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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2015 Feb;185(2):347–355. doi: 10.1016/j.ajpath.2014.10.015

Human Mannose-Binding Lectin Inhibitor Prevents Myocardial Injury and Arterial Thrombogenesis in a Novel Animal Model

Vasile I Pavlov 1, Ying S Tan 1, Erin E McClure 1, Laura R La Bonte 1, Chenhui Zou 1, William B Gorsuch 1, Gregory L Stahl 1,∗
PMCID: PMC4305178  PMID: 25482922

Abstract

Myocardial infarction and coagulation disorders are leading causes of disability and death in the world. An important role of the lectin complement pathway in myocardial infarction and coagulation has been demonstrated in mice genetically deficient in lectin complement pathway proteins. However, these studies are limited to comparisons between wild-type and deficient mice and lack the ability to examine reversal/inhibition of injury after disease establishment. We developed a novel mouse that expresses functional human mannose-binding lectin (MBL) 2 under the control of Mbl1 promoter. Serum MBL2 concentrations averaged approximately 3 μg/mL in MBL2+/+Mbl1−/−Mbl2−/− [MBL2 knock in (KI)] mice. Serum MBL2 level in MBL2 KI mice significantly increased after 7 (8 μg/mL) or 14 (9 μg/mL) days of hyperglycemia compared to normoglycemic mice (P < 0.001). Monoclonal antibody 3F8 inhibited C3 deposition on mannan-coated plates in MBL2 KI, but not wild-type, mice. Myocardial ischemia/reperfusion in MBL2 KI mice revealed that 3F8 preserved cardiac function and decreased infarct size and fibrin deposition in a time-dependent manner. Furthermore, 3F8 prevented ferric chloride–induced occlusive arterial thrombogenesis in vivo. MBL2 KI mice represent a novel animal model that can be used to study the lectin complement pathway in acute and chronic models of human disease. Furthermore, these novel mice demonstrate the therapeutic window for MBL2 inhibition for effective treatment of disease and its complications.

The innate immune system plays an important role in host defense. The complement system, as a part of the innate immune system, is involved in protection against pathogens.1 The complement cascade can be activated/initiated through three distinct pathways: classic, alternative, and lectin. Lectin pathway (LP) activation is initiated by the presence of specific structures on microorganisms (bacterial, fungal, and some viral) binding to IgM or by changes in glycosylation patterns on compromised cells.2–5 There are several pattern recognition molecules that may be involved in LP activation, such as mannose-binding lectin (MBL) 2 (Mbl1 and Mbl2 in mice), ficolins (1, 2, and 3), and collectin 11.6,7 These pattern recognition molecules are associated with MBL-associated serine proteases (1, 2, and 3) and can directly activate C4 and C2 in the LP.3,4,7–17 MBL plays a significant role as an initiation molecule that recognizes endogenous ligands after oxidative stress and tissue injury, ultimately leading to vascular wall remodeling, thrombogenesis, and other cellular injuries.2,5,18–22

Studying the role of MBL in animal models of human disease has been limited to comparison of wild-type (WT) mice to Mbl-null mice, as well as reconstitution of Mbl-null mice with human recombinant MBL2. There are no inhibitors to both murine Mbl1 and Mbl2, and this prevents the study of the therapeutic window in animal models of human disease. Furthermore, the lack of inhibitors to murine Mbl1 and Mbl2 does not allow the study of disease succession or reversal of outcomes after MBL complex activation in disease models. To circumvent these limitations and to advance the field, we generated a novel human MBL2-expressing mouse that lacks murine Mbl1 and Mbl2. We report that MBL2+/+Mbl1−/− Mbl2−/− (MBL2 KI) mice produce functional human MBL2 and display LP activity similar to WT mice. Furthermore, anti-MBL2 (clone 3F8) monoclonal antibody (mAb) in the MBL2 KI mouse significantly protects the ischemic/reperfused murine myocardium from loss of myocardial function, decreases myocardial infarct size, and prevents myocardial fibrin deposition and occlusive thrombogenesis in vivo.

Materials and Methods

Animals

All procedures were reviewed and conducted according to the institute's Animal Care and Use Committee. All experiments were performed under the standards and principles set forth in the Guide for the Care and Use of Laboratory Animals.23

MBL2-Expressing Mice

Appropriate gene-targeted mouse CH57BL/6 embryonic stem cells were generated to make a MBL2 cDNA knock-in mouse in the Mbl1 locus using homologous recombination. Briefly, human MBL2 cDNA open reading frame sequence (approximately 0.75 kb) was amplified from a human MBL2 cDNA clone from GenBank (http://www.ncbi.nlm.nih.gov/nuccore/NM_000242.2; Accession number NM_000242). The final targeting vector (Supplemental Figure S1) was obtained by standard molecular cloning. Aside from the homology arms and cDNA, the final vector also contained flippase recognition target sequences flanking the neomycin expression cassette (ie, used for positive selection of the electroporated embryonic stem cells) and a diphtheria toxin expression cassette (ie, used for negative selection of the embryonic stem cells). The final vector was confirmed by restriction digestion and sequencing analysis. The restriction enzyme, NotI, was used for linearizing the final vector before electroporation. Male chimeras were bred with flippase deleter mice, and germ-line transmission was identified in four heterozygotes (two males and two females). The MBL2+/− mice were bred with CH57BL/6 Mbl-null mice (B6.129S4-Mbl1tm1Kata Mbl2tm1Kata/J; The Jackson Laboratory, Bar Harbor, ME) and offspring screened by PCR. After successful breeding strategies, the MBL2+/+ Mbl1−/− Mbl2−/− (MBL2 KI) mouse line was generated and expanded.

CH57BL/6 (WT) mice (8 to 12 weeks old; Taconic Farms, Hudson, NY) were used as background controls for MBL2 KI mice. Serum MBL2 concentrations in MBL2 KI mice were measured in male and female mice, as previously described.24 Serum MBL2 concentrations were also measured after 7 or 14 days of hyperglycemia as described.25 C3 deposition on mannan-coated 384-well plates (M7504; Sigma, St. Louis, MO) was analyzed as previously described, but with modifications.24 Briefly, 2% mouse sera from WT or MBL2 KI mice were incubated on mannan-coated plates in the presence of phosphate-buffered saline (PBS; vehicle), 10 μg/mL anti-human MBL2 mAb (clone 3F8), or 100 mmol/L N-acetylglucosamine (GlcNAc; Sigma). After incubation for 30 minutes at 37°C, the plates were washed (4×) and mouse C3 deposition was detected using goat anti-mouse C3 (MP Biomedicals, Santa Ana, CA) and a donkey anti-goat IR Dye 800–conjugated secondary antibody (Rockland Immunochemicals, Gilbertsville, PA), as previously described.24 Integrated intensity was measured for each well (LiCor CLX, Lincoln, NE), and each experiment was performed in triplicate six times.

Pharmacodynamic studies for the inhibition of sera MBL2 in the MBL2 KI mice were investigated. Briefly, 50 μL of mouse blood was collected by venipuncture and mAb 3F8 was given i.p. at 30, 100, 300, or 600 μg per mouse. Serial blood samples (50 μL) were then taken daily for up to 7 days. MBL2 concentrations were measured as described.24

Murine MI/R Model

The murine myocardial ischemia and reperfusion (MI/R) model was performed as described previously.22,26 After 45 minutes of ischemia, the myocardium was allowed to reperfuse for 5 minutes, 1 hour, or 4 hours. ECG changes (ie, ST segment) were monitored and used to establish ischemia and reperfusion. After reperfusion and echocardiography, the chest cavity was opened and the heart was excised and fixed overnight in 10% neutral-buffered formalin or processed for infarct size.

The following groups of MBL2 KI mice were studied in the MI/R protocols: i) sham MI/R (two WT and two MBL2 KI mice) or MI/R treated with ii) PBS (control) or 3F8 (i.v.) as follows: iii) at time of reperfusion (REP; 100 μg 3F8 per mouse), iv) after 15 minutes of reperfusion (100 μg 3F8 per mouse), v) after 30 minutes of reperfusion (100 μg 3F8 per mouse), or vi) 1 hour before ischemia (100 μg 3F8 per mouse).

Infarct Size Measurements

Myocardial infarct size was performed with modifications, as described.18,19,22,27 After 4 hours of reperfusion, the mice were anesthetized with sodium pentobarbital. The chest cavity was opened, and the heart was decompressed by cutting all venous supply. The left anterior descending coronary artery ligation was retightened. The heart was flushed retrograde through the thoracic aorta with 500 μL PBS and then perfused with 200 μL 5% Brilliant Blue G (Acros; Fisher Scientific, Pittsburgh, PA). The hearts were excised and divided into cross sections from base to apex into 1-mm slices with a coronal acrylic matrix (Roboz; Surgical Instrument Co. Inc., Gaithersburg, MD). Sections were incubated in 1% triphenyltetrazolium chloride (Acros) at 37°C for 15 minutes. Each section of the heart was placed between two Gold Seal cover glasses (Erie Scientific LLC, Portsmouth, NH). Images were taken with a Nikon SMZ800 stereoscopic zoom microscope and digital SPOT Insight camera (Diagnostic Instruments, Inc., Sterling Heights, MI), and areas were calculated with ImageJ software version 1.48 (NIH, Bethesda, MD). Infarct size was determined by calculating the total areas of the left ventricular free wall, infarcted tissue, nonischemic tissue, and the ischemic area at risk. The area at risk was not significantly different between groups (data not shown).

Echocardiography

Transthoracic echocardiography was used to evaluate cardiac function, as described.19,20 Echocardiography was performed 4 hours after reperfusion with a Philips Sonos 5500 instrument (Philips Medical Systems, Andover, MA) with a 7- to 15-MHz probe. The mice were anesthetized with sodium pentobarbital and placed on a heating table in a supine position. Ejection fraction (EF) was evaluated by long- and short-axis area measurements of the left ventricle during systole and diastole, as described.19,20

Ferric Chloride Coagulation Model

The mouse ferric chloride coagulation model was used as described.21,28,29 Isoflurane-anesthetized mice were placed in a supine position, and the right common carotid artery was exposed by blunt dissection. Carotid artery blood flow was measured with a Doppler flow probe (Transonic Systems Inc., Ithaca, NY), as described.21 Whatman filter paper saturated with 3.5% ferric chloride (FeCl3) was applied to either side of the carotid artery, proximal to the Doppler flow probe. The filter papers were removed after 3 minutes, and blood flow was measured continuously for 30 minutes. The mice were treated with PBS (control; N = 3) or 3F8 (300 μg; i.p.; 3 hours before carotid artery flow measurements; N = 4).

Histological and Immunohistochemical Analysis

Myocardial sections (5 μm thick) were obtained and deparaffinized using EZ-DeWax Solution (Biogenex, Fremont, CA). After a brief rinse, the sections were subjected to antigen retrieval by heating the slides in sodium citrate buffer (10 mmol/L, pH 6.0) to 85°C for 30 minutes. Endogenous peroxidase was blocked with 3% H2O2 in methanol/PBS (1:1 ratio) for 15 minutes, followed by 1-hour incubation with goat anti-mouse IgG Fab fragments (MP Biomedicals, Solon, OH) (1:100 in PBS/0.05% Triton X-100). The slides were rinsed in PBS, followed by a 30-minute incubation with normal goat serum (Vector Laboratories, Burlingame, CA) (1:2 in PBS/0.05% Triton X-100). Mouse β-fibrin was localized with mAb 59D8 (a gift from Dr. Charles Esmon (Oklahoma Medical Research Foundation, Oklahoma City, OK)30; 1:500 in PBS/0.05% Triton X-100) and goat anti-mouse IgG horseradish peroxidase conjugated (Jackson ImmunoResearch, West Grove, PA) (1:700 in PBS/0.05% Triton X-100 for 30 minutes). The sections were incubated with diaminobenzidine, and fibrin deposition was visualized with a Nikon Eclipse E400 microscope and analyzed using SPOT imaging software version 5.1 (Diagnostic Instruments, Inc.).

Mouse β-fibrin deposition analysis was performed by ImageJ software. Because fibrin deposition was localized in the left ventricle, the percentage area of fibrin deposition was acquired by measuring the total area positively stained for fibrin within the ischemic/reperfused left ventricle using the region of interest tool in ImageJ software.

Statistical Analysis

All statistical analysis was performed with SigmaPlot 12.5 software (SPSS, Chicago, IL). Data are presented as means ± SEM. Student's t-test, one-way analysis of variance, and the Student-Newman-Keuls test were used to demonstrate significance between groups. A one-way repeated-measures analysis of variance and Student-Newman-Keuls test were used to demonstrate significance between the two groups over time. P < 0.05 was considered statistically significant.

Results

MBL2-Expressing Mouse Model

Functional serum MBL2 expression was evaluated in male and female MBL2 KI mice using mannan-coated plates (Figure 1A). MBL2 levels in males and females averaged 3.1 ± 0.2 μg/mL. There was no significant difference between male and female serum MBL2 levels. To evaluate the ability of MBL2 to respond as an acute phase protein under control of the Mbl1 promoter, we measured MBL2 after hyperglycemia induction in male mice. MBL2 levels were significantly increased after 7 days [7.7 ± 1.1 μg/mL (range, 4.2 to 11.7 μg/mL); N = 7] or 14 days [8.6 ± 0.7 μg/mL (range, 5.9 to 11.0 μg/mL); N = 8] of hyperglycemia compared to normoglycemic [3.2 ± 0.3 μg/mL; (range, 1.2 to 6.4 μg/mL); N = 28] male mice (P < 0.001). There was no significant difference in serum MBL2 levels after 7 or 14 days of hyperglycemia.

Figure 1.

Figure 1

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A: Serum mannose-binding lectin 2 (MBL2) concentration in male and female MBL2 KI mice. No significant difference is found between the groups. B: C3 deposition onto mannan-coated plates. Summary of C3 deposition for control (vehicle), 10 μg/mL 3F8, and 100 mmol/L N-acetylglucosamine [GlcNAc; in wild-type (WT) and MBL2 KI sera]. Numbers within the bars represent number of experiments. C: Pharmacodynamics of 3F8 in MBL2 KI mice. Baseline MBL2 levels were obtained at day 0, followed by anti-MBL2 mAb 3F8 treatment (30, 100, 300, or 600 μg per mouse, i.p.). 3F8 antibody dose dependently inhibits circulating MBL2 levels at a dose of 100 μg per mouse and higher. Data are means ± SEM (B). ∗P < 0.05 compared to respective control (vehicle; B).

Functional evaluation of LP activation (ie, MBL2 complexed with mouse complement proteins) was evaluated ex vivo in WT and MBL2 KI mice by observing C3 deposition on mannan-coated plates. WT and MBL2 KI sera deposited similar levels of mouse C3 on mannan-coated plates, suggesting equivalent LP activation (Figure 1B). C3 deposition was significantly inhibited by 100 mmol/L GlcNAc in WT or hMBL KI sera and demonstrates the complete inhibition of mouse Mbl1 and Mbl2 in WT mice, as well as the human MBL2 complex in the MBL2 KI mice. In sharp contrast, anti-MBL2 mAb 3F8 did not inhibit mouse C3 deposition in WT sera. However, C3 deposition from MBL2 KI sera was significantly inhibited by mAb 3F8 to the same level as GlcNAc. These data demonstrate that the LP was activated in the serum of MBL2 KI mice to the same extent as WT mice and demonstrate the first biological available to inhibit total MBL2 in a mouse model.

The pharmacodynamics of 3F8-inhibited MBL2 in MBL2 KI mice were evaluated. Anti-MBL2 mAb 3F8 (30, 100, 300, or 600 μg per mouse, i.p.) was given after a baseline sera sample was obtained. 3F8 antibody dose dependently inhibited circulating MBL2 levels in a time-dependent manner (Figure 1C). At a dose of 100 μg per mouse and higher, complete inhibition of functional MBL2 levels was obtained at 24 hours. At a dose of 300 or 600 μg per mouse, MBL2 was functionally inhibited for up to 3 days. Complete inhibition of MBL2 levels for up to 1 week was observed at 600 μg per mouse. These data demonstrate that complete inhibition of MBL2 over various time spans can be obtained using a single dose of 3F8.

3F8 Preserves Myocardial Function

Myocardial function after MI/R was evaluated in MBL2 KI mice treated with 3F8 mAb. Untreated MBL2 KI (control) mice had a myocardial EF of approximately 40% after MI/R (Figure 2A), which is significantly decreased from sham MI/R mice. In contrast, treating MBL2 KI mice immediately at reperfusion (REP) with 100 μg 3F8 per mouse significantly prevented MI/R-induced loss of cardiac function, and the EF of the REP group was not significantly different from the sham group. Furthermore, treating MBL2 KI mice with 100 μg 3F8 per mouse after 15 or 30 minutes of reperfusion also significantly protected against loss of cardiac function compared to the control; however, the magnitude of cardiac protection was significantly lower than that observed when treatment was given immediately at reperfusion (REP group). The data demonstrate a 3F8-induced protection from MBL2-induced loss of cardiac function after MI/R, and the therapeutic window decreases as reperfusion time increases.

Figure 2.

Figure 2

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A: Myocardial ejection fraction (EF) measurements. EF was assessed in sham myocardial ischemia and reperfusion (MI/R) (sham) mice and in mice after 45 minutes of ischemia and 4 hours of reperfusion. Summary of murine myocardial EF data for control [phosphate buffered saline (PBS), i.v.] or 3F8 treated (100 μg per mouse, i.v.) at 0 (REP), 15 (15 minutes REP), or 30 (30 minutes REP) minutes after reperfusion. The number of experiments is identified within the bars. B: Representative images of myocardial slices after MI/R (45 minutes or 4 hours) and tetrazolium chloride staining in control (PBS, i.v.) or 3F8 treatment (100 μg per mouse, i.v.) at 0 (REP), 15 (15 minutes REP), or 30 (30 minutes REP) minutes after reperfusion. Brilliant Blue G dye (blue) denotes the nonischemic area, whereas red and white dyes demonstrate the area at risk (ischemic tissue). White (unstained) tissue denotes infarcted tissue, and red denotes viable tissue. C: Percentage of infarcted left ventricle. The percentage of the infarcted area was calculated from the weight of the area at risk (AAR), area of infarct, and area of noninfarct. The AAR was not significantly different between groups (data not shown). The number of experiments is identified within the bars. Data are means ± SEM (A and C). ∗P < 0.05 compared to control (15 or 30 minutes of reperfusion), ∗∗P < 0.01 compared to control (15 or 30 minutes of reperfusion), and ∗∗∗P < 0.001 compared to sham, REP (15 or 30 minutes of reperfusion) (A); ∗P < 0.05 compared to 15- and 30-minute REP groups, ∗∗∗P < 0.001 compared to all treated groups (C).

3F8 Reduces Infarct Size

To determine whether 3F8 protects the myocardium from infarction and reperfusion injury, we measured infarct size. We observed that i.v. treatment with 100 μg 3F8 per mouse, at 0, 15, or 30 minutes after reperfusion, significantly protects MBL2 KI mice from myocardial infarction compared with the control (Figure 2, B and C). Myocardial sections from base to apex from representative myocardial slices from the four groups of mice studied are presented in Figure 2B. Similar to the EF data (Figure 2A), myocardial infarction size was significantly smaller if the treatment started at reperfusion (0 minutes of reperfusion; REP) and infarct size increased if treatment was delayed (Figure 2C). Thus, anti-MBL2 antibody preserves myocardial function, as well as myocardial tissue, from MI/R injury within a short therapeutic window.

3F8 Prevents Fibrin Deposition within Myocardium

Hearts were evaluated for β-fibrin deposition after MI/R, because recent studies demonstrate a link between LP activation and coagulation in vivo.21,22,31 β-Fibrin deposition was increased within the left ventricular free wall that was subjected to ischemia/reperfusion in control mice after 5 minutes of reperfusion and decreased after 1 hour of reperfusion (Figure 3A). Mouse β-fibrin deposition was mainly vascular and perivascular at 5 minutes of reperfusion, and by 1 hour, β-fibrin was mainly within the interstitium (Figure 3B). Compared to control, 3F8-treated mice had significantly less left ventricular β-fibrin deposition at both time points (Figure 3, A and C). These data support the hypothesis that inhibiting MBL2 reduces β-fibrin formation during reperfusion of the ischemic myocardium, in addition to the cardioprotection observed in myocardial EF and infarct size.

Figure 3.

Figure 3

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A: Representative micrographs of fibrin staining (black) at 5 minutes and 1 hour of reperfusion in MBL2 KI mice treated with phosphate-buffered saline (control) or 3F8 (100 μg per mouse, i.v.) before myocardial ischemia and reperfusion (MI/R). Arrows denote ischemic/reperfused area and where fibrin staining would be located. B: Representative micrographs of fibrin staining (brown) at 5 minutes and 1 hour of reperfusion in MBL2 KI mice treated with PBS (control). C: The percentage of fibrin deposition within the ischemic/reperfused left ventricle after 5 minutes or 1 hour of reperfusion. Data are means ± SEM (C). N = 3 to 5 per group (C). ∗P < 0.05 compared to 1-hour control; ∗∗∗P < 0.001 compared to respective time control and also comparing 1-hour to 5-minute control. Original magnifications: ×1 (A); ×20 (B).

3F8 Prevents Occlusive Arterial Thrombogenesis

Our previous studies have demonstrated that Mbl-null mice do not develop an occlusive thrombus in response to ferric chloride compared to WT mice.21,22,31 MBL2 KI mice (PBS treated; control) developed an occlusive thrombus within 15 minutes after ferric chloride application (Figure 4). In contrast, treatment of MBL2 KI mice with 300 μg 3F8 completely inhibited ferric chloride–induced thrombogenesis. These results demonstrate that 3F8 significantly prevents occlusive arterial thrombogenesis in vivo in response to ferric chloride in the MBL2 KI mouse. These data further demonstrate that all functional MBL in the MBL2 KI mouse are of human origin. Furthermore, the murine serine proteases associated with the MBL2 complex are sufficient to induce thrombogenesis in vivo and are completely inhibited by anti-MBL2 mAb.

Figure 4.

Figure 4

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3F8 inhibits ferric chloride–induced thrombogenesis in vivo. Wild-type mice were treated with phosphate-buffered saline (control) or 3F8 (300 μg per mouse, i.p.) 3 hours before the application of ferric chloride to the carotid artery. Blood flow was continuously monitored in both groups for 30 minutes after ferric chloride application. Data are means ± SEM. ∗∗∗P < 0.001 compared to control.

Discussion

Studying the role of complement in human disease by using animal models has been fraught with many challenges. First, most complement inhibitors designed for use in humans do not cross-react with the same complement components in other species. Second, the use of complement-deficient animals relies on comparison with complement-sufficient/WT littermates or by reconstitution with recombinant or isolated proteins to re-establish the WT phenotype in the deficient animals. Third, with regard to the study of the LP, unlike humans, which have one functional MBL2 protein, most animal species have two functional MBL molecules (eg, Mbl1 and Mbl2). Regarding these limitations, it then becomes either impossible or cost prohibitive to establish chronic animal models of human disease or to establish a disease and then evaluate the effectiveness of complement inhibition on disease stability and/or reversal of injury. To address these limitations, we generated a humanized mouse that expresses MBL2 under the control of the Mbl1 locus and then removed both murine Mbl forms (Mbl1 and Mbl2) by breeding with the Mbl-null mouse, so that only MBL2 remained.

The MBL2 KI mice display a phenotype that is similar, if not identical, to a WT mouse. C3 deposition on mannan-coated plates was inhibited by specific inhibition of MBL2 with mAb 3F8 in MBL2 KI serum, whereas WT serum was not inhibited. The magnitude of LP inhibition by 3F8 was the same as GlcNAc and, thus, demonstrates that only MBL2 is present in the MBL2 KI mouse. Pharmacodynamic studies demonstrated that a single dose of mAb 3F8 inhibited MBL2 for at least 7 days. Because mAb 3F8 is a mouse anti-human MBL2 inhibitory antibody, we believe that repeat doses of 3F8 will unlikely establish an immune response and that MBL2 could be inhibited for many weeks, if not months, in the MBL2 KI mouse. Thus, the combined use of MBL2 KI mice and mAb 3F8 makes an ideal pair of reagents to study the long-term actions of MBL2 in chronic murine models of human disease.

Baseline serum MBL2 expression in the MBL2 KI mouse averages approximately 3 μg/mL, which is approximately half the concentration observed for Mbl1 in the inbred CH57BL/6J mouse.6 We designed the targeting vector for MBL2 insertion to the Mbl1 locus, because this promoter region reportedly mimics that of the MBL2 locus and displays acute phase activity, whereas the Mbl2 locus maintains constitutively expressed Mbl2.6 Serum MBL2 expression significantly increased over time in a hyperglycemia model (streptozocin injection), such that serum MBL2 levels doubled over 7 to 14 days of hyperglycemia. Thus, we demonstrate that the Mbl1 promoter region up-regulates MBL2 expression in the MBL2 KI mice during stress.

We have previously demonstrated that MBL is the initiating molecule that activates complement and the LP after MI/R.19 We demonstrate in the present study that MBL2 inhibition protects the ischemic/reperfused myocardium from loss of EF and myocardial infarction, further demonstrating that these mice lack functional murine Mbl1 and Mbl2. The greatest protection of myocardial function (EF) and structure was observed when 3F8 was given at the time of reperfusion. However, the therapeutic window in MI/R is much shorter in murine MI/R compared to the 18-hour therapeutic window observed in murine stroke.32 Furthermore, clinical studies conducted in the Assessment of Pexelizumab in Acute Myocardial Infarction trial showed no benefit from C5 inhibition by pexelizumab.33 We believe that the failure of the Assessment of Pexelizumab in Acute Myocardial Infarction trial was due to late administration of the drug (median of 2.8 hours after onset of pain) and continuous formation of the terminal complement complex. Because low MBL2 levels are associated with decreased injury in humans undergoing stroke or myocardial infarction, our data suggest that anti-MBL2 therapy should be given early in the reperfusion phase of myocardial reperfusion to maximize outcomes.34,35

Specific interactions between the coagulation and complement systems exist (Table 1). Until recently, these complex interactions have been demonstrated in vitro using reductionist models. The ferric chloride model of occlusive thrombosis is a widely used model for drug design, target validation, drug discovery, and evaluation of inhibitors for platelet function, thrombin, and specific actions of almost all proteins involved in coagulation and thrombolysis; thus, it is an appropriate model for evaluation of anti-coagulation preclinical studies.61 We demonstrated a central role of the MBL complex in ferric chloride–induced thrombogenesis in vivo.21,22,31 Herein, 3F8 significantly inhibited ferric chloride—induced thrombus formation. 3F8 also inhibited β-fibrin deposition after MI/R. Interestingly, β-fibrin deposition within the reperfused myocardium moved over time from a vascular/perivascular to interstitial location and suggests significant alterations to vascular permeability after MI/R that is also inhibited by anti-MBL2 mAb. Additional studies for this hypothesis are warranted. Thus, MBL2 inhibition, which also inhibits MBL-associated serine protease 1 and 2 function within the MBL complex, protects the myocardium from tissue inflammation, intravascular coagulation, and cellular injury after MI/R.18,19

Table 1.

Summary of the Interactions between the Complement and Coagulation Systems

Substrate Factor Reference
Activates/Cleaves
 MASP1 Factor XIII, fibrinogen, and thrombin 36–38
 MASP2 Thrombin (through prothrombin) 39
 MBL, MASP1, and MASP2 Fibrin 36,40
 C5a TF and tPA (through PAI-1) 41–44
 C3a and C5a TF (through TNF-α and IL-6) 45–49
 C3b Fibrin 50,51
 MAC (C5-9) TF 52
 Xa C3 and C5 53
 Plasmin C3 and C5 54
 Thrombin C3, C5, and DAF 54,55
 Platelets C3 56,57
 Fibrin Complement 58
Inactivates
 Heparin Classic and alternative complement pathways 59,60
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DAF, decay-accelerating factor; MASP, mannose-binding lectin (MBL)-associated serine proteases; PAI, plasminogen activator inhibitor; TF, tissue factor; TNF, tissue necrosis factor; tPA, tissue plasminogen activator.

In conclusion, we developed a novel humanized MBL2-expressing mouse under the control of the Mbl1 promoter, which displays acute phase activity. By using this novel mouse, we expanded the findings of a role for MBL in MI/R by showing MBL-mediated β-fibrin deposition and also a short therapeutic window. This novel mouse will advance the field by being able to establish disease and then observing if MBL2 inhibition reverses injury or halts its progression. Furthermore, for the first time, chronic murine models of disease can be effectively evaluated for LP contribution. Moreover, the MBL2 KI mouse will be effective for drug design, target validation, and drug discovery for the LP.

Acknowledgment

We thank Margaret Morrissey for technical assistance during the course of these studies.

Footnotes

Supported by NIH grants 5R01HL056086-17 and 5R01AI089781-03 (G.L.S.).

Disclosures: G.L.S. is listed as inventor on a patent on the use of monoclonal antibody 3F8.

Supplemental Data

Supplemental Figure S1

Final MBL2 targeting vector. The human mannose-binding lectin (MBL) 2 cDNA open reading frame sequence (KI) was placed within the 5′ homology arm (approximately 5.5 kb, containing partial exon 2) and the 3′ homology arm (approximately 3.3 kb, containing partial exon 5). Diphtheria toxin (DTA) and neomycin (Neo) cassettes were inserted for selection purposes. Flippase recognition target (Frt) was inserted for removal of the Neo cassette. NotI was used to linearize the vector for electroporation. LHA, long homologous arm; SHA, short homologous arm.

mmc1.pdf (14.3KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure S1

Final MBL2 targeting vector. The human mannose-binding lectin (MBL) 2 cDNA open reading frame sequence (KI) was placed within the 5′ homology arm (approximately 5.5 kb, containing partial exon 2) and the 3′ homology arm (approximately 3.3 kb, containing partial exon 5). Diphtheria toxin (DTA) and neomycin (Neo) cassettes were inserted for selection purposes. Flippase recognition target (Frt) was inserted for removal of the Neo cassette. NotI was used to linearize the vector for electroporation. LHA, long homologous arm; SHA, short homologous arm.

mmc1.pdf (14.3KB, pdf)

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