MR imaging of tropomyosin-4 antibody targeting synthetic phenotype vascular smooth muscle cells in vitro

Objective: To isolate, culture, and identify the synthetic phenotype vascular smooth muscle cells (VSMC) and identify the specific marker protein (tropomyosin-4, TPM-4) of synthetic phenotype. To employ the immune molecular imaging technique to develop MRI of probe targeted with TPM-4 antibody VSMC in vitro. Methods: The synthetic phenotype VSMC and endothelial cells (EC) were isolated and cultured in vitro, respectively. Immunocytochemistry (ICC) staining for a-smooth muscle actin (SMA) and VIII factor was performed for cell identification, respectively. The high expression level of TPM-4 protein was tested by immunofluorescence double staining. The MRI molecular probe was built by chemical cross-linking, TPM-4 conjunctive probe (TPM4-USPI0) as the experimental group, IgG conjunctive probe (IgG-USPIO) as the negative group, non-conjunctive probe (USPIO) as the control group, and PBS as the blank group. The synthetic VSMC were incubated with probes within experimental group, negative group, control group, respectively, and EC were incubated with experimental group as another control group. Prussian blue staining was employed to analyze the specific-targeting and MTT assay was used to test bioactivity of the probe under different concentrations (0, 5, 10, 20, 40 μg/ml) in vitro. 7.0 T MRI scanner was used to detect the magnetic properties. With 7.0 T MRI scanner, the T₂WI images of different probes labeled synthetic VSMC and different concentration gradient (1 × 10³, 5 × 10³, 1 × 10⁴, 5 × 10⁴) TPM4-USPIO labeled cells were obtained and analyzed. T₂ signal and MTT data among groups were compared using single factor analysis of variance (ANOVA) and LSD test. Results: The synthetic phenotype of VSMC were isolated and cultured successfully, and the VSMC could express the TPM-4 protein. The synthetic phenotype VSMC had a high level of the protein expression. The probe was made successfully. The T₂ relaxivity of TPM4-USPI0 and IgG-USPIO were 0.0350 × 10⁶, 0.0316 × 10⁶ mol/s, respectively, with high stability as USPIO (0.0292 × 10⁶ mol/s). Prussian blue staining results showed that the experimental group probe could specifically bind to the synthetic VSMC. MTT results showed that iron concentration within 40 μ/ml or less had no effect on VSMC proliferation activity. The T₂WI of experimental group showed lower signal than the control group. The T₂ relaxivity was (116.67 ± 2.08) ms, which was less than the control group [(217.67 ± 2.52), (219.33 ± 2.08) ms, respectively] and the blank group [(205.33 ± 1.53) ms] (F = 1670.43, P < 0.01). The T₂ relaxivity of the different concentration gradient labeled cells (1 × 10³, 1 × 10⁴, 1 × 10⁵) were (184.33 ± 2.08), (169.67 ± 1.15), (116.67 ± 2.08 ) ms, respectively (F = 684.35, P < 0.01). No significant difference of the T₂WI gradual signal dim was found between cells with the same order concentration (P > 0.05). Conclusions: The synthetic phenotype of VSMC can be obtained by PDGF-BB treatment. TPM4-USPIO probe is efficient, specific and targeted at combination with synthetic VSMC. The T₂WI signal changed obviously under high field MRI scanner, which provides a new way for molecular imaging research. (authors)