Establishing a Link between Endothelial Cell Metabolism and Vascular Behaviour in a Type 1 Diabetes Mouse Model

Abstract

Background/aims: Vascular complications contribute significantly to the extensive morbidity and mortality rates observed in people with diabetes. Despite well known that the diabetic kidney and heart exhibit imbalanced angiogenesis, the mechanisms implicated in this angiogenic paradox remain unknown. In this study, we examined the angiogenic and metabolic gene expression profile (GEP) of endothelial cells (ECs) isolated from a mouse model with type1 diabetes mellitus (T1DM).

Methods: ECs were isolated from kidneys and hearts of healthy and streptozocin (STZ)-treated mice. RNA was then extracted for molecular studies. GEP of 84 angiogenic and 84 AMP-activated Protein Kinase (AMPK)-dependent genes were examined by microarrays. Real time PCR confirmed the changes observed in significantly altered genes. Microvessel density (MVD) was analysed by immunohistochemistry, fibrosis was assessed by the Sirius red histological staining and connective tissue growth factor (CTGF) was quantified by ELISA.

Results: The relative percentage of ECs and MVD were increased in the kidneys of T1DM animals whereas the opposite trend was observed in the hearts of diabetic mice. Accordingly, the majority of AMPK-associated genes were upregulated in kidneys and downregulated in hearts of these animals. Angiogenic GEP revealed significant differences in Tgfβ, Notch signaling and Timp2 in both diabetic organs. These findings were in agreement with the angiogenesis histological assays. Fibrosis was augmented in both organs in diabetic as compared to healthy animals.

Conclusion: Altogether, our findings indicate, for the first time, that T1DM heart and kidney ECs present opposite metabolic cues, which are accompanied by distinct angiogenic patterns. These findings enable the development of innovative organ-specific therapeutic strategies targeting diabetic-associated vascular disorders.

Keywords: Carbohydrate and lipid metabolism; Cell sorting; Endothelium metabolism; Genomics; Micro and macrovascular complications.

Fig. 1.

Body weight (A) and glycemia (B) in T1DM (STZ-treated) and healthy (CTR) mice for 10 weeks after STZ administration. Week −1 in glycemia graph refers to glycemia one week before STZ administration. (*p< 0.05 STZ vs Control).

Fig. 2.

(A) Number of CD31-positive cells in kidney and heart assessed by FACS-based ECs isolation from of control and T1DM animals. Values are in percentage of total number of cells. (B) Representative plots showing the discrimination of endothelial cells (CD31+) isolated from the left ventricle and kidneys of control and STZ-induced diabetic mice after exclusion of hematopoietic (CD45+) and erytroid (TER119+) cells. (C) Microvessel density in kidney and heart of STZ-treated and control animals by immunohistocheminstry against CD31. Values correspond to the number of vessels per total tissue area. p value ≤ 0.05. Images magnification x200.

Fig. 3.

The antiangiogenic gene expression profile analysis in heart (A) and kidney (B) of control and T1DM animals. Values represent fold change of STZ-treated vs control animals. Red spots are upregulated genes; Green spots are downregulated genes.

Fig. 4.

Fibrosis analyses in heart and kidney of control and T1DM animals. (A) Sirius red histological staining of STZ-treated and control mice. Graphs illustrate the quantification of red stained area, shown in histological images. Magnification X200. (B) CTGF quantification by ELISA assay in both organs of STZ-treated and control mice.

Fig. 5.

AMPK signaling gene expression profile analysis in kidney (A) and heart (B) of control and T1DM animals. Values represent fold change of STZ-treated vs control animals. Red spots are upregulated genes; Green spots are down regulated genes.