DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells.

Using DNA microarray screening (GeneFilter 211, Research Genetics, Huntsville, AL) of mRNA from primary human umbilical vein endothelial cells (HUVEC), we identified 52 genes with significantly altered expression under shear stress [25 dynes/cm(2) for 6 or 24 h (1 dyne = 10 microN), compared with matched stationary controls]; including several genes not heretofore recognized to be shear stress responsive. We examined mRNA expression of nine genes by Northern blot analysis, which confirmed the results obtained on DNA microarrays. Thirty-two genes were up-regulated (by more than 2-fold), the most enhanced being cytochromes P450 1A1 and 1B1, zinc finger protein EZF/GKLF, glucocorticoid-induced leucine zipper protein, argininosuccinate synthase, and human prostaglandin transporter. Most dramatically decreased (by more than 2-fold) were connective tissue growth factor, endothelin-1, monocyte chemotactic protein-1, and spermidine/spermine N1-acetyltransferase. The changes observed suggest several potential mechanisms for increased NO production under shear stress in endothelial cells.

Shear stress differentially regulates PGHS-1 and PGHS-2 protein levels in human endothelial cells.

The secretion of prostacyclin (PGI2) by endothelial cells is regulated by shear stress. Prostaglandin H synthase (PGHS) is considered to be a key limiting enzyme in the synthesis of PGI2 from arachidonic acid. Endothelial cells were cultured in the presence of 4, 15, or 25 dyn/cm2 shear stress using a parallel plate flow chamber to assess the effect of shear stress on both PGHS isoforms, PGHS-1 and PGHS-2. In cells exposed to 4, 15, or 25 dyn/cm2 shear stress PGHS-1 and PGHS-2 protein levels initially decreased. The decrease was followed by a sustained increase for PGHS-1 but only a transient increase for PGHS-2. The duration of the PGHS-2 increase depended on the magnitude of the shear stress. The effect of altering shear stress levels on PGHS protein levels in cells preconditioned to either 4, 15, or 25 dyn/cm2 shear stress for 48 h was also studied. Changing shear stress levels effected PGHS-2 but not PGHS-1. Increases in shear stress levels from 4 to 15 or 25 dyn/cm2 caused a decrease in PGHS-2. In contrast, decreases in shear stress levels from 15 or 25 to 4 dyn/cm2 caused PGHS-2 to increase. There was a continual decrease in PGHS-2 when the shear stress was changed from 15 to 25 or 25 to 15 dyn/cm2. In summary, the regulation of PGHS-2 by shear stress is dependent upon the magnitude of the shear stress, whereas the regulation of PGHS-1 protein levels seems to be independent of the shear stress magnitude. The regulation of PGHS-1 and PGHS-2 protein levels by shear stress indicates that these proteins play an important role in the maintenance of cardiovascular homeostasis as regulators of PGI2 production.

Characterization of two baboon surfactant protein A genes.

The gene encoding surfactant protein A (SP-A) is expressed in type II pneumonocytes and is developmentally and hormonally regulated in fetal lung tissue. SP-A is encoded by a single-copy gene in rabbits, dogs, rats, and mice. By contrast, the human genome contains two similar genes, hSP-A1 and hSP-A2, which are differentially regulated during development and differentially regulated by adenosine 3',5'-cyclic monophosphate (cAMP) and glucocorticoid treatment of human fetal lung in culture. In the present study, we have isolated and characterized baboon genomic clones containing two highly similar SP-A genes. Restriction mapping of these clones, together with Southern analysis of genomic DNA, indicates that these comprise two distinct baboon SP-A genes. Sequence comparison of DNA upstream of the transcription initiation sites and within the 3'-untranslated regions encoded by exon VI indicates that one of the baboon SP-A genes (bSP-A1) is more similar to hSP-A1, whereas the other (bSP-A2) is more similar to hSP-A2. Primer extension analysis of baboon lung mRNA indicates that both baboon SP-A genes utilize conserved transcription initiation sites. Reverse transcriptase-polymerase chain reaction analysis of RNA isolated from lung tissues of fetal baboons of 160 days gestational age indicates that both bSP-A1 and bSP-A2 are expressed in baboon fetal lung and that mRNA transcripts of bSP-A1 and bSP-A2 genes are primarily comprised of sequences encoded by exons I and III-VI. However, minor transcripts of the bSP-A1 gene containing exon II and exon II plus an extension also were detected. The presence of two SP-A genes in the baboon suggests that duplication of the SP-A gene occurred > 26.5 million years ago, before divergence of the baboon lineage from the man-gorilla-chimpanzee clade.

Characterization of mRNA transcripts and organization of human SP-A1 and SP-A2 genes.

In the present study, we have characterized the mRNA transcripts and intron-exon organization of the human surfactant protein (SP)A1 and SP-A2 genes. By primer extension analysis of mRNA isolated from human fetal lung explants using an oligonucleotide primer to exon II (as delineated in the SP-A1 gene), a minimum of nine primer extended transcripts was observed. Rapid amplification of cDNA ends was used to amplify the primer extended transcripts for sequence analysis. Sequence analysis of 47 full-length primer extended cDNAs and comparison with the sequences of the genes encoding SP-A1 and SP-A2 revealed four different classes of transcripts of the SP-A2 gene and five different classes of transcripts of the gene encoding SP-A1. A major difference between SP-A2 and SP-A1 mRNA transcripts is that SP-A2 transcripts are always comprised of sequences contained within six exons; the extra exon in SP-A2 (exon II of VI) encodes additional 5'-untranslated sequence and is located between exons I and II of SP-A1. By contrast, the majority of transcripts of the SP-A1 gene are comprised of sequences contained within five exons. In the cases of both SP-A1 and SP-A2 genes, a small proportion of the mRNA transcripts contain sequences present in alternate exons. In addition, the majority of the SP-A1 mRNA transcripts are initiated 5 bp downstream of the transcription initiation site of SP-A2. In our companion paper [McCormick and Mendelson. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L367-L374, 1994], we report that the SP-A1 and SP-A2 genes are differentially regulated during development and by adenosine 3',5'-cyclic monophosphate and glucocorticoids in human fetal lung in culture.

Human SP-A1 and SP-A2 genes are differentially regulated during development and by cAMP and glucocorticoids.

Expression of the surfactant protein A (SP-A) gene is lung specific, developmentally induced, and regulated by adenosine 3',5'-cyclic monophosphate (cAMP) and glucocorticoids. Humans have two highly similar genes encoding SP-A (SP-A1 and SP-A2). In the companion paper [S.M. McCormick, V. Boggaram, and C.R. Mendelson Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L354-L366, 1994] we report that SP-A1 and SP-A2 RNA transcripts are alternatively spliced at their 5' ends, resulting in nine different primer-extended transcripts. In the present study, primer extension was used to assess the relative levels of expression of the SP-A1 and SP-A2 genes in human adult lung tissue and in fetal lung tissues maintained in organ culture in the absence or presence of dibutyryl (DB)cAMP (1 mM) and dexamethasone (Dex, 10(-4) M). Primer extension and Northern analysis were used to assess the effects of these agents on the levels of expression of these genes. In human adult lung tissue, 65% of the SP-A mRNA transcripts were derived from the SP-A2 gene, whereas only 35% were from SP-A1. On the other hand, in lung tissue from a 28-wk gestation neonate, only SP-A1 mRNA transcripts were detected, and, in midgestation fetal lung cultured in control medium, 65% of the SP-A mRNA was found to be SP-A1 and 35% was SP-A2.(ABSTRACT TRUNCATED AT 250 WORDS)

To what extent would women prefer chorionic villus sampling to amniocentesis for prenatal diagnosis?

Sixty-six pregnant women and 46 doctors were interviewed about their preferences for chorionic villus sampling (CVS) or amniocentesis for prenatal diagnosis in a hypothetical situation where the indication was late maternal age. The standard gamble method was used to calculate each individual's degree of preference for one procedure over the other (utility) expressed in terms of the risk of miscarriage associated with the preferred procedure that would be tolerated in order to have that procedure. Utilities for each group were calculated and compared. Pregnant women nominated a median utility for CVS of a miscarriage rate of 0.9%, while doctors nominated a median utility for CVS of a miscarriage rate of 1.2%. The difference between these utilities was not statistically significant. The method described in this study can enable potential consumers of a new procedure to provide the minimum 'clinically important difference' between a new procedure and an existing procedure necessary for calculation of the sample size in a controlled clinical trial.