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CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis

View Article: PubMed Central - PubMed

ABSTRACT

The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with 111InCl3 in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases.

No MeSH data available.


CTHRSSVVC-phage is internalized by macrophages. CTHRSSVVC-phage is internalized by J774A.1 mouse macrophage but not human endothelial cells (HCAECs). J774A.1 cells (A) or HCAECs (B) were incubated at 37 °C with CTHRSSVVC or insertless Fd-tet phages for 8 h to allow phage internalization. Membrane bound phages were removed by cell washing, internalized phages (red) and cell nucleus (blue) revealed by cell permeabilization, and phage staining using an anti-bacteriophage antibody. Non-permeabilized cells indicate the successful removal of cell surface bound phages. Magnification = 100×.
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ijms-17-01383-f006: CTHRSSVVC-phage is internalized by macrophages. CTHRSSVVC-phage is internalized by J774A.1 mouse macrophage but not human endothelial cells (HCAECs). J774A.1 cells (A) or HCAECs (B) were incubated at 37 °C with CTHRSSVVC or insertless Fd-tet phages for 8 h to allow phage internalization. Membrane bound phages were removed by cell washing, internalized phages (red) and cell nucleus (blue) revealed by cell permeabilization, and phage staining using an anti-bacteriophage antibody. Non-permeabilized cells indicate the successful removal of cell surface bound phages. Magnification = 100×.

Mentions: Encouraged by our results that the CTHRSSVVC-phage targets CD163 positive cells, we then probed whether this peptide could participate in receptor-mediated internalization. Selected peptides that target membrane bound receptors can be internalized by cells in a receptor-dependent manner [15,24]. We have therefore evaluated the capability of phage displaying the CTHRSSVVC peptide for internalization by macrophages. In these experiments, we selected a CD163+ mouse derived macrophage like monocyte cell line (J774A.1 cells) to perform our assay [25]. As a control, we used human coronary artery endothelial cells (HCAECs) because they do not express CD163 receptors. Cells were seeded on microtiter wells and incubated with CTHRSSVVC or Fd-tet insertless negative control phages for 8 h. The cells were then washed to remove all surface bound phage and internalized phage particles visualized by immunofluorescence using permeabilized cells. We found that CTHRSSVVC-phage was efficiently internalized by the J774A.1 cells (Figure 6A) but not by HCAECs (Figure 6B). Because macrophages are professional phagocytic cells, we observed that both phages (CTHRSSVVC and insertless Fd-tet) were internalized to a certain extent by the J774A.1 cells in sharp contrast to the human coronary endothelial cells. However, it is important to note that the CTHRSSVVC-phage was internalized by J774A.1 cells to a significantly larger extent as compared to the negative insertless control phage. In all cases, non-permeabilized cells were negative, indicating that no surface bound phage remained after the washing steps. These experimental results, taken together, suggest that CTHRSSVVC synthetic peptide is also internalized by CD163+ macrophages through receptor-mediated endocytosis (Unpublished data).


CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis
CTHRSSVVC-phage is internalized by macrophages. CTHRSSVVC-phage is internalized by J774A.1 mouse macrophage but not human endothelial cells (HCAECs). J774A.1 cells (A) or HCAECs (B) were incubated at 37 °C with CTHRSSVVC or insertless Fd-tet phages for 8 h to allow phage internalization. Membrane bound phages were removed by cell washing, internalized phages (red) and cell nucleus (blue) revealed by cell permeabilization, and phage staining using an anti-bacteriophage antibody. Non-permeabilized cells indicate the successful removal of cell surface bound phages. Magnification = 100×.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037663&req=5

ijms-17-01383-f006: CTHRSSVVC-phage is internalized by macrophages. CTHRSSVVC-phage is internalized by J774A.1 mouse macrophage but not human endothelial cells (HCAECs). J774A.1 cells (A) or HCAECs (B) were incubated at 37 °C with CTHRSSVVC or insertless Fd-tet phages for 8 h to allow phage internalization. Membrane bound phages were removed by cell washing, internalized phages (red) and cell nucleus (blue) revealed by cell permeabilization, and phage staining using an anti-bacteriophage antibody. Non-permeabilized cells indicate the successful removal of cell surface bound phages. Magnification = 100×.
Mentions: Encouraged by our results that the CTHRSSVVC-phage targets CD163 positive cells, we then probed whether this peptide could participate in receptor-mediated internalization. Selected peptides that target membrane bound receptors can be internalized by cells in a receptor-dependent manner [15,24]. We have therefore evaluated the capability of phage displaying the CTHRSSVVC peptide for internalization by macrophages. In these experiments, we selected a CD163+ mouse derived macrophage like monocyte cell line (J774A.1 cells) to perform our assay [25]. As a control, we used human coronary artery endothelial cells (HCAECs) because they do not express CD163 receptors. Cells were seeded on microtiter wells and incubated with CTHRSSVVC or Fd-tet insertless negative control phages for 8 h. The cells were then washed to remove all surface bound phage and internalized phage particles visualized by immunofluorescence using permeabilized cells. We found that CTHRSSVVC-phage was efficiently internalized by the J774A.1 cells (Figure 6A) but not by HCAECs (Figure 6B). Because macrophages are professional phagocytic cells, we observed that both phages (CTHRSSVVC and insertless Fd-tet) were internalized to a certain extent by the J774A.1 cells in sharp contrast to the human coronary endothelial cells. However, it is important to note that the CTHRSSVVC-phage was internalized by J774A.1 cells to a significantly larger extent as compared to the negative insertless control phage. In all cases, non-permeabilized cells were negative, indicating that no surface bound phage remained after the washing steps. These experimental results, taken together, suggest that CTHRSSVVC synthetic peptide is also internalized by CD163+ macrophages through receptor-mediated endocytosis (Unpublished data).

View Article: PubMed Central - PubMed

ABSTRACT

The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with 111InCl3 in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases.

No MeSH data available.