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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

Phages displaying the peptide sequence CTHRSSVVC bind to human atherosclerotic lesions. (A) hematoxylin–eosin (HE) staining to human samples. Bar, 50 µm in all panels; (B) Phage recovery from CX7C peptide phage library after each round of biopanning in atheroma from each patient. Error bars indicate standard error of mean (SEM); (C) Phage Overlay Assay to atheroma and normal donor carotid tissue samples (n = 3, each group), figure shows representative slides of immunohistochemical staining for CTHRSSVVC-phage (CD163-mimic) and CVQLNSLPC-phage (Galectin-4 mimic) in the normal carotid (top) and atherosclerotic lesion from patient 2 (bottom), with a strong positively stained to lipid core region (hemorrhage, thrombus and cholesterol crystals) are indicated by the star (*). Bar, 100 µm in all panels; (D) Quantification of binding phage in human atheroma and normal carotid (n = 3, each group), 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. Statistical difference was determined by one-way analysis of variance followed by the Kruskal–Wallis test, and it was accepted at a value of * p < 0.05, vs. normal carotid.
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ijms-17-01383-f001: Phages displaying the peptide sequence CTHRSSVVC bind to human atherosclerotic lesions. (A) hematoxylin–eosin (HE) staining to human samples. Bar, 50 µm in all panels; (B) Phage recovery from CX7C peptide phage library after each round of biopanning in atheroma from each patient. Error bars indicate standard error of mean (SEM); (C) Phage Overlay Assay to atheroma and normal donor carotid tissue samples (n = 3, each group), figure shows representative slides of immunohistochemical staining for CTHRSSVVC-phage (CD163-mimic) and CVQLNSLPC-phage (Galectin-4 mimic) in the normal carotid (top) and atherosclerotic lesion from patient 2 (bottom), with a strong positively stained to lipid core region (hemorrhage, thrombus and cholesterol crystals) are indicated by the star (*). Bar, 100 µm in all panels; (D) Quantification of binding phage in human atheroma and normal carotid (n = 3, each group), 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. Statistical difference was determined by one-way analysis of variance followed by the Kruskal–Wallis test, and it was accepted at a value of * p < 0.05, vs. normal carotid.

Mentions: We have used a phage display library (labeled as CX7C) in which seven amino acid long random peptides are displayed on the surface of a filamentous phage fused to the minor pIII coat protein [8]. Given the heterogeneity of atherosclerosis disease, and our goal to develop target-specific peptides that bind to molecular markers which are common to most atherosclerotic lesions, phage display selection was performed in tandem using specimens obtained from three different patients whose atheroma had distinct inflammation characteristics to the lipid core region, (Figure 1A, Table 1). Although the initial histologic analyses were not complete, all elements of the intima, including those from the atherosclerotic lesion and endothelium, were exposed to the procedures.


CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis
Phages displaying the peptide sequence CTHRSSVVC bind to human atherosclerotic lesions. (A) hematoxylin–eosin (HE) staining to human samples. Bar, 50 µm in all panels; (B) Phage recovery from CX7C peptide phage library after each round of biopanning in atheroma from each patient. Error bars indicate standard error of mean (SEM); (C) Phage Overlay Assay to atheroma and normal donor carotid tissue samples (n = 3, each group), figure shows representative slides of immunohistochemical staining for CTHRSSVVC-phage (CD163-mimic) and CVQLNSLPC-phage (Galectin-4 mimic) in the normal carotid (top) and atherosclerotic lesion from patient 2 (bottom), with a strong positively stained to lipid core region (hemorrhage, thrombus and cholesterol crystals) are indicated by the star (*). Bar, 100 µm in all panels; (D) Quantification of binding phage in human atheroma and normal carotid (n = 3, each group), 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. Statistical difference was determined by one-way analysis of variance followed by the Kruskal–Wallis test, and it was accepted at a value of * p < 0.05, vs. normal carotid.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01383-f001: Phages displaying the peptide sequence CTHRSSVVC bind to human atherosclerotic lesions. (A) hematoxylin–eosin (HE) staining to human samples. Bar, 50 µm in all panels; (B) Phage recovery from CX7C peptide phage library after each round of biopanning in atheroma from each patient. Error bars indicate standard error of mean (SEM); (C) Phage Overlay Assay to atheroma and normal donor carotid tissue samples (n = 3, each group), figure shows representative slides of immunohistochemical staining for CTHRSSVVC-phage (CD163-mimic) and CVQLNSLPC-phage (Galectin-4 mimic) in the normal carotid (top) and atherosclerotic lesion from patient 2 (bottom), with a strong positively stained to lipid core region (hemorrhage, thrombus and cholesterol crystals) are indicated by the star (*). Bar, 100 µm in all panels; (D) Quantification of binding phage in human atheroma and normal carotid (n = 3, each group), 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. Statistical difference was determined by one-way analysis of variance followed by the Kruskal–Wallis test, and it was accepted at a value of * p < 0.05, vs. normal carotid.
Mentions: We have used a phage display library (labeled as CX7C) in which seven amino acid long random peptides are displayed on the surface of a filamentous phage fused to the minor pIII coat protein [8]. Given the heterogeneity of atherosclerosis disease, and our goal to develop target-specific peptides that bind to molecular markers which are common to most atherosclerotic lesions, phage display selection was performed in tandem using specimens obtained from three different patients whose atheroma had distinct inflammation characteristics to the lipid core region, (Figure 1A, Table 1). Although the initial histologic analyses were not complete, all elements of the intima, including those from the atherosclerotic lesion and endothelium, were exposed to the procedures.

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&minus;/&minus;) 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 &lt; 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&minus;/&minus;) 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 &gt;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