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. 2020 Jan-Dec:29:963689720946277.
doi: 10.1177/0963689720946277.

Integrins α4β1 and αVβ3 are Reduced in Endothelial Progenitor Cells from Diabetic Dyslipidemic Mice and May Represent New Targets for Therapy in Aortic Valve Disease

Alexandru Filippi  1 , Alina Constantin  1 , Nicoleta Alexandru  1 , Geanina Voicu  1 , Cristina Ana Constantinescu  1 , Daniela Rebleanu  1 , Madalina Fenyo  1 , Dan Simionescu  2 , Agneta Simionescu  1   2 , Ileana Manduteanu  1 , Adriana Georgescu  1
Affiliations

Integrins α4β1 and αVβ3 are Reduced in Endothelial Progenitor Cells from Diabetic Dyslipidemic Mice and May Represent New Targets for Therapy in Aortic Valve Disease

Alexandru Filippi et al. Cell Transplant. 2020 Jan-Dec.

Abstract

Diabetes reduces the number and induces dysfunction in circulating endothelial progenitor cells (EPCs) by mechanisms that are still uncovered. This study aims to evaluate the number, viability, phenotype, and function of EPCs in dyslipidemic mice with early diabetes mellitus and EPC infiltration in the aortic valve in order to identify possible therapeutic targets in diabetes-associated cardiovascular disease. A streptozotocin-induced diabetic apolipoprotein E knock-out (ApoE-/-) mouse model was used to identify the early and progressive changes, at 4 or 7 days on atherogenic diet after the last streptozotocin or citrate buffer injection. Blood and aortic valves from diabetic or nondiabetic ApoE-/- animals were collected.EPCs were identified as CD34 and vascular endothelial growth factor receptor 2 positive monocytes, and the expression levels of α4β1, αVβ3, αVβ5, β1, αLβ2, α5 integrins, and C-X-C chemokine receptor type 4 chemokine receptor on EPC surface were assessed by flow cytometry. The number of CD34 positive cells in the aortic valve, previously found to be recruited progenitor cells, was measured by fluorescence microscopy. Our results show that aortic valves from mice fed 7 days with atherogenic diet presented a significantly higher number of CD34 positive cells compared with mice fed only 4 days with the same diet, and diabetes reversed this finding. We also show a reduction of circulatory EPC numbers in diabetic mice caused by cell senescence and lower mobilization. Dyslipidemia induced EPC death through apoptosis regardless of the presence of diabetes, as shown by the higher percent of propidium iodide positive cells and higher cleaved caspase-3 levels. EPCs from diabetic mice expressed α4β1 and αVβ3 integrins at a lower level, while the rest of the integrins tested were unaffected by diabetes or diet. In conclusion, reduced EPC number and expression of α4β1 and αVβ3 integrins on EPCs at 4 and 7 days after diabetes induction in atherosclerosis-prone mice have resulted in lower recruitment of EPCs in the aortic valve.

Keywords: EPC; aortic valve; endothelial progenitor cells; integrins; recruitment.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
EPC phenotype and viability. (A) The vast majority of EPCs identified as CD34+/VEGFR+ cells were also CD133+ corresponding to the early EPC phenotype normally found in circulation. (B). Seven-day long atherogenic diet and not diabetes-induced EPCs cell death as measured by propidium iodide staining. EPC: endothelial progenitor cell; VEGFR: vascular endothelial growth factor receptor.
Figure 2.
Figure 2.
EPC numbers and alterations. EPCs from atherogenic diet-fed animals suffered apoptosis, as shown by the elevated cleaved caspase-3 levels in STZ7 and CIT7 groups (A). However, circulatory EPC counts were reduced in diabetic animal groups (STZ4 and STZ7) compared with control groups (CIT4 and CIT7) (B) as diabetic EPCs show lower but not significantly modified levels of the chemotaxis-mediating CXCR4 (C) and the TRF2 levels seemed to be higher in EPCs from diabetic animals, but no statistical significance was noted among groups (D) (*P < 0.05, **P < 0.01 in one-way ANOVA and # P < 0.05 in two-way ANOVA). ANOVA: analysis of variance; CIT: citrate buffer; CXCR4,: C-X-C chemokine receptor type 4; EPC: endothelial progenitor cell; STZ: streptozotocin.
Figure 3.
Figure 3.
Relative expression levels of integrins. Diabetes reduced the levels of α4β1 (A) and αVβ3 (D) integrins and had no significant effect on the relative expression level of αLβ2 (B) and αVβ5 (C) dimers and β1 (E) and α5 (F) monomers (two-way analysis of variance, ### P < 0.001, ## P < 0.01).
Figure 4.
Figure 4.
EPC recruitment in the aortic valve. Bright-field image with whole aortic valve section (A). Fluorescence microscopy images from sections incubated with anti-CD34 antibodies (green) and 4′,6-diamidino-2-phenylindole (blue) showing representative microscopic fields for the CIT4 (C), CIT7 (D), STZ4 (E), and STZ7 (F) groups. Arrows show CD34+ cells. Bar graph showing percent CD34-possitive cells for the groups analyzed (B) (one-way analysis of variance, *P < 0.05). CIT: citrate buffer; EPC: endothelial progenitor cell; STZ: streptozotocin.
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