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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.


Related in: MedlinePlus

CD163 expression and CTHRSSVVC-phage overlay by different assays co-localize in atheroma and spleen tissues. (A) CTHRSSVVC-phage overlay localization and expression of CD163 in atherosclerotic tissue particularly in areas rich in macrophages (indicated by the star (*)). Phages bound to tissue were revealed using an anti-bacteriophage antibody. No significant staining was observed with the insertless phage Fd-tet. Bar, 50 µm (left panels) and 20 µm (right panels); (B) CTHRSSVVC (CD163-mimic) or CVSSTLLRC (PlGF-mimic) phage localization (green) and CD163 expression (red) in spleen tissue observed by immunofluorescence and visualized by confocal microscopy. Phages bound to tissue were revealed using an anti-bacteriophage antibody. The bottom panels show the merge of the phage overlay (upper panels) and CD163 expression (middle panels). Bright yellow (as exemplified by the arrows) indicates co-localization. Note that the co-localization with CVSSTLLRC-phage was not as complete as with the other peptide. Bar, 100 µm in all panels.
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ijms-17-01383-f002: CD163 expression and CTHRSSVVC-phage overlay by different assays co-localize in atheroma and spleen tissues. (A) CTHRSSVVC-phage overlay localization and expression of CD163 in atherosclerotic tissue particularly in areas rich in macrophages (indicated by the star (*)). Phages bound to tissue were revealed using an anti-bacteriophage antibody. No significant staining was observed with the insertless phage Fd-tet. Bar, 50 µm (left panels) and 20 µm (right panels); (B) CTHRSSVVC (CD163-mimic) or CVSSTLLRC (PlGF-mimic) phage localization (green) and CD163 expression (red) in spleen tissue observed by immunofluorescence and visualized by confocal microscopy. Phages bound to tissue were revealed using an anti-bacteriophage antibody. The bottom panels show the merge of the phage overlay (upper panels) and CD163 expression (middle panels). Bright yellow (as exemplified by the arrows) indicates co-localization. Note that the co-localization with CVSSTLLRC-phage was not as complete as with the other peptide. Bar, 100 µm in all panels.

Mentions: The phage overlay studies showed that three peptides had low reactivity in atherosclerotic lesions (Table 1), whereas the CTHRSSVVC-phage peptide presented strong positivity in the same sample and lipid core region from three patients (Figure 1C, Table 1), although a simple background for phages could also be observed (Figure 1C). As a proof of specificity, control experiments demonstrated no reactivity between CTHRSSVVC-phage peptide and normal carotid samples, and between negative control insertless phage (Fd-tet) and atherosclerotic carotid samples. The analysis of the results from bioinformatic and functional assays (phage overlay) showed that CTHRSSVVC-phage peptide mimic proteins with relevant roles in atherosclerosis progression and had the strongest reactivity against atherosclerotic lesions. The CTHRSSVVC-phage peptide was selected for further studies (Figure 1C). Quantification of binding phage in human atheroma and normal carotid was carried out in twelve samples that were divided in four different groups: (1) Atheroma with CTHRSSVVC-phage; (2) Atheroma with Fd-phage; (3) Normal carotid with CTHRSSVVC-phage; and (4) Normal carotid with Fd-phage (Figure 1D, Table 3). CD163 expression and CTHRSSVVC-phage overlay were co-localized in atheroma and spleen tissues, respectively. The significant overlap in tissue staining by CTHRSSVVC-phage (Figure 2A) and anti-CD163 (Figure 2A) were observed in atherosclerotic lesions, particularly, in areas rich in macrophages (Figure 2A, indicated by the star (*)). No significant staining was detected when the Fd-tet insertless phage (Figure 2A) or control antibody were utilized. However, single background populations for phages were observed. We have further modified the phage overlay protocol, and it was done by immunofluorescence and fluorescence microscopy to allow for better overlap of the images. Consequently, the merger of the two channels would indicate a perfect co-localization of phage and CD163 staining. Indeed, as shown in Figure 2B, CTHRSSVVC-phage overlay (green) and CD163 immunostaining (red) overlap produced an almost entirely yellow image. The co-localization observed when the phage displaying a different peptide (CVSSTLLRC) was incubated with the spleen tissue (Figure 2B) was less complete. When the tissues were immunostained with the thyroid tissue (negative control), the reactions resulted in negative observation (Figure 3).


CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis
CD163 expression and CTHRSSVVC-phage overlay by different assays co-localize in atheroma and spleen tissues. (A) CTHRSSVVC-phage overlay localization and expression of CD163 in atherosclerotic tissue particularly in areas rich in macrophages (indicated by the star (*)). Phages bound to tissue were revealed using an anti-bacteriophage antibody. No significant staining was observed with the insertless phage Fd-tet. Bar, 50 µm (left panels) and 20 µm (right panels); (B) CTHRSSVVC (CD163-mimic) or CVSSTLLRC (PlGF-mimic) phage localization (green) and CD163 expression (red) in spleen tissue observed by immunofluorescence and visualized by confocal microscopy. Phages bound to tissue were revealed using an anti-bacteriophage antibody. The bottom panels show the merge of the phage overlay (upper panels) and CD163 expression (middle panels). Bright yellow (as exemplified by the arrows) indicates co-localization. Note that the co-localization with CVSSTLLRC-phage was not as complete as with the other peptide. Bar, 100 µm in all panels.
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Related In: Results  -  Collection

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ijms-17-01383-f002: CD163 expression and CTHRSSVVC-phage overlay by different assays co-localize in atheroma and spleen tissues. (A) CTHRSSVVC-phage overlay localization and expression of CD163 in atherosclerotic tissue particularly in areas rich in macrophages (indicated by the star (*)). Phages bound to tissue were revealed using an anti-bacteriophage antibody. No significant staining was observed with the insertless phage Fd-tet. Bar, 50 µm (left panels) and 20 µm (right panels); (B) CTHRSSVVC (CD163-mimic) or CVSSTLLRC (PlGF-mimic) phage localization (green) and CD163 expression (red) in spleen tissue observed by immunofluorescence and visualized by confocal microscopy. Phages bound to tissue were revealed using an anti-bacteriophage antibody. The bottom panels show the merge of the phage overlay (upper panels) and CD163 expression (middle panels). Bright yellow (as exemplified by the arrows) indicates co-localization. Note that the co-localization with CVSSTLLRC-phage was not as complete as with the other peptide. Bar, 100 µm in all panels.
Mentions: The phage overlay studies showed that three peptides had low reactivity in atherosclerotic lesions (Table 1), whereas the CTHRSSVVC-phage peptide presented strong positivity in the same sample and lipid core region from three patients (Figure 1C, Table 1), although a simple background for phages could also be observed (Figure 1C). As a proof of specificity, control experiments demonstrated no reactivity between CTHRSSVVC-phage peptide and normal carotid samples, and between negative control insertless phage (Fd-tet) and atherosclerotic carotid samples. The analysis of the results from bioinformatic and functional assays (phage overlay) showed that CTHRSSVVC-phage peptide mimic proteins with relevant roles in atherosclerosis progression and had the strongest reactivity against atherosclerotic lesions. The CTHRSSVVC-phage peptide was selected for further studies (Figure 1C). Quantification of binding phage in human atheroma and normal carotid was carried out in twelve samples that were divided in four different groups: (1) Atheroma with CTHRSSVVC-phage; (2) Atheroma with Fd-phage; (3) Normal carotid with CTHRSSVVC-phage; and (4) Normal carotid with Fd-phage (Figure 1D, Table 3). CD163 expression and CTHRSSVVC-phage overlay were co-localized in atheroma and spleen tissues, respectively. The significant overlap in tissue staining by CTHRSSVVC-phage (Figure 2A) and anti-CD163 (Figure 2A) were observed in atherosclerotic lesions, particularly, in areas rich in macrophages (Figure 2A, indicated by the star (*)). No significant staining was detected when the Fd-tet insertless phage (Figure 2A) or control antibody were utilized. However, single background populations for phages were observed. We have further modified the phage overlay protocol, and it was done by immunofluorescence and fluorescence microscopy to allow for better overlap of the images. Consequently, the merger of the two channels would indicate a perfect co-localization of phage and CD163 staining. Indeed, as shown in Figure 2B, CTHRSSVVC-phage overlay (green) and CD163 immunostaining (red) overlap produced an almost entirely yellow image. The co-localization observed when the phage displaying a different peptide (CVSSTLLRC) was incubated with the spleen tissue (Figure 2B) was less complete. When the tissues were immunostained with the thyroid tissue (negative control), the reactions resulted in negative observation (Figure 3).

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.


Related in: MedlinePlus