Cardio-omentopexy requires a cardioprotective innate immune response to promote myocardial angiogenesis in mice

Abstract

Objective: The pedicled greater omentum, when applied onto stressed hearts using omentopexy, has been shown to be protective in humans and animals. The mechanisms underlying cardioprotection using omentopexy remain elusive. This study examined whether macrophage-mediated angiogenesis accounts for the cardioprotective effect of omentopexy in mice.

Methods: C57BL/6 mice were subjected to minimally invasive transverse aortic constriction for 6 weeks and subsequent cardio-omentopexy for 8 weeks. Control mice underwent the same surgical procedures without aortic constriction or cardio-omentopexy.

Results: Transverse aortic constriction led to left ventricular concentric hypertrophy, reduced mitral E/A ratio, increased cardiomyocyte size, and myocardial fibrosis in the mice that underwent sham cardio-omentopexy surgery. The negative effects of transverse aortic constriction were prevented by cardio-omentopexy. Myocardial microvessel density was elevated in the mice that underwent aortic constriction and sham cardio-omentopexy surgery, and cardio-omentopexy further enhanced angiogenesis. Nanostring gene array analysis uncovered the activation of angiogenesis gene networks by cardio-omentopexy. Flow cytometric analysis revealed that cardio-omentopexy triggered the accumulation of cardiac MHCII^loLyve1+TimD4+ (Major histocompatibility complex class II^low lymphatic vessel endothelial hyaluronan receptor 1+ T cell immunoglobulin and mucin domain conataining 4+) resident macrophages at the omental-cardiac interface. Intriguingly, the depletion of macrophages with clodronate-liposome resulted in the failure of cardio-omentopexy to protect the heart and promote angiogenesis.

Conclusions: Cardio-omentopexy protects the heart from pressure overload-elicited left ventricular hypertrophy and dysfunction by promoting myocardial angiogenesis. Cardiac MHCII^loLyve1+TimD4+ resident macrophages play a critical role in the cardioprotective effect and angiogenesis of cardio-omentopexy.

Keywords: AXL, AXL receptor tyrosine kinase; Akt, protein kinase B; CD45, lymphocyte common antigen; CD64, cluster of differentiation 64; COP, cardio-omentopexy; Calm1, calmodulin 1; Cdh5, cadherin 5; Clodro, clodronate-liposomes; Crk, proto-oncogene c-Crk; Ctnnb1, catenin β1; Ctnnd1, catenin delta 1; Cybb, cytochrome B-245 beta chain; Cyfip1, cytoplasmic FMR1 interacting protein 1; ECM, extracellular matrix; F4/80, F4/80 antigen; HCM, hypertrophic cardiomyopathy; HSP89aa1, heat shock protein 89aa1; Hippo, hippocampal; Itpr2, inositol 1,4,5-trisphosphate receptor type 2; Kdr, kinase insert domain receptor; Kras, kirsten rat sarcoma virus; LV, left ventricle; Ly6Clo, lymphocyte antigen-6Clow; Ly6G, lymphocyte antigen 6 complex locus G6D; Lyve1, lymphatic vessel endothelial hyaluronan receptor 1; MHCIIlo, major histocompatibility complex class IIlow; Ncf1, neutrophil cytosolic factor 1; Nck2, NCK adaptor protein 2; Nckap1H, NCK-associated protein 1H; Nos3, nitric oxide synthase 3; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; PI3K, phosphoinositide-3-kinase; Plcg1, phospholipase Cγ1; Plcg2, 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase γ2; Prkaca, protein kinase cAMP-activated catalytic subunit α; Prkacb, protein kinase cAMP-activated catalytic subunit β; Prkca, protein kinase Cα; Ptk2, protein tyrosine kinase 2; Ptk2b, protein tyrosine kinase 2β; Rac1, Rac family small GTPase 1; Rock2, Rho associated coiled-coil containing protein kinase 2; Src, proto-oncogene tyrosine-protein kinase Src; TAC, transverse aortic constriction; TGF, transforming growth factor; TimD4, T cell immunoglobulin and mucin domain conataining 4; VEGF-A, vascular endothelial growth factor A; Vav1, Vav guanine nucleotide exchange factor 1; WGA, wheat germ agglutinin; angiogenesis; cardiac hypertrophy; cardio-omentopexy; iB4, biotinylated-isolectin B4; mTOR, mammalian target of rapamycin; macrophages.

Graphical abstract

Cardio-omentopexy ameliorates TAC-induced diastolic dysfunction via angiogenesis in mice.

Figure 1

Experimental protocol. A, Effects of cardio-omentopexy (COP) on the heart in a murine model of transverse aortic constriction (TAC). In the TAC+COP group, C57BL/6 mice were subjected to TAC for 6 weeks and subsequently COP for 8 weeks. The transverse aorta was constricted between the innominate and left common carotid arteries using a 7-0 prolene suture ligature tied against a 25-gauge blunted needle. The pedicled greater omentum with the right gastroepiploic artery was transferred to the heart through the diaphragm. Mice in TAC and COP groups underwent TAC or COP alone. Control mice underwent all surgical procedures without the TAC and/or connection of the greater omentum with the heart. B, Effects of macrophage depletion with clodronate-liposome (Clodro) on the cardioprotective effect of COP. The geometry and function of the left ventricle was evaluated with echocardiography (Echo). Flow cytometry (flow) was conducted to analyze macrophage subsets in mouse hearts, and mouse hearts received histopathological examination (histo) to determine cardiomyocyte size, fibrosis, and microvessel density.

Figure 2

Cardio-omentopexy (COP) reduced transverse aortic constriction (TAC)-induced left ventricular hypertrophy and diastolic dysfunction. A, Representative echocardiographic M-mode images of the left ventricle; (B) anterior wall thickness at end systole and anterior wall thickness at end diastole; (C) posterior wall thickness at end systole and posterior wall thickness at end diastole; (D) left ventricular mass; and (E) mitral E/A ratio. Control mice were subjected to sham TAC surgery for 6 weeks and subsequent sham COP surgery for 8 weeks. COP mice were subjected to sham TAC surgery for 6 weeks and subsequent COP for 8 weeks. TAC mice underwent TAC for 6 weeks and subsequent sham COP surgery for 8 weeks. TAC+COP mice were subjected to TAC for 6 weeks and subsequent COP for 8 weeks. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Larger extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. ∗P < .05 versus control; #P < .05 versus TAC (n = 12-13 mice per group).

Figure 3

Cardio-omentopexy (COP) reduced transverse aortic constriction (TAC)-elicited increases in heart weight and left ventricular weight. A, gross pathology; (B) heart weight:body weight ratio; (C) heart weight:tibia length ratio; (D) left ventricular weight:body weight ratio; and (E) left ventricular weight:tibia length ratio. The mice of control, COP, TAC, and TAC+COP groups were treated as described in Figure 2. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. ∗P < .05 versus control; #P < .05 versus TAC (n = 12 mice per group).

Figure 4

Cardio-omentopexy (COP) decreased the transverse artic constriction (TAC)-induced increases in cardiomyocyte size and myocardial fibrosis in C57BL/6 mice. A, Representative heart sections of TAC and TAC+COP mice stained with wheat germ agglutinin (WGA); B, quantification of cardiomyocyte surface area; C, representative heart sections stained with Masson's trichrome; and D, quantification of myocardial fibrosis. The mice of control, COP, TAC, and TAC+COP groups were treated as described in Figure 2. Scale bar = 50 μm. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Larger extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. ∗P < .05 versus control; #P < .05 versus TAC (n = 8-10 sections per group).

Figure 5

The effect of transverse aortic constriction (TAC) and cardio-omentopexy (COP) on microvessel density and gene expression profiling of mouse hearts. A, representative heart sections stained with isolectin B4 showing microvessels; B, quantification of microvessel density; C, global significance scores of pathways identified in fibrosis panel analysis. Only the most affected pathways, or pathways with a score >1.5 or less than −1.5, are shown; D, fold change of genes associated with angiogenesis. The mice of control, COP, TAC, and TAC+COP groups were treated as described in Figure 2. Scale bar = 400 μm. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. Values in panels (C) and (D) are for the TAC+COP group (n = 3) relative to the TAC group (n = 3). ECM, Extracellular matrix; mTOR, mammalian target of rapamycin; PDGF, platelet-derived growth factor; PI3K-Akt, phosphoinositide-3-kinase-protein kinase B; Hippo, hippocampal; MHC, major histocompatibility complex; TGF, transforming growth factor. ∗P < .05 versus control; #P < .05 versus TAC (n = 8 sections per group).

Figure 6

Cardio-omentopexy (COP) induces the accumulation of cardiac macrophages (MФs) at the myocardial-omentum junction. A, Representative images of MФ CD68 staining at the omental-myocardial junction (interface) in a mouse heart subjected to transverse aortic constriction (TAC) followed by COP; (B) flow cytometric analysis of total MФs (CD64-positive, F4/80-positive) in the mouse hearts of interface and remote regions; and (C) flow cytometric analysis of resident TimD4-positive MФs (MHCII-positive, TimD4-positive) in the mouse hearts of interface and remote regions. The mice of control, COP, TAC, and TAC+COP groups were treated as described in Figure 2.

Figure 7

Depletion of macrophages with clodronate-liposomes (Clodro) impaired the cardioprotective effect of cardio-omentopexy (COP) in the mice subjected to transverse aortic constriction (TAC). A, CD68-stained heart sections showing macrophages in mouse hearts; and (B) quantification of left ventricular wall thickness; (C) left ventricular mass; (D) mitral E/A ratio; (E) wheat germ agglutinin-stained heart sections; (F) quantification of cardiomyocyte size; (G) Masson's trichrome-stained heart sections; (H) quantification of myocardial fibrosis; (I) isolectin B4-stained heart sections; and (J) quantification of microvessel density in mouse hearts. The mice in the TAC+COP+phosphate-buffered saline (PBS) group were subjected to TAC for 6 weeks and subsequent COP for 8 weeks and given PBS during 8 weeks of COP. The animals in the TAC+COP+Clodro group were subjected to TAC for 6 weeks and subsequent COP for 8 weeks and injected with Clodro during 8 weeks of COP. Scale bar = 400 μm. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Larger extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. PBS, Phosphate-buffered saline; AWd, anterior wall at end diastole; AWs, anterior wall at end systole; PWd, posterior wall at end diastole; PWs, posterior wall at end systole. ∗P < .05 versus TAC+COP+PBS (n = 8-10 per group).

Figure 8

Key study methods, results, and implications. A, Left ventricular diastolic function (mitral E/A ratio) measured from echocardiography; B, changes in myocardial angiogenesis (microvessel density measured from the biotinylated-isolectin B4-stained mouse hearts); C, effects of macrophage depletion with clodronate-liposome on myocardial angiogenesis in the mice subjected to transverse aortic constriction (TAC) and cardio-omentopexy (COP). Control mice were subjected to sham TAC surgery for 6 weeks and subsequent sham COP surgery for 8 weeks. COP mice were subjected to sham TAC surgery for 6 weeks and subsequent COP for 8 weeks. TAC mice underwent TAC for 6 weeks and subsequent sham COP surgery for 8 weeks. TAC+COP mice were subjected to TAC for 6 weeks and subsequent COP for 8 weeks. The lower and upper borders of the box represent the lower and upper quartiles (25th percentile and 75th percentile). The middle horizontal line represents the median. The lower and upper whiskers represent the minimum and maximum values of nonoutliers. Larger extra dots represent outliers.

Figure E1

Validation of mouse model of cardiac hypertrophy using transverse aortic constriction (TAC) according to gross pathology and fibrosis. The aortic arch of C57BL/6 mice was constricted between the innominate artery and left common carotid artery for 6 weeks. Sham mice underwent all surgical procedures except constriction of the aorta. A, Echocardiographic image showing aortic banding in a mouse; (B) validation of TAC-induced cardiac hypertrophy; and (C) Masson's trichrome-stained heart sections showing fibrosis elicited by TAC.

Figure E2

Transverse aortic constriction (TAC) and cardio-omentopexy. A, Schematic of the cardio-omentopexy procedure after TAC; and (B) the photograph of cardio-omentopexy in a mouse.

Figure E3

Left ventricular hypertrophy in C57BL/6 mice subjected to transverse aortic constriction (TAC) for 6 weeks. A, anterior wall at end diastole; (B) anterior wall at end systole; (C) posterior wall at end diastole; (D) posterior wall at end systole; and (E) left ventricular mass. Sham mice underwent all surgical procedures without constriction of the aorta. Parasternal short axis 2-chamber view-guided M-mode images of the left ventricle were used to quantitate left ventricular wall thickness and left ventricular mass. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Larger extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. ∗P < .05 versus sham (n = 31-32 mice per group).

Figure E4

Echocardiographic parameters of mouse hearts 6 weeks after transverse aortic constriction (TAC). A, Heart rate; (B) left ventricular internal diameter at end diastole; (C) left ventricular internal diameter at end systole; (D) fractional shortening; (E) left ventricular volume at end diastole; (F) left ventricular volume at end systole; (G) ejection fraction; (H) mitral E/A ratio. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. There were no differences in all echocardiographic parameters between TAC and sham groups (n = 31-32 mice per group).

Figure E5

Heart rate (A) and systolic function (B) of mouse hearts 8 weeks after cardio-omentopexy (COP). Control mice were subjected to sham transverse aortic constriction (TAC) surgery for 6 weeks and subsequent sham COP surgery for 8 weeks. COP mice were subjected to sham TAC surgery for 6 weeks and subsequent COP for 8 weeks. TAC mice underwent TAC for 6 weeks and subsequent sham COP surgery for 8 weeks. TAC+COP mice were subjected to TAC for 6 weeks and subsequent COP for 8 weeks. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. There were no differences in heart rate and ejection fraction between groups (n = 12-13 mice per group).

Figure E6

Physical weights and bone length of experimental groups at termination. A: body weight; B: tibia length; C: the ratio of lung weight/body weight: D: the ration of lung wright/tibia length. Control mice were subjected to sham transverse aortic constriction (TAC) surgery for 6 weeks and subsequent sham TAC surgery for 8 weeks. COP mice were subjected to sham TAC surgery for 6 weeks and subsequent cardio-omentopexy (COP) for 8 weeks. TAC mice underwent TAC for 6 weeks and subsequent sham COP surgery for 8 weeks. TAC+COP mice were subjected to TAC for 6 weeks and subsequent COP for 8 weeks. Lung tissue weight was normalized to body weight or tibia length. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. There were no differences in body weight, tibia length, the ratios of both lung weight/body weight and lung weight/tibia length between groups (n = 12-13 mice per group).

Figure E7

Quantification of microvessel density in mouse hearts stained with CD31 antibodies. Control mice were subjected to sham transverse aortic constriction (TAC) surgery for 6 weeks and subsequent sham cardio-omentopexy (COP) surgery for 8 weeks. COP mice were subjected to sham TAC surgery for 6 weeks and subsequent COP for 8 weeks. TAC mice underwent TAC for 6 weeks and subsequent sham COP surgery for 8 weeks. TAC+COP mice were subjected to TAC for 6 weeks and subsequent COP for 8 weeks. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of nonoutliers. Larger extra dots represent outliers. P values were determined using 2-way repeated measures analysis of variance followed by post hoc analysis using Mann–Whitney test for comparison between 2 groups. ∗P < .05 versus control; #P < .05 versus COP; &P < .05 versus TAC (n = 10 sections per group).

Figure E8

Evidence for omental fusion to myocardium in mouse. Displayed are histologic sections of control versus transaortic constriction (TAC) murine hearts followed by cardio-omentopexy. Tissue sections were stained with wheat germ agglutinin (WGA; green), which stains glycoproteins on cell membranes and matrix, to visualize cross cellular sectional area and connective tissue.

Figure E9

Candidate pathways by which the omentum might protect the heart after cardio-omentopexy. The omentum might secrete paracrine factors to mobilize cardioprotective endothelial cells (ECs) during angiogenesis. Alternatively, such factors might trigger reparative macrophages (Mɸs) in the heart. It is unclear if such macrophages originate from the omentum, the circulation, or are resident in the heart. Another scenario is that the omentum might mobilize protective progenitor cells (PCs). CM, Cardiomyocyte.