**3. Microvascular changes**

DR initially is a disorder of retinal capillaries that later propagates to the larger vessels. In the early stages of DR, microvascular lesions are characterized by development of microaneurysms, capillary leakage resulting in intraretinal hemorrhages, hard exudates, retinal edema, and also capillary occlusion resulting in ischemia and cotton wool spots formation. More advanced stages of DR are associated with vascular changes such as vein beading, loop formation, intraretinal microvascular abnormalities (IRMA). DR progression leads to neovascularization, intravitreal hemorrhages, expanding of fibrous tissue, causing retinal traction and detachment. Exudative or ischemic forms of the sight threatening diabetic maculopathy may develop in any stage of DR [16].

Long before any clinically visible alterations occur, histological and pathophysiological changes in the wall of the vessels develop, involving thickening of the basement membrane, loss of pericytes, disturbance of endothelial cell functions. A crucial role in the progress of the disease is played by pericytes, developmentally originating from mesoderm. Pericytes are located along the endothelial cell tube, embracing with their cytoplasmic processes endothelial cells and providing mechanical support for the capillary wall [17, 18]. Pericytes are known as specialized contractile cells and function in the capillaries such as smooth muscle cells in the larger vessels, controlling vascular tone, and perfusion pressure [18, 19]. Pericytes are encased in a basement membrane (BM) that is continued with the endothelial BM (**Figure 1A**). The pericyte-endothelial cell interface is mainly divided by the BM. However, it was demonstrated that pericyte and endothelial cell plasma membranes contact across the BM fenestra [20]. There are different types of contacts described between endothelial cells and pericytes:

*Pathophysiology of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.100588*

#### **Figure 1.**

*Blood-retina barrier. (A) Schematic transverse section of capillary showing the endothelium, basement membrane, pericyte, and gap junction. (B) Schematic drawing of the proteins linked with tight junctions between endothelial cells. JAMs – junction adhesion molecules, ZO-1 – zonula occludens-1. (Modified from Ueno [29]).*

peg-and-socket junctions, adhesion plaques, and gap junctions. In peg-and-socket contacts, cytoplasmic fingers of the pericytes interposed into the deep endothelial cell invaginations and, as assumed, support anchorage [21]. Adhesion plaques at the pericyte and endothelial cell plasma membrane serve as a mechanical binding among two cells, which allows the contraction or relaxation of the pericyte to be conveyed to the endothelial cell and thereby to affect capillary diameter [22]. Gap junctions are supposed to permit a direct connections between the cytoplasm of pericyte and endothelial cell [23]. It was proposed that ionic currents, the passage of small molecules and nucleotides, occur between endothelial cells and pericytes through the gap junctions [23, 24]. Moreover, it was shown that pericytes suppress capillary endothelial cell proliferation when cells are co-cultured in physical contact with each other, probably via gap junctions [25]. Interactions between endothelium and pericytes are also regulated by cell adhesion molecules, produced by both cell types, imbalance of which may cause leakage of the BRB during the early stages of DR [26]. Thereby, pericytes in the capillaries are closely associated with endothelial cells and regulate each other functions. Total cytoplasmic areas of the pericytes enveloping capillary and the cytoplasmic areas of the endothelial cells covering these capillaries comprise an average 1:1 ratio in human, which is much higher than that in other tissues [27, 28]. The cause for this high ratio is the necessity for an exceedingly high barrier function in the retina itself in order to prevent an extra fluid accumulation that could result in vision impairment. It seems that pericyte coverage in capillaries correlates positively with endothelial barrier characteristics in different tissues, and greater pericytes density apparently provides better integrity for the vasculature [27]. The BRB comprises the inner BRB (iBRB) and the outer BRB (oBRB). The iBRB is formed by the continuous lining of endothelial cells, tight junctions (zonula occludens) between adjacent endothelial cells and interconnecting pericytes. Tight junction proteins between apical regions of retinal pigment epithelial cells are structural components of the oBRB [29, 30]. The tight junctions are composed of integral membrane proteins, namely: claudin, occludin, junction adhesion molecules, and a number of accessory proteins such as zonula occludens −1 (ZO-1), ZO-2, ZO-3 (**Figure 1B**) [29, 31]. Pericytes are supposed to maintain the integrity of the iBRB by induction of expression of occludin and other junction proteins [30]. The early feature of DR is loss of pericytes, induced

by high glucose levels that has been shown in a row of experiments. Naruse and colleagues demonstrated that high concentration of glucose inhibited proliferation of retinal capillary pericytes in the culture [32]. In particular, fluctuating glucose levels increased pericyte apoptosis in vitro [33]. Since pericytes are important compound of the capillary wall and maintain a capillary structure, loss of them results in localized outpouching of the microvessel wall. This process is linked with microaneurysms development, which is the earliest clinical sign of DR. Progressive pericyte apoptosis in complex with hypoxia causes dilation of the capillaries, venous caliber abnormalities such as venous beading and venous loops. Microaneurysms and dilated capillaries are usually incompetent and leaky [16]. Pericyte loss is accompanied by dysfunction and apoptosis of endothelial cells as well. Endothelial cells play an important role in the regulation of capillary permeability and tone. These cells are responsible for metabolism of BM, coagulation balance, migration, and adhesion of leucocytes to the vessel wall, production of ET-1 [34]. It was demonstrated in vitro that endothelium in high glucose conditions secreted more BM material such as collagen and fibronectin IV, and overexpression of these products remained detectable even after endothelial cells were returned to normal glucose exposure [35, 36]. Thickening of BM in the early phase of DR may prevent endothelium-pericytes contacts that increases pericyte apoptosis due to deprivation of nourishment, while endothelium, losing control of proliferation from pericyte is involved in the formation of new vessels in later stages of retinopathy. Thickened BM reduces a diameter of affected vessels and facilitates capillary obliteration. Dolgov and colleagues demonstrated weakening of endothelial intercellular gap junctions in the vessels during DR [37]. It was shown an increased apoptosis in cultured endothelial cells exposed by high glucose levels [38]. Furthermore, high glucose affects functions of endothelium indirectly by increased production of vasoactive agents and growth factors in other cells [39]. Thereby, DR progression leads to pericyte and endothelium pronounced disappearance, thickening of BM, and formation of acellular capillaries (tubes formed by basement membrane only), capillary occlusion, and ischemia. Non-perfusion in some capillaries induces hypoxia, dilatation, and increased intracapillary pressure in others. Thereby, loss of pericytes impaired functions and later apoptosis of endothelium resulted in progressive retinal ischemia and BRB breakdown. BRB disintegration may occur at the level of both the iBRB and oBRB, causing accumulation of intraretinal fluid and plasma proteins first of all in the inner and outer plexiform layers of the retina, which is visible ophthalmoscopically as intraretinal hemorrhages, retinal edema, and hard exudates. Fluid accumulation in the macular region can cause a macular edema leading to neuronal distortion and visual impairment [16]. Diabetic macular edema may be focal or diffuse. Focal edema is mainly caused by leakage from microaneurysms, whereas diffuse macular edema is a result of generalized leakage from dilated capillaries throughout the posterior pole, which is coupled with occlusion of capillaries. Diabetic maculopathy can associate with ischemia as well, due to mostly capillary obliteration, which is the main cause of visual impairment in this case. Progressive vessel occlusion increases retinal hypoxia and leads to the formation of significant non-perfusion areas in the retina, cotton wool spots, or soft exudates and intraretinal microvascular abnormalities (IRMA). Cotton wool spots develop in cases of retinal arteriole occlusion and focal ischemia, which courses blockage of axoplasmic current and accumulation of large spheroidal axon swellings ("cystoid bodies") and intra-axonal organelles in the retinal nerve-fiber layer [40]. IRMA is a tortuous collateral vessel located midway between arteries and veins. It is hypothesized that IRMAs are either dilated preexisting capillaries or newly formed vessels developing due to obliteration of capillaries and ischemia. IRMAs practically have no leakage and usually do not cross major large vessels [41]. In response to tissue hypoxia, vascular endothelial growth factor (VEGF) is released and stimulates angiogenesis. New vessels usually emerge

*Pathophysiology of Diabetic Retinopathy DOI: http://dx.doi.org/10.5772/intechopen.100588*

from venous part of the retinal vasculature and grow uncontrolled [42]. If these vessels break the inner limiting membrane, they are defined as retinal neovascularization. New vessels penetrate through the inner limiting membrane, proliferate along the posterior hyaloid. They are fragile and may tear leaking blood into the retina and vitreous. Subsequently, a fibrovascular scar tissue grows from the retinal surface into the vitreous cavity. Fibrous tissue retraction may course tractional retinal detachment and vision loss [43, 44].
