Patterns of living beta-actin movement in wounded human coronary artery endothelial cells exposed to shear stress.

We previously demonstrated that physiologic levels of shear stress enhance endothelial repair. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate (Albuquerque et al., 2000, Am. J. Physiol. Heart Circ. Physiol. 279, H293-H302). However, the patterns and movements of beta-actin filaments responsible for cell motility and translocation in human coronary artery endothelial cells (HCAECs) have not been previously investigated under physiologic flow. HCAECs transfected with beta-actin-GFP were cultured on type I collagen-coated coverslips. Confluent cell monolayers were subjected to laminar shear stress of 12 dynes/cm(2) for 18 h in a parallel-plate flow chamber to attain cellular alignment and then wounded by scraping with a metal spatula and subsequently exposed to a laminar shear stress of 20 dynes/cm(2) (S-W-sH) or static (S-W-sT) conditions. Time-lapse imaging and deconvolution microscopy was performed during the first 3 h after imposition of S-W-sH or S-W-sT conditions. The spatial and temporal dynamics of beta-actin-GFP motility and translocation during wound closure in HCAEC monolayers were analyzed under both conditions. Compared with HCAEC under S-W-sT conditions, our data show that HCAEC under S-W-sH conditions demonstrated greater beta-actin-GFP motility, filament and clumping patterns, and filament arcs used during cellular attachment and detachment. These findings demonstrate intriguing patterns of beta-actin organization and movement during wound closure in HCAEC exposed to physiological flow.

Shear stress enhances human endothelial cell wound closure in vitro.

Repair of the endothelium occurs in the presence of continued blood flow, yet the mechanisms by which shear forces affect endothelial wound closure remain elusive. Therefore, we tested the hypothesis that shear stress enhances endothelial cell wound closure. Human umbilical vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were cultured on type I collagen-coated coverslips. Cell monolayers were sheared for 18 h in a parallel-plate flow chamber at 12 dyn/cm(2) to attain cellular alignment and then wounded by scraping with a metal spatula. Subsequently, the monolayers were exposed to a laminar shear stress of 3, 12, or 20 dyn/cm(2) under shear-wound-shear (S-W-sH) or shear-wound-static (S-W-sT) conditions for 6 h. Wound closure was measured as a percentage of original wound width. Cell area, centroid-to-centroid distance, and cell velocity were also measured. HUVEC wounds in the S-W-sH group exposed to 3, 12, or 20 dyn/cm(2) closed to 21, 39, or 50%, respectively, compared with only 59% in the S-W-sT cells. Similarly, HCAEC wounds closed to 29, 49, or 33% (S-W-sH) compared with 58% in the S-W-sT cells. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate. These results suggest that physiological levels of shear stress enhance endothelial repair.

Placental expression of glucose transporter proteins 1 and 3 in growth-restricted fetal rats.

OBJECTIVE: The purpose of this study was to determine the effect of intrauterine growth restriction on the placental expression of glucose transporter proteins. STUDY DESIGN: Intrauterine growth restriction was induced by bilateral uterine artery ligation in the pregnant rat at a gestational age of 19 days (term is 21.5 days). Maternal rats were killed and fetuses were delivered by hysterotomy on gestational days 20 and 21. Control fetuses from mothers that had been subjected to a sham operation were studied simultaneously. Glucose transporter protein 1 and glucose transporter protein 3 messenger ribonucleic acid was quantified by reverse transcriptase-polymerase chain reaction amplification. Glucose transporter protein 1 and glucose transporter protein 3 densities in placental membranes were also assessed by Western blotting and by immunohistochemical analysis. RESULTS: Glucose transporter protein 1 messenger ribonucleic acid, expressed as a multiple of the matched sham control value, was unchanged on both days 20 and 21 of gestation. Glucose transporter protein 3 messenger ribonucleic acid was also unchanged. Western blotting demonstrated no change in expression of glucose transporter protein 1 or glucose transporter protein 3 on either day 20 or 21 of gestation. Immunohistochemical staining patterns for glucose transporter protein 1 and glucose transporter protein 3 on the syncytiotrophoblastic membranes were similar between the growth-restricted group and the sham control group. CONCLUSION: Placental expression of glucose transporter proteins in the pregnant rat is unchanged with uteroplacental insufficiency.

Localization and quantification of glucose transporters in liver of growth-retarded fetal and neonatal rats.

To determine whether altered transport of glucose into the hepatocyte may be an important factor contributing to abnormal hepatic glucose metabolism in the intrauterine growth-retarded (IUGR) fetus and newborn, we measured glucose transport (glucose uptake, GLUT protein, and mRNA) and localization of GLUT protein in liver of control (sham operated) and IUGR fetal (day 20) and postnatal (1, 4, 14, and 21 days) rats. GLUT-1 and -2 proteins were localized to the hepatocyte. Glucose uptake and GLUT-1 protein and mRNA levels were increased in IUGR fetal and neonatal liver. GLUT-2 protein and mRNA levels were low in IUGR and control fetal liver. After birth, GLUT-2 abundance did not differ from controls. Run-on experiments showed that the rate of transcription of GLUT-1 and -2 did not differ between IUGR and control rats. However, the transcription rate of GLUT-1 decreased with age, and the GLUT-2 transcription rate increased with age. These studies indicate that the metabolic and physiological factors that cause IUGR also alter glucose transporter expression in fetal liver.

Modulation of glucose transport in fetal rat lung: a sexual dimorphism.

Male fetuses exhibit delayed lung maturation and surfactant production in comparison with female fetuses. This delay may be related to sex hormone effects: estrogen enhances and androgens delay lung development. The uptake of glucose, an important precursor for surfactant synthesis, may be differently affected by estrogen and androgens. In these studies we determined the effects of these two hormones on glucose transport (glucose uptake, glucose transporter [Glut] 1 protein, and mRNA) and hexokinase activity in lung tissue of fetal rats. On Day 20 of gestation (term = 21.5 d) lung tissue was harvested from female and male fetal rats, minced into explants, and cultured for 24 h. Basal glucose uptake, measured in the absence of sex hormones, was 37% higher (P < 0.05) in female compared with male lungs. Explants were washed and cultured for an additional 3 h or 24 h in either estradiol or dihydrotestosterone (DHT) at 0, 1, 10, or 100 nM. Twenty-four-hour treatment with estradiol in both male and female explants increase 2-deoxyglucose uptake, Glut 1 protein, and mRNA levels (P < 0.05). However, explants from male fetuses were not as responsive to estradiol treatment as were those from females (P < 0.05). Treatment for 24 h with DHT decreased 2-deoxyglucose uptake, Glut 1 protein, and mRNA levels in females and males (P < 0.05). There was no difference in response between females and males. Short-term incubation (3 h) with sex hormones had no effect on glucose uptake. However, 3-h treatment with estradiol did increase Glut 1 mRNA levels (P < 0.05). Hexokinase activity was not affected by estradiol or DHT treatment. These findings indicate that estradiol and DHT differentially regulate glucose uptake in fetal rat lung tissue. This regulation of substrate supply (glucose) by estradiol and DHT may be another mechanism for the sexual dimorphism observed in lung development and surfactant synthesis.

Intrauterine growth retardation alters mitochondrial gene expression and function in fetal and juvenile rat skeletal muscle.

Uteroplacental insufficiency alters the anabolic metabolism of the fetus, resulting in intrauterine growth retardation (IUGR). The metabolic and physiologic factors that cause IUGR have long standing consequences after birth. Postnatal growth and glucose metabolism are altered in the IUGR infant. Skeletal muscle is an important component of growth and metabolizes up to 70% of i.v. glucose. The ability of skeletal muscle to metabolize glucose is affected by ATP availability. We hypothesized that gene expression and function of proteins involved in mitochondrial ATP production and distribution would be altered in juvenile IUGR muscle. To test this hypothesis, we used a model of IUGR, induced by bilateral uterine artery ligation in the pregnant rat, that mimics uteroplacental insufficiency in the human. RT-PCR was used to measure the mRNA levels of three important mitochondrial proteins; NADH-ubiquinone-oxireductase subunit 4L(ND-4L), subunit C of the F1F0-ATP synthase (SUC), and adenine nucleotide translocator 1 (ANT1) in IUGR and control rats in fetal and juvenile life. In the fetus, mRNA levels of all three proteins were significantly increased in IUGR skeletal muscle. In contrast, in juvenile animals, mRNA levels of all three proteins were significantly decreased. mRNA levels of other metabolically important proteins, glucose-6-phosphate dehydrogenase and carnitine-palmitoyl-transferase II, were not significantly altered in IUGR juvenile animals. To assess if decreased gene expression is associated with altered mitochondrial function, we measured the mitochondrial NAD+/NADH ratio in d 21 juvenile control and IUGR muscle. At d 21, decreased gene expression if ND-4L, SUC, and ANTI is associated with a decreased mitochondrial NAD+/NADH ratio. The results of our study suggest that the metabolic alterations associated with uteroplacental insufficiency in the rat result in altered fetal and postnatal muscle mitochondrial mRNA expression as well as altered postnatal mitochondrial function.

Measurement of GLUT mRNA in liver of fetal and neonatal rats using a novel method of quantitative polymerase chain reaction.

Transfer of glucose into the hepatocyte is mediated by glucose transporters (GLUTs). GLUT mRNA levels are usually measured by Northern blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) is often used to measure RNA abundance. However, this method is only semiquantitative and has no internal control during first-strand synthesis. We designed a method of coreverse transcription and PCR amplification using bovine rhodopsin as an internal control for both cDNA synthesis and amplification. As part of the validation of this technique, we determined that there was no nonspecific amplification of bovine GLUTs by rhodopsin primers, that there were no differences in amplification due to different regions of the Glut gene amplified, and that there were no secondary structure effects on amplification. We applied our modified method of RT-PCR to measure the ontogeny of GLUT expression in liver of fetal and postnatal rats (d20 fetuses and d1, d4, d14, and d21 juvenile rat pups). GLUT 1 mRNA quantity decreased whereas GLUT 2 increased with age. We were able to detect small quantities of GLUT 3 in fetal liver and of GLUT 5 in postnatal liver. This method of RT-PCR provides an internal control and allows measurement of mRNA levels in small quantities of tissue, making it ideal for use in the fetus and any system in which mRNA levels are low.

Altered hepatic gene expression of enzymes involved in energy metabolism in the growth-retarded fetal rat.

Intrauterine growth retardation (IUGR) resulting from placental insufficiency is a common complication of pregnancy. Bilateral uterine artery ligation of the pregnant rat is a model which mimics intrauterine growth retardation in the human. IUGR rat fetuses have altered hepatic energy and redox states, with reduced fetal hepatic ATP/ADP ratio, increased cytosolic NAD+/NADH ratio, and decreased mitochondrial NAD+/NADH ratio. These critical changes in energy metabolism contribute to IUGR. The effects of these changes at the molecular level are largely unknown. To address these effects we compared hepatic mRNA populations of IUGR and normal fetuses and neonates using mRNA differential display, a polymerase chain reaction-based method for assaying transcriptional differences under various conditions. We isolated and sequenced 18 cDNA products whose mRNA levels were elevated in IUGR compared with normal fetal and neonatal liver. These analyses demonstrated that NADH-ubiquinone oxireductase subunit 4L mRNA (ND-4L) was significantly increased in liver of IUGR fetuses and neonates. This suggested that IUGR may be associated with altered expression of genes involved in the generation of ATP and NADH. Therefore, we measured mRNA levels of adenine-nucleotide translocator-2 (ANT-2), glucose-6-phosphate dehydrogenase (G6PD), mitochondrial malate dehydrogenase (MMD), ornithine transcarbamylase (OTC), and phosphofructokinase-2 (PFK-2) using a semiquantitative reverse transcriptase-polymerase chain reaction-based technique. In the IUGR fetus, ND-4L, ANT-2, G6PD, and MMD mRNA levels were significantly elevated; PFK-2 mRNA levels were unchanged, and OTC levels were decreased. In the IUGR newborn rat, mRNA levels of all 6 enzymes were increased suggesting that the metabolic state of the growth retarded newborn remains abnormal after birth. Uteroplacental insufficiency affects the immediate and long-term metabolic milieu of the growth retarded animal, and forces specific adjustments, including the expression of mRNA encoding enzymes involved with hepatic energy production.

The effects of severe maternal diabetes on glucose transport in the fetal rat.

We mimicked the condition of severe maternal diabetes by administering high doses of streptozotocin (STZ) to the pregnant rat to determine the effects of increased glucose availability on fetal glucose transport and to assess whether a relationship might exist between glucose transport and altered fetal growth. Fetuses of STZ-treated pregnant rats were growth retarded (3.86 +/- 0.13 vs. 5.29 +/- 0.06 g), hyperglycemic (30.0 +/- 1.0 vs. 5.5 +/- 0.5 mM/liter), and hyperinsulinemic (1263.8 +/- 138.3 vs. 817.9 +/- 116.7 pM/liter). Glucose uptake, Glut 1 messenger RNA (mRNA), and Glut 1 protein were greater in STZ-treated fetal brain than in controls (50%, 83%, and 50%, respectively; P < 0.05). Glut 3 mRNA levels in STZ-treated and control fetal brain were equivalent and significantly less than levels of Glut 1. Glucose uptake in muscle of STZ fetuses was 70% greater than control values (P < 0.05). Glut 1 mRNA levels were increased by 93% in STZ fetal muscle (P < 0.05). Neither Glut 3 nor Glut 4 mRNA could be detected in STZ-treated and control fetal muscle. Glut 1 protein levels were increased by 70% in STZ-treated fetal muscle compared to control muscle (P < 0.05). These observations indicate that altered glucose transport per se does not directly contribute to fetal growth retardation with maternal STZ diabetes. Perturbations in other physiological and metabolic factors may contribute to the pathogenesis of fetal growth retardation in STZ-induced diabetes during pregnancy.

The effect of insulin and insulin-like growth factor-I on glucose transport in normal and small for gestational age fetal rats.

In a model of asymmetric small for gestational age (SGA) fetal growth retardation, we have previously found that glucose transport is decreased in lung (an organ whose growth is restricted) and unaffected in brain (growth is normal). The SGA model alters a number of physiological and metabolic factors that may decrease glucose transport, thereby causing growth retardation. Specifically, insulin and insulin-like growth factor-I (IGF-I) concentrations are diminished in SGA fetuses. We hypothesized that the specific modulation by these factors of gene expression of a glucose transporter, Glut-1, is impaired. We performed bilateral uterine arterial ligation in pregnant rats on day 19 of gestation (term = 21.5 days) and obtained fetal brain, lung, and skeletal muscle on day 20. Lung and muscle explants and monolayers of glial cells and type II pneumocytes were cultured in the presence or absence of insulin or IGF-I for 24 h. Glucose uptake and levels of Glut-1 protein and mRNA were similar in brains of SGA and control fetuses and were not affected by treatment with insulin or IGF-I. Treatment with insulin or IGF-I increased glucose uptake and levels of Glut-1 protein and mRNA in a dose-dependent manner in lung and muscle from control fetuses. However, the response in SGA lung was not as great as that in controls. SGA muscle demonstrated no significant response to either hormone. These findings suggest that changes in glucose transport modulation might contribute to the development of asymmetric growth retardation, and that maintenance of normal transporter function and expression in brain may play a role in sparing its growth.

Glucose regulates glut 1 function and expression in fetal rat lung and muscle in vitro.

The mechanisms that regulate cellular glucose transport (glucose uptake, Glut 1 protein, and mRNA) in the fetus are not known. We attempted to define the effects of glucose availability alone in vitro on glucose transport in fetal rat lung and muscle. On day 20 of gestation (term = 21.5 days), lung and muscle tissues were harvested from normal fetal rats, minced into explants, and cultured for 24 h in standard culture medium (lung, 28 mM; muscle, 5.5 mM glucose). Explant cultures were washed and cultured for an additional 1 or 24 h in medium containing one of four concentrations of glucose: 1) glucose free, 2) low glucose, 3) high glucose, and 4) standard. Twenty-four-hour, but not 1-h, treatment of fetal lung and muscle in vitro with low concentrations of glucose increased 2-deoxyglucose uptake and Glut 1 protein and mRNA levels (P < 0.05). Culture in high glucose medium for 24 h, but not 1 h, decreased 2-deoxyglucose uptake and Glut 1 protein and mRNA levels (P < 0.05). Culture in glucose-free medium for 24 h up-regulated glucose transport in lung and down-regulated glucose transport in muscle, indicating that regulation of fetal glucose transport may be tissue specific. These findings differ from our studies of in vivo models of altered fetal growth and abnormal glucose availability. Maternal bilateral uterine artery ligation limits glucose availability to the fetus, and glucose transport is down-regulated. Low glucose in vitro has the opposite effect. Maternal diabetes increases glucose availability to the fetus, and glucose transport is up-regulated. High glucose in vitro does the opposite. We conclude that while glucose alone in vitro affects its uptake by the cell, other factors that are altered in these in vivo conditions act in concert with glucose to regulate glucose transport in the fetus.

Differential effects of short and long durations of insulin-induced maternal hypoglycemia upon fetal rat tissue growth and glucose utilization.

We studied the effects of short and long durations of insulin-induced maternal hypoglycemia upon in vivo glucose utilization of several fetal tissues in the rat. Osmotic minipumps filled with insulin were implanted in pregnant rats on d 15 or 18 of gestation (term 21.5 d), and radiolabeled 2-deoxyglucose was used to measure relative glucose utilization rates (rGU) of fetal liver, lung, muscle, kidney, heart, placenta, and brain on d 20 of gestation after 2 or 5 d of hypoglycemia. Maternal plasma glucose concentrations decreased within 24 h of pump placement and remained less than controls throughout gestation. Fetal plasma glucose and insulin concentrations on d 20 were equally reduced after 2 and 5 d of hypoglycemia. Both 2 and 5 d of hypoglycemia were associated with significant reductions in the rGU of fetal liver, lung, and muscle. Reductions in fetal kidney rGU also occurred after 2 and 5 d of hypoglycemia but to a smaller degree. rGU of fetal heart was reduced after 2 d of hypoglycemia, but was normal after 5 d of hypoglycemia. Both 2 and 5 d of hypoglycemia were associated with increased rGU of fetal brain. Five d, but not 2 d of hypoglycemia resulted in decreased fetal weight on d 20 of gestation. However, at term, newborn pups delivered of hypoglycemic mothers weighed significantly less than controls regardless of the timing of minipump placement. Liver, lung, and carcass of these growth-retarded pups weighed less than control tissues, whereas kidney, heart, and brain weights were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)