`Vascular Thrombosis in Type II
`Diabetes Mellitus
`
`JOHN A. COLWELL
`
`One prevailing point of view is that many indi-
`
`viduals with established type II diabetes have
`gone through years of impairment of glucose
`tolerance and hyperinsulinism before the ap-
`pearance of fasting hyperglycemia (1,2). Peripheral insu-
`lin resistance plus a pancreatic p-cell insulin secretory
`defect presumably wage a battle over the years that
`results ultimately in clinically apparent type II diabetes
`(3). If this sequence of events is correct, many individuals
`with type II diabetes have had years of hyperinsulinism,
`in contrast to their age- and sex-matched control sub-
`jects. Further, there may be a disproportionate elevation
`of plasma proinsulin levels in individuals with IGT and
`frank diabetes (4,5).
`These individuals are at a high risk for cardiovascular
`events, and this increased risk does not appear to be
`completely explained by the association of IGT or type II
`diabetes with such classical risk factors as hypertension,
`hypercholesterolemia, or cigarette smoking (6). These
`observations have led to numerous studies directed at
`determining the pathogenesis of accelerated cardiovas-
`cular disease in diabetes; a daunting challenge, for
`atherosclerosis in nondiabetic individuals is an extraor-
`dinarily complex process (7). The early events appear to
`involve macrophage adherence to endothelium, followed
`by macrophage migration to the subendothelial space.
`Here, macrophages may be transformed into foam cells.
`
`From the Ralph H. Johnson Department of Veterans Affairs Medical Center,
`and the Division of Endocrinology, Diabetes, and Metabolism, the Medical
`University of South Carolina, Charleston, South Carolina.
`Address correspondence and reprint requests to John A. Colwell, M.D.,
`Ph.D., Division of Endocrinology, Diabetes, and Metabolism, Medical Univer-
`sity of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.
`Received for publication 1 October 1992 and accepted 7 October 1992.
`Type II, non-insulin-dependent diabetes mellitus; IGT, impaired glucose
`tolerance; HDL, high-density lipoprotein; VLDL, very-low-density lipoprotein;
`LDL, low-density lipoprotein; PAI-1, plasminogen activator inhibitor-1; t-PA,
`tissue-plasminogen activator; type I, insulin-dependent diabetes mellitus;
`DCCT, Diabetes Control and Complications Trial.
`
`A variety of growth factors and cytokines may be re-
`leased from damaged endothelium, macrophages, and
`smooth muscle cells. Platelets may adhere to the site of
`macrophage attachment and release thromboxane and
`growth factors. Smooth muscle cells may proliferate and
`migrate, accompanied by thrombus formation, vascular
`encroachment, and occlusion.
`In diabetes, added contributors to this process are
`numerous (8,9). Many investigators have focused on
`processes that may result from hyperglycemia—an em-
`inently logical (albeit elusive) approach. Provocative find-
`ings include subtle endothelial damage, which may
`accompany increased glucose concentrations in in vitro
`systems, effects of glycated lipoproteins on macrophage
`or endothelial function, an increased susceptibility of
`glycated lipoproteins to oxidation, a litany of effects of
`oxidized (glycoxidized?) lipoproteins on vascular cell
`biology, and increased vascular wall levels of irreversible
`advanced glycation products (8,9). Good evidence sug-
`gests that individuals with IGT and hyperinsulinism may
`have quantitative alterations of plasma lipoproteins that
`may contribute to accelerated atherosclerosis. Further, a
`constellation of plasma lipoprotein changes, which the-
`oretically would accelerate atherosclerosis, often are
`found in people with established type II diabetes, includ-
`ing an increase in plasma triglyceride levels and lowered
`HDL-cholesterol concentrations (10). The former may
`reflect increased VLDL production by the liver and/or a
`decrease in peripheral metabolism, and may be mani-
`fested in the ultracentrifuge by a population of small
`VLDLs and of intermediate-density lipoproteins. These
`lipoproteins may be particularly atherogenic. Although
`plasma LDL cholesterol levels may or may not be ele-
`vated, evidence is increasing that a family of small,
`dense LDL particles may be present in diabetes and
`potentiate atherosclerosis. Such changes also may be
`found in the insulin-resistance syndrome (syndrome X),
`which is observed often in individuals with IGT (11).
`To complicate matters, individuals with diabetes ap-
`
`DIABETES, VOL. 42, JANUARY 1993
`
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`Ex. 1048, p. 1 of 4
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`
`
`pear to have an increased tendency toward vascular
`thrombosis. Longitudinal data in individuals with diag-
`nosed type II diabetes indicate that major vascular
`events such as myocardial infarction and vascular death
`may occur at a rate of 5-7% per year, even in individuals
`who have had no known previous cardiovascular event
`(12). This extraordinarily high cardiovascular event rate is
`at least three to four times that seen in comparable
`individuals without diabetes. In addition to clinically rec-
`ognizable vascular events, individuals with type II diabe-
`tes often have unrecognized vascular occlusive disease
`of the coronary and peripheral vascular systems. The net
`effect of all of this is an increased recognition by diabe-
`tologists, cardiologists, vascular surgeons, epidemiolo-
`gists, and health professionals in general of the incred-
`ible toll that diabetes extracts from the large vessel
`system.
`A hypercoagulable state may exist in diabetes. For
`instance, subtle alterations of plasma levels of protein C
`and antithrombin III have been noted and may relate to
`glycemic regulation with insulin (9). It has long been
`recognized that the endothelial protein, von Willebrand
`Factor, may be found in excess in the plasma of diabetic
`subjects and may relate to the endothelial damage and
`increased platelet adhesiveness reported in diabetes. An
`avalanche of studies describes a variety of alterations of
`platelet function in diabetes (13,14). Clinical trial data in
`diabetic and nondiabetic subjects indicate that low-dose
`aspirin therapy to block the platelet release reaction is
`clearly indicated as a secondary prevention strategy
`(15). Further, this may prove to be a wise primary
`prevention approach in the general population (16). A
`suggestive finding is the elevation of plasma fibrinogen
`levels in diabetes, particularly in individuals with poor
`glycemic regulation. A physiological rationale for this
`finding has been provided by studies that show in-
`creased fibrinogen synthesis and turnover in diabetes,
`related to insulin deficiency and correctable by insulin
`administration (17,18).
`In recent years, attention has been directed to the
`fibrinolytic system in diabetes, with the expectation that
`alterations in the balance between fibrin deposition to
`form a clot and lysis of that clot could help explain
`thrombotic events in diabetes (19,20,21). The perspec-
`tive by Schneider et al. (this issue, p. 1-7) is an elegant
`review of original work from their laboratory and from
`other researchers who have been active in this rapidly
`changing field of investigation. Schneider et al., working
`in an exceptionally strong environment for research in
`diabetes and its complications at The Washington Uni-
`versity School of Medicine, have materially added to our
`knowledge of the role that alterations of the fibrinolytic
`system could play in accelerated atherosclerosis and
`thrombosis in diabetes mellitus. What is important and
`new about this work? It provides in vitro evidence to
`support the concept that insulin and proinsulin may
`accelerate vascular thrombosis via actions on the liver
`and on the endothelium. Specifically, when exposed to
`high concentrations of insulin, human hepatoma cells
`release increased amounts of PAI-1, related to an in-
`crease in PAI-1 mRNA expression, probably related to
`
`DIABETES, VOL. 42, JANUARY 1993
`
`J.A. COLWELL
`
`decreased degradation. By using porcine aortic endo-
`thelial cells, a good model for atherosclerosis in man,
`they found that insulin augmented endothelial PAI-1
`synthesis, probably by enhancing translation of PAI-1
`mRNA. Proinsulin, in concentrations near those that may
`be observed in type II diabetes, led to an increase in
`PAI-1 activity, even in the presence of a marked excess
`of insulin, indicating an insulin-independent pathway.
`PAI-1 mRNA expression and synthesis were increased
`concordantly. If these events are operative in vivo, they
`would support the concept that a diminution in clot lysis,
`mediated through inhibition of tissue plasminogen acti-
`vator by PAI-1, may contribute to thrombosis in diabetes.
`Other regulators of PAI-1 release have been shown in
`in vitro systems. Activated macrophages may release
`cytokines such as tumor necrosis factor and interleu-
`kin-1. These cytokines have been shown to suppress
`t-PA mRNA and to induce transcription of PAI-1 in
`endothelial cells (23,24). Lipoproteins also may affect this
`system. Endothelial production of PAI-1 is increased after
`incubation with VLDL (25), whereas LDL induces PAI-1
`synthesis in hepatocytes (26). Thus, critical interrelation-
`ships at the site of the atherosclerotic lesions may occur
`that involve not only insulin and proinsulin but also
`activated macrophages and altered lipoprotein metabo-
`lism in diabetes. Further research is needed in diabetic
`subjects to continue to unravel the precise sequence of
`events and involved mechanisms in this very complex
`system.
`What is the clinical implication of this work? Of course,
`it can be hazardous to transfer in vitro studies to clinical
`medicine, particularly in a disease as heterogeneous as
`diabetes mellitus. Nevertheless, the point of view that this
`system could be of importance is supported by numer-
`ous observations. Insulin resistance, coupled with in-
`creased plasma immunoreactive insulin levels, may be
`observed early in the natural history of type II diabetes,
`and these people appear to be at an increased risk for
`thrombotic events. Many studies have shown increased
`plasma PAI-1 levels in people with hyperinsulinism and in
`type II diabetes (19-21). As noted, plasma proinsulin/
`insulin ratios may be increased in people with IGT and in
`those with frank type II diabetes (4,5). Thus, one can
`visualize a scenario in which hyperinsulinemia and/or
`hyperproinsulinemia could contribute to vascular throm-
`bosis in type II diabetes via effects on endothelial and/or
`hepatic PAI-1 synthesis and release.
`On the other hand, many unexplained issues remain.
`As already noted, the pathogenesis of accelerated ath-
`erosclerosis in diabetes is incredibly complicated, and it
`is probably simplistic to speculate that an imbalance in
`one system, such as the fibrinolytic system, is the major
`contributor. Further, many conditions are associated with
`hyperinsulinism, such as obesity, certain forms of type II
`diabetes, acromegaly, and insulinoma, that do not ap-
`pear to have accelerated atherosclerosis. Insulin admin-
`istration to individuals with type II diabetes and marked
`elevations of fasting plasma glucose (i.e., >11.1 mM
`[200 mg/dl]) will usually improve the atherogenic lipid
`profile, protein glycation, and perhaps glycoxidation—
`processes that, at this stage of diabetes, may be more
`
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`
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`critical for accelerating the process of atherosclerosis
`than is the PA system. Evidence to date does not indicate
`an effect of insulin on plasma PAI-1 because levels are
`usually normal in type I patients on insulin (21) and do not
`rise with acute administration of insulin (19). Indeed, the
`hypothesis that insulin is atherogenic when administered
`to diabetic subjects remains controversial (27), and is not
`supported by available clinical trial data in type II dia-
`betic subjects (28).
`Proinsulin may be a very different matter. Many immu-
`noreactive insulin assays apparently cross-react to some
`degree with insulin. Therefore, studies that show hyper-
`insulinemia may have been measuring both hormones.
`Researchers with specific proinsulin assays report an
`increase in the ratio of proinsulin to insulin in type II
`diabetes (4,5). Further, proinsulin therapy in one clinical
`trial was associated with several cardiovascular events,
`so the trial was stopped. However, increased major
`vascular events are expected in a type II diabetic popu-
`lation, and unless one follows appropriate control groups
`simultaneously, one cannot be sure about the specific
`relationship to proinsulin therapy.
`What is the practicing physician who sees diabetic
`patients to conclude? Should he/she be concerned about
`the use of insulin for type II patients who have inadequate
`glycemic control on sulfonylureas? Will this lead to an
`increase in vascular events? What about insulin use in
`type I patients? Ongoing studies, such as the DCCT (30)
`and the UK Prospective Diabetes Trial (31), will give
`excellent information on the benefits and risks of various
`forms of therapy in diabetes. However, neither trial is
`directed at exploring the risks and benefits of the inten-
`sive use of insulin in type II patients with hyperglycemia,
`despite oral agents or standard insulin therapy. Recently,
`the Department of Veterans Affairs started a feasibility
`trial of standard insulin therapy versus intensive insulin
`treatment in such a population (12). It is only through
`clinical trials of this nature that informed choices can be
`made by physicians who treat diabetic patients. We
`urgently need additional studies in other stages of type II
`diabetes to be able to balance the benefits and risks of
`various forms of therapy in diabetes as regards cardio-
`vascular disease. Studies such as those by Schneider et
`al. (this issue, p. 1-7) add important new information to
`help us understand the pathogenesis of accelerated
`atherosclerosis and thrombosis in diabetes. From such
`an understanding, appropriate questions for clinical trials
`may be asked, and revised standards for clinical care
`should emerge.
`
`ACKNOWLEDGMENTS
`This work was supported by the Research Service of the
`Department of Veterans Affairs.
`
`REFERENCES
`1. Warram JH, Martin BC, Krolewski AS, Soeldner JS, Kahn CR: Slow
`glucose removal rate and hyperinsulinemia precede the develop-
`ment of type II diabetes in the offspring of diabetic parents. Ann
`Intern Med 113:909-15, 1990
`2. Haffner SM, Stern MP, Hazuda HP, Mitchell BD, Patterson JK:
`Cardiovscular risk factors in confirmed prediabetic individuals: does
`the clock for coronary heart disese start ticking before the onset of
`
`clinical diabetes? JAMA 263:2893-98, 1990
`3. Porte D Jr: Banting lecture 1990: p-cells in type II diabetes mellitus.
`Diabetes 40:166-80, 1991
`4. Ward WK, LaCava EC, Paquette TL, Beard JC, Wallum BJ, Porte D:
`Disproportionate elevation of immunoreactive proinsulin in type II
`(non-insulin-dependent) diabetes mellitus and in experimental insu-
`lin resistance. Diabetologia 30:698-702, 1987
`5. Nagi DK, Hendra TJ, Ryle AJ, Cooper TM, Temple RC, Clark PMS,
`Schneider AE, Hales CN, Yudkin JS: The relationships of concen-
`trations of insulin, intact proinsulin and 32-33 split proinsulin with
`cardiovascular risk factors in type II (non-insulin-dependent) dia-
`betic subjects. Diabetologia 33:532-37, 1990
`6. Consensus Panel: American Diabetes Association Consensus
`Statement: role of cardiovascular risk factors in prevention and
`treatment of macrovascular disease in diabetes. Diabetes Care
`12:573-79, 1989
`7. Ross R: The pathogenesis of atherosclerosis: an update. N Engl J
`Med 314:488-500, 1986
`8. Lopes-Virella MF (Ed.): Proceedings of the XIV International Diabe-
`tes Federation Satellite Symposium on Macrovascular Complica-
`tions of Diabetes Mellitus. Diabetes 41 (Suppl. 2)1:119, 1992
`9. Colwell JA, Lyons TJ, Klein RL, Lopes-Virella MF: New Concepts
`About the Pathogenesis of Atherosclerosis and Thrombosis in
`Diabetes Mellitus. In The Diabetic Foot. 5th ed. Levin MJ, Ed. St.
`Louis, MO, Mosby, 1993, p. 79-114
`10. Lopes-Virella MFL, Stone PG, Colwell JA: Serum high density
`lipoprotein in diabetic patients. Diabetologia 13:285-91, 1977
`11. Brunzell JD, Chair A: Lipoprotein pathophysiology and treatment. In
`Ellenberg and Rifkin's Diabetic Mellitus. Rifkin H, Porte D Jr, Eds.
`New York, Elsevier, 1990
`12. Abraira C, Emanuele N, Colwell J, Henderson W, Comstock J, Levin
`S, Nuttal F, Sawin C, and the VA Cooperative Study Group:
`Glycemic control and complications in type II diabetes: design of a
`feasibility trial. Diabetes Care 15:1560-71, 1992
`13. Colwell JA, Lopes-Virella MF, Winocour PD, Halushka PV: New
`Concepts about the Pathogenesis of Atherosclerosis in Diabetes
`Mellitus. In The Diabetic Foot. 4th ed. Levin ME, O'Neal LW, Eds. St.
`Louis, MO, Mosby, 1988, p. 51-70
`14. Colwell JA, Winocour PD, Lopes-Virella MF: Platelet function and
`platelet interactions in atherosclerosis and diabetes mellitus. In
`Diabetes Mellitus: Theory and Practice. Rifkin H, Porte D, Eds. New
`York, Elsevier, 1989, p. 249-56
`15. Antiplatelet Trialists' Collaboration: Secondary prevention of vascu-
`lar disease by prolonged antiplatelet treatment. Br Med J 296:320-
`31, 1988
`16. Steering Committee of the Physicians' Health Study Research
`Group: Final report on the aspirin component of the ongoing
`Physicians' Health Study. N EnglJ Med 321:129-35, 1989
`17. Jones RL, Peterson CM: The fluid phase of coagulation and the
`accelerated atherosclerosis of diabetes mellitus. Diabetes 30
`(Suppl. 2):33-38, 1981
`18. De Feo P, Gaisano MG, Haymond MW: Differential effects of insulin
`deficiency on albumin and fibrinogen synthesis in humans. J Clin
`Invest 88:833-40, 1991
`19. Juhan-Vague I, Alessi MC, Vague P: Increased plasma plasmino-
`gen activator inhibitor 1 levels: a possible link between insulin
`resistance and atherothrombosis. Diabetologia 34:457-62, 1991
`20. Gough SCL, Grant PJ: The fibrinolytic system in diabetes mellitus.
`Diabetic Med 8:898-905, 1991
`21. Walmsley D, Hampton KK, Grant PJ: Contrasting fibrinolytic re-
`sponses in type I (insulin dependent) and type II (non-insulin
`dependent) diabetes. Diabetic Med 8:954-59, 1991
`23. van Hindsberg VWM, Kooistra T, van den Berg EA, Princen HMG,
`Fiers W, Emeis JJ: Tumour necrosis factor increases the production
`of plasminogen activator inhibitor in human endothelial cells in vitro
`and in rats in vivo. Blood 72:1467-73, 1988
`24. Emeis JJ, Kooistra T: Interleukin 1 and lipopolysaccharide induce an
`inhibitor of tissue-type plasminogen activator in vivo and in cultured
`endothelial cells. J Exp Med 163:1260-66, 1986
`25. Stiko-Rahm A, Wiman B, Hamsten A, Nilsson J: Secretion of
`plasminogen activator inhibitor 1 from cultured human umbilical vein
`endothelial cells is induced by very low density lipoprotein. Athero-
`sclerosis 10:1067-73, 1990
`26. Latron Y, Alessi MC, Anfosso F, Nalbone G, Lafont H, Portugal H,
`Juhan-Vague I: Effect of low density lipoproteins on secretion of
`plasminogen activator inhibitor (PAI-1) by human endothelial cells
`and hepatoma cells. Fibrinolysis 4 (Suppl. 2):82-83, 1990
`27. Colwell JA, Lopes-Virella ML, Mayfield R, Sens D (Eds.): Workshop
`on insulin and atherogenesis. MetabolisnT\2 (Suppl. 1):1—91.1985
`28. University Group Diabetes Program: A study of the effects of
`hypoglycemic agents on vascular complications in patients with
`adult-onset diabetes. II. Mortality results. Diabetes 19 (Suppl. 2):
`789-830, 1970
`
`10
`
`DIABETES, VOL. 42, JANUARY 1993
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`IPR2015-01496
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`Ex. 1048, p. 3 of 4
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`
`
`29. Galloway JA, Hooper SA, Spradlin CT, Howey DC, Frank BH,
`Bowsher RR, Anderson JH: Biosynthetic human pronsulin: Review of
`chemistry, in vitro and in vivo receptor binding, animal and human
`pharmacology studies, and clinical trial experience. Diabetes Care
`15:666-92, 1992
`
`30. The DCCT Research Group: The Diabetes Control and Complica-
`tions Trial (DCCT): update. Diabetes Care 13:427-33, 1990
`31. UK Prospective Diabetes Study Group: UK Prospective Diabetes
`Study (UKPDS). VIII. Study design, progress and performance.
`Diabetologia 34:877-90, 1991
`
`J.A. COLWELL
`
`DIABETES, VOL. 42, JANUARY 1993
`
`11
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`IPR2015-01496
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`Ex. 1048, p. 4 of 4
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