Purpose The incidence of Diabetic retinopathy (DR), a microvascular manifestation of diabetes, is worldwide rapidly rising. Biomarkers of DR are urgently needed, as vision loss can be prevented with early diagnosis and treatment. Here, we introduce a novel biomarker of early diabetic micro-vasculopathy. The dynamics of growth factor expression is of paramount interest. Using our unique molecular imaging approach, we investigate early endothelial changes in retinas of living diabetic animals.

Methods Molecular imaging probes were custom made by covalently conjugating carboxylated fluorescent microspheres (MSs, 2μm) with mouse IgG, anti-VEGFR-2, or VEGF. MSs (6x10⁸) were injected into the tail vein of each animal. MS interaction in the fundus vessels was studied using a scanning laser ophthalmoscope (SLO, HRA2). 30min after MS injection, rats were perfused with PBS and rhodamine-labeled conA. Flatmounts were prepared for ex vivo evaluation of accumulated MS and leukocytes. Western blotting was used to quantify the amount of VEGFR-2 in retinal vessels of normal and diabetic animals. Diabetes was induced by intraperitoneal injection of Streptozotocin in male Long-Evans rats (180-200g).

Results The number of a-VEGFR-2-Ab and VEGF molecules on our imaging probes was quantified by flow-cytometry, to be 27453 and 27094, respectively. Western blot analysis showed significantly higher VEGFR-2 expression in diabetic retinas (P=0.04). Immunohistochemistry showed no VEGFR-2 staining in normal retinal

vessels, while retinal capillaries were distinctively positive for VEGFR-2. In vivo molecular imaging showed significantly higher a-VEGFR-2-Ab-conjugated imaging probes in diabetic retinas (P=0.02),

compared to normal controls. Nearly all VEGFR-2 signal was detected in retinal capillaries.

Conclusions The present work introduces a novel biomarker of early diabetic microvasculopathy. The pattern and dynamic of VEGFR-2 in diabetic retinal capillaries might herald the progression of the diseases into the proliferative stage. Our molecular imaging approach to in vivo VEGFR-2 detection could be further developed to detect subclinical signs of diabetic retinopathy in humans, impacting clinical management of DR.