Joseph R. Williamson

Hyperglycemic Pseudohypoxia and Diabetic Complications

Vasodilation and increased blood flow are characteristic early vascular responses to acute hyperglycemia and tissue hypoxia. In hypoxic tissues these vascular changes are linked to metabolic imbalances associated with impaired oxidation of NADH to NAD+ and the resulting increased ratio of NADH/NAD+. In hyperglycemic tissues these vascular changes also are linked to an increased ratio of NADH/NAD+, in this case because of an increased rate of reduction of NAD+ to NADH. Several lines of evidence support the likelihood that the increased cytosolic ratio of free NADH/NAD+ caused by hyperglycemia, referred to as pseudohypoxia because tissue partial pressure oxygen is normal, is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an important role in the pathogenesis of diabetic complications. These effects of hypoxia and hyperglycemia-induced pseudohypoxia on vascular and neural function are mediated by a branching cascade of imbalances in lipid metabolism, increased production of superoxide anion, and possibly increased nitric oxide formation.

Selective inhibition of the inducible nitric oxide synthase by aminoguanidine

Abstract Overproduction of the free radical nitric oxide (NO) has been implicated in the pathogenesis of a variety of inflammatory and immunologically mediated diseases as well as complications of diabetes. In the present study we have demonstrated that aminoguanidine selectively inhibits the cytokine-inducible isoform of NO synthase which appears to be responsible for the excess production of NO linked to these disease states. By using organ, cell and enzyme-based measurements we have shown that aminoguanidine is equipotent to NG-monomethyl-L-arginine (L-NMA) as an inhibitor of the cytokine-induced isoform of NO synthase but is 10 to 100-fold less potent as an inhibitor of the constitutive isoform. Thus, aminoguanidine may be useful as a selective inhibitor of the inducible NO synthase in the treatment of disease states characterized by the pathological overproduction of NO.

Aminoguanidine, a Novel Inhibitor of Nitric Oxide Formation, Prevents Diabetic Vascular Dysfunction

Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO·) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 β-induced 1) nitrite formation (an oxidation product of NO·) and cGMP accumulation by the rat β-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is ∼ 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO· in the pathogenesis of diabetic vascular complications.

Prevention of Diabetic Vascular Dysfunction by Guanidines: Inhibition of Nitric Oxide Synthase Versus Advanced Glycation End-Product Formation

This study was undertaken to compare the ability of two guanidine compounds (aminoguanidine and methylguanidine), with different in vitro effects on NO synthase activity and AGE formation, to inhibit diabetic vascular dysfunction developing early after the onset of diabetes. In rats with STZ-induced diabetes of 5-wk duration, regional vascular [125I]albumin permeation was increased about two- to threefold in ocular tissues, sciatic nerve, and aorta; in general, both guanidine compounds normalized albumin permeation in diabetic rats without affecting it in controls. Methylguanidine was only ∼7% as effective as aminoguanidine as an inhibitor of AGE formation from L-lysine and G6P; both compounds were poor inhibitors of AR. Methylguanidine was ∼1–5% as potent as aminoguanidine and L-NMMA as an inhibitor of the cytokine- and endotoxin-inducible isoform of NO synthase. In contrast, the potency of methylguanidine as an inhibitor of the constitutive isoform of NO synthase was comparable to that of aminoguanidine, and both guanidine compounds were much less effective than L-NMMA. These observations suggest a role for a relative or absolute increase in NO production in the pathogenesis of early diabetic vascular dysfunction and raise the possibility that inhibition of diabetic vascular functional changes by aminoguanidine may reflect inhibition of NO synthase activity rather than, or in addition to, prevention of AGE formation.

Prevention of Hemodynamic and Vascular Albumin Filtration Changes in Diabetic Rats by Aldose Reductase Inhibitors

This study investigated hemodynamic changes in diabetic rats and their relationship to changes in vascular albumin permeation and increased metabolism of glucose to sorbitol. The effects of 6 wk of streptozocin-induced diabetes and three structurally different inhibitors of aldose reductase were examined on 1) regional blood flow (assessed with 15-μm 85Sr-labeled microspheres) and vascular permeation by 125I-Iabeled bovine serum albumin (BSA) and 2) glomerular filtration rate (assessed by plasma clearance of 57Co-labeled EDTA) and urinary albumin excretion (determined by radial immunodiffusion assay). In diabetic rats, blood flow was significantly increased in ocular tissues (anterior uvea, posterior uvea, retina, and optic nerve), sciatic nerve, kidney, new granulation tissue, cecum, and brain. 125I-BSA permeation was increased in all of these tissues except brain. Glomerular filtration rate and 24-h urinary albumin excretion were increased 2- and 29-fold, respectively, in diabetic rats. All three aldose reductase inhibitors completely prevented or markedly reduced these hemodynamic and vascular filtration changes and increases in tissue sorbitol levels in the anterior uvea, posterior uvea, retina, sciatic nerve, and granulation tissue. These observations indicate that early diabetes-induced hemodynamic changes and increased vascular albumin permeation and urinary albumin excretion are aldose reductase-linked phenomena. Discordant effects of aldose reductase inhibitors on blood flow and vascular albumin permeation in some tissues suggest that increased vascular albumin permeation is not entirely attributable to hemodynamic changes. We hypothesize that 1) increases in blood flow may reflect impaired contractile function of smooth muscle cells in resistance arterioles and 2) increases in vascular 125I-BSA permeation and urinary albumin excretion reflect impaired vascular barrier functional integrity in addition to increased hydraulic conductance secondary to microvascular hypertension associated with decreased vascular resistance.

Current status of capillary basement-membrane disease in diabetes mellitus.

(All are verbatim summaries) Siperstein. M. D.;-linger. R. H.; and Madison, L. L. (Dept. of Intern. Med., Univ. of Texas Southwestern Med. Sch., and VA Hosp. , Dallas, Tx.): STUDIES OF MUSCLE CAPILLARY BASEMENT MEMBRANES IN NORMAL SUBJECTS, DIABETIC AND PREDIABETIC PATIENTS. J. Clin. Invest. 47:1968, 1973. A technique is described for the measurement of muscle capillary basement membranes by electron microscopic examination of needle biopsies of the quadriceps muscle. With this procedure it has been possible to obtain an objective evaluation of the significance of capillary basement membrane hypertrophy in diabetic microangiopathy. The results of such studies of muscle capillary basement membrane thickness in 50 normal, 5 1 diabetic, and 30 prediabetic patients have demonstrated the following. First, that the average capillary basement membrane width of diabetic patients is over twice that of normal subjects; moreover, such basement membrane thickening is a very constant finding among overtly diabetic patients, in that approximately 98 per cent of individual diabetic subjects demonstrated this lesion. The degree of basement membrane thickening in diabetic patients is, however, unrelated to age, weight, severity, or duration of diabetes. Second, capillary basement membrane hypertrophy has been found in approximately 50 per cent of patients who are genetically prediabetic but who have not yet demonstrated evidence of the manifest carbohydrate disturbances of diabetes mellitus. Third, in contrast to the results obtained in genetically diabetic patients, subjects with severe hyperglycemia due to causes other than genetic diabetes only infrequently show basement membrane hyper-

Experimental allergic encephalomyelitis in the rat is inhibited by aminoguanidine, an inhibitor of nitric oxide synthase

This study assessed the role of de novo nitric oxide (NO) production in the pathogenesis of experimental allergic encephalomyelitis (EAE) by using aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS), which preferentially inhibits the cytokine- and endotoxin-inducible isoform of NOS versus the constitutive isoforms consisting of endothelial and neuronal NOS. The maximum clinical severity of EAE and the duration of illness were significantly reduced or totally inhibited by twice daily subcutaneous injection of 100 mg/kg body weight AG. Histochemical staining for NADPH diaphorase, which detects enzymatic activity of NOS, revealed positive reactivity in untreated EAE rats both in parenchymal blood vessel walls and in anterior horn cell neurons, while normal rats and rats with EAE treated with AG showed predominantly the neuronal positivity. Moreover, this NADPH staining pattern was further supported by the immunohistochemical findings that endothelial NOS (eNOS) expression was increased in blood vessels in the inflamed lesions of untreated EAE rats and that inducible NOS (iNOS) was detected in some inflammatory cells, while treatment with AG could significantly reduce both iNOS and eNOS production. These results suggest that: (i) both iNOS and eNOS are upregulated in inflamed areas of the rat central nervous system in EAE; (ii) increased NO production plays a role in the development of clinical signs in EAE; and (iii) selective inhibitors of iNOS and/or eNOS may have therapeutic potential for the treatment of certain autoimmune diseases.

Increased Vascular Permeability in Spontaneously Diabetic BB/W Rats and in Rats With Mild Versus Severe Streptozocin-Induced Diabetes: Prevention by Aldose Reductase Inhibitors and Castration

125I-labeled albumin permeation (IAP) has been assessed in various tissues in spontaneously diabetic insulin-dependent female BB/W rats and in male Sprague-Dawley rats with severe or mild forms of streptozocin-induced diabetes (SS-D and MS-D, respectively). In BB/W diabetic rats and in rats with SS-D, indices of IAP were significantly increased in tissues and vessels predisposed to diabetic vascular disease in humans, including the eyes (anterior uvea, posterior uvea, and retina), sciatic nerve, aorta, kidney, and new vessels formed after induction of diabetes. No evidence of increased IAP was observed in heart, brain, testes, or skeletal muscle in BB/W or SS-D rats. In MS-D rats, indices of IAP were increased only in the kidney and in new vessels formed after the onset of diabetes. Marked tissue differences were observed in the effects of two structurally different aldose reductase inhibitors (sorbinil and tolrestat) and of castration on diabetes-induced increases in IAP and in tissue levels of polyols in SS-D rats. Both aldose reductase inhibitors and castration completely prevented diabetes-induced increases in IAP in new vessels and in sciatic nerve in BB/W and SS-D rats. Both aldose reductase inhibitors also markedly decreased IAP in the anterior uvea (∼85%), posterior uvea (∼65-75%), retina (∼65-70%), and kidney (∼70-100%); castration reduced IAP in the anterior uvea (∼55%), kidney (∼50%), and retina (∼30%) but had no effect on the posterior uvea. The diabetes-induced increases in IAP in the aorta were reduced only slightly (∼20%) by aldose reductase inhibitors and castration. In SS-D rats, tissue polyol levels were markedly increased in sciatic nerve, retina, anterior uvea, and posterior uvea but not in the aorta. Castration significantly reduced polyol levels only in the sciatic nerve and retina, whereas both aldose reductase inhibitors markedly decreased polyol levels in anterior and posterior uvea and in sciatic nerve and retina. These observations demonstrate that 1) the most characteristic functional alteration in vessels in human diabetics, i.e., increased vascular permeability, is evident in rats with diabetes of only 3 wk duration; 2) the most marked increases in IAP in the diabetic rat occur in vessels associated with clinically significant vascular disease in human diabetics; 3) even very mild diabetes is associated with increased IAP in the kidney and in new vessels induced by angiogenesis in the diabetic milieu; 4) diabetes-induced increases in IAP in all tissues examined are aldose reductase-linked phenomena and, with the exception of the posterior uvea, are modulated by sex steroids; and 5) the extent to which diabetes-induced increases in IAP are modulated by sex steroids and reduced or completely prevented by aldose reductase inhibitors varies markedly in different vascular beds.

Differences in pericyte contractile function in rat cardiac and skeletal muscle microvasculatures

Abstract In order to evaluate contractile activity of pericytes, rat hearts and hindlimbs were perfused separately with angiotensin (1 μg/ml), norepinephrine (1 μg/ml), or vasopressin (10 −3 U/ml) for 3 min, fixed by perfusion, then processed for electron microscopy and morphometry. An electronic planimeter was employed to quantify configurational alterations (buckling) of endothelium between pericyte processes which provided an index of pericyte contraction (PCI). Inherent in this technique is the assumption that contracting pericytes distort and convolute endothelial cell membranes (increasing the PCI) abutting pericytes, but do not affect endothelial configuration elsewhere around the capillary circumference. PCI values were virtually identical in control hearts and skeletal muscle and in low-flow hindlimb perfusions (101.2 ± 0.3% (SD), 101.2 ± 0.7%, and 102.3 ± 0.8%, respectively). All three vasoactive agents increased perfusion pressure significantly in both hearts and hindlimbs with one exception (norepinephrine produced a slight pressure drop in hearts). While perfusion with vasoactive agents had no effect on the cardiac PCI, skeletal PCI values were markedly elevated for all three vasoactive agents (124.1 ± 8.2, 118.7 ± 4.9, and 120.2 ± 6.8% for angiotensin, norepinephrine, and vasopressin, respectively). Marked buckling of endothelium in apposition with pericytes and the absence of such changes elsewhere in the vessel wall were documented in morphologic studies of drug-perfused skeletal muscle. In both control hearts and hindlimbs and drug-perfused hearts, endothelial configuration in apposition with pericytes did not differ from the rest of the vessel. These observations, together with ultrastructural and morphometric data documenting much more extensive interaction between pericytes and endothelium in skeletal than in cardiac muscle, strongly suggest that pericytes in rat hindlimb skeletal muscle constrict in response to selected vasoactive agents while those in cardiac muscle do not.

Pericyte-endothelial relationships in cardiac and skeletal muscle capillaries

Abstract In order to elucidate the structure and function of pericytes, we have utilized electron microscopy and morphometric techniques to examine pericyte ultrastructure and to characterize their relationship to endothelial cells in rat hearts and rat and human skeletal muscle. Ultrastructural features of pericytes (in all three tissues) and their distribution along capillaries (84 and 95% of sectioned vessels from rat cardiac and skeletal muscle, respectively) were similar. On the other hand, pericyte secondary processes in heart and skeletal muscle differed markedly; those in skeletal muscle were larger and encircled capillaries to a greater extent than in rat hearts (20 vs 10% of vessel circumferences, respectively) and their interactions with endothelium were more intimate and complex. In both rat and human skeletal muscle, pericyte secondary processes penetrate deeply into endothelial cells and interdigitate with them extensively; likewise, endothelial evaginations protrude deeply into pericytes. These phenomena are rarely observed in rat cardiac muscle. Both myelinated and unmyelinated axons are closely associated with cardiac capillaries and pericytes, but are virtually absent from skeletal muscle microvasculatures. These differences documented in pericyte-endothelial topological relationships in heart and skeletal muscle support the concept that pericyte function may vary in different tissues.