Expression and regulation of endothelin precursor mRNA in avascular human amnion.

Posttranslational processing of preproendothelin in endothelial cells gives rise to endothelin, a 21 amino acid polypeptide that is a potent vasoconstrictor. Endothelin production is believed to be mediated principally by transcriptional mechanisms. Previously, preproendothelin mRNA expression has been detected only in vascular endothelial tissue and cells. In this study, we found that preproendothelin mRNA is expressed in an avascular human tissue, namely, amnion, an extraembryonic fetal membrane. Preproendothelin mRNA was not detected in avascular chorion laeve tissue (also an extraembryonic fetal membrane), in the highly vascularized fetal trophoblast, or in maternal uterine tissues. Furthermore, we found that preproendothelin gene expression is retained in human amnion cells maintained in primary monolayer culture. Using the amnion cells in primary monolayer culture to investigate the regulation of preproendothelin mRNA expression, we found that epidermal growth factor (EGF) and interleukin-1 (IL-1) act to stimulate preproendothelin mRNA levels; in addition, the induction of preproendothelin mRNA by either of these agents is enhanced upon simultaneous treatment with cycloheximide. These findings are indicative that preproendothelin gene expression in amnion is regulated positively by EGF and IL-1 and that inhibition of protein synthesis leads to superinduction of preproendothelin mRNA. In human umbilical cord endothelial cells, neither IL-1 nor EGF stimulate preproendothelin mRNA expression but inhibition of protein synthesis does lead to increased levels of preproendothelin mRNA. The amnion, therefore, provides a useful system for expansion of our understanding of the tissue specific expression and regulation of preproendothelin mRNA.

Effects of protamine, heparinase, and hyaluronidase on endothelial permeability and surface charge.

We undertook studies in the isolated perfused rat lung to determine 1) the effects of endothelial charge neutralization with the polycation protamine sulfate on microvascular permeability, lung water, and anionic ferritin binding to the endothelium and 2) the role of heparan sulfate and hyaluronate, negatively charged cell surface glycosaminoglycans, on permeability. Capillary permeability was determined by tissue 125I-albumin accumulation in isolated perfused rat lungs. In control lungs the 5-min albumin uptake was 0.50 +/- 0.05 cm3.s-1.g dry tissue-1 X 10(-3). It was increased by 132 +/- 7.8% (P less than 0.001) by protamine (0.08 mg/ml) and 65 +/- 12% (P less than 0.01) by heparinase (5 U/ml), whereas hyaluronidase (25 NFU/ml) was without effect. In control lungs total water was 4.83 +/- 0.15 ml g/dry tissue. Protamine increased lung water 12 +/- 2% (P less than 0.05). Heparinase caused a 9 +/- 3% increase (P less than 0.05), and hyaluronidase had no effect. Electron microscopy demonstrated that protamine increased anionic ferritin binding to the surface of endothelial cells. We conclude that protamine sulfate neutralization of negative charge in the pulmonary microcirculation leads to increased microvascular permeability. Heparin sulfate may be responsible for this charge effect.

Detection of intracardiac left-to-right shunting in adults: a prospective analysis of the variability of the standard indocyanine green technique in patients without shunting.

Intracardiac left-to-right shunting may be detected and quantitated by an oximetric analysis of blood from the right-sided cardiac chambers and prominent early recirculation of indocyanine green after it is injected into the central venous circulation and sampled from a systemic artery. Although the variability of oximetric measurements has been established in patients without shunting and a range of normal has been determined for the technique, the variability of the indocyanine green method among persons without shunting has not been clarified; as a result, a range of normal for indocyanine green has not been established. In 66 adult patients in whom hydrogen inhalation (an extremely sensitive technique for detecting even very small intracardiac left-to-right shunts) revealed no shunting, indocyanine green curves were generated by injection into the pulmonary artery as blood was sampled from a systemic artery. In these patients the percentage left-to-right shunt (that is, the percentage of pulmonary blood flow reaching the lungs through an intracardiac shunt) (determined with the equation of Carter et al) ranged from -9% to +26% (+7 +/- 8%, mean +/- standard deviation). There was no definable relation between the percentage left-to-right shunt and the indicator dilution measurement of cardiac output. Thus, these data establish a range of normal for the indocyanine green technique of detecting and measuring intracardiac left-to-right shunting. If this technique is to be used reliably to detect shunting, its results must demonstrate a percentage shunt in excess of +26%.

Myocyte and endothelial injury with ischemia reperfusion in isolated rat hearts.

We determined the time course of ischemic injury, the effects of reperfusion, and the protective effects of prostacyclin, oxygen radical scavengers, and diltiazem on myocardial myocyte and endothelial cell functions in isolated rat hearts. Left ventricular power and coronary microvascular permeability were used as indexes of myocyte and endothelial cell function, respectively. Neither 5- nor 10-min ischemia reperfusion significantly changed power or permeability. However, with reperfusion following 20 and 30 min of ischemia, power was reduced 50 and 60% and permeability increased 70 and 90%. In 30-min ischemic hearts the ischemia-induced increase in permeability was apparent after 4 min reperfusion and further exacerbated at 20 min. Hypoxic reperfusion did not prevent increased permeability. Prostacyclin or a combination of superoxide dismutase, catalase, and mannitol also did not prevent increased permeability, and the radical scavengers did not ameliorate depressed power. In contrast, perfusion with diltiazem during ischemia reperfusion blunted the reduction in power and prevented the increase in permeability. We conclude that ischemia reperfusion causes similar time course of injury to myocytes and endothelial cells; reperfusion contributes to endothelial injury, and diltiazem affords protection to both cell types.

The effects of hyaluronidase on interstitial hydration, plasma protein exclusion, and microvascular permeability in the isolated perfused rat heart.

Hyaluronic acid, a principal glycosaminoglycan of the cardiac interstitium, may have a role in interstitial hydration, interstitial plasma protein exclusion and microvascular transport process (Wiederhielm, 1976b). We have investigated whether hyaluronidase reduces myocardial hyaluronate concentrations and thereby alters these several physical aspects in the isolated rat heart. Studies were conducted in ischemic, as well as aerobic hearts because of the reported therapeutic efficacy of the enzyme in myocardial ischemia. Two hours of perfusion with hyaluronidase significantly reduced myocardial hyaluronate content. Additionally, hyaluronidase decreased interstitial volume of both aerobic and otherwise edematous ischemic hearts, and prevented ischemic induced increased coronary vascular resistance in ischemic hearts. However, hyaluronidase did not effect the albumin interstitial exclusion volume or microvascular albumin and sorbitol exchange in aerobic hearts. In ischemic hearts, the enzyme did not prevent nor enhance the increase in microvascular permeability which occurred. We conclude that hyaluronate is neither a determinant of interstitial protein exclusion nor microvascular permeability, but plays an important role in interstitial hydration.

Hyaluronidase reversal of increased coronary vascular resistance in ischemic rat hearts.

Testicular hyaluronidase prevents increased coronary vascular resistance (CVR) during prolonged myocardial ischemia. The mechanism is unknown, but edema and contracture both have been suggested to increase CVR. Additionally, the extent of contracture has been inversely related to ATP levels. Therefore, isolated perfused ischemic rat hearts were treated with hyaluronidase, following a 25% increase in CVR, to determine whether 1) increased CVR was reversed, 2) edema or contracture was reduced, and 3) tissue ATP levels were increased. Three hours of low-flow ischemia decreased coronary flow (CF) from 17.4 +/- 0.13 to 12.6 +/- 0.2 ml X min-1 X g dry tissue-1. During the subsequent 2 h of ischemia, CF of vehicle-treated hearts continued to decline to 8.0 +/- 0.76 ml X min-1 X g dry tissue-1, whereas CF of hyaluronidase-treated hearts increased to 15.6 +/- 1.17 ml X min-1 X g dry tissue-1. These changes in CF persisted during postischemic perfusion. Furthermore, restoration of coronary vascular resistance by hyaluronidase was associated with a 19% reduction in tissue water compared with control ischemic hearts but not with a reduction in cardiac contracture or an increase in tissue ATP. These results suggest that treatment of ischemic hearts with hyaluronidase reverses increased CVR through a reduction in tissue edema.