Pathogenesis of Diabetic Microangiopathy: An Overview ANTHONY
H.
BARNEIT,
B.SC.,
M.D.,
F.R.c.P.,
Birmingham,
United Kingdom
Major susceptibility factors for diabetic microangiopathy include duration of disease and probably quality of metabolic control. The mechanism of development of microangiopathy is incompletely understood but appears to involve functional abnormalities within the microcirculation, enhanced glucose metabolism, hemostatic abnormality, and genetic susceptibility. This article reviews the factors believed to be involved in pathogenesis and attempts to draw these together by suggesting a sequence of pathogenic interactions that could result. in the microvascular changes seen in susceptible target organs. Possibilities for therapeutic intervention based on these pathogenic mechanisms are discussed. A small pilot trial of an oral hypoglycemic agent, gliclazide, is reported, providing evidence for a specific action of this drug on thromboxane synthesis and platelet aggregation. This is independent of glycemic control and may in part be mediated by a fall in lipid peroxides.
From the University of Birmingham, Birmmgham, United Kingdom. Requests for reprints should be addressed to Anthony H. Barnett. M.D., Department of Medicine. East Birmingham Hospital, Bordesley Green East, Birmingham B9 5ST, United Kingdom.
D
iabetic microangiopathy accounts for much of the morbidity and mortality of diabetes. Our understanding of its pathogenesis is still not complete, although recent research has cast new light on the problem and has suggested possible therapeutic options. The major susceptibility factors for microangiopathy are duration of disease and probably quality of metabolic control [l-4]. These associations do not, however, explain the mechanism of development, which probably reflects many factors, including functional abnormalities within the microcirculation, enhanced glucose metabolism via different pathways of nonglycolytic glucose metabolism, hemostatic abnormality, and genetic susceptibility [51.
GENETICSUSCEPTIBILITY Although microvascular disease cannot occur without the metabolic abnormality of diabetes, the time of appearance and severity of complications show wide individual variation. In insulin-dependent diabetes, about one-third of subjects develop severe microangiopathic complications with poor long-term prognosis, whereas the remainder tend to run a more benign course. Indeed, some patients never develop clinically evident complications, even after 30-40 years of the disease. Both identicaltwin studies [6] and studies looking at immunogenetic associations with retinopathy [7] have suggested a genetic component. Our own group has described associations between diabetic retinopathy and various human leukocyte antigen (HLA)encoded markers on chromosome 6, such as HLADR4 and an allotype of the fourth component of complement C4B3 [8], and an association with a phenotype of the immunoglobulin heavy chain markers on chromosome 14, for both insulin-dependent [9] and non-insulin-dependent diabetes [lo]. Genetic markers may be used to predict which diabetic patients will be most susceptible to complications, but they cannot operate in the absence of diabetes itself. They may act as modulators that determine when damage will occur, or they may affect the end-organ response to abnormal metabolism. Variation in genetic susceptibility may explain, at least in part, the variable rate of development of complications in patients with apparently similar blood glucose control.
June 24, 1991
The Amencan Journal of Medicine
Volume 90 (suppl 6A)
6A-67S
SYMPOSIUM
ON GLICWIDE
/ BARNETT
MECHANISMOF DEVELOPMENTOF MICROANGIOPATHY
diabetic patients. Interestingly, serum immunoglobulin G (IgG) from diabetics had fluorescence characteristics similar to those of the long-lived tisThe mechanism of development of microangiopasue proteins, and the strength of the fluorescence thy is still incompletely understood. However, it was related to the degree of retinopathy [19]. This appears to involve development of capillary basefluorescence is similar in those proteins whose ment membrane thickening, nonenzymatic glycoamino groups have been modified by reaction with sylation of long-lived tissue proteins, abnormalities malonylaldehyde or other peroxide degradation of endothelial cells and platelets, and prostaglandin products to form aminoimino propene adducts [ZO]. abnormalities, and perhaps increased damage by These reactive aldehydes are formed during the free radicals. metabolism of prostaglandins and by lipid peroxidaCAPILLARYBASEMENTMEMBRANETHICKENING tion, possibly as a result of free radical attack. Free radicals are violently reactive chemical speCapillary basement membrane thickening is the cies with an unpaired electron in their structure. histologic hallmark of microangiopathy. Type IV They are produced during the course of normal collagen seems to be the major structural element metabolism and are usually eliminated rapidly by and heparan sulfate the major proteoglycan, toantioxidants and other enzymes. In some diseases, gether with laminin and fibronectin [ll-131. Hepaparticularly where inflammation or ischemia are ran sulfate is produced by the endothelial cell and is fundamental (e.g., rheumatoid arthritis, diabetes), highly negatively charged. It produces a regular there is evidence of excessive free radical activity. latticework of anionic sites that hinder the filtration Polyunsaturated fatty acids, which are constituents of negatively charged proteins such as albumin [14]. of cell membranes, are particularly susceptible to In diabetes there is impaired synthesis of proteofree radical attack, as are both tissue and extracelglycans and an increase in hydroxylysine and its lular proteins-particularly those with a large proglycosidically linked disaccharide units. Such alterportion of disulfide bridges, such as IgG or albumin. ations lead to abnormal packing of the peptide Linkage of carbohydrates to proteins (glycation) chains, promoting excessive leakiness of the memalso appears to increase susceptibility of proteins to brane [15]. Basement membrane thickening and free radical attack [21]. Interestingly, arachidonic leakiness contribute to abnormalities in the microacid, which is a precursor of prostaglandins, concirculation that are now well described. tains a number of methylene interrupted double bonds, which are particularly prone to free radical NONENZYMATICGLYCOSYLATION attack (see below). In the presence of hyperglycemia, glucose chemiThe possibility of increased free radical activity cally attaches to protein, nonenzymatically, to form in diabetes can be inferred both from studies of flua stable product (ketoamine). In the presence of orescent proteins and by examining the products of prolonged hyperglycemia, further reactions occur lipid peroxidation. Several groups have reported very slowly in long-lived tissue proteins, leading to increased lipid peroxidation in diabetes [22,23], and production of advanced glycosylation end-products we have shown increased diene conjugated lipids (a (AGE) [16]. AGE are resistant to degradation, the process characteristically induced by free radicals) final reaction being irreversible. AGE can be iden- to be associated with retinopathy [24]. Both free tified by a characteristic brown pigment fluoresradicals and their lipid hydroperoxide products cence (“protein browning”) [17]. Studies have con- have been reported to be directly cytotoxic to vasfirmed a relationship between collagen browning cular endothelial cells [25]. In addition, they stimuand microangiopathy [Ml. late cyclooxygenase and hence prostaglandin synthesis while at the same time inhibiting prostacyFREERADICALACTIVITY,ANTIOXIDANTSTATUS, clin production [25-2’71. AND THE POLYOLPATHWAY Why should diabetic patients have increased free radical activity? Normally, free radicals are rapidly There are alternative mechanisms that may be eliminated by antioxidants, such as reduced glutainvolved in inducing protein fluorescence in longthione and vitamins C and E. Diabetic patients, lived nonenzymatically glycosylated proteins. Until however, have lower concentrations of these norrecently, fluorescence had been described only in mally protective compounds [28-311. This diminproteins with a long half-life, such as collagen. Simiished antioxidant reserve may be due to competilar changes in short-lived proteins could also have tion for reduced nicotinamide adenine dinucleotide pathogenic importance. Albumin and immunoglobulins are deposited on endothelial cell basement phosphate (NADPH), which is a cofactor required membrane in diabetic patients. We have recently to recycle the oxidized free radical scavengers back investigated the fluorescence of serum proteins in to their effective reduced form (“redox cycling”). 6A-68S
June 24, 1991
The American Journal of Medicine
Volume 90 (suppl 6A)
I
I
G,ucose
7%)
NADPH
)
Sorbito,
ft
NADP+
)
Fr”ctose
NADH+
NAD
Figure 1. Sorbitol pathway.
NADPH is produced from the hexose monophosphate shunt, and one source of competition for NADPH comes from the sorbitol pathway (Figure 1). This pathway, converting glucose to sorbitol, has been implicated in the pathogenesis of many diabetic complications. In the presence of intracellular hyperglycemia, glucose is converted by the enzyme aldose reductase to sorbitol. This is further degraded by sorbitol dehydrogenase to fructose (this is the rate-limiting step). During conversion of glucose to sorbitol, NADPH is consumed. The mechanism of action of aldose reductase inhibitors in the context of prevention and treatment of complications is still unclear and is probably unrelated to osmotic stress. It has been proposed that increased flux through the polyol pathway, which is preventable by aldose reductase inhibitors, causes increased NADPH utilization and leaves the tissue less able to resist oxidative stress [32].
ENDOTHELIALCELLS,HEMOSTASIS,AND PROSTAGLANDINS Endothelial cells not only produce proteoglycans and have aldose reductase activity, but they are also responsible for the formation of Factor VIII, plasminogen activator, and prostacyclin. Studies of these various factors implicate endothelial cell dysfunction in the pathogenesis of microangiopathy. Factor VIII may be increased in patients with retinopathy [33], thus promoting microthrombus formation. Prostacyclin is a powerful vasodilator and inhibits platelet aggregation. Studies have shown decreased prostacyclin production from vascular walls in animal models of diabetes [34] and reduced circulating prostacyclin concentrations in diabetic patients [30]. Plasminogen activator, which converts plasminogen to plasmin and acts to promote fibrinolysis, has also been reported to be low in diabetes [35]. Other studies have also suggested reduced fibrinolysis in diabetes [36,37]. Other hemostatic abnormalities are well described, particularly those of platelet function. Thromboxane Aa is produced in the platelets and is a powerful vasoconstrictor and promotes further platelet aggregation. An imbalance between thromboxane and prostacyclin, in favor of thromboxane, June 24,
is described in diabetes. Thromboxane Aa release is increased in platelets taken from patients with vascular complications [38] even when isolated from diabetic plasma [39]. This suggests that there is an intrinsic abnormality of control via phospholipase Aa at the membrane of these platelets, which leads to increased activity and the tendency to increased formation of thromboxanes [40]. Interestingly, increased phospholipase A2 activity has been linked to lowered levels of intraplatelet vitamin E (an important antioxidant) and may be a consequence of increased membrane lipid peroxidation [31]. Platelets also carry other powerful mediators of the microcirculation, such as serotonin and plateletderived growth factors [41,42]. Increased platelet aggregation in diabetic patients can be detected in vitro by measuring platelet response to aggregating agents or in vivo by measuring proteins released from the platelet on aggregation, such as pthromboglobulin and platelet factor 4. Both techniques have detected increased platelet reactivity in diabetic microangiopathy [41-441. Data from our own unit illustrate this as well (Table I). I 1 TABLE
I
In Vivo Platelet Aggregation Parameters (Mean + SD) in a Group of Insulin-Dependent Diabetics with Varying Degrees of Diabetic Retinopathy Versus Matched Controls Diabetic Complications
BTG (rig/ml)
Control (n = 26)
None (n = 23)
18i. 14*
49 + 42
62 ? 32
72 ? 46
61 k 42
65 c2 93 1.7 1.3
5.0 60 fIt 67 3.5 105+ 24 23+18 2.5-t 0.4 11Ii 13
2.2 6ot64 c 1.8 ‘$3! ;;
2.79 62 i2 16 2.4 102.4+ 22
2.5: 0.5 10-k 16
2.52 0.6 122 15
;;w;y
2.4 1422 2.4 11
AT Ill MicroAgg (%) Fib (g/L) FPA(rig/ml)
892 18 12 2 lit
2.2+ 0.4 728
104 + 22
16 ? 114 2.5t 0.7 142 14
Mild (n = 18)
Severe (n = 55)
All
ZO? 16
All = all diabetic groups considered together; BTG = P-thromboglobulin; PF4 = platelet factor 4; MicroAgg = mrcroaggregates; Fib = fibrinogen; FPA = frbrinopeptide A; AT III = antithrombrn III. *p
H.
BARNEIT,
B.SC.,
M.D.,
F.R.c.P.,
Birmingham,
United Kingdom
Major susceptibility factors for diabetic microangiopathy include duration of disease and probably quality of metabolic control. The mechanism of development of microangiopathy is incompletely understood but appears to involve functional abnormalities within the microcirculation, enhanced glucose metabolism, hemostatic abnormality, and genetic susceptibility. This article reviews the factors believed to be involved in pathogenesis and attempts to draw these together by suggesting a sequence of pathogenic interactions that could result. in the microvascular changes seen in susceptible target organs. Possibilities for therapeutic intervention based on these pathogenic mechanisms are discussed. A small pilot trial of an oral hypoglycemic agent, gliclazide, is reported, providing evidence for a specific action of this drug on thromboxane synthesis and platelet aggregation. This is independent of glycemic control and may in part be mediated by a fall in lipid peroxides.
From the University of Birmingham, Birmmgham, United Kingdom. Requests for reprints should be addressed to Anthony H. Barnett. M.D., Department of Medicine. East Birmingham Hospital, Bordesley Green East, Birmingham B9 5ST, United Kingdom.
D
iabetic microangiopathy accounts for much of the morbidity and mortality of diabetes. Our understanding of its pathogenesis is still not complete, although recent research has cast new light on the problem and has suggested possible therapeutic options. The major susceptibility factors for microangiopathy are duration of disease and probably quality of metabolic control [l-4]. These associations do not, however, explain the mechanism of development, which probably reflects many factors, including functional abnormalities within the microcirculation, enhanced glucose metabolism via different pathways of nonglycolytic glucose metabolism, hemostatic abnormality, and genetic susceptibility [51.
GENETICSUSCEPTIBILITY Although microvascular disease cannot occur without the metabolic abnormality of diabetes, the time of appearance and severity of complications show wide individual variation. In insulin-dependent diabetes, about one-third of subjects develop severe microangiopathic complications with poor long-term prognosis, whereas the remainder tend to run a more benign course. Indeed, some patients never develop clinically evident complications, even after 30-40 years of the disease. Both identicaltwin studies [6] and studies looking at immunogenetic associations with retinopathy [7] have suggested a genetic component. Our own group has described associations between diabetic retinopathy and various human leukocyte antigen (HLA)encoded markers on chromosome 6, such as HLADR4 and an allotype of the fourth component of complement C4B3 [8], and an association with a phenotype of the immunoglobulin heavy chain markers on chromosome 14, for both insulin-dependent [9] and non-insulin-dependent diabetes [lo]. Genetic markers may be used to predict which diabetic patients will be most susceptible to complications, but they cannot operate in the absence of diabetes itself. They may act as modulators that determine when damage will occur, or they may affect the end-organ response to abnormal metabolism. Variation in genetic susceptibility may explain, at least in part, the variable rate of development of complications in patients with apparently similar blood glucose control.
June 24, 1991
The Amencan Journal of Medicine
Volume 90 (suppl 6A)
6A-67S
SYMPOSIUM
ON GLICWIDE
/ BARNETT
MECHANISMOF DEVELOPMENTOF MICROANGIOPATHY
diabetic patients. Interestingly, serum immunoglobulin G (IgG) from diabetics had fluorescence characteristics similar to those of the long-lived tisThe mechanism of development of microangiopasue proteins, and the strength of the fluorescence thy is still incompletely understood. However, it was related to the degree of retinopathy [19]. This appears to involve development of capillary basefluorescence is similar in those proteins whose ment membrane thickening, nonenzymatic glycoamino groups have been modified by reaction with sylation of long-lived tissue proteins, abnormalities malonylaldehyde or other peroxide degradation of endothelial cells and platelets, and prostaglandin products to form aminoimino propene adducts [ZO]. abnormalities, and perhaps increased damage by These reactive aldehydes are formed during the free radicals. metabolism of prostaglandins and by lipid peroxidaCAPILLARYBASEMENTMEMBRANETHICKENING tion, possibly as a result of free radical attack. Free radicals are violently reactive chemical speCapillary basement membrane thickening is the cies with an unpaired electron in their structure. histologic hallmark of microangiopathy. Type IV They are produced during the course of normal collagen seems to be the major structural element metabolism and are usually eliminated rapidly by and heparan sulfate the major proteoglycan, toantioxidants and other enzymes. In some diseases, gether with laminin and fibronectin [ll-131. Hepaparticularly where inflammation or ischemia are ran sulfate is produced by the endothelial cell and is fundamental (e.g., rheumatoid arthritis, diabetes), highly negatively charged. It produces a regular there is evidence of excessive free radical activity. latticework of anionic sites that hinder the filtration Polyunsaturated fatty acids, which are constituents of negatively charged proteins such as albumin [14]. of cell membranes, are particularly susceptible to In diabetes there is impaired synthesis of proteofree radical attack, as are both tissue and extracelglycans and an increase in hydroxylysine and its lular proteins-particularly those with a large proglycosidically linked disaccharide units. Such alterportion of disulfide bridges, such as IgG or albumin. ations lead to abnormal packing of the peptide Linkage of carbohydrates to proteins (glycation) chains, promoting excessive leakiness of the memalso appears to increase susceptibility of proteins to brane [15]. Basement membrane thickening and free radical attack [21]. Interestingly, arachidonic leakiness contribute to abnormalities in the microacid, which is a precursor of prostaglandins, concirculation that are now well described. tains a number of methylene interrupted double bonds, which are particularly prone to free radical NONENZYMATICGLYCOSYLATION attack (see below). In the presence of hyperglycemia, glucose chemiThe possibility of increased free radical activity cally attaches to protein, nonenzymatically, to form in diabetes can be inferred both from studies of flua stable product (ketoamine). In the presence of orescent proteins and by examining the products of prolonged hyperglycemia, further reactions occur lipid peroxidation. Several groups have reported very slowly in long-lived tissue proteins, leading to increased lipid peroxidation in diabetes [22,23], and production of advanced glycosylation end-products we have shown increased diene conjugated lipids (a (AGE) [16]. AGE are resistant to degradation, the process characteristically induced by free radicals) final reaction being irreversible. AGE can be iden- to be associated with retinopathy [24]. Both free tified by a characteristic brown pigment fluoresradicals and their lipid hydroperoxide products cence (“protein browning”) [17]. Studies have con- have been reported to be directly cytotoxic to vasfirmed a relationship between collagen browning cular endothelial cells [25]. In addition, they stimuand microangiopathy [Ml. late cyclooxygenase and hence prostaglandin synthesis while at the same time inhibiting prostacyFREERADICALACTIVITY,ANTIOXIDANTSTATUS, clin production [25-2’71. AND THE POLYOLPATHWAY Why should diabetic patients have increased free radical activity? Normally, free radicals are rapidly There are alternative mechanisms that may be eliminated by antioxidants, such as reduced glutainvolved in inducing protein fluorescence in longthione and vitamins C and E. Diabetic patients, lived nonenzymatically glycosylated proteins. Until however, have lower concentrations of these norrecently, fluorescence had been described only in mally protective compounds [28-311. This diminproteins with a long half-life, such as collagen. Simiished antioxidant reserve may be due to competilar changes in short-lived proteins could also have tion for reduced nicotinamide adenine dinucleotide pathogenic importance. Albumin and immunoglobulins are deposited on endothelial cell basement phosphate (NADPH), which is a cofactor required membrane in diabetic patients. We have recently to recycle the oxidized free radical scavengers back investigated the fluorescence of serum proteins in to their effective reduced form (“redox cycling”). 6A-68S
June 24, 1991
The American Journal of Medicine
Volume 90 (suppl 6A)
I
I
G,ucose
7%)
NADPH
)
Sorbito,
ft
NADP+
)
Fr”ctose
NADH+
NAD
Figure 1. Sorbitol pathway.
NADPH is produced from the hexose monophosphate shunt, and one source of competition for NADPH comes from the sorbitol pathway (Figure 1). This pathway, converting glucose to sorbitol, has been implicated in the pathogenesis of many diabetic complications. In the presence of intracellular hyperglycemia, glucose is converted by the enzyme aldose reductase to sorbitol. This is further degraded by sorbitol dehydrogenase to fructose (this is the rate-limiting step). During conversion of glucose to sorbitol, NADPH is consumed. The mechanism of action of aldose reductase inhibitors in the context of prevention and treatment of complications is still unclear and is probably unrelated to osmotic stress. It has been proposed that increased flux through the polyol pathway, which is preventable by aldose reductase inhibitors, causes increased NADPH utilization and leaves the tissue less able to resist oxidative stress [32].
ENDOTHELIALCELLS,HEMOSTASIS,AND PROSTAGLANDINS Endothelial cells not only produce proteoglycans and have aldose reductase activity, but they are also responsible for the formation of Factor VIII, plasminogen activator, and prostacyclin. Studies of these various factors implicate endothelial cell dysfunction in the pathogenesis of microangiopathy. Factor VIII may be increased in patients with retinopathy [33], thus promoting microthrombus formation. Prostacyclin is a powerful vasodilator and inhibits platelet aggregation. Studies have shown decreased prostacyclin production from vascular walls in animal models of diabetes [34] and reduced circulating prostacyclin concentrations in diabetic patients [30]. Plasminogen activator, which converts plasminogen to plasmin and acts to promote fibrinolysis, has also been reported to be low in diabetes [35]. Other studies have also suggested reduced fibrinolysis in diabetes [36,37]. Other hemostatic abnormalities are well described, particularly those of platelet function. Thromboxane Aa is produced in the platelets and is a powerful vasoconstrictor and promotes further platelet aggregation. An imbalance between thromboxane and prostacyclin, in favor of thromboxane, June 24,
is described in diabetes. Thromboxane Aa release is increased in platelets taken from patients with vascular complications [38] even when isolated from diabetic plasma [39]. This suggests that there is an intrinsic abnormality of control via phospholipase Aa at the membrane of these platelets, which leads to increased activity and the tendency to increased formation of thromboxanes [40]. Interestingly, increased phospholipase A2 activity has been linked to lowered levels of intraplatelet vitamin E (an important antioxidant) and may be a consequence of increased membrane lipid peroxidation [31]. Platelets also carry other powerful mediators of the microcirculation, such as serotonin and plateletderived growth factors [41,42]. Increased platelet aggregation in diabetic patients can be detected in vitro by measuring platelet response to aggregating agents or in vivo by measuring proteins released from the platelet on aggregation, such as pthromboglobulin and platelet factor 4. Both techniques have detected increased platelet reactivity in diabetic microangiopathy [41-441. Data from our own unit illustrate this as well (Table I). I 1 TABLE
I
In Vivo Platelet Aggregation Parameters (Mean + SD) in a Group of Insulin-Dependent Diabetics with Varying Degrees of Diabetic Retinopathy Versus Matched Controls Diabetic Complications
BTG (rig/ml)
Control (n = 26)
None (n = 23)
18i. 14*
49 + 42
62 ? 32
72 ? 46
61 k 42
65 c2 93 1.7 1.3
5.0 60 fIt 67 3.5 105+ 24 23+18 2.5-t 0.4 11Ii 13
2.2 6ot64 c 1.8 ‘$3! ;;
2.79 62 i2 16 2.4 102.4+ 22
2.5: 0.5 10-k 16
2.52 0.6 122 15
;;w;y
2.4 1422 2.4 11
AT Ill MicroAgg (%) Fib (g/L) FPA(rig/ml)
892 18 12 2 lit
2.2+ 0.4 728
104 + 22
16 ? 114 2.5t 0.7 142 14
Mild (n = 18)
Severe (n = 55)
All
ZO? 16
All = all diabetic groups considered together; BTG = P-thromboglobulin; PF4 = platelet factor 4; MicroAgg = mrcroaggregates; Fib = fibrinogen; FPA = frbrinopeptide A; AT III = antithrombrn III. *p
