therapeutic potential of endothelin receptor antagonists in diabetes

Chakrabarti, Cukiernik, Mukherjee & ChenTherapeutic potential of endothelin receptor antagonists in diabetes

Monthly Focus: Oncologic, Endocrine & Metabolic

Therapeutic potential of endothelinreceptor antagonists in diabetes

Subrata Chakrabarti, Mark Cukiernik, Suranjana Mukherjee &Shali Chen

Department of Pathology, The University of Western Ontario, London,Ontario, Canada

Endothelins (ETs) are widely distributed in the body and perform severalvascular and non-vascular functions. Experimental evidence indicates thatabnormalities of the ET system occur in several organs affected in chronicdiabetic complications. Furthermore, ET antagonists were found to preventstructural and functional changes in the target organs of chronic diabeticcomplications in animal models. Abnormalities of plasma ET levels havealso been demonstrated in human diabetes. This review discusses the roleof ET in the pathogenesis of chronic diabetic complications. The currentexperimental evidence suggests that ET antagonism may potentiallyrepresent an adjuvant therapeutic tool in the treatment of chronic diabeticcomplications.

Keywords: cardiomyopathy, diabetic complications, endothelins, endothelinantagonism, nephropathy, neuropathy, retinopathy

Exp. Opin. Invest. Drugs (2000) 9(12):2873-2888

1. Introduction

During last few years considerable evidence has accumulated to suggestthat endothelins (ET) are of importance in several disease processes [1-4].ETs are 21 amino acid peptides, the action of which are mediated viaG-protein coupled receptors. ET-1, the first ET discovered, is the mostpotent known vasoconstrictor [2,3]. In addition, ETs have several othervascular and non-vascular functions. Due to their widespread tissue distri-bution and vast number of actions, they are of significance in thedevelopment of several diseases affecting micro- and macrovasculature.Although ETs have some roles in the regulation of insulin secretion, themain importance of ETs in diabetes lies with their role in the pathogenesisof chronic diabetic complications, which will be the focus of this review. Anumber of biochemical abnormalities may occur, as a result of increasedglucose levels, in diabetes. These changes may affect cellular function in avariety of ways [4]. Metabolic and functional abnormalities of the cells in thediabetic milieu leading to the abnormalities of the ET system may be ofsignificant importance in the development of structural and functionallesions. These abnormalities may affect multiple cellular constituents of thetissues. Alteration of the ETs in diabetes may in turn affect other vasoactiveor functionally important factors with profound consequences. In thisreview we briefly discuss ETs and their receptors, followed by an outline ofthe pathologies and the pathogenesis of major chronic diabetic complica-tions. Finally, we highlight the role of ETs in these complications.

28732000 © Ashley Publications Ltd. ISSN 1354-3784

Review

1. Introduction

2. Endothelin system

3. Pathological changes inchronic diabeticcomplications

4. Pathogenesis of chronicdiabetic complications

5. Expert opinion

Acknowledgements

Bibliography

http://www.ashley-pub.com

Expert Opinion on Investigational Drugs

http://www.ashley-pub.com

2. Endothelin system

2.1 Endothelins and endothelin receptors

The ET family comprises three unique isoformsknown as ET-1, ET-2 and ET-3, all of which show ahigh degree of homology at the amino acid sequencelevel. ETs all contain 21 amino acid residues and havetwo intramolecular disulphide bonds [2,3,5]. ETs, firstdiscovered as endothelial products, have since beenshown to have widespread cellular localisation [2,3,5].All ETs have several important functions in variousorgan systems. In addition to maintaining the bloodflow in various organs, they modulate cell

proliferation and differentiation. ET-1 sensitises theblood vessels to the action of noradrenaline,serotonin and angiotensins [2,3,5,6]. They playimportant roles in cardiac, renal and endocrinefunctions. In the heart, ET-1 causes positiveinotrophic and chronotrophic effects, shortens actionpotential and stimulates cardiac hypertrophy. ET-1causes renal vasoconstriction, stimulates mitogenesisof the mesangial cells, reduces renin release and Na+

reabsorption. ETs stimulate aldosterone, catechola-mine, luteinising hormone releasing hormone,oxytocin, adrenocorticotrophic hormone andgonadotrophin secretion and inhibit thyroglobulin

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Invest. Drugs (2000) 9(12)

2874 Therapeutic potential of endothelin receptor antagonists in diabetes

Table 1: Description of non-peptide endothelin antagonists currently in clinical development.

Drug name Manufacturer Country/clinicalstatus

Indications Ref.

ETA & ETB antagonists

L-749329L-751281L-754142L-749805

Merck & Co., Inc. US/RT Emesis, hypertension, brain haemorrhage [25]

A-182086 Abbott Laboratories US/NDR Hypertension, cardiac failure [26]

BosentanRO 47-0203/001RO 47-8634RO 48- 5033RO 64-1056

Actelion Ltd.Roche Holding Ltd.

Switzerland/C3Switzerland/C3Switzerland/C1Germany/DR

Pulmonary hypertension, congestive heartfailure, cardiac failure, migraine,cerebrovascular disease, coronary arterydisease, inflammatory bowel disease

[18]

J-104132BQ4508-2L-753037

Banyu PharmaceuticalBanyu PharmaceuticalMerck &Co.

US/C2US/C1US/C1

Cardiac failure, hypertension, hypertension,cardiac failure

[27]

SB-209670SK&F-66861

SmithKline Beecham plc. UK/C2UK/DR

Renal failure, migraine, restenosis [28]

ETA antagonists

BMS 182874 Bristol-Myers Squibb Co. US/discontinued Hypertension [30]

EnrasentanSB-217242

SmithKline Beecham UK/C2 Chronic obstructive pulmonary disease,hypertension, cardiac failure

[29]

Tarasentan (Atrasentan)A-127222A-147627ABT-627

Abbott International Ltd. US/C2US/C1

Prostate cancer, hypertension [31]

TBC11251 (Sitaxsentan)IPI-I1040IPI-1251TBC-11241

Texas Biotechnology Co. US/C2US/DR

Cardiac failure, chronic obstructivepulmonary disease, hypertension, brainhaemorrhage

[32]

RO 485695 Roche Holding Ltd. Switzerland/C2 Cardiac failure [18]

A-20637(ET-3 antagonist)

Abbott Laboratories US/NDR Reperfusion injury, restenosis, renal failure [33]

C1: Phase I; C2: Phase II; C3: Phase III; DR: Discovery; NDR: No development reported; RT: Research tool.

and prolactin release [5,6]. Furthermore, they arebelieved to function as neuromodulators and act asimportant autocrine and paracrine growth factors [5].ET-1 is involved in the induction of several proto-oncogenes including c-fos, c-jun and c-myc [5-7].

In humans, the ET-1 gene is located on chromosome6, whereas ET-2 and ET-3 genes are located onchromosome 1 and 20, respectively [8]. IncreasedET-1 levels are believed to be exclusively due toregulation of gene transcription, as there are noknown intracellular storage mechanisms for ET-1[5,6,9]. The expression of ET-1 mRNA can be inducedby a variety of growth factors and cytokines, includingthrombin, transforming growth factor (TGF)-β,tumour necrosis factor-α (TNF-α), immunoglobin 1and insulin [10-12]. Other vasoactive substances thatcan increase ET-1 expression include noradrenaline,angiotensin II, vasopressin and bradykinin [13].Inhibitory factors include endothelium-derived nitricoxide (NO), heparin via inhibition of PKC and prosta-cyclin [14,15]. ETs are secreted in the abluminal side ofthe endothelium and circulating ET-1 is rapidlycleared from the blood by kidney, liver and lung [16].

ETs interact with a group of well characterisedreceptors, known as ETA, ETB and ETC, of which onlyETA and ETB are found in mammals [5,6,17]. The ETAreceptor has a greater affinity for ET-1 than ET-2 and agreater affinity for ET-2 than ET-3, while ETB receptorhas shown equal affinities for all three ETs [6,17,18].Both ET receptors, when activated, stimulatephospholipase C activity and generate inositoltrisphosphate and diacylglycerol leading to Ca2+

mobilisation. ETB receptors in some cells are alsolinked to inhibitory G-proteins. Activation of such apathway may lead to cyclic AMP inhibition andNa+-H+-antiporter activation. [5,6,17]. ETA receptorsare found primarily in vascular smooth muscle cells,where they mediate vasoconstriction and mitogenesis[19]. The ETB receptor has dual functions and hasbeen shown to cause both vasoconstriction andvasodilatation [17,18]. Activation of the ETB receptorcan cause an endothelium-dependent vasodilatation,primarily through the release of NO [20]. However, theETB receptor has further been shown to causevasoconstriction [21]. Hence, in vivo response of ET-1may be variable depending on the number andactivity of ETA or ETB receptors. Moreover, in diabetesthese receptors may show tissue-specific andduration-dependent alteration, further modulatingeffects of ET.

2.2 ET antagonism

Due to their widespread biological activity, theimportance of ETs is increasingly being identified inseveral disease processes. ETs are thought to playimportant pathophysiological roles in many disordersaffecting heart and microvasculature, such as conges-tive heart failure, systemic and pulmonaryhypertension, cerebral vasospasm, migraine, acuterenal failure and chronic complications of diabetes.ET receptor antagonists are, therefore, potentialtherapeutic agents to counteract pathogenetic effectsof ETs.

Several peptide and non-peptide ET antagonists withtheir potential clinical uses [18,22-38] are described inTable 1 and Table 2. Many compounds act via aspecific receptor (ETA or ETB), while others worknon-selectively on both receptors with variousaffinity. As discussed in the above section, both ETAand ETB receptors are responsible for theET-mediated pathophysiological changes. Thetherapeutic target in each disease process may varydepending on the type of ET receptors involved in themediation of the particular pathophysiological event.In addition, the blockade of one receptor may becompensated by the other, due to the possible cross-talk between the two receptors. Hence, in someinstances the dual ETA/ETB receptor antagonists mayhave more therapeutic potential over the selectivereceptor antagonists [18]. In addition, somenon-peptide ET antagonists have the clinicaladvantages over the shortcomings of the peptidegroups. As evident from Table 1, some of the drugsare in Phase II and Phase III stages of clinical trials.Hence it is possible that ET antagonists could be atherapeutic approach or adjuvant-targeted approachfor several disease processes.

In the following sections we discuss the pathophysio-logical changes in chronic diabetic complications andidentify the role of ETs in the pathogenesis of some ofthese changes. The data from preclinical studies areinstrumental in establishing potential therapeuticroles of ETs in the treatment of chronic diabeticcomplications.

3. Pathological changes in chronic diabeticcomplications

Chronic complications of diabetes affect several targetorgans, producing a variety of lesions. Although thereare some similarities in the pathogenetic mechanisms,


Chakrabarti, Cukiernik, Mukherjee & Chen 2875

the pathological changes and their functional signifi-cance in various target organs of diabeticcomplications vary enormously. In this section we willoutline major pathological features of chronic diabeticcomplications in various organs.

3.1 Retinopathy

Diabetes is the most important systemic diseasecausing blindness [39,40]. Among the several riskfactors for the development of diabetic retinopathy,poor glycaemic control is the primary pathogeneticfactor leading to clinical retinopathy and othercomplications of diabetes [40-43].

Diabetic retinopathy is classified in various progres-sive stages, namely non-proliferative (background)retinopathy, pre-proliferative (severe or advancedbackground) retinopathy and proliferative retino-pathy. Background retinopathy consists of capillarymicroangiopathy, retinal haemorrhages and exudate,macular oedema and soft exudate (cotton wool spots)[40]. Venous dilatation and beading, profuse retinalhaemorrhages and exudate, widespread capillarynon-perfusion and intraretinal microvascularabnormalities are features of pre-proliferative retino-pathy. Patients with such lesions are prone to developproliferative retinopathy. Neovascularisation is thecharacteristic feature of the proliferative stage. Thesenew vessels may lead to bleeding and tractionalretinal detachment.

Capillary basement membrane thickening is an earlyand characteristic change in diabetic retinopathy, aswell as diabetic complications in other organs [4]. At

the cellular level, the loss of capillary pericytes byapoptosis has been described as one of the earliesthistopathological abnormalities in retinal capillaries[44,45]. Several lesions, such as microvascular flowalteration, or endothelial cell proliferation may occuras a consequence of pericyte dropout [4]. Endothelialcell loss and subsequent proliferation leads to theformation of microaneurysms and subsequentneovascularisation [46,47]. In addition, rheologicalperturbation also occurs in the retina in diabetesaffecting several anti- and procoagulant factors[47,48].

Functional abnormalities involving retinal blood flow,increased permeability and alteration of retinalelectrophysiological properties have been widelystudied. In human diabetes, retinal blood flow isinitially decreased, followed by an increase whenbackground retinopathy is present [4]. Assessments ofthe retinal blood flow in the diabetic rat have resultedin discrepancies as to whether blood flow increases ordecreases [50,51]. Various pharmaceutical agents haveshown to prevent retinal blood flow abnormalities[52-60]. Hard exudate, seen in background retino-pathy, is formed as a result of increased permeability.Vascular endothelial growth factor (VEGF) hasrecently been established as an important mediator ofincreased retinal vascular permeability, acting via aPKC-dependent mechanism [61]. Retinal functionalabnormalities in early diabetic retinopathy furtherinclude abnormalities of the electroretinogramsuggesting early neuronal functional abnormalitiesand prolongation of the latencies of the visual evokedpotentials [49-53].



Table 2: Description of peptide endothelin antagonists currently in clinical development.

Drug name Manufacturer Country/clinical status Indications Ref.

ETA & ETB antagonists

PD-145065 Pfizer, Inc. US/C1US/DR

Hypertension, pancreatitis [34]

PD-156252 Parke-Davis Co. US/NDR Hypertension [35]

TAK-044 TAP Pharmaceuticals Takedachemicals Ind. Ltd.

US/C2Japan/C2

Renal failure, hypertension,myocardial infarction

[36]

ETA antagonists

BQ-123 Merck & Co. Inc. BanyuPharmaceutical

US/RTJapan/C1(discontinued)

Hypertension, peripheralvascular disease, hypertension

[37]

BQ-788 Banyu Pharmaceutical Japan/discontinued Hypertension [37]

FR-139317FK-139317

Fujisawa Pharm. Co. Japan/discontinued Cardiovascular disease,inflammation, renal disease,hypertension

[38]

C1: Phase I; C2: Phase II; C3: Phase III; DR: Discovery; NDR: No development reported; RT: Research tool.

3.2 Nephropathy

Diabetes is the most important cause of renal failure inindustrialised countries [66]. It affects approximately30% of Type 1 diabetic patients [67]. Microalbuminuriais the earliest clinical marker of renal affection indiabetes and is associated with glomerular hyperfiltra-tion [68]. As renal damage progresses, the patientsdevelop macroalbuminuria and reduced glomerularfiltration rate.

Characteristic pathological features of diabeticnephropathy consist of thickening of glomerularcapillary basement membrane, mesangial matrixexpansion and tubulointerstitial fibrosis. However,earlier in diabetes there is renal enlargement due tocellular hypertrophy affecting both the glomeruli andtubules. With increasing duration of diabetes, thepatients develop arteriolosclerosis. The glomerularfiltration rate continues to decline due to continuedmesangial matrix expansion and glomerulosclerosiswith obliteration of the filtration area due to increasedproduction of extracellular matrix (ECM) proteins andtheir decreased degradation. The tubulointerstitialfibrosis worsens. In the later stage, patients developcharacteristic nodular accumulations of ECM proteins,i.e., Kimmelstiel-Wilson nodules [69-72]. Clinically theovert nephropathy manifests as proteinuria in thenephrotic range, hypertension and other features ofrenal failure. Similar to other chronic complications, ithas been demonstrated that high blood glucose levelis a key factor leading to the development of diabeticnephropathy [42,43,73]. It has further been shown thattight blood glucose control may even reverse thestructural changes in the kidneys [74]. Systemichypertension, which leads to high intraglomerularhypertension and hyperfiltration, is another risk factorin the progression of nephropathy, as it promotestissue destruction and sclerosis. In recent years, TGF-βhas been established as a major factor producing renallesions in diabetes [75].

3.3 Neuropathy

About 60 - 70% of diabetics have mild to severe formsof nerve damage. The severe form of diabetic nervedisease is a major cause of lower extremity amputa-tion [74]. Diabetic neuropathy can affect both somaticand autonomic nerves, causing a variety of symptoms.Diabetic neuropathy can broadly be classified asmononeuropathy or polyneuropathy. Mononeuro-pathies can involve isolated single nerves or mayaffect multiple nerves (mononeuritis multiplex).These neuropathies may affect peripheral or cranial

nerves [76,77]. Polyneuropathies can affect sensory,motor or autonomic nervous system. Acute sensoryneuropathy clinically manifests as a painful conditionwith complete recovery. Proximal motorneuropathies, also known as amyotrophy, aremanifested as acute onset of pain and weakness ofproximal muscle. Chronic sensorimotor polyneuro-pathy is the commonest type of neuropathy. It ismanifested as progressive gloves and stockinganaesthesia, paresthesia or hyperasthesia, impairedbalance, proprioception and vibration. Althoughmotor weakness is not pronounced, wasting of smallmuscle and loss of reflex activity is also manifested.Foot ulceration and other neuropathic changes maysubsequently develop. Electrophysiologicallyimpaired nerve conduction velocity is a key feature.Autonomic neuropathy may produce bladder, bowelor gastric motility problems and postural hypotension[76,77].

3.4 Cardiovascular complications:atherosclerosis, hypertension, cardiomyopathy

Both atherosclerosis and hypertension occur at amuch higher rate in the diabetic population comparedwith their non-diabetic counterpart [78]. In addition,insulin resistance, if present, accelerates atheroscle-rosis [79]. Progressive atherosclerotic occlusion maylead to significant loss of blood supply to the organs.Along with hyperglycaemia, abnormalities of lipidmetabolism may have additional effects.

Cardiac complications are a major cause of morbidityand mortality in the diabetic population. Diabeticindividuals are two- to four-times more likely to haveheart disease compared with their non-diabeticcounterparts and 75% of diabetes-related deaths aredue to heart disease [80]. Three major aspects ofcardiac involvement in diabetes include coronaryatherosclerosis, diabetic cardiomyopathy andautonomic neuropathy. In addition, for poorlyunderstood reasons, diabetic patients developcongestive cardiac failure more readily and havesignificantly worse prognosis than their non-diabeticcounterparts once they develop coronary disease[81-83]. Clinically, 40 - 50% of diabetics withoutpreviously known cardiac disease manifestabnormalities of left ventricular mechanical function,primarily affecting diastolic properties. These changesmanifest clinically as failure of left ventricular contrac-tile function and prolonged relaxation [81-83].Pathological findings include cardiomegaly andmyocardial fibrosis. Myocardial hypertrophy,



interstitial and perivascular fibrosis, myocyte necrosisas well as thickening of the capillary basementmembrane are some other abnormal structuralfindings in the heart in diabetes [81-83]. Cardiacautonomic neuropathy may manifest as prolongationof R-R interval in the electrocardiogram [84].

4. Pathogenesis of chronic diabeticcomplications

Studies in animals, as well as major epidemiologicalstudies in human diabetes, have shown that hypergly-caemia is the primary insult in the pathogenesis ofchronic diabetic complications [4,85-87]. Severalmetabolic perturbations may be occur secondary tohyperglycaemia, leading to cellular dysfunction. It ishowever to be pointed out that although there aresome general similarities as to the occurrence of theseabnormalities, there are several tissue-specificabnormalities. This review will briefly comment onsome of these pathways.

4.1 Polyol pathway and its consequences

Augmented polyol pathway activity is an importantmechanism in the pathogenesis of chronic diabeticcomplications. Secondary to hyperglycaemia, aportion of the intracellular glucose is reduced tosorbitol by the enzyme aldose reductase (AR). Sorbitolis further oxidised to fructose by sorbitol (polyol)dehydrogenase (SD). Accumulated sorbitol may leadto osmotic injury and cell death [85,86]. Otherconsequences of the polyol pathway activation, otherthan the direct accumulation of sorbitol, may be ofimportance [85-88]. The depletion of other osmolytes,secondary to the intracellular accumulation ofsorbitol, may be associated with the perturbed cellularand metabolic functions [85-88]. AR uses NADPH as aco-factor and SD uses NAD+ as hydrogen acceptor.Hence, AR and SD stochiometrically oxidise NADPand increase NADH/NAD+ ratios, causing a redoximbalance and a state of pseudohypoxia in the targetorgans of diabetic complications [4,85]. Increasedglycolysis may further contribute to the increasedNADH/NAD+ ratio [52,85]. This change may favourincreased free radical generation, inhibition of fattyacid oxidation, increased DAG synthesis, increasedformation of reduced glutathione [89-90], increasedprostaglandin synthesis, decreased NO synthesis anddefective DNA repair [52,85]. Increased DAGsynthesis further activates PKC [4,6,8,52]. However,controversies exist regarding the development of

‘pseudohypoxia’ in some organs such as the retina[91,92].

4.2 Oxidative damage

Increased oxidative stress due to free radicals and NOgeneration have been suggested to play a significantrole in the pathogenesis of diabetic complications [93].Elevated glucose in a cell-free system can generatefree radicals [94]. The hydroxyl radical produced byglucose auto-oxidation has been shown to damageproteins [95]. Hyperglycaemia may induce andactivate various lipoxygenase enzymes, promotingthe interaction of NO with superoxide anionsproducing peroxynitrite and hydroxyl radicals [93,96].Peroxinitrate is toxic to the endothelial cell [97]. Inaddition, the anti-oxidant defence activities arereduced in experimental galactosaemic and diabeticanimals. GSH levels, Ca2+Mg2+-ATPase activity andNa+K+-ATPase activities were markedly diminished inboth diabetic and galactose-fed animals; the latter is ahyperhexosaemic normohormonal model of diabeticcomplication [89,90,98]. Non-enzymatic glycationmay lead to direct oxidative stress as well as the inacti-vation of superoxidase dismutase (SOD) [4,46,93,99].The administration of vitamins C and E was successfulin normalising the anti-oxidant defence mechanismsof diabetic rats. Oxidative damage may further affectseveral metabolic pathways. It has recently beendemonstrated that hyperglycaemia-induced increasedproduction of superoxides may lead to PKC activa-tion, increased non-enzymatic glycation, sorbitolaccumulation and NF-κB activation in endothelialcells [100].

4.3 DAG-PKC pathway

PKC exists as several distinct isoforms, with differentenzymatic properties, functions and distributions.Based on their Ca2+ and phorbol ester sensitivitiesthey have been classified into three subgroups:classical (α,ρ,γ), novel (δ,ε,η,θ), atypical (ι ,λ) and theexistence of a fourth group (µ) has been recentlyconsidered [101,102]. Increased PKC activities areseen in several target organs of diabetic complications[4,87]. High glucose levels lead to a de novo increasein DAG synthesis, which is a potent PKC activator[4,87]. PKC activation may be further activated via thePI3 kinase pathway [103]. It has been demonstratedthat hyperhexosaemia induces increased DAG levelsand PKC activation in the retina and aorta of bothdiabetes and galactosaemia, as well as decreasedNa+K+-ATPase and Ca2+Mg2+-ATPase activity in these



tissues [89,60,104,105]. Augmented PKC anddecreased Na+K+-ATPase activity in the retina of thestreptozotocin (STZ)-induced diabetic rat can beprevented by treatment with a specific inhibitor of theβ-isoform of PKC [60,106]. PKC influences severalimportant vascular functions such as blood flow andpermeability [4,87,107].

PKC isoforms have been shown to be an importantregulator of several factors responsible for cellsurvival and growth such as ET-1, VEGF, platelet-derived growth factor (PDGF), epidermal growthfactor (EGF), insulin-like growth factor (IGF) andfibroblast growth factor (FGF) [4,87,101]. IncreasedPKC may lead to increased or decreased NO genera-tion in a tissue-specific way [4,85,87,108]. Thesegrowth factors are of further importance in mediatingthe later effects of diabetic retinopathy such asendothelial cell proliferation and neovascularisation[4,85,87,109].

4.4 Non-enzymatic glycation

Non-enzymatic modification of tissue proteins byphysiological hexoses in vivo is an importantsecondary mechanism in the pathogenesis of chronicdiabetic complications [4,110]. Glucose, glucose-6-phosphate, trioses and fructose generated asend-products of the polyol pathway, as well asproducts of the pentose phosphate pathway, take partin non-enzymatic glycation of proteins [4,111].Advanced glycation end-products (AGEs) may furtherform by strong glycating dicarbonyl compounds suchas 3-dioxyglucosone, methylglyoxal and glyoxals[112]. AGEs accumulate in the tissue over time andsugar concentrations both in ageing and diabetespatients, being augmented in the latter [4,110].

The mechanisms by which AGEs may causepathological changes include intracellular alterationof protein function, interference with ECM functionand abnormalities secondary to the increasedcytokine and free radical formation through interac-tions with several AGE specific receptors [4,110,113].Auto-oxidation of glucose and AGE can producereactive oxygen species [4,114]. AGE and AGEreceptor interaction may lead to oxidative stress andactivation of NF-κB [116]. In vascular endothelial cells,AGE formation may affect the gene expression ofthrombomodulin, ET-1 and modify growth factorssuch as VEGF and bFGF [4,116,117]. In the microvas-culature, AGEs may induce permanent abnormalitiesof ECM proteins [118]. AGEs alter signal transduction

pathways and the levels of soluble signals such ascytokines, hormones and free radicals, which candirectly affect protein and DNA functions in targettissues [4,116]. AGE formation has been causallyrelated to defective vasodilator response to NO [119].Anti-oxidant treatments have been shown to inhibitreactive oxygen species as well as glucose-inducedAGE formation [4,120].

4.5 Role of endothelins in chronic diabeticcomplications

ETs have been implicated in a variety of vasculardysfunctions, including those observed in patientswith diabetes mellitus. Clinically, patients with Type 1diabetes exhibited elevated abnormalities of plasmaET-1 levels, ranging from increased, unchanged todecreased. Similar data have been obtained fromType 2 diabetics [121]. Variability of the durationand/or the level of metabolic control, effects of insulininjection and the methodologies used for ET measure-ment have been suggested as possible contributingfactors [121]. It is not clear how plasma ET-1 levelscorrelate with occurrence of diabetic complications.On the other hand, as ETs act as a paracrine orautocrine factors, plasma levels of the peptides maynot be a good indicator of its biological activity. Invitro studies have shown that high glucose can causeincreased production of ET-1 in cultured endothelialcells [122,123]. Insulin also stimulates ET-1 productionin endothelial cells [124].

Several of the aforementioned metabolic abnormali-ties may alter ET production and/or action in diabetes.As outlined earlier, high glucose causes PKC activa-tion via increased DAG synthesis. An increased ratioof NADH/NAD+, due to an augmented polyolpathway, also favours DAG synthesis [87]. Activationof PKC has been demonstrated in several organs indiabetes [4,87]. Specifically, PKC-β isoform is ofimportance in this respect [4,60,87]. PKC-β overex-pressing mice have been shown to produce lesionshistologically similar to those seen in the myocardiumin diabetes [125]. However, several other mechanismsmay be of importance in augmented ET-1 expressionin diabetes. ET-1 interacts with other potent vasoac-tive substances such as NO and VEGF [126-128]. VEGFis increased in diabetes secondary to PKC activationand in turn, may increase ET-1 expression [123].Non-enzymatic glycation and oxidative stress maylead to reduced NO production and subsequentincreased ET expression [93]. Recently it has furtherbeen demonstrated that AGE formation in the



endothelial cells can increase ET-1 mRNA expressionvia NF-κB [129].

ETs are present and widely distributed throughoutvarious tissues in the body. In ocular tissues ET-1 ispresent in several areas including the optic nerve,vascular and extravascular sites in the retina and theuveal tract [130-132]. Along with other investigators,we have demonstrated increased ET-1 and ET-3immunoreactivities in the retina of STZ-induced ratsand the spontaneously diabetic BB/W rat [130-132].Blockage of the ETA receptor, utilising a specificblocker, BQ-123, increases retinal blood flow inSTZ-induced diabetic rats [133]. Furthermore,blocking of ET-converting enzyme-1 (ECE-1) byphosphoramidon normalised diabetes-inducedreduced retinal blood flow [133]. Using laser Dopplersonography, we have recently demonstratedincreased resistivity index (RI), a marker for vasocon-striction in the retinal bed in both STZ-induceddiabetic and galactose-fed rats after one month offollow-up. These changes were prevented by a dualET-receptor blocker bosentan [134,135]. Simultane-ously, ET-1, ET-3 and ETA mRNA levels wereincreased in hyperhexosaemic rats. After six monthsof follow-up, ETB receptor mRNA was also elevated inthe retina of hyperhexosaemic animals and the RIvalues were not different from the non-diabeticcontrols [134,135]. These observations may be indica-tive of a duration-dependent, differential activation ofvarious components of the ET-system, accounting forvariable alteration in retinal blood flow.

Recently, we have demonstrated that after one monthof hyperhexosaemia in diabetic and galactose-fedrats, increased production of collagen and fibronectinmRNA can be prevented by bosentan treatment. In thesame study, after six months of follow-up,hyperhexosaemia-induced increased retinal capillarybasement membrane thickening was prevented by ETreceptor antagonism [136].

Increased plasma ET-1 levels have been demonstratedin diabetic patients with retinopathy [137]. However,in a recent report, vitreous humour ET-1 levels werefound to be reduced in patients with proliferativediabetic retinopathy [138].

In the kidney, both ET-1 and ET-3 show widespreadtissue distribution [139]. From a physiologicalperspective, ETs have roles in the regulation of renalblood flow, GFR, sodium and water reabsorption. ETsare expressed in the endothelium, epithelium as wellas in the mesangium in the glomeruli, in the tubular

epithelium and in the collecting ducts and vasa recta.ET-binding sites have been localised in these cells andin interstitial cells. ET system has been demonstratedto be altered in several disease processes affecting thekidneys [5,139].

In the kidney of diabetic rats, increased ET-1 mRNAand increased renal ET-1 clearance in association withproteinuria has been demonstrated [140]. IncreasedETA receptors are present in the kidneys of diabeticrabbits [141]. The long-term consequences ofET-peptides may involve cellular changes requiringdifferential gene expression and contribute tolong-term nuclear signalling [5-7,142]. It has beendemonstrated that diabetes-induced increasedexpression of glomerular α1(I), α1(III), α1(IV)collagen, laminin B1 and B2, TNF-α, PDGF, TGF-βand basic FGF can be completely blocked bytreatment with an ETA receptor antagonist [142].Mitogenesis induced by the increased production ofETs may be altered under hyperglycaemic conditions.Previous studies have implicated ETs in having aregulatory link with the components of the ECM, sincerat mesangial cells showed that ET-1 can increase theproduction of ECM components such as laminin andcollagen α1 (IV) [143].

In the heart, ET-1 is produced by both myocytes andendothelial cells and high affinity ET-1 binding sitesare present in the cardiac myocytes [144-146]. ET-1evokes positive inotropic and chronotropic effectsand prolongation of action potential [147,148].Hypoxia and ischaemia are two important upregula-tors of ET-1 expression in the heart [149]. Severalinvestigators have shown that ET-1 mRNA andreceptor binding are elevated in the rat heart indiabetes, even after a few weeks duration [149,151].Furthermore, a duration-dependant alteration ofchronotropic and ionotropic response to ET-1 has alsobeen demonstrated in the isolated atria from thediabetic rat [152]. We have demonstrated that after sixmonths of diabetes, myocardial cell death and focalscarring of the myocardium are associated withincreased expression of ET-1 and both ETA and ETBreceptor mRNA as well as increasedET-immunoreactivity and ET receptor density. Inaddition, a similar increased mRNA expression for twoECM proteins was detectable. The increased ECMprotein mRNA, myocardial scarring as well asincreased apoptosis were completely prevented bytreatment of diabetic animals with ET-receptorantagonist bosentan. This study provided directevidence as to the importance of the ET system in the



pathogenesis of diabetic heart disease [153]. SinceET-1 is produced by the endothelial cells and smoothmuscle cells contain ET receptors, increased produc-tion of ET-1 in diabetes leads to mitogenesis andsmooth muscle cell proliferation and acceleratedatherosclerosis. Increased plasma ET-1 levels havebeen demonstrated in diabetic patients with athero-sclerosis and hypertension [154,155].

Impaired phosphoinositide metabolism in peripheralnerves may lead to reduced activity of PKC andNa+K+-ATPase [156]. This is in sharp contrast to theretinal findings, where an activation of PKC has beenestablished [4,87]. Peripheral nerves from diabeticanimals show reduced DAG levels [156,157]. Concep-tually impaired phosphoinositides, in diabetes, mayaffect ET receptor-mediated signal transduction. Inspite of this, PKC inhibitors prevent diabetes-inducedreduced neuronal Na+K+-ATPase [158]. These datademonstrate that biochemical changes in hypergly-caemia may vary in target organs of diabeticcomplications, depending on the tissue microenviron-ment. Nerve conduction velocity deficit and reducedendoneurial blood flow in STZ-induced diabetic ratswere prevented by a specific ETA antagonist, as well

as by a blockade of both ETA and ETB receptors[159,160]. It is interesting to note that in these experi-ments, only specific ETA antagonist treatment lead to areduction in the systemic blood pressure [159]. Wehave demonstrated that immunoreactive ET-1 andET-3 are increased in the peripheral nerve in diabetes[161]. However, no data are yet available as towhether ET blockade is beneficial in preventing laterchanges in diabetic neuropathy such as nerve fibreloss.

Complex interactions have been demonstratedamong various vasoactive factors in diabetes, whichare of importance in the pathogenesis of diabeticcomplications. As outlined in the earlier sections,metabolic alterations in diabetes, such as augmentedpolyol pathway, PKC activation, non-enzymaticglycation, as well as oxidative damage may lead,directly or indirectly, to the generation and/or activa-tion of several vasoactive factors, which may furtherinteract with each other (Figure 1). There are variouslines of evidence demonstrating interaction betweenPKC and vasoactive factors in diabetes. ETs beingpeptides interact with PKC and NO [5,6]. ET upregula-tion may be caused by hyperglycaemia-induced PKC



Glucose

Non-enzymaticglycation

PKC Oxidative stressPolyol pathway

Endothelin

NAD/NADH VEGF NONF- B

Figure 1: Mechanisms leading to alterations of endothelins in diabetes.NAD/NADH: Nicotine adenine dinucleotide; NF-κB: Nuclear factor-κB; NO: Nitric oxide; PKC: Protein kinase C; VEGF: Vascularendothelial growth factor.

κ

activation [4,5,6]. Conversely, ET-1 is involved in theinduction of AP-1 regulatory sites, which in turnactivates PKC [7,162,163]. ET-1 facilitates PKC translo-cation from the cytosol to the membrane andincreases DAG production [162,163]. It has beendemonstrated in cerebral capillaries that ET-1st imulates Na+K+-ATPase act ivi ty by aPKC-dependent pathway [164]. Furthermore, PKCactivation in a tissue selective fashion may increase ordecrease NO [4,85,87]. It has further been shown thatα-tocopherol (a PKC inhibitor) can prevent diabetes-induced endothelial relaxation [165].

ET-1 has a positive feedback regulatory effect on NOsynthesis, which has an inhibitory effect on ET-1synthesis [5,6,123,126]. A co-stimulatory interactionfurther exists between ET-1, ET-3 and VEGF [123,128].

PKC is an important regulator of VEGF production inthe target organs of diabetic complications and inhibi-tion of the β-isoform of PKC has been shown toprevent VEGF expression and diabetes-inducedincreased permeability in the retina [61]. VEGF alsoincreases preproET mRNA [127,128]. It has furtherbeen shown that in endothelial cells, both ET-1 andET-3 stimulate VEGF production via a PKC-dependantmechanism [127,128]. Of relevance to this review, indiabetes, activation of the polyol pathway limits NOsynthesis, hence limiting the inhibitory effect of NOon ET-1, resulting in increased ET-1 production [85].In human umbilical vein endothelial cells (HUVECs)we have demonstrated that high glucose levels as wellas VEGF or NO synthase inhibition causes increasedexpression of ET-1 mRNA and protein [123]. Suchglucose-induced ET-1 increases were howeverprevented by VEGF-neutralising antibodies and PKCinhibition [123]. These interactions probably representregulatory mechanisms controlling the availability ofthese peptides. For example, increased ET-1 in earlydiabetes may lead to hypoxia and subsequent releaseof VEGF producing increased permeability. In thesame context, as VEGF induces endothelial cell prolif-eration and ET-1 causes smooth muscle cellproliferation, together they may have important rolesin diabetic micro- and macroangiopathy. Among theother factors, as discussed previously, ET-1 increasesbFGF and TGF-β mRNA expression which in turninduce NO formation [166,167]. NO on the other hand,has a stimulatory effect on VEGF synthesis [168].

5. Expert opinion

Data gathered to date suggest that the pathogenesis ofchronic diabetic complication is indeed a complexphenomenon. Several factors may simultaneously beactivated in response to hyperglycaemia and anintricate interplay occurs among such factors. Vasoac-tive factors like ET, due to their multiple functionalcapabilities, may play significant roles as effectormolecules in chronic diabetic complications. PKCactivation, non-enzymatic glycation, oxidativedamage, as well as polyol pathway activation may allin part contribute to the alteration of ETs. ETs in turnmay effect other vasoactive factors. Evidence gatheredfrom multiple animal experiments in several laborato-ries, indeed, indicates that ETs are of importance inthe pathogenesis of several chronic diabetic compli-cations. ET antagonism, based on the present data, isindeed a potential therapeutic modality in thetreatment of chronic diabetic complications such asretinopathy, nephropathy, neuropathy and cardiovas-cular complications. These data must be furtherconfirmed by additional long-term studies in experi-mental animal models, which should include bothfunctional and structural analyses. However, ultimatetherapeutic efficacy of ET antagonism in diabeteswould depend on availability of safe ET-antagonistswith their therapeutic efficacy validated bywell-conducted clinical trials.

Acknowledgements

The authors acknowledge the grant support from theCanadian Diabetes Association in honour of FlorenceLangille and from the Heart and Stroke Foundation ofOntario.

Bibliography

1. BENIGNI A, PERICO N, REMUZZI G: Endothelin antago-nists and renal protection. J. Cardiovasc. Pharmacol.(2000) 35(Suppl. 2):S75-78.

2. YANAGISAWA M, KURIHARA H, KIMURA S et al.: A novelpotent vasoconstrictor peptide produced by vascularendothelial cells. Nature (1988) 332:411-415.

3. INOUE A, YANAGISAWA M, KIMURA S et al.: The humanendothelin family: three structurally and pharmaco-logically distinct isopeptides predicted by threeseparate genes. Proc. Natl. Acad. Sci. USA (1989)86:2863-2867.



4. KING GL, BROWNLEE M: The cellular and molecularmechanisms of diabetic complications. Endocrinol.Metab. Clin. North Am. (1996) 25:255-270.

5. RUBANYI GM, POLOKOFF MA: Endothelins: Molecularbiology, biochemistry, pharmacology, physiology andpathophysiology. Pharmacol. Rev. (1994) 46:325-414.

6. LEVIN ER: Endothelins. N. Engl. J. Med. (1995)333:356-363.

7. SIMONSON MS, JONES JM, DUNN MJ: Differential regula-tion of fos and jun gene expression and AP-1 Ciselement activity by endothelin isopeptides: Possibleimplications for mitogenic signalling by endothelin. J.Biol. Chem. (1992) 267:8643-8649.

8. INOUE A, YANAGISAWA M, KIMURA S et al.: The humanendothelin family: Three structurally and pharmaco-logically distinct isopeptides predicted by threeseparate genes. Proc. Natl. Acad. Sci. USA (1989)86:2863-2867.

9. KIRCHENGAST M MUNTER K: Endothelin-1 andendothelin receptor antagonists in cardiovascularremodelling. PSEBM (1999) 221:312-324.

10. EMORI T, HIRATA Y, IMAI T et al.: Cellular mechanismsof thrombin on endothelin-1 biosynthesis and releasein bovine endothelial cells. Biochem. Pharmacol. (1992)44:2409-2411.

11. KURIHARA H, YOSHIZUMI M, SUGIYAMA T et al.:Transforming growth factor beta stimulates theexpression of endothelin mRNA from vascularendothelial cells. Biochem. Biophys. Res. Commun. (1989)159:1435-1440.

12. MARSDEN PA, BRENNER BM: Transcriptional regulationof the endothelin gene by TNF alpha. Am. J. Physiol.(1992) 262:854-861.

13. MIYAUCHI T, MASAKI T: Pathophysiology of endothelinin the cardiovascular system. Ann. Rev. Physiol. (1999)61:391-415.

14. EMORI T, HIRATA Y, IMAI T, EGUCHI S, KANNO K,MARUMO F: Cellular mechanism of natriureticpeptides-induced inhibition of endothelin-1 biosyn-thesis in rat endothelial cells. Endocrinology (1993)133:2474-2480.

15. IMAI T, HIRATA Y, EMORI T, MARUMO F: Heparin has aninhibitory effect on endothelin-1 synthesis andrelease by endothelial cells. Hypertension (1993)21:353-358.

16. GASCI S, WAGNER OF, VIERHAPPER H, NOWOTNY, P,WALDHAUSL W: Regional hemodynamic effects andclearance of endothelin-1 in humans: renal andperipheral tissue may contribute to the overalldisposal of the peptide. J. Cardiovasc. Pharmacol. (1992)19:176-180.

17. SAKURAI T, YANAGISAWA M, MASAKI T: Molecularcharacterization of endothelin receptors. TrendsPharmacol. Sci. (1992) 13:103-108.

18. ROUX S, BREU V, ERIEL SI, CLOZEL M: Endothelinantagonism with bosentan: a review of potentialapplications. J. Mol. Med. (1999) 77:364-376.

19. OHLSTEIN EH, DOUGLAS SA: Endothelin-1 modulatesvascular smooth muscle structure and vasomotion:implications in cardiovascular pathology. Drug. Dev.Res. (1993) 29:108-128.

20. DE NUCCI G, THOMAS R, D’ORLEANS-JUSTE P et al.:Pressor effects of circulating endothelin are limited byits removal in the pulmonary circulation and by therelease of prostacyclin and endothelium derivedrelaxing factor. Proc. Natl. Acad. Sci. USA (1988)85:9797-9800.

21. SEO B, OEMAR BS, SIEBENMANN R, VON SEGESSER L,LUSCHER TF: Both ETA and ETB receptors mediatecontraction to endothelin-1 in human blood vessels.Circulation (1994) 89:1203-1208.

22. DOUGLAS SA: Clinical development of endothelinreceptor antagonists. Trends Pharmacol. Sci. (1997)18(11):408-412.

23. FILEP JG: Endothelin receptor antagonists: Newperspectives in endothelin research. Drugs Today(1995) 31(3):155-171.

24. SCHIFFRIN EL: Endothelin: role in hypertension. Biol.Res. (1998) 31(3):199-208.

25. CODY WL, HE JX, DEPUE PL et al.: Structure-activityrelationships of the potent combinedendothelin-A/endothelin-B receptor antagonistsAc-DDip16-Leu-Asp-Ile-Trp21:Development ofendothelin-B receptor selective antagonists. J. Med.Chem. (1995) 38(15):2809-2819.

26. AMBERG W, HERGENRODER S, HILLEN H et al.:Discovery and synthesis of (S)-3-[2-(3,4-dimethoxyphenyl) ethoxy]-2-(4,6-dimethylpyrimidin-2-yloxy)-3,3-diphenylpropionic acid (LU-302872), anovel orally active mixed ETA/ETB receptor antago-nist. J. Med. Chem. (1999) 42(16):3026-3032.

27. OKADA M, IKEDA T, OHTA H et al.: J-104132, a potent,orally active, mixed ETA/ETB receptor antagonist,attenuated salt-sensitive hypertension. Jpn. J.Pharmacol. (1999) 79(Suppl. I):281.

28. ELLIOTT JD, EZEKIEL M, GELLAI M, DOUGLAS SA,OHLSTEIN EH: Antihypertensive effect of theendothelin receptor antagonist SB-209670. Faseb J.(1994) 8(5):A40.

29. BECK GR, JR., DOUGLAS SA, ELLIOT JD, OHLSTEIN EH:Agonist-dependent inhibition by peptide and nonpeptide endothelin receptor antagonists in the rabbitisolated pulmonary artery. J. Cardiovasc. Pharmacol.(1995) 26(Suppl. 3):S385-S388.

30. NABOKOV AV, AMANN K, WESSELS S, MUNTER K,WAGNER J, RITZ E: Endothelin receptor antagonistsinfluence cardiovascular morphology in uremic rats.Kidney Int. (1999) 55(2):512-519.

31. OPGENORTH TJ, ADLER AL, CALZADILLA SV, CHIOU WJ:Pharmacological characterization of A-127722: Anorally active and highly potent ET (A)-selective



receptor antagonist. J. Pharmacol. Exp. Ther. (1996)276(2):473-481.

32. WU C, CHAN MF, STAVROS F, RAJU B, OKUN I, CASTILLORS: Structure-activity relationships of N2-aryl-3-(isoxazolysulfamoyl)-2-thiophenecarboxamides asselective endothelin receptor-A antagonists 1. J. Med.Chem. (1997) 40(11):1682-1689.

33. LIU G, HENRY KJ, SZCZEPANKIEWICZ BG et al.: Highlyselective endothelin antagonists for ETa receptor:synthesis and structure-activity relationships. ACS213th. San Francisco, USA (1997):MEDI193.

34. BATTISTINI B, WARNER TD, FOURNIER A, VANE JR:Comparison of PD-145065 and Ro-46-2005 as antago-nists of contractions of guinea-pig airways induced byendothelin-1 or IRL-1620. Eur. J. Pharmacol. (1994)252(3):341-345.

35. CODY WL, HE JX, REILY MD et al.: Design of a potentcombined pseudopeptide endothelin-A/endothelin-Breceptor antagonist, Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21 (PD 156252): examination of itspharmacokinetic and spectral properties. J. Med.Chem. (1997) 40(14):2228-2240.

36. AWANE Y, KUSUMOTO K, KUBO K, KAWATA A, KIKUCHIT, WAKIMASU M: Pharmacological profile in vitro of anew endothelin receptor antagonist, TAK-044. Jpn. J.Pharmacol. (1994) 64(Suppl. 1):AO337.

37. LOVE MP, FERRO CJ, HAYNES WG et al.: Endothelinreceptor antagonism in patients with chronic heartfailure. Cardiovasc. Res. (2000) 47(1):166-172.

38. MIYAHARA T, KOIZUMI T, KUBO K et al.: Endothelinreceptor blockade attenuates air embolization-induced pulmonary hypertension in sheep. Eur. J.Pharmacol. (1999) 385(2-3):163-169.

39. MAZZE RS, SINNOCK P, DEEB L, BRIMBERRY JL: Anepidemiological model for diabetes mellitus in theUnited States: five major complications. Diab. Res. Clin.Prac. (1985) 1:185-191.

40. BLOM ML, GREEN WR, SCHALBAT AP: Diabetic retino-pathy: a review. Del. Med. J. (1994) 66:379-388.

41. KLEIN R, KLEIN BEK, MOSS SE, DAVIS MD, DEMET DL:Glycosylated hemoglobin predicts the incidence andprogression of diabetic retinopathy. JAMA (1998)260:2864-2871.

42. DIABETES CONTROL AND COMPLICATIONS TRIALRESEARCH GROUP: The effect of intensive treatment ofdiabetes on the development of long-term complica-tions in insulin-dependent diabetes mellitus. N. Engl. J.Med. (1993) 329:977-986.

43. SANTIAGO JV: Lessons from the diabetes control andcomplication trial. Diabetes (1993) 42:1549-1554.

44. KUWABARA T, COGAN DG: Retinal vascular patterns VI.Mural cells of the retinal capillaries. Arch. Ophthalmol.(1963) 69:492.

45. MIZUTANI M, KERN TS, LORENZI M: Accelerated death ofretinal microvascular pericytes in human and

experimental diabetic retinopathy. J. Clin. Invest. (1996)97:2883-2890.

46. KERN TS, ENGERMAN RL: Comparison of retinal lesionsin alloxan-diabetic rats and galactose-fed rats. Curr. EyeRes. (1994) 13:863-867.

47. PORTA M: Endothelium: the main actor in the remodel-ling of the retinal microvasculature in diabetes.Diabetologia (1996) 39:739-744.

48. CERIELLO A: Coagulation activation in diabetesmellitus: the role of hyperglycemia and therapeuticprospects. Diabetologia (1993) 36:1119-1125.

49. CERIELLO A, DELL RUSSO P, ZUCCOTTI C et al.:Decreased antithrombin III activity in diabetes may bedue to non-enzymatic glycosylation. A preliminaryreport. Thromb. Haemostas. (1983) 50:633-634.

50. SHIBA T, BURSELL SE, CLERMONT A, SPORTSMAN R,HEATH W, KING GL: Protein kinase C (PKC) activationis a causal factor for the alteration of retinal blood flowin diabetes of short duration. Invest. Ophthalmol. Vis. Sci.(1991) 32(Suppl.):785-789.

51. TILTON RG, CHANG K, ALLISON W, WILLIAMSON JR:Comparable diabetes induced increases in retinalblood flow assessed with conventional versusmolecular (3H-Desmethylimiprimine) microspheres.Invest. Ophthalmol. Vis. Sci. (1992) 33(Suppl.):1048-1052.

52. PUGLIESE G, TILTON RG, WILLIAMSON JR: Glucose-induced metabolic imbalances in the pathogenesis ofdiabetic vascular disease. Diabetes Metab. Rev. (1991)7:35-59.

53. TILTON RG, CHANG K, NYENGAARD JR, VAN DM, IDO Y,WILLIAMSON JR: Inhibition of sorbitol dehydrogenase.Effects on vascular and neural dysfunction instreptozocin-induced diabetic rats. Diabetes (1995)44:234-242.

54. TILTON RG, CHANG K, HASAN KS et al.: Prevention ofdiabetic vascular dysfunction by guanidines. Inhibi-tion of nitric oxide synthase versus advancedglycation end-product formation. Diabetes (1993)42:221-232.

55. TILTON RG, CHANG K, PUGLIESE G et al.: Prevention ofhemodynamic and vascular albumin filtrationchanges in diabetic rats by aldose reductase inhibitors.Diabetes (1989) 38:1258-1270.

56. HASAN KS, CHANG K, ALLISON W et al.: Glucose-inducedincreases in ocular blood flow are prevented byaminoguanidine and L-NMMA, inhibitors of nitricoxide synthase. Invest. Ophthalmol. Vis. Sci. (1993)34(Suppl.):1127-1133.

57. WILLIAMSON JR, CHANG K, ALLISON W, KILO C:Endoneurial blood flow changes in diabetic rats.Diabet. Med. (1993) 10(Suppl. 2):49S-51S.

58. KUNISAKI M, BURSELL SE, CLERMONT AC et al.: Vitamin Eprevents diabetes-induced abnormal retinal bloodflow via the diacylglycerol-protein kinase C pathway.Am. J. Physiol. (1995) 269:E239-E246.



59. CLERMONT AC, BRITTIS M, SHIBA T, MCGOVERN T, KINGGL, BURSELL SE: Normalization of retinal blood flow indiabetic rats with primary intervention using insulinpumps. Invest. Ophthalmol. Vis. Sci. (1994) 35:981-990.

60. ISHII H, JIROUSEK MR, KOYA D et al.: Amelioration ofvascular dysfunctions in diabetic rats by an oral PKCbeta inhibitor. Science (1996) 272:728-731.

61. AIELLO LP, BURSELL SE, CLERMONT A et al.: Vascularendothelial growth factor-induced retinalpermeability is mediated by protein kinase C in vivoand suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes (1997) 46:1473-1480.

62. HOTTA N, KOH N, SAKAKIBARA F et al.: Effect ofpropionyl-L-carnitine on oscillatory potentials inelectroretinogram in streptozotocin-diabetic rats. Eur.J. Pharmacol. (1996) 311:199-206.

63. HOTTA N, KOH N, SAKAKIBARA F et al.: Effect of analdose reductase inhibitor on abnormalities ofelectroretinogram and vascular factors in diabeticrats. Eur. J. Pharmacol. (1997) 326:45-51.

64. SEGAWA M, HIRATA Y, FUJIMORI S, KADA K: Thedevelopment of electroretinogram abnormalities andpossible roles of polyol pathway activity in diabetichyperglycemia and galactosemia. Metabolism (1988)37:454-459.

65. MACGREGOR LC, MATCHINSKY FM: Treatment with analdose reductase inhibitor or with myo-inositolarrests deterioration of electroretinogram of diabeticrats. J. Clin. Invest. (1985) 76:887-889.

66. HELD PJ, PORT FK, BLAGG CR, AGODA LYC: United SatesRenal Data System 1990 annual report. Am. J. KidneyDis. Suppl. (1990) 2:1-106.

67. ANDERSEN AR, CHRISTIANSEN JS, ANDERSON JK,KREINER S, DEKERT T: Diabetic neuropathy in Type I(insulin-dependent) diabetes: An epidemiologicalstudy. Diabetologia (1983) 25:496-501.

68. BREYER JA, BAIN RP, EVANS JK et al.: Predictors of theprogression of renal insufficiency in patients withinsulin-dependent diabetes and overt diabetic nephro-pathy. Kidney Int. (1996) 50:1651-1658.

69. MOGENSEN CE, CHRISTENSEN CK, VITTINGHUS E: Thestages in diabetic renal disease. With emphasis in thestage of incipient diabetic neuropathy. Diabetes (1983)32:64-78.

70. COLTRAN RS, KUMAR V, COLLINS: The Kidney. In:Robbins Pathologic Basis of Disease (6th Edition). ColtranRS, Kumar V, Collins T (Eds.), WB Sanders, Toronto, Canada(1999):966-968.

71. STEFFES M, PLUTH R, SCHMIDT D, MCCRERY R, BASGENJ, MAUER M: Glomerular and mesangial cell number ininsulin dependent diabetes mellitus (IDDM). Proc. Eur.Diabetic Nephropathy Study Group (1996):27.

72. EPSTEIN FH: Pathophysiology of Progressive nephro-pathies. N. Engl. J. Med. (1998) 339:1448-1456.

73. United Kingdom prospective diabetic study. (1996)Lancet 352:837-853.

74. DIABETES CONTROL AND COMPILATIONS RESEARCHGROUP: Effect of intensive therapy. Kidney Int. (1995)47:1703-1720.

75. SHARMA K, JIN Y, GUO J, ZIYADEH FN: Anti-TGF-βantibody attenuates hypertrophy and extracellularmatrix gene expression. Diabetes (1996) 45:522-530.

76. THOMAS PK: Diabetic neuropathy: models,mechanisms and mayhem. Can. J. Neuro. Sci. (1992)19:1.

77. BOULTON AJM: Pathogenesis of diabetic neuropathy.In: The Diabetes Annual. Marshall SM, Home PD, AlbertiKGMM, Krall LP (Eds.), Elsevier Science Publishers BV(1993) 11:192.

78. EPSTEIN M, SOWERS JR: Diabetes mellitus andhypertension. Hypertension (1992) 19:403-418.

79. HEDBLAD B, NILSSON P, JANZON L, BERGLUND G:Relation between insulin resistance and carotidintima-media thickness and stenosis in non-diabeticsubjects. Results from a cross-sectional study inMalmo, Sweden. Diabet. Med. (2000) 17(4):299-307.

80. International Diabetes Federation Triennial Report.1991-1994. IDF Brussels (1994).

81. SAVAGE MP, KROLEWSKI AS, KENIEN GG, LEBEIS MP,CHRISTLIEB AR, LEWIS SM: Acute myocardial infarctionin diabetes mellitus and significance of congestiveheart failure as a prognostic factor. Am. J. Cardiol.(1988) 62:665-669.

82. SHEHADEH A, REGAN TJ: Cardiac consequences ofdiabetes mellitus. Clin. Cardiol. (1995) 18:301-305.

83. LEWINTER MM: Diabetic cardiomyopathy: an overview.Cor. Artery Dis. (1996) 7:95-98.

84. ZHANG WX, CHAKRABARTI S, GREEN DA, SIMA AA:Diabetic autonomic neuropathy in BB rats and effect ofARI treatment on heart rate variability and vagusstructure. Diabetes (1990) 39:613-618.

85. WILLIAMSON JR, CHANG K, FRANGOS M et al.: Perspec-tives in diabetes. Hyperglycemic pseudohypoxia anddiabetic complications. Diabetes (1993) 42:801-813.

86. GREENE DA, STEVENS MJ: The sorbitol-osmotic andsorbitol-redox hypotheses. In: Diabetes Mellitus. Lippin-cott Raven Publishers, Philadelphia, USA (1996):801-809.

87. KOYA D, KING GL: Protein kinase C activation and thedevelopment of diabetic complications. Diabetes (1998)47:859-866.

88. STEVENS MJ, HOSAKA Y, MASTERSON JA, JONES SM,THOMAS TP, LARKIN DD: Downregulation of thehuman taurine transporter by glucose in culturedretinal pigment epithelial cells. Am. J. Physiol. (1999)277:E760-E771.

89. KERN TS, KOWLURU RA, ENGERMAN RL: Abnormalitiesof retinal metabolism in diabetes or galactosemia:ATPases and glutathione. Invest. Ophthalmol. Vis. Sci.(1994) 35:2962-2967.

90. KOWLURU RA, KERN TS, ENGERMAN RL: Abnormalitiesof retinal metabolism in diabetes or experimental



galactosemia. IV. Antioxidant defense system. FreeRadic. Biol. Med. (1997) 22:587-592.

91. WINKLER BS, ARNOLD MJ, BRASSELL MA, SLITER DR:Glucose dependence of glycolysis, hexose monophos-phate shunt activity, energy status and the polyolpathway in retinas isolated from normal (nondiabetic) rats. Invest. Ophthalmol. Vis. Sci. (1997) 38:62-71.

92. OBROSOVA IG, SONE H, MASTERSON JA: Evaluation ofβ1-adrenoceptor antagonist and antioxidant therapyon diabetes-induced changes in retinal NAD(P)-Redoxstatus: Evidence against pseudohypoxia? Diabetes(1998) 47(Suppl. 1):A40.

93. NADLER J, WINER L: Free radicals, nitric oxide anddiabetic complications. In: Diabetes Mellitus. LeRoith D etal. (Eds.) , Lippincott-Raven, Philadelphia, USA(1996):840-848.

94. WOLFF SP, CRABBE MJC, THORNALLEY PJ: The autoxida-tion of glyceraldehyde and other simple monosaccha-rides. Experientia (1984) 40:244.

95. HUNT JV, DEAN RT, WOLFF SP: Hydroxyl radicalproduction and autoxidative glycosylation. Glucoseautoxidation as the cause of protein damage in theexperimental glycation model of diabetes mellitus andageing. Biochem. J. (1988) 256:205-212.

96. JIANG ZY, WOOLLARD AC, WOLFF SP: Hydrogenperoxide production during experimental proteinglycation. FEBS Lett. (1990) 268:69-71.

97. THORNALLEY PJ, WOLFF SP, CRABBE MJ, STERN A: Theoxidation of oxyhaemoglobin by glyceraldehyde andother simple monosaccharides. Biochem. J. (1984)217:615-622.

98. TAGAMI S, KONDO T, YOSHIDA K, HIROKAWA J,OHTSUKA Y, KAWAKAMI Y: Effect of insulin onimpaired antioxidant activities in aortic endothelialcells from diabetic rabbits. Metab. Clin. Exp. (1992)41:1053-1058.

99. ARAI K, MAGUCHI S, FUJII S, ISHIBASHI H, OIKAWA K,TANIGUCHI N: Glycation and inactivation of humanCu-Zn-superoxide dismutase. Identification of the invitro glycated site. J. Biol. Chem. (1987) 262:16969-16972.

100. NISHIKAWA T, EDELSTEIN D, DU XL, YAMAGISHI S,MATSUMURA T, KANEDA Y: Normalizing mitochon-drial superoxide production blocks three pathways ofhyperglycaemic damage. Nature (2000) 404:787-790.

101. NEWTON AC: Regulation of protein kinase C. Curr.Opin. Cell Biol. (1997) 9:161-167.

102. JOHANNES FJ, PRESTLE J, EIS S, OBERHAGEMANN P,PFIZENMAIER K: PKC is a novel, atypical member of theprotein kinase C family. J. Biol. Chem. (1994)269:6140-6148.

103. LE GOOD JA, ZIEGLER WH, PAREKH DB, ALESSI DR,COHEN P, PARKER PJ: Protein kinase C isotypescontrolled by phosphoinositide 3 kinase through theprotein kinase PDK1. Science (1998) 281:2042-2045.

104. SHIBA T, INOGUCHI T, SPORTSMAN JR, HEATH WF,BURSELL S, KING GL: Correlation of diacylglycerol level

and protein kinase C activity in rat retina to retinalcirculation. Am. J. Physiol. (1993) 265:E783-E793.

105. XIA P, INOGUCHI T, KERN TS, ENGERMAN RI, OATES PJ,KING GL: Characterization of the mechanism for thechronic activation of DAG-PKC pathway in diabetesand hypergalactosemia. Diabetes (1994) 43:1122-1129.

106. KOWLURU RA, KERN TS, ENGERMAN RL, ARMSTRONG D:Abnormalities of retinal metabolism in diabetes orexperimental galactosemia. III. Effects of antioxi-dants. Diabetes (1996) 45:1233-1237.

107. LYNCH JJ, FERRO TJ, BLUMENSTOCK FA, BROCKENAUERAM, MALIK AB: Increased endothelial albuminpermeability mediated by protein kinase C activation.J. Clin. Invest. (1990) 85:1991-1998.

108. SHARMA K, DANOFF TM, DEPIERO A, ZIYADEH FN:Enhanced expression of inducible nitric oxidesynthase in murine macrophages and glomerularmesangial cells by elevated glucose levels: possiblemediation via protein kinase C. Biochem. Biophys. Res.Commun. (1995) 207:80-88.

109. BLAKESLEY VA, LEROTHER D: The role of growthfactors in the pathogenesis of diabetic vascularcomplications. In: Diabetes Mellitus. LeRoith D, Taylor SI,Olefsky M (Eds.), Lippincott-Raven, Philadelphia, USA(1996):824-831.

110. VLASSARA H: Recent progress in advanced glycationend products and diabetic complications. Diabetes(1997) 46(Suppl. 2):S19-S25.

111. TAKAGI Y, KASHIWAGI A, TANAKA Y, ASAHIAN T,KIKKAWA R, SHIGETA Y: Significance of fructose-induced protein oxidation and formation of advancedglycation end product. J. Diabetes Complications (1995)9:89-91.

112. GLOMB MA, MONNIER VM: Mechanism of proteinmodification by glyoxal and glycolaldehyde, reactiveintermediates of the Maillard reaction. J. Biol. Chem.(1995) 270:10017-10026.

113. STITT AW, LI YM, GARDINER TA, BUCALA R, ARCHER DB,VLASSARA H: Advanced glycation end products (AGEs)co-localize with AGE receptors in the retinal vascula-ture of diabetic and of AGE-infused rats. Am. J. Pathol.(1997) 150:523-531.

114. STITT AW, HE C, VLASSARA H: Characterization of theadvanced glycation end-product receptor complex inhuman vascular endothelial cells. Biochem. Biophys.Res. Commun. (1999) 256:549-556.

115. MOHAMED AK, BIERHAUS A, SCHIEKOFER S,TRITSCHLER H, ZIEGLER R, NAWROTH PP: The role ofoxidative stress and NF-kappaB activation in latediabetic complications. Biofactors (1999) 10:157-167.

116. ESPOSITO C, GERLACH H, BRETT J: Endothelial receptormediated binding of glucose modified albumin isassociated with increased monolayer permeabilityand modulation of cell surface coagulant properties. J.Exp. Med. (1992) 170:1387-1397.

117. YAMAGISHI SI, YONEKURA H, YAMAMOTO Y et al.:Advanced glycation end products-driven angiogenesis



in vitro. Induction of the growth and tube formation ofhuman microvascular endothelial cells throughautocrine vascular endothelial growth factor. J. Biol.Chem. (1997) 272:8723-8730.

118. HAITOGLOU CS, TSILIBARY EC, BROWNLEE M,CHARNOIS AS: Altered cellular interaction betweenendothelial cells and non-enzymatically glucosylatedlaminin/type IV collagen. J. Biol. Chem. (1992)267:12404-12407.

119. BUCALA R, TRACEY KJ, CERAMI A: Advanced glycosyla-tion products quench nitric oxide and mediatedefective endothelium-dependent vasodilation inexperimental diabetes. Diabetologia (1991) 87:432.

120. GIARDINO I, EDELSTEIN D, BROWNLEE M: BCL-2 expres-sion or antoxidants prevent hyperglycemia-inducedformation of intracellular advanced glycation endproducts in bovine endothelial cells. J. Clin. Invest.(1996) 97:1422-1428.

121. HOPFNER RL, GOPALAKRISHNAN V: Endothelin:emerging role in diabetic vascular complications.Diabetologia (1999) 42:1383-1394.

122. YAMAUCHI T, OHNAKA K, TAKAYANAGI R, UMEDA F,NAWATA H: Enhanced secretion of endothelin_1 byelevated glucose levels from cultured bovine aorticendothelial cells. FEBS Lett. (1990) 267:16-18.

123. CHEN S, APOSTOLOVA M, CHERIAN G, CHAKRABARTI S:Interaction of endothelin-1 with vasoactive factors inmediating glucose-induced increased permeability inendothelial cells. Lab. Invest. (2000) 80:1311-1321.

124. HU RM, LEVIN ER, FRANK HJL: Insulin stimulatesproduction and secretion of endothelin from bovineendothelial cells. Diabetes (1993) 42:351-358.

125. WAKASAKI H, KOYA D, SCHOEN FJ et al.: Targetedoverexpression of protein kinase C2 isoform inmyocardium causes cardiomyopathy. Proc. Natl. Acad.Sci. USA (1997) 94:9320-9325.

126. VANHOUTTE PM: Endothelin-1. A matter of life andbreath. Nature (1994) 368:693-694.

127. MATSUURA A, KAWASHIMA S, YAMOCHI W et al.:Vascular endothelial growth factor increasesendothelin-converting enzyme expression in vascularendothelial cells. Biochem. Biophys. Res. Commun. (1997)235:713-716.

128. PEDRAM A, RAZANDI M, HU RM, LEVIN ER: Vasoactivepeptides modulate vascular endothelial cell growthfactor production and endothelial cell proliferationand invasion. J. Biol. Chem. (1997) 27:17097-17103.

129. QUEHENBERGER P, BIERHAUS A, FASCHING P,MUELLNER C, KLEVESATH M, HONG M: Endothelin 1transcription is controlled by nuclear factor-κB inAGE-stimulated cultured endothelial cells. Diabetes(2000) 49:1561-1570.

130. CHAKRABARTI S, SIMA AAF: Endothelin-1 andEndothelin-3 like immunoreactivity in the eyes ofdiabetic and non-diabetic BB/W rats. Diab. Res. Clin.Prac. (1997) 37:109-120.

131. CHAKRAVARTHY U, DOUGLASAJ, BAILIE R, MCKIBBEN B,ARCHER DB: Immunoreactive endothelin distributionin ocular tissue. Invest. Ophthalmol. Vis. Sci. (1997)35:2448-2454.

132. CHAKRABARTI S, GAN XT, MERRY A, KARMAZYN M, SIMAAAF: Augmented retinal endothelin-1, endothelin-3,EndothelinA and EndothelinB gene expression inchronic diabetes. Curr. Eye Res. (1998) 17:301-307.

133. TAKAGI C, BURSELL SE, LIN YW et al.: Regulation ofretinal hemodynamics in diabetic rats by increasedexpression and action of endothelin-1. Invest.Ophthalmol. Vis. Sci. (1996) 37:2504-2518.

134. DENG DX, EVANS T, MUKHERJEE K, DOWNEY D,CHAKRABARTI S: Diabetes induced dysfunction in theretina: role of endothelins. Diabetologia (1999)42:1228-1234.

135. EVANS T, DENG DX, MUKHERJEE K, DOWNEY D,CHAKRABARTI S: Endothelins, their receptors andretinal vascular dysfunction in galactose-fed rats.Diabetes Res. Clin. Pract. (2000) 48:75-85.

136. EVANS T, DENG DX, CHEN S, CHAKRABARTI S:Endothelin receptor blockade prevents augmentedextracellular matrix component mRNA expressionand capillary basement membrane thickening in theretina of diabetic and galactose fed rats. Diabetes (2000)49:662-666.

137. KAWAMURA M, OHGAWARA H, NARUSE M et al.:Increased plasma endothelin in NIDDM patients withretinopathy. Diabetes Care (1992) 15:1396-1397.

138. OGATA M, NARUSE M, IWASAKI N et al.: Immunoreactiveendothelin levels in the vitreous fluid are decreased indiabetic patients with proliferative retinopathy. J.Cardiovasc. Pharmacol. (1998) 31(Suppl. 1):S378-S379.

139. SIMONSON MS: Endothelins: multifunctional renalpeptides. Physiol. Rev. (1993) 73:375-411.

140. TURNER NC, MORGAN PJ, HAYNES AC et al.: Elevatedrenal endothelin-1 clearance and mRNA levels associ-ated with albuminuria and nephropathy innon-insulin dependent diabetes mellitus studies inobese fa/fa Zucker rats. Clin. Sci. (1997) 93:565-571.

141. KHAN MA, DASHWOOD MR, THOMPSON CS, MUMTAZFH, MIKHAILIDIS DP, MORGAN RJ: Time-dependentup-regulation of endothelin-A receptors anddown-regulation of endothelin-B receptors and nitricoxide synthase binding sites in the renal medulla of arabbit model of partial bladder outlet obstruction:potential clinical relevance. BJU Int. (1999)84(9):1073-1080.

142. NAKAMURA T, EBIHARA I, FUKUI M, TOMINO Y, KOIDAH: Effect of a specific endothelin-A receptor antagoniston mRNA levels for extracellular matrix componentsand growth factors in diabetic glomeruli. Diabetes(1995) 44:895-899.

143. ISHIMURA E, SHOUJI S, NISHIZAWA Y, MORII H,KASHGARIAN M: Regulation of mRNA expression forECM by cultured rat mesangial cells. J. Am. Soc. Nephrol.(1991) 2:546.



144. SUZUKI T, KUMAZAKI T, MITSUI Y: Endothelin-1 isproduced and secreted by neonatal rat cardiacmyocytes in vitro. Biochem. Biophys. Res. Commun.(1993) 191:823-830.

145. CHUA BH, CHUA CC, DIGLIO CA, SIU BB: Regulation ofendothelin-1 mRNA by angiotensin II in rat heartendothelial cells. Biochem. Biophys. Acta (1993)1178:201-206.

146. FUKUCHI M, GIAID A: Expression of endothelin-1 andendothelin-converting enzyme-1 mRNAs and proteinsin failing human hearts. J. Cardiovasc. Pharmacol. (1998)31(Suppl. 1):S421-423

147. HU JR, VON HARSDORF R, LANG RE: Endothelin haspotent inotropic effects in rat atria. Eur. J. Pharmacol.(1988) 158:275-278.

148. ISHIKAWA T, YANAGISAWA M, KIMURA S, GOTO K,MASAKI T: Positive chronotropic effects of endothelin,a novel endothelium-derived vasoconstrictor peptide.Pflugers Arch. (1988) 413:108-110.

149. KARMAZYN M: The role of endothelins in cardiacfunction in health and disease. In: Myocardial Ichaemia:Mechanisms, Reperfusion, Protection. Karmazyn M (Ed.),Birkhauser, Basel, Switzerland (1996):209-230.

150. LIN YW, DUH E, JIANG Z: Expression ofpreproEndothelin-1 mRNA in streptozotocin-induceddiabetic rats. Diabetes (1996) 45(Suppl. 2):48A.

151. VESCI L, MATTERA GG, TOBIA P, CORSICO N, CALVANI M:Cardiac and renal endothelin-1 binding sites instreptozotocin-induced diabetic rats. Pharmacol. Res.(1995) 32:363-367

152. LIEU AT, REID JJ: Changes in the responsiveness toendothelin-1 in isolated atria from diabetic rats. Eur. J.Pharmacol. (1994) 261:33-42.

153. CHEN S, EVANS T, MUKHERJEE K, KARMAZYN M,CHAKRABARTI S: Diabetes-induced myocardialstructural changes: Role of endothelin-1 and itsreceptors. J. Mol. Cell Cardiol. (2000) 32(9):1621-1629.

154. FERNANDEZ AC, PATINO R, IBARRA J et al.: Endothelin inhypertension and diabetes mellitus. In: Endothelin inCardiovascular Diseases. Luscher TF (Ed.), Springer, BerlinHeidelberg, New York, USA (1995):157-170.

155. PERFETTO F, TARQUINI R, DE LEOBARDIS V et al.:Vascular damage and not hypertension per seinfluences endothelin-1 plasma levels in patients withnon insulin dependent diabetes mellitus. Recent Prog.Med. (1997) 88:317-320.

156. SIMA AAF, SUGIMOTO K: Experimental diabeticneuropathy: an update. Diabetologia (1999) 42:773-788.

157. ZHU X, EICHBERG J: 1,2-diacylglycerol content and itsarachidonyl-containing molecular species arereduced in sciatic nerve from streptozotocin-induceddiabetic rats. J. Neurochem. (1990) 55:1087-1090.

158. HERMENEGILDO C, FELIPO V, MINANA MD, ROMERO FJ,GRISOLIA S: Sustained recovery of Na+/K+-ATPaseactivity in sciatic nerve of diabetic mice by

administration of H7 or callphostin C, inhibitors ofprotein kinase C. Diabetes (1993) 42:257.

159. CAMERON NE, DINES KC, COTTER MA: The potentialcontribution of endothelin-1 to neurovascularabnormalities in streptozotocin diabetic rats.Diabetologia (1994) 37:1209-1215.

160. STEVENS EJ, TOMLINSON DR: Effects of endothelinreceptor antagonism with bosentan on peripheralnerve function in experimental diabetes. Br. J.Pharmacol. (1995) 115:373-379.

161. DENG D, EVANS T, XU G, SIMA AAF, CHAKRABARTI S:Endothelin-1 and its receptors in the pathogenesis ofneuronal and retinal functional abnormalities in thediabetic rats. Proceedings of the 58th Scientific Sessions onDiabetes. Chicago, USA (1998):0559.

162. TERTIN-CLARY C, EUDE I, FOURNIER T, PARIS B,BREUILLER-FOUCHE M, FERRE F: Contribution ofprotein kinase C to ET-1-induced proliferation inhuman myometrial cells. Am. J. Physiol. (1999) 276(3 Pt1):503-511.

163. CATALAN RE, MARTINEZ AM, ARAGONES MD et al.:Endothelin-stimulated phosphoinositide turnoverand protein kinase C translocation in rat synapto-somes. Biochem. Mol. Biol. Int. (1996) 38:7-14.

164. KAWAI N, YAMAMOTO T, YAMAMOTO H, MCCARRONRM, SPATZ M: Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-C1-cotransport throughETA receptors and protein kinase C-dependentpathway in cerebral capillary endothelium. J.Neurochem. (1995) 65:1588-1596.

165. ROSEN P, BALLHAUSEN T, BLOCH W, ADDICKS K:Endothelial relaxation is disturbed by oxidative stressin the diabetic rat heart: influence of tocopherol asantioxidant. Diabetologia (1995) 38:1157-1168.

166. FAURE V, COURTOIS Y, GOUREAU O: Differentialregulation of nitric oxide synthase-II mRNA by growthfactors in rat, bovine and human retinal pigmentedepithelial cells. Ocul. Immunol. Inflamm. (1999) 7:27-34.

167. HASDAI D, HOLMES DR, JR., RICHARDSON DM, IZHAR U,LERMAN A: Insulin and IGF-I attenuate the coronaryvasoconstrictor effects of endothelin-1 but not ofsarafotoxin 6c. Cardiovasc. Res. (1998) 39:644-650.

168. GHISO N, ROHAN RM, AMANO S, GARLAND R, ADAMISAP: Suppression of hypoxia-associated vascularendothelial growth factor gene expression by nitricoxide via cGMP. Invest. Ophthalmol. Vis. Sci. (1999)40:1033-1039.

Subrata Chakrabarti† , Mark Cukiernik, Suranjana Mukherjee & ShaliChen† Author for correspondenceDepartment of Pathology, DSB 4011, The University of WesternOntario, London, Ontario, CanadaTel.: +1 519 663 3381; Fax: +1 519 663 2930;E-mail: [email protected]



therapeutic potential of endothelin receptor antagonists in diabetes

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