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Modulation of cardiac macrophages by phosphatidylserine-presenting liposomes improves infarct repair
Associated Data
Abstract
Herein we investigated a new strategy for the modulation of cardiac macrophages to a reparative state, at a predetermined time after myocardial infarction (MI), in aim to promote resolution of inflammation and elicit infarct repair. The strategy employed intravenous injections of phosphatidylserine (PS)-presenting liposomes, mimicking the anti-inflammatory effects of apoptotic cells. Following PS-liposome uptake by macrophages in vitro and in vivo, the cells secreted high levels of anti-inflammatory cytokines [transforming growth factor β (TGFβ) and interleukin 10 (IL-10)] and upregulated the expression of the mannose receptor—CD206, concomitant with downregulation of proinflammatory markers, such as tumor necrosis factor α (TNFα) and the surface marker CD86. In a rat model of acute MI, targeting of PS-presenting liposomes to infarct macrophages after injection via the femoral vein was demonstrated by magnetic resonance imaging (MRI). The treatment promoted angiogenesis, the preservation of small scars, and prevented ventricular dilatation and remodeling. This strategy represents a unique and accessible approach for myocardial infarct repair.
An important goal in cardiology is to minimize infarct size and improve healing after myocardial infarction (MI). Following MI, resident and recruited macrophages remove necrotic and apoptotic cells, secrete cytokines, and modulate angiogenesis at the infarct site (1). As such, the macrophage is a primary responder cell involved in the regulation of post-MI infarct wound healing at multiple levels (2). According to recent studies, different subsets of macrophages are responsible for these different activities; during the early inflammatory phase (phase 1), proinflammatory macrophages dominate the injury site and phagocytose apoptotic/necrotic myocytes and other debris, whereas during inflammation resolution (phase 2), the dominant subsets are the reparative macrophages, which propagate infarct repair (3, 4). The duration and extent of the early inflammatory phase have major implications on infarct size and ventricle remodeling (5).
Herein, we conceived a previously undescribed strategy for controlling the duration and extent of the inflammatory phase following MI, in aim to reduce myocardium damage, preserve infarct size, and prevent ventricle remodeling. Our strategy exploits the principle underlying the anti-inflammatory effects of apoptotic cells, which are known to actively suppress inflammation by inhibiting the release of proinflammatory cytokines from macrophages while augmenting the secretion of anti-inflammatory cytokines, such as transforming growth factor β (TGFβ) and Interleukin 10 (IL-10) as well as the expression of the mannose receptor, CD206 (6, 7). Macrophages recognize the apoptotic cells via surface ligands, among them phosphatidylserine (PS), and “silently” clear the cells (8–10). In humans, apoptosis after MI occurs mainly in the border zones and in the remote areas of ischemia (11), thus its effects on inflammation resolution are considered to be minor.
Our strategy is based on exogenous administration of PS-presenting liposomes, designed to mimic the apoptotic cells in terms of PS presentation on their surface and their anti-inflammatory effects. We examined liposome uptake by peritoneal and cardiac macrophages, in vitro and in vivo, and verified the consequent upregulation in anti-inflammatory responses, by measuring the profile of cytokine secretion and surface marker expressions. The effects of i.v. injections of PS-presenting liposomes on angiogenesis, infarct size, and ventricle remodeling were examined in a rat model of acute MI.
Results
Phosphatidylserine (PS)-Presenting Liposomes.
The liposomes were constructed to present the “death signal” PS on their surface available for ligation by the PS receptor (PSR) on macrophages. The presence of PS on liposome surface was validated by Fluorescence Activated Cell Sorter (FACS) analysis. Fluorescein isothiocyanate (FITC)-annexin V was bound to 98% of the PS-presenting liposomes (Fig. 1A), while staining was minor in PS-lacking liposomes (11.21%, probably nonspecific staining) (Fig. 1B). Additionally, zeta potential measurements revealed a net surface charge of (-98.6 ± 11.3 mV) on PS-presenting liposomes, while that on the PS-lacking liposomes was less negative (-21.43 ± 0.46 mV), due to the greater fraction of phosphatidylcholine (PC) on the surface of the latter liposomes. In PS-lacking liposomes, the PC fraction constituted 74% by mass, while in PS-presenting liposomes it was 36.5%. Thus, the PS-presenting liposomes dispersed uniformly in physiological medium due to repulsion forces (Fig. 1C), while the PS-lacking liposomes formed aggregates (Fig. 1D). By cryotransmission electron microscope (cryo-TEM) analysis (Fig. 1 E and F), most liposomes had few lamellas. No differences in outer shape or in size of liposomes constructed with or without PS were observed. The liposomes had a particle size of 1.2 ± 0.3 μm for efficient uptake by macrophages (Fig. S1) (12).
Liposome features. (A and B) FACS analysis of PS-presenting (A) and PS-lacking (B) liposomes; the numbers on pictures represent the percentage liposomes with bound Annexin V on their surface. (C and D) Light microscope images of PS-presenting (C) and PS-lacking (D) liposomes. Bar = 32 μm. (E and F) Cryo-TEM pictures of PS-presenting (E) and PS-lacking (F) liposomes. Bar = 200 nm.
Uptake of PS-Presenting Liposomes by Peritoneal Macrophages and Immunomodulation.
The in vitro uptake mechanism of PS-presenting liposomes was initially studied in cultures of peritoneal macrophages because they represent an accessible cell type compared to cardiac macrophages; their isolation protocol is simpler and reproducible (see SI Methods). The peritoneal macrophages were treated with cytochalasin B (CY), which inhibits particle phagocytosis but not the PS-receptor-mediated endocytosis (13), prior to administration of FITC-bovine serum albumin (BSA) encapsulating liposomes. The treatment with CY had no effect on the uptake of PS-presenting liposomes by macrophages, while it reduced by a factor of almost 20 the uptake of PS-lacking liposomes (Fig. 2A), indicating that the uptake of PS-presenting liposomes occurs via the PS-receptor-mediated endocytosis pathway.
In vitro/in vivo uptake of PS-presenting liposomes by peritoneal macrophages (F4/80 positive) (A and B) and immunomodulation (C–F). (A) In vitro uptake of (FITC-BSA)-liposomes by adhered macrophages, pretreated for 2 h with 10 μM of cytochalasin B (CY) prior to incubation with liposomes for 1 h. After uptake, the cells were detached and analyzed by FACS for F4/80+ cells that have uptaken fluorescent (FITC) liposomes (n = 3 wells per each group). (B) In vivo macrophage uptake of PS-presenting liposomes (n = 3) and PS-lacking liposomes (n = 3) containing FITC-BSA; the liposomes were injected i.p. to mice and 3 h later the peritoneal cells were lavaged and analyzed by FACS. (C–F) Immunomodulation of macrophages by PS-presenting liposomes. A proinflammatory response was induced in peritoneal macrophages by i.p. injection of lipopolysacharide (LPS, 250 μg) into mice. Three h later, the animals were randomly divided into three groups: those injected with only LPS (LPS group, n = 3); LPS followed by i.p. injection of PS-presenting liposomes (LPS+PS group, n = 3); or LPS followed by i.p. injection of PS-lacking liposomes (LPS+PC, n = 3). An additional group consisted of nontreated (NT, n = 3) animals (with no LPS activation). Three h after treatment with liposomes, peritoneum lavage was analyzed by FACS (cells) and ELISA (lavage fluid). (C and D) FACS analyses for the relative expression of CD86 (C) and CD206 (D) out of the F4/80 positive macrophages. (E and F) ELISA of lavage fluids for secretion levels of (E) IL-10 and (F) TGFβ.
The in vivo uptake of PS-presenting liposomes encapsulating FITC-(BSA) 3 h after i.p. injection into mice peritoneum was confirmed and quantified by FACS analysis of the lavaged macrophages. The macrophages (identified by their CD11b marker) uptook threefold more of the PS-presenting liposomes than PS-lacking ones (Fig. 2B). The difference in liposome uptake is attributed to the specific engulfment of the PS-presenting liposomes through the PS receptor on macrophages.
Next, we examined whether the uptake of PS-presenting liposomes by macrophages can mimic the modulating effect of apoptotic cell uptake and elicit anti-inflammatory responses in activated macrophages. For that, a proinflammatory response was induced in macrophages by injecting a strong inducer of proinflammatory responses—lipopolysaccharide (LPS), into the peritoneum of mice. Fig. 2 C and D shows that following the in vivo uptake of PS-presenting liposomes, the state of the lavaged macrophages changed from proinflammatory (treated with LPS) to anti-inflammatory, as judged by the significant decrease in CD86 expression—a surface marker for proinflammatory macrophages (Fig. 2C, p < 0.05) and the increase in CD206 mannose receptor expression—a surface marker for reparative macrophages (Fig. 2D, p < 0.05) (14, 15). By contrast, the uptake of PS-lacking liposomes did not affect the macrophage state, and LPS-treated macrophages maintained their proinflammatory phenotype induced by the LPS.
The results were further supported by the ELISAs performed on the peritoneal lavage fluids for determining cytokine secretion levels. Fig. 2 E and F shows significantly greater secretion levels of the anti-inflammatory cytokines, TGFβ and IL-10, from LPS-activated macrophages following the uptake of PS-presenting liposomes, and lower levels after the uptake of PS-lacking liposomes, compared with the nontreated mice. These findings validate that PS-presenting liposomes can mimic apoptotic cells and therefore promote a “silent” anti-inflammatory clearance.
Effect of PS-Presenting Liposomes on Macrophage Population at the Infarct.
Because the treatment time with PS-presenting liposomes after MI is an important parameter in the proposed strategy, we performed an analysis to identify and quantify the macrophage numbers and their subpopulation ratio, proinflammatory vs. reparative, at the infarct at different times after MI induction in mice. The percentage of cardiac macrophages (F4/80 positive cells out of total cardiac cells, following enzymatic digestion of the heart) showed a trend of increasing level starting 2 d after MI (Fig. S2A). Analysis of the proinflammatory macrophage subset (CD86-positive out of F4/80 positive cells) and reparative macrophage subset (CD206 out of F4/80 positive cells) revealed a trend of change in the initial ratio of reparative/proinflammatory subpopulations (0.85) in favor of the reparative macrophage subset (1.39) 4 d after MI induction (Fig. S2B).
An intramyocardial injection of PS-presenting liposomes, PS-lacking liposomes, or saline immediately after MI had no significant effect on the total macrophage number at the infarct (Fig. S2A). However, the macrophage subset ratio at the infarct changed after treatment with PS-presenting liposomes as reflected by the anti-inflammatory cytokine secretion profiles of IL-10 (Fig. 3A) and TGFβ (Fig. 3B); there was an increase in their secretion level from macrophages isolated as early as 3 d after MI and treatment with PS-presenting liposomes compared to their levels in animals treated with PS-lacking liposomes or saline. The same trend was seen in cardiac macrophages isolated 4 d after MI with the respective treatments (Fig. S2C and D). Concomitantly with the increase in anti-inflammatory responses, a trend of decrease in the secretion level of the proinflammatory cytokine TNFα was noticed 3 d after MI, in macrophages isolated from PS-presenting liposomes treated mice compared with PS-lacking liposomes or saline-treated mice (Fig. 3C). Taken together, these results indicate that the treatment with PS-presenting liposomes can induce cardiac macrophages to secrete anti-inflammatory cytokines as early as 3 d after treatment, 1 d earlier than without treatment.
Immunomodulation of cardiac macrophages after MI. MI was induced in mice, followed by intramyocardial (into the infarct) injections of either PS-presenting liposomes (PS lip) (n = 15), PS-lacking liposomes (PC lip) (n = 15), saline (n = 25), or no treatment (NT) (n = 10). Three days later, macrophages were isolated from the infarcted hearts, cultured for 24 h and the collected culture medium was analyzed by the respective ELISAs using antibodies against IL-10 (A), TGFβ (B), and TNFα (C). * denotes statistically significant difference.
Tracking Liposomes in Infarct after i.v. Administration.
The ability of PS-presenting liposomes to target the infarct after i.v. injection and be engulfed by cardiac macrophages was investigated with liposomes containing iron-oxide (see SI Methods). The liposomes were injected through the femoral vein, 48 h after MI induction in rats. MRI scans of the hearts 4 d later (Fig. 4A) reveal the presence of resident and/or infiltrating macrophages at the infarct that had taken up PS-presenting liposomes and accumulated in the infarct (detected by the dark dots–arrows).
In vivo uptake and accumulation of PS-presenting liposomes in cardiac macrophages after i.v. administration to rats. MI was induced in rats and 48 h later, PS-presenting liposomes entrapping iron-oxide or saline were injected through the femoral vein. Four days later, the rats were examined by MRI (A) to evaluate macrophage accumulation at the infarct. The dark areas in the coronal sections represent macrophages, which have uptaken PS-presenting liposomes containing the iron oxide. (B) Histology/immuno-histochemistry of cross-sections from hearts excised from mice treated with i.v. injections of PS-presenting liposomes containing iron oxide, 48 h after MI. Four days after liposome injection, the animals were sacrificed and hearts were fixated and sliced for histology and immuno-staining for ED1 (a marker for resident macrophages, brown color) and iron oxide (blue color). Bar = 500 μm.
To validate the above, cross-sections from the treated hearts were immuno-stained for ED-1 (macrophage marker, brown stain) and for iron (blue stain). The overlapping picture (right panel) reveals colocalization of the two stains, indicating targeting and uptake of liposomes by macrophages at the infarct (Fig. 4B). By contrast, in the control group, sporadic weak staining for endogenous iron (red blood cells) and macrophages was seen, but no colocalization of the stains.
Effect of i.v. Administration of PS-Presenting Liposomes on Cardiac Function and Structure.
The finding that PS-presenting liposomes can be targeted to the infarct after i.v. administration and be taken up by cardiac macrophages prompted us to test the efficacy of this strategy to improve infarct repair after MI. The treatment was performed 48 h after MI induction because at this time point the macrophages are found in greater numbers at the infarct than immediately after MI. The rats were randomly subjected to an injection into the femoral vein of either PS-presenting liposomes (n = 10), PS-lacking liposomes (n = 10), or saline (n = 10). Echocardiography studies were performed 1 d after MI, prior to treatment, to validate infarction and 1 mo later, to examine the effect of treatment on infarct repair. In addition, the rat hearts were examined by histochemistry for angiogenesis and by postmortem morphometric analysis for dimensions of LV remodeling.
Treatment with PS-presenting liposomes by i.v. administration enhanced angiogenesis at the infarct compared to infarcts treated with PS-lacking liposomes or saline. This is revealed when comparing the vessel density at the infarct 4 weeks after MI, evaluated by immuno-staining of both lectin (Fig. S3A) marking the endothelial cells in blood vessels and α-smooth muscle actin (SMA, Fig. S3B), marking the pericytes and arterioles that surround matured blood vessels. Quantitatively, it is observed that the mature blood vessel density is 1.5-fold greater in rats treated with PS-presenting liposomes compared to animals treated with PS-lacking liposomes or saline (Fig. 5A). It is also seen that PS-lacking liposomes did have some effect on angiogenesis compared to saline; however, it was not as distinct as PS-presenting liposomes.
Effect of i.v. injected PS-presenting liposomes on angiogenesis and LV remodeling after MI. Rats were subjected to MI and 48 h later were injected i.v. with either PS-presenting liposomes (n = 10) (PS lip), PS-lacking liposomes (n = 10) (PC lip), or saline (n = 10). Four weeks later, the harvested hearts were sectioned and stained with antibodies against α-SMA and lectin for detecting mature blood vessels (Fig. S3A) and Masson’s trichrome to allow morphometric measurements of scar thickness and expansion index (Fig. S3B). (A) The vessel density (#/mm2) was determined from five different fields in each slide, randomly selected from the α-SMA and lectin-immuno-stained cross-sectioned slides, using Cell* software (Olympus Soft imaging Solutions GmbH). (B) Expansion index and relative infarct size determined from morphometric analysis. (C) Results of echocardiography study for measured LVED and LVES areas. Bonferroni post test was performed.
Morphometric analyses on Masson’s tri-chrome-stained cross-sections (Fig. S4) reveal the presence of thinner scars in rats treated with saline or PS-lacking liposomes compared to those treated with PS-presenting liposomes. The expansion index and scar thickness (Fig. 5B) showed significant improvements after treatment with PS-presenting liposomes compared to saline treatment.
Left ventricle end systolic and diastolic areas (LVES area and LVED area) (Fig. 5C) were expanded in the groups treated with PS-lacking liposomes or saline compared to the group treated with PS-presenting liposomes. In the latter group, the left ventricle wall maintained its area size, indicating that the treatment with PS-presenting liposomes is capable of preventing the LV remodeling associated with MI.
Discussion
Herein, we investigated a previously undescribed strategy for modulating macrophages to an anti-inflammatory state at a predetermined time after MI, in aim to promote resolution of inflammation and preserve small infarct size and LV dimensions. In conceiving the strategy, several criteria guided us: the use of natural, well-defined and safe materials; the treatment should be minimally invasive; and the principles underlying the strategy to resolve inflammation should mimic the anti-inflammatory effects of apoptotic cell clearance by macrophages (16).
In humans, apoptosis after MI is not as significant as necrotic cell death, which may explain why the inflammation responses after MI are frequently so excessive, causing massive tissue damage (17). Recognizing the anti-inflammatory effects of apoptotic cells, we hypothesized that the exogenous application of PS-presenting liposomes as apoptotic-mimicking particles would act to resolve the inflammation after MI while providing a safe a-cellular, reproducible, and accessible approach. Thus, we designed liposomes carrying the apoptotic signal PS on their surface, sized 1-μm, suitable for i.v. administration as well as for uptake by macrophages. An initial proof-of-concept study to prove the capability of PS-presenting liposomes to modulate macrophage activity was performed with peritoneal macrophages due to their greater accessibility. Peritoneal and cardiac macrophages share a similar machinery of apoptotic cell recognition; they both respond to the “eat me” signal presented as phosphatidylserine on apoptotic cell surface, and as a result they secrete similar patterns of cytokines. Thus, the studies with peritoneal macrophages may represent faithfully the behavior of cardiac macrophages toward apoptotic cells. We confirmed that the uptake of PS-presenting liposomes by macrophages occurs via PS-receptor endocytosis since inhibition of the regular nonspecific phagocytosis by cytochalasin B did not affect their uptake.
The uptake of PS-presenting liposomes by LPS-activated peritoneal macrophages upregulated the anti-inflammatory phenotype, as reflected by the enhanced expression of CD206 (Mannose receptor) and the greater secretion levels of the anti-inflammatory cytokines TGFβ and IL-10. Concomitantly, we noted downregulation in the expression of the proinflammatory marker CD86. The anti-inflammatory macrophage phenotype after uptake of PS-presenting liposomes mimicked that of activated macrophages after the uptake of apoptotic cells (14, 15). Furthermore, our studies show that the uptake of PS-presenting liposomes was effective in reversing the strong proinflammatory effects of LPS on macrophages by upregulating the expression of anti-inflammatory markers and cytokine secretion. These factors have been previously shown to contribute to the immunosuppressive effects of apoptotic cells in vivo, as shown in IL-10-deficient mice (18).
Importantly, the PS-presenting liposomes were capable of modulating the macrophage activation state at the infarct site. An intramyocardial injection of PS-presenting liposomes after MI induced the anti-inflammatory phenotype in macrophages on site already at day 3 after treatment, while with no treatment or when treated with PS-lacking liposomes this event occurred later after MI, from day 4 and onwards. The induced anti-inflammatory phenotype was accompanied by elevated secretion levels of IL-10 and TGFβ and decreased secretion of the proinflammatory cytokine TNFα. These results prompted us to test whether this strategy would be valuable for infarct therapy.
The choice to administer the liposomes for infarct therapy by i.v. injection was made because this represents a more clinically relevant treatment modality. The liposomes were injected 2 d after MI, at a time point where the number of macrophages at the infarct begins to increase and the cells become influential. By MRI and immuno-histochemistry, we confirmed that the PS-presenting liposomes targeted the cardiac macrophages at the infarcted heart after i.v. injection. The precise uptake mechanism is yet unclear; either the liposomes were targeted to the infarct where they were uptaken by resident/recruited macrophages, or while in circulation, the liposomes were uptaken by circulating macrophages, which subsequently migrated to the infarct site.
The i.v. administration of PS-presenting liposomes affected the extent of angiogenesis, infarct size, and LV remodeling after MI. Angiogenesis leading to the formation of mature blood vessels was greater after treatment with PS-presenting liposomes compared to PS-lacking liposomes (1.5-fold higher vessel density) although it did not reach a statistical significance. The echocardiography study revealed that LV end systolic and diastolic areas (LVES area and LVED area) were preserved in the group treated with PS-presenting liposomes compared to the controls, PS-lacking liposomes, and saline-treated groups. All these effects are in corroboration with the reparative activities of the macrophage subpopulation in phase 2. These cells have been shown to release proangiogenic factors such as vascular endothelial growth factor (VEGF), participate in the fusion of endothelial tip cells, and serve as bridge cells (7). Their effect on infarct size preservation can be attributed to the salvation of the remaining myocardial tissue by the enhanced angiogenesis as well as by downregulating the excessive inflammation and secretion of the anti-inflammatory cytokines, such as IL-10 and TGF-β.
Collectively, our results are promising, indicating that the modulation of recruited or resident cardiac macrophages by applying PS-presenting liposomes is feasible and with consequences leading to attenuation in left ventricle remodeling and prevention of heart dilatation. With respect to translation into the clinics, the strategy of using autologous apoptotic cells to treat chronic heart failure has been clinically tested (ACCLAIM trial) showing some benefit for the treatment (19). Yet, there are concerns using autologous apoptotic cells because this treatment can ameliorate autoimmune diseases, for example via the release of autoantigens. The use of well-defined PS-presenting liposomes abrogates the concerns associated with apoptotic cell treatment, while still benefiting from their anti-inflammatory effect and beneficial effects on infarct therapy.
Methods
Preparation and Characterization of Liposomes.
The liposomes were prepared as described (20) from a lipid mixture (Avanti Polar Lipids) of phosphatidylserine (PS), phosphatidylcholine (PC), and cholesterol (CH) at 1∶1∶1.33 molar ratios, respectively (PS-presenting liposomes), or from PC and CH (1.5∶1 molar ratio) (PS-lacking liposomes). The degree of PS exposure on liposomes was assessed by (i) binding of FITC-annexin V to PS on liposome surface and analysis by FACS and (ii) measuring the zeta potential of liposome preparations using Zeta Plus particle size analyzer (Brookhaven Instruments Corporation).
SI Text provides the details for preparation method of liposomes encapsulating fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA, Sigma Chemical Co.) or iron-oxide nanoparticle solution (Endorem), as well as details of liposome evaluation by cryotransmission electron microscope (Cryo-TEM).
Uptake Studies by Peritoneal Macrophages.
The online data supplement provides details of the methods for isolation and cultivation of macrophages from the peritoneum of Balb/c mice, as well as the in vitro and in vivo uptake experiments.
Induction of Myocardial Infarction (MI).
The animal studies were performed in accordance with the Animal Care and Use Committee guidelines of Tel Aviv University, which conforms to the policies of the American Heart Association. MI induction was attained by permanently occluding the left main coronary artery (LAD) with an intramural suture (6-0 polypropylene), as previously described (21).
Evaluation of Macrophage Population after MI and Treatment.
Twenty g Balb/c female mice (Harlan Lab) underwent MI induction and were randomly divided into four groups (15 each); three groups received an intramyocaridal (into the infarct) injection of a solution of either PS-presenting liposomes (0.03 M), PS-lacking liposomes (0.03 M) or saline (35-μL). The fourth group remained untreated. At various time points after MI induction and treatment, hearts were harvested and underwent three 10-min cycles of enzymatic digestion using a cocktail of Dispase II and Trypsin-EDTA (22). The isolated cells were analyzed by FACS for the percentage of total macrophages (F4/80 positive), the fraction of proinflammatory macrophages (F4/80 positive cells expressing the proinflammatory marker CD86) and the fraction of reparative macrophages (F4/80 positive cells expressing the mannose receptor CD206) (14, 15).
The isolated macrophages were cultured in 24-well tissue culture plates, 500,000 cells/well, in medium composed of RPMI 1640 medium, supplemented with 2 mM L-Glutamine, 10% (v/v) fetal bovine serum (FBS) and 100 U/mL Penicillin, 1 μg/mL Streptomycin, 2.5 U/mL Nystatin, for 24 h (all materials from Biological Industries). The collected culture medium was analyzed for secretion of TNFα, IL-10 and TGFβ by their respective ELISAs (R&D Systems Inc.) according to manufacturer’s instructions.
Tracking Liposomes at Infarct After i.v. Administration.
The uptake of i.v. injected PS-presenting liposomes by cardiac macrophages was investigated in a rat model of acute MI (23). Forty-eight h after MI, the SD rats were injected through the femoral vein with 0.03 M of PS-presenting liposomes containing iron oxide (2 μg/mL) (n = 4) or saline as a control (n = 4). Four days after injection, the rats were examined by MRI using a 0.5-T GE iMRI machine with a specially constructed animal probe. Image sequences included T1 spin echo and T2* gradient echo. Following imaging, the rats were sacrificed and their hearts were harvested and examined by immunohistochemical staining for ED1 in macrophages (Serotec) and for iron (Sigma-Aldrich).
I.v. Injections of PS-Presenting Liposomes and Echocardiography Studies.
Forty-eight h after MI induction in female Sprague–Dawley rats (Harlan Laboratories), the survivors were subjected to injection through the femoral vein of 150 μL of either a 0.06 M solution of PS-presenting liposomes (n = 10) or PS-lacking liposomes (n = 10). The control group was injected with saline (n = 10).
Echocardiography studies were performed 24 h after MI induction (baseline echocardiogram) and after 1 mo. The rats were anesthetized, their chests were shaved, and they were placed on their backs; images were obtained by placing the transducer against the chest on the left anterior side. Echocardiograms were performed with a commercially available echocardiography system equipped with 12.5 MHz phased transducer (Hewlett Packard). The heart was first imaged in the 2D mode in the parasternal long and short axis views of the left ventricle. M-mode images were obtained at the level below the tip of the mitral valve leaflets at the level of the papillary muscles. All measurements were averaged for three consecutive cardiac cycles and performed by an experienced technician blinded to the treatment group.
Immunohistochemical and Morphometric Analysis.
One month after treatment, the hearts were arrested with 15% KCl, perfused with 4% formaldehyde (15 mmHg) for 20 min, and then sectioned into 3–4 transverse slices and parallel to the atrioventricular ring. Each slice was fixed with 10% buffered formalin, embedded in paraffin, and sectioned into 5-μm slices. Serial sections were immuno-labeled with following antibodies: anti- α-smooth muscle actin (SMA; monoclonal from Sigma-Aldrich) against pericytes and arterioles and lectin (Sigma-Aldrich) for detection of endothelial cells. The immuno-stained sections were viewed under Olympus light microscope (BX61, Motorized System Microscope) connected to an Olympus (DD71) digital capture system. The vessel density (#/mm2) was determined from three or five different fields in each slide, randomly selected from the α-SMA and lectin-immuno-stained cross-sectioned slides, using Cell* software (Olympus Soft imaging Solutions GmbH).
Postmortem morphometric analysis was performed on heart slices obtained 5 mm from the heart’s apex. Five-μm thick cross-sections were subjected to Masson’s trichrome staining for nuclei, cytoplasm, and collagen visualization. The slides were photographed and analyzed with planimetry software (Sigma Scan Pro version 5). The following parameters were measured: average wall thickness (mm) (averaged from three measurements of septum thickness), average scar thickness (mm) (averaged from three measurements of scar thickness), LV cavity area (mm2), and whole LV area (mm2). Relative scar thickness was calculated as average scar thickness divided by average wall thickness. Infarct expansion index was calculated as follows: [LV cavity area/whole LV area]/ relative scar thickness.
Statistical Analysis.
Statistical analysis was performed with GraphPad Prism version 5.03 for Windows (GraphPad Software). All variables are expressed as mean ± SEM from at least three independent experiments. One way ANOVA studies were performed in liposome uptake experiment, activation in vivo, macrophage activation mechanism after MI, vessel and macrophage density, and morphometric analysis. Two-way ANOVA studies were performed in the activation of macrophage in vitro experiment as well as in the functional experiment. P < 0.05 was considered statistically significant.
Acknowledgments.
We thank Prof. Ron N. Apte and Dr. Alon Monsonego for their critical reading of the paper and useful comments, Emil Ruvinov for his help with the statistics, and Radka Holbova and Dr. Yael Mardor for excellent technical assistance. The research was supported by grants from the Israel Science Foundation (1368/08), the European Union FWP7 (INELPY), and the Israel Ministry of Science, Culture and Sport. This work was performed in partial fulfillment of the requirements of Tamar Harel-Adar for a PhD degree in the Department of Biotechnology Engineering, Ben-Gurion University of the Negev. Prof. Cohen holds the Claire and Harold Oshry Professor Chair in Biotechnology.
Footnotes
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1015623108/-/DCSupplemental.