Adenosine Receptors and Vascular Inflammation

Abstract

Epidemiological studies have shown a positive correlation between poor lung function and respiratory disorders like asthma and development of adverse cardiovascular events. Increased adenosine (AD) levels are associated with lung inflammation which could lead to altered vascular responses and systemic inflammation. There is relatively little known about the cardiovascular effects of adenosine in a model of allergy. We have shown that A₁ adenosine receptors (AR) are involved in altered vascular responses and vascular inflammation in allergic mice. Allergic A₁wild type mice showed altered vascular reactivity, increased airway responsiveness and systemic inflammation. Our data suggests that A₁ AR is pro-inflammatory systemically in this model of asthma. There are also reports of the A_2B receptor having anti-inflammatory effects in vascular stress; however its role in allergy with respect to vascular effects hasn’t been fully explored. In this review, we have focused on the role of adenosine receptors in allergic asthma and the cardiovascular system and its possible mechanism (s) of action.

1. Generation of adenosine and its metabolism

Adenosine is a ubiquitous purine nucleoside with multiple physiological effects. It has several regulatory functions including relaxation of the vascular smooth muscle, neuromodulation, regulation of inotropy, chronotropy amongst others [1]. Under normal physiological conditions, adenosine is formed by the intracellular conversion of S-adenosyl-L methionine (SAM) to S-adenosyl-L-homocysteine (SAH), which is then converted to adenosine and homocysteine by SAH-hydrolase [2, 3]. Adenosine can also be produced extra-cellulary through successive dephosphorylation of ATP; ATP is dephosphorylated to ADP and then AMP which is converted to adenosine via ecto-5’-nucleotidases [4, 5]. Adenosine, thus, produced can be further converted to inosine by the enzyme adenosine deaminase (ADA) and finally is broken down to uric acid which is excreted in urine. Adenosine can be phosphorylated to AMP via the enzyme adenosine kinase. Extra-cellular concentrations of adenosine are elevated several fold during periods of increased metabolic demand, injury or stress such as ischemia [6] and inflammation.

2. Adenosine receptors

Adenosine acts on specific cell surface purinergic receptors to induce various physiological effects, and thus far, four subtypes have been identified; namely A₁, A_2A, A_2B and A₃. All four adenosine receptors are G-protein coupled and the effects that adenosine produces depends on both the receptor subtype that is activated as well as organ/tissue in which the receptor is present. A₁ and A₃ receptors are coupled to G_i while the A_2A and A_2B receptors are coupled to G_s. A₁ and A_2A have a high affinity for adenosine, while A_2B is low affinity and A₃ is intermediate [7, 8]. The A₁ receptor signals through G_i/G_o-proteins and its activation leads to an inhibition of adenylyl cyclase (AC) which causes a lowering of cyclic AMP (cAMP). Signaling through the A₁ receptor can also lead to activation of IP₃/DAG through the PLCβ-III pathway [9]. The A_2A receptor signals through the G_s pathway and its activation leads to stimulation of AC resulting in increased production of cAMP. The A_2A receptor is mainly anti-inflammatory and is involved in coronary vasodilation as well as vascular smooth muscle relaxation to various degrees depending on the species [10-12]. The A_2B receptor signals through G_s/q and its activation can either result in increased cAMP, IP₃/DAG and Ca²⁺ levels. Lastly, the A₃ receptor signals through G_i and is negatively coupled to AC [13].

3. Asthma

Asthma is a chronic lung disease characterized by inflammation and airway hyper-reactivity. There are over 20 million people diagnosed with asthma, with 9 million under the age of 18 years. Allergic asthma accounts for about half of these numbers. The disease consists of episodic events which manifest as hyper-responsiveness of the airways to common triggers like dust, pollen, cold air, exercise, cigarette smoke and other pollutants, resulting in bronchospasm and difficulty in breathing. Airway obstruction occurs due to inflammation, excessive mucus production, and overactive bronchial smooth muscle. Initially, the inflammation is acute in nature but eventually becomes chronic with the progression of the disease, and long term effects of asthma are linked to the inflammatory component of the disease. Disease management consists of anti-inflammatory drugs, leukotriene modifiers and bronchodilators.

4. Adenosine and asthma

Adenosine has long been implicated in the pathogenesis of asthma [14]. Inhalation of adenosine was shown to be a potent bronchoprovocant in asthmatic patients [15]. Inosine, the deaminated metabolite of adenosine does not produce bronchoconstriction [16] suggesting that the effect on bronchial smooth muscle is specific to adenosine and possibly involves adenosine receptors. Increases in adenosine levels correspond to increased airway inflammation and tissue damage, and asthmatic patients also have significantly higher levels of adenosine in bronchoalveolar lavage fluid (BAL) and exhaled breath condensate than normal healthy subjects [17, 18]. Adenosine causes degranulation of mast cells which leads to the release of histamine and subsequent cascade of events including H₁ receptor mediated bronchoconstriction and inflammatory response [19, 20]. This mast-cell mediated response is believed to occur through the A_2B receptor although this has not been conclusively proven as yet [21].

Mice that lack the adenosine deaminase (ADA) enzyme have large amounts of adenosine in their lungs and severe lung inflammation [22]. In fact, these mice were unable to survive beyond three weeks due to respiratory distress ADA deficient mice demonstrated the presence of eosinophilia in the lungs extensive mast cell degranulation and increased levels of serum IgE [23], indicating a strong correlation between chronic elevation of adenosine levels and increased lung inflammation. There is evidence that adenosine causes recruitment of inflammatory cells to the lung [24] and amplification of the inflammatory response [25].

In allergic mice, adenosine among other inflammatory mediators from allergic lung and activated leukocytes are released [16, 26]. This adenosine may be involved in further release of chemotactic and inflammatory mediators in the lung and systemic circulation by acting on its receptors present on different cells including mast cells, eosinophils, neutrophils and other inflammatory cells [25, 27-29]. Experimentally induced temporary elevations in lung adenosine levels through the inhalation of adenosine (resulting from breakdown of adenosine 5'-monophosphate) have been shown to cause an increase in infiltration of eosinophils in patients with asthma [30].

5. Reactive airway diseases and cardiovascular complications

There is epidemiological evidence indicating that people suffering from asthma, chronic obstructive pulmonary disease (COPD) and reactive airway diseases are at an increased risk for developing cardiovascular disease (CVD) [31-33]. There is an association between atherosclerosis and stroke with reactive airway diseases. One study found that adult-onset asthma was associated with increased carotid atherosclerosis in women [34]. Impaired lung function has been found to be a risk factor for CVD [35]. Bronchial hyper-responsiveness to methacholine is associated with increased carotid intima-media thickness [36]. One of the leading causes for hospitalizations and deaths occurring in COPD patients is cardiovascular events [37].

Recent evidence in animal models indicates that CV complications associated with asthma are independent of asthma therapy and could be a result of asthma itself. Myocardial ischemia-reperfusion injury was enhanced in a rabbit model of systemic allergy and asthma [38] and allergic inflammation in the airways enhanced neutrophil recruitment to the myocardium and severity of ischemia-reperfusion in a murine model [39]. Allergic mice have poor vascular responses and systemic inflammation, with adenosine aerosol exacerbating these effects [40]; Table 1). In our study, we found that use of an A₁ AR antagonist ameliorated some of these effects that lead to the hypothesis that A₁ AR receptor is involved in proinflammatory effects systemically in a mouse model of allergic asthma.

Table 1.

Vascular responses to adenosine in Balb-C mice aorta [40]

Group	Response to 10-4 M adenosine (%)
Control	21.44±3.94
Allergic	7.02±2.94*
Allergic + adenosine	-4.45±3.8 (contraction)*#

^*P<0.05 compared to control

^#P<0.05 compared to allergic tissues.

+ Relaxation; - contraction

6. Adenosine receptors in asthma and vasculature

Adenosine has a well established role in the control of vascular tone, and its effects are exerted through the activation of four ARs [41]. A₁ and A₃ ARs have been shown to be involved in vasoconstriction [9, 42-44] whereas A_2A and A_2B ARs cause vasorelaxation [45-47]. Adenosine and other inflammatory mediators are released from allergic lung and activated leukocytes [16, 26]. Adenosine that is generated in allergic lungs may be involved in the release of further chemotactic and inflammatory mediators in the lung and systemic circulation by acting on its receptors present on different cells including mast cells, eosinophils, neutrophils and other inflammatory cells [25, 27-29]. This has been confirmed by recent observations where experimentally induced temporary elevations in lung adenosine levels through the inhalation of adenosine (resulting from breakdown of adenosine 5'-monophosphate) have been shown to cause an increase in infiltration of eosinophils in patients with asthma [30].

The four ARs have different modulatory roles in asthma, and the cardiovascular system [48-50]. Many reports suggest that the A₁ AR is involved in direct bronchoconstrictor effects of adenosine. The expression of A₁ AR was found to be elevated in a rabbit model of asthma and the use of an A₁ antisense nucleotide to inhibit this upregulation resulted in blunted bronchoconstriction to adenosine [51, 52]. Use of a selective A₁ antagonist (L-97) produced significant reduction of airway hyper-responsiveness in allergic rabbits by blocking these receptors [53]. A recent study reported that the expression of A₁ AR is significantly elevated in airway smooth muscle in asthmatic patients [54]. Other than the effects on airway smooth muscle, the A₁ AR has been implicated in increased mucin production in human tracheal cells [55] and in mediating monocyte phagocytosis [56]. All these studies suggest a strong role for the A₁ AR in asthma, both in airway obstruction and inflammation.

A₁ ARs also have systemic effects and are involved in blood pressure regulation [57] and vasoconstriction [9, 42-44]. Recent studies suggest a role for A₁ AR in altered peripheral vascular reactivity and increased systemic inflammation [40]; Table 1 and Figs. 1-2). In this study, Balb/C (A₁ wild-type: A₁WT) mice were divided into 3 groups: control or non allergic (A₁WT CON), ragweed sensitized mice (A₁WT SEN) and ragweed sensitized mice further challenged with a single aerosol of adenosine (A₁WT SEN+AD) were studied.

Figure 1.

Systemic inflammatory markers in plasma A) IL-5 B) IL-13 C) TNF-α in A₁WT non-allergic control (A₁WT CON), A₁WT allergic (A₁WT SEN), A₁WT allergic further challenged with adenosine (A₁WT SEN+AD) mice [40] *P<0.05 compared to WT CON; #P<0.05 compared to WT SEN

Figure 2.

Plasma levels of IL-5 in A₁KO non-allergic control (A₁KO CON), A₁KO allergic (A₁KO SEN), A₁KO allergic further challenged with adenosine (A₁KO SEN+AD) mice (unpublished observation; IL-13 and TNF-α were not detected in A₁KO plasma)

The A_2B AR has been found to play a protective role in vasculature against arterial injury (vascular stress) and is involved in the regulation of TNF-alpha, which contributes to the anti-inflammatory actions of adenosine[58]. In A_2B receptor–null mice, the expression of vascular adhesion molecules was increased, as also the levels of pro-inflammatory cytokines [59, 60]. A_2B antagonism also produced further impairment of adenosine mediated aortic vasorelaxation in allergic mice [40].

Despite these data, there is little information specifically regarding the role of lung inflammation (as seen in asthma) in the development of cardiovascular disease, in relation to the vascular and systemic effects of adenosine. Systemic inflammation, endothelial dysfunction and altered vascular reactivity appear to be associated with asthma. Adenosine plays an important role in these effects and exogenously administered adenosine to increase the lung levels experimentally beyond the allergen exposure exacerbates observed outcomes.

Recent work from our lab [61] using A₁ AR knock-out (A₁AR KO) mice has shown the involvement of A₁ ARs in systemic and vascular inflammation. Data showed that the allergic A₁WT mice (A₁WT SEN and A₁WT SEN+AD groups) had lower adenosine-mediated aortic relaxation to 5’-N-ethylcarboxamidoadenosine (NECA; non-selective adenosine analog) (Table 2) and higher contraction to 2-chloro-N6-cyclopentyladenosine (CCPA; selective A₁ AR agonist), with increased protein expression of A₁ AR in the aorta. There were also significantly higher levels of inflammatory markers in the plasma, and in aortic tissue. In contrast, no impairment in adenosine-mediated vascular responses was observed in A₁ AR KO (Table 2; allergic and non-allergic controls: control or non allergic A₁KO CON, ragweed sensitized A₁KO SEN and ragweed sensitized further challenged with a single aerosol of adenosine A₁KO SEN+AD) aortic tissues. There were also significantly lower (or undetectable) levels of inflammatory markers in aortic tissue in allergic KO mice. These data implicate the involvement of A₁ AR in the deleterious systemic effects in allergic mice that may occur as a result of inflammation. The exact signaling mechanism(s) of the A₁ AR remains to be elucidated.

Table 2.

Vascular responses to 5’-N-ethylcarboxamidoadenosine (NECA; non-selective AR agonist) in A₁ wild-type and knockout Balb/C mice aorta [61]

Group	Response to 10-5 M NECA (% relaxation)
A1WT Control	48.64±2.98
A1WT Allergic	28.08±5.06*
A1WT Allergic + adenosine	17.4±5.53*
A1KO Control	43.0±2.45
A1KO Allergic	48.48±1.93
A1KO Allergic + adenosine	48.2±1.47

^*P<0.05 compared to WT control

The A_2A receptor has been reported to have anti-inflammatory and protective role in the lung, especially lung injury A_2A KO mice were shown to have higher levels of pro-inflammatory cytokines and extensive tissue damage when subjected to an inflammatory insult that were less in the corresponding A_2A WT mice [62].

Our lab has reported that A_2A KO mice have higher oxidative stress that leads to impaired tracheal relaxation [63]. There have also been reports that the A_2A AR protects against myocardial ischemia reperfusion injury [64]. In some preliminary data from our lab (unpublished), we have also observed that allergic A_2A KO mice have poor aortic relaxation and increased airway response (whole body plethysmography) compared to WT. All these findings lead us to speculate that the A_2A AR may protect against vascular inflammation.

Reports have suggested that activation of A₃ receptor by inosine [65] as well as adenosine [66] leads to mast-cell induced increase in vascular permeability and extravasation. A₃ receptors can also have anti-inflammatory effects [67]. The A₃ AR also increases mucin production in asthma [68] and has been shown to be involved in bronchoconstriction and eosinophilia [69, 70]. Other reports suggest an anti-inflammatory role for A₃ receptors in diseases like rheumatoid arthritis [71]. The role of the A₃ receptor thus could possibly be both pro- and ant-inflammatory. How this receptor behaves in the vasculature in allergic mice remains to be seen.

7. Possible pathway for adenosine-induced vascular changes in asthma

Based on the literature and some of our data we hypothesize a speculative pathway for lung inflammation leading to systemic effects (Fig 3). We found that IL-13 levels were highest in allergic mice [40]. A review of the literature shows a close relationship between IL-13 and adenosine in lung inflammation [72]. IL-13 is an important mediator in asthma and has been implicated in lung inflammation and airway remodeling. It is a product of mast cell degranulation and is also released from T-helper 2 (Th2) cells. Adenosine and IL-13 interact with each other positively as shown in studies done with asthmatic mice [72].These authors found that IL-13 caused a progressive increase in adenosine accumulation, inhibited the activity of adenosine deaminase, and augmented the expression of the A₁, A_2B, and A₃ ARs. Their findings suggest that adenosine signaling contributes to and influences the severity of IL-13 and Th2 -mediated disorders such as asthma.

Figure 3.

Possible pathway for adenosine receptors as both pro- and anti-inflammatory effectors in vascular inflammation in asthma. “??” indicates unknown or possible mediators.

IL-13 signaling occurs through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. This cytokine acts on its receptor (via JAK-1) and leads to phosphorylation of STAT6, which induces the transcription of several genes including 5-lipoxygenase (LO). 5-LO, via the arachidonic acid pathway, produces inflammatory leukotrienes (LTs) which are very important mediators of IL-13 induced lung injury [73]. Recent studies have also suggested a proatherosclerotic role for LTs with LTB₄ acting as a potent leukocyte chemoattractant that amplifies inflammation in atherosclerosis [74].

IL-13 and IL-4 act synergistically with TNF-α to increase eotaxin levels in the lungs. Eotaxin increases lung eosinophilia in asthma and is very important in amplification of eosinophilic inflammation. Though eosinophilia is considered to be a characteristic hallmark of asthmatic inflammation, eosinophils are not found in any significant amounts in atherosclerotic lesions. Eotaxin may have a novel role in atherosclerosis independent of eosinophils. Haley et al. have reported that eotaxin and its receptor, CCR3, were overexpressed in the inflammatory infiltrate of human atheroma [75]. CCR3 was predominantly expressed on macrophages and they suggest eotaxin may participate in mast cell activation or recruitment. In addition to this, in our most recent study [61] we have found significantly higher levels of IL-5 in aortic tissue of allergic A₁WT mice. This may further support a role for IL-5 induced eotaxin effects in vasculature.

In conclusion, adenosine mediated effects in asthma may extend beyond the lung and have consequences both systemically and in the vasculature. It is possible that inflammation underlies these additional effects and there is evidence so far for the A₁ AR having a pro-inflammatory role. There remains much to be learnt in this field, with each adenosine receptor's role not yet delineated. Such studies would help to further the potential of adenosine agonists and antagonists as therapeutic agents for systemic and vascular inflammation from a number of disorders including hypertension, diabetes, rheumatoid arthritis and many other complications.

ABBREVIATIONS

AC

Adenylyl cyclase

ACh

Acetylcholine

ADA

Adenosine deaminase

AD

Adenosine

ADP

Adenosine diphosphate

AR

Adenosine receptor

AK

Adenosine kinase

AMP

Adenosine monophosphate

BAL

Bronchoalveolar lavage

cAMP

Cyclic adenosine monophosphate

CCPA

2-chloro-N6-cyclopentyladenosine

CGS

21860 2-p-(2-carboxyethyl)phenethylamino-5' N- ethylcarboxy amidoadenosine hydrochloride

COPD

Chronic obstructive pulmonary disease

CV

Cardiovascular

CVD

Cardiovascular disease

DAG

Diacylglycerol

DPCPX

1,3-Dipropyl-8-cyclopentylxanthine

IP₃

Inositol triphosphate

LO

Lipoxygenase

LT

Leukotriene

KO

Knockout

NECA

N-ethylcarboxamide-adenosine

SAH

S-Adenosyl-L-homocysteine

SAH-hydrolase

S-Adenosyl-L-homocysteine-hydrolase

SAM

S-Adenosyl-L methionine

WT

Wild type