Specialized pro-resolving lipid mediators in cardiovascular disease, diagnosis, and therapy

Abstract

Persistent inflammation is the key aggravator in many cardiovascular diseases, including atherosclerosis, aneurysm, injury/reperfusion, thrombosis, and neointimal hyperplasia following surgical or percutaneous interventions. Resolution is an active process orchestrated by specialized pro-resolving lipid mediators (SPMs) which tamp down acute inflammatory signals, promote healing and facilitate a return to homeostasis. SPMs are endogenously derived from poly-unsaturated fatty acids, and their biologic activity is mediated via specific G-protein coupled receptor binding. The potency of SPM in regulating the inflammatory response has encouraged investigation into their therapeutic and diagnostic use in cardiovascular pathologies. Herein we describe the translational groundwork which has established the synthesis and interactions of SPM in cardiovascular and hematologic cells, the therapeutic effects of SPM in animal models of cardiovascular disease, and some early technologies that harness and attempt to optimize SPM delivery and “resolution pharmacology”. Further studies are required to precisely determine the mechanisms of resolution in the cardiovascular system and to determine the clinical settings in which SPM can be utilized to optimize patient outcomes.

Graphic Abstract

Vascular injury, inflammation, resolution, and repair. Injury to EC and underlying vSMC is the initiating event. The expression of cytokines and cell adhesion molecules is increased, resulting in leukocyte recruitment and the production of proinflammatory mediators. PUFA such as arachidonic acid (AA) are converted by lipoxygenases (LO) and cyclooxygenases (COX) into proinflammatory lipid mediators such as prostaglandin (PG) and leukotriene (LT). These in turn promote vascular permeability, platelet aggregation, further leukocyte recruitment, and augmentation of inflammatory cytokines. A lipid mediator class switch occurs in which LO and COX begin to convert PUFA (e.g. DPA, DHA, EPA) into SPM. SPM downregulate inflammatory processes, reducing cytokine production and promote clearance of debris and tissue repair via M1-M2 macrophage transition. If resolution is delayed or insufficient, inflammatory signals persist resulting in dysfunctional VSMC, fibrosis, and exaggerated neointimal hyperplasia.

1.1. Background

1.1.1. Traditional Paradigm of Cardiovascular Disease and Treatment

Acute and chronic inflammation play a major etiologic role in cardiovascular disease (atherosclerosis, aneurysmal disease, venous thrombosis, ischemia-reperfusion). Pathologic inflammation occurs in numerous clinical scenarios, both in native disease progression (e.g. atheromatous plaque growth, rupture, thrombosis, dissection, etc.) and iatrogenic settings (e.g. angioplasty, stenting, bypass surgery, etc.). Diet, exercise, and lipid lowering medications can slow progression or stabilize atherosclerotic disease, but invasive interventions are often necessary to treat symptoms or prevent catastrophic consequences. These procedures generally result in an acute-on-chronic inflammatory response. Following reperfusion of ischemic tissue, the resulting cascade often worsens the extent of local injury. If resolution does not occur promptly, protracted inflammation leads to further end-organ damage, recurrent disease and downstream clinical events. Current therapeutics used in the setting of vascular interventions have focused on the use of cytotoxic, anti-inflammatory, or anti-thrombotic drugs which are nonspecific and may cause undesired effects on the endothelium and immune system, possibly even increasing long-term mortality[1]. These agents can delay physiologic healing, placing areas of vascular injury at increased risk of thrombosis. While recent trials have demonstrated the potential benefits of altering the inflammatory response in cardiovascular disease, they have also shown the consequences of inhibiting upstream immunologic mechanisms, including fatal infectious complications[2]. Harnessing resolution mechanisms may represent an important new opportunity to modulate the vascular inflammatory response without impairing host defense and healing or increasing bleeding risk, first termed “resolution pharmacology” by Peretti et al[3]. This review will summarize the relevant biochemistry, cell biology, and translational studies that underpin the development of resolution pharmacology for cardiovascular disease.

1.1.2. Biology of Vascular Injury, Inflammation, and Remodeling

Numerous types of insults (metabolic, hemodynamic, traumatic, etc.) and risk factors integrate at the vasculature and lead to inflammation. The underlying key step in many cardiovascular pathologies is injury to the endothelial cell (EC) barrier, with variable damage to underlying matrix and vascular smooth muscle cells (vSMC), and exposure to circulating blood elements. This leads to a cascade of signals promoting thrombosis and inflammation. Apoptotic EC and vSMC, as well as recruitment of platelets, coagulation proteins, and leukocytes create a rapid influx of inflammatory cytokines (e.g. IL-1B, IL-6, TNF-a) and chemokines (e.g. MCP-1) as well as reactive oxygen species (ROS) to the site of injury. Proliferative growth factors are released from cellular elements and extracellular matrix (e.g. PDGF, FGF), causing vSMC activation to a state of increased migration, proliferation, resistance to apoptosis, expression of pro-inflammatory cytokines and synthesis of vascular matrix proteins. Failure to resolve this acute inflammatory process in a timely fashion leads to disease progression, clinical events, resistance to therapies, and/or recurrence of disease post-intervention, such as neointimal hyperplasia (NIH) leading to vessel narrowing [4].

1.1.3. Biochemistry of resolution

Resolution has been identified as an active process in the timeline of inflammation, involving a complex network of specific pro-resolving mediators, receptors, and downstream pathways. Researchers have investigated this framework to explore mechanisms of disease as well as potential therapeutic or diagnostic tools[5–8]. These studies identified distinct classes of specialized pro-resolving lipid mediators (SPM), endogenously derived from polyunsaturated fatty acids (PUFA) through molecular profiling in small animal models of self-limited inflammation. Precursor PUFAs for SPMs include n-6 arachidonic acid (AA) and the n-3 docosapentaenoic acid (DPA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). These SPM families include E- and D- series resolvins, protectins, maresins, and lipoxins, many of which have been produced via total organic synthesis[9–11]. The biologic activity of SPM are carried out by various GPCRs, including ALX/FPR2, DRV1/GPR32, DRV2/GPR18, ERV1/ChemR23, GPR37, GPR101 and LGR6[12–15]. Importantly, ongoing research has continued to uncover new SPM classes, mediators and receptors suggesting biochemical redundancy, time- and tissue-specificity in the resolution response to various stimuli[12]. There also exist non-lipid pro-resolving mediators, such as Annexin A1, a peptide which acts on ALX/FPR2[16]. This would be expected in a process so fundamental to organismal health. Whereas SPM share key defining biologic properties such as improving the “resolution-index” in small animal models of sterile peritonitis [17] we refer to them as a broad category in this review, while recognizing that specific SPMs may play critical roles in a tissue, time, and disease context.

In physiologic resolving inflammatory exudates, cell-cell interactions lead to the generation of active signals that limit leukocyte, namely PMN, accumulation and promote the clearance of apoptotic cells and cellular debris[18]. SPMs coordinate these interactions by enhancing M1-M2 phenotypic polarization in macrophages, promoting lipoxygenase (LO) activity, and upregulating SPM receptor expression[19–22]. The biochemical mechanisms underlying the observed “lipid mediator class switch” from pro-inflammatory mediators (e.g. prostaglandins, leukotrienes) to SPM within resolving exudates are of considerable interest and are actively being defined[19,21].

A deficiency or lack of resolution signals may thus contribute to many disease states and is of particular interest in the pathogenesis of cardiovascular disease. If the inflammatory cycle is left unchecked, a detrimental cycle of leukocyte recruitment, saturation of cytokines and growth factors, vSMC stimulation and proliferation leads to intimal hyperplasia and stenosis[4,23–26].

1.2. SPM Biosynthesis and Activity in the Cardiovascular System

1.2.1. Vascular Biosynthesis

The PUFA precursors AA, EPA, DPA and DHA are made available at local sites of injury through edema and the generation of tissue exudates. The conversion of these precursors into lipid mediators is carried out by lipoxygenase (LO) and cyclooxygenase (COX) enzymatic activity. These processes are likely carried out through the exchange of biochemical metabolites between cells, including leukocytes, platelets, and vascular cells, i.e. ECs and vSMCs. Early in the inflammatory response, AA-derived pro-inflammatory prostaglandins and leukotrienes account for the majority of synthesized lipid mediators. A lipid mediator class switch is then responsible for the subsequent synthesis of SPMs utilizing the same enzyme systems acting on a broader array of substrates including the n-3 PUFA. Subcellular localization of the key enzyme 5-LO appears to be one critical mechanism in this class switch that yields SPMs[22]. These mediators are synthesized locally and active transiently, as they are rapidly metabolized by available enzymes such as eicosanoid oxidoreductases and prostaglandin dehydrogenase[27]. The specific origins of SPMs in the vasculature have yet to be fully elucidated, but likely involve cell-cell interactions with exchange of intermediates. LXA₄ was discovered intraluminally in a study of coronary angioplasty in humans[28]. Recent studies demonstrated that freshly harvested non-diseased human artery segments and cultured vascular cells are capable of producing D-series resolvins and maresins when exposed to precursors such as DHA, in the absence of intervening leukocytes or platelets [29]. Conditioned medium from vascular cells treated with DHA supplementation reduced leukocyte adhesion on cytokine activated target cells, and this action was carried out by the receptors ALX/FPR2 and GPR32. In the murine mesenteric microcirculation, LXA₄ is produced by platelet-leukocyte and leukocyte-endothelial interactions as a protective response to vascular insult such as ischemia reperfusion[30]. A similar response is seen in the cerebral and renal microvasculature following ischemic insult[31,32]. These studies provide evidence that the vasculature is capable of endogenous production of SPM to counteract and balance acute inflammation.

Importantly, several relevant drugs have been implicated in the biosynthesis of lipid mediators, including aspirin and statin. Oral administration of aspirin acetylates and alters the active site of COX-2. This inhibits the production of proinflammatory prostaglandins and promotes the production of aspirin-triggered (AT) forms of SPM such as 15-epi-lipoxin, a potent antiinflammatory and pro-resolving mediator[33] and AT-RvD1[27]. These stereoisomers have prolonged bioavailability and may account for some of the effects of aspirin. Atorvastatin increases the production of the 13-series resolvins (RvTs) via S-nitrosylation of COX-2, which improved survival in a mouse model of sepsis [34]. Conversely agents such as COX-2 inhibitors are “resolution-toxic”.

1.2.2. Endothelial Cells

In the cardiovascular system, ECs form the protective barrier between circulating blood elements and underlying vascular architecture, maintaining laminar blood flow, and regulating platelet, leukocyte, and other macromolecular activity. When this barrier is breached or activated, a thrombo-inflammatory response is initiated, which in an evolutionary sense may be protective against pathogens or traumatic exsanguination, but in chronic situations can lead to deleterious effects. Endothelial cells interact with a number of SPMs, such as RvD1, RvD2, MaR1, LXA₄, and PD1 (Table I). The downstream effects include reduction of proinflammatory cytokines, adhesion molecule expression, and leukocyte-EC interaction, carried out by various SPM receptors, including ALX/FPR2, DRV1/GPR32, DRV2/GPR18, and ERV1/ChemR23 [9,29,35–41].

Table I.

Summary of effects of SPM on various cardiovascular and inflammatory cell types

Cell Type	SPM Studied	Effects	References
Endothelial Cell	RvD1, RvD2, MaR1, LXA4, PD1	Reduce proinflammatory cytokine production, adhesion molecule expression, leukocyte-EC adhesion	[9,35–38]
vSMC	RvD1, RvD2, RvE1, MaR1, LXA4	Reduced proliferation, migration, vasoconstriction	[23,35,42,43]
Platelets	RvD1, RvE1, MaR1	Enhanced hemostasis, reduced aggregation and inflammatory cytokine secretion	[36,56–58]
Macrophages	RvD1, RvD2, ATRvD1, AT-RvD3, ATPD1, AT-LXB4, MaR1	Enhanced M2 polarization, efferocytosis	[19,20,47,52–55,64]
Neutrophils	RvD2, RvD4, RvE1	Reduced NET release, recruitment	[42,47–51]

1.2.3. Vascular Smooth Muscle Cells

vSMCs also play a critical role in cardiovascular disease. Physiologically, they constitute the majority of the vessel wall, providing mechanical integrity, regulating vascular tone, and contributing to both functional and dysfunctional vessel wall healing. In the resting state, vSMCs are quiescent and highly organized, surrounded by other architectural proteins in the extracellular matrix. In response to injury and the resultant availability of inflammatory cytokine and growth factors (e.g. TNF-α, IL-1, IL-6, PDGF, FGF), vSMCs assume an activated state prone to migration, proliferation, and secretion of proinflammatory factors. When this inflammatory state is left unchecked, neointimal hyperplasia is promoted and ultimately leads to stenosis of the affected blood vessel. In vitro, treatment with RvD1, RvD2, and MaR1 reduce leukocyte-VSMC interactions via downregulation of cell adhesion molecules and other proinflammatory genes[23,35]. Treatment with RvD1, RvD2, RvE1 and LXA₄ attenuate vSMC migration, as well as proliferation to a modest degree, after exposure to promigratory and proliferative growth factors[23,42,43] (Table I). These effects are mediated at least in part by receptor-mediated activation of the cyclic AMP/protein kinase A pathway[44].

SPMs are also able to modulate the contractility of vSMC. RvE1 reduced vSMC hyperreactivity and calcium hypersensitivity induced by IL-1 and TNF-α[45], and pretreatment with RvE1, RvD1 or RvD2 resulted in significant inhibition of constriction in human pulmonary arteries and rat thoracic aortas in response to a thromboxane A₂ mimetic[46].

1.2.4. Circulating Cells

Neutrophil recruitment is the hallmark of any acute inflammatory response, promoting the local production of inflammatory cytokines and radical oxygen species. Treatment with RvD1, RvD2, RvD4, and RvE1 reduces neutrophil recruitment and neutrophil extracellular trap (NET) activity [42,47–51] (Table I). Subsequently, monocytes are the second wave of leukocyte recruitment and transition to macrophages in tissue. Macrophages assist with tissue repair and clearance of apoptotic ECs, vSMCs, and neutrophils. Initially macrophages are activated into their M1 phenotype, which increase the production of proinflammatory cytokines and radical oxygen intermediates in an attempt to augment the inflammatory response and clear pathogens. SPM (e.g. RvD1, RvD2, AT-RvD1, AT-RvD3, AT-PD1, AT-LXB₄, MaR1) encourage transition to the M2 phenotype, which is associated with clearance of apoptotic cells, wound healing and tissue repair [19,20,47,52–55] (Table I).

Platelet recruitment and aggregation is the initial response to cardiovascular events such as injury and plaque rupture when subendothelial vascular structures are exposed to the blood. They are also critical in the formation of pathologic thromboses. Treatment with various SPM produce different effects on platelet activity and function. MaR1 enhances the hemostatic function of platelets but reduces inflammatory activity[56]. RvD1 reduces platelet activation even in response to pro-aggregation stimuli such as ADP[57,58]. RvE1 similarly blocks ADP-mediated signals to reduce platelet aggregation [36] (Table I). Platelet interaction with leukocytes in vascular injury or thrombosis leads to enhanced production of MaR1, potentially promoting resolution pathways[59].

1.3. SPM in Cardiovascular Disease

1.3.1. Atherosclerosis

Impaired resolution of vascular inflammation plays a key role in atherosclerosis. Correlation between a lack of resolution mechanisms and atherosclerosis was first reported by Merched and colleagues, who demonstrated that mice deficient in 12/15-lipoxygenase activity had accelerated atherosclerosis[9]. Work by Ho and colleagues demonstrated that human patients with peripheral arterial disease had lower levels of the circulating SPM aspirin-triggered lipoxin A₄ (ATL; 15-epi-LXA₄)[60]. Since then, a deficiency in SPMs has been demonstrated in patients with coronary artery disease and cerebrovascular disease[52,61,62]. In a study of patients with known cardiovascular disease with significantly reduced levels of circulating RvD_{n-3 DPA}, in vitro supplementation of RvD_{n-3 DPA} with these patients’ blood resulted in significant dose-dependent reduction in platelet and leukocyte activation[63]. In a mouse model, deletion of ChemR23/ERV1 (receptor for SPM RvE1) caused proatherogenic signaling in macrophages, impaired efferocytosis, and increased plaque size and core necrosis[53].

SPMs have been identified in hyperlipidemic mouse atherosclerotic lesions, and a lack of SPM is associated with plaque instability. In these mice, treatment with RvD1, RvD2, and MaR1 improve plaque phenotype and increase fibrous cap thickness[64,65] (Table II). A study of ALX/FPR2 (receptor for RvD1) knockout mice revealed that these mice developed unstable atherosclerotic plaques with reduced collagen[40]. The same group demonstrated that treatment with ATL reduced atheroprogression in ApoE deficient mice through ALX/FPR2 signaling, and that this benefit was lost in ALX/FPR2 deficient mice[66]. In a rabbit model of periodontitis and high cholesterol diet, treatment with topical RvE1 reduced periodontitis and diminished atherogenesis with less overall arterial plaque, lower intima/media ratio, and decreased leukocyte infiltration[67].

Table II.

Summary of in vivo models demonstrating effects of SPM in cardiovascular disease

Model Type	Species	SPMs studied	Effects	References
Atherosclerosis	Mouse, Rabbit	RvE1, RvD1, RvD2, MaR1	Reduced atherosclerosis, thicker fibrous cap, enhanced plaque stability, reduced calcifications	[53,64–69]
Myocardial IR	Mouse, Rat	RvD1, RvE1	Reduced size of infarct, improved myocardial recovery	[78–81]
Cerebral IR	Mouse	15-Epi-LXA4, LXA4, RvD1, RvD2, MaR1	Reduced inflammation, mortality	[31,82–86]
Visceral IR	Mouse	RvD1–3, AT-RvD1, PD1, LXA4, 15-Epi-LXA4	Reduced kidney, hepatic, lung, visceral injury and inflammation	[30,87–92]
Hind-limb ischemia	Mouse	LXA4, RvD2	Improved skeletal muscle recovery	[51,93]
Aneurysm	Mouse	RvD1, RvD2	Decreased aneurysm formation	[48,54,72]
Venous Thrombosis	Mouse	RvD2, RvD4	Reduced thrombus burden	[49,73]

A hallmark of advanced atherosclerosis is plaque calcification. Calcification is the deposition of calcium and phosphate into calcium-phosphate crystals due to the phenotypic transformation of vSMCs into osteoblast-like cells. Vascular calcification can occur in different patterns (e.g. intimal versus medial) but is generally associated with clinical disease progression and can present major therapeutic challenges. Currently there is no pharmacologic therapy that effectively targets this process. In a mouse model of vascular calcification, RvE1 and chemerin stimulation of ChemR23 resulted in reduced phosphate-induced calcification in vSMCs[68,69].

1.3.2. Aortic Aneurysmal Disease

Aortic aneurysmal disease is dilation of the aorta at risk for rupture and life-threatening hemorrhage. The underlying pathophysiology is chronic inflammation within the aortic wall associated with increased protease activity and reduced stability of structural elements. Accumulation of leukocytes (lymphocytes and monocytes/macrophages) in the aortic wall leads to loss of structural integrity and depletion of vSMC. The only available treatment is surgery, as there are no pharmacologic treatments yet identified that affect the progression of aneurysmal disease. Several studies have demonstrated that PUFA supplementation in animal models of aortic aneurysm reduce inflammation and matrix degeneration[70]. In a mouse model of elastase induced aortic aneurysm, treatment with RvD1 and RvD2 promoted M2 polarization of macrophages and reduced the activity of matrix metalloproteinases (MMP), and attenuated aneurysm formation and progression[54] (Table II). In a mouse model of angiotensin II (AT-II) induced aortic aneurysm, treatment with RvD1 inhibited neutrophil extracellular trap phagocytosis (NETosis), reduced markers of inflammation, and decreased aneurysm progression[48]. Pillai et al demonstrated the clinical relevance of resolution biochemistry in aortic aneurysmal disease by revealing time dependent changes in circulating lipid mediators in patients undergoing aortic aneurysm repair[71]. Petri et al demonstrated that aneurysmal human aortas had significantly reduced levels of ALX/FPR2, and deletion of either ALX/FPR2 or the lipoxin-synthesizing 12/15 lipoxygenase resulted in enhanced aneurysm formation and increased aneurysmal leukocyte recruitment in a mouse model of abdominal aortic aneurysm[72]. The same study also demonstrated that pre-treatment with AT-LXA₄ prior to UV light exposure resulted in pro-resolution signaling (inhibition of p38 phosphorylation) in murine neutrophils, but this was not seen in ALX/FPR2 deficient neutrophils.

1.3.3. Venous Thrombotic Disease

Deep vein thrombosis (DVT) is the formation of a blood clot in the deep veins of the legs or the pelvis as a result of some combination of impaired flow, injury to the blood vessel and hypercoagulability. Following the acute event, chronic inflammation and downstream venous remodeling often leads to post-thrombotic syndrome in the affected limb. This involves venous valvular insufficiency causing pain, swelling, dermatitis, and chronic wounds. SPM participate in the acute phase of DVT clearance and may modulate the severity of inflammation and reduce the likelihood or severity of post-thrombotic syndrome. In vitro, RvE1 reduces platelet activation in response to stimulants such as ADP[57,58] (Table II). In a mouse model of burn injury, RvD2 reduced dermal vessel thrombosis[73]. The coagulation process leads to a temporal production of SPM that is likely important for subsequent clearance[74]. More recently, treatment with RvD4 reduced thrombus burden and neutrophil recruitment and increased pro-resolving monocytes and the synthesis of other D-series resolvins in a mouse model of IVC thrombosis[49].

1.3.4. Ischemia/Reperfusion Injury

In ischemic disease, anoxia and deprivation of critical metabolic substrates leads to infarct. Without reperfusion or the development of significant collateral circulation, the injured cells die and are replaced with dysfunctional scar tissue. With reperfusion (spontaneous or percutaneous coronary intervention), ischemia is halted, but additional injury and tissue damage occurs as a result of reperfusion and exposure to radical oxygen species (ROS). This injury is mediated by microvascular obstruction, myocardial edema, and exposure to inflammatory factors[75–77]. With unchecked inflammation as a result of reperfusion, the extent of injury extends beyond the initial area affected by ischemia, resulting in increased morbidity and mortality. In murine models of cardiac[50,78–81], cerebral[31,82–86], liver[55,87–89], lung[90], kidney[91,92], visceral[30], and hind leg[51,93] ischemia, pre-treatment with SPM (RvD1–3, AT-RvD1, RvE1, PD1, LXA₄, AT-LXA₄) has reduced tissue injury by enhancing cell protection pathways and reducing the harmful effects of ROS (Table II).

Following myocardial ischemia, persistent inflammation leads to myocyte injury and worse heart failure. In a model of ischemic cardiomyopathy with ALX/FPR2 gene knockout mice, the loss of ALX/FPR2 (RvD1 receptor) resulted in magnified obesogenic cardiomyopathy and renal inflammation[94]. In a separate study, treatment with RvD1 promoted resolution and improved ventricular myocyte recovery and function[79]

1.3.5. Vascular Injury and Remodeling

Invasive interventions for cardiovascular disease entail mechanical injury or detouring focal areas of disease to improve or restore circulation. These include angioplasty, stenting, endarterectomy, thrombectomy, and bypass surgery. Unfortunately, these interventions necessitate acute injury on diseased tissue riddled with chronic inflammation. The severity of the resultant acute inflammatory response ultimately affects vessel healing and long-term clinical outcomes, resulting in excessive scarring and narrowing of the vessel lumen in 50% or more of patients in 2–3 years[4,24,95–100]. A current hypothesis is that locally generated SPMs are critical in resolving vascular inflammation after injury, and they represent an important mechanism that may be deficient in patients requiring cardiovascular interventions[6,11,36].

In a rabbit model of femoral artery angioplasty, intraarterial delivery of RvD1 or RvD2 led to reduced NIH at one month[23] (Table III). In murine models of carotid artery ligation, neointimal hyperplasia (NIH) was significantly reduced with systemic (intra-peritoneal) delivery of RvD2 or MaR1[47]. In a mouse model of femoral artery wire injury, treatment with intraperitoneal RvE1 or oral supplementation with eicosapentanoic acid (EPA), the precursor to E-series resolvins, attenuated NIH through multiple mechanisms, including reduced neutrophil recruitment, suppression of t-cell traffic through RANTES suppression, and M2 macrophage polarization[101].

Table III.

Summary of in vivo models demonstrating effects of SPM in neointimal hyperplasia

Model Type	Species	SPMs studied	Effects	References
Artery ligation	Mouse	RvD2, MaR1, AT-LXA4	Decreased neointima	[43,47]
Angioplasty	Rat, Rabbit	RvD1, RvD2, PD1iso	Decreased neointima	[23,42,101–103]
Vein Bypass	Rabbit, Rat (synthetic graft)	RvD1, AT-RvD1	Reduced vein graft hyperplasia, reduces inflammation and promotes vascular regeneration	[104,105]

Wu et al demonstrated reduced NIH (50–60%) in a rat model of carotid artery angioplasty with RvD1 using perivascular pluronic gel or a biodegradable scaffold delivery[42]. Similar reductions in NIH in this model have been demonstrated with intravenous delivery of RvD1 and PD1iso[102]. In contrast to local delivery, oral supplementation of RvD1 reduced leukocyte recruitment and markers of acute inflammation, but did not affect downstream NIH in this model [103].

In a rabbit model of carotid artery bypass with autologous vein graft, Wu et al also demonstrated reduced NIH with perivascular RvD1 administration (Figure 1) [104]. In a rat model of aortic bypass with prosthetic polycaprolactone electrospun vascular graft, direct loading of AT-RvD1 into the graft fibers reduced inflammation and promoted vascular tissue regeneration[105]. Though much work remains to elucidate the optimal dosing, timing, and method of delivery, these studies provide encouraging evidence that SPMs may represent a new class of agents to improve vessel healing and reduce restenosis.

Figure 1:

Local delivery of SPM to improve vascular graft healing. Bypass grafts are subject to an acute inflammatory response that can lead to intimal hyperplasia, particularly at the anastomoses. The cartoon (A) depicts two methods of perivascular delivery of SPM using a gel or a thin film device, illustrated in panel B as applied to rabbit vein grafts in-vivo. PLGA films (“wraps”) were created with a thin bi-layered design for unidirectional release and loaded with 1 μg of RvD1. Delivery of RvD1 with gel or wrap decreased rabbit vein graft hyperplasia at 28 days post-implantation, while vehicle controls had no effect (C,D).

Adapted with permission from ref 104

1.4. Clinical Application

1.4.1. Human Research

Many studies have investigated the relationship between omega-3 PUFAs and cardiovascular health[106]. Randomized clinical trials testing omega-3 PUFA supplementation have demonstrated variable results[107,108]. A randomized trial conducted in Italy from 1993–1995 demonstrated that oral supplementation of n-3 PUFA in patients who recently suffered myocardial infarction reduced the relative risk of death, nonfatal myocardial infarction or stroke by 10%[109]. More recently, the REDUCE-IT trial demonstrated that twice daily oral supplementation of 2g icosapent ethyl, a highly purified EPA ethyl ester, significantly lowered the risk of ischemic events, including cardiovascular death, in patients with elevated triglyceride levels despite statin therapy[110]. On the other hand, the VITAL trial demonstrated that 1g of marine omega-3 fatty acid once daily did not reduce the risk of major cardiovascular events[111].

Variations in dosing, composition and formulation may affect the interpretation of these results. Most of these studies tested lower doses of marine oil supplements and focused on major clinical endpoints including myocardial infarction, stroke, and mortality.

With the discovery of SPM and their precursors, more contemporary studies have been able to study the effect of oral PUFA and SPM supplementation on the overall lipid and inflammatory profile of patients with vascular disease. Oral supplementation with omega-3 fatty acids increases circulating plasma levels of SPM and their intermediates [112,113]. The OMEGA-PAD-I trial demonstrated that 1-month daily supplementation with 4.4 g of fish oil in patients with peripheral arterial disease resulted in a decrease in triglycerides and an increase in the omega-3 index and pathway markers SPM production, such as 5-hydroxyeicosapentaenoate (5-HEPE), 15-HEPE, 18-HEPE, and 4-HDHA, but without a concomitant increase in circulating SPM levels or changes in systemic inflammatory markers[112]. Secondary analysis of this data demonstrated a strong correlation between the observed increase in omega-3 index and production of SPM production pathway markers[114]. The OMEGA-PAD-II trial continued the experiment for 3 months, and demonstrated significant increases in the SPMs lipoxin A₄, and RvE3, although there were no changes in markers of systemic inflammation or clinical indicators of disease severity[113]. The OMEGA-SPM trial studied the effect of an oral supplement containing both marine oils and mono-hydroxylated SPM precursors, such as 17-HDHA and 18-HEPE, in PAD patients and found a shift in lipid profile favoring SPM over PG which was associated with an increase in phagocytic activity in circulating monocytes and macrophages, as well as a reduction in several pro-inflammatory markers on circulating monocytes[115].

1.4.2. Challenges to Clinical Application

The application of SPM as either therapeutics or diagnostic tools faces several challenges. SPM are unstable molecules which are rapidly metabolized by ubiquitously present dehydrogenases and reductases[27], limiting their durability. They are synthesized and active locally, hence the physiologic relevance of circulating plasma levels is not known. From a pharmacologic standpoint, the synthesis of many SPM is complex with unstable intermediates. Optimal pharmacologic dosing and duration of treatment for SPM therapeutics is unclear. With the multitude of enzymes and targets present in the biosynthesis and interactions of SPM, it is unclear what formulation might best promote resolution in the human physiology.

Clinical and in vivo studies demonstrate that the nutritional approach to supplement resolution mechanisms presents a variety of challenges. The balance between omega-3 and omega-6 metabolomes and their utilization of substrate and biosynthetic enzymes are complex and may involve direct competition and the production of varying downstream prostanoids and SPM [6,11,116–118]. The resultant lipid mediator isomers may have effects of varying potency or may not interact equally (or even competitively) with the analogous receptor[29,57,119,120]. Individual patient characteristics such as age and underlying disease (e.g. CAD, HTN, DMII, smoking) may adversely impact the pathways of lipid mediator synthesis and resolution biology[121]. Tissue specific context is also critically important for synthesis, degradation, and biologic activity of SPM. Thus, oral administration of precursors rather than active SPM may not achieve adequate local concentrations of lipid mediators[122]. In cases where localized disease is of interest, targeted delivery methods may lead to better outcomes.

Bioavailability and stability are important considerations for therapeutics and remain a key challenge for bioactive lipids. Several synthetic analogs of SPM and SPM receptor agonists have been described in an attempt to replicate the bioactivity of SPM with improved durability and simpler synthesis. To date four generations of LXA₄ analogs have been produced which have reduced metabolic inactivation and improved stability and potency, including the BLXA₄-ME analog which has been developed into an oral rinse to treat gingivitis[123]. An analog of RvE1 known as RX-10045, is being used in a stable aqueous micellar formulation for the topical treatment of corneal inflammation[124]. Analogs of other SPM have also been produced, including benzo-diacetylenic-17R-RvD1-methyl ester (BDA-RvD1) and DRV1/GPR32 agonists [125,126]. Further studies are required to determine their in-vivo potency.

Direct administration of SPM to areas of vascular disease may address the issue of achieving clinically significant local concentrations. As an example, investigators directly injected an SPM cocktail in a human blister model and demonstrated reduced PMN counts as a marker of enhanced resolution[21]. We developed a biodegradable poly(lactic-co-glycolic acid) thin film for controlled, directional release of RvD1. Use of this drug-eluting device was associated with reduced neointimal hyperplasia in a rabbit bypass grafting model (Figure 1) [104]. Shi et al directly loaded AT-RvD1 into the fibers of a prosthetic polycaprolactone electrospun vascular graft, which resulted in reduced inflammation and promoted vascular tissue regeneration in a rat model of aortic bypass[105]. These methods of local SPM delivery are relevant to surgical settings with direct exposure of the vessel of interest. Other carriers, such as nanoparticles, may provide protection of SPM from enzymatic degradation, and could be developed for intravascular administration or as coatings on catheter-based devices such as balloons or stents[127]. For example, in a mouse model of ischemic stroke, neutrophil membrane-derived nanovesicles loaded with RvD2 were administered intravenously to target and treat the acute inflammatory response following brain ischemia and reperfusion, which resulted in reduced inflammation and improved neurologic function[86]. Fredman et al developed a collagen-IV targeted nanoparticle containing Ac2–26, an Annexin A1 mimetic that activates the FPR2/ALX receptor, which was able to improve plaque stability, reduced oxidative stress, and decreased necrosis of the core in a mouse model of atherosclerosis[16].

Though there are some studies demonstrating relative deficiency of certain SPM in cardiovascular disease states, biomarkers correlating with improved resolution and clinical outcomes are yet to be identified[52,60–63]. This is an important need in the clinical arena, as it would provide a therapeutic resolution target analogous to the use of C-reactive protein for cardiovascular anti-inflammatory trials[2,128]. Circulating plasma levels of SPM have been measured in various studies [118,52,114,86] and are often indexed to either total prostaglandins or leukotriene B4. In a study of patients with known cardiovascular disease with significantly reduced levels of circulating RvD_{n-3 DPA}, in vitro mixing of RvD_{n-3 DPA} with these patients’ blood resulted in significant dose-dependent reduction in platelet and leukocyte activation[63]. Further work is required to validate such measurements in the context of clinical settings of inflammatory disease. As researchers elucidate the pharmacodynamics, pharmacokinetics, and expression of resolution receptors and enzymes in the cardiovascular system, more directed approaches to translating resolution biology to cardiovascular disease can take place.

1.5. Conclusion

Inflammation is central to cardiovascular diseases, and responses to therapeutic interventions. The identification of biochemical pathways that actively mediate resolution offers new opportunities for monitoring disease progression, treatment responses, and therapeutics. SPM and their receptors are critical drivers of resolution, supported by a strong foundation of basic and translational science. Much work is required to precisely identify the molecular and cellular pathways of resolution involved in specific cardiovascular pathologies. Acute clinical settings such as thrombosis, reperfusion, invasive procedures, or surgery may offer the optimal initial opportunities to leverage SPM or their analogues as therapeutics. The development of novel drug delivery platforms and improved SPM formulations is ongoing and heralds future clinical applications for the cardiovascular patient.

Article Highlights:

• SPM are generated locally, resolve inflammation, and promote tissue repair

• Biologic activity of SPM is mediated via G-protein coupled receptor interactions

• SPM are beneficial in animal models of cardiovascular disease

• SPM-based therapeutics will require novel formulation and delivery platforms

• More research is needed into mechanisms of resolution in cardiovascular disease

Table of Abbreviations

SPM

Specialized pro-resolving lipid mediator

EC

Endothelial cell

vSMC

Vascular smooth muscle cell

PUFA

Polyunsaturated fatty acid

EPA

Eicosapentaenoic acid

DPA

Docosapentaenoic acid

DHA

Docosahexaenoic acid

RvD#

D-series resolvin #

RvE#

E-series resolvin #

Mar1

Maresin1

GPCR

G-protein coupled receptor

LO

Lipoxygenase

COX

Cyclooxygenase

AA

Arachidonic acid

PDGF

Platelet derived growth factor

FGF

Fibroblast growth factor

PMN

Polymorphonucelar cells/neutrophils

MCP-1

Monocyte chemoattractant protein-1

IL-#

Interleukin-#

LXA₄

Lipoxin A₄

PD1

Protectin D1

NIH

Neointimal hyperplasia

AT

Aspirin triggered

CV

Cardiovascular

AMP/ADP

Adenosine mono/di phosphate

HEPE

Hydroxyeicosapentaenoate

HDHA

Hydroxydocosahexaenoic acid

IR

Ischemia/reperfusion