Damage to the vascular endothelium of diabetic patients can be assessed by analysing blood samples for the number of circulating endothelial cells with mitochondrial DNA deletions.

We report that diabetic patients with both (a) persistent hyperglycaemia, indicated by HbAlC > 9% of total haemoglobin (Hb) and (b) clinically evident microor macro-vascular disease have a significant increase in the number of detached vascular endothelial cells circulating in their blood relative to age-matched diabetic controls without vascular disease. We find that circulating endothelial cells (CECs) with a characteristic 4977 base pair (bp) deletion in their mitochondrial DNA (mt DNA) were particularly numerous in diabetic patients with early signs of (a) nephropathy (proteinuria without renal failure) or (b) peripheral vascular disease. Increased levels of CECs have previously been identified (i) in patients suffering from Mediterranean spotted fever, a disease induced by Rickettsia conorii, a bacterial parasite of endothelial cells [ I ] , (ii) in smokers [2], (iii) in diabetics [3] and (iv) in non-diabetics with peripheral vascular disease (PVD)[4]. With ethical permission, 15ml of citrate-treated blood were obtained from diabetic patients using vacutainers and diluted three-fold with phosphate-buffered saline (PBS, pH 7.4). The diluted blood was carefully layered onto a cushion of Histopaque-I077 (Sigma) and centrifuged at 800g for 30 min at room temperature. The mononuclear cell band, free of erythrocytes and containing mostly lymphocytes, monocytes and circulating endothelial cells, was drawn off and diluted ten-fold with PBS. After thorough mixing, the cells were harvested by centrifugation at 400 g for 20 min at 4OC. The supernatant was carefully discarded and the cells resuspended in 1 ml of PBS. Three densities of Percoll ( I ,060 g/ml, 1.070 g/ml and 1.082 g/ml) were then made up as series of stepped gradients [5]. 0.5 ml of the mononuclear cell fraction was layered on top and centrifuged at 800 g for 30 min at 4°C. 0.5 ml of the same mononuclear fraction was retained for PCR. Cells with buoyant densities in fractions (a) <1.060 g/ml (b) >1.060 to <1.070 g/ml (c) >1.070 to <1.082 g/ml and (d) >1.082 g/ml were isolated, washed with PBS and analysed by PCR. Typical results obtained from a diabetic patient with peripheral vascular disease are shown in Fig 1. PCR analysis of fraction (a) using primers DELI (np 8345-8364) and DEL2 (np 13827-13808), specific for the 4977 bp human mt DNA deletion, revealed a product identical in size to the deletion detected in cultured endothelial cells following a hyperglycaemic insult [6]. Percoll fraction (a) had both the highest number of CECs and the highest level of the 4977 bp deletion. Fraction (b) contained fewer CECs but the highest levels of lymphocytes. A further PCR product (approximately 800 bp, Fig 1) was detected mainly in fractions (c) and (d) of both normal controls and diabetics and might be associated with maturing reticulocytes. We are currently investigating this possibility. The presence of CECs in the blood of patients with PVD was confirmed by specific immunofluorescent labelling of endothelial cells (Fig 2) followed by analysis using a fluorescenceactivated cell sorter (FACS) [8]. The results, presented as plots of fluorescence intensity (FLI) versus forward scatter are shown in Fig 2. It is evident that a population of cells exists in the mononuclear cell fraction which has a relatively high fluorescence intensity and a moderately large forward scatter (Fig 2A, encircled area). This profile appears to be very similar to that obtained when 1 x lo5 HUVE cells were added to the mononuclear cell fraction from a non-diabetic age-matched control immediately prior to immunostaining (Fig 2B). This population of cells was absent from the same non-diabetic control in the absence of added HUVE cells (Fig 2C) and is also absent from a diabetic patient with no clinically evident vascular disease (Fig 2D). Blood from diabetic patients with microvascular complications such as nephropathy and retinopathy was also analysed. The intensity of the PCR product obtained after analysis of cells from the <1.060 g/ml Percoll fraction (representing the purest fraction of CECs) was scored as strong (+++) (See lane 3, Fig 1 for an example of a strong intensity band), intermediate (++) (50% intensity of the strong band shown in Fig 1) or weak (+) (< 20% intensity of the strong band in Figl). The results of a preliminary survey are presented in Table 1. All patients studied had a HbAlC > 9%. Patients with PVD or nephropathy (proteinuria without renal failure) appeared to have highest levels of both CECs and the 4977 bp mt DNA deletion. Both non-diabetic and diabetic controls without clinically evident vascular disease had no detectable CECs. It remains to be established: (a) if CECs are the only type of cells present in the blood which harbour the 4977 bp mt DNA deletion (b) if the 4977 bp deletion generated by hyperglycaemia is a key event in the aetiology of diabetic vascular complications. Two observations are relevant: (i) The 4977bp deletion removes several mitochondrial genes including one which codes for subunit 6 of the mitochondrial ATP-synthase [9]. Thus, as the level of 4977bp deletion increases in a cell, the level of ATP synthesis by oxidative phosphorylation should fall. (ii) When the 4977bp deletion reaches high levels in ex vivo cultured endothelial cells (there are several hundred copies of mtDNA per cell), they detach from their substratum [6]. This observation could explain the origin of CECs in vivo in patients with persistent hyperglycaemia. In conclusion, we have demonstrated for the first time the presence of a major deletion in the mt DNA (one third of the total mt genome) in CECs obtained from diabetics with vascular complications. C Markers: a, 50615 I7 bp b. I018 hp c, 1636 bp d, 2036 bp b