Heparan Sulfate Proteoglycans in Diabetes.

Diabetes is a complex disorder responsible for the mortality and morbidity of millions of individuals worldwide. Although many approaches have been used to understand and treat diabetes, the role of proteoglycans, in particular heparan sulfate proteoglycans (HSPGs), has only recently received attention. The HSPGs are heterogeneous, highly negatively charged, and are found in all cells primarily attached to the plasma membrane or present in the extracellular matrix (ECM). HSPGs are involved in development, cell migration, signal transduction, hemostasis, inflammation, and antiviral activity, and regulate cytokines, chemokines, growth factors, and enzymes. Hyperglycemia, accompanying diabetes, increases reactive oxygen species and upregulates the enzyme heparanase that degrades HSPGs or affects the synthesis of the HSPGs altering their structure. The modified HSPGs in the endothelium and ECM in the blood vessel wall contribute to the nephropathy, cardiovascular disease, and retinopathy seen in diabetes. Besides the blood vessel, other cells and tissues in the heart, kidney, and eye are affected by diabetes. Although not well understood, the adipose tissue, intestine, and brain also reveal HSPG changes associated with diabetes. Further, HSPGs are significantly involved in protecting the ß cells of the pancreas from autoimmune destruction and could be a focus of prevention of type I diabetes. In some circumstances, HSPGs may contribute to the pathology of the disease. Understanding the role of HSPGs and how they are modified by diabetes may lead to new treatments as well as preventative measures to reduce the morbidity and mortality associated with this complex condition.

Proteoglycans and Diabetes.

BACKGROUND: Most proteoglycans are heterogeneous molecules composed of a protein core with glycosaminoglycans (GAGs) attached. GAGs are highly negatively charged molecules that readily bind to enzymes, growth factors, cytokines etc. and as such have many functions. The role played by proteoglycans in diabetes has only recently been investigated. METHODS: The importance of proteoglycans and the effects of diabetes on proteoglycans are discussed. Possible strategies for reducing diabetic complications associated with preventing proteoglycan destruction are examined. RESULTS: Proteoglycans are altered in the endothelium, vascular wall, kidney, retina, heart, gut epithelial cells, bone and cartilage with diabetes. A decrease in proteoglycans, associated with hyperglycemic conditions, is reported to be due to a decrease in proteoglycan synthesis or an increase in destruction. Destruction may be a result of an upregulation of enzymes that degrade GAGs or destruction by reactive oxygen species. Several studies suggest that upregulation of heparanase and its destruction of heparan sulfate proteoglycans may be responsible for many of the complications associated with diabetes particularly in the kidney and blood vessels leading to chronic kidney disease, atherosclerosis and acute coronary syndrome. Preliminary studies suggest that administration of GAGs may be beneficial in reducing or delaying the harmful consequences of diabetes in the kidney and retina. CONCLUSIONS: Changes in proteoglycans are partially responsible for diabetic complications. Recent studies demonstrate that administration of GAGs may reduce or delay diabetic complications. Further studies are required to understand the alterations in proteoglycans associated with diabetes, and the protective potential of administered GAGs.

Repeated Oral or Subcutaneous LMWH Has similar Antithrombotic Activity in a Rat Venous Thrombosis Model: Antithrombotic Activity Correlates With Heparin on Endothelium When Orally Administered.

Low-molecular-weight heparins (LMWHs) endure as important drugs for thromboprophylaxis. Although clinical use relies on the subcutaneous (SC) route, our previous studies show that single-dose orally administered LMWHs have antithrombotic activity. Since thromboprophylaxis requires long-term treatment, we examined antithrombotic effects of subacute oral LMWHs in a rat venous thrombosis model and compared results to SC or single-dose oral administration. We measured LMWH in endothelium and plasma, weight change and complete blood counts (CBC). Oral LMWH tinzaparin (3 × 0.1 mg/kg/12 or 24 hours) or reviparin (3 × 0.025 mg/kg/24 hours) significantly decreased thrombosis compared to saline. In the subacute study (60 × 0.1 mg/kg/12 hours), oral or SC tinzaparin significantly reduced thrombosis compared to saline but not to single or 3 × 0.1 mg/kg/12 hours oral tinzaparin. Antithrombotic effects were similar between oral and SC administration. LMWH was found on endothelium following oral but not SC administration. Endothelial concentrations were significantly correlated with incidence of stable thrombi ( P = 0.021 and 0.04 for aortic and vena cava endothelium respectively, χ2 test) and total thrombi ( P = 0.003 for vena cava endothelium). Anti-Xa activity was significantly greater for oral or SC LMWH than saline and significantly greater for SC versus oral LMWH. Values for CBCs were within normal ranges (mean ± 2 SD). There was no evidence of bleeding. Weight gain was similar between groups. In conclusion, subacute oral and SC LMWH have similar antithrombotic effects. Antithrombotic activity with oral administration is correlated with endothelial LMWH concentrations but not with plasma anticoagulant activity.

Effect of oral administration of unfractionated heparin (UFH) on coagulation parameters in plasma and levels of urine and fecal heparin in dogs.

The effects of heparin administration, by the oral route, were evaluated in dogs. In single and multiple dose studies (single 7.5 mg/kg, multiple 3 × 7.5 mg/kg per 48 h), plasma, urine, and fecal samples were collected at various times up to 120 h after oral administration of unfractionated heparin. Changes in plasma and urine anti-Xa activity, plasma and urine anti-IIa activity, plasma activated partial thromboplastin time (APTT) and antithrombin (ATIII), and chemical heparin in urine and feces were examined with time. There was support for heparin absorption, with significant differences in APTT, heparin in plasma as determined by anti-Xa activity (Heptest) in the single dose study and plasma anti-Xa activity, anti-IIa activity and ATIII; and chemical heparin in urine in the multiple dose study. No clinical evidence of bleeding was detected in any dog during the studies. Oral heparin therapy may be applicable for thromboembolic disease in animals. Further studies are warranted to determine the effects of oral heparin at the endothelial level in the dog.

Les effets de l'administration d'héparine, par voie orale, furent évalués chez des chiens. Dans des études suite à une administration unique et à des administrations multiples (unique 7,5 mg/kg; multiples 3 × 7,5 mg/kg par 48 h), des échantillons de plasma, d'urine et de fèces furent prélevés à différents intervalles jusqu'à 120 h suivant l'administration orale d'héparine non-fractionnée. On examina dans le temps les changements de l'activité anti-Xa dans le plasma et l'urine, l'activité anti-IIa dans le plasma et dans l'urine, le temps de thromboplastine partielle (APTT) et d'antithrombine (ATIII) activé par le plasma, et l'héparine dans l'urine et les fèces. Il y a évidence d'absorption d'héparine, avec des différences significatives de l'APTT, de l'héparine plasmatique tel que déterminé par l'activité anti-Xa (Heptest) dans l'étude à dose unique et dans l'activité anti-Xa plasmatique, l'activité anti-IIa et l'ATIII; et de l'héparine chimique dans l'urine lors de l'étude à doses multiples. Aucune évidence clinique de saignement ne fut détectée chez les chiens au courant des études. Une thérapie orale à l'héparine pourrait être applicable pour des maladies thromboemboliques chez les animaux. Des études supplémentaires sont nécessaires afin de déterminer les effets d'administration d'héparine orale au niveau endothélial chez les chiens.(Traduit par Docteur Serge Messier).

Glycosaminoglycans, hyperglycemia, and disease.

SIGNIFICANCE: Diabetes is a widespread disease with many clinical pathologies. Despite numerous pharmaceutical strategies for treatment, the incidence of diabetes continues to increase. Hyperglycemia, observed in diabetes, causes endothelial injury resulting in microvascular and macrovascular complications such as nephropathy, retinopathy, neuropathy, and increased atherosclerosis. RECENT ADVANCES: Proteoglycans are chemically diverse macromolecules consisting of a protein core with glycosaminoglycans (GAGs) attached. Heparan sulfate proteoglycans are important compounds found on the endothelial cell membrane and in the extracellular matrix, which play an important role in growth regulation and serve as a reservoir for cytokines and other bioactive molecules. Endothelial cells are altered in hyperglycemia by a reduction in heparan sulfate and upregulation and secretion of heparanase, an enzyme that degrades heparan sulfate GAGs on proteoglycans. Reactive oxygen species, increased in diabetes, also destroy GAGs. CRITICAL ISSUES: Preservation of heparan sulfate proteoglycans on endothelial cells may be a strategy to prevent angiopathy associated with diabetes. The use of GAGs and GAG-like compounds may increase endothelial heparan sulfate and prevent an increase in the heparanase enzyme. FUTURE DIRECTIONS: Elucidating the mechanisms of GAG depletion and its significance in endothelial health may help to further understand, prevent, and treat cardiovascular complications associated with diabetes. Further studies examining the role of GAGs and GAG-like compounds in maintaining endothelial health, including their effect on heparanase, will determine the feasibility of these compounds in diabetes treatment. Preservation of heparan sulfate by decreasing heparanase may have important implications not only in diabetes, but also in cardiovascular disease and tumor biology.

Repeated doses of oral and subcutaneous heparins have similar antithrombotic effects in a rat carotid arterial model of thrombosis.

Although heparins are usually injected intravenously or subcutaneously, antithrombotic activity is observed in rat models following single oral heparin doses. Since repetitive dosing is usually needed for thromboprophylaxis, study objectives were to determine whether repetitive oral heparin prevented arterial thrombosis and to compare effectiveness to subcutaneous administration. Wistar rats were given subcutaneous or oral unfractionated heparin ([UFH] 1 mg/kg per 48 h), low-molecular-weight heparin ([LMWH] tinzaparin, 0.1 mg/kg per 12 h), or saline for 30 days. On the last day, thrombosis was initiated by placing 30% FeCl(3)-soaked filter paper on the distal carotid. Subsequent flow measurements, for a 60-minute period, included recorded time of initial thrombus formation (time till thrombus begins [TTB]), and time until carotid occlusion (time till occlusion [TTO]). The formed thrombus was dried and weighed. The activated partial thromboplastin time (aPTT), anti-factor Xa, and antithrombin activity were determined from the plasma. Both oral and subcutaneous heparins significantly increased TTB and TTO. Time of initial thrombus formations were 12.6 ± 1.1, 21.2 ± 2.2, 25.3 ± 3.9, 21.7 ± 3.1, and 21.3 ± 1.7 minutes and TTOs were 29.3 ± 3.6, 54.8 ± 4.0, 60.0 ± 0.3, 56.7 ± 3.3, and 58.3 ± 1.7 minutes (mean ± SEM) for control, subcutaneous UFH, oral UFH, subcutaneous LMWH, and oral LMWH, respectively. Thrombus weight was 2.52 ± 0.29 g in control and was reduced to 43%, 23%, 33%, and 28% of control weight for subcutaneous UFH, oral UFH, subcutaneous LMWH, and oral LMWH, respectively. Thrombus weight was significantly less for oral compared to subcutaneous UFH. The aPTT for oral UFH, and anti-factor Xa activity in the LMWH-treated groups were significantly greater than control (two-tailed t tests). These findings confirm that orally administered heparins are absorbed. Repeated treatment with oral heparin showed similar antithrombotic activity compared to subcutaneous heparin. Oral heparin use for arterial thromboprophylaxis should be further investigated.

dGlucose is linked to renal function changes in diabetes.

AIMS: This study examines if dglucose, two-hour postprandial (2 hPP) minus fasting glucose (F), predicts glycemic control better than F or 2 hPPglucose. METHODS: F and 2 hPPglucose, and renal function variables; BUN, serum creatinine (Scr), and estimated GFR (eGFR), were obtained from 56 insulin treated diabetic adults. 2 hPP-F(d) was calculated. Variables were compared when 2 hPPglucose was <200 (n=23) or >200 mg/dL (n=33). Correlation coefficients were calculated for F, 2 hPP or 2 hPP-F(d) renal function variables versus those for glucose. RESULTS: Variables differed significantly between F and 2 hPP (t-test, p<0.05) for all patients and when 2 hPPglucose was < or >200 mg/dL, except dBUN at <200 mg/dL. When F, 2 hPP or 2 hPP-F(d) variables between 2 hPPglucose< and >200 mg/dL were compared, dScr was significant (p=0.0327). Correlation coefficients between dglucose and dScr or deGFR, were significant for all patients (r=0.420, p=0.0013, and r=-0.434, p=0.0008, respectively) and for 2 hPPglucose >200 mg/dL (r=0.523, p=0.0018 and r=-0.513, p=0.0023, respectively) but not 2 hPPglucose <200 mg/dL. When dglucose increased by 100 mg/dL, dScr increased by 0.08 and 0.11 mg/dL, and deGFR decreased by 2.73 and 3.73 mL/min for all patients and >200 mg/dL, respectively. CONCLUSIONS: dGlucose better predicts renal function changes than F or 2 hPPglucose. Postprandial hyperglycemia (<200 mg/dL) control is crucial for renal protection in diabetes.

Changes in cultured endothelial cell glycosaminoglycans under hyperglycemic conditions and the effect of insulin and heparin.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) contain glycosaminoglycan (GAG) chains made primarily of heparan sulfate (HS). Hyperglycemia in diabetes leads to endothelial injury and nephropathy, retinopathy and atherosclerosis. Decreased HSPG may contribute to diabetic endothelial injury. Decreased tissue HS in diabetes has been reported, however, endothelial HS changes are poorly studied. OBJECTIVE: To determine total GAGs, including HS, in endothelium under hyperglycemic conditions and the protective effect of insulin and heparin. METHODS: Confluent primary porcine aortic endothelial cells (PAECs) were divided into control, glucose (30 mM), insulin (0.01 unit/ml) and glucose plus insulin treatment groups for 24, 48 and 72 hours. Additionally, PAECs were treated with glucose, heparin (0.5 microg/ml) and glucose plus heparin for 72 hours. GAGs were isolated from cells and medium. GAG concentrations were determined by the carbazole assay and agarose gel electrophoresis. RESULTS: GAGs were significantly increased only in control and glucose plus insulin groups at 72 versus 24 hours. Glucose decreased cell GAGs and increased medium GAGs, and insulin alone decreased cell GAGs at all times compared to control. In the glucose plus insulin group, cell GAGs were less than control at 24 hours, and greater than glucose or insulin alone at 48 and 72 hours while GAGs in medium were greater than control at all times and glucose at 72 hours. Heparin increased GAGs in glucose treated cells and medium. CONCLUSION: High glucose and insulin alone reduces endothelial GAGs. In hyperglycemic conditions, heparin or insulin preserves GAGs which may protect cells from injury. Insulin is an effective diabetic therapy since it not only lowers blood glucose, but also protects endothelium.

Movement of heparins across rat gastric mucosa is dependent on molecular weight and pH.

PURPOSE: Movement of unfractionated (UFH) and low molecular weight heparins (LMWHs) through gastric mucosa was compared to determine effect of molecular weight on absorption. METHODS: Rat gastric mucosa, mounted in an Ussing chamber, was bathed in oxygenated Kreb's buffer, containing mannitol on the mucosal (lumen) at pH 7.4 or 4, and glucose on the serosal side (circulation) at pH 7.4. Heparins (10 mg/ml) were added to the mucosal side. Potential difference (PD), resistance, and short circuit current (Isc), were determined. Buffers and tissues were extracted to measure heparin by gel electrophoresis. RESULTS: PD increased on heparin addition and following a lag period, that was longer for UFH at pH 7.4 and LMWHs at pH 4.0, returned to baseline. Isc increased slightly for UFH at pH 4.0 but significantly for LMWHs at pH 7.4. More UFH or LMWHs were recovered from serosal buffers at pH 4.0 and pH 7.4 respectively. Results suggest UFH and LMWHs cross gastric mucosa faster, and active transport is involved, at pH 4.0 and pH 7.4, respectively. CONCLUSIONS: Decreasing heparin size, increases movement through gastric mucosa at mucosal buffer pH 7.4 but not pH 4.0. The stomach environment may favor UFH absorption while the intestine environment favors LMWH absorption.

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 microg/ml) for 7 days. To help define the mechanism of endothelial damage, cells were also exposed to H(2)O(2) (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 [(35)S]labeled extracellular matrix of bovine corneal endothelial cells and analyzing released radioactive products by gel filtration and beta-scintillation. Heparanase mRNA was found in high-glucose- and H(2)O(2)-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 H(2)O(2)-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.

An in vitro study with an ussing chamber showing that unfractionated heparin crosses rat gastric mucosa.

Heparin, traditionally given parenterally, is used to treat and prevent thrombosis. Our previous results suggest that orally administered unfractionated heparin (UFH) is absorbed and has antithrombotic effects. However, there is little evidence indicating the site and mechanism of heparin absorption. Our aim was to determine whether the stomach is an absorption site. Rat gastric mucosa was mounted in an Ussing chamber, and UFH was added to the mucosal buffer at pH 7.4. Potential difference (PD), resistance (R), and short circuit current (I(sc)) across the mucosa were determined comparing the mucosal to the serosal side. Mucosal and serosal buffers and tissue were analyzed for chemical heparin and anticoagulant activity, antifactor Xa (anti-Xa) and antifactor IIa (anti-IIa) activity. The PD became more negative on UFH addition. Following a lag period, PD returned to the resting level. Changes in R followed those in PD, whereas I(sc) did not change. Heparin was found in the serosal and mucosal buffer and tissue. Heparin in the serosal buffer had anti-Xa and anti-IIa activity. Decreasing the pH of the mucosal buffer to 4.0, decreased the lag period for PD. Decreasing the concentration of UFH resulted in less pronounced changes in PD and less heparin in the serosal buffer. Changes in PD suggest that heparin moves across the mucosa. Presence of heparin in the serosal buffer and mucosal tissue, indicate that heparin crosses rat gastric mucosa. A stable I(sc) indicates passive diffusion contributes to heparin movement. The stomach could be a site for oral heparin absorption.

Evidence for the absorption of heparin by rat stomach.

Antithrombotic activity and heparin with endothelium are observed in rats when heparin is administered by the oral route. Peak endothelial concentrations at 6 min suggest rapid absorption. To identify the site of absorption, stomach and duodenum were isolated by tying the pyloric sphincter of male Wistar rats and heparin (unfractionated bovine lung, 60 mg/kg) was administered by stomach tube or injected into the duodenum. Heparin in plasma and aortic endothelium, collected within 15 min, was determined by densitometry following agarose gel electrophoresis with toluidine blue staining. Heparin was recovered in 5 of 10 endothelial (0.136+/-0.068 microg/cm(2)) and 6 of 10 plasma (0.06+/-0.02 microg/ml) samples when administered in the stomach and in 0 of 9 endothelial and 2 of 9 plasma (0.02+/-0.02 microg/ml) samples when injected into the duodenum. To further study heparin distribution, stomach layers were separated and analysed 15 min and 4h following heparin administration by stomach tube. Heparin was recovered in muscle and mucosal layers as well as washes indicating that heparin passes through stomach tissue. Heparin was also recovered from the portal vein, endothelium and lung. These results indicate that heparin is absorbed following oral administration and that the stomach is an important site of absorption.

EDHF-mediated rapid restoration of hypotensive response to acetylcholine after chronic, but not acute, nitric oxide synthase inhibition in rats.

Several in vitro studies have shown that endothelium-dependent vasodilatation is maintained by endothelium-derived hyperpolarizing factor (EDHF) or prostacyclin in vessels isolated from endothelial nitric oxide synthase knockout mice. Since this has not been addressed by in vivo studies, we sought to define the magnitude and the onset time of this compensation by recording blood pressure responses to endothelium-dependent vasodilators in rats treated acutely or chronically with the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME). Groups of male Sprague-Dawley rats were given plain water (control) or L-NAME (0.7 mg/ml) in drinking water for 1 day, 5 days, 3 wks or 6 wks. Dose-dependent hypotensive responses to acetylcholine, bradykinin and sodium nitroprusside were determined in anesthetized rats before and after acute intravenous infusion of either L-NAME or a combination of apamin plus charybdotoxin that would selectively inhibit EDHF. Acute L-NAME treatment increased the mean arterial pressure and inhibited acetylcholine- and bradykinin-induced fall in blood pressure in control but not in chronic L-NAME treated rats. The endothelium-dependent hypotensive responses to acetylcholine and bradykinin were restored in rats treated with L-NAME after a time period of 24 h along with increased sensitivity to sodium nitroprusside and reduced plasma nitrate+nitrite levels. While apamin+charybdotoxin pretreatment inhibited the responses to acetylcholine and bradykinin in both acute and chronic L-NAME treated groups, it was more pronounced in the latter group. In conclusion, chronic inhibition of nitric oxide synthase results in the development of a compensatory hypotensive response to acetylcholine within 24 h and this is mediated by EDHF.

In vivo antithrombotic synergy of oral heparin and arginine: endothelial thromboresistance without changes in coagulation parameters.

On the basis of suggested clinical efficacy in an uncontrolled study in ninety-seven patients with unstable angina, an animal study was conducted to investigate antithrombotic synergy between orally administered heparin and arginine. A rat venous thrombosis model tested the difference in thrombus formation when heparin (7.5 mg/kg) and arginine (113 mg/kg) were administered, alone or in combination, by stomach tube with a minimum of 20 rats/group. Oral heparin, arginine, and heparin plus arginine reduced thrombus formation by 50%, 75%, and 90%, respectively, when compared to saline administration. Heparin was recovered from endothelium, yet there was little or no observable plasma anticoagulant activity. An orally administered low-molecular-weight anticoagulant glycosaminoglycan mixture, sulodexide (7.5 mg/kg), showed an 88% reduction in stable thrombus formation when administered alone but showed no synergy with oral arginine. A 28-day study with oral sulodexide (2.9 mg/kg) and arginine (43.9 mg/kg), 20 rats/group, showed antithrombotic activity with minimal anticoagulant activity indicating suitability for long term treatment. These findings suggest the endothelial localization of heparin and a synergistic antithrombotic effect for orally administered heparin and arginine.

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

BACKGROUND: Uncontrolled 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. OBJECTIVES: To determine if high glucose as well as heparanase cause endothelial cell injury and if insulin, heparin and bFGF protect cells from this injury. METHODS: Cultured porcine aortic endothelial cells were treated with high glucose (30 mM) and/or insulin (1 U/ml) and/or heparin (0.5 microg/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. RESULTS: A 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. CONCLUSION: Endothelial 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.

Increased plasma anti-Xa activity and recovery of heparin from urine suggest absorption of orally administered unfractionated heparin in human subjects.

Although heparin is not generally administered orally, the results of studies involving rats suggest that heparin is absorbed, with low levels in plasma but extensive distribution to the endothelium. To determine whether evidence of absorption after oral administration can also be demonstrated in human subjects, we administered unfractionated porcine heparin in a single dose of 1000 U/kg to 6 healthy human subjects. Plasma anticoagulant activity was monitored between 5 minutes and 72 hours after administration, and chemical heparin concentrations were determined in 24-hour urine samples for as long as 120 hours after administration. Plasma anticoagulant activity, determined by anti-Xa activity, increased as soon as 5 minutes after heparin administration, peaked at 120 minutes, and was still increased 72 hours after administration. Values were significantly greater 15 minutes to 48 hours after administration compared with values before administration (paired t test). Mean activated partial thromboplastin time and Heptest values in subjects given heparin were greater than those in controls 15 and 30 minutes and 5 to 120 minutes after administration, respectively. Heparin was recovered from urine as long as 120 hours after administration (the latest time point at which samples were collected); greater amounts were excreted between 48 and 120 hours than before 48 hours. Recovery from both plasma and urine suggest that unfractionated heparin administered orally is absorbed in human subjects, is widely distributed, and is found in the body at least 120 hours after administration. Because heparin is readily bound to endothelium, recovery from plasma and urine likely reflect considerable amounts with endothelium, as has been observed in other species.

Tissue distribution of the low molecular weight heparin, tinzaparin, following administration to rats by the oral route.

Heparins are antithrombotic drugs given by intravenous and subcutaneous routes. However, we have observed that heparins have antithrombotic activity in a rat model when administered orally despite low plasma levels, with low molecular weight heparins (LMWHs) being effective at lower single doses than unfractionated heparins (UFH). Since LMWHs may have other pharmaceutical uses and little is known regarding the pharmacokinetics of oral LMWHs, our objectives were to determine the distribution of the LMWH tinzaparin (Logiparin) following oral dosing. To study distribution at different doses, 0.025-15 mg/kg tinzaparin was given by stomach tube to rats. Gut and non-gut tissues were sampled 4 h later. In a time course study, plasma and tissue samples were collected at eight time points within 24 h after oral administration (60 mg/kg, 4 rats/time interval). Accumulated urine and faeces were collected over 4 and 24 h using metabolic cages. Gut tissue and washes, faeces, urine and non-gut tissue were extracted and analysed for heparin by agarose gel electrophoresis with toluidine blue staining. Activated partial thromboplastin time (APTT) and anti-Xa activity, by Heptest and chromogenic assay, estimated plasma tinzaparin concentrations. Stomach and lung tinzaparin concentrations demonstrated a dose-effect. Peak concentrations in tissue and washes of stomach, duodenum, jejunum, ileum and colon were at 6-30, 15-30, 30 min, 2 and 4 h, respectively. Amounts found at peak times in combined tissue and washes accounted for 46% and 0.5% in stomach (15 min) and colon (4 h), respectively. Tinzaparin was recovered from liver, lung, endothelial samples, and urine at 24 h, but not in faeces. Non-significant increases were seen in APTT and the Heptest, however, anti-Xa activity was significantly greater than control at all times examined, peaking at 2 h. No bleeding was observed. Results are consistent with oral absorption of tinzaparin with wide tissue distribution, likely on endothelium with little in plasma, as previously observed for UFH. Oral administration of LMWHs should be further studied.

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.

Dextran sulfate protects porcine but not bovine cultured endothelial cells from free radical injury.

Previous studies demonstrated that the polyanion dextran sulfate (DS) protects rat coronary and porcine aortic endothelium (PAE) from oxygen-derived free radical (OFR) injury due to hydrogen peroxide (H2O2) or xanthine/xanthine oxidase (X/XO). To determine if DS has a similar protective effect in bovine aortic endothelium (BAE) and bovine brain microvascular endothelium (BBME), H2O2 or X/XO was added to confluent cultures. Cell injury was assessed 1 d later by measuring the percentage of viable cells (by trypan blue exclusion) and the release of lactate dehydrogenase (LDH) into the medium. After H2O2 doses of 6.0 mM for BAE and BBME and 0.8 mM for PAE, and after X doses of 10 microM and XO doses of 0.3 U/mL for all cell types, approximately 50% of cells were viable. Cultures were pretreated with DS (0.001 to 500 microg/mL) 24 to 26 h prior to H2O2 or X/XO exposure. Pretreatment at concentrations of 0.5, 5, and 50 microg/mL significantly increased the percentage of viable cells and reduced LDH release in cultures of PAE, but not BAE or BBME, treated with H2O2. Similarly, pretreatment with DS concentrations of 5 and 50 microg/mL significantly increased the percentage of viable cells and reduced LDH release in cultures of PAE, but not BAE or BBME, treated with X/XO. Thus, DS protected porcine but not bovine endothelium. Catalase (10 U/mL) increased the percentage of viable cells and reduced LDH release in H2O2-treated BAE and BBME, suggesting that DS likely acts by a different mechanism and does not neutralize H2O2. These results suggest that the protective effect of DS on OFR-injured endothelium is species-dependent.

Tissue distribution of [14C]sucrose octasulfate following oral administration to rats.

PURPOSE: Aluminum sucrose octasulfate (SOS) is used clinically to prevent ulcers. Under physiologic conditions, the sodium salt of this drug can be formed. Our objective was to determine whether sodium SOS was absorbed when administered orally. In addition to furthering our understanding of aluminum SOS, this study also aimed to clarify how other polyanionic drugs, such as heparin and low-molecular-weight heparins, are absorbed. METHODS: [14C]-labeled and cold sodium SOS (60 mg/kg) were given to rats by stomach tube. Radioactivity was counted in gut tissue, gut washes, and nongut tissue (i.e., lung, liver, kidney, spleen, endothelial, and plasma samples) at 3 min, 6 min, 15 min, 30 min, 60 min, 4 h, and 24 h, and in urine and feces accumulated over 4 h and 24 h. RESULTS: Peak radioactivity was found in the tissue and washes of the stomach, ileum, and colon at 6 min, 60 min, and 4 h, respectively, showing progression through the gut. Gut recovery accounted for 84% of the dose at 6 min but only 12% of the dose at 24 h, including counts from feces. Radioactivity was recovered from nongut tissue (averaging 8.6% of the dose) and accumulated urine (18% of the dose at 24 h). When total body distribution was considered, the recovery of radioactivity was greater for the endothelium than for plasma (peak percentage of the dose was 65% at 15 min, 20% at 3 min, 5% from 20 to 240 min for the vena cava, aortic endothelium, and plasma, respectively). CONCLUSIONS: Results indicate that sodium SOS is absorbed, agreeing with previous studies demonstrating the oral absorption of other sulfated polyanions. Endothelial concentrations must be considered when assessing the pharmacokinetics of these compounds. The measured plasma drug concentrations reflect the much greater amounts of drug residing with the endothelium.