Apo AI Nanoparticles Delivered Post Myocardial Infarction Moderate Inflammation | Circulation Resear

Introduction

Meet the First Author, see p 1345

The global prevalence of severe secondary complications after acute myocardial infarction (AMI), including heart failure, are continuing to rise secondary to obesity, type 2 diabetes (T2D), and an aging population.1,2 Current therapy for AMI is directed toward restoration of coronary blood flow (reperfusion) through medications or revascularization procedures including via percutaneous coronary intervention and coronary artery bypass grafting. Although reperfusion strategies are successful in limiting injury to the heart, patients with AMI remain at increased risk for secondary events, including heart failure both in the short-term (weeks) and long-term (months-years).3 Indeed, cardiac tissue recovery postinfarction is dependent, not only on the size of the acute infarct but also on the quantitative characteristics of the reparative response.

One of the earliest responses to ischemia is the infiltration of immune cells into the damaged myocardium where they play a key role in reparative pathways necessary for cardiac recovery. However, unbalanced or exaggerated inflammation also contributes to adverse fibrotic remodeling and cardiomyocyte apoptosis, aggravating tissue damage.4,5 Studies have shown that neutrophils initiate and amplify the cardiac inflammatory response including by attracting the proinflammatory monocyte subtype and that limiting their abundance or function improves cardiac repair.5,6 However, neutrophil depletion in experimental AMI results in aggravation of adverse remodeling and impaired cardiac function.7 These data highlight that limitation of inflammation is important to optimal postischemic heart recovery but that maintaining inflammatory signaling is also necessary for tissue healing. A large number of preclinical studies in post–myocardial infarction models have demonstrated protective effects of anti-inflammatory therapies that target inflammatory integrins, chemokines, or cytokines.8 Although the clinical translation of these targeted anti-inflammatory interventions remains challenging, the recent Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) trial examining the IL (interleukin)-1β inhibitor, canakinumab, showed reduction of both recurrent cardiovascular events and hospitalization for heart failure in patients with a previous AMI.9,10

HDL (high-density lipoprotein) particles and their constituents have recently been identified as a potential therapeutic approach to improve heart recovery in animal models of myocardial infarction11,12 and have demonstrated immune-modulatory capacity in other contexts.13–16 We have recently shown that a single intravenous dose of human Apo AI (apolipoprotein AI) reconstituted with phosphatidylcholine (n-apo AI [Apo AI nanoparticles]; CSL111) delivered immediately postcardiac ischemia at the onset of reperfusion improved heart function in mice.17 This benefit was mediated, at least in part, through enhanced cardiac glucose uptake, which would be expected to limit ischemic injury under hypoxic conditions through promotion of ATP production via glycolysis.17,18 Beyond this mechanism, previous studies have reported multiple actions of HDLs which may benefit postischemic heart remodeling, including anti-inflammatory properties. Clinical and preclinical studies indicate that HDL reduced neutrophil and monocyte activation as well as ex vivo adhesion to endothelial cells via reduction of the surface expression of the key integrin CD11b.19–21 An in vivo study in mice also provided evidence that HDL delivered before ischemia-reperfusion reduces the number of neutrophils within ischemic cardiac tissue.11 However, it is unknown whether HDL delivered in a therapeutic context after myocardial infarction is sufficient to modulate the acute postischemic inflammatory response for beneficial outcomes. In particular, the mechanisms by which HDL modulates systemic and cardiac inflammation remain undefined. Furthermore, no study has examined effects of HDL treatment on the postischemic inflammatory response in the setting of T2D where levels of inflammation are higher and the function of HDL particles is impaired.

Our study demonstrates that a single injection of n-apo AI delivered immediately after myocardial infarction reduces the systemic and cardiac inflammatory response in metabolically healthy and insulin-resistant mice. These observations are in the context of n-apo AI mediated improvement in cardiac function, as demonstrated in our previous study of identical experimental design.17 We further report direct actions of n-apo AI on myocardium and leukocytes, with binding of n-apo AI to neutrophils and monocytes. Our human data further explore the clinical translational relevance of the anti-inflammatory effects of n-apo AI in patients with T2D.

Methods

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Mice

All animal research was conducted in accordance with the National Health and Medical Research Council of Australia guidelines and was approved by the Alfred Medical Research and Education Precinct Animal Ethics committee.

Wild-type (WT; C57Bl/6) mice were procured from the Alfred Medical Research and Education Precinct Animal Services. SR-BI (scavenger receptor BI) knockout (B6;129S-Scarb1tm1Kri/J, Scarb1−/−) mice were initially procured from Jackson Research Laboratory and then maintained on a mixed C57Bl6/J;129S1 background, with heterozygote mice bred together to obtain the Scarb1−/− and WT littermate mice. All mice used in this study were 8-to-10-week-old males and were fed either a standard chow (14 MJ/kg; 8% fat, 21% protein, 71% carbohydrate; Specialty Feeds) or a high-fat diet (HFD; 19 MJ/kg; 43% fat, 21% protein, 36% carbohydrates; SF04-001; Specialty Feeds) for 8 weeks. HFD-induced obesity and insulin resistance of mice were assessed, as previously published.22 Only male animals were studied to align results with our previous work.17

Primary end point was the effect of n-apo AI on the number of leukocytes recruited into the ischemic heart. Animals were randomized to surgery and treatment groups (random assignment, Excel), and analysis of data was performed blinded. Power calculations to determine group sizes were based on variance measures from our previous studies or literature using a power of 0.8 and alpha 0.05.14,17 Criteria of exclusion were used for the HFD (blood glucose and insulin levels, fat mass>20% body weight) and recovery postsurgery (>15% body weight loss, inactivity). No animals were excluded in this study.

N-apo AI Preparation

n-apo AI (CSL111; CSL Behring) are a complex composed of human Apo AI purified from plasma and soybean phosphatidylcholine in a molar ratio of 1:150 to form particles resembling nascent HDL.23 Each vial was reconstituted with 50 mL sterile 0.9% sodium chloride solution to a final concentration of 25 mg Apo AI/ml. Details on fluorescent labeling of n-apo AI in the Data Supplement.

Coronary Artery Occlusion and Reperfusion Surgery

Myocardial ischemia surgery was performed as previously described.17 Briefly, mice received an intraperitoneal injection of anesthetic (ketamine [100 mg/kg], xylazine [20 mg/kg]) together with atropine (0.96–1.2 mg/kg) and carprofen (5 mg/kg) and a ligation of the left anterior descending coronary artery which was performed and then released 30 minutes later to commence reperfusion. Sham control animals underwent the same surgical procedure without coronary artery occlusion.

Humans

Blood samples were from a previous clinical trial approved by the Alfred Hospital Ethics Committee, performed in accordance with the Declaration of Helsinki, and with informed written consent from all participants (https://www.clinicaltrials.gov; Unique identifier: NCT00395148).21,24 Twelve fasting male patients with T2D received both n-apo AI (CSL111; 80 mg/kg of Apo AI) and a saline placebo as an intravenous infusion over 4 hours on separate occasions in a randomized cross-over design study (Participant Baseline Characteristics in Table IA in the Data Supplement). The primary end point for this clinical study was plasma glucose whilst other metabolic parameters, including cholesterol efflux capacity and inflammatory markers, were among the secondary and exploratory measures.21,24 Blood from participants was collected at 4 and 72 hours postinfusion commencement for a complete blood count (Alfred Health Clinical Haematology).

Statistical Analysis

All data were tested for normality using a Shapiro-Wilk test. When all groups passed the test for normality, a parametric test was used. This, depending on the number of groups involved or whether there were repeated measurements, involved an unpaired or paired Student t test, 1-way ANOVA, or 2-way ANOVA. When at least one of the groups did not pass the test for normality a nonparametric alternative was used, namely Mann-Whitney test in place of unpaired Student t test, Wilcoxon matched-pairs signed-rank test in place of paired Student t test, or Kruskal-Wallis test in place of 1-way ANOVA. Given the absence of a nonparametric equivalent to a 2-way ANOVA, data were log-transformed and in all instances achieved normality (using a Shapiro-Wilk test), and a 2-way ANOVA was applied on the transformed data. Only statistically significant differences (P<5.0×10−2) are shown in figures. Statistical analyses were conducted using GraphPad Prism (v7.04). More details can be found in the Data Supplement.

Additional methods can be found in the Data Supplement.

Results

N-apo AI Limits the Postischemic Inflammatory Response in Metabolically Healthy and Insulin-Resistant Mice

To study the effect of n-apo AI on the postischemic acute inflammatory response, we replicated the same experimental conditions of our previous study in which we demonstrated improvement in postischemic cardiac function with n-apo AI treatment.17 Mice were fed with either a normal chow diet (metabolically healthy) or an HFD (insulin-resistant) and then received a single intravenous bolus of either saline (control) or n-apo AI (CSL111, 80 mg/kg) at the onset of reperfusion after surgically induced myocardial ischemia. Flow cytometry showed that n-apo AI reduced the number of circulating leukocytes (CD45+ cells) 1 day after myocardial ischemia-reperfusion in both chow and HFD mice (Figure 1A, P=4.15×10−3). Analysis of leukocytes reservoirs, the bone marrow and the spleen, showed that more leukocytes were detected in the spleen of chow and HFD mice treated with n-apo AI compared to saline (Figure 1B, P=5.38×10−3), but no statistical difference with treatment was observed in the bone marrow (Figure 1C). Importantly, a single bolus of n-apo AI decreased the number of leukocytes recruited into the ischemic left ventricle (LV) 1 day postischemia in both diet groups (Figure 1D, P=2.51×10−2).

Figure 1. n-apo AI (apo AI nanoparticles) limits the postischemic inflammatory response in metabolically healthy and insulin-resistant mice. Number of leukocytes quantified by flow cytometry after surgically induced myocardial ischemia-reperfusion in chow-fed and high-fat diet (HFD)–fed mice treated with either saline or n-apo AI (CSL111, 80 mg/kg). Total number of leukocytes (CD45+ cells) in (A) blood, (B) spleen, (C) bone marrow, and (D) heart (left ventricle) 1 d post–ischemia-reperfusion (n=12–18/group). Number of (E) neutrophils, (F) T lymphocytes, (G) B lymphocytes, and (H) monocytes measured in left ventricle 24 h (neutrophils), 3 d (T and B lymphocytes), or 5 d (monocytes) post–ischemia-reperfusion (n=5–13/group). I, Ratio of Ly6CLow to Ly6CHigh monocytes in left ventricle 5 d after ischemia-reperfusion (n=6–8/group). White bar: saline, gray bar: n-apo AI. Data presented as mean±SEM and analyzed using a 2-way ANOVA on (A, D, E, H, and I) normal or (B, C, F, and G) log-transformed data; P values for treatment effect, no diet, or interaction effect. N per group is detailed in Table II in the Data Supplement.

Compared with saline, n-apo AI reduced recruitment of the entire spectrum of inflammatory cells to the LV: neutrophils at 1 day postischemia (Figure 1E, P=2.71×10−2), T lymphocytes and B lymphocytes at 3 days (Figure 1F through 1G, P=4.94×10−3 and P=2.71×10−2, respectively), and monocytes at 5 days (Figure 1H, P=2.54×10−3). Five days after ischemia-reperfusion, the ratio of anti-inflammatory (Ly6CLow) to proinflammatory (Ly6CHigh) inflammatory monocyte subtypes (Ly6CLow/Ly6CHigh) was increased in the LV of mice treated with n-apo AI compared with saline (Figure 1I, P=4.12×10−2). These results suggest that n-apo AI may modulate the recruitment or the differentiation of monocyte subtypes in favor of the anti-inflammatory population (Ly6CLow).

Before myocardial infarction, HFD-induced insulin-resistant mice presented signs of monocytosis when compared with chow-fed mice, although the total leukocyte number was not statistically different (Figure IA through IG in the Data Supplement). Post-MI, the number of circulating leukocytes, including monocytes, was elevated but not statistically different between dietary groups (Figure 1A).

N-apo AI Reduces Postischemic Plasma Levels of Cardiac Troponin-I in Metabolically Healthy And Insulin-Resistant Mice

Compared with saline, n-apo AI treatment reduced plasma levels of the clinical biomarker of cardiac injury, cardiac troponin-I, 24 hours post-MI in both chow and HFD mice (Figure 2, P=1.01×10−2).

Figure 2. n-apo AI (apo AI nanoparticles) reduces postischemic plasma levels of cardiac troponin-I in metabolically healthy and insulin-resistant mice. Plasma levels of cardiac troponin-I measured 24 h after surgically induced myocardial ischemia-reperfusion in chow-fed and high-fat diet (HFD)–fed mice treated with either saline or n-apo AI (CSL111, 80 mg/kg; n=6/group except n=5/HFD+n-apo AI). White bar: saline, gray bar: n-apo AI. Data presented as mean±SEM and analyzed using a 2-way ANOVA, P value for treatment effect, no diet, or interaction effect.

Post-MI, plasma levels of cardiac troponin-I were not statistically different in chow and HFD mice (Figure 2, no diet effect) and, as described above, the inflammatory response was also not statistically different between dietary groups (Figure 1). Thus, mice for the rest of the study were on a normal chow diet.

N-apo AI Modulates the Recruitment of Leukocytes into the Ischemic LV

Myocardial infarction elicited a large increase in the levels of the chemokines that specifically attract neutrophils (CXCL1 [chemokine (C-X-C motif) Ligand], CXCL2, and CXCL5) and monocytes (CCL2, chemokine [(C-C motif) Ligand 2]) in the ischemic left ventricle (Figure 3A through 3D). One day post–ischemia-reperfusion, the levels of these CXCL1, CXCL2, CXCL5, and CCL2 chemokines were strongly reduced in left ventricles of mice treated with n-apo AI compared with those treated with saline (Figure 3A through 3D, P=2.04×10−2, P=1.25×10−2, P=2.02×10−2, and P=1.45×10−2, respectively). By flow cytometry, no statistical difference was observed with treatment on the level of circulating neutrophil CXCR2 (C-X-C chemokine Receptor 2), the receptor for CXCL1, CXCL2, and CXCL5, at 1 day post-MI (Figure 3E). Similarly, there was no statistical difference in the level of CCR2 (C-C chemokine Receptor 2) on monocytes, the receptor for CCL2, at 3 days postsurgery with n-apo AI treatment compared with saline (Figure 3F). However, n-apo AI reduced the level of integrin CD11b on the proinflammatory monocyte subtype Ly6CHigh at 1 day postischemia and on the anti-inflammatory monocyte subtype Ly6CLow at 3 days (Figure 3G, P=4.46×10−2 and P=1.85×10−2, respectively). There was no statistical difference in CD11b surface expression on neutrophils 1 day post-MI (Figure 3G). These results highlight a dual action of n-apo AI on chemokine production in ischemic cardiac tissue as well as on monocyte adhesion and migration, supporting a cooperative limitation of the inflammation in the left ventricle.

Figure 3. n-apo AI (apo AI nanoparticles) modulates recruitment of neutrophils and monocytes in ischemic heart tissue. Levels of expression of chemokines (A) CXCL1 (chemokine [C-X-C motif] Ligand 1), (B) CXCL2, (C) CXCL5, and (D) CCL2 (chemokine [C-C motif] Ligand 2) in left ventricles of mice treated with saline or n-apo AI (80 mg/kg) 1 d post–myocardial infarction (MI) or sham-surgery control (n=4/SHAM+saline, n=5/SHAM+n-apo AI, n=11/MI groups for A and C, n=10/MI groups for B and D). E, Surface expression of CXCR2 (C-X-C chemokine Receptor 2) on circulating neutrophils at 1 d post-MI (n=10/saline, n=11/n-apo AI) and (F) CCR2 (C-C chemokine Receptor 2) surface expression on monocyte subtypes at 3 d post–ischemia-reperfusion (n=7/group). G, Surface expression of CD11b on circulating neutrophils and monocyte subtypes 1 and 3 d post-MI (n=10/saline, n=11/n-apo AI for day 1; n=7/group for day 3). Expression measured by flow cytometry as the median intensity fluorescence (MFI) of the marker and expressed as percentage of control (saline). White bar: saline, gray bar: n-apo AI. Data presented as mean±SEM and analyzed using an unpaired Student t test for all comparisons except within SHAM groups where saline and n-apo AI groups were compared using a Mann-Whitney test.

N-apo AI Enters Ischemic Cardiac Tissue

To explore the potential for a direct effect of n-apo AI on cardiac tissue, we studied its distribution after intravenous injection in mice subjected to ischemia-reperfusion or sham-surgery. The plasma level of n-apo AI was still measurable 1 day postinjection and then significantly declined 3 and 5 days later and was not detectable 14 days postinjection (Figure 4A). One day postcardiac ischemia-reperfusion, we measured 6-fold more n-apo AI in the left ventricle and 5-fold more in the spleen than in the quadriceps (Figure 4B; P=1.21×10−4 and P=2.41×10−2, respectively). Almost no n-apo AI was found in the right ventricle, and no n-apo AI was detectable in bone marrow (Figure 4B). Interestingly, we detected more n-apo AI in the left ventricle and in the spleen of mice subjected to ischemia compared with sham-operated mice (Figure 4C and 4D, P=1.39×10−3 and P=2.17×10−2, respectively) but no statistical difference in the quadriceps (Figure 4E), right ventricle (Figure 4F), or plasma (Figure 4A).

Figure 4. n-apo AI (apo AI nanoparticles) enters the ischemic left ventricle and to the spleen.A, Plasma level of n-apo AI measured post–myocardial infarction (MI) or sham-surgery and n-apo AI injection (80 mg/kg; n=3–6/group). B, Quantity of apo AI per mg of left ventricle (LV), spleen, quadriceps, right ventricle (RV), or per femur bone marrow (BM) 1 d post-MI and n-apo AI injection and expressed as a percentage of LV (n=5–8/group). Apo AI quantification in (C) LV, (D) spleen, (E) quadriceps, and (F) right ventricle (RV) 1 d post-MI or sham-surgery and n-apo AI or saline injection (n=5–13/group). White bar: saline, gray bar: n-apo AI. G, Visualization of labeled n-apo AI–Cy5.5 in RV and LV 1 d postcardiac ischemia-reperfusion: n-apo AI–Cy5.5 in pink, endothelial cells in green, matrix extra-cellular in red, nuclei in blue. H, Colocalization of n-apo AI–Cy5.5 and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in LV 1 d post-MI: n-apo AI–Cy5.5 in pink, nuclei in blue, TUNEL in green. White arrows indicate apoptotic nuclei. I, Visualization of Ly6C+ neutrophils and n-apo AI–Cy5.5 in LV 1 d postcardiac ischemia-reperfusion: n-apo AI–Cy5.5 in pink, nuclei in blue, neutrophils in green. White arrows indicate neutrophils. Data presented as mean±SEM. A, Two-way ANOVA, P=2.24×10−11 for time effect, no group (surgery), or interaction effect; (B) One-way ANOVA and post hoc Tukey multiple comparisons test, all pairwise comparisons (10 tests); (C, D, E, and F) unpaired Students t test; groups with no detectable values were excluded from statistical analysis. N per group is detailed in Table II in the Data Supplement.

N-apo AI was labeled with fluorochrome Cy5.5 (n-apo AI–Cy5.5) to allow histochemical visualization, and Cy5.5 labeled albumin (albumin-Cy5.5) was used as a negative control. N-apo AI–Cy5.5 (80 mg/kg, delivered intravenously at the start of reperfusion) was present within some cardiomyocytes in the infarct and peri-infarct regions of the LV 1 day post–ischemia-reperfusion but could not be visualized in right ventricles (Figure 4G). No staining was observed in either ventricle with the Cy5.5 labeled albumin control. In the LV, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining colocalized with n-apo AI–Cy5.5 staining, suggesting that n-apo AI was localized to cells undergoing apoptosis (Figure 4H). Neutrophils were also found to colocalize with n-apo AI–Cy5.5 (Figure 4I). Thus, taken together, these data indicate that n-apo AI enters ischemic and inflamed cardiac tissue, and interacts with apoptotic cardiomyocytes and leukocytes.

N-apo AI Binds to Neutrophils and Monocytes

We used n-apo AI–Cy5.5 (or negative control albumin-Cy5.5) to study direct interactions between leukocytes and n-apo AI by flow cytometry. Importantly when albumin-Cy5.5 was injected into mice subjected to ischemia-reperfusion or sham-surgery, there were no Cy5.5+ leukocytes in the blood, spleen, bone marrow, or left ventricle (Figure II in the Data Supplement).

One day post–ischemia-reperfusion and n-apo AI–Cy5.5 injection, 83.4±4.8% (mean±SEM) of the circulating neutrophils and 95.3±0.5% of the circulating monocytes were positive for n-apo AI–Cy5.5, whereas <8% of T cells were Cy5.5+ (Figure 5A). The median fluorescence intensity (MFI) of n-apo AI–Cy5.5 was also higher for circulating monocytes than neutrophils, implying that monocytes bound more n-apo AI than neutrophils (Figure 5B, P=2.65×10−5). With regard to monocyte subtypes, more proinflammatory monocytes (Ly6CHigh) were positive for n-apo AI–Cy5.5, and the MFI was higher compared to anti-inflammatory monocytes (Ly6CLow; Figure 5A and 5B, P=3.15×10−6 and P=9.07×10−6, respectively). The preferential binding of n-apo AI for monocytes and particularly for the Ly6CHigh monocytes was also observed in the spleen and bone marrow (Figure 5C through 5F). Importantly, monocytes and neutrophils recruited into ischemic left ventricles were also positive for n-apo AI–Cy5.5 at 3 days post–ischemia-reperfusion and n-apo AI–Cy5.5 injection. In LV, 91.2±0.9% of the neutrophils and 72.0±3.4% of the monocytes bound n-apo AI indicating that neutrophils showed higher binding for n-apo AI than monocytes (Figure 5G, P=9.09×10−6). At 3 days post-MI, the percentage of proinflammatory monocytes positive for n-apo AI–Cy5.5 was higher than the anti-inflammatory monocytes (Figure 5G, P=2.45×10−6); however, the MFI of n-apo AI–Cy5.5 was similar in the 2 subtypes (Figure 5H). Confocal microscopy observations of bone marrow–derived neutrophils incubated ex vivo with n-apo AI–Cy5.5 confirmed clear binding between n-apo AI and leukocyte (Figure 5I).

Figure 5. n-apo AI (apo AI nanoparticles) and leukocytes interaction post–myocardial infarction (MI). Percentage of (A) circulating leukocytes, (C) spleen-derived leukocytes (E) bone marrow–derived leukocytes, and (G) left ventricle-derived leukocytes that are positive for n-apo AI–Cy5.5 at 1 or 3 d post–myocardial infarction (MI) and n-apo AI–Cy5.5 injection (80 mg/kg). Median fluorescence intensity (MFI) of n-apo AI–Cy5.5 of (B) circulating, (D) spleen-derived, (F) bone marrow–derived, and (H) left ventricle–derived leukocytes 1 or 3 d post–ischemia-reperfusion and n-apo AI–Cy5.5 injection (n=8/group for A–F, n=7/group for G and H). Dark gray bar: T cells, white bar: neutrophils, gray bar: monocytes. I, Visualization of bone marrow–derived neutrophils incubated ex vivo with either albumin-Cy5.5 or n-apo AI–Cy5.5 (nuclei in blue, albumin or n-apo AI in red). Data presented as mean±SEM. One-way ANOVA and post hoc Tukey multiple comparisons test, all pairwise comparisons (3 tests) when comparing 3 groups; Unpaired Student t test when comparing 2 groups.

Interestingly, we observed greater interactions between n-apo AI and circulating, spleen-derived, and bone marrow–derived leukocytes 1 day after a surgically induced myocardial infarction compared with sham-surgery (Figure IIA through IIC in the Data Supplement). In the left ventricle, n-apo AI/neutrophil interactions were enhanced in MI compared with sham-operated mice, but no statistical differences were observed with respect to monocytes, potentially due to the early collection time (1 day postsurgery; Figure IID in the Data Supplement).

N-apo AI/Monocyte Interactions Are Partially Dependant on SR-BI

It has previously been reported that the anti-inflammatory effects of HDL particles on human monocytes and neutrophils require SR-BI.19,20 Thus, to determine whether SR-BI is involved in n-apo AI/leukocyte interactions, surface expression of SR-BI on circulating leukocytes was measured by flow cytometry. One day postischemia, >90% of monocytes and neutrophils, but <7% of T cells, were positive for SR-BI (Figure 6A). Interestingly, the percentage of proinflammatory monocytes (Ly6CHigh) positive for SR-BI was higher than the anti-inflammatory monocytes (Ly6CLow; Figure 6B, P=1.66×10−3), and the proinflammatory monocytes (Ly6CHigh) also showed a higher level of expression of SR-BI (expressed as MFI) compared with anti-inflammatory monocytes (Ly6CLow; Figure 6D, P=4.09×10−5). These results align with our data on n-apo AI/leukocyte binding, particularly with regard to monocyte subtypes (Figure 5), and supports (albeit inconclusively) a mechanistic interaction between SR-BI monocyte surface expression and n-apo AI binding. To more directly determine whether SR-BI mediates n-apo AI/monocyte interactions, bone marrow–derived monocytes from WT or SR-BI knockout (Scarb1−/−) mice were incubated in vitro with n-apo AI–Cy5.5 for 1 day. For both WT and Scarb1−/− mice, about 95% of monocytes and subtypes were positive for n-apo AI–Cy5.5 (Figure 6E and 6F). However, we observed a 22.2±2.3% reduction in MFI for n-apo AI–Cy5.5 in Scarb1−/−-derived monocytes, as well as the Ly6Chigh and Ly6CLow monocyte subtypes compared with WT (Figure 6G, P=2.36×10−3 for monocytes and Figure 6HP=1.09×10−4 for subtypes). These data indicate that in absence of SR-BI, n-apo AI can still bind to monocyte, but each individual immune cell carries less n-apo AI, highlighting the partial role of SR-BI in n-apo AI/monocyte interactions.

Figure 6. SR-BI (scavenger receptor BI) is involved in n-apo AI (apo AI nanoparticles)/monocyte interactions.A and B, Percentage of circulating leukocytes that expressed SR-BI and (C and D) median fluorescence intensity (MFI) of SR-BI for each leukocyte population at 1 d post–myocardial infarction and n-apo AI injection (80 mg/kg, or saline control; n=11/group). E and F, Percentage of wild-type (WT) or Scarb1−/−-derived monocyte and subtypes binding to n-apo AI after 1 d of incubation (n=5/WT, N=6/Scarb1−/−). G and H, MFI of n-apo AI–Cy5.5 on total and monocyte subtypes after 1 d of incubation (n=5/WT, N=6/Scarb1−/−). White bar: saline or WT, gray bar: n-apo AI, hatched bar: Scarb1−/−. Data presented as mean±SEM. A, C, E, and G, Unpaired Student t test, (B, D, F, and H) 2-way ANOVA on normal or log-transformed data for (B), P value for cell effect, no treatment or interaction effect for (B, D, and F), P value for genotype effect, no cell or interaction effect for (H).

N-apo AI Infusion in Patients With T2D Reduces the Number of Circulating Leukocytes

We investigated whether anti-inflammatory effects of n-apo AI observed in our preclinical model could translate into humans. The same n-apo AI preparation with weight-adjusted dosing was used in our human trial, where 12 fasting unmedicated male patients with T2D received both n-apo AI (CSL111, 80 mg/kg of Apo AI, 4-hour intravenous infusion) or a saline placebo on separate occasions in a randomized cross-over design study (Table I in the Data Supplement). 21,24 At completion of the infusion (4 hours), the number of circulating leukocytes was reduced with n-apo AI compared with saline, and this effect was still evident 72 hours later (Figure 7A, P=1.61×10−2 and P=4.88×10−3 respectively). In terms of leukocyte subpopulations, n-apo AI decreased the number of circulating monocytes both immediately and 72 hours postinfusion (Figure 7B, P=1.37×10−2 and P=1.63×10−2, respectively), whereas neutrophils were reduced at 72 hours (Figure 7C, P=1.59×10−2) and lymphocytes immediately postinfusion (Figure 7D, P=4.88×10−4). There was no statistical difference in the number of eosinophils or basophils with n-apo AI intervention compared to placebo (Table IB in the Data Supplement). These results demonstrate a rapid anti-inflammatory effect of n-apo AI in humans, supporting translational relevance of our preclinical data.

Figure 7. n-apo AI (apo AI nanoparticles) infusion in patients with type 2 diabetes (T2D) reduces the number of circulating leukocytes. Number of circulating (A) leukocytes, (B) monocytes, (C) neutrophils, and (D) lymphocytes in male patients with T2D who received both n-apo AI (80 mg/kg) or a saline placebo on separate occasions in a randomized cross-over design study (n=12/group).21,24 White bar: saline, gray bar: n-apo AI. Data presented as mean±SEM. A and D, Wilcoxon matched-pairs signed-rank test; (B and C) paired Student t test, n-apo AI vs saline, for each time point.

Discussion

In the current study, we demonstrate that a single bolus of n-apo AI delivered immediately after cardiac ischemia-reperfusion reduces systemic and cardiac inflammation in both metabolically healthy and insulin-resistant mice. We further report direct actions on both the ischemic myocardium and leukocytes with direct binding to neutrophils and monocytes. The reduction in inflammation with the n-apo AI intervention was also associated with lower plasma levels of cardiac troponin-I, a clinical diagnostic biomarker of MI, which has been associated with infarct size and cardiac functional recovery.25,26 These results align with our previous study in which we used identical experimental conditions to the current study and demonstrated that n-apo AI significantly reduces infarct size and improves postischemic left ventricular structure and function, in part, through modulation of cardiac glucose metabolism.17 We now demonstrate that n-apo AI also modulates the postischemic inflammatory response, contributing to the recovery of cardiac tissue and ventricular function. These preclinical results are supported by our clinical data showing an anti-inflammatory effect of n-apo AI infusion in patients with T2D.

N-apo AI and Leukocyte Activity

Our study shows a reduction in the number of leukocytes recruited into ischemic cardiac tissue after treatment with n-apo AI. This was due to a lower number of leukocytes being mobilized from the spleen into the circulation, in association with direct interactions of n-apo AI with both leukocytes and cardiomyocytes. Previously, it has been shown that infusion of n-apo AI (CSL111) or transgenic overexpression of apo AI can reduce myelopoiesis in mouse models of atherosclerosis.13,14 However, despite clear interactions between n-apo AI and bone marrow–derived leukocytes, no statistical difference in the number of leukocytes in the bone marrow was observed with n-apo AI in our study. This likely relates to the AMI context where there is a greater acute inflammatory stimulus than atherosclerosis. Moreover, it has been shown that splenic leukocytes are the major contributor to the increase in blood leukocytes following an AMI.27 This aligns with our data that suggest an acute effect of n-apo AI on the mobilization of leukocytes from the spleen in the setting of AMI. Moreover, n-apo AI treatment reduced chemokine expression in the ischemic left ventricle, limiting attraction of immune cells to the heart. In addition, there was a reduction in the surface expression of the CD11b integrin, known to be involved in endothelial adhesion and transmigration, on circulating proinflammatory monocytes at 1 day postischemia, followed by a reduction of CD11b on anti-inflammatory monocytes at 3 days postischemia. This chronology aligns with the known cascade of recruitment of monocyte subtypes postischemia where proinflammatory monocytes are first recruited into cardiac tissue for clearance of dead cells around 3 days postischemia, followed by anti-inflammatory monocytes to limit inflammation at day 5. Moreover, our previous clinical studies demonstrated that peripheral blood monocyte CD11b expression, neutrophil adhesion to a fibrinogen matrix, and plasma level of integrin sVCAM-1 (soluble Vascular Cell Adhesion Molecule-1) were reduced with n-apo AI infusion compared with placebo in unmedicated patients with T2D.21,24 These results support pleiotropic anti-inflammatory effects of n-apo AI including on leukocyte activity, which would be expected to reduce their recruitment into cardiac tissue.

HDL has been shown in vitro to inhibit macrophage polarization from the anti-inflammatory subtype (M2) toward the proinflammatory subtype (M1) by inducing expression of M2-macrophage markers, as well as by reducing expression of M1-associated inflammatory markers.28,29 Circulating proinflammatory monocytes are also known to differentiate into anti-inflammatory monocytes to then be recruited to tissue where they contribute to tissue repair.30 Our data suggest that n-apo AI may positively modulate differentiation into the anti-inflammatory monocyte subtype. Indeed, early post–ischemia-reperfusion, we observed a preferential binding of n-apo AI to the proinflammatory monocyte subtype which was followed by a reduction of proinflammatory monocytes within cardiac tissue 5 days post–ischemia-reperfusion. Despite our lack of direct evidence, our new data are consistent with previous studies, reinforcing the possible role of n-apo AI in increasing polarization of the proinflammatory toward the anti-inflammatory monocyte subtype during acute MI.

Our study mostly focuses on neutrophils and monocytes; however, given that the n-apo AI intervention modulated cardiac levels of T and B lymphocytes and that recent studies have highlighted a significant role of lymphocytes in postischemic cardiac recovery,31,32 deeper analysis of the effect of n-apo AI on lymphocytes would be interesting to pursue in future studies.

Role of SR-BI in n-apo AI/Leukocyte Interactions

The n-apo AI used in this study can interact with known receptors for Apo AI including SR-BI, ABCA1, and ABCG1 (ATP-Binding Cassette transporters A1 and G1). Here, we demonstrated that interactions between n-apo AI and monocytes are dependent on SR-BI but not exclusively. We observed a link between in vivo SR-BI surface expression on leukocytes and n-apo AI/leukocyte interactions. We also measured ex vivo a 22.2±2.3% reduction of n-apo AI/monocyte interaction in Scarb1−/− compared with WT mice, thus implicating SR-BI, as well as other receptors. Translating these ex vivo findings into a physiological context remains complex due to the composition of n-apo AI changing after infusion into the circulation. Our previous human study demonstrated that within 4 hours of infusion of n-apo AI, both HDL-C (HDL cholesterol) and Apo AI levels were increased compared to placebo.24 It has also been demonstrated in humans that postinfusion, n-apo AI interacts with native HDL via spontaneous fusion and fission events.33 This suggests a possible enrichment of n-apo AI with other lipoproteins and lipids, increasing the number of possible ligand/receptor interactions and signaling pathways. S1P (sphingosine-1-phosphate), a major lipid mediator of HDL particles known to contribute to anti-inflammatory effect of HDL,11,34 may also have a role in n-apo AI anti-inflammatory effects demonstrated in this current study. Further studies are required to determine the role of SR-BI and potentially of S1P in the postischemic anti-inflammatory effects of n-apo AI.

Inflammation and Cardiac Tissue Preservation

This study demonstrates an anti-inflammatory effect of n-apo AI in the setting of AMI. Postischemic cardiac inflammation has been clearly linked to multiple aspects of cardiac repair, including fibrotic remodeling and revascularization. We have previously shown that animals treated with n-apo AI in an identical study design had reduced cardiac interstitial fibrosis and increased capillary density, which aligns with the anti-inflammatory effect demonstrated in this current study.17 These results are supported by recent preclinical studies where recombinant HDL also improved cardiac function and remodeling with reduction of cardiac fibrosis in the setting of diabetic cardiomyopathy and heart failure.35,36 Interestingly, our previous study also showed that n-apo AI protected cardiac tissue from ischemic injury by directly and rapidly increasing cardiac glucose uptake and utilization in cardiomyocytes. It is possible that by preserving cardiac cell metabolism through supporting glycolysis, n-apo AI may salvage myocardial cells by reducing their susceptibility to injury and indirectly reducing the inflammatory response. However, the current study also demonstrates direct effects of n-apo AI on leukocytes, with binding and subsequent modulation of inflammatory cell activity (CD11b expression on monocytes). N-apo AI also modulated the postischemic egress of leukocytes from the spleen. The potential of n-apo AI in the context of postischemic heart recovery is broadened by its multiple actions that may benefit postischemic heart recovery, which beyond stimulation of glycolytic cardiomyocyte metabolism and limitation of inflammation, extends to vasodilation and actions in potentially reducing and stabilizing coronary plaques including cholesterol efflux/reverse cholesterol transport, antithrombotic, and antioxidative properties.

Therapeutic Potential and Clinical Relevance

Decades of research have identified and tested anti-inflammatory therapies for the management of atherosclerotic coronary disease, with no success until recently.8–10 Broad anti-inflammatory interventions using glucocorticoids or NSAIDS have had mixed results in the acute phase post-MI.37,38 Targeted anti-inflammatory interventions against chemokines or integrins, such as CCL2 or CD18, have also been underwhelming and raised concerns about clinical translation as chemokine-mediated signaling is also important for anti-inflammatory and repair processes.39–42 However, new discoveries targeting the proinflammatory IL-1R (IL-1 receptor) or IL-1β have raised hopes for the management of postischemic cardiac inflammation. In the majority of preclinical AMI studies, both the IL-1R antagonist (anakinra) and the anti-IL-1β antibody (canakinumab) have been associated with clear beneficial outcomes.43–45 Recently, canakinumab showed benefit in a randomized, double-blind clinical trial of 10 000 patients with previous AMI where recurrent cardiovascular events and hospitalization for heart failure were reduced.9,10 These promising results provide positive support for long-term anti-inflammatory approaches for cardiovascular diseases. In the setting of myocardial infarction, one of the major challenges for anti-inflammatory therapies is in the timing of delivery. Targeting the acute phase postischemia to manage the acute unbalanced inflammatory response is crucial for cardiac tissue healing. In most studies, including with canakinumab,10 treatments were administrated weeks after the cardiac ischemic event and their potential effects on the acute inflammatory response are unknown. Our new therapeutic approach using n-apo AI delivered immediately after cardiac ischemia acts rapidly on multiple mechanisms controlling the cardiac postischemic inflammatory response. Our preclinical data demonstrate an overall anti-inflammatory effect of n-apo AI in association with beneficial outcomes including reduced infarct size and improved cardiac function, as demonstrated in our previous study.17 The clinical relevance of our findings is supported by our human data showing reduction in the number of circulating leukocytes after n-apo AI infusion. A current phase 3 clinical trial using n-apo AI (https://www.clinicaltrials.gov; Unique identifier: NCT03473223, CSL112, a reformulation of CSL111 with identical amounts of the active component [apo AI]) will answer critical questions regarding the efficacy of CSL112 on reducing recurrent major adverse cardiovascular events in patients presenting with acute coronary syndrome.

Nonstandard Abbreviations and Acronyms

AMI

acute myocardial infarction
ApoA-I

apolipoprotein AI
HDL

high-density lipoprotein
HDL-C

HDL cholesterol
HFD

high-fat diet
LV

left ventricle
MI

myocardial infarction
N-apo AI

apo AI nanoparticles
S1P

sphingosin-1-phosphate
SR-BI

scavenger receptor BI
T2D

type 2 diabetes

Acknowledgments

We thank Agus Salim for his advice on statistical analyses.

Sources of Funding

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (APP103652, B.A. Kingwell; 1059454 B.A. Kingwell); the Operational Infrastructure Support Scheme of the Victorian State Government. CSL Ltd provided partial financial support as well as the n-apo AI (Apo AI nanoparticles;

CSL111) to the Baker Institute (B.A. Kingwell and A.L. Richart as named investigators) under a nonrestrictive Materials Transfer Agreement but had no role in either development of study design, data acquisition, or interpretation of data.

Disclosures

During this project, B.A. Kingwell held an advisory board position with CSL Ltd, and her Institution received financial support for research unrelated to the current study, as well as partial support for the current project (B.A. Kingwell and A.L. Richart as named investigators) and modest travel reimbursement. Since completing this work, B.A. Kingwell has accepted an employment contract with CSL Ltd. A.J. Murphy and A.L. Richart currently receive financial support from CSL Ltd for research unrelated to the current study. S.A. Didichenko and A.V. Navdaev are employees of CSL Behring. The other authors report no conflicts.

Supplemental Materials

Expanded Materials & Methods

Online Figures I–III

Online Table I–II

Footnotes

The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.120.316848.

For Sources of Funding and Disclosures, see page 1435.

Correspondence to: Adele Richart, Baker Heart and Diabetes Institute, PO Box 6492, Melbourne, Victoria 3004, Australia, Email adele.richart@baker.edu.au
Bronwyn Kingwell, CSL Limited, Bio 21 Institute, 30 Flemington Rd, Melbourne, Victoria 3010 Australia, Email bronwyn.kingwell@csl.com.au

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