Linda M. Hiebert

The observation of heparin on endothelium after injection

Abstract Heparin was demonstrated by the microelectrophoresis procedure of Jaques & Wollin in endothelial powders obtained after acetone extraction of Hautchen preparations after injection of 1,000 units in rats. Concentrations of heparin calculated for the endothelium was 30 to 7,500 times greater than the concentration of heparin in blood at a given time with a comparable dose. An electron micrograph of preparations stained with Ruthenium Red taken at five minutes after intravenous injection of heparin, demonstrated a marked increase in electron density, as compared to normal endothelium, at the cell membrane and in vesicles near the luminal surface of the cell.

Vascular sequestration of heparin.

Abstract Heparin was administered intravenously and by intratracheal instillation to rats. With 3 H-heparin, radioactivity accumulated in endothelium and vessel wall of aorta and vena cava. These vessels were also removed from rats receiving higher doses of cold heparin, extracted for mucopolysaccharides and heparin identified and measured by microelectrophoresis. This procedure revealed the presence of more heparin on the endothelium of the aorta than of the vena cava. Despite the presence of high radioactivity in the vessel wall denuded of endothelium, no heparin was obtained by the chemical extraction, suggesting a heparin metabolizing activity in the endothelium and/or vessel wall. Heparin was also administered intravenously to rats and by intratracheal instillation to mice. The drug was identified by light and electron microscopy in the endothelial tissue of rat aorta and of mouse pulmonary vessels.

Heparin protects cultured arterial endothelial cells from damage by toxic oxygen metabolites

Toxic oxygen metabolites can damage endothelial cells and may play an important role in the initiation and progression of atherosclerotic lesions. Since the antithrombotic drug heparin, interacts with endothelium, we wished to determine if heparin would protect endothelial cells from free radical injury. Endothelial cell injury was produced by the addition of xanthine and xanthine oxidase to cultured cells and assessed by changes in cell viability and release of lactate dehydrogenase (LDH) to the media. Pretreatment with heparin 24 h prior to addition of xanthine and xanthine oxidase significantly decreased cell damage. We suggest that heparin (and related compounds) can protect endothelium from free radical damage, and is therefore prophylactic for ischemic and inflammatory injury, and the development and progression of atheroma.

Heparanase upregulation in high glucose-treated endothelial cells is prevented by insulin and heparin.

Heparan sulfate proteoglycans on the endothelial cell surface and extracellular matrix play an important role in vascular homeostasis. Previous studies have shown that the quantity of heparan sulfate is reduced in kidney and other organs in diabetes. The objectives of this study were to determine if heparanase is induced by high glucose in endothelial cells and if heparin and/or insulin or basic fibroblast growth factor (bFGF) affect this upregulation. Cultured porcine aortic endothelial cells in M199 medium were treated with high glucose (30 mM) and/or bFGF (1 or 10 ng/ml) or high glucose plus insulin (1 U/ml) and/or heparin (0.5 μg/ml) for 7 days. To help define the mechanism of endothelial damage, cells were also exposed to H2O2 (0.1 mM) for 1 day or mannitol (30 mM) for 7 days. Heparanase mRNA was detected by reverse transcription polymerase chain reaction. Heparanase activity was measured by incubating cell lysates with [35S]labeled extracellular matrix of bovine corneal endothelial cells and analyzing released radioactive products by gel filtration and β-scintillation. Heparanase mRNA was found in high-glucose– and H2O2-treated cells; however, it was not found in control cells, mannitol- or high glucose plus insulin– and/or heparin-treated cells, or fresh porcine tissue. Heparanase activity was only found in high-glucose– and H2O2-treated cells. As well, bFGF did not prevent heparanase mRNA upregulation by high glucose. From these observations, we concluded that heparanase upregulation by high glucose is prevented by insulin and/or heparin but not bFGF. Reactive oxygen species, but not changes in osmolarity, may be involved in the upregulation of heparanase.

Endothelial cell injury by high glucose and heparanase is prevented by insulin, heparin and basic fibroblast growth factor

BackgroundUncontrolled hyperglycemia is the main risk factor in the development of diabetic vascular complications. The endothelial cells are the first cells targeted by hyperglycemia. The mechanism of endothelial injury by high glucose is still poorly understood. Heparanase production, induced by hyperglycemia, and subsequent degradation of heparan sulfate may contribute to endothelial injury. Little is known about endothelial injury by heparanase and possible means of preventing this injury.ObjectivesTo determine if high glucose as well as heparanase cause endothelial cell injury and if insulin, heparin and bFGF protect cells from this injury.MethodsCultured porcine aortic endothelial cells were treated with high glucose (30 mM) and/or insulin (1 U/ml) and/or heparin (0.5 μ g/ml) and /or basic fibroblast growth factor (bFGF) (1 ng/ml) for seven days. Cells were also treated with heparinase I (0.3 U/ml, the in vitro surrogate heparanase), plus insulin, heparin and bFGF for two days in serum free medium. Endothelial cell injury was evaluated by determining the number of live cells per culture and lactate dehydrogenase (LDH) release into medium expressed as percentage of control.ResultsA significant decrease in live cell number and increase in LDH release was found in endothelial cells treated with high glucose or heparinase I. Insulin and/or heparin and/or bFGF prevented these changes and thus protected cells from injury by high glucose or heparinase I. The protective ability of heparin and bFGF alone or in combination was more evident in cells damaged with heparinase I than high glucose.ConclusionEndothelial cells injured by high glucose or heparinase I are protected by a combination of insulin, heparin and bFGF, although protection by heparin and/or bFGF was variable.

Orally administered dextran sulfate is absorbed in HIV-positive individuals

Preliminary in vivo studies suggested that oral dextran sulfate was poorly absorbed, but investigations were limited by inadequate methods for measuring the drug in the body. To determine absorption in HIV-positive subjects, hydrogenated dextran sulfate, average molecular weight 8000 (Usherdex 8), was orally administered in a short-term (single dose, 4 g/day for 5 days, 7 subjects) and in a long-term study (1 g, 4 times per day for 29 to 335 days, 8 subjects), which was a continuation of the short-term study with the inclusion of an additional subject. When an agarose gel electrophoresis technique with toluidine blue staining was used, the drug was recovered from plasma (67%, peak 2.2 microg/mL) and circulating peripheral blood lymphocyte (PBL) samples (50%, peak 333 microg/L blood) obtained at 5 and 15 minutes and 1, 3, 6, and 24 hours after the first days dose and from plasma (56%) and PBL samples (38%) obtained 5 minutes after administration on 4 subsequent days in the short-term study. In the long-term study, the drug was found in plasma (67%, peak 2.4 microg/mL) and PBL samples (25%, peak 126 microg/L blood) obtained at monthly visits within 4 hours of the last dose. The drug was found in all urine samples from all subjects in both studies (short-term study, 24-hour samples up to 4 days after the final dose; long-term study, monthly samples within 4 hours of the last dose). In the long-term study, bone marrow preparations from 3 subjects showed metachromatic inclusions present in reticular cells when the cells were stained with toluidine blue, indicating the presence of sulfated polyanions. A significant rise in activated partial thromboplastin time and a drop in platelet count (P < .025) were demonstrated, with thrombocytopenia developing in 3 patients. Mild-to-moderate gastrointestinal disturbances were experienced by 6 subjects in the short-term study and by all subjects in the long-term study. One subject experienced mild central nervous system symptoms in the short-term study. These results indicate that dextran sulfate is absorbed after oral administration; therefore, further studies on its efficacy, particularly in the early stages of the disease, along with additional observations on its toxicity, are warranted.

Antithrombotic activity of oral unfractionated heparin.

Although heparin is believed to be poorly absorbed orally, we recently demonstrated that oral heparin rapidly enters the circulation, with most of the drug being taken up by endothelium. To determine the effective antithrombotic dose of oral heparin, we induced thrombosis by applying 10% formalin in 65% methanol to exposed rat jugular vein. Saline or heparin, at doses ranging from 3.25 to 60 mg/kg, was immediately placed in the stomach; 4 h later, the vein was inspected for a thrombus. A dose-dependent decrease in thrombosis was observed with oral heparin. Although there was little change in anticoagulant activity as measured by the activated partial thromboplastin time (APTT) of plasma samples taken 4 h after administration, a significant dose effect was demonstrated by regression analysis. Heparin could be demonstrated chemically in 52% of plasma samples and in 38% of aortic or vena caval endothelial samples. A significant dose effect was observed in aortic endothelial heparin concentrations, with amounts 1,000-fold that determined in plasma. These results indicate that oral heparin exhibits antithrombotic activity in a dose-dependent manner, with low levels in plasma.

Orally administered heparins prevent arterial thrombosis in a rat model.

Our previous studies demonstrated that orally administered heparins prevent thrombosis in a rat jugular vein thrombosis model, where bovine unfractionated heparin (UFH) and the low molecular weight heparin tinzaparin reduced thrombotic incidence by 50% at 7.5 and 0.1 mg/kg, respectively. Our objectives were to determine if similar antithrombotic effects of oral heparin could be observed in an arterial thrombosis model. In this model, filter paper soaked in 30% ferric chloride was applied to the exposed rat carotid artery. A flowmeter recorded blood flow over a 60 min period determining time when the thrombus began forming (TTB) and time till occlusion (TTO). Immediately following, the thrombus was removed, dried and weighed 24 h later. Bovine UFH (7.5 mg/kg), tinzaparin (0.1 mg/kg) or saline was administered by stomach tube at 2, 5 and 25 h prior to thrombus initiation. TTB was significantly increased when UFH was given at 5 and 25 h but not 2 h prior, and when tinzaparin was given at 5 but not 2 or 25 h prior compared to rats given oral saline. TTO was significantly increased for both UFH and tinzaparin when given 5 and 25 h but not 2 h prior (one-way ANOVA). There was no difference in TTO and TTB between UFH and tinzaparin treated groups. A trend in reduction in thrombus weight was observed for UFH at 5 and 25 h prior and tinzaparin at 5 h prior to thrombus initiation (one-way ANOVA). Although no significant changes were observed in activated partial thromboplastin times, Heptest or anti-Xa activity from plasma of heparin treated rats, endothelial heparin concentrations were significantly greater than controls for UFH at 5 h and for tinzaparin at 2, 5, and 24 h. Thus, heparins administered by the oral route are effective antithrombotic agents in arterial as well as venous models.

The uptake of heparin by liver sinusoidal cells in normal and atherosclerotic rabbits

Abstract Sections of liver from normal and atherosclerotic rabbits, treated 5 to 8 weeks with daily subsequent heparin or saline injections, were examined. Both paraffin and epon sections stained with toluidine blue were observed. Metachromatic inclusions indicative of heparin were found in cells lining the liver sinusoids of heparin treated rabbits but not in similar cells of saline treated rabbits. Electronmicroscopic examination of sections adjacent to the thick epon sections indicated that these metachromatic inclusions were present in vacuoles of endothelial cells as well as Kupffer cells. This observation supports the existing evidence that the liver along with the reticuloendothelial system and endothelial cells play a role in the distribution of administered heparin.

Tissue distribution and antithrombotic activity of unlabeled or 14C-labeled porcine intestinal mucosal heparin following administration to rats by the oral route.

Distribution and antithrombotic activity of orally administered unfractionated porcine heparin were studied. [14C]Heparin was prepared by de-N-acetylation of porcine mucosal heparin followed by re-N-acetylation, using [14C]acetic anhydride. [14C]Heparin and (or) cold heparin (60 mg/kg) were administered by stomach tube to male Wistar rats. Blood, all levels of gut and gut contents, liver, lung, spleen, kidney, and aortic and vena caval endothelium were collected under deep anesthesia at 3, 6, 15, 30, and 60 min and 4 and 24 h (6 rats/group) after administration. Urine and feces were collected at 24 h, using metabolic cages. In three additional rats, drugs were administered in gelatin capsules. Tissues listed above and tongue, esophagus, trachea, brain, heart, thymus, bile ducts, vena caval and aortic walls, ureters, bladder, samples of muscle, skin, hair, and bone marrow were collected at 24 h. Radioactivity and chemical heparin, measured by agarose gel electrophoresis, were observed in all tissues examined as well as gut washes, plasma, urine, and feces. Radiolabel recovered was confirmed to be heparin by autoradiograms of gradient polyacrylamide electrophoretic gels. [14C]Heparin and chemical heparin in gut tissue suggest a transit time of 4 h. Porcine or bovine heparin (7.5 mg/kg), administered by stomach tube, decreased the incidence of thrombosis induced by applying 10% formalin in 65% methanol to the exposed jugular vein of rats. Heparin isolation from non-gut tissue, endothelium, urine, and plasma and the observed antithrombotic effect are consistent with oral bioavailability.