Effects of protein kinase C inhibition and activation on proliferation and apoptosis of bovine retinal pericytes.

AIMS/HYPOTHESIS: Drop-out of capillary pericytes occurs early and selectively in diabetic retinopathy. High glucose concentrations decrease replication and increase apoptosis of cultured pericytes. Since glucose activates protein kinase C, we investigated the effects of modulating this intracellular mediator on replication, cell cycle and apoptosis of cultured bovine retinal pericytes. METHODS: Pericytes cultured in 5.6 or 28 mmol/l glucose were exposed to a protein kinase C activator (phorbol 12-myristate 13-acetate) and/or a selective inhibitor of its beta2 isoform (LY379196). Cells were counted after 7 days. Proliferation by the tetrazolium to formazan assay and DNA synthesis by 5-bromo-2'-deoxyuridine incorporation were measured at day 4. Cell cycle by flow cytometry and apoptosis by ELISA were assessed at day 2. RESULTS: High glucose reduced pericyte replication and increased apoptosis. Protein kinase C activation increased proliferation, while inhibition of its beta2 isoform decreased it. Cell cycle was accelerated by protein kinase C activation and delayed by inhibition. Apoptosis was enhanced by protein kinase C inhibition and reduced by activation. CONCLUSIONS/INTERPRETATION: Protein kinase C inhibition amplifies the anti-proliferative and pro-apoptotic effects of high glucose on cultured pericytes, whereas stimulation reduces apoptosis and promotes proliferation both in physiological glucose and high glucose. Protein kinase C inhibition, proposed for the treatment of diabetic macular edema and proliferative retinopathy, might accelerate pericyte dropout in earlier stages when these cells are still present in retinal capillaries.

Failure of angiotensin II and insulin to stimulate transforming growth factor-beta1. Release from cultured bovine retinal pericytes.

BACKGROUND: Activation of the renin-angiotensin system (RAS) may induce cardiovascular and renal fibrosis in hypertension and diabetes. This fibrogenic effect is mainly mediated by Transforming Growth Factor-B1 (TGF-B1), a multifunctional citokyne released by endothelial, vascular smooth muscle and renal mesangial cells, that is able to increase extracellular matrix deposition. Retinal capillary pericytes have functions similar to those of mesangial cells, including ability to synthesize and release TGF-B1 and produce extracellular matrix. An intraocular RAS was described in the human eye and may produce effects similar to those observed in the heart and kidney, which could be mediated by TGF-B1. In particular, TGF-B1 might be involved in thickening of the capillary basement membrane in diabetic microangiopathy. We therefore aimed at evaluating the possible effects of Angiotensin-II on TGF-B1 secretion by cultured retinal pericytes (BRP). METHODS: BRP cultures were incubated with Angiotensin-II or insulin (known to play a permissive effect on TGF-B1 release from mesangial cells) or Angiotensin-II + insulin at final concentrations of 10-10, 10-8, 10-6, 10-4 mol/L. RESULTS: Baseline TGF-B1 concentrations in the supernatants of pericyte cultures were 6 139 +/- 1 919 pg/mL/106 cells; no changes of TGF-B1 concentrations resulted from adding increasing amounts of Ang II, insulin or both. CONCLUSIONS: Though confirming that cultured bovine retinal pericytes spontaneously release TGF-B1, Angiotensin-II did not produce any stimulatory effects of in our experimental system

Coronary reactive hyperaemia after nitric oxide inhibition in the anaesthetized goat.

In the dog it has been shown that, while the inhibition of the endothelial release of nitric oxide reduces the duration, the total hyperaemic flow and the peak flow of the acetylcholine and myogenic coronary vasodilator responses, in the reactive hyperaemia the peak is not affected. The difference has been attributed to the different time required by the coronary blood flow to reach its maximum: long enough when acetylcholine is given or myogenic vasodilatation is elicited, this time is very short in the reactive hyperaemia. Thus it has been argued that only when the time to the peak of a hyperaemic response is sufficiently long, the increased shear stress acting on the coronary endothelium at the beginning of the hyperaemia can enhance the maximum value of the vasodilatation. Such an effect is impaired by NO-inhibition. Since in the goat the time to the peak of the coronary reactive hyperaemia is much longer than in the dog (10-14 s vs 3-4 s), the present study aimed at investigating whether the same effect caused by the NO-inhibition on the maximum flow of the acetylcholine and myogenic hyperaemic responses in the dog, can also be obtained in the goat for the peak flow of the coronary reactive hyperaemia. Experiments performed in anaesthetised goats showed that NO-inhibition reduces the duration of the reactive hyperaemia without affecting the maximum hyperaemic flow. It is suggested that in the reactive hyperaemia the large predominance of metabolic factors prevents the shear stress from playing a role in enhancing the peak flow.