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Laser microdissection-based analysis of mRNA expression in human coronary arteries with intimal thickening.

ABSTRACT: Intimal thickening is an early phase of atherosclerosis characterized by differentiation of plaque smooth muscle cells (SMCs) from a contractile to a synthetic phenotype. We used laser microdissection (LMD) plus real-time RT-PCR to quantify mRNAs for calponin-1 and smoothelin, markers of the contractile phenotype, and for serum response factor (SRF), a regulator of SMC differentiation, in intimal and medial SMCs of human coronary arteries with intimal thickening. RNA expression was also analyzed by ISH and protein expression was detected by IHC. LMD plus RT-PCR found similar levels of SRF mRNA in intimal and medial SMCs, while medial mRNA levels for calponin-1 and smoothelin were higher. ISH confirmed that smoothelin mRNA levels in media exceeded those in intima, whereas SRF mRNA levels were similar at both sites. For calponin-1 and smoothelin, protein levels mirrored respective mRNA levels. By contrast, more medial than intimal SRF protein was present. Our results indicate that intimal SMCs exhibit a largely synthetic phenotype, perhaps reflecting lower intimal levels of SRF protein; ISH and LMD plus real-time RT-PCR provide comparable results; as a valuable alternative to ISH, LMD plus RT-PCR allows parallel measurement of several transcripts; and tissue gene expression studies must measure both protein and mRNA levels.

SUBMITTER: Stolle K

PROVIDER: S-EPMC3957817 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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Laser microdissection-based analysis of mRNA expression in human coronary arteries with intimal thickening.

Stolle Katrin K Weitkamp Benedikt B Rauterberg Jürgen J Lorkowski Stefan S Cullen Paul P

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 20041101 11

Intimal thickening is an early phase of atherosclerosis characterized by differentiation of plaque smooth muscle cells (SMCs) from a contractile to a synthetic phenotype. We used laser microdissection (LMD) plus real-time RT-PCR to quantify mRNAs for calponin-1 and smoothelin, markers of the contractile phenotype, and for serum response factor (SRF), a regulator of SMC differentiation, in intimal and medial SMCs of human coronary arteries with intimal thickening. RNA expression was also analyzed ...[more]

PMID: 15505346

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Project description:BackgroundThe endometrium prepares for implantation under the control of steroid hormones. It has been suggested that there are complicated interactions between the epithelium and stroma in the endometrium during menstrual cycle. In this study, we demonstrate a difference in gene expression between the epithelial and stromal areas of the secretory human endometrium using microdissection and macroarray technique.MethodsThe epithelial and stromal areas were microdissected from the human endometrium during the secretory phase. RNA was extracted and amplified by PCR. Macroarray analysis of nearly 1000 human genes was carried out in this study. Some genes identified by macroarray analysis were verified using real-time PCR.ResultsIn this study, changes in expression <2.5-fold in three samples were excluded. A total of 28 genes displayed changes in expression from array data. Fifteen genes were strongly expressed in the epithelial areas, while 13 genes were strongly expressed in the stromal areas. The strongly expressed genes in the epithelial areas with a changes >5-fold were WAP four-disulfide core domain 2 (44.1 fold), matrix metalloproteinase 7 (40.1 fold), homeo box B5 (19.8 fold), msh homeo box homolog (18.8 fold), homeo box B7 (12.7 fold) and protein kinase C, theta (6.4 fold). On the other hand, decorin (55.6 fold), discoidin domain receptor member 2 (17.3 fold), tissue inhibitor of metalloproteinase 1 (9 fold), ribosomal protein S3A (6.3 fold), and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (5.2 fold) were strongly expressed in the stromal areas. WAP four-disulfide core domain 2 (19.4 fold), matrix metalloproteinase 7 (9.7-fold), decorin (16.3-fold) and tissue inhibitor of metalloproteinase 1 (7.2-fold) were verified by real-time PCR.ConclusionsSome of the genes we identified with differential expression are related to the immune system. These results are telling us the new information for understanding the secretory human endometrium.

Dataset Information

Laser microdissection-based analysis of mRNA expression in human coronary arteries with intimal thickening.

Publications

Laser microdissection-based analysis of mRNA expression in human coronary arteries with intimal thickening.

Similar Datasets

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