mice with angiotensin II-induced hypertension (15). Using flow cytometry, we found that angiotensin II infusion increases intracellular accumulation of IsoLG-protein-adducts in antigen presenting dendritic cells (DCs) and this was confirmed by mass spectrometry analysis (15). In addition to mice receiving angiotensin II infusion, accumulation of IsoLG-protein adducts has been found in other models of hypertension including deoxycorticosterone acetate (DOCA)–salt, L-Nitroarginine methyl ester (L-NAME)/high salt, as well as in transgenic mice overexpressing smooth muscle specific NADPH oxidase subunit p22^phox (15, 24, 25).

Importantly, IsoLG-protein adducts increase in human hypertension. We found that patients with resistant hypertension had increased levels of plasma F₂-isoprostanes compared to well-controlled and normotensive subjects (23.2 ± 11.5 vs. 18.3 ± 10.3 vs. 11.6 ± 7.0 pg/mL, p<0.05 respectively; adjusted for BMI, HbA1C, LDL, and statin use. IsoLGs and F2-isoprostanes are co-produced in the isoprostane pathway of lipid peroxidation (17, 19, 26). We also found that the IsoLG-protein adduct content in monocytes of humans with hypertension was 3-fold greater than those without hypertension (15). In addition, we observed a similar increase in IsoLG-protein adducts in a small population (< 0.5%) of circulating CD83⁺ which were CD19⁺ B cells (15). These studies suggest that IsoLGs are increased in hypertension and may serve as biomarkers of this disease.

IsoLG-protein adducts as mediators of hypertension:

There is compelling evidence that IsoLG-protein adducts mediate inflammation and contribute to hypertension. As mentioned above, IsoLGs once formed react with lysine residues on proteins forming lactam adducts and cross-linked proteins. Zagol-Ikapitte and Amarnath et al. have synthesized several compounds that scavenge IsoLGs in vitro at a rate more than 2 orders of magnitude greater than the potency of the reaction with the ε-amine of lysine (27, 28). One such compound is 2-hydroxybenzylamine (2-HOBA), and we have demonstrated that it prevents tissue accumulation of IsoLG-protein adducts in hypertension (15). We found that pre-treatment with 2-HOBA and several other IsoLG scavengers including 5-methyl-2-hydroxybenzylamine (5-Me-2-HOBA) and pentylpyridoxamine (PnPM) prevent hypertension with no apparent toxicity, while related compounds that exhibit low reactivity with IsoLGs such as N-methyl-2-HOBA (N-Me-2-HOBA), and 4-HOBA do not (15, 27). Importantly, the IsoLG scavengers demonstrated slow rate constants for reactions with malondialdehyde (MDA), another lipid modification, and these rates were unrelated to their ability to lower blood pressure (15). Of note, the effects of IsoLG scavengers on preventing hypertension cannot be attributed to inhibition of cyclooxygenase enzymes since Zagol-Ikapitte and Amarnath et al. demonstrated that none of these compounds inhibit cyclooxygenase enzymes (27). While these studies suggest that IsoLGs mediate hypertension and provide a potential therapeutic target, no studies demonstrating a lowering of already established hypertension have been done. This is especially important since it would mimic the clinical setting. In addition, studies describing the effects of infused IsoLGs on blood pressure have not been performed.

Mechanisms of IsoLG-mediated immune activation and hypertension:

Many potential mechanisms may underlie the role of IsoLG induced inflammation and hypertension. Specifically, IsoLGs might induce inflammation and hypertension, however, to date very few studies have been done. As mentioned above, IsoLGs are extremely reactive and covalently adduct to protein lysine residues which undergo further reactions to form extensive protein-protein and DNA cross-links (20, 29). The cellular fate of these cross-linked proteins is not clearly understood, however, Davies et al. have found that they are poorly degraded by the proteosome depending on the number of adducts that are present on the protein, and can inhibit the ability of the proteosome to degrade normal protein substrates (30). Proteosome inhibition induces NF-κB activation, which has been implicated in the pathogenesis of hypertension (31, 32). Thus, IsoLG-crosslinked proteins, could potentially promote an NF-κB-mediated inflammatory response leading to hypertension.

In 2014, we published a new mechanism underlying immune activation in hypertension mediated by IsoLGs. We found that hypertension causes a striking increase in formation of IsoLG-modified proteins in DCs which correlated with an increase in surface expression of B7 ligands CD80 and CD86 (15) (Figure 2). These B7 ligands interact with co-stimulatory molecules on T cells including CD28 and were previously shown to be critical for hypertension (33). This co-stimulatory signal, together with antigen presentation and cytokine production are required in the classical T cell activation pathway. We found that DCs that accumulate IsoLG-protein adducts produce pro-inflammatory cytokines IL-1β, IL-6 and IL-23 and induce T cell proliferation and production of TNF-α, IFN-γ and IL-17A which contribute to hypertension (5, 15, 16). Importantly, adoptive transfer of DCs from hypertensive mice prime hypertension to sub-pressor doses of angiotensin II, and this can be prevented if the donor mice are treated with the IsoLG scavenger 2-hydroxybenzylamine (2-HOBA) (15). Thus, IsoLG-adducted proteins may be presented by DCs as neoantigens that activate T cells. Activated T cells infiltrate the kidney and vasculature and release cytokines that promote vascular dysfunction and sodium/water retention, leading to hypertension (5).

Figure 2: Hypertensive stimuli leading to immune cell activation via IsoLG-protein adduct formation.

Hypertensive stimuli including angiotensin II, sympathetic outflow and excess dietary salt lead to increased NADPH oxidase-dependent production of reactive oxygen species (ROS) by antigen presenting myeloid derived cells. This leads to oxidation of arachidonic acid and formation of IsoLG-protein adducts. These are immunogenic and activate antigen presenting cells to produce inflammatory cytokines including IL 1b, IL6 and IL23. These myeloid cells activate T cell to proliferate and produce inflammatory cytokines including IL-17A, TNF-α and IFN-γ which lead to hypertension.

Numerous studies have found that oxidation leads to protein immunogenicity via mechanisms that are not well defined. While ROS production is increased in other cell types including vascular smooth muscle and endothelial cells, previous studies indicate that in the absence of T cells or the cytokine IL-17, hypertensive stimuli have minimal effects on vascular ROS production in vivo (5). This suggests that the adaptive immune system plays a very important role in signaling ROS production in these cells. Thus, it is conceivable that superoxide production in DCs might be causative in promoting neoantigen formation and ultimately T cell activation, which then leads to ROS production in vascular cells. Mice lacking either Nox2, or p22^phox in DCs are protected against development of hypertension and IsoLG-protein adducts, suggesting that the NADPH oxidase is a major source of ROS that ultimately lead to the formation of IsoLGs (10, 15, 34). We found that ex vivo treatment of DCs with 1mM tert-butyl hydroperoxide (t-BHP) for 30 minutes led to IsoLG formation and surface expression of CD86 in DCs. T-BHP treated DCs promoted T cell proliferation and this was prevented by co-treatment with 2-HOBA. Adoptive transfer of t-BHP treated DCs augmented blood pressure elevation in response to low dose angiotensin II in recipient mice (15). These results confirm the concept that oxidative stress and IsoLG formation in DCs is sufficient to activate T cells and promote hypertension.

In addition to processing and presentation of intracellular IsoLG-protein adducts formed in response to increase oxidative stress to CD8⁺ T cells, we find that DCs likely process extracellular antigens and present them CD4⁺ T cells. Wu et al. found that DCs pulsed with aortic proteins from mice with vascular specific overexpression of the NADPH oxidase subunit p22^phox had increased production of cytokines. Specifically, augmented levels of IL-1β, IL-6, TGF-β1, and GM-CSF were observed as were activated T cells of syngeneic mice (35). In addition, we found that exposing DCs to IsoLG-modified renal homogenates enhances their ability to drive CD8⁺ proliferation, likely through cross-presentation (15). Examination of T cell receptor usage revealed that an oligo-clonal population of CD8+ T cells in the kidney contributes to hypertension (36).

Another potential mechanism by which IsoLGs may contribute to inflammation in hypertension is through danger-associated molecular patterns (DAMPs) due to their injurious effect on forming protein-protein and DNA cross-links (20, 29). Antigen presenting cells recognize DAMPS via pattern recognition receptors and these have been implicated in hypertension and vascular dysfunction (37-41). However, the specific role of IsoLGs acting as DAMPS in hypertension remains to be elucidated.

In addition, IsoLG-adducted and crosslinked proteins could break immune tolerance and elicit an autoantibody response to normally non-immunogenic self-antigens. Previous studies have found that natural IgM antibodies are produced against lipid oxidation-specific epitopes which are thought to play an important protective role in preventing inflammatory reactions induced by the oxidatively modified lipids that they recognize (42, 43). These oxidation-specific epitopes are important targets of innate immunity in cardiovascular disease (42, 44). It is not known whether antibodies against IsoLGs contribute to hypertension.

Role of Angiotensin II in Promoting Isolevuglandin Formation:

The renin-angiotensin system plays a physiological role in controlling blood pressure and its dysregulation contributes to the development of hypertension (45). Angiotensin II, a key molecule in the renin-angiotensin system, is used as a hypertensive stimulus to induce experimental hypertension. It raises blood pressure through various mechanisms including vasoconstriction and secretion of aldosterone (45). Angiotensin II further acts on the kidneys to promote fluid retention/sodium reabsorption and on the brain to increase sympathetic out flow, fluid intake, and sodium appetite (46). In addition, recent studies have found that angiotensin II regulates the immune system (5). We recently found that angiotensin II-induced hypertension leads to production of NADPH oxidase-dependent superoxide in DCs resulting in increased lipid peroxidation IsoLG-protein adduct formation (15). Interestingly, we found that angiotensin II had a minimum effect on inducing IsoLG-protein adduct formation in DCs in vitro. These studies suggest that in vivo, angiotensin II may exert its effects on DCs indirectly, through other mechanism including augmentation of sympathetic outflow and increased sodium retention.

Renal Sympathetic Mechanisms and IsoLG-protein Adduct Formation in Hypertension:

In a recent study, we found that the kidney is a likely site for activation of DCs in hypertension. We discovered that hypertension induces formation of IsoLG-modified proteins in DCs, and that renal denervation prevents this (47). Moreover, DCs from mice that had undergone renal denervation could not confer hypertension to recipients (47). These data indicate that renal sympathetic nerves serve as a link between activation of the central nervous system and peripheral inflammation in hypertension.

There are two potential mechanisms that could lead to IsoLG-protein adduct accumulation in DCs. First, it is possible that DCs might themselves be activated by sympathetic nerves, which in turn could drive NADPH oxidase within DCs. We have tested the effect of several neurotransmitters on IsoLG-protein adduct formation in DCs and have found that norepinephrine, a major neural transmitter of efferent sympathetic nerves, dose dependently increases IsoLG-protein adduct formation in DCs in vitro. Further studies are needed to determine the specific adrenergic receptor involved in IsoLG-protein adduct formation in DCs. Other neurotransmitters released from sympathetic nerves, such as neuropeptide Y and substance P, have no effect on IsoLG-protein adduct formation in DCs (47).

The second mechanism by which the nervous system may induce IsoLG-protein adduct accumulation in DCs is by its effects on other cell types. The sympathetic nervous system may in fact activate NADPH oxidase in diverse cell types. Importantly, we have evidence that renal sympathetic nerves induce high levels of superoxide production in endothelial cells in the kidney (47). DCs intimately interact with endothelial cells, and in fact, the passage of monocytes through endothelial cells leads to their transformation to DCs (48). It is therefore possible that ROS generated by endothelial cells lead to extracellular accumulation of IsoLG-protein adducts that are then phagocytosed by DCs. Another scenario is that hydrogen peroxide produced by endothelial cells could activate the NADPH oxidase in DCs. This is known to occur in the case of vascular NADPH oxidase (49). Further research is needed to examine the specific mechanism by which sympathetic outflow induces IsoLG-protein adduct formation in DCs.

Excess Dietary Salt and its Role in Promoting IsoLG-protein Adduct Formation:

Extensive evidence demonstrates that excess dietary Na⁺ is epidemiologically linked to hypertension. Throughout most of human history, Na⁺ consumption was less than 0.6-0.8 g/day (50). The AHA recommends a maximum of 2.3 g/day of Na⁺ intake. However, the Center for Disease Control (CDC) recently reported that Americans consume more than double this amount, and less than 10% of the U.S. population observes the recommended guidelines. In the US, the average Na+ intake is 4 grams per day (50, 51). A meta-analysis by He et al estimated that modest reductions in Na⁺ intake would lower blood pressure and reduce the annual number of new cases of coronary heart disease and stroke in the U.S. by 20% (52). However, accomplishing these dietary goals is difficult, as more than 70% of dietary Na+ is added to food prepared outside of the home (53). Thus, the impact of excess dietary salt intake remains an area of intense interest, with unsolved questions and barriers hampering success of general recommendations on dietary salt intake.

Traditionally, interstitial Na⁺ has been considered to be in equilibrium with plasma. Moreover, a vast amount of literature suggests that increased Na⁺ intake results in volume retention and increased cardiac output leading to hypertension. However, there is growing evidence that Na⁺ can accumulate in the interstitium without commensurate increases in interstitial water, leading to concentrations exceeding those of plasma (54-56). Such high concentrations of Na+ polarize immune cells towards an inflammatory phenotype, enhancing interleukin (IL)-17 production (11, 57, 58). Likewise, high salt intake increases the percent of circulatory pro-inflammatory intermediate monocytes (59). These studies suggest that elevated Na+ increases inflammation and contributes to hypertension. Moreover, they suggest that therapeutic strategies to reduce tissue Na⁺ may reduce inflammation and hypertension.

Studies in our laboratory have found that elevated Na+ contributes to inflammation and hypertension by increasing IsoLG-protein adduct formation in antigen presenting cells. DOCA-salt hypertension is associated with activation of monocyte derived DCs leading to production of IsoLG-protein adducts (15). Recently, we found that elevated Na⁺ is a potent stimulus for IsoLG-protein adduct formation in DCs in mice. This occurs through amiloride sensitive Na⁺ transporters (13). The increased intracellular Na+ is exchanged for calcium (Ca²⁺) via the Na⁺/Ca²⁺ exchanger. Ca²⁺ activates protein kinase C (PKC) which in turn phosphorylates the NADPH oxidase subunit p47^phox. This leads to activation of the NADPH oxidase, increased superoxide and derivative reactive oxygen species (ROS) production (13). Thus, diuretics such as amiloride may confer anti-inflammatory actions directly on immune cells.

Our studies indicate that the increased superoxide production in DCs is associated with accumulation of IsoLG-protein adducts (15). Exposure of DCs to elevated Na⁺ (190 mM NaCl) but not an equiosmolar concentration of mannitol increased formation of IsoLG-protein adducts when compared to the normal physiological concentrations (150 mM NaCl) (13). The high salt-induced formation of IsoLG-protein adducts is superoxide and NADPH oxidase-dependent since it was prevented in mice lacking the p22^phox docking subunit of this enzyme complex in DCs. This increase in Na+ concentrations was associated with increased expression of CD86, a B7 ligand which indicates maturation and has been shown to be essential for hypertension (13, 33). High salt also increased DC production of proinflammatory cytokine IL-1β which contributes to priming T cells for production of IL-17A and mediates hypertension (13, 60, 61). We have previously shown that scavenging of IsoLGs prevents hypertension-induced production of IL-1β by DCs (15). Importantly, DCs exposed to high salt induced pro-hypertensive cytokine production in primed T cells and sensitized mice to a normally suppressor dose of angiotensin II leading to hypertension (13). These effects were prevented by scavenging of IsoLGs during exposure of DCs to high salt suggesting that high salt-induced activation of DCs is dependent on the formation of IsoLGs (13). While these studies are informative about the effect of elevated Na⁺ on immune activation and hypertension, they are only in vitro and the in vivo mechanisms by which tissue accumulation of salt activates immune cells are still not known.

Conclusions and future perspectives:

IsoLG-protein adducts play and important role in promoting inflammation and hypertension. Scavenging of IsoLGs blunts inflammation and the hypertensive response to various stimuli including angiotensin II, catecholamines, and excess salt (Figure 2). The specific peptides altered by IsoLGs in hypertension are not known and future research efforts should be directed toward this goal. Despite ongoing efforts by many laboratories, immunogenic peptides have not been identified for many diseases known to be caused by T cells. The involvement of the proteasome in the processing of IsoLG-adducted proteins also needs to be investigated. The proteasome plays a diverse role in the immune system. The ubiquitin-proteasome system assists with maintenance of cellular homeostasis by degrading molecules responsible for numerous processes. In antigen presenting cells, the proteasome facilitates the presentation of endogenously generated antigens into MHC-I. Interestingly, augmented auto-inflammation correlates with proteasome levels in autoimmune disease (62, 63). Importantly, Bortezomib, a proteasome inhibitor FDA approved for the treatment of multiple myeloma, reduced the expression of DC activation markers CD86, CD80, CD40, and CD83. Moreover, DCs treated with a proteasome inhibitor were unable to activate T-cells (64, 65). Given these findings, proteasome inhibition has been proposed as a therapy for autoimmunity and may be a novel therapeutic for hypertension and its cardiovascular complications. This is important because the current anti-hypertensive drugs are not sufficient to prevent end-organ damage. Even among patients with successful control of blood pressure, the cardiovascular risk remains greater than that of the normal population. The currently available drugs can be categorized into 5 classes including diuretics, adrenergic receptor antagonists, calcium channel blockers and drugs that block the renin/angiotensin system. Data from large clinical trials indicate that from 20 to 30% of patients have persistently elevated blood pressure despite taking 3 or more of these drugs (66). And yet no new drugs have been developed in the past 3 decades that fall outside of these categories.

Our research indicates that many forms of hypertension, including angiotensin II, DOCA-salt, and norepinephrine-induced hypertension are dependent on immune cell activation. This suggests that a common mediator, such as ROS may be responsible for linking the various hypertensive stimuli to inflammation. However, antioxidants have proven ineffective in reducing cardiovascular risk in large clinical trials. This might be in part because their rate constants (about 10⁴ M/sec) is far below the rate constant of reactions of free radicals with endogenous antioxidants (10⁹ to 10¹⁰ M/sec). For example, 1600 IU of vitamin E is required to suppress isoprostane levels (the precursor of IsoLGs) in humans (67). These very high doses have adverse effects in several clinical trials and therefore should not be used (68). Thus, scavenging IsoLGs, which are downstream of ROS may provide a more targeted approach for treatment of prevention of hypertension and cardiovascular disease.