Increased expression of basement membrane collagen in human diabetic retinopathy.

Abstract

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 (ft-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. (J. Clin. Invest. 1994. 93:438-442).

Effect of Combined Antisense Oligonucleotides Against High-Glucose–and Diabetes-Induced Overexpression of Extracellular Matrix Components and Increased Vascular Permeability

“…These results indicate that the translation initiation site for each of the three ECM components is an effective target site for simultaneous inhibition of expression of the three ECM components and that the excess vascular permeability in the diabetic rat retinas is, at least in part, due to altered composition of the retinal vascular basement membrane. Several laboratories including ours have previously demonstrated increased synthesis of basement membrane components in cultured endothelial cells grown in high glucose medium (26 -28), in tissues of diabetic rats (6), and in retinal vessels of patients with diabetes (7,8). These studies have established that both high glucose and diabetes increase synthesis of fibronectin, collagen IV, and laminin (6 -8,26,27,29 -34).…”

Diabetic retinopathy: Some cellular, molecular and therapeutic considerations

“…165 Increased synthesis of collagen IV, fibronectin and laminin by endothelial cells and pericytes under the influence of hyperglycaemia and AGEs adds to the bulk of the basement membrane. [166][167][168] Furthermore advanced glycation of long-lived matrix proteins impedes their degradation by catabolic enzymes78 leading to a net increase in basement membrane thickness?5 In diabetes the thickened basement membrane becomes cross-linked, rigid and poorly compliant and may physically restrict capillary contraction and dilatation. Exaggeration of heparin sulphate proteoglycan synthesis also adds to the structural and biochemical complexity of the basement membrane affecting its ligation of key vasogenic, heparin-binding growth factors such as VEGF and bFGF.…”

Transforming growth factor-β receptor and fibronectin expressions in aortic smooth muscle cells in diabetic rats

“…Reactions were performed in 10 mmol/l TrisHCl, pH 8.3, containing 1.5 mmol/l MgCl 2 , 50 mmol/l KCl, 0.2 mmol/l each of dATP, dGTP, dCTP and dTTP, 20 pmol of sense and antisense primers and 2.5 units of Taq polymerase (Perkin Elmer Cetus, Norwalk, Conn., USA). PCR primers were prepared at nucleotide sequences 511-531 (sense), 1329-1350 (antisense) of human TGF-b type I receptor [14], at 868-893 (sense), 1348-1371 (antisense) of rat TGF-b type II receptor [18] and at 1-18 (Exon 1)(sense), 1014-1032 (Exon 3)(antisense) of human b -actin [19]. PCR conditions were as follows: 30 cycles for TGF-b receptors or 25 cycles for b -actin at 94°C (1 min), 60°C (2 min) and 72°C (3 min), followed by 10 min at 72°C.…”

“…The protocol of RT-PCR was designed to measure the level of TGF-b receptors relative to the expression of an endogenous internal standard gene (b-actin) [19,20]. To quantify the mRNA expressions, TGF-b receptors and b -actin cDNAs generated in the same RT reaction were amplified in separate tubes containing increasing volumes of the RT reaction (0.75, 1.5, 3.0 and 6.0 m l) to document amplification in the linear region for each cDNA [19].…”