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In diabetes there is a long latency between the onset of hyperglycemia and the appearance of structural microangiopathy. Because Ly6Clow patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. We found that in wild-type mice, diabetes reduced PMo in the general circulation but increased by fourfold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1−/− mice, a model of absence of PMo, there was no increase in leukostasis, and at 6 months of diabetes, the number of retinal acellular capillaries almost doubled compared with diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculoprotective, and housekeeping activities, as well as profound suppression of genes related to inflammation and apoptosis. Promigratory CXCR4 was no longer upregulated at longer duration when retinal acellular capillaries begin to increase. Thus, after a short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes and document within the natural history of diabetic retinopathy processes of protection and repair that can provide novel paradigms for prevention.

In diabetes there is a long latency between onset of hyperglycemia and appearance of structural microangiopathy. Because Ly6Clow patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. We found that, in wild-type mice, diabetes reduced PMo in the general circulation but increased by 4-fold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1-/- mice ─ a model of absence of PMo ─ there was no increase in leukostasis at all; and at 6 months of diabetes the number of retinal acellular capillaries almost doubled when compared to diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculo-protective, and housekeeping activities; as well as profound suppression of genes related to inflammation and apoptosis. Pro-migratory CXCR4 was no longer upregulated at longer duration, when retinal acellular capillaries begin to increase. Thus, after short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes, and document within the natural history of diabetic retinopathy processes of protection-repair that can provide novel paradigms for prevention.

Endothelial progenitor cells (EPCs) enter the systemic circulation in response to cues related to vascular damage and need for neovascularization. Thus, EPCs could become readily accessible informers of vascular status and enable the survey of vascular pathologies during preclinical stages. To identify EPC changes with biomarker potential, we investigated whether discrete EPC abnormalities were associated with early nonproliferative diabetic retinopathy (NPDR). Two EPC subtypes with different functions have been characterized to date—one solely committed to the endothelial lineage and the other carrying both endothelial and monocytic markers. We found that only the latter, colony-forming units (CFU)-Hill cells, manifested abnormalities in type 1 diabetic patients with NPDR compared with control subjects. The abnormalities consisted in an increased number of colonies formed in vitro and downregulation of the molecules that facilitate homing at sites of vascular injury. The abnormalities were absent in type 1 diabetic patients free of retinopathy and other complications, despite long diabetes duration, but were detected in some of the patients without clinical retinopathy after short diabetes duration. CFU-Hill cells are potential informers of diabetic microangiopathy but may be preempted from carrying out reparative functions if the molecular abnormalities compromise interactions with the damaged vascular wall.

The roles of transforming growth factor (TGF)-β in extracellular matrix production and vascular remodeling, coupled with increased TGF-β expression and signaling in diabetes, suggest TGF-β as an important contributor to the microangiopathy of diabetic retinopathy and nephropathy. To investigate whether increased TGF-β signaling could be a therapeutic target for preventing retinopathy, we used a pharmacologic approach (SM16, a selective inhibitor of the type 1 TGF-β receptor activin receptor–like kinase 5, orally active) to inhibit the increased, but not the basal, Tgf-β signaling in retinal vessels of diabetic rats. At the level of vascular gene expression, 3.5 months' diabetes induced minimal changes. Diabetes + SM16 for 3 weeks caused widespread changes in gene expression poised to enhance vascular inflammation, thrombosis, leakage, and wall instability; these changes were not observed in control rats given SM16. The synergy of diabetes and SM16 in altering gene expression was not observed in the lung. At the level of vascular network morphology, 7 months' diabetes induced no detectable changes. Diabetes + SM16 for 3 weeks caused instead distorted morphology and decreased density. Thus, in diabetes, retinal vessels become dependent on a small increase in TGF-β signaling via activin receptor–like kinase 5 to maintain early integrity. The increased TGF-β signaling may protect against rapid retinopathy progression and should not be a target of inhibitory interventions.

PURPOSE: We seek to identify pathogenic mechanisms for diabetic retinopathy that can become therapeutic targets beyond hyperglycemia and hypertension. We investigated if a defective myogenic response of retinal arteries to increased perfusion pressure, which exposes capillaries to increased pressure and flow, is associated with the onset of clinical retinopathy. METHODS: We examined prospectively the incidence of retinopathy in type 1 diabetic individuals tested 4 years earlier for the retinal arterial myogenic response, and in a cross-sectional study the prevalence of defective myogenic response in type 1 patients who had diabetic retinopathy. Among these, we contrasted early-onset (after 15 ± 2 years of diabetes, E-DR; n = 5) to late-onset (after 26 ± 3 years of diabetes, L-DR; n = 7) retinopathy. We measured the myogenic response using a laser Doppler blood flowmeter after a change in posture from sitting to reclining, which increases retinal perfusion pressure. RESULTS: Five of seven participants who 4 years prior had a defective myogenic response had now developed clinical retinopathy; as compared with only one of six participants who 4 years prior had a normal response (P = 0.10). In the cross-sectional study, all participants had normal retinal hemodynamics at steady state. In response to the postural change, only the E-DR group showed defective myogenic response (P = 0.005 versus controls, P = 0.02 versus L-DR) and abnormally high retinal blood flow (P = 0.016 versus controls). CONCLUSIONS: In type 1 diabetic patients, a defective myogenic response of retinal arteries to pressure is not required for the development of clinical retinopathy, but is prominently associated with an accelerated onset of retinopathy.

OBJECTIVE Prevention of diabetic retinopathy would benefit from availability of drugs that preempt the effects of hyperglycemia on retinal vessels. We aimed to identify candidate drug targets by investigating the molecular effects of drugs that prevent retinal capillary demise in the diabetic rat. RESEARCH DESIGN AND METHODS We examined the gene expression profile of retinal vessels isolated from rats with 6 months of streptozotocin-induced diabetes and compared it with that of control rats. We then tested whether the aldose reductase inhibitor sorbinil and aspirin, which have different mechanisms of action, prevented common molecular abnormalities induced by diabetes. The Affymetrix GeneChip Rat Genome 230 2.0 array was complemented by real-time RT-PCR, immunoblotting, and immunohistochemistry. RESULTS The retinal vessels of diabetic rats showed differential expression of 20 genes of the transforming growth factor (TGF)-β pathway, in addition to genes involved in oxidative stress, inflammation, vascular remodeling, and apoptosis. The complete loop of TGF-β signaling, including Smad2 phosphorylation, was enhanced in the retinal vessels, but not in the neural retina. Sorbinil normalized the expression of 71% of the genes related to oxidative stress and 62% of those related to inflammation. Aspirin had minimal or no effect on these two categories. The two drugs were instead concordant in reducing the upregulation of genes of the TGF-β pathway (55% for sorbinil and 40% for aspirin) and apoptosis (74 and 42%, respectively). CONCLUSIONS Oxidative and inflammatory stress is the distinct signature that the polyol pathway leaves on retinal vessels. TGF-β and apoptosis are, however, the ultimate targets to prevent the capillary demise in diabetic retinopathy.

Aims/hypothesis Abnormalities in retinal haemodynamics have been reported in patients with type 1 diabetes in advance of clinical retinopathy. These abnormalities could therefore be useful as early markers or surrogate endpoints for studying the microangiopathy. Since the DCCT, the increased focus on good glycaemic control is changing the natural history of diabetic retinopathy. Based on this, the aim of this study was to investigate whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities. Methods We measured retinal haemodynamics by laser Doppler flowmetry in 33 type 1 diabetic individuals with no or minimal retinopathy (age 30 ± 7 years, duration of diabetes 8.8 ± 4.6 years, 9% showing microaneurysms), and 31 age- and sex-matched non-diabetic controls. The study participants were not taking vasoactive medications, and blood glucose at the time of haemodynamic measurements was required to be between 3.8 and 11.1 mmol/l. Results HbA1c was 7.5 ± 1.2% and blood glucose 7.7 ± 2.8 mmol/l in these type 1 diabetic individuals, indicating relatively good glycaemic control. Retinal blood speed, arterial diameter and blood flow were not different between the diabetic individuals and the matched controls. Conclusions/interpretation Type 1 diabetic patients with no or minimal retinopathy who maintain relatively good glycaemic control do not show abnormalities of the retinal circulation at steady state, even after several years of diabetes. In such patients it may be necessary to test the vascular response to challenges to uncover any subtle abnormalities of the retinal vessels.

Diabetic retinal microangiopathy is characterized by increased permeability, leukostasis, microthrombosis, and apoptosis of capillary cells, all of which could be caused or compounded by activation of complement. In this study, we observed deposition of C5b-9, the terminal product of complement activation, in the wall of retinal vessels of human eye donors with 9 ± 3 years of type 2 diabetes, but not in the vessels of age-matched nondiabetic donors. C5b-9 often colocalized with von Willebrand factor in luminal endothelium. C1q and C4, the complement components unique to the classical pathway, were not detected in the diabetic retinas, suggesting that C5b-9 was generated via the alternative pathway, the spontaneous activation of which is regulated by complement inhibitors. The diabetic donors showed a prominent reduction in the retinal levels of CD55 and CD59, the two complement inhibitors linked to the plasma membrane by glycosylphosphatidylinositol anchors, but not in the levels of transmembrane CD46. Similar complement activation in retinal vessels and selective reduction in the levels of retinal CD55 and CD59 were observed in rats with a 10-week duration of streptozotocin-induced diabetes. Thus, diabetes causes defective regulation of complement inhibitors and complement activation that precede most other manifestations of diabetic retinal microangiopathy. These are novel clues for probing how diabetes affects and damages vascular cells.

To reconstruct the events that may contribute to the accelerated death of retinal vascular cells in diabetes, we investigated in situ and in vitro the activation of nuclear factor-kappaB (NF-kappaB), which is triggered by cellular stress and controls several programs of gene expression. The retinal capillaries of diabetic eye donors showed an increased number of pericyte nuclei positive for NF-kappaB, when compared with nondiabetic donors, whereas endothelial cells were negative. Microvascular cell apoptosis and acellular capillaries were increased only in the diabetic donors with numerous NF-kappaB-positive pericytes. Likewise, high glucose in vitro activated NF-kappaB in retinal pericytes but not in endothelial cells, and increased apoptosis only in pericytes. Studies with NF-kappaB inhibitors suggested that in pericytes, basal NF-kappaB has prosurvival functions, whereas NF-kappaB activation induced by high glucose is proapoptotic. Pericytes exposed to high glucose showed increased expression of Bax and of tumor necrosis factor-alpha, which were prevented by the NF-kappaB inhibitors and mimicked by transfection with the p65 subunit of NF-kappaB, and failed to increase the levels of the NF-kappaB-dependent inhibitors of apoptosis. Colocalization of activated NF-kappaB and Bax overexpression was observed in the retinal pericytes of diabetic donors. A proapoptotic program triggered by NF-kappaB selectively in retinal pericytes in response to hyperglycemia is a possible mechanism for the early demise of pericytes in diabetic retinopathy.

For several decades the pathobiology of diabetic retinopathy has been the object of conjecture and hypotheses. Indeed, very little was known about the cellular events triggered by diabetes in the retina and about the processes underlying the microangiopathy. In the last few years there has been a concerted effort to acquire such information, and the work has targeted not just the retinal vessels but more comprehensively the retina. The picture emerging is one of multiplicity: multiple cell types in the retina are affected early by diabetes and multiple processes are operative in the microangiopathy. The main abnormalities captured to date are altered expression of several genes, apoptosis, microthrombosis, and proinflammatory changes. The new information needs to be integrated into temporal and mechanistic sequences and further expanded but it is beginning to provide a framework for the cellular dimension of diabetic retinopathy.

In diabetic retinopathy, capillary nonperfusion and eventual obliteration can lead to retinal ischemia and sight-threatening neovascularization. The occurrence of retinal microthrombosis in human diabetes has long been suspected and occasionally observed but never systematically demonstrated. We used trypsin digestion to isolate the intact vascular network from retinas obtained postmortem from nine diabetic donors (age 64 ± 11 years, duration of diabetes 6 ± 4 years; mean ± SD) and eight age-matched nondiabetic donors. Topographically matched sectors (each one-sixth of retina) of diabetic and nondiabetic retinas were tested sequentially with antibodies to fibrin cross-linking factor XIII and platelet glycoprotein (GP)-IIIa to identify fibrin-platelet thrombi. In some trypsin digests, we also examined vascular cell apoptosis. The retina from a nondiabetic donor, 24 years of age, who had died of trauma, was used to exclude confounding influences caused by the postmortem period. When compared with those of nondiabetic donors, the retinas of diabetic donors showed double the number of capillary segments with colocalized immunostaining for factor XIII and GPIIIa (P = 0.02). The total area of the positive segments was fourfold greater in the diabetic than in the nondiabetic donors (P = 0.02) and correlated with the duration of diabetes (r = 0.71, P < 0.05). Large thrombi were six times more frequent in the diabetic donors (P = 0.03), and there was a significant topographical association of microthrombosis with apoptotic cells in both diabetic and nondiabetic vessels (P = 0.0001). Hence, diabetes of short duration was found to be associated with a greater than normal number and size of platelet-fibrin thrombi in the retinal capillaries. These thrombi can contribute to capillary obliteration and retinal ischemia and may be a practical target for early drug intervention.

Mesangial cell abnormalities in spontaneously hypertensive rats before the onset of hypertension. To identify kidney biosynthetic abnormalities that may precede the onset of hypertension, we studied the expression of flbronectin (FN) and collagen IV (Coll IV) in young SHR (4 weeks of age) whose systolic blood pressure was normal and similar to that of age-matched control WKY rats. In isolated glomeruli the level of FN protein assessed by immunoblotting tended to be lower in the SHR than in the WKY rats. By Northern analysis the FN/actin mRNA ratio was significantly lower in glomeruli from SHR (0.56 ± 0.47) than in glomeruli from WKY rats (2.0 ± 0.8). These abnormalities were maintained in vitro since the expression of FN was significantly lower in SHR than in WKY cultured mesangial cells (FN/actin mRNA ratio=0.84 ± 0.46 vs. 1.9 ± 0.7, P = 0.029). No differences in Coll IV mRNA or protein levels were observed in SHR glomeruli and mesangial cells when compared with WKY rats. The levels of aortic FN and Coll IV mRNAs were not different in SHR and WKY rats. In addition, mesangial cells from SHR showed a significantly higher growth rate than those from WKY. The biosynthetic and proliferative abnormalities observed in the SHR mesangial cells appear to reflect genetic characteristics, and could provide novel insights into cellular mechanisms linking the genetics of hypertension with predisposition to glomerular pathology.

The histological lesions of diabetic micro-angiopathy have a long latency, but vascular cell function may be affected at early stages of the process. Rats with experimental galactosaemia develop a diabetic-like retinopathy in the absence of other metabolic abnormalities characteristic of diabetes mellitus; basement membrane thickening is measurable in their retinal vessels after 7 months of galactose feeding. To examine the course of biosynthetic changes relevant to the process, retinal expression of collagen IV and fibronectin were compared in rats fed a 30% galactose diet or a control diet for 5 or 9 weeks. Total retinal RNA was studied by reverse transcription-polymerase chain reaction; the fibronectin primers encompassed the alternatively spliced EIIIA exon. The levels of alpha 1 (IV) collagen and fibronectin mRNAs were measured relative to an internal standard (beta-actin mRNA). The proportion of EIIIA+ to EIIIA- fibronectin transcripts was similar in the retinas of control and galactose-fed rats, which, however, showed increased levels of both fibronectin and collagen IV mRNAs in the presence of unchanged beta-actin mRNA levels. An upward trend was detected by 5 weeks of galactose feeding; and after 9 weeks the fibronectin/actin ratio was 1.2 +/- 0.3 vs 0.8 +/- 0.2 in controls (p = 0.015) and the collagen IV/actin ratio was 1.3 +/- 0.3 vs 0.9 +/- 0.2 in controls (p = 0.04). Thus, hyperhexosaemia of a few weeks' duration is a perturbation sufficient to increase the synthesis of basement membrane components in the retina. The search for additional early biosynthetic changes should assist in reconstructing the pathogenesis of hexose-induced retinal microangiopathy.

Impaired activity of the pentose phosphate pathway of glucose metabolism caused by hereditary deficiency of its key regulatory enzyme glucose-6-phosphate dehydrogenase (G6PD) has consequences that may worsen or attenuate the course of diabetic complications. Decreased availability of NADPH can predispose to oxidative stress and endothelial dysfunction, but can also limit the activity of the polyol pathway and cholesterol synthesis. Reduced availability of pentose phosphates for nucleic acid synthesis could impair cell proliferation. We sought to learn in which direction G6PD deficiency affects diabetic retinopathy.We enrolled patients who were G6PD-deficient or -sufficient with type 1 diabetes of duration 15 years or longer for whom HbA(1c) records were available for at least the previous 3 years. Renal failure and smoking were exclusion criteria. For each participant seven standard field colour photographs were obtained of each eye, and retinopathy was graded in a masked fashion.The clinical characteristics of the 19 G6PD-deficient patients studied (age 42 ± 9 years, diabetes duration 24 ± 6 years, average HbA(1c) over 3 years 6.7 ± 0.8%) were similar to those of the 35 G6PD-sufficient patients. Almost 90% of patients in both groups had retinopathy; however, proliferative retinopathy was noted solely among G6PD-deficient patients (28%, p = 0.0036 vs G6PD-sufficient). The G6PD-deficient patients also showed a trend for increased frequency of microalbuminuria.The data suggest that G6PD deficiency accelerates the microvascular complications of diabetes, and that among the consequences of G6PD deficiency those that can enhance the damage caused by diabetes outweigh those that could be protective.

To identify events and mechanisms that might contribute to the poor reversibility of diabetic complications, we examined whether diabetes or high glucose induces changes in gene expression and whether such changes outlast the presence of the metabolic abnormalities. The study focused on fibronectin because the increased amounts of this glycoprotein found in diabetic tissues and thickened basement membranes are as yet unexplained. In streptozotocin-induced diabetic rats, fibronectin mRNA levels were increased to 304 +/- 295% of control (mean +/- SD) in the kidney cortex (P less than 0.02), and to 271 +/- 273% of control in the heart (P less than 0.02), while actin mRNA levels remained unchanged. Elevation of fibronectin mRNA persisted for weeks after restoration of near-normoglycemia. In cultured human endothelial cells, high glucose-induced overexpression of fibronectin and collagen IV remained detectable after replating and multiple cell divisions in the absence of high glucose. Cells shifted to normal-glucose medium after prolonged exposure to high glucose also exhibited a proliferative advantage over cells chronically maintained in normal glucose. Thus, diabetes increases fibronectin expression in tissues that are known targets of the complications, and the effect is not readily reversible. The in vitro studies suggest that hyperglycemia may be responsible for these events through induction of self-perpetuating changes in gene expression.

The polyol pathway is a two-step metabolic pathway in which glucose is reduced to sorbitol, which is then converted to fructose. It is one of the most attractive candidate mechanisms to explain, at least in part, the cellular toxicity of diabetic hyperglycemia because (i) it becomes active when intracellular glucose concentrations are elevated, (ii) the two enzymes are present in human tissues and organs that are sites of diabetic complications, and (iii) the products of the pathway and the altered balance of cofactors generate the types of cellular stress that occur at the sites of diabetic complications. Inhibition (or ablation) of aldose reductase, the first and rate-limiting enzyme in the pathway, reproducibly prevents diabetic retinopathy in diabetic rodent models, but the results of a major clinical trial have been disappointing. Since then, it has become evident that truly informative indicators of polyol pathway activity and/or inhibition are elusive, but are likely to be other than sorbitol levels if meant to predict accurately tissue consequences. The spectrum of abnormalities known to occur in human diabetic retinopathy has enlarged to include glial and neuronal abnormalities, which in experimental animals are mediated by the polyol pathway. The endothelial cells of human retinal vessels have been noted to have aldose reductase. Specific polymorphisms in the promoter region of the aldose reductase gene have been found associated with susceptibility or progression of diabetic retinopathy. This new knowledge has rekindled interest in a possible role of the polyol pathway in diabetic retinopathy and in methodological investigation that may prepare new clinical trials. Only new drugs that inhibit aldose reductase with higher efficacy and safety than older drugs will make possible to learn if the resilience of the polyol pathway means that it has a role in human diabetic retinopathy that should not have gone undiscovered.

Previously studied inhibitors of aldose reductase were largely from two chemical classes, spirosuccinamide/hydantoins and carboxylic acids. Each class has its own drawbacks regarding selectivity, in vivo potency, and human safety; as a result, the pathogenic role of aldose reductase in diabetic retinopathy remains controversial. ARI-809 is a recently discovered aldose reductase inhibitor (ARI) of a new structural class, pyridazinones, and has high selectivity for aldose versus aldehyde reductase. To further test the possible pathogenic role of aldose reductase in the development of diabetic retinopathy, we examined the retinal effects of this structurally novel and highly selective ARI in insulinized streptozotocin-induced diabetic rats. ARI-809 treatment was initiated 1 month after diabetes induction and continued for 3 months at a dose that inhibited the polyol pathway in the retina of diabetic rats to a similar extent as sorbinil, a poorly selective hydantoin ARI previously shown to prevent retinopathy in this model. ARI-809 improved survival, inhibited cataract development, normalized retinal sorbitol and fructose, and protected the retina from abnormalities that also occur in human diabetes: neuronal apoptosis, glial reactivity, and complement deposition. Because ARI-809 is a novel chemotype highly selective for aldose reductase, these results support the notion that aldose reductase is the key relay that converts hyperglycemia into glucose toxicity in neural and glial cell types in the retina.

The prevention of diabetic retinopathy requires drugs that leverage the benefits of glycemic control without adding the burden of side effects. Aspirin at dosages of 1–1.5 g/day has prevented manifestations of diabetic retinal microangiopathy in a clinical trial as well as in studies with dogs. Because lower and safer doses of aspirin could be used if its beneficial effects on retinopathy were due to antithrombotic effects, we compared the effects of a selective antiplatelet drug (clopidogrel) to those of aspirin in streptozotocin-induced diabetic rats. Clopidogrel did not prevent neuronal apoptosis, glial reactivity, capillary cell apoptosis, or acellular capillaries in the retina of diabetic rats. Aspirin, at doses yielding serum levels (

The polyol (sorbitol) pathway of glucose metabolism is activated in many cell types when intracellular glucose concentrations are high, and it can generate cellular stress through several mechanisms. The role of the polyol pathway in the pathogenesis of diabetic retinopathy has remained uncertain, in part because it has been examined preferentially in galactose-induced retinopathy and in part because inhibition studies may not have achieved full blockade of the pathway. Having observed that the streptozotocin-induced diabetic rat accurately models many cellular processes characteristic of human diabetic retinopathy, we tested in the diabetic rat if documented inhibition of the polyol pathway prevents a sequence of retinal vascular abnormalities also present in human diabetes. An inhibitor of aldose reductase, the rate-limiting enzyme in the pathway, prevented the early activation of complement in the wall of retinal vessels and the decreased levels of complement inhibitors in diabetic rats, as well as the later apoptosis of vascular pericytes and endothelial cells and the development of acellular capillaries. Both rat and human retinal endothelial cells showed aldose reductase immunoreactivity, and human retinas exposed to high glucose in organ culture increased the production of sorbitol by a degree similar to that observed in the rat. Excess aldose reductase activity can be a mechanism for human diabetic retinopathy.

We tested the hypothesis that the apoptosis of inner retina neurons and increased expression of glial fibrillary acidic protein (GFAP) observed in the rat after a short duration of diabetes are mediated by polyol pathway activity. Rats with 10 weeks of streptozotocin-induced diabetes and GHb levels of 16 ± 2% (mean ± SD) showed increased retinal levels of sorbitol and fructose, attenuation of GFAP immunostaining in astrocytes, appearance of prominent GFAP expression in Müller glial cells, and a fourfold increase in the number of apoptotic neurons when compared with nondiabetic rats. The cells undergoing apoptosis were immunoreactive for aldose reductase. Sorbinil, an inhibitor of aldose reductase, prevented all abnormalities. Intensive insulin treatment also prevented most abnormalities, despite reducing GHb only to 12 ± 1%. Diabetic mice, known to have much lower aldose reductase activity in other tissues when compared with rats, did not accumulate sorbitol and fructose in the retina and were protected from neuronal apoptosis and GFAP changes in the presence of GHb levels of 14 ± 2%. This work documents discrete cellular consequences of polyol pathway activity in the retina, and it suggests that activation of the pathway and “retinal neuropathy” require severe hyperglycemia and/or high activity of aldose reductase. These findings have implications for how to evaluate the role of the polyol pathway in diabetic retinopathy.

IGF-I promotes the survival of multiple cell types by activating the IGF-I receptor (IGF-IR), which signals downstream to a serine/threonine kinase termed Akt. Because in diabetes vascular and neural cells of the retina undergo accelerated apoptosis, we examined IGF-I synthesis and signaling in the human and rat diabetic retina. In retinas obtained postmortem from six donors aged 64 ± 8 years with a diabetes duration of 7 ± 5 years, IGF-I mRNA levels were threefold lower than in the retinas of six age-matched nondiabetic donors ( P = 0.005). In the retinas of rats with 2 months9 duration of streptozotocin-induced diabetes, IGF-I mRNA levels were similar to those of control rats, but after 5 months of diabetes they failed to increase to the levels recorded in age-matched controls ( P < 0.02). Retinal IGF-I expression was not altered by hypophysectomy, proving to be growth-hormone independent. IGF-IR levels were modestly increased in the human diabetic retinas ( P = 0.02 vs. nondiabetic retinas) and were unchanged in the diabetic rats. Phosphorylation of the IGF-IR could be measured only in the rat retina, and was not decreased in the diabetic rats (94 ± 18% of control values). In the same diabetic rats, phosphorylation of Akt was 123 ± 21% of control values. There was not yet evidence of increased apoptosis of retinal microvascular cells after 5 months of streptozotocin-induced diabetes. Hence, in the retina of diabetic rats, as in the retina of diabetic human donors, IGF-I mRNA levels are substantially lower than in age-matched nondiabetic controls, whereas IGF-IR activation and signaling are not affected, at least for some time. This finding suggests that in the diabetic retina, the activation of the IGF-IR is modulated by influences that compensate for, or are compensated by, decreased IGF-I synthesis.

Diabetes of even short duration accelerates the death of capillary cells and neurons in the inner retina by a process consistent with apoptosis. We examined whether the process is accompanied by changes in the expression of endogenous regulators of apoptosis. In postmortem retinas of 18 diabetic donors (age 67 +/- 6 years, diabetes duration 9 +/- 4 years) the levels of pro-apoptotic Bax were slightly, but significantly, increased when compared with levels in 20 age-matched nondiabetic donors (P = 0.04). In both groups, Bax localized to vascular and neural cells of the inner retina. Neither pro-apoptotic Bcl-X(S), nor pro-survival Bcl-X(L) appeared affected by diabetes. The levels of these molecules could not be accurately quantitated in lysates of retinal vessels because of variable degrees of glial contamination. However, studies in situ showed in several pericytes, the outer cells of retinal capillaries, intense Bax staining often in conjunction with DNA fragmentation. Bovine retinal pericytes exposed in vitro to high glucose levels for 5 weeks showed elevated levels of Bax (P = 0.03) and increased frequency of annexin V binding, indicative of early apoptosis. Hence, human diabetes selectively alters the expression of Bax in the retina and retinal vascular pericytes at the same time as it causes increased rates of apoptosis. The identical program induced by high glucose in vitro implicates hyperglycemia as a causative factor in vivo, and provides a model for establishing the role of Bax in the accelerated death of retinal cells induced by diabetes.

To reconstruct the mechanisms for the vasoobliteration that transforms diabetic retinopathy into an ischemic retinopathy, we compared the occurrence of cell death in situ in retinal microvessels of diabetic and nondiabetic individuals. Trypsin digests and sections prepared from the retinas of seven patients (age 67 +/- 7 yr) with .9 +/- 4 yr of diabetes and eight age- and sex-matched nondiabetic controls were studied with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction which detects preferentially apoptotic DNA fragmentation. The count of total TUNEL+ nuclei was significantly greater in the microvessels of diabetic (13 +/- 12 per one-sixth of retina) than control subjects (1.3 +/- 1.4, P = 0.0016), as were the counts of TUNEL+ pericytes and endothelial cells (P < 0.006). The neural retinas from both diabetic and nondiabetic subjects were uniformly TUNEL-. Retinal microvessels of rats with short duration of experimental diabetes or galactosemia and absent or minimal morphological changes of retinopathy, showed TUNEL+ pericytes and endothelial cells, which were absent in control rats. These findings indicate that (a) diabetes and galactosemia lead to accelerated death in situ of both retinal pericytes and endothelial cells; (b) the event is specific for vascular cells; (c) it precedes histological evidence of retinopathy; and (d) it can be induced by isolated hyperhexosemia. A cycle of accelerated death and renewal of endothelial cells may contribute to vascular architectural changes and, upon exhaustion of replicative life span, to capillary obliteration.

Basement membrane thickening is the most prominent and characteristic feature of early diabetic microangiopathy. Unknown is not only the causative process but also whether the thickening reflects increased synthesis of specific components. Because collagen type IV is uniquely present in basement membranes and represents their predominant structural element, we studied its expression in retinas obtained postmortem from five patients with 8 +/- 3 yr of diabetes and six nondiabetic controls. The collagen IV transcript proved to be rare in adult human retina and undetectable by Northern analysis. We thus identified a set of primers and conditions to detect the transcript by the reverse transcriptase polymerase chain reaction and to measure its level relative to an endogenous internal standard (beta-actin mRNA). In the diabetic patients the levels of collagen IV mRNA were increased twofold over levels in controls, whereas the actin mRNA levels were similar in the two groups. Hence, the collagen IV/actin ratio was 0.53 +/- 0.15 in diabetic samples and 0.24 +/- 0.09 in control samples (P = 0.004). These results indicate that diabetes induces a twofold increase in the expression of collagen IV by the cells that synthesize basement membranes in the adult retina (vascular cells). Insofar as high ambient glucose in vitro elicits the same effect, it may be proposed that basement membrane thickening in diabetes results from enhanced synthesis of specialized component molecules sustained by hyperglycemia.

The nature of the process leading to the acellular nonperfused capillaries of diabetic microangiopathy remains unknown. Because these capillaries manifest thickened basement membranes, we asked whether the process causing deposition of excess extracellular matrix in diabetes modifies cell-matrix interactions in a direction that would compromise cell renewal. In 44 individual isolates of human umbilical vein endothelial cells we observed that high glucose concentrations (30 mM) induce coordinate increases in the levels of mRNAs encoding fibronectin and the fibronectin-specific integrin receptor alpha 5 beta 1 as well as in the cognate proteins. Expression of the integrin subunit alpha 3, component of the alpha 3 beta 1 polyspecific receptor for fibronectin, laminin, and collagen, was also up-regulated by high glucose. Overexpression of integrins correlated with increased cell attachment to exogenous fibronectin and laminin as well as to complex matrix. Moreover, cells exhibited firmer steady-state adhesion to their own matrix. To correlate these in vitro observations with events in human diabetic retinopathy we measured integrin levels in retinal trypsin digests prepared from 10 patients with 8.2 +/- 1.6 (mean +/- SE) years of diabetes and 10 age- and sex-matched nondiabetic controls. Microvessels of diabetic patients showed increased immunostaining for beta 1 integrin (P = 0.025) when compared with control microvessels. These data show that high glucose and diabetes increase integrin expression and thus alter the interaction of vascular endothelial cells with their basement membranes in the direction of firmer cell-matrix adhesion. This could compromise the migration and replication critical to the reendothelialization process and contribute to microvascular occlusion.

Purpose The current approach to the prevention of diabetic retinopathy relies on intensive anti-diabetes treatment and is only partially successful. A marker of retinopathy risk would enable strategies of surveillance, screening of adjunct drugs, and targeted drug interventions. The authors sought to identify early abnormalities of retinal vessels that are not prevented by the current therapeutic approach. Methods Retinal thickness (an informer of vascular permeability) and hemodynamic parameters at baseline and longitudinally were measured in 27 subjects (age, 32 ± 9 years [mean ± SD]) with well-controlled type 1 diabetes of 12.4 ± 6.4 years' duration and no retinopathy, and in 27 control subjects. In a subset of 17 patients and 11 controls, the hemodynamic response to reclining, a postural change that increases retinal perfusion pressure, was measured. Results Baseline foveal thickness and hemodynamic parameters were similar in the diabetic and control subjects. Foveal thickness increased over 12 months in the diabetic subjects, from 217 ± 22 μm to 222 ± 20 μm (P = 0.0036), remaining however within the normal range. Reclining uncovered in 47% of diabetic subjects (P = 0.016 compared with controls) an absent myogenic response (i.e., unchanged or increased arterial diameter instead of the normal decrease). The patterns were repeatable. Only the diabetic group with defective vasoconstriction showed widening arterial diameter over 12 months, a change presaging vascular dilatation in diabetic retinopathy. Conclusions Defective myogenic response to pressure was the first detectable abnormality of retinal vessels in subjects with well-controlled type 1 diabetes. Because of its selective occurrence, interpretability in individual patients, and pathogenic potential, the abnormality deserves evaluation as a risk marker for retinopathy.

To identify agents and mechanisms responsible for the thickened basement membranes characteristic of diabetic angiopathy we examined the effects of high glucose (30 mM) on the expression of genes related to extracellular matrix composition and turnover and investigated whether the changes induced by high glucose were mimicked and sustained by activation of protein kinase C or A. In human umbilical vein endothelial cells high glucose increased fibronectin, collagen IV, tissue plasminogen activator (tPA), and plasminogen activator-inhibitor 1 (PAI-1) mRNA levels 2-fold but did not affect type IV and interstitial collagenase expression. Acute treatment with phorbol esters resulted in increased collagen IV, tPA, PAI-1, and interstitial collagenase mRNAs; the type IV collagenase mRNA levels were instead suppressed to 50% of control. Upon longer exposure to phorbol esters (48 h) suppression of fibronectin and PAI-1 mRNAs also occurred. Intracellular elevation of cAMP led to over-expression of fibronectin and type IV collagenase and potentiated the effects of phorbol esters on collagen IV, tPA, and interstitial collagenase expression. The mRNA changes induced by high glucose occurred in the absence of protein kinase C activation or cAMP elevation. These studies indicate that events other than activation of protein kinase C or A bridge high ambient glucose to changes in endothelial cell gene expression that may contribute to diabetic angiopathy.

Whether the increased capillary permeability characteristic of diabetes extends to the blood-brain barrier is presently unclear. We have examined in streptozotocin-diabetic rats the permeability of the blood-brain barrier at the level of 12 discrete brain regions employing 3 intravenous tracers of different molecular weight: sucrose, insulin and horseradish peroxidase. In animals killed 5 min after tracer injection both the sucrose and the inulin spaces were similar to controls. In order to assess whether more prolonged circulation of tracers would uncover leakage, we studied brain spaces at longer intervals after tracer injections. When inulin was allowed to circulate for 15 min prior to killing, animals with 4 weeks of diabetes (but not 2 weeks) exhibited larger inulin spaces at the level of the medio-basal hypothalamus (p less than 0.01), medio-dorsal hypothalamus (p less than 0.05) and periaqueductal gray (p less than 0.01). Horseradish peroxidase, even after 75 min of perfusion, remained confined in both diabetic and control animals to central nervous system areas devoid of blood-brain barrier. Thus, after a relatively short duration of diabetes the blood-brain barrier manifests an increased permeability. It is subtle, limited to some brain regions and selective for low molecular weight tracers.

In an attempt to elucidate the mechanism of action of phenformin, eleven juvenile-onset, insulin-requiring diabetic subjects underwent four different treatment regimens during standard breakfast tests. These four treatments were: control (no insulin or phenformin); insulin alone (15 U regular insulin administered subcutaneously one-half hour before breakfast); phenformin alone (50 mg of the timed-release capsule given twice daily for three days before the study and two and one-half hours before breakfast on the day of study); and phenformin plus insulin (in the amounts and at the times stated above). Phenformin was found to decrease postprandial hyperglycaemia significantly when compared with control values, and its addition to insulin further decreased the postprandial glucose rise below that found with insulin alone (p less than 0.005). These effects were associated with a reduction in early (30-min) postprandial hyperglucagonaemia (p less than 0.05). Triglyceride levels, gastrin secretion, growth hormone levels, and increments of alpha-amino nitrogen were not affected by phenformin. Thls, suppression of postprandial hyperglucagonaemia may be an additional mechanism in the reduction of postprandial hyperglycaemia after phenformin.

We studied the contribution of alpha- and beta-adrenergic receptor activation to the cardiovascular, metabolic, and hormonal effects of dopamine. At a concentration of 1.5 mug/kg.min, the infusion of dopamine in 12 normal volunteers was associated with a transient but significant rise in pulse rate, which was prevented by propranolol. Venous plasma glucose did not change throughout the experiments, and a mild increase in plasma free fatty acid levels observed during the administration of dopamine alone was antagonized by propranolol. In contrast, neither the beta-adrenergic blocker, propranolol, nor the alpha-adrenergic blocker, phentolamine, was effective in inhibiting the dopamine-induced rise in plasma glucagon (from 82+/-9 to 128+/-14 pg/ml; P < 0.005) and serum insulin (from 7.5+/-1 to 13+/-1.5 muU/ml; P < 0.005) or its suppression of plasma prolactin (from 8.5+/-1 to 5.2+/-0.8 ng/ml; P < 0.001). Although serum growth hormone levels did not change during the infusion of dopamine alone, an obvious rise occurred in three subjects during the combined infusion of propranolol and dopamine. Whereas some metabolic and cardiovascular effects of dopamine are mediated through adrenergic mechanisms, these observations indicate that this is not the case for the effects of this catecholamine on glucagon, insulin, and prolactin secretion, and thus provide further support for the theory of a specific dopaminergic sensitivity of these hormonal systems in man.

To examine whether abnormal pancreatic alpha-cell function found in human diabetes mellitus may represent a selective insensitivity to glucose, plasma glucagon responses to hyperglycemia and elevation of plasma free fatty acid levels (both known suppressors of glucagon secretion) were compared in juvenile-onset, insulin-requiring diabetic subjects, and in normal nondiabetic subjects. In the latter, both elevation of plasma free fatty acid levels induced by heparin administration of hyperglycemia produced by intravenous infusion of glucose resulted in a comparable 30--40% suppression of circulating glucagon levels (P less than 0.01). In the diabetic subjects, glucagon suppression by hyperglycemia (less than 20%) was less than that occurring in normal subjects (P less than 0.01), even when accompanied by infusion of supraphysiologic amounts of insulin. However, suppression of glucagon levels by elevation of plasma free fatty acids in the diabetic group was similar to that found in normal subjects and of comparable magnitude to that due to hyperglycemia in the normal subjects. These results thus demonstrate a selective impairment of the diabetic alpha-cell response to glucose and provide further evidence for the presence of an abnormal alpha-cell glucoreceptor in human diabetes mellitus.

To study the individual effects of glucagon and growth hormone on human carbohydrate and lipid metabolism, endogenous secretion of both hormones was simultaneously suppressed with somatostatin and physiologic circulating levels of one or the other hormone were reproduced by exogenous infusion. The interaction of these hormones with insulin was evaluated by performing these studies in juvenile-onset, insulin-deficient diabetic subjects both during infusion of insulin and after its withdrawal. Infusion of glucagon (1 ng/kg-min) during suppression of its endogenous secretion with somatostatin produced circulating hormone levels of approximately 200 pg/ml. When glucagon was infused along with insulin, plasma glucose levels rose from 94 +/- 8 to 126 +/- 12 mg/100 ml over 1 h (P less than 0.01); growth hormone, beta-hydroxy-butyrate, alanine, FFA, and glycerol levels did not change. When insulin was withdrawn, plasma glucose, beta-hydroxybutyrate, FFA, and glycerol all rose to higher levels (P less than 0.01) than those observed under similar conditions when somatostatin alone had been infused to suppress glucagon secretion. Thus, under appropriate conditions, physiologic levels of glucagon can stimulate lipolysis and cause hyperketonemia and hyperglycemia in man; insulin antagonizes the lipolytic and ketogenic effects of glucagon more effectively than the hyperglycemic effect. Infusion of growth hormone (1 mug/kg-h) during suppression of its endogenous secretion with somastostatin produced circulating hormone levels of approximately 6 ng/ml. When growth hormone was administered along with insulin, no effects were observed. After insulin was withdrawn, plasma beta-hydroxybutyrate, glycerol, and FFA all rose to higher levels (P less than 0.01) than those observed during infusion of somatostatin alone when growth hormone secretion was suppressed; no difference in plasma glucose, alanine, and glucagon levels was evident. Thus, under appropriate conditions, physiologic levels of growth hormone can augment lipolysis and ketonemia in man, but these actions are ordinarily not apparent in the presence of physiologic levels of insulin.

To test the hypothesis that cerebral capillaries, which share the embroyologic and morphologic characteristics of retinal capillaries, might have the same abnormal permeability in diabetic patients, we investigated the growth hormone response to a small amount of peripherally administered dopamine (1.5 microgram/kg.min). Consistent with the known exclusion of systemic dopamine from brain parenchyma, no rise was observed in 12 normal subjects. In 10 of 12 juvenile-onset, insulin-dependent diabetic patients, however, a substantial growth hormone rise occurred (peak value, 19.2 +/- 3.0 ng/ml [mean +/- SE]). Comparision of metabolic and cardiovascular responses to the infusion in both groups did not suggest that higher circulating levels of dopamine had been achieved in the diabetics. Other growth hormone stimuli (apomorphine in decreasing amounts, glucagon, and graded physical exercise) failed to indicate that hypothalamic hypersensitivity could account for the consistent rise. We postulate that an abnormal permeability of the blood-brain barrier in the diabetic patients permitted exposure of the hypothalamic structures regulating growth hormone secretion to a greater fraction of the infused dopamine.