Figure 4.
Figure 4. Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines relevant to WBC recruitment. ECs were seeded on the collagen gel in the absence (EC/Gel) or presence (EC/SMC) of SMCs. SMCs were also embedded in the collagen gels without coculture of ECs (NC/SMC). The cells cultured on Petri dishes were used as controls (Control). In additional experiments, EC/Gel and EC/SMC were treated with IL-1β for 4 hours. (A) Specificity of ECs and SMCs. ECs and SMCs were purified from their cocultures, and their specificity was examined by Western blot analysis of their lysates using antibodies against PECAM-1 and SMα-actin as markers for ECs and SMCs, respectively. (B-C) Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines. The mRNA levels of selected adhesion molecules and chemokines in ECs (B) and SMCs (C) from different experimental conditions were determined by RT-PCR analysis, as described in “Materials and methods.” Amplification of cDNA was performed in parallel samples using human GAPDH primers. Results are representative of triplicate experiments with similar results. (D) Coculture with SMCs increased ICAM-1, VCAM-1, and E-selectin expressions on ECs. ECs isolated from EC/SMC or EC/Gel were analyzed by flow cytometry, as described in “Materials and methods.” The EC/SMC shows a shift of fluorescence intensity profile for ICAM-1, VCAM-1, and E-selectin, as compared with the EC/Gel. ECs incubated with only FITC-conjugated antibody were used as blank controls. Results are representative of duplicate experiments with similar results. (E) Coculture of ECs and SMCs increased the release of chemokines into conditioned media and collagen gels. The soluble forms of the chemokines in both the media and collagen gels of EC/Gel, NC/SMC, and EC/SMC experiments were determined by ELISA assay, as described in “Materials and methods.” The collagen gel incubated with only the media was used as negative controls (NC/Gel). The results shown are mean ± SEM from 3 independent experiments. *P < .05 for EC/SMC versus NC/Gel, EC/Gel, and NC/SMC samples.

Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines relevant to WBC recruitment. ECs were seeded on the collagen gel in the absence (EC/Gel) or presence (EC/SMC) of SMCs. SMCs were also embedded in the collagen gels without coculture of ECs (NC/SMC). The cells cultured on Petri dishes were used as controls (Control). In additional experiments, EC/Gel and EC/SMC were treated with IL-1β for 4 hours. (A) Specificity of ECs and SMCs. ECs and SMCs were purified from their cocultures, and their specificity was examined by Western blot analysis of their lysates using antibodies against PECAM-1 and SMα-actin as markers for ECs and SMCs, respectively. (B-C) Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines. The mRNA levels of selected adhesion molecules and chemokines in ECs (B) and SMCs (C) from different experimental conditions were determined by RT-PCR analysis, as described in “Materials and methods.” Amplification of cDNA was performed in parallel samples using human GAPDH primers. Results are representative of triplicate experiments with similar results. (D) Coculture with SMCs increased ICAM-1, VCAM-1, and E-selectin expressions on ECs. ECs isolated from EC/SMC or EC/Gel were analyzed by flow cytometry, as described in “Materials and methods.” The EC/SMC shows a shift of fluorescence intensity profile for ICAM-1, VCAM-1, and E-selectin, as compared with the EC/Gel. ECs incubated with only FITC-conjugated antibody were used as blank controls. Results are representative of duplicate experiments with similar results. (E) Coculture of ECs and SMCs increased the release of chemokines into conditioned media and collagen gels. The soluble forms of the chemokines in both the media and collagen gels of EC/Gel, NC/SMC, and EC/SMC experiments were determined by ELISA assay, as described in “Materials and methods.” The collagen gel incubated with only the media was used as negative controls (NC/Gel). The results shown are mean ± SEM from 3 independent experiments. *P < .05 for EC/SMC versus NC/Gel, EC/Gel, and NC/SMC samples.

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