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

Animals

Apolipoprotein E knock-out (ApoE^−/−) mice from the breeding colony of Taconi were bred in our facility at the Institute of Cellular Biology and Pathology (ICBP) “Nicolae Simionescu”, kept under a 12 h light:12 h dark cycle, with food and water ad libitum. All experimental protocols were approved by the Ethics Committee of ICBP “Nicolae Simionescu” and by the national authority in charge, ANSVSA.

Male, 12-weeks-old, ApoE^−/− mice were injected intraperitoneally (i.p.) for five consecutive days with 55 mg/kg of body weight of STZ (Sigma-Aldrich, St. Louis, MO, USA) in citrate buffer (final citrate concentration 20.7 mM), pH 4.5, or with an equivalent volume of citrate buffer (CIT, pH 4.5) as recently described by Tucureanu et al ²¹ . After the last i.p. injection, the diet was switched from standard chow to atherogenic diet (standard chow supplemented with 1% cholesterol and 15% butter) for 4 days (STZ4 and CIT4 groups, 8 and 7 animals, respectively) or 7 days (STZ7 and CIT7 groups, 8 animals each) when the animals were sacrificed. Consequently, four experimental groups of mice were established: STZ4, STZ7, and appropriate controls such as CIT4 and CIT7. After profound surgical anesthesia was induced with a ketamine and xylazine mixture (100 mg/10 mg/kg body weight) via i.p. injection, blood was collected on 5 mM EDTA through a ventricular puncture, mice were perfused with phosphate-buffered saline (PBS; pH 7.2), and the aortic valves were collected from each animal by dissection.

The biochemical and echocardiographic parameters, as well as the aortic valve histology of these animals, were presented at length in our recent article ²¹ . This model of STZ-induced diabetes on a background of diet-induced atherosclerosis recapitulates the major aspects of valve pathology within 7 days of atherogenic diet after the last STZ injection.

Flow Cytometry Analysis of Circulating Endothelial Progenitor Cells

The mononuclear cell fractions were obtained from the whole blood of each mouse by density gradient centrifugation using Histopaque-1077 (density 1.077 g/ml; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. The remaining erythrocytes were lysed by a 5-min incubation with ammonium-chloride-potassium (ACK) lysing buffer (Life Technologies, Waltham, MA, USA), and unspecific binding was blocked by washing with 2% fetal bovine serum (FBS) in PBS. Cells were numbered and aliquoted at 2 × 10⁵/sample. All samples, except the autofluorescence and isotype control samples, were incubated with both anti-CD34-AlexaFluor488 (FAB65181G; R&D Systems, Minneapolis, MN, USA) and anti-VEGFR2 allophycocyanin (APC) (FAB4432A; R&D Systems, MN, USA) antibodies for the identification of EPCs as previously described by Georgescu et al ^{22 –24} . In addition, each sample was also incubated with anti-CD133-phycoerythrin (PE) (BZ-141204; Biolegend, San Diego, CA, USA), anti-CXCR4-PE (FAB21651P-100; R&D Systems, MN, USA), anti-α_Lβ₂ integrin (unconjugated ab13219; Abcam, Cambridge, UK), anti-α₅ integrin-PE (103905; Biolegend, San Diego, CA, USA), anti-β₁-PE (FAB2405P; R&D Systems, MN, USA), anti-α4β1 (unconjugated BZ-103705; Biolegend, San Diego, CA, USA), anti-α_Vβ₃-PE (sc7312; Santa Cruz, Dallas, TX, USA), anti-caspase3 (unconjugated 9664; Cell Signaling Technology, Leiden, The Netherlands), anti-TRF2 (unconjugated MA141001; Thermo Fisher Scientific, Waltham, MA, USA), anti-α_Vβ₅ (unconjugated sc81632; Santa Cruz, Dallas, TX, USA) antibodies, or propidium iodide. Where needed conjugated secondary antibodies such as goat anti-mouse-PE (ab7002; Abcam), goat anti-rat-PE (A10545; Invitrogen Waltham, MA, USA), and goat anti-rabbit-PE (F0110; R&D Systems, MN, USA) were also added after a wash. Samples were measured using a Gallios Beckman Coulter flow cytometer (ex: 488 nm, em: 525 nm BP for Alexa Fluor 488; em: 575 nm BP for PE; and ex: 635 nm, em: 660 nm BP for APC), and data were analyzed using Flowing Software 2 (Turku University, Finland). For the gating strategy see Supplemental Fig. 1.

Immunohistological Examination of Aortic Valve Leaflets

The heart specimens were cryoprotected in solutions containing increasing concentrations of glycerol (5%, 10%, 20%, and 50%), washed in 3% sucrose, snap-frozen in liquid nitrogen, and mounted in OCT compound (NEG-50, Thermo Scientific, Waltham, MA, USA). Serial cryostat sections, 5-µm thick (Leica CM1850, IL, USA), containing the three aortic valvular leaflets were collected on poly-l-lysine-treated slides. The sections were fixed in cold acetone for 20 min at −20 °C, washed, and incubated in 0.1% Sudan Black B in ethanol for 1 min for autofluorescence reduction. After blocking in 3% bovine serum albumin (BSA) for 30 min, the sections were incubated overnight at 4 °C with anti-CD34-AlexaFluor488 (FAB65181G; R&D Systems, MN, USA). The next day, the sections were incubated for 5 min with 0.4 µg/ml 4′,6-diamidino-2-phenylindole (DAPI); coverslips were mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) and after curing the mountant for 24 h at 4 °C, the coverslips were sealed with acrylic nail polish. Bright-field images, as well as DAPI and CD34-AlexaFluor488 fluorescence images, were acquired with a fluorescence microscope (Olympus IX81, Shinjuku, Tokyo, Japan), using the same exposure for all sections. All cells, as indicated by the DAPI-stained nuclei and the CD34 positive cells were counted automatically using an ImageJ 1.48v Macro requiring the user to select the leaflet area from bright-field images. The CD34 positive cells (EPCs) were defined as regions of more than 500 contiguous pixels each having a fluorescence intensity of more than the mean intensity + 2 standard deviations of the pixels’ grayscale intensity in that image.

ApoE^−/− Mice with Diabetes and High-Fat Diet, a Model for Diabetic Dyslipidemia

In this experimental model, mean glycemia values for CIT control groups were 144.3 ± 5.2 mg/dl for CIT4 and 144.3 ± 5.2 mg/dl for CIT7, while for STZ groups, these were 219.1 ± 18.2 mg/dl for STZ4 and 271.8 ± 25.8 mg/dl for STZ7 as previously published ²¹ . Also, the duration of the atherogenic diet of 4 or 7 days significantly interacted with diabetes to increase plasma cholesterol and triglyceride concentration and induce early molecular and functional changes in aortic heart valves ²¹ .

Number and Viability of Circulating Endothelial Progenitor Cells Decline in ApoE^−/− Mice with Diabetes and High-Fat Diet

EPCs phenotype was tested by flow cytometry, where EPCs were identified as CD34/VEGFR double-positive monocyte-derived cells. Another marker for circulatory EPCs (early EPCs) described in the literature is the stem-cell marker CD133 and, as expected, the majority (more than 94% for all groups) of EPCs were also CD133 positive cells (see Fig. 1A). Further, the cell count and viability of EPCs from the atherogenic diet-fed control (CIT4 and CIT7 groups) and diabetic animals (STZ4 and STZ7 groups) were studied. EPCs’ viability, as measured by propidium iodide staining, dropped from 95.4% at 4 days to 66.5% at 7 days in the CIT groups (one-way analysis of variance [ANOVA], Bonferroni posttest, **P < 0.01) and from 87.4% to 64.3% in the STZ groups (one-way ANOVA, Bonferroni posttest, *P < 0.05; Fig. 1B), suggesting that the diet length is the main source of variance. This reduction in viability was potentially caused by apoptosis as shown by the higher cleaved Caspase 3 levels in EPCs from the 7-day groups, regardless of diabetes (two-way ANOVA, ^# P < 0.05; Fig. 2A). However, the cell death was not mirrored by the EPCs’ count, as diabetes alone, with no source of variation from diet’s length, reduced the number of circulatory EPCs to about two-thirds of the ones observed in citrate controls, even at 4 days after the last STZ injection (0.16 and 0.15% EPC cells in CIT groups vs 0.10 and 0.11% in STZ groups at 4 and 7 days, respectively) (two-way ANOVA, ^# P < 0.05, Fig. 2B). This is probably caused by a drop in EPC’s mobilization from bone marrow as suggested by a possible reduction in CXCR4 expression in cells from diabetic animals (our results showed a reduction by 42.1% at 4 days and by 50.8% at 7 days; however, these results were not statistically significant) (two-way ANOVA, P = 0.08; Fig. 2C). We also examined TRF2 levels, as indicative of cell senescence, and not only EPCs in all groups showed a higher level of this marker compared with other monocytes, but EPCs from diabetic animals had double TRF2 levels compared with the nondiabetic controls; however, no statistically significant modifications were seen between the treatment groups (see Fig. 2D).

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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.

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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.

Expression of α4β1 and αVβ3 Integrins on Circulating Endothelial Progenitor Cells Diminishes in ApoE^−/− Mice with Diabetes and High-Fat Diet

In our flow cytometry experiments, the α₄β₁ integrin expression level was significantly decreased in EPCs from diabetic animals (STZ4 and STZ7 groups compared with CIT4 and CIT7 groups) (two-way ANOVA, ^### P < 0.001, Fig. 3A). In EPCs of diabetic origin, we also found a decrease in α_Vβ₃ integrin expression level (two-way ANOVA, ^## P < 0.01, Fig. 3D). Also, by applying flow cytometry analysis, we found no significant changes in the other integrins tested, α₅ and β₁ monomers and α_Vβ₅ and α_Lβ₂ integrins (Fig. 3B, C, E, F).

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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).

Recruitment of CD34 Positive Cells in Aortic Valve Leaflets Decreases in ApoE^−/− Mice with Diabetes and High-Fat Diet

To evaluate whether if the altered integrin expression observed was associated with altered EPC recruitment in the aortic valve (Fig. 4A, C-F), we assayed the CD34 positive cell counts between investigated experimental groups (STZ4, CIT4 and STZ7, CIT7). We observed that the 7-day atherogenic diet led to a higher CD34 positive cell number in the control group (CIT7), while diabetes (STZ7 group) significantly reduced that number to values observed at earlier time points (CIT4 and STZ4 groups; one-way ANOVA, Bonferroni post-test, *P < 0.05; see Fig. 4B).

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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.