The PCR products were electrophoresed on a 1% agarose gel and visualized with GreenSafe (NZYTech?, Lisbon, Portugal) using UV light. Table Domperidone 3 Oligonucleotide sequences and amplicon sizes in conventional and quantitative real-time PCR. qPCR results. In conclusion, decreased expression of TGFRs might suggest a reduction in TGF-1 participation in the MMP/TIMP imbalance throughout CVeD progression. Further studies about molecular events in the varicose vein wall are required and should take into consideration the venous anatomical region and CVeD clinical progression. = 29) and between the CEAP2C3 and CEAP4C6 groups (= 31). Therefore, differences regarding sex, age, BMI, and pregnancies presented 0.05. Table 1 Participants demographic and clinical features. Data shown in this table refer to the total number of participants from which subgroups were selected (in order to achieve demographic and clinical feature equivalence) for further analyses. and (Figure 1). The absence of and gene expression was reconfirmed after using umbilical arteries and the PC3 prostate cell line cDNA as positive controls. Also, and were excluded from further qPCR analyses due to very low gene expression in the cDNA pools. Open in a separate window Figure 1 PCR analysis of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and transforming growth factor- receptors (TGFRs) in cDNA pools of varicose and healthy veins. Amplification of -actin housekeeping gene was used as a control of the cDNA synthesis. Umbilical arteries or the PC3 prostate cell line was used as a positive control for the amplification of in varicose veins. Figure 2 presents the comparison between healthy and CVeD veins from the tibiotarsal region. The gene expression of (= 0.006), (= 0.010), (= 0.026), ( 0.001), and (= 0.001) was significantly decreased in the CEAP2C3 veins when compared to controls. Similarly, the gene expression of ( 0.001) and (= 0.019) was significantly decreased in the CEAP4C6 veins when compared to controls. Open in a separate window Figure 2 MMP, TIMP, and TGFR gene expression in healthy, CEAP2C3, and CEAP4C6 veins (from the tibiotarsal junction). Their gene expression was determined by qPCR and after normalization with -actin housekeeping gene. All results are expressed as fold expression. Error bars indicate mean standard error of the mean (SEM), 0.05. Comparisons between the CEAP2C3 and CEAP4C6 vein groups (Figure 3) from different anatomic harvest sites (tibiotarsal, saphenofemoral, and tributaries) were also performed. No significant differences in MMP, TIMP, and TGFR gene expression were found between the two CVeD groups from the tibiotarsal junction. From the saphenofemoral junction, only gene expression was significantly lower in the CEAP4C6 veins (= 0.027). Finally, in varicose tributary veins, (= 0.002) and (= 0.050) gene expressions were significantly increased, while gene expression (= 0.002) was significantly decreased in the CEAP4C6 group. Open in a separate window Open in a separate window Figure 3 MMP, TIMP, and TGFR gene expression in the CEAP2C3 and CEAP4C6 veins (from three different regions). Their gene expression was determined by qPCR after normalization with -actin housekeeping gene. All results are expressed as fold expression. Error bars indicate mean SEM, 0.05. 3.2. MMP, TIMP, and TGFR Immunoreactivity in Healthy and Varicose Vein Walls Qualitative results of IHC analysis are shown in Figure 4 and Table 2. Positive immunostaining for MMP2, TIMP2, and TGFR2 was more consistently found in both intima and media layers of varicose and healthy veins. A closer look at the staining intensity revealed that MMP2 was decreased in all.28/2009, 26 February 2009). decreased gene expression Domperidone of and and increased gene expression of and were found in advanced clinical stages. Most immunohistochemistry results for tunica intima were coherent with qPCR results. In conclusion, decreased expression of TGFRs might suggest a reduction in TGF-1 participation in the MMP/TIMP imbalance throughout CVeD progression. Further studies about molecular events in the varicose vein wall are required and should take into consideration the venous anatomical region and CVeD clinical progression. = 29) and between the CEAP2C3 and CEAP4C6 groups (= 31). Therefore, differences regarding sex, age, BMI, and pregnancies presented 0.05. Table Domperidone 1 Participants demographic and clinical features. Data shown in this table refer to the total number of participants from which subgroups were selected (in order to achieve demographic and clinical feature equivalence) for further analyses. and (Figure 1). The absence of and gene expression was reconfirmed after using umbilical arteries and the PC3 prostate cell line cDNA as positive controls. Also, and were excluded from further qPCR analyses due to very low gene expression in the cDNA pools. Open in a separate window Figure 1 PCR analysis of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and transforming growth factor- receptors (TGFRs) in cDNA pools of varicose and healthy veins. Amplification of -actin housekeeping gene was used as a control of the cDNA synthesis. Umbilical arteries or the PC3 prostate cell line was used as a positive control for the amplification of in varicose veins. Figure 2 presents the comparison between healthy and Domperidone CVeD NAV3 veins from the tibiotarsal region. The gene expression of (= 0.006), (= 0.010), (= 0.026), ( 0.001), and (= 0.001) was significantly decreased in the CEAP2C3 veins when compared to controls. Similarly, the gene expression of ( 0.001) and (= 0.019) was significantly decreased in the CEAP4C6 veins when compared to controls. Open in a separate window Figure 2 MMP, TIMP, and TGFR gene expression in healthy, CEAP2C3, and CEAP4C6 veins (from the tibiotarsal junction). Their gene expression was determined by qPCR and after normalization with -actin housekeeping gene. All results are expressed as fold expression. Error bars indicate Domperidone mean standard error of the mean (SEM), 0.05. Comparisons between the CEAP2C3 and CEAP4C6 vein groups (Figure 3) from different anatomic harvest sites (tibiotarsal, saphenofemoral, and tributaries) were also performed. No significant differences in MMP, TIMP, and TGFR gene expression were found between the two CVeD groups from the tibiotarsal junction. From the saphenofemoral junction, only gene expression was significantly lower in the CEAP4C6 veins (= 0.027). Finally, in varicose tributary veins, (= 0.002) and (= 0.050) gene expressions were significantly increased, while gene expression (= 0.002) was significantly decreased in the CEAP4C6 group. Open in a separate window Open in a separate window Figure 3 MMP, TIMP, and TGFR gene expression in the CEAP2C3 and CEAP4C6 veins (from three different regions). Their gene expression was determined by qPCR after normalization with -actin housekeeping gene. All results are expressed as fold expression. Error bars indicate mean SEM, 0.05. 3.2. MMP, TIMP, and TGFR Immunoreactivity in Healthy and Varicose Vein Walls Qualitative results of IHC analysis are shown in Figure 4 and Table 2. Positive immunostaining for MMP2, TIMP2, and TGFR2 was more consistently found in both intima and media layers of varicose and healthy veins. A closer look at the staining intensity revealed that MMP2 was decreased in all tunicae, TIMP2 was decreased in the intima and media, and TGFR2 was slightly decreased.
