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The effect of alloxan-induced diabetes on tissue plasminogen activator activity and plasmin inhibition in the rat
THROMBOSISRESEARCH 23; 421-434,1981 0049-3848/81/160421-14$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.
THE EFFECT OF ALLOXAN-INDUCED ACTIVATOR
ACTIVITY
A. Smokovitis, Department
DIABETES ON TISSUE PLASMINOGEN
AND PLASMIN
INHIBITION IN THE =T
W. Auerswald,
of Medical University
Physiology,
B. R. Binder School of Medicine,
of Vienna,.Austria
(Received20.2.1981;in revised form 6.6.1981. Accepted by Editor M. Samama. Received in final form by ExecutiveEditorialOffice 20.8.1981)
ABSTRACT In alloxan-diabetic rats the plasminogen activator activity (PAA) showed a tissue- and time-dependent pattern of changes in key organs. In arteries of the lung, heart, and kidney, and in the intima of the aorta the PAA was markedly enhanced on the 4th day after the induction of diabetes. Thereafter, up to the 12th week post induction of diabetes, the PAA in arteries of the lung showed a gradual return to the normal, while the intima of the aorta at the nonbranching regions exhibited a fluctuation in the PAA; at the branching regions the PAA was constantly increased as it was also in the controls. In arteries of the myocnrdium and the kidney (particularly of the renal cortex) the PAA was impaired. In caudal vena cava the PAA was unchanged throughout experimentation. Also, no difference was noted between diabetic rats and control rats in the PAA of the epicardiur.1, endocardium, serosa of the lung and kidney and adventitia of the aorta. Tissue plasmin inhibition showed no significant difference between diabetic rats and controls.
Supported by a grant from the Austrian of Scientific Research (No. 4074)
Fund for the Promotion
Key words: Alloxan-diabetes. Tissue fibrinolysis. Branching regions. 421
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INTRODUCTION
Blood fibrinolysis in diabetics has drawn the attention of many investigators (l-lo), but studies dealing with tissue fibrinolysis are very limited, although a defective vascular fibrinolytic system could be involved in the diabetic angiopathy. Superficial vessels in diabetics frequently show a reduced PAA (7, 8, ll), while the mechanism of release of plasminogen activator might also be defective (2, 7, 8, 12). In experimental animal models the fibrinolytic response to different physiological or pathophysiological stimuli was found markedly varying from one tissue to another (13, 14). Therefore, the possible tissue variation in the fibrinolytic response to the diabetic state was studied in rats until the 12th week post induction of diabetes.
MATERIALS
AND METHODS
Seventy Sprague-Dawley male rats, weighing 200 to 210 g, were injected subcutaneously with alloxan (Loba-Chemie, Vienna, Austria) at the dose of 10 mg / 100 g after 24 h fast. Alloxan was dissolved in saline immediately before injection. Thirty rats injected with saline served as controls. Rats were injected at about 4 pm; at that time the diabetogenic effect of chemicals inducing diabetes was found to be the highest (15). All of the animals were provided a regular commercial rat chow diet and water ad libitum. Rats were occasionally housed for two hours in metabolic cages for urine collection to test for glucosuria. Blood samples were taken for gl,ucos~ determination from the tail. Glucose was determined enz:?.\atically (Boehringer Mannheim Biochemica Test Kit). Sixty of the alloxan-injected rats developed severe diabetes, six rats showed only a slight diabetic symptomatology, while in four rats alloxan had not any diabetogenic effect (for details see results). Ten severely diabetic rats died during the first week post induction of diabetes. The remainder of the severely diabetic rats (fifty) as well as the controls (thirty) were sacrificed in groups 4 days, 2, 3, 4, 5, 6, 8, and 12 weeks post injection of alloxan or saline. The rats which developed slight diabetes were sacrificed 4 weeks and 8 weeks after administration of alloxan; by the 8th week rats with slight diabetes showed spontaneous recovery of the diabetic symptoms. Four alloxan-treated rats which developed no diabetes were sacrificed in the 8th week post injection. The .animals were always sacrificed at about 11 am. Specimens from the thoracic aorta, caudal vena cava, lung, heart, and kidney taken immediately after the rats were sacrificed, were
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423
washed slightly in saline and immediately frozen and stored at -70~ C. Sections of specimens (whole organs and isolated tissue layers or blood vessels) taken from alloxan-treated rats and controls were made from the same anatomical area of each .organ and studied histochemically under the same experimental conditions for detection of plasminogen activator activity and plasmin inhibition as described before (16-18). For this purpose, plasminogen-rich and plasminogen-free fibrinogen (Organon-Teknika, The Netherlands), thrombin (Leo, Denmark), and plasmin (Novo, Denmark) were used. Forty sections were studied from each specimen. The evaluation of the PAA was made by taking into consideration the incubation period and the extent of lysis. The PAA of each section was evaluated according to the following grades: grade I, well demarcated foci of lysis; grade II, small areas of lysis; grade III, large areas of lysis. 1, 2, and 3 points were allotted to grades I, II, and III, respectively. The mean of the points scored for each set of the sections was taken as a measure of the PAA of the specimen. Student's t-test for unpaired observations was used for the statistical analysis. RESULTS In 86% of the rats, alloxan at the injected dose and time of the day induced severe diabetes: hyperglycemia (Fig. I), glucosuria, polyuria, weight loss (Table I, Fig. 21, polydypsia and polyphagia. Glucosuria, polyuria, and polydypsia were observed from the first day following alloxan injection. No attempt was made to ameliorate the disease; ten rats died during the first week post induction of diabetes. In 8.4% of the alloxan-injected rats only a slight diabetic symp$omatology was developed (nonfqsting glucose in plasma 210 - 20 mg/ 100 ml, ip controls 125 - 6 mg / 100 ml; p < 0.001. The values are mean - S.D.). Rats with slight diabetes showed spontaneous recovery eight weeks after injection of alloxan (all parameters studied showed no difference to those in controls). In the remainder 5.6% of the rats alloxan had not any diabetogenic effect. After cataracts exhibited rats from
eight weeks, 80% of the severely diabetic rats had in one eye and by the 12th week 85% of the rats cataracts bilaterally and were blind. None of the the other groups had cataracts at this age.
Severely diabetic rats manifested a significant increase in kidney size and weight, while size and weight of the heart were decreased compared to controls (Table I). On the 4th day after the induction of severe diabetes an increased PAA was seen in arteries of lung, kidney, and myocardium and in the intima of aorta compared to controls (pO.O5). Thereafter up to the 12th week post induction of severe diabetes, the pattern of changes in PAA was different in the different organs studied:
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In the caudal vena cava no changes in the PAA were seen throughout experimentation compared to controls (p > 0.05). Also, no difference was noted between severely diabetic rats and control rats in the PAA of the epicardium, endocardium, serosa of the lung and kidney and adventitia of the aorta (p > 0.05). In lung the initially increased arterial PAA continued to be slightly elevated until the sixth week compared to controls (p < 0.05); from the 8th week on the PAA showed no difference from that in controls (p > 0.05). The changes of the PAA in the intima of the aorta, and in arteries of renal cortex and myocardium in severely diabetic rats as well as the PAA in corresponding controls are given in the Fig. 3. In the intima of the aorta the initially enhanced PAA (on the 4th day) was concomitant with damaged, detached endothelial cells. After two to three weeks, the aortic intima at
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FIBRINOLYSISIN DIABETICRATS
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the nonbranching regions showed a decreased PAA compared to controls (p l O.OOl), while after four to six weeks an apparently regenerated endothelium produced an increased PAA compared to controls (p < 0.001). From the 8th week on the aortic PAA showed almost no difference from that in controls (p > 0.05) (Fig. 3). At the branching regions of the aorta the PAA was always higher than at the nonbranching regions in diabetic rats and controls. It should be pointed out that the decreased PAA during the course of diabetes concerned only the nonbranching regions of the aorta; at the branching regions the PAA was almost unchanged compared to controls (p > 0.05). In kidney, particularly in the cortex, and in myocardium, after the initially increased PAA, the arteries showed a decreased PAA two to twelve weeks.after the induction of diabetes compared to controls (p < 0.001) (Fig. 3). However, in 15% of the kidney specimens necrotic areas were seen eight to twelve weeks post induction of diabetes. Arteries in necrotic areas exhibited increased PAA compared to the arteries in control specimens (p < 0.001). Also, some arteries (mainly larger arteries) in renal cortex and myocardium which showed thickened walls exhibited enhanced PAA compared to arteries in control specimens (p < 0.001). However, in all these cases the number of arteries with abnormal morphology and enhanced PAA was limited and did not change statistically
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FIG. 2 Changes in body weight during 12 weeks after induction of severe alloxan-diabetes in Sprague-Dawley rats.
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FIBRINOLYSIS IN DIABETIC RATS
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the pattern of decreased arterial PAA seen in specimens of kidney and myocardium two to twelve weeks post induction of diabetes. Tissues from rats, which exhibited only a slight diabetic symptomatology and recovered spontaneously two months later, showed a pattern of PAA comparable to that in controls (p ' 0.05). In alloxan-injected rats which did not develop diabetes at all the tissue PAA showed no difference from that in controls (p > 0.05). No significant change in the tissue plasmin inhibition was noted in rats injected with alloxan (diabetic or no diabetic). Evaluation and comparison of plasminogen activator activity and plasmin inhibition were made separately in
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Changes in plasminogen activator activity (PAA) in the intima of the nonbranching regions of the aorta (_), and in arteries of the renal cortex (bd) and myocardium (--_) during 12 weeks after induction of severe alloxan-diabetes in Sprague-Dawley rats. Closed symbols indicate the PAA in diabetic rats and open symbols in contrcls. The values of PAA were estimated on whole organ sections.
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FIBRINOLYSIS IN DIABETIC RATS
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isolated tissue layers or blood vessels and in whole organ sections from alloxan-injected and control rats. In every case isolated tissue layers or isolated blood vessels showed higher PAA than the same tissue layers or vessels in corresponding whole organ sections in agreement with our previous studies (14, 17-18).
DISCUSSION -
The same dose of alloxan injected the same time of the day into Sprague-Dawley rats induced in 86% of the rats severe diabetes, in 7% slight diabetes, while in 7% of the rats alloxan had not any diabetogenic effect. Differences in the response to alloxan were ascribed to differences in pancreatic i3-cells destruction and recovery (19). Also, a circadian variation in the effect of diabetes-inducing factors in animals has been reported (15); furthermore, the genetic background can influence the susceptibility to alloxan-induced diabetes and the recovery from diabetes (20). The diabetic state induced by alloxan should be accompanied by a low blood insulin level (21), a high blood pressure (21, and changes in free fatty acids and triglycerides in the 221, blood (19, 23). The body weight decreased dramatically in severely diabetic rats, while size and weight of kidneys were significantly increased, a fact also seen by others in experimental diabetes (19, 21, 24-25) as well as in human diabeties (26-27). In contrast, the size and weight of the heart were markedly reduced in severely diabetic rats. Tissue PAA, however, did not exhibit a uniform pattern of changes. In arteries of lung, heart, kidney, and in the intima of aorta of severely diabetic rats the PAA showed a varying and time-dependent response (Fig. 3); in caudal vena cava, on the other hand, as well as in epicardium, endocardium, serosa of lung and kidney and in the adventitia of aorta no change in the PAA was seen. The fact that rats injected with alloxan showed changes in tissue PAA only in case severe diabetes has been developed,probably means that these changes are not due to a direct effect of alloxan on the tissue PAA. Furthermore, since the tissue plasmin inhibition showed no significant difference between diabetic rats and controls, the observed changes in PAA should be due to differences in the concentration of plasminogen activator. Glucose has been reported to induce increase in blood fibrinolytic activity in animals and humans (28-29). Although the level of glucose in the blood was significantly increased throughout the course of severe diabetes, the tissue PAA showed no obvious correlation with blood glucose level. Such a correlation could neither be established between the tissue PAA pattern and the other parameters mentioned above, since the pattern of PAA was dependent on the tissue studied as well
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as on the time after Ltic inticcc:on of iia!Jetes. The increased PAA seen in most organs studied early after the induction of diabetes is in correlation with the reported increase in blood fibrinolytic activity shortly after alloxan During the course of the induced injection into rats (30). severe diabetes the following differences in PAA patterns are of particular interest: (a) between PAA in arteries and veins; (b) between PAA in large and small arteries (aorta and arteries in lung, myocardium or renal cortex); and (c) between PAA in arteries in different tissues (in lung on the one hand and in myocardium and renal cortex on the other) up to at least twelve weeks post induction of diabetes. In agreement with our previous studies in Wistar rats and normal and diabetic Sprague-Dawley other animal species (31), rats showed a constantly increased PAA at the branching regions of the aorta compared to that in nonbranching regions. The same observation has also been made in the normal human aorta (32, It is of particular interest that the PAA at the branching 33). regions remained increased even when the PAA at the nonbranching regions of the aorta in diabetic rats has been markedly decreased. A similar observation has been made in genetically hypercholesterolemic rats (34). Based on all these observations, a new hypothesis has been developed (35) relating the increased PAA with the initiation of atherosclerosis at the branching regions of the aorta which are predilection regions for atherosclerosis. The increased arterial PAA seen in some specimens of kidney with necrosis might be due to the fact that arteries in necrotic areas of the kidney from alloxan-diabetic rats are occluded by endothelial cell proliferation (36) and in proliferated endothelial cells the PAA is increased (31). Also, increased PAA in sclerotic arteries of the renal cortex might be attributed to increased adventitial PAA as seen in arterioThe enhanced PAA found in some sclerotic aorta (37-38). arteries of the myocardium with thickened walls might possibly be explained in the same way. The results of the present study indicate that differences in tissue (vascular) fibrinolytic response to diabetes in humans as well as differences in the response between large and small vessels are possible. Also, durin.3 the induction of the disease the fibrinolytic state might be different from that in the course of the established disease. Furthermore, the vascular fibrinolytic activity might depend upon the severity of the disease. Interestingly, in some rats which developed slight diabetes followed by spontaneous recovery two months post injection, the arterial PAA was found to be normal in the renal cortex and myocardium. These findings are in agreement with observations made in humans (7-8). Rats showing spontaneous recovery from alloxan diabetes were found to have regenerated B-cells (19). Also, in the rat model, pancreatic transplantation prevented histological changes induced by diabetes (39).
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The impaired arterial PAA in kidney and heart of severely diabetic rats is of particular interest. Diabetic state in humans accelerates the development of atherosclerosis in the arteries of the heart and kidney (29, 40). Increased deposits of fibrin in diabetes mellitus as well as accumulation of fibrinogen within the vessel walls in diabetic microangiopathy have been reported (41-44). Impaired PAA ans prostacyclin production (45-46) in arteries, associated with platelet hyperreactivity and other humoral changes (47-48) as well as with hypertension (491, might be involved in the evolution of the diabetic angiopathy. REFERENCES 1.
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21. IAMS, S.G. and WEXLER, B.S. Alloxan diabetes in spontaneously hypertensive rats: gravimetric, metabolic and histopathological alterations. Brit. J. Exp. Path. 58, 177-199, 22.
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THE EFFECT OF ALLOXAN-INDUCED ACTIVATOR
ACTIVITY
A. Smokovitis, Department
DIABETES ON TISSUE PLASMINOGEN
AND PLASMIN
INHIBITION IN THE =T
W. Auerswald,
of Medical University
Physiology,
B. R. Binder School of Medicine,
of Vienna,.Austria
(Received20.2.1981;in revised form 6.6.1981. Accepted by Editor M. Samama. Received in final form by ExecutiveEditorialOffice 20.8.1981)
ABSTRACT In alloxan-diabetic rats the plasminogen activator activity (PAA) showed a tissue- and time-dependent pattern of changes in key organs. In arteries of the lung, heart, and kidney, and in the intima of the aorta the PAA was markedly enhanced on the 4th day after the induction of diabetes. Thereafter, up to the 12th week post induction of diabetes, the PAA in arteries of the lung showed a gradual return to the normal, while the intima of the aorta at the nonbranching regions exhibited a fluctuation in the PAA; at the branching regions the PAA was constantly increased as it was also in the controls. In arteries of the myocnrdium and the kidney (particularly of the renal cortex) the PAA was impaired. In caudal vena cava the PAA was unchanged throughout experimentation. Also, no difference was noted between diabetic rats and control rats in the PAA of the epicardiur.1, endocardium, serosa of the lung and kidney and adventitia of the aorta. Tissue plasmin inhibition showed no significant difference between diabetic rats and controls.
Supported by a grant from the Austrian of Scientific Research (No. 4074)
Fund for the Promotion
Key words: Alloxan-diabetes. Tissue fibrinolysis. Branching regions. 421
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INTRODUCTION
Blood fibrinolysis in diabetics has drawn the attention of many investigators (l-lo), but studies dealing with tissue fibrinolysis are very limited, although a defective vascular fibrinolytic system could be involved in the diabetic angiopathy. Superficial vessels in diabetics frequently show a reduced PAA (7, 8, ll), while the mechanism of release of plasminogen activator might also be defective (2, 7, 8, 12). In experimental animal models the fibrinolytic response to different physiological or pathophysiological stimuli was found markedly varying from one tissue to another (13, 14). Therefore, the possible tissue variation in the fibrinolytic response to the diabetic state was studied in rats until the 12th week post induction of diabetes.
MATERIALS
AND METHODS
Seventy Sprague-Dawley male rats, weighing 200 to 210 g, were injected subcutaneously with alloxan (Loba-Chemie, Vienna, Austria) at the dose of 10 mg / 100 g after 24 h fast. Alloxan was dissolved in saline immediately before injection. Thirty rats injected with saline served as controls. Rats were injected at about 4 pm; at that time the diabetogenic effect of chemicals inducing diabetes was found to be the highest (15). All of the animals were provided a regular commercial rat chow diet and water ad libitum. Rats were occasionally housed for two hours in metabolic cages for urine collection to test for glucosuria. Blood samples were taken for gl,ucos~ determination from the tail. Glucose was determined enz:?.\atically (Boehringer Mannheim Biochemica Test Kit). Sixty of the alloxan-injected rats developed severe diabetes, six rats showed only a slight diabetic symptomatology, while in four rats alloxan had not any diabetogenic effect (for details see results). Ten severely diabetic rats died during the first week post induction of diabetes. The remainder of the severely diabetic rats (fifty) as well as the controls (thirty) were sacrificed in groups 4 days, 2, 3, 4, 5, 6, 8, and 12 weeks post injection of alloxan or saline. The rats which developed slight diabetes were sacrificed 4 weeks and 8 weeks after administration of alloxan; by the 8th week rats with slight diabetes showed spontaneous recovery of the diabetic symptoms. Four alloxan-treated rats which developed no diabetes were sacrificed in the 8th week post injection. The .animals were always sacrificed at about 11 am. Specimens from the thoracic aorta, caudal vena cava, lung, heart, and kidney taken immediately after the rats were sacrificed, were
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washed slightly in saline and immediately frozen and stored at -70~ C. Sections of specimens (whole organs and isolated tissue layers or blood vessels) taken from alloxan-treated rats and controls were made from the same anatomical area of each .organ and studied histochemically under the same experimental conditions for detection of plasminogen activator activity and plasmin inhibition as described before (16-18). For this purpose, plasminogen-rich and plasminogen-free fibrinogen (Organon-Teknika, The Netherlands), thrombin (Leo, Denmark), and plasmin (Novo, Denmark) were used. Forty sections were studied from each specimen. The evaluation of the PAA was made by taking into consideration the incubation period and the extent of lysis. The PAA of each section was evaluated according to the following grades: grade I, well demarcated foci of lysis; grade II, small areas of lysis; grade III, large areas of lysis. 1, 2, and 3 points were allotted to grades I, II, and III, respectively. The mean of the points scored for each set of the sections was taken as a measure of the PAA of the specimen. Student's t-test for unpaired observations was used for the statistical analysis. RESULTS In 86% of the rats, alloxan at the injected dose and time of the day induced severe diabetes: hyperglycemia (Fig. I), glucosuria, polyuria, weight loss (Table I, Fig. 21, polydypsia and polyphagia. Glucosuria, polyuria, and polydypsia were observed from the first day following alloxan injection. No attempt was made to ameliorate the disease; ten rats died during the first week post induction of diabetes. In 8.4% of the alloxan-injected rats only a slight diabetic symp$omatology was developed (nonfqsting glucose in plasma 210 - 20 mg/ 100 ml, ip controls 125 - 6 mg / 100 ml; p < 0.001. The values are mean - S.D.). Rats with slight diabetes showed spontaneous recovery eight weeks after injection of alloxan (all parameters studied showed no difference to those in controls). In the remainder 5.6% of the rats alloxan had not any diabetogenic effect. After cataracts exhibited rats from
eight weeks, 80% of the severely diabetic rats had in one eye and by the 12th week 85% of the rats cataracts bilaterally and were blind. None of the the other groups had cataracts at this age.
Severely diabetic rats manifested a significant increase in kidney size and weight, while size and weight of the heart were decreased compared to controls (Table I). On the 4th day after the induction of severe diabetes an increased PAA was seen in arteries of lung, kidney, and myocardium and in the intima of aorta compared to controls (p
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In the caudal vena cava no changes in the PAA were seen throughout experimentation compared to controls (p > 0.05). Also, no difference was noted between severely diabetic rats and control rats in the PAA of the epicardium, endocardium, serosa of the lung and kidney and adventitia of the aorta (p > 0.05). In lung the initially increased arterial PAA continued to be slightly elevated until the sixth week compared to controls (p < 0.05); from the 8th week on the PAA showed no difference from that in controls (p > 0.05). The changes of the PAA in the intima of the aorta, and in arteries of renal cortex and myocardium in severely diabetic rats as well as the PAA in corresponding controls are given in the Fig. 3. In the intima of the aorta the initially enhanced PAA (on the 4th day) was concomitant with damaged, detached endothelial cells. After two to three weeks, the aortic intima at
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the nonbranching regions showed a decreased PAA compared to controls (p l O.OOl), while after four to six weeks an apparently regenerated endothelium produced an increased PAA compared to controls (p < 0.001). From the 8th week on the aortic PAA showed almost no difference from that in controls (p > 0.05) (Fig. 3). At the branching regions of the aorta the PAA was always higher than at the nonbranching regions in diabetic rats and controls. It should be pointed out that the decreased PAA during the course of diabetes concerned only the nonbranching regions of the aorta; at the branching regions the PAA was almost unchanged compared to controls (p > 0.05). In kidney, particularly in the cortex, and in myocardium, after the initially increased PAA, the arteries showed a decreased PAA two to twelve weeks.after the induction of diabetes compared to controls (p < 0.001) (Fig. 3). However, in 15% of the kidney specimens necrotic areas were seen eight to twelve weeks post induction of diabetes. Arteries in necrotic areas exhibited increased PAA compared to the arteries in control specimens (p < 0.001). Also, some arteries (mainly larger arteries) in renal cortex and myocardium which showed thickened walls exhibited enhanced PAA compared to arteries in control specimens (p < 0.001). However, in all these cases the number of arteries with abnormal morphology and enhanced PAA was limited and did not change statistically
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the pattern of decreased arterial PAA seen in specimens of kidney and myocardium two to twelve weeks post induction of diabetes. Tissues from rats, which exhibited only a slight diabetic symptomatology and recovered spontaneously two months later, showed a pattern of PAA comparable to that in controls (p ' 0.05). In alloxan-injected rats which did not develop diabetes at all the tissue PAA showed no difference from that in controls (p > 0.05). No significant change in the tissue plasmin inhibition was noted in rats injected with alloxan (diabetic or no diabetic). Evaluation and comparison of plasminogen activator activity and plasmin inhibition were made separately in
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isolated tissue layers or blood vessels and in whole organ sections from alloxan-injected and control rats. In every case isolated tissue layers or isolated blood vessels showed higher PAA than the same tissue layers or vessels in corresponding whole organ sections in agreement with our previous studies (14, 17-18).
DISCUSSION -
The same dose of alloxan injected the same time of the day into Sprague-Dawley rats induced in 86% of the rats severe diabetes, in 7% slight diabetes, while in 7% of the rats alloxan had not any diabetogenic effect. Differences in the response to alloxan were ascribed to differences in pancreatic i3-cells destruction and recovery (19). Also, a circadian variation in the effect of diabetes-inducing factors in animals has been reported (15); furthermore, the genetic background can influence the susceptibility to alloxan-induced diabetes and the recovery from diabetes (20). The diabetic state induced by alloxan should be accompanied by a low blood insulin level (21), a high blood pressure (21, and changes in free fatty acids and triglycerides in the 221, blood (19, 23). The body weight decreased dramatically in severely diabetic rats, while size and weight of kidneys were significantly increased, a fact also seen by others in experimental diabetes (19, 21, 24-25) as well as in human diabeties (26-27). In contrast, the size and weight of the heart were markedly reduced in severely diabetic rats. Tissue PAA, however, did not exhibit a uniform pattern of changes. In arteries of lung, heart, kidney, and in the intima of aorta of severely diabetic rats the PAA showed a varying and time-dependent response (Fig. 3); in caudal vena cava, on the other hand, as well as in epicardium, endocardium, serosa of lung and kidney and in the adventitia of aorta no change in the PAA was seen. The fact that rats injected with alloxan showed changes in tissue PAA only in case severe diabetes has been developed,probably means that these changes are not due to a direct effect of alloxan on the tissue PAA. Furthermore, since the tissue plasmin inhibition showed no significant difference between diabetic rats and controls, the observed changes in PAA should be due to differences in the concentration of plasminogen activator. Glucose has been reported to induce increase in blood fibrinolytic activity in animals and humans (28-29). Although the level of glucose in the blood was significantly increased throughout the course of severe diabetes, the tissue PAA showed no obvious correlation with blood glucose level. Such a correlation could neither be established between the tissue PAA pattern and the other parameters mentioned above, since the pattern of PAA was dependent on the tissue studied as well
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as on the time after Ltic inticcc:on of iia!Jetes. The increased PAA seen in most organs studied early after the induction of diabetes is in correlation with the reported increase in blood fibrinolytic activity shortly after alloxan During the course of the induced injection into rats (30). severe diabetes the following differences in PAA patterns are of particular interest: (a) between PAA in arteries and veins; (b) between PAA in large and small arteries (aorta and arteries in lung, myocardium or renal cortex); and (c) between PAA in arteries in different tissues (in lung on the one hand and in myocardium and renal cortex on the other) up to at least twelve weeks post induction of diabetes. In agreement with our previous studies in Wistar rats and normal and diabetic Sprague-Dawley other animal species (31), rats showed a constantly increased PAA at the branching regions of the aorta compared to that in nonbranching regions. The same observation has also been made in the normal human aorta (32, It is of particular interest that the PAA at the branching 33). regions remained increased even when the PAA at the nonbranching regions of the aorta in diabetic rats has been markedly decreased. A similar observation has been made in genetically hypercholesterolemic rats (34). Based on all these observations, a new hypothesis has been developed (35) relating the increased PAA with the initiation of atherosclerosis at the branching regions of the aorta which are predilection regions for atherosclerosis. The increased arterial PAA seen in some specimens of kidney with necrosis might be due to the fact that arteries in necrotic areas of the kidney from alloxan-diabetic rats are occluded by endothelial cell proliferation (36) and in proliferated endothelial cells the PAA is increased (31). Also, increased PAA in sclerotic arteries of the renal cortex might be attributed to increased adventitial PAA as seen in arterioThe enhanced PAA found in some sclerotic aorta (37-38). arteries of the myocardium with thickened walls might possibly be explained in the same way. The results of the present study indicate that differences in tissue (vascular) fibrinolytic response to diabetes in humans as well as differences in the response between large and small vessels are possible. Also, durin.3 the induction of the disease the fibrinolytic state might be different from that in the course of the established disease. Furthermore, the vascular fibrinolytic activity might depend upon the severity of the disease. Interestingly, in some rats which developed slight diabetes followed by spontaneous recovery two months post injection, the arterial PAA was found to be normal in the renal cortex and myocardium. These findings are in agreement with observations made in humans (7-8). Rats showing spontaneous recovery from alloxan diabetes were found to have regenerated B-cells (19). Also, in the rat model, pancreatic transplantation prevented histological changes induced by diabetes (39).
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The impaired arterial PAA in kidney and heart of severely diabetic rats is of particular interest. Diabetic state in humans accelerates the development of atherosclerosis in the arteries of the heart and kidney (29, 40). Increased deposits of fibrin in diabetes mellitus as well as accumulation of fibrinogen within the vessel walls in diabetic microangiopathy have been reported (41-44). Impaired PAA ans prostacyclin production (45-46) in arteries, associated with platelet hyperreactivity and other humoral changes (47-48) as well as with hypertension (491, might be involved in the evolution of the diabetic angiopathy. REFERENCES 1.
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34. SMOKOVITIS, A., AUERSWALD, W. and BINDER, B.R. Plasminogenaktivatoraktivitgt der Aorta bei genetisch hypercholesterintiischen Ratten; Wirkung von Streptozotocin. In: Fibrinolvse, Thrombose, HZnostase. E. Deutsch, K. Lechner (Eds.). Stuttgart-New York: F.K. Schattauer Verlag, 1980. pp. 743-746. 35. SMOKOVITIS, A. A new hypothesis: Possible mechanisms in the involvement of the increased plasminogen activator activity in branching regions of the aorta in the initiation of atherosclerosis. Thromb. Haemost. 43, 141-146, 1980. 36. WEXLER, B.C. Histopathologic diabetes in arteriosclerotic Diabetes 2, 324-336, 1970.
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in
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46. SILBERBAUER, K., SCHERNTHANER, G., SINZINGER, H., PIZAKATZER, H. and WINTER, M. Decreased vascular prostacyclin in juvenile-onset diabetes. N. Engl. J. Med. 300, 366-367, 1979. 47. PACKHAM, M.A. Methods for detection of hypersensitive platelets. Thromb. Haemost. 40, 175-195, 1978. 48. COLWELL, J-A., HALUSHKA, P.V., SARJI, K-E., LOPES-VIRELLA M.F. and SAGEL, J. Vascular disease in diabetes. Pathophysiological mechanisms and therapy. . Arch. Intern. Med. 139, 225-230, 1979. 49. McMILLAN, D.E. Diabetic angiopathy. Its lessons in vascular physiology. Amer. Heart J. 96, 401-406, 1978.