C.G. Schalkwijk

Plasma concentration of C-reactive protein is increased in type I diabetic patients without clinical macroangiopathy and correlates with markers of endothelial dysfunction: evidence for chronic inflammation.

Summary Moderately increased plasma concentrations of C-reactive protein are associated with an increased risk of cardiovascular disease. C-reactive protein, its relation to a low degree of inflammatory activation and its association with activation of the endothelium have not been systematically investigated in Type I (insulin-dependent) diabetes mellitus. C-reactive protein concentrations were measured in 40 non-smoking patients with Type I diabetes without symptoms of macrovascular disease and in healthy control subjects, and in a second group of Type I diabetic patients (n = 60) with normo- (n = 20), micro- (n = 20) or macroalbuminuria (n = 20). Differences in glycosylation of α1-acid glycoprotein were assayed by crossed affinity immunoelectrophoresis. Activation of the endothelium was measured with plasma concentrations of endothelial cell markers. The median plasma concentration of C-reactive protein was higher in Type I diabetic patients compared with healthy control subjects [1.20 (0.06–21.64) vs 0.51 (0.04–9.44) mg/l; p < 0.02]. The Type I diabetic subjects had a significantly increased relative amount of fucosylated α1-acid glycoprotein (79 ± 12 % vs 69 ± 14 % in the healthy control subjects; p < 0.005), indicating a chronic hepatic inflammatory response. In the Type I diabetic group, log(C-reactive protein) correlated significantly with von Willebrand factor (r = 0.439, p < 0.005) and vascular cell adhesion molecule-1 (r = 0.384, p < 0.02), but not with sE-selectin (r = 0.008, p = 0.96). In the second group of Type I diabetic patients, increased urinary albumin excretion was associated with a significant increase of von Willebrand factor (p < 0.0005) and C-reactive protein (p = 0.003), which were strongly correlated (r = 0.53, p < 0.0005). Plasma concentrations of C-reactive protein were higher in Type I diabetic patients without (clinical) macroangiopathy than in control subjects, probably due to a chronic hepatic inflammatory response. The correlation of C-reactive protein with markers of endothelial dysfunction suggests a relation between activation of the endothelium and chronic inflammation. [Diabetologia (1999) 42: 351–357]

Altered expression of genes related to blood―retina barrier disruption in streptozotocin-induced diabetes

Disruption of the blood-retina barrier (BRB) is an early phenomenon in preclinical diabetic retinopathy (PCDR). Two vascular permeability pathways may be affected, the paracellular pathway involving endothelial cell tight junctions, and the endothelial transcellular pathway mediated by endocytotic vesicles (caveolae). The relative contribution of both pathways to vascular permeability in PCDR is unknown. We compared transcription levels in entire rat retina of genes related to these pathways between control conditions and after 6 and 12 weeks of streptozotocin-induced diabetes, as well as in bovine retinal endothelial cells (BRECs) exposed to VEGF and bovine retinal pericytes (BRPCs), using real-time quantitative RT-PCR. To confirm endothelial-specificity, immunohistochemical staining was performed in rat retina, and mRNA transcript levels were compared between BRECs and BRPCs. mRNA and protein of most paracellular transport-related genes were specifically expressed by retinal endothelial cells, whereas vesicle transport-related mRNA and proteins were present in various retinal cell types, including endothelial cells. Expression of selected endothelial cell tight junction genes and particularly that of occludin and claudin-5 was reduced in the diabetic retina and in BRECs after exposure to VEGF. Expression of 6 out of 11 vesicular transport-related genes was upregulated after induction of diabetes. Of these, only plasmalemma vesicle-associated protein (PV-1) was exclusively expressed in BRECs and not in BRPCs. PV-1 transcription was markedly induced in diabetic retina and by VEGF in BRECs. Caveolin-1 immunostaining was primarily found in the retinal vasculature, and its mRNA levels in BRECs were highly abundant and VEGF-inducible. Whereas the endothelial tight junction genes occludin and claudin-5 showed a transient downregulation, we observed long-term upregulation in diabetic retina and VEGF-induced expression in BRECs of the vesicular transport-related genes caveolin-1 and PV-1. The altered gene expression profiles observed in this study suggest a transient induction of the paracellular pathway and prolonged involvement of transcellular endothelial transport mechanisms in the increased permeability of retinal capillaries in PCDR.

Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina

Aims/hypothesisReferred to as CCN, the family of growth factors consisting of cystein-rich protein 61 (CYR61, also known as CCN1), connective tissue growth factor (CTGF, also known as CCN2), nephroblastoma overexpressed gene (NOV, also known as CCN3) and WNT1-inducible signalling pathway proteins 1, 2 and 3 (WISP1, −2 and −3; also known as CCN4, −5 and −6) affects cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. AGEs formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesised that pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression.Materials and methodsCCN gene expression levels were studied at the mRNA and protein level in retinas of control and diabetic rats using real-time quantitative PCR, western blotting and immunohistochemistry at 6 and 12xa0weeks of streptozotocin-induced diabetes in the presence or absence of aminoguanidine, an AGE inhibitor. In addition, C57BL/6 mice were repeatedly injected with exogenously formed AGE to establish whether AGE modulate retinal CCN growth factors in vivo.ResultsAfter 6xa0weeks of diabetes, Cyr61 expression levels were increased more than threefold. At 12xa0weeks of diabetes, Ctgf expression levels were increased twofold. Treatment with aminoguanidine inhibited Cyr61 and Ctgf expression in diabetic rats, with reductions of 31 and 36%, respectively, compared with untreated animals. Western blotting showed a twofold increase in CTGF production, which was prevented by aminoguanidine treatment. In mice infused with exogenous AGE, Cyr61 expression increased fourfold and Ctgf expression increased twofold in the retina.Conclusions/interpretationCTGF and CYR61 are downstream effectors of AGE in the diabetic retina, implicating them as possible targets for future intervention strategies against the development of diabetic retinopathy.

The new cardioprotector Monohydroxyethylrutoside protects against doxorubicin-induced inflammatory effects in vitro

Besides its cardiotoxic effect, doxorubicin also elicits inflammatory effects in vivo. 7-Monohydroxyethylrutoside (monoHER) has recently been used as a protector against doxorubicin-induced cardiotoxicity in vivo. It is not known yet whether monoHER can also protect against doxorubicin-induced inflammatory effects. The aim of the present study was (1) to illustrate the inflammatory effects of doxorubicin in vitro and (2) to evaluate a possibly protective effect of monoHER. In order to demonstrate the inflammatory effects of doxorubicin and the possible protection of monoHER, proliferating human umbilical cord vascular endothelial cells (HUVECs) were incubated with different concentrations of doxorubicin ranging from 12.5 to 600u2009nM with(out) 200u2009μM monoHER. Resting (confluent) HUVECs were incubated with (0.5–25u2009μM) doxorubicin with(out) monoHER (0.2–1.2u2009mM) and the viability of endothelial cells and their propensity to adhere to neutrophils were measured 24u2009h after treatment. The localisation of adhered neutrophils was determined with immunofluorescence microscopy. To further characterise the mechanism of doxorubicin-induced neutrophil adhesion, the expression of the HUVECs surface adhesion molecules was determined after doxorubicin treatment. Doxorubicin decreased the viability and proliferation capacity of HUVECs in a concentration-dependent manner. The proliferating HUVECs were much more sensitive to doxorubicin (IC50=60.0±20.8u2009nM) than resting cells (LC50=4.0±0.3u2009μM). Doxorubicin also increased the adhesion of neutrophils reaching a plateau value at a doxorubicin concentration of ⩾0.4μM (P=0.0113). The induced neutrophil adhesion was accompanied by overexpression of VCAM and E-selectin but not ICAM. Although monoHER did not reverse the effect of doxorubicin on the proliferation of endothelial cells, it significantly protected resting HUVECs against the cytotoxic effect of doxorubicin (⩽25u2009μM, P<0.0015). In addition, monoHER completely protected against the stimulatory effect of doxorubicin on neutrophil adhesion, and inhibited the doxorubin-induced expression of VCAM and E-selectin on the surface of treated HUVECs. This study illustrates that monoHER, which protects against doxorubicins cardiotoxic effect, can also protect against doxorubicin-induced inflammatory effects. These data prompt further investigation about the possible link between doxorubicin-induced inflammatory effects and its cardiotoxicity in vivo.

Amadori‐albumin correlates with microvascular complications and precedes nephropathy in type 1 diabetic patients

Background Amadori‐albumin, a major glycated protein, is involved in experimental hyperglycaemia‐induced microvascular complications, and is associated with advanced nephropathy in Type I diabetic patients in humans. Our aim was to assess the association of Amadori‐albumin with early nephropathy and with retinopathy in Type I diabetic patients and the involvement of chronic low‐degree inflammation therein.

N(epsilon)-(carboxymethyl)lysine depositions in intramyocardial blood vessels in human and rat acute myocardial infarction: a predictor or reflection of infarction?

Objective—Advanced glycation end products (AGEs), such as Nϵ-(carboxymethyl)lysine (CML), are implicated in vascular disease. We previously reported increased CML accumulation in small intramyocardial blood vessels in diabetes patients. Diabetes patients have an increased risk for acute myocardial infarction (AMI). Here, we examined a putative relationship between CML and AMI. Methods and Results—Heart tissue was stained for CML, myeloperoxidase, and E-selectin in AMI patients (n=26), myocarditis patients (n=17), and control patients (n=15). In AMI patients, CML depositions were 3-fold increased compared with controls in the small intramyocardial blood vessels and predominantly colocalized with activated endothelium (E-selectin–positive) both in infarction and noninfarction areas. A trend of increased CML positivity of the intima of epicardial coronary arteries did not reach significance in AMI patients. In the rat heart AMI model, CML depositions were undetectable after 24 hours of reperfusion, but became clearly visible after 5 days of reperfusion. In line with an inflammatory contribution, human myocarditis was also accompanied by accumulation of CML on the endothelium of intramyocardial blood vessels. Conclusions—CML, present predominantly on activated endothelium in small intramyocardial blood vessels in patients with AMI, might reflect an increased risk for AMI rather than being a result of AMI.

Homocysteine-lowering treatment with folic acid plus vitamin B6 lowers urinary albumin excretion but not plasma markers of endothelial function or C-reactive protein: further analysis of secondary end-points of a randomized clinical trial.

Background Hyperhomocysteinaemia is an independent risk factor for atherosclerosis and is thought to induce its effects through causing endothelial dysfunction. We studied the effect of homocysteine‐lowering treatment with folic acid plus vitamin B6 on urinary and plasma markers of endothelial function, and on plasma C‐reactive protein, a marker of chronic inflammation.