Caspase-1 mediates hyperlipidemia-weakened progenitor cell vessel repair

Abstract

Caspase-1 activation senses metabolic danger-associated molecular patterns (DAMPs) and mediates the initiation of inflammation in endothelial cells. Here, we examined whether the caspase-1 pathway is responsible for sensing hyperlipidemia as a DAMP in bone marrow (BM)-derived Stem cell antigen-1 positive (Sca-(1+)) stem/progenitor cells and weakening their angiogenic ability. Using biochemical methods, gene knockout, cell therapy and myocardial infarction (MI) models, we had the following findings: 1) Hyperlipidemia induces caspase-1 activity in mouse Sca-(1+) progenitor cells in vivo; 2) Caspase-1 contributes to hyperlipidemia-induced modulation of vascular cell death-related gene expression in vivo; 3) Injection of Sca-1+ progenitor cells from caspase-1(-/-) mice improves endothelial capillary density in heart and decreases cardiomyocyte death in a mouse model of MI; and 4) Caspase-1(-/-) Sca-(1+) progenitor cell therapy improves mouse cardiac function after MI. Our results provide insight on how hyperlipidemia activates caspase-1 in Sca-(1+) progenitor cells, which subsequently weakens Sca-(1+) progenitor cell repair of vasculature injury. These results demonstrate the therapeutic potential of caspase-1 inhibition in improving progenitor cell therapy for MI.

Figure 1

Hyperlipidemia increases caspase-1 activity in Sca-1⁺ progenitor cells. A. Experiment design. Wild-type (WT) mice and ApoE^−/− mice were fed with either chow diet or high fat (HF) for 12 weeks (w) before their bone marrows (BM) were collected for fluorescence activated cell sorter (FACS) analysis. B. After gating mononuclear cells from the BM, Sca-1⁺ stem cells were gated from the mononuclear cells. C. Among Sca-1⁺ stem cell populations in the BM, caspase-1 activity was measured. Gating of caspase-1 positive (Casp1⁺) cells in Sca-1⁺ population of mouse BM was shown in the left. Quantification was shown in the right.

Figure 2

Flow chart of experiment design that was used for cDNA microarray analysis.

Figure 3

Caspase-1 contributes to hyperlipidemia-induced gene changes related to vascular cell death. A. Volcano plots of apoliprotein E deficient (ApoE^−/−) mice versus Wild type (WT) mice aorta DNA expression comparisons (blue), with the overlay of Caspase-1 (Casp1)^−/−/ApoE^−/− versus ApoE^−/− comparisons (red) are depicted by estimated fold change (FC) (log2 FC, x-axis) and statistical significance (−log10 P value, y-axis). B. Cooperation between Caspase1 and hyperlipidemia is shown by Log2 FC/Log2 FC plot comparing gene expression value for Caspase1/ApoE DKO versus ApoE KO (x-axis), and parallel ApoE KO versus WT (y-axis). C. Venn diagram shows the profile of two gene expression comparisons. Among 23,470 mapped genes, a total of 6,745 genes were significantly changed induced by hyperlipidemia and 2,541 genes were significantly changed caused by Caspase1 deletion. Among the changed genes, there are 969 genes changed in condition of hyperlipidemia and reversed by Caspase1 deletion. D. The heat-maps represent the z-score of the expression level of top 50 reversed genes (hyperlipidemia increased or decreased genes which are down-regulated or up-regulated by deletion of Caspase1). E. Core analysis with Ingenuity pathway analysis (IPA) shows that the major molecular and cellar functional pathways are cellular growth and proliferation and cell death. F. The network show the connection of the caspase-1 reserved genes associated with apoptosis and necrosis of endothelial cells.

Figure 4

Caspase-1 contributes to hyperlipidemia-induced gene changes related to heart dysfunction. A. Tox analysis with Ingenuity pathway analysis (IPA) shows that the clinical pathology endpoints of these reversed genes. Heart hypertrophy and heart failure are the top endpoints associated with cardiovascular disease. Heat-maps showing the expression level of the involved genes in each endpoint are listed in the right.

Figure 5

Caspase-1^−/− Sca-1⁺ progenitor cell therapy improves cardiac function after MI. A. Myocardiac infarction (MI) and cell therapy model. Schematic representation of experimental plan including high fat diet feeding, cardiac function monitoring with echocardiography, cell therapy with purified Sca-1+ bone marrow cells followed by immunohistochemistry and flow cytometry analyses. B. The CellVue^R NIR780 fluorescence-labeled cells were traced to heart after intravenous injection. The CellVue^R NIR780 fluorescence-labeled purified Sca-1⁺ bone marrow cells (2 × 10⁶ cells/mouse) were traced to mouse heart after cell therapy (n=4 for tracer group, n=2 for non-cell tracer group). CT: no cell therapy control; cell: cell therapy C. M-mode Echocardiography. Representative M-mode echocardiographs of control mice and myocardiac infarcted mice. D. Cardiac functions measured with echocardiography. The cardiac function measurements of control mice, myocardial infarction (MI) mice, mice receiving wild-type (WT) Sca-1⁺ BM cells and mice receiving caspase-1(Casp1)^−/− Sca-1⁺ BM cells. The numbers shown indicate the numbers of mice in the group. E. Heart, Lung, Liver weight/body weight. The ratios of heart weight/body weight, lung weight/body weight and liver weight/body weight of mice receiving cell therapy and control mice.

Figure 6

Caspase-1^−/− Sca-1⁺ progenitor cell therapy increases IB4+ capillary density and decreases TUNEL+ cardiomyocytes. A. Capillary density detected with IB4 staining for endothelial cells in neovasculature. Histochemical analysis of heart cross sections showed that cell therapy with Sca-1⁺ BM cells from casp-1^−/− mice increases IB4+ capillary density (endothelial cells) in comparison to the cell therapy with Sca-1⁺ BM cells from wild type (WT) mice. B. TUNEL Assay for detecting cell death in myocardial infarcted heart. Histochemical analysis of heart cross sections showed that the cell therapy with Sca-1⁺ BM cells from casp-1^−/− mice decreases TUNEL+ cardiomyocytes in comparison to the cell therapy with Sca-1⁺ BM cells from WT mice.

Figure 7

A new working model that caspase-1 inhibition improves Sca-1⁺ stem cell therapy for myocardial infarction. Hyperlipidemia activates caspase-1 activation in Sca-1⁺ stem cells and vascular cells in aorta. Activation of caspase-1 upregulates proinflammatory gene expression and promotes pyroptosis and apoptosis presumably in Sca-1⁺ stem cells, endothelial cells and cardiomyocytes and causes cardiac dysfunction. Inhibition/depletion of caspase-1 improves survival of Sca-1⁺ stem cells/progenitor cells, and cardiomyocytes, promotes angiogenesis, and improves cardiac function after myocardial infarction.