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Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies., (© 2024. The Author(s).)

Purpose: Long-term clinical studies have now shown that tight control of blood pressure in type 2 diabetes reduces the risk of diabetes-related death and common diabetic complications, including diabetic retinopathy. However, the mechanisms by which hypertension enhances diabetic microvascular disease, especially diabetic retinopathy, are poorly understood. We developed an experimental model of hypertension in diabetic rats and studied the early ultrastructural changes in retinal capillaries under these conditions., Methods: Hypertension was induced in diabetic BioBreeding (BB) rats by unilateral nephrectomy, weekly subcutaneous mineralocorticoid and 0.9% oral saline. Serial blood pressures and ultrastructural features of retinal capillaries were recorded in four groups: normotensive Wistar rats, normotensive diabetic rats, hypertensive Wistar rats and hypertensive diabetic rats., Results: A significant and sustained increase in systolic blood pressure occurred in both groups of nephrectomised rats. There was a significant increase in the number of caveolae (i) in both pericytes and endothelial cells in animals with hypertension and diabetes together compared with all other groups and (ii) in pericytes in animals with diabetes alone. The number of direct contacts between pericytes and endothelial cells was reduced in diabetic and hypertensive diabetic animals. Hypertension and diabetes had an interactive effect in producing retinal capillary basement membrane thickening., Conclusions: In the BB rat hypertension and diabetes have an interactive effect in increasing the number of caveolae in both endothelial cells and pericytes. We speculate that this may be a reflection of changes in calcium and nitric oxide metabolism in these animals.