Endothelial progenitor dysfunction in the pathogenesis of diabetic retinopathy: treatment concept to correct diabetes-associated deficits

Abstract

Progressive obliteration of the retinal microvessels is a characteristic of diabetic retinopathy and the resultant retinal ischemia can lead to sight-threatening macular edema, macular ischemia and ultimately preretinal neovascularization. Bone marrow derived endothelial progenitor cells (EPCs) play a critical role in vascular maintenance and repair. There is still great debate about the most appropriate markers that define an EPC. EPCs can be isolated using cell sorting by surface phenotype selection or in vitro cell culture. For freshly isolated cells, EPC cell sorting is heavily dependent on the surface markers used; EPCs can also be isolated by in vitro propagation of heterogeneous mixtures of cells in culture using adhesion to specific substrates and cell growth characteristics. in vitro isolation enables consistent reproducibility and using this approach at least two distinct types of EPCs with different angiogenic properties have been identified from adult peripheral and umbilical cord blood; early EPCs (eEPCs) and late outgrowth endothelial progenitor cells (OECs). Emerging studies demonstrate the potential of these cells in revascularization of ischemic/injured retina in animal models of retinal disease. Since ischemic retinopathies are leading causes of blindness, they are a potential disease target for EPC-based therapy. In this chapter, we summarize the current knowledge about EPCs and discuss the possibility of cellular therapy for treatment of diabetic macular ischemia and the vasodegenerative phase of diabetic retinopathy. We also report current pharmacological options that can be utilized to correct diabetes associated defects in EPCs so as to enhance the therapeutic utility of these cells.

Fig. 1

Adult stem cells of the bone marrow. The bone marrow hosts at least two known types of adult stem cells, the mesenchymal stem cells (MSC) and the hematopoietic stem cells (HSC); the most prominent adult stem cell in the bone marrow. The HSC can give rise to the hematopoietic progenitor cells (HPC) which in turn give rise to the lymphoid progenitor cell, the myeloid progenitor cells, and likely the EPC. The precise origin of the EPC is under debate as this cell may directly arise from the HSC or from the HPC. The bone marrow microenvironment is composed of bone marrow stromal cells (which are the source of SDF-1), adipocytes, and cells of the bone matrix, osteoblasts and osteoclasts. The vessels within the bone marrow, composed of pericytes and endothelium, function to provide a barrier between the hematopoietic compartment and the circulatory system. Figure adapted from Domen, et al. [5]

Fig. 2

Mobilization and homing are two closely related processes. Mobilization involves the exodus of HSC/HPC from the bone marrow into the circulation while homing is the “opposite” of this event. HSC mobilize from the endosteal niche, move to the vascular niche, and ultimately into the circulation. This normally occurs when stress induces changes of SDF-1 levels in the bone marrow. The mechanism of stress-induced mobilization as occurs following irradiation or G-CSF-induced mobilization is not fully known, but is, in part, accomplished by the upregulation of proteases such as MMP-2, MMP-9, cathepsin-G and elastase. These proteases cleave niche retention signals like membrane-bound stem cell factor (mSCF), SDF-1, VCAM-1 and osteopontin (Opn). Gradients of fibroblast growth factor 4 (FGF-4) also regulate mobilization. For homing events, key steps are needed. Upon reaching the bone marrow vasculature, SDF-1-stimulated circulating HSC/HPC express integrins such as very late antigen 4 (VLA-4) and hyaluronan binding–cellular adhesion molecule (CD44). These integrins, in turn, interact with vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-and P-selectins expressed on bone marrow endothelial cells which slows down the circulating HSC/HSP in a process known as “rolling.” Following rolling, firm adhesion and subsequent endothelia transmigration into the hematopoietic compartment is mainly accomplished by VLA-4 interactions. Once extravasated the cells, the cells migrate along extravascular hematopoietic cords toward specific niches through as SDF-1 gradient or receding oxygen gradient originating from the supporting osteoblastic or endothelia niches. BMEC: Bone marrow microvascular endothelial cell. Figure adapted from Wilson and Trumpp [10]

Fig. 3

Common methods of precursor isolation. Culture of eEPCs includes a 5-day process wherein non-adherent MNCs give rise to the EPC colony. OECs are derived from adherent MNCs cultured on collagen for 21 days in endothelial growth conditions and demonstrate typical cobblestone morphology

Fig. 4

Morphology of Early and Late Outgrowth EPCs. (A) Typical colony of early EPCs which develop into spindle shaped cells by day 7 (B). Early EPCs do not proliferate readily, which leads to the typical sub-confluent appearance. In contrast, late outgrowth ECs (C) show rapid growth as indicated by the dividing cell (black arrow) and display the typical endothelial cobblestone morphology and proliferate abundantly (D)

Fig. 5

Schematic of the hypothetic diabetic retinopathy progress. In physiological conditions, CD34⁺ EPCs contribute to routine blood vessel maintenance through eNOS activation and NO-mediated stimulation of CD14⁺ EPCs. In diabetes, initially, cytokines like stem cell factor (SCF), monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) released by dysfunctional CD34⁺ EPCs initiate CD14⁺ EPC-mediated aberrant vascular repair resulting in retinal ischemia. This phase is referred to as non proliferative diabetic retinopathy (NPDR). The vasodegenerative phase of diabetic retinopathy associated with reduced reparative function of EPCs evolves in the proliferative diabetic retinopathy (PDR). This phase is characterized by pathological neovascularization seen in the diabetic retina

Fig. 6

Release of EPCs is reduced in a diabetic environment. In a non-diabetic environment ischemic injury results in the release of growth factors at the site of injury which stimulate the release of EPCs from bone marrow. EPCs then migrate to the site of injury and initiate blood vessel repair (angiogenesis). In a diabetic environment ROS, proinflamatory and antiangiogenic factors are increased above non-diabetic levels while NO is reduced. This results in a blunted response to ischemic injury and marginal repair at the site of injury