Biomedical subjects

A Sandra

Publications and source records attributed to A Sandra.

At least 55 records · Page 3Linked to original sources

PubMed1990

Microarterial synthetic graft repair: interstitial cellular components.

Although the histologic changes occurring during healing on the lumen surface of large vessel synthetic vascular grafts have been well characterized, the cells populating the interstices of microvascular grafts have not been examined in detail. Since microvascular grafts are required to provide vascular continuity under quite different physiological and hemodynamic conditions as compared with large vessel grafts, these interstitial cells within the synthetic graft material may also vary as a function of graft size. Monoclonal antibodies, light microscopy, and scanning and transmission electron microscopy were used in this study to identify the cells present within the 30-microns pores of 1-mm diameter polytetrafluoroethylene and replamineform silicone rubber grafts. Identified cells included few capillary endothelial cells enclosing erythrocytes, rare proliferating endothelial cells, few macrophages, rare foreign body giant cells, and a majority of fibroblasts. There was no evidence of smooth muscle cells or myofibroblasts within the interstices of these microvascular prostheses 12 weeks after implantation in the rabbit central ear artery. The graft types differed by the presence of foreign body giant cells and more densely packed collagen between cells in the replamineform silicone rubber graft interstices.

Animals↗

PubMed1990

Insulin differentially alters transcapillary movement of intravascular IGFBP-1, IGFBP-2 and endothelial cell IGF-binding proteins in the rat heart.

Insulin-like growth factor binding-proteins 1 and 2 (IGFBP-1, IGFBP-2) and endothelial cell IGF binding proteins (ECBP) were individually perfused through isolated beating rat hearts in the absence and presence of insulin. Insulin caused an increased movement of IGFBP-1 from the vascular space to tissues of the heart. Subendothelial content of IGFBP-1 was 110%, 126% (p less than .01) and 132% (p less than 0.05) of control hearts when perfused with 1, 10 and 100 ng/ml insulin, respectively. . In contrast, insulin treatment was associated with a decrease in ECBP content in cardiac tissue, being 83%, 62% (p less than 0.005) and 73% (p less than 0.05) of control when perfused with 1, 10 and 100 ng/ml insulin. The efflux of IGFBP-2 from the intravascular space was unaffected by insulin. The subendothelial tissue distribution of the transported binding proteins was not changed by insulin perfusion, with IGFBP-1 and IGFBP-2 localizing predominantly in cardiac muscle and ECBP having greater affinity for connective tissue elements. We conclude that in the perfused rat heart, insulin can differentially alter transcapillary movement of IGFBP-1, IGFBP-2 and endothelial cell IGF-binding proteins. Such insulin-facilitated changes could potentially mediate nutrient-dependent transport of IGF-I and IGF-II to peripheral tissues.

Animals↗

PubMed1990

Transcapillary permeability and subendothelial distribution of endothelial and amniotic fluid insulin-like growth factor binding proteins in the rat heart.

Insulin-like growth factor (IGF) binding proteins (IGFBP) were purified from conditioned media of cultured bovine endothelial cells (ECBP) and from human amniotic fluid (IGFBP-1), and then labeled by radioiodination. 125I-ECBP and 125I-IGFBP-1 were perfused through isolated beating rat hearts for 1 and 5 min, and the hearts fixed and analyzed for 125I-BP content and distribution. One to 4% of the perfused 125I-ECBP and 125I-IGFBP-1 crossed the capillary boundary. The ECBPs predominantly localized as intact 125I-BP in connective tissue elements of the heart with less 125I-BP in cardiac muscle. The ratio of 125I-ECBP in connective tissue: muscle (normalized to percent vol of these compartments) was greater than or equal to 10:1. In contrast, the IGFBP-1 had a greater affinity for cardiac muscle with ratios of 125I-IGFBP-1 in connective tissue:muscle of approximately 1:2. When 125I-IGF-I, in the absence of any BPs, was perfused through the hearts approximately 3-5% left the microcirculation and was found in subendothelial tissues. 125I-IGF-I localized primarily to cardiac muscle with a distribution of connective tissue:cardiac muscle of about 1:3. The findings in the isolated perfused heart were confirmed in intact animals. After 125I-IGFBP-1 was injected into anesthetized rats and allowed to circulate for 5 min, substantial radioactivity was associated with the heart. As in the isolated heart, the IGFBP-1 preferentially localized to cardiac muscle with a connective tissue:cardiac muscle ratio of 1:3. We conclude that IGFBPs produced by endothelial cells and the IGFBP-1 contained in amniotic fluid can cross the capillary boundaries of the rat heart, and that the ECBPs preferentially localize in connective tissue elements of the myocardium, whereas IGFBP-1 predominantly localizes in cardiac muscle.

Amniotic Fluid↗

PubMed1990

Tissue localization of perfused endothelial cell IGF binding protein is markedly altered by association with IGF-I.

Perfused endothelial cell IGF binding proteins (ECBP) have been previously demonstrated to leave the microcirculation of the rat heart and distribute primarily in connective tissue elements of the heart. In the present study, ECBP have been crosslinked to IGF-I and the biologically inactive [1-27,gly4,38-70]-hlGF-I, an analog of IGF-I lacking the type I IGF receptor domain. The crosslinked ECBPs were perfused through the isolated rat heart and their tissue distributions determined. Both [ECBP-Analog] and [ECBP-IGF-I] left the microcirculation of the heart. [ECBP-Analog] preferentially localized in connective tissue elements with a muscle:connective tissue ratio of approximately 1:6, similar to the tissue distribution of perfused ECBP. In contrast, the [ECBP-IGF-I] complexes localized in cardiac muscle with a muscle to connective tissue ratio of approximately 3:1, virtually identical to the tissue distribution of IGF-I when the IGF-I is perfused through the heart in the absence of any binding proteins. We conclude that 1) ECBP in the presence of IGF will cross capillary boundaries and 2) the tissue distribution of [ECBP-IGF-I] is dictated by the IGF-I molecule.

Animals↗

PubMed1989

Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line.

Differentiated rat L6 skeletal muscle cell cultures maintained in glucose-deficient medium containing 25 mM xylose displayed a rapid, reversible, time- and concentration-dependent 3-5-fold increase in glucose transport activity. Glucose deprivation in the continuous presence of insulin (24 h) resulted in an overall 9-10-fold stimulation of glucose transport activity. In contrast, acute (30 min) and chronic (24 h) insulin treatment of L6 cells maintained in high glucose (25 mM)-containing medium resulted in a 1.5- and 4-fold induction of glucose transport activity, respectively. Acute glucose deprivation and/or insulin treatment had no significant effect on the total amount of glucose transporter protein, whereas the long-term insulin- and glucose-dependent regulation of glucose transport activity directly correlated with an increase in the cellular expression of the glucose transporter protein. In situ hybridization of the L6 cells demonstrated a 3-, 4-, and 6-fold increase in glucose transporter mRNA induced by glucose deprivation, insulin, and glucose deprivation plus insulin treatments, respectively. Similarly, Northern blot analysis of total RNA isolated from glucose-deprived, insulin, and glucose-deprived plus insulin-treated cells resulted in a 4-, 3-, and 9-fold induction of glucose transporter mRNA, respectively. The continuous presence of insulin in the medium, either in the presence or absence of glucose, resulted in a transient alteration of the glucose transporter mRNA. The relative amount of the glucose transporter mRNA was maximally increased at 6-12 h which subsequently returned to the basal steady-state level within 48 h. These data demonstrate a role for insulin and glucose in the overall regulation of glucose transporter gene expression which may account for the alteration of glucose transporter activity of muscle tissue observed in pathophysiological states such as type II diabetes mellitus.

Animals↗

PubMed1989

Preparation of collagen gel matrices for light and electron microscopy.

Cells grown on type I hydrated collagen gels require special techniques for sample preparation and processing in order to optimize the removal of all reagents from the collagen matrix and prevent artifactual shrinkage. This method includes cutting out a small block of the collagen gel, postfixation, and transfer to a scintillation vial for further processing. These additional steps ensure that all sides of the block will come in contact with solutions and reduces the possibility of reagent trapping within the collagen matrix. Additionally, in our study the collagen gel and endothelial cells that form a monolayer on the surface are oriented to allow the microtomist greater assurance of cutting true cross sections, thus saving time and increasing reproducibility. The dehydration sequence is also modified, with an increase in the times and additional steps, especially in the higher concentrations of dehydrant.

Animals↗

PubMed1988

Insulin, insulin-like growth factors, and vascular endothelium.

Endothelial cells form the intimal lining of the entire vascular system. The vascular endothelium is continuously and directly bathed by components of the bloodstream and represents the initial fixed anatomical surface with which these components come in contact. In the past decade, the methodologies for studying endothelial cell functions have markedly advanced, enabling direct and detailed study of the vascular endothelium. From such studies, it is now apparent that the vascular endothelium represents an extraordinarily complex network of cells demonstrating a multitude of distinct anatomic, metabolic, and immunologic properties critical to such processes as angiogenesis, atherosclerosis, thrombosis, neoplasia, and a variety of metabolic disorders including homocystinuria and diabetes mellitus. This report will focus on the interactions of insulin and the insulin-like growth factors (IGFs) with vascular endothelium, based on studies with cultured endothelial cells, isolated microvessels, and perfused organ systems. Data will be presented relevant to the following concepts: (1) endothelial cells, in culture and in vivo, have specific receptors for insulin, IGF-I, and IGF-II; (2) insulin, IGF-I, and IGF-II have both distinct and overlapping functions in cultured endothelial cells; (3) cultured endothelial cells process receptor-bound insulin, IGF-I, and IGF-II, by distinct processes; (4) in vivo, capillary endothelial receptors are integrally involved in the transport of intact insulin to subendothelial sites of insulin action; and (5) vascular endothelium has specialized cellular features that are likely to contribute to the unique interactions of endothelial cells with insulin and the IGFs.

Animals↗

PubMed1988

IGF receptors in myocardial capillary endothelium: potential regulation of IGF-I transport to cardiac muscle.

Beating rat hearts were perfused with 125I-IGF-II alone or 125I-IGF-II and unlabeled IGF-II or insulin, then prepared for radioautography. Maximal 125I-IGF-II grain counts over capillaries were decreased in a dose-dependent manner by unlabeled IGF-II but were unaffected by coperfusion with insulin. To determine a potential role for capillary receptors in the transfer of circulating IGF to cardiac muscle, the effects of sequential loss of capillary IGF binding sites was determined. For IGF-I, loss of capillary binding sites by trypsin perfusion was accompanied by proportional decreases in the subsequent appearance of IGF-I in cardiac muscle. In contrast, similar decrements of capillary IGF-II binding did not affect muscle levels of IGF-II. We conclude that capillary endothelium of the intact heart possesses distinct IGF-I and IGF-II binding sites, with the capillary IGF-I binding sites being of potential importance in the transfer of vascular IGF-I to subendothelial cardiac muscle.

Animals↗

PubMed1988

Differential sensitivity of the insulin receptor to proteolysis after beta-adrenergic stimulation.

The cellular mechanism by which the specific binding of [125I]insulin to intact rat adipocytes is inhibited by isoproterenol has been studied. By exposing control and isoproterenol-treated cells to trypsin (0-150 micrograms/ml for 20 min at 4 degrees C) and measuring the intact insulin receptor pool following detergent solubilization, a differential sensitivity to proteolysis of the cell membrane receptor was observed. At low trypsin concentration (less than 30 micrograms/ml), approximately 40% of the specific insulin binding in isoproterenol-treated cells was insensitive to proteolysis as compared to control cells. At higher levels of trypsin (50-150 micrograms/ml) both groups displayed similar levels of trypsin-insensitive receptors which, at the highest trypsin concentration, accounted for 10% of the total receptors in intact cells. Detergent-solubilized receptors from isoproterenol-treated cells, on the other hand, exhibited the same sensitivity to trypsin proteolysis as solubilized receptors from control cells. The time course of the onset and reversal of the isoproterenol-induced binding alteration in intact adipocytes has been analyzed by mild trypsinization (20 micrograms/ml). Results indicated that insulin receptors resistant to trypsin under these conditions mediated the decreased surface binding and were re-expressed on the cell surface upon removal of isoproterenol. Experiments in which adipocytes were fractionated into plasma membrane and Golgi-enriched fractions indicated that the loss of surface insulin binding was not accompanied by a decrease in the proportion of receptors in the adipocyte plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1988

Vascular transport of insulin to rat cardiac muscle. Central role of the capillary endothelium.

Using intact, beating hearts, we have assessed the interaction of insulin with capillary endothelium and the subsequent appearance of insulin in cardiac muscle. Rat hearts were perfused with 125I-insulin (10(-10) M) alone or in combination with unlabeled insulin (10(-9)-10(-5) M). 125I grains (shown to represent greater than 90% intact insulin) over both capillary endothelium and cardiac muscle decreased in a dose-dependent manner when hearts were co-perfused with labeled insulin and increasing concentrations of unlabeled insulin. Perfusion of 125I-desoctapeptide (DOP) insulin, a low affinity insulin analogue, with unlabeled insulin (10(-9)-10(-5) M) had no effect on the appearance of 125I-DOP insulin over microvessel endothelium and muscle. When capillary receptors were first destroyed by trypsin treatment or blocked by anti-receptor antibodies, the appearance of 125I-insulin in cardiac muscle decreased proportional to the inhibition of insulin binding to the capillary receptors. We conclude that insulin binding to capillary endothelial receptors is a central step in the transport of intravascular insulin to rat cardiac muscle.

Animals↗

PubMed1987

Effect of chronic isoproterenol exposure on insulin binding and insulin-stimulated hexose transport in isolated rat adipocytes.

The effect of chronic exposure of isolated rat adipocytes to the beta-adrenergic agonist isoproterenol has been studied with respect to insulin binding and insulin-stimulated hexose uptake. Isoproterenol exposure led to a progressive decrease in both the number of surface insulin receptors and the stimulation of hexose uptake. The effect on insulin binding was reversible by removal of the beta-agonist within an hour of its addition. Later exposures of adipocytes to isoproterenol resulted in an irreversible cellular defect by leading to a progressive inability of the cells to regain their normal level of insulin-stimulated hexose uptake and insulin binding.

Adipose Tissue↗

PubMed1986

Arachidonic acid inhibition of insulin action and phosphoinositide turnover in fat cells.

Incubation of isolated rat adipocytes with 1 microM arachidonic acid (20:4) coupled to equimolar amounts of bovine serum albumin (BSA) results in the cellular uptake of the fatty acid and a subsequent inhibition of insulin-stimulated antilipolysis and lipogenesis without altering glucose transport. These effects are apparently not mediated at the insulin receptor level since insulin binding is not altered in arachidonate-enriched fat cells. In addition, effects on antilipolytic and lipogenic are not specific for arachidonic acid. Oleic or palmitic acid can mimic these effects in both insulin-stimulated and PGE2-stimulated cells. Adipocyte enrichment with 20:4, however, specifically inhibits the insulin-stimulated turnover of phosphoinositides. The latter can be specifically prevented by preincubation with ibuprofen. These results suggest that the level of intracellular arachidonate may play a major role in modulating insulin-stimulated phosphoinositide turnover and thereby indirectly regulate certain aspects of insulin action which involve lipid metabolism.

Adipose Tissue↗

PubMed1986

Effects of vesicle-adipocyte interaction on regulation of insulin receptor recycling.

The effect of interacting isolated rat adipocytes with small, unilammelar vesicles on insulin receptor internalization and processing was studied. Treatment of freshly isolated cells with vesicles containing phosphatidylcholine and phosphatidylserine followed by incubation in 35 mM Tris-containing buffer considerably reduced the chloroquine-induced increase in cell-associated 125I-insulin and significantly inhibited the time and insulin dependent loss of surface insulin receptors. The internal receptor pool, as measured by insulin binding to detergent solubilized adipocytes, was relatively smaller in vesicle-treated cells. Concomitant with a slower rate of receptor internalization, insulin-sensitive hexose uptake also demonstrated significantly slower kinetics of decreased response with time. These results support the conclusion that pretreatment of fat cells with phospholipid vesicles inhibits normal insulin receptor cycling.

Adipose Tissue↗

PubMed1985

Receptors for insulin-like growth factor-I (IGF-I) in myocardial capillary endothelium of the intact perfused heart.

Isolated intact, beating hearts were perfused with HPLC-pure [125]-IGF-I (1 ng/ml) alone or [125]-IGF-I (1 ng/ml) plus varying concentrations of unlabeled IGF-I (10-3,000 ng/ml) or unlabeled insulin (1,000-100,000 ng/ml). After 1 min of perfusion with peptides, the hearts were rapidly fixed, sectioned and analyzed for radioautographic [125I] grain counts. Greater than 90% of [125I] grains were shown to represent intact [125I]-IGF. Maximal grain counts over capillaries occurred after perfusion with [125I]-IGF-I alone and decreased in a dose-dependent manner when unlabeled IGF-I was coperfused. Coperfusion of [125I]-IGF-I with unlabeled insulin also decreased 125I grains over capillaries but less potently than unlabeled IGF-I. EM radioautography demonstrated that [125I]-IGF-I grains were localized over capillary endothelial cells. Thus, specific IGF-I receptors are present in the capillary endothelium of the intact heart and have properties similar to IGF-I receptors in cultured capillary endothelial cells.

Adipose Tissue↗

PubMed1985

Morphological alterations in cultured endothelial cells induced by arachidonic acid.

The addition of arachidonic acid (20:4), but not other fatty acids, including the structurally similar eicosapentaenoic acid (20:5), induced specific morphological changes in cultured endothelial cells derived from bovine aorta and pulmonary artery. Cells exhibited a time- and dose-dependent change from their normal, epithelioid morphology to become elongated, polygonal, and spindle-shaped. Cells isolated from aorta appeared more sensitive to these changes than those from pulmonary artery. The effect was observed as early as 12 h after exposure to 20:4, required 48 h for maximal expression, and could be reversed in 2-5 h after change to normal media. The morphological alteration was not observed in cells treated with leukotrienes or PGE2. When cells were pretreated with ibuprofen, aspirin, or indomethacin to block prostaglandin synthesis and then exposed to 20:4, the dose-response effect was shifted to the left. This increased sensitivity to 20:4 suggests either a direct effect of 20:4 on cell morphology or an indirect effect due to metabolites of 20:4 which are not dependent on the cyclooxygenase pathway.

Animals↗

PubMed1985

Phospholipid-induced inhibition of insulin-stimulated glucose transport in isolated adipocytes: interactions of phospholipids with inhibitors of glucose transport and insulinmimetic agents.

Upon interaction with phospholipid vesicles containing phosphatidylserine, isolated rat adipocytes demonstrate an inhibition of insulin-stimulated hexose uptake. In order to elucidate the mechanism of this effect, adipocytes were treated with agents, alone or in combination with vesicles, which affected the insulin-sensitive response at the receptor and post-receptor level. The effect of vesicles at a maximal inhibitory concentration proved to be non-additive with dexamethasone, suggesting that vesicles may act in a manner similar to this agent. In contrast, fat cells treated with vesicles and N-ethylmaleimide (NEM) or trypsin at submaximally effective concentrations demonstrate a partially additive inhibition of insulin-stimulated 2-deoxyglucose uptake. Vesicle treatment of adipocytes before stimulation with agents which mimic insulin, such as Con A and H2O2, demonstrates the same effects as insulin with respect to hexose uptake. These results support the contention that vesicles inhibit insulin action at least partially at the post-receptor level, and may directly interfere with the hexose transport site.

Adipose Tissue↗

PubMed1984

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte.

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific D-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.

Adipose Tissue↗

PubMed1984

The isolated sinoatrial node cell in primary culture from the newborn rat.

We prepared primary cell cultures of the sinus node region from newborn rat hearts. Sinoatrial node cells were easily distinguished from the other cardiac muscle cells and nonmuscle cells in culture by size, configuration, and rapid, attenuated spontaneous contractions (185.0 +/- 8/min, mean +/- SEM). The spontaneously contracting sinoatrial node cells were extremely sensitive to acetylcholine and norepinephrine, responding to concentrations at least 1000-fold less than other cardiac muscle cells. These same sinoatrial node cells in culture were fixed and precisely relocated by either subsequent scanning or transmission electron microscopy. The ultrastructural features of these sinoatrial node cells in culture were similar to those observed in the cells of intact sinus node sections from the source hearts. This study is the first to present single, spontaneously active, neonatal sinoatrial node cells maintained in vitro with morphological and functional properties desirable for physiological investigations.

Animals↗