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BackgroundThe term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs).MethodsHuman blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types.ResultsOur data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs.ConclusionsThis study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

We examined the ability of pyridoxamine (PM), an inhibitor of formation of advanced glycation end products (AGEs) and lipoxidation end products (ALEs), to protect against diabetes-induced retinal vascular lesions. The effects of PM were compared with the antioxidants vitamin E (VE) and R-␣-lipoic acid (LA) in streptozotocin-induced diabetic rats. Animals were given either PM (1 g/l drinking water), VE (2,000 IU/kg diet), or LA (0.05%/kg diet). After 29 weeks of diabetes, retinas were examined for pathogenic changes, alterations in extracellular matrix (ECM) gene expression, and accumulation of the immunoreactive AGE/ALE N -(carboxymethyl)lysine (CML). Acellular capillaries were increased more than threefold, accompanied by significant upregulation of laminin immunoreactivity in the retinal microvasculature. Diabetes also increased mRNA expression for fibronectin (2-fold), collagen IV (1.6-fold), and laminin ␤ chain (2.6-fold) in untreated diabetic rats compared with nondiabetic rats. PM treatment protected against capillary drop-out and limited laminin protein upregulation and ECM mRNA expression and the increase in CML in the retinal vasculature. VE and LA failed to protect against retinal capillary closure and had inconsistent effects on diabetes-related upregulation of ECM mRNAs. These results indicate that the AGE/ALE inhibitor PM protected against a range of pathological changes in the diabetic retina and may be useful for treating diabetic retinopathy.

The essential role of VEGF in blood vessel formation in the embryo and in the adult is well established (1, 2). A single VEGF gene gives rise, by alternative splicing, to multiple isoforms (VEGF 121 , VEGF 165 , and VEGF 189 in humans versus VEGF 120 , VEGF 164 , and VEGF 188 in the mouse) that differ in molecular mass, solubility, and receptor binding (3). VEGF 120 lacks exons 6 and 7, encoding extracellular matrix binding structures, and is therefore the most soluble. VEGF 188 contains all exons and avidly binds to the cell surface and extracellular matrix. VEGF 164 lacks only exon 6 and has intermediate properties. The VEGF isoforms bind to several receptors: VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and neuropilin-1 (NP-1) (3). NP-1 is a semaphorin receptor involved in neuron guidance. As a VEGF 164-specific coreceptor for VEGFR-2, NP-1 also affects angiogenesis (4) and enhances the angiogenic activity of VEGF 164 (5). To define the differential role of the VEGF isoforms in vivo, mice expressing single VEGF isoforms were generated. Impaired myocardial angiogenesis has been demonstrated in VEGF 120/120 mice (expressing VEGF 120) (6). Here we report that loss of VEGF 164 (in VEGF 120/120 and VEGF 188/188 mice) impaired retinal arterial development, whereas loss of VEGF 164 and VEGF 188 (in VEGF 120/120 mice) led to dysregulated vessel outgrowth and patterning in the retina. These observations suggest possible mechanisms for the distinct roles of the VEGF isoforms in retinal vascular patterning and arterial endothelial cell specification. Methods Generation of transgenic mice. Targeted mutagenesis was achieved by homologous and Cre/lox P-mediated sitespecific recombination in embryonic stem (ES) cells. The strategy to generate VEGF 120/120 mice (deletion of exons 6 and 7) has been described previously (6). Targeting vectors used to generate VEGF 164/164 mice and VEGF 188/188 mice were constructed by replacing the genomic sequence with a cDNA containing the fused

Retinopathy is a major complication of diabetes mellitus and this condition remains a leading cause of blindness in the working population of developed countries. As diabetic retinopathy progresses a range of neuroglial and microvascular abnormalities develop although it remains unclear how these pathologies relate to each other and their net contribution to retinal damage. From a haemodynamic perspective, evidence suggests that there is an early reduction in retinal perfusion before the onset of diabetic retinopathy followed by a gradual increase in blood flow as the complication progresses. The functional reduction in retinal blood flow observed during early diabetic retinopathy may be additive or synergistic to proinflammatory changes, leucostasis and vasoocclusion and thus be intimately linked to the progressive ischaemic hypoxia and increased blood flow associated with later stages of the disease. In the current review a unifying framework is presented that explains how arteriolar dysfunction and haemodynamic changes may contribute to late stage microvascular pathology and vision loss in human diabetic retinopathy.