**2.3. Time course of hyperglycemic injury**

At cellular level, hyperglycemic damage occurs within a few days and induces compensatory and repair mechanisms that may have consequences in the cell population. Vascular endothelium covers a huge surface in the body and possesses a huge capacity to compensate for any damage that occurs over longer periods, thus changes in vascular function may occur with a delay.

In experimental models, glucose levels are often above 20–30 mmol/L and vascular dysfunction develops over weeks or within a few months [37]. The development of hyperglycemiainduced endothelial cell damage is neither instantaneous *in vitro*, it usually takes a few days of exposure to high glucose levels to induce a significant increase in the mitochondrial membrane potential and oxidant production [35, 38]. Hyperglycemia-induced ROS production induces RNA and DNA damage that may be responsible for the reduced proliferation rate observed in endothelial cells [39]. Reduced proliferation and senescence occur after more than 10 doublings of endothelial cells exposed to 25 mmol/L glucose *in vitro* [25].

On the other hand, diabetic vascular complications occur after years of hyperglycemic exposure and poor glycemic control accelerates the development of the disease [40, 41]. Although complications usually first appear some years after clinical diagnosis, retinopathy and nephropathy were often present (in 10–37% of patients) at the time of clinical diagnosis or within the first year after diagnosis [42]. Glucose levels that induce endothelial damage are moderately elevated in most patients due to improved diabetes care and diabetes selfmanagement education and support (DSME/S) [43, 44].

Endothelial cell senescence and reduced proliferation are the dominant features in diabetes, still pathological proliferation of blood vessels occurs in diabetic retinopathy [45]. This controversy is explained by the fact that progressive retinal angiogenesis is preceded by a series of events that is characterized by reduced cell proliferation and stimulates neovascularization in the retina [45]. Proliferative diabetic retinopathy is not the primary pathogenic response to hyperglycemia but a compensatory response to retinal hypoxia. Diabetic retinopathy starts with the loss of two cell types of the retinal capillaries: the endothelial cells and the vessel supporting pericytes and the earliest pathologic signs are acellular, nonperfused capillary segments in the retina [45]. Pericyte loss may precede the endothelial damage in the retina and it is caused by angiotensin II overexpression induced by oxidative stress in diabetes. However, the increased number of migrating pericytes and loss of pericytes from the straight parts of capillaries may also occur as a result of hypoxia, and thus might be a consequence of prior endothelial damage. On the other hand, the loss of pericytes results in reduced proliferation of stalk endothelial cells leading to fewer phalanx cells and promotes hypoxia in the retina. Hypoxia is the main stimulus of uncontrolled proliferation in diabetic vessels and both angiotensin II and vascular endothelial growth factor (VEGF) are involved in the neovascularization. In the pathological angiogenesis, not only the retinal endothelial cells take part but also the bone marrow-derived progenitor cells that may explain how enhanced proliferation capacity replaces the cell loss at the later stage.
